CA2395654A1 - Nucleic acids, proteins, and antibodies - Google Patents
Nucleic acids, proteins, and antibodies Download PDFInfo
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- CA2395654A1 CA2395654A1 CA002395654A CA2395654A CA2395654A1 CA 2395654 A1 CA2395654 A1 CA 2395654A1 CA 002395654 A CA002395654 A CA 002395654A CA 2395654 A CA2395654 A CA 2395654A CA 2395654 A1 CA2395654 A1 CA 2395654A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antogonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
Description
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
~~ TTENANT LES PAGES 1 A 340 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
Nucleic Acids, Proteins, and Antibodies [1] This application refers to a "Sequence Listing" that is provided only on electronic media in computer readable form pursuant to Administrative Instructions Section 801(a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and Achninistrative Instructions Sections 801 to 806, and is hereby incorporated in its entirety.
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
~~ TTENANT LES PAGES 1 A 340 NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:
Nucleic Acids, Proteins, and Antibodies [1] This application refers to a "Sequence Listing" that is provided only on electronic media in computer readable form pursuant to Administrative Instructions Section 801(a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and Achninistrative Instructions Sections 801 to 806, and is hereby incorporated in its entirety.
[2] The Sequence ~L,isting is provided as an electronic file (PJZ05 seqList.txt, 825,391 bytes in size, created on January 13, 2001) on four identical compact discs (CD-R), labeled "COPY 1," "COPY 2," "COPY 3," and "CRF." The Sequence Listing complies with Annex C of the Administrative Instructions, and may be viewed, for example, on an IBM-PC
machine rumzing the MS-Windows operating system by using the V viewer software, version 2000 (see World Wide Web URL: http:l/www.fileviewer.com).
Field of the Iyavehtion [3] The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic '1 methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention.
The present invention further relates to methods andlor compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. .
Background of the Invention [4] The immune system is an intricate network of cells, tissues and soluble molecules that function to protect the body from invasion by foreign substances and pathogens. The major cells of the immune system are lymphocytes, including B cells and T
cells, and myeloid cells, including, basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages and dendritic cells. In addition to these cellular components of the immune system, soluble molecules- such as antibodies, complement proteins, and cytokines- circulate in lymph and blood plasma, and play important roles in immunity.
machine rumzing the MS-Windows operating system by using the V viewer software, version 2000 (see World Wide Web URL: http:l/www.fileviewer.com).
Field of the Iyavehtion [3] The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic '1 methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention.
The present invention further relates to methods andlor compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. .
Background of the Invention [4] The immune system is an intricate network of cells, tissues and soluble molecules that function to protect the body from invasion by foreign substances and pathogens. The major cells of the immune system are lymphocytes, including B cells and T
cells, and myeloid cells, including, basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages and dendritic cells. In addition to these cellular components of the immune system, soluble molecules- such as antibodies, complement proteins, and cytokines- circulate in lymph and blood plasma, and play important roles in immunity.
[5] The immune system can be subdivided into the acquired and innate immune systems. The cells of the innate immune system (e.g., neutrophils, eosinophils, basophils, mast cells) are not antigen specific and their action is notenhanced by repeated exposure to the same antigen. The cells of the acquired immune system (B and T cells) are antigen specific. Repeated exposure of B and T cells to an antigen results in improved immune repsonses (memory responses) produced by these cell types. The cells and products of the acquired immune system can recruit components of the innate system to mount a focused immune response. For a more extensive review of the immune system, see Fundamental Immunolo~y, 4th edition, Ed. William Paul, Lippincott-Raven Pub. (1995). ~ a [6] An immune response is seldom carried out by a single cell type, but rather requires the coordinated efforts of several cell types. In order to coordinate an immune response, it is.
necessary that cells of the immune system communicate with each other and with other cells of the body. Communication between cells may be made by cell-cell contact, ~
between membrane bound molecules on each cell, or by the interaction of soluble components of the immune system with cellular receptors. Signaling between cell types may have one or more of a variety of consequences, including activation, proliferation, differentiation, and apoptosis. Activation and differentiation of immune cells may result in the expression or secretion of polypeptides, or other molecules, which in turn affect the function of other cells .
andlor molecules of the immune system.
necessary that cells of the immune system communicate with each other and with other cells of the body. Communication between cells may be made by cell-cell contact, ~
between membrane bound molecules on each cell, or by the interaction of soluble components of the immune system with cellular receptors. Signaling between cell types may have one or more of a variety of consequences, including activation, proliferation, differentiation, and apoptosis. Activation and differentiation of immune cells may result in the expression or secretion of polypeptides, or other molecules, which in turn affect the function of other cells .
andlor molecules of the immune system.
[7] Molecules which stimulate or suppress immune system function are known as immunomodulators. These molecules, which include endogenous proteins (e.g., cytokines, cytokine receptors, and intracellular signal transduction molecules), molecules derived from . microorganisms, and synthetic agents, may exert their modulatory effects at one or more stages of the immune response, such as antigen recognition, stimulation of 'cytokine production and release, and/or activation/differentiation of lymphocytes and myeloid cells.
Immunomodulators may enhance (immunoprophylaxis, immunostimulation), restore (immunosubstitution, immunorestoration) or suppress (immunosuppression, immunodeviation) immunological functions or activities.
Immunomodulators may enhance (immunoprophylaxis, immunostimulation), restore (immunosubstitution, immunorestoration) or suppress (immunosuppression, immunodeviation) immunological functions or activities.
[8] Immunomodulatory compounds. have many important applications in clinical practice..' For example, immunosuppressing agents (which attenuate or prevent unwanted immune responses) can be used to prevent tissue're~ection during organ transplantation, to prevent Rh hemolytic disease of the newborn, or to treat autoimmune disorders.
A
mechanism of action common to many immunosuppressants is the inhibition of T
cell activation and/or differentiation. Antilymphocyte antibodies have also been used to attenuate immune system functions. Currently-used immunosuppressive agents can produce a number of side effects which limit their use. Among the' most serious secondary effects include ' kidney and liver toxicity, increased risk of infection, hyperglycemia, neoplasia, and osteoporosis (see, e.g., Freeman, Clin. Biochem. 24(1):9-14 (1991); Mitchison, Dig.
Dis.ll(2):78-101 (1993)). . , [9] Immunostimulants, ~ which enhance the activity of immune cells and molecules, comprise another class of immunomodulatory agents with important clinical applications.
Such applications include, for example, the treatment of immunodeficiency disorders (e.g.
AIDS and severe combined immunodeficierlcy), chronic infectious diseases (e.g.
viral hepatitis, papillomavirus, and herpesvirus), and cancer. An important class of endogenous immunostimulants is the cytokines. These soluble signaling molecules are produced by a number of cell types, and . are critical to the regulation of the immune response.
Immunostimulatory mechanisms can include proliferation, differentiation and/or activation of immune cells or progenitors of immune cells. For example, interleukin-2 (IL-2) binds to IL-2 receptors on T lymphocytes and induces proliferation and differentiation.
Another cytokine, interferon alpha, stimulates the immune system through a variety of mechanisms, including_~
activation of macrophages, T lymphocytes, and natural killer cells. Interferon alpha also induces the expression of antiviral proteins (see Chapter 50, The Pharmacolo icg al Basis of Therapeutics, 9th Edition, Eds. Hardman, Limbird, Molinoff, Ruddon, and Gilman; McGraw Hill (1996)). Limitations of current immunostimulant therapies include anaphylaxis, pulmonary edema, and renal toxicity, to name a few.
A
mechanism of action common to many immunosuppressants is the inhibition of T
cell activation and/or differentiation. Antilymphocyte antibodies have also been used to attenuate immune system functions. Currently-used immunosuppressive agents can produce a number of side effects which limit their use. Among the' most serious secondary effects include ' kidney and liver toxicity, increased risk of infection, hyperglycemia, neoplasia, and osteoporosis (see, e.g., Freeman, Clin. Biochem. 24(1):9-14 (1991); Mitchison, Dig.
Dis.ll(2):78-101 (1993)). . , [9] Immunostimulants, ~ which enhance the activity of immune cells and molecules, comprise another class of immunomodulatory agents with important clinical applications.
Such applications include, for example, the treatment of immunodeficiency disorders (e.g.
AIDS and severe combined immunodeficierlcy), chronic infectious diseases (e.g.
viral hepatitis, papillomavirus, and herpesvirus), and cancer. An important class of endogenous immunostimulants is the cytokines. These soluble signaling molecules are produced by a number of cell types, and . are critical to the regulation of the immune response.
Immunostimulatory mechanisms can include proliferation, differentiation and/or activation of immune cells or progenitors of immune cells. For example, interleukin-2 (IL-2) binds to IL-2 receptors on T lymphocytes and induces proliferation and differentiation.
Another cytokine, interferon alpha, stimulates the immune system through a variety of mechanisms, including_~
activation of macrophages, T lymphocytes, and natural killer cells. Interferon alpha also induces the expression of antiviral proteins (see Chapter 50, The Pharmacolo icg al Basis of Therapeutics, 9th Edition, Eds. Hardman, Limbird, Molinoff, Ruddon, and Gilman; McGraw Hill (1996)). Limitations of current immunostimulant therapies include anaphylaxis, pulmonary edema, and renal toxicity, to name a few.
[10] The discovery of new human immunomodulatory ' polynucleotides, the polypeptides encoded by them, and antibodies that immunospecifically bind these polypeptides, satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, prevention and/or prognosis of disorders of the immune system, including, but not limited to, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura and multiple sclerosis), immunodeficiencies (e.g., X-linked agammaglobulinemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, and ataxia telangiectasia), chronic infections (e.g., HIV, viral hepatitis, and herpesvirus), and neoplastic disorders.
Additionally, immunomodulatory molecules would be useful as agents to boost immune responsiveness to pathogens or to suppress immune reactions, for example as is necessary in conjunction with organ transplantation.
Summary of the Invention [1l] The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention.
The present invention further relates to methods and/or compositions for inhibiting. or enhancing the production and function of the polypeptides of the present invention.
Detailed Description . . Tables [12] Table 1A summarizes some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifier (SEQ ID
NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, "Clone ID
NO:Z", for a cDNA clone related to each contig sequence disclosed in Table.lA. The third column provides a unique contig identifier, "Contig ID:" for each of the contig sequences disclosed in Table 1A. The fourth column provides the sequence identifier, "SEQ ID
NO:X", for each of the contig sequences disclosed in Table 1A. , The fifth column, "ORF (From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineate the preferred open reading frame (ORF~ that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1A
as SEQ ID.
NO:Y (column 6). Column 7 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y).
Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); ~ specifically, the Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE
(Wisconsin Package v10.0, Genetics Computer~Group (GCG), Madison, Wisc.). This method returns a measure of the probability that a given residue is found on the surface of the protein.
Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 1A as "Predicted Epitopes". In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1A. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8, "Tissue Distribution" shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention. The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed~in the other tissues and/or cell libraries tested. For those identifier codes in which the first two letters are not "AR", the second number in column 8 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO:X) was_identified in the tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are "AR" designate inforrraation generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse transcription. .
After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe , hybridized to each of the gene targets represented on the ,array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after ','[array code]:" represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal andlor diseased tissues) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression. Column 9 provides the chromosomal location of polynucleotides corresponding to SEQ ID ~NO:X.
Chromosomal .
location was determined by fording exact 'matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database.
Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Merldelian Inheritance in Man (Online Mendelian Inheritance in .Man, OMIMTM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins. University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location ~of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM
identification number is disclosed in column 10 labeled "OMIM Disease References)". A key to the OMIM reference identification numbers is provided in Table 5.
[13] Table 1B summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic .-...--.-.-._ 6 sequences (SEQ ID NO:B). The first column provides a unique clone identifier, "Clone ID
NO:Z", for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the correspondixig row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" for a fragment of the BAC clone identified in column four of the corresponding row of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
[14] Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, "Clone ID
NO:Z", corresponding to a cDNA clone disclosed in Table 1A. The second column provides the unique contig identifier, "Contig ID:" corresponding to contigs in Table 1A
and allowing for correlation with the information in Table 1A. The third column provides the sequence identifier, "SEQ ID NO:X", for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM") as further described below.
The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, "Score/Percent Identity", provides a quality score or the percent identity, of the hit disclosed in columns five and six.
Columns 8 and 9, "NT From" and "NT To" respectively, delineate the polynucleotides in "SEQ ID
NO:X" that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID
NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
[15] Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, "Clone ID", for a cDNA clone related to contig sequences disclosed in Table 1A.. The second- column provides the sequence identifier, "SEQ ID NO:X", for contig sequences disclosed in Table 1A. The third column provides the unique contig identifier, "Contig ID:", for contigs disclosed in Table 1A. The fourth column provides a unique integer 'a' where 'a' is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth column provides a unique integer 'b' where 'b' is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14.
For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded'from the invention. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequences) having the accession numbers) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC
clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC
clone).
[16]. Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1A, column 8. Column 1 provides the tissue/cell source identifer code disclosed in Table~lA, Column ~8. Columns 2-5 provide a description of the tissue or cell source.
Codes corresponding to diseased tissues are indicated in column 6 with the word "disease". The use of the word "disease" in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the "disease"
designation may still be derived from sources directly or indirectly involved in a disease-state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where - r the tissue/cell source is a library, column 7 identifies the vector used to generate the library.
[17] Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1A, column 10. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance..in Man (Online Mendelian Inheritance in Man, OMIM.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/)..
Column 2 provides diseases associated with the cytologic band disclosed in Table 1A, column 9, as determined using the Morbid Map database.
[18] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present 'application.
[19] Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.
[20] Table 8 provides a physical characterization of clones encompassed by the invention. The first column provides the unique clone identifier, "Clone ID
NO:Z", for certain cDNA clones of the invention, as described in Table 1A. The second column provides the size of the cDNA insert contained in the corresponding cDNA clone.
Definitions [21] The following definitions are provided to facilitate understanding of certain terms used throughout.this specification.
[22] In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated.
polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term "isolated" does not refer to genomic or cDNA
libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or, other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention. .
[23] As used herein, a "polynucleotide" refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid sequence contained in SEQ ID NO:X (as described in~column 3 of Table 1A) or the complement thereof; a cDNA sequence contained in Clone ID NO:Z (as described in column 2 of Table 1A and contained within a library deposited with the ATCC); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B or a fragment or variant thereof; or a nucleotide coding sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA
sequence, including the 5' and 3' untranslated sequences, the coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
[24] In the present invention, "SEQ ID NO:X" was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing --all or most of the sequence for SEQ ID NO:X is deposited at Human Genome Sciences, Inc.
(HGS) in a catalogued and archived library. As shown, for example, in column 2 of Table 1A, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID NO:Z). Each Clone ID is unique to an individual clone and the Clone ID is all the ' information needed to retrieve a given clone from the HGS library.
Furthermore, certain clones disclosed in this application have been deposited with the ATCC on October 5, 2000, having the ATCC designation numbers PTA 2574 and PTA 2575; and on January 5, 2001, having the depositor reference numbers TS-l, TS-2, AC-l, and AC-2. In addition to the individual cDNA clone deposits, most of the cDNA libraries from which the clones were derived were deposited at the American Type~Culture Collection (hereinafter "ATCC").
Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID NO:Z to determine the 'library source by reference to Tables 6 and 7. Table 7 lists the deposited .
cDNA libraries by name and links each library to an ATCC Deposit. Library names contain four characters, for example, "HTWE." The name of ~a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example "HTWEP07". As mentioned below, Table 1A correlates the Clone ID names with SEQ ID NO:X. Thus, starting with an SEQ ID NO:X, one can use Tables 1, 6 and 7 to determine the corresponding Clone ID, which library it came from and Which ATCC deposit the library is contained in.
Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCC is located at 10g01 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC deposits were made pursuant to the terms .
of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
[25] In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000; 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
[26] A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein), the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID NO:Z (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones deposited with the ATCC, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1B or the complement thereof. 't Stringent hybridization conditions" refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisod'ium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ~g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
[27] Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an ove .might incubation at 37 degree C in a solution comprising 6X
SSPE (20X SSPE
= 3M NaCI; 0.2M NaH2P04; 0.02M EDTA, pH 7.4),_ 0.5% SDS, 30% forrnamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE, 0.1%
SDS.
Additionally, immunomodulatory molecules would be useful as agents to boost immune responsiveness to pathogens or to suppress immune reactions, for example as is necessary in conjunction with organ transplantation.
Summary of the Invention [1l] The present invention relates to novel proteins. More specifically, isolated nucleic acid molecules are provided encoding novel polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides and/or polypeptides, and antibodies. The invention further relates to diagnostic and therapeutic methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention.
The present invention further relates to methods and/or compositions for inhibiting. or enhancing the production and function of the polypeptides of the present invention.
Detailed Description . . Tables [12] Table 1A summarizes some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifier (SEQ ID
NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, "Clone ID
NO:Z", for a cDNA clone related to each contig sequence disclosed in Table.lA. The third column provides a unique contig identifier, "Contig ID:" for each of the contig sequences disclosed in Table 1A. The fourth column provides the sequence identifier, "SEQ ID
NO:X", for each of the contig sequences disclosed in Table 1A. , The fifth column, "ORF (From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineate the preferred open reading frame (ORF~ that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1A
as SEQ ID.
NO:Y (column 6). Column 7 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y).
Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); ~ specifically, the Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE
(Wisconsin Package v10.0, Genetics Computer~Group (GCG), Madison, Wisc.). This method returns a measure of the probability that a given residue is found on the surface of the protein.
Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 1A as "Predicted Epitopes". In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1A. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8, "Tissue Distribution" shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention. The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed~in the other tissues and/or cell libraries tested. For those identifier codes in which the first two letters are not "AR", the second number in column 8 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO:X) was_identified in the tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are "AR" designate inforrraation generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse transcription. .
After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe , hybridized to each of the gene targets represented on the ,array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after ','[array code]:" represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal andlor diseased tissues) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression. Column 9 provides the chromosomal location of polynucleotides corresponding to SEQ ID ~NO:X.
Chromosomal .
location was determined by fording exact 'matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database.
Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Merldelian Inheritance in Man (Online Mendelian Inheritance in .Man, OMIMTM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins. University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location ~of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM
identification number is disclosed in column 10 labeled "OMIM Disease References)". A key to the OMIM reference identification numbers is provided in Table 5.
[13] Table 1B summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic .-...--.-.-._ 6 sequences (SEQ ID NO:B). The first column provides a unique clone identifier, "Clone ID
NO:Z", for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the correspondixig row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" for a fragment of the BAC clone identified in column four of the corresponding row of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
[14] Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, "Clone ID
NO:Z", corresponding to a cDNA clone disclosed in Table 1A. The second column provides the unique contig identifier, "Contig ID:" corresponding to contigs in Table 1A
and allowing for correlation with the information in Table 1A. The third column provides the sequence identifier, "SEQ ID NO:X", for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM") as further described below.
The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, "Score/Percent Identity", provides a quality score or the percent identity, of the hit disclosed in columns five and six.
Columns 8 and 9, "NT From" and "NT To" respectively, delineate the polynucleotides in "SEQ ID
NO:X" that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID
NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
[15] Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, "Clone ID", for a cDNA clone related to contig sequences disclosed in Table 1A.. The second- column provides the sequence identifier, "SEQ ID NO:X", for contig sequences disclosed in Table 1A. The third column provides the unique contig identifier, "Contig ID:", for contigs disclosed in Table 1A. The fourth column provides a unique integer 'a' where 'a' is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth column provides a unique integer 'b' where 'b' is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14.
For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded'from the invention. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequences) having the accession numbers) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC
clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC
clone).
[16]. Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1A, column 8. Column 1 provides the tissue/cell source identifer code disclosed in Table~lA, Column ~8. Columns 2-5 provide a description of the tissue or cell source.
Codes corresponding to diseased tissues are indicated in column 6 with the word "disease". The use of the word "disease" in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the "disease"
designation may still be derived from sources directly or indirectly involved in a disease-state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where - r the tissue/cell source is a library, column 7 identifies the vector used to generate the library.
[17] Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1A, column 10. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance..in Man (Online Mendelian Inheritance in Man, OMIM.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/)..
Column 2 provides diseases associated with the cytologic band disclosed in Table 1A, column 9, as determined using the Morbid Map database.
[18] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present 'application.
[19] Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.
[20] Table 8 provides a physical characterization of clones encompassed by the invention. The first column provides the unique clone identifier, "Clone ID
NO:Z", for certain cDNA clones of the invention, as described in Table 1A. The second column provides the size of the cDNA insert contained in the corresponding cDNA clone.
Definitions [21] The following definitions are provided to facilitate understanding of certain terms used throughout.this specification.
[22] In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state. For example, an isolated.
polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term "isolated" does not refer to genomic or cDNA
libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or, other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention. .
[23] As used herein, a "polynucleotide" refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid sequence contained in SEQ ID NO:X (as described in~column 3 of Table 1A) or the complement thereof; a cDNA sequence contained in Clone ID NO:Z (as described in column 2 of Table 1A and contained within a library deposited with the ATCC); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B or a fragment or variant thereof; or a nucleotide coding sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA
sequence, including the 5' and 3' untranslated sequences, the coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
[24] In the present invention, "SEQ ID NO:X" was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing --all or most of the sequence for SEQ ID NO:X is deposited at Human Genome Sciences, Inc.
(HGS) in a catalogued and archived library. As shown, for example, in column 2 of Table 1A, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID NO:Z). Each Clone ID is unique to an individual clone and the Clone ID is all the ' information needed to retrieve a given clone from the HGS library.
Furthermore, certain clones disclosed in this application have been deposited with the ATCC on October 5, 2000, having the ATCC designation numbers PTA 2574 and PTA 2575; and on January 5, 2001, having the depositor reference numbers TS-l, TS-2, AC-l, and AC-2. In addition to the individual cDNA clone deposits, most of the cDNA libraries from which the clones were derived were deposited at the American Type~Culture Collection (hereinafter "ATCC").
Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID NO:Z to determine the 'library source by reference to Tables 6 and 7. Table 7 lists the deposited .
cDNA libraries by name and links each library to an ATCC Deposit. Library names contain four characters, for example, "HTWE." The name of ~a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example "HTWEP07". As mentioned below, Table 1A correlates the Clone ID names with SEQ ID NO:X. Thus, starting with an SEQ ID NO:X, one can use Tables 1, 6 and 7 to determine the corresponding Clone ID, which library it came from and Which ATCC deposit the library is contained in.
Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCC is located at 10g01 University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC deposits were made pursuant to the terms .
of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
[25] In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000; 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
[26] A "polynucleotide" of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein), the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID NO:Z (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones deposited with the ATCC, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1B or the complement thereof. 't Stringent hybridization conditions" refers to an overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x SSC (750 mM NaCI, 75 mM trisod'ium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ~g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
[27] Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an ove .might incubation at 37 degree C in a solution comprising 6X
SSPE (20X SSPE
= 3M NaCI; 0.2M NaH2P04; 0.02M EDTA, pH 7.4),_ 0.5% SDS, 30% forrnamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE, 0.1%
SDS.
In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).
[28] Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
[29] Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the setluence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
[30] The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
[31] The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, ~ i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous-_ _ _ _.
[28] Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
[29] Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the setluence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide," since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
[30] The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
[31] The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, ~ i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous-_ _ _ _.
research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslatiori natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, .ADP-ribosylation, amid~ation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, inethylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND .MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press', New York, pgs. 1-12 (1983); Seifter et al., Meth.
Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[32] "SEQ ID NO:X" refers to a polynucleotide sequence described, for example, in Tables lAor 2, while "SEQ ID NO:Y" refers to a polypeptide.sequence described in column 6 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 4 of Table 1A.
The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. "Clone ID NO:Z" refers to a cDNA clone described in column 2 of Table 1A.
[33] "A polypeptide having functional activity" refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
[34] The polypeptides of the invention can be assayed for functional activity (e.g.
biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay immunomodulatory polypeptides (including fragments and variants) of the invention for activity using assays as described in Examples 21, 22, 23, 29, 32, 33, 35, 40, and 45.
[35] "A polypeptide having biological activity" refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without, dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
[36] Table 1A summarizes some of the polynucleotides encompassed by the invention (including contig sequences (SEQ ID NO:X) and clones (Clone ID NO:Z) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby.
(See, for instance, PROTEINS - STRUCTURE AND .MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press', New York, pgs. 1-12 (1983); Seifter et al., Meth.
Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[32] "SEQ ID NO:X" refers to a polynucleotide sequence described, for example, in Tables lAor 2, while "SEQ ID NO:Y" refers to a polypeptide.sequence described in column 6 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 4 of Table 1A.
The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. "Clone ID NO:Z" refers to a cDNA clone described in column 2 of Table 1A.
[33] "A polypeptide having functional activity" refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
[34] The polypeptides of the invention can be assayed for functional activity (e.g.
biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay immunomodulatory polypeptides (including fragments and variants) of the invention for activity using assays as described in Examples 21, 22, 23, 29, 32, 33, 35, 40, and 45.
[35] "A polypeptide having biological activity" refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without, dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
[36] Table 1A summarizes some of the polynucleotides encompassed by the invention (including contig sequences (SEQ ID NO:X) and clones (Clone ID NO:Z) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby.
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[37] The first column in Table 1A provides the gene number in .the application corresponding to the clone identifier. The second column in Table 1A provides a unique "Clone ID NO:Z" for a cDNA clone related to each contig sequence disclosed in Table 1A.
This clone ID references the cDNA clone which contains at least the 5' most sequence of the assembled contig and at least a portion of SEQ ID NO:X was determined by directly sequencing 'the referenced clone. The reference clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein. ' .
[38] The third column in Table 1A provides a unique "Contig ID" identification for each contig sequence. The fourth column provides the "SEQ ID NO:" identifier for each of the contig polynucleotide sequences disclosed in Table 1A. The fifth column, "ORF (From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence '.'SEQ ID NO:X" that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1A, column 6, as SEQ ID NO:Y.
Where the nucleotide position number "To" is lower than the nucleotide position number "From", the preferred ORF is the reverse complement of the referenced polynucleotide sequence.
[39] The sixth column in Table 1A provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated by "ORF
(From-To)". Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. .
[40] . Column 7 in Table 1A lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y); as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN (Version 3.11 for the Power Macintosh, DNASTAR, Inc., 1228 South Park Street Madison, WI). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted. epitopes as described in Table 1A. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may ~vary_slightly.
[41] Column 8 in Table 1A provides an expression profile and library code:
count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1A, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention.
The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4.
For those identifier codes in which the first two letters are not "AR", the second number in column 8 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the tissue/cell source.
Those tissue/cell source identifier codes in which the first two letters are "AR" designate.
information generated using DNA array technology. Utilizing this-technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse transcription.
After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal' generated from each array was used to normalize gene expression between the different hybridizations. The value presented after "[array code]:" represents the mean of the duplicate values, following background subtraction and probe~normalization. One of skill in the ark could routinely use this information to identify normal and/or diseased tissues) which show a predominant expression pattern of the corresponding polynucle0tide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
[42] .Column 9 in Table 1A provides a chromosomal map location for certain polynucleotides of the invention. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the ~NCBI (National Center for Biotechnology Information) UniGene database. Each sequence in the UniGene database is assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often. available for one or more sequences) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster. ~ , [43] A modified version of the computer program BLASTN (Altshul et al., J.
Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993)) was used to search the UniGene database for EST or cDNA sequences that contain exact or near-exact matches to a polynucleotide sequence of the invention (the 'Query'). A
sequence from the UniGene database (the 'Subject') was said to be an exact match if it contained a segment of 50 nucleotides in length such that 48 of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same UniGene cluster, and mapping data was available for this cluster, it is indicated iri Table 1A under the heading "Cytologic Band". Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.
[44] Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database .of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIMTM (supra). If the putative chromosomal location of a polynucleotide of the invention (Query sequence) was associated with a disease in the Morbid Map database, an OMIM reference identification number was noted in column 10, Table 1A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference identification numbers (column 1), and provides a description of the associated disease in Column 2.
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z NO:X ID: NO:B From-To HMSKFl3 12 708207 AC023891 339 1-32 HMSKF13 12 708207 AC023891 340 . 1-169 HMWDR50 19 667595 AB014080 348 l-276 195-494.
HUFAK35 25 636241 AC018433 353' 1-2744 .~ 1504-1816 . 5670-5784 _ 1856-1951 . 3205-3305 HE8PY29 45 . 887862 AC009948 365 1-439 HLTEH39 116 943641. AC004204 383 1-1285 [45] Table 1B summarizes additional polynucleotides .encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, "Clone ID .
NO:Z", fox a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" for a fragment of the BAC clone identified in column four of the corresponding row of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
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w [46] Table 2 further characterizes certain encoded polypeptides of the invention, by providing the results of comparisons to protein and protein family databases.
The first column provides a unique clone identifier, "Clone ID NO:", corresponding to a cDNA' clone disclosed in Table 1A. The second column provides the unique contig.
identifier, "Contig ID:" which allows correlation with the information in Table 1A. The third column provides the sequence identifier, "SEQ ID NO:", for the contig polynucleotide sequences.
The fourth column provides the 'analysis method by which the homology/identity disclosed in the Table was determined. The"fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five.
Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM"), as described below.
[47] The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Table 1A, column 3 (e.g., SEQ ID NO:X or the 'Query' sequence) was, used to search against the NR database. The computer program BLASTX
was used to compare a 6-frame translation of the Query sequence to the NR
database (for information about the BLASTX algorithm please see Altshul et al., J. Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring 'Subject') is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring 'Subject' is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than 1.0e-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by the number of~Query amino acids in the HSP
and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.
[48] . The PFAM database, PFAM version 2.1, (Sonnhammer et al., Nucl. Acids Res., 26:320-322, 1998)) consists of a series of multiple sequence alignments; one alignment for each protein family. Each .multiple sequence alignment is converted into a probability model called a Hidden Markov Model, or HMM; that represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin et al., Biological sequence analysis: probabilistic models of proteins and r2ucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER
version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1A) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2, and the database accession number of the PFAM hit is provided in column 6.
Column 7 provides the score returned by HMMER version 1.8 for the alignment.
Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.
[49] As mentioned, columns 8 and 9 in Table 2, "NT From" and "NT To", delineate the polynucleotides of "SEQ ID NO:X" that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2.
Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.
[50] The nucleotide sequence SEQ ID, NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X
are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the ~cDNA contained in Clone ID NO:Z.
These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A.
[51] Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. ' The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9%
identical to the actual DNA sequence (for example, one base insertion or deletion in an .
open reading frame of over 1000 bases).
[52] Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA
containing cDNA
Clone ID NO:Z (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and having depositor reference numbers TS-1, TS-2, AC-l, and AC-2;
and/or as set forth, for example, in Table 1A, 6 and 7). The nucleotide sequence of each~deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X.
[S3] The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
RACE Protocol Fog Recovery of Full-Lefzgth Genes [54] , Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M.A.; et al., Proc. Nat'1.
Acad.
Sci. USA, 85:8998-9002_ (1988). A cDNA clone missing either the 5' or 3' end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor., The following briefly describes a modification of this original 5' RACE
procedure. Poly A+ or total RNA is reverse transcribed with Superscript II
(Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA
is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL).
Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI, SaII and CIaI) at the 5' end and a primer containing just these restriction sites. This double-stranded cDNA is PCR
amplified for~40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose geI
and the region of gel containing cDNA products the predicted size of missing protein-coding DNA
is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or. SaII, and ligated to a plasmid such as pBluescript SKII
(Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts.
Correct 5' ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial .
kits are used to amplify and recover 3' ends.
(55] Several quality-controlled kits are commercially available for purchase.
Similar reagents and methods to those above are supplied in kit form from Gibco/BRL
for both 5' and 3' RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure. are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
[56] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library double-stranded DNA: ~ An.. asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
RNA Ligase PYOtocol Fog Gehe~atiug The 5' o~ 3' Eud Seqaceuces To Obtaita Full Leugth Genes [57] Once a gene of interest is identified, several methods are available for the identification of the 5' or 3' portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5' and 3' RACE.
While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5' or 3' end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5' RACE
is available for generating the missing 5' end of a desired full-length gene.
(This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):,1683-1684 (1993)).
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA
presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR. amplify the 5' portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA
oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide.' The first strand synthesis -reaction can then be used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence. of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the relevant gene'.
[58] , The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCC (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and receiving ATCC designation numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth, for example, in Table 1A, Table 6, or Table 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Table 7. These deposits are referred to as "the depos2ts" herein.
The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A (Clone ID NO:Z). A clone which is isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire coding region of a human gene ' or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC Deposits by use of a sequence (or portion thereof) described in, for example Tables lAor 2 by procedures hereinafter further described, and others apparent to those skilled in the art.
[59] Also provided in Table 7 is the name of the vector which contains the cDNA
clone, Each vector is routinely used in the art. The following additional information is provided for convenience. -[60] ~ Vectors Lambda Zap (LT.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988);
Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17.9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037. pBS
contains an ampicillin resistance gene~and pBK contains a neomycin resistance gene.
Phagemid pBS
may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK
may be excised from the Zap Express vector. Both phagemids may be transformed into E.
coli strain XL-1 Blue, also available from Stratagene. ---- _----.-..-.
[61] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3:0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897.
All Sport vectors contain an ampicillin resistance gene and may be transformed into E.
coli strain DHlOB, also available from Life Technologies. See, for instance, Gruber, C.' E., et al., Focus 15:59- (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR°2.1, which'is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, CIarIc, J.
M., Nuc. Acids Res. 16.9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (1991).
[62] The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID NO:Y, and/or the deposited clone (Clone ID NO:Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or. primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of ~genomic material.
[63] Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in Clone ID NO:Z, using information from the sequences disclosed herein or the clones deposited with the ATCC. For .example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
[64] The polypeptides of the invention can be prepared in any suitable manner:
Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
[65] The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often -.
advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple ~histidine residues, or an additional sequence for stability during recombinant production.
[66] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
[67] ~ The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence contained in Clone ID NO:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ
ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B.
Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID,NO:B as defned in column 6 ofTable 1B are also encompassed by the invention. The present invention further encompasses a-polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, andlor the cDNA contained in Clone ID NO:Z. .
[68] Moreover, representative examples of polynucleotides of.the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in Table 1B column 6, or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in Table 1B column 6, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, "or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as B~AC
ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and' have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed' by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[69J Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated _ 192 in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAG ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides~
and polypeptides are also encompassed by the invention.
[70] Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in column 6 of Table 1B
which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X
(see Table 1B, column 2) and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[71j Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1B column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in the same row of Table 1B
column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in the same row of Table 1B column 6, wherein sequentially delineated sequences in the table (i.e.
corresponding to those exons located closest to each other) are directly contiguous in a 5' to 3' orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID
NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[72j In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B, and the polynucleotide sequence of SEQ ID
NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that.bind these polypeptides are also encompassed by the invention.
[73] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ ID NO:X
(e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s). and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID NO:Z. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[74] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1B.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[75] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. 'Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[76] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the, 3' 10 polynucleotides of , one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[77] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides,.nucleic acids, and polypeptides are also encompassed by the invention.
[78] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.
[79] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of _ another sequence in column 6 are directly contiguous. Nucleic acids which hybridize to the complement of these 20~ contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the.
invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[80] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 corresponding to the same Clone ID NO:Z (see Table 1B, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of .
these 20 lower stringency conditions, are also encompassed by the invention.
Polypeptides -encoded by these polynucleotides andlor nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments andwariants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[81] In specific embodiments, polynucleotides of the invention comprise, of alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[82] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B is directly contiguous with the 5'..l0.polynucleotides of the next sequential exon delineated in Table 1B, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides andlor nucleic acids, other polynucleotides~ and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[83] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior ~to conception of the present invention. ~ Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fourth column of Table 1A, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus 15 of SEQ
ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14. More specifically, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides~of the invention do not consist of at least one, two, three, four,. five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including fox example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through.
these accessions are hereby incorporated by reference in their entirety.
SEQ
EST Disclaimer Clone NO: Contig ID
Range NO: Z X ID: of a Accession #'s Range of b HTTEK47 11 5736491 - 396 15 - AA400005, AW205244, AW 134723, 410 and AI689951.
HMSKF13 12 7082071 - 387 15 - AA148393, AC023891, and AC023891.
HLHCT68 I3 7647451 - 425 15 - 891150, H67895, AI205078, 439 Z250I2, C052I2, ' 230134, AA194359, AC010344, AC010344, AC010344, AC008496, and AC008496.
HKAFF33 14 9742001 - 175 15 - AA335761, T58784, AA053162, 189 AA220207, F07425, N34268, AA535485, C16914, F11628, F00945, AA380834, AA321294, AA437332, AA132565, T27675, AA527306, H11697, H63653, W24084, AW374230, AA158146, AL035930, AA369234, AW407037, AA361916, W24054, AA977833, AI910189, AA358563, AA385962, T28025, N42582, AA336109, AA321393, AI910249, AI696864, AA305941, AW179215, H98493, AW385684, AA522541, AW062331, W44848, AA188398, AA535767, AA159383, C18543, AA053691, AA371996, AW384708, AA133506, AW404398, AI910244, AI565209, ' ~ AI114563, AI910095, N24326, AA382286, H95866, H68295, AW361810, M24045, 233459, AJ001977, L38251, M24046, D83957, X99704, ~D50852, D64145, X76189, U41420, 247377, U07230, AF220291, D49820, D83030, D64146, D64151, D64152, D64147,.M24097, L20091, M84386, AF115462, D83031, 283247, X73518, X96582,'Y09156, X82122, X78343, D31817, M28172, M84174, X87841, U06487, LJ06695, Y11843, L54059, M99388, U41386, X67818, X70856, X70857, Y18552, M21963, U60218, M24096, AJ005590, D83956, M28206, M26432, X58536, D64148, 215144, AJ010748, M84173, D50854, AJ001975, AJ001976, AJ001973, AJ001974, AJ272049, M26429, D83029, AF118891, AJ133100, E02883, M24030, AJ238694, AJ133267, AJ251755, AP000508, AF017331, AF015557, AF038574, AF201475, AF144.665, AF102689, U61274, M12679, U06835, M26712, M28160, D64149, U56246, Y10520, D84394, M11799, U50088, AF198649, Y18649, AJ131853, U56260, AF028596, U60324, -M63454, M16272, M26430, U11267, M32320, D49552, AJ010749, U09853, X83394, M16273, M54883, AP000507, E01341, M12967, 539758, X03945, AR008238, AJ245869, U60423, X55711, U38976, U41833, AF215919, D89334, U81012, Y18658, AF042290, AF115461, AF117767, AJ132660, Y11229, AJ133472, AJ249725, AF061862, ' AF205535, AF205539, AF213682,w X83003, --AC004204, U74386, U17572, AF115460, M24036, M24035, M24034, X61710, U04244, U64801, X81363, U88407, M24048, X83005, U74387, A93993, U76397, AF017317, AF065641, AF054010, AF061868, U70581, U90241, U91331, AF102568, AF110264, AF110261, AF098266, AF176080, AF220289, I14657, Y15745, AJ010323, AJ133476, AJ249164, Y14767, Y17065, AF061858, U96787, AF019568, AF015555, AF036553, AF145761, AF145763, AF104219;
AF179632, AF189726, AF108425, U05581, AF017321, X75953, L42280, U05580, M24044, L20088, D85761, Y09058, D50300, M77774, M77778, M77777, D25275, M94052, M94053, M94051, L22028, M24040, M24039, M24032, U04243, L22649, L36318, L36591, U29083, U29480, L17005, U63653, D38526, X97321, X13116, M30679, AC004182, M24038, Y08994, U36492, M28207, L42024, U38975, AJ242661, L42345, X60253, X13115, M28204, M15470, 246810, 249112, 279751, U11261, U11263, U60322, U05582, M24037, X 61706, AF168611, U05579, I14637, I14638, and I14642.
HTLAQ18 15 11931141 - 820 15 - AA442624, AI382107, AW269016, 834 AI286158, AA719238, AI001168, AI280133, AW339870, AA009701, AA932363, AI783808, AA587120, . AI241143, AI266558, AI584159, AI370392, AW161202, AI499325, AI702343, AI494201, AI818728, W19236, AI491904, AI537516, AI916720, AI621341, AW059828, H89138, AA938181, AI357283, AI499581, AI817253, AI174819, AA749425, AI859644, AW020419, AI680369, AW080531, AI539260, AI273856, AI434760, AW083168, AA493923, AI801325, AL047344, AI690620, AI366959;
AW058275, AW022494, AW020288, AI630474, AA769285, AW081110, AA768550, AW022542, AW020144, AA587590, AI634919, AI289791, AI360816, AA837290, AI345396, AI344935, AI656270, AW193299, AW189563, AA420722, AI312210, AI334893, AI611728, AA928539, AI208112, AI343091, AI401697, AI289436, AI865116, AI335476, AI311892, AI582871, AI336503, AI500706, AI345370, AL047152, AA888196, AI828806, AI687568, AL121270, AI538850, AL041772, AI582434, AI336565, AI440511, AI421662, N29277, AI340552, AI345471, AI954721, AI347569, AW191003, AL048499, AI961590, AA555145, AA504514, AL079910, AI050881, AI349279, AI440457, AW020381, AI538885, AI590755, AA65~1924, AL133054, S79832, AF022363, AL110218, AF132676, AF061836, D00174, AF205861, U87620, AF1'14168, Y10823, 577771, AL137292, X00861, A08456, A31057, AF111849, AF026124, I77092, A18777, AF028823, AF177401, . AF085809, AF120268, AR038854, AR050959, D44497, AL122111,.AL117583, M79462, A08910, A08911, A08909, X52128, A08907, A08908, U95739, AC004686, AC007392, AC009233, A70386, AL137523, U49908, . Y18680, I18355, AL133099, I34392, AB022159, AC009501, E08516, AF200416, AF013249, AF036941, A27171, AF146568, AF022857, AF022858, AJ131955, A07588, AR012379, AL137284, AB007812, AL080162, AL080057, AL133010, AC002540, AC007172, AC002287, AC007458, 561953, AF090934, 536676, U76419, AF054988, AR059958, AL117457, AL133014, M64936, AF098162, AC004837, AC018767, AF035161, X72387, AF074604, A65336, E04233, U96683, AL080150, AF100931, A20553, AF082526, AL049300, AF113699, AR029490, D83989, AF111851, AL050277, AL133016, AL110228, Y08616, U95114, U75604, AF201468,108319, AF169154, AF113690, AF110329, AL109672, AL133640, AF126488, AL110158, AL133062, AL133047, AL137662, AL117432, AL133565, E02221, AF108357, E06743, AC005968, AF161699, AF113689, X00474, AL137495, I32738, AL050092, AF124435, E01963, AC004399, AC003032, AC005353, AL096776, AF179633, AC005057, AP000161, AP000020, AL034374, AC005091, AL022147, AC005048, AC002416, 294277, AC004690, AC008067, AC006222, AC008014, AL022170, AC007056, AC005291, AC007298, U66059, AF115410, AR016469, X73361., and AB026995.
HLYAW 16 9765311 - 383 15 - AI124815, AW402317, AW402511, AW405194, F13067, AI826037, AA160317, AW408434, AW403392, AA381090, AA224068, AA381088, AL039796,, AA381030, D82221, AA102697, AA263135, AW402322, AA663896, D82177, AW327936, AA336651, D82189, AA295685, AA401643, H23327, W40489, AA227277, AA151951, AL138056, AW402698, AW402264, U46382, AW401505, AW401719, C18310, T29764, AA344828, AA310808, T52219, AW408440, AW403793, AW402754, D56208, AA159230, T52124, AA295712, AA380938, AA351420, AI359260, AW403872, AA449607, AA121088, 819052, AW402402, AA075796, AA100848, AW239462, 820954, F07582, AW402383, AA348993, AA157351, AA350486, AA173111, AA313847, AW401548, AW405127, AI750810, AA381116, F05537, AA152039, AA299668, AW403070, AW402855, F06315, AW405549, AA352960, AA348075., AW403114, AW408321, AW401393, AW404182, AI365653, AW273557, AA381988, AW385684, AA378689, AW402401;
AW402424, AW405525, AA381052, AA381239, AA160101, AA296474, AA158M09, W44826, AA129140, AA005245, N44225, AA160714, AA488534, F06589, AA055685, AA352502, AW404586, AA081491, AA853431, AA143626, AA159125, AA358919, AA083156, F06198, AI214149, AW403253, AI214126, 854504, AA143468, AA263158, AI696864, AA085102, W52004, T10924, AA916617, AA160070, ' AA102181, AA312514, 830668, AA305941, C16914, U96942, 246808, X75953, X64366, M24038, L42282, L42283, AJ131118, X90391, Y13029, Y09118, U64801, M15470, D50290, D50291, U75533, M19757, M24075, U14943, U11263, X84725, L09736, M84694, M84383, M84384, M24037, E03438, U11261, U17572, L41353, D31816, Y08995, M24035, M95530, X61710, X61706, M19756, X81363, E02885, M58636, L41925, U03698, L42345, M80670, E03437, 215143, Y08994, M68964, M32319, AC004182, U11265, D50300, M28205, E02884, D50299, L04696, U17107, U30904, U80945, U30936, L42281, L41214, AF170577, L40599, M84381, M84385, AJ133267, 222651, L04695, E02248, X61709, D44499, U03027, U03859, U06862, L11603, L19937, U50710, U70528, M84382, L41086, D44501, U90566, L38504, D49824, AF022783, L11666, U04244, AJ250917, U90563, U90564, U90565, M84380, M63454, U11267, X03664, M33573, U31971, M32320, L20090, AJ245869, X13116, M30679, X60253, U09864, D50295-, M28203, AF115461, AF118895, U11262, U11266, L07950, X73578, L19923, U27608, D8~3043, AF115460, L11571, L11570, D50296, AF016641, U36392, L09735, U01848, U35431, D50293, L22027, L33923;
AB008102, 233453, D44500, M86403, D14343, AF026218, D50294, AF118892, D25275, M24039, L32862, AJ249724, ' AF071767, U04243, L22028, AF056981;
U90561, M24045, M94053, Y09157, M32318, X61707, AF130734, M24036, L36591, AJ133471, AF190276, Y17193, X03665, X60255, X60693, X60254, L36318, AF065644, AJ132659, AF118893, AF118894, L42280, L20089, X13115, AF002268, E01341, M12967, M11799, S39758, X03945, Y12378, U90560, U90562, AR008238, U80670, AF028595, D87665, M54883, M24033, U50091, M12678, AJ243433, U93915, U58643, X55711, AF226838, Y18552, AJ007603, D83956, M22794, U58315, L22649, U05581, D85761, M77778, M77777, M77776, M77775, X99734, Y09058, M24032, U42837, D85762, U63653, AF017334, AF18185'7, AF181844, M94052, M94051, M24040, U21053, U29083, U29480, AF118891, X64454, U52169, U36492, L42024, AF208430, AF072765, AF189013, AFI08428, X86704, U05580, U05582, L20086, AF198652, Y11228, X90390, AF105029, AF181846, AJ007605, X99704, Y18288, Y18648, AF115464, 246810, AJ238702, AJ006020, AF023442, AF135550, M77774, A93992, AF096631, D64149, and D64150.
HMCFY36 17 9436381 - 302 15 - AW408434, AW402317, 003945, 316 AW327936, AW405194, AI696864, AW402511, AA263135, T68508, AW402322, AA663896, AA294911, AW405525, AW401505, AA352603, AW401548, AW239462, AA366365, AW402264, AW401719, AA361489, AA158146, AA147151, 018310, . AA488534, AA361477, D82189, AA159413, AA158109, AI124815, AA112349, AA151951, AA160714, AA477506, AW403872, AL039796, AA372227, 019056, AA263158, . AA121088, AA581164, AA385962, AW402402, AA100848, AA335761, AA151891, W24084, F11628, H1I697, 834651, W24054, AA220207, N42582, AA361916, D82221, AW403392, AI359260, T75330, AA160037, D82177, T28025, X94481, L76091, AB030573, X96473, 246808, L20090, E03438, U06697, AJ005590, E02248, M80670, E03437, Y08994, M68964, L41925, M32319, AF004370, L76931, U58110, U31334, Y13029, D50300, D50291, U75533, M19757, U14943, M28205, U03698, M95530, AJ002151, U28759, L41086, U32660, U11263, 215143, M24037, L41214, U16309, D31816, M84380, U15639, M33573, U05581; U16298, Y15840, L31798, X84725, M24035, U70528, L36591, M84382, AF035648, AF035649, D50295, U09864, U29083, U96942, AB030574, U37325, X13116, AJ271160, L22028, L42024, U34810, U83580, M29864, AF118891, U15638, AF035647, E02885, X90391, D50290, M58636, L42282, M24075, L42345, L42146, AB032598, AF118894, AJ223282, U15640, U14756, U11261, U11267, X75953, X64366, Y08995, L11571, M24038, AJ131118, X61710, X61706, U04244, U64801, L42283, M32320, U90560, U05577, X90390, U50091, ' Y15841, AB032097, AB036049, AB036050, X86704, X95410, L76094, U18659, L42280, ~.J74387, U18660, D44499, U11262, U17572, AB032094, AB032095, AB032096, AB032599, AB036051, AJ002676, X94.480, L76088, L76089, L76090, L42506, AF033351, M83193, M83191, AJ238702, L07950, U05575, U03027, U03859; L11570, D25275, M94053, L09735, L22027, M24039, L11603, -- - L19937, X61709, U04243, L36318, -AF016641, L42281, X81363, AJ250917, D49824, M28203, X94574, U74386, AB032933, U05583, D44501, D14343, U11266, M83195, M83194, M83192, M86403, U05582, AB030575, X94482, X99734, U32678, L76093, Y09058, D50293, D50294, D50296, M77774, M77778, M77777, M77776, M94052, M94051, L09736, L11666, M84694, U31971, U50710, U80945, L22649, U29480, U30936, U36392, M19756, M84384, U63653, . AF056981, X60693, U90566, M15470, X13115, M24045, U50086, L42296, Y14205, D44500, D50693, L20088, L20089, U06862, M24036, M24040, M24032, U17107, L38504, L41353, AF170577, M84381, L32862, U90561, AB032093, X87268, L76935, L76934, U18790, L75942, L76932, L76933, L36979, U90562, X60255, M24048, MZ8204, AF118895, AF118892, D84394, U05585, Y09118, U11265, U05578, E02884, Y09157, D50299, L04696, L04695, X61707, U30904, AF115461, L40599, M84383, X60254, and 222651.
HMSPF61 18 12265441 - 644 15 - D80195, D80193, D59927, D51423, 658 D59619, D80210, D81030, D80391, D80240, D80253, T03269, D80227, D80196, D80219, D80043, . D58283, D80188, D80022, D59889, D80366, D51799, D80038, F13647, D59275, 075259, D80045~ 015076, D80378, 014014, D50995, D80134, D81026, D59467, D59787, D80949, D80168, D80212, AA285331, 014227, 014429, D50979, T11417, D80164, D58253, T11051, D80166, D59695, D59859, D80269, D80268, D80064, D59502, D80024, D57483, D59610, AW178893, D80241, D81111, D52291, 014331, AW 177440, D51060, , AW178775, 014389, AW369651, AA305409, AW179328, AW378532, D51079, Ar905856, AW352158, D51097, AW178762, AW360834, AW17750I, D80522, AW17751I, D800I4, D51022, AI557751, AW360811, AW378540, AA305578, AW366296, AW375405, AW378534, AW377671, D80251, AW360817, D80248, AW375406, AW179220, AW179332, AW377672, AW179023, AW178905, AA514188, AW177505, AW360841, AW352I71, AW377676, AW352170, AW177731, AW178907, AW179019, AW179024, AA514186, 005695, D80133, AW178906, AW179020, AW177456, D80258, AW367967, AW179329, AW178980, .
AW177733, AW378528, AW178908, AW179018, AW178774, AW178914, AW378543, AW378525, D80132, D80302, AW178781, AW352174, D80439, AW178911, AW352163, D80247, AA809122, T48593, AL048680, D51103, AA03.3512, D80157, AW367950, AW378533, 006015, D45260, -- - D58246, D59503, AI557774, - AI525923, 003092, H67854, H67866, AW360855, AW378542, AI525917, AI535959, AI525920, AA514184, 014973, AI525227, 014046, 005763, AI525235, A67220, A25909, D34614, D26022, AR025207, Y17188, A62300, X671S5, A62298, X68127, D88547, A85396, AB012117, AR066482, A85477, A78862, A86792, D89785, U87250, A84916, I19525, AJ132110, A44171, X93549, X82626, AR018138, AF13.5125, I18371, S69292, AR064240, AF058696, AR008278, Y12724, AR051191, AB028859, 578798,X89963, AR008430, AR062871,115997, AR037157, A80951, X92518, AR051190, A43601, X60736, A94995, AR008443, A20702, AR062872, AR017907, A43189, AR062873, A43188, A20700, AR023705, A84772, A84776, A84773, A84775, AR067731, AR067732, A58522, A91750, A84774, I50126, I50I32, I50I28, I50133, A4S456, AR016514, Y09669, AR066488, A82595, AR060138, A26615, AR052274, AR060385, A30438, AB002449, AR066487, A43192, A43190, AR038669, 232749, AR066490, D50010, X55486, D88507,118367, AB033111, Y17187, AB012121, M60510, A70867, A63261, AR016691, AR016690, U46128, AR000511, AR008408, AR060133, D13509, A64136, A68321, I14842, AR054175, AB023656, . U87247, AF123263, AR008277, and AR008281.
HMWDR50 19 6675951 - 565 15 = AA005245, AA021087, AA313847, 579 W44826, AI750810, .AA243092, AW408321, AW405I27, AW401393, AAI76I92, AW405549, 017443, AAOS2922, AW403070, W56226, AW402424, AA129140, 827263, AA013055, W60367, AA299668, AA348993, N35264, T52117, AA381239, AA381052, AA296474, AA378689, F05537, AA853431, AW404182, AW401666, AA381988, ' AI214149, AI214126, AA152039, AW404586, AA058454, AA130130, N25137, AA100680, AA092258, AA158109, AA160037, 854504, M21533, AB014080, AC006139, AP000514, M16714, AJ251960, AF117228, L76290, AJ245567, Y18g51, AB012686, AJ271225, AB005048, X64880, AJ223972, AB012685, AF055066, K02883, AB023056, AP000519, M20022, M30681, X00492, M31183, AF101046, 293949, 293776, X55710, AF148862, AJ002533, M32505, X87678, M32506, X87680, L78455, L78934, L79943, AF111103, 255558, 258713, AF111102;
258696; U02976, U02977, U41837, AF004918, X03210, M18964, X03443, X01652, AB014080, AB014080, AC006139, and AC006139.
HSSJM44 20 11751001 - 2355 15 - AA633868, AI953254, AA858030, 2369 AI377035, AA600028, AI948432, AI347033, AI8198S2, AI953812, AI274863, AA913660, AI221391, AA402834, AI682227, AA313382, AI393729, 843673, T07940, T75200, AA402685, 246198, H10173, T34442, AA594521, AW05091~9, T07941, AI150326, AA921803, AA775669, AI301221, AI394223, AI869224, 241820, AW074819, 813072, AI424149, AA357096, AA383450, AI335517, AW302961, AI611494, AI348865, AI224202, AA368045, AW302995, and AI252073.
HPTXN38 21 9741951 - 350 15 - AW327936, AI696864, AW408434, AA263135, AW402317, AW385684, AA970332, AA294911, AW402511, AW239462, C03945, AA527306, AA190717, AA158146, AW405194, T68508, AA366365, AA663896, AW402322, AA112349, AA321294, AA361489, AA385962, AW401505, AA335761, AA352603, F00945, . AW351546, AW402698, AA147151, AA305941, 'AW405525, AA988615, AW401548, H11697, AA243206, AW293543, AW402264, AW401719, AA159413, AA158109, C18310, AI365653, AA527504, AA581164, AA220207, W24084, AA377064, AA372227, D82189, AA304603, AA477506, N42582, 834651, AA337991, AA160714, T10924, H98493, AI124815, AA151951, F11628, W24054, AA367387, AW403,872, AA263158, C04018, AA100848, AA121088, AA361916, T28025, AW407037, AW392650, AA321393, AL035930, AA126083, AI359260, D82221, AW403392, AA160037, L76091, AB030573, D50291, M19757, U14943, AJ223282, U16309, X94481, AB032598, L31798, AB032097, U11263, L20090, M24037, U75533, X96473, U16298, U32660, L76090, D50290, M24075, D50300, AF035647, L76935, AB032599, L76089, U18660, 246808, E03438, U18659, . AB036049, AB036050, X95410, X86704, .
L76931, AB030575, AF004370, AB032095, U06697, AJ005590, U58110, L76934, U31334, Y15841, AB032094, X99734, U11261, M80670, M68964, L41925, M32319, X94482, Y08995, X61706, U28759, AB032096, L76088, X84725, L76095, AF035649, AJ002151, X90391, L42282, Y08994, Y14205, X87268, L76094, X61710,-E02248, E03437, X86703, U18790, X64366, AJ131118, L42283, L22028, L36591, AB030574, AB032093, L76933, L36979, D50693, U70528, M84382, U29083, X75953, AJ002676, X94480, _ AF033351, L76930, U11267, L42280, M94053, U04243, AF035648, M29864, M28205, M32320, L42024, AF056981, U34618, L76932, U29880, U32678, U18789, L42146, L41214, M24035, U90562, U64801, 215143, M58636, U15639, M83191, U90561, U96942, AB036051, L42506, L75942, L20089, U06862, Y13029, M33573, M77774, M77777, M77776, L76096, U90560, M24038, _ - AB032933, AJ238702, L09735, U09864, U74387, L76093, X61709, M84384, M77778, U63653, D14343, X60693, U83580, D50295, U31971, M19756, L41086, M83194, M24048, U50710, AF115461, L09736, L36318, X60255, E02885, AF115460, M83193, X60254, L38504, U05581, U03027, U03859, L11603, L19937, M84694, M84383, Y09058, M94052, M94051, L22649, U29480, M84381, Y09118, AJ250917, U37324, M62852, -U15638, D49824, U90566, L42296, M83195, D83043, AJ251003, AJ006978, X13115, M24045, D25275, M24039, M86403, L20088, D50299, U30936, L42281, M28203, U37323, M14013, U05575, U50091, Y09157, L33923, AB008102, U43337, U17572, M15470, . ' AF118892, U11265, L42144, L40182, X73578, U37325, Y15840, M29865, M24040, - M24032, L04695, U17107, U30904, U03698, M95530, U05582, X81363, D44501, X61707, M32318, A28264, U15640, U04244, U11262, U11266, L20086, AF026218, U18661, L42345, Y14606, L07950, AJ237703, U05587, . L11571, L11570, D50296, L04696, U36392, L40599, AF016641, M77775, M84385, M84380, 233453, D44500, AF118895, and X94574.
HRKPA05 22 9742011 - 136 15 - AA158146, H63653, AA437332, 150 N42582, AA053162, H11697, N34268, AA977833, AA190717, AA053691, AA535767, T58784, AA133506, AA188398, W24054, W24084, ' F07425, AA321294, W44848, and N24326.
HTGFY58 23 9741981 = 289 15 - AW327936, AW408434, AI696864, 303 T68508, AA294911, AW402317, AW402511, AW385684, C03945, AW239462, AW402322, AW405194, AA663896, AW385181, AA243206, AA147151, F00945, AA190717, AA321294, AW402698, AA385962, AA581164, AA159413, AW402264, AW401719, AA335761, AA361477, D82189, AI124815, AA263158, AA367387, H11697, AA361916, 834651, W24084, W24054, N42582, AA151891, F11628, T28025, D82221, AW403392, AI359260, T75330, AB032093, L76933, L36979, X87268, U16309, L31798, U28759, AF035648, AF035649, U34618, U29880, U31334, X96473, AB032598, E02885, M58636, U15640,U90566, L76930, U14756,.U83580, U15638, AF035647, L75942, L76932, L76094, _ U18660, U11265, E02884, L04696, L04695, U309.04, D50295, 215143, U32678, X94481, L76091, X94574, X86703, 222651, U32660, L20089, U06862, L40599, D50299, L42345, AJ002676, X94480, X99734, AF033351, AB030573, L76090, L42506, U15639, M86403, U09864, D50290, L36591, X90391, -L42282, U30936, M24075, M84381, 246808, U96942, L42146, AJ223282, AB036049, AB036050, AB036051, X95410, AB008102, X7S953, D50300, U27I07, X64366, AJ131118, U64801, L42283, U06697, U90564, U18790, AF056981, AF004370, L22028, M24038, U75533, Y08994, M84385, M29864, AJ005590, AB032096, L76088, U11263, L76096, AJ238702, U90561, AJ002151, E02248, M80670, M24037, E03437, M68964, L41925, L42281, M32319, U90563, U90565, M32318, AB032097, D44500, L33923, D25275, U11267, M83192, L20090, U04244, D50293, U507.10, E03438, M24039, M32320, L32862, U74387, AF130734, D44501, U14943, U16298, D87665, M83191, D50291, L11666, U80945, M19757, M84384, Y09157, X61707 D44499, U11266, X99735, M15470, M83193, U90559, L42280, L36318, U03027, U03859, DS0294, D50296, L22027, L11603, L19937, X61709, U36392, M11799, AJ250917, AJ133267, D49824, U18659, U11262, M28203, M83195, M83194, L42145, L42144, L40182, M63454, L07950, L11570, AF016641, Y09058, M94052, M94053, M94051, U042.43, L22649, U29480, L41086, L20088, X61710, U70528, AJ237703, M84382, M84383, U31971, M24040, M24032, AF105226, X55711, U11261, D50710, L09735, L42296, Y08995, X61706, U29083, M19756, M29865, X60254, M59840, U37330, U05575, U50086, U90562, AJ251003, Y15841, AJ271160, AJ006978, X60255, Y14205, Y09118, U90558, L42024, D50693, U05580, X64454, and U21053.
HTXJBO1 24 11245961 - 987 15 - AA988703, AA675914, AA303646, AA675909, AW367626, AW058625, N40329, T54177, AA352261, AA595003, AA565043, AA079556, U95729, AF021222, U95731, AF021221, AF021223, AF021224, X9;1625, U69978, U95730, U95732, U95733, U95734, AF021225, AF021226, AF055384, L14848, AB000878, AB000882, AP000506, U65416, AC006046, AP000507, AJ249394, AC004180, D84394, AF055385, AF055387, X92841, Y16807, Y18113, AF045598, U56944, Y16811, U56947, U56948, AF055388, Y16804, AB015600, U56942, U56943, U56945, Y16803, Y16809, AF097406, U56949, U56955, Y16801, Y16805, Y18111, Y18114, Y18116, U56941, Y16808, Y18112, Y18115, U56946, U56952, U56954, Y16810, AF097403, Y16806, Y18117, Y18118, ,, U56940, U56950, AF097404, U56951, U56953, Y18110, AF045600, AF045597, AF045599, AF055386, AF045601, AF045596, AF045604, AJ242442, AJ242439, Y16802, AJ242438, AF045603, AF055389, AF'045602, AJ242441, AJ242440, AJ242443, AF055390, AB014083,. AC004208, AC004207, AC004170, AF055066, AB023058, AP000521, AP000515, AL022723, AJ242444, AJ242445, AC004200, AP0005.19, AB023056, AB003604, AB003602, AB003603, AB003601, AB003600, AB003599, AB003606, AB003607, AJ251156, AB003605, .
ABb03608, AJ251158, AJ251160, U69624, U69625, U69628, U69623, U69630, U69626, U69629, U69627, L29409, U69631, L2941.1, L29410, L29408, L29406, L29407, U69622, AB003614, AB003609, AB003612, AB003610, AB003611, AB003616, AJ251159, AJ251157, AJ251I61, AF106643, AF085013, AF106637, AF106655, AF085029, AF136159, AF106634, AF106640, AF106649, AF085037, AF011879, AF106646, AF136158, AF106633, AFI06651, AF011839, AF106639, AF106648, AF011836, AF011848, AF079414, AF085020, AF011845, AF106642, AF085024, AFOI1887, AF011842, AF011860, AF136157, AF106632, AFI0664I, AF106635, AF106638, AF011847, AF011853, AF106647, U69965, U69968, U69967, U69977, U69964, U69971, U69966, and U69969.
HUFAK35 25 636241 1 - 209 15 - AA449607, AA351420, AA295712, AW402698, AA401643, AL138056, N44225, H23327, AW385684, D56208, AW402402, AW403793, AW402754, AAISI95I, D82221, AW403114, AW403392, AW393618, AA380938, AW403872, AW405525, AA336651, AA160317, AW401548, AW402264, AW401719, AW401505, D82177, C18310, AW408440, AA310808, AI365653, . AW402322, D82189, AW239462, ' AW405194, AW402511, AW402317, AW408434, AA962801, AL039796, T29764, AA263135, AW327936, AI359260, AA350486, F07582, AA381116, AA075796, AA100680, AA132653~ AA083156, AA348075, AA129140, AW402855, AW402401, AA227277, AAI59230, 819052, AA081491, AA143626, U46382, F06589, AA055685, W52004, 854504, AA005245, AA352534, AA358919, AA970332, F06198, AA295230, AA160070, AF039198, AJ272049, U44064, AJ001977, AJ001975, AJ001976, AJ001973, . - AJ001974, D83957, 222752, AP000508, D84394, AC004204, Y14624, AF223221, AF221124, M17567, M17566, Y16411, AJ133474, AF036554, AF147701, AJ245869, AJ133100, AF105226, M16273, M16272, AF223219, AJ238694, D50710, U90558, .
~
Y15746, Y17064, Y18538, U56246, 8552, . ~ D83956, AJ238702, AF056981, U90561, E01341, M12967, 539758, X03945, AR008238, U90563, U90564, U90565, _ U90566, D50693, AF017330, AF015556, Y18533, AF145466, AJ249163, U56259, Y18660, AF036552, AF062587, AF.015558, U90562, AJ251755, D50709, M54883, M11799, M59865, AF019567, AF145760, AF220290, AF015554, AF104218, AF016304, AF173007, L24491, U90560, U89946, U90179, AP000507, M63454, X00495, D77998, AB005048, AJ223972, L41086, X55711, AJ005199, AF065646, I14616, U31373, AF101046, AC004182, AJ245567, AJ271225, AJ133267, AJ133475, AF105240, L76290, D64150, U89947, U90178, X03664, X03665, M16007, D50853, M84172, M99389, U60321, AF017323, U90559, J03027, 272423, AF042289, A93992, AF181857, AF072765, AF105029, AJ251960, K02883, AF117228, AJ250917, X99704, AJ251541, AJ271626, Y14684, AF002271, AF036556, U38975, AJ242661, D83741, D64149, AC004172, AC004193, U06835, U58643, AF115462, Y10520, 246810, D89333, U93915, AF017328, AF081275, AF017334, AF205536, AF226838, 293949, 293776, 272422, 297370, L20091, D83031, X73518, X78343, X87841, M99388, U06695, X67818, X55710, M15497, AL022723, AJ238151~ AF148863, U38976, L47206, L47231, L76288, L76291, L76289, 296924, 272007, AF076476, 280228, 275172, 249112, 279751, D64146, D64152, D83029, AF115464, L540S9~ U70580, AF096631, Y18648, AJ131852, AJ007603, M22794, U67748, U67330, U55022, AF002273, AF061863, AF072767, AF056483, U52175, U88254, AF108428, AF184216, AF208430, D38526, X97321, 283247, X76I89, X82I2Z, M28172, M24030, Y11843, U41420, L38251, X70856, AF072763, U90244, AF189013, . AF102563, AF170578, U59701, AF055066, I14588, AF002275, AF034409, AF135540, M26712, M24097, AB008136, 280227, ' Y15842, U80670, D49820, AB032095, AP000519, 233459, D64147, D64151, D64148,'AJ007605, U76396, AF054011, U58315, U90240, AF188886, U76394, AC018433, AC004172, AC004182, AC004204, and AC004204.
HWHKC91 26 9741911 - 419 15 - AA663896, AA263135, AL039796, AW401505, C18310, AW401719, AW402264, AW402317, AW402322, AW405194, AI124815, AW402511, AW403872, AW408440, AW402402, AW239462, AW408434, AA401643, AA160317, D82221, AA151951, AA310808, AW403793, D82189, AW402698, AW401548, AW402754, H23327, AA227277, AA100680, AW402383, AA548636, AA121088, AW327936, AI826037, AA224068, AA449607, AW405525, AW403392, AA102697, AA075796, AA158109, AA173111, AW385684, AA132653, AI365653, AW401393, AW408321, AW405127, AI7508I0, AW403114, 820954, D82177, AA160714, AA313847, AI359260, F13067, W52004, T29764, T52124, AA488534, AA381090, AA100848, AW405549, W40489, 819052, AW402424, D56208, AW403070, AA381088, AA058454, AA380938, T72958, AL138056, AA083156, AA005245, AA159413, AA381030, AA127128, AA336651, AA352960, AI214126, AI214149, AA152039, AA295712, N44225, AA351420, T52219, AA350486, AA344828, AA299668;
F07582, AA378689, AA295685, AA381116, AA381239, AA159230, AA853431, AW404182, AW393618, F05537, AA348993, AA157351, AA381988, W44826, AA160037, AA296474, AA348075, AA381052, AA352502, AA962801, AW402855, F06315, U46382, AA160101, AW402401, AA776025, F06589, AA358919, AW273557, AA143652, AA055685, AW404586, AA081491, T10924, AW403253, C19056, F06198, AA143468, AA352603, AA361477, 854504, AA129140, AA085102, AA160070, AA294911, ' AA295230, AA916617, AA143626, AA431065, AA102181, AA319533, 830668, T75330, T71226, AA527306, AW009172, AA372227, C16914, D44499, U03027, U03859, L11603, L19937, Y13029, L22027, D49824, D50294, L11666, U50710, AJ250917, D44500, U06862, D50293, U70528, M84382, L32862, X61709, M28203, D44501, U80945, U11262, U11266, L07950, M24033, L11571, L11570, D50296, AF016641, U36392, AF170577, D50295, L42281, M95530, M84385, U17572, D31816, U03698, M84380, X81363, U11265; E02884, D50299, L04696, U17107, LT30904, U30936, L40599, U04243, M84381, 222651, Y09118, L41214, L04695, M94053, 246808, U01848, U35431, U09864, L22649, M24039, X64454, AJ245869, D85762, M33573, M24036, U21053, Y09058, M24035, L36318, U64801, E03438, M94052, . M94051, L09736, M84694, M84383, U96942, U11261, D50290, 215143, M24075, D85761, M77775, M24032, AF 115461, Y08995, X61710, X61706, D2527S, L4234S, U11267, E02885, M58636, X13116, M30679, M24040, U29480~ U31971, M32320, L41925, Y13567, X91749, M24034, U04245, U21052, U290S7, L41353, M32317, M19756, M84384, X75953, M80670, E0343.7, Y08994, M68964, D50291, U75533, X64366, X90391, M24038, L42283, M32319, M19757, L17005, U88407, U14943, L42024, L36591, AJ131118, L42282, AF022783, M28205, AF189017, X90390, X84725, M63454, X60253, U04244, L09735, L22028, U29083, U11263, M24037, U90563, U90564, U90565, X60255, X60693, M77778, M77777, M77776, U36492, E02248, D50300, X99704, 246810, L42280, D83956, X60254, L19923, U636S3, U56246, Y18552, L33923, Y09157, M32318, X61707, AF118892, AF130734, D83043, M86403, AF118894, AF115460, AB008102, X70856, M77774, M84172, D64150, D64152, L20089, AF118893, X73578, U27608, M28172, M99389, D64151, L41086, M15470, 246809, 215144, D50853, U06695, U41420, L38251, D50854, M84173, M84174, D64146, M24045, M24097, L20088, U90566, 233453, X82I22, . D64148, D83029, AF118896, L38504, L20090, AP000507, X67818, D64149, X83394, AF026218, X77658, 283247, X76189, AJ010748, AF168611, E02883, L24373, U41386, X97321, U05581, AJ133267, X70857, AF115464, Y11843, X73518, X87841, M24030, D49552, AJ010749, ~
' 233459, D64I47, D87665, U44064, D84394, AF118895, M99388, D83031, X78343, D38526, X96582, Y09156, D31817, U098S3, and U06487.
HTSFJ40 27 11728261 - 108015 - W28953, AA313257, AI401170, 1094 H19139, 854508, H10122, H08285, 859784, F08505, AA490183, AI270117, AL046409, AI284640, ' 243765, AI963720, RS2605;
AW088846, AI431303, AI334443, AW 193265, AA682912, ' AW303196, AI305766, AI613280, AW3013S0, AW419262, AA720702, AA470969, AL042853, AW327868, AA610491, AA857486, AL045053, AL044940, AL041690, AW265385, AL138455, AI350211, AL138265, AI623720, AI345654, AI110770, AW072923, AI890348, AL037683, AA468022, AW276827, AI754658, AA581903, AA483223, AW407578, AL046205, AI307608, AW238278, AI281881, AI133164, AL119691, AA101689, AI754336, AL038474, AA877817, AA577906, AL134972, AW270270, AI801482, AA551503, AI355206, AI821271, AI538852, AW274349, AI457397, AA623002, AW410400, AW408717, AA515909, . AI345681, AI345675, AA680243, AA503473, AI732120, AL042420, AA908687, AW438643, AL038705, AI679782, AL048626, AI537506, AW028429, AA522942, AA523837, AI289067, AI559705, AI6I9997, AA584167, AW302903, AI754253, AW021207, AW304584, AI375710, AI064864, AA587256, AW406755, AW268300, AA533333, F08180, AAS31372, AL120687, AW083402, F36273, AW004911, AW274346, AA631507, AW088202, AA846876, AI345518, AI688846, AA507824, AA719292, AL038785, AL119984, AA521399, AI341664, AI254615, AA446657, AI469968, AA601355, AI568678, AW193432, AA526787, AL079645, AA599920, AI312309, AA491831, AI888518, AI962050, AI625244, AI814735, AW338086, AA806796, AA649642, AI708009, AA126450, AA521323, AW021583, AW302013, AI061334, AI696962, AI744995, AA493471, AL042753, AL046457, AW163293, AA523815, AA491284, AA6640I5, AI270559, AA610493, AA846935, AI583283, AA503475, AA469451, AW 162049, H71429, AI471481, AW271904, AI929531, AI339850, AI805363, AA525824, AI192631, AA482711, AA569167, AA613345, AA244357, AIS70261, AI633025, AI305547, ,' , AI345157, AW029038, AA639248, AI358571, AL043009, AI610159, AI053672, AI298710, AI053623, AL048925, AW276435, AI281697, AI890918, AA810269, AA713815, AI732865, AA493708, AL039958, AA584581, AI246119, AA569471, AW062724, AA780515, AA828042, AW 131249, AI249997, AA679514, AL120269, NS3150, AI368745, AF015149, AC005529, AC005902, D83989, AL022323, AL139054, I51997, AC0062$5, AG006120, AC005777, XS4180, U18391, U18394, AC004808, AL020993, AC005257, AC007216, AC006271, AF196779, U18395, AC007541, X55926, U57009, 298745, AC004989, AC004491, Y18000, AL121748, AC004019, AC005037, AF015156, AF015151, AL020995, U18393, AC005632, AC006030, U95742, U18392, AC005242, AC004832, AC002310, AL035697, U02531, AC004963, AC007014, AC007191, US7007, X54175, X54181, AL080242, 286061, AC004519, AL031319, AC006441, AC007052, X54178, U57008, AC007677, AC004650, AC005678, X54179, AC006501, AC016025, AL035659, . AC005244, 269917, AC004686, AC003003, AC004453, AC005019, AF015147, AC005387,. AL122021, AC002347, AC003664, AC005154, X54176, X53550, AL031577, U67801, AC005821, AC000041, X55927, U18398, U57006, AL035420, AL021808, AC005216, AC004816;
AC004884, AL049745, AL022721, AC006101, AF015148, AC005829, AP000049, U02532, AC006006, AL121892, AC005306, ' AL049874, AP000311, AC005291, AC002395;
AC005859, AF027390, AL136295, AL080243, AC004638, AC003962, AC008372, X55931, AC004236, AC007283, AC000052, AC004690, 298742, AL031777, AC004987, AC003075, U18399, AL031257, AC006128, AC007688, AC004751, AL031428, AL031311, AC005527, AC006017, AC006208, AC005899, AC007051, AC004493, AL049712, AP000131, AP000209, AL133485, 249816, AB020859, AP000552, -- - AC002429, AC006539, AL034548, w-- AJ003I47, AC00715I, X54177, X55923, AC007663, 298051, AC005261, AL035668, AC002128, AP000248, AP000095, AL034351, AC005740, AP000555, U62293, U63721, AC005694, U67231, AC002385, U18387, AP000459, AC007488, AC006373, AC006482, AC007043, AP000239, AL049775, AC005247, AC004053, AC007666, AF020503, AC008115, AL022315, AF024533, AC004381, AC005726, AC006211, AP000365, AL008718, AL031587, AL031289, U18396, AL023575, X88791, AC004099, AC004593, AC003957, - , ~ AC000066, AC005081, AF041427, AC005486, AC005699, AL049760, AD000092, AC008168, AL096701, AC005217, AL050097, AL031054, 283840, AL121591, 294279, AC007298, AC002401, U63630, U66059, AP000501, AL078474, AC004948, 299716, AC005913, AL022163, AC009247, AL022400, AL109753, 222650, - AC005971, AC006251, U85195, U91321, AC005763, AC002400, AL022302, AC006549, AP000036, AC002126, AC006213, U67233, AC002300, AC004998, AP000959, AF029308, AC003007, AF067844, AL035072, U91328, AL121653, AJ010770, X55925, AC005102, AL035071, AL023882, U69730, AE000658, L78833, U67832, and AC006040.
HEON059 28 7413611 - 848 15 - W37916, AW369323, AL138197, 862 AA292636, AA789205, AA278688, AI077497, AI670821, AW117287, AA608906, AA382777, AA724269, AA278680, AA827227, AI1.60796, AA600253, AI990171, AA625768, W37874, AA421286, AA292637, AA844108, . AA917528, AW316905, AA768446, and AF117210.
HEMBZ62 29 10787971 - 119615 - AW 138235, AW070706, 813025, . 1210 AA346549, and AA317149.
- HWBA018 30 7511251 - 383 15 - AL359711, AL359711, AL359711, 397 AL109947, AL109947, AL109947, AL136222, and AL 136222. .
HTLEN77 31 11470241 - 119015 - T89857, T89583, AL121270, 1204 AI349772, - AI064830, AL121365, AL047042, AL045500, AL036396, AW117882, AW238730, AL119791, AI687376, AW268253, AI636456, AI349004, AI433976, AI868831, AL036274, AL047763, AI285735, AL038778, AW301409, AL135661, AI43645,6, AL036802, AI475371, AW103371, AI275175, AI909666, AL040243, AW071349, AI433157, AI815383, AI521012, AL036146, AI697137, AI687728, AI349645, AI635461, AI250293, AI702406, AI53871.6, AW195957, AI439087, AW162071, AI678302, AI568870,, AI564719, AL036759, AI620284, AI340582, AL119049, AW169653, AI349933, AI866608, AI445432, AW089572, AI625079, AW2741-92, AI699857, -- -AW071417, AI440426, AI590128, AL046849, AI281779, AI500553, AI863014, AL038605, AI500077, AI857296, AI498579, AI366S49, AI580190, AW074993, AI567351, AL119748, AI439745, AI312152, AI345735, AL04S903, AI349937, AI207510, AI906328, AW068845, AI540832, AI613017, AI249257, AL120854, AI920968, AW 166645, AI343112, AI702433, AI497733, AWI48320, AI800453, AI349614, AI800433, AI679724, AI686926, AW087445, AW080838, AI690751, AW303152, AI873731, AI349256, AI567632, AI758437, AI610307, AI282655, AI631107, AL120736, AI597750, AI907070, AI499463, AI499393, AI818683, AI682743, AI608667, AI673256, AA613907, AI633419, AI866002, AI349598, AI866780, AL048871, AI499131, AI281773, AL040169, AL043326, AI921379, AI440239, AI560012, AA572758, AI628205, AI597918, AA640779, AI690835, AI469532, AI568854, AW 132121, AI568855, AI969601, AI224992, AI609592, AL038779, AI866887, AL036980, AW074869, AI475134, AW268768, AI348897, AI434281, AI682841, AI271786, AI800411, AI680113, AI889203, AI874109, AW301300, AI580984, AI934036, AI671679, AI889839, AI583316, AI251485, AI909662, AW167776, AI753683, AI813914, AI493248, AI969567, AW235035, AI919058, AI281762, AW085799, AI334902, AI149592, AI811863, AI459811, AI862142, AI635942, AI687415, AI818206, AI952114, AL036240, AL047041, AI802542, AI343059, AI687465;
AW026882, AI269696, AI492540, AI570384, AA528822, AL036631, AI345860, AI610645, AA585422, AI696398, AI609580, AL042753, AI539771, AI696846; AI307466, AI366991, AI500659, AA508692, AI445025, AL044207, AI349226, AI687375, AW104724, AI591311, AI624668, AI754897~ AI345744, AI569616, AL121014, AI684265, AI624859, AI612913, AI340519, AI536685, AI446606, AI872711, AA938383, AI318569, AW075351, AI620868, AI907061, AW 118557, AI859733, AI799305, AW302965, AI609331, AW269097, AI309401, AI446628, AW183130, AI432969, AI432229,'AL036247, AI690426, AI679764, AI636445, AW302992, AW002342, AI220734, AI619502, 568736, I48979, AL050393, AF090900, L31396, L31397, 578214, AF090934, A93016, AF125949, AF113013, AF078844, AL080060, Y11587, AF090901, I89947, AF118064, AF118070, AF113694, AL133016, AF113691, AL133640, AF090943, AL117457, AL133606, AF104032, AL117460; AF090903, AL137527, AL110196,.
AR059958, AL050146, AL049938, AL049452, AL110221, AF113690, AF113689, A1242859, AL050149, AL122050, AB019565, AF090896, I89931, AF113676, i142766, X84990, AL050I08, A08916, AFI06862, ALI33075, AL050116, AF113677, AL049314, AL122093, A08913, AF113019, AL049466, AL133557, AL096744, AF017152, AL137283, AL122121, AL080124, AL080137, AL050277, AL133565, E03348, AL137557, AL133093, Y16645, . I48978, AF158248, AFI13699, AL137459, . AR011880, Y11254, X63574, AJ000937, AL133080, U91329, E07361, AFl 11851, AFI7740I, AL049430, ALI22123, X82434, . ~ AF091084, AL050138, AF125948, AF146568, AF097996, AL117394, AL110225, E07108, AF079765, AL049300, AL137550, I49625, AL117583, U00763, AL133560, AF017437, A65341, S61953, A08910, AL117585, X70685, E02349, AJ238278, AL050024, AC007390, AL117435, AL049464, AL049382, A08912, AF067728, AF118094, I33392, A58S24, A58523, AF183393, AL133113, AL137538, U72620, A77033, A77035, 282022, AC002467, E05822, A08909, AL122110, AL137271, AL122098, AF091512, A03736, A12297, AL049283, AF061943, X72889, AL137648, AL137463, U80742, AC006371,103321, AC004686, AF087943, AC005940, Y09972, I09360, AC004987, X96540, I42402, AL080127, X93495, AL110197, X57961, AL096776, U35846, X65873, AC004093, AC006336, AL080159, U95739, U67958, I00734, AL137521, E00617, E00717, E00778, AL022147, AC004690, AF111112, AL110280, X98834, AC002464, AC007172, AJ012755, AL133014, AL133104, I26207, AR013797; U49908, AL133568, AC007298, A93350, E08263, E08264, I1'7767, AL133072, AL022165, L13297, I66342, AL137S23, AL137560, E15569, AR000496, U39656, U58996, AF026816, AL035587, Y14314, AL137533, X83508, AC006944, AL122049, AF153205, AC004200, AF061981, AL034374, 237987, AL133077, AL137476, and AF119337.
HTADZ74 32 11728251 - 128315 - AI735624, AW192167, AI307682, 1297 AF077346, and AC007278.
HELHB88 33 12267041 - 345215 - AI671143, AI580905, AI632369, 3466 AA773823, AW370310, AA773263, AA313515, AA985I66, AL121I53, AI382884, AA181724, AI7S0406, AI469456, AA024936, AA155593, AA024853, AL120063, T78993, T78788, T82245, AA155607, AI750407, AL134372, AA670080, L25219, AA247838, AI525585,.
AF1i4487, AF114488, AF064243, AF064244, U61166, AF127798, AF132672, AF132478, AF132481, AP000311, AP000117, AP000193, AP000049, AP000116, AP000050, AP000048, ' AP000308, AP000115, and AWS94325.
HE9TD31 34 8158451 - 941 15 - AI475682, AI439613, AA815076, 955 AB033082, ~
AF
132480, and AF132479.
HGBDG55 35 8158581 - 522 15 - AA368408~ and AL360268.
HOUHL51 36 l 1264801 - 760 1S - AA431822, AI341790, AW295199, 774 AI656610, AA037543, AW292290, AA431419, AA974280, AA815270, AA037457, AI651702, .
AA583011, AI208605, AI419858, AA620408, AA417333, AA417321, W28051, AA251183, AI917695, W28536, AI557751, D80258, T11417, D80064, D59627, C06015, D81026, C15076, D59467, C14331, D80164, D800I4, D59787, C14227, AI535686, D81030, D59503, F13647, D80166, AI557774, C14389, D51799, AA809122, D59859, D80212, D58283, D59619, D80210, D80240, D51423, D80022, DS0995, D80195, D80253, D58246, D50979, D80391, D59275, D80038, D80043, D80227, D80T88, D59502, D80219, D57483, D59889, D80196, D80269, D80168, D80522, AA305578, AA514184, D80439, C14014, D80268, D80133, D80045, AA305409, D80247, D80366, D51022, D80157, D80251, D51250, H67854, 221582, C03092, H67866, AI024754, AI525920, C14973, C05763, T02974, D59551, AI525227, AI525235, D45273, AI535961, C16955, 233452, A84916, A62298, A62300, A82595, AR018138, AR008278, AJ132110, AR060385, AB028859, AF058696, AB002449, AR008277, AR008281, I14842, AR054175, and I79511.
HEOPP67 37 8276301 - 436 15 - AA641653, 299396, AF181972, 450 and AF181973.
HKAOV71 38 8276791 - 743 ,15 - AF123303, and AF004161.
HFIJC31 39 8281481 - 519 15 - AA885328, N79858, and AI184184.
533 ' HDQID90 40 8319761 - 953 15 - AA767219, AI809238, AI219470, 967 AA767092, AA114887, T71487, AA464762, AA504439, 225261, N87679, D57415, AA278335, AW300598, W46278, AA669095, D56990, . D54675; AI797687, AI948608, AA909071, AW236181, AI718165, AB033082, AF132480, AF132479, andAC002350.
HKACD80 41 12065941 - 193715 - AI191318, AI978812, AA586860, 1951 AA805184;
AI628509, AI582366, W73797, W73745, AI620297, AW083832, AW239293,_ AW386876, AI056600, AI056739, AI362766, AI494212, AI077551, AA935678, AI348675, . AI358232, AA251769, AA968828, AA659758, AI891139, C06060, AA746268, AA251926, AA506524, H58621, D81244, AA291462, H58622, AW193598, 838144, 867182, AI919497, AI250032, AA604444, AI567397, AA905208, AA836253, AA551675, AW087829, AI364618, AI421662, 857498, AW080157, AW166086, AI813914, 859996, AL043152, AI680498, AI469754, AL079794, AI540179, AI554821, AI874261, AL119511, . AL045413, AL119399, AL042382, AI560545~, AI366900, AW262491, AW262042, AI954504, AI089970, AI677646, AI679164, AI,119457, AI868204, AI431909, AI453328, AI538085, AI680221, AI056328, AI536923, AW162194, AI553926, AI567302, AI538908, ~AI525934, AI921~746, AI799199, AI582871, AW129947, AW020381, AI805671, AA69333I, AA514684, AI687134, AI280747, AI184903, AI696714, AI609409, AW089932, AI812107; AL037454, AI564426, AI470293, AW148876, AI783.569, AI648458, AI673278, AI891084, AI865906, AI628015, AL042544, AI818353, AI819663, AW079572, AI887163, AI950688, AI355277, AI801152, AI702527, AL042191, AI203903, AW089006, AI696583, AI536563, AI798373, AA848053, AI633125, AI439745, AI698391, AL037104, AI538850, N25033, AW160905, AI610667, AI520785, AW080346, AI520862, AI921371, AI863382, AI918435, AI475815, AW150778, AI623941, AI690748, AI818562, AI469532, AI249946, AI499890, AW149869, AI961286, AI345688, AI624548, AI524179, AL037081, AI691131, AI581139, AI889213, AI374987, AA808175, AI539260, AI623179, AA832154, AI824557, AW074605, AI890391, AA190341, AI524654, AI587114, AI564290, AW084447, AW104641, AI336575, AI453767, AI886206, AI287326, AW082532, AI499131, AW090102, AA693355, AI498067, AL043168, AI368579, AWI62118, AW152415, AI744243, AA868961, AI826941, AI7984.56, AI627874, AI801592, AI872154, ALI18781, AI249877, AI583578, AI590423, AW 169653, AI922996, AI633061, AI309769, W45039, AI866127, AW088591, AW025279, AI520809, AI887620, AW189332, AI247193, AW118553, AW129616, AW301344, AW160916, AI863002, AI972070, AI612852, AI921479, AI401697, AI890223, AI141400, AL045453, AI245279, AI684021, AI433611, AI345415, AW151785, AI635032, AI690813, AW085639, AI816292, AI610690, AA731241, AW132001, AI359787, AI473451, AI921464, AI9I7963, AW236186, AI244105, AW089844, AI564723, AI927233, AW104724, AA857847, AF086313, U77594, AF090900, AL137530, A21625, Y11587, AF200464, AF111851; AF102578, U02475, AF090934, AF159615, AB025103, AF114784, 213966, AC002471, AC005374, AF076464, AL133084, X79812, I33392, L04859', X97332, AL049314, A77033, A77035, AF141289, AL133047, AF139986, AL080140, AF038847, S36676, U58653, A65340, AF100931, AF069506, AF084644, AF084645, AR068466, I32738, AF061795, AF151685, AL137523, AL133061, AF113019, X80340, AL137271, E01314, U35846, ALI37292, AR022283, AL137284, AL050143, S78453, AF113677, AF026008, AF081197, AF081195, AL050149, A07588, AF182215, AL133640, X84990, AB007812, 554890, AL080124, AL136884, X62580, AL133062, AL080126, AL117435, A58524, A58523, A08907, A32826, A30330, A32827, A30331, AF000167, U70981, AF094480, AL137529, AL137711, AF044323, AFI06657, U72621, AL137294, AL096728, 148978, AR029580, Y10655, AF119336, AL035587, U73682, AF061981, AF019298, AF029750, AF047716, AL133070, AF031147, AF077051, AF167995, I89947, E12580, AL080234, S77771, AL133075, AL137558, AF036941, A12297, A08910, A03736, A08909, AF067728, A91160, X99971, X65873, A91162, AL137550, AL034417, AL049996, A08908, L10353, AF115392, U58996, AL117416, AF017152, 237987, . ~ AL110221, AL122045, AF032666, A18777, ' E00984, I04527, AL122110, U92992, AL133067, AL049382, E12579, AL137459, AL137547, U75604, AR038854, AL117648, X82434, AL 137554, A08912, AF026124, U62807, AL096751, I68732, A08911, AL009179, AF078844, AF14S233, A65341, X60786, AL117587, AF114818, AF079763, . ' AF126488, AJ005870, AL137660, AL137254, X93328, AL137479, X00861, AF169154, . X99257, AF168992, AL133088, Y13350, U90884, AL080110, I80062, AF111849, AL122098, AF000301, AL080162, AL050116, AL137533, AF060555, AL0801S0, 576508, AL133049, AF106862, 297214, X72889, ElSS82, A08913, AJ1319S5, AL122104, 282022, U67082, AL137S38, AL122100, AF090886, AF118558, AF124728, AL137256, AL137534, AF039138, AF039137, AF030513, AR034821, AL137480, AF107018, AR064250, X15132, AL137627, X82397, AC004383, AL137478, AR012379, AL137560, AR059958, M27260, 582852, AL117443, AL050138, AF054831, U49908, AR050959, U75378, A15345, X61970; and AC004594.
HTEED80 42 849075 1 - 129915 - W87655, AA465429, AA203572, 1313 AI479983, AI540082, AA192438, AA836236, AA465358, AA829419, AA507269, AW290970, AI796504, AA904433, AA368409, AA688079, AW189971, AA601527, AA393948, F36549, W87656, AA320120, AI862710, F30605, AA196706, and AJ242978.
HE8TI39 43 12227701 - 280715 - AI681280, AA251688, W60548, 2821 Z4S463, H86769, F07768, H86760, 242543, AW339546, 815292, 244339, H8676S, H86774, H86962, AA091738, 858217, F06562, AW377760, and 821166.
HSDJH12 44 11639161 - 788 15 - AA428452, AA134294, T83462, 802 AI219740, AA010048, AI478566, and AI990289.
HE8PY29 4S 887862 1 - 741 15 - AI271550, AI753504, AA809220, 755 AW081079, W78099, AW386283, AI264068, AI219556, AW082138, AA455733, AI382746, AA548778, AA431230, AF092137, AF100751, AF040252, AC009948, AC009948, and AC009948.
HNTMB90 46 9109341 - 718 15 - 299396, AL038837, AL037051, 732 AL036725, AA631969, AL039074, AW392670, AL036418, AL039085, AL039564, AL036858, AL039156; AL039108, AL038509, AL039109, AL039128, AL036,924, AL037094, AL039659, '- AL038531, AL036196, AL039625, AL039648, - AL045337, AL036767, AL119497, AL037082, AL037526, AL036190, AL038447, AL119483, AL037639, AW372827, AL039678, AW363220, AL039629, AW384394,~
AL1194S7, AL039423, AL036238, AL039150, AL119319, AL040992, AL119324, AL042909, U46350, AL038520, AL119484, AL119391, AL037077, AL119443, AL119522, U46351, AL1193S5, AL119363, AL037726, U46341, U46347, LJ46349; AL134533, AL039410, AL038851, AL119341, AL134531, AL119335, AL119418, AL119396, AL039386, AL036998, AL036733, AL119496, AL037615, AL119439, AL036268, AL037085, AL119401, AL119444, AL037205, AL037027, AL037178, AL0453S3, U46346, AL036679, AL0426I4, AL036765;
AL042965, AL042975, AL134528, AL134538, AL042984, AL036191, AL119399, AL134920, AL042544, U46345, AL036719, AL043003, AL043019, AL042542, AL042551, AL0424S0, AL043029, AL134542, AL134532, AL037021, AL037054, AL036836, AL036158, AL119464, AL036774, AL036886, AL036999, AL036964, AR066494, AR060234~, AR023813, - AR064707, A81671, AR069079, AB026436, AR054110, and AR064706. -HMKCH92 47 9109361 - 789 1 S T78768, 813299, 814933, AB003592, D87248, ACOI8833, AC034I92, AC026206, AC026206, AC022381, and AC022381.
HTELV86 48 11264991 - 1572 15 - AW241703, AA923237, AA382531, 1586.
AA918449, AA738033, AA019310, 014331, AA305409, D80164, D51060, D51799, D80166, DS9619, D80210, D80240, 014014, D81030, D59859, D80212, D59502,'C14389, - D80219, 015076, D51423, D59275;
D80253, D50979, AA514186, D80188, D80439, .
D80022, D80366, D80195, D58283, D80133, D80391, D8024.8, D59787, D59467, D80043, D80227, D81026, D59610, D57483, D80024, AIS57751, D80196, D80269, AA809122, D59889, DS1022, D80522, D80268, D50995, D59927, AA305578, D80038, AA514188, D80193, D80045, D80247, b80241, D80251, AW360811, D80378, AI535686, AW177440, N71180, DS1103, D80302, AW178893, 014407, D80157, AW377671, AW375405, T03269, AW178906, AW366296, DS1759, . f AW360817, 014344, AW179328, AW375406, AA853033, AW378534, AW 179332, AW377672, AW179023, AW178905, AW020592, AW378532, N71199, AW378528, AW 178762, H67854, N81164, 006015, AI345688, AI696583, AW377676, AW352171, AW352170, AW177731, AI272244, AW178907, AW179019, AW179024, T48593, AW020397, AA749449, D51250, N25033, AI918408, D45260, _ 'AI557241, AW360834, AI612885, AW179020, AW178775, AW075382, AW022168, D81111, AW022593, AI525653, AW 177456,, AW 195253, AW
178980, AI915795, AW178914, AW074057, AW178774, AW023351, AW177733, AL120696, AW 178908, AW 179018, AA505147, AW080076, 003092, AW019-988, AI261815, AI280082, D59695, AW 177728, AW022981, AI557238, AW021182, AA099519, D59627, AW020480, AW023863, . AW020328, AW021178, AW020425, AI590043, AW022826, AI345010, AW022299, AW189802, AI538247"
AW021059, AI582912, AA857847, AI282673, AI696340, AI0940.92, AI469764, AI473471, AW021561, AI539260, AW264009, AI698391, AI918554, AI961309, AW378543, AI061180, AW378525, H67866, AW022760, AW352163, D80134, D80949, AI525923, AI289310, AI889147, N49165, AW192300, AI567971, D80132, AI224373, AW021909, AW020406, AW023469, D5I079, AA127461, AI866510, AI547227, AA975952, AW302990, AA868486, AW020710, AW021693, AA641818, AI469516, AW079432, AI866461, ' U35371, X99043, A62298, A84916, AR018138, A82595, A62300, Y11505, U79457, Y17188, 282022, A30438, AB028859, AJ132110, AR008278, Y17187, AF058696, AR060385, AB002449, S68736, I50133, X82626, X67155, A94995, D26022, Y12724, A25909, A91160, A67220, D89785, .
A78862, D34614, AR008443, I50128, I50126, I50132, Y08991, AF022857, AF022858, X68127, AR008277, AR008281, AR066488, AR016514, A45456, D88547, AR060138, A26615, AR052274, X92070, AF074604, AL133558, I48978, S63521, Y09669, A43192, A43190, AR038669, 573498, 583440, U46128, I14842, AR066487, U80919, AR054175, AR016691, AR016690, A51774, _ E12580, AF126247, AF113694, AL122049, ' X59812, AF090901, A08910, A08909, AF118558, AL137641, A08907, A08908, D44497, AF031147, E06788, E06790, . AR068466, E06789, I52013, U53505, AF195092, X98066 AF131773, AF093119, I46765, AF081825, AF137367, A63261, 575997, AF117959, D50010, AL049382, U61971, U61970, AF044323, AR025207, AF180525, AL080126, I48979, AF143957, I25049, AF016271, AR005011, AL137479, AR038854, AL008706, AF097996, AR050959, " " - - X66871, AL080234, AF177401, w~ AL096728, I00734, A31057, 536676, AL133088, AL133081, AL137459, E00617, E00717, E00778, AF139986, AL133049, AF057300, AF057299, AC004470, AL049276, AF211175, AL137259, A17115, A18079, AL079293, , AF078844, AF113019, AF038562, AR005195, I33392, AL110296, 569510, AF040723, Y11435, U92992, AR008408, AL122104, X54971, I25048, AJ005690, M92439, AF192522, AL032822, AL02I393, I29004, X66417, AL137657, I28326, AF051325, E01314, Y14314, AL133665, X97332, AF119336, AF090943, A65340, AL137560, and ALl 17626.
HNTAF23 49 9109471 - 239 15 - AW392670, AL119355, AW372827, 253 U46341, AL119483, AL119319, U46349, AL119457, AL119324, AW384394, AL119497, AW363220, 299396, AL134920, AL134524, AL119484, AL119363, AL119391, U46350, - , U46347, U46351, AL119443, AL119444, AI142137, AL119341, AL134538, AL119439, AL119522, AL119396, U46346, AL043029, AL037205, AL1,34531, AL119335, AL042614, - . AL119399, AI142139, AL119496, AL134533, AL119418, U46345, AL0430I1, AL042984, AL043019, AL042544, AL042896, AL043033, AL042965, AL042975, AL042450, AL042542, AL043003, AL119464, AL042551, AB026436, A81671, AR060234, AR054110, AR066494, AR069079, and AR043113.
HNBUTOi 50 10253751 - 100915 -'1023AA279757, AI632246, AI478566, AA977612, AI219740, AA716656, AA687260, AI801069, AA071046, AI985849, AW370598, AA630617, AW370599, AW370625, AA134295, AW390691, AII43764, D30955, AI990289, T79236, AW370620, AA352142, AA074442, AW071043, and AI744728.
HEMFI21 51 11282661 - 221515 - AI670810, AI720056, AW 195755, AW268679, AI400941, AI867849, AA053882, AI672024, AI880208, AW196438, AI655564, AI682042, AA034417, W27229, AW376127, AA425562, AI972I98, AA883340, AA132258, AI584045, AA770253, AW137059, AA132362, AA132257, AA425357, T62545, AW243732, AA491390, AI915665, AA721474, AA483037, AI269187, AA724043, AA346646, AW390324, N22655, AW377734, AC006042, and AL078581, , HMSOL52 52 9211261 - 129015 - AI911515, AI360955, AW028045, 1304 AI796049, AI609712, AW195544, AI184337, AI470056, AI361065, N34939, AI017177, AI038779, AI440241, AI65145~1, AA789292, AA854683, AI765258, AI702748, and AA384884.
HHGAE47 53 9221941 - 705 15 - AW025529, AW026010, AA657904, AA662803, AA886335, AA158820, AI475932, AW050607, AI885090, AI056120, AI244837, AA485566, AI375435, AA922036, AA878578;
- AA643750, AI056614, AW449834, AW197722, AI393408, AA485405, AI560410, AA161103, AI749095, AI720931, AW058170, AI446208, N57590, AI268967, AI832600, AI913781, AA910277, N52768, AI277003, AI914599, AI192693; N57604, AI673692, AW050712, AA631339, N52783, and AI919380.
HMCGL45 54 922195 1 - 114115 - AW025529, AI475932, AW026010, 1155 .
AA886335, AA662803, AI056120, . AW050607, AI885090, AI375435, AW449834, AA161103, AI244837, AA878578, AA922036, AW19.7722, AI056614, AI393408, AW058170, AA643750, AI560410, AI749095, AI720931, AI446208, N52768, AI913781, AI277003, AI268967, AA910277, AI914599, AI192693, N52783, AW050712, AA485405, AI673692, AA631339, AA657904, AA485566, AA158820, AI619710, AI560351, AI919380, . ' ~ N57590; AI832600, N57604, T25136, ' AW 198090, AI499963, AW023338, AI638644, AI890214, AI538850, N75779, AI633125, AI686817, AI499570, AI500061, AI860027, AI473536, AI925164, AW163834, AI690813, AW 162194, AA641818, AI684244, AI469505, AI376425, AI802542, AI673363, AI670009, AI498067, AW082532, AI433157, .
AI886055, AW080700, AI702073, AI884318, AW 128834, AI633198, AI289310, AI620056, AI590043, AW103928, AI890907, AW152182, AI589428, AI961589, AI679550, AI961414, AI698391, AI479292, AI701097, AI570861, AI147292, AW169604, AL045413, AW081383, AI345688, AI245008, AI539800, AI950729, AI927233, AL079799, AI491775, AI872423, AI538980, AI288050, AI635634, AI440239, AA805434, AW 161579, AW151893, AW148363, AI866465, AI538564, AI571439, AW083374, AL041150, AI973152, AI445611, AL047100, AI538116, AI687362, AI281757, AI241923, AA580663, AI440399, AI095003, AW022808, AI540674, AI954475, AI499890; AI471282, AI648494, AI927755, AI621341, AI445829, AI432030, AI915291, AI932503, AI341690, AI613038, AI866040, AW073865, AI699823, AW190194, AI559619, AL037582, AL037602, AI627988, AI685005, AL039086, AI348901, 841605, AI932794, AI345415, AI636588, AW163554, AI572096, AI612852, AW050998, AI580436, AI627893, AI568138, AA587590, AI869377, AL046466, AL118781, AI819545, AI270183, AI912434, AI567513, AI524179, AA830709, AW051088, I48978, I48979, AL137550, AF061573, A15345, I89947, I68732, A65340, AL117460, A77033, A77035, X63162, AF153205, AR029490, AL023657, D16301, AL117587, U78525, AL050366, E12747, AF067728, AF115392, AF090900, AR038854, A76335, E02349, 223.
AF102578, I33392, A58524, A58523, A08910, A08909, A65341, AJ005690, AL080159, A08908, 282022, A58545, AL137294, AL080074, AL137533, A52563~
AL137479, U37359, X70685, A93350, AF047716, A03736, AL122100, A08913, U49908, AL122110, X82434, AF183393, AF094480, - . ' 237987, AL050393, A08912, Y10080, S54890, A08911, A08907, AL133049, AF113690, AF100931, AL137271, U35846, . AL096744, 213966, Y14314, AR034821, S'76508, AF026816, AL136884, AF215669, AL049347, AR068753,. AL050149, AF177401, AF039138, AF039137, AF146568, AL117435, X72889, X53587, AL050155, AL133112, AL133637, AB031 I47, U55017, X67688, AF090903, AL137480, D83032, AL050277, AF097996, AF200464, AL117457, AL133665, - AL080163, 297214, AL133623, AR020905, -AF017437, AL122049, AF113699, AL110158, AF158248, AL137656, AL133010, A21103, - AF111849, AL110221, AF061981, AL080148, X81464, L04849, A18777, 536676, AL137530,_I25049., E01614, E13364, AF115410, AC002471,'AC005374, S75997, I89931, AF145233, AL049339, A07588, AF087943, U95114, AL080110, AL080139, AL137529, L04504, 577771, AL117392, I49625, AF107847, AL117416, AL137459, Y09972, AF058921, I09499, AL137476, AL137537, AF114170, AL137526, AF090943, AL133558, E05822, I33391, Y11254, AJ000937, AL133080, AL122104, AF141289, . ~ U58996, AL133075, AL050116, S53987, AL137488, U72621, AF030513, AL133113, AL133072, AF032666, AJ012755, X83508, AF061943, A17115, A18079, A08916, AF106945, I89934, AL133067, U83980, Y10823, U90884, AL117585, X66862, X66871, AL050024, S82852, AF17665.1, X84990, I03321, U96683, AL137256, _ AF180525, A12297, AL137521, AF106862, AF057300, AF057299, AL110225, AL110218, AR011880, A18788, AF113694, AB016226, A08456, A86558, A76337, AF118092, E12806, AF026124, AF061795, AF151685, U73682, I32738, AF090901, AL122093, .
AL050092, AL050138, and AL137292.
HEOQN14 55 923752 1 - 103115 - AI014538, AWb06457, AI479414, 1045 AI805243, AI290929, AI129301, AI872459, AI601146, AI708870, AI973043, AI540074, AI186894, AI682389, AI654747, AA460832, AI392777, AA405714, AA649837, AI356090, AI358510, AW294364, AA954900, AA991687, AI540589, AI953865, AA977875, AW19b678, 861326, 854477, AW009738, AA724308, AW297100, 854409, AA627570, AA504833, AA489470, H08185, 808582, AA778454, AI810108, 241744, R43473,.AA765208, AI698394, 239824, H19140, . 241120, F03843, AA701889, AA159318, AW408231, AA404221, H84256, AW131981, AI401170, AA405779, AI475002, F01761, AW189730, H84262, F04422, AA404687, AA502309, AA371650, H29188, AA581151, AA477301, AA749407, AA477302, and AI144326.
HSOBC04 56 9272801 - 120615 - AA115298, AI741325, AI688227, 1220 AI819333, AA452504, -AI925664, AI742595, AI174530, AA115338, AI567500, AA563582, AA461615, AI142563, AA80'7844, N94422, AI095261, AA569395, W58424, AA687480, AA479551, AA582573, AI081428, AA779677, AI280806;
N24393, AA988617, AI863187, AA834079, AW302361, AI362861, AW273442, AA150123, AA553678, AI752480, AI312661, W52661, AI298150, AA463418, W72509, AA024450, W72139, AI037968, W79868, AI028169, AA477651, H39596, W02690, AI198327, AI952450, AA926794, AI087245;
W74236, AW004736, AI334346, AI870989, H98040, AI689546, AI332748, W76066, AA150031, 840403, AI349417, AA595996, W80872, H99144, AW166280, W52767, AA496878, H25985, AI357863, 855375, AA363023, AW104147, AA378409, AI979074, AI376184, AI687489, T32290, N26307, H97338, N95244, W77880, . AI917258, W256b4, AI536791, AA024802;
AA577352, AA328156, AA359865, AA367475, AA461442, AA358275, W80763, T09474, AA987427, AI611160, AA888165, AA595303, AI918172, W30769, AI201782, AA187662, W21074, AA411955, AA935961, AA090719, AA411956, AA451977, AI371307, . ' AW074526, N79974, AI635472, N39751, AI612934, AA478489, AA102215, AI802295, AI750502, AA496836, AL133116, L07063, and AC012192.
HTXKL86 57 12122831 - 148615 - AI014538, AW006457, AI401170, 1500 AI479414, AI805243, AI290929, AI129301, AI872459, AI601146, AI708870, W28953, AI973043, AI540074, AI186894, AI682389, AI810108, AI654747, AA460832, AI392777, AA405714, AA649837, H10122, AI356090, AA313257, AW294364, AI358510, AI540589, AA954900, AA991687, H19139, H08285, AW408231, AI953865, AA371650, 854508, AA977875, AW009738, AW297100, 859784, 854477, AW407594, F07194, AA724308, F08180, _ . F08505, 243765, AA627570, H08185, 852605, AA489470, F07185, AW190678, AW407965, 808582, F05493, AA504833, AA778454, 843473, AI698394, AA765208, 813670, H19140, 245409, 239824, AA461135, 813641, 241120, F03843, AW 131981, AA701889, AA159318, AA404221, H84256, AI475002, AA405779, AW189730, F01761, AA502309, 861326, H84262, AA404687, F04422;
854409, H29188; AA749407, AA581151, AA477301, 241744, AA477302, AI144326, and AW513623.
HUJCT05 58 11254081 - 162515 - AW268357, AI889091, C15588, 1639 AI949350, AI056961, AI124874, 839133, AA887911, AW023386, AC003962, and AF155116.
HHFOC79 60 9354061 - 102415 - AI569931, AA450162, AA405198, 1038 H26214, H14443, N28528, 873380, AW015358, 848456, AI750978, AA358230, 219130, AA359395, AA744173, H26831, and AC008745.
HMTBB 61 11252651 - 576 15 - AA582539, AI963340, AI097093, 17 590 AA286856, AI761614, AI149781, AI460219, AI032670, AI636161, AI819154, AI089302, H12042, AI811219, H05308, T95010, AA836993, AW271462, 837000, AI001803, AA904906, AA743196, AI015200, AA453607, FO5000, AA578803, AI241466~ AI033193, AA330970, F03322, F01968, AA037601, T75492, N47542, AW 183219, AI288171, AA054759, F01965, AA651907, AL119324, AL119457, AL042544, AL119399, AW392670, . AL119484, AL119439, U46347, AL119443, U46351, AL134530, AL134519, AL119391, AL119319, AL119418, AW372827, U46350, AW363220, AW384394, AL119522, 299396, AL119497, AL119363, AL119355, U46349, AL119444, U46341, AL037205, AL119483, AL119396, AL119401, AL119341, AL119464, AL134525, AL043003, U46346, AL119335, AL134528, AL119496, AL134538, U46345, AL043019, AI142132, AL042542, AL042614, AL042450, AL042984, AL042965, AL042975, AL043029, AL042551, AL122084, AL049611, AB026436, AR060234, AR066494, AR054110, A81671, AR043113, AR069079, and AW612030.
HKGDE58 62 11252641 - 133215 - AW271462, AA582539, AI963340, 1346 AI097Q93, AI460219, AA286856, AI761614, AI149781, AI032670, AI819154, AI089302, AI636161, AA448686, H12042, AI811219, AA287162, , AA836993, T95.010, AI001803,.
AA904906, -AA743196, AI015200, 837000, H05308, AA453607, F05000, AA578803, AI241466, AI033193, F03322, AA037601, AA330970, F01968, 813858, H12041, T75492, 246111, F07044, T94956, H05358, N47542, AA053290, AW183219, T75535, AA454139, F05720, AI288171, AA651907, AA054759, F01965, F05717, AL122084, and AL049611.
HCHMW40 63 11295601 - 906 15 - AI732539, AI791495, AI791325, 920 AA709067, AW082062, AI791964, AI732667, AA505923, AW057561, AI909857, AI909862, AA601601, AI909853, AW151270, AI597748, AA938181, .
AA079317, AI017427, AI285826, AI473547, AA689584, AL036241, AI251221,, AI571699, AA836392, AI624279, AI343091, AW403717, AI624304, AI366992, AI310332, AW193299, AI289436, AI499325, AI289791, AA764900, AI590755, AA860080, AI866127, AI860897, - AI744256, AA713657, AI440238, AA427700, AI378123, AW021662, AI619754, AA713781, AW090387, AI702343, AI344819, AF094480, I28326, AF106657, A20553, L12407, AF007142, AF076633, AL133016, AF100781, A26498, A27631, AF090900, AR055519, AF028823, AL137298, A44314, U78525, I29004, X66417, AL080060, A23630, M19658, and E13998.
HE8QZ34 64 10868071 - 107015 - 835313, AA210809, W28575, 1084 AA112126, 886156, AA286753, AW405566, AA385668, AA384297, H25863, H50786, 825032, 246079, AA334931, AA490204, and ' AW367213.
HE8TM80 65 11857701 - 143815 - AW368592, AL040968, AW390796, AW361341, AA125853, AW361336, 856714, AW368596, AA127005, AA436573, .
' AI610191, AL040878, H53723,'AW386767, 872151, AA662674, H06566, AI620201, AW272525, AA428869, AA876119, AA643689, AI810630, T70821, AA126031, AI24~268, AI525912, AI624158, AA715437, AI139291, AA506178, AA366775, AW295598, AA071351, AI635219, AI628041, AA126626, AI223312, AI831145, AW272772, AA223723, AI393334, AA101504, AA125839, AA609710, AA166669, AA969727, AA813942, AA627552, AA588589, ' AA569083, T70552, AI520735, AI654578, AW173734, AW245101, AI128760, AI589990, AA436546, AW377757, AA653217,.AA909871, N25030, H98122, AA706136, AA182573, AI612136, AI612151, AW301938, AW074868, AI308036, AA281089, H84055, H29320, T90829, H06511, AW338571, AW292509, 856868, AI474847, AI682762, AW028456, AA307834, AA969587, 810267, and AF191018.
HHEFL55 66 957539 1 - 127315 - AW411262, AW239479, AA306419, 1287 C75208, .
AA311414, H06122, W38676, AA297279, T50005, AA302953, AW192348, AA334000, AA151050, AW383613, AW383609, AW383535, AA226372, and AA938031.
HWLEY40 67 11720211 - 142215 - AI014538, AW006457, AI479414, 1436 AI805243, AI129301, AI401170, AI290929, AI872459, AI601146, AI708870, W28953, AI973043, AI540074, AI682389, AI810108, AI392777, AI186894, AA460832, AA405714, AA649837, H10122, AW294364, AA313257, AI654747, AI358510, AI540589, AA954900, H19139, AA991687, H08285, AW408231, AI953865, AA371650; AI356090, AW009738, 854508, . AA977875, 859784, AW407594, F07194, F08180, F08505, 243765, AA489470, 854477, AA627570, 852605, AW297100, F07185, ---- -- - AA724308, AW407965, AA778454, 808582, F05493, AA504833, 843473, AI698394, AA765208, 813670, H08185, 245409, AW190678, 239824, AA461135, H19140, AW131981, 813641, AA701889, AA159318, AA404221, H84256, AI475002, AW189730, AA405779, F01761, H84262, AA404687, 241120, F03843, F04422, 861326, H29188, AA749407, AA581151, AA477301, AA502309, 854409, AA477302, AI144326, and 241744.
HHAWC08 68 10881461 -461 15 -475 H06763, F13201, T75396, N41533, AA310599, N26380, AW403968, AW247011, ~
W07220, N43834, N44897, AA322203, ' N25010, and AR0096 48.
HCEHD66 69 9591601 -1311 15 - _ 1325 AI968437, AI824971, AW104052, AI762197, - AI598138, AI088543, AI492390, AI827280, AA058923, AW007187, AW 135225, AI391466, AI808139, AA534403, AW028554, AI369729, AA460467, AA135928, AW006062, AW138526, AA507443, AI479413, AI400940, AW 137272, AW381735, AA135929, AA085774, W81153, . AA918755, C03738, 885039, AI472852, AI937792, AI867512, AA599118, N62215, AI864402, H41491, N62216, D44882, H14329, W30972~ H40979, AA463408, AI744140, H40980, N62166, AA319197, AI766568, N62223, AF186409, AF020184, L27421, L27420, and AC006241.
HMSGF27 70 12066831 - 156615 - AI191318, AI978812, AA586860, 1580 AI628509, ~
AA805184, W73797, AI582366, W73745, AW239293, AI620297, AW083832, AW386876, AI056600, AI056739, AI362766, AI494212, AI077551, AA935678, AI348675, AI358232, AA251769, AA968828, AA659758, AI891139, C06060, 867182, AA746268, AA251926, AA506524, H58621, D81244, AA291462, H58622, AW193598, AI919497, AI250032, AA604444, AI567397, AA905208, AA836253, 857498, AI525934, AA551675, AI364618, AI421662, AW080157, AW087829, AI813914, 838144, AW166086, AI680498, AI523628, 859996, AI554821, AI874261, AL045413, AI469754, AI366900, AW262491, AI540i79, AI560545, AW262042, AI954504, AI089970, AI677646, AL043152, AI679164, AI431909, AI868204, AI453328, AI538085, AI680221, AW089006, AI056328, AI470293, AI567302, AW162194, AI799199, AI690748, AI921746, AA741027, AI553926, AI58287i, AI805671, AI280747, AI536563, AI687134, AA514684, AI184903, AI610667, AI609409, AW089932, AI812107, AW020381, AI355277, AW148876, AI564426, AI628015, AL037454, AI648458, AW079572, AI891084, AI375702, AI865906, . AI818353, AI950688, AI439745, AI783569, AI801152, AW080346, AI673278, AL042191, AI538908, AI863382, AA848053, AI633125, AI520785, AL037104, AI698391, AW160905, AI538850, AI520862, AI917963, AI432570, AW151136, AI624548, AI918435, AI702527, AI499890, AI818562, AI623941, AW149869, AI469532, AI336575, AI961286, AI249946, AI345688, AL037081, AI581139, AI889213, AI824557, AA808175, AI623179, AW074605, AI890391, AW084447, AI453767, AI524179, AI287326, AI587114, AW169653, AW104641, AI886206, AI499131, AI368579, AI819663, AW082532, AW090102, AI866127, AI374987, AI498067, AI744243, AI802244, AI798456, AI826941, AI801592, AI520809, AI436438, AI872154, AI203903, .
. AI921479, AI249877, AI922996, AI583578, AW 189332, AI590423, AI524654, AL040011, . AI309769, AW152415, AI633061, AW088591, AW301344, N25033, AL118781, AI890223,,AI247193, AI887620, AW160916~
AA868961, AW 129616, AW 151785, AA916133, AI684021, AI635032, AI916419, W45039, AI691131, AA911767, AI433611, AI345415, AW025279, AIS64290, AW085639, AI816292, AI610690, AI690813, AI687424, AW132001, AI677796, AW 118553, AI564723, AI624529, AI620075, AI619631, AW 104724, AI4734S
1, AI972070, AI564719, AI950729; AI624604, AA857847, AW263796, AI589428, AI866469, AW118518, AW08938,7, AI798373, AA693331, AI445611, AI697372, AI359787, AI696398, AW088628, AIS72017, AI921464, . AW129456, AI758694, AI573032, AI922707,' AI696714, AI491852, AW 190042, AW166870, AI921633, AW089844, AI474.107, AW169234, AI927233, AF086313, AF090900, AL137S30, AF111851, AF200464, - - AF102578, AF090934, U02475, AF159615, AB025103, 213966, X79812, I33392, AL049314, A77033, A77035, AF139986, AL080140, 536676, A65340, AF038847, AF100931, AF069506, I32'738, AL137523, AF141289, AF061795, AF151685, A21625, AF113019, X80340, AL137271, E01314, - U35846, AL137292, A32826, A32827, I89947, . - AL133084, AC002471, AC005374, I48978, AL133070, AL050149, 578453, AF113677, AL137284, AF081197, AF08M
195, AF182215, AL133640, X84990, A~007812, AF076464, A07588, AL080124, AL136884, X62580, AL133062, AL080126, AL117435, A58524, A58523, A08907, U86379, AF094480,-AF1'14784, AL137529, AL137711, 577771, AF044323, AF1066S7, U72621, AL137294, 554890, A30330, A3033I, Y10655, I68732, AL03S587, AF117657, AF061981, AR068466, AF031147, AF036941, AR029580, AL133075, AL137558, A12297, A03736, A08910, L04859, X97332, A08909, AF067728, X65873, AL137550, AL049996, A08908, AF115392, U58996, AL117416, AF017152, 237987, AL110221,~AL122045, AL133047, . AF032666, AF026008, A18777, AL122110, AL133067, AL049382, A08913, AL137459, AF047716, AF195092, AL050143, AL137547, AR038854, AL117648, X82434, L10353, LT70981, AF026124, A08912, AL096751, AL133061, AF078844, U92992, AF119336, AF145233, A65341, AL117587, AF114818, AF079763, U73682, AL137660, U58653, AL137479, A08911, AF169154, I89931, X99257, AF051325, Y13350, U90884, E15582, AF111849, AL137554, AL122098, AF000301, AL080162, AL050116, AL137533, , AL133049, S76508, AF106862, 297214, X72889, AR022283, L24896, AF167995, I49625, L13297, X60786, AL034417, 282022, AL137538, AL122100, AF090886, AL137256, AF084644, AF039138, AF039137, AF084645, AF030513, AR034821, AL137480, AF019298, AL050366, AL022315, AL137478, AL133088, AL137560, E12580, AL080110, AR059958, S82852, A91160, M27260, AL080150, AL050138, A91162, U49908, AL133623, AL096728, I89934, A15345, AR020905, AL133637, A86558, AL122050, AL122104, AL080234, AF118558, AF199027, U57352, AF090901, AF137367, U72620, AL137627, AF118094, AL080159, AF118090, AL117443, X72387, AL122123, X53587, AL133665, AC007056, X81464, AF022813, 561953, A57389, X66871, AL050277, AC002464, E03671, E02349, AL117460, AL137574, I09499, AL080148, AL110228, I48979, AF077051, AR016469, AB019565, X98066, AL049339, X87582, U67958,, A83556, I46765, X55446, AF126488, AF112208, and AJ003118.
HBGMG39 71 971414 1 - 471 15 - AA024454, AA024670, AI458409, 485 AI740930, AI742565, AI743686, AI066465, AI168481, AI379125, AI569972, AW069135, AI497641, AW192429, AW084071, AW339039, AW102701, AI061450, AI693756, AI991329, AW003414, AI342244, AI870883, AW007899, AI609020, AI453165, AI248142, AI129686, AI992036, AI761292, AI623708, AI864435, AW151858, AI362058, AW044270, AI378430, AW008808, AI479128, AL036585, AI923881, AI963067, AI740972, AI,045227, AI351617, AI334039, AI452903, AI700412,. AI475537, AI469546, AI187911, AI066744, AI190677, AI812069, AI08123-1, AI050026, AI890929, AA551905, AI080156, AI554840, AW073739, AI926062, AA577674, AW 190499, AW250073, AI524714, AI884727, AW168902, AI096904, _ ~AI369151, AI858574, AI422058, AA304774, --w- AI890721, AI052823, AI683215, AI087154, . AA682804, AW080685, AW004991, AA912478, AW079985, AA054275, AA461453, AI668789, AA970861, AA010790, AW085591, AI819302, AI217784, AI017023, AI584163, AI627434, AA709077, AA984939, AA446048, AI564659, AI000961, AI095374, AI581899, AA532435, AA864689, AW087352, AW057507, AW132118, AA744639, N53410, AA969103, AA918799, AA862362, AI672825, AA779825, AA931463, - AI193638, AW191953, N20167, AI569968, AW 150032, AA612750, AI619904, AA429899, W02'113, AA969104, W48745,.
AA827905, AW 190663, AA780072, N50640, , - AA533847, W32574, AI640737, AI587533, AW182184, AI933617, AW073566, AI469425, AL041483, AA765028, AI952283, AI961508, AI095341, AA937508,'N48059, AA228075, AI869852, N78603, AA485535, AA506502, AA877348, AA252354, . AA010101, AA011219, T23065, AW444850, - AI917215, AA506948, AW297832, AW204879, N53257, AI917614, AI167689, AI937775, AI858564, AW043696, AW168023, AI263865, AI885655, AA450098, AW139189, AA883859, E12258, AF153686, E12259, E12260, and AL390719.
HTTCT34 72 10975981 - 623 15 - C75208, AA311414, AW411262, 637 H06122, W38676, AA334000, AA297279, T50005, AA302953, AW383613, AW383609, AW192348, AW239479, AW383535, and AA151050.
HAWAM69 73 943104 1 - 190115 - AA430300, AA541688, AA776700, ~ 1915 AW385785, AA679037, AA573270, AA126614, AL045796, AA682186, AI268236, AI963606, AW192904, AI926591, AI924827, AI922590, AI032288, AI375804, AA705172, ~
AW081541, AA694514, AI1308 83, N25288, AA931725,.AI800450, AI270687, AI366906, AW058362, AI683319, AA436891, W69578, AI597744, AI446542, 859176, AW453004, AI911821, AA687634, AI095665, AI130013, W69579, AA722782, AI191864, AI587015, AA398533, AA676733, AI476374, AA115447, AA554327, AAT59328, AW242281, . AA042956, AI139766, AA135916, AA886732, AA664356, AA358590,.H71919, AI565897, AW304844, AA916086, AA618576, AA363371, 244808, AA430199, AI370031, AA320329, AA393105, AW452852, AA135927, AI004140, AA135926, AA042816, H44791, T35731, AI865731, AA813424, 842647, 827785, T32691, AI934183, AA11.5446, AI857286, AW008428, AI631988, AA678468, AW075384, H44790, AI569918, - AI918635, AA603858, AA601518, AI745618, ' H42641, AI445766, 827874, AI939990, AA677131, AW364938, AI569374, AW029062, C01947, and AA732827.w -' HMTAV95 74 614936 1 - 395 15 - AB023187, AL137000, AL137000, 409 and AL137000.
_ _ HTEON29 76 11265221 - 119015 - AW004028, AI968030, AW237673, AA432290, AI143780, AW138422, AA428635, AA112090, AI14379'1;
and AA861634.
HHFGP83 77 11748261 - 263515 - AW027467, AW021001, AI679358, ' 2649 AA628405, AW002474, AA142868, AI720221, AI559580, AI952940, AI971163, AA126704, AW189486, AI086066, AI338366, AI632310, AW089385, W92463, AI372019, AI335800, N67118, AW130513, AI339087, W72626, AI962685, AI811098, AA043282, AI268440, AA054560, AI564240, AI623726, AA748136, AA012896, AA716090, AA047297, AA652890, AI869611, AI199091, AA938386, AA708870, AA760924, AA043925, AA582637, AA056075, AA017551, AI202160, AI916439, AA618223, AA632478, AA975496, AA249580, AI422146, AA932810, AA628573, AA628559, ~
' . AW 195978, D81448, AW 188932, AI857354, AI914554, AW193055, AA046731, 875666, AI873760, AI127156, AW023470, AA043406, AW023472, AI690518, AA043200, AA054620, AA468866, AW382445, AA047158, C01055, W76609, AA043281, AI918561, AI570316, AA338995, AI653300, AI917733, W27493, AL133599, AF004840, and AF090866.
HTEKS20 78 11234581 - 106315 - AI936596, AA868353, AI797296, 1077 AA725553, AI221970, AA428462, AA429551, AA431190, AA629305, AA629047, AI073397, ' AW235895, AI123443, AI808267, AA609412, AI914363, AA953895, AI214385, AA431516, AA911681, AA781953, AI825106, AA298758, AI215028, AA909534, AA723768, D10393, and 563991.
HCHATO1 79 12022141 - 383515 - AI357645, AW372245, AW372243, AI870876, AL079756, W80383, AA143521, ~
AI934507, AI497785, AA131786, AA143522, .
AA775429, N73794, AI190675, AA570709, AL041943, AW026848, AA746031, AI950978, AI422318, AA131772, AI885729, . AW026326, AA635171, AA934699,~
AA316540; AI246491, AI160357, AA487855, .
' AI041954, H11648, AI660052, AW152198, AI285004, AW028295, AI561088, AW166539, AI635148, AI094048, ' AW072627, AA464017, N24219, AA458881, ' AI094041., AI092047, AA464078, AA588471, AI804530, AI348338, AA487856, H99677, AI186100, H99573, AI025641, AA909894, N32879, AI081468, AW273806, AA872312, W78982, AA740804, M78765, AW337818, .
AI160858, AA977238, AA627981, AA985314, AA653069, AI610235, AI288191, AA492020, AI937558, AI357858, AW273022, H23489, N28433, AA018338, AW050885, AW365055, W01009, AI494441, AA348323, AA953253, AA484024, H30484, AA4S9071, AW025210, H90615, H90519, AA732761, F10250, N41828, AW117618, AW024678, AW079303, N47599, AA150812, 241805, 246179, AA864205, AA463943, D51145, AA464727, H12176, C16600, AA641974, T31584, AI886471, N73825, 808891, N45481, H20834, AI270409, F12634, AI991925, AA976247, - 809511, N76390, N68976, AA342119, 843266, AI802239, T90454, T78049, AI648496, AI001885, T31949, AI468389, AA046221, 843775, AA740939, AI655751, AA477480, AW139948, T31978, 817167, AA496370, 843146, 809622, 825017, AA496647, AA319007, AA348324;
T35092, . H46223, H61245, AA046114, 808999, . , 809784, AW071171, N99421, AI'085437, H61246, N84366, AA641917, AA648218, --- ~ ' , AA342120, T27314, AI042591, AB014S76,.
AA872361, AW517691, AW591480, and .
AW614740.
HAPNZ77 80 11719621 - 152215 - AI829331, AI307338, AA831880, 1536 AI379982, AA768459, AW270258, AW271904, AI345I57, AI431303, AL042756, AI755214, AA584489, AW265385, AI354423, AI963720, AL038936, AL040054, AI754105, AW021917, AI754567, AI733856, AW419389, AA402129, AW419262, AA680243, AA410788, AA634837, AI613459, AI926728, AW023672, AL041894, AI625604, AI282253, AA455483, AW327868, AW193265, AW069227, AA574442, AI064864, AA521323, AI244127, AA629992, AL046409, AI284640, AW327624, T05834, AI192631, AL048626, . AI054333, A I305766, AI613280, AA013168, . . AI962030, AI732120, AW075979, AA630854, AI753488, AA521399, AL079734, AA659832, AA019973, AI061313, AI336054, AI885572, AW270256; AA644090, H05940, AI799607, AA613627, AL118991, AI345654, AI587583, AA526625, AI291823, AI587565, AI003611, AI754767, AW166611, AA533176, - AW020088, AA833875, AA833896, a AA176605, AI332615, AI675615, A3039809, AW 157456, AA526326; AW275432, AW162697, AW007759, AA857486, AW023111, AI279417, AL046746, AL037683, AA634786, AI306232, AI017251, AW303196, AA864603, AI307201, AI457397, T49633, AI251104, AI290405, AA502991, AA904275, AI753113, AA491814, AW062724, AW439558, AA226584, AW301350, - AI696793, AW274I91, AW190505, AI133102, AL041706, AI867058, AW274349, AA904137, AI888468, AA513851, AW302903, AI270117, AA613227, AI016704, -- ------ AI570943, AI377413, AW022934, AI110770, AW131457, AA262752, AI224583, AA491284, D58782, AA779783, AW021583, AW304584, AI564185, AI272052, AW410354, H68343, AI890348, AL133668, AA394271, AA610509, AI040051, AW082117, AW117740, AI366555, AA086318, AI090334, AI870197, AW02I I6I, AA908687, AI696962, AC005859, AC003046, AL096791, AC005783, AC005939, AC003030, U73023, AC007312, AC005502, ' AC004841, AP000503, AC006001, AF053356, AC004417, AL109798, AC004590, AL139054, AL022329, AC004837, AC003971, AC003958, AL022476, . - AC006249, 285987, I51997, AF134726, AC006115, AC005701, AF146191, AC005037, AC009509, AL078581, L44140, AC005225, AC008078, AC012627, AL121595, 275888, 246936, AC007277, X53550, U57005, AP000697, AC007447, X55925, AC005211, X54177, AL021579, U80017, X54178, 275887, AC004895, AL022322, AC004139, AL035461, AL049636, AF111169, AF109907, AP000501, AC005102, AL109527, 298941, AC005251, AC010206, .
- ~ ., M37551, AC005490, U18392, U07561, AC012085, 273963, U18387, AC006442, AC007686, AC007999, U02532, AF077058, AL121603, AC007731, AL049699, AC006511, AC007204, AC006088, -AL035468, AL024507, AC005224, AC008115, AC005833, AC005630, AC004854, AL024498, AC006271, AL008627, AF064861, AC006948, AC004985, AC007685, AC007376, U02531, AC005829, 273358, L31848, AL031652, AL135,744, AF015147, AL079339, AC004002, U47924, AL034420, AC002470, AC004655, 298946, AL110502, AC007536, AL132987, AC007030, AC005500, AC003107, AP000210, AP000132, AC002504, AC006064, AC006006, U57006, AC005800, AC000118, ACQ05049, AC005772, AL049869, AC006251, AF015157, AC006388, AF149774, _ AL023807, AL080276, AL121655, AC005899, 282244, AC005821, X54176, 282182, ' AC000082, AC005015, AJ003147, AC00$394, AC004477, AC002115, AL031228, AL049757, 299943, AL021155, 284469, _ AF088219, AC005206, AC002558, ~ .
AC004876, AL096802, AP000359, AP000354, D83989, AF117829, AC007461, AC005013, 299715, AC007055, L77570;
AC004240, AC004686, AC005874, AF134471, AC005214, AF191544, AL050341, AC005005, AC012599, AC005756, AC008372, , AC005379, AC004253, AC002119, AC007226, AC008041, AC006027, -- AC004878, U57008, AL024509, AC004408, AF196779, AD000092, AC006536, AL022401, AL133245, AC005667, 293017, AC004821, AC008009, AC004106;
AL049759, AC007298, AL035587, AF196969, AF036405, AL031666, AC005901,.
AC004019, AF165142, AC004458, AL078477, 281370, AL132718, AC002094, AL049540, AF160252, 269667, AC009275, AC000029, AC006367, AL021918, AC004859, AC005480, AC005239, AL031433, AC002430, -AL034429, U62317, AC005751, 280232, AC005678, AC007546, AL121653, AC007384, AC005696, AC006600, AL021393, AL136295, AC002492, 283826, AL022'726, AF003626, X55931, AL021917, AC005089, AC004087, U67801, AC004992, AL022311, and AL034418.
HSIA078 81 12223431 - 198815 - AA527435, AW195324, AI653000, AW051613, AA514619, AI675204, AI652532, AA435717, AI659333, AI796596, AI273289;
AI880669, AI826786, AA889355, AI174916, AW004627, AA377072, AA255838, AA397980, AA430523, AI565825, AI435476, AW001866, N52904, AA430608, AI760594, AA298640, W69756; AA594479, AI149418, AI911011, AI871818, N71537, AI089421, . . - AA400874, AI038591, AA854839, AI565867, ' AI131012, AI144119, H65663, AW044396, N47230, AI732273, AW079534, AA847967, AW027678, AL044698, AA224892, T36269, AA009702, AI668849, AW 182206, AA011130, N78511, AI676028, AA968449, AI984040, AA207018, AA658246, N73670, AI937659, 853598, AA453038, AA904224, AW293549, 848261, AA775033, H52314, ' 838289, H48428, AW083969, AA588654, F10880, AA578060, AW298073, W25831, . . AA889378, AA483944, AC002302, X62260, AC002288, AL035588, AC007425, AF181896, AC004.216, AP000280, AP000038, AF003528, AL033525, AC009498, AC007676, AP000107, AC006080, AC005704, AC005332, AC005011, AL022328, AL118497, AC007221, AC005213, AL132987, U69570, 248484, AB020858, AC004382, AL049753, AC004874, AG006023, AC003976, AL035668, AL035671, AL139165, K03021, ' AC005771, AC004894, D83402, AC005004, AC005183, AC004129, AC004671, AC006354, AC005046, AC005161, AL117338, AC005184, AC006599, AF042484, AC004100, AJ006345, AC005235, AC004875, AC003693, AC004982, AL021877, AL009183, AC002546, 283822, 275741, AC002541, AC004098, U61375, AC007637, AC004061, AC005922, AL049821, 295114, AC006952, AC007529, AF095703, AL021395, AL080286, AL035696, AL117436, AC006973, 282246, AP000466, AL078638, AC007680, 284484, AP000030, AL035663, AP000252, AC011605, AL035427, AC012088, 286062, AC000378, AL035106, AL117375, AC004984, AFOOI550, AC006360, AP000134, AP000212, AC007392, AL022159, 296074, AC011594, AC004530, AC008012, AL132718, AC005060, AC007543, AC004015, AC008069, AL133162, AF001548, AL031390, AL135746, AC009263;
AB014079, AC007156, AC007198, AC004889, AL031123, AP000228, AL024508, AP000140, 268324, AP000146, AC005533, AL035633, AL031737, AC000119, AC004838, AC004616, AC007450, AP000088, AC005086, AC006398, AC006511, AC009275, AC004055, AC004882, AL021707, AC002563, AP000299, AC006139, AC005553, AF047825, 297876, 284480, AL020989, AP000514, AC004888, AC007314, AB020865, AL117347, AC006367, AC005562, AP000045, AP000113, AC005393, ACOOS690, AL049839, AL034425, AC007617, 275957, AC005529, ' AC005701, U95740, AF152365, AC012083, AL021407, AC000072, AC011700, AL031120,'AC005538, L77569, AC004381, AL109914, AL00863'S, AC009464, AL032056, U91319, AL110120, AC007455, AL022329, . 284488, AL035072, AC006213, AL022162, AC006196, AL118512, AL023806, AP000536, AL121866, ACOOS587, AL033384, AC006998, AC007262, AF107258, AL034555, AL021451, AC006064, AL050350, 274409, and AC004968.
HWBEG18 82 909798 1 - 940 15 - AA906863, AW408789, AW452373, AA352977, AA732349, AI269653, AI492098, and AB020653.
HFCBB56 83 12043221 - 209015 - AA491955, AA053463, AI620992, AW005974, F13749, AW408767, AA864271, . AW162288, AA565232, AA501867, AW265688, AI493583, AA744094, AA904211, AW316599, AI224619, AA019S42, AA339423, AI696793, AA878492, AW081610, AI492579, AI623364, AI445373, AA630535, AA513983, AI243793~
AI889426, AW054936, AI678867, AI049955, AA828834, AI190.648, AA721645, AI278372, AW236219, AI633386, AI797998, AA744048, AA669155, AA572983, AA862312, AI889995, AI636734, AI368862, AA283081, AI279417, AW407889, AI312090, AI049630, AI689019, AL042667, AL042670, AW337805, AI887755, AA832016, AA833896, AL049869, 284469, AL122020, AC004821, 295114, 283822, AL022315, AC002477, AC004815, AC007298, AL034429, AC005800, _ AC005736, AL031311, AL009172, .
AC012384, AL034400, AF053356, AC007435, AC002312, AC009516, AC004185, AC004921, AL031657, AC005670, AF165926, AC020663, AL031680, AP000501, AL133245, AC002549, AC005697, AC003664, AC005755, AL022322, AC002430, AC005722, AP000509, AC002395, AC005088, AC007384, AC005972, AC009509, 295113, AC006441, AL133243, - AL035423, AC000025, AC016025, AF117829, AC000379, D84394, AL139054, AC005527, AC003043, AL031681, AC005531, AP000704, AC004659, AC004476, AF001550, AC011311, AC005529, AL031431, AL022238, AC004494, AC007880, AC007242, AC005089, AL022324, .
AL0312$0, AF064861, AL024507~
AC005952, AC005837, AL031848, AC004382, AC007785, AC007934, AC005484, AL049829, AC005412, AC005726, AC002091, AC004383, AF111168, - AC002300, AC000035, AP000210, AP000132, AF109907, AC004699, AC009946, AP000251, AL008629, AC006285, AP000155, AC002369, AC0.05324, AC005037, AC006160, AL031666, AC004675, 298047, AC007684, r ' AC016831, AL049843, AL049709, 274739, AP000030, AC005390, 294056, AL049538, "
AF196970, AC007308, AC004895, _ ~ AC002350, AL035555, AC003048, AC007066, AL121658, AC005746, AC002528, 283844, 299127, AL121603, AC005632, AC004585, ACOQ5480, 286090, AL121655, AC005042, AF196969, AC004552, AP000098, AL031293, AC002472, AC005969, AF088219, AC004231, 307848, AC004020, AC004796, AC005377, AC002381, AL031588, AP000500, AL133353, AC002378, 275887, AL022313, 268324, AC005535, AC003962, AL031587, AC004148, AL021939, AC005871, AC005880, AC007690, AC007664, AC004884, 295125, AC005399, D83253, AC005304, AL035458, AC005332, AL035684, AL008631;
AC004167, AF030453, AC006454, AL022476, AC002565, AC004099, AC005669, AL031591, AL031432, AC002492, U91321, AP000088, AC004865, AL031427, AC009542, AP000150, AF003626, AC004685, AC009247, AC007546, AC005300, AC005829, AC006146, AC006001, AC006023, AP000014, AC007312, AC007993, AC005730, AC009330, AP000009, 283846, AC012085, AC005808, AL034554, 284466, AC004593, 298742, AC004859, AL022316, AL035409, AC008372, AC005902, AF031078, AL109952, AL133448, AP000352, AC003957, AF205588, AP000116, AC004816, AC007687, AF030876, AC005901, AC000394, ACOQ7011, AC002351, AC018633, AC005839, AF047825, AC005695, ~ -- - --AC005180, AC007528, AL031117, AC004149, AF067844, AL022320, AL121595, AP000689, AC006450, AL033527, AL020997, AC005562, AC006064, and AL035587.
HEMCL65 84 9109001 - 453 15 - AI902552, and AI902562.
HMAMB94 85 12253431 - 244015 - AW369653, AW024321, AA766018, AA905970, AI652025, AA158332, AI627492, AW068035, AA158041, 225180, W40230, AI493579, AI536877, AA191353, W45464, AW175724, AA670064, AA190444, AA248856, AA247921, AL121448, AI926451, AI983662, W60091, AW068830, 890961, .
890977, 890972, and AA309409.
HCQCI06 86 9150001 - 119015 - AI123952, AI751915, AA343597, 1204 W79144, . AA345718, AA304549, AA256582, W81545, . D45547, AL117664, AC068763, AC068763, and AC069223.
HWLFG75 87 12276421 - 202615 - AI356559, AW 163067, AI937030, 2040 AI652337, AW028706, AW157098, AW028808, AA443325, AA004795, AW005140, - AW173645, 860229, AA442531, AI274924, . AI810652, AI924004, AI572794, AI336556, AI672253, AI147260, AI872258, AI347103, AA467751, AA724594, QI280850, 852646, AA536110, H16834, AW450707, AW444512, AI376913, AA468349, AI807962, AA927875, 842625, AA609873, W28566, AI918962, AA578362, AA578062, 817389, C18386, 815375, AI016851, 860462, H16941, AI423739, AA467933, AA740299, AA025666, 842116, AA978110, AI423740, AW117517, AI886594, AA443338, AL040243, AI275175, AL119748, AL121270, AI857296, AI433157, AL045500, AI433976, AW071349, AL047763, AI702406, AI250293, AI436456, AL048871, AW 117882, AI620284, AW 195957, AL119791, AI064830, AI568870, AI863014, AI702433, AI499463, AI538716, AI439087, AI349933, AI678302, AI633419, AI868831, AI440239, AW074993, AW162071, AW235035, AW071417, AW274192, AI349256, AI866608, AI349645, AI690835, AI498579, AI613017, AI349004, AI349772, AW301409, AL135661, AI699857, AI540832, AI440426, AW103371, AI628205, AI281779, AI500659, AL036146, AL036396, AI340582, AI521012, AI500077, AW169653, AI249257, AI567351, AI500553, AW238730, AW268253, AI224992, AI312152, AI345735, AI349937, AW089572, AL120736;
AI366549, AI564719, AL047042, AI568854, AI597918, AI866002, AI673256, AI281773, AI590128, AI539771, AL038605, AI497733, AI969601, AI636456, AL036802, AW087445, AL119049, AW068845, AI282655, AI690751, AI612913, AW148320, AI800453, AI800433, AI3431I2, AI909666, AI567632, AI635461, AI687728, AI686926, AI800411, AI682841, AW3031S2, AI348897, AI610645, AI349614, AI207510, AI697137, AL121365, AI597750, AI921379, ' AI679724, AI758437, AA640779, AI866887, AI432229, AA613907, AI625079, AI499393, AW301300, AA572758, AL036759, AI475371, AL036980, AW074869, AL036274, ' . AI815383, AI610307, AI570384, AI631107, AI608667, AI818683, AI969567, AI434281, AI687376, AI285735, AL046849, AI282903, AL040169, AL043326, AI873731, AI920968, AI445432, AI469532, AI889203, AI580190, AL038779, AI811863, AI680113, AI906328, AW085799, AI889839, AI671679, AI874109, AI620868, AI753683, AL120854, AI934036, ' AI609592, AI569616, AI583316, AW166645, AW080838, AI439745, AI446606, AI255071, AI343059, AI866780, AI919058, AW002342, AL038778, AI475134, AI687415, AI345860, AI952114, AI872711, AW168384, AA585422, ' AI580984, AL047041, AI269696, AI149592, AI907070, AI610756, AW1'67776, AI345111, AI799199, AI499131, AW 196141, AI499512, AI907061, AI909662, AI349598, AI307466, AI539153, AL080060, I48979, Y11587, AL133016, S68736, AF090901, AF113691, I89947, AL133640, AL110221, AL137527, A93016, AR059958, 578214, AL049452, AL050393, AF090934, AF104032, AF113013, , ' AF118064, AF118070, L31396, L31397, AF113689, AF078844, AF090900, AF113676, AF090903, AL049938, AF125949, AF113694, AF090943, AJ242859, AL133606, AL117460, ' . AL050146, AL110196, AF113690, AL117457, AL050149, AF106862, X84990, I89931, AF090896, A08916, AL050116, AL050108, AL050138, U42766, AL049466, AL133075, AB019565, AL049314, E03348, AL122050, AF113677, A08913, AF113019, AL080137, . AF017152, AL080124, AL137283, AL133557, AL122093, AL096744, AL133080, AL050277, AR011880, I48978, U91329, AL133093, AL122121, E07361, AF158248, X63574, ' AL049430, AF113699, AL137557, AL133565, Y16645, Y11254, AL137459, X82434, AL122123, AJ000937, AF091084, AC002467, AF111851, AF177401, AL117394, AF097996, AF146568, AL117583, AF125948, AL110225, AL021393, AF079765, AF017437, I49625, 561953, I33392, AL049382, A08912, U80742, U00763, AL050024, AL117585, E07108, AL133560, AL137550, AF095901~
A65341, AF000145, A08910, AC002464, AL049464,.
AJ238278, E02349p AL049300, AC007390, AC006371, AL117435, AF067728, X70685, ' X72889, 282022, AL122098, AF183393, AC006222, AF118094, AF001552, A58524, A58523, AL137271, U72620, AL122110, A08909, I03321, U62317, AL122049, AL022147, AL137463, AL096776, AC004200, ' -- I09360, AL137538, AL133113, ~- ~ AL049283, 239 .
E05822, A03736, A77033, A77035, AC005057, AL031281, AL080127, AC007172, AC010077, 299297, AF061943, A12297, U35846, AF087943, AL137648, AC007043, A21625, X96540, AC004690, . ~ A08911, AF110520, AL078630, AL049557, AC007458, AL078602, X65873, U95738, AP000344, L13297, AC009233, AL110197, AF 177767, AC007298, AL031346, X93495, AC004686, AC006336, AL022165, AL133072, AC005291, AC004093, U67958, AL080159, A21622, AF130342, AJ012755, AP000520, U66059, U95739, and AW514074.
HBODA38 88 9234561 - 188215 - AA196401, W79841, AA112831, 1896 AA197134, .
C05212, W19681, AA195812, 225012, _ AI004215, 230134, AA194641, AI809925, AA258550, 228480, F29133, AA699576, F20387, AI129717, AA194359, AI079487, AA195355, AA195094, AA931319, F34387, 219343, F36761, H67895, AA258604, AI378821, F19321, AI089231, 891150, AI702453, AI288969, AI313118, AI351843, ' F31038, N56278, AI381419, AA195356, . AA131264, AA195051, AA192095, AI264911, and AA086284.
HCYBK19 89 12253241 - 276415 - AI829726, AI809562, AI927757, 2778 AI927758, AI927768, AW297770, AA165336, AI638072, AI149852, AW298053, AI365991, AA904547, H15885, AA985065, AI359235, AW207262, AI470080, AI421572, AA305419, AA985170, AW341104, A1423550, AI362528, AI453702, AI654827, AA927292, AI365149, AI365324, H91991, D81295, AI797740, 862753, AI357382, AI953516, AA909430, H01576, AA969956, AA379840, H52748, 837302, AA905264, 243289, 818887, 868272, .
H04859, AA746048, T31617, H04764, 244077, T78758, T32807, D60323, AA634475, H01477, AI193962, H00918, D81446, M86147, AA983881, AA318875, N64328, AA165369, D62629, 239361, T16370, F03272, H00919, N34301, H46107, AW590162, AW593404, AW593449, AW593464, and AW593480.
HWNCY05 90 12230311 - 118115 - W40569, AW025860, D63226, . 1195 AA334307, AL038838, AL037436, AL037323, AL038983, AI142134, AL134524, AL038822, AL037727, AL038532, AL037343, AL040617, AL044186, AL041238, AL047012, AL037335, AL037435, -AL044125, AL044037, AL047170, AL040463, 3 AL045684, AL040625, AL047219, AL044162, AL043677, AL040193, AL043467, AL040510, AL040621, AL043538, AL041752, AL047183, . AL043496, AL040464, AL043923, AL043814, AL037443, AL040576, AL043845, AL040839, AL045753, AL041635, AL041133, AL041347, . ' AL040294, AL041324, AL046442, AL044064, . AL041459, AL041577, AL040322, AL043492, AL041602, AL041098; AI:044074, AL046850, AL040052, AL040768, AL040119, AL046994, AL041730, AL041523, AL043627, AL046914, AL040472, AL041374, AL045817, AL041096, AL043848, AL043570, AL042135, AL040444, AL041163, AL047057, AL037295, AL045671, AL039316, AL041159, AL038761, AL041955, AL046392, AL040075, AL04_1292, AL041358, AL044272, AL041296, AL041142, AL044258, AL041346, AL041086, AL0411~68, AL045920, AL040149, AL040529, AL040148, AL040332, AL042898, AL,046330, AL049018, AL041197, AL040458, AL044187, AL041246, AL047036, AL041233, AL045990, AL039643, AL040128, AL040571, AL040553, AL040745, AL040370, AL044274, AL079878, AI,039338, AL041277, AL040342; AL040155, AL045328, AL044199, AL041131, AL041186, AL040285, AL040414, AL039360, AL041051, AL040091, AL039744, AL037341, AL044165, AL040090, AL045989, AL040168, AL042096, AL043941, AL079852, AL041227, AL043775, AL046327, AL044201, AL040253, AL045857, AL040082, AL039432, AL043444, AL040329, AL038745, AL041140, AL044529, AL037279, AL041278, AL040263, AL045725, AL040255, AL040238, AL047037, AL039915, AL043612, 228355, AL049069, AL134110, AL047163, D61254, AA585101, T11028, AL043537, AI546945, AL041210, T23957, T23985, AL045211, AA585439, AL046147, AI526194, AA585476, AI525556, AI547295, 016300, AI541365, 230131, AA174170, AL045327, AI541374, AI525431, AI541523, AI541514, 829445, AL041344, AL540967, AI541390, 016305, AI546999, AI541508, D57491, T23~888, AL080031, AI557238, AI526073, AI557731, AI526140, T41289, AI557262, AI525306, 829218, AI541535, AI535660; AI557799, D55233, 014723, AA585453, AI546828, AA585356, AI546875, AI547006, AI525320, AI556967, ' AI541205, AI535639, 829177, AI541013, 828735, AI546855, AI541509, 015189, AI526184, AI541534, AI526176, AI557734, AI526144, AI541017, AI525316, AA283326, AI546891, AI557727, AI536138, AI541533, AI557787, AL038651, AI541307, AI526186, 828895, AC006928, AR064707, I05558, I18895, I08396,.Y16359, E13740, E03627, AR062871, A25909, I48927, I84553, I84554, A81878, A60212, A60209, A60210, A60211;
. . . A02712, A77094, A77095,.AJ244003, y AJ244004, AJ244005, AF082186,-AR031566, A35536, I08389, A98767, A02135, A04663, A93963, A93964, AR062872, I63120, AR017907, AR062873, I00682, A11623, E00609, I06859, A11178, E01007, A18050, A23334, A75888, I70384, A60i 11, A23633, AR007512, I15717, I15718, A90655, I08395, D13316, AR043601, A95051~, A18053, A64973, A20702, A43189, A43188, A20700, A98420, A98423, A98432, A98436, A98417, A98427, M28262, A85395, AR038855, A85476, A35537, D78345, A02136, A04664, A11245, A11624, AR037157, AR054109, I13349, A10361, A91750, A02710, E14304, A07700, A13392, AR031488, I13521, I52048, I44531, I21869, A91965, I44516, A70040, E16678, AJ244007, I44681, X83865, A84772, A84776, A84773, A84775, A84774, .
AR067731, AR067732, A58522, A22738, I62368, A86792, A93016, I01995, I25027, I26929, I44515, I26928, I26930, I26927, I08051, AF149828, I03331, E12615, AR035193, A92133, A13393, A27396, AR027100, I49890,:I28266, A82653, E16636, A24783, A24782, A95117, AB025273, I60241, I60242, D50010, A58524, A58523, AJ238010, A20699, E00696, E00697, E03813, I66482, AR009151, I66483, I66484, AR027099, I66487, Y09813, AR051957, AR038762, 166485, I66498, 166497, I66496, AR038066, I66486, AR066494, AR051652, 232836, AR051651, AR008429, AJ230935, X07299, AJ230902, AJ230951, AJ231009, A70872, AJ230972, E17098, D13509, X81969, AJ231028, U94592, AJ230867, AR022273, I66495~ I66494, A22734, AJ230845, A70869, E12584,119525, A93923, D17247, A06631, AR035975~ AR035977, 560422, A93916, AR063812, I18302, Y14219, A24548, A24546, - A93931, A16035, A85203,133632, AL133053, AR035974, AR035976, AR035978, AL122101, E03654, AR023813, AR054723, AL133074, AL133049, and A22739.
HTXNN68 91 11433001 - 151615 - AA287304, AI418151, AA885205, 1530 N32677, AA147402, AI630898, AI362039, AW075796, AA905465, and D86967.
HWWFWO 92 933671 1 - 880 ~ 15 AA287304, AI418151, AA885205, - 894 N32677, 6 AI630898, AA147402, AI362039, AW075796, - ~ AA905465, AL134533, AW392670, AL134531, AI142139,.AL119418, AL119483, AL119319, AL119443, AL119324, AL119522, U46350, AL119457, AL134920, AW372827, AW363220, AL119484, AL119391, AL134538, AL134902, AL119341, 299396, AW384394, AL119497~ AL119363, AL119355, AL119396, AL119496, D86967, AR069079, AB026436, AR066494, AR060234, and A81671.
HOSDR06 93 12295581 - 217015 - AI942417, AI800288, AI830283, 2184 AW384845, AA442721, AI637544, AA252612, AA305619, AA224269, N63098, AI670786, AA811590, N80213, AA431396, AI498460, AA371384, N30797, AI280008, AA298474, 224818, AA432386, AI770070, AA364533, AA280152, AA297828, F13799, AA875925, N93459, AI110673, AA090893, AA029327, AA825662, - ----~ ---- F30025, AA029328, N41572, AA298473, AI768963, AI949001, 833662, AW105155, AI377278, AA018569, AW195563, AW377767, AI215536, AI268149, AA280153, ' AA804204, and D38552.
HSDHB12 95 941973 1 - 624 15 - AA447298.
HTTJW49 96 948107 1 - 648 15 - At1199865, AI769428, AI061340, 662 AW268880, AA707168, AI970984, AI884812, AW444872, AI479954, AI356088, AI701720, AI765045, AA722812, AW236544, AA410516, AI267987, AA005114, AI298592, AI865503, AI633370, AA878382, AW389168, AF118838, Y17571, AF164632, AF164526, AF164527, AC004458, AF164528, and AF 164525.
HELDR74 97 12189821 - 142815 - AI741422, AW249482, AA573909, AA085764, AW272801, AI052311, AA490620, AI683396, AA151131, AI700257, AA310938, AI284596, AA961817, AA862960, AW073675, 887485, AI828443;
AI925221, - AI969547, AW001375, AI521481, N24896, AI925228, AI695515, AA609182, AA151130, AI245859, AA490809, AA040451, AW 139250, AI970384, AI961068, T67610, . - ~ . AA923298, AA513675, AW027490, T96070, AI624751, T67494, AI936161, AW196036, AI917354, AA679554, N36317, AA302588, AI932690, AW250249, 888163, T72363, AI796143, W32439, AA582049, AI539047, W45013, and AF113795.
HOEET48 98 963290 1 - 146615 - AI797684, AW239200, AA456267, 1480 AI478733, AI751749, AI990902, AA427646, AI379565, . AI970534, W95460, AA788855;
AA405402, AW068453, AW294114, AI751750, AA594137, AA947297, AW177719, AI057073, AA427487, AI341112, AA232452, AA041304, AW068711, H73236, AA041328, W95567, AW167569, AA853047, AI652166,.
W02069, H74164, 834003, AI341381, AW176526, AA580289, D30965, D31176, ' AA367502, and AR035969.
HBODE51 99 11889981 - 258015 = AW083201, AA056328, AA404251, AA404701, AA610014, AA535370, . AA479540, W26963, AI803179, AA693610, AA167788, AA477641, T30626, AW118235, AA148356, 820037, T10239, W40184, AA677785, AA780412, T33085, T15444, 236286, F37344, AA166644, 243182, .
AW367537, AI699497, AA258857, AA480033, W28626, AI625089, F27424, 861287, AI569618, AW376308, AA324131, AW337372, T10238, 241030, AA319764, 819966, AW058317, AW268504, AA091588, F04174, AI807608, AA249876, AI758553, AI887457, AA665925, 854586, AW009070, AA641818, AW166832, AI627360, T80262, AI619820, AI698391, AA767924, AI918435, AI553645, AI636507~ AI244343, AI872423, AW238688, AI524179; AI913330, AI824458, AI348609, AI889189, AI918809, AI559752, AI247293, AI138221, AI648494, AW148350, _ AL036634, AI499890, AI479292, AI818350, AI800159, AI927233, AI612913, AI445611, AI470717, AI287616, AI452560, AA80454.1, AI433647, AI613270, AL042745, AL042744, W45039, AW089638, A99218, Y14494, A99220, AJ012755, AF087943, A23327;
AL133623, AF131821, 213966, AF090900, AF129131, AL137271, AF141289, AL110223, A84109, X83544, AF172319, X69026, X60759, AF115410, AL137478, Y10183, and AJ005690.
HE8UT58 100 973153 1 - 109015 - ~AL079991, AW409774, AA404732, -AA782257, AZ808909, AA099.036, AA677654, AA405418, AW341107, AW409667, AA659407, AA503263, AI433410, AI885266, AI341147, AA905366, AI692534, AI027623, AA468344, AA923181, AA778251, AI681129, AA533117, AI986154, AA099037, AI955317, AI276678, H29006, AA404602, AI347168, AA371047, AI420387, AW135263, AA868124, T12623, H09118, AA430277, AA365338, T08839, AA813337, H29109;
H11272, AW137535, AB014.534, AF116574, AF116573, and AC032004.
HFXJI27 101 971046 1 - 427 15 - AI335925, AI890750, AA187638, 441 N39062, AI268071, D79411, N75622, N22381, H13284, 843578, F10497, 826373, AI625385, AI220697, 821997, AA932204, AI493179, AI373111, AA909963, AI476063, AA909961, AI888010, AI619918, AA976881, AW339040, AI432041, AA015789, AI049514, AI355877, AI888471, N59174, AI679618, AI373318, AA670204, AI358468, AI336216, AI266210, AA947133, 833792, AW081307, AA610540, AA928678, AA922947, AA922946, AI817788, AI245931, AA948492, AA044223, T63607, AI433872, AW269052, AI587384, AA827770, AW182646, AI127888, H89128, AI268557, D62713, T63317, 835227, AA018661, AW023089, N67.948, T62565;
H01498, 821841, AW151373, D29213, AW022288;
AA678512, and AB023187.
HETH038 102 11245981 - 139315 - AA675914, AW058625, AA595003, 1407, AA565043,,AW192302, T54086, AI287312, AA988703, AW438698, AA675910, AI990695, N40329, AA079650, T54177, AI824376, AI675850, AA079556, AA303646, N71782, AW367626, AA675909, T29568,, H69835, C01666, AA352261, AI969951, AA077925, AA441887, L14848, AF055384, . AB015600, U56947, U56949, U56955, Y16801, Y16809, Y16811, Y16807, Y18114, Y16804, U56944, U56948, Y18113, Y18116, . U56943, U56945, Y16803, AF097406, U56942, Y18111, Y18115, X91625, Y18117, U56952, Y16808, Y16805, U69978, Y18112, U56941, U56946, Y16806, Y18118, U56953, U56954, Y16810, AF097403, AF097404, U56940, US6950, U56951, Y18110, AF045597, AF055385, U9S731, AF021223, AF021224, U95730, U95732, U95733, U95734, AF021225, AF021226, U95729, AF055387, AF021222, AF021221, AF055386, AF055389, AF055388, AC004180, X92841, AJ249394, AC006046, AB000882, AB000878, AP000506, U65416, AC004170, D84394, ' AP000507, Y16802, AB023058, AJ242442, AP000521, AL022723, AF045598, AF055066, AJ242438, AJ242439, AF045600, AF045599, AJ242441, AF055390, AF045596, AF045604, AF045601, AC004208, AB014083, AJ242440, AJ242443, AP000515, AF045603, AF045602, U69625, U69628, U69623, CT69624, U69629, U69626, U69631, AJ242444, L29410, L29409, AJ242445, L29408, U69630, U69627, L29411, L29406, L29407, U69622, AC004207, AC004200, AP000519, AB023056, AB003601, AB003600, AB003599, AB003606, AB003605, AB003608, AJ251160, AB003604, AB003602, AB003603, AB003607, AJ251156, AJ251158, AB003609, AB003612, AJ251157, AB003614, AB003610, AB003611, AB003616, AJ251159, AJ251161, AF106651, AF011836, AF085020, AF011845, AF136158, AF106633, AF106642, AF085024, ~
. AF011887, AF011860, AF106639, AF106648, AF011839, AF011848, AF079414, AF011842, AF0850I3, AF106637, AFI06655, AF085029, . AF106643, AF136159, AF106634, AF106640, ' AF106649, AF085037, AF411879, AF106646, AF106635, AF011853, AF136157, AF106638, AF106641, AF011847, AF106632, AF106647, U69965, U699'71, U69969, U69966, U69975, U69964, U69977, U69968, U69967, AB000880, AF085022, AF085014, AF085050, AF085038, AF08S046, AF085018, and LT69973.
HTEIL07 103 953803 1 - 445 15 - H55431, and AL031843.
HDPTC44 104 553453 1 - 561 15 - AF010446, U22963, AF03146_9, 575 AF010447, AF073485, AF073486, AJ132011, U94989, AF010448, AF035672, and AF068692.
HHPDE40 105 552112 1 - 582 15 - AW449998, U60319, AF079407, 596 292910, AR036572, U91328, Y09800, AF079409, AF079408, and Y09801.
HCESP56 106 827671 1 - 500 15 - AW247740, AW247029, AW204207, , 514 W39269, AA32S536, 814422, W52568, Y16752, AL022170, and 265186.
HH_FGZ38 107 11171921 - 154615 - AA372117, AI864976, AI468754, 1560 AA133546, AA160339, AW449104, AI206956, . AW374444, AA595656, AA278441, AI767157, AI263438, AI859910, AA216640, AA070240, T59738, and AA311762.
HAHEF22 108 910996 1 - 872 15 - AA447298.
HCUEV29 109 816065 1 - 491 15 ; AW410192, AI570209, AA583494, AW087991, AW337550, T30350, T24722, AW246233, and AL031283.
HBGQN46 110 945370 1 - 888 15 - AA479990, AW293680, AI473367, 902 AI184880, AI086741, AI081183, AI214276, AA742259, AA836754, 855060, AA251267, AI285889, AI813391, AW297831, AI819671, AF038458, and AF038458.
HMQCE42 111 806815 1 - 194 15 - D56208, N44225, AA348075, 208 AA3~51420, 819052, AA449607, H23327;
AA227277, AA081491, AA102697, 854504, AA083156, .
246810, D38526, D49552, AJ010749, X96582, X97321, Y09156, X83394, 233459, D64147, U09853, X70857, M28160, D11383, _ AJ001977 D83957, and AP000508.
HEQAY32 112 12072061 - 259915 - AA459604, AA703415, AA703508, 2613 AI337830, AA993540, AI924185, AA398958, AA047107, AI581879, 88$084, H08326, AA399541, AA047244, AI479681,.AI570681, AW007699, AA826082, 850486, AA742413, AI278602, AA810070, AW129039, AI346375, AI636166, AI468327, 244337, AW248520, AI817167, AI188457, AI923575; AI480234, AI184221, AI289600, AW005514, AI696876, AA864258, AW248475, AI804579, AW000853, AI091327, AI192782, AW 104750, AI299237, AA766769, AI124027, AI309629, AA450027, AI378094, AA829783, AA505829, AA421949, AI721136, AI923569, T79992, AA317334, AI269575, AA878088, 850042, H08229, AA852854, N27053, T36249, 245648, AA454095, AA878089, M78847, AI523984, AA740396, H08868, AI366112, AA322162, AW138078, F04540, AI824732, AA373622, AI864722, AI383526, F04098, AW009838, AA825440, 243430, F02816, F07842, 850380, AI983715, AA383971, T24108, AI979211, AA402090, AW271173, AA335352, 814266, AL110339, AW080157, W45039, AI359744, AA713657, H03560, AA582025, AI553645, AL036673, AI640375, AI824576, AI287449, AA102339, AI871660, AI582900, AI743505, AI40169,7, AW007284, AB033004, AF104222;
AC006529, AF124728,~AL137294, AW514348, and AW778993.
HOEFI09 113 974194 1 - 426 15 - C 18310, AW402322, AW405194, ' 440 AW402317, AW408434, AW401505, AL039796, AW239462, D82221, AW402511, AA158109, AA160317, AA263135, AI124815, AA310808, AW327936, D82189, AW401719, AW402264, AW402698, AA160714, AA151951, , AW403872, AA663896, AW402402, D82177, AA100848, AA100680; AW405525, 819052, AA102697, AW401548, AW403793, AA224068, AW402754, AW408440, AA401643, W40489, F13067, AI826037, AW403392, AA381090, H23327, AA121088, AW402383, AA449607, T52124, AA380938, . AA381088, AA160037, AA159230, AA336651, 812066, AI359260, AL138056, AA157351, AW403114, AA295712, AA381030, AA351420, T29764, AA548636;
AA058454, AW402855, AI365653, N44225, AA295685, AA227277, AA350486, F07582, T72958, AA127128, AA159413, AW402401, AA160101, D56208, AA112349, W52004, AA152039, AA344828, AA348075, .
AW2735.57, F06315, AA143652, AA102181, AA488534, AI696864, T52219, H23377, AA159125, F06589, AA358919, AW403253, AA085102, U46382, AA263158, AA143468, AA143626, 859764, AA916617, 830668, AA295230, T75330, Y13029, U96942, 246808, L36591, L22028, L36318, M94052, M94051, M24040, Y09058, M24032, L22649, U29083, M94053, D44501, U04243, U11262, U11266, U09864, L42024, L07950, U21053, L11571, L11570, D50296, AF016641, U36392, M84380, M24036, U04244, X64454, L20088, AB032096, X61709, U29480, L42280, D50300, U56246, U16298, AJ223282, X94574, AB032097, U58110, L76931, D44500, X99735, U17572, U11267, L76093, D31816, M95530, D50294, L22027, L 11666, D44499, D50295, Y18552, L17005, U03698, M32320, X81363, AF170577, U80945, U05578, L32862, L413~3, D83956, D50293, U03027, U03859, L11603, L19937, L42281, M24039, U05575, X13116, M30679, L41925, 215143, AJ245869, M24035, U90558, AJ002676, D49824, M80670, E03437, Y08994, M68964, M32319, U31334, Y09118, L09736, M84694, U50710, L41214, X94480, AF033351, L42506, AJ250917, E03438, X91749, M24034, U21052, Y13567, L76088, M84383, AJ006020, M28203, U70528, L76094, M84382, U04245, U29057, M32317, U88407, M28205, X90391, X84725, M84384, M33573, L42345, U32678, AB036051, M19756, AF161881, X94481, X75953, AF189017, X64366, M24038, L42282, . L42283, D83030, AF118894, AJ131118, D50290, M24075, X60255, X60693, U648b1, y X90390, D50291, M19757, AF022783, AF115460, M77775, X77658, L24373, M15470, M28204, D25275, U01848, U35431, X60253, M84386, D85761, U11263, U14943, AF118895, U75533, X96582, Y09156, L38504, L54059, U90566, D85762, M24037, X13115, U18789, X60254, L20089, U90559, U31971, U90563, U90564, U90565, U11261, L09735, L20090, M84381, M77778;
M77777, M77776, AJ133267, E02885, M58636, U05580, D50068, U11265, Y08995, X61710, - X61706, X58536, D50299, U17107, U30936, M24045, M84385, X03664, U63653, D83043, E02884, L04696, U30904, L40599, U05576, U05577, D14343, M29864, AJ223602, U34810, AF115461, Y09157, X61707, L41086, D50710, M26432, M59840, U06862, L04695, AJ237703, X99704, AB032094, AJ002675, AJ003063, AJ002677, L76935, 222651, U41832, M32318, U05582, AF130734, AF118893, M77774, AP000507, M29865, D64149, and X60251.
HCEOR02 115 12276801 - 209415 - AW292158, AI933139, AA325666, AA317428, and AI887071.
HLTEH39 116 943641 1 - 170 15 - AA970332, AA361489, AA158146, AW402698, AW405525, AW402322, AW385684, AW401505, C18310, AW401548, AW239462, AW402264, AW401719, AA294911, AA151951, AA663896, AA263135, AA352603, AA243206, C03945, AA527306, AW351546, AW405194, AW402317, AW408434, AI696864, AW327936, AA160714, AA477506, AA190717, AA305941, AA367387, AI365653, AA366365, AA527504, AA158109, T68508, AA337991, AA372227, AA321294, AA159413, AL039796, AA100848, AW403872, AA112349, AA385962, AA581164, F00945, H98493, AA335761, AW402402, AA121088, AA127128, AA310808, AA100680,.T10924, T71226, AA401643, 834651, AA352534, AA058454, AA126083, AA227277, N42582, W24084, W24054, AA304603, H11697, AW402383, H23327, AA361916, AA220207, AW403793, . AA449607, F11628, AW402754, AA492406, AA503610, T55088, U15638, D83030, M84386, X96582, Y09156, L54059, M26432, X58536, U43337, M77775, U36492, U15640, U14756, AF035647, D64148, L76088, L20091, D83031, AJ010748, M99388, AF168611, X67818, X60251, U18789, AB032096, AB032097, AJ223282, X94482, U29480, U74387, U18661, U06835, D64149, .
233459, D64147, L42280, Y09058, M94052, M94053, M94051, U04243, L22649, L36318, L17005, X78343, X87841, X70857, Y10520, X60249, X60248, M24096, M28160, X94574, AB032091, AB032094, AB032095, U38976, AF033351, AF115464, 246810, AF076476, - , 280227, X99704, U83580, D64151, D83029, L20088, M77774, M77778, M77777, M77776,.
M24040, M24032, M24033, 215144, M84381, .
LT63653; D49552, D50854, AJ010749, 283247, X76189, X97321, U09853, M84173, M84174, . Y11843, X83394, X95410, M28207, M26712, M28206, M26430, AB036049, AB036050, AB036051, X94480, X87268, L76090, L42506, AJ242661, L76931, U58110, D38526, D64145, M11886, Y13029, U32660, M29865, D44499, D44501, U11262, U11266, M83193, M83191, M83194, L07950,1VI86403, AF118894, D85762, U03027, U03698, U03859, L11571, L11570, D50294, D50296, . D50299, D31816, L22027, M24039, M95530, L11666, L11603, L19937, X61709, U80945, AF016641, U30936, U36392, M84384, X73518, M24030; U38975, AJ250917, D49824, U05584, M28203, AJ238694, U15639, AB032093, AB032599, D83741, AJ002676, AJ002677, X86704, U34810, L76093, L76930, L76094, L76089, U16298, L75942, L76932, L76933, L36979, L76225, D25275, U74386, AB032933, D64146, D64152, D14343, U11261, U11265, U17572, M83195, U05577, U50837, L20089, E02884, D85761, Y08995, X90390, X91749, U06862, M24036, L04696, L09736, M24034, L04695, M84694, X61710, X61706, U17107, U21052, U30904, U29083, L41353, L42281, AF170577, L40599, X81363, M84380, M84385, U88407, X82122, M28172, U06695, U41420, AF115462, L38251, X70856, 222651, M21963, U59654, M28204, AJ251755, 246809, AB032598, U18660, AB030575, AJ002675, X99735, AJ223602, .. AJ003063, U32678, U59647, -.- D50852, M26429, AJ272049, 272007, AB008136, AJ237703, 279751, 247377, U07230, D49820, D50710, D44500, Y09118, Y13567, M83192, U05576, X64454, D50290, D50293, U04245, U21053, U29057, ~AC004204, and AC004204.
HRODF07 117 12220931 - 129915 - AW238558, AI278521, AW238227, AW084017, AW265370, AW440242, AW238497, AW238306, AW238292, AW082445, AI355144, AW082458, AW238597, AW079466, AW366290, AW381175, AW366622, AW276887, AW270776, AW438489, AW270547, AW270610, AW438542, AI821931, AW270648, AW265248, AA610773, AW276814, AW277207, AA729444, AW276866, AW270597, AI732543, AW276913, AW2651.12, AW26517.0, AW438551, AW270550, AA501809, AW276884, AJ243667, AF077374, and AI355145.
Code Description issue Organ Cell DiseaseVector Line .
AR022 a Heart a Heart AR023 a Liver a Liver AR024 a mammary glanda mammar land AR025 a Prostate a Prostate AR026 a small intestinea small intestine AR027 a Stomach a Stomach AR028 Blood B cells Blood B cells .
AR029 Blood B cells Blood B cells activated activated AR030 Blood B cells Blood B cells resting restin AR031 Blood T cells Blood T cells activated activated AR032 Blood T cells Blood T cells resting resting AR033 brain brain AR034 breast breast . ' AR035 breast cancer breast cancer AR036 Cell Line CAOV3Cell Line AR037 cell line PA-1 cell line AR038 cell line transformedcell line transformed AR039 colon colon AR040 colon (9808co65R)colon (9808co65R) AR041 colon (9809co15)colon (9809co15) AR042 colon cancer colon cancer AR043 colon cancer colon cancer (9808co64R) (9808co64R) AR044 colon cancer colon cancer 9809co14 9809co14 AR045 corn clone S corn clone AR046 com clone 6 corn clone AR047 com clone2 corn clone2 AR048 corn clone3 corn clone3 AR049 Corn Clone4 Corn Clone4 ' ARO50 Donor II B CellsDonor II
24hrs B Cells 24hrs ARO51 Donor II B CellsDonor II
72hrs B Cells 72hrs AR052 Donor II B-CellsDonor II
24 hrs. B-Cells ' hrs.
AR053 Donor. II B-CellsDonor II
72hrs B-Cells 72hrs AR054 Donor II RestingDonor II
B Cells Resting B
Cells ARO55 Heart Heart AR056 Human Lung (clonetech)Human Lung (clonetech) AR057 Human Mammary Human Mammary (clontech) (clontech) -- AR058 Human Thymus ' Human Thymus (clonetech) (clonetech) AR059 Jurkat (unstimulated)Jurkat (unstimulated) AR060 Kidne Kidne AR06.1Liver Liver AR062 Liver(Clontech)Liver(Clontech) AR063 Lymphocytes Lymphocytes chronic lymphocytic chronic lymphocytic leukaemia leukaemia AR064 Lymphocytes Lymphocytes diffuse large B cell lymphomadiffuse large B cell lymphoma AR065 Lymphocytes Lymphocytes follicular lym homa follicular 1 m homa AR066 normal breast normal breast AR067 Normal Ovarian Normal Ovarian (4004901) ' (4004901) AR068 Normal Ovary Normal Ovary AR069 Normal Ovary Normal Ovary AR070 Normal Ovary Normal Ovary 98066005 ..
AR071 Ovarian Cancer Ovarian Cancer AR072 Ovarian Cancer Ovarian Cancer (97026001)' (97026001) -AR073 Ovarian Cancer Ovarian Cancer ' .
(97076029) (97076029) AR074 Ovarian Cancer Ovarian Cancer (98046011) (98046011) AR075 Ovarian Cancer Ovarian Cancer (98066019) (98066019) AR076 Ovarian Cancer - Ovarian ~ ~ Cancer (98076017) (98076017) AR077 Ovarian Cancer Ovarian Cancer (98096001) (98096001) AR078 ovarian cancer ovarian cancer AR079,Ovarian Cancer Ovarian Cancer AR080 Ovarian Cancer Ovarian Cancer AR081 Ovarian Cancer Ovarian Cancer AR082 ovarian cancer ovarian cancer ' AR083 Ovarian Cancer Ovarian Cancer AR084 Ovarian Cancer Ovarian Cancer AR085 Ovarian Cancer Ovarian Cancer ._....- 98076045 ..
_ AR086 ovarian cancer ovarian cancer AR087 Ovarian Cancer Ovarian Cancer AR088 Ovarian cancer Ovarian cancer 3rd C00 3rd AR089 Prostate Prostate AR090 Prostate (clonetech)Prostate (clonetech) AR091 rostate cancer rostate cancer AR092 prostate cancer,#15176prostate cancer #15176 AR093 prostate cancerprostate #15509 cancer #15509 AR094 prostate cancerprostate #15673 cancer #15673 AR095 Small IntestineSmall Intestine (Clontech) (Clontech) AR096 S Teen S leen AR097 Thymus T cells Thymus T
activated cells activated AR098 Thymus T cells Thymus T
resting cells restin AR099 Tonsil Tonsil AR100 Tonsil geminal . Tonsil center geminal -centroblast center centroblast AR101 Tonsil germinalTonsil germinal center B center B
cell cell AR102 Tonsil lym h Tonsil 1 node m h node AR103 Tonsil memory Tonsil memory B cell B
cell AR104 Whole Brain Whole Brain AR105 Xeno raft ES-2 Xeno raft AR106 Xeno raft SW626Xeno raft H0004 Human Adult Human Adult Spleen Uni-ZAP
Spleen S leen XR
H0008 Whole 6 Week Uni-ZAP
Old XR
Emb o.
H0009 Human Fetal Uni-ZAP
Brain XR
H0011 Human Fetal Human Fetal Kidne Uni-ZAP
Kidne Kidne XR
H0012 Human Fetal Human Fetal Kidne Uni-ZAP
Kidne Kidne XR
H0013 ' Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old XR
Embryo Emb o H0014 Human Gall BladderHuman Gall Gall Bladder Uni-ZAP
Bladder XR
H0015 Human Gall Bladder,Human Gall Gall Bladder Uni-ZAP
fraction II Bladder - XR
' ' H0020 Human HippocampusHuman Brain Uni-ZAP
Hi ocam us XR
H0024 Human Fetal Human Fetal Lun Uni-ZAP
Lun~ III Lun XR
H0025 Human Adult Human Adult Lymph Lambda Lymph L m h Node Node ZAP II
Node H0028 Human Old Ova Human Old Ovac Bluescri Ovar t H0030 Human Placenta Uni-ZAP
XR
H0031 Human Placenta Human PlacentaPlacenta Uni-ZAP
XR
H0032 Human Prostate Human ProstateProstate Uni-ZAP
XR
H0033 Human Pituitar Human Pituitary Uni-ZAP
XR
H0036 Human Adult Human Adult Small Uni-ZAP
Small Small Int. XR
r Intestine Intestine H0038 Human Testes Human TestesTestis Uni-ZAP
XR
H0039 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor Tumor XR
H0040 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor Tumor XR
H0041 Human Fetal Human Fetal Bone Uni-ZAP
Bone Bone XR
H0042 Human Adult Human Adult Lung Uni-ZAP
Pulmonary Pulmona XR
H0045 Human Esophagus,Human Esophagus,Esophagus diseaseUni-ZAP
Cancer cancer XR
H0046 Human EndometrialHuman EndometrialUterus diseaseUni-ZAP
Tumor Tumor XR
H0048 Human Pineal Human Pineal Uni-ZAP
Gland Gland XR
H0050 Human Fetal Human Fetal Heart Uni-ZAP
Heart Heart XR
H0051 Human HippocampusHuman Brain Uni-ZAP
Hi ocam us XR
H0052 Human CerebellumHuman CerebellumBrain ~ Uni-ZAP
XR
H0053 Human Adult Human Adult Kidney Uni-ZAP
Kidney Kidne XR
H0056 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein, Vein Endothelialvein XR
Endo, remake Cells H0057 Human Fetal Uni-ZAP
S Teen XR
H0059 Human Uterine Human UterineUterus diseaseLambda Cancer Cancer ZAP II
, H0060 Human Macro Human Macro Blood Cell Bluescri hake ha a Line t H0063 Human Th mus Human Th Th mus Uni-ZAP
mus XR
H0068 Human Skin TumorHuman Skin Skin diseaseUni-ZAP
Tumor XR
H0069 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells T-Cells Line XR
H0076.Human Membrane Human MembraneBlood Cell Uni-ZAP
Bound Bouiid Pol Line XR
Pol comes comes H0081 Human Fetal Human Fetal Skin Uni-ZAP
Epithelium Skin XR
(Skin) H0083 HUMAN JURKAT Jurkat Cells Uni-ZAP
MEMBRANEBOUND XR
POLYSOMES
H0085 Human Colon Human Colon Lambda ZAP II
H0086 Human epithelioidEpithelioid Sk Muscle diseaseUni-ZAP
sarcoma Sarcoma, XR
muscle H0087 Human Th mus Human Th Bluescri mus t H0090 Human T-Cell T-Cell L T-Cell diseaseUni-ZAP
L m homa m homa XR
H0099 Human Lung Cancer,Human Lung Lung pBluescript subtracted Cancer HO100 Human Whole.SixHuman Whole Embryo Uni-ZAP
Week Six XR
Old Embr o Week Old Embr o HO101 Human 7 Weeks Human Whole Embryo Lambda Old _ ~ 7 ZAP II
Emb o, subtractedWeek Old Emb o H0102 Human Whole Human Whole Embryo Bluescript 6 Week Six p Old Embr o (II),Week Old subt Embryo H0105 Human Fetal Human Fetal Heart ' pBluescript Heart, Heart subtracted -H0109 Human Macrophage,Macrophage Blood Cell pBluescript Line subtracted HOl Human Placenta,Human PlacentaPlacenta pBluescript l l subtracted H0112 Human ParathyroidHuman ParathyroidParathyroid pBluescript Tumor, subtractedTumor H0116 Human Thymus Human ThymusThymus pBluescript Tumor, subtracted Tumor H0123 Human Fetal Human Fetal Brain Uni-ZAP
Dura Mater Dura XR
Mater H0124 Human Human Sk Muscle diseaseUni-ZAP
XR
Rhabdom osarcomaRhabdom osarcoma H0125 Cem cells cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line XR
treated Treated Cem, Jurkat, Raji, and Su t H0130 LNCAP untreatedLNCAP Cell -ProstateCell Uni-ZAP
Line Line XR
H0131 LNCAP + o.3nM LNCAP Cell Prostate Cell Uni-ZAP
81881 Line Line XR
H0134 Raji Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line XR
treated Treated Cem,, Jurkat, Ra i, and Su t H0135 Human Synovial Human SynovialSynovium Uni-ZAP
Sarcoma XR
Sarcoma H0139 Activated T-Cells,Activated Blood Cell Uni-ZAP
4 hrs. T-Cells Line XR
H0141 Activated T-Cells,Activated Blood Cell Uni-ZAP
12 hrs. T-Cells Line XR
H0144 Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early XR
Stage Human Stage Human H0149 7 Week Old EarlyHuman Whole Embryo Uni-ZAP
Stage 7 XR
Human, subtractedWeek Old Emb o H0150 Human E idid E idid mis Testis Uni-ZAP
mus XR
H0156 Human Adrenal Human AdrenalAdrenal diseaseUni-ZAP
Gland XR
Tumor Gland Tumor Gland H0159 Activated T-Cells,Activated Blood Cell Uni-ZAP
8 hrs., T-Cells Line XR
libation 2 H0161 Activated T-Cells,Activated Blood Cell Uni-ZAP
24 hrs., T-Cells Line XR
ligation 2 H0163 Human Synovium Human SynoviumSynovium Uni-ZAP
XR
H0165 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer, XR
Sta a B2 Cancer, sta a B2 H0169 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer, XR
Stage C fractionCancer, sta a C
H0170 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old XR
Human Earl 5ta -a Human H0171 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old , XR
Human, II Early Stage Human H0178 Human Fetal Human Fetal Brain Uni-ZAP
Brain Brain XR
H0179 Human Neutro Human NeutroBlood Cell Uni-ZAP
hil hil Line XR
H0180 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer Breast Cancer H0181 Hnman Primary Human PrimaryBreast diseaseUni-ZAP
~ Breast I ~ XR
Cancer Breast Cancer H0182Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer Breast Cancer H0184Human Colon Human Colon Liver diseaseLambda Cancer, ZAP II
metasticized Cancer, metasticized to live to liver H0187Restin~ T-Cell T-Cells Blood Cell Lambda Line ZAP II
H0188Human Normal Human NormalBreast Uni-ZAP
Breast XR
Breast H0189Human Resting Human Blood Cell Uni-ZAP
Line XR
Macrophage Macrophage/Monoc ytes H0191Human Activated' Human Blood Cell Uni-ZAP
Line XR
Macrophage (LPS),Macrophage/Monoc, thiour tes H0192Cem Cells, cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line XR
treated, subtraTreated Cem, .lurkat, Ra'i, and Su t H0194Human Cerebellum,Human Cerebellum, Brain pBluescript subtracted H0196Human Cardiomyopathy,Human Heart Uni-ZAP
~ XR
subtracted Cardiom o ath H0204Human Colon Human Colon Colon pBluescript Cancer, subtracted Cancer H0205Human Colon Human Colon Colon pBluescript Cancer, differential Cancer H0208Early Stage Human Fetal Lung pBluescript Human Lung, Lurig subtracted H0212Human Prostate,Human ProstateProstate pBluescript subtracted H0213Human Pituitary,Human Pituitary Uni-ZAP
XR.
subtracted H0214Raji cells, CyclohexamideBlood Cell pBluescript cyclohexamide Line treated, subtractedTreated Cem, Jurkat, Ra'i, and Su t H0224Activated T-Cells,Activated Blood Cell Uni-ZAP
12 hrs, T-Cells Line XR
subtracted H0225Activated T-Cells,Activated Blood Cell Uni-ZAP
l2hrs, T-Cells Line XR
, differentiall ex ressed H0231Human Colon, Human Colon Bluescri subtraction t H0235Human colon Human Colon Liver pBluescript cancer, metaticized Cancer, metasticized, to liver, subtraction to liver H0244Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old XR
Embr o, subtractedEmbryo H0247Human Membrane Human MembraneBlood Cell Uni-ZAP
Bound Line XR
- Polysomes- EnzymeBound Polysomes -Subtraction H0250Human ActivatedHuman Monocytes Uni-ZAP
' XR
Monoc tes H0251Human ChondrosarcomaHuman Cartilage diseaseUni-ZAP
XR
Chondrosarcoma H0252Human OsteosarcomaHuman Bone -- diseaseUni-ZAP
-w XR
-Osteosarcoma H0253 Human adult Human Adult Testis Uni-ZAP
testis, large Testis XR
inserts H0254 Breast Lymph Breast LymphLymph Uni-ZAP
node cDNA Node Node XR
library H0255 breast lymph Breast LymphLymph Lambda node CDNA Node Node ZAP II
librar H0257 HL-60, PMA 4H HL-60 Cells,Blood Cell Uni-ZAP
PMA Line XR
stimulated H0261 H. cerebellum, Human CerebellumBrain Uni-ZAP
Enzyme XR
subtracted H0263 human colon Human Colon Colon diseaseLambda cancer ZAP II
Cancer H0264 human tonsils Human TonsilTonsil Uni-ZAP
XR
H0265 Activated T-CellT-Cells Blood Cell Uni-ZAP
Line XR
( l2hs)/Thiouridine (abelledEco H0266 Human MicrovascularHMEC Vein Cell Lambda Line ZAP II
Endothelial Cells, fract.
A
H0268 Human UmbilicalHUVE Cells UmbilicalCell Lambda Vein Line ZAP II
Endothelial vein Cells, fract.
A
H0271 Human Neutrophil,Human NeutrophilBlood Cell Uni-ZAP
- Line XR
Activated Activated H0274 Human Adult Human Adult Spleen Uni-ZAP
Spleen, XR
fractionII S Teen H0280 K562 + PMA (36 K562 Cell cell lineCell ~ ZAP Ex hrs) line Line ress H0284 Human OB MG63 Human Bone Cell ~ Uni-ZAP
control Line XR
fraction I Osteoblastoma MG63 cell line H0286 Human OB MG63 Human Bone Cell Uni-ZAP
treated Line XR
( 10 nM E2) Osteoblastoma .
fraction I
MG63 cell line H0290 Human OB HOS Human Bone Cell Uni-ZAP
treated Line XR
( 1 nM E2) fractionOsteoblastoma I HOS
cell line H0292 Human OB HOS Human Bone Cell Uni-ZAP
treated Line XR
( 10 nM E2) Osteoblastoma fraction I H05 cell line H0294 Amniotic Cells Amniotic Placenta Cell Uni-ZAP
- TNF Cells - ' Line XR
induced TNF induced H0300 CD34 positive CD34 PositiveCord Blood ZAP Express cells (Cord Cells Blood) H0305 CD34 positive CD34 PositiveCord Blood ZAP Express . cells (Cord Cells Blood) H0306 CD34 depleted CD34 DepletedCord Blood ZAP Express Buffy Coat (Cord Blood) Buffy Coat (Cord Blood) H0309 Human Chronic Synovium, Synovium diseaseUni-ZAP
Synovitis Chronic XR
Synovitis/
Osteoarthritis H0316 HUMAN STOMACH Human StomachStomach Uni-ZAP
XR
H0318 HUMAN B CELL Human B CellLymph . diseaseUni-ZAP
' Node XR
LY MPHOMA Lymphoma H0327human corpus Human CorpusBrain Uni-ZAP
colosum , Callosum XR
H0328human ovarian Ovarian CancerOvar ~ diseaseUni-ZAP
cancer XR
H0329DermatofibrosarcomaDermatofibrosarcom. Skin diseaseUni-ZAP
Protuberance a Protuberans XR
H0331Hepatocellular HepatocellularLiver diseaseLambda Tumor Tumor ZAP II
H0333HemangiopericytomaHemangiopericytomBlood diseaseLambda a vessel ZAP II
H0334Kidne cancer Kidne CancerKidne diseaseUni-ZAP
XR
H0341Bone Marrow Bone Marrow Bone MarrowCell Uni-ZAP
Cell Line Cell Line XR
(RS4;11) Line RS4;11 H0342Lin ual G rus Lin ual G Brain Uni-Za rus XR
H0343stomach cancer Stomach Cancer diseaseUni-ZAP
(human) - XR
S383A (human) H0344Adipose, tissueAdipose - Uni-ZAP
(human) 6825A XR
(human) H034SSKIN Skin - 4000868HSkin Uni-ZAP
XR
H0349human adult Human Adult Liver pCMVSport liver cDNA Liver 1 Libra ' H0351Glioblastoma GlioblastomaBrain diseaseUni-ZAP
XR
H0352wilm"s tumor Wilm"s Tumor diseaseUni-ZAP
XR.
H0354Human Leukoc Human LeukocBlood Cell CMVS ort tes tes Line 1 .
H0355Human Liver Human Liver, ~ pCMVSport normal Adult 1 H0361Human rejected Human Rejected diseasepBluescript ' kidney Kidne ' -H0366I~428 cell lineL428 ZAP Ex ress H0369H. Atrophic Atrophic Uni-ZAP
Endometrium Endometrium XR
and m ometrium H0370H. Lymph node Lymph node diseaseUni-ZAP
breast with XR
Cancer Met. Breast Cancer H0372Human Testes Human TestesTestis CMVS ort H0373Human Heart Human Adult Heart CMVS ort Heart 1 H0375Human Lun~ Human Lun CMVS ort H0376Human Spleen Human Adult Spleen pCMVSport . S Teen 1 H0388Human Rejected Human Rejected. diseasepBluescript Kidney, Kidne 704 re-excision H0390Human Amygdala Human Amygdala diseasepBluescript De cession, De cession re-excision H0391H. Meniingima, Human Meningimabrain p5 ortl H0392H. Meningima, Human Meningimabrain pSportl Ml H0393Fetal Liver, Human Fetal Liver Bluescri subtraction Liver t II
H0394A-14 cell line Redd-Sternber~ ZAP Ex cell ' ress H0395AI-CELL LINE Redd-Sternber ZAP Ex cell ress H0396LI Cell line ' Redd-SternberQ ZAP Ex cell ress H0402CD34 depleted CD34 DepletedCord Blood ZAP Express Buffy Coat Buffy Coat . (Cord (Cord Blood), Blood) re-excision ' __. H0403H. Umbilical HUVE Cells UmbilicalCell Uni-ZAP
._. I Vein Line XR
.
_ __.~
- Endothelial vein Cells, IL4 induced H0409 H. Striatum Human Brain,Brain pBluescript Depression, subtracted Striatum Depression H0411 H Female Bladder,Human FemaleBladder pSportl Adult ~
Adult Bladder H0412 Human umbilicalHUVE Cells UmbilicalCell pSportl vein Line, endothelial vein cells, IL-4 induced H0413 Human UmbilicalHUVE Cells UmbilicalCell pSportl Vein Line Endothelial vein Cells, uninduced H0415 H. Ovarian Tumor,Ovarian Tumor,Ovary diseasepCMVSport II, 2.0 OV5232 ~ OV5232 $0416 Human Neutrophils,Human NeutrophilBlood Cell ~ pBl,uescript - Line Activated, re-excisionActivated ' H0421 Human Bone Marro4v,Bone Marrow pBluescript re-excision H0422 T-Cell PHA 16 T-Cells Blood Cell 5 ortl hrs Line H0423 T-Cell PHA 24 T-Cells Blood Cell S ortl hrs ' Line H0424 Human Pituitary,Human Pituitary Bluescript subt IX
H0427 Human Adipose Human Adipose, pSportl left hi ti oma H0428 Human Ovary Human Ovary Ovary pSportl Tumor H0431 H. Kidney Medulla,Kidney medullaKidney pBluescript re-excision H0433 Human UmbilicalHUVE Cells UmbilicalCell pBluescript Vein Line Endothelial vein cells, frac B, re-excision H0434 Human Brain, Human Brain, pBluescript striatum, re-excision Striatum H0435 Ovarian Tumor Ovarian Tumor,Ovary pCMVSport 10-3-95 2.0 H0436 Restino T-Cell T-Cells Blood Cell S ortl Libr ,II Line H0437 H Umbilical HUVE Cells UmbilicalCell Lambda Vein Line ZAP II
Endothelial vein .
Cells, frac A, re-excision H0438 H. Whole Brain Human Whole ZAP Express #2, re- Brain excision #2 H0439 Human Eosino Eosino hits Bluescri hits t H0~I4.1H. Kidney Cortex,Kidney cortexKidney pBluescript subtracted H0442 H. Striatum Human Brain,Brain pBluescript Depression, , subt II ~ Striatum De cession H0444 Spleen metasticSpleen, MetasticSpleen diseasepSportl melanoma malignant melanoma H0445 Spleen, ChronicHuman Spleen,Spleen diseasepSportl CLL
1 m hoc tic leukemia H0455 H. Striatum Human Brain,Brain pBluescript Depression, subt Striatum De cession H0457 Human Eosino Human Eosino- - S ortl hits hits H0459 CD34+cells, CD34 positive pCMVSport II, cells 2.0 H0477 Human Tonsil, Human TonsilTonsil S ortl Lib 3 H0478 Salivary Gland,Human SalivarySalivary pSportl Lib 2 Gland Qland -H0483 Breast Cancer Breast Cancer pSportl cell line, Cell MDA 36 line, MDA
H0484 Breast Cancer Breast Cancer pSportl Cell line, Cell angiogenic line, Angiogenic, H0485 Hodgkin"s LymphomaHodgkin"s ' diseasepCMVSport I 2.0 Lymphoma I
H0486 Hodgkin"s LymphomaHodgkin"s diseasepCMVSport II 2.0 L m homa II
H0487 Human Tonsils, Human Tonsils CMVS ort lib I 2.0 H0488 Human Tonsils, Human Tonsils CMVS ort Lib 2 2.0 H0489 Crohn"s DiseaseIleum Intestine diseaseS ortl H0492 HL-60, RA 4h, HL-60 Cells,Blood Cell Uni-ZAP
Subtracted RA Line XR
stimulated for 4H
H0494 Keratinoc to Keratinocyte CMVS ort 2.0 H0497 HEL cell line HEL cell HEL , pSportl line ' 92.1.7 H0505 Human AstrocyteHuman Astrocyte pS ortl H0506 Ulcerative ColitisColon Colon pSportl H0509 Liver, HepatomaHuman Liver,Liver diseasepCMVSport 3.0 He atoma, anent 8 H0510 Human Liver, Human Liver,Liver pCMVSport normal 3.0 normal, patient # 8 H0518 pBMC stimulatedpBMC stimulated pCMVSport w/ poly 3.0 I/C with of I/C
H0519 NTERA2, controlNTERA2, pCMVSport 3.0 ' Teratocarcinoma cell line H0520 NTERA2 + retinoicNTERA2, ~ pSportl acid, 14 days Teratocarcinoma cell line H0521 Primary DendriticPrimary Dendritic pCMVSport Cells, 3.0 lib 1 cells H0522 Primary DendriticPrimary Dendritic pCMVSport 3.0 cells,frac 2 cells H0529 Myoloid ProgenitorTF-1 Cell pCMVSport Cell Line; 3.0 Line Myoloid progenitor cell line H0538 Merkel Cells Merkel cellsL m h S ortl node H0539 Pancreas Islet Pancreas Pancreas diseasepSportl Cell Tumor Islet Cell Tumour H0542 T Cell hel er Hel er T CMVS ort I cell 3.0 H0543 T cell hel er Hel er T CMV S ort II cell 3.0 H0544 Human endometrialHuman endometrial pCMVSport . 3.0 stromal cells stromal cells H0545 Human endorrietrialHuman endometrial pCMVSport 3.0 stromal cells-treatedstromal cells-treated with ro~esterone with roe H0546Human endometrialHuman endometrial pCMVSport ~ 3.0 stromal cells-treatedstromal cells-treated with estradiol with estra H0547NTERA2 teratocarcinomaNTERA2, pSportl cell line+retinoicTeratocarcinoma acid (14 da s) cell line H0549H. Epididiymus,Human ' Uni-ZAP
caput & XR
corpus Epididiymus, caput and co us H0550H. Epididiymus,Human ' Uni-ZAP
cauda XR
Epididiymus, cauda H0551Human Thymus Human Thymus ' pCMVSport Stromal 3.0 Cells Stromal Cells H0553Human Placenta Human Placenta CMVS ort 3.0 H0555Rejected Kidney,Human RejectedKidney diseasepCMVSporf lib 4 3.0 Kidne -H0556Activated T- T-Cells Blood Cell Uni-ZAP
Line XR
cell( 12h)/Thiouridine-re-excision H0559HL-60, PMA 4H, HL-60 Cells,Blood Cell Uni-ZAP
re- PMA Line XR
excision ' stimulated H0560KMH2 KMH2 CMVS ort 3.0 H0561L428 L428 CMVS ort 3.0 H0567Human Fetal Human Fetal pCMVSport Brain, Brain 2.0 normalized A5002F
H0570Human Fetal Human Fetal pCMVSport Brain, ~ Brain 2.0 normalized C500H
H0571Human Fetal Human Fetal pCMVSport Brain, Brain 2.0 normalized C500HE
H0574Hepatocellular HepatocellularLiver diseaseLambda Tumor; re- ZAP II
- excision Tumor _ H0575Human Adult Human Adult Lung Uni-ZAP
. XR
Pulmonar ;re-excisionPulmonar H0576Resting T-Cell;T-Cells Blood Cell Lambda re- Line ZAP II
excision H0579Pericardium Pericardium Heart S ortl H0580Dendritic cells,Pooled dendritic pCMVSporf3.0 pooled cells H0581Human Bone Marrow,Human Bone Bone Marrow pCMVSport 3.0 treated ' Marrow H0583B Cell 1 m homaB Cell L B Cell diseaseCMVS ort m homa 3.0 H0584Activated T-cells,Activated Blood Cell Uni-ZAP
24 T=Cells Line XR
hrs,re-excision H0585Activated T-Cells,l2Activated Blood Cell Uni-ZAP
T-Cells Line XR
hrs,re-excision H0586Healing groin healing groingroin diseasepCMVSport wound, 6:5 3.0 hours post incisionwound, 6.5 hours ost incision H0587Healing groin Groin-2/19/97groin diseasepCMVSport wound; 7.5 3.0 hours ost incision H0590Human adult Human Adult Small Uni-ZAP
small 5ma11 Int. XR
intestine,re-excisionIntestine -- Human T-cell T-Cell L T=Cell diseaseUni-ZAP
H0591 m homa XR
lym homa;re-excision H0592 Healing groin HGS wound diseasepCMVSport wound - healing 3.0 zero hr post-incisionproject; ' abdomen (control) H0593 Olfactory -.Olfactory pCMVSport epithelium 3.0 epithelium;nasalcavityfrom roof of left nasal cacit H0594 ' Human Lung Human Lung Lung diseaseLambda Cancer;re- Cancer ZAP II
excision H0595 Stomach cancer Stomach Cancer diseaseUni-ZAP
~ - XR
(human);re-excision5383A (human) H0596 Human Colon Human Colon Colon Lambda Cancer;re- ZAP II
excision Cancer H0597 Human Colon; Human Colon Lambda re-excision ZAP II
H0598 Human Stomach;re-Human StomachStomach Uni-ZAP
XR
excision H0599 Human Adult Human Adult Heart Uni-ZAP
Heart;re- Heart XR
excision . H0600Healing AbdomenAbdomen diseasepCMVSport 3.0 wound;70&90 min post incision H0601 Healing AbdomenAbdomen diseasepCMVSport 3.0 Wound;l5 days post incision H0604 Human Pituitary,Human Pituitary pBluescript re-excision H0606 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer;re-excisionBreast Cancer H0608 H. Leukoc tes, H.Leukoc CMVS ort control tes 1 H0615 Human Ovarian Ovarian CancerOvary diseaseUni-ZAP
Cancer XR
Reexcision H0616 Human Testes, Human TestesTestis Uni-ZAP
Reexcision XR
H0617 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer ReexcisionBreast Cancer H0618 Human Adult Human Adult Testis Uni-ZAP
Testes, Testis XR
Lar a Inserts, Reexcision H0619 Fetal Heart ' Human FetalHeart Uni-ZAP
Heart XR
H0620 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney; Kidney XR
Reexcision H0622 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor; XR
Reexcision Tumor .
H0623 Human UmbilicalHuman UmbilicalUmbilical . Uni-ZAP
Vein; XR
Reexcision Vein Endothelialvein Cells H0624 12 Week Early Twelve Week Embryo Uni-ZAP
Stage Old XR
Human II; ReexcisionEarly Stage Human H0625 Ku 812F Baso Ku 812F Baso S ortl hils Line hils H0626 Saos2 Cells; Saos2 Cell ~ pSportl Untreated' Line; , Untreated H0628 Human Pre-DifferentiatedHuman Pre- Uni-ZAP
XR
Adipocytes Differentiated Adi oc tes H0629 Human Leukoc Human Normalized CMVS ort te, control 1 #2 leukocyte H0630 Human Human Normalized , pCMVSport Leukocytes,normalizedleukocyte control #4 H063T Saos2, DexamethosomeSaos2 Cell pSportl Line;
Treated Dexamethosome Treated H0632 Hepatocellular HepatocellularLiver Lambda Tumor;re- ZAP II
excision Tumor H0633 Lung Carcinoma TNFalpha diseasePSportl A549 activated TNFalpha activatedA549--Lung Carcinoma H063q.Human Testes Human TestesTestis diseaseUni-ZAP
Tumor, re- XR
excision Tumor H0635 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells, T-Cells Line XR
re-excision H0638 CD40 activated.monocyteCD40 activated pSportl dendridic cellsmonocyte dendridic cells -H0640 Ficolled Human Ficolled Other Stromal Human Cells, UntreatedStromal Cells, -Untreated H0641 LPS activated LPS activated pSportl derived dendritic cellsmonocyte derived dendritic cells H0642 Hep G2 Cells, Hep GZ Cells Other lambda Libra H0643 He G2 Cells, . He G2 Cells Other PCR Libra H0644 Human Placenta Human PlacentaPlacenta Uni-ZAP
(re- XR
excision) H0645 Fetal Heart, Human Fetal Heart Uni-ZAP
re-excision Heart XR
H0646 Lung, Cancer Metastatic pS ortl (4005313 P
A3): Invasive squamous Poorly cell lung Differentiated carcinoma, _ -Lung poorly di Adenocarcinoma, ' H0647 Lung, Cancer Invasive diseasepSportl (4005163 poorly B7): Invasive, differentiated Poorly Diff. lung Adenocarcinoma,adenocarcinoma Metastatic H0648 Ovary, Cancer: Papillary - diseasepSportl (4004562 Cstic B6) Papillary neoplasm Serous of low Cystic Neoplasm,malignant Low potentia Malignant Pot H0649 Lung, Normal: Normal Lung pSportl (4005313 Bl) H0650 B-Cells B-Cells CMVS ort 3.0 H0651 Ovary, Normal: Normal Ovary ~ pSportl (9805C040R) H0652 Lung, Normal: Normal Lung pSportl (4005313 B1) H0653 5tromal Cells Stromal Cells pSportl H0654 Lung, Cancer: Metastatic Other (4005313 A3) Invasive Squamous Poorly-~ cell lung differentiated Carcinoma Metastatic poorly lung adenoc dif ' H0656 B-cells (unstimulated)B-cells pSportl (unstimulated) H0657 B-cells (stimulated)B-cells (stimulated) S ortl H0658 Ovary, Cancer 9809C332- Ovary diseasepSportl Poorly &
(9809C332): differentiateFallopian Poorly differentiated Tubes adenocarcinoma H0659 Ovary, Cancer Grade II Ovary diseasepSportl Papillary (15395A1F): Carcinoma, Grade II Ovary Pa illar Carcinoma H0660 Ovary, Cancer: Poorly differentiated~ diseasepSportl (15799A1F) Poorlycarcinoma, ovary differentiated carcinoma H0661 Breast, Cancer:Breast cancer diseasepSportl (4004943 ~
A5) H0662 Breast, Normal:Normal BreastBreast pSportl -(400552282) #4005522(B2) H0663 Breast, Cancer:Breast CancerBreast diseasepSportl (4005522 -A2) #4005522(A2) H0664 Breast, Cancer:Breast CancerBreast diseasepSportl ~
(9806C012R) ' H0665 Stromal cells Stromal cells S ortl 3.88 3.88 H0666 Ovary, Cancer: Ovarian Cancer, diseasepSportl (4004332 A2) Sample .
#4004332A2 -H0667 5tromal cells(HBM3.18).Stromal pSportl cell(HBM
3.18) H0668 stromal cell stromal cell p5portl clone 2.5 clone 2.5 H0670 Ovary, Cancer(4004650Ovarian Cancer pSportl -A3): Well-Differentiated4004650A3 Micropapillary Serous Carcinoma H0671 Breast, Cancer:Breast Cancer- pSportl (9802C020E) Sample #
H0672 Ovary, Cancer: Ovarian Ovary pSportl (4004576 A8) Cancer(4004576A8) H0673 Human Prostate Human ProstateProstate Uni-ZAP
Cancer, .~ XR
Sta~e B2; re-excisionCancer, stage H0674 Human Prostate Human ProstateProstate U ni-ZAP
Cancer, XR
Sta a C; re-excissionCancer, stage C
H0675 Colon, Cancer: Colon Cancer pCMVSport 3.0 (9808C064R) 9808C064R
H0677 TNFR de enerateB-Cells PCRII
oliQo H0682 Serous Papillaryserous papillary pCMVSport 3.0 Adenocarcinoma adenocarcinoma , (9606G304SPA3B) H0683 Ovarian Serous Serous papillary pCMVSport Papillary 3.0 Adenocarcinoma adenocarcinoma, stage 3C
(9804601 H0684 Serous PapillaryOvarian Cancer-Ovaries ' pCMVSport 3.0 Adenocarcinoma 98106606 H0685 Adenocarcinoma Adenocarcinoma pCMV Sport of of 3.0 Ovary, Human Ovary, Human Cell Line, Cell # OVCAR-3 Line, # OVCAR-H0686 Adenocarcinoma Adenocarcinoma pCMVSport of of 3.0 Ovary, Human ~ Ovary, Cell Line Human Cell Line # SW-626 H0687 Human normal Human normal, Ovary pCMVSport 3.0 ovar (#96106215)ova (#96106215) H0688 Human Ovarian Human Ovarian, pCMVSport 3.0 Cancer(#98076017)cancer(#98076017), mRNA from Maura Ru H0689 Ovarian Cancer Ovarian Cancer, pCMVSport 3.0 #98066019 H0690 Ovarian Cancer,Ovarian Cancer,, pCMVSport # 3.0 97026001 #97026001 H0691 Normal Ovary, normal ovary, pCMVSport 3.0 #97106208 #97106208 H0693 Normal ProstateNormal Prostate pCMVSport 3.0 #ODQ3958EN Tissue #
H0694 Prostate gland Prostate prostate pCMVSport gland, 3.0 adenocarcinoma adenocarcinoma,gland mod/diff, gleason H0695 mononucleocytesmononucleocytes pCMVSport from 3.0 patient from patient at Shad Grove Hos it N0006 Human Fetal Human Fetal .
Brain Brain ~' Human Hippocampus,Human rescreened ~ Hi ocam us 50002 Monocyte activatedMonoc te-activatedblood Cell Uni-ZAP
Line XR
50003 Human OsteoclastomaOsteoclastomabone diseaseUni-ZAP
XR
50005 Heart Heart-left Heart CDNA
ventricle 50007 Early Stage Human Fetal Uni-ZAP
Human Brain Brain XR
50010 Human Amygdala Amygdala Uni-ZAP
XR
S0011 STROMAL - Osteoclastomabone diseaseUni-ZAP
XR
OSTEOCLASTOMA
50022 Human OsteoclastomaOsteoclastoma. ~ Uni-ZAP
XR
Stromal Cells Stromal Cells -unam lifted 50024 Human Kidney Human Kidney Medulla -unam lifted Medulla 50026 Stromal cell stromal cellBone Cell Uni-ZAP
TF274 marrow Line XR
S0027 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
serum Line XR
treated artery 50028 Smooth muscle,controlSmooth musclePulmanaryCell Uni-ZAP.
Line XR
ante S0029 brain stem Brain stem brain Uni-ZAP
XR
50031 5 final cord S final cords final Uni-ZAP
cord XR
50032 Smooth muscle-ILbSmooth musclePulmanaryCell Uni-ZAP
. ' Line XR
induced arter - -- ' ~ - ' 50036 Human SubstantiaHuman Substantia Uni-ZAP
Nigra XR
NiQra 50037 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
ILlb Line XR
induced ~ artery 50038 Human Whole Human Whole - . ZAP Express Brain #2 - Brain Olioo dT > l.SKb#2 S0040 Adipocytes Human Adipocytes Uni-ZAP
XR
from Osteoclastoma SOO~kIProstate BPH rostate BPH Prostate diseaseUni-ZAP
~ XR
50045 Endothelial Endothelial endothelialCell Uni-ZAP
cells-control cell Line XR
cell-lun S0046 Endothelial-inducedEndothelial endothelialCell Uni-ZAP
' ~ cell Line XR
cell-lung S0049 Human Brain, Human Brain, Uni-ZAP
Striatum XR
Striatum 50050 Human Frontal Human Frontal diseaseUni-ZAP
Cortex, XR
Schizophrenia Cortex, , Schizo hrenia 50051 Human Human ' diseaseUni-ZAP
XR
Hypothalmus,SchizophrenHypothalamus, is Schizo hrenia S0052 neutro hils human neutroblood Cell Uni-ZAP
control hits Line XR
S0053 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS L_ XR
ine induced induced S0106 STRIATUM BRAIN diseaseUni-ZAP
XR
DEPRESSION
S0114 Aner is T-cell AnerQic T-cell Cell ~ ~ Uni-ZAP
Line XR
S0116 Bone marrow Bone marrow Bone Uni-ZAP
marrow XR
50118 Smooth muscle Smooth musclePulmanaryCell Uni-ZAP
control 2 Line XR
artery' 50122 Osteoclastoma-normalizedOsteoclastomabone diseasepBluescript A
50126 Osteoblasts Osteoblasts Knee Cell Uni-ZAP
Line XR
S0132 Epithelial-TNFaAirway Epithelial Uni-ZAP
and INF XR
induced 50134 A o totic T-cella o totic Cell Uni-ZAP
cells Line XR
SO1~0 eosino hil-II,Seosino hil lun Cell Uni-ZAP
induced Line XR
S0142 Macrophage-oxLDLmacrophage- blood Cell Uni-ZAP
Line XR
oxidized LDL
treated S01~14Macrophage (GM-CSFMacrophage LJni-ZAP
(GM- XR
treated) CSF treated) 50146 rostate-edited rostate BPH Prostate Uni-ZAP
XR
S0148 Normal ProstateProstate rostate Uni-ZAP
XR
50150 LNCAP rostate LNCAP Cell ProstateCell Uni-ZAP
cell line . Line Line XR
50152 PC3 Prostate PC3 prostate Uni-ZAP
cell line cell XR
line 50174 Prostate-BPH Human Prostate~ pBluescript subtracted II
BPH ' ' S0182 Human B Cell Human B- ' Uni-ZAP
8866 Cell 8866 XR
S0188 Prostate,BPH, Human Prostate diseasepSportl Lib 2 BPH
50190 Prostate BPH,LibHuman Prostate S ortl 2, subtracted BPH
S0192 Synovial FibroblastsSynovial pSportl Fibroblasts (control) 50194 Synovial by S novial S ortl oxia Fibroblasts S0196 Synovial IL-1/TNFSynovial pSportl Fibroblasts stimulated S0206 Smooth Muscle- Smooth musclePulmanaryCell pBluescript HASTE Line normalized arter 50210 Messan~ial cell,Messanoial S ortl frac 2 cell 50212 Bone Marrow Bone Marrow - p5portl Stromal Cell, untreatedStromal -Cell,untreated S0214 Human Osteoclastoma,Osteoclastomabone diseaseUni-ZAP
re- XR
excision S0216 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line XR
induced induced 50218 Apoptotic T-cell,apoptotic Cell Uni-ZAP
re- cells Line XR
excision S0222 H. Frontal H. Brain, Brain diseaseUni-ZAP
Frontal XR
cortex,epileptic;re-Cortex, Epileptic' excision S0242 Synovial FibroblastsSynovial p5portl Fibroblasts (Ill/TNF), subt 50250 Human OsteoblastsHuman OsteoblastsFemur diseaseCMVS ort II 2.0 50260 S final Cord, S final cords final ~ Uni-ZAP
re-excision cord XR
S0276 Synovial hypoxia-RSFSynovial Synovial pSportl fobroblasts subtracted (rheumatoid)tissue S0278 H Macrophage Macrophage ~ Uni-ZAP
(GM-CSF (GM- XR
treated), re-excisionCSF treated) 50280 Human Adipose Human Adipose Uni-ZAP
Tissue, XR
re-excision Tissue ~
50282 Brain Frontal Brain frontalBrain' Lambda Cortex, re- cortex ZAP II
excision 50294 Larynx tumor Larynx tumorLarynx,vocal diseasepSportl ' cord .
S0300 Frontal lobe,dementia;re-Frontal LobeBrain Uni-ZAP
XR
excision dementia/Alzheimer' 's S0306 Larynx normal Larynx normal pSportl #10 261-S0308 S leen/normal S Teen normal S ortl 50312 Human _ Human diseasep5portl osteoarthritic;fractionosteoarthritic II
cartila a S0314 Human Human diseasepSportl osteoarthritis;fractionosteoarthritic I
cartila a S0316 Human Normal Human Normal pSportl Cartila~e,FractionCartila a I
S0322 Siebben Pol Siebben Pol S ortl osis osis 50328 Palate carcinomaPalate carcinomaUvula diseaseS ortl 50330 Palate normal Palate normalUvula S ortl S0332 Pharynx carcinomaPharynx carcinomaHypopharynx 'pSportl ~
S0342 Adipocytes;re-excisionHuman Adipocytes , Uni-ZAP
from Osteoclastoma XR
S0344 Macrophage-oxLDL;macrophage- blood Cell Uni-ZAP
re- oxidized Line XR
excision LDL
treated S0346 Human Amygdala;re-Amygdala Uni-ZAP
excision XR
S0350 Phar nx CarcinomaPhar nx carcinomaH o har diseaseS ortl nx 50352 Larynx CarcinomaLar nx carcinoma disease5 ortl S0354 Colon Normal Colon NormalColon S ortl II
50356 Colon CarcinomaColon CarcinomaColon diseaseS ortl .
50358 Colon Normal Colon NormalColon S ortl III
S0360 Colon Tumor Colon Tumor Colon diseaseS ortl II' 50364 Human uadrice Quadrice 5 ortl s s muscle 50366 Human Soleus Soleus Muscle S ortl S0370 Larynx carcinomaIILarynx carcinoma diseaseSportl ' 50372 Larynx carcinomaLarynx carcinoma disease5portl III ' 50374 Normal colon Normal colon 5 ortl 50376 Colon Tumor Colon Tumor diseaseS ortl S0378 Pancreas normalPancreas ' pSportl PCA4 Normal No PCA4 No 50380 Pancreas Tumor Pancreas diseasepSportl PCA4 Tu Tumor PCA4 Tu S0382 Lar nx carcinomaLar nx carcinoma diseaseS ortl IV
S0384 Ton ue carcinomaTon~ue carcinoma disease5 ortl S0388 Human Human . diseaseUni-ZAP
Hypothalamus,schizophreHypothalamus, XR
nia, re-excisionSchizo hrenia 50390 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
control; arter Line XR
re-excision S0392 Salivary Gland Salivary pSportl gland;
normal 50400 Brain; normal Brain; normal S ortl - 50402 Adrenal Gland,normalAdrenal gland; ~ . pSportl normal S0404 Rectum normal Rectum, normal pSportl S0406 Rectum tumour Rectum tumour pSportl 50408 Colon, normal Colon, normal S ortl S0410 Colon, tumour Colon, tumour S ortl ' S0414 Hippocampus, Hippocampus, Other Alzheimer Alzheimer Subtracted Subtracted 50418 CHME Cell Line;treatedCHME Cell pCMVSport 5 Line; 3.0 hrs treated S0420 CHME Cell CHME Cell p5portl Line,untreated line, ~ untreatetd S0422 Mo7e Cell Line Mo7e Cell pCMVSport GM-CSF Line 3.0 treated (lng/ml)GM-CSF treated (1n /ml) 50424 TF-1 Cell Line TF-1 Cell pSportl GM-CSF Line Treated GM-CSF Treated S0426 Monocyte activated;Monocyte-activatedblood Cell Uni-ZAP
~ re- Line XR
excision S0428 Neutrophils human iieutrophilsblood Cell Uni-ZAP
control; re- Line XR
excision S0432 Sinus piniformisSinus piniformis pSportl Tumour Tumour 50434 Stomach Normal Stomach Normal~ diseaseS ortl S0436 Stomach Tumour Stomach Tumour~ diseaseS ortl 50438 Liver Normal Liver Normal pSportl MetSNo MetSNo 50440 Liver Tumour Liver Tumour S ortl Met 5 Tu S0442 Colon Normal Colon Normal S ortl S0444 Colon Tumor Colon Tumour~ diseaseS ortl S0446 Ton~ue Tumour Ton~ue Tumour S ortl S0452 Th mus Th mus S ortl 50456 Ton~ue Normal Tongue Normal - S ortl 50458 Thyroid Normal Thyroid normal pSportl (SDCA2 Nod S0460 Th roid Tumour Thyioid Tumour S ortl _50462Th roid Th roiditisTh roid Th 5 ortl roiditis S0466 Lar nx Tumor La nx Tumor diseaseS ortl S0468 Ea.h .926 cell Ea.h .926 S ortl line cell line S0470 Adenocarcinoma PYFD diseaseS ortl S0474 Human blood Platelets Blood Other platelets latelets S0665 Human Amygdala;Amygdala ' Uni-ZAP
re- . XR
excission S3012 Smooth Muscle Smooth musclePulmanaryCell pBluescript Serum artery Line Treated, Norm S3014 Smooth muscle, Smooth musclePulmanaryCell pBluescript serum arter Line induced,re-exc S6016 H. Frontal Cortex,H. Brain, Brain diseaseUni-ZAP
E ile tic ~ Frontal XR
Cortex, E
ile tic 56024 Alzheimers, Alzheimer"s/SpopgyBrain diseaseUni-ZAP
spongy than a XR
than a 56026 Frontal Lobe, Frontal LobeBrain ~ Uni-ZAP
Dementia dementialAlzheimer' XR
.
,s S6028 Human Manic Human Manic Brain diseaseUni-ZAP
- Depression de ression XR
Tissue ~ tissue T0002 Activated T-cellsActivated Blood Cell pBluescript T-Cell, Line SK-PBL fraction T0003 Human Fetal Human Fetal Luna Lun Bluescri t SK-T0004 Human White Human White Bluescri Fat Fat t SK-T0006 Human Pineal Human Pinneal pBluescript Gland Gland SK-T0008 Colorectal TumorColorectal diseasepBluescript Tumor SK-T0010 Human Infant Human Infant Other Brain Brain T0023 Human PancreaticHuman Pancreatic diseasepBluescript Carcinoma Carcinoma SK-T0040 HSC172 cells SA172 Cells Bluescri t SK-T0041 Jurkat T-cell Jurkat T-cell Bluescri Gl ' hase t SK-T0042 Jurkat T-Cell, Jurkat T-Cell Bluescri S Base Line t SK-T0048 Human Aortic Human Aortic _ pBluescript ' SK-Endothelium Endothilium T0049 Aorta endothelialAorta endothelial pBluescript cells + SK-TNF-a cells T0060 Human White Human White Bluescri Adi ose Fat t SK-T0067 Human Thyroid Human Thyroid Bluescri t SK-T0068 Normal Ovary, Normal Ovary,~ pBluescript SK-Premeno ausal Premeno ausal T0069 Human Uterus, Human Uterus, pBluescript normal SK-normal T0071 Human Bone MarrowHuman Bone pBluescript SK-Marrow T0082 Human Adult Human Adult Bluescri Retina Retina t SK-T0109 Human (HCC) pBluescript cell line SK-liver (mouse) metastasis, remake TO110 Human colon,.carcinoma pBluescript SK-(HCC) cell line, remake T0114 Human (Caco-2) pBluescript cell line, SK-adenocarcinoma, colon, remake L0002 Atrium cDNA
library Human heart L0004 ClonTech HL
1065a L0005 Clontech human aorta of A+ mRNA (#6572) L0021 Human adult (K.Okubo) L0022 Human adult lung 3"
directed MboI
cDNA .
L0040 Human colon'mucosa L0055 Human rom eloc to L0065 Liver HepG2 cell line.
L0105 Human aorta aorta polyA+
(TFu'iwara) L0109 Human brain brain cDNA
L0142 Human placenta placenta ~ ' cDNA
(TFu'iwara) L0157 Human fetal ~ brain brain (TFu'iwara) L0163 Human heart heart cDNA
(YNakamura) L0194 Human pancreaticpancreatic Patu cancer cancer cell line Patu 8988t 8988t L0351 Infant brain, _ BA, M13-Bento Soares .
derived L0352 Normalized infant BA, M13-brain, Bento Soares derived L0361 Stratagene ovary ovary Bluescript SK
(#937217) L0362 Stratagene ovarian . Bluescript cancer SK-(#937219) L0364 NCI CGAP_GC5 perm cell Bluescri tumor t SK-L0366 Strata ene schizoschizo hrenic Bluescri brain brain t SK-S11 S-11 frontal lobe L0369 NCI CGAP_AA1 adrenal adenomaadrenal Bluescri land t SK-L0370 Johnston frontalooled frontalbrain Bluescri cortex lobe t SK-L0371 NCI CGAP_Br3 breast tumorbreast Bluescri t SK-L0372 NCI CGAP Col2 colon tumor colon Bluescri t SK-L0373 NCI CGAP_Coll tumor colon Bluescri t SK-L0374 NCI CGAP_Co2 tumor colon Bluescript SK-L0375 NCI CGAP_ICid6 kidney tumorkidney Bluescript SK-L0376 NCI CGAP_Larl larynx lar nx Bluescri t SK-L0378 NCI CGAP_Lul lun tumor lun ~ Bluescri t 5K-L0381 NCI_CGAP_HN4 squamous pharynx Bluescript cell ~ SK-carcinoma L0382 NCI CGAP_Pr25 a ithelium rostate Bluescri (cell line) t SK-L0383 NCI_CGAP_Pr24 invasive prostate Bluescript tumor (cell SK-line) L0384 NCI CGAP_Pr23 rostate tumorrostate Bluescri t SK-L0385 NCI CGAP_Gasl oastric tumorstomach Bluescri t SK-L0386 NCI CGAP_HN3 squamous , tongue luescript cell SK-B
carcinoma from base of ton ue L0387 NCI CGAP_GCBO germinal tonsil Bluescript center B- 1 SK-cells L0393 B, Human Liver t1 1 tissue L0415 b4HB3MA CotB-HAP-Ft Lafmid BA
L0438 normalized infanttotal brain brain lafmid brain BA
cDNA
L0439 Soares infant 4vhole Lafmid brain 1NIB brain BA
L0455 Human retina retina eye lambda cDNA . gtl0 randomly primed sublibrar L0462.WATM1 - lambda ~tl l L0471 Human fetal Lambda heart, ZAP
Lambda ZAP Ex Ex ress ress L0475 KG1-a Lambda ' KG1-a Lambda Zap Zap Express cDNA Express library (Stratagene) L0477 HPLA CCLee placenta Lambda ZAP II
L0480 Stratagene cat#937212 Lambda ( 1992) , ZAP, pBluescript SK(-) L0483 Human ancreatic Lambda islet ZAPII
L0485 STRATAGENE Humanskeletal leg muscle Lambda skeletal musclemuscle ZAPII
cDNA
libra , cat.
#936215.
L0499 NCI CGAP_HSC2 stem cell bone pAMPI
34+/38+ marrow L0512 NCI_CGAP_Ov36 borderline ovary pAMPl ovarian carcinoma L0517 NCI CGAP_Prl AMP10 L0518 NCI CGAP_Pr2 AMP10 L0519 NCI CGAP Pr3 AMP10 L0520 NCI_CGAP_Alvl alveolar pAMPlO
rhabdomyosarcoma L0521 NCI_CGAP_Ewl Ewina"s sarcoma AMP10 L0526~NCI_CGAP_Prl2 metastatic pAMPlO
prostate , bonelesion L0527 NCI_CGAP_Ov2 ovar AMP10 L0528 NCI CGAP_Pr5 prostate pAMPlO
L0529 NCI_CGAP_Pr6 prostate pAMPlO
L0533 NCI_CGAP HSC1 stem cells bone marrow AMP10 L0534 Chromosome?Fetalbrain brain pAMPlO
Brain cDNA Library L0535 NCI_CGAP_Br5 infiltratingbreast pAMPlO
ductal carcinoma L0540 NCI_CGAP_PrlO invasive prostate pAMPlO
prostate tumor L0542 NCI_CGAP_Prl normal prostaticprostate pAMPlO
l a ithelial cells L0543 NCI_CGAP_Pr9 normal prostaticprostate pAMPlO
a ithelial cells L0545 NCI_CGAP_Pr4.l prostatic prostate pAMPlO
, intraepithelial neoplasia - high grade L0546 NCI_CGAP PrlB stroma rostate AMP10 L0547 NCI_CGAP_Prl6 tumor prostate pAMPlO
L0549 NCI_CGAP_HN10 carcinoma pAMPlO
in situ from retromolar tri one LO551 NCI_CGAP_HN7 noi'mal squamous pAMPlO
epithelium, floor of -mouth L0558 NCI_CGAP_Ov40 endometrioidovary pAMPlO
ovarian metastasis L0564 Jia bone marrowbone marrow Bluescri stroma stroma t L0565 Normal Human Bone Hip pBluescript Trabecular Bone Cells LO581 Strata~ene liver liver Bluescri (#937224) t SK
L0586 HTCDL1 pBluescript SK(-) L0588 Stratagene endothelial pBluescript cell SK-L0590 Stratagene fibroblast pBluescriptf SK-(#937212) L0591 Stratagene HeLa' pBluescript cell s3 SK-L0592 Stratagene hNT _ ~pBluescript neuron SK-(#937233) L0593 Stratagene pBluescript SK-neuroepithelium (#937231) L0594 Stratagene , , pBluescript SK-neuroepithelium L0595 Stratagene NT2 neuroepithelialbrain pBtuescript neuronal cells SK-recursor937230 -- ---L0596 Stratagene colon colon pBluescript SK-(#937204) L0597 Stratagene corneal cornea pBluescript stroma SK-(#937222) L0598 Morton Fetal cochlea ear Bluescri Cochlea t SK-L0599 Strataaene lun - IunQ Bluescri (#937210) t SK-L0600 Weizmann Olfactoryolfactory nose pBluescript epithelium SK-E ithelium L0601 Stratagene pancreas pancreas pBluescript SK-(#937208) L0602 Pancreatic Isletancreatic ' ancreas Bluescri islet t SK-L0603 Stratagene placenta placenta pBluescript , SK-(#937225) L0604 Stratagene muscle. muscle skeletal pBluescript muscle L0605 Stratagene fetalfetal spleenspleen pBluescript spleen , SK-' (#937205) L0606 NCI CGAP L m5 follicular 1 m h Bluescri I m homa node t SK-L0607 NCI_CGAP_Lym6 mantle cell lymph pBluescript node SK-I m homa L0608 Stratagene lunglung carcinomalung NCI-H69 pBluescript carcinoma SK-.
L0611 Schiller meningiomameningioma brain pBluescript SK-(Strata~ene) L0617 Chromosome 22 pBluescriptIIKS
exon L0619 Chromosome 9 pBluescriptIIKS
exon II
L0622 HMl pcDNAII
(Invitro en) L0623 HM3 pectoral pcDNAII
muscle (after mastectom ) (Invitro en) L0627 NCI CGAP Col bulk tumor colon CMV-SPORT2 -L0628 NCI CGAP Ovl ovar bulk ova CMV-SPORT2 tumor L0629 NCI_CGAP_Mel3 metastatic bowel pCMV-SPORT4 (skin melanoma rimar to bowel ) L0630 N.CI CGAP CNS1 substantia brain CMV-SPORT4 niQra L0631 NCI CGAP Br7 breast CMV-SPORT4 L0635 NCI_CGAP PNS dorsal root peripheral , pCMV-SPORT4 1 ganglion nervous system L0636 NCI-CGAP_Pitl four pooled brain pCMV-SPORT6 pituitary adenomas L0637 NCI_CGAP_Brn53 three pooledbrain pCMV-SPORT6 menin~iomas L0638 NCI_CGAP_Brn35 tumor, 5 brain pCMV-SPORT6 pooled.(see descri tion) L0639 NCI_CGAP_Bm52 tumor, 5 brain pCMV-SPORT6 pooled (see description) L0640 NCI_CGAP_Brl8 four pooled breast pCMV-SPORT6 high-grade tumors, ' ' ' including two rims L0641 NCI CLAP Col7 juvenile colon pCMV-SPORT6 I granulosa tumor .
L0642 NCI-CGAP_Col8 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0643 NCI_CGAP_Col9 moderately , colon pCMV-SPORT6 differentiated' adenocarcinoma L06a4 NCI_CGAP_Co20 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0646 NCI_CGAP_Col4 moderately- colon pCMV-SPORT6 differentiated adenocarcinoma L0647 NCI_CGAP_5ar4 five pooled connective pCMV-SPORT6 sarcomas, tissue including m xoid Ii -osarcoma L0648 NCI_CGAP_Eso2 ,squamous esophagus pCMV-SPORT6 cell -carcinoma "
L0649 NCI_CGAP_GU1 2 pooled genitourinary pCMV-SPORT6 high-grade transitionaltract cell tumors L0650 NCI CGAP_Kidl3 2 pooled kidney pCMV-Wilms" S PORT6 tumors, one primary and one metast L0651 NCI CGAP_Kid8 renal cell kidne CMV-SPORT6 tumor L0652 NCI_CGAP_Lu27 four pooled lung pCMV-SPORT6 poorly-differentiated adenocarcinomas L0653 NCI_CGAP_Lu28 two pooled lung pCMV-SPORT6 squamous cell carcinomas L0654 NCI CGAP Lu31 lung, CMV-SPORT6 cell line L0655 NCI_CGAP_Lyml2 lymphoma, lymph pCMV-SPORT6 node follicular mixed _ small and lar a cell L0656 NCI CGAP Ov38 normal a ovar CMV-SPORT6 ithelium L0657 NCI_CGAP_Ov23 tumor, 5 ovary pCMV-SPORT6 pooled (see descri tion) L0658 NCI_CGAP Ov35 tumor, 5 ovary pCMV-SPORT6 pooled (see descri tion) L0659 NCI CGAP Panl adenocarcinomaancreas CMV-SPORT6 L0661 NCI_CGAP_Me115 malignant skin pCMV-SPORT6 melanoma, metastatic to lymph ' node L0662 NCI_CGAP_Gas4 poorly differentiatedstomach pCMV-SPORT6 adenocarcinoma with.si~net r L0663 NCI_CGAP_Ut2 moderately- uterus pCMV-SPORT6 . ' differentiated endometrial adenocarcino - L0664 NCI CGAP Ut3 poorly-differentiateduterus pCMV-I I
endometrial -adenocarcinoma, L0665 NCI_CGAP_Ut4 serous papillaryuterus pCMV-SPORT6 carcinoma, high grade, 2 ooled t L0666 NCI_CGAP_Ut1 well-differentiateduterus pCMV-SPORT6 endometrial adenocarcinoma, L0667 NCI_CGAP_CMLl myeloid cells,whole pCMV-SPORT6 . 18 blood pooled CML , cases, BCR/ABL rearra L0717 Gessler Wilms SPORT1 tumor L0718 Testis 5 SPORT1 L0720 PN001-Normal prostate pSportl Human Prostate L0731 Soares_pregnant uterus T7T3-Pac uterus_ p NbHPU
L0738 Human colorectal T7T3D
cancer L0740 Soares melanocytemelanocyte pT7T3D
~
2NbHM (Pharmacia) with a modified olylinker L0741 Soares adult brain pT7T3D
brain N264HBSSY (Pharmacia) with a modified.
of linker L0742 Soares adult brain pT7T3D
brain N2b5HB55Y (pharmacia) with a modified of linker L0743 Soares breast breast pTTT3D
2NbHBst (Pharmacia) ' with a modified of linker L0744 Soares breast breast pT7T3D
3NbHBst (Pharmacia) with a modified of linker L0745 Soares retina retina eye pT7T3D
N2b4HR
(Pharmacia) with a modified of linker L0746 Soares retina retina eye pT7T3D
N2b5HR
(Pharmacia) with a modified olylinker L0747 Soares fetal_heart heart pT7T3D
NbHH
19W , ~ (Pharmacia) with a modified of linker L0748 Soares fetal Liver . pT7T3D
liver spleen and 1 NFLS Spleen (Pharmacia) with a modified .
olylinker L0749 Soares_fetal Liver pTTT3D
liver_spleen and 1NFLS S1 Spleen (Pharmacia) with a modified of linker L0750 Soares fetal lung pTTT3D
lung_NbHLl I
9W (Pharmacia) with a modified olylinker L0751 5oares ovary ovarian tumorovary pTTT3D
tumor NbHOT . (Pharmacia) with a modified olylinker L0752 Soares_parathyroid_tumorparathyroid parathyroid pTTT3D
tumor NbHPA gland (Pharmacia) with a modified polylinker L0753 Soares_pineal_gland_N3H. pineal pTTT3D
gland PG
(Pharmacia) with a modified of linker L0754 Soares placenta placenta pTTT3D
(Pharmacia) with a modified of linker L0755 Soares_placenta_8to9wee, placenta pTTT3D
ks 2NbHP8to9W (Pharmacia) with a modified of linker L0756 Soares_multiple_sclerosismultiple pTTT3D
sclerosis 2NbHMSP lesions (Pharmacia) with a modified polylinker V TYPE
L0757 Soares senescentsenescent pTTT3D
fibrobla fibroblast sts_NbHSF (Pharmacia) ' with a modified polylinker V_TYPE
L0758 Soares_testis pTTT3D-Pac NHT
(Pharmacia) with a modified polylinker L0759 Soares total - pTTT3D-Pac fetus_Nb2H
F8 9w _ (Pharmacia) with a modified of linker LO'761NCI_CGAP_CLL1 B-cell, chronic pTTT3D-Pac lymphotic . (Pharmacia) leukemia with a modified of linker L0762 NCI_CGAP_Brl.l breast pTTT3D-Pac (Pharmacia) with a modified olylinker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (Pharrnacia) with a modified of linker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac (Pharmacia) with a modified olylinker L0765 NCI_CGAP_Co4 colon pT7T3D-Pac (Pharmacia) with a modified of linker L0766 NCI_CGAP_GCB germinal pT7T3D-Pac 1 center B
cell (Pharmacia) with a modified polylinker L0767 NCI_CGAP_GC3 pooled germ pT7T3D-Pac cell tumors (Pharmacia) with a modified of linker L0768 NCI_CGAP_GC4 pooled germ pT7T3D-Pac cell tumors (Pharmacia) with a modified of linker L0769 NCI_CGAP_Brn25 anaplastic brain pT7T3D-Pac , oligodendroglioma (Pharmacia) with a modified of linker L0770 NCI_CGAP_Brn23 glioblastomabrain ~ pTTT3D-Pac (pooled) (Pharmacia) with a modified of linker L0771 NCI_CGAP_Co8 adenocarcinomacolon pTTT3D-Pac (Pharmacia) with a modified olylinker L0772 NCI_CGAP_ColO colon tumor colon pT7T3D-Pac RER+
(Pharmacia) with a modified of linker L0773 NCI_CGAP_Co9 colon tumor colon pTTT3D-Pac RER+
(Pharmacia) 4vith a modified of linker L0774 NCI_CGAP_Kid3 ' kidney pT7T3D-Pac (Pharmacia) with a modified of linker L0775 NCI_CGAP_KidS 2 pooled kidney pTTT3D-Pac tumors (clear cell . (Pharmacia) type) .- with a modified polylinker L0776NCI CGAP_LuS. carcinoid lung pT7T3D-Pac (Pharmacia) with a modified of linker L0777Soares_NhHMPySlPooled humanmixed pTTT3D-Pac (see melanocyte, below) (Pharmacia) fetal ' heart, and with a pregnant modified of linker L0778Barstead pancreas pancreas pT7T3D-Pac ' _ HPLRB 1 (Pharmacia) with a modified of linker L0779Soares_NFL_T_GBC pooled pT7T3D-Pac (Pharmacia) with a modified polylinker L0780Soares_NSF_F8_9W_OT pooled pT7T3D-Pac PA_P_S 1 (Pharmacia) with a modified olylinker L0782NCI_CGAP_Pr21 normal prostateprostate pT7T3D-Pac (Pharmacia) with a modified of linker L0783~ NCI_CGAP_Pr22normal prostateprostate pT7T3D-Pac (Pharmacia) _ ' with a modified of linker L0785Barstead spleen spleen pT7T3D-Pac (Pharmacia) with a modified of linker L0786Soares_NbHFB whole pT7T3D-Pac ~ brain (Pharmacia) with a modified - polylinker L0787.NCI_CGAP_Subl ' pT7T3D-Pac (Pharmacia) with a modified of linker L0788NCI_CGAP_Sub2 ~ pTTT3D-Pac (Pharmacia) with a modified of linker L0789NCI_CGAP_Sub3 pT7T3D-Pac .
(Pharmacia) with a modified of linker L0790NCI_CGAP_5ub4 pTTT3D-Pac (Pharmacia) with a modified of linker ' L0791NCI_CGAP_SubS . pT7T3D-Pac (Pharmacia) with a modified olylinker L0792 NCI CGAP_Sub6 p T7T3D-Pac (Pharmacia) with a modified olylinker L0793 NCI_CGAP_Sub7 pT7T3D-Pac (Pharmacia) with a modified polylinker L0794 NCI_CGAP_GC6 pooled germ , pTTT3D-Pac cell tumors (Pharmacia) with a modified of linker L0796 NC1_CGAP_Brn50 medulloblastomabrain pT7T3D-Pac (Pharmacia) with a modified of linker L0800 NCI_CGAP_Col6 colon tumor,colon pTTT3D-Pac RER+
(Pharmacia) with a modifted of linker L0803 NCI_CGAP_Kidl1 kidney pTTT3D-Pac (Pharmacia) with a modified of linker L0804 NCI_CGAP_Kidl2 2 pooled kidney pT7T3D-Pac tumors (clear cell (Pharmacia) type) with a modified polylinker L0805 NCI_CGAP_Lu24 carcinoid lung pT7T3D-Pac (Pharmacia) with a modified of linker L0806 NCI_CGAP_Lul9 squamous lung pT7T3D-Pac cell carcinoma, (Pharmacia) poorly differentiated with a (4 modified of linker L0807 NCI_CGAP_Ovl8 fibrotheoma ovary pT7T3D-Pac (Pharmacia) with a modified of linker L0808 Barstead prostate prostate pT7T3D-Pac BPH
- HPLRB41 ' (Pharmacia) ' with a modified olylinker L0809 NCI_CGAP_Pr28 prostate pTTT3D-Pac (Pharmacia) - with a modified polylinker L2251 Human fetal Fetahlung lung OMIM Description Reference 106300 Anl losin s and litis 108800 Atrial se tal defect, secundum ty a 1201'60 Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420, 120160 Osteo orosis, idio athic, 166710 120160 Ehlers-Danlos s ndrome, a VIIA2, 130060 120160 Marfan syndrome, aty ical 120290 OSMED s ndrome, 215150 120290 Stickler s drome, a II, 184840 120810 C4 deficient 120820 C4 deficient 126650 Chloride diarrhea, con enital, Finnish e, 214700 126650 Colon cancer 129900 EEC s ndrome-1 142857 Pem higoid, susce tibility to 142858 ~ Be llium disease, chronic,. susce tibili to 145001 H a arath roidism-'aw tumor s ndrome 150270 La n eal adductor aral sis 150292 E idermol sis bullosa, Herlitz 'unctional e, 226700 154276 Mali ant h erthermia susce tibili 3 154705 Marfan s drome, a II
1.67250, Pa et disease of bone 170261 Bare 1 in hoc to s ndrome, a I, due to TAP2 deficient 173360 Thrombo hilia due to excessive lasmino en activator inhibitor 173360 Hemorrha is diathesis due to PAIL deficient 177900 Psoriasis susce tibili -1 179450 Ra eed sensitivi 183600 S lit hand/foot malformation, a 1 187680 . 6-merca to urine sensitivi 193300 Renal cell carcinoma 193300 von Hi el-Lindau s ndrome 201910 Adrenal h a lasia, con enital, due to 21-h drox lace deficient 208250 Jacobs s ndrome 217000 C2 deficient 222100 Diabetes mellitus, insulin-de endent-1 227646 Fanconi anemia, ty a D
23 3100 Renal lucosuria 235200 Hemochromatosis 248611 Ma 1e s ru urine disease, a Ib 253260 Biotinidase deficient 256550 Sialidosis, a I
256550 Sialidosis, a II
278720 Xeroderma i entosum, ou C
600202 Dyslexia, s ecific, 2 600261 Ehlers-Danlos-like s ndrome 600995 Ne hrotic s ndrome, idio atliic, steroid-resistant 601154 Cardiom o ath , dilated, 1E
601253 Muscular d stro h , limb- irdle, a IC
601652 Glaucoma 1A, rima o en an 1e, 'uvenile-onset, 137750 601868 Deafness, autosomal dominant 13 602011 Pancreatic endocrine tumors 602136 Refsum disease, infantile, 266510 602136 Zellwe er s ndrome-l, 214100 _60_2136 Adrenoleukod stro h , neonatal, 202370 602280 Retinitis i mentosa-14, 600132 602447 Corona arte disease, susce tibili to .
602475 ~ Ossification of osterior lon itudinal 1i ament of s ine Polynzccleotide and Polypeptide Variants [84] The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID
NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A, the nucleotide sequence as defined in columns 8 and 9'of Table'2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, the nucleotide sequence as defned in column 6 of Table 1B, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1B, the cDNA sequence contained in Clone ID NO:Z, and/or nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in Clone ID NO:Z.
[85] The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in column 7 of Table 1A, a polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded,by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined in column 6 of Table 1B, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a polypeptide sequence encoded by the cDNA
sequence contained in Clone ID NO:Z. ' [86] ' "Variant" , refers to a polynucleotide or polypeptide differing from the.
polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
[87] Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence.
selected from the group consisting of: (a) a nucleotide sequence described in SEQ ID NO:X
or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence in SEQ
ID NO:X or the cDNA in Clone ID NO:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID
NO:Z; (c) a nucleotide sequence in SEQ ID NO:X ,or the cDNA in Clone ID NO:Z
which encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a polypeptide;
(e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ID
NO:Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the .
cDNA in Clone ID NO:Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by 'the cDNA in Clone ID NO:Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above. .
[88] The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, -95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ
ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA
contained in Clone ID NO:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of~SEQ ID NO:Y, a nucleotide sequence encoding a polypeptide 281 .
sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in Clone ID
NO:Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in column 7 of Table 1A or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A or the complementary strand thereto, andlor polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
[89] In a preferred embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucl.eotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides. In another preferred embodiment, polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[90] In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (b) the amino acid sequence of a mature form of a polypeptide having the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) the amino acid sequence of a biologically_active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z.
' [91] The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the polypeptide encoded by the~nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B, the amino acid sequence as defined in column 7 of Table 1A, an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also .
provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
[92] By a nucleic acid having a nucleotide sequence at least; for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include, up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to in Table 1A or~2 as the ORF (open reading frame), or any fragment specified as described herein.
[93] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least ~0%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a _ .___. .
nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match.
between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
In a sequence alignment the query and subject sequences are both DNA
sequences. An RNA sequence can be compared by converting U's to T's~ The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group, Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
[94] ,If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which .
are not matched/aligned, as a percent of the total.bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity,. calculated by the above FASTDB program using the specified parameters, to arnve at a final percent identity score.
This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
[95] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent.l0% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
[96] By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino. acid sequence at least 95% identical to a query amino acid sequence, up to.
5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
[97] As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence identified in column 6) or Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof; the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B, or .a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in Clone ID
NO:Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM
0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
[98] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results:
This is because .the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated.at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-tenninal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent .identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N-and C- terminal residues of the subject sequence.
[99] For example, a 90 amino acid residue subject sequence is aligned with a residue query sequence to determine percent identity. The deletion occurs ' at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%: In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query: In this case the percent identity__ _~_ calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequnce are manually corrected for. No.~other manual corrections are to made for the purposes of the present invention.
[100] The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred., Moreover, polypeptide variants in which less than 5.0, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
[101] Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can,vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
[102] Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant I~GF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).) [103] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J, Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1 a. They used random mutagenesis to generate over 3,500 individual IL-1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." In fact, only 23 unique amino acid sequences, ,out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
[104] Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will.
likely be retained when less than the majority of 'the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N-or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
[105] Thus, the invention further includes polypeptide variants which show a functional activity (e.g., biological activity) of the polypeptides of the invention.
Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules knowmin the art so as have little effect on activity.
[106] The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%,.97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide. having the amino acid sequence of an N
and/or C
terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use .
the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA
expression in specific tissues (e.g.,'normal or diseased tissues); and (4) i~
situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).
[107] Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in fact, encode a polypeptide having functional activity. By a polypeptide having "functional activity" is meant, a-polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein of the invention. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an anti-polypeptide of the_invention antibody], immunogenicity. (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and' ability to bind to a receptor or ligand for a polypeptide of the invention.
[108] The functional activity of the . polypeptides, and fragments, variants and derivatives of the invention, can be assayed by various methods.
[109] For example, in one embodiment where one is assaying for the ability, to bind or compete . with a full-length polypeptide of the present invention for binding to an anti-polypetide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked 'immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions,' immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, inimunoflubrescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
[110] In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, _, _._ _ and affinity blotting. See generally, Phizicky et al:, Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a polypeptide of the present invention to bind to a substrates) of the polypeptide of the invention can be routinely assayed using techniques known in the art.
[111] In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants and derivatives thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention. _ [112] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in Clone ID NO:Z, the nucleic acid sequence referred to in Table 1A (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 2 (e.g,. the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides "having functional activity." In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the ai~t that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This. is because the ,skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
[113] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al.; "Deciphering the Message in Protein Sequences:
Tolerance to Amino-Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
[114] The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural. selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
[115] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
[116] ~ As the authors state, these two strategies have revealed that proteins are .
surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu;
replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and. His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one.or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the~polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, serum albumin (preferably human serum albumin) or a fragment thereof, or leader or secretory sequence, or .a sequence facilitating purification, or, (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)).
Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
[117] For example, polypeptide variants containing .amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation.. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. IW munol. 2:-33-1-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
[118] . A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid.sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course it is highly preferable for a polypeptide to have an amino acid sequence which comprises the amino acid sequence of a polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ
_ ID NO:X, an 'amino acid sequence encoded by the portion of SEQ ID NO:X as defned in columnns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ
ID NO:X, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z
which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
[119] In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid'sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the~mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID.NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in Clone ID
NO:Z or fragments thereof; wherein the fragments or variants have. 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynzacleotide and Polypeptide Fragmefzts [120], The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a "polynucleotide fragment" refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in Clone ID NO:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA
contained in Clone ID NO:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ
ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a pol'ynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1B or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto.
[121] The polynucleotide fragments of the invention are Preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt~ or at least about 150 nt in length. A fragment "at least 20 nt in length," for examfle, is intended to include 20 or more contiguous bases from the cDNA sequence contained in Clone ID
NO:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context "about" includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have 'uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length ) are also encompassed by the invention.
[122] Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number l.-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 12'01-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, . 1751-1800, 1801-1850, 1851.-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101- , 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750; 5751-5800, 5801-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400,. 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context "about"
includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein.
Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions ' or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides. ' [123] Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of,_.a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500; 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001.-3050, 3051-3.100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601- .
3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751=5800, 5801-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in Clone ID NO:Z, or the complementary strand thereto.
In this context "about" includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity).
More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[124] Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic .acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1B column 6. Additional, representative examples.of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic. acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand of a sequence delineated in column 6 of Table 1B. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID
NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1B, column.
6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that .
bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and po~ypeptides are also encompassed by the invention.
[125] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode.
these polypeptides, and antibodies that bind these polypeptides _are also encompassed by the invention.
[126] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ
ID NO:X (e.g., as, defined in Table 1A or 1B) or fragments or , variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[127] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of.Table 1B, and the polynucleotide sequence. of SEQ TD NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[128] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' ~10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[129] In additional specific embodiments, polynucleotides of the.invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are.
also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[130] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the. sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention: Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also . encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[131] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one ~of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments,. the 3' 10 polynucleotides, of one of the sequences delineated in column 6,of Table 1B is directly contiguous with the 5' 10 polynucleotides of the next sequential exon delineated in Table 1B, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic vacids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids;- and polypeptides are also encompassed by the invention.
[132] In the present invention, a "polypeptide fragment" refers to an amino acid sequence which is a portion of that. contained in SEQ ID NO:Y, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID
NO:X, a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid sequence encoded by the cDNA contained in Clone ID NO:Z. Protein (polypeptide) fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460,.461-__ _.
480, 481-500, 501-520, .521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, ,1061-1080, 1081-1100, 110.1-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, '761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241=1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about" includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
[133] Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues ofva complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[134] Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form.
Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
[135] The present invention further provides polypeptides having_one or more residues deleted from the amino terminus of the amino acid, sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and' 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1B, and/or a polypeptide encoded by the cDNA contained in Clone ID NO:Z). , In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention. , [136] The present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ
ID
NO:X as defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by the cDNA
contained in Clone ID NO:Z). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-l, and where n corresponds to the position of amino . acid residue in a polypeptide of the invention.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[137] In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA contained in Clone ID NO:Z, and/or the complement thereof, where n and m are integers as described above.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[138] Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g.; biological activities, ability to multimerize, ability to ,bind a ligand) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities'can readily be determined by routine methods described herein and otherwise known in the art. rt is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[139] The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[140] ~ Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in Clone ID NO:Z, or the polyrlucleotide sequence as defined in column 6 of Table 1B, maybe analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by .a _...
polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y
and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2) or the clDNA contained in Clone ID NO:Z may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, WI 53715 USA; http://www.dnastar.com~. _ [141] Polypeptide regions that may be routinely obtained using the DNASTAR
computer algorithm include, but are not limited to, Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions;
Eisenberg alpha-and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., 1, 2, 3 or 4) of the features set out above.
[142] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR
analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
['143] Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g.
biological activity) of the polypeptide sequence of Which the amino acid sequence is a fragment. By a. polypeptide displaying ',a "functional activity" is meant a polypeptide capable of one or more known functional activities associated with a full-length protein, such as, fox example, biological activity; antigenicity, immunogenicity, and/or multimerization, as described herein:
[144] ' Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
[145] In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or moxe of the antigenic fragments of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding-these polypeptides are also encompassed'by the invention.
[146] The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y;
a.
polypeptide sequence encoded by SEQ ID NO:X or the 'complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B
as defined in column 6 of Table 1B or the complement thereto; the polypeptide sequence encoded by the cDNA contained in Clone ID, Nb:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ~ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in Clone ID NO:Z
under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or . alternatively, under lower stringency hybridization conditions defined supra.
[147] The texm "epitopes," as used herein, refers to poxtions of a polypeptide having.
antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An "immunogenic epitop.e," as used herein, is defined as a portion of a pxotein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term "antigenic epitope," as used herein, is. de _fined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[148] Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,21 l.) [149] In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least l I, at least 12, at least 13, at Ieast I4,.at Ieast 15, at least 20, at least 25, at. least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, r60, 65, 70, 7~, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes~
are useful; for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
[150] Non-limiting examples of epitopes of poiypeptides that can be used to generate antibodies of the invention'include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portions) of SEQ ID
NO:Y specified in column 7 of Table 1A. These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index which is included in_ the DNAStar suite of computer programs.
By "comprise" it is intended that a polypeptide contains at least,one, two, three, four, five, six or more of the portions) of SEQ ID NO:Y shown in column 7 of Table 1A, but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the~poxtion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterolgous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein.
In particular embodiments, epitope portions'of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in column 7 of Table 1A.
[151] Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. See,. for instance, Sutcliffe et al., supra;
Wilson et al., supra; Chow et al., Proc. Natl. Acad.. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes. include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a Garner protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at , .
the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
[152] Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supf~a; Wilson~et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 pg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. . Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed ~to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, fox instance, by adsorption to the peptide on. a solid support and elution of the selected antibodies according to methods well known in the arE.
,.
[153] As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including. fragments or variants thereofj, may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1; CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides.
By way of another non-limiting example, polypeptides and/or antibodies of the present 'invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S.
Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Patent 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide. or human serum albumin polypeptide).
Polynucleotides encoding fusion proteins of the invention are also .encompassed by the invention.
[154] Such fusion proteins as those described above may facilitate purification and may increase half life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobuliris. See, e.g., vEP
394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin).
conjugated to an FcRn'binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure _due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-(1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Fusion Proteins [155] Any polypeptide of the present invention can be used to generate fusion proteins.
For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as targeting~molecules once fused to other proteins:
[156] Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linleer sequences.
[157] In certain preferred embodiments, proteins of the invention axe fusion proteins comprising an amino acid ' sequence that is an N and/or C- terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence of the invention.
Polynucleotides encoding these proteins are also encompassed by the invention.
[158] Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
[159] As one of skill in the~art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, cari be combined with hetero.logous polypeptide sequences. For example, the polypeptides of the present invention may .be fused with heterologous polypeptide sequences, 'for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), or albumin (including, but not limited to, native ~or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses,fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A
0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
In drug discovery, fox example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); I~. Johanson et al., J. Biol.
Chem. 270:9459-9471 (1995).
[164] Moreover, the polypeptides- of the present invention can be fused to marker sequences, such as a polypeptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as 308.
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag; corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).
[161] Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well ; as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793;
5,811,238;.5;830,721; 5,834,252;
and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);
and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each. of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:x and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to.random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be~recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
[162] Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. .
Recombinant and Synthetic Production of PolYpeptides of the Invention [163] The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective.
In the latter case, viral propagation generally will occur only in complementing host cells.
[164] The polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, .such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
[165] The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, .the E. coli lac, trp, phoA
and tac promoters, the .SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[166] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G41 ~, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178));
insect cells such as Drosophila S2 and Spodoptera S~ cells; animal cells such a~s CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
[167] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNHl6a, pNHlBA, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3', pDR540, pRITS available from Pharmacia Biotech, Inc.
Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG
available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD 1, pTEFl/Zeo, pYES2lGS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, PHIL-S1, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA).
Other suitable vectors will be readily apparent to the skilled artisan.
[168J Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the marine myeloma cell line, NSO) which are glutamine synthase negative.
Glutarnine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: W087/04462;
W086/05807;
W089/01036; W089/10404; and W091/06657, which are hereby incorporated in their .
entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a.GS expression system in marine myeloma cells is described in Bebbington et al., Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechhol.
Prog. 11:1 (1995) which are herein incorporated by reference.
[169] The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have charactei'istics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
[170] Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated J
transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
[171] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequence's via .homologous recombination (see, e.g., US Patent Number 5,641,670, issued June 24, ,1997; International Publication Number WO
96/29411;
International Publication Number WO 94/12650; Koller et al., P~oc. Natl. Acad.
Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 34:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[172] Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
[173] Polypeptides of the present invention can also be recovered from:
products 'purified from natural sources, including bodily fluids, tissues and cells, Whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques , from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypep'tides of the present invention may be glycosylated or may be non-glycosylated. 'In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes._ _ Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eulcaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
[174] In.one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eulcaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol ~s its sole carbon source. A main step in the methanol metabolization pathway is.the oxidation of methanol to formaldehyde using OZ. This reaction is catalyzed by the enzyme alcohol. oxidase. In order to metabolize methanol as its sole carbon source, Piclzia pasto~is must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for OZ. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of.the two-alcohol oxidase genes (AOXI ) is highly active. In the presence of methanol, alcohol oxidase produced from the AOXI gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, 3.F., et al., Nucl. Acids Res. 15:3859-76 (1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the ADXI
regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
[175] In one example, the plasmid vector pPIC9K is used to express DNA
encoding a polypeptide of the invention, as set forth herein, in a Piclaea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J.
Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOXI
promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
[176] Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDI, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as.
long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required. ' .
[177] In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
[178] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic y material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/,or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/2941 l, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[179] In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-(1984)).. For example, a polypeptide corresponding to a fragment of a polypeptide can, be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl ammo acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[180] The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, arnidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited; to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin; etc.
[181] Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or G-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein. .
[182] Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (lzih iz3h izsh 131I), carbon (14C), sulfur (35S), tritium (3H), indium (lln, nzln m3mln msmln technetium 9~Tc 99mTc thallium 2°iTi 6s ), ( , ), ( ), gallium ( Ga, Ga), alladium losPd mol bdenum 99Mo xenon lssxe fluorine (18F) is3Sm l~~Lu ls9Gd p ( )~ Y ( )~ ( )> > > > >
ia9pm~ IaoLa~ ms~,b~ is6Ho~ 9o.Y~ 4~sc~ iasRe~ issRe~ i4aPr~ nose and 9~Ru.
[183] In specific embodiments, a polypeptide of the present invention or fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, l~~Lu, 9oY, is6Ho, and is3Sm, to polypeptides.
In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 11n. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is g°Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al., Clin Cancer Res. ,4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
[184] As mentioned, the proteins of the invention may be modified by either natural processes; such as posttranslational processing, or by chemical modification techniques which are well known in the. art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide.
Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by~ synthetic methods. Modifications include acetylation, acylation, 'ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide .derivative, covalent attachment'of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York ' (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol.
182:626-646 (1990); Rattan et al., Ann: N.Y. Acad. Sci. 663:48-62 (1992)).
[185] Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may. be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
[186] The polymer may be of any molecular weight, and may be branched or . unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000; 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
a [187] As noted above, the polyethylene glycol may have a branched structure.
. Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575;
h Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 1$:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
[188] The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of . the protein. There are anumber of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl~ chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulflrydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
[189] . As suggested above, polyethylene glycol may be attached to proteins,via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, ' histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
[190] . One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino 'groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
[191] As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein. either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S.
Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
[192] One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene . glycol (MPEG) using tresylchloride (C1SOZCHZCF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[193] Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U..S. Patent No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by refe~'ence. Pegylated protein products produced. using the reaction chemistries set out herein are included within the scope of the invention.
[194] The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For 'example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule.
Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carner Sys. 9:249-304 (1992).
[195] The polypeptides of the invention can be recovered and purified_from chemical synthesis and recombinant cell cultures by standard methods which include, but are not ' .
limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose -chromatography,. hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification. , ' [196] The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, thewpresent invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the ._ polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
[197]' Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID
NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns ~
and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence.
In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In , additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
320 _ ..
[198] As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
[199] ' . Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of. the invention contact one another in solution; In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ
ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in Clone ID NO:Z). . In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent .associations are the consequence of chemical or recombinant manipulation. -Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable 'of forming covalently associated multimers, such as for example, osteoprotegerin (see; e.g., International Publication NO:
WO
98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two, or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incozporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
[200] Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the .culture supernatant using techniques known in the art.
[201] Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S.
patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention. .
[202] In another example, proteins of the invention are associated by interactions.
between Flag~ polypeptide sequence contained ~in fusion proteins of the invention containing Flag~ polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag~ fusion proteins of the invention and anti-Flag~. antibody.
[203] The multirtiers of the invention may be generated using chemical techniques known in the art: For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C- .
terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its _ entirety).
[204] Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced' recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, .
polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a ~olypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader' sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant , techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can.be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Antibodies [205] Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR)~which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in Clone ID
No:Z, and/or an epitope, o~ the present invention) as determined, by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or~subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgGl . In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.
[206] Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions) alone~'or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable regions) with a hinge region, CH1; CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, 'horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
[207] The antibodies of the present invention may be monospecific, bispecific, trispecific_or_ of..greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; I~ostelny et al., J. Immunol. 148:1547-1553 (1992).
[208] Antibodies of the present invention may be described or specified in terms of the epitope(s) or portions) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portions) may be specified as described herein, e.g.; by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures.. Preferred epitopes of the invention include the predicted epitopes shown iri column 7 of Table 1A, as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
[209] . Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least-70%, at .
least 65%, at least,60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit honiologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less thazi 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s)~ of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10-2 M, 10-z M, 5 X 10-3 M, 10-3 M, 5 X 10-4 M, 10-4 M, 5. X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6M, 5 X 10-' M, 10' M, 5 X 10-8 M, 10-8 M, X 10-9 M, 10-9 M, 5 X 10-1° M; 10-1° M, 5 X 10-" M, 10-11 M, 5 X
10-12 M, 10-12 M, 5 X
10-13 M, 10-13 M, 5 X 10-14 M, 10-la M, 5 X 10-15 M, or 10-15 M.
[210] The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
[211] Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features' both receptor-specific 'antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitatiorl followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
[212] The invention also features receptor-specific antibodies .which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, fox example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT
publication WO
96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J.
Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998);
Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);
Carlson et al., J. Biol. Chem..272(17):11295-11301 (1997); Taryrnan et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
[213] ' Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention; including both in vitro and ifa vivo diagnostic arid therapeutic methods. For example, the antibodies have utility iri immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
incorporated by reference herein in its entirety.
[214] As discussed in more detail below, the antibodies of the present invention maybe used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT
publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995;
and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.
[215] The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation,~
~amidation, derivatization by known protectinglblocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but-not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical amino acids.
[216] The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc.
to induce the production of sera containing polyclonal antibodies specific for the antigen.
Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic . polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, .
dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
[217] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. ' 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-(Elsevier, N.Y., 1981). (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through -hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not=
the method by which it is produced.
[218] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known. in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones. - -[219] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein; preferably, the hybridonia is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
[220] Another well known method for producing both polyclonal and monoclonal human B~ cell lines is transformation using Epstein Barr Virus (EBV).
Protocols for generating EBV-transformed B cell lines are commonly known imthe art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of .B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived -from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues.
Tissues are generally made into single cell suspensions prior to EBV
transformation.
Additionally, steps may be taken to either physically remove or inactivate T
cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
[221] In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, ,clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV
lines may become monoclonal or polyclonal as a, result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and II~R-4). Thus, the present invention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
[222] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
[223] For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display .antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.
Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic et ~, al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994);
PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737;
W0.92/01047; WO. 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.
Patent Nos. 5,698,426; 5;223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
[224] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
[225] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a rriurine monoclonal antibody and a human immunoglobulin constant region. Methods for, producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 .
(1986); Gillies ~ et al., (1989) J. Immuriol. Methods 125:191-202; U.S. Patent Nos.
'5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in ,their . entirety. Humanized antibodies are antibody molecules from non-human -species antibody that binds the desired antigen having one or more _complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5)-:489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), .and chain shuffling (IJ.S.
Patent No. 5,565,332).
[226] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above fusing antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111;
and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91110741; each of which is incorporated herein by reference in its entirety.
[227] . Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and. light chain ' immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells; Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
The modified embryonic stem cells are expanded and microinjected into blastocysts to -produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a~selected~antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can'b~e obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes -. harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int.
Rev.
Immunol. 13:65-93 (1995). For ~a detailed ~ discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT~publications WO 98/24893; WO 92/01047; WO 96/34096;
WO
96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923;
5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771;
5,939,598;
6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharni (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
[228] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Biotechnology 12:899-903 (1988))., [229] Further, antibodies to the polypeptides of the invention can, in tum, be utilized to generate anti-idiotype antibodies that '"mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J.
7(5):437-444; (1989) and Nissinoff, J.. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of.the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of .such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
[230] Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Cherl et al., Hum. Gene Ther. 5:595-601 (1994);
Marasco, W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev.
Microbiol.
51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999);
Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999);
Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein.
Pol~nucleotides Encoding Antibodies [231] The~invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having, the amino acid sequence of SEQ
ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, andlor to a polypeptide encoded by the cDNA contained in Clone ID
NO:Z.
[232] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in I~utmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the. synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
[233] Alternatively, a polynucleotide encoding an antibody maybe generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR
amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, , e.g., a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be ~ cloned into replicable cloning vectors using any method well known in the art.
[234]' Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create, amino acid substitutions, deletions, and/or insertions.
[235] In a specific embodiment, the amino acid sequence of the heavy andlor light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the. art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human, framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (I998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
[236] In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., ' Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody, molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as~ those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
[237] Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No: 4,946,778; Bird, Science 242:423- 42 (1988); Huston et al., Proc. Natl.
Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge;
resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Methods of Producing Antibodies [238] The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include,.
but- are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
[239] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g.,. a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro. recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light,chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.
Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
-, [240] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. . Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
[241] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when. transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) _ . . containing antibody coding sequences; ,plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e:g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole ~'ecombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human , cytomegalovirus, is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
[242] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by~ adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so. that the cloned target gene product can be released from the GST moiety.
[243] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugipe~da cells. The antibody coding sequence may be cloned individually into non-essential regions . 338 (for example the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
[244] In mammalian host cells, a number of viral-based expression systems .may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)).
Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences.
Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)). . .
[245] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
To this end, 'eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2; BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
[246] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express fihe antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites; etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then. are switched to a selective .media. The selectable marker in the recombinant plasmid confers.
resistance to the selection and allows. cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
[247] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad.
Sci. USA
48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci.~USA 77:357 (1980);
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.
Biochem=. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their entireties.
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[37] The first column in Table 1A provides the gene number in .the application corresponding to the clone identifier. The second column in Table 1A provides a unique "Clone ID NO:Z" for a cDNA clone related to each contig sequence disclosed in Table 1A.
This clone ID references the cDNA clone which contains at least the 5' most sequence of the assembled contig and at least a portion of SEQ ID NO:X was determined by directly sequencing 'the referenced clone. The reference clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein. ' .
[38] The third column in Table 1A provides a unique "Contig ID" identification for each contig sequence. The fourth column provides the "SEQ ID NO:" identifier for each of the contig polynucleotide sequences disclosed in Table 1A. The fifth column, "ORF (From-To)", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence '.'SEQ ID NO:X" that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1A, column 6, as SEQ ID NO:Y.
Where the nucleotide position number "To" is lower than the nucleotide position number "From", the preferred ORF is the reverse complement of the referenced polynucleotide sequence.
[39] The sixth column in Table 1A provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated by "ORF
(From-To)". Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. .
[40] . Column 7 in Table 1A lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y); as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN (Version 3.11 for the Power Macintosh, DNASTAR, Inc., 1228 South Park Street Madison, WI). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted. epitopes as described in Table 1A. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may ~vary_slightly.
[41] Column 8 in Table 1A provides an expression profile and library code:
count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1A, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention.
The first number in column 8 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4.
For those identifier codes in which the first two letters are not "AR", the second number in column 8 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the tissue/cell source.
Those tissue/cell source identifier codes in which the first two letters are "AR" designate.
information generated using DNA array technology. Utilizing this-technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse transcription.
After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal' generated from each array was used to normalize gene expression between the different hybridizations. The value presented after "[array code]:" represents the mean of the duplicate values, following background subtraction and probe~normalization. One of skill in the ark could routinely use this information to identify normal and/or diseased tissues) which show a predominant expression pattern of the corresponding polynucle0tide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
[42] .Column 9 in Table 1A provides a chromosomal map location for certain polynucleotides of the invention. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the ~NCBI (National Center for Biotechnology Information) UniGene database. Each sequence in the UniGene database is assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often. available for one or more sequences) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster. ~ , [43] A modified version of the computer program BLASTN (Altshul et al., J.
Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993)) was used to search the UniGene database for EST or cDNA sequences that contain exact or near-exact matches to a polynucleotide sequence of the invention (the 'Query'). A
sequence from the UniGene database (the 'Subject') was said to be an exact match if it contained a segment of 50 nucleotides in length such that 48 of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same UniGene cluster, and mapping data was available for this cluster, it is indicated iri Table 1A under the heading "Cytologic Band". Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.
[44] Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database .of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIMTM (supra). If the putative chromosomal location of a polynucleotide of the invention (Query sequence) was associated with a disease in the Morbid Map database, an OMIM reference identification number was noted in column 10, Table 1A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference identification numbers (column 1), and provides a description of the associated disease in Column 2.
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z NO:X ID: NO:B From-To HMSKFl3 12 708207 AC023891 339 1-32 HMSKF13 12 708207 AC023891 340 . 1-169 HMWDR50 19 667595 AB014080 348 l-276 195-494.
HUFAK35 25 636241 AC018433 353' 1-2744 .~ 1504-1816 . 5670-5784 _ 1856-1951 . 3205-3305 HE8PY29 45 . 887862 AC009948 365 1-439 HLTEH39 116 943641. AC004204 383 1-1285 [45] Table 1B summarizes additional polynucleotides .encompassed by the invention (including cDNA clones related to the sequences (Clone ID NO:Z), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, "Clone ID .
NO:Z", fox a cDNA clone related to each contig sequence. The second column provides the sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column provides a unique contig identifier, "Contig ID:" for each contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" for a fragment of the BAC clone identified in column four of the corresponding row of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
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w [46] Table 2 further characterizes certain encoded polypeptides of the invention, by providing the results of comparisons to protein and protein family databases.
The first column provides a unique clone identifier, "Clone ID NO:", corresponding to a cDNA' clone disclosed in Table 1A. The second column provides the unique contig.
identifier, "Contig ID:" which allows correlation with the information in Table 1A. The third column provides the sequence identifier, "SEQ ID NO:", for the contig polynucleotide sequences.
The fourth column provides the 'analysis method by which the homology/identity disclosed in the Table was determined. The"fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR
hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five.
Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as "NR"), or a database of protein families (herein referred to as "PFAM"), as described below.
[47] The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Table 1A, column 3 (e.g., SEQ ID NO:X or the 'Query' sequence) was, used to search against the NR database. The computer program BLASTX
was used to compare a 6-frame translation of the Query sequence to the NR
database (for information about the BLASTX algorithm please see Altshul et al., J. Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring 'Subject') is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring 'Subject' is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than 1.0e-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by the number of~Query amino acids in the HSP
and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.
[48] . The PFAM database, PFAM version 2.1, (Sonnhammer et al., Nucl. Acids Res., 26:320-322, 1998)) consists of a series of multiple sequence alignments; one alignment for each protein family. Each .multiple sequence alignment is converted into a probability model called a Hidden Markov Model, or HMM; that represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin et al., Biological sequence analysis: probabilistic models of proteins and r2ucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER
version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1A) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2, and the database accession number of the PFAM hit is provided in column 6.
Column 7 provides the score returned by HMMER version 1.8 for the alignment.
Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.
[49] As mentioned, columns 8 and 9 in Table 2, "NT From" and "NT To", delineate the polynucleotides of "SEQ ID NO:X" that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2.
Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.
[50] The nucleotide sequence SEQ ID, NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X
are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the ~cDNA contained in Clone ID NO:Z.
These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A.
[51] Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. ' The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9%
identical to the actual DNA sequence (for example, one base insertion or deletion in an .
open reading frame of over 1000 bases).
[52] Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA
containing cDNA
Clone ID NO:Z (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and having depositor reference numbers TS-1, TS-2, AC-l, and AC-2;
and/or as set forth, for example, in Table 1A, 6 and 7). The nucleotide sequence of each~deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X.
[S3] The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
RACE Protocol Fog Recovery of Full-Lefzgth Genes [54] , Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M.A.; et al., Proc. Nat'1.
Acad.
Sci. USA, 85:8998-9002_ (1988). A cDNA clone missing either the 5' or 3' end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor., The following briefly describes a modification of this original 5' RACE
procedure. Poly A+ or total RNA is reverse transcribed with Superscript II
(Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA
is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL).
Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI, SaII and CIaI) at the 5' end and a primer containing just these restriction sites. This double-stranded cDNA is PCR
amplified for~40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose geI
and the region of gel containing cDNA products the predicted size of missing protein-coding DNA
is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or. SaII, and ligated to a plasmid such as pBluescript SKII
(Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts.
Correct 5' ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial .
kits are used to amplify and recover 3' ends.
(55] Several quality-controlled kits are commercially available for purchase.
Similar reagents and methods to those above are supplied in kit form from Gibco/BRL
for both 5' and 3' RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure. are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
[56] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library double-stranded DNA: ~ An.. asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
RNA Ligase PYOtocol Fog Gehe~atiug The 5' o~ 3' Eud Seqaceuces To Obtaita Full Leugth Genes [57] Once a gene of interest is identified, several methods are available for the identification of the 5' or 3' portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5' and 3' RACE.
While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5' or 3' end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5' RACE
is available for generating the missing 5' end of a desired full-length gene.
(This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):,1683-1684 (1993)).
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA
presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR. amplify the 5' portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can then be ligated to an RNA
oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide.' The first strand synthesis -reaction can then be used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence. of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the relevant gene'.
[58] , The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCC (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and receiving ATCC designation numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth, for example, in Table 1A, Table 6, or Table 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Table 7. These deposits are referred to as "the depos2ts" herein.
The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A (Clone ID NO:Z). A clone which is isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire coding region of a human gene ' or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC Deposits by use of a sequence (or portion thereof) described in, for example Tables lAor 2 by procedures hereinafter further described, and others apparent to those skilled in the art.
[59] Also provided in Table 7 is the name of the vector which contains the cDNA
clone, Each vector is routinely used in the art. The following additional information is provided for convenience. -[60] ~ Vectors Lambda Zap (LT.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988);
Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17.9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037. pBS
contains an ampicillin resistance gene~and pBK contains a neomycin resistance gene.
Phagemid pBS
may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK
may be excised from the Zap Express vector. Both phagemids may be transformed into E.
coli strain XL-1 Blue, also available from Stratagene. ---- _----.-..-.
[61] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3:0, were obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897.
All Sport vectors contain an ampicillin resistance gene and may be transformed into E.
coli strain DHlOB, also available from Life Technologies. See, for instance, Gruber, C.' E., et al., Focus 15:59- (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR°2.1, which'is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, CIarIc, J.
M., Nuc. Acids Res. 16.9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (1991).
[62] The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID NO:Y, and/or the deposited clone (Clone ID NO:Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or. primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of ~genomic material.
[63] Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in Clone ID NO:Z, using information from the sequences disclosed herein or the clones deposited with the ATCC. For .example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
[64] The polypeptides of the invention can be prepared in any suitable manner:
Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
[65] The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often -.
advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple ~histidine residues, or an additional sequence for stability during recombinant production.
[66] The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
[67] ~ The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence contained in Clone ID NO:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ
ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B.
Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID,NO:B as defned in column 6 ofTable 1B are also encompassed by the invention. The present invention further encompasses a-polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, andlor the cDNA contained in Clone ID NO:Z. .
[68] Moreover, representative examples of polynucleotides of.the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in Table 1B column 6, or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in Table 1B column 6, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, "or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as B~AC
ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and' have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed' by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[69J Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated _ 192 in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAG ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides~
and polypeptides are also encompassed by the invention.
[70] Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof.
Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in column 6 of Table 1B
which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X
(see Table 1B, column 2) and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1B which correspond to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
[71j Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1B column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in the same row of Table 1B
column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strands) of the sequences delineated in the same row of Table 1B column 6, wherein sequentially delineated sequences in the table (i.e.
corresponding to those exons located closest to each other) are directly contiguous in a 5' to 3' orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1B, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID
NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[72j In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B, and the polynucleotide sequence of SEQ ID
NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that.bind these polypeptides are also encompassed by the invention.
[73] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID
NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ ID NO:X
(e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s). and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID NO:Z. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[74] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments, the delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1B.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[75] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. 'Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[76] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the, 3' 10 polynucleotides of , one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[77] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
Additionally, fragments and variants of the above-described polynucleotides,.nucleic acids, and polypeptides are also encompassed by the invention.
[78] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.
[79] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of _ another sequence in column 6 are directly contiguous. Nucleic acids which hybridize to the complement of these 20~ contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the.
invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[80] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 corresponding to the same Clone ID NO:Z (see Table 1B, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of .
these 20 lower stringency conditions, are also encompassed by the invention.
Polypeptides -encoded by these polynucleotides andlor nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments andwariants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[81] In specific embodiments, polynucleotides of the invention comprise, of alternatively consist of, a polynucleotide sequence in which the 3' 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifer SEQ ID
NO:X (see Table 1B, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[82] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B is directly contiguous with the 5'..l0.polynucleotides of the next sequential exon delineated in Table 1B, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides andlor nucleic acids, other polynucleotides~ and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[83] Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior ~to conception of the present invention. ~ Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fourth column of Table 1A, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus 15 of SEQ
ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14. More specifically, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides~of the invention do not consist of at least one, two, three, four,. five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including fox example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequences) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through.
these accessions are hereby incorporated by reference in their entirety.
SEQ
EST Disclaimer Clone NO: Contig ID
Range NO: Z X ID: of a Accession #'s Range of b HTTEK47 11 5736491 - 396 15 - AA400005, AW205244, AW 134723, 410 and AI689951.
HMSKF13 12 7082071 - 387 15 - AA148393, AC023891, and AC023891.
HLHCT68 I3 7647451 - 425 15 - 891150, H67895, AI205078, 439 Z250I2, C052I2, ' 230134, AA194359, AC010344, AC010344, AC010344, AC008496, and AC008496.
HKAFF33 14 9742001 - 175 15 - AA335761, T58784, AA053162, 189 AA220207, F07425, N34268, AA535485, C16914, F11628, F00945, AA380834, AA321294, AA437332, AA132565, T27675, AA527306, H11697, H63653, W24084, AW374230, AA158146, AL035930, AA369234, AW407037, AA361916, W24054, AA977833, AI910189, AA358563, AA385962, T28025, N42582, AA336109, AA321393, AI910249, AI696864, AA305941, AW179215, H98493, AW385684, AA522541, AW062331, W44848, AA188398, AA535767, AA159383, C18543, AA053691, AA371996, AW384708, AA133506, AW404398, AI910244, AI565209, ' ~ AI114563, AI910095, N24326, AA382286, H95866, H68295, AW361810, M24045, 233459, AJ001977, L38251, M24046, D83957, X99704, ~D50852, D64145, X76189, U41420, 247377, U07230, AF220291, D49820, D83030, D64146, D64151, D64152, D64147,.M24097, L20091, M84386, AF115462, D83031, 283247, X73518, X96582,'Y09156, X82122, X78343, D31817, M28172, M84174, X87841, U06487, LJ06695, Y11843, L54059, M99388, U41386, X67818, X70856, X70857, Y18552, M21963, U60218, M24096, AJ005590, D83956, M28206, M26432, X58536, D64148, 215144, AJ010748, M84173, D50854, AJ001975, AJ001976, AJ001973, AJ001974, AJ272049, M26429, D83029, AF118891, AJ133100, E02883, M24030, AJ238694, AJ133267, AJ251755, AP000508, AF017331, AF015557, AF038574, AF201475, AF144.665, AF102689, U61274, M12679, U06835, M26712, M28160, D64149, U56246, Y10520, D84394, M11799, U50088, AF198649, Y18649, AJ131853, U56260, AF028596, U60324, -M63454, M16272, M26430, U11267, M32320, D49552, AJ010749, U09853, X83394, M16273, M54883, AP000507, E01341, M12967, 539758, X03945, AR008238, AJ245869, U60423, X55711, U38976, U41833, AF215919, D89334, U81012, Y18658, AF042290, AF115461, AF117767, AJ132660, Y11229, AJ133472, AJ249725, AF061862, ' AF205535, AF205539, AF213682,w X83003, --AC004204, U74386, U17572, AF115460, M24036, M24035, M24034, X61710, U04244, U64801, X81363, U88407, M24048, X83005, U74387, A93993, U76397, AF017317, AF065641, AF054010, AF061868, U70581, U90241, U91331, AF102568, AF110264, AF110261, AF098266, AF176080, AF220289, I14657, Y15745, AJ010323, AJ133476, AJ249164, Y14767, Y17065, AF061858, U96787, AF019568, AF015555, AF036553, AF145761, AF145763, AF104219;
AF179632, AF189726, AF108425, U05581, AF017321, X75953, L42280, U05580, M24044, L20088, D85761, Y09058, D50300, M77774, M77778, M77777, D25275, M94052, M94053, M94051, L22028, M24040, M24039, M24032, U04243, L22649, L36318, L36591, U29083, U29480, L17005, U63653, D38526, X97321, X13116, M30679, AC004182, M24038, Y08994, U36492, M28207, L42024, U38975, AJ242661, L42345, X60253, X13115, M28204, M15470, 246810, 249112, 279751, U11261, U11263, U60322, U05582, M24037, X 61706, AF168611, U05579, I14637, I14638, and I14642.
HTLAQ18 15 11931141 - 820 15 - AA442624, AI382107, AW269016, 834 AI286158, AA719238, AI001168, AI280133, AW339870, AA009701, AA932363, AI783808, AA587120, . AI241143, AI266558, AI584159, AI370392, AW161202, AI499325, AI702343, AI494201, AI818728, W19236, AI491904, AI537516, AI916720, AI621341, AW059828, H89138, AA938181, AI357283, AI499581, AI817253, AI174819, AA749425, AI859644, AW020419, AI680369, AW080531, AI539260, AI273856, AI434760, AW083168, AA493923, AI801325, AL047344, AI690620, AI366959;
AW058275, AW022494, AW020288, AI630474, AA769285, AW081110, AA768550, AW022542, AW020144, AA587590, AI634919, AI289791, AI360816, AA837290, AI345396, AI344935, AI656270, AW193299, AW189563, AA420722, AI312210, AI334893, AI611728, AA928539, AI208112, AI343091, AI401697, AI289436, AI865116, AI335476, AI311892, AI582871, AI336503, AI500706, AI345370, AL047152, AA888196, AI828806, AI687568, AL121270, AI538850, AL041772, AI582434, AI336565, AI440511, AI421662, N29277, AI340552, AI345471, AI954721, AI347569, AW191003, AL048499, AI961590, AA555145, AA504514, AL079910, AI050881, AI349279, AI440457, AW020381, AI538885, AI590755, AA65~1924, AL133054, S79832, AF022363, AL110218, AF132676, AF061836, D00174, AF205861, U87620, AF1'14168, Y10823, 577771, AL137292, X00861, A08456, A31057, AF111849, AF026124, I77092, A18777, AF028823, AF177401, . AF085809, AF120268, AR038854, AR050959, D44497, AL122111,.AL117583, M79462, A08910, A08911, A08909, X52128, A08907, A08908, U95739, AC004686, AC007392, AC009233, A70386, AL137523, U49908, . Y18680, I18355, AL133099, I34392, AB022159, AC009501, E08516, AF200416, AF013249, AF036941, A27171, AF146568, AF022857, AF022858, AJ131955, A07588, AR012379, AL137284, AB007812, AL080162, AL080057, AL133010, AC002540, AC007172, AC002287, AC007458, 561953, AF090934, 536676, U76419, AF054988, AR059958, AL117457, AL133014, M64936, AF098162, AC004837, AC018767, AF035161, X72387, AF074604, A65336, E04233, U96683, AL080150, AF100931, A20553, AF082526, AL049300, AF113699, AR029490, D83989, AF111851, AL050277, AL133016, AL110228, Y08616, U95114, U75604, AF201468,108319, AF169154, AF113690, AF110329, AL109672, AL133640, AF126488, AL110158, AL133062, AL133047, AL137662, AL117432, AL133565, E02221, AF108357, E06743, AC005968, AF161699, AF113689, X00474, AL137495, I32738, AL050092, AF124435, E01963, AC004399, AC003032, AC005353, AL096776, AF179633, AC005057, AP000161, AP000020, AL034374, AC005091, AL022147, AC005048, AC002416, 294277, AC004690, AC008067, AC006222, AC008014, AL022170, AC007056, AC005291, AC007298, U66059, AF115410, AR016469, X73361., and AB026995.
HLYAW 16 9765311 - 383 15 - AI124815, AW402317, AW402511, AW405194, F13067, AI826037, AA160317, AW408434, AW403392, AA381090, AA224068, AA381088, AL039796,, AA381030, D82221, AA102697, AA263135, AW402322, AA663896, D82177, AW327936, AA336651, D82189, AA295685, AA401643, H23327, W40489, AA227277, AA151951, AL138056, AW402698, AW402264, U46382, AW401505, AW401719, C18310, T29764, AA344828, AA310808, T52219, AW408440, AW403793, AW402754, D56208, AA159230, T52124, AA295712, AA380938, AA351420, AI359260, AW403872, AA449607, AA121088, 819052, AW402402, AA075796, AA100848, AW239462, 820954, F07582, AW402383, AA348993, AA157351, AA350486, AA173111, AA313847, AW401548, AW405127, AI750810, AA381116, F05537, AA152039, AA299668, AW403070, AW402855, F06315, AW405549, AA352960, AA348075., AW403114, AW408321, AW401393, AW404182, AI365653, AW273557, AA381988, AW385684, AA378689, AW402401;
AW402424, AW405525, AA381052, AA381239, AA160101, AA296474, AA158M09, W44826, AA129140, AA005245, N44225, AA160714, AA488534, F06589, AA055685, AA352502, AW404586, AA081491, AA853431, AA143626, AA159125, AA358919, AA083156, F06198, AI214149, AW403253, AI214126, 854504, AA143468, AA263158, AI696864, AA085102, W52004, T10924, AA916617, AA160070, ' AA102181, AA312514, 830668, AA305941, C16914, U96942, 246808, X75953, X64366, M24038, L42282, L42283, AJ131118, X90391, Y13029, Y09118, U64801, M15470, D50290, D50291, U75533, M19757, M24075, U14943, U11263, X84725, L09736, M84694, M84383, M84384, M24037, E03438, U11261, U17572, L41353, D31816, Y08995, M24035, M95530, X61710, X61706, M19756, X81363, E02885, M58636, L41925, U03698, L42345, M80670, E03437, 215143, Y08994, M68964, M32319, AC004182, U11265, D50300, M28205, E02884, D50299, L04696, U17107, U30904, U80945, U30936, L42281, L41214, AF170577, L40599, M84381, M84385, AJ133267, 222651, L04695, E02248, X61709, D44499, U03027, U03859, U06862, L11603, L19937, U50710, U70528, M84382, L41086, D44501, U90566, L38504, D49824, AF022783, L11666, U04244, AJ250917, U90563, U90564, U90565, M84380, M63454, U11267, X03664, M33573, U31971, M32320, L20090, AJ245869, X13116, M30679, X60253, U09864, D50295-, M28203, AF115461, AF118895, U11262, U11266, L07950, X73578, L19923, U27608, D8~3043, AF115460, L11571, L11570, D50296, AF016641, U36392, L09735, U01848, U35431, D50293, L22027, L33923;
AB008102, 233453, D44500, M86403, D14343, AF026218, D50294, AF118892, D25275, M24039, L32862, AJ249724, ' AF071767, U04243, L22028, AF056981;
U90561, M24045, M94053, Y09157, M32318, X61707, AF130734, M24036, L36591, AJ133471, AF190276, Y17193, X03665, X60255, X60693, X60254, L36318, AF065644, AJ132659, AF118893, AF118894, L42280, L20089, X13115, AF002268, E01341, M12967, M11799, S39758, X03945, Y12378, U90560, U90562, AR008238, U80670, AF028595, D87665, M54883, M24033, U50091, M12678, AJ243433, U93915, U58643, X55711, AF226838, Y18552, AJ007603, D83956, M22794, U58315, L22649, U05581, D85761, M77778, M77777, M77776, M77775, X99734, Y09058, M24032, U42837, D85762, U63653, AF017334, AF18185'7, AF181844, M94052, M94051, M24040, U21053, U29083, U29480, AF118891, X64454, U52169, U36492, L42024, AF208430, AF072765, AF189013, AFI08428, X86704, U05580, U05582, L20086, AF198652, Y11228, X90390, AF105029, AF181846, AJ007605, X99704, Y18288, Y18648, AF115464, 246810, AJ238702, AJ006020, AF023442, AF135550, M77774, A93992, AF096631, D64149, and D64150.
HMCFY36 17 9436381 - 302 15 - AW408434, AW402317, 003945, 316 AW327936, AW405194, AI696864, AW402511, AA263135, T68508, AW402322, AA663896, AA294911, AW405525, AW401505, AA352603, AW401548, AW239462, AA366365, AW402264, AW401719, AA361489, AA158146, AA147151, 018310, . AA488534, AA361477, D82189, AA159413, AA158109, AI124815, AA112349, AA151951, AA160714, AA477506, AW403872, AL039796, AA372227, 019056, AA263158, . AA121088, AA581164, AA385962, AW402402, AA100848, AA335761, AA151891, W24084, F11628, H1I697, 834651, W24054, AA220207, N42582, AA361916, D82221, AW403392, AI359260, T75330, AA160037, D82177, T28025, X94481, L76091, AB030573, X96473, 246808, L20090, E03438, U06697, AJ005590, E02248, M80670, E03437, Y08994, M68964, L41925, M32319, AF004370, L76931, U58110, U31334, Y13029, D50300, D50291, U75533, M19757, U14943, M28205, U03698, M95530, AJ002151, U28759, L41086, U32660, U11263, 215143, M24037, L41214, U16309, D31816, M84380, U15639, M33573, U05581; U16298, Y15840, L31798, X84725, M24035, U70528, L36591, M84382, AF035648, AF035649, D50295, U09864, U29083, U96942, AB030574, U37325, X13116, AJ271160, L22028, L42024, U34810, U83580, M29864, AF118891, U15638, AF035647, E02885, X90391, D50290, M58636, L42282, M24075, L42345, L42146, AB032598, AF118894, AJ223282, U15640, U14756, U11261, U11267, X75953, X64366, Y08995, L11571, M24038, AJ131118, X61710, X61706, U04244, U64801, L42283, M32320, U90560, U05577, X90390, U50091, ' Y15841, AB032097, AB036049, AB036050, X86704, X95410, L76094, U18659, L42280, ~.J74387, U18660, D44499, U11262, U17572, AB032094, AB032095, AB032096, AB032599, AB036051, AJ002676, X94.480, L76088, L76089, L76090, L42506, AF033351, M83193, M83191, AJ238702, L07950, U05575, U03027, U03859; L11570, D25275, M94053, L09735, L22027, M24039, L11603, -- - L19937, X61709, U04243, L36318, -AF016641, L42281, X81363, AJ250917, D49824, M28203, X94574, U74386, AB032933, U05583, D44501, D14343, U11266, M83195, M83194, M83192, M86403, U05582, AB030575, X94482, X99734, U32678, L76093, Y09058, D50293, D50294, D50296, M77774, M77778, M77777, M77776, M94052, M94051, L09736, L11666, M84694, U31971, U50710, U80945, L22649, U29480, U30936, U36392, M19756, M84384, U63653, . AF056981, X60693, U90566, M15470, X13115, M24045, U50086, L42296, Y14205, D44500, D50693, L20088, L20089, U06862, M24036, M24040, M24032, U17107, L38504, L41353, AF170577, M84381, L32862, U90561, AB032093, X87268, L76935, L76934, U18790, L75942, L76932, L76933, L36979, U90562, X60255, M24048, MZ8204, AF118895, AF118892, D84394, U05585, Y09118, U11265, U05578, E02884, Y09157, D50299, L04696, L04695, X61707, U30904, AF115461, L40599, M84383, X60254, and 222651.
HMSPF61 18 12265441 - 644 15 - D80195, D80193, D59927, D51423, 658 D59619, D80210, D81030, D80391, D80240, D80253, T03269, D80227, D80196, D80219, D80043, . D58283, D80188, D80022, D59889, D80366, D51799, D80038, F13647, D59275, 075259, D80045~ 015076, D80378, 014014, D50995, D80134, D81026, D59467, D59787, D80949, D80168, D80212, AA285331, 014227, 014429, D50979, T11417, D80164, D58253, T11051, D80166, D59695, D59859, D80269, D80268, D80064, D59502, D80024, D57483, D59610, AW178893, D80241, D81111, D52291, 014331, AW 177440, D51060, , AW178775, 014389, AW369651, AA305409, AW179328, AW378532, D51079, Ar905856, AW352158, D51097, AW178762, AW360834, AW17750I, D80522, AW17751I, D800I4, D51022, AI557751, AW360811, AW378540, AA305578, AW366296, AW375405, AW378534, AW377671, D80251, AW360817, D80248, AW375406, AW179220, AW179332, AW377672, AW179023, AW178905, AA514188, AW177505, AW360841, AW352I71, AW377676, AW352170, AW177731, AW178907, AW179019, AW179024, AA514186, 005695, D80133, AW178906, AW179020, AW177456, D80258, AW367967, AW179329, AW178980, .
AW177733, AW378528, AW178908, AW179018, AW178774, AW178914, AW378543, AW378525, D80132, D80302, AW178781, AW352174, D80439, AW178911, AW352163, D80247, AA809122, T48593, AL048680, D51103, AA03.3512, D80157, AW367950, AW378533, 006015, D45260, -- - D58246, D59503, AI557774, - AI525923, 003092, H67854, H67866, AW360855, AW378542, AI525917, AI535959, AI525920, AA514184, 014973, AI525227, 014046, 005763, AI525235, A67220, A25909, D34614, D26022, AR025207, Y17188, A62300, X671S5, A62298, X68127, D88547, A85396, AB012117, AR066482, A85477, A78862, A86792, D89785, U87250, A84916, I19525, AJ132110, A44171, X93549, X82626, AR018138, AF13.5125, I18371, S69292, AR064240, AF058696, AR008278, Y12724, AR051191, AB028859, 578798,X89963, AR008430, AR062871,115997, AR037157, A80951, X92518, AR051190, A43601, X60736, A94995, AR008443, A20702, AR062872, AR017907, A43189, AR062873, A43188, A20700, AR023705, A84772, A84776, A84773, A84775, AR067731, AR067732, A58522, A91750, A84774, I50126, I50I32, I50I28, I50133, A4S456, AR016514, Y09669, AR066488, A82595, AR060138, A26615, AR052274, AR060385, A30438, AB002449, AR066487, A43192, A43190, AR038669, 232749, AR066490, D50010, X55486, D88507,118367, AB033111, Y17187, AB012121, M60510, A70867, A63261, AR016691, AR016690, U46128, AR000511, AR008408, AR060133, D13509, A64136, A68321, I14842, AR054175, AB023656, . U87247, AF123263, AR008277, and AR008281.
HMWDR50 19 6675951 - 565 15 = AA005245, AA021087, AA313847, 579 W44826, AI750810, .AA243092, AW408321, AW405I27, AW401393, AAI76I92, AW405549, 017443, AAOS2922, AW403070, W56226, AW402424, AA129140, 827263, AA013055, W60367, AA299668, AA348993, N35264, T52117, AA381239, AA381052, AA296474, AA378689, F05537, AA853431, AW404182, AW401666, AA381988, ' AI214149, AI214126, AA152039, AW404586, AA058454, AA130130, N25137, AA100680, AA092258, AA158109, AA160037, 854504, M21533, AB014080, AC006139, AP000514, M16714, AJ251960, AF117228, L76290, AJ245567, Y18g51, AB012686, AJ271225, AB005048, X64880, AJ223972, AB012685, AF055066, K02883, AB023056, AP000519, M20022, M30681, X00492, M31183, AF101046, 293949, 293776, X55710, AF148862, AJ002533, M32505, X87678, M32506, X87680, L78455, L78934, L79943, AF111103, 255558, 258713, AF111102;
258696; U02976, U02977, U41837, AF004918, X03210, M18964, X03443, X01652, AB014080, AB014080, AC006139, and AC006139.
HSSJM44 20 11751001 - 2355 15 - AA633868, AI953254, AA858030, 2369 AI377035, AA600028, AI948432, AI347033, AI8198S2, AI953812, AI274863, AA913660, AI221391, AA402834, AI682227, AA313382, AI393729, 843673, T07940, T75200, AA402685, 246198, H10173, T34442, AA594521, AW05091~9, T07941, AI150326, AA921803, AA775669, AI301221, AI394223, AI869224, 241820, AW074819, 813072, AI424149, AA357096, AA383450, AI335517, AW302961, AI611494, AI348865, AI224202, AA368045, AW302995, and AI252073.
HPTXN38 21 9741951 - 350 15 - AW327936, AI696864, AW408434, AA263135, AW402317, AW385684, AA970332, AA294911, AW402511, AW239462, C03945, AA527306, AA190717, AA158146, AW405194, T68508, AA366365, AA663896, AW402322, AA112349, AA321294, AA361489, AA385962, AW401505, AA335761, AA352603, F00945, . AW351546, AW402698, AA147151, AA305941, 'AW405525, AA988615, AW401548, H11697, AA243206, AW293543, AW402264, AW401719, AA159413, AA158109, C18310, AI365653, AA527504, AA581164, AA220207, W24084, AA377064, AA372227, D82189, AA304603, AA477506, N42582, 834651, AA337991, AA160714, T10924, H98493, AI124815, AA151951, F11628, W24054, AA367387, AW403,872, AA263158, C04018, AA100848, AA121088, AA361916, T28025, AW407037, AW392650, AA321393, AL035930, AA126083, AI359260, D82221, AW403392, AA160037, L76091, AB030573, D50291, M19757, U14943, AJ223282, U16309, X94481, AB032598, L31798, AB032097, U11263, L20090, M24037, U75533, X96473, U16298, U32660, L76090, D50290, M24075, D50300, AF035647, L76935, AB032599, L76089, U18660, 246808, E03438, U18659, . AB036049, AB036050, X95410, X86704, .
L76931, AB030575, AF004370, AB032095, U06697, AJ005590, U58110, L76934, U31334, Y15841, AB032094, X99734, U11261, M80670, M68964, L41925, M32319, X94482, Y08995, X61706, U28759, AB032096, L76088, X84725, L76095, AF035649, AJ002151, X90391, L42282, Y08994, Y14205, X87268, L76094, X61710,-E02248, E03437, X86703, U18790, X64366, AJ131118, L42283, L22028, L36591, AB030574, AB032093, L76933, L36979, D50693, U70528, M84382, U29083, X75953, AJ002676, X94480, _ AF033351, L76930, U11267, L42280, M94053, U04243, AF035648, M29864, M28205, M32320, L42024, AF056981, U34618, L76932, U29880, U32678, U18789, L42146, L41214, M24035, U90562, U64801, 215143, M58636, U15639, M83191, U90561, U96942, AB036051, L42506, L75942, L20089, U06862, Y13029, M33573, M77774, M77777, M77776, L76096, U90560, M24038, _ - AB032933, AJ238702, L09735, U09864, U74387, L76093, X61709, M84384, M77778, U63653, D14343, X60693, U83580, D50295, U31971, M19756, L41086, M83194, M24048, U50710, AF115461, L09736, L36318, X60255, E02885, AF115460, M83193, X60254, L38504, U05581, U03027, U03859, L11603, L19937, M84694, M84383, Y09058, M94052, M94051, L22649, U29480, M84381, Y09118, AJ250917, U37324, M62852, -U15638, D49824, U90566, L42296, M83195, D83043, AJ251003, AJ006978, X13115, M24045, D25275, M24039, M86403, L20088, D50299, U30936, L42281, M28203, U37323, M14013, U05575, U50091, Y09157, L33923, AB008102, U43337, U17572, M15470, . ' AF118892, U11265, L42144, L40182, X73578, U37325, Y15840, M29865, M24040, - M24032, L04695, U17107, U30904, U03698, M95530, U05582, X81363, D44501, X61707, M32318, A28264, U15640, U04244, U11262, U11266, L20086, AF026218, U18661, L42345, Y14606, L07950, AJ237703, U05587, . L11571, L11570, D50296, L04696, U36392, L40599, AF016641, M77775, M84385, M84380, 233453, D44500, AF118895, and X94574.
HRKPA05 22 9742011 - 136 15 - AA158146, H63653, AA437332, 150 N42582, AA053162, H11697, N34268, AA977833, AA190717, AA053691, AA535767, T58784, AA133506, AA188398, W24054, W24084, ' F07425, AA321294, W44848, and N24326.
HTGFY58 23 9741981 = 289 15 - AW327936, AW408434, AI696864, 303 T68508, AA294911, AW402317, AW402511, AW385684, C03945, AW239462, AW402322, AW405194, AA663896, AW385181, AA243206, AA147151, F00945, AA190717, AA321294, AW402698, AA385962, AA581164, AA159413, AW402264, AW401719, AA335761, AA361477, D82189, AI124815, AA263158, AA367387, H11697, AA361916, 834651, W24084, W24054, N42582, AA151891, F11628, T28025, D82221, AW403392, AI359260, T75330, AB032093, L76933, L36979, X87268, U16309, L31798, U28759, AF035648, AF035649, U34618, U29880, U31334, X96473, AB032598, E02885, M58636, U15640,U90566, L76930, U14756,.U83580, U15638, AF035647, L75942, L76932, L76094, _ U18660, U11265, E02884, L04696, L04695, U309.04, D50295, 215143, U32678, X94481, L76091, X94574, X86703, 222651, U32660, L20089, U06862, L40599, D50299, L42345, AJ002676, X94480, X99734, AF033351, AB030573, L76090, L42506, U15639, M86403, U09864, D50290, L36591, X90391, -L42282, U30936, M24075, M84381, 246808, U96942, L42146, AJ223282, AB036049, AB036050, AB036051, X95410, AB008102, X7S953, D50300, U27I07, X64366, AJ131118, U64801, L42283, U06697, U90564, U18790, AF056981, AF004370, L22028, M24038, U75533, Y08994, M84385, M29864, AJ005590, AB032096, L76088, U11263, L76096, AJ238702, U90561, AJ002151, E02248, M80670, M24037, E03437, M68964, L41925, L42281, M32319, U90563, U90565, M32318, AB032097, D44500, L33923, D25275, U11267, M83192, L20090, U04244, D50293, U507.10, E03438, M24039, M32320, L32862, U74387, AF130734, D44501, U14943, U16298, D87665, M83191, D50291, L11666, U80945, M19757, M84384, Y09157, X61707 D44499, U11266, X99735, M15470, M83193, U90559, L42280, L36318, U03027, U03859, DS0294, D50296, L22027, L11603, L19937, X61709, U36392, M11799, AJ250917, AJ133267, D49824, U18659, U11262, M28203, M83195, M83194, L42145, L42144, L40182, M63454, L07950, L11570, AF016641, Y09058, M94052, M94053, M94051, U042.43, L22649, U29480, L41086, L20088, X61710, U70528, AJ237703, M84382, M84383, U31971, M24040, M24032, AF105226, X55711, U11261, D50710, L09735, L42296, Y08995, X61706, U29083, M19756, M29865, X60254, M59840, U37330, U05575, U50086, U90562, AJ251003, Y15841, AJ271160, AJ006978, X60255, Y14205, Y09118, U90558, L42024, D50693, U05580, X64454, and U21053.
HTXJBO1 24 11245961 - 987 15 - AA988703, AA675914, AA303646, AA675909, AW367626, AW058625, N40329, T54177, AA352261, AA595003, AA565043, AA079556, U95729, AF021222, U95731, AF021221, AF021223, AF021224, X9;1625, U69978, U95730, U95732, U95733, U95734, AF021225, AF021226, AF055384, L14848, AB000878, AB000882, AP000506, U65416, AC006046, AP000507, AJ249394, AC004180, D84394, AF055385, AF055387, X92841, Y16807, Y18113, AF045598, U56944, Y16811, U56947, U56948, AF055388, Y16804, AB015600, U56942, U56943, U56945, Y16803, Y16809, AF097406, U56949, U56955, Y16801, Y16805, Y18111, Y18114, Y18116, U56941, Y16808, Y18112, Y18115, U56946, U56952, U56954, Y16810, AF097403, Y16806, Y18117, Y18118, ,, U56940, U56950, AF097404, U56951, U56953, Y18110, AF045600, AF045597, AF045599, AF055386, AF045601, AF045596, AF045604, AJ242442, AJ242439, Y16802, AJ242438, AF045603, AF055389, AF'045602, AJ242441, AJ242440, AJ242443, AF055390, AB014083,. AC004208, AC004207, AC004170, AF055066, AB023058, AP000521, AP000515, AL022723, AJ242444, AJ242445, AC004200, AP0005.19, AB023056, AB003604, AB003602, AB003603, AB003601, AB003600, AB003599, AB003606, AB003607, AJ251156, AB003605, .
ABb03608, AJ251158, AJ251160, U69624, U69625, U69628, U69623, U69630, U69626, U69629, U69627, L29409, U69631, L2941.1, L29410, L29408, L29406, L29407, U69622, AB003614, AB003609, AB003612, AB003610, AB003611, AB003616, AJ251159, AJ251157, AJ251I61, AF106643, AF085013, AF106637, AF106655, AF085029, AF136159, AF106634, AF106640, AF106649, AF085037, AF011879, AF106646, AF136158, AF106633, AFI06651, AF011839, AF106639, AF106648, AF011836, AF011848, AF079414, AF085020, AF011845, AF106642, AF085024, AFOI1887, AF011842, AF011860, AF136157, AF106632, AFI0664I, AF106635, AF106638, AF011847, AF011853, AF106647, U69965, U69968, U69967, U69977, U69964, U69971, U69966, and U69969.
HUFAK35 25 636241 1 - 209 15 - AA449607, AA351420, AA295712, AW402698, AA401643, AL138056, N44225, H23327, AW385684, D56208, AW402402, AW403793, AW402754, AAISI95I, D82221, AW403114, AW403392, AW393618, AA380938, AW403872, AW405525, AA336651, AA160317, AW401548, AW402264, AW401719, AW401505, D82177, C18310, AW408440, AA310808, AI365653, . AW402322, D82189, AW239462, ' AW405194, AW402511, AW402317, AW408434, AA962801, AL039796, T29764, AA263135, AW327936, AI359260, AA350486, F07582, AA381116, AA075796, AA100680, AA132653~ AA083156, AA348075, AA129140, AW402855, AW402401, AA227277, AAI59230, 819052, AA081491, AA143626, U46382, F06589, AA055685, W52004, 854504, AA005245, AA352534, AA358919, AA970332, F06198, AA295230, AA160070, AF039198, AJ272049, U44064, AJ001977, AJ001975, AJ001976, AJ001973, . - AJ001974, D83957, 222752, AP000508, D84394, AC004204, Y14624, AF223221, AF221124, M17567, M17566, Y16411, AJ133474, AF036554, AF147701, AJ245869, AJ133100, AF105226, M16273, M16272, AF223219, AJ238694, D50710, U90558, .
~
Y15746, Y17064, Y18538, U56246, 8552, . ~ D83956, AJ238702, AF056981, U90561, E01341, M12967, 539758, X03945, AR008238, U90563, U90564, U90565, _ U90566, D50693, AF017330, AF015556, Y18533, AF145466, AJ249163, U56259, Y18660, AF036552, AF062587, AF.015558, U90562, AJ251755, D50709, M54883, M11799, M59865, AF019567, AF145760, AF220290, AF015554, AF104218, AF016304, AF173007, L24491, U90560, U89946, U90179, AP000507, M63454, X00495, D77998, AB005048, AJ223972, L41086, X55711, AJ005199, AF065646, I14616, U31373, AF101046, AC004182, AJ245567, AJ271225, AJ133267, AJ133475, AF105240, L76290, D64150, U89947, U90178, X03664, X03665, M16007, D50853, M84172, M99389, U60321, AF017323, U90559, J03027, 272423, AF042289, A93992, AF181857, AF072765, AF105029, AJ251960, K02883, AF117228, AJ250917, X99704, AJ251541, AJ271626, Y14684, AF002271, AF036556, U38975, AJ242661, D83741, D64149, AC004172, AC004193, U06835, U58643, AF115462, Y10520, 246810, D89333, U93915, AF017328, AF081275, AF017334, AF205536, AF226838, 293949, 293776, 272422, 297370, L20091, D83031, X73518, X78343, X87841, M99388, U06695, X67818, X55710, M15497, AL022723, AJ238151~ AF148863, U38976, L47206, L47231, L76288, L76291, L76289, 296924, 272007, AF076476, 280228, 275172, 249112, 279751, D64146, D64152, D83029, AF115464, L540S9~ U70580, AF096631, Y18648, AJ131852, AJ007603, M22794, U67748, U67330, U55022, AF002273, AF061863, AF072767, AF056483, U52175, U88254, AF108428, AF184216, AF208430, D38526, X97321, 283247, X76I89, X82I2Z, M28172, M24030, Y11843, U41420, L38251, X70856, AF072763, U90244, AF189013, . AF102563, AF170578, U59701, AF055066, I14588, AF002275, AF034409, AF135540, M26712, M24097, AB008136, 280227, ' Y15842, U80670, D49820, AB032095, AP000519, 233459, D64147, D64151, D64148,'AJ007605, U76396, AF054011, U58315, U90240, AF188886, U76394, AC018433, AC004172, AC004182, AC004204, and AC004204.
HWHKC91 26 9741911 - 419 15 - AA663896, AA263135, AL039796, AW401505, C18310, AW401719, AW402264, AW402317, AW402322, AW405194, AI124815, AW402511, AW403872, AW408440, AW402402, AW239462, AW408434, AA401643, AA160317, D82221, AA151951, AA310808, AW403793, D82189, AW402698, AW401548, AW402754, H23327, AA227277, AA100680, AW402383, AA548636, AA121088, AW327936, AI826037, AA224068, AA449607, AW405525, AW403392, AA102697, AA075796, AA158109, AA173111, AW385684, AA132653, AI365653, AW401393, AW408321, AW405127, AI7508I0, AW403114, 820954, D82177, AA160714, AA313847, AI359260, F13067, W52004, T29764, T52124, AA488534, AA381090, AA100848, AW405549, W40489, 819052, AW402424, D56208, AW403070, AA381088, AA058454, AA380938, T72958, AL138056, AA083156, AA005245, AA159413, AA381030, AA127128, AA336651, AA352960, AI214126, AI214149, AA152039, AA295712, N44225, AA351420, T52219, AA350486, AA344828, AA299668;
F07582, AA378689, AA295685, AA381116, AA381239, AA159230, AA853431, AW404182, AW393618, F05537, AA348993, AA157351, AA381988, W44826, AA160037, AA296474, AA348075, AA381052, AA352502, AA962801, AW402855, F06315, U46382, AA160101, AW402401, AA776025, F06589, AA358919, AW273557, AA143652, AA055685, AW404586, AA081491, T10924, AW403253, C19056, F06198, AA143468, AA352603, AA361477, 854504, AA129140, AA085102, AA160070, AA294911, ' AA295230, AA916617, AA143626, AA431065, AA102181, AA319533, 830668, T75330, T71226, AA527306, AW009172, AA372227, C16914, D44499, U03027, U03859, L11603, L19937, Y13029, L22027, D49824, D50294, L11666, U50710, AJ250917, D44500, U06862, D50293, U70528, M84382, L32862, X61709, M28203, D44501, U80945, U11262, U11266, L07950, M24033, L11571, L11570, D50296, AF016641, U36392, AF170577, D50295, L42281, M95530, M84385, U17572, D31816, U03698, M84380, X81363, U11265; E02884, D50299, L04696, U17107, LT30904, U30936, L40599, U04243, M84381, 222651, Y09118, L41214, L04695, M94053, 246808, U01848, U35431, U09864, L22649, M24039, X64454, AJ245869, D85762, M33573, M24036, U21053, Y09058, M24035, L36318, U64801, E03438, M94052, . M94051, L09736, M84694, M84383, U96942, U11261, D50290, 215143, M24075, D85761, M77775, M24032, AF 115461, Y08995, X61710, X61706, D2527S, L4234S, U11267, E02885, M58636, X13116, M30679, M24040, U29480~ U31971, M32320, L41925, Y13567, X91749, M24034, U04245, U21052, U290S7, L41353, M32317, M19756, M84384, X75953, M80670, E0343.7, Y08994, M68964, D50291, U75533, X64366, X90391, M24038, L42283, M32319, M19757, L17005, U88407, U14943, L42024, L36591, AJ131118, L42282, AF022783, M28205, AF189017, X90390, X84725, M63454, X60253, U04244, L09735, L22028, U29083, U11263, M24037, U90563, U90564, U90565, X60255, X60693, M77778, M77777, M77776, U36492, E02248, D50300, X99704, 246810, L42280, D83956, X60254, L19923, U636S3, U56246, Y18552, L33923, Y09157, M32318, X61707, AF118892, AF130734, D83043, M86403, AF118894, AF115460, AB008102, X70856, M77774, M84172, D64150, D64152, L20089, AF118893, X73578, U27608, M28172, M99389, D64151, L41086, M15470, 246809, 215144, D50853, U06695, U41420, L38251, D50854, M84173, M84174, D64146, M24045, M24097, L20088, U90566, 233453, X82I22, . D64148, D83029, AF118896, L38504, L20090, AP000507, X67818, D64149, X83394, AF026218, X77658, 283247, X76189, AJ010748, AF168611, E02883, L24373, U41386, X97321, U05581, AJ133267, X70857, AF115464, Y11843, X73518, X87841, M24030, D49552, AJ010749, ~
' 233459, D64I47, D87665, U44064, D84394, AF118895, M99388, D83031, X78343, D38526, X96582, Y09156, D31817, U098S3, and U06487.
HTSFJ40 27 11728261 - 108015 - W28953, AA313257, AI401170, 1094 H19139, 854508, H10122, H08285, 859784, F08505, AA490183, AI270117, AL046409, AI284640, ' 243765, AI963720, RS2605;
AW088846, AI431303, AI334443, AW 193265, AA682912, ' AW303196, AI305766, AI613280, AW3013S0, AW419262, AA720702, AA470969, AL042853, AW327868, AA610491, AA857486, AL045053, AL044940, AL041690, AW265385, AL138455, AI350211, AL138265, AI623720, AI345654, AI110770, AW072923, AI890348, AL037683, AA468022, AW276827, AI754658, AA581903, AA483223, AW407578, AL046205, AI307608, AW238278, AI281881, AI133164, AL119691, AA101689, AI754336, AL038474, AA877817, AA577906, AL134972, AW270270, AI801482, AA551503, AI355206, AI821271, AI538852, AW274349, AI457397, AA623002, AW410400, AW408717, AA515909, . AI345681, AI345675, AA680243, AA503473, AI732120, AL042420, AA908687, AW438643, AL038705, AI679782, AL048626, AI537506, AW028429, AA522942, AA523837, AI289067, AI559705, AI6I9997, AA584167, AW302903, AI754253, AW021207, AW304584, AI375710, AI064864, AA587256, AW406755, AW268300, AA533333, F08180, AAS31372, AL120687, AW083402, F36273, AW004911, AW274346, AA631507, AW088202, AA846876, AI345518, AI688846, AA507824, AA719292, AL038785, AL119984, AA521399, AI341664, AI254615, AA446657, AI469968, AA601355, AI568678, AW193432, AA526787, AL079645, AA599920, AI312309, AA491831, AI888518, AI962050, AI625244, AI814735, AW338086, AA806796, AA649642, AI708009, AA126450, AA521323, AW021583, AW302013, AI061334, AI696962, AI744995, AA493471, AL042753, AL046457, AW163293, AA523815, AA491284, AA6640I5, AI270559, AA610493, AA846935, AI583283, AA503475, AA469451, AW 162049, H71429, AI471481, AW271904, AI929531, AI339850, AI805363, AA525824, AI192631, AA482711, AA569167, AA613345, AA244357, AIS70261, AI633025, AI305547, ,' , AI345157, AW029038, AA639248, AI358571, AL043009, AI610159, AI053672, AI298710, AI053623, AL048925, AW276435, AI281697, AI890918, AA810269, AA713815, AI732865, AA493708, AL039958, AA584581, AI246119, AA569471, AW062724, AA780515, AA828042, AW 131249, AI249997, AA679514, AL120269, NS3150, AI368745, AF015149, AC005529, AC005902, D83989, AL022323, AL139054, I51997, AC0062$5, AG006120, AC005777, XS4180, U18391, U18394, AC004808, AL020993, AC005257, AC007216, AC006271, AF196779, U18395, AC007541, X55926, U57009, 298745, AC004989, AC004491, Y18000, AL121748, AC004019, AC005037, AF015156, AF015151, AL020995, U18393, AC005632, AC006030, U95742, U18392, AC005242, AC004832, AC002310, AL035697, U02531, AC004963, AC007014, AC007191, US7007, X54175, X54181, AL080242, 286061, AC004519, AL031319, AC006441, AC007052, X54178, U57008, AC007677, AC004650, AC005678, X54179, AC006501, AC016025, AL035659, . AC005244, 269917, AC004686, AC003003, AC004453, AC005019, AF015147, AC005387,. AL122021, AC002347, AC003664, AC005154, X54176, X53550, AL031577, U67801, AC005821, AC000041, X55927, U18398, U57006, AL035420, AL021808, AC005216, AC004816;
AC004884, AL049745, AL022721, AC006101, AF015148, AC005829, AP000049, U02532, AC006006, AL121892, AC005306, ' AL049874, AP000311, AC005291, AC002395;
AC005859, AF027390, AL136295, AL080243, AC004638, AC003962, AC008372, X55931, AC004236, AC007283, AC000052, AC004690, 298742, AL031777, AC004987, AC003075, U18399, AL031257, AC006128, AC007688, AC004751, AL031428, AL031311, AC005527, AC006017, AC006208, AC005899, AC007051, AC004493, AL049712, AP000131, AP000209, AL133485, 249816, AB020859, AP000552, -- - AC002429, AC006539, AL034548, w-- AJ003I47, AC00715I, X54177, X55923, AC007663, 298051, AC005261, AL035668, AC002128, AP000248, AP000095, AL034351, AC005740, AP000555, U62293, U63721, AC005694, U67231, AC002385, U18387, AP000459, AC007488, AC006373, AC006482, AC007043, AP000239, AL049775, AC005247, AC004053, AC007666, AF020503, AC008115, AL022315, AF024533, AC004381, AC005726, AC006211, AP000365, AL008718, AL031587, AL031289, U18396, AL023575, X88791, AC004099, AC004593, AC003957, - , ~ AC000066, AC005081, AF041427, AC005486, AC005699, AL049760, AD000092, AC008168, AL096701, AC005217, AL050097, AL031054, 283840, AL121591, 294279, AC007298, AC002401, U63630, U66059, AP000501, AL078474, AC004948, 299716, AC005913, AL022163, AC009247, AL022400, AL109753, 222650, - AC005971, AC006251, U85195, U91321, AC005763, AC002400, AL022302, AC006549, AP000036, AC002126, AC006213, U67233, AC002300, AC004998, AP000959, AF029308, AC003007, AF067844, AL035072, U91328, AL121653, AJ010770, X55925, AC005102, AL035071, AL023882, U69730, AE000658, L78833, U67832, and AC006040.
HEON059 28 7413611 - 848 15 - W37916, AW369323, AL138197, 862 AA292636, AA789205, AA278688, AI077497, AI670821, AW117287, AA608906, AA382777, AA724269, AA278680, AA827227, AI1.60796, AA600253, AI990171, AA625768, W37874, AA421286, AA292637, AA844108, . AA917528, AW316905, AA768446, and AF117210.
HEMBZ62 29 10787971 - 119615 - AW 138235, AW070706, 813025, . 1210 AA346549, and AA317149.
- HWBA018 30 7511251 - 383 15 - AL359711, AL359711, AL359711, 397 AL109947, AL109947, AL109947, AL136222, and AL 136222. .
HTLEN77 31 11470241 - 119015 - T89857, T89583, AL121270, 1204 AI349772, - AI064830, AL121365, AL047042, AL045500, AL036396, AW117882, AW238730, AL119791, AI687376, AW268253, AI636456, AI349004, AI433976, AI868831, AL036274, AL047763, AI285735, AL038778, AW301409, AL135661, AI43645,6, AL036802, AI475371, AW103371, AI275175, AI909666, AL040243, AW071349, AI433157, AI815383, AI521012, AL036146, AI697137, AI687728, AI349645, AI635461, AI250293, AI702406, AI53871.6, AW195957, AI439087, AW162071, AI678302, AI568870,, AI564719, AL036759, AI620284, AI340582, AL119049, AW169653, AI349933, AI866608, AI445432, AW089572, AI625079, AW2741-92, AI699857, -- -AW071417, AI440426, AI590128, AL046849, AI281779, AI500553, AI863014, AL038605, AI500077, AI857296, AI498579, AI366S49, AI580190, AW074993, AI567351, AL119748, AI439745, AI312152, AI345735, AL04S903, AI349937, AI207510, AI906328, AW068845, AI540832, AI613017, AI249257, AL120854, AI920968, AW 166645, AI343112, AI702433, AI497733, AWI48320, AI800453, AI349614, AI800433, AI679724, AI686926, AW087445, AW080838, AI690751, AW303152, AI873731, AI349256, AI567632, AI758437, AI610307, AI282655, AI631107, AL120736, AI597750, AI907070, AI499463, AI499393, AI818683, AI682743, AI608667, AI673256, AA613907, AI633419, AI866002, AI349598, AI866780, AL048871, AI499131, AI281773, AL040169, AL043326, AI921379, AI440239, AI560012, AA572758, AI628205, AI597918, AA640779, AI690835, AI469532, AI568854, AW 132121, AI568855, AI969601, AI224992, AI609592, AL038779, AI866887, AL036980, AW074869, AI475134, AW268768, AI348897, AI434281, AI682841, AI271786, AI800411, AI680113, AI889203, AI874109, AW301300, AI580984, AI934036, AI671679, AI889839, AI583316, AI251485, AI909662, AW167776, AI753683, AI813914, AI493248, AI969567, AW235035, AI919058, AI281762, AW085799, AI334902, AI149592, AI811863, AI459811, AI862142, AI635942, AI687415, AI818206, AI952114, AL036240, AL047041, AI802542, AI343059, AI687465;
AW026882, AI269696, AI492540, AI570384, AA528822, AL036631, AI345860, AI610645, AA585422, AI696398, AI609580, AL042753, AI539771, AI696846; AI307466, AI366991, AI500659, AA508692, AI445025, AL044207, AI349226, AI687375, AW104724, AI591311, AI624668, AI754897~ AI345744, AI569616, AL121014, AI684265, AI624859, AI612913, AI340519, AI536685, AI446606, AI872711, AA938383, AI318569, AW075351, AI620868, AI907061, AW 118557, AI859733, AI799305, AW302965, AI609331, AW269097, AI309401, AI446628, AW183130, AI432969, AI432229,'AL036247, AI690426, AI679764, AI636445, AW302992, AW002342, AI220734, AI619502, 568736, I48979, AL050393, AF090900, L31396, L31397, 578214, AF090934, A93016, AF125949, AF113013, AF078844, AL080060, Y11587, AF090901, I89947, AF118064, AF118070, AF113694, AL133016, AF113691, AL133640, AF090943, AL117457, AL133606, AF104032, AL117460; AF090903, AL137527, AL110196,.
AR059958, AL050146, AL049938, AL049452, AL110221, AF113690, AF113689, A1242859, AL050149, AL122050, AB019565, AF090896, I89931, AF113676, i142766, X84990, AL050I08, A08916, AFI06862, ALI33075, AL050116, AF113677, AL049314, AL122093, A08913, AF113019, AL049466, AL133557, AL096744, AF017152, AL137283, AL122121, AL080124, AL080137, AL050277, AL133565, E03348, AL137557, AL133093, Y16645, . I48978, AF158248, AFI13699, AL137459, . AR011880, Y11254, X63574, AJ000937, AL133080, U91329, E07361, AFl 11851, AFI7740I, AL049430, ALI22123, X82434, . ~ AF091084, AL050138, AF125948, AF146568, AF097996, AL117394, AL110225, E07108, AF079765, AL049300, AL137550, I49625, AL117583, U00763, AL133560, AF017437, A65341, S61953, A08910, AL117585, X70685, E02349, AJ238278, AL050024, AC007390, AL117435, AL049464, AL049382, A08912, AF067728, AF118094, I33392, A58S24, A58523, AF183393, AL133113, AL137538, U72620, A77033, A77035, 282022, AC002467, E05822, A08909, AL122110, AL137271, AL122098, AF091512, A03736, A12297, AL049283, AF061943, X72889, AL137648, AL137463, U80742, AC006371,103321, AC004686, AF087943, AC005940, Y09972, I09360, AC004987, X96540, I42402, AL080127, X93495, AL110197, X57961, AL096776, U35846, X65873, AC004093, AC006336, AL080159, U95739, U67958, I00734, AL137521, E00617, E00717, E00778, AL022147, AC004690, AF111112, AL110280, X98834, AC002464, AC007172, AJ012755, AL133014, AL133104, I26207, AR013797; U49908, AL133568, AC007298, A93350, E08263, E08264, I1'7767, AL133072, AL022165, L13297, I66342, AL137S23, AL137560, E15569, AR000496, U39656, U58996, AF026816, AL035587, Y14314, AL137533, X83508, AC006944, AL122049, AF153205, AC004200, AF061981, AL034374, 237987, AL133077, AL137476, and AF119337.
HTADZ74 32 11728251 - 128315 - AI735624, AW192167, AI307682, 1297 AF077346, and AC007278.
HELHB88 33 12267041 - 345215 - AI671143, AI580905, AI632369, 3466 AA773823, AW370310, AA773263, AA313515, AA985I66, AL121I53, AI382884, AA181724, AI7S0406, AI469456, AA024936, AA155593, AA024853, AL120063, T78993, T78788, T82245, AA155607, AI750407, AL134372, AA670080, L25219, AA247838, AI525585,.
AF1i4487, AF114488, AF064243, AF064244, U61166, AF127798, AF132672, AF132478, AF132481, AP000311, AP000117, AP000193, AP000049, AP000116, AP000050, AP000048, ' AP000308, AP000115, and AWS94325.
HE9TD31 34 8158451 - 941 15 - AI475682, AI439613, AA815076, 955 AB033082, ~
AF
132480, and AF132479.
HGBDG55 35 8158581 - 522 15 - AA368408~ and AL360268.
HOUHL51 36 l 1264801 - 760 1S - AA431822, AI341790, AW295199, 774 AI656610, AA037543, AW292290, AA431419, AA974280, AA815270, AA037457, AI651702, .
AA583011, AI208605, AI419858, AA620408, AA417333, AA417321, W28051, AA251183, AI917695, W28536, AI557751, D80258, T11417, D80064, D59627, C06015, D81026, C15076, D59467, C14331, D80164, D800I4, D59787, C14227, AI535686, D81030, D59503, F13647, D80166, AI557774, C14389, D51799, AA809122, D59859, D80212, D58283, D59619, D80210, D80240, D51423, D80022, DS0995, D80195, D80253, D58246, D50979, D80391, D59275, D80038, D80043, D80227, D80T88, D59502, D80219, D57483, D59889, D80196, D80269, D80168, D80522, AA305578, AA514184, D80439, C14014, D80268, D80133, D80045, AA305409, D80247, D80366, D51022, D80157, D80251, D51250, H67854, 221582, C03092, H67866, AI024754, AI525920, C14973, C05763, T02974, D59551, AI525227, AI525235, D45273, AI535961, C16955, 233452, A84916, A62298, A62300, A82595, AR018138, AR008278, AJ132110, AR060385, AB028859, AF058696, AB002449, AR008277, AR008281, I14842, AR054175, and I79511.
HEOPP67 37 8276301 - 436 15 - AA641653, 299396, AF181972, 450 and AF181973.
HKAOV71 38 8276791 - 743 ,15 - AF123303, and AF004161.
HFIJC31 39 8281481 - 519 15 - AA885328, N79858, and AI184184.
533 ' HDQID90 40 8319761 - 953 15 - AA767219, AI809238, AI219470, 967 AA767092, AA114887, T71487, AA464762, AA504439, 225261, N87679, D57415, AA278335, AW300598, W46278, AA669095, D56990, . D54675; AI797687, AI948608, AA909071, AW236181, AI718165, AB033082, AF132480, AF132479, andAC002350.
HKACD80 41 12065941 - 193715 - AI191318, AI978812, AA586860, 1951 AA805184;
AI628509, AI582366, W73797, W73745, AI620297, AW083832, AW239293,_ AW386876, AI056600, AI056739, AI362766, AI494212, AI077551, AA935678, AI348675, . AI358232, AA251769, AA968828, AA659758, AI891139, C06060, AA746268, AA251926, AA506524, H58621, D81244, AA291462, H58622, AW193598, 838144, 867182, AI919497, AI250032, AA604444, AI567397, AA905208, AA836253, AA551675, AW087829, AI364618, AI421662, 857498, AW080157, AW166086, AI813914, 859996, AL043152, AI680498, AI469754, AL079794, AI540179, AI554821, AI874261, AL119511, . AL045413, AL119399, AL042382, AI560545~, AI366900, AW262491, AW262042, AI954504, AI089970, AI677646, AI679164, AI,119457, AI868204, AI431909, AI453328, AI538085, AI680221, AI056328, AI536923, AW162194, AI553926, AI567302, AI538908, ~AI525934, AI921~746, AI799199, AI582871, AW129947, AW020381, AI805671, AA69333I, AA514684, AI687134, AI280747, AI184903, AI696714, AI609409, AW089932, AI812107; AL037454, AI564426, AI470293, AW148876, AI783.569, AI648458, AI673278, AI891084, AI865906, AI628015, AL042544, AI818353, AI819663, AW079572, AI887163, AI950688, AI355277, AI801152, AI702527, AL042191, AI203903, AW089006, AI696583, AI536563, AI798373, AA848053, AI633125, AI439745, AI698391, AL037104, AI538850, N25033, AW160905, AI610667, AI520785, AW080346, AI520862, AI921371, AI863382, AI918435, AI475815, AW150778, AI623941, AI690748, AI818562, AI469532, AI249946, AI499890, AW149869, AI961286, AI345688, AI624548, AI524179, AL037081, AI691131, AI581139, AI889213, AI374987, AA808175, AI539260, AI623179, AA832154, AI824557, AW074605, AI890391, AA190341, AI524654, AI587114, AI564290, AW084447, AW104641, AI336575, AI453767, AI886206, AI287326, AW082532, AI499131, AW090102, AA693355, AI498067, AL043168, AI368579, AWI62118, AW152415, AI744243, AA868961, AI826941, AI7984.56, AI627874, AI801592, AI872154, ALI18781, AI249877, AI583578, AI590423, AW 169653, AI922996, AI633061, AI309769, W45039, AI866127, AW088591, AW025279, AI520809, AI887620, AW189332, AI247193, AW118553, AW129616, AW301344, AW160916, AI863002, AI972070, AI612852, AI921479, AI401697, AI890223, AI141400, AL045453, AI245279, AI684021, AI433611, AI345415, AW151785, AI635032, AI690813, AW085639, AI816292, AI610690, AA731241, AW132001, AI359787, AI473451, AI921464, AI9I7963, AW236186, AI244105, AW089844, AI564723, AI927233, AW104724, AA857847, AF086313, U77594, AF090900, AL137530, A21625, Y11587, AF200464, AF111851; AF102578, U02475, AF090934, AF159615, AB025103, AF114784, 213966, AC002471, AC005374, AF076464, AL133084, X79812, I33392, L04859', X97332, AL049314, A77033, A77035, AF141289, AL133047, AF139986, AL080140, AF038847, S36676, U58653, A65340, AF100931, AF069506, AF084644, AF084645, AR068466, I32738, AF061795, AF151685, AL137523, AL133061, AF113019, X80340, AL137271, E01314, U35846, ALI37292, AR022283, AL137284, AL050143, S78453, AF113677, AF026008, AF081197, AF081195, AL050149, A07588, AF182215, AL133640, X84990, AB007812, 554890, AL080124, AL136884, X62580, AL133062, AL080126, AL117435, A58524, A58523, A08907, A32826, A30330, A32827, A30331, AF000167, U70981, AF094480, AL137529, AL137711, AF044323, AFI06657, U72621, AL137294, AL096728, 148978, AR029580, Y10655, AF119336, AL035587, U73682, AF061981, AF019298, AF029750, AF047716, AL133070, AF031147, AF077051, AF167995, I89947, E12580, AL080234, S77771, AL133075, AL137558, AF036941, A12297, A08910, A03736, A08909, AF067728, A91160, X99971, X65873, A91162, AL137550, AL034417, AL049996, A08908, L10353, AF115392, U58996, AL117416, AF017152, 237987, . ~ AL110221, AL122045, AF032666, A18777, ' E00984, I04527, AL122110, U92992, AL133067, AL049382, E12579, AL137459, AL137547, U75604, AR038854, AL117648, X82434, AL 137554, A08912, AF026124, U62807, AL096751, I68732, A08911, AL009179, AF078844, AF14S233, A65341, X60786, AL117587, AF114818, AF079763, . ' AF126488, AJ005870, AL137660, AL137254, X93328, AL137479, X00861, AF169154, . X99257, AF168992, AL133088, Y13350, U90884, AL080110, I80062, AF111849, AL122098, AF000301, AL080162, AL050116, AL137533, AF060555, AL0801S0, 576508, AL133049, AF106862, 297214, X72889, ElSS82, A08913, AJ1319S5, AL122104, 282022, U67082, AL137S38, AL122100, AF090886, AF118558, AF124728, AL137256, AL137534, AF039138, AF039137, AF030513, AR034821, AL137480, AF107018, AR064250, X15132, AL137627, X82397, AC004383, AL137478, AR012379, AL137560, AR059958, M27260, 582852, AL117443, AL050138, AF054831, U49908, AR050959, U75378, A15345, X61970; and AC004594.
HTEED80 42 849075 1 - 129915 - W87655, AA465429, AA203572, 1313 AI479983, AI540082, AA192438, AA836236, AA465358, AA829419, AA507269, AW290970, AI796504, AA904433, AA368409, AA688079, AW189971, AA601527, AA393948, F36549, W87656, AA320120, AI862710, F30605, AA196706, and AJ242978.
HE8TI39 43 12227701 - 280715 - AI681280, AA251688, W60548, 2821 Z4S463, H86769, F07768, H86760, 242543, AW339546, 815292, 244339, H8676S, H86774, H86962, AA091738, 858217, F06562, AW377760, and 821166.
HSDJH12 44 11639161 - 788 15 - AA428452, AA134294, T83462, 802 AI219740, AA010048, AI478566, and AI990289.
HE8PY29 4S 887862 1 - 741 15 - AI271550, AI753504, AA809220, 755 AW081079, W78099, AW386283, AI264068, AI219556, AW082138, AA455733, AI382746, AA548778, AA431230, AF092137, AF100751, AF040252, AC009948, AC009948, and AC009948.
HNTMB90 46 9109341 - 718 15 - 299396, AL038837, AL037051, 732 AL036725, AA631969, AL039074, AW392670, AL036418, AL039085, AL039564, AL036858, AL039156; AL039108, AL038509, AL039109, AL039128, AL036,924, AL037094, AL039659, '- AL038531, AL036196, AL039625, AL039648, - AL045337, AL036767, AL119497, AL037082, AL037526, AL036190, AL038447, AL119483, AL037639, AW372827, AL039678, AW363220, AL039629, AW384394,~
AL1194S7, AL039423, AL036238, AL039150, AL119319, AL040992, AL119324, AL042909, U46350, AL038520, AL119484, AL119391, AL037077, AL119443, AL119522, U46351, AL1193S5, AL119363, AL037726, U46341, U46347, LJ46349; AL134533, AL039410, AL038851, AL119341, AL134531, AL119335, AL119418, AL119396, AL039386, AL036998, AL036733, AL119496, AL037615, AL119439, AL036268, AL037085, AL119401, AL119444, AL037205, AL037027, AL037178, AL0453S3, U46346, AL036679, AL0426I4, AL036765;
AL042965, AL042975, AL134528, AL134538, AL042984, AL036191, AL119399, AL134920, AL042544, U46345, AL036719, AL043003, AL043019, AL042542, AL042551, AL0424S0, AL043029, AL134542, AL134532, AL037021, AL037054, AL036836, AL036158, AL119464, AL036774, AL036886, AL036999, AL036964, AR066494, AR060234~, AR023813, - AR064707, A81671, AR069079, AB026436, AR054110, and AR064706. -HMKCH92 47 9109361 - 789 1 S T78768, 813299, 814933, AB003592, D87248, ACOI8833, AC034I92, AC026206, AC026206, AC022381, and AC022381.
HTELV86 48 11264991 - 1572 15 - AW241703, AA923237, AA382531, 1586.
AA918449, AA738033, AA019310, 014331, AA305409, D80164, D51060, D51799, D80166, DS9619, D80210, D80240, 014014, D81030, D59859, D80212, D59502,'C14389, - D80219, 015076, D51423, D59275;
D80253, D50979, AA514186, D80188, D80439, .
D80022, D80366, D80195, D58283, D80133, D80391, D8024.8, D59787, D59467, D80043, D80227, D81026, D59610, D57483, D80024, AIS57751, D80196, D80269, AA809122, D59889, DS1022, D80522, D80268, D50995, D59927, AA305578, D80038, AA514188, D80193, D80045, D80247, b80241, D80251, AW360811, D80378, AI535686, AW177440, N71180, DS1103, D80302, AW178893, 014407, D80157, AW377671, AW375405, T03269, AW178906, AW366296, DS1759, . f AW360817, 014344, AW179328, AW375406, AA853033, AW378534, AW 179332, AW377672, AW179023, AW178905, AW020592, AW378532, N71199, AW378528, AW 178762, H67854, N81164, 006015, AI345688, AI696583, AW377676, AW352171, AW352170, AW177731, AI272244, AW178907, AW179019, AW179024, T48593, AW020397, AA749449, D51250, N25033, AI918408, D45260, _ 'AI557241, AW360834, AI612885, AW179020, AW178775, AW075382, AW022168, D81111, AW022593, AI525653, AW 177456,, AW 195253, AW
178980, AI915795, AW178914, AW074057, AW178774, AW023351, AW177733, AL120696, AW 178908, AW 179018, AA505147, AW080076, 003092, AW019-988, AI261815, AI280082, D59695, AW 177728, AW022981, AI557238, AW021182, AA099519, D59627, AW020480, AW023863, . AW020328, AW021178, AW020425, AI590043, AW022826, AI345010, AW022299, AW189802, AI538247"
AW021059, AI582912, AA857847, AI282673, AI696340, AI0940.92, AI469764, AI473471, AW021561, AI539260, AW264009, AI698391, AI918554, AI961309, AW378543, AI061180, AW378525, H67866, AW022760, AW352163, D80134, D80949, AI525923, AI289310, AI889147, N49165, AW192300, AI567971, D80132, AI224373, AW021909, AW020406, AW023469, D5I079, AA127461, AI866510, AI547227, AA975952, AW302990, AA868486, AW020710, AW021693, AA641818, AI469516, AW079432, AI866461, ' U35371, X99043, A62298, A84916, AR018138, A82595, A62300, Y11505, U79457, Y17188, 282022, A30438, AB028859, AJ132110, AR008278, Y17187, AF058696, AR060385, AB002449, S68736, I50133, X82626, X67155, A94995, D26022, Y12724, A25909, A91160, A67220, D89785, .
A78862, D34614, AR008443, I50128, I50126, I50132, Y08991, AF022857, AF022858, X68127, AR008277, AR008281, AR066488, AR016514, A45456, D88547, AR060138, A26615, AR052274, X92070, AF074604, AL133558, I48978, S63521, Y09669, A43192, A43190, AR038669, 573498, 583440, U46128, I14842, AR066487, U80919, AR054175, AR016691, AR016690, A51774, _ E12580, AF126247, AF113694, AL122049, ' X59812, AF090901, A08910, A08909, AF118558, AL137641, A08907, A08908, D44497, AF031147, E06788, E06790, . AR068466, E06789, I52013, U53505, AF195092, X98066 AF131773, AF093119, I46765, AF081825, AF137367, A63261, 575997, AF117959, D50010, AL049382, U61971, U61970, AF044323, AR025207, AF180525, AL080126, I48979, AF143957, I25049, AF016271, AR005011, AL137479, AR038854, AL008706, AF097996, AR050959, " " - - X66871, AL080234, AF177401, w~ AL096728, I00734, A31057, 536676, AL133088, AL133081, AL137459, E00617, E00717, E00778, AF139986, AL133049, AF057300, AF057299, AC004470, AL049276, AF211175, AL137259, A17115, A18079, AL079293, , AF078844, AF113019, AF038562, AR005195, I33392, AL110296, 569510, AF040723, Y11435, U92992, AR008408, AL122104, X54971, I25048, AJ005690, M92439, AF192522, AL032822, AL02I393, I29004, X66417, AL137657, I28326, AF051325, E01314, Y14314, AL133665, X97332, AF119336, AF090943, A65340, AL137560, and ALl 17626.
HNTAF23 49 9109471 - 239 15 - AW392670, AL119355, AW372827, 253 U46341, AL119483, AL119319, U46349, AL119457, AL119324, AW384394, AL119497, AW363220, 299396, AL134920, AL134524, AL119484, AL119363, AL119391, U46350, - , U46347, U46351, AL119443, AL119444, AI142137, AL119341, AL134538, AL119439, AL119522, AL119396, U46346, AL043029, AL037205, AL1,34531, AL119335, AL042614, - . AL119399, AI142139, AL119496, AL134533, AL119418, U46345, AL0430I1, AL042984, AL043019, AL042544, AL042896, AL043033, AL042965, AL042975, AL042450, AL042542, AL043003, AL119464, AL042551, AB026436, A81671, AR060234, AR054110, AR066494, AR069079, and AR043113.
HNBUTOi 50 10253751 - 100915 -'1023AA279757, AI632246, AI478566, AA977612, AI219740, AA716656, AA687260, AI801069, AA071046, AI985849, AW370598, AA630617, AW370599, AW370625, AA134295, AW390691, AII43764, D30955, AI990289, T79236, AW370620, AA352142, AA074442, AW071043, and AI744728.
HEMFI21 51 11282661 - 221515 - AI670810, AI720056, AW 195755, AW268679, AI400941, AI867849, AA053882, AI672024, AI880208, AW196438, AI655564, AI682042, AA034417, W27229, AW376127, AA425562, AI972I98, AA883340, AA132258, AI584045, AA770253, AW137059, AA132362, AA132257, AA425357, T62545, AW243732, AA491390, AI915665, AA721474, AA483037, AI269187, AA724043, AA346646, AW390324, N22655, AW377734, AC006042, and AL078581, , HMSOL52 52 9211261 - 129015 - AI911515, AI360955, AW028045, 1304 AI796049, AI609712, AW195544, AI184337, AI470056, AI361065, N34939, AI017177, AI038779, AI440241, AI65145~1, AA789292, AA854683, AI765258, AI702748, and AA384884.
HHGAE47 53 9221941 - 705 15 - AW025529, AW026010, AA657904, AA662803, AA886335, AA158820, AI475932, AW050607, AI885090, AI056120, AI244837, AA485566, AI375435, AA922036, AA878578;
- AA643750, AI056614, AW449834, AW197722, AI393408, AA485405, AI560410, AA161103, AI749095, AI720931, AW058170, AI446208, N57590, AI268967, AI832600, AI913781, AA910277, N52768, AI277003, AI914599, AI192693; N57604, AI673692, AW050712, AA631339, N52783, and AI919380.
HMCGL45 54 922195 1 - 114115 - AW025529, AI475932, AW026010, 1155 .
AA886335, AA662803, AI056120, . AW050607, AI885090, AI375435, AW449834, AA161103, AI244837, AA878578, AA922036, AW19.7722, AI056614, AI393408, AW058170, AA643750, AI560410, AI749095, AI720931, AI446208, N52768, AI913781, AI277003, AI268967, AA910277, AI914599, AI192693, N52783, AW050712, AA485405, AI673692, AA631339, AA657904, AA485566, AA158820, AI619710, AI560351, AI919380, . ' ~ N57590; AI832600, N57604, T25136, ' AW 198090, AI499963, AW023338, AI638644, AI890214, AI538850, N75779, AI633125, AI686817, AI499570, AI500061, AI860027, AI473536, AI925164, AW163834, AI690813, AW 162194, AA641818, AI684244, AI469505, AI376425, AI802542, AI673363, AI670009, AI498067, AW082532, AI433157, .
AI886055, AW080700, AI702073, AI884318, AW 128834, AI633198, AI289310, AI620056, AI590043, AW103928, AI890907, AW152182, AI589428, AI961589, AI679550, AI961414, AI698391, AI479292, AI701097, AI570861, AI147292, AW169604, AL045413, AW081383, AI345688, AI245008, AI539800, AI950729, AI927233, AL079799, AI491775, AI872423, AI538980, AI288050, AI635634, AI440239, AA805434, AW 161579, AW151893, AW148363, AI866465, AI538564, AI571439, AW083374, AL041150, AI973152, AI445611, AL047100, AI538116, AI687362, AI281757, AI241923, AA580663, AI440399, AI095003, AW022808, AI540674, AI954475, AI499890; AI471282, AI648494, AI927755, AI621341, AI445829, AI432030, AI915291, AI932503, AI341690, AI613038, AI866040, AW073865, AI699823, AW190194, AI559619, AL037582, AL037602, AI627988, AI685005, AL039086, AI348901, 841605, AI932794, AI345415, AI636588, AW163554, AI572096, AI612852, AW050998, AI580436, AI627893, AI568138, AA587590, AI869377, AL046466, AL118781, AI819545, AI270183, AI912434, AI567513, AI524179, AA830709, AW051088, I48978, I48979, AL137550, AF061573, A15345, I89947, I68732, A65340, AL117460, A77033, A77035, X63162, AF153205, AR029490, AL023657, D16301, AL117587, U78525, AL050366, E12747, AF067728, AF115392, AF090900, AR038854, A76335, E02349, 223.
AF102578, I33392, A58524, A58523, A08910, A08909, A65341, AJ005690, AL080159, A08908, 282022, A58545, AL137294, AL080074, AL137533, A52563~
AL137479, U37359, X70685, A93350, AF047716, A03736, AL122100, A08913, U49908, AL122110, X82434, AF183393, AF094480, - . ' 237987, AL050393, A08912, Y10080, S54890, A08911, A08907, AL133049, AF113690, AF100931, AL137271, U35846, . AL096744, 213966, Y14314, AR034821, S'76508, AF026816, AL136884, AF215669, AL049347, AR068753,. AL050149, AF177401, AF039138, AF039137, AF146568, AL117435, X72889, X53587, AL050155, AL133112, AL133637, AB031 I47, U55017, X67688, AF090903, AL137480, D83032, AL050277, AF097996, AF200464, AL117457, AL133665, - AL080163, 297214, AL133623, AR020905, -AF017437, AL122049, AF113699, AL110158, AF158248, AL137656, AL133010, A21103, - AF111849, AL110221, AF061981, AL080148, X81464, L04849, A18777, 536676, AL137530,_I25049., E01614, E13364, AF115410, AC002471,'AC005374, S75997, I89931, AF145233, AL049339, A07588, AF087943, U95114, AL080110, AL080139, AL137529, L04504, 577771, AL117392, I49625, AF107847, AL117416, AL137459, Y09972, AF058921, I09499, AL137476, AL137537, AF114170, AL137526, AF090943, AL133558, E05822, I33391, Y11254, AJ000937, AL133080, AL122104, AF141289, . ~ U58996, AL133075, AL050116, S53987, AL137488, U72621, AF030513, AL133113, AL133072, AF032666, AJ012755, X83508, AF061943, A17115, A18079, A08916, AF106945, I89934, AL133067, U83980, Y10823, U90884, AL117585, X66862, X66871, AL050024, S82852, AF17665.1, X84990, I03321, U96683, AL137256, _ AF180525, A12297, AL137521, AF106862, AF057300, AF057299, AL110225, AL110218, AR011880, A18788, AF113694, AB016226, A08456, A86558, A76337, AF118092, E12806, AF026124, AF061795, AF151685, U73682, I32738, AF090901, AL122093, .
AL050092, AL050138, and AL137292.
HEOQN14 55 923752 1 - 103115 - AI014538, AWb06457, AI479414, 1045 AI805243, AI290929, AI129301, AI872459, AI601146, AI708870, AI973043, AI540074, AI186894, AI682389, AI654747, AA460832, AI392777, AA405714, AA649837, AI356090, AI358510, AW294364, AA954900, AA991687, AI540589, AI953865, AA977875, AW19b678, 861326, 854477, AW009738, AA724308, AW297100, 854409, AA627570, AA504833, AA489470, H08185, 808582, AA778454, AI810108, 241744, R43473,.AA765208, AI698394, 239824, H19140, . 241120, F03843, AA701889, AA159318, AW408231, AA404221, H84256, AW131981, AI401170, AA405779, AI475002, F01761, AW189730, H84262, F04422, AA404687, AA502309, AA371650, H29188, AA581151, AA477301, AA749407, AA477302, and AI144326.
HSOBC04 56 9272801 - 120615 - AA115298, AI741325, AI688227, 1220 AI819333, AA452504, -AI925664, AI742595, AI174530, AA115338, AI567500, AA563582, AA461615, AI142563, AA80'7844, N94422, AI095261, AA569395, W58424, AA687480, AA479551, AA582573, AI081428, AA779677, AI280806;
N24393, AA988617, AI863187, AA834079, AW302361, AI362861, AW273442, AA150123, AA553678, AI752480, AI312661, W52661, AI298150, AA463418, W72509, AA024450, W72139, AI037968, W79868, AI028169, AA477651, H39596, W02690, AI198327, AI952450, AA926794, AI087245;
W74236, AW004736, AI334346, AI870989, H98040, AI689546, AI332748, W76066, AA150031, 840403, AI349417, AA595996, W80872, H99144, AW166280, W52767, AA496878, H25985, AI357863, 855375, AA363023, AW104147, AA378409, AI979074, AI376184, AI687489, T32290, N26307, H97338, N95244, W77880, . AI917258, W256b4, AI536791, AA024802;
AA577352, AA328156, AA359865, AA367475, AA461442, AA358275, W80763, T09474, AA987427, AI611160, AA888165, AA595303, AI918172, W30769, AI201782, AA187662, W21074, AA411955, AA935961, AA090719, AA411956, AA451977, AI371307, . ' AW074526, N79974, AI635472, N39751, AI612934, AA478489, AA102215, AI802295, AI750502, AA496836, AL133116, L07063, and AC012192.
HTXKL86 57 12122831 - 148615 - AI014538, AW006457, AI401170, 1500 AI479414, AI805243, AI290929, AI129301, AI872459, AI601146, AI708870, W28953, AI973043, AI540074, AI186894, AI682389, AI810108, AI654747, AA460832, AI392777, AA405714, AA649837, H10122, AI356090, AA313257, AW294364, AI358510, AI540589, AA954900, AA991687, H19139, H08285, AW408231, AI953865, AA371650, 854508, AA977875, AW009738, AW297100, 859784, 854477, AW407594, F07194, AA724308, F08180, _ . F08505, 243765, AA627570, H08185, 852605, AA489470, F07185, AW190678, AW407965, 808582, F05493, AA504833, AA778454, 843473, AI698394, AA765208, 813670, H19140, 245409, 239824, AA461135, 813641, 241120, F03843, AW 131981, AA701889, AA159318, AA404221, H84256, AI475002, AA405779, AW189730, F01761, AA502309, 861326, H84262, AA404687, F04422;
854409, H29188; AA749407, AA581151, AA477301, 241744, AA477302, AI144326, and AW513623.
HUJCT05 58 11254081 - 162515 - AW268357, AI889091, C15588, 1639 AI949350, AI056961, AI124874, 839133, AA887911, AW023386, AC003962, and AF155116.
HHFOC79 60 9354061 - 102415 - AI569931, AA450162, AA405198, 1038 H26214, H14443, N28528, 873380, AW015358, 848456, AI750978, AA358230, 219130, AA359395, AA744173, H26831, and AC008745.
HMTBB 61 11252651 - 576 15 - AA582539, AI963340, AI097093, 17 590 AA286856, AI761614, AI149781, AI460219, AI032670, AI636161, AI819154, AI089302, H12042, AI811219, H05308, T95010, AA836993, AW271462, 837000, AI001803, AA904906, AA743196, AI015200, AA453607, FO5000, AA578803, AI241466~ AI033193, AA330970, F03322, F01968, AA037601, T75492, N47542, AW 183219, AI288171, AA054759, F01965, AA651907, AL119324, AL119457, AL042544, AL119399, AW392670, . AL119484, AL119439, U46347, AL119443, U46351, AL134530, AL134519, AL119391, AL119319, AL119418, AW372827, U46350, AW363220, AW384394, AL119522, 299396, AL119497, AL119363, AL119355, U46349, AL119444, U46341, AL037205, AL119483, AL119396, AL119401, AL119341, AL119464, AL134525, AL043003, U46346, AL119335, AL134528, AL119496, AL134538, U46345, AL043019, AI142132, AL042542, AL042614, AL042450, AL042984, AL042965, AL042975, AL043029, AL042551, AL122084, AL049611, AB026436, AR060234, AR066494, AR054110, A81671, AR043113, AR069079, and AW612030.
HKGDE58 62 11252641 - 133215 - AW271462, AA582539, AI963340, 1346 AI097Q93, AI460219, AA286856, AI761614, AI149781, AI032670, AI819154, AI089302, AI636161, AA448686, H12042, AI811219, AA287162, , AA836993, T95.010, AI001803,.
AA904906, -AA743196, AI015200, 837000, H05308, AA453607, F05000, AA578803, AI241466, AI033193, F03322, AA037601, AA330970, F01968, 813858, H12041, T75492, 246111, F07044, T94956, H05358, N47542, AA053290, AW183219, T75535, AA454139, F05720, AI288171, AA651907, AA054759, F01965, F05717, AL122084, and AL049611.
HCHMW40 63 11295601 - 906 15 - AI732539, AI791495, AI791325, 920 AA709067, AW082062, AI791964, AI732667, AA505923, AW057561, AI909857, AI909862, AA601601, AI909853, AW151270, AI597748, AA938181, .
AA079317, AI017427, AI285826, AI473547, AA689584, AL036241, AI251221,, AI571699, AA836392, AI624279, AI343091, AW403717, AI624304, AI366992, AI310332, AW193299, AI289436, AI499325, AI289791, AA764900, AI590755, AA860080, AI866127, AI860897, - AI744256, AA713657, AI440238, AA427700, AI378123, AW021662, AI619754, AA713781, AW090387, AI702343, AI344819, AF094480, I28326, AF106657, A20553, L12407, AF007142, AF076633, AL133016, AF100781, A26498, A27631, AF090900, AR055519, AF028823, AL137298, A44314, U78525, I29004, X66417, AL080060, A23630, M19658, and E13998.
HE8QZ34 64 10868071 - 107015 - 835313, AA210809, W28575, 1084 AA112126, 886156, AA286753, AW405566, AA385668, AA384297, H25863, H50786, 825032, 246079, AA334931, AA490204, and ' AW367213.
HE8TM80 65 11857701 - 143815 - AW368592, AL040968, AW390796, AW361341, AA125853, AW361336, 856714, AW368596, AA127005, AA436573, .
' AI610191, AL040878, H53723,'AW386767, 872151, AA662674, H06566, AI620201, AW272525, AA428869, AA876119, AA643689, AI810630, T70821, AA126031, AI24~268, AI525912, AI624158, AA715437, AI139291, AA506178, AA366775, AW295598, AA071351, AI635219, AI628041, AA126626, AI223312, AI831145, AW272772, AA223723, AI393334, AA101504, AA125839, AA609710, AA166669, AA969727, AA813942, AA627552, AA588589, ' AA569083, T70552, AI520735, AI654578, AW173734, AW245101, AI128760, AI589990, AA436546, AW377757, AA653217,.AA909871, N25030, H98122, AA706136, AA182573, AI612136, AI612151, AW301938, AW074868, AI308036, AA281089, H84055, H29320, T90829, H06511, AW338571, AW292509, 856868, AI474847, AI682762, AW028456, AA307834, AA969587, 810267, and AF191018.
HHEFL55 66 957539 1 - 127315 - AW411262, AW239479, AA306419, 1287 C75208, .
AA311414, H06122, W38676, AA297279, T50005, AA302953, AW192348, AA334000, AA151050, AW383613, AW383609, AW383535, AA226372, and AA938031.
HWLEY40 67 11720211 - 142215 - AI014538, AW006457, AI479414, 1436 AI805243, AI129301, AI401170, AI290929, AI872459, AI601146, AI708870, W28953, AI973043, AI540074, AI682389, AI810108, AI392777, AI186894, AA460832, AA405714, AA649837, H10122, AW294364, AA313257, AI654747, AI358510, AI540589, AA954900, H19139, AA991687, H08285, AW408231, AI953865, AA371650; AI356090, AW009738, 854508, . AA977875, 859784, AW407594, F07194, F08180, F08505, 243765, AA489470, 854477, AA627570, 852605, AW297100, F07185, ---- -- - AA724308, AW407965, AA778454, 808582, F05493, AA504833, 843473, AI698394, AA765208, 813670, H08185, 245409, AW190678, 239824, AA461135, H19140, AW131981, 813641, AA701889, AA159318, AA404221, H84256, AI475002, AW189730, AA405779, F01761, H84262, AA404687, 241120, F03843, F04422, 861326, H29188, AA749407, AA581151, AA477301, AA502309, 854409, AA477302, AI144326, and 241744.
HHAWC08 68 10881461 -461 15 -475 H06763, F13201, T75396, N41533, AA310599, N26380, AW403968, AW247011, ~
W07220, N43834, N44897, AA322203, ' N25010, and AR0096 48.
HCEHD66 69 9591601 -1311 15 - _ 1325 AI968437, AI824971, AW104052, AI762197, - AI598138, AI088543, AI492390, AI827280, AA058923, AW007187, AW 135225, AI391466, AI808139, AA534403, AW028554, AI369729, AA460467, AA135928, AW006062, AW138526, AA507443, AI479413, AI400940, AW 137272, AW381735, AA135929, AA085774, W81153, . AA918755, C03738, 885039, AI472852, AI937792, AI867512, AA599118, N62215, AI864402, H41491, N62216, D44882, H14329, W30972~ H40979, AA463408, AI744140, H40980, N62166, AA319197, AI766568, N62223, AF186409, AF020184, L27421, L27420, and AC006241.
HMSGF27 70 12066831 - 156615 - AI191318, AI978812, AA586860, 1580 AI628509, ~
AA805184, W73797, AI582366, W73745, AW239293, AI620297, AW083832, AW386876, AI056600, AI056739, AI362766, AI494212, AI077551, AA935678, AI348675, AI358232, AA251769, AA968828, AA659758, AI891139, C06060, 867182, AA746268, AA251926, AA506524, H58621, D81244, AA291462, H58622, AW193598, AI919497, AI250032, AA604444, AI567397, AA905208, AA836253, 857498, AI525934, AA551675, AI364618, AI421662, AW080157, AW087829, AI813914, 838144, AW166086, AI680498, AI523628, 859996, AI554821, AI874261, AL045413, AI469754, AI366900, AW262491, AI540i79, AI560545, AW262042, AI954504, AI089970, AI677646, AL043152, AI679164, AI431909, AI868204, AI453328, AI538085, AI680221, AW089006, AI056328, AI470293, AI567302, AW162194, AI799199, AI690748, AI921746, AA741027, AI553926, AI58287i, AI805671, AI280747, AI536563, AI687134, AA514684, AI184903, AI610667, AI609409, AW089932, AI812107, AW020381, AI355277, AW148876, AI564426, AI628015, AL037454, AI648458, AW079572, AI891084, AI375702, AI865906, . AI818353, AI950688, AI439745, AI783569, AI801152, AW080346, AI673278, AL042191, AI538908, AI863382, AA848053, AI633125, AI520785, AL037104, AI698391, AW160905, AI538850, AI520862, AI917963, AI432570, AW151136, AI624548, AI918435, AI702527, AI499890, AI818562, AI623941, AW149869, AI469532, AI336575, AI961286, AI249946, AI345688, AL037081, AI581139, AI889213, AI824557, AA808175, AI623179, AW074605, AI890391, AW084447, AI453767, AI524179, AI287326, AI587114, AW169653, AW104641, AI886206, AI499131, AI368579, AI819663, AW082532, AW090102, AI866127, AI374987, AI498067, AI744243, AI802244, AI798456, AI826941, AI801592, AI520809, AI436438, AI872154, AI203903, .
. AI921479, AI249877, AI922996, AI583578, AW 189332, AI590423, AI524654, AL040011, . AI309769, AW152415, AI633061, AW088591, AW301344, N25033, AL118781, AI890223,,AI247193, AI887620, AW160916~
AA868961, AW 129616, AW 151785, AA916133, AI684021, AI635032, AI916419, W45039, AI691131, AA911767, AI433611, AI345415, AW025279, AIS64290, AW085639, AI816292, AI610690, AI690813, AI687424, AW132001, AI677796, AW 118553, AI564723, AI624529, AI620075, AI619631, AW 104724, AI4734S
1, AI972070, AI564719, AI950729; AI624604, AA857847, AW263796, AI589428, AI866469, AW118518, AW08938,7, AI798373, AA693331, AI445611, AI697372, AI359787, AI696398, AW088628, AIS72017, AI921464, . AW129456, AI758694, AI573032, AI922707,' AI696714, AI491852, AW 190042, AW166870, AI921633, AW089844, AI474.107, AW169234, AI927233, AF086313, AF090900, AL137S30, AF111851, AF200464, - - AF102578, AF090934, U02475, AF159615, AB025103, 213966, X79812, I33392, AL049314, A77033, A77035, AF139986, AL080140, 536676, A65340, AF038847, AF100931, AF069506, I32'738, AL137523, AF141289, AF061795, AF151685, A21625, AF113019, X80340, AL137271, E01314, - U35846, AL137292, A32826, A32827, I89947, . - AL133084, AC002471, AC005374, I48978, AL133070, AL050149, 578453, AF113677, AL137284, AF081197, AF08M
195, AF182215, AL133640, X84990, A~007812, AF076464, A07588, AL080124, AL136884, X62580, AL133062, AL080126, AL117435, A58524, A58523, A08907, U86379, AF094480,-AF1'14784, AL137529, AL137711, 577771, AF044323, AF1066S7, U72621, AL137294, 554890, A30330, A3033I, Y10655, I68732, AL03S587, AF117657, AF061981, AR068466, AF031147, AF036941, AR029580, AL133075, AL137558, A12297, A03736, A08910, L04859, X97332, A08909, AF067728, X65873, AL137550, AL049996, A08908, AF115392, U58996, AL117416, AF017152, 237987, AL110221,~AL122045, AL133047, . AF032666, AF026008, A18777, AL122110, AL133067, AL049382, A08913, AL137459, AF047716, AF195092, AL050143, AL137547, AR038854, AL117648, X82434, L10353, LT70981, AF026124, A08912, AL096751, AL133061, AF078844, U92992, AF119336, AF145233, A65341, AL117587, AF114818, AF079763, U73682, AL137660, U58653, AL137479, A08911, AF169154, I89931, X99257, AF051325, Y13350, U90884, E15582, AF111849, AL137554, AL122098, AF000301, AL080162, AL050116, AL137533, , AL133049, S76508, AF106862, 297214, X72889, AR022283, L24896, AF167995, I49625, L13297, X60786, AL034417, 282022, AL137538, AL122100, AF090886, AL137256, AF084644, AF039138, AF039137, AF084645, AF030513, AR034821, AL137480, AF019298, AL050366, AL022315, AL137478, AL133088, AL137560, E12580, AL080110, AR059958, S82852, A91160, M27260, AL080150, AL050138, A91162, U49908, AL133623, AL096728, I89934, A15345, AR020905, AL133637, A86558, AL122050, AL122104, AL080234, AF118558, AF199027, U57352, AF090901, AF137367, U72620, AL137627, AF118094, AL080159, AF118090, AL117443, X72387, AL122123, X53587, AL133665, AC007056, X81464, AF022813, 561953, A57389, X66871, AL050277, AC002464, E03671, E02349, AL117460, AL137574, I09499, AL080148, AL110228, I48979, AF077051, AR016469, AB019565, X98066, AL049339, X87582, U67958,, A83556, I46765, X55446, AF126488, AF112208, and AJ003118.
HBGMG39 71 971414 1 - 471 15 - AA024454, AA024670, AI458409, 485 AI740930, AI742565, AI743686, AI066465, AI168481, AI379125, AI569972, AW069135, AI497641, AW192429, AW084071, AW339039, AW102701, AI061450, AI693756, AI991329, AW003414, AI342244, AI870883, AW007899, AI609020, AI453165, AI248142, AI129686, AI992036, AI761292, AI623708, AI864435, AW151858, AI362058, AW044270, AI378430, AW008808, AI479128, AL036585, AI923881, AI963067, AI740972, AI,045227, AI351617, AI334039, AI452903, AI700412,. AI475537, AI469546, AI187911, AI066744, AI190677, AI812069, AI08123-1, AI050026, AI890929, AA551905, AI080156, AI554840, AW073739, AI926062, AA577674, AW 190499, AW250073, AI524714, AI884727, AW168902, AI096904, _ ~AI369151, AI858574, AI422058, AA304774, --w- AI890721, AI052823, AI683215, AI087154, . AA682804, AW080685, AW004991, AA912478, AW079985, AA054275, AA461453, AI668789, AA970861, AA010790, AW085591, AI819302, AI217784, AI017023, AI584163, AI627434, AA709077, AA984939, AA446048, AI564659, AI000961, AI095374, AI581899, AA532435, AA864689, AW087352, AW057507, AW132118, AA744639, N53410, AA969103, AA918799, AA862362, AI672825, AA779825, AA931463, - AI193638, AW191953, N20167, AI569968, AW 150032, AA612750, AI619904, AA429899, W02'113, AA969104, W48745,.
AA827905, AW 190663, AA780072, N50640, , - AA533847, W32574, AI640737, AI587533, AW182184, AI933617, AW073566, AI469425, AL041483, AA765028, AI952283, AI961508, AI095341, AA937508,'N48059, AA228075, AI869852, N78603, AA485535, AA506502, AA877348, AA252354, . AA010101, AA011219, T23065, AW444850, - AI917215, AA506948, AW297832, AW204879, N53257, AI917614, AI167689, AI937775, AI858564, AW043696, AW168023, AI263865, AI885655, AA450098, AW139189, AA883859, E12258, AF153686, E12259, E12260, and AL390719.
HTTCT34 72 10975981 - 623 15 - C75208, AA311414, AW411262, 637 H06122, W38676, AA334000, AA297279, T50005, AA302953, AW383613, AW383609, AW192348, AW239479, AW383535, and AA151050.
HAWAM69 73 943104 1 - 190115 - AA430300, AA541688, AA776700, ~ 1915 AW385785, AA679037, AA573270, AA126614, AL045796, AA682186, AI268236, AI963606, AW192904, AI926591, AI924827, AI922590, AI032288, AI375804, AA705172, ~
AW081541, AA694514, AI1308 83, N25288, AA931725,.AI800450, AI270687, AI366906, AW058362, AI683319, AA436891, W69578, AI597744, AI446542, 859176, AW453004, AI911821, AA687634, AI095665, AI130013, W69579, AA722782, AI191864, AI587015, AA398533, AA676733, AI476374, AA115447, AA554327, AAT59328, AW242281, . AA042956, AI139766, AA135916, AA886732, AA664356, AA358590,.H71919, AI565897, AW304844, AA916086, AA618576, AA363371, 244808, AA430199, AI370031, AA320329, AA393105, AW452852, AA135927, AI004140, AA135926, AA042816, H44791, T35731, AI865731, AA813424, 842647, 827785, T32691, AI934183, AA11.5446, AI857286, AW008428, AI631988, AA678468, AW075384, H44790, AI569918, - AI918635, AA603858, AA601518, AI745618, ' H42641, AI445766, 827874, AI939990, AA677131, AW364938, AI569374, AW029062, C01947, and AA732827.w -' HMTAV95 74 614936 1 - 395 15 - AB023187, AL137000, AL137000, 409 and AL137000.
_ _ HTEON29 76 11265221 - 119015 - AW004028, AI968030, AW237673, AA432290, AI143780, AW138422, AA428635, AA112090, AI14379'1;
and AA861634.
HHFGP83 77 11748261 - 263515 - AW027467, AW021001, AI679358, ' 2649 AA628405, AW002474, AA142868, AI720221, AI559580, AI952940, AI971163, AA126704, AW189486, AI086066, AI338366, AI632310, AW089385, W92463, AI372019, AI335800, N67118, AW130513, AI339087, W72626, AI962685, AI811098, AA043282, AI268440, AA054560, AI564240, AI623726, AA748136, AA012896, AA716090, AA047297, AA652890, AI869611, AI199091, AA938386, AA708870, AA760924, AA043925, AA582637, AA056075, AA017551, AI202160, AI916439, AA618223, AA632478, AA975496, AA249580, AI422146, AA932810, AA628573, AA628559, ~
' . AW 195978, D81448, AW 188932, AI857354, AI914554, AW193055, AA046731, 875666, AI873760, AI127156, AW023470, AA043406, AW023472, AI690518, AA043200, AA054620, AA468866, AW382445, AA047158, C01055, W76609, AA043281, AI918561, AI570316, AA338995, AI653300, AI917733, W27493, AL133599, AF004840, and AF090866.
HTEKS20 78 11234581 - 106315 - AI936596, AA868353, AI797296, 1077 AA725553, AI221970, AA428462, AA429551, AA431190, AA629305, AA629047, AI073397, ' AW235895, AI123443, AI808267, AA609412, AI914363, AA953895, AI214385, AA431516, AA911681, AA781953, AI825106, AA298758, AI215028, AA909534, AA723768, D10393, and 563991.
HCHATO1 79 12022141 - 383515 - AI357645, AW372245, AW372243, AI870876, AL079756, W80383, AA143521, ~
AI934507, AI497785, AA131786, AA143522, .
AA775429, N73794, AI190675, AA570709, AL041943, AW026848, AA746031, AI950978, AI422318, AA131772, AI885729, . AW026326, AA635171, AA934699,~
AA316540; AI246491, AI160357, AA487855, .
' AI041954, H11648, AI660052, AW152198, AI285004, AW028295, AI561088, AW166539, AI635148, AI094048, ' AW072627, AA464017, N24219, AA458881, ' AI094041., AI092047, AA464078, AA588471, AI804530, AI348338, AA487856, H99677, AI186100, H99573, AI025641, AA909894, N32879, AI081468, AW273806, AA872312, W78982, AA740804, M78765, AW337818, .
AI160858, AA977238, AA627981, AA985314, AA653069, AI610235, AI288191, AA492020, AI937558, AI357858, AW273022, H23489, N28433, AA018338, AW050885, AW365055, W01009, AI494441, AA348323, AA953253, AA484024, H30484, AA4S9071, AW025210, H90615, H90519, AA732761, F10250, N41828, AW117618, AW024678, AW079303, N47599, AA150812, 241805, 246179, AA864205, AA463943, D51145, AA464727, H12176, C16600, AA641974, T31584, AI886471, N73825, 808891, N45481, H20834, AI270409, F12634, AI991925, AA976247, - 809511, N76390, N68976, AA342119, 843266, AI802239, T90454, T78049, AI648496, AI001885, T31949, AI468389, AA046221, 843775, AA740939, AI655751, AA477480, AW139948, T31978, 817167, AA496370, 843146, 809622, 825017, AA496647, AA319007, AA348324;
T35092, . H46223, H61245, AA046114, 808999, . , 809784, AW071171, N99421, AI'085437, H61246, N84366, AA641917, AA648218, --- ~ ' , AA342120, T27314, AI042591, AB014S76,.
AA872361, AW517691, AW591480, and .
AW614740.
HAPNZ77 80 11719621 - 152215 - AI829331, AI307338, AA831880, 1536 AI379982, AA768459, AW270258, AW271904, AI345I57, AI431303, AL042756, AI755214, AA584489, AW265385, AI354423, AI963720, AL038936, AL040054, AI754105, AW021917, AI754567, AI733856, AW419389, AA402129, AW419262, AA680243, AA410788, AA634837, AI613459, AI926728, AW023672, AL041894, AI625604, AI282253, AA455483, AW327868, AW193265, AW069227, AA574442, AI064864, AA521323, AI244127, AA629992, AL046409, AI284640, AW327624, T05834, AI192631, AL048626, . AI054333, A I305766, AI613280, AA013168, . . AI962030, AI732120, AW075979, AA630854, AI753488, AA521399, AL079734, AA659832, AA019973, AI061313, AI336054, AI885572, AW270256; AA644090, H05940, AI799607, AA613627, AL118991, AI345654, AI587583, AA526625, AI291823, AI587565, AI003611, AI754767, AW166611, AA533176, - AW020088, AA833875, AA833896, a AA176605, AI332615, AI675615, A3039809, AW 157456, AA526326; AW275432, AW162697, AW007759, AA857486, AW023111, AI279417, AL046746, AL037683, AA634786, AI306232, AI017251, AW303196, AA864603, AI307201, AI457397, T49633, AI251104, AI290405, AA502991, AA904275, AI753113, AA491814, AW062724, AW439558, AA226584, AW301350, - AI696793, AW274I91, AW190505, AI133102, AL041706, AI867058, AW274349, AA904137, AI888468, AA513851, AW302903, AI270117, AA613227, AI016704, -- ------ AI570943, AI377413, AW022934, AI110770, AW131457, AA262752, AI224583, AA491284, D58782, AA779783, AW021583, AW304584, AI564185, AI272052, AW410354, H68343, AI890348, AL133668, AA394271, AA610509, AI040051, AW082117, AW117740, AI366555, AA086318, AI090334, AI870197, AW02I I6I, AA908687, AI696962, AC005859, AC003046, AL096791, AC005783, AC005939, AC003030, U73023, AC007312, AC005502, ' AC004841, AP000503, AC006001, AF053356, AC004417, AL109798, AC004590, AL139054, AL022329, AC004837, AC003971, AC003958, AL022476, . - AC006249, 285987, I51997, AF134726, AC006115, AC005701, AF146191, AC005037, AC009509, AL078581, L44140, AC005225, AC008078, AC012627, AL121595, 275888, 246936, AC007277, X53550, U57005, AP000697, AC007447, X55925, AC005211, X54177, AL021579, U80017, X54178, 275887, AC004895, AL022322, AC004139, AL035461, AL049636, AF111169, AF109907, AP000501, AC005102, AL109527, 298941, AC005251, AC010206, .
- ~ ., M37551, AC005490, U18392, U07561, AC012085, 273963, U18387, AC006442, AC007686, AC007999, U02532, AF077058, AL121603, AC007731, AL049699, AC006511, AC007204, AC006088, -AL035468, AL024507, AC005224, AC008115, AC005833, AC005630, AC004854, AL024498, AC006271, AL008627, AF064861, AC006948, AC004985, AC007685, AC007376, U02531, AC005829, 273358, L31848, AL031652, AL135,744, AF015147, AL079339, AC004002, U47924, AL034420, AC002470, AC004655, 298946, AL110502, AC007536, AL132987, AC007030, AC005500, AC003107, AP000210, AP000132, AC002504, AC006064, AC006006, U57006, AC005800, AC000118, ACQ05049, AC005772, AL049869, AC006251, AF015157, AC006388, AF149774, _ AL023807, AL080276, AL121655, AC005899, 282244, AC005821, X54176, 282182, ' AC000082, AC005015, AJ003147, AC00$394, AC004477, AC002115, AL031228, AL049757, 299943, AL021155, 284469, _ AF088219, AC005206, AC002558, ~ .
AC004876, AL096802, AP000359, AP000354, D83989, AF117829, AC007461, AC005013, 299715, AC007055, L77570;
AC004240, AC004686, AC005874, AF134471, AC005214, AF191544, AL050341, AC005005, AC012599, AC005756, AC008372, , AC005379, AC004253, AC002119, AC007226, AC008041, AC006027, -- AC004878, U57008, AL024509, AC004408, AF196779, AD000092, AC006536, AL022401, AL133245, AC005667, 293017, AC004821, AC008009, AC004106;
AL049759, AC007298, AL035587, AF196969, AF036405, AL031666, AC005901,.
AC004019, AF165142, AC004458, AL078477, 281370, AL132718, AC002094, AL049540, AF160252, 269667, AC009275, AC000029, AC006367, AL021918, AC004859, AC005480, AC005239, AL031433, AC002430, -AL034429, U62317, AC005751, 280232, AC005678, AC007546, AL121653, AC007384, AC005696, AC006600, AL021393, AL136295, AC002492, 283826, AL022'726, AF003626, X55931, AL021917, AC005089, AC004087, U67801, AC004992, AL022311, and AL034418.
HSIA078 81 12223431 - 198815 - AA527435, AW195324, AI653000, AW051613, AA514619, AI675204, AI652532, AA435717, AI659333, AI796596, AI273289;
AI880669, AI826786, AA889355, AI174916, AW004627, AA377072, AA255838, AA397980, AA430523, AI565825, AI435476, AW001866, N52904, AA430608, AI760594, AA298640, W69756; AA594479, AI149418, AI911011, AI871818, N71537, AI089421, . . - AA400874, AI038591, AA854839, AI565867, ' AI131012, AI144119, H65663, AW044396, N47230, AI732273, AW079534, AA847967, AW027678, AL044698, AA224892, T36269, AA009702, AI668849, AW 182206, AA011130, N78511, AI676028, AA968449, AI984040, AA207018, AA658246, N73670, AI937659, 853598, AA453038, AA904224, AW293549, 848261, AA775033, H52314, ' 838289, H48428, AW083969, AA588654, F10880, AA578060, AW298073, W25831, . . AA889378, AA483944, AC002302, X62260, AC002288, AL035588, AC007425, AF181896, AC004.216, AP000280, AP000038, AF003528, AL033525, AC009498, AC007676, AP000107, AC006080, AC005704, AC005332, AC005011, AL022328, AL118497, AC007221, AC005213, AL132987, U69570, 248484, AB020858, AC004382, AL049753, AC004874, AG006023, AC003976, AL035668, AL035671, AL139165, K03021, ' AC005771, AC004894, D83402, AC005004, AC005183, AC004129, AC004671, AC006354, AC005046, AC005161, AL117338, AC005184, AC006599, AF042484, AC004100, AJ006345, AC005235, AC004875, AC003693, AC004982, AL021877, AL009183, AC002546, 283822, 275741, AC002541, AC004098, U61375, AC007637, AC004061, AC005922, AL049821, 295114, AC006952, AC007529, AF095703, AL021395, AL080286, AL035696, AL117436, AC006973, 282246, AP000466, AL078638, AC007680, 284484, AP000030, AL035663, AP000252, AC011605, AL035427, AC012088, 286062, AC000378, AL035106, AL117375, AC004984, AFOOI550, AC006360, AP000134, AP000212, AC007392, AL022159, 296074, AC011594, AC004530, AC008012, AL132718, AC005060, AC007543, AC004015, AC008069, AL133162, AF001548, AL031390, AL135746, AC009263;
AB014079, AC007156, AC007198, AC004889, AL031123, AP000228, AL024508, AP000140, 268324, AP000146, AC005533, AL035633, AL031737, AC000119, AC004838, AC004616, AC007450, AP000088, AC005086, AC006398, AC006511, AC009275, AC004055, AC004882, AL021707, AC002563, AP000299, AC006139, AC005553, AF047825, 297876, 284480, AL020989, AP000514, AC004888, AC007314, AB020865, AL117347, AC006367, AC005562, AP000045, AP000113, AC005393, ACOOS690, AL049839, AL034425, AC007617, 275957, AC005529, ' AC005701, U95740, AF152365, AC012083, AL021407, AC000072, AC011700, AL031120,'AC005538, L77569, AC004381, AL109914, AL00863'S, AC009464, AL032056, U91319, AL110120, AC007455, AL022329, . 284488, AL035072, AC006213, AL022162, AC006196, AL118512, AL023806, AP000536, AL121866, ACOOS587, AL033384, AC006998, AC007262, AF107258, AL034555, AL021451, AC006064, AL050350, 274409, and AC004968.
HWBEG18 82 909798 1 - 940 15 - AA906863, AW408789, AW452373, AA352977, AA732349, AI269653, AI492098, and AB020653.
HFCBB56 83 12043221 - 209015 - AA491955, AA053463, AI620992, AW005974, F13749, AW408767, AA864271, . AW162288, AA565232, AA501867, AW265688, AI493583, AA744094, AA904211, AW316599, AI224619, AA019S42, AA339423, AI696793, AA878492, AW081610, AI492579, AI623364, AI445373, AA630535, AA513983, AI243793~
AI889426, AW054936, AI678867, AI049955, AA828834, AI190.648, AA721645, AI278372, AW236219, AI633386, AI797998, AA744048, AA669155, AA572983, AA862312, AI889995, AI636734, AI368862, AA283081, AI279417, AW407889, AI312090, AI049630, AI689019, AL042667, AL042670, AW337805, AI887755, AA832016, AA833896, AL049869, 284469, AL122020, AC004821, 295114, 283822, AL022315, AC002477, AC004815, AC007298, AL034429, AC005800, _ AC005736, AL031311, AL009172, .
AC012384, AL034400, AF053356, AC007435, AC002312, AC009516, AC004185, AC004921, AL031657, AC005670, AF165926, AC020663, AL031680, AP000501, AL133245, AC002549, AC005697, AC003664, AC005755, AL022322, AC002430, AC005722, AP000509, AC002395, AC005088, AC007384, AC005972, AC009509, 295113, AC006441, AL133243, - AL035423, AC000025, AC016025, AF117829, AC000379, D84394, AL139054, AC005527, AC003043, AL031681, AC005531, AP000704, AC004659, AC004476, AF001550, AC011311, AC005529, AL031431, AL022238, AC004494, AC007880, AC007242, AC005089, AL022324, .
AL0312$0, AF064861, AL024507~
AC005952, AC005837, AL031848, AC004382, AC007785, AC007934, AC005484, AL049829, AC005412, AC005726, AC002091, AC004383, AF111168, - AC002300, AC000035, AP000210, AP000132, AF109907, AC004699, AC009946, AP000251, AL008629, AC006285, AP000155, AC002369, AC0.05324, AC005037, AC006160, AL031666, AC004675, 298047, AC007684, r ' AC016831, AL049843, AL049709, 274739, AP000030, AC005390, 294056, AL049538, "
AF196970, AC007308, AC004895, _ ~ AC002350, AL035555, AC003048, AC007066, AL121658, AC005746, AC002528, 283844, 299127, AL121603, AC005632, AC004585, ACOQ5480, 286090, AL121655, AC005042, AF196969, AC004552, AP000098, AL031293, AC002472, AC005969, AF088219, AC004231, 307848, AC004020, AC004796, AC005377, AC002381, AL031588, AP000500, AL133353, AC002378, 275887, AL022313, 268324, AC005535, AC003962, AL031587, AC004148, AL021939, AC005871, AC005880, AC007690, AC007664, AC004884, 295125, AC005399, D83253, AC005304, AL035458, AC005332, AL035684, AL008631;
AC004167, AF030453, AC006454, AL022476, AC002565, AC004099, AC005669, AL031591, AL031432, AC002492, U91321, AP000088, AC004865, AL031427, AC009542, AP000150, AF003626, AC004685, AC009247, AC007546, AC005300, AC005829, AC006146, AC006001, AC006023, AP000014, AC007312, AC007993, AC005730, AC009330, AP000009, 283846, AC012085, AC005808, AL034554, 284466, AC004593, 298742, AC004859, AL022316, AL035409, AC008372, AC005902, AF031078, AL109952, AL133448, AP000352, AC003957, AF205588, AP000116, AC004816, AC007687, AF030876, AC005901, AC000394, ACOQ7011, AC002351, AC018633, AC005839, AF047825, AC005695, ~ -- - --AC005180, AC007528, AL031117, AC004149, AF067844, AL022320, AL121595, AP000689, AC006450, AL033527, AL020997, AC005562, AC006064, and AL035587.
HEMCL65 84 9109001 - 453 15 - AI902552, and AI902562.
HMAMB94 85 12253431 - 244015 - AW369653, AW024321, AA766018, AA905970, AI652025, AA158332, AI627492, AW068035, AA158041, 225180, W40230, AI493579, AI536877, AA191353, W45464, AW175724, AA670064, AA190444, AA248856, AA247921, AL121448, AI926451, AI983662, W60091, AW068830, 890961, .
890977, 890972, and AA309409.
HCQCI06 86 9150001 - 119015 - AI123952, AI751915, AA343597, 1204 W79144, . AA345718, AA304549, AA256582, W81545, . D45547, AL117664, AC068763, AC068763, and AC069223.
HWLFG75 87 12276421 - 202615 - AI356559, AW 163067, AI937030, 2040 AI652337, AW028706, AW157098, AW028808, AA443325, AA004795, AW005140, - AW173645, 860229, AA442531, AI274924, . AI810652, AI924004, AI572794, AI336556, AI672253, AI147260, AI872258, AI347103, AA467751, AA724594, QI280850, 852646, AA536110, H16834, AW450707, AW444512, AI376913, AA468349, AI807962, AA927875, 842625, AA609873, W28566, AI918962, AA578362, AA578062, 817389, C18386, 815375, AI016851, 860462, H16941, AI423739, AA467933, AA740299, AA025666, 842116, AA978110, AI423740, AW117517, AI886594, AA443338, AL040243, AI275175, AL119748, AL121270, AI857296, AI433157, AL045500, AI433976, AW071349, AL047763, AI702406, AI250293, AI436456, AL048871, AW 117882, AI620284, AW 195957, AL119791, AI064830, AI568870, AI863014, AI702433, AI499463, AI538716, AI439087, AI349933, AI678302, AI633419, AI868831, AI440239, AW074993, AW162071, AW235035, AW071417, AW274192, AI349256, AI866608, AI349645, AI690835, AI498579, AI613017, AI349004, AI349772, AW301409, AL135661, AI699857, AI540832, AI440426, AW103371, AI628205, AI281779, AI500659, AL036146, AL036396, AI340582, AI521012, AI500077, AW169653, AI249257, AI567351, AI500553, AW238730, AW268253, AI224992, AI312152, AI345735, AI349937, AW089572, AL120736;
AI366549, AI564719, AL047042, AI568854, AI597918, AI866002, AI673256, AI281773, AI590128, AI539771, AL038605, AI497733, AI969601, AI636456, AL036802, AW087445, AL119049, AW068845, AI282655, AI690751, AI612913, AW148320, AI800453, AI800433, AI3431I2, AI909666, AI567632, AI635461, AI687728, AI686926, AI800411, AI682841, AW3031S2, AI348897, AI610645, AI349614, AI207510, AI697137, AL121365, AI597750, AI921379, ' AI679724, AI758437, AA640779, AI866887, AI432229, AA613907, AI625079, AI499393, AW301300, AA572758, AL036759, AI475371, AL036980, AW074869, AL036274, ' . AI815383, AI610307, AI570384, AI631107, AI608667, AI818683, AI969567, AI434281, AI687376, AI285735, AL046849, AI282903, AL040169, AL043326, AI873731, AI920968, AI445432, AI469532, AI889203, AI580190, AL038779, AI811863, AI680113, AI906328, AW085799, AI889839, AI671679, AI874109, AI620868, AI753683, AL120854, AI934036, ' AI609592, AI569616, AI583316, AW166645, AW080838, AI439745, AI446606, AI255071, AI343059, AI866780, AI919058, AW002342, AL038778, AI475134, AI687415, AI345860, AI952114, AI872711, AW168384, AA585422, ' AI580984, AL047041, AI269696, AI149592, AI907070, AI610756, AW1'67776, AI345111, AI799199, AI499131, AW 196141, AI499512, AI907061, AI909662, AI349598, AI307466, AI539153, AL080060, I48979, Y11587, AL133016, S68736, AF090901, AF113691, I89947, AL133640, AL110221, AL137527, A93016, AR059958, 578214, AL049452, AL050393, AF090934, AF104032, AF113013, , ' AF118064, AF118070, L31396, L31397, AF113689, AF078844, AF090900, AF113676, AF090903, AL049938, AF125949, AF113694, AF090943, AJ242859, AL133606, AL117460, ' . AL050146, AL110196, AF113690, AL117457, AL050149, AF106862, X84990, I89931, AF090896, A08916, AL050116, AL050108, AL050138, U42766, AL049466, AL133075, AB019565, AL049314, E03348, AL122050, AF113677, A08913, AF113019, AL080137, . AF017152, AL080124, AL137283, AL133557, AL122093, AL096744, AL133080, AL050277, AR011880, I48978, U91329, AL133093, AL122121, E07361, AF158248, X63574, ' AL049430, AF113699, AL137557, AL133565, Y16645, Y11254, AL137459, X82434, AL122123, AJ000937, AF091084, AC002467, AF111851, AF177401, AL117394, AF097996, AF146568, AL117583, AF125948, AL110225, AL021393, AF079765, AF017437, I49625, 561953, I33392, AL049382, A08912, U80742, U00763, AL050024, AL117585, E07108, AL133560, AL137550, AF095901~
A65341, AF000145, A08910, AC002464, AL049464,.
AJ238278, E02349p AL049300, AC007390, AC006371, AL117435, AF067728, X70685, ' X72889, 282022, AL122098, AF183393, AC006222, AF118094, AF001552, A58524, A58523, AL137271, U72620, AL122110, A08909, I03321, U62317, AL122049, AL022147, AL137463, AL096776, AC004200, ' -- I09360, AL137538, AL133113, ~- ~ AL049283, 239 .
E05822, A03736, A77033, A77035, AC005057, AL031281, AL080127, AC007172, AC010077, 299297, AF061943, A12297, U35846, AF087943, AL137648, AC007043, A21625, X96540, AC004690, . ~ A08911, AF110520, AL078630, AL049557, AC007458, AL078602, X65873, U95738, AP000344, L13297, AC009233, AL110197, AF 177767, AC007298, AL031346, X93495, AC004686, AC006336, AL022165, AL133072, AC005291, AC004093, U67958, AL080159, A21622, AF130342, AJ012755, AP000520, U66059, U95739, and AW514074.
HBODA38 88 9234561 - 188215 - AA196401, W79841, AA112831, 1896 AA197134, .
C05212, W19681, AA195812, 225012, _ AI004215, 230134, AA194641, AI809925, AA258550, 228480, F29133, AA699576, F20387, AI129717, AA194359, AI079487, AA195355, AA195094, AA931319, F34387, 219343, F36761, H67895, AA258604, AI378821, F19321, AI089231, 891150, AI702453, AI288969, AI313118, AI351843, ' F31038, N56278, AI381419, AA195356, . AA131264, AA195051, AA192095, AI264911, and AA086284.
HCYBK19 89 12253241 - 276415 - AI829726, AI809562, AI927757, 2778 AI927758, AI927768, AW297770, AA165336, AI638072, AI149852, AW298053, AI365991, AA904547, H15885, AA985065, AI359235, AW207262, AI470080, AI421572, AA305419, AA985170, AW341104, A1423550, AI362528, AI453702, AI654827, AA927292, AI365149, AI365324, H91991, D81295, AI797740, 862753, AI357382, AI953516, AA909430, H01576, AA969956, AA379840, H52748, 837302, AA905264, 243289, 818887, 868272, .
H04859, AA746048, T31617, H04764, 244077, T78758, T32807, D60323, AA634475, H01477, AI193962, H00918, D81446, M86147, AA983881, AA318875, N64328, AA165369, D62629, 239361, T16370, F03272, H00919, N34301, H46107, AW590162, AW593404, AW593449, AW593464, and AW593480.
HWNCY05 90 12230311 - 118115 - W40569, AW025860, D63226, . 1195 AA334307, AL038838, AL037436, AL037323, AL038983, AI142134, AL134524, AL038822, AL037727, AL038532, AL037343, AL040617, AL044186, AL041238, AL047012, AL037335, AL037435, -AL044125, AL044037, AL047170, AL040463, 3 AL045684, AL040625, AL047219, AL044162, AL043677, AL040193, AL043467, AL040510, AL040621, AL043538, AL041752, AL047183, . AL043496, AL040464, AL043923, AL043814, AL037443, AL040576, AL043845, AL040839, AL045753, AL041635, AL041133, AL041347, . ' AL040294, AL041324, AL046442, AL044064, . AL041459, AL041577, AL040322, AL043492, AL041602, AL041098; AI:044074, AL046850, AL040052, AL040768, AL040119, AL046994, AL041730, AL041523, AL043627, AL046914, AL040472, AL041374, AL045817, AL041096, AL043848, AL043570, AL042135, AL040444, AL041163, AL047057, AL037295, AL045671, AL039316, AL041159, AL038761, AL041955, AL046392, AL040075, AL04_1292, AL041358, AL044272, AL041296, AL041142, AL044258, AL041346, AL041086, AL0411~68, AL045920, AL040149, AL040529, AL040148, AL040332, AL042898, AL,046330, AL049018, AL041197, AL040458, AL044187, AL041246, AL047036, AL041233, AL045990, AL039643, AL040128, AL040571, AL040553, AL040745, AL040370, AL044274, AL079878, AI,039338, AL041277, AL040342; AL040155, AL045328, AL044199, AL041131, AL041186, AL040285, AL040414, AL039360, AL041051, AL040091, AL039744, AL037341, AL044165, AL040090, AL045989, AL040168, AL042096, AL043941, AL079852, AL041227, AL043775, AL046327, AL044201, AL040253, AL045857, AL040082, AL039432, AL043444, AL040329, AL038745, AL041140, AL044529, AL037279, AL041278, AL040263, AL045725, AL040255, AL040238, AL047037, AL039915, AL043612, 228355, AL049069, AL134110, AL047163, D61254, AA585101, T11028, AL043537, AI546945, AL041210, T23957, T23985, AL045211, AA585439, AL046147, AI526194, AA585476, AI525556, AI547295, 016300, AI541365, 230131, AA174170, AL045327, AI541374, AI525431, AI541523, AI541514, 829445, AL041344, AL540967, AI541390, 016305, AI546999, AI541508, D57491, T23~888, AL080031, AI557238, AI526073, AI557731, AI526140, T41289, AI557262, AI525306, 829218, AI541535, AI535660; AI557799, D55233, 014723, AA585453, AI546828, AA585356, AI546875, AI547006, AI525320, AI556967, ' AI541205, AI535639, 829177, AI541013, 828735, AI546855, AI541509, 015189, AI526184, AI541534, AI526176, AI557734, AI526144, AI541017, AI525316, AA283326, AI546891, AI557727, AI536138, AI541533, AI557787, AL038651, AI541307, AI526186, 828895, AC006928, AR064707, I05558, I18895, I08396,.Y16359, E13740, E03627, AR062871, A25909, I48927, I84553, I84554, A81878, A60212, A60209, A60210, A60211;
. . . A02712, A77094, A77095,.AJ244003, y AJ244004, AJ244005, AF082186,-AR031566, A35536, I08389, A98767, A02135, A04663, A93963, A93964, AR062872, I63120, AR017907, AR062873, I00682, A11623, E00609, I06859, A11178, E01007, A18050, A23334, A75888, I70384, A60i 11, A23633, AR007512, I15717, I15718, A90655, I08395, D13316, AR043601, A95051~, A18053, A64973, A20702, A43189, A43188, A20700, A98420, A98423, A98432, A98436, A98417, A98427, M28262, A85395, AR038855, A85476, A35537, D78345, A02136, A04664, A11245, A11624, AR037157, AR054109, I13349, A10361, A91750, A02710, E14304, A07700, A13392, AR031488, I13521, I52048, I44531, I21869, A91965, I44516, A70040, E16678, AJ244007, I44681, X83865, A84772, A84776, A84773, A84775, A84774, .
AR067731, AR067732, A58522, A22738, I62368, A86792, A93016, I01995, I25027, I26929, I44515, I26928, I26930, I26927, I08051, AF149828, I03331, E12615, AR035193, A92133, A13393, A27396, AR027100, I49890,:I28266, A82653, E16636, A24783, A24782, A95117, AB025273, I60241, I60242, D50010, A58524, A58523, AJ238010, A20699, E00696, E00697, E03813, I66482, AR009151, I66483, I66484, AR027099, I66487, Y09813, AR051957, AR038762, 166485, I66498, 166497, I66496, AR038066, I66486, AR066494, AR051652, 232836, AR051651, AR008429, AJ230935, X07299, AJ230902, AJ230951, AJ231009, A70872, AJ230972, E17098, D13509, X81969, AJ231028, U94592, AJ230867, AR022273, I66495~ I66494, A22734, AJ230845, A70869, E12584,119525, A93923, D17247, A06631, AR035975~ AR035977, 560422, A93916, AR063812, I18302, Y14219, A24548, A24546, - A93931, A16035, A85203,133632, AL133053, AR035974, AR035976, AR035978, AL122101, E03654, AR023813, AR054723, AL133074, AL133049, and A22739.
HTXNN68 91 11433001 - 151615 - AA287304, AI418151, AA885205, 1530 N32677, AA147402, AI630898, AI362039, AW075796, AA905465, and D86967.
HWWFWO 92 933671 1 - 880 ~ 15 AA287304, AI418151, AA885205, - 894 N32677, 6 AI630898, AA147402, AI362039, AW075796, - ~ AA905465, AL134533, AW392670, AL134531, AI142139,.AL119418, AL119483, AL119319, AL119443, AL119324, AL119522, U46350, AL119457, AL134920, AW372827, AW363220, AL119484, AL119391, AL134538, AL134902, AL119341, 299396, AW384394, AL119497~ AL119363, AL119355, AL119396, AL119496, D86967, AR069079, AB026436, AR066494, AR060234, and A81671.
HOSDR06 93 12295581 - 217015 - AI942417, AI800288, AI830283, 2184 AW384845, AA442721, AI637544, AA252612, AA305619, AA224269, N63098, AI670786, AA811590, N80213, AA431396, AI498460, AA371384, N30797, AI280008, AA298474, 224818, AA432386, AI770070, AA364533, AA280152, AA297828, F13799, AA875925, N93459, AI110673, AA090893, AA029327, AA825662, - ----~ ---- F30025, AA029328, N41572, AA298473, AI768963, AI949001, 833662, AW105155, AI377278, AA018569, AW195563, AW377767, AI215536, AI268149, AA280153, ' AA804204, and D38552.
HSDHB12 95 941973 1 - 624 15 - AA447298.
HTTJW49 96 948107 1 - 648 15 - At1199865, AI769428, AI061340, 662 AW268880, AA707168, AI970984, AI884812, AW444872, AI479954, AI356088, AI701720, AI765045, AA722812, AW236544, AA410516, AI267987, AA005114, AI298592, AI865503, AI633370, AA878382, AW389168, AF118838, Y17571, AF164632, AF164526, AF164527, AC004458, AF164528, and AF 164525.
HELDR74 97 12189821 - 142815 - AI741422, AW249482, AA573909, AA085764, AW272801, AI052311, AA490620, AI683396, AA151131, AI700257, AA310938, AI284596, AA961817, AA862960, AW073675, 887485, AI828443;
AI925221, - AI969547, AW001375, AI521481, N24896, AI925228, AI695515, AA609182, AA151130, AI245859, AA490809, AA040451, AW 139250, AI970384, AI961068, T67610, . - ~ . AA923298, AA513675, AW027490, T96070, AI624751, T67494, AI936161, AW196036, AI917354, AA679554, N36317, AA302588, AI932690, AW250249, 888163, T72363, AI796143, W32439, AA582049, AI539047, W45013, and AF113795.
HOEET48 98 963290 1 - 146615 - AI797684, AW239200, AA456267, 1480 AI478733, AI751749, AI990902, AA427646, AI379565, . AI970534, W95460, AA788855;
AA405402, AW068453, AW294114, AI751750, AA594137, AA947297, AW177719, AI057073, AA427487, AI341112, AA232452, AA041304, AW068711, H73236, AA041328, W95567, AW167569, AA853047, AI652166,.
W02069, H74164, 834003, AI341381, AW176526, AA580289, D30965, D31176, ' AA367502, and AR035969.
HBODE51 99 11889981 - 258015 = AW083201, AA056328, AA404251, AA404701, AA610014, AA535370, . AA479540, W26963, AI803179, AA693610, AA167788, AA477641, T30626, AW118235, AA148356, 820037, T10239, W40184, AA677785, AA780412, T33085, T15444, 236286, F37344, AA166644, 243182, .
AW367537, AI699497, AA258857, AA480033, W28626, AI625089, F27424, 861287, AI569618, AW376308, AA324131, AW337372, T10238, 241030, AA319764, 819966, AW058317, AW268504, AA091588, F04174, AI807608, AA249876, AI758553, AI887457, AA665925, 854586, AW009070, AA641818, AW166832, AI627360, T80262, AI619820, AI698391, AA767924, AI918435, AI553645, AI636507~ AI244343, AI872423, AW238688, AI524179; AI913330, AI824458, AI348609, AI889189, AI918809, AI559752, AI247293, AI138221, AI648494, AW148350, _ AL036634, AI499890, AI479292, AI818350, AI800159, AI927233, AI612913, AI445611, AI470717, AI287616, AI452560, AA80454.1, AI433647, AI613270, AL042745, AL042744, W45039, AW089638, A99218, Y14494, A99220, AJ012755, AF087943, A23327;
AL133623, AF131821, 213966, AF090900, AF129131, AL137271, AF141289, AL110223, A84109, X83544, AF172319, X69026, X60759, AF115410, AL137478, Y10183, and AJ005690.
HE8UT58 100 973153 1 - 109015 - ~AL079991, AW409774, AA404732, -AA782257, AZ808909, AA099.036, AA677654, AA405418, AW341107, AW409667, AA659407, AA503263, AI433410, AI885266, AI341147, AA905366, AI692534, AI027623, AA468344, AA923181, AA778251, AI681129, AA533117, AI986154, AA099037, AI955317, AI276678, H29006, AA404602, AI347168, AA371047, AI420387, AW135263, AA868124, T12623, H09118, AA430277, AA365338, T08839, AA813337, H29109;
H11272, AW137535, AB014.534, AF116574, AF116573, and AC032004.
HFXJI27 101 971046 1 - 427 15 - AI335925, AI890750, AA187638, 441 N39062, AI268071, D79411, N75622, N22381, H13284, 843578, F10497, 826373, AI625385, AI220697, 821997, AA932204, AI493179, AI373111, AA909963, AI476063, AA909961, AI888010, AI619918, AA976881, AW339040, AI432041, AA015789, AI049514, AI355877, AI888471, N59174, AI679618, AI373318, AA670204, AI358468, AI336216, AI266210, AA947133, 833792, AW081307, AA610540, AA928678, AA922947, AA922946, AI817788, AI245931, AA948492, AA044223, T63607, AI433872, AW269052, AI587384, AA827770, AW182646, AI127888, H89128, AI268557, D62713, T63317, 835227, AA018661, AW023089, N67.948, T62565;
H01498, 821841, AW151373, D29213, AW022288;
AA678512, and AB023187.
HETH038 102 11245981 - 139315 - AA675914, AW058625, AA595003, 1407, AA565043,,AW192302, T54086, AI287312, AA988703, AW438698, AA675910, AI990695, N40329, AA079650, T54177, AI824376, AI675850, AA079556, AA303646, N71782, AW367626, AA675909, T29568,, H69835, C01666, AA352261, AI969951, AA077925, AA441887, L14848, AF055384, . AB015600, U56947, U56949, U56955, Y16801, Y16809, Y16811, Y16807, Y18114, Y16804, U56944, U56948, Y18113, Y18116, . U56943, U56945, Y16803, AF097406, U56942, Y18111, Y18115, X91625, Y18117, U56952, Y16808, Y16805, U69978, Y18112, U56941, U56946, Y16806, Y18118, U56953, U56954, Y16810, AF097403, AF097404, U56940, US6950, U56951, Y18110, AF045597, AF055385, U9S731, AF021223, AF021224, U95730, U95732, U95733, U95734, AF021225, AF021226, U95729, AF055387, AF021222, AF021221, AF055386, AF055389, AF055388, AC004180, X92841, AJ249394, AC006046, AB000882, AB000878, AP000506, U65416, AC004170, D84394, ' AP000507, Y16802, AB023058, AJ242442, AP000521, AL022723, AF045598, AF055066, AJ242438, AJ242439, AF045600, AF045599, AJ242441, AF055390, AF045596, AF045604, AF045601, AC004208, AB014083, AJ242440, AJ242443, AP000515, AF045603, AF045602, U69625, U69628, U69623, CT69624, U69629, U69626, U69631, AJ242444, L29410, L29409, AJ242445, L29408, U69630, U69627, L29411, L29406, L29407, U69622, AC004207, AC004200, AP000519, AB023056, AB003601, AB003600, AB003599, AB003606, AB003605, AB003608, AJ251160, AB003604, AB003602, AB003603, AB003607, AJ251156, AJ251158, AB003609, AB003612, AJ251157, AB003614, AB003610, AB003611, AB003616, AJ251159, AJ251161, AF106651, AF011836, AF085020, AF011845, AF136158, AF106633, AF106642, AF085024, ~
. AF011887, AF011860, AF106639, AF106648, AF011839, AF011848, AF079414, AF011842, AF0850I3, AF106637, AFI06655, AF085029, . AF106643, AF136159, AF106634, AF106640, ' AF106649, AF085037, AF411879, AF106646, AF106635, AF011853, AF136157, AF106638, AF106641, AF011847, AF106632, AF106647, U69965, U699'71, U69969, U69966, U69975, U69964, U69977, U69968, U69967, AB000880, AF085022, AF085014, AF085050, AF085038, AF08S046, AF085018, and LT69973.
HTEIL07 103 953803 1 - 445 15 - H55431, and AL031843.
HDPTC44 104 553453 1 - 561 15 - AF010446, U22963, AF03146_9, 575 AF010447, AF073485, AF073486, AJ132011, U94989, AF010448, AF035672, and AF068692.
HHPDE40 105 552112 1 - 582 15 - AW449998, U60319, AF079407, 596 292910, AR036572, U91328, Y09800, AF079409, AF079408, and Y09801.
HCESP56 106 827671 1 - 500 15 - AW247740, AW247029, AW204207, , 514 W39269, AA32S536, 814422, W52568, Y16752, AL022170, and 265186.
HH_FGZ38 107 11171921 - 154615 - AA372117, AI864976, AI468754, 1560 AA133546, AA160339, AW449104, AI206956, . AW374444, AA595656, AA278441, AI767157, AI263438, AI859910, AA216640, AA070240, T59738, and AA311762.
HAHEF22 108 910996 1 - 872 15 - AA447298.
HCUEV29 109 816065 1 - 491 15 ; AW410192, AI570209, AA583494, AW087991, AW337550, T30350, T24722, AW246233, and AL031283.
HBGQN46 110 945370 1 - 888 15 - AA479990, AW293680, AI473367, 902 AI184880, AI086741, AI081183, AI214276, AA742259, AA836754, 855060, AA251267, AI285889, AI813391, AW297831, AI819671, AF038458, and AF038458.
HMQCE42 111 806815 1 - 194 15 - D56208, N44225, AA348075, 208 AA3~51420, 819052, AA449607, H23327;
AA227277, AA081491, AA102697, 854504, AA083156, .
246810, D38526, D49552, AJ010749, X96582, X97321, Y09156, X83394, 233459, D64147, U09853, X70857, M28160, D11383, _ AJ001977 D83957, and AP000508.
HEQAY32 112 12072061 - 259915 - AA459604, AA703415, AA703508, 2613 AI337830, AA993540, AI924185, AA398958, AA047107, AI581879, 88$084, H08326, AA399541, AA047244, AI479681,.AI570681, AW007699, AA826082, 850486, AA742413, AI278602, AA810070, AW129039, AI346375, AI636166, AI468327, 244337, AW248520, AI817167, AI188457, AI923575; AI480234, AI184221, AI289600, AW005514, AI696876, AA864258, AW248475, AI804579, AW000853, AI091327, AI192782, AW 104750, AI299237, AA766769, AI124027, AI309629, AA450027, AI378094, AA829783, AA505829, AA421949, AI721136, AI923569, T79992, AA317334, AI269575, AA878088, 850042, H08229, AA852854, N27053, T36249, 245648, AA454095, AA878089, M78847, AI523984, AA740396, H08868, AI366112, AA322162, AW138078, F04540, AI824732, AA373622, AI864722, AI383526, F04098, AW009838, AA825440, 243430, F02816, F07842, 850380, AI983715, AA383971, T24108, AI979211, AA402090, AW271173, AA335352, 814266, AL110339, AW080157, W45039, AI359744, AA713657, H03560, AA582025, AI553645, AL036673, AI640375, AI824576, AI287449, AA102339, AI871660, AI582900, AI743505, AI40169,7, AW007284, AB033004, AF104222;
AC006529, AF124728,~AL137294, AW514348, and AW778993.
HOEFI09 113 974194 1 - 426 15 - C 18310, AW402322, AW405194, ' 440 AW402317, AW408434, AW401505, AL039796, AW239462, D82221, AW402511, AA158109, AA160317, AA263135, AI124815, AA310808, AW327936, D82189, AW401719, AW402264, AW402698, AA160714, AA151951, , AW403872, AA663896, AW402402, D82177, AA100848, AA100680; AW405525, 819052, AA102697, AW401548, AW403793, AA224068, AW402754, AW408440, AA401643, W40489, F13067, AI826037, AW403392, AA381090, H23327, AA121088, AW402383, AA449607, T52124, AA380938, . AA381088, AA160037, AA159230, AA336651, 812066, AI359260, AL138056, AA157351, AW403114, AA295712, AA381030, AA351420, T29764, AA548636;
AA058454, AW402855, AI365653, N44225, AA295685, AA227277, AA350486, F07582, T72958, AA127128, AA159413, AW402401, AA160101, D56208, AA112349, W52004, AA152039, AA344828, AA348075, .
AW2735.57, F06315, AA143652, AA102181, AA488534, AI696864, T52219, H23377, AA159125, F06589, AA358919, AW403253, AA085102, U46382, AA263158, AA143468, AA143626, 859764, AA916617, 830668, AA295230, T75330, Y13029, U96942, 246808, L36591, L22028, L36318, M94052, M94051, M24040, Y09058, M24032, L22649, U29083, M94053, D44501, U04243, U11262, U11266, U09864, L42024, L07950, U21053, L11571, L11570, D50296, AF016641, U36392, M84380, M24036, U04244, X64454, L20088, AB032096, X61709, U29480, L42280, D50300, U56246, U16298, AJ223282, X94574, AB032097, U58110, L76931, D44500, X99735, U17572, U11267, L76093, D31816, M95530, D50294, L22027, L 11666, D44499, D50295, Y18552, L17005, U03698, M32320, X81363, AF170577, U80945, U05578, L32862, L413~3, D83956, D50293, U03027, U03859, L11603, L19937, L42281, M24039, U05575, X13116, M30679, L41925, 215143, AJ245869, M24035, U90558, AJ002676, D49824, M80670, E03437, Y08994, M68964, M32319, U31334, Y09118, L09736, M84694, U50710, L41214, X94480, AF033351, L42506, AJ250917, E03438, X91749, M24034, U21052, Y13567, L76088, M84383, AJ006020, M28203, U70528, L76094, M84382, U04245, U29057, M32317, U88407, M28205, X90391, X84725, M84384, M33573, L42345, U32678, AB036051, M19756, AF161881, X94481, X75953, AF189017, X64366, M24038, L42282, . L42283, D83030, AF118894, AJ131118, D50290, M24075, X60255, X60693, U648b1, y X90390, D50291, M19757, AF022783, AF115460, M77775, X77658, L24373, M15470, M28204, D25275, U01848, U35431, X60253, M84386, D85761, U11263, U14943, AF118895, U75533, X96582, Y09156, L38504, L54059, U90566, D85762, M24037, X13115, U18789, X60254, L20089, U90559, U31971, U90563, U90564, U90565, U11261, L09735, L20090, M84381, M77778;
M77777, M77776, AJ133267, E02885, M58636, U05580, D50068, U11265, Y08995, X61710, - X61706, X58536, D50299, U17107, U30936, M24045, M84385, X03664, U63653, D83043, E02884, L04696, U30904, L40599, U05576, U05577, D14343, M29864, AJ223602, U34810, AF115461, Y09157, X61707, L41086, D50710, M26432, M59840, U06862, L04695, AJ237703, X99704, AB032094, AJ002675, AJ003063, AJ002677, L76935, 222651, U41832, M32318, U05582, AF130734, AF118893, M77774, AP000507, M29865, D64149, and X60251.
HCEOR02 115 12276801 - 209415 - AW292158, AI933139, AA325666, AA317428, and AI887071.
HLTEH39 116 943641 1 - 170 15 - AA970332, AA361489, AA158146, AW402698, AW405525, AW402322, AW385684, AW401505, C18310, AW401548, AW239462, AW402264, AW401719, AA294911, AA151951, AA663896, AA263135, AA352603, AA243206, C03945, AA527306, AW351546, AW405194, AW402317, AW408434, AI696864, AW327936, AA160714, AA477506, AA190717, AA305941, AA367387, AI365653, AA366365, AA527504, AA158109, T68508, AA337991, AA372227, AA321294, AA159413, AL039796, AA100848, AW403872, AA112349, AA385962, AA581164, F00945, H98493, AA335761, AW402402, AA121088, AA127128, AA310808, AA100680,.T10924, T71226, AA401643, 834651, AA352534, AA058454, AA126083, AA227277, N42582, W24084, W24054, AA304603, H11697, AW402383, H23327, AA361916, AA220207, AW403793, . AA449607, F11628, AW402754, AA492406, AA503610, T55088, U15638, D83030, M84386, X96582, Y09156, L54059, M26432, X58536, U43337, M77775, U36492, U15640, U14756, AF035647, D64148, L76088, L20091, D83031, AJ010748, M99388, AF168611, X67818, X60251, U18789, AB032096, AB032097, AJ223282, X94482, U29480, U74387, U18661, U06835, D64149, .
233459, D64147, L42280, Y09058, M94052, M94053, M94051, U04243, L22649, L36318, L17005, X78343, X87841, X70857, Y10520, X60249, X60248, M24096, M28160, X94574, AB032091, AB032094, AB032095, U38976, AF033351, AF115464, 246810, AF076476, - , 280227, X99704, U83580, D64151, D83029, L20088, M77774, M77778, M77777, M77776,.
M24040, M24032, M24033, 215144, M84381, .
LT63653; D49552, D50854, AJ010749, 283247, X76189, X97321, U09853, M84173, M84174, . Y11843, X83394, X95410, M28207, M26712, M28206, M26430, AB036049, AB036050, AB036051, X94480, X87268, L76090, L42506, AJ242661, L76931, U58110, D38526, D64145, M11886, Y13029, U32660, M29865, D44499, D44501, U11262, U11266, M83193, M83191, M83194, L07950,1VI86403, AF118894, D85762, U03027, U03698, U03859, L11571, L11570, D50294, D50296, . D50299, D31816, L22027, M24039, M95530, L11666, L11603, L19937, X61709, U80945, AF016641, U30936, U36392, M84384, X73518, M24030; U38975, AJ250917, D49824, U05584, M28203, AJ238694, U15639, AB032093, AB032599, D83741, AJ002676, AJ002677, X86704, U34810, L76093, L76930, L76094, L76089, U16298, L75942, L76932, L76933, L36979, L76225, D25275, U74386, AB032933, D64146, D64152, D14343, U11261, U11265, U17572, M83195, U05577, U50837, L20089, E02884, D85761, Y08995, X90390, X91749, U06862, M24036, L04696, L09736, M24034, L04695, M84694, X61710, X61706, U17107, U21052, U30904, U29083, L41353, L42281, AF170577, L40599, X81363, M84380, M84385, U88407, X82122, M28172, U06695, U41420, AF115462, L38251, X70856, 222651, M21963, U59654, M28204, AJ251755, 246809, AB032598, U18660, AB030575, AJ002675, X99735, AJ223602, .. AJ003063, U32678, U59647, -.- D50852, M26429, AJ272049, 272007, AB008136, AJ237703, 279751, 247377, U07230, D49820, D50710, D44500, Y09118, Y13567, M83192, U05576, X64454, D50290, D50293, U04245, U21053, U29057, ~AC004204, and AC004204.
HRODF07 117 12220931 - 129915 - AW238558, AI278521, AW238227, AW084017, AW265370, AW440242, AW238497, AW238306, AW238292, AW082445, AI355144, AW082458, AW238597, AW079466, AW366290, AW381175, AW366622, AW276887, AW270776, AW438489, AW270547, AW270610, AW438542, AI821931, AW270648, AW265248, AA610773, AW276814, AW277207, AA729444, AW276866, AW270597, AI732543, AW276913, AW2651.12, AW26517.0, AW438551, AW270550, AA501809, AW276884, AJ243667, AF077374, and AI355145.
Code Description issue Organ Cell DiseaseVector Line .
AR022 a Heart a Heart AR023 a Liver a Liver AR024 a mammary glanda mammar land AR025 a Prostate a Prostate AR026 a small intestinea small intestine AR027 a Stomach a Stomach AR028 Blood B cells Blood B cells .
AR029 Blood B cells Blood B cells activated activated AR030 Blood B cells Blood B cells resting restin AR031 Blood T cells Blood T cells activated activated AR032 Blood T cells Blood T cells resting resting AR033 brain brain AR034 breast breast . ' AR035 breast cancer breast cancer AR036 Cell Line CAOV3Cell Line AR037 cell line PA-1 cell line AR038 cell line transformedcell line transformed AR039 colon colon AR040 colon (9808co65R)colon (9808co65R) AR041 colon (9809co15)colon (9809co15) AR042 colon cancer colon cancer AR043 colon cancer colon cancer (9808co64R) (9808co64R) AR044 colon cancer colon cancer 9809co14 9809co14 AR045 corn clone S corn clone AR046 com clone 6 corn clone AR047 com clone2 corn clone2 AR048 corn clone3 corn clone3 AR049 Corn Clone4 Corn Clone4 ' ARO50 Donor II B CellsDonor II
24hrs B Cells 24hrs ARO51 Donor II B CellsDonor II
72hrs B Cells 72hrs AR052 Donor II B-CellsDonor II
24 hrs. B-Cells ' hrs.
AR053 Donor. II B-CellsDonor II
72hrs B-Cells 72hrs AR054 Donor II RestingDonor II
B Cells Resting B
Cells ARO55 Heart Heart AR056 Human Lung (clonetech)Human Lung (clonetech) AR057 Human Mammary Human Mammary (clontech) (clontech) -- AR058 Human Thymus ' Human Thymus (clonetech) (clonetech) AR059 Jurkat (unstimulated)Jurkat (unstimulated) AR060 Kidne Kidne AR06.1Liver Liver AR062 Liver(Clontech)Liver(Clontech) AR063 Lymphocytes Lymphocytes chronic lymphocytic chronic lymphocytic leukaemia leukaemia AR064 Lymphocytes Lymphocytes diffuse large B cell lymphomadiffuse large B cell lymphoma AR065 Lymphocytes Lymphocytes follicular lym homa follicular 1 m homa AR066 normal breast normal breast AR067 Normal Ovarian Normal Ovarian (4004901) ' (4004901) AR068 Normal Ovary Normal Ovary AR069 Normal Ovary Normal Ovary AR070 Normal Ovary Normal Ovary 98066005 ..
AR071 Ovarian Cancer Ovarian Cancer AR072 Ovarian Cancer Ovarian Cancer (97026001)' (97026001) -AR073 Ovarian Cancer Ovarian Cancer ' .
(97076029) (97076029) AR074 Ovarian Cancer Ovarian Cancer (98046011) (98046011) AR075 Ovarian Cancer Ovarian Cancer (98066019) (98066019) AR076 Ovarian Cancer - Ovarian ~ ~ Cancer (98076017) (98076017) AR077 Ovarian Cancer Ovarian Cancer (98096001) (98096001) AR078 ovarian cancer ovarian cancer AR079,Ovarian Cancer Ovarian Cancer AR080 Ovarian Cancer Ovarian Cancer AR081 Ovarian Cancer Ovarian Cancer AR082 ovarian cancer ovarian cancer ' AR083 Ovarian Cancer Ovarian Cancer AR084 Ovarian Cancer Ovarian Cancer AR085 Ovarian Cancer Ovarian Cancer ._....- 98076045 ..
_ AR086 ovarian cancer ovarian cancer AR087 Ovarian Cancer Ovarian Cancer AR088 Ovarian cancer Ovarian cancer 3rd C00 3rd AR089 Prostate Prostate AR090 Prostate (clonetech)Prostate (clonetech) AR091 rostate cancer rostate cancer AR092 prostate cancer,#15176prostate cancer #15176 AR093 prostate cancerprostate #15509 cancer #15509 AR094 prostate cancerprostate #15673 cancer #15673 AR095 Small IntestineSmall Intestine (Clontech) (Clontech) AR096 S Teen S leen AR097 Thymus T cells Thymus T
activated cells activated AR098 Thymus T cells Thymus T
resting cells restin AR099 Tonsil Tonsil AR100 Tonsil geminal . Tonsil center geminal -centroblast center centroblast AR101 Tonsil germinalTonsil germinal center B center B
cell cell AR102 Tonsil lym h Tonsil 1 node m h node AR103 Tonsil memory Tonsil memory B cell B
cell AR104 Whole Brain Whole Brain AR105 Xeno raft ES-2 Xeno raft AR106 Xeno raft SW626Xeno raft H0004 Human Adult Human Adult Spleen Uni-ZAP
Spleen S leen XR
H0008 Whole 6 Week Uni-ZAP
Old XR
Emb o.
H0009 Human Fetal Uni-ZAP
Brain XR
H0011 Human Fetal Human Fetal Kidne Uni-ZAP
Kidne Kidne XR
H0012 Human Fetal Human Fetal Kidne Uni-ZAP
Kidne Kidne XR
H0013 ' Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old XR
Embryo Emb o H0014 Human Gall BladderHuman Gall Gall Bladder Uni-ZAP
Bladder XR
H0015 Human Gall Bladder,Human Gall Gall Bladder Uni-ZAP
fraction II Bladder - XR
' ' H0020 Human HippocampusHuman Brain Uni-ZAP
Hi ocam us XR
H0024 Human Fetal Human Fetal Lun Uni-ZAP
Lun~ III Lun XR
H0025 Human Adult Human Adult Lymph Lambda Lymph L m h Node Node ZAP II
Node H0028 Human Old Ova Human Old Ovac Bluescri Ovar t H0030 Human Placenta Uni-ZAP
XR
H0031 Human Placenta Human PlacentaPlacenta Uni-ZAP
XR
H0032 Human Prostate Human ProstateProstate Uni-ZAP
XR
H0033 Human Pituitar Human Pituitary Uni-ZAP
XR
H0036 Human Adult Human Adult Small Uni-ZAP
Small Small Int. XR
r Intestine Intestine H0038 Human Testes Human TestesTestis Uni-ZAP
XR
H0039 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor Tumor XR
H0040 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor Tumor XR
H0041 Human Fetal Human Fetal Bone Uni-ZAP
Bone Bone XR
H0042 Human Adult Human Adult Lung Uni-ZAP
Pulmonary Pulmona XR
H0045 Human Esophagus,Human Esophagus,Esophagus diseaseUni-ZAP
Cancer cancer XR
H0046 Human EndometrialHuman EndometrialUterus diseaseUni-ZAP
Tumor Tumor XR
H0048 Human Pineal Human Pineal Uni-ZAP
Gland Gland XR
H0050 Human Fetal Human Fetal Heart Uni-ZAP
Heart Heart XR
H0051 Human HippocampusHuman Brain Uni-ZAP
Hi ocam us XR
H0052 Human CerebellumHuman CerebellumBrain ~ Uni-ZAP
XR
H0053 Human Adult Human Adult Kidney Uni-ZAP
Kidney Kidne XR
H0056 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein, Vein Endothelialvein XR
Endo, remake Cells H0057 Human Fetal Uni-ZAP
S Teen XR
H0059 Human Uterine Human UterineUterus diseaseLambda Cancer Cancer ZAP II
, H0060 Human Macro Human Macro Blood Cell Bluescri hake ha a Line t H0063 Human Th mus Human Th Th mus Uni-ZAP
mus XR
H0068 Human Skin TumorHuman Skin Skin diseaseUni-ZAP
Tumor XR
H0069 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells T-Cells Line XR
H0076.Human Membrane Human MembraneBlood Cell Uni-ZAP
Bound Bouiid Pol Line XR
Pol comes comes H0081 Human Fetal Human Fetal Skin Uni-ZAP
Epithelium Skin XR
(Skin) H0083 HUMAN JURKAT Jurkat Cells Uni-ZAP
MEMBRANEBOUND XR
POLYSOMES
H0085 Human Colon Human Colon Lambda ZAP II
H0086 Human epithelioidEpithelioid Sk Muscle diseaseUni-ZAP
sarcoma Sarcoma, XR
muscle H0087 Human Th mus Human Th Bluescri mus t H0090 Human T-Cell T-Cell L T-Cell diseaseUni-ZAP
L m homa m homa XR
H0099 Human Lung Cancer,Human Lung Lung pBluescript subtracted Cancer HO100 Human Whole.SixHuman Whole Embryo Uni-ZAP
Week Six XR
Old Embr o Week Old Embr o HO101 Human 7 Weeks Human Whole Embryo Lambda Old _ ~ 7 ZAP II
Emb o, subtractedWeek Old Emb o H0102 Human Whole Human Whole Embryo Bluescript 6 Week Six p Old Embr o (II),Week Old subt Embryo H0105 Human Fetal Human Fetal Heart ' pBluescript Heart, Heart subtracted -H0109 Human Macrophage,Macrophage Blood Cell pBluescript Line subtracted HOl Human Placenta,Human PlacentaPlacenta pBluescript l l subtracted H0112 Human ParathyroidHuman ParathyroidParathyroid pBluescript Tumor, subtractedTumor H0116 Human Thymus Human ThymusThymus pBluescript Tumor, subtracted Tumor H0123 Human Fetal Human Fetal Brain Uni-ZAP
Dura Mater Dura XR
Mater H0124 Human Human Sk Muscle diseaseUni-ZAP
XR
Rhabdom osarcomaRhabdom osarcoma H0125 Cem cells cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line XR
treated Treated Cem, Jurkat, Raji, and Su t H0130 LNCAP untreatedLNCAP Cell -ProstateCell Uni-ZAP
Line Line XR
H0131 LNCAP + o.3nM LNCAP Cell Prostate Cell Uni-ZAP
81881 Line Line XR
H0134 Raji Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line XR
treated Treated Cem,, Jurkat, Ra i, and Su t H0135 Human Synovial Human SynovialSynovium Uni-ZAP
Sarcoma XR
Sarcoma H0139 Activated T-Cells,Activated Blood Cell Uni-ZAP
4 hrs. T-Cells Line XR
H0141 Activated T-Cells,Activated Blood Cell Uni-ZAP
12 hrs. T-Cells Line XR
H0144 Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early XR
Stage Human Stage Human H0149 7 Week Old EarlyHuman Whole Embryo Uni-ZAP
Stage 7 XR
Human, subtractedWeek Old Emb o H0150 Human E idid E idid mis Testis Uni-ZAP
mus XR
H0156 Human Adrenal Human AdrenalAdrenal diseaseUni-ZAP
Gland XR
Tumor Gland Tumor Gland H0159 Activated T-Cells,Activated Blood Cell Uni-ZAP
8 hrs., T-Cells Line XR
libation 2 H0161 Activated T-Cells,Activated Blood Cell Uni-ZAP
24 hrs., T-Cells Line XR
ligation 2 H0163 Human Synovium Human SynoviumSynovium Uni-ZAP
XR
H0165 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer, XR
Sta a B2 Cancer, sta a B2 H0169 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer, XR
Stage C fractionCancer, sta a C
H0170 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old XR
Human Earl 5ta -a Human H0171 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old , XR
Human, II Early Stage Human H0178 Human Fetal Human Fetal Brain Uni-ZAP
Brain Brain XR
H0179 Human Neutro Human NeutroBlood Cell Uni-ZAP
hil hil Line XR
H0180 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer Breast Cancer H0181 Hnman Primary Human PrimaryBreast diseaseUni-ZAP
~ Breast I ~ XR
Cancer Breast Cancer H0182Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer Breast Cancer H0184Human Colon Human Colon Liver diseaseLambda Cancer, ZAP II
metasticized Cancer, metasticized to live to liver H0187Restin~ T-Cell T-Cells Blood Cell Lambda Line ZAP II
H0188Human Normal Human NormalBreast Uni-ZAP
Breast XR
Breast H0189Human Resting Human Blood Cell Uni-ZAP
Line XR
Macrophage Macrophage/Monoc ytes H0191Human Activated' Human Blood Cell Uni-ZAP
Line XR
Macrophage (LPS),Macrophage/Monoc, thiour tes H0192Cem Cells, cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line XR
treated, subtraTreated Cem, .lurkat, Ra'i, and Su t H0194Human Cerebellum,Human Cerebellum, Brain pBluescript subtracted H0196Human Cardiomyopathy,Human Heart Uni-ZAP
~ XR
subtracted Cardiom o ath H0204Human Colon Human Colon Colon pBluescript Cancer, subtracted Cancer H0205Human Colon Human Colon Colon pBluescript Cancer, differential Cancer H0208Early Stage Human Fetal Lung pBluescript Human Lung, Lurig subtracted H0212Human Prostate,Human ProstateProstate pBluescript subtracted H0213Human Pituitary,Human Pituitary Uni-ZAP
XR.
subtracted H0214Raji cells, CyclohexamideBlood Cell pBluescript cyclohexamide Line treated, subtractedTreated Cem, Jurkat, Ra'i, and Su t H0224Activated T-Cells,Activated Blood Cell Uni-ZAP
12 hrs, T-Cells Line XR
subtracted H0225Activated T-Cells,Activated Blood Cell Uni-ZAP
l2hrs, T-Cells Line XR
, differentiall ex ressed H0231Human Colon, Human Colon Bluescri subtraction t H0235Human colon Human Colon Liver pBluescript cancer, metaticized Cancer, metasticized, to liver, subtraction to liver H0244Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old XR
Embr o, subtractedEmbryo H0247Human Membrane Human MembraneBlood Cell Uni-ZAP
Bound Line XR
- Polysomes- EnzymeBound Polysomes -Subtraction H0250Human ActivatedHuman Monocytes Uni-ZAP
' XR
Monoc tes H0251Human ChondrosarcomaHuman Cartilage diseaseUni-ZAP
XR
Chondrosarcoma H0252Human OsteosarcomaHuman Bone -- diseaseUni-ZAP
-w XR
-Osteosarcoma H0253 Human adult Human Adult Testis Uni-ZAP
testis, large Testis XR
inserts H0254 Breast Lymph Breast LymphLymph Uni-ZAP
node cDNA Node Node XR
library H0255 breast lymph Breast LymphLymph Lambda node CDNA Node Node ZAP II
librar H0257 HL-60, PMA 4H HL-60 Cells,Blood Cell Uni-ZAP
PMA Line XR
stimulated H0261 H. cerebellum, Human CerebellumBrain Uni-ZAP
Enzyme XR
subtracted H0263 human colon Human Colon Colon diseaseLambda cancer ZAP II
Cancer H0264 human tonsils Human TonsilTonsil Uni-ZAP
XR
H0265 Activated T-CellT-Cells Blood Cell Uni-ZAP
Line XR
( l2hs)/Thiouridine (abelledEco H0266 Human MicrovascularHMEC Vein Cell Lambda Line ZAP II
Endothelial Cells, fract.
A
H0268 Human UmbilicalHUVE Cells UmbilicalCell Lambda Vein Line ZAP II
Endothelial vein Cells, fract.
A
H0271 Human Neutrophil,Human NeutrophilBlood Cell Uni-ZAP
- Line XR
Activated Activated H0274 Human Adult Human Adult Spleen Uni-ZAP
Spleen, XR
fractionII S Teen H0280 K562 + PMA (36 K562 Cell cell lineCell ~ ZAP Ex hrs) line Line ress H0284 Human OB MG63 Human Bone Cell ~ Uni-ZAP
control Line XR
fraction I Osteoblastoma MG63 cell line H0286 Human OB MG63 Human Bone Cell Uni-ZAP
treated Line XR
( 10 nM E2) Osteoblastoma .
fraction I
MG63 cell line H0290 Human OB HOS Human Bone Cell Uni-ZAP
treated Line XR
( 1 nM E2) fractionOsteoblastoma I HOS
cell line H0292 Human OB HOS Human Bone Cell Uni-ZAP
treated Line XR
( 10 nM E2) Osteoblastoma fraction I H05 cell line H0294 Amniotic Cells Amniotic Placenta Cell Uni-ZAP
- TNF Cells - ' Line XR
induced TNF induced H0300 CD34 positive CD34 PositiveCord Blood ZAP Express cells (Cord Cells Blood) H0305 CD34 positive CD34 PositiveCord Blood ZAP Express . cells (Cord Cells Blood) H0306 CD34 depleted CD34 DepletedCord Blood ZAP Express Buffy Coat (Cord Blood) Buffy Coat (Cord Blood) H0309 Human Chronic Synovium, Synovium diseaseUni-ZAP
Synovitis Chronic XR
Synovitis/
Osteoarthritis H0316 HUMAN STOMACH Human StomachStomach Uni-ZAP
XR
H0318 HUMAN B CELL Human B CellLymph . diseaseUni-ZAP
' Node XR
LY MPHOMA Lymphoma H0327human corpus Human CorpusBrain Uni-ZAP
colosum , Callosum XR
H0328human ovarian Ovarian CancerOvar ~ diseaseUni-ZAP
cancer XR
H0329DermatofibrosarcomaDermatofibrosarcom. Skin diseaseUni-ZAP
Protuberance a Protuberans XR
H0331Hepatocellular HepatocellularLiver diseaseLambda Tumor Tumor ZAP II
H0333HemangiopericytomaHemangiopericytomBlood diseaseLambda a vessel ZAP II
H0334Kidne cancer Kidne CancerKidne diseaseUni-ZAP
XR
H0341Bone Marrow Bone Marrow Bone MarrowCell Uni-ZAP
Cell Line Cell Line XR
(RS4;11) Line RS4;11 H0342Lin ual G rus Lin ual G Brain Uni-Za rus XR
H0343stomach cancer Stomach Cancer diseaseUni-ZAP
(human) - XR
S383A (human) H0344Adipose, tissueAdipose - Uni-ZAP
(human) 6825A XR
(human) H034SSKIN Skin - 4000868HSkin Uni-ZAP
XR
H0349human adult Human Adult Liver pCMVSport liver cDNA Liver 1 Libra ' H0351Glioblastoma GlioblastomaBrain diseaseUni-ZAP
XR
H0352wilm"s tumor Wilm"s Tumor diseaseUni-ZAP
XR.
H0354Human Leukoc Human LeukocBlood Cell CMVS ort tes tes Line 1 .
H0355Human Liver Human Liver, ~ pCMVSport normal Adult 1 H0361Human rejected Human Rejected diseasepBluescript ' kidney Kidne ' -H0366I~428 cell lineL428 ZAP Ex ress H0369H. Atrophic Atrophic Uni-ZAP
Endometrium Endometrium XR
and m ometrium H0370H. Lymph node Lymph node diseaseUni-ZAP
breast with XR
Cancer Met. Breast Cancer H0372Human Testes Human TestesTestis CMVS ort H0373Human Heart Human Adult Heart CMVS ort Heart 1 H0375Human Lun~ Human Lun CMVS ort H0376Human Spleen Human Adult Spleen pCMVSport . S Teen 1 H0388Human Rejected Human Rejected. diseasepBluescript Kidney, Kidne 704 re-excision H0390Human Amygdala Human Amygdala diseasepBluescript De cession, De cession re-excision H0391H. Meniingima, Human Meningimabrain p5 ortl H0392H. Meningima, Human Meningimabrain pSportl Ml H0393Fetal Liver, Human Fetal Liver Bluescri subtraction Liver t II
H0394A-14 cell line Redd-Sternber~ ZAP Ex cell ' ress H0395AI-CELL LINE Redd-Sternber ZAP Ex cell ress H0396LI Cell line ' Redd-SternberQ ZAP Ex cell ress H0402CD34 depleted CD34 DepletedCord Blood ZAP Express Buffy Coat Buffy Coat . (Cord (Cord Blood), Blood) re-excision ' __. H0403H. Umbilical HUVE Cells UmbilicalCell Uni-ZAP
._. I Vein Line XR
.
_ __.~
- Endothelial vein Cells, IL4 induced H0409 H. Striatum Human Brain,Brain pBluescript Depression, subtracted Striatum Depression H0411 H Female Bladder,Human FemaleBladder pSportl Adult ~
Adult Bladder H0412 Human umbilicalHUVE Cells UmbilicalCell pSportl vein Line, endothelial vein cells, IL-4 induced H0413 Human UmbilicalHUVE Cells UmbilicalCell pSportl Vein Line Endothelial vein Cells, uninduced H0415 H. Ovarian Tumor,Ovarian Tumor,Ovary diseasepCMVSport II, 2.0 OV5232 ~ OV5232 $0416 Human Neutrophils,Human NeutrophilBlood Cell ~ pBl,uescript - Line Activated, re-excisionActivated ' H0421 Human Bone Marro4v,Bone Marrow pBluescript re-excision H0422 T-Cell PHA 16 T-Cells Blood Cell 5 ortl hrs Line H0423 T-Cell PHA 24 T-Cells Blood Cell S ortl hrs ' Line H0424 Human Pituitary,Human Pituitary Bluescript subt IX
H0427 Human Adipose Human Adipose, pSportl left hi ti oma H0428 Human Ovary Human Ovary Ovary pSportl Tumor H0431 H. Kidney Medulla,Kidney medullaKidney pBluescript re-excision H0433 Human UmbilicalHUVE Cells UmbilicalCell pBluescript Vein Line Endothelial vein cells, frac B, re-excision H0434 Human Brain, Human Brain, pBluescript striatum, re-excision Striatum H0435 Ovarian Tumor Ovarian Tumor,Ovary pCMVSport 10-3-95 2.0 H0436 Restino T-Cell T-Cells Blood Cell S ortl Libr ,II Line H0437 H Umbilical HUVE Cells UmbilicalCell Lambda Vein Line ZAP II
Endothelial vein .
Cells, frac A, re-excision H0438 H. Whole Brain Human Whole ZAP Express #2, re- Brain excision #2 H0439 Human Eosino Eosino hits Bluescri hits t H0~I4.1H. Kidney Cortex,Kidney cortexKidney pBluescript subtracted H0442 H. Striatum Human Brain,Brain pBluescript Depression, , subt II ~ Striatum De cession H0444 Spleen metasticSpleen, MetasticSpleen diseasepSportl melanoma malignant melanoma H0445 Spleen, ChronicHuman Spleen,Spleen diseasepSportl CLL
1 m hoc tic leukemia H0455 H. Striatum Human Brain,Brain pBluescript Depression, subt Striatum De cession H0457 Human Eosino Human Eosino- - S ortl hits hits H0459 CD34+cells, CD34 positive pCMVSport II, cells 2.0 H0477 Human Tonsil, Human TonsilTonsil S ortl Lib 3 H0478 Salivary Gland,Human SalivarySalivary pSportl Lib 2 Gland Qland -H0483 Breast Cancer Breast Cancer pSportl cell line, Cell MDA 36 line, MDA
H0484 Breast Cancer Breast Cancer pSportl Cell line, Cell angiogenic line, Angiogenic, H0485 Hodgkin"s LymphomaHodgkin"s ' diseasepCMVSport I 2.0 Lymphoma I
H0486 Hodgkin"s LymphomaHodgkin"s diseasepCMVSport II 2.0 L m homa II
H0487 Human Tonsils, Human Tonsils CMVS ort lib I 2.0 H0488 Human Tonsils, Human Tonsils CMVS ort Lib 2 2.0 H0489 Crohn"s DiseaseIleum Intestine diseaseS ortl H0492 HL-60, RA 4h, HL-60 Cells,Blood Cell Uni-ZAP
Subtracted RA Line XR
stimulated for 4H
H0494 Keratinoc to Keratinocyte CMVS ort 2.0 H0497 HEL cell line HEL cell HEL , pSportl line ' 92.1.7 H0505 Human AstrocyteHuman Astrocyte pS ortl H0506 Ulcerative ColitisColon Colon pSportl H0509 Liver, HepatomaHuman Liver,Liver diseasepCMVSport 3.0 He atoma, anent 8 H0510 Human Liver, Human Liver,Liver pCMVSport normal 3.0 normal, patient # 8 H0518 pBMC stimulatedpBMC stimulated pCMVSport w/ poly 3.0 I/C with of I/C
H0519 NTERA2, controlNTERA2, pCMVSport 3.0 ' Teratocarcinoma cell line H0520 NTERA2 + retinoicNTERA2, ~ pSportl acid, 14 days Teratocarcinoma cell line H0521 Primary DendriticPrimary Dendritic pCMVSport Cells, 3.0 lib 1 cells H0522 Primary DendriticPrimary Dendritic pCMVSport 3.0 cells,frac 2 cells H0529 Myoloid ProgenitorTF-1 Cell pCMVSport Cell Line; 3.0 Line Myoloid progenitor cell line H0538 Merkel Cells Merkel cellsL m h S ortl node H0539 Pancreas Islet Pancreas Pancreas diseasepSportl Cell Tumor Islet Cell Tumour H0542 T Cell hel er Hel er T CMVS ort I cell 3.0 H0543 T cell hel er Hel er T CMV S ort II cell 3.0 H0544 Human endometrialHuman endometrial pCMVSport . 3.0 stromal cells stromal cells H0545 Human endorrietrialHuman endometrial pCMVSport 3.0 stromal cells-treatedstromal cells-treated with ro~esterone with roe H0546Human endometrialHuman endometrial pCMVSport ~ 3.0 stromal cells-treatedstromal cells-treated with estradiol with estra H0547NTERA2 teratocarcinomaNTERA2, pSportl cell line+retinoicTeratocarcinoma acid (14 da s) cell line H0549H. Epididiymus,Human ' Uni-ZAP
caput & XR
corpus Epididiymus, caput and co us H0550H. Epididiymus,Human ' Uni-ZAP
cauda XR
Epididiymus, cauda H0551Human Thymus Human Thymus ' pCMVSport Stromal 3.0 Cells Stromal Cells H0553Human Placenta Human Placenta CMVS ort 3.0 H0555Rejected Kidney,Human RejectedKidney diseasepCMVSporf lib 4 3.0 Kidne -H0556Activated T- T-Cells Blood Cell Uni-ZAP
Line XR
cell( 12h)/Thiouridine-re-excision H0559HL-60, PMA 4H, HL-60 Cells,Blood Cell Uni-ZAP
re- PMA Line XR
excision ' stimulated H0560KMH2 KMH2 CMVS ort 3.0 H0561L428 L428 CMVS ort 3.0 H0567Human Fetal Human Fetal pCMVSport Brain, Brain 2.0 normalized A5002F
H0570Human Fetal Human Fetal pCMVSport Brain, ~ Brain 2.0 normalized C500H
H0571Human Fetal Human Fetal pCMVSport Brain, Brain 2.0 normalized C500HE
H0574Hepatocellular HepatocellularLiver diseaseLambda Tumor; re- ZAP II
- excision Tumor _ H0575Human Adult Human Adult Lung Uni-ZAP
. XR
Pulmonar ;re-excisionPulmonar H0576Resting T-Cell;T-Cells Blood Cell Lambda re- Line ZAP II
excision H0579Pericardium Pericardium Heart S ortl H0580Dendritic cells,Pooled dendritic pCMVSporf3.0 pooled cells H0581Human Bone Marrow,Human Bone Bone Marrow pCMVSport 3.0 treated ' Marrow H0583B Cell 1 m homaB Cell L B Cell diseaseCMVS ort m homa 3.0 H0584Activated T-cells,Activated Blood Cell Uni-ZAP
24 T=Cells Line XR
hrs,re-excision H0585Activated T-Cells,l2Activated Blood Cell Uni-ZAP
T-Cells Line XR
hrs,re-excision H0586Healing groin healing groingroin diseasepCMVSport wound, 6:5 3.0 hours post incisionwound, 6.5 hours ost incision H0587Healing groin Groin-2/19/97groin diseasepCMVSport wound; 7.5 3.0 hours ost incision H0590Human adult Human Adult Small Uni-ZAP
small 5ma11 Int. XR
intestine,re-excisionIntestine -- Human T-cell T-Cell L T=Cell diseaseUni-ZAP
H0591 m homa XR
lym homa;re-excision H0592 Healing groin HGS wound diseasepCMVSport wound - healing 3.0 zero hr post-incisionproject; ' abdomen (control) H0593 Olfactory -.Olfactory pCMVSport epithelium 3.0 epithelium;nasalcavityfrom roof of left nasal cacit H0594 ' Human Lung Human Lung Lung diseaseLambda Cancer;re- Cancer ZAP II
excision H0595 Stomach cancer Stomach Cancer diseaseUni-ZAP
~ - XR
(human);re-excision5383A (human) H0596 Human Colon Human Colon Colon Lambda Cancer;re- ZAP II
excision Cancer H0597 Human Colon; Human Colon Lambda re-excision ZAP II
H0598 Human Stomach;re-Human StomachStomach Uni-ZAP
XR
excision H0599 Human Adult Human Adult Heart Uni-ZAP
Heart;re- Heart XR
excision . H0600Healing AbdomenAbdomen diseasepCMVSport 3.0 wound;70&90 min post incision H0601 Healing AbdomenAbdomen diseasepCMVSport 3.0 Wound;l5 days post incision H0604 Human Pituitary,Human Pituitary pBluescript re-excision H0606 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer;re-excisionBreast Cancer H0608 H. Leukoc tes, H.Leukoc CMVS ort control tes 1 H0615 Human Ovarian Ovarian CancerOvary diseaseUni-ZAP
Cancer XR
Reexcision H0616 Human Testes, Human TestesTestis Uni-ZAP
Reexcision XR
H0617 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast XR
Cancer ReexcisionBreast Cancer H0618 Human Adult Human Adult Testis Uni-ZAP
Testes, Testis XR
Lar a Inserts, Reexcision H0619 Fetal Heart ' Human FetalHeart Uni-ZAP
Heart XR
H0620 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney; Kidney XR
Reexcision H0622 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor; XR
Reexcision Tumor .
H0623 Human UmbilicalHuman UmbilicalUmbilical . Uni-ZAP
Vein; XR
Reexcision Vein Endothelialvein Cells H0624 12 Week Early Twelve Week Embryo Uni-ZAP
Stage Old XR
Human II; ReexcisionEarly Stage Human H0625 Ku 812F Baso Ku 812F Baso S ortl hils Line hils H0626 Saos2 Cells; Saos2 Cell ~ pSportl Untreated' Line; , Untreated H0628 Human Pre-DifferentiatedHuman Pre- Uni-ZAP
XR
Adipocytes Differentiated Adi oc tes H0629 Human Leukoc Human Normalized CMVS ort te, control 1 #2 leukocyte H0630 Human Human Normalized , pCMVSport Leukocytes,normalizedleukocyte control #4 H063T Saos2, DexamethosomeSaos2 Cell pSportl Line;
Treated Dexamethosome Treated H0632 Hepatocellular HepatocellularLiver Lambda Tumor;re- ZAP II
excision Tumor H0633 Lung Carcinoma TNFalpha diseasePSportl A549 activated TNFalpha activatedA549--Lung Carcinoma H063q.Human Testes Human TestesTestis diseaseUni-ZAP
Tumor, re- XR
excision Tumor H0635 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells, T-Cells Line XR
re-excision H0638 CD40 activated.monocyteCD40 activated pSportl dendridic cellsmonocyte dendridic cells -H0640 Ficolled Human Ficolled Other Stromal Human Cells, UntreatedStromal Cells, -Untreated H0641 LPS activated LPS activated pSportl derived dendritic cellsmonocyte derived dendritic cells H0642 Hep G2 Cells, Hep GZ Cells Other lambda Libra H0643 He G2 Cells, . He G2 Cells Other PCR Libra H0644 Human Placenta Human PlacentaPlacenta Uni-ZAP
(re- XR
excision) H0645 Fetal Heart, Human Fetal Heart Uni-ZAP
re-excision Heart XR
H0646 Lung, Cancer Metastatic pS ortl (4005313 P
A3): Invasive squamous Poorly cell lung Differentiated carcinoma, _ -Lung poorly di Adenocarcinoma, ' H0647 Lung, Cancer Invasive diseasepSportl (4005163 poorly B7): Invasive, differentiated Poorly Diff. lung Adenocarcinoma,adenocarcinoma Metastatic H0648 Ovary, Cancer: Papillary - diseasepSportl (4004562 Cstic B6) Papillary neoplasm Serous of low Cystic Neoplasm,malignant Low potentia Malignant Pot H0649 Lung, Normal: Normal Lung pSportl (4005313 Bl) H0650 B-Cells B-Cells CMVS ort 3.0 H0651 Ovary, Normal: Normal Ovary ~ pSportl (9805C040R) H0652 Lung, Normal: Normal Lung pSportl (4005313 B1) H0653 5tromal Cells Stromal Cells pSportl H0654 Lung, Cancer: Metastatic Other (4005313 A3) Invasive Squamous Poorly-~ cell lung differentiated Carcinoma Metastatic poorly lung adenoc dif ' H0656 B-cells (unstimulated)B-cells pSportl (unstimulated) H0657 B-cells (stimulated)B-cells (stimulated) S ortl H0658 Ovary, Cancer 9809C332- Ovary diseasepSportl Poorly &
(9809C332): differentiateFallopian Poorly differentiated Tubes adenocarcinoma H0659 Ovary, Cancer Grade II Ovary diseasepSportl Papillary (15395A1F): Carcinoma, Grade II Ovary Pa illar Carcinoma H0660 Ovary, Cancer: Poorly differentiated~ diseasepSportl (15799A1F) Poorlycarcinoma, ovary differentiated carcinoma H0661 Breast, Cancer:Breast cancer diseasepSportl (4004943 ~
A5) H0662 Breast, Normal:Normal BreastBreast pSportl -(400552282) #4005522(B2) H0663 Breast, Cancer:Breast CancerBreast diseasepSportl (4005522 -A2) #4005522(A2) H0664 Breast, Cancer:Breast CancerBreast diseasepSportl ~
(9806C012R) ' H0665 Stromal cells Stromal cells S ortl 3.88 3.88 H0666 Ovary, Cancer: Ovarian Cancer, diseasepSportl (4004332 A2) Sample .
#4004332A2 -H0667 5tromal cells(HBM3.18).Stromal pSportl cell(HBM
3.18) H0668 stromal cell stromal cell p5portl clone 2.5 clone 2.5 H0670 Ovary, Cancer(4004650Ovarian Cancer pSportl -A3): Well-Differentiated4004650A3 Micropapillary Serous Carcinoma H0671 Breast, Cancer:Breast Cancer- pSportl (9802C020E) Sample #
H0672 Ovary, Cancer: Ovarian Ovary pSportl (4004576 A8) Cancer(4004576A8) H0673 Human Prostate Human ProstateProstate Uni-ZAP
Cancer, .~ XR
Sta~e B2; re-excisionCancer, stage H0674 Human Prostate Human ProstateProstate U ni-ZAP
Cancer, XR
Sta a C; re-excissionCancer, stage C
H0675 Colon, Cancer: Colon Cancer pCMVSport 3.0 (9808C064R) 9808C064R
H0677 TNFR de enerateB-Cells PCRII
oliQo H0682 Serous Papillaryserous papillary pCMVSport 3.0 Adenocarcinoma adenocarcinoma , (9606G304SPA3B) H0683 Ovarian Serous Serous papillary pCMVSport Papillary 3.0 Adenocarcinoma adenocarcinoma, stage 3C
(9804601 H0684 Serous PapillaryOvarian Cancer-Ovaries ' pCMVSport 3.0 Adenocarcinoma 98106606 H0685 Adenocarcinoma Adenocarcinoma pCMV Sport of of 3.0 Ovary, Human Ovary, Human Cell Line, Cell # OVCAR-3 Line, # OVCAR-H0686 Adenocarcinoma Adenocarcinoma pCMVSport of of 3.0 Ovary, Human ~ Ovary, Cell Line Human Cell Line # SW-626 H0687 Human normal Human normal, Ovary pCMVSport 3.0 ovar (#96106215)ova (#96106215) H0688 Human Ovarian Human Ovarian, pCMVSport 3.0 Cancer(#98076017)cancer(#98076017), mRNA from Maura Ru H0689 Ovarian Cancer Ovarian Cancer, pCMVSport 3.0 #98066019 H0690 Ovarian Cancer,Ovarian Cancer,, pCMVSport # 3.0 97026001 #97026001 H0691 Normal Ovary, normal ovary, pCMVSport 3.0 #97106208 #97106208 H0693 Normal ProstateNormal Prostate pCMVSport 3.0 #ODQ3958EN Tissue #
H0694 Prostate gland Prostate prostate pCMVSport gland, 3.0 adenocarcinoma adenocarcinoma,gland mod/diff, gleason H0695 mononucleocytesmononucleocytes pCMVSport from 3.0 patient from patient at Shad Grove Hos it N0006 Human Fetal Human Fetal .
Brain Brain ~' Human Hippocampus,Human rescreened ~ Hi ocam us 50002 Monocyte activatedMonoc te-activatedblood Cell Uni-ZAP
Line XR
50003 Human OsteoclastomaOsteoclastomabone diseaseUni-ZAP
XR
50005 Heart Heart-left Heart CDNA
ventricle 50007 Early Stage Human Fetal Uni-ZAP
Human Brain Brain XR
50010 Human Amygdala Amygdala Uni-ZAP
XR
S0011 STROMAL - Osteoclastomabone diseaseUni-ZAP
XR
OSTEOCLASTOMA
50022 Human OsteoclastomaOsteoclastoma. ~ Uni-ZAP
XR
Stromal Cells Stromal Cells -unam lifted 50024 Human Kidney Human Kidney Medulla -unam lifted Medulla 50026 Stromal cell stromal cellBone Cell Uni-ZAP
TF274 marrow Line XR
S0027 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
serum Line XR
treated artery 50028 Smooth muscle,controlSmooth musclePulmanaryCell Uni-ZAP.
Line XR
ante S0029 brain stem Brain stem brain Uni-ZAP
XR
50031 5 final cord S final cords final Uni-ZAP
cord XR
50032 Smooth muscle-ILbSmooth musclePulmanaryCell Uni-ZAP
. ' Line XR
induced arter - -- ' ~ - ' 50036 Human SubstantiaHuman Substantia Uni-ZAP
Nigra XR
NiQra 50037 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
ILlb Line XR
induced ~ artery 50038 Human Whole Human Whole - . ZAP Express Brain #2 - Brain Olioo dT > l.SKb#2 S0040 Adipocytes Human Adipocytes Uni-ZAP
XR
from Osteoclastoma SOO~kIProstate BPH rostate BPH Prostate diseaseUni-ZAP
~ XR
50045 Endothelial Endothelial endothelialCell Uni-ZAP
cells-control cell Line XR
cell-lun S0046 Endothelial-inducedEndothelial endothelialCell Uni-ZAP
' ~ cell Line XR
cell-lung S0049 Human Brain, Human Brain, Uni-ZAP
Striatum XR
Striatum 50050 Human Frontal Human Frontal diseaseUni-ZAP
Cortex, XR
Schizophrenia Cortex, , Schizo hrenia 50051 Human Human ' diseaseUni-ZAP
XR
Hypothalmus,SchizophrenHypothalamus, is Schizo hrenia S0052 neutro hils human neutroblood Cell Uni-ZAP
control hits Line XR
S0053 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS L_ XR
ine induced induced S0106 STRIATUM BRAIN diseaseUni-ZAP
XR
DEPRESSION
S0114 Aner is T-cell AnerQic T-cell Cell ~ ~ Uni-ZAP
Line XR
S0116 Bone marrow Bone marrow Bone Uni-ZAP
marrow XR
50118 Smooth muscle Smooth musclePulmanaryCell Uni-ZAP
control 2 Line XR
artery' 50122 Osteoclastoma-normalizedOsteoclastomabone diseasepBluescript A
50126 Osteoblasts Osteoblasts Knee Cell Uni-ZAP
Line XR
S0132 Epithelial-TNFaAirway Epithelial Uni-ZAP
and INF XR
induced 50134 A o totic T-cella o totic Cell Uni-ZAP
cells Line XR
SO1~0 eosino hil-II,Seosino hil lun Cell Uni-ZAP
induced Line XR
S0142 Macrophage-oxLDLmacrophage- blood Cell Uni-ZAP
Line XR
oxidized LDL
treated S01~14Macrophage (GM-CSFMacrophage LJni-ZAP
(GM- XR
treated) CSF treated) 50146 rostate-edited rostate BPH Prostate Uni-ZAP
XR
S0148 Normal ProstateProstate rostate Uni-ZAP
XR
50150 LNCAP rostate LNCAP Cell ProstateCell Uni-ZAP
cell line . Line Line XR
50152 PC3 Prostate PC3 prostate Uni-ZAP
cell line cell XR
line 50174 Prostate-BPH Human Prostate~ pBluescript subtracted II
BPH ' ' S0182 Human B Cell Human B- ' Uni-ZAP
8866 Cell 8866 XR
S0188 Prostate,BPH, Human Prostate diseasepSportl Lib 2 BPH
50190 Prostate BPH,LibHuman Prostate S ortl 2, subtracted BPH
S0192 Synovial FibroblastsSynovial pSportl Fibroblasts (control) 50194 Synovial by S novial S ortl oxia Fibroblasts S0196 Synovial IL-1/TNFSynovial pSportl Fibroblasts stimulated S0206 Smooth Muscle- Smooth musclePulmanaryCell pBluescript HASTE Line normalized arter 50210 Messan~ial cell,Messanoial S ortl frac 2 cell 50212 Bone Marrow Bone Marrow - p5portl Stromal Cell, untreatedStromal -Cell,untreated S0214 Human Osteoclastoma,Osteoclastomabone diseaseUni-ZAP
re- XR
excision S0216 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line XR
induced induced 50218 Apoptotic T-cell,apoptotic Cell Uni-ZAP
re- cells Line XR
excision S0222 H. Frontal H. Brain, Brain diseaseUni-ZAP
Frontal XR
cortex,epileptic;re-Cortex, Epileptic' excision S0242 Synovial FibroblastsSynovial p5portl Fibroblasts (Ill/TNF), subt 50250 Human OsteoblastsHuman OsteoblastsFemur diseaseCMVS ort II 2.0 50260 S final Cord, S final cords final ~ Uni-ZAP
re-excision cord XR
S0276 Synovial hypoxia-RSFSynovial Synovial pSportl fobroblasts subtracted (rheumatoid)tissue S0278 H Macrophage Macrophage ~ Uni-ZAP
(GM-CSF (GM- XR
treated), re-excisionCSF treated) 50280 Human Adipose Human Adipose Uni-ZAP
Tissue, XR
re-excision Tissue ~
50282 Brain Frontal Brain frontalBrain' Lambda Cortex, re- cortex ZAP II
excision 50294 Larynx tumor Larynx tumorLarynx,vocal diseasepSportl ' cord .
S0300 Frontal lobe,dementia;re-Frontal LobeBrain Uni-ZAP
XR
excision dementia/Alzheimer' 's S0306 Larynx normal Larynx normal pSportl #10 261-S0308 S leen/normal S Teen normal S ortl 50312 Human _ Human diseasep5portl osteoarthritic;fractionosteoarthritic II
cartila a S0314 Human Human diseasepSportl osteoarthritis;fractionosteoarthritic I
cartila a S0316 Human Normal Human Normal pSportl Cartila~e,FractionCartila a I
S0322 Siebben Pol Siebben Pol S ortl osis osis 50328 Palate carcinomaPalate carcinomaUvula diseaseS ortl 50330 Palate normal Palate normalUvula S ortl S0332 Pharynx carcinomaPharynx carcinomaHypopharynx 'pSportl ~
S0342 Adipocytes;re-excisionHuman Adipocytes , Uni-ZAP
from Osteoclastoma XR
S0344 Macrophage-oxLDL;macrophage- blood Cell Uni-ZAP
re- oxidized Line XR
excision LDL
treated S0346 Human Amygdala;re-Amygdala Uni-ZAP
excision XR
S0350 Phar nx CarcinomaPhar nx carcinomaH o har diseaseS ortl nx 50352 Larynx CarcinomaLar nx carcinoma disease5 ortl S0354 Colon Normal Colon NormalColon S ortl II
50356 Colon CarcinomaColon CarcinomaColon diseaseS ortl .
50358 Colon Normal Colon NormalColon S ortl III
S0360 Colon Tumor Colon Tumor Colon diseaseS ortl II' 50364 Human uadrice Quadrice 5 ortl s s muscle 50366 Human Soleus Soleus Muscle S ortl S0370 Larynx carcinomaIILarynx carcinoma diseaseSportl ' 50372 Larynx carcinomaLarynx carcinoma disease5portl III ' 50374 Normal colon Normal colon 5 ortl 50376 Colon Tumor Colon Tumor diseaseS ortl S0378 Pancreas normalPancreas ' pSportl PCA4 Normal No PCA4 No 50380 Pancreas Tumor Pancreas diseasepSportl PCA4 Tu Tumor PCA4 Tu S0382 Lar nx carcinomaLar nx carcinoma diseaseS ortl IV
S0384 Ton ue carcinomaTon~ue carcinoma disease5 ortl S0388 Human Human . diseaseUni-ZAP
Hypothalamus,schizophreHypothalamus, XR
nia, re-excisionSchizo hrenia 50390 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
control; arter Line XR
re-excision S0392 Salivary Gland Salivary pSportl gland;
normal 50400 Brain; normal Brain; normal S ortl - 50402 Adrenal Gland,normalAdrenal gland; ~ . pSportl normal S0404 Rectum normal Rectum, normal pSportl S0406 Rectum tumour Rectum tumour pSportl 50408 Colon, normal Colon, normal S ortl S0410 Colon, tumour Colon, tumour S ortl ' S0414 Hippocampus, Hippocampus, Other Alzheimer Alzheimer Subtracted Subtracted 50418 CHME Cell Line;treatedCHME Cell pCMVSport 5 Line; 3.0 hrs treated S0420 CHME Cell CHME Cell p5portl Line,untreated line, ~ untreatetd S0422 Mo7e Cell Line Mo7e Cell pCMVSport GM-CSF Line 3.0 treated (lng/ml)GM-CSF treated (1n /ml) 50424 TF-1 Cell Line TF-1 Cell pSportl GM-CSF Line Treated GM-CSF Treated S0426 Monocyte activated;Monocyte-activatedblood Cell Uni-ZAP
~ re- Line XR
excision S0428 Neutrophils human iieutrophilsblood Cell Uni-ZAP
control; re- Line XR
excision S0432 Sinus piniformisSinus piniformis pSportl Tumour Tumour 50434 Stomach Normal Stomach Normal~ diseaseS ortl S0436 Stomach Tumour Stomach Tumour~ diseaseS ortl 50438 Liver Normal Liver Normal pSportl MetSNo MetSNo 50440 Liver Tumour Liver Tumour S ortl Met 5 Tu S0442 Colon Normal Colon Normal S ortl S0444 Colon Tumor Colon Tumour~ diseaseS ortl S0446 Ton~ue Tumour Ton~ue Tumour S ortl S0452 Th mus Th mus S ortl 50456 Ton~ue Normal Tongue Normal - S ortl 50458 Thyroid Normal Thyroid normal pSportl (SDCA2 Nod S0460 Th roid Tumour Thyioid Tumour S ortl _50462Th roid Th roiditisTh roid Th 5 ortl roiditis S0466 Lar nx Tumor La nx Tumor diseaseS ortl S0468 Ea.h .926 cell Ea.h .926 S ortl line cell line S0470 Adenocarcinoma PYFD diseaseS ortl S0474 Human blood Platelets Blood Other platelets latelets S0665 Human Amygdala;Amygdala ' Uni-ZAP
re- . XR
excission S3012 Smooth Muscle Smooth musclePulmanaryCell pBluescript Serum artery Line Treated, Norm S3014 Smooth muscle, Smooth musclePulmanaryCell pBluescript serum arter Line induced,re-exc S6016 H. Frontal Cortex,H. Brain, Brain diseaseUni-ZAP
E ile tic ~ Frontal XR
Cortex, E
ile tic 56024 Alzheimers, Alzheimer"s/SpopgyBrain diseaseUni-ZAP
spongy than a XR
than a 56026 Frontal Lobe, Frontal LobeBrain ~ Uni-ZAP
Dementia dementialAlzheimer' XR
.
,s S6028 Human Manic Human Manic Brain diseaseUni-ZAP
- Depression de ression XR
Tissue ~ tissue T0002 Activated T-cellsActivated Blood Cell pBluescript T-Cell, Line SK-PBL fraction T0003 Human Fetal Human Fetal Luna Lun Bluescri t SK-T0004 Human White Human White Bluescri Fat Fat t SK-T0006 Human Pineal Human Pinneal pBluescript Gland Gland SK-T0008 Colorectal TumorColorectal diseasepBluescript Tumor SK-T0010 Human Infant Human Infant Other Brain Brain T0023 Human PancreaticHuman Pancreatic diseasepBluescript Carcinoma Carcinoma SK-T0040 HSC172 cells SA172 Cells Bluescri t SK-T0041 Jurkat T-cell Jurkat T-cell Bluescri Gl ' hase t SK-T0042 Jurkat T-Cell, Jurkat T-Cell Bluescri S Base Line t SK-T0048 Human Aortic Human Aortic _ pBluescript ' SK-Endothelium Endothilium T0049 Aorta endothelialAorta endothelial pBluescript cells + SK-TNF-a cells T0060 Human White Human White Bluescri Adi ose Fat t SK-T0067 Human Thyroid Human Thyroid Bluescri t SK-T0068 Normal Ovary, Normal Ovary,~ pBluescript SK-Premeno ausal Premeno ausal T0069 Human Uterus, Human Uterus, pBluescript normal SK-normal T0071 Human Bone MarrowHuman Bone pBluescript SK-Marrow T0082 Human Adult Human Adult Bluescri Retina Retina t SK-T0109 Human (HCC) pBluescript cell line SK-liver (mouse) metastasis, remake TO110 Human colon,.carcinoma pBluescript SK-(HCC) cell line, remake T0114 Human (Caco-2) pBluescript cell line, SK-adenocarcinoma, colon, remake L0002 Atrium cDNA
library Human heart L0004 ClonTech HL
1065a L0005 Clontech human aorta of A+ mRNA (#6572) L0021 Human adult (K.Okubo) L0022 Human adult lung 3"
directed MboI
cDNA .
L0040 Human colon'mucosa L0055 Human rom eloc to L0065 Liver HepG2 cell line.
L0105 Human aorta aorta polyA+
(TFu'iwara) L0109 Human brain brain cDNA
L0142 Human placenta placenta ~ ' cDNA
(TFu'iwara) L0157 Human fetal ~ brain brain (TFu'iwara) L0163 Human heart heart cDNA
(YNakamura) L0194 Human pancreaticpancreatic Patu cancer cancer cell line Patu 8988t 8988t L0351 Infant brain, _ BA, M13-Bento Soares .
derived L0352 Normalized infant BA, M13-brain, Bento Soares derived L0361 Stratagene ovary ovary Bluescript SK
(#937217) L0362 Stratagene ovarian . Bluescript cancer SK-(#937219) L0364 NCI CGAP_GC5 perm cell Bluescri tumor t SK-L0366 Strata ene schizoschizo hrenic Bluescri brain brain t SK-S11 S-11 frontal lobe L0369 NCI CGAP_AA1 adrenal adenomaadrenal Bluescri land t SK-L0370 Johnston frontalooled frontalbrain Bluescri cortex lobe t SK-L0371 NCI CGAP_Br3 breast tumorbreast Bluescri t SK-L0372 NCI CGAP Col2 colon tumor colon Bluescri t SK-L0373 NCI CGAP_Coll tumor colon Bluescri t SK-L0374 NCI CGAP_Co2 tumor colon Bluescript SK-L0375 NCI CGAP_ICid6 kidney tumorkidney Bluescript SK-L0376 NCI CGAP_Larl larynx lar nx Bluescri t SK-L0378 NCI CGAP_Lul lun tumor lun ~ Bluescri t 5K-L0381 NCI_CGAP_HN4 squamous pharynx Bluescript cell ~ SK-carcinoma L0382 NCI CGAP_Pr25 a ithelium rostate Bluescri (cell line) t SK-L0383 NCI_CGAP_Pr24 invasive prostate Bluescript tumor (cell SK-line) L0384 NCI CGAP_Pr23 rostate tumorrostate Bluescri t SK-L0385 NCI CGAP_Gasl oastric tumorstomach Bluescri t SK-L0386 NCI CGAP_HN3 squamous , tongue luescript cell SK-B
carcinoma from base of ton ue L0387 NCI CGAP_GCBO germinal tonsil Bluescript center B- 1 SK-cells L0393 B, Human Liver t1 1 tissue L0415 b4HB3MA CotB-HAP-Ft Lafmid BA
L0438 normalized infanttotal brain brain lafmid brain BA
cDNA
L0439 Soares infant 4vhole Lafmid brain 1NIB brain BA
L0455 Human retina retina eye lambda cDNA . gtl0 randomly primed sublibrar L0462.WATM1 - lambda ~tl l L0471 Human fetal Lambda heart, ZAP
Lambda ZAP Ex Ex ress ress L0475 KG1-a Lambda ' KG1-a Lambda Zap Zap Express cDNA Express library (Stratagene) L0477 HPLA CCLee placenta Lambda ZAP II
L0480 Stratagene cat#937212 Lambda ( 1992) , ZAP, pBluescript SK(-) L0483 Human ancreatic Lambda islet ZAPII
L0485 STRATAGENE Humanskeletal leg muscle Lambda skeletal musclemuscle ZAPII
cDNA
libra , cat.
#936215.
L0499 NCI CGAP_HSC2 stem cell bone pAMPI
34+/38+ marrow L0512 NCI_CGAP_Ov36 borderline ovary pAMPl ovarian carcinoma L0517 NCI CGAP_Prl AMP10 L0518 NCI CGAP_Pr2 AMP10 L0519 NCI CGAP Pr3 AMP10 L0520 NCI_CGAP_Alvl alveolar pAMPlO
rhabdomyosarcoma L0521 NCI_CGAP_Ewl Ewina"s sarcoma AMP10 L0526~NCI_CGAP_Prl2 metastatic pAMPlO
prostate , bonelesion L0527 NCI_CGAP_Ov2 ovar AMP10 L0528 NCI CGAP_Pr5 prostate pAMPlO
L0529 NCI_CGAP_Pr6 prostate pAMPlO
L0533 NCI_CGAP HSC1 stem cells bone marrow AMP10 L0534 Chromosome?Fetalbrain brain pAMPlO
Brain cDNA Library L0535 NCI_CGAP_Br5 infiltratingbreast pAMPlO
ductal carcinoma L0540 NCI_CGAP_PrlO invasive prostate pAMPlO
prostate tumor L0542 NCI_CGAP_Prl normal prostaticprostate pAMPlO
l a ithelial cells L0543 NCI_CGAP_Pr9 normal prostaticprostate pAMPlO
a ithelial cells L0545 NCI_CGAP_Pr4.l prostatic prostate pAMPlO
, intraepithelial neoplasia - high grade L0546 NCI_CGAP PrlB stroma rostate AMP10 L0547 NCI_CGAP_Prl6 tumor prostate pAMPlO
L0549 NCI_CGAP_HN10 carcinoma pAMPlO
in situ from retromolar tri one LO551 NCI_CGAP_HN7 noi'mal squamous pAMPlO
epithelium, floor of -mouth L0558 NCI_CGAP_Ov40 endometrioidovary pAMPlO
ovarian metastasis L0564 Jia bone marrowbone marrow Bluescri stroma stroma t L0565 Normal Human Bone Hip pBluescript Trabecular Bone Cells LO581 Strata~ene liver liver Bluescri (#937224) t SK
L0586 HTCDL1 pBluescript SK(-) L0588 Stratagene endothelial pBluescript cell SK-L0590 Stratagene fibroblast pBluescriptf SK-(#937212) L0591 Stratagene HeLa' pBluescript cell s3 SK-L0592 Stratagene hNT _ ~pBluescript neuron SK-(#937233) L0593 Stratagene pBluescript SK-neuroepithelium (#937231) L0594 Stratagene , , pBluescript SK-neuroepithelium L0595 Stratagene NT2 neuroepithelialbrain pBtuescript neuronal cells SK-recursor937230 -- ---L0596 Stratagene colon colon pBluescript SK-(#937204) L0597 Stratagene corneal cornea pBluescript stroma SK-(#937222) L0598 Morton Fetal cochlea ear Bluescri Cochlea t SK-L0599 Strataaene lun - IunQ Bluescri (#937210) t SK-L0600 Weizmann Olfactoryolfactory nose pBluescript epithelium SK-E ithelium L0601 Stratagene pancreas pancreas pBluescript SK-(#937208) L0602 Pancreatic Isletancreatic ' ancreas Bluescri islet t SK-L0603 Stratagene placenta placenta pBluescript , SK-(#937225) L0604 Stratagene muscle. muscle skeletal pBluescript muscle L0605 Stratagene fetalfetal spleenspleen pBluescript spleen , SK-' (#937205) L0606 NCI CGAP L m5 follicular 1 m h Bluescri I m homa node t SK-L0607 NCI_CGAP_Lym6 mantle cell lymph pBluescript node SK-I m homa L0608 Stratagene lunglung carcinomalung NCI-H69 pBluescript carcinoma SK-.
L0611 Schiller meningiomameningioma brain pBluescript SK-(Strata~ene) L0617 Chromosome 22 pBluescriptIIKS
exon L0619 Chromosome 9 pBluescriptIIKS
exon II
L0622 HMl pcDNAII
(Invitro en) L0623 HM3 pectoral pcDNAII
muscle (after mastectom ) (Invitro en) L0627 NCI CGAP Col bulk tumor colon CMV-SPORT2 -L0628 NCI CGAP Ovl ovar bulk ova CMV-SPORT2 tumor L0629 NCI_CGAP_Mel3 metastatic bowel pCMV-SPORT4 (skin melanoma rimar to bowel ) L0630 N.CI CGAP CNS1 substantia brain CMV-SPORT4 niQra L0631 NCI CGAP Br7 breast CMV-SPORT4 L0635 NCI_CGAP PNS dorsal root peripheral , pCMV-SPORT4 1 ganglion nervous system L0636 NCI-CGAP_Pitl four pooled brain pCMV-SPORT6 pituitary adenomas L0637 NCI_CGAP_Brn53 three pooledbrain pCMV-SPORT6 menin~iomas L0638 NCI_CGAP_Brn35 tumor, 5 brain pCMV-SPORT6 pooled.(see descri tion) L0639 NCI_CGAP_Bm52 tumor, 5 brain pCMV-SPORT6 pooled (see description) L0640 NCI_CGAP_Brl8 four pooled breast pCMV-SPORT6 high-grade tumors, ' ' ' including two rims L0641 NCI CLAP Col7 juvenile colon pCMV-SPORT6 I granulosa tumor .
L0642 NCI-CGAP_Col8 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0643 NCI_CGAP_Col9 moderately , colon pCMV-SPORT6 differentiated' adenocarcinoma L06a4 NCI_CGAP_Co20 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0646 NCI_CGAP_Col4 moderately- colon pCMV-SPORT6 differentiated adenocarcinoma L0647 NCI_CGAP_5ar4 five pooled connective pCMV-SPORT6 sarcomas, tissue including m xoid Ii -osarcoma L0648 NCI_CGAP_Eso2 ,squamous esophagus pCMV-SPORT6 cell -carcinoma "
L0649 NCI_CGAP_GU1 2 pooled genitourinary pCMV-SPORT6 high-grade transitionaltract cell tumors L0650 NCI CGAP_Kidl3 2 pooled kidney pCMV-Wilms" S PORT6 tumors, one primary and one metast L0651 NCI CGAP_Kid8 renal cell kidne CMV-SPORT6 tumor L0652 NCI_CGAP_Lu27 four pooled lung pCMV-SPORT6 poorly-differentiated adenocarcinomas L0653 NCI_CGAP_Lu28 two pooled lung pCMV-SPORT6 squamous cell carcinomas L0654 NCI CGAP Lu31 lung, CMV-SPORT6 cell line L0655 NCI_CGAP_Lyml2 lymphoma, lymph pCMV-SPORT6 node follicular mixed _ small and lar a cell L0656 NCI CGAP Ov38 normal a ovar CMV-SPORT6 ithelium L0657 NCI_CGAP_Ov23 tumor, 5 ovary pCMV-SPORT6 pooled (see descri tion) L0658 NCI_CGAP Ov35 tumor, 5 ovary pCMV-SPORT6 pooled (see descri tion) L0659 NCI CGAP Panl adenocarcinomaancreas CMV-SPORT6 L0661 NCI_CGAP_Me115 malignant skin pCMV-SPORT6 melanoma, metastatic to lymph ' node L0662 NCI_CGAP_Gas4 poorly differentiatedstomach pCMV-SPORT6 adenocarcinoma with.si~net r L0663 NCI_CGAP_Ut2 moderately- uterus pCMV-SPORT6 . ' differentiated endometrial adenocarcino - L0664 NCI CGAP Ut3 poorly-differentiateduterus pCMV-I I
endometrial -adenocarcinoma, L0665 NCI_CGAP_Ut4 serous papillaryuterus pCMV-SPORT6 carcinoma, high grade, 2 ooled t L0666 NCI_CGAP_Ut1 well-differentiateduterus pCMV-SPORT6 endometrial adenocarcinoma, L0667 NCI_CGAP_CMLl myeloid cells,whole pCMV-SPORT6 . 18 blood pooled CML , cases, BCR/ABL rearra L0717 Gessler Wilms SPORT1 tumor L0718 Testis 5 SPORT1 L0720 PN001-Normal prostate pSportl Human Prostate L0731 Soares_pregnant uterus T7T3-Pac uterus_ p NbHPU
L0738 Human colorectal T7T3D
cancer L0740 Soares melanocytemelanocyte pT7T3D
~
2NbHM (Pharmacia) with a modified olylinker L0741 Soares adult brain pT7T3D
brain N264HBSSY (Pharmacia) with a modified.
of linker L0742 Soares adult brain pT7T3D
brain N2b5HB55Y (pharmacia) with a modified of linker L0743 Soares breast breast pTTT3D
2NbHBst (Pharmacia) ' with a modified of linker L0744 Soares breast breast pT7T3D
3NbHBst (Pharmacia) with a modified of linker L0745 Soares retina retina eye pT7T3D
N2b4HR
(Pharmacia) with a modified of linker L0746 Soares retina retina eye pT7T3D
N2b5HR
(Pharmacia) with a modified olylinker L0747 Soares fetal_heart heart pT7T3D
NbHH
19W , ~ (Pharmacia) with a modified of linker L0748 Soares fetal Liver . pT7T3D
liver spleen and 1 NFLS Spleen (Pharmacia) with a modified .
olylinker L0749 Soares_fetal Liver pTTT3D
liver_spleen and 1NFLS S1 Spleen (Pharmacia) with a modified of linker L0750 Soares fetal lung pTTT3D
lung_NbHLl I
9W (Pharmacia) with a modified olylinker L0751 5oares ovary ovarian tumorovary pTTT3D
tumor NbHOT . (Pharmacia) with a modified olylinker L0752 Soares_parathyroid_tumorparathyroid parathyroid pTTT3D
tumor NbHPA gland (Pharmacia) with a modified polylinker L0753 Soares_pineal_gland_N3H. pineal pTTT3D
gland PG
(Pharmacia) with a modified of linker L0754 Soares placenta placenta pTTT3D
(Pharmacia) with a modified of linker L0755 Soares_placenta_8to9wee, placenta pTTT3D
ks 2NbHP8to9W (Pharmacia) with a modified of linker L0756 Soares_multiple_sclerosismultiple pTTT3D
sclerosis 2NbHMSP lesions (Pharmacia) with a modified polylinker V TYPE
L0757 Soares senescentsenescent pTTT3D
fibrobla fibroblast sts_NbHSF (Pharmacia) ' with a modified polylinker V_TYPE
L0758 Soares_testis pTTT3D-Pac NHT
(Pharmacia) with a modified polylinker L0759 Soares total - pTTT3D-Pac fetus_Nb2H
F8 9w _ (Pharmacia) with a modified of linker LO'761NCI_CGAP_CLL1 B-cell, chronic pTTT3D-Pac lymphotic . (Pharmacia) leukemia with a modified of linker L0762 NCI_CGAP_Brl.l breast pTTT3D-Pac (Pharmacia) with a modified olylinker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (Pharrnacia) with a modified of linker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac (Pharmacia) with a modified olylinker L0765 NCI_CGAP_Co4 colon pT7T3D-Pac (Pharmacia) with a modified of linker L0766 NCI_CGAP_GCB germinal pT7T3D-Pac 1 center B
cell (Pharmacia) with a modified polylinker L0767 NCI_CGAP_GC3 pooled germ pT7T3D-Pac cell tumors (Pharmacia) with a modified of linker L0768 NCI_CGAP_GC4 pooled germ pT7T3D-Pac cell tumors (Pharmacia) with a modified of linker L0769 NCI_CGAP_Brn25 anaplastic brain pT7T3D-Pac , oligodendroglioma (Pharmacia) with a modified of linker L0770 NCI_CGAP_Brn23 glioblastomabrain ~ pTTT3D-Pac (pooled) (Pharmacia) with a modified of linker L0771 NCI_CGAP_Co8 adenocarcinomacolon pTTT3D-Pac (Pharmacia) with a modified olylinker L0772 NCI_CGAP_ColO colon tumor colon pT7T3D-Pac RER+
(Pharmacia) with a modified of linker L0773 NCI_CGAP_Co9 colon tumor colon pTTT3D-Pac RER+
(Pharmacia) 4vith a modified of linker L0774 NCI_CGAP_Kid3 ' kidney pT7T3D-Pac (Pharmacia) with a modified of linker L0775 NCI_CGAP_KidS 2 pooled kidney pTTT3D-Pac tumors (clear cell . (Pharmacia) type) .- with a modified polylinker L0776NCI CGAP_LuS. carcinoid lung pT7T3D-Pac (Pharmacia) with a modified of linker L0777Soares_NhHMPySlPooled humanmixed pTTT3D-Pac (see melanocyte, below) (Pharmacia) fetal ' heart, and with a pregnant modified of linker L0778Barstead pancreas pancreas pT7T3D-Pac ' _ HPLRB 1 (Pharmacia) with a modified of linker L0779Soares_NFL_T_GBC pooled pT7T3D-Pac (Pharmacia) with a modified polylinker L0780Soares_NSF_F8_9W_OT pooled pT7T3D-Pac PA_P_S 1 (Pharmacia) with a modified olylinker L0782NCI_CGAP_Pr21 normal prostateprostate pT7T3D-Pac (Pharmacia) with a modified of linker L0783~ NCI_CGAP_Pr22normal prostateprostate pT7T3D-Pac (Pharmacia) _ ' with a modified of linker L0785Barstead spleen spleen pT7T3D-Pac (Pharmacia) with a modified of linker L0786Soares_NbHFB whole pT7T3D-Pac ~ brain (Pharmacia) with a modified - polylinker L0787.NCI_CGAP_Subl ' pT7T3D-Pac (Pharmacia) with a modified of linker L0788NCI_CGAP_Sub2 ~ pTTT3D-Pac (Pharmacia) with a modified of linker L0789NCI_CGAP_Sub3 pT7T3D-Pac .
(Pharmacia) with a modified of linker L0790NCI_CGAP_5ub4 pTTT3D-Pac (Pharmacia) with a modified of linker ' L0791NCI_CGAP_SubS . pT7T3D-Pac (Pharmacia) with a modified olylinker L0792 NCI CGAP_Sub6 p T7T3D-Pac (Pharmacia) with a modified olylinker L0793 NCI_CGAP_Sub7 pT7T3D-Pac (Pharmacia) with a modified polylinker L0794 NCI_CGAP_GC6 pooled germ , pTTT3D-Pac cell tumors (Pharmacia) with a modified of linker L0796 NC1_CGAP_Brn50 medulloblastomabrain pT7T3D-Pac (Pharmacia) with a modified of linker L0800 NCI_CGAP_Col6 colon tumor,colon pTTT3D-Pac RER+
(Pharmacia) with a modifted of linker L0803 NCI_CGAP_Kidl1 kidney pTTT3D-Pac (Pharmacia) with a modified of linker L0804 NCI_CGAP_Kidl2 2 pooled kidney pT7T3D-Pac tumors (clear cell (Pharmacia) type) with a modified polylinker L0805 NCI_CGAP_Lu24 carcinoid lung pT7T3D-Pac (Pharmacia) with a modified of linker L0806 NCI_CGAP_Lul9 squamous lung pT7T3D-Pac cell carcinoma, (Pharmacia) poorly differentiated with a (4 modified of linker L0807 NCI_CGAP_Ovl8 fibrotheoma ovary pT7T3D-Pac (Pharmacia) with a modified of linker L0808 Barstead prostate prostate pT7T3D-Pac BPH
- HPLRB41 ' (Pharmacia) ' with a modified olylinker L0809 NCI_CGAP_Pr28 prostate pTTT3D-Pac (Pharmacia) - with a modified polylinker L2251 Human fetal Fetahlung lung OMIM Description Reference 106300 Anl losin s and litis 108800 Atrial se tal defect, secundum ty a 1201'60 Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420, 120160 Osteo orosis, idio athic, 166710 120160 Ehlers-Danlos s ndrome, a VIIA2, 130060 120160 Marfan syndrome, aty ical 120290 OSMED s ndrome, 215150 120290 Stickler s drome, a II, 184840 120810 C4 deficient 120820 C4 deficient 126650 Chloride diarrhea, con enital, Finnish e, 214700 126650 Colon cancer 129900 EEC s ndrome-1 142857 Pem higoid, susce tibility to 142858 ~ Be llium disease, chronic,. susce tibili to 145001 H a arath roidism-'aw tumor s ndrome 150270 La n eal adductor aral sis 150292 E idermol sis bullosa, Herlitz 'unctional e, 226700 154276 Mali ant h erthermia susce tibili 3 154705 Marfan s drome, a II
1.67250, Pa et disease of bone 170261 Bare 1 in hoc to s ndrome, a I, due to TAP2 deficient 173360 Thrombo hilia due to excessive lasmino en activator inhibitor 173360 Hemorrha is diathesis due to PAIL deficient 177900 Psoriasis susce tibili -1 179450 Ra eed sensitivi 183600 S lit hand/foot malformation, a 1 187680 . 6-merca to urine sensitivi 193300 Renal cell carcinoma 193300 von Hi el-Lindau s ndrome 201910 Adrenal h a lasia, con enital, due to 21-h drox lace deficient 208250 Jacobs s ndrome 217000 C2 deficient 222100 Diabetes mellitus, insulin-de endent-1 227646 Fanconi anemia, ty a D
23 3100 Renal lucosuria 235200 Hemochromatosis 248611 Ma 1e s ru urine disease, a Ib 253260 Biotinidase deficient 256550 Sialidosis, a I
256550 Sialidosis, a II
278720 Xeroderma i entosum, ou C
600202 Dyslexia, s ecific, 2 600261 Ehlers-Danlos-like s ndrome 600995 Ne hrotic s ndrome, idio atliic, steroid-resistant 601154 Cardiom o ath , dilated, 1E
601253 Muscular d stro h , limb- irdle, a IC
601652 Glaucoma 1A, rima o en an 1e, 'uvenile-onset, 137750 601868 Deafness, autosomal dominant 13 602011 Pancreatic endocrine tumors 602136 Refsum disease, infantile, 266510 602136 Zellwe er s ndrome-l, 214100 _60_2136 Adrenoleukod stro h , neonatal, 202370 602280 Retinitis i mentosa-14, 600132 602447 Corona arte disease, susce tibili to .
602475 ~ Ossification of osterior lon itudinal 1i ament of s ine Polynzccleotide and Polypeptide Variants [84] The present invention is directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID
NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A, the nucleotide sequence as defined in columns 8 and 9'of Table'2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, the nucleotide sequence as defned in column 6 of Table 1B, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1B, the cDNA sequence contained in Clone ID NO:Z, and/or nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in Clone ID NO:Z.
[85] The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in column 7 of Table 1A, a polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded,by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined in column 6 of Table 1B, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a polypeptide sequence encoded by the cDNA
sequence contained in Clone ID NO:Z. ' [86] ' "Variant" , refers to a polynucleotide or polypeptide differing from the.
polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
[87] Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence.
selected from the group consisting of: (a) a nucleotide sequence described in SEQ ID NO:X
or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence in SEQ
ID NO:X or the cDNA in Clone ID NO:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID
NO:Z; (c) a nucleotide sequence in SEQ ID NO:X ,or the cDNA in Clone ID NO:Z
which encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a polypeptide;
(e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ID
NO:Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the .
cDNA in Clone ID NO:Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by 'the cDNA in Clone ID NO:Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above. .
[88] The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, -95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ
ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA
contained in Clone ID NO:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of~SEQ ID NO:Y, a nucleotide sequence encoding a polypeptide 281 .
sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in Clone ID
NO:Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in column 7 of Table 1A or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in column 7 of Table 1A or the complementary strand thereto, andlor polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
[89] In a preferred embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucl.eotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides. In another preferred embodiment, polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[90] In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (b) the amino acid sequence of a mature form of a polypeptide having the amino acid sequence of SEQ ID
NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) the amino acid sequence of a biologically_active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z.
' [91] The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the polypeptide encoded by the~nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B, the amino acid sequence as defined in column 7 of Table 1A, an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also .
provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
[92] By a nucleic acid having a nucleotide sequence at least; for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include, up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to in Table 1A or~2 as the ORF (open reading frame), or any fragment specified as described herein.
[93] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least ~0%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a _ .___. .
nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match.
between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
In a sequence alignment the query and subject sequences are both DNA
sequences. An RNA sequence can be compared by converting U's to T's~ The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty=30, Randomization Group, Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
[94] ,If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results.
This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which .
are not matched/aligned, as a percent of the total.bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent identity,. calculated by the above FASTDB program using the specified parameters, to arnve at a final percent identity score.
This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
[95] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent.l0% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
[96] By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino. acid sequence at least 95% identical to a query amino acid sequence, up to.
5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
[97] As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence identified in column 6) or Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof; the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B, or .a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in Clone ID
NO:Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM
0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
[98] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results:
This is because .the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated.at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-tenninal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
This percentage is then subtracted from the percent .identity, calculated by the above FASTDB
program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N-and C- terminal residues of the subject sequence.
[99] For example, a 90 amino acid residue subject sequence is aligned with a residue query sequence to determine percent identity. The deletion occurs ' at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%: In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query: In this case the percent identity__ _~_ calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequnce are manually corrected for. No.~other manual corrections are to made for the purposes of the present invention.
[100] The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred., Moreover, polypeptide variants in which less than 5.0, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
[101] Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can,vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
[102] Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant I~GF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).) [103] Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J, Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1 a. They used random mutagenesis to generate over 3,500 individual IL-1 a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that "[m]ost of the molecule could be altered with little effect on either [binding or biological activity]." In fact, only 23 unique amino acid sequences, ,out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
[104] Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will.
likely be retained when less than the majority of 'the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N-or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
[105] Thus, the invention further includes polypeptide variants which show a functional activity (e.g., biological activity) of the polypeptides of the invention.
Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules knowmin the art so as have little effect on activity.
[106] The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%,.97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide. having the amino acid sequence of an N
and/or C
terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use .
the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA
expression in specific tissues (e.g.,'normal or diseased tissues); and (4) i~
situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).
[107] Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in fact, encode a polypeptide having functional activity. By a polypeptide having "functional activity" is meant, a-polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein of the invention. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an anti-polypeptide of the_invention antibody], immunogenicity. (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and' ability to bind to a receptor or ligand for a polypeptide of the invention.
[108] The functional activity of the . polypeptides, and fragments, variants and derivatives of the invention, can be assayed by various methods.
[109] For example, in one embodiment where one is assaying for the ability, to bind or compete . with a full-length polypeptide of the present invention for binding to an anti-polypetide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked 'immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions,' immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, inimunoflubrescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
[110] In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, _, _._ _ and affinity blotting. See generally, Phizicky et al:, Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a polypeptide of the present invention to bind to a substrates) of the polypeptide of the invention can be routinely assayed using techniques known in the art.
[111] In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants and derivatives thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention. _ [112] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in Clone ID NO:Z, the nucleic acid sequence referred to in Table 1A (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 2 (e.g,. the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides "having functional activity." In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the ai~t that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This. is because the ,skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
[113] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al.; "Deciphering the Message in Protein Sequences:
Tolerance to Amino-Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
[114] The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural. selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
[115] The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
[116] ~ As the authors state, these two strategies have revealed that proteins are .
surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu;
replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and. His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one.or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the~polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, serum albumin (preferably human serum albumin) or a fragment thereof, or leader or secretory sequence, or .a sequence facilitating purification, or, (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)).
Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
[117] For example, polypeptide variants containing .amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation.. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. IW munol. 2:-33-1-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
[118] . A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid.sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course it is highly preferable for a polypeptide to have an amino acid sequence which comprises the amino acid sequence of a polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ
_ ID NO:X, an 'amino acid sequence encoded by the portion of SEQ ID NO:X as defned in columnns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ
ID NO:X, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z
which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
[119] In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid'sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the~mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID.NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in Clone ID
NO:Z or fragments thereof; wherein the fragments or variants have. 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polynzacleotide and Polypeptide Fragmefzts [120], The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a "polynucleotide fragment" refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in Clone ID NO:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA
contained in Clone ID NO:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ
ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a pol'ynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1B or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID
NO:B as defined in column 6 of Table 1B or the complementary strand thereto.
[121] The polynucleotide fragments of the invention are Preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt~ or at least about 150 nt in length. A fragment "at least 20 nt in length," for examfle, is intended to include 20 or more contiguous bases from the cDNA sequence contained in Clone ID
NO:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context "about" includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have 'uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length ) are also encompassed by the invention.
[122] Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number l.-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 12'01-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, . 1751-1800, 1801-1850, 1851.-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101- , 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750; 5751-5800, 5801-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400,. 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context "about"
includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein.
Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions ' or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides. ' [123] Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of,_.a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1400, 1401-1450, 1451-1500; 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2900, 2901-2950, 2951-3000, 3001.-3050, 3051-3.100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601- .
3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751=5800, 5801-5850, 5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in Clone ID NO:Z, or the complementary strand thereto.
In this context "about" includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity).
More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
[124] Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic .acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1B column 6. Additional, representative examples.of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic. acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand of a sequence delineated in column 6 of Table 1B. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1B, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID
NO:A (see Table 1B, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1B, column.
6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that .
bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and po~ypeptides are also encompassed by the invention.
[125] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1B, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode.
these polypeptides, and antibodies that bind these polypeptides _are also encompassed by the invention.
[126] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ
ID NO:X (e.g., as, defined in Table 1A or 1B) or fragments or , variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[127] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of.Table 1B, and the polynucleotide sequence. of SEQ TD NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
[128] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' ~10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[129] In additional specific embodiments, polynucleotides of the.invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are.
also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[130] In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one of the. sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention: Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also . encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
[131] In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3' 10 polynucleotides of one ~of the sequences delineated in column 6 of Table 1B and the 5' 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments,. the 3' 10 polynucleotides, of one of the sequences delineated in column 6,of Table 1B is directly contiguous with the 5' 10 polynucleotides of the next sequential exon delineated in Table 1B, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic vacids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids;- and polypeptides are also encompassed by the invention.
[132] In the present invention, a "polypeptide fragment" refers to an amino acid sequence which is a portion of that. contained in SEQ ID NO:Y, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID
NO:X, a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid sequence encoded by the cDNA contained in Clone ID NO:Z. Protein (polypeptide) fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460,.461-__ _.
480, 481-500, 501-520, .521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, ,1061-1080, 1081-1100, 110.1-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, '761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241=1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about" includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
[133] Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues ofva complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[134] Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form.
Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
[135] The present invention further provides polypeptides having_one or more residues deleted from the amino terminus of the amino acid, sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and' 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1B, and/or a polypeptide encoded by the cDNA contained in Clone ID NO:Z). , In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention. , [136] The present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ
ID
NO:X as defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by the cDNA
contained in Clone ID NO:Z). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-l, and where n corresponds to the position of amino . acid residue in a polypeptide of the invention.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[137] In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA contained in Clone ID NO:Z, and/or the complement thereof, where n and m are integers as described above.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[138] Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g.; biological activities, ability to multimerize, ability to ,bind a ligand) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities'can readily be determined by routine methods described herein and otherwise known in the art. rt is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
[139] The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
[140] ~ Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in Clone ID NO:Z, or the polyrlucleotide sequence as defined in column 6 of Table 1B, maybe analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by .a _...
polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y
and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2) or the clDNA contained in Clone ID NO:Z may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, WI 53715 USA; http://www.dnastar.com~. _ [141] Polypeptide regions that may be routinely obtained using the DNASTAR
computer algorithm include, but are not limited to, Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions;
Eisenberg alpha-and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., 1, 2, 3 or 4) of the features set out above.
[142] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR
analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
['143] Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g.
biological activity) of the polypeptide sequence of Which the amino acid sequence is a fragment. By a. polypeptide displaying ',a "functional activity" is meant a polypeptide capable of one or more known functional activities associated with a full-length protein, such as, fox example, biological activity; antigenicity, immunogenicity, and/or multimerization, as described herein:
[144] ' Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
[145] In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or moxe of the antigenic fragments of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding-these polypeptides are also encompassed'by the invention.
[146] The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y;
a.
polypeptide sequence encoded by SEQ ID NO:X or the 'complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B
as defined in column 6 of Table 1B or the complement thereto; the polypeptide sequence encoded by the cDNA contained in Clone ID, Nb:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ~ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in Clone ID NO:Z
under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or . alternatively, under lower stringency hybridization conditions defined supra.
[147] The texm "epitopes," as used herein, refers to poxtions of a polypeptide having.
antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An "immunogenic epitop.e," as used herein, is defined as a portion of a pxotein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term "antigenic epitope," as used herein, is. de _fined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[148] Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,21 l.) [149] In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least l I, at least 12, at least 13, at Ieast I4,.at Ieast 15, at least 20, at least 25, at. least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, r60, 65, 70, 7~, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes~
are useful; for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
[150] Non-limiting examples of epitopes of poiypeptides that can be used to generate antibodies of the invention'include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portions) of SEQ ID
NO:Y specified in column 7 of Table 1A. These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index which is included in_ the DNAStar suite of computer programs.
By "comprise" it is intended that a polypeptide contains at least,one, two, three, four, five, six or more of the portions) of SEQ ID NO:Y shown in column 7 of Table 1A, but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the~poxtion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterolgous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein.
In particular embodiments, epitope portions'of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in column 7 of Table 1A.
[151] Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. See,. for instance, Sutcliffe et al., supra;
Wilson et al., supra; Chow et al., Proc. Natl. Acad.. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes. include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a Garner protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at , .
the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
[152] Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supf~a; Wilson~et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 pg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. . Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed ~to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, fox instance, by adsorption to the peptide on. a solid support and elution of the selected antibodies according to methods well known in the arE.
,.
[153] As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including. fragments or variants thereofj, may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1; CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides.
By way of another non-limiting example, polypeptides and/or antibodies of the present 'invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S.
Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Patent 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide. or human serum albumin polypeptide).
Polynucleotides encoding fusion proteins of the invention are also .encompassed by the invention.
[154] Such fusion proteins as those described above may facilitate purification and may increase half life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobuliris. See, e.g., vEP
394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin).
conjugated to an FcRn'binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure _due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-(1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Fusion Proteins [155] Any polypeptide of the present invention can be used to generate fusion proteins.
For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as targeting~molecules once fused to other proteins:
[156] Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linleer sequences.
[157] In certain preferred embodiments, proteins of the invention axe fusion proteins comprising an amino acid ' sequence that is an N and/or C- terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence of the invention.
Polynucleotides encoding these proteins are also encompassed by the invention.
[158] Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
[159] As one of skill in the~art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, cari be combined with hetero.logous polypeptide sequences. For example, the polypeptides of the present invention may .be fused with heterologous polypeptide sequences, 'for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), or albumin (including, but not limited to, native ~or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses,fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A
0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
In drug discovery, fox example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); I~. Johanson et al., J. Biol.
Chem. 270:9459-9471 (1995).
[164] Moreover, the polypeptides- of the present invention can be fused to marker sequences, such as a polypeptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as 308.
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag; corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).
[161] Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well ; as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793;
5,811,238;.5;830,721; 5,834,252;
and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999);
and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each. of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:x and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to.random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be~recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
[162] Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. .
Recombinant and Synthetic Production of PolYpeptides of the Invention [163] The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective.
In the latter case, viral propagation generally will occur only in complementing host cells.
[164] The polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, .such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
[165] The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, .the E. coli lac, trp, phoA
and tac promoters, the .SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[166] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G41 ~, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178));
insect cells such as Drosophila S2 and Spodoptera S~ cells; animal cells such a~s CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
[167] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNHl6a, pNHlBA, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3', pDR540, pRITS available from Pharmacia Biotech, Inc.
Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG
available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD 1, pTEFl/Zeo, pYES2lGS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, PHIL-S1, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA).
Other suitable vectors will be readily apparent to the skilled artisan.
[168J Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the marine myeloma cell line, NSO) which are glutamine synthase negative.
Glutarnine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: W087/04462;
W086/05807;
W089/01036; W089/10404; and W091/06657, which are hereby incorporated in their .
entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal antibodies using a.GS expression system in marine myeloma cells is described in Bebbington et al., Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechhol.
Prog. 11:1 (1995) which are herein incorporated by reference.
[169] The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have charactei'istics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
[170] Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated J
transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
[171] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequence's via .homologous recombination (see, e.g., US Patent Number 5,641,670, issued June 24, ,1997; International Publication Number WO
96/29411;
International Publication Number WO 94/12650; Koller et al., P~oc. Natl. Acad.
Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 34:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[172] Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
[173] Polypeptides of the present invention can also be recovered from:
products 'purified from natural sources, including bodily fluids, tissues and cells, Whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques , from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypep'tides of the present invention may be glycosylated or may be non-glycosylated. 'In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes._ _ Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eulcaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
[174] In.one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eulcaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol ~s its sole carbon source. A main step in the methanol metabolization pathway is.the oxidation of methanol to formaldehyde using OZ. This reaction is catalyzed by the enzyme alcohol. oxidase. In order to metabolize methanol as its sole carbon source, Piclzia pasto~is must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for OZ. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of.the two-alcohol oxidase genes (AOXI ) is highly active. In the presence of methanol, alcohol oxidase produced from the AOXI gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, 3.F., et al., Nucl. Acids Res. 15:3859-76 (1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the ADXI
regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
[175] In one example, the plasmid vector pPIC9K is used to express DNA
encoding a polypeptide of the invention, as set forth herein, in a Piclaea yeast system essentially as described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins and J.
Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOXI
promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
[176] Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDI, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-Sl, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as.
long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required. ' .
[177] In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
[178] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic y material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/,or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/2941 l, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
[179] In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-(1984)).. For example, a polypeptide corresponding to a fragment of a polypeptide can, be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl ammo acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[180] The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, arnidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited; to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin; etc.
[181] Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or G-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein. .
[182] Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (lzih iz3h izsh 131I), carbon (14C), sulfur (35S), tritium (3H), indium (lln, nzln m3mln msmln technetium 9~Tc 99mTc thallium 2°iTi 6s ), ( , ), ( ), gallium ( Ga, Ga), alladium losPd mol bdenum 99Mo xenon lssxe fluorine (18F) is3Sm l~~Lu ls9Gd p ( )~ Y ( )~ ( )> > > > >
ia9pm~ IaoLa~ ms~,b~ is6Ho~ 9o.Y~ 4~sc~ iasRe~ issRe~ i4aPr~ nose and 9~Ru.
[183] In specific embodiments, a polypeptide of the present invention or fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, l~~Lu, 9oY, is6Ho, and is3Sm, to polypeptides.
In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 11n. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is g°Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art - see, for example, DeNardo et al., Clin Cancer Res. ,4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
[184] As mentioned, the proteins of the invention may be modified by either natural processes; such as posttranslational processing, or by chemical modification techniques which are well known in the. art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide.
Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by~ synthetic methods. Modifications include acetylation, acylation, 'ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide .derivative, covalent attachment'of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York ' (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol.
182:626-646 (1990); Rattan et al., Ann: N.Y. Acad. Sci. 663:48-62 (1992)).
[185] Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may. be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
[186] The polymer may be of any molecular weight, and may be branched or . unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000; 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
a [187] As noted above, the polyethylene glycol may have a branched structure.
. Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575;
h Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 1$:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
[188] The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of . the protein. There are anumber of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl~ chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulflrydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
[189] . As suggested above, polyethylene glycol may be attached to proteins,via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, ' histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
[190] . One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino 'groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
[191] As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein. either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S.
Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
[192] One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene . glycol (MPEG) using tresylchloride (C1SOZCHZCF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[193] Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U..S. Patent No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by refe~'ence. Pegylated protein products produced. using the reaction chemistries set out herein are included within the scope of the invention.
[194] The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For 'example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule.
Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carner Sys. 9:249-304 (1992).
[195] The polypeptides of the invention can be recovered and purified_from chemical synthesis and recombinant cell cultures by standard methods which include, but are not ' .
limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose -chromatography,. hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification. , ' [196] The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, thewpresent invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the ._ polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
[197]' Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID
NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns ~
and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence.
In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In , additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
320 _ ..
[198] As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
[199] ' . Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of. the invention contact one another in solution; In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ
ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in Clone ID NO:Z). . In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent .associations are the consequence of chemical or recombinant manipulation. -Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable 'of forming covalently associated multimers, such as for example, osteoprotegerin (see; e.g., International Publication NO:
WO
98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two, or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incozporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
[200] Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the .culture supernatant using techniques known in the art.
[201] Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S.
patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention. .
[202] In another example, proteins of the invention are associated by interactions.
between Flag~ polypeptide sequence contained ~in fusion proteins of the invention containing Flag~ polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag~ fusion proteins of the invention and anti-Flag~. antibody.
[203] The multirtiers of the invention may be generated using chemical techniques known in the art: For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C- .
terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its _ entirety).
[204] Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced' recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, .
polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a ~olypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader' sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant , techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can.be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Antibodies [205] Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR)~which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in Clone ID
No:Z, and/or an epitope, o~ the present invention) as determined, by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or~subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgGl . In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.
[206] Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions) alone~'or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable regions) with a hinge region, CH1; CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, 'horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
[207] The antibodies of the present invention may be monospecific, bispecific, trispecific_or_ of..greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; I~ostelny et al., J. Immunol. 148:1547-1553 (1992).
[208] Antibodies of the present invention may be described or specified in terms of the epitope(s) or portions) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portions) may be specified as described herein, e.g.; by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures.. Preferred epitopes of the invention include the predicted epitopes shown iri column 7 of Table 1A, as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
[209] . Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least-70%, at .
least 65%, at least,60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit honiologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less thazi 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s)~ of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10-2 M, 10-z M, 5 X 10-3 M, 10-3 M, 5 X 10-4 M, 10-4 M, 5. X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6M, 5 X 10-' M, 10' M, 5 X 10-8 M, 10-8 M, X 10-9 M, 10-9 M, 5 X 10-1° M; 10-1° M, 5 X 10-" M, 10-11 M, 5 X
10-12 M, 10-12 M, 5 X
10-13 M, 10-13 M, 5 X 10-14 M, 10-la M, 5 X 10-15 M, or 10-15 M.
[210] The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
[211] Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features' both receptor-specific 'antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitatiorl followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
[212] The invention also features receptor-specific antibodies .which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, fox example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT
publication WO
96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J.
Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998);
Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);
Carlson et al., J. Biol. Chem..272(17):11295-11301 (1997); Taryrnan et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
[213] ' Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention; including both in vitro and ifa vivo diagnostic arid therapeutic methods. For example, the antibodies have utility iri immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
incorporated by reference herein in its entirety.
[214] As discussed in more detail below, the antibodies of the present invention maybe used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT
publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995;
and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.
[215] The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation,~
~amidation, derivatization by known protectinglblocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but-not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical amino acids.
[216] The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc.
to induce the production of sera containing polyclonal antibodies specific for the antigen.
Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic . polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, .
dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
[217] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. ' 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-(Elsevier, N.Y., 1981). (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through -hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not=
the method by which it is produced.
[218] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known. in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones. - -[219] Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein; preferably, the hybridonia is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
[220] Another well known method for producing both polyclonal and monoclonal human B~ cell lines is transformation using Epstein Barr Virus (EBV).
Protocols for generating EBV-transformed B cell lines are commonly known imthe art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of .B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived -from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues.
Tissues are generally made into single cell suspensions prior to EBV
transformation.
Additionally, steps may be taken to either physically remove or inactivate T
cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
[221] In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, ,clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV
lines may become monoclonal or polyclonal as a, result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and II~R-4). Thus, the present invention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
[222] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
[223] For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display .antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.
Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic et ~, al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994);
PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737;
W0.92/01047; WO. 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.
Patent Nos. 5,698,426; 5;223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
[224] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
[225] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a rriurine monoclonal antibody and a human immunoglobulin constant region. Methods for, producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 .
(1986); Gillies ~ et al., (1989) J. Immuriol. Methods 125:191-202; U.S. Patent Nos.
'5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in ,their . entirety. Humanized antibodies are antibody molecules from non-human -species antibody that binds the desired antigen having one or more _complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5)-:489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), .and chain shuffling (IJ.S.
Patent No. 5,565,332).
[226] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above fusing antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111;
and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91110741; each of which is incorporated herein by reference in its entirety.
[227] . Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and. light chain ' immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells; Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
The modified embryonic stem cells are expanded and microinjected into blastocysts to -produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a~selected~antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can'b~e obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes -. harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int.
Rev.
Immunol. 13:65-93 (1995). For ~a detailed ~ discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT~publications WO 98/24893; WO 92/01047; WO 96/34096;
WO
96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923;
5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771;
5,939,598;
6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharni (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
[228] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Biotechnology 12:899-903 (1988))., [229] Further, antibodies to the polypeptides of the invention can, in tum, be utilized to generate anti-idiotype antibodies that '"mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J.
7(5):437-444; (1989) and Nissinoff, J.. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of.the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of .such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
[230] Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Cherl et al., Hum. Gene Ther. 5:595-601 (1994);
Marasco, W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev.
Microbiol.
51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999);
Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999);
Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein.
Pol~nucleotides Encoding Antibodies [231] The~invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having, the amino acid sequence of SEQ
ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, andlor to a polypeptide encoded by the cDNA contained in Clone ID
NO:Z.
[232] The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in I~utmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the. synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
[233] Alternatively, a polynucleotide encoding an antibody maybe generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR
amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, , e.g., a cDNA clone from a cDNA library that encodes the antibody.
Amplified nucleic acids generated by PCR may then be ~ cloned into replicable cloning vectors using any method well known in the art.
[234]' Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create, amino acid substitutions, deletions, and/or insertions.
[235] In a specific embodiment, the amino acid sequence of the heavy andlor light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the. art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human, framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (I998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
[236] In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., ' Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody, molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as~ those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
[237] Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No: 4,946,778; Bird, Science 242:423- 42 (1988); Huston et al., Proc. Natl.
Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge;
resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Methods of Producing Antibodies [238] The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include,.
but- are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
[239] Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g.,. a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro. recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light,chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.
Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
-, [240] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. . Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
[241] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when. transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) _ . . containing antibody coding sequences; ,plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e:g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole ~'ecombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human , cytomegalovirus, is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
[242] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by~ adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so. that the cloned target gene product can be released from the GST moiety.
[243] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugipe~da cells. The antibody coding sequence may be cloned individually into non-essential regions . 338 (for example the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
[244] In mammalian host cells, a number of viral-based expression systems .may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)).
Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences.
Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)). . .
[245] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
To this end, 'eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2; BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
[246] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express fihe antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites; etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then. are switched to a selective .media. The selectable marker in the recombinant plasmid confers.
resistance to the selection and allows. cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
[247] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad.
Sci. USA
48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci.~USA 77:357 (1980);
O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.
Biochem=. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their entireties.
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Claims (24)
1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:
(a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA sequence contained in Clone ID NO:Z, which is hybridizable to SEQ ID
NO:X;
(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(c) a polynucleotide encoding a polypeptide fragment of a polypeptide encoded by SEQ ID NO:X or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X;
(d) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(e) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(f) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X, having biological activity;
(g) a polynucleotide which is a variant of SEQ ID NO:X;
(h) a polynucleotide which is an allelic variant of SEQ ID NO:X;
(i) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y;
(j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A
residues or of only T residues.
(a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA sequence contained in Clone ID NO:Z, which is hybridizable to SEQ ID
NO:X;
(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(c) a polynucleotide encoding a polypeptide fragment of a polypeptide encoded by SEQ ID NO:X or a polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X;
(d) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(e) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X;
(f) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X, having biological activity;
(g) a polynucleotide which is a variant of SEQ ID NO:X;
(h) a polynucleotide which is an allelic variant of SEQ ID NO:X;
(i) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y;
(j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A
residues or of only T residues.
2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a protein.
3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:Y
or the polypeptide encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X.
or the polypeptide encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z, which is hybridizable to SEQ ID NO:X.
4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:X or the cDNA
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X.
sequence contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X.
5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
7. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.
8. A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim 1.
9. A recombinant host cell produced by the method of claim 8.
10. The recombinant host cell of claim 9 comprising vector sequences.
11. An isolated polypeptide comprising an amino acid sequence at least 90%
identical to a sequence selected from the group consisting of:
(a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z, having biological activity;
(c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(d) a polypeptide, epitope of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(e) a full length protein of SEQ ID NO:Y or the encoded sequence contained in cDNA
Clone ID NO:Z;
(f) a variant of SEQ ID NO:Y;
(g) an allelic variant of SEQ ID NO:Y; or (h) a species homologue of the SEQ ID NO:Y.
identical to a sequence selected from the group consisting of:
(a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z, having biological activity;
(c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(d) a polypeptide, epitope of SEQ ID NO:Y or the encoded sequence contained in cDNA Clone ID NO:Z;
(e) a full length protein of SEQ ID NO:Y or the encoded sequence contained in cDNA
Clone ID NO:Z;
(f) a variant of SEQ ID NO:Y;
(g) an allelic variant of SEQ ID NO:Y; or (h) a species homologue of the SEQ ID NO:Y.
12. The isolated polypeptide of claim 11, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
13. An isolated antibody that binds specifically to the isolated polypeptide of claim 11.
14. A recombinant host cell that expresses the isolated polypeptide of claim 11.
15. A method of making an isolated polypeptide comprising:
(a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
(a) culturing the recombinant host cell of claim 14 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.
16. The polypeptide produced by claim 15.
17. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polynucleotide of claim 1.
18. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
(a) determining the presence or absence of a mutation in the polynucleotide of claim 1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.
19. A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the polypeptide of claim 11 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
(a) determining the presence or amount of expression of the polypeptide of claim 11 in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.
20. A method for identifying a binding partner to the polypeptide of claim 11 comprising:
(a) contacting the polypeptide of claim 11 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
(a) contacting the polypeptide of claim 11 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.
21. The gene corresponding to the cDNA sequence of SEQ ID NO:Y.
22. A method of identifying an activity in a biological assay, wherein the method comprises:
(a) expressing SEQ ID NO:X in a cell;
(b) isolating the supernatant;
(c) detecting an activity in a biological assay; and identifying the protein in the supernatant having the activity.
(a) expressing SEQ ID NO:X in a cell;
(b) isolating the supernatant;
(c) detecting an activity in a biological assay; and identifying the protein in the supernatant having the activity.
23. The product produced by the method of claim 20.
24. A method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 11.
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| US17906500P | 2000-01-31 | 2000-01-31 | |
| US60/179,065 | 2000-01-31 | ||
| US18062800P | 2000-02-04 | 2000-02-04 | |
| US60/180,628 | 2000-02-04 | ||
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| US60/184,664 | 2000-02-24 | ||
| US18635000P | 2000-03-02 | 2000-03-02 | |
| US60/186,350 | 2000-03-02 | ||
| US18987400P | 2000-03-16 | 2000-03-16 | |
| US60/189,874 | 2000-03-16 | ||
| US19007600P | 2000-03-17 | 2000-03-17 | |
| US60/190,076 | 2000-03-17 | ||
| US19812300P | 2000-04-18 | 2000-04-18 | |
| US60/198,123 | 2000-04-18 | ||
| US20551500P | 2000-05-19 | 2000-05-19 | |
| US60/205,515 | 2000-05-19 | ||
| US20946700P | 2000-06-07 | 2000-06-07 | |
| US60/209,467 | 2000-06-07 | ||
| US21488600P | 2000-06-28 | 2000-06-28 | |
| US60/214,886 | 2000-06-28 | ||
| US21513500P | 2000-06-30 | 2000-06-30 | |
| US60/215,135 | 2000-06-30 | ||
| US21688000P | 2000-07-07 | 2000-07-07 | |
| US21664700P | 2000-07-07 | 2000-07-07 | |
| US60/216,880 | 2000-07-07 | ||
| US60/216,647 | 2000-07-07 | ||
| US21749600P | 2000-07-11 | 2000-07-11 | |
| US21748700P | 2000-07-11 | 2000-07-11 | |
| US60/217,487 | 2000-07-11 | ||
| US60/217,496 | 2000-07-11 | ||
| US21829000P | 2000-07-14 | 2000-07-14 | |
| US60/218,290 | 2000-07-14 | ||
| US22096300P | 2000-07-26 | 2000-07-26 | |
| US22096400P | 2000-07-26 | 2000-07-26 | |
| US60/220,963 | 2000-07-26 | ||
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| US22526800P | 2000-08-14 | 2000-08-14 | |
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| US22526600P | 2000-08-14 | 2000-08-14 | |
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| US22575700P | 2000-08-14 | 2000-08-14 | |
| US22575900P | 2000-08-14 | 2000-08-14 | |
| US22451800P | 2000-08-14 | 2000-08-14 | |
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| US60/225,268 | 2000-08-14 | ||
| US60/225,267 | 2000-08-14 | ||
| US60/225,270 | 2000-08-14 | ||
| US60/225,213 | 2000-08-14 | ||
| US60/225,757 | 2000-08-14 | ||
| US60/225,266 | 2000-08-14 | ||
| US60/225,447 | 2000-08-14 | ||
| US60/225,214 | 2000-08-14 | ||
| US60/225,759 | 2000-08-14 | ||
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| US22627900P | 2000-08-18 | 2000-08-18 | |
| US22686800P | 2000-08-22 | 2000-08-22 | |
| US22718200P | 2000-08-22 | 2000-08-22 | |
| US22668100P | 2000-08-22 | 2000-08-22 | |
| US60/227,182 | 2000-08-22 | ||
| US60/226,279 | 2000-08-22 | ||
| US60/226,681 | 2000-08-22 | ||
| US60/226,868 | 2000-08-22 | ||
| US22700900P | 2000-08-23 | 2000-08-23 | |
| US60/227,009 | 2000-08-23 | ||
| US22892400P | 2000-08-30 | 2000-08-30 | |
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| US23141400P | 2000-09-08 | 2000-09-08 | |
| US60/231,414 | 2000-09-08 | ||
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| US23196800P | 2000-09-12 | 2000-09-12 | |
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| US60/237,037 | 2000-10-02 | ||
| US23993500P | 2000-10-13 | 2000-10-13 | |
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Publications (1)
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Family
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| CA002398411A Withdrawn CA2398411A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
| CA002393002A Withdrawn CA2393002A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
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| CA002394841A Withdrawn CA2394841A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
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| CA002393912A Withdrawn CA2393912A1 (en) | 2000-01-31 | 2001-01-17 | Nucleic acids, proteins, and antibodies |
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| CA (37) | CA2392757A1 (en) |
| WO (48) | WO2001055319A2 (en) |
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