CA2395827A1 - Nucleic acids, proteins, and antibodies - Google Patents

Abstract

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Description

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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 Administrative Instructions Sections 801 to 806, and is hereby incorporated in its entirety.

[2] The Sequence Listing is provided as an electronic file (PJZ04 seqList.txt, 930,050 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 running the MS-Windows operating system by using the V viewer software, version 2000 (see World Wide Web URL: http://www.fileviewer.com).
Field of the Ifzvehtioya [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 methods useful for diagnosing, treating, preventing and/or prognosing disorders related to these novel polypeptides. The invention further rebates 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.
Background of the Invention [4] The human endocrine system is a collection of specialized organs and body tissues that produce, store, and secrete hormones. Endocrine organs are important in regulating the body's growth and development, controlling the function of various tissues, supporting pregnancy and other reproductive functions, and regulating metabolism.

[5] Endocrine organs are sometimes called ductless glands because they have no ducts connecting them to specific body parts. The hormones they secrete are released into the bloodstream. The primary glands that make up the human endocrine system are the hypothalamus, the pituitary gland, the thyroid gland, the parathyroid glands, the islets of the pancreas (which produce insulin), the adrenal glands, the ovaries and the testes. During pregnancy, the placenta also acts as an endocrine gland in addition to its other functions.
Hormones secreted by these glands control physical growth, sexual function, metabolism, and other functions.

[6] Most hormones are 'proteins composed of amino acid chains of varying length.
Others are steroids, fatty r substances derived from cholesterol. Very , small amounts of hormones can trigger very large responses in the body. Hormones bind to receptors on a cell's surface or inside a cell. The binding of a hormone to a receptor speeds up, slows down, or in some other way alters the cell's function. Ultimately, hormones control the function of entire organs. They control growth and development, reproduction, and sexual characteristics. They influence the way the body uses and stores energy. Hormones also control the volume of fluid and the levels of salt and sugar in the blood. . Some hormones affect only one or two organs, while others affect the whole body. For example, thyroid-stimulating hormone is produced in the pituitary gland. and affects only the thyroid gland. In contrast, thyroid hormone is produced in the thyroid gland, but it affects cells throughout the body..Insulin, produced by the islet cells of the pancreas, affects the metabolism of glucose, protein, and fat' - - -- throughout the body.

[7] Endocrine system or hormone imbalance disorders can be classified as disturbances of hormone production, or insensitivities of target tissues to hormonal effects.
The first type, called production disorders, are 'divided into hypofunction disorders , (insufficient activity) and hyperfunction disorders (excess activity).
Hypofunction disorders can have a variety of causes, including malformations in the endocrine gland itself. More commonly, hypofunction is caused by disease or injury. Hypofunction can also result, when target tissue is unable to respond to hormones. Hyperfunction can be caused by glandular tumors that secrete hormone without responding to feedback controls. In addition, some autoimmune conditions create antibodies that have the side effect of stimulating hormone production. Infection of an endocrine gland can have the same result. Many of the endocrine disorders that affect adults also affect children but may produce different symptoms.

[8] The pituitary gland, a pea-sized gland at the base of the brain, produces 'a number of hormones, such as corticotropin, thyroid-stimulating hormone, follicle-stimulating hormone, and luteinizing hormone, which control the function of other endocrine glands and stimulate these glands to produce other hormones.

[9] Luteinizing hormone (lutropin, LH), and follicle-stimulating hormone (follitropin, FSH), as well as the related placental hromone, chorionic gonadotropin (choriogonadotropin, CG), are referred to as gonadotropic hormones because of their actions on gonadal cells. The primary physiological effect of the gonadotropins is the promotion of gametogenesis and/or gonadal 'steroid production. Quantitative analyses of plasma LH and FSH
levels, as determined by radioimmunoassays and/or bioassays specific for each hormone, are useful in the diagnoses of several reproductive disorders. In both males and females, low or undetectable levels of LH and FSH are indicative of hypogonadotropic hypogonadism and suggest hypothalamic disease or pituitary failure.

[10] Growth hormone, another pituitary hormone, ensures growth during childhood. In pituitary dwarfism, the pituitary gland produces inadequate amounts of growth hormone, which cause abnormally slow growth and short stature with normal proportions.
Most short children, however, have normally functioning pituitary glands and are short because their growth spurt is late or their parents are relatively short. . .

[11] If the pituitary gland malfunctions before puberty, growth is delayed, sexual characteristics do not develop, and the thyroid and adrenal glands function inadequately.
After puberty; pituitary malfunction can cause decreased sex~drive, impotence, and shrinking of the testes. Inadequate pituitary, function is called hypopituitarism. In children, hypopituitarism may lie caused by a noncancerous pituitary tumor (craniopharyngioma), an injury, or an infection, or it may have no identifiable cause (idiopathic hypopituitarism).
Rarely, hypopituitarism occurs as part of Hand-Schiiller-Christian disease, which affects small areas of bone and lungs and the function of the pituitary gland.

[12] In panhypopituitarism, the production of all pituitary hormones decreases or stops.
This disorder can result when the entire pituitary gland is damaged.

[13] Sometimes only one piW itary hormone is missing. For example, if only luteinizing hormone is missing (isolated luteinizing hormone deficiency), the testes develop and produce sperm, because these functions are controlled by follicle-stimulating hormone, but the testes do not produce enough testosterone. Testosterone is the hormone that stimulates the development of male secondary sexual characteristics, such.as deepening of the voice, growth of facial hair, and maturation of the penis. Consequently, boys who have isolated luteinizing hormone deficiency do not develop these characteristics.
Abnormally long arms and legs may be another symptom of this deficiency.

[14] The thyroid gland produces thyroid hormone, which controls the speed at which the body's chemical functions proceed (metabolic rate). Thyroid hormones are the best known iodine-containing compounds with biological function. They have two functions in the human: as a crucial determinant of normal development (especially of the central nervous system) and in adults, to maintain metabolic homeostasis. Thyroid disorders are common with two general presentations: changes in the shape and/or size of the gland, or changes in secretion of hormones from the gland.

[15] Thyroid nodules and goiter, the most common endocrinopathies, can be caused by benign or malignant tumors. A goiter may exist if the thyroid gland is underactive (producing too little thyroid hormone) or overactive (producing too much). An enlarged thyroid gland present at birth is called congenital goiter. Some children have Pendred's syndrome, a hereditary condition that combines deaf mutism and congenital goiter.

[16] Hypothyroidism, or myXedema when severe, results when the thyroid gland cannot produce adequate amounts of thyroid hormone for the body's needs. Worldwide, hypothyroidism is most often the result of iodine deficiency. In nonendemic areas where iodine is sufficient, chronic autoimmune thyroiditis (Hashimoto's' thyroiditis) accounts for the majority of cases. Hypothyroidism present at birth is known as congenital hypothyroidism and is the most common .preventable cause of mental retardation in the world. Diagnosis and early intervention with thyroid hormone replacement prevent the development of cretinism. Hypothyroidism that begins in childhood (juvenile hypothyroidism) slows growth, sometimes resulting in disproportionately short limbs. Tooth development is delayed. Hypothyroidism. that begins in adolescence (adolescent hypothyroidism) is similar to that in adults, except that it may delay puberty. Symptoms include a hoarse voice, slowed speech, droopy eyelids, a puffy face, hair loss, dry skin, a slow pulse, and weight gain.

[17] Hyperthyroidism results from overactivity of the thyroid gland. In 'newborns, the most common cause of hyperthyroidism is neonatal Graves' disease, a potentially life-threatening illness that can occur in infants whose mothers have or have had Graves' disease.
Graves' disease, a form of hyperthyroidism, is an autoimmune disorder in which the body .
produces antibodies that timulate the thyroid gland. In pregnant women, these antibodies cross the placenta and stimulate the fetus' thyroid gland. Graves' disease in the mother may result in stillbirth, miscarriage, or premature birth. In a newborn, symptoms of an overactive thyroid gland--poor weight gain, fast heart rate, high blood pressure, nervousness or irritability, vomiting, and diarrhea--may start within several days after birth. The enlarged thyroid gland (goiter) can press on the windpipe and interfere with breathing.
The high thyroid hormone levels cause the fast heart rate, which can lead to heart failure. Bulging eyes, common in the adult foi-~n of the disorder, also occur in newborns. Graves' disease is potentially fatal if not recognized and treated appropriately.

[18] Islet cells of the pancreas synthesize and secrete insulin, the primary hormone responsible for controlling the uptake, utilization, arid storage of cellular nutrients.-One of the , most common diseases of the endocrine system is diabetes mellitus. Most patients can be classified as having either insulin-dependent diabetes mellitus (type I
diabetes) or non-.
,insulin-dependent diabetes.mellitus (type II diabetes). Virtually all forms of diabetes mellitus are due to a decrease in the circulating concentration of insulin and a decrease in the response of peripheral tissues to insulin. These,abnormalities lead to alterations in the metabolism of charbohydrates, lipids, ketones, and amino'~acids; the central feature of the syndrome is hyperglycemia.

[19] The two adrenal glands produce several types of hormones. The inner part (medulla). of each gland produces epinephrine and norepinephrine, which are responsible for the fight-or-flight reaction to danger and emotional stress. The outer part (adrenal cortex) produces two classes of steroids: the corticosteroids and the androgens. The actions of the . corticosteroids are generally described as glucocorticoids (carbohydrate metabolism-regulating) and mineralocorticoid (electrolyte balance-regulating). In humans, hydrocortisone (cortisol) is the main glucocorticoid, and aldosterone is the main mineralocorticoid. The effects of corticosteroids are numerous and diverse and include: alterations in carbohydrate, protein, and lipid metabolism; maintenance of fluid and electrolyte balance;
and preservation of normal function of the cardiovascular system, the immune system, the kidney, skeletal muscle, the endocrine system, and the nervous system.

[20] Under normal conditions, the hypothalamus produces corticotropin-releasing hormone (CRH) which regulates secretion of adrenocorticotropic hormone (ACTH) from the pituitary gland. ACTH stimulates the adrenal cortex to secrete glucocorticoids, mineralocorticoids and weak androgens such as . androstenedione and dehydroepiandrosterone, which can then be converted peripherally into more potent androgens. . . . ' [21] The testes have two main functions: synthesizing testosterone, the primary male sex hormone (androgen), and producing sperm. The testes may be underactive --a condition called male hypogonadism -- because the pituitary gland does not secrete the hormones that are needed to stimulate the testes or because there is a problem within the testes. When the testes are underactive, androgen production is deficient. Growth and sexual development may be retarded, sperm production is low, and the penis may be small. The symptoms vary depending on the age at which the androgen deficiency begins.

[22] Estrogens and progestins are endogenous hormones that produce numerous physiological actions. In women, these include, developmental effects, neuroendocrine actions involved in the control of ovulation, the cyclical preparation of the reproductive tract for fertilization and implantation, and major actions on mineral, charbohydrate, protein, and lipid metabolism. The ovaries are the principal source of circulating estrogen in premenopausal women. In men and postmenopausal women, the principal source of estrogen is adipose tissue stroma where estrone is synthesized from dehydroepiandrosterone, secreted by the adrenal cortex: Progesterone is secreted by the ovary mainly from the corpus luteum during the second half of the menstrual cycle. Progesterone is important for the maintenance of pregnancy. .

[23] Examples of proteins that may play a therapeutic and/or diagnostic role in diseases and/or disorders of the endocrine system andlor diseases andlor disorders related to hormone imbalance include, for example, adrenocorticotropic hormone, alpha-amylase, androgen recptor, calcitonin, estrogen receptor, growth hormone, insulin, luteinizing liormone,, thyroid hormone. Growth hormone is approved for replacement therapy in growth hormone deficient children. Recombinant somatropin (HUMATROPETM) is identical to the hormone isolated from human pitutaries. Corticosteroids (e.g., prednisone, fludrocortisone or cortisol) are useful in treatment of Addison's disease. Hypothyroidism is treated by replacing the deficient thyroid hormone, using one of several different oral preparations.
The preferred form is synthetic thyroid hormone, T4. Gonadotropins are increasingly being used for treatment of infertility. In males with hypogonadotropic hypogonadism, gonadotropin therapy can be used to achieve fertility. CG therapy can be used for cases of cryptorchidism (the failure of one or both testes to .descend into the scrotum) that are not a result of anatomical blockage. Antagonists of CG are being explored for use in long term contraception methods in females. Insulin is the mainstay for treatmen of virtually all type I
diabetes mellitus patients and many type II diabetes mellitus patients.
Accordingly, there exists a need to identify and utilize novel proteins associated with the endocrine system and/or hormones and/or hormone antagonists.

[24] The discovery ~of novel human endocrine system associated polynucleotides and the polypeptides encoded by them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, prevention and/or prognosis of diseases and/or disorders of the endocrine system or hormone imbalance, including, but not limited to reproductive disorders (e.g. male and female infertility, impotence, and premature labor), as well as disorders and/or cancers of endocrine tissues, including, but not limited to, the pancreas (e.g., diabetes mellitus, diabetes insipidus, and congenital pancreatic agenesis); the adrenal glands (e.g., Addison's Disease, Cushing's Syndrome, ~ and pheochromocytomas);
ovaries (e.g., ovarian cancer, polycystic ovary syndrome); the pituitary .gland (e.g., hyper-, hypopituitarism, pituitary dwarfism, and pituitary adenoma); the thyroid (e.g., hyper-, hypothyroidism, Plummer's Disease, and Grave's Disease); the parathyroid gland (e.g., hyper-, hypoparathyroidism); the hypothalamus, and testes (e.g., Klinefelter's Syndromes and testicular cancer).
Summary of the Invention [25] 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 Desc~iptioa Tables [26] 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 1A. 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 J
of SEQ ID NO:X that delineate the preferred open reading frame (ORF) that encodes the amino acid sequence shown imthe 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 gxeater 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 he 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 dGTP, 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 and/or 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 finding 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 Mendelian Inheritance_ in Man (Online Mendelian Inheritance in Man, OMIMTM. McI~usick-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.

[27] 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", 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 corresponding row of the table. The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" fox 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).

[28] 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 columw. 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. .

[29] 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 iri an identified BAC
clone). .

[30] 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 identifier code disclosed in Table 1A, Column $. _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 laclting 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 the tissue/cell source is a library, column 7 identifies the vector used to generate the library.

[31] 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.

[32] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application.

[33] Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.

[34] 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 [35] ~ The following definitions are provided to facilitate understanding of certain terms, used throughout this specification.

[36] 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 cbntained 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.

[37] 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 legion, 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).

[38] ~ 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, 200.1, having the depositor reference numbers TS-1, TS-2, AC-1, 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 L
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 1~O: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 10801 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. , [39] 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.Skb, 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 flanlcing gene(s).

[40] 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. "Stringent hybridization conditions"
refers to an overnight incubation at 42 degree C in a solution comprising 50% formainide, Sx SSC (750 mM NaCl, 75 mM trisodiurn citrate),'S0 mM sodium phosphate (pH 7.6), Sx 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.

[41] ~ 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 overnight 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% formamide, 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).

[42] 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 bf the hybridization conditions described above, due to problems with compatibility.

[43] Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract 'of a cDNA shown in the sequence 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).

[44] 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.

[45] The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptidebonds, 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 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)).

[46] "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.

[47] "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 fox a polypeptide.

[48] 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 endocrine-associated polypeptides (including fragments and variants) of the, invention for activity using assays as described in Examples 34, 52, and 57. .

[49] "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 exit, 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).

[50] 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.

~

c~

OA

U

."., do.~

o ~
~.

o ~

-N '"'" "~

~

-~' C 01 a1 N

r y O O ~ O
~ rx x .~ Zs p o ~, U d d ~~ x ~ UjN ,~N c~
A ~
o H i ~ co .
as ~
~

rn ,L, a o H:~' p 0 0 ~ op a x x . x x a a,, O N l~O N ~ M M

O O ~--~N ~,O ,-iN

G ~ _ a ~ ~
' _j, ~ ~ ; _j~J,~ - , U ~Uv~cn ~ -o'U

o C'7~ ~' E-"
-O O O ~ O o O O O ~ O
~ .~

~ O O
h N

O N ~O ~ o N c ~''pp ~OI~N d-- "~ ~ N ~1o n "'' d1 '' l 01 ,-aN N " ~ M ,-," ,--, y ~

~ c~ ~0~,~,~, bU~.,~ -A.0 , b~~D
W - b ~ ~

'' C7 a d d ~ E H d v~ c5d ri,~ d -~

~ , ~aA N N

p o O [-i d' h N O O O
O

p ~ N N ~ due'~ dN" d0"
p ' G> O i i i , , i ~ . ~ ,-~ ,...,M - N ,-~ N

m ~ ~ x -p a M ~ M ~ M M

. O ,-~ ~ . ~ ' ~n W

~z . 0 ~ pp M
~

~ A O 0 r,, N O

M N

O H..~ ~ N tn N ~ o o V

~ .

~ - M
~ ~ N ,~ p -~ . ~ x W fi, x ~

x U _ a z '~ _ N M

_ is ~ ~ p M N~ N (vjN cal,-i~ ,-i,-,i,-i,-i '-.i ~ ~

~"'C7 ~ O ~Nx ~''O ~ N ~ ~ ~ ~ ~ ~ N O M

d ~ ~ ,,~o ~ d a o 0 0 0 0 0 0 0 0 0 0 0 x ~ ~ N a x x a a ~ ~,~ ~ ~ x x.x ~ M

N ~-~ ~O
N ~ M ~.rjN c~jcV N ,-m'',-'~"~"~"' .

0oOv ~ ~ iV,=-il~~ ,.:-.~p~~jUj ~no0d'00 .

O p O p ~ O O ~ p ~ ~ ~ 0o ' N ~ ~ dM'~ ~
d Ncf' GYr~ O O ~ a O O O O O ueO O O O O O
O O

1 ~'f'~. . . rn!T!~T'!!'N~T'!!'i'~
1 1 1 !T!
.

M I~ M
d'V7 N M r . ~ ~ ~ , ' ~iO ~ ~ "
~ ~

_''~ ~ Q., ,~d ~' ~

, ~ ~ a~d P., U~ C~ ~ ~'0 0 0 ,~U..V~ C~7~ 0 0 ~ 4r O ~o ~ O ~ N ~ M

~ v7.- N
-aN d-ooA ,--~
,~

_~ ~ _ccs_c~ dD_~_cis~ ' _cC' ~
~

~ a. d d d C7d H a,d a, N
~

N ~ N' ~ N

n .
c N
.-v ~ M M ~

N ~ ~ ' O ~ ~ i M
N ~ N N ~

O

N

O ~,pN '"'~

C~,_, 00,~

N

.

x a x .

-i ,-i,-ir;,...;,~ .-i,--i,-a.-i,-a,-i,~ ,-r~ ,~ ,-i ,-.iM N N

op vp t~01N ui vpt~M a; M ~nN ~ ,-;t~ N O ON~O d-M '"'ippV7 M ' ~O00 M ~p ~pt N

O ~ \OV V1 \O n O O O p O O p O O O O O O O ~ O O d '~''O O
x x x x ~ ~ ~ a ~ ~ a a x x x x x , , ,~ x ~

-; ,-i,~,-;,-i,-i,.;,-i,-i,-i,-i~--~,-i,-i,-;,-i,-i~~ ~ ~!~iN

'-i O cn~ 01Q~ ~ ,=iv0\O Q\ O ~O,opd1 \DM ~ Ov N
~O d' ~O~pd'N ~Ol~O l~ oa~ 01 01 v' ~ N ~ d'~ '-' M O O O ~OV1 l~l~oo~ I~~'~ ~ON 7 t d" O O ~ O
O O O O O O O O O I~ N O LY O

O O O O O O p p O ., G7~

~ 0_O~ M M
~ 00 Ql . OM 00V1 O ~ ,-' ~ 0o ~' -~i'~
j, cC O !_~. , ~ ~ O

O O O O

M ~D00001~ ~ ~ 00 .

O b~A>, ~ O ~ ~ i.

a, d c7 d a;d a E-~

l0 M

M N

d' N d' r 01 ~p i O. N

~ C/7 -; 00 N CV N ,-i"~,-i'".',~~" ~ ,-i,-i,-i~;,-i,-i,-i,-ir.;,-.i~ ~ M N

p d: ,:.; M p CVV1O d'V'j'~~ ~ ,--i\D cVI~V~ N O O ~ Qv.
"" . ~ O V7V'1 ' ~
~

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l~

O O O O O O O O O O O O O O O O O ~ ~.]O
O

v~ x x ~ x x ~ ~ ,~a ~ ~ a .-7~-.a~ o i i i ~ ~ ; i i ~

N N N N "'-~,- r-,_;,- ,-"",;, ,- ,-;,-i.-ir.-,--r-i,-i~-~ cn cV

.-;O o001O ~ ~'O a1 M ~pood' ~yN a\ (~
I~

V~I~ M l~~r1l~ N ,.-~O oo~.,.~M ~O ~Dl~d1 d'V1~ ~ N 00~

O t~ d't~~ ~ d',-,O O O O N trll~d'.~ ~ l~ l~d' O O ~

x x x x x x x x x ~ ~ ''~
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a a ~ ~ .aa a ~
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O ,.r ~ ~,N

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N

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l~ O

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N
~ ,-;~ ,~ N N N N ~ ~ ,._.;,-i ,-i O ~.~ ~ ~ ~ ~ ~ O oo ~ ~ N
v'7 00~ Q O N d O d-l~ N N

' ~ ~D ~ ~ ~l~l '~~ ~, ~r~

~ N

,_;,-i,-; ~ ,-. N N N N r-i,-i~-i ~ ,-i ~ \p ~ ~

M O ~ 00 ~f' ~0'Cf01~ 01 O O ' ~

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o ~ o. x ~ ''~~ ~ ~ x a -i \O ~ ~ ,-i \p,-;N ~ ~.rjd'00 -' ~ ~

~Dt~ N d'O ' V~ ~ M d' ~ d- N d' N
' o M

N l~ M ood ~ ~ o I~ N N N N ~i cri~ ~ ,-~N N N cn ~ ~' ~' ~ ~ a ~

t~' ~ E-'d H ~ 'C.7'C~d ~ ~ ~ ~ ~ ~ ~ ~ G~
"' O O ~ O ~ O O ~ ~ (~'J'~''. O O O _ f~.'Q.O V O O O O O
O 'U .'"'.'' N M ~ OvO M ~ ~' O ~."'O d.~ ~''O O O +~O ~ .'~ oo O
N d- p~ +~ ~ .~ '' O

~ ,-i~ O '-,M ,-it~',~'N O N M d'~ Ir1~ N M M ~ M V1 ,~

N N M M ~ d'd'~n ~0'~~O N N N .N.-~,-.,N d'o0,-~N N N M

~ , ~ ~ ~ ~ i ~ , ~ i ~ i . ~ i ~ ~ ~ ~ , i ~
O s..~ a..~ ~ ccs~ ~ O O O ~ ~ ~ s. ~,c~O J,O ~ G.

n v _ _ ~ _ Q _ _ v _ _ ~ _ ~
7 ~ 7 7 ~ 7 ~

p.,e E-~~ .-.~C ~ C , A,~ ~ C U ~ ~ C d P U

~ ~ ~ ~

d. ~ d.

M N M N

d1 N . ~ N . . .

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M

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r~ ~ '-1 N

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_ ,-, ~ r-, r;

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pp~"'Q~1~ ~ ~ ~ ~ ~ ~ O ~ ~ N ~ o0 ~ ~ 0 O ~D~ I~l~I~M ~Oo V1 o O M ~' O N ~O d' .~
O O o [~
l~ M [~

O O O O O O O O O O O O O x O o ~ O O
x x x a ~ a a a x ~ ~ x x a , a a a N ,~

..-i,-i,.._;,~,-;r-.i'-i.-;.-iri -iri.-i~ ~ N --i~-i M SriO ~ W n ~i\Da ~ ooI~ M ~ _ rid' ~ O h '"'V1 ~D~pd't~ O N ~ ~ d'~ ~ ~ W M V1 ~O01~
N

o O N O V1~ ~Ol~ oou'1~p ~OI~t ~ O O ~ON O . o t~ O oo l~

x x a x ~ ,~,~ ~ a a x a , x '~ x x x x x ~
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d\~ , .,o0O

. 01 ~ N
i i a ~,~ ~
d ry ~ o o a o ~, ' +

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o M N

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M

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.

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O D1~pI~ v'iN d; O ~ ~ ~ N ~ o d j O ~ l~~ l~ , O pr t~

~ O d ~ O ~ d ~ ., d O O O O ~ O O p M

; ~ x a a ,a a x ~ ~~ M

~ N ~,~ N

N N -i~ ~ ,-i ~O'"''".',-i,-i ~_ M 0 ~ ~ O i M

~p~ ~ ~ ~ d 0 ~ N . ~ ~ ~ ~ ~
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~

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N

t~

c~ ' cC ' ~''~, C ~ ~ ~:>, _ , d , C7_ ' _ d ~' H ~ H C~

~ H E-~

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O

d' _ v7 r., N N p\.

~-0c~i ~ c~~ ~ ~ 'n j, j~ j, d d d d d x x H a a ~
E-, N O M
, N M N

M N M N

M M

O ~ O ~ V'1 ,-i ,-, ~ i i M M
'1 ue ~ ' M ~ ~ O' d ~ , ' N ~ N ~ N

-.~ ,-.w N
- -N
. O . .

N N

~ ~ r N d d' -p ," U1~O v1 ,.._, Q1 ~ d100 z ~ w ~ ' a ~ r~ .

.

x x x x ~

N M ~ ~.

a - ~ O ~ N

M ~,,~N N N .--iri ~ ,_;ri'-ir;,--~ri~-.i~ ~ ~ '~~ 01 d'a\ s0 d' ~ M ~ ,=i N d'~ ~ a1 ~ l~~' O ~ N O
p o p ~ ~ O O O p O O p O O O O O ~ O O
x a x a a x x x x x x ~ ,~ a ~ x a ~ ~'~ o d' M M N N ~ . ,-ir; ,-~rir;~ ~ ~ ~ .-i,-a M ~ .-;
,-i d'cn l~ M cVv'7~O O ~i'i ""-' p y dN'dN'OvW N oNo~ M O ~p ~DO ~O d-V'1 ~ NpI N
0oI~ ,-~~ N M d'O ,-,,--~,-~~ ~Oo0~O . I~ O O N O
7 ~ ~l o ~lx x x'p x x '~~..W-.a,-a~ l~~l ~.7 N d~ N
.-, oo d' , ~-,N N ' r1M d tiDN ~ ~~' , tn ~ v~, ' d v~H o . ~' ~ ~j o. o o ~, ~ O x O
d- O ~ O O
O N
vo'p N N ~ op~ hod-O O ~ ~ O ~ ~. j,~
H a,a, a a.~~ H t7a t7 d' N V1~D M

M N M M N

~ M ' O N
- ~"'i ~'~ 00 N M ~n N ' ~ d ~ ~
N .. N N
N

due'N. d'~ N

N
Cf'M ' N M M
O

x -~s o ,...;

o p d o x ~

~

. O i C.
c M ,-N p~ O

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_ _ _ ' n ' N i N N ~ ~

; ~ ; ir ,: o ~ ; ~ ..Wit' , o O w- y,O ~ ~ ~ ',~ ~ oo' ~ ~ ~ oo ~ ~ ~ P.o0 go t n a c a ~ ~ ~ ~ . a ~ ~,, ' ~ H
' ~, H ~, ;~.~ H

~ ~, d d ~ o o d H o U o o ~ U o o d 0 0 0 0 0 ~-'~ o ~ o 1-'~ o~ ~ o '~-'~ o~~ o .~"'O ',-,'~ '~"'~ ~ ~"'' O i-' O .'"' M M O O . 0l V ,-i V~~
~

N d' ,-~N ,- W~a\~D ,.-m-,v~ ,-.-,M ~ ~ ,-,,-~M a1~~,-,N
d' ,--~ -, ~,~ s~,~ ~ ~,~ ~ ~ ~ ~ ~.a.:cd ~ d4 ~ Q.,m a~ b0~ Q.w too ~ c~ ~ a c~ ~ a c~ a c~c~ ~ ~ ~ w ~ ~ ~ a a t~ ~ O~ O d- ~ N M

t~~ ~ oo v~ 00 00 00 M M M M N M M ' M

\D ~D --~ ~ d' d' dl V1 V1 , Q 00 00 V1 , M

~ i i i i ~ i 1f~ ~ ~--i O

M M ~ 00 00 tI~

N M M N

N M ~ d' ~ ~ ~
, v~ V1 V~ tn N V~ v~ V7 O

N ~ .

~ ~ ~ M

\D M M ~ O
O

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N

x ~, O O ~ '~ M

.,M
O Mp ,,:.; ,-;~ ~ ,-;~ O ~ N d'M cricnN
~ O N O M O . v>ooN ipp N
~ x x O O ~ O ~ O
~ M x x ~ ~"'~ x x a a M
'".',-i~--i'-! M d'Wit'criM
xx cn N

..
d1~ O ,-iv'i0 '-~~ co' cnO ~O
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a p o p p x ,,~O l~ o ~
f~ tf~ !'T'!rn. !"T~ tf~ rnO . . O
1 . 1 1 p 1 .

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M .. ,~l~ -'~ N

O 0 a ,-,,-1,~~ ~ ~ p~O N M
.

M by V1. ~ ~

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..at a H a, ~ o o ~ 0 ~ o o "

0 0 0 0 o ~ ~ o ~ 0 0 ~ ~, p . , .~ ~ ~ o d-d-o ' w '- o. ,--. o ~
M N N

O ..rr ~ d'~'~~ ~n ~ ~ ~ .-aN M
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~id w w d E -,~
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tnO i '"' ; O .

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y ~ , ~ N

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N V1 M ~ M

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N N

N

~

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x x x ~. _ ~
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N N N N ,-i ,~ ~ ~ ri ,_; ~ ,-i r-i ,-i ,~ ,-i r.; .-i ,-i ,-a ri ,~ ,-i Ti ~
r"' ,-i O ~p l~ oo ~ ~ N '~ M ,_; ~D d' d' oo ~ O oo N ~ V'i d' d1 t O p O O O O ~ O O ~ O O O ~ O ~. ~. ~ O O O 'O O O O O
x ,~ ,~ ~ x x .~ x x ,~ x x x ~ x x ~ a ~, ,~ a a a x x a N (wj N N N .--i r'' '-a ,~ ,_; ~ ~ .-i ,~ ri ~ ~-i ri ,.~ ,-i ~-.i ,-i ~ r-i ~ ,--.i N O ~ N O N '"'. ~ V7 \~ ~p d' d' ~j ~ ,-i \O, M V1 ~ ~-i d- ~O v1 a\ d- v1 Ov N op O N d' .~ ~ ~p ~O a1 ~ t~ O oo ~D ~ V1 O ,-, ~ l~ v1 O ~ M ~ N O d' O O' N ~..~ N d' c~ ~ I~ oo two ~ t~
O~OOO,.,O~,.,~O.,Op,.O~.,""~p'"'OT'.'OT"pT'.O,"O',~0,,000000~0 O ~ N , ~ I~ M
' , cVd' - .. ..

N O ,"2,' O ~ O O O

d a d d ~ . ~

r-, ~ ~;~ a; u;

~ ~ 0 ~ ~ ' h h n , n d o . o o ~ 0 - o o ~ x x d x ~ ~ a ~

N O ~ N
M O ~

O ooO M ~ -~~--y' M ~D
M ~O ~ O

J, ~ ~ ~ l'~~ _ _J,~C_cd, ~ ~ o0 v~
~ ~ ~ ~ d d ~' d o O O a c5c~ d d C7 0 0 ~ U ~ a~ ~ i O O d, d,~ O '~'O O O O O O ~ M O O O C/7O FJ~

'-' ,~ N ''' ~O pptnpa '"'M
N d' M ~
'' d't ~ M ~ N ,-,M n ~ ~ ,-r ~ p~,-.-yp[w , ~ ~ ~.,~.b13b_A ~..,, _cd_~,~ ~ ~ ~ _cd'_~ p O v_ W., O

cW~ H E-~d d v~ a,d C7 d ~ U~ d d C7 a.~a.x H a, M N, M N M N N

' p . ,~ 00 M l0 -- ~''~ ~ N

W p ~ i i ' i i ~ M N M
' ' r ~
, ~ M ~ d. ' ~ Ifs M ~ M ~ M M
, O
.

O d' d' ~ '~t' 00 ~O ~p ~ ~ o ~ N
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~ ~ , [51] 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.

[52] 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.

[53] 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.

[54] 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., 122.8 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.

[55] 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 tvvo 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 art could routinely use this information to identify normal and/or 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.

[56] . 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.

[57] 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 in 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.

[58] 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 HACCH94 49 847143 AL161458 467 ~ . 1-1140 HWHPO29 57 857383 AL049779 469 ' 1-210 _ _ . ,~ . 2599-2753 . 3548-3655 HADGE44 59 ~ 864257 AC015844 470 1-127 HADGE44 59 864257. AC015844 .472 1-1104 416=535 ' 1184-1340 ' ' ' HBGMZ39 82 947112 AC019337 484 ~ 1-1991 _ 4360-4485 HMAJI~74 99 966324 AC073670 491 1-1071 , r ' 2386-2508 HMTAV95' 104 . 614936 AL137000 494 1-510 [59~ 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", 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 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 x x w x x ___ - (60] _ Tabs 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 ~protei_n_~database (herein referred to as "NR"), or a database of protein families (herein .
referred to as "PFAM"), as described below.
[61] 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 HS,Ps. 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.
[62] 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., Durbiri et al., Biological sequence analysis: probabilistic models of proteins and nucleic 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.
[63] 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.
[64] 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 iri 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.
[65] 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).
[66] 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-1, 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. - .
[67] 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 For Recovery of Full Length Geyaes [68] Partial cDNA .clones can be made full-length by utilizing the rapid amplification of cDNA ends (R.ACE) procedure described in Frohman, M.A., et al., Proc.
Nat'1. Acad.

Sci. USA, 85:8998-9002 (1988). A cDNA clone missing either the S' 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 S' 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 S' 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 gel 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 Xhol or SaII, and ligated to a plasmid such as pBluescript SKIT
(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 S' ends are confirmed by comparing this sequence with the putatively identified homologue and ovexlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3' ends.
[69] Several quality-controlled kits are commercially available for purchase.
Similar reagents and methods to those above are supplied in kit form from GibcoBRL for both S' 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.
[70] An alternative to generating S' 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 Protocol For Generating The 5' or 3' End Sequences To Obtain Full Lehgth Getzes ' <
[71] Once a gene of interest is identified, several methods are available for the identification of the 5' or 3' portions. of the genie 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 equenced 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.

[72] The present invention also relates to vectors oi- 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-l, 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 deposits" 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.
[73] ~ 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. .
[74] ~ Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(IJ.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 Expxess vector. Both phagemfds may be transformed into E.
coli strain XL-1 Blue, also available from Stratagene.
139 .

[75] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 arid 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 DH10B, 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, Clark, J.
M., Nuc. Acids Res. 16: 9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (1991).
[76] - The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID NO:Y, andlor 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.
[77] Also provided in the present invention are allelic variants, orthologs, andlor 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 malting suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the~desired homologue.
[78] 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.
[79] The polypeptides may be iri 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.
[80] 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.
[81] 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 defined in column 6 of Table 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, and/or the cDNA contained in Clone ID NO:Z.
[82] 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 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 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.
(83] 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 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.
[84] Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, tvo, 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 1-B, 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.

[85] Moreover, representative examples of polynucleotides ofthe 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 axons 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 identifed 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.lB, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID
NO:A (se'e 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.
[86] ~ 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, cplumn 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.

[87] 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 sequences) 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.
[88] In further specific embodiments, polynucleotides of the invention comprise, or alternatively.consis~ 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.
[89] 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.
[90] In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3' 1'0 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. ' [91] 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 calumn 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 andlor nucleic acids, other polynucleot'ides 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, arid polypeptides are also encompassed by the invention., [92] 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 palypeptides 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. .
[93] 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.
[94] 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 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. , (95] ' In specific embodiments, polynucleotides of the invention comprise, or 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 lB,.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. .
[96] 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' 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 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.
[97] 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 ZS to the fnal 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 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 ari 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' tlwough .
these accessions are hereby incorporated by reference in their entirety.

SEQ

ID EST Disclaimer Clone NO: Contig ID

Range NO: Z X ID: of a Accession #'s ' Range of b HCFAT05 12 11557051 = 214315 - AA811374, AL133816, AI095381, 2157 M38217, M55515, L23499, M85217, X16001, M30312, M31744, AR050270, U38240, U38182, U96110, and U45979.

HMSKF 13 9793231 - 406 15 - AA148393.

HLHCT68 14 10774571 - 699 15 - 891198, H67940, AI205078, 713 891150, AW360811, D80391, D59.787, AW177440, _ D80022, C14389, D59859, D58283, C14331, D80164, D59619, D80210, D80240, AA305409, D80043, D80166, D80195, D59467, D51423, D51799, D59275, D80253, D59502, D80227, AI525909, D59927, .

AA305578, D80193, D80038, T03269, D51097, AW 179328, AW375406, AW378528, AW178906, AW378534, AW375405, AW179332, AW377672, T48593, AW179023, AW178905, AW178762, AW366296, AW378532, AW377676, AW352170, AI557644, A62300, A62298, AF058696, A84916, AB028859, AJ132110, AR018138, AR008278, Y17188, AR008443, A94995, D26022, D88547, X67155, A67220, Y12724, D89785, A78862, D34614, A25909, X82626, ' and AR008382.

HTLAQ18 15 12077911 - 798 15 - AA442624, AI382107, AW269016, 812 AI286158, _ AA719238, AI001168, AI280133, AW339870, AA009701, AA932363, and AL133054.

HEQAY32 16 11523321 - 108115 - AA459604, AA703415, AA703508, 1095 AI337830, AA993540, AI924185, AA398958, AA047107, AI581879, AA399541, AI479681, AA047244, AW007699, AI570681, AA826082, AA742413, AI278602, AA810070, AI346375, AI468327, AA766769, AI378094, AI721136, AA317334, AI269575, 850486, AA878088, H08229, T36249, AA878089, M78847, ' H08868, AA322162, AW138078, F04540, AI824732, AI383526, F04098, F02816, 850380, AA383971, W45039, AA713657, AW080157, H03560, AA582025, AI553645, AL036673, AI559752, AI640375, AI824576~

AI554386, AI287449, AI245008, AI871660, AI743505, AW007284, AA836253, AI590624, AI689157, AI469270, and AB033004.

HSSJM44 17 11786491 - 144315 - AA633868, AI953254, AA858030, 1457 AI377035, AA600028; AI948432, AI347033, AI819852, AI953812, AI274863, AA913660, AI221391, AA402834, AI682227, AA313382, AI393729, 843673, T75200, AA402685, H10173, 246198, AA594521, T34442, AW050919, AI150326, AA921803, AA775669, AI301221, AI394223, AI869224, 241820, AW074819, AI424149, AA357096, 813072, AA383450, _T07941, and AA368045.

_ HAHEF22 18 11750901 - 2189 15 - AA447298, AI979303, AI627654, 2203 AI762005, AI627344, AW138446, AI308065, AI636384, AI825690, AW025834, W67420, AI190111, AI921024, W67304, AW082705, AW204142, AA052901, AF086254, and AW771199.

HCEOR02 19 11796531 - 2105 15 - AW292158, AI933139, AA325666, AA317428, and AI887071.

HWLiJL2820 11788261 - 1655 15 - AW137674, AI800157, AI417831, AW 170428, AI887643, AA417227, AA765913, AI146511, AI811175, AI096783, AI683981, AA582233, AI749223, AI672942, AI523825, AA417373, W94311, AA488273, AI498202, AI458507, AA148361, H17540, AI347438, F24450, AA152345, AA70903I, , AI445386, AA312283, 239534, 844715, 861365, AI206473, F04042, AA740156, D20247, T85309, F33808, H06862, AA937493, T85523, AL135661, AI312428, AW162071, AI538716, AW301409, AI433157, AA640779, AL036980, AI800453, , AL079963, AW087445, AA572758, AL036146, AI800433, AI554245, AI539808, .

AW301505, AI280747, AL045500, AI611348, AW302992, AI684265, AW 169653, AI334884, AI340582, AI475394, AI564723, AL036396, AW074993, AI340627, AI349933, AI349614, AW071417, AI348897, AI610362, AL119791, AI318280, AI269862, AI312152, AI345735, AI349937, AI500077, AI702406, AA225339, AI829327, AA938383, AI567612, AI497733, AI866798, AI567351, AW268253, AA508692, AI612885, AW022682, , AW075084, AW020693, AW089572, AI590118, AI247193, AI307708, AW071349, AW023590, AI569579, AI340519, AI307520, AL038778, AL121365, AW238730, AI621209, - AL040243, AI648684, AI627988, AW150578, AW020561, AISOI325, AI251221, AI537273, AI349645, AI440426, AL036802, AL045266, AW 148320, AW 169527, AI349004, ' AI420521., AI680388, AI564247, AI282281, AW075351, AI925156, AI7~8437, AI869367, AI784252, AI868831, AI608936, AW075413, AI570781, AW193134, AI874166, AA613907, AW074869, AI274728, AI537677, AW129916, AI349957, AI4397I7, AI433384, AI890806, AW302988, AI687728, AI955906, . AA528822, AW268220, AI431424, AI950664, AL039086, AI343112, AI521012, AW105601, AL036673, AI306613, AI433976;
AL044207, AW301300, AI281779, AI349598, AI434468, ' AI608667, AL036664, AI589267, AW068845, AW403717, AW302965, AI344928, AI468872, AI57I909, AI922075, AWI49227, . ' AI569583, AI920968, AI572787, AI270707, AI818683, AI499393, AI500659, AI284131, AI499285, AI349256, AI345347~
AI597918, --- AI313320, AI343030, AI252023, AI434020, AI633419, AI500706, AI445237, AI862144, AI312146, AI312339, AI873731, AI345258, AI434256, AI30940I, AI690835, AI564719, AL120854, AI934036, AI469532, AI475371, AW103371, AA012905, AW071380, AI634224, AI340603, AI580190, AL040169, AA427700, AA833760, AI620284, AI310925, AI524671, AI312399, AI632033, AI274508, AI383919, AI311159, AI679758, AI687415,.

AW087838, AI921176, AI620866, AI446606, AI334450, AI817244, AI683099, AI689420, ' ' AI362637, AI631107, AL036631, AI696626, AI345674, AI906328, AI349226, AW268072, AW080402, AI932794, AI683684, AI207510, AI696846, AF044574, I48979, AL050138, AF118064, AF113690, AL117460, AF113013, 148978, AF090934, AF118070, AF113694, AF090943, AL050108, AF090900, AL050393,.

AL117457, AJ242859, AL050146, A08916, AF090896, AL133606, S78214, I89947, - - - AL133640, A08913, AF090903, ~AL133016, ' ~ AF078844, A08910, Y16645, I89931, AF125948, AL049452, I49625, AL117435, AL049938, AJ238278, AL137550, AL122050, AF090901, AL049464, AL110196, U00763, U42766, X63574, U35846, AF113691, AL080060, Y11587, Y11254, AF146568, AF017437, A08909, AL110221, AL122093, AF118094, A65341, AF125949, AL110225, AL133S65, AF091084, AF113689, AFI13677, A93016, AF111851,103321, X72889, L31396, AF113699, A77033, A77035, AL137459, AF177401, S68736, X96540;
AR011880, X82434, A58524, A58523, AL133557, L31397, AL049466, AL133560, AL133075, AF104032, AL080124, AL137527, A12297, ALI17583, AL0501I6; ABOI9565, ~ A03736, ' AL137557, AL049314, AL1I7585, E07108, AL050149, AL117394, AL122123, AL133093, AL050277, AL133080, X70685, AR059958, X84990, AF113676, AF158248, AL049300, AL137463, AF113019, E03348, AL050024, AL049382, AL137271, E02349, 282022, AF183393', AF079765, AL122121, AL137560, AL122098, AF017152, AJ000937, U80742, AL080137, AF106862, AL122110, E07361, M30514, AL049430, U91329, AF097996, AL137538, AL096744, X93495, I33392, AL133113, U72620, AL080127, AL137648, X65873; AL049283, AL137521, I09360, U67958, AF061943, AL137283, AF087943, AL110197, AF026124, A08912, AF067728, AL080159, 561953, AJ012755, E08263, E08264, X98834, I42402, AL133072, AL122049, E15569, AF026816, Y09972, I26207, Y14314, I17767, AR038969, A93350, AF119337, AL050172, AL110280, AL137556, 100734, AL1330I4, U49908, E00617, E00717, E00778, A90832, US8996, AL133098, AL133077, Y07905, AR000496, U39656, AF111112, AF153205, AF185576,.AF079763, AL133568, E05822, AF000145, A07647, U68387, AF057300, AF057299, AL133104, AF003737, 272491, U96683, AL137526, AL137523, AL137533, X53587, AC004093, AR038854, AL080074, AL117440, AF111849, A45787, AL137476, L19437, AF008439, AF100931, AF162270, U78525, AL137292, AF061573, AL133067, E04233, AF106827, AF061795, AF151685, AL137478, AL122111, _ ' . X92070, 237987, AL137300, AL133031, I96214, L13297, L3011'7, X87582,,E08631, X83508, A08911, AR013797, AJ006417, AF081197, AL050092, AL137480, AF095901, AF132676, AF061836, AF000301, AL117432, E02221, and AL080158.

HLMD095 21 9283441 - 469 15 - AC020641.

HBGQN46 22 10899091 - 1254 15 - AW297831, AI813391, AA479990, 1268 AI819671, AW293680, AI473367, AA478944, AI184880, AI086741, AI081183, AI214276, AA742259, ' AA237023, AA251267, AA836754, 855060, AI285889, AI589267, AF038458, and AI990375.

HRODF07 23 11522571 - 1159 15 - AW238558, AW238227, AW084017, 1.173 AI278521, AW265370, ~W440242, AW238497; AW238292, AW238306, AW082445, AI355144, AW082458, . AW238597, AW079466, AW366290, AW381175, AW366622, AJ243667, and AF077374.

HNHCI32 24 8616731 - 586 15 - AF112462, and AR035954.

HPTVL90 25 11472521 - 1157 15 - AI742586, AI472051, AA994212, 1171 AI823636, AI738645, AI808204, AI742084, AI458889, AI694439, AI570505, AI005161, AI126407, AW166260, AI819324, AI689840, AI564776, AI802140, AA044826, AI379936, AW418896, AI01$575, AI708063, AA044874, N33369, AI798664, AA983545, t1A996109, AI268684, AI948596, AI051014:, AI498702, AW295897, AI625938, AI948603, H92520, AW139306, AA317357, AI521441, AI021975, AW135452, AW242712, AI264270, AI373261, AI244808, H77889, AI985535, 800296, AA677354, T99948, N35293, AA938619, H58937, AA917514, AI393979, AW044210, AI768427, AA779970, W25595, AW072204, AW020604_, AA883635, AI470848, AW351592, H92521, AI208112, AA335622, AI955919, AI308950, AI919367, H58984, AF126781, AI085468, and AW614124.

HNGFK28 27 11033261 - 1074 15 - AW177440, T03269, C14389, ' 1088 AW178893, D58283, D59859, D80022, C14331, D80166, AW179328, D80195, D80193, D59927, , D59467, D51423, D59619, D80210, D51799, ' D80391, D80164,.D59275, D80240, D80253, D80043, D59787, D80227, D59502, D8~1030, D59610, D80212, D80196, D80188, D80219, C15076, D80269, D57483, D80038, D80366, C14429, AA305409, AW378532, D50979, '~ D59889, D50995, D80241, D80378, ' AW178775, D80024, AW360811, AW178762, D51022, D80045, AW 177501, AW352117, D80251, AW177511, AW377671, C14014, AW375405, D51060, AW1764fr7, AW352158, AA305578, D81026, D80248, C75259, AW179332, AW366296, AW360844, AW360817, AW375406, AW378534, AI905856, AW377672, AW 179023, _ - AW178905, AA514188, D80522, AW178906, AA514186, D80133, AW360841, AW352171,, AW377676, AW352170, AW 177731, AW178907, AW179019, AW179024, D51097, AW179329, AW177505, AW179020, D58253, AW 178909, AW 177456, AW378540, AW178980, AW177733, AW378528, AW 178908, AW 178754, AW
179018, . ~ AW360834, D80268, D80134, AW 179004, C05695, AW1789.14, AW378525, F13647, D80302, D80439, AA285331, D80247, AW178911, T48593, AW352174, AW352163, AW178774, AW378543, AW177728, C14227;

AW 178983, D80132, D81111, AW352120, D80064, D51103, AW178781, D80157, AW367950, D51759, AW177723, T11417, AW178986, AW367967, DS8246, C06015, AW378533, AW177734, D45260, AI557751, D58101, D59503, C03092, H67854, AW378542, H67866, AW378539, AA8b9122, AI535850, D80314, AI525923, AI557774, AW360855, AI525917, T03116, D59317, D80258, D80014, AA514184, C14975, AI525920, AI525913, AI535959, AI535686, AI525227, AI525925, Y17188, A78862, D34614, A84916, A67220, D89785, A62300, A62298, D26022, AJ132110, X67155, . ~ - A25909, Y12724, D88547, AR018138, X82626, AF058696, AR008278, AR025207, AB028859, A94995, AR008443, I50126;

I50132, I50128, I50133, AB012117, , AR066488, A82595, AR016514, AR060138, , A45456, A26615, AR052274, Y09669, AR060385, A85396, AR066482, A44171, AB002449, AR066487, X68127, A43192, A43.190, AR038669, A30438, A85477, D50010, I19525, A86792, X93549, Y17187, AR066490, I18367, U87250, D88507, A63261, A70867, AR008408, AR062872, D13509, AR060133, AR016691, AR016690, U46128, A64136, A68321, I14842, AR054175, AF135125, AF'123263, AR050680, U79457, X93535, U87247, and AR008382.

HSHAX84 29 604956 1 - 4$8 15 - AC020850, AC034242, and AC022123.

HWHHWS 31 11124451 - 141815 - AW192697, AI336274, AI041819, ~ 1432 AA808430, 4 ~-~ ---- AA780044, AI740785, AI073467, - - AA883903, AI022809, AW157214, AI871953, AIS21233, AW009412, AA554447, AA443442, AA689601, AA8867.25, AIS89839, AA863294, AI034240, AA595998, AI669406, AA932317, AI986294, AW 152255, AI925407, AA806498, AI887353, AA978039, AI338458, AI042442, AA443308, AI537869, AA890338, AA992867, ' . AI139401, AI085301, AI263340, AA917926, N58734, AI348041, AA854105, AI742678, AI080542, AA011230, AA937977, AA582S60, 897517, AA047368, AA844312, W21953, AA838761, N65985, N38922, HS9433, W40446, AI813359, AA022870, T58241, N58822, AI929215, AI025841, AI050672, AI810556, AA450114, AW 172366, AA724684, AW119135, W39201, H70636, AI127132, AI140234, N72645, H66260, H70698, AI141369, W80431, AI869659, AA844036, AI184041, 897516, AA026877, AA535528, AI8235~8, 851009, AA724350, N93947, AI468130, AI193365, AW140087, AI942223, W80S61, N80674, AW196307, AA995087; AA598544, AI500188;
AA022869, AA890043, H9I080, AA910599, AW262527, AA229969, AA057027, N75027, AW0062I0, W05627, C21470, AA055012, H69720, AA999870, N90260, AA890022, N94250, AA137079, AA036917, H47150, AA846564, AA463838, AA026818, AI277999, W07723, and AA460271.

HTXGF27 32 12259901 - 1309 15 - AI935291, AI565606, AW055263, 1323 AA827998, AA134925, 871442, W73084, W37623, AI376157, AW071733, AA760869, _ AW084636, AW084135, W37624, N40260, AA621956, AI261507, AI024926, AA576858;

AA782442, AI753564, W73188, AA457140, W37568, AI362706, AI088401, N80584, AA100989, AI870558, AA552108, AA464487, AA577315, AA135041, H99262, N42314, AA099421, AA917378, AI955891, AW009052, AI865605, AI811602, W37443, AA506577, N28234, AI564918, W00732;

H56180, AI784357, AW407317, AI571677, W07706, AA297089, AI018764, T64405, . . 871095, F19530, AA628651, AA297614, H56396, AA588512, T64219, AI29I707, 839750, AA622681, 836668, AI564962, AA814874, AI868306, AW247018, C00495, AA082510, AI535639, AI557241, AI541346, AL117567, AF151851, AB002453, and AW472879.

HCUGR38 33 1093062'1 - 575 15 -HCUGN19 34 10949231 - 876 15 - AI990886, AI660160, AA937166, 890 AI871379, AI740633, AI927423, AI867197, AI380503, AI744631, AI4S7940, AI300806, AI588959, AI928256, AW237763, AI765531, AI830241, AI800Z08, AA535256, AI969915, AW301155, ' . AW316942, AW071230, AI762594, AW300271, AW058189, H44971, AI829069, 1s4 AA828477, AA804639, AA953355, 883518, 883503, AW138552, AI870155, AA204689, AA505286, H01355, H01309, AA767789, AI092045, 832005, 865688, H28671, AW196042, AA306658, H45024, AA207090, AA135022, 831122, 865687, and AW137350.

HPTGB43 35 7264601 - 492 15 - AA573537, AI160878, AA885566, 506 AA861893, AA433946, AA568688, AA811995, AA442766, AA838602, AW168298, AI283278, AI015935, AA782093, AI472891, AI199547, AA573777, AI085835, AI669114, AI690776, T61807, AA652483, AA086353, AW204782, AI004311, AI566973, AA501347, AI281530, AA876623, AI589871, AW138834, AW245578, AA613669, AA714315, AA8133,27, AA507560, N52392, AA492250, AI221056, AA446582,'AI083879, AI304532, AI304454, AA531050, AA720002, AI304455, AA720021, N54353, AA418354, AI937637, . AI573258, AI382182, AI557530, AI244679, AI557529, AA305352, N30214, H85048, AA918480, AA230200, AI571328, AI859175, AI264256, F21642; H65466, AI99111.2, H91692, C00330, T73417, N63050, H93999, ' AW300104, AI885837, AA134203, T71348, AI808444, N32117, AI985397, AA855127, AA431169, T55136, AA291581, 230219, T73332, H72870, H77954, N63300, AI141454, AA293323, AA432189, AW150864;
T61353, T73347, AA229346, T72864, N88648, ' AI251780, AA641099, AA625514,.850529, AW245568, H78357, T98646, F 15963, AA430.753, H72469, AA313563, H85040, AA446709, AA938645, AA522761, AF113123, and AR009500.

HEON059 36 12183431 - 1160 15 - AA789205, AI077,497, AI670821, 1174 W37915, .

AW1I7287, AW369323, AA608906, AA827227, AA600253, AI160796, AA724269, AA278680, AL138197, AA292636, AI990171, AA421286, AA844108, W37874, AA292537, AA768446, AA917528, AA278688, AA625767, AI972106, AA298808, AA382777, AW316905, AA625768, andAF117210.

HHAMF38 37 11998451 - 2129 15 - AW002967, AW027779, AI803049, AW194946, AW182921,.AI983694, AA978351, AI219858, AI658726, AI669330, AW418951, AI984802, AA128041, AI942355,.

AI038656, AI697774, AI694647, T74734, AI830735, AI922255, AI658577, AW129197, M77987, AI918192, T48344, AA128086, AA970231, T50347, AA702280, AI191395, AA912726, AI364931, AW450850, D45721, AI570573, AI224891, AA836277, AI459038, AI433889, AF211943, AF187014, U13395, AF187015, AC009044, and AF212843.

HFIJC31 38 11540631 - 1143 15 - AW369756, AI767361, AW062278, 1157 .

AA885328, AA452837, AA452978, AW016065, AI005282, AI263850, AA514551, N79858, N62955, AI674818, and AI184184.

HNTBF75 39 8367011 - 402 15 - AI659901, AF078850, and U81186.

HSHIA89 40 11484111 - 911 15 - AA314747, AW204099, AW301249, AA249613, AI261562, AA775742, AA609749, AI446434, and J02649:

HNGLG06 44 11623991 - 593 15 - AA654505.

HHAME78 48 11806681 - 545 15 - AA429728, H84670, AW440575, 559 W00611, T60510, AA430753, AA625514, AA446709, W00555, AW245568, H91789, H64869, H78356, H72469, AA313563, H95032, N56787, N78252, H73548, AI885837, 220777, _ 886214, T72288, 850625, AI991112, AI573258, T72293, AI382182, N40867, AI589871, T71510, AA134.202, AA305352, N63300, N72798, AW168298, H47484, _ ~ AW245578, N30214, T98698, AI244679, N63050, H84633, AA714315, AA507560, AA230291, T73332, AA442766, AA613669, N76319, AR009500, AF113123, and A74991.

HACCH94 49 8471431 - 1399 15 - AI093369, AW292321, AA972431, 1413 N40174, AA746376, AA130392, AA286750, AA287684, 871586, 871568, 871587, H03136, H03946, 871567, AI471079, H97311, AA365025, AF039686, AF118670, AR034800, AF081916, AL161458, and AL161458.

HTEED80 50 11659941 - 884 15 = AA465429, AA203572, AA192438, 898 AI479983, AA829419, W87655, AA836236, AW290970, AI796504, AA904433, AA368409, AW189971, AA601527, AA688079, F36549, AA320120, W87656, AI540082, AA393948, F30605, AA507269, AA465358, AI476046, AI500061, AI697304, AW002838, AI564160, AI801793, AI858874, AI801605, AI491775, AI679916, AW161579, AW118508, AW162189, AI521040, AI800384, AI564765, AI697324, AI658566, AI379711, AI623736, AI5.82932, AW058243, AI569328, AI597805, AI521560, AW081343, AI567128~
AI758528, AI684145, AI254226, AI696626, AI469516, AI167353, AI915291, AW085373, AI924971, AI470648, AI689404, AI452857, AW088899, AI274527, AW059713, AI572418, AI963193, AW 118518, AI573060, AI368579, AI863047, .

AI624953, AI362522, AW150453, AI287489, AI452707, AA830821, AW076093, AI633125, AI554427, AI089782, AI698391, AW129722, AI815232, AI826225, AI582871, AI560545, AI537303, AI915295, AI889189, AW078767, AI922215, AI431975, AI886753, AI887268, AI609589, AI811192, AI630876, W74529, AI634219, AL046931, AA555145, AI249946, AA807088, AI536638, AI633427, AI590048, .

AW087824, AI567360, AW193635, AW051088, AI865942, AI434432, AI656270, AI383804, AI801232, AI611743, AI699020, AI567582, AI926041, AI690472, AI367705, AI280521, AW022102, AI679550, AI630749, AI800380, AI828731, AW161098, AI918554, AW265004, AI590630, AI677797, AI636456, AI572892, AI608807, AI689614, AI520931, AW080327, AW024374, AW149876, AI655932, AA617645, AI590999, AI801325, AI680162, AW021373, AI91.2510, AI801152, AI681985, AI621086, AI521538, AI654329, AW083804, AI819976, AI758272, AI500662, AI951222, AW050522, AI538764, AI804983, . AI589993, AW079334, AI365256, AI050666, AW085786, AI307736, AI886055, AI564500, N57346, AI799344, AI537643, AI472536, AI749373, AI684021, AW029401, AI701074, AW051048, AI280637, AI653782, AW058207, AI632997, AI096771, AI926367, AI432218, AI868204, AI244380, AI469532, - AW251034, AL036631, AI805769, AI434242, AI491897, F37364, AW078689, N33175, AI469738, AI446124, AI679174, AI249877, AI963216, AA568405, AI799235, AI866786, AW 161202, AI539771, AI932638, AI925196, AI635942, AI559737, AI609127, N74355, AI680066, AL046449, AW 152182, AI680194, AI699154, AW074459, AW118398, . , AW084097, AI918408, AI818977, AA514684, AI952542, AI610307, AI611354, AW235482, AI435641, AI620003, AI863014, AI583578, AI005511, AI473536, AI620714, AI758613, AI872914, AW079159, AI340603, AI923989, AI698265, AI285735, AW087862, AW080079, AI673267, AI801213, AI345745, AI612723, AI373617, I89947, AR013797, X82434, AB019565, A08910, A08909, 148978, AF113019, AF125949, A08913, AL137459, AF120268, 577771, AF106862, X72387, A93350, A77033, A77035, AL137271, Y16258, Y16257, E02756, Y16256, AL049382, AF019298, Y14314, AL122045, AF026008, U35846, L78810, A18777, A65341, AL133077, AL122110, X59414, U75370, A03736, AJ006417, AL132985, AF026816, I89931, Y16645, X56039, AJ000937, AF183393; AF185614, I49625, AF111112, AL050277, AL096744, AL096751, AL133629, Y10823, AL122050, AF087943, . AL137560, AL133640, AF155119, X92070, AF026124, AF177401, AL137488, AL117435, AL137550, AL137539, 561953, AL049465, Y10655, AL133104, AF113690, AL049324, ' AL137286, U67958, AL133070, AF141289, U00763, S68736, AL137527, A08907, AL110171, AF119337, AL133069, AF114170, - AL137712, A08912, AL137555, AL133072, AL122121, A08911, AR050959, U80742, AF067420, Z82022~ AL137648, 237987, AL133568, A08916, X98'834, AL133075, AR038854, S78214, A08908, AL137557, I33392, AL117626, Y10080, AL133081, AL049460, AF090903, AL050138, AB029065, S76508, AL110280, I89934, AL035458, AF012536, AF117959, AF113677, I48979, AF118094, AF067728, AL080159, X62580, AL133645, AL137536, AF162270, AF047443, AL110226, AL117394, AL122123, U77594, A93016, A90832, AF090934, A57389, D83989, L10353, AL050024, AL110196, AL137523, AL122111, AL080110, E12580, AL137538, AL117585, AF017152, AL080127, I26207, U87620, I30339,130334, AL133016, Y08616, AL133014, Y14634, AL049426, AF085809, AF079765, AJ012582, AL133560, AF097996, AL049464, E12579, AB028451, X84990, AF090900, X86693, AL080137, AF090901, AR068182, AL137480, AF102578, AL137659, AJ012755,,AC002382, AL137300, I00734, L13297, AL137276;
AF067790, AJ001838, AL137556, AF038847, AB025103, L30117, AF109155, J05032, U30290, L31396, E00617, E00717, E00778, AL137660, AL137254, AL137521, L31397, AF104032, AC006112, AC007298, E15569, AL080124, AR054987, AL137429, AF113689, AF118064, E05822, AL137640, AL110197, U96683, AL110221, AF090886, AL117457, AL050116, AR038969, AF158248, AF146568, AL137273, AL049452, A58524, A58523,189944, AF113013, E15582, AF078844, AF207750, AF082526, I09360, AL049938, AL133080, AR000496, X83544, U39656, 272491, AL117583, I46765, M86826, AF017790, AL122098, AB007812, AL117649, Y09972, X57084; AL137656, AL133113, I03321, ' AF057300, and AF057299.

HDPND85 51 12148441 - 143115 - AW001572, W58459, AI125225, 1445 AI803690, AI673434, AW050614, AW131630, . AW050632, AI378033, AW452081, AA908673, AI129923, AI214596, AA448177, .

W58347, W58514, AA809706, AA575928, AI200014, AI620164, AI221658, W58472, AW371786, AW371789, AA573435, AW371791, AA723851, AI090712, AI695504, D60924, AW39237,1, D80993, D80994, AA877467, T49181, AI356759, AI361294, AI422493, AW382253,~AA055320, AI699226, T49180, AW351512, T66827, AA864184, AA865889, AA906223, and AC003042.

HP CB73 52 115617,11 - 251815 - AA056098, and M91498.

HNHNR36 53 11263111 - 400 15 - AA834169.

. 702 HAPQS74 55 11612591 - 515715 - AW070235, AA481601, AW237310, AI004312, AA287561, AI401739, AA889952, AI743497, AI905471, AI627341, AA918798, AI198899, AI807434, AW004921, AA287878, AA040134, AA291780, AA040087, AI090624, AI700634, AA725407, N46832, AW105397, H98067, AI278308, AI935486, AI082491, AI288962, N27578, AA856998, AA282679, AI288866, AW192623, AI300312, AA125850, AA125849, AI628481, AA614418, AA587213, AI080488, N20307, N79110, AA158618, AI625517, AA781779, AA568542, AA746161, AA861320, AA461403, H29530, AA969724, AW293072, N28375, N62348, AA456558, AA705967, AA860980, N78359, AA766163, - AI493380, AA911302, AA041513, AW341264, AI207883, AA952942, AA854582, AI620161, AA412433,.AA687380, AI277221, AA046252, AA307431, 851543, AA041460, 851544, AA291710, 863409, AA412432, AI369523, AW027768, 813217, AA282568, H83725, AA046371, AA904380, AA836571, AA341565, AA507137, AA737593, AA122226, AA884022, AI970236, 'AA978278, AA331618, AW087431;

AA844030, AI150822, AA252450, N62593, 831018, D60624, 840301, 830963, AA121104, AA776175, AI762334, AA918838, C21414, AF136450, and AW474 364.

HKABI68 56 12253041 - 1122 15 - _ 1136 AI199122, AI936969, AI199133, AA872611, AI039903, AI612797, AW249217, AI889785, AA570270, AA640054, AW264062, AA887493, AI379630, AA161210, . AW054653, AA863073, AI039925, AA425116, AA858167, AA723658, AW404684, AA894363, AW246763, AA812532, AW272085, AA534535, AW006467, AA769574, AI937269, AW009237, AI342862, AA724160, AA724161, AI672916, AA987192, AA076633, AI802598, AA534492, AA315785, AA666289, AA335559, AI537849, AA115832, AI961872, AA918899, N94606, AA429231, AI206495, AW068540, AI217629, AI990066, AI080701, AI264200, AI266063, AI085598, AI797655, AA280727, AA283026, AI783775, AA551979, T08490, AA282973, W23813, 238447, AI908114, AA258858, N75525, 242191, AI927900, AW062261, AA463743, 849551, AA336855; D20630, and AA130867.

HWHP029 57 8573831 - 657 15 - AL049779, and AL049779.

HTFOB57 58 11888591 - 1290 15 - U66685, AI126698, AI992159, 1304 AA604377, AI355573, AA523723~ AA917648, AI589741, AI684462, AA045146, AA085954, AA085953, AA812594, AI80'0531, AA281007, AA719856, AA705854, AI620440, AA977873, AA428564, W47347, H59649, AI342347, AI028246, AA515298, AA045255, 880339, AA535613, AI129.155, AA536067, AA862715, AW006676, AI864034, AI905741, AW404049, AA552289, AI184189, AI346779, AA309643, AA429520, AA846640, F24595, AA747728, H02423, AW088429, H11197, w ~ H59609, AA628329, H02321, AA335316, AI014512, W4745'1, AA991326, AA192275, AA335655, AA195630, AW002298, AA437120, 807941, AW069632, AI271~814, .

T12458, AA811669, AA552246, AA281173, 806392, F25492, AA775242, AA931765, 806391, F19391, T59746, 831819, AA732918, T85620, T30711, AW376621, AA317259, 831832, W81195, AA969767, AI245151, AW129052, T93959, T16101, AA926731, AI189541, AA923760, AA195609, 829713, AI474712, W81263, AA317516, .

F34410, T59628, AI073952, AW 193134, AW082623, AI583611, AI050666, AI624529, AA464646, AI608807, AI539723, AI582912, AI952542, AA835966, AI697324, AI921438, AI678411, AI887163, AI277008, AW168791, AI783861, AI583054, AI890057, AI536563, AI301046, AI926593, AI567637, AW265004, AI677797, AI819958, AI624604, AW 105429, AI355613, AI689420, AI679550, AI419374, AI718161, AW168485, AI561199, AI355277, ' AI244380, AW409775, F37006, AL038575, AW074142, AI950664, AI473652, AI791396;

AI669639, AW2?7066, AI932638, AW409772, AI805688, AW243705, AI824361, AI521100, AW152469, AI805638, AI912288, AI572892, AA830821, AW183621, AI888671, AW021091, AI825964, AI371228, AW263804, AI288285, AI890806, AW131999, AW088899, AW129271, AI586931, AW078767, AI567582, AI589428, AI634450, AI677983, AI285439, AA908294, AW192052, AI872072, AI480118, AW058341, AL048312, AI499279, AI636719, AI251876, AI918554, AW 151847, AI918637, AI623363, AI886055, AI478282, AI597805, AW083804, AI884469, AI886022, AI366549, AW089258, AI955906, AI636788, AI566630, AI686597, AW168650, AA494167, AI568138, AW073898, AW172981, AI499986, , AI590686, AW087938, AI951222, AW079334, AI570966, AI863321, AI701897, AW 130711, AI620287, AL039086, AW081268, AW162194, AW082532, AI491710, AI610671, AI859644, AI890574, ' AI612747, AL036638, AI431962, AI471429, ,_ AI919147, AI8681.63, AW103628, AI568886, AI539089, AI758748, AI680504, AW088605, AI623682, AW167924, AI500504, AI345688, AI804586, AA760851, AI305157, AI611743, AI151016, AI915295, AI431979, AI266558, AW007955, AI470293, AI539780, N74355, AF213381, Y16258, Y16257, E02756, Y16256, Y08769, U90884, U77594, AF008439, AL117578, AL133014, AL122098, 577771, AF031147, AJ238278;
AF026124, .

AF090900, AF137367, AL137711, 579832, AL110222, AF022363, A18777, X66862, J05032, I89947, I48978, AL133558;

AL049314, A08913, AF185576, AR038854, AB025103, A08912, A27171, A08910, AL137476, I89931, A08909, AL049460, AL117585, E02221, I49625, A0890$, U96683, 576508, AL050277, AF114170, X80340, . I89934, I22272, I26207, I09499, X66975, D83989, AB019565, AL080060, AF012536, AJ242859, 583440, AL122118, AR068182, AL117432, E15569, AL080124, I17544, A08916, AL137658, AF106862, AF113694, AR019470, AL137530, AL050172, AF057300, AF057299, AF104032, AL110280, AC006371, A08907, A86558, AL049466, E01812, X93495, AL133072, AF055917, I00734, AF100931, AL137276, AJ001838, AL137283, AF118094, S69510, AF113676, E00617, E00717, E00778, AF158248, AL137660, AL122121, E15582, AF169154, AF067790, A57389, U92068, AL133640, AF176651, AF159615, AB007812, AL080127, AF044323, AF146568, X63410, AF106657, AF002672, AL050092, AL133565, A08911, I89944, AF119336; AF094480, AL137459, A12297, AL137641, AL137523, AF162270, E02152, AF113019, AL137556, AF090934, A65340, X79812, U95114, U58996, AF210052, X00861, AL137521, AL080126, AL137665, AF113689, AL133645, AF120268, AF125949, 568736, X72889, 561953, A21103, S36676, . , A65341, AI.110196, AR000496, I33392, U39656, AL133.081, AL117583, AF151109, AF047716, 237987, AF090886, U87620, I30339, I30334, AF180525, AL133010, AB008792, AF102578, 297214, M92439, AB008791, AF111112, E03348, AF028823, L19437, Y11587, AF215669, E04233, AL137478, E03349, AL080159, AL117626, ~

11851, E12806, L30117, AL117416, AR059958, AF017790, AL137574, U42031, AF061795, Y14314, AF151685, AL133016, AL023657, AF199027, A90832, AL117440, AF061981, AL137495, U78525, I32738, AL080148, E01614, E13364, AJ006417, AC002467, Y10080, S63521, Y16645, AL13'7538, X59414, U00686, AF040751, X72387, U72621, AL137294, AF042090, . AF195092, AL110171, AF003737, U67958, X06146, X70685, AF001215, AL133624, I03321, E08631, X54971, AL137547, U35846, AF000145, U91329, AC007298, AF078844, AL117648, AL133104, AL133067, AL122049, AL137526, X76228, L13297, AF022813, AF067728, AF118070, AF065135, A83556, and AF051325.

HADGE44 59. 8642571 - 448 15 - N63063, ALb41916, AI908941, 462 AA029504, AB020629, AC005922, AC015844, AC015844, and AC015844.

HBGDA14 61~ 10759971 - 879 15 -HSDJL45 62 1137808i - 1392 15 - AA577135, AA295867, and AI526193.

HDPRN38 63 11639981 - 1829 15 - AI765848, AI435448, AI458176, , 1843 AI268603, . AA232176, AA677741, AA232159, AI245247, H84466, AI470359, AI922359, AI039989, ' AW069705, AI521773,'AI382545, AW149957, AA526438, AL046519, H84467, ' AI609972, AW026305, AI421950, AL119284, AI419337, 282214, AC004263, AL049636, AC005182, AC005519, AC009516, AC004646, AC002425, AC004900, L78810, AP000510, AL031584, AC002395, AC005829, AP000081, AC005823, AF124730, AP000501, AC004686, AL117694, AC006285, AL022238, AL049553, AL031311, U95739, AC006581, AL049776, AL035405, AL008582, AL132712, AC006441, AL022326, AL023553, AC007193, AC004953, AL031667, AL109952, AC007216, AL023879, AC007099, AC003110, AC007225, AC020663, AP000961, AL031295, AF196779, AC005952, AL008732, AC008039, AC004997, AC005305, AC006004, 284486, AC001231, AC005500, AL023807, AC007731, AC008038, AC002331, AC004797, AC004477, AC005071, AP000556, AL035659, AC004520, AF109907, L35532, AC003043, AC005661, 299716, AC007536, AC004999, AC005229, . AC005225, AC004805, AF139813, AF207550, AC003108, AF064861, AC005031, AC005081, AC012384, AL121754, AL022328, AF111167, AC004659, AL049830, AP000466, AF111168, AC003070, AC005778, AC007637, AC005839, 284484, AC004228, 281364, 286090, L44140, AF001552, AL049569, AC005940, AP000355, AL020995, ' AC006948, AC005730, U73643, AC006211, AC006379, AC002350; 297630, AC005015, L78833, U91321, AP000552, AL023883,, AB003151, AC005913, AL031643, AC005562, AL035422, 285996, AF107045, AC006257, AF053356, AC005722, AC004156, AL049795, 298200, AC005695;

AC005740, AL035691, AC006064, AC005295, AC004743, AC000035, AL109628, AC016830, U52111, AC009399, AC004929, AL049576, AC005993, AC007283, AC005255, 294721, AC005529, U91322, AC004196, AC002302, AC004019, AC005158, AL034549, 298946, AC006449, AC002375, AJ246003, AC00?371, AC006312, . AL049539, AC008044, AL021155, AL031985, AP000350~ AL034402, AC003025, AL022312, 268279, AL079295, AC005800, AC005585, AP000359, and AC004217.

HETKH30 64 11145081 - 1020 15 - AA436699, AA442502, H25167, 1034 AA306355, H11845, and AA584520.

' HE9RE2165 11787981 - 1618 15 - AI589113, AL120391, AA224260, 1632 AA326268, w AW 136392, 883178, T78371, AA317066, AW162314, AA601376, AW410844, AA533660, AW265468, AL121039, AI702049, AA610644, AW162332, AW148821, AI609992, AW072963, AW419201, AI572680, AA425283, AI439525, AI433952, AI744963, AA584360, AA525818, AI064968, AA493464, AI065031, AL133861, AW162762, AW020094, AL044966, AA779599, AI242236, AI744890, N99245, AA315052, AW021399, AW 192,930, M77888, AW272619, AI285651, AL138262, AI445699, AA199578, AI567676, AA362670, AI734076, AW057760, AA525753, AW148964, AI734075, AA640305, AI133355, AA112864, AA524604, AA507623, AA676462, AW028376, AI905408, AI520984, AI052366, AI683079, 892703, AI538404, AL038533, AW272389, AL133216, AF098786, AF162762, AL009181, AC004594, AL049830, AC004673, AC007677, AC005069, AL139054, 293928, AC005726, AC007386, AC006227, AC000353, U91325, AP000100, AL021918, AC004915, AC007968, AL096712, U95743, AC007207, AC005690, AB026898, AL117258, AB023054, . AC004453, AC002072, AL121593, AC007172, U80460, AL035588, AP000263, 282201, AL049759, AC005520, AL109627, AL034423, AC005538, AC002386, AP000036, AJ229041, AL121653, AL133396, AL031585, ' 292844, AC004526, AC007395, AC007539, AL022345, AE000661, AL033521, AC005323, 282203, AL133162, 298200, AL024498, AC012384, AC006213, AC006017, AL022396, AL132641, AC005696, AC005539, AC007567, AL049869, AF207550, AC005066, AL035414, AC004881, AL034402, AC005828, AC003041, AL121580, AC006208, AF042091, AC006080, AC001231, AL031432, AC004125, D87675, AC004552, AC007551, AC007262, AL022165, AC007151, U91321, AJ229042, 295152, AF186194, AL021921, AC004106, AC006141, AL021877, U95738, AC004659, AC004944, AC006238, AL121754, AC004966, AC008044, AC005244, AC006487, AC006948, AC004129, AL021392, AC006143, AP000099, AC002040, AC006966, AC003964, U91327, AL050308, AP000260, AL121652, AC002452, AC003024, AC009294, AL031121, AC000120, AC007748, AC004048, AL021391, AC006466, AF152365, AC004072, AC005386, AL132712, AL033527, AL133321, AL031587, AL022238, AC003969, AF051976, ' AC003048, and AL031665.

HNTMB90 66 11547921 - 164415 - AW242587, and AA496178.

HMKCH92 67 910936 1 - 789 15 - T78768, 813299, 814933, AB003592, D87248, AC018833; AC034192, AC026206, AC026206, AC02_2381, and AC022381.

HTELV86 68 11653591 - 149015 - AW241703, AA923237, AA382531, ' 1504 AA918449, AA738033, AA019310, AA853033, AI696583, AI272244, AA749449, N25033, AI918408, AW075382, AW195253, AI915795, AL120696, AA505147, AW080076, AI261815, AI280082, AI590043, AI345010, AI538247, AI582912, AA857847, AI282673, AI696340, AI094092, AI469764, AI473471, AI698391, AW264009, AI918554, AI961309, AI289310, AI889147, N49165, AW192300, AI567971, AI224373, AI547227, AA975952, AA868486, AI469516, AW079432, AA693355, AW020693, AIS40606, AI619820, AW 131228, AI699175, AI801589, AI580436, AI370623, AI587588, AI241678, AI282822, AW083572, AW059828, AI537274, AW074057, AI345005, AI568293, AI868180, AL039274, AI340627, AI688848, AI114703, AI628325, N98597, AW089844, AW151974, AI473536, AL045997, AI345014, AI612885, AI538850, AI613314, AA056265, AI282346, AI673140, AW238753, AI799681, AI473208, AW302990, N29277, AI584130, AI687568, AI419826, AI799189, AW16I579, AI638644, AW411043, AW073907, AI658566, AA417278, AA729782, AL120056, AA042949, AI252023, AI683568, AW 188525, N68309, AI351959, AI499570, AL036954, AI758270, AI345026, AI816976, AI569440, AI699823, AW372903, W45537, AI318280, AA503384, AI583558, AI254042, AL048644, AI889189, AI065145, AW 151132, AI282720, AI669864, AI554428, AA835966, AI446405, T99953, AW020932, AI345261, AL037602, AI538692, 848064, AI439527, AI241884, .

AA693331, AA834534, AL037582, 841605, .

AI371228, AW189270, AW023871, AI858465, AI871933, AI889168, AW068845, AI468959, AI499279, AA827164, W33163, AL119828, U35371, X99043, X92070, AF074604, AL133558, I48978, 563521, 583440, U80919, E12580, 573498, AF126247, AF113694, X59812, AF090901, A08910, A08909, AF118558, AL137641, A08907, A08908, AF031147, AR068466, E06788, E06790, E06789, I52013, U53505, AF195092, X98066, AF131773, AF093119,146765, AF081825, AF137367, 575997, AF117959, AL049382, U61971, U61970, AF044323, AF180525, AL080126, I48979, AF143957, I25049, AF016271, AR005011, AF097996, AL137479, AR038854, AL008706, AR050959, X66871, AL080234, AF177401, AL096728, I00734, A31057, 536676, AL133088, AL133081, AL137459, E00617, E00717, E00778, AF139986, AF057300, AF057299, AL049276, AF211175, AL137259, AF038562, AL079293, AF078844, AF113019, I33392, A.L110296, 569510, AF040723, Y11435, U92992, AR005195, AL122104, X54971, I25048, AJ005690, M92439, AF192522, I29004, X66417, AL137657, I28326, AF051325, E01314, Y14314, AL133665, X97332, AF119336, AF090943, A65340, AL137560, AL117626, AF054988, AF162782, AL050172, AL137534, AL080140, AL137488, AF140224, AF090432, AL122123, I89947, M84133, AF131814, AL049339, Y16645, AR066485, D44497, AF102166, AL080110, AF094480, AL137529, E07108, . AL050149, A52563, AL080163, AL137267, A90832, X06146, I80062, AL133104, . AF141315, AF118094, AR000496, U39656, AF069506, M85165, X52128, M96857, AJ006039, M27260, U37359, AL122045, AF125949, AJ010277, 582852, U91329, AL133560~ AR020905, AL137526, AF082526, X79812, AL049314, AF118092, and AL050280.

HNTAF23 69 12170371 - 267015 - ~ AI378342, T'65383, AI298501, 2684 AA235292, AI312700, AA815287, 860713, T65460, AA904336, AI286061, AI868696, AA557339, F09466, AA234847, AL042166, 239008, AA077472, 860712, and 817631.

HTXJD74 70 12126831 - 195815 - AI884928, AW299727, AW204926, AA933627, AI471959, AI860951, AI674548, AA648384, AW134703, AI817454, AW207053, AI801449, AI927200, AI741288, . AI283846, AI360406, AI969837, AI359870, AI471951, N70264, W57964, W57938, AI928115, AI023464, AI824946, W79288, , AA242781, AI479588, AI962494, AI246231, W04200, AA778582, AI094509, AI248982, AA242806, AI093921, AA806316, AA962783, AA255447, AI086106, AW440004, AI867514, AA030013, AA143002, AA256554, 815486, AA973189, AI277037, AA029757, H01787, . , AA913805, T96295, AA581087, AA142852, AI991436, AA348729, AI766737, AW369326, AI918290, AI817,745, N90613, AI902506, AI740457, AA303510; AI902496, AA758159, H01038, AW298344, C00431, AA910879, AA843339, AI902495, AI902509, W 19941, AW452907, AI902510, AI610042, AF114436, AF151638, U21660, AF114430, 250026, AF151639, AF040261, 250024, AF114437, AF114431, AF114434, AF114432, AF114433, AF114435, and AF040266.

HAUAI17 71 11538741 - 110315 - AI742586, AA994212, AI472051, 1117 AI823636, AI738645, AI808204, AI458889, AI694439, AI742084, AI570505, AI005161, AW166260, AI126407, AI819324, AI689840, AI802140, . AI564776, AA044826, A3379936;
AI018575, ' AW418896, AI708063, AA044874, N33369, AI798664, AA983545, AA996109, AI268684, - ---w AI948596, AI051014, AI498702, - -' AI625938, -AW295897, AI948603, AI521441, AI021975, AA317357, AI264270, AI373261, H92520, AW 139306, AI244808, H77889, 800296, AI985535, AW135452, AW242712, AA677354, AA938619, N35293, H58937, AA917514, T99948, AI393979, AA779970, AI768427, W25595, AW044210, AA883635, AW072204, AW351592, AI470848, AW020604, H92521, AA335622, AI955919, AI208112, AI308950, H58984, AI919367, and AF126781.

HNHNP81 72 9283781 - 604 15 - D51060, 014014, D58283, AA305409, D80253, D80024, D80166, D59619, D80210, -, D80240, D8036'6, AA514186, 014389, D80043, D81030, D80133, D80247, D59859, D80212, D51799, D80164, D80219, D51423, D80022, D80391, D59787, D80195, D80188, D80248, 014331, D59502, D59467, D57483, D59275, D59610, D80227, D81026, D50995, D80196, D80439, D80251, D80269, D59889, -- . ~ D51022, D50979, D80268, D80522, 015076, D59927, AA305578, D80038, D80193, D80045, D80241, AA514188, AW360811, D80378, AW 177440, D80302, 005695, 014429, AW178893, AW377671, AW375405, D80157, T03269, 075259, AW178906, AW179328, AW366296, AW360844, AW360817, D59373, AW375406, D51103, AW378534, AW179332, AW377672, AW 179023, AW 178905; D51759, AW360841, AW378532, D58253, AWI77731, AW177501, AW 177511, D80132, D80134, AW352171, AW377676, AW352170, AW 178907, AW378528, AW178762, AW179019, .

AW 179024, D51250, AW 176467, AW 178983, AW177505, D81111, D59653, AW179020, AW 178775, AW367967, AW369651, AW178909, T48593, AW177456, AW179329, AW 178980, AW 178914, AW
177733, AW 178908,~AW 178754, AW
179018, 006015, AW352158, AW352117, D80949, AW178774, D59695, D52291, D45260, AW352120, D59627, D51079, AW179004, AW179012, AW378525, AW352163, F13647, AW360834, T11417, D80064, 221582, D80168, 014298, AW378543, AW352174, D80258, AW177728, H67854, 014227, AW179009, AI525923, AW367950, AW178911, AW177722, D59503, AI910186, AW378540, 003092, H67866, AA809122, D51221, AW 178781, D58101, AI905856, D58246, AW177508, 014407, D80228, AI525917, 014077, AW178986, AW 177497, T03116, AI535686, AI535850, D59317, D51213, D80014, D59474, AW 177734, AI525920, D45273, AW 177723, 014973, 014344, AW378533, AA514184, D59551, 014957, AI525215, D60010, w " AI525235; D60214, AI525227, 014046, AI557774, AI557751, AI525912, T03048, AI525925, D51097,'AI525242, AA285331, AW378542, AI525222, AW378539, 230160, 016955, 005763, 233452, T02974, D51053, AW360855, H67858, AI525237;
004682, D50981, T02868, 013958, AI525928, D80314, . AI525238, A62298, AB028859, AR018138, AJ132110, A62300, AR008278, A84916, AF058696, A82595, AR060385, AB002449, D89785, X67155, Y17188, A94995, D26022, Y12724, A25909, A67220, A78862, D34614, AR008443, I50126, I50132, I50128, I50133, D88547, AR066488, AR016514, AR060138, A45456, A26615, AR052274, X82626, Y09669, A43192, A43190, AR038669;

AR066487, A30438, I14842, AR025207, AR054175, D50010, Y17187, AR066490, AR008277, AR008281, A63261, I18367, _ AR008408, AR062872, A70867, AR016691, AR016690, U46128, D13509, A64136, A68321, AR060133, AB012117, I79511, ' X68127, U79457, AF123263, AR032065, 282022, A63887, and AR008382.

HFIDL68 73 9284751 - 516 15 - AI375172.

HCHON59 74 12007551 - 2205 15 - AW299683, AA315514, AW 152065, AA305116, AI961078, AA210776, 856767, AA622988, W04752, AW236710, 889462, 825303, AA781427, 896008, 818161, N76920, 824760, 219446, AA694165, AA740627, AW074408, AA345403, AA348726, T86393, AI076025, T99947, . AA875871, AA337010, AI224620, H17194, AI916251, T79510, AI077381, AA363905, AA749291, AA004316, AA815288, 889463, AW044490, 817355, AA054853, T98732, H17900, AW085956, AA211903, AA716584, AW297867, 225140, 220433, 895999, AA133996, H52630, A74519, and AW662221.

HPMKL,5675 9347341 - 1189 15 - AA035425, W67511, AA035426, 1203 AA035119, AA058394, W68123, ~AA053291, AA126828, AA127310, AA035375, AL358178, AL358178, and AL356421.

HCHNJ32 76 11540581 - 856 15 - AI589871, AI991112, AI573258, 870 AW168298, AI382182, N63300, AW245568, AW245578, N30214, AI244679, AA625514, AA442766, AI015935, AA714315, AI669114, AA613669~

. AA433946, AA568688, AI808444, N63050, AA813327, AI885837, AI281530, N52392, AA811995, AA492250, AA507560, AI221056, AI083879, AA313563, AA72Q021, AA446582, AI557529, AI085835, AI557530, AA305352, AA430753, AI304532, AA838602, AI690776, AA573777, AA446709, AI304455, AA782093, T73332, AI566973, AA885566, AA573537, AI283278, AA720002, AA861893, AI304454, H72469, W00555, AI199547, W00611., AI160878, AI472891, AA531050, AI937637, H95032, H78356, N32117, N54353, N78252, AI571328, AW204782, AI004311, H64869, . ~ T61807, AA876623, AA418354, H65466, 850529, AW138834, F21642, AA086353, . AI859175, AI264256, AA652483, H85048, AA501347, T61353, T60510, T55136, AA134203, H91692, 230219, AA431169, AA855127, 850625, AI985397, AI141454, AI858739, H93999, H72870, H73548, H91789, AA291581, AW150864, AA293323, T73417, 886214, AA918480, T98646, AW300104, AI382568, AA432189, T72288, H77954, T73347, C00330, AI251780, AA478304, N59791, T72293, T71510, . AA230200, AW207071, F15963, AI918444, AA478363, AA134202, N88648, AA429728, 220777, N72798, AI927580, N56787, AI954738, AA974081, AA641099, T73399, T71348, H84670, AA541321, T98698, AI126088, AW440575, H78357, AI567583, T72864, 220778, N40867, AA983562, AA229346, H47484, T71167, AA230291, T72848, H84633, AW050725, AI401699, AI560514, H85040, AI470293, AW026633, AI689614, AW081186, AA418240, AI469157, AI583054, AI654137, AI886715, AI244105, AI815239, AI446110, AI583578, AI433198, AI370322, AI440444, AI270039, AI362525, AI934276, AA974245, AW088131, AW 103431, AI479903, ~AI678762, AA937558, AI805688, AW409638, AI241792, AI918424, AI500039, AA766618, AI358042, AI446124, AWI52415, AI076774, AW081252, AA631001, AA938645, AW009066, AI371243, AI801536, AA599588, AF113123, AR009500, A74991, A74992, AB025103, AL080127, X51671, AL049959, U31991, E12579, E12888, AF094480, M84133, .

AF106934, AL137534, AL050170, AF182215, AF108357, AL022165, X99257, AL122111, U94316, U92068, 568736, AB026995, AB007812, AL137561, AR016802, AL049460, AF029750, X62773, AF109906, AF012536, A57389, AC009286, J00983, ' AF107018, AL031281, A32826, A30330, A32827, A30331, AL137556, AL137268, X53587, AF105427, AF033653, 569510, AR068182, AC004213, 561953, AL137659, 298744, AL035258, AC005992, U75378, AF035161, X00861, AL035407, AF017104, .

and AF111112.

H7TSC21 77 11443151 - 581 15 - AA077171, AR028895, AR028903, AC006466, 223273, 223272, AR028893, AR028894, AR028901, AR028902, D17516, AR028892, AR028900, D82935, AR028890, L16506, AR028891, 223279, 223274, AR028898, 223275, D14908, 223282, AR028899; D14909, D16465, L16680, AR028889, AR028888, AR028897, and D 17290.

-- HPMLD30 78 12234861 - 286015 - AA653812, 578429, E03539, - ~ 2874 and X01966.

___ HOFMN17 80 943323 1 - 778 15 - AA683020, AW409699, AI809532, 792 AI453056, AA778576, AW005786, AI042586, AI359703, H91861, AW205217, H91914, AB020628, AF111097, AF111098, U72487, AF081144, AF081146, U78105, AF111099, AF081145, and AF081147.

733 , HBGMZ39 82 947112 1 - 588 15 - AI820661, AI791493, AA989356, 602 AI791282, AI732537, AI792053, AW207804, 822360, 872427, AA505927, 822019, 872474, AC008537, AC008537, AC008537, AC019337, AC019337, and AC019337.

HFRAG17 83 11280111 - 139415 - AA084699.

HBXAB78 86 948357 1 - 407 15 - AC073541, and AC022123.

HTGBN65 87 11272721 - 767 15 - .

HDPBQ32 89 949191 1 - 290615 - AL041780, AA447233, W63594, 2920 AI479623, AL120043, AI862014, AW370548, AW370546, AA833890, AA528106, AW342135, AA767439, AI884921, AA810497, AI479859, AW246967, AI598172, - ~ AA316311, AI862137, AA808946, - AW438512, AW250167, AW014235, N48194, N31847, AA565004, AI147687, AI421531, W94723, AA709397, AI041972, AI860296, AW029341, AI311132, AI097206, AI124091, AI363367, AI093332, AW273088, AI017231, AA658369, AA669438, AA488942, W94478, AA436731, AI439831, AI986078, W26165, AI864954, AI866181, AA648310, AW274809, AA635499, H10274, AA630795, AW316730, AW440901, W04336, AL041779, AA658353, AA767423, AW105681, AW002817, ' AA976719, H38610, AA452736, W88618, AI573056, AI017009, AI192454, T33808, AA.042879, AA878793, AI818364, AI435523, AA485399, AI280334, AI669596, N23334, ~AI630924, AI349078, AAO$6333, AL135653, AI360950, AW262423, AI374697, AI568362, AA782533, AI908446, AAS81018, T69944, AA2,62554, N23946, AA366444, AA027924, AA452551, AA358621, F37326, AL134964, 811755, AA761233, AA358622, AI206778, AW262418, 823435, H37936, AW298228, H86266, AW272102, AA100347, AA626778, ' H38589, N64738, T06960, T53997, AI814020, AA988901, H11262, T64797, AI033988, C00287, F27815, AA215782, 817275, F05786, . ~ AW079229, AI525067, 807905, T24060, AA299962, H10218, W27761, F12185, AI352630, D56196, 812465, N91751, AA193243, AI018166, 807852, AA424815, H38851, AA370279, T82106, 851860, T81790, 241898, F11035, F02033, AA093862, AA918426, AA043004, AA371442, N24549, AA351515, H86265, AA805378, 841949, T55253, T72550, AA485560, H11263, 837351, 844220, F01399, T93582, 841392, AW238018, AA027858, AW088974, T70019, AA918403, AA356909, AA034107, N74170, AA832430, N5334'1, AW070570, AA632032, AW238023, N87429, AL040495, 854254, .

' ~ T10580, AI828485, W26617, AA495855, AW 137399, 224796, AI873580, F00149, AW024740, AA192580, AI086408, AI799910, AA585140, W27789, AW 131985, AW406062, AA629234, N63605, AA639515, AA885541, . N56490, AA377723, AA583734, AA627268, AI908889, AI371383, and N83787.

HEQAP17 90 9493581 - 807 15 - 821 AI131555, AI769466, AA215577, AW190975, AA258335, AA258499, AL044652, S63848, Y17793, and A49045.

HWLLB11 91 9548491 - 731 15 - 745 AI745636, and AA102414.

HNTEF53 92 9548521 - 234215 - 2356AA557324, AI655577, AI696732, AI923200, AA863360, AW262723, AI697332, AW275990, AI436648, AW276183, 856515, AI362521, 853456, 853457, D62878, AA337301, AA652746, AW264444, 856123, AA319338, D79346, D79250, N56346, AA886832, and AL138223.

HNTND64 93 9548711 - 392 I5 - 406 AC025090, and AC025090.

HFPFA83 94 9556141 - 723 15 - 737 C14389, C15076, D59467, D58283, D50979, D80522, D80164, D80166, D80195, D80043, D80227, D81030, D59275, D59502, D80188, D59859, D80022, C14331, D51423, D59619, D80210, D51799, D80391, D80240, D80253, D80038, D80269, D59787, D80193, D59610, D80212, D80196, D8b219, D81026, D59927, D57483, D80378, AW177440, D80366, D80251, AA305409, AA305578, D59889, D50995, D80024, D80241, D51022, D80045, C14429, D51060, C75259, T03269, AW178893, AW179328, AA514188, AW378532, D80248, C14014, AW377671, D5,1250, AW369651, AW178762, AW178775, AW177501, D80134, AW177511, AA514186, D80133, AW 176467, D58253, AW360811, AW352117, C05695, AW375405, AW352158, D80268, AI9I0186, D80132, AW366296, AW178906, AW360844, AW360817, AW375406, AW378534, AW179332, AW377672, AW179023, AW178905, D80302, D59627, AI905856, AW378540, AW352171, . D80258, D80439, AW377676, AW352170, AW177731, AW178907, AW179019, AW 179024, D59373, D80247, AW 177505, AW 179020, AW360841, AW 178909, AW 177456, AW 179329, AW
178980, AW 177733, AW378528, AW 178908, AW178754, AW179018, AW352174, 221582, AW360834, D51103, AW179004, AW179012, C06015, AW178914, AW378525, AW367967, D80157, AW 177722, D51759, AW 177728, AW179009, AA285331, AW178774, w AW 178911, D51097, AW378543, AW352163, D58101, D80064, D58246, D80014, D59503, AW178983, AW352120, AW178781, T48593, AI535850, AW 177723, T11417, D59653, AA809122, AW177508, D4S260, D59317, C14975, AW378533, AW367950, F13647, D81111, H67854; C03092, C14227, H67866, AI557774, AI525923, AW177497, T02974, AI557751, AW178986, T03116, C14298, D45273, D52291, AW177734, D59474, AI525917, AT525227, DS9695, D60010, C14973, AI535961, C14344, C14407, AI535686, C14957, DS 1221, D59551, AI525920, AA514184, AI525242, D60214, . T03048, C14046, AI525912, AI525235, C16955, AI525925, AI525222, D80168, AW378542, AW378539, AI52S215, AI525237, C05763, 233452, AI525928, AW360855, T02868, D51213, D3145'8, H67858, AR018138, AJ132110, A84916, A62300, A62298, AR008278, AF058696, AB028859, X67155, Y17188, D26022, A25909, A67220, D89785,'A78862, D34614, . D88547, I82448, Y12724, X82626, AR025207, AR016808, A82595, AR060385, A94995,' AB002449, AR008443, AB012117, I50126, I50132, I50128, I50133, AR066488, AR016514, AR060138, A45456, A26615, AR052274, A85396, AR066482, A44171, A85477, 119525, A86792, Y09669, A43192, A43190, AR038669, AR066490, U87250, AR066487, X93549, I14842, A30438, I18367, D88507, AR054175, D50010, Y17187, A63261, AR008277, AR008281, AR008408, AR062872, A70867, AR016691, AR016690, U46128, D13509, I79511, A64136, A68321, AR060133, X68127, AF135125, U79457, AF123263, AB023656, AR032065, AB033111, X93535, and AR008382.

H7TSA07 95 11286291 - 521 15 - AA077171, AR028893, AR028901, AR028894, AR028902, D82935, AC006466, L16506, L16680, AR028891, 223274, 223282, AR028899, D14909, D16465, AR028888, 223272, 223275, D17290, AR028892, AR028900, D17516, AR028895, AR028903, AR028890, 223279, AR028898, D14908;

223273, AR028889, and AR028897.

HNTDM29 96 11563561 - 241415 - AI568492, AI367139, AI536953, 2428 AW409812, AI871439, AW182297, AW015353, AA682919, AA683020, N810S4, AI420931, AI453277, AA772469~ AA427474, AA323620, AA292140, AI085790, D52745, AA291970, N73672, AAS58818, AW409699, AW235674, AI970122, N78513, W07864, AI758415, AW439042, AA291908, 241587, AI809532, AI453056, AA715060, AA778576, AW005786, AI042586, AI359703,~H91861, C00934, AW205217, H91914, AB020628, --- ~ AF111097, AF111098, U72487, AF081144, AF081146, AF 111099, U78105, AF081145, __and_AF081147.

HBCBT19 97 11522331 - 1193 15 - AW194384, AI921843, AW025219, AI688167, AA861776, W58728, AI189664, AI627470, AA131986, AW316534, AI024367, AI200318, AA969204, AA505874, AI077983, AA805925, AI401269, AA026664, AW075217, AI299373, W27452, AI676036, AI890667, AW118903, AA131681, AA831151, 871771, AI453474, AW372475, AA501987, AA366467, AW294217, AW294214, AA918168, H29874, AA861349, AI149118, AI769603, AI,138671, AA844743, ' AI769366, AI373905, H92161, T82046, AI471771, AW235611, AI262961, N92383, AW272266, AA806045, AI697728, 871811, 860094, 853357, AA987344, T71625, AA335794, AA007217, AI939498, AI024365, AA635601, AA766069, AW243229, W24578, AA026712, AI267768, AI572256, H92160, AI885357, andAW172330.

HMCFA91 98 11513471 - 1280 15 - AW194384, AI921843, AW025219, AI688167, AA861776, W58728, AI189664, AI627470, AA131986, AW316534, AI024367, AI200318, AA969204, AA505874, AI077983, AA805925, AI401269, AA026664, AW075217, AI299373, W27452, AI676036, AI890667, AW118903, AA131681, AW294217, AW294214, 871771, AI453474, . AW372475, AA366467, AA501987, AA918168, H29874, AI149118, AA844743, AI138671, AA861349, AI769603, AA831151, AI769366, AI373905, H92161, T82046, AI471771, AI262961, AW235611, N92383, AA806045, AW272266, AI697728, 871811, ' AA987344, 860094, 853357, T71625, AA335794, AA0072I7, AI939498, AI024365, AA635601, AA766069, AW243229, W24578, AA026712, AI267768, AI572256, H92160, AI885357, AW172330, D80253, D59859, D51423, D51799, C14389, D80227, D57483, D80366, D80188, D59889, D80166, D59619, D80210, D80240, D81030, D80269, D58283, D80212, D80022, D59275, C14331, D80038, D50979, D80219, D80024, D80195, D59467, D80391, D80164, D80043, D59787, D59502, -D59610, AA305409, D80378, D80196, C14014, D59927, C15076, D80251, D51022, AA305578, D50995, D80193, D81026, D80045, D80241, D51060, D80248, AW177440, AA514186, AA514188, C14429, AW360811, AW178893, D80133, D80522, D80268, T03269, AW375405, AW179328, D58253, AW378532, AW366296, AW377671, AW360817, AW375406, D80439, AW378534, AW179332, AW377672, AW179023, ~AW178905, AW177501, AW177511, D80134, AW179019, D80302, AW178762, D80247, - -- AW378528, D51250, AW352171, - AW377676, AW352170, AW177731, AW178775, AW178907, AW177733, AW178906, AW 179024, D80132, D80157, AW369651, AW 179020, D51079, AW 177456, AW 179329, AW178980, AW178908, D51759, AW179018, D51103, D80949, AW178914, AW378525, C06015, AI525920, D80168, D45260, AI905856, AW178774, AW178911, AW378543, H67854, AW352163, AWl'77728, C03092, T48593, AW178781, AW378540, H67866, D80014, AW360834, AA809122, AI525923, AI525917, T11417, F13647, D58246, D81111, D80064, AW367950, D51221, T03116, D59627, D59474, D80258, D59317, C14973, C14227, D59503, C14344, AW378533, AR008278, A62298, AR018138, ' A84916, A62300, AJ132110, AB028859, AF058696, A67220, X67155, Y17188, D26022, A25909, D89785, A78862, I734614, Y12724, A82595, AB002449, AR060385, D88547, A94995, X82626, AR008443, I50126, I50132, I50128, I50133, I82448, AR025207, AR0541.75, AR016514, AR060138, A45456, A26615, AR052274, AR066488, A43192, A43190, AR038669, Y09669, AR066487, I14842, A30438, AR008277, AR008281, Y17187, A63261, AR066490, D50010, AB012117, I18367, AR062872, A70867, AR016691, AR016690, U46128, AR008408, A85396, A64136, A68321, AR066482, A44171, X64588, I79511, A85477, I19525, A86792, D88507, D13509, X68127, AR060133, X93549, and AF123263.

HMAJK74 99 9663241 - 1073 15 - AC073670.

HSLHC11 100 9663251 - 244 15 - AA585098, T18597, 845895, ' 258 828735, 829445, AA585325, AA585476, 829218, 828892, AA170832, 829657, 828967, 828965, AA283326, D60765, AA585101, D53447, 232887, D59751, C15406, D54897, D53161, C16315, D60844, D57491, C16293, 828895, D53472, AI557734, D61.254, 232822, C15069, AA585439, 228355, AI557262, AI546875, AI546999, C16294, C15120, D52835, AI541383, A1557864, AI541356, D59436, AA585155, D61185, C16292, AI557763, AI547006, AI547250, AI526140, 829179, AI541365, AI525500, AI55774Q, AI546971, AI541374, AI525306, AI541307, AI535660, D571'86, AI546945, D55233, AI541205, C16300, AI525431, AI546921, AI526078, C16305, AI526194, AI557731, AI557787, AI525316, C15762, AI526184, AI557727, AI541535, AI547039, AI525556, AI540967, AI541517, AI541013, 230131, AA585356, AI536138, AI541346, C15737, . C16296, AI526016, D60730, 829177, AI557807, AI546996, 829262, AI526191, 829172, AI546891, AI541523, C16290, AI557718, AI525339, AI535639, AI557758, AI557084, AI541527, AI547202, AI557408, AI557809, C14208, AI526109, AI525320, AI557155, AI547196, T19407, AI540903, AI557602, AI541034, AI525856, AI526073, 233559, T41289, AA514191, AI540974, AI526113, AI547137, D54850, AI557264, AI541321, AI525286, AI526180, AI556967, .

AA585453, C14723, AI541510, AA174170, AA585430, AI557808, AI557279, AI526024, T41329, AI546829, AA585117, D59458, AI546954, AI546831, AI546901, AI557533, AI526112, AI540920, AI526158, AI526195, AI541345, AI557039, C14322,'AI526117, AI524890, AI524904, AI557799, AI541514, AI557852, AI547189, AI541422, C14391, AI525656, AI540944, AI557786, AI525332, AI541415, AI546828, AI541515, AI541027, AR062871, AR038855, A25909, Y09813, X55486, A20702, AR062872, AR062873, A20700, A43189, A43188, AR017907, AJ244005, Y16359, A98420, A98423, A98432, A98436, A98417, A98427, AF082186, AR038762, 232836, D50010, D13509, AJ244004, AR054723, X81969, . D78345, AJ244003, X82786, X76012, AR031365, AC005913, AJ244006, AJ243486, A98767, A93963, A93964, I63120, AR003381, AR031358, AR008443, and AR017826.

HSXEJ10 101 1_1653_05_1 - 330 15 -HBGMA34 102 11263101 - 243 15 - C 16292, D79576, and AA773146.

HE8NI24 103 971296 1 - 737 15 - AA883367, AA332611, AA732890, 751 AI283442, AI6'73342, AI631153, AI200800, AI910962, T11417, D80258, D59503, D80014, D81111, C14227, D80064, AI557751, D58246, C06015, AA514184, AI535959, AW178893, .

AW178907, AW375405, AW17744b, AI535686, AW360834, AW178908, AW360811, D80314, AA809122, D80251, D80253, C03092, D80247, D80043, AA285331, AW176467, C14389;
AW179328, T48593, AW375406, D80439, AW378534, AW179332, D58283, AW377672, AW179023, AW178905, D59859, D80022, C14331, D80166, AW177731, D80195, AA305578, D80193, D59927, T03269, D59467, D51423, D59619, AW378528, D80210, AW178906, D51799, D80391, D80164~ D59275, AW178762, D80240, D80038, AW179019, D59787, D80227, AW378533, D59502, AA305409, AW378532, F13647, D45260, AW178914,'AW378542, AW360855, AW377676, I50126, I50132, I50128, I50133, AF123263, A70867, D88547, AR062872, AR066488, AR016514, A62300, D50010, X82626, AR066487, Y17187, AR060138, A84916, A45456, A67220, D89785, A62298, Y09669, Y17188, AB028859, A82595, A78862, D34614, A94995, D26022, AR060385, A30438, AJ132110, AR018138, A26615, AR052274, A43192, AR008278, X67155, Y12724, A63261, A43190, AR038669, AF058696, A25909, X68127, AR008443,.AB002449, AR025207, AR016691, AR016690, and U46128.

HMTAV95 104 6149361 - 395 15 - AB023187, AL137000, AL137000, 409 and AL137000.

HHFGP83 105 11809591 - 223715 - AW027467, AA628405, AW021001, AI679358, AW002474, AA142868, AI720221, AI559580, AI971163, AI952940, AI086066, AW189486, AA126704, AI338366, AI632310, AW089385, W92463, AI339087, AI372019, AI335800, N67118, AW130513, AI811098, AI962685, W72626, AA043282, AA054560, AI268440, AA716090, AI564240, AI623726, AA938386, AA012896, AA047297, AA652890, AI869611, AI199091, ~ AA708870, _ AA760924, AA748136, AA043925, AA056075, AA582637, AA017551, AI202160, AI916439, AA618223, AA632478, AA975496, AA249580, AI422146, AA932810, AA628573, AA628559, D81448, AW195978, AW188932, AI914554, AI857354, AW 193055, AA046731, 875666, AI873760, AI127156, AW023470, AA043406, AW023472, AA043200, AI690518, AA054620, AA468866, AW382445, AA047158, 001055, W76609, AA043281, AI918561, AI570316, AA338995, AI653300, AI917733, AL133599, AF004840, and AF090866.

HDPVY34 106 11591361 - 227615 - U66680, AI651024, AW338289, 2290 AA099357, AI476130, AI651541, AI082697, AI240339, T84930, AA101906, AI634187, AI457597, - AI457313, AI696793, AW327624, AI963720, AL041450, AI598003, AI678785, AW193265, AA984191, AI559645, AI380617, F29989, , AA653139, AI431303, AI368745, AI619997, AI338426, AI244254, AI270117, AI923052, AW 162288, AI278972, AA610509, AI613280, AA469451, AI537020, AI821881, AA574442, AI821918, AI733856, AI866487, AW088581, T05834, AA679266, AI564185, AA827231, .

AW089589, F13749, AA563770, T92055, AI342622, AW008169, AW 157492, AI610920, F36273, AA167055, AI708005, AI358229, AW419262, AI753365, AI270524, AI287921, AI350211, AL046898, AL120282, AI090334, AI654285, AA303054, AI821892, AA805841, AW27~1904, AA584489, 891911, AI251104, AI148404, AI610159, AI079901, AA613624, AW089950, AI244758, AI581486, AI634691, AI862716, AW406755, AA904137, AI252483, AW088202, AW 193432, AI246796, AI365988, T57767, AA507991, AW007662, F32710, AW117740, T50676, AA955031, AI952900, AI688846, AA669840, y_ AA772906, AW303254, AI085719, AW302983, AC005922, AC005495, AD000813, AL049694, 293244, AC009501, AC005081, U89335, AL121655, AC000353, AL022165, AC002996, AC006120, AC008372, AL121653, AC006115, AL021397, AC002492, AC003109, AL121603, AC004814, AC007390, AL022336, AC004921, AL050332, AC005015, U50871, AC006285, AJ010770, AL049697, AC002470, AC003029, AC007731, AC005874, AF134471, AF111169, AE000658, AC002301, AC007406, AC003690, AC000026, AC003108, AL022323, AL049733, AC005277, AC002536, AL079295, AP000045, AC007421, AC004854, AC004883, AC005156, AL034420, AC007162, AC004797, AL031846, AC006449, AC005886, AC004099, AL049557, AL034548, AL035361, U85195, AC004887, AC0.07388, D84394, AC004905, Y18000, AC005288, U95743, AC006360, AC006261, AC002394, AF196779, AC004383, AC007225, AC004813, AC002390, AC004890, AC005279, AL035420, AC005242, U47924, AC007030, AC005722, AF196969, AC005257, AL022320, AL008582, AC006141, AC005330, AL033521, U95090, AL031229, AC005255, AC005829, AC000124, AL135744, AL122021, U82668, AC006111, AL034555, AC006001, AL049757, AC005011, AF207550, AC005520, AP000556, AL049829, 285986, AP000152, 273496, AC006211, AC004812, AF134726, AC008040, AL049830, AC004996, AC005280, U95742, AC004647, AC006480, AC004987, AC003688, AC004534, AC005225, AC005512, 299716, AC004230;

' AC007688, AL035072, AC001228, AC005399, AL080243, AC005100, AC000052, AC004087, AC007226, AC006509, AC002464, AC007216, . .

AC005412, AC005182, AC003957, AC002116, AL049780, AC005726, AC004690, AC005071, AL049776, AC004263, 283840, AC006552, AL031681, AL009179, AC004802, AL049869, AC006336, AC004257, AL031283, AC004765, AC007277, AL078477, AL032822, AC002565, AC004084, AL034429, AP000089, AC004966, AL117339, AL109613, AC004686, AL035703, AC005531, AL035403, AC003035, AC004841, AC008394, AC005519, AC004975, AJ011930, AC005779, AC005530, AC005209, AC005037, AL096701, AC004031, AC004865, AC005913, AC007055, AF152365, AC007308, AP0001I6, AP000247, AP000501, AL022721, AC005914, AC007686, AC007021, AC004408, AP000114, AP000046, AC003982, AP000557, AL034451, AC004858, AC003010, AP000552, AL021154, 298884, tAC005962, 284466, AC005212, AC005920, U51280, 285994, AC007263, D83253, AC005696, AL008718, AL031432, AL031774, AP000502, AC004458, AL109984, AC005996, 285987, AL031728, 283826, AC007666, AC005291, AL133445, AC009263, AF045555, AP000518, D87675, AC005694, 293783, AC002115, AL020997, AC002558, AL021808, AC004859, AL034449, ACOb4552, and AC005537.

HAGGS65 107 11404881 - 693 15 - AA029504, AA448354, AI076474, , 707 AI076494, AC005922, AB020629, and AF213393.

HE9TK49 108 11264871 - 144115 - 014331, 014389, 015076, D80212, ' 1455. D80268, D80439, D81026, 014429, D59502, D80391, D59787, D80227, D59927, D59619, D80210, D80240, D80022, D80166, D80196, D80045, D80269, AI557751, D81030, D50995, AA809122, D80219, AA305409, D80164, D58283, D80195, D51022, D59859, D80248, D59467, D51423, D51799, D59275, D80253, D80043, D51060, D80193, D80133, D80366, D80188, D50979, D80522, D80247, 014014, AA305578, D80038, D59610, D57483, AA514186, AA514188, D59889, D80024, D80378, AW360811, D80157, AW177440, D51759, D80302, D80241, D80251, T03269, AW178893, AW178906, D51103, 003092, AW377671, AW375405, D59373, D59653, AW366296, AW360844, AW179328, AW360817, 075259, 006015, AW375406, AW378534, AW179332, AW377672, AW179023, AW178905, AW177731, AW378532, T48593, AW177501, AW177511, 005695, AW179020, AW178986, AW378528, AW178762, AW352171, D45260, 014344, AW377676, D80132, AW352170, AW178907, 014407, AW178908, AW179019, AW179024, D80134, AW360834, D51250, AW360841, D58246, D80949, AW177505, AW178775, AW178909, AW177456, AW378533, H67854, AW 179004, T03116, AW 179329, H67866, AW 178980; AW 176467, AW
177733, AW178754, AW179018, AW352158, F13647, AW352117, AW352120, D59503, T11417, 014227, AI535686, AW178914, AW367950, AI525923, AW178774, D80258, D59317, AW 179009, AW 179012, D80064, AW 178911, D80228, AW378525, AW177722, AW352163, 014973, D81111, AI535959, AI52591.7, AW177734, D80014, N66429, D80168, D51221, D59695, AW378543, AW378540, AW177728, D59474, AI525920, D60010, AI910186, D58101, AA514184, D59627, D51213, D59551, AI905856, 014957, AW 178781, D60214, AI525227, C 14046, AI557774, AI525235, AA285331, AI525242, . . 014077, AI525912, AI525925, AI525215, AW378539, D45273, 016955, AW378542, C05763, 233452, T02974, D51053, 221582, . AI525222, C14298, C06084, T02868, AW360855, AI525237, H67858, AF126965, AF126966, AC004590, AF124351, AB012043, ,. AF134985, AF134986, AF125161, AF027984, AJ012569, A30438, A82595, A62298, A84916, AR018138, Y17188, A62300, AB028859, AJ132110, AR008278, AF058696, AR060385, AB002449, Y17187, X82626, A94995, I50126, X67155, I50132, I50128, 150133, D26022, Y12724, A25909, A67220, D89785, A78862, D34614, AR066488, AROI65I4, AR008277, AR00828I, AR060138, Y09669, A45456, A26615, AR052274, AR008443, AR054175, I14842, AR016691, AR016690, LJ46128, X68127, D88547, A43192, A43190, AR038669, AR066487, X64588, A63261, D50010, AR062872, A70867, AR025207, AR008408, A64136, A68321, I79511, I18367, D13509,.

A8066133, AF123263, and AR032065.

HTLDW37 109 12066891 - 107115 - AI732328, AA587744, AA371876, , 1085 AI076774, AI814025, AI859464, AI267185, AA693331, AI811603, AI918554, AI889147, AW083168, AI436429, AI491904, N67350, AI688847, AI432110, AI401699, AI005511, AI307569, AI521551, AL135607, AA504514, AI383804, AI553669, AA856697, AI494201, N36182, AA514684, AL048644, AW263804, AI582912, AI292193, AI679959, AI580290, AI133489, AI349957, AI478533, AI635079, AI702301, AW020397, AW149876, AA835966, AA857847, AI446405, AI345005, AI612885, AL038541, AI340627, AI702527, AL038463, 841605, AW020693, AI560545, AI371228, AI345143, AI690472, AI345736, AA939199, AI468959, AW081186, AI307736, AI540606, N99092, N71180, AI348847, AI336633, AI393006, AA019646;
N75771, AI349279, AI690946, AI345527, AI242736, AA493923, AA042949, AI250627, N22406, AA494167, AW071380, AA056265, AI887775, AI334893, AI310606, AI349186, ~AI538218, AI340653, AW168503, AI590423, . AI804505, AI623834, AL120831, W45537, AW079432, AI250646, H89138, AI582871, AA468418, AI343131, AW079771, AW236186, AL045421, AI307210, AI307734, AI307708, AI336513, AI312325, AW071395, AI500659, AI336662, AI336634, AI868204, AI340659, AW071377, AI311159, AI348895, AW411351, AI313320, AI307446, AI0506'66, AI336495, AI340644, AI633061, AI313352, AI310920, AI345397, AL049085, AI307503, AI334930, AI309443, AI373276, AI349622, AW021373, AI307520, AI345026, AI336585,, AI340664, AI310592, AI307542, AI349266,.

AI334452, AI349787, AI445237, AW084151, - -- AI344938, AW151138, AA614517, AI348969, AI345370, AI312146, AI811192, AI312339, AI309431, AI340537, AI345156, AI345739, AI345674, AW083572, AW022102, AI366959, AI345258, AI889168, AI440263, AI345737, AI312143, AI344935, AA579232, AW079334, AI349637, AI307459, AI343140, AW068845, AI866465, AI310575, AI334920, v ~ AI434256, AA176980, AI336654, AI349971, AI345014, AI174910, AW081255, AI815232, AW022494, AI345639, AW071276, AI307507, AI348879, W33163, AI684013, AI310927, AW020288, AI343030, AI311604, AI345562, AI307578, AI336488, AI349805, AI335235, AI336565, AI471909, AI349955, AI271234, AI307456, AI312210, AI307454, AI273856, AI313346, AW075093, AW079736, AI311440, AI648408, AI379711, AI312432, AI310930, AI340533, AI312271, AA833760, AW020710, AI345817, AI241678, AI312357, AI349269, AI312333, N27632, AI310945, AI340552, AW303089, AI584130, AI307735, AW190808, AI345471, AA062896, AI340511, AA455772, AI312431, W48671, AI312168, AI334895, AI468873, AI002285, AI312237, AI312144, AI784214, AI799189, AW191003, AW162189, AI312353, AI349738, AI659555, AB014607, AL080158, . U96683, X70685, AF087943, AL049324, _ AF016271, 577771, X86693, AL110196, 100734, U53505, A07647, E00617, E00717, ~

E00778, A18777, L13297, E01314, 575997, L40386, AF113013, X72624, E12579, AJ242859, X52128, AF113676, AL137555, A08913,189931, AF030165, U90884, 272491, A08912, E01963, A08910, I89934, I49625, A08909, AL080127, X99717, A08911, . AR038854, A08907, A08908, AF158248, A27171, AR068466, S76508, AF061573, AF100931, 563521, AF111851~
U68233, I92592, AF113694, I48978, AF022813, AF058921, AL133558, AL117583, X92070, J05032, X54971, AF036268, A12297, AL133606, AL137292, 222828, AL110159, AL137526, E12580, X55446, AL117649, AL080137, AL137527, AR053103;
AL050208, AF026124, AL080162, AF125948, Y08616, ' X06146, X59414, U02567, X72387, AL137539, AL050015, AF012536, AF118064, AL133093, AL122050, AL122111, AF094480, AF047443, AL133568, 553987, AF180525, AF085809, AJ012755, AF200464, AR054987, I41145, AF078844, AL133067, AL049464, X80340, U57715, Y10080, AL050280, ' AB025103, AF114818, E01812, AJ238278, L31396, L31397, X62773, A90832, AL137476, E01573, E02319, E02152, AL137276, U75932, E02914, AF067790, X60769, AF090943, AL133636, X99257, AF069506, AF142672, U68387, U57352, AL080126, X65873, I89944, AF082324, E15582, I22272, AR034830, I96214, AF175903, 536676, AF176651, AL110197, AL133098, AF044221, AF162270, AL050108, AC006112; Y08769, AL080086,189947, AF031903, AF017437, 578453, Y10823, AF067728, X87582, AF051325, AF079763, U30290, AF199027, AL050143, U00763, U35846, AJ006417, AL137665, X57961, AL110171, AF000167, X63410, M19658, AJ001838, AF118070, AF065135, L40363, M30514, AL133624, 237987, M69222, U49434, AL122093, X66975, U58653, AL117435, AL137641, X63574, AF019298, AF008439, AL049452, AF201468, X72889, AP000083, X00861, U75378, U66274, 561953, AB019565, Y11435, AF113690, A57389, AF118094, AL137298, AB026995, AL050024, U92068, AF038847, AL133640, AF155119, L30117, AF159148, AL080234, ~. AL137548, S69510, M27260,'AL122045, AJ010277, AL110228, S68736, AL137273, AL050155; A52563, AJ012582, AL096751, AF137367, AL137521, U91329, AF182215, AL122123, AR011880, AL035458, AC004093, AL137712, Y10655, AF028823, AL137283, X76228, AF215669, AL049314, AF036941, AF106827, I42402, I17767, and AR059883.

HNFCS26 110 8994061 - 1687 15 - H70763, AW376414, AW403498, 1701 AW205281, AW204171, AW450761, AI571894, AF114491, AC004262, AC005327, AC004999, AF053356, AC004841, AL021707, AC003010, AC004834, AC007151, M22403, AC007663, and AL034400.

HEMCL65 111 11979081 - 2754 15 - AI813787, AA156022, AI803963, 2768 W88875, AI184863, W16503, AA156150, AA876225, AA746200; N78762, AI265775, AA011319, AA630260, 006489, W88669, AI278949, AA302765, W76132, AA335675, AI903080, 884531, AI217602, AA335358, AI352263, 020950, AA010931, AI903083, N93972,~

T05694, AI366745, AA341407, T59150, AW294276, AA303624, AA302509, W40341, H62113, 816422, T59188, 853174, AA302764, AI265773, AI902552, H51927, 899429, H61208, AI129190, AA327257, T48993, AI926445, 899441, AA358843, 884975, AI902562, 885924, and T87725.

HMAMB94 112 12047081 - 2358 15 - AW369653, AW024321, AA766018, AA905970, AI652025, AA158332, AW068035, AI627492, AA158041, AI493579, 225180, W40230,,AI536877, AA191353, W45464, AW175724, AA670064, AA190444;

AA247921, AL121448, AA248856, AI926451, AI983662, W60091, AW068830, 890961, 890977, 890972, and AA309409.

HKMLN95 113 11957971 - 2002 15 - AA551127, AI692457, AI765517, 2016 AI749951, AI631959, AI129348, AI949762, AI672100, AI950134, AI609235, AI692456, AI65,1144, AW189207, AI935651, AA868261, AI151427, AA044198, W63627, AI521732, AI949853, AA161274, AW300441, AI708643, AA868518, AI962729, AI150783, AI015909, AA595810, AI281874, AI819752, AI479243, AI745688, AI341421, AW027973, AW022195, AA132312, H99174, AA429830, AA070213, N52408, AA856798, AI913890, AI745679, AI554270, AA442125, AA554278, AA161275, AW016589, AA702375, N24457, AA969821, AA070298, AI635327, AA699477, AI458226, AA043064, AI982949, AW439708, AI687133, AW272645, AW177545, AA946996, AW341771, AW177556, AI342767, 899590, N95053, AW402507, AI074359, AI630618, H84183, AA557498, 825323, AA446257, 843298, AW243239, AI583569, AW 194714, AA551069, 892184, AA557798, AI433955, AA714014, N66644, AI824194, 887671, T57956, AA313194, AI208421, AI921595, T57874, AA027072, AA156655, 887665, AI370681, 814400, AA352103, 887659, AA860614, AI140574, 824026, N58584, 887672, 238717, AI870045, AW151040, 884296, AA542839, AI277638, 892288, AA307482, AI954284, AI472463, N67635, AA442124, 818926, 884303, AI632684, N72814, AI472552, 242525, 884309, T94235, R26~521, AA091407, AA876334, T26330, AA565557, AA609829, N53150, AF078850, U81186, AF064635, ' AW473112, AW473746, AW474308, AW517836, AW611546, and AW611946.

HCQCI06 114 10826331 - 198215 - AI827458, AI627268, AI654812, 1996 AI123952, .

AI093158, AI399783, AI419376, AI338834, AI308129, AI800234, AI082388, AW022777, AI751915, AA256582, AI675002, AW015280, W78050, AI962653; AA405577, AA405578, AI247176, H28492, AA636051, AI14105,1, AI751916, AI633550, AA318231, AA143272, AI081761, AI819602, D62975, AI379282, AA852344, AA343597, AI143558, AA625818, AI378667, W79144, AA770096, AI867823, W81546, AA938549, AI421066, AA897387, AI219502, AI925096, AI088303, D45547, . AI886278, AA345718, AW006261, AA304549, AA628653, AA328200, D79724, W81545, D80253, D51423, D57483, D58283, D80043, D59889, D59859, D80188, D80366, D80166, D59619, 014429, D80210, D51799, D80240, D80212, D80195, D80391, D80219, D80022, D80227, D80196, D81030, 014331, D59927, D80024, D80269, D80038, D59610, D80193, D59502, D59275, D50995, 014014, D80241, D80045, 075259, D59467, D50979, D59787, D80378, T03269, D80164, 015076, ~

AA305409, C14389, D51060, AI557751, --- - D80134, D58253, AW 178893, - - D51250, D80268, F13647, D81111, D51079, D81026, D80949, D51022, D80251, AW179328, D80168, AW377671, C14227, AW378532, AW177440, AA305578, AW178775, C14407, D80522, 221582, D80248, AW369651, AW352158, AI910186, AW178762, AI905856, AA514188, D59695, D52291, C05695, D80133, AA514186, C14298, D51097, D80064, AW177501, AW177511, AW360811, AA285331, AW360834, D80247, AW352117, AW176467, AW375405, AW378540, AW179024, AW366296, AW360844, AW360817, AW375406, AW378534, AW179332, AW377672, AW179023, AW178905, AA809122, D80132, AW 177505, D80439, D59373, D80302, AW3S2171, AW377676, AW178906, AW3S2170, AW177731, AW178907, ~

AW 179019, AW179018, AW360841, AW179020, AW179220, AW178909, T11417, AW177456, AW179329, AW178980, AW177733, AW378528, AW178908, AW178754, D80014, AW352174, AW179004, D80157, AW 179012, D51103, AW 178914, D45260, AW378525, D51759, AW 177722, AW177728, AW179009, AW178774, AW178911, AW378543, AW367967, - AW352163, C06015, AW178983, D58246, AW352120, T02974, T03116, AI557774, AW178781, T48593, H678S4, D59653, AI535961, D58101, AW177723, D80258, AI535686, D59503, C14344, D51213, C14975, D59627, AL117664, A62298, A84916, A62300, AR018I38, AJ132110, Y17I88, X67155, A78862, A67220, D89785, A25909, D26022, D34614, X82626, D88547, AR025207, X68127, AR008278, AF058696, AB028859, A82595, I82448, Y12724, AB012117, A30438, A85396, 282022, AR066482, A44171, Y17187, A85477,119525, A86792, AR060385, X93549, AF135125, A94995, U87250, AB002449, AR008443, AR008277, AR008281, U79457, I50126, I50132,I50128,I50133, AR066488, AR016S14, AR060138, A45456, A26615, AR052274, U46128, AR016691, AR016690, Y09669, A43192, A43190, AR038669, , X64588, AR066487, AR054175, AR066490, D88507, I14842, I18367, D50010, AR064240, A63261, AR008408, AB033111, AR062872, A70867, A64136, A68321, D13509, AR060133, I79511, A63887, U87247, AB023656, 232749, AF123263, AR032065, X93535, and AR008382.

HBODA38 l15 12017271 - 262615 - AA196401, W79841, AA112831, 2640 AA197134, C05212, W19681, AA195812, 225012, . AI004215, 230134, AA194641, AI809925, AA193095, AA258550, 228480, F29133, AA699576, F20387, AI129717, AA194359, AI079487, AA195355, AA195094, AA931319, F34387, 219343, F36761, H67895, AA258604, AI378821, F1932I, AI089231, R9I 150, AI702453, AI288969, AI313118, AI351843, F31038, N56278, AI381419, AA195356, AA131264, AA195051, AA192095, AI264911, and AA086284.

HCYBK19 116 11786091 - 191215 - AI829726, AI809562, AI927757, 1926 AI927758, AI927768, AA165336, AW297770, AI638072, AI149852, AW298053, AI365991, AA904547, H15885, AA985065, AW207262, AI470080, AA305419, AA985170, AI423550, AA927292, AI654827, AI365324, AI365149, AW341104, AI797740, D81295, AI953516, AA379840, AA969956, H52748, H01576,R37302, AA905264, 243289, 818887, H04859, T31617, H04764, AA634475, T78758, 244077, H01477, H00918, D81446, M86147, AA983881, AI359235, AI421572, AA318875, AA165369, 239361, AI357382, AA909430, F03272, N34301, AA746048, T61118, T61231, T99075, N76169, W47509, AA022553, AA022563, AA0226I9, AA022756, AA037432, AA037536, AA040799, AA041237, AA135434, AA135851, AA187282, AA526958, AA534796, AA808710, AA811857, AA917399, AA932446, AA976524, AA994096, AI096731, AI097577, 001224, AA486848, AA487210, AA599808, 220518, AA775018, AA815473, AA868002, AA9164.85, AA954587, AI077798, AI04183.7, 240389, 243773, 244465, F01533, F03038, AI337051, AI337103, AI361239, AI365422, AI393007, AI418804, AI422355, AI566775, AI140741, AI167671, AI168446, AI217880, AI659801,.AI638311, AI253554, AI263824, AI340097, AI672005, AI672254, AI689887, AI766177, AI699568, AI824694, AI825633, AI857559, AI859594, AI870796, AI870807, AI890982, AI810442, AI816854, AI951417, AI955206, AI968385, AW002654, AW003862, AW006897, AW055000, AW084952, AW090376, AW075616, , AW167474, AW196370, AW197118, AW263060, AW274972, AW299439, AW513094, AW628101, AW768855, and AW779887. ' HOFNH30 117 9283651 - 362 15 - AF186380, and AF127138.

HWMEV63 118 9311541 - 440 15 - D13626, and AC078816.

HBXBG65 119 9327801 - 521 15 - 836281, AF094480, and AF094479.

HSDHB12 120 941973I - 624 15 - AA447298.

HLWAR77 121 9474841 - 127515 - AA449919, AA449920, and AF119815.

HE8AM92 122 11659911 - 193315 - AW195380, N23786, AI807492, 1947 AI734217, AA425224, AA634119, AI668657, AI088774, AI805290, AI693531, W20302, AI344107, AI830204, AA071290, AI023328, AI446315, AI764987, AI367900, AW069189, AI088165, AA541267, AA779556, AA228107, AI040754, AA552601, AA314127, 854083, AI263054, AA521303, H29680, AA046045, AI870566, AA122313, AI921148, AA815201, AA884602, 860682, AW297058, H08571, AI361541, 860683, AA747446, AI284342, T66056, AA873444, AA122312, AA386170, AA399256, AW242164, 819696, 818168, F13183, D52689, 243231, AI093433, N36074, T77025, AA151813, T65297, AA350782, AI309309, AA576719, 854134, D25599, AW004629, 836565, N46213, AI620774, T33549, AA811454, C20572, 812223, AA337811, F02683, F10790, AI421110, F03793, F12174, 834437, AA888883, AI289652, AW105294, F07164, F08075, AI355753, H29594, AA328526, T36239, AA975250, 850027, F06393, F11810, 840964, 839944, 848971, 841712, N36942, F07553, AA642020, AW367327, AA279402, AI354930, AI244963, AI092767, AW008997, 844252, 850366, AI335889, AI460092, AI766428, AA806386, AA334776, AI358493, . AA331654, AA774074, 824870, F05321, F05907, AA577350, AI569329, AI273806, AI358182, F03445, F05235, 844573, F05322, N69072, F01488, AA902193, AI284343, AA046154, AI335808, AI022959, F05267, 814569, AW069641, AI471308, F01581, D61303, AA425500, T25071, AI283001, T24915, AA071293, AA984503, F02156, AA748545, T3I990, F01582, FOI520, AI699076, 858045, AA412231, N90680, T10067, AA169608, AW371978, AJ243721, and AF182814.

HWLXT48 123 11522821 - 734 15 - AW449I48, AA932449, AW245862, AA306873, N44651, AA305881, AA088641, N31513, AA906040, 825850, 888'663, AI07340I, AI824292, AW248398, AA13717I, AW189250, AI952811, AL136295, and AF044127.

HBCPT10 124 11523251 - 941 15 - AW449148, AA932449, AA306873, ~

AW245862, N44651, AA305881, AA088641, N31513, AA906040, 825850, 888663, AI073401, AI824292, AW248398, AA137171, AL136295, and AF044127.

HCHAS12 125 11719701 - 1248 15 - AI539386, AA868538, AI190303, 1262 AI2I9986, AI345954, AA988977, AI309975, AI338679, AI200426, AI720044, AI827995, AI807471, N37092, AA932930, W74439, AW268605, AI202768, AI829710, AI436105, AI808710, AI148589, AI091541, AA865258, AI332422, AA883578, AI830140, AI222787, AA435561, AI536845, AA436117, AI742423, AI476645, AA976254, AI684261, AI991280, AI040961, . AI204236, AI09I532, AW001083, AA776717, AI911731, AI807161, AI798704, AW 195890, AA758038, AA884231, AA906270, AA884285, AW084515, AI286196, -'---- AA917965, AI149711, AI203679, AA740667, AA923373, AI936554, AA846787, AI167652, AA843421, AA994527, AW391262, AI142135, AA393148, AI083755, AA456074, AW043785, AI833323, AA725406, AI393567, AI243219, AW269733, AW304042, AI243370, AA910051, AI694334, AI200425, AI291760, AI167246, AI679583, AA757222, AI027967, AI688130, AI031908, AW188625, AI311479, AI025986, AA758549, AI318065, AI150116, AI799122, AW 166483, AI091504, N29346, AI935008, AA897637, AI241349, W58740, AI347903, AA972041, AA962659, AA884703, AA748637, AI807973, AA996162, AA971938, AI024835, AI243421, AW183835, AI858097, AI798180, AI276559, AI083851, AI493367, N40362, AA875917, AA410432, AA505880, AI743644, AA305510, AA938552, AA904368, AI284271, N27547, AI911350, AI377383, 823891, AI187351, AA905015, AI214377, AW082764, AW371415, -. AA740333, AA843427, AA954270, AW085014, AI971480, AI689289, AA954722, N26589, AI216245, AA977877, AI220007, AA777492, W00472, 876814, AA815444, AA099550, 865987, AA969436, AA412317, AA455577, AA861386, AA815351, AI168675, AI698618, AI160545, AI762065, AA305179, AA952918, AI269132, W79084, AA835882, AI205806, AI243242, AI223152, 865986, AI198119, AA970621, AA999722, AI025228, H02479, AI215980, AW082794, AA927156, 883423, AA912408, AA928243, AI289227, AW137925, AA890I54, AA758323, AA932728, 863480, AA305134, AI216504, 871169, 823890, AA972542, H72396, H12460, AA885425, AA628621, AI272123, 889052, AW135447, AI749504, AA724803, - AA775373, 863278, AA877346, H01351, H01218, 875983, F37219, AA970616~

AI826276, AI189999, H59895, 876230, 831547, AI001978, H724,21, N39659, 802720, 873757, 873883, 873670, AA483809, 863227, AA885048, AW 104374, AA295914, H02478, AI885432, AI784120, AI149979, AA442745, 873969, 837557, H03862, 826664, H17430, AA853105, AA832163, 825112, 876229, H00834, AA833569, H03308, 879832, 836714, 831066, D60944, AI417747, AA579968, AA305581, AA429715, . AA705903, 876442, AI559375, AF151810, and AF039696. ' HAPBS07 126 9673251 - 742 15 - AA284453, AA481096, AA465418, 756 W17165, AW405741, AW404541, AA304808, AR028677, AF067174, arid AC007556.

HWLPR94 127 11948151 - 1514 15 - AI978918, AA609992, AW188672, AA284453, AW405741, AW404541, AA608938, AA481041, AA481096, AA304808, AA465418, AA287021, W 17165, AI982593, AI540484, AA640217,---AI041126, AI358701, AW059769, AR028677, AF067174, and AC007556_.

HAPOI67 128 971184 1 - 219015 - H70763, H70762~, AW376414, . 2204 AW403498, AI571894, AW450761, AF114491, AC004262, and AC005327.

HCBME31 130 971321 1 - 147015 - AW385618, AW385612, AW385608, AI394057, AA031354, AW293177, AA031353, AA369669, AI174266, AW403498, AW376414, AA157797, AW376997, AW376921, AF114491, and AC004262.

HJMBP48 131 11358671 - 142315 - U66688, AI420480, AI656922, 1437 AI168486, AA116008, AA116009, AW015363, AI083583, AI680780, 889046, AI825488, N22021, H67248, AA357634, AA719140, AA309016, AI989486, AI380719, AA524463, H49149, AA190206, AA585380, AI554218, AI6792.11, AI251830, AI306613, AI640379, AI702073, AI612750, AI867042, AI866002, AI680457, AI566630, AI927092, AA830821, AW079159, AI80S638, AI249877, AW 193274, AW002174, AI624668, AI919345, AI890784, AI285431, AI860537, AI919107, AW083804, AI366549, AI699862, AI636719, AI699011, AI952920, AI308032, AW 104146, AI344785, AI766980, AC568854, AI250293, AW087938, N74355, AI241819, AI932949, AW088899, AW088134, AW192226, AI264741, AI828731, AW089179, AW074161, AI696819, AI680280, AA847936, AI539711, AI566507, AI570966, AI446373, AI498579, AI816954, AI952064, AW085667, AI365256, AW085786, AI432040, AI636619, AI591407, AI619716, AI888944, AI612759, AW151729, . AI280661, AI5376I7, AI610429, AI570I42, AW130863, AI682779, AI244380, AI890182, AI610645, AI860045, AW163823, AI859464, AI805688, AI611743, AI696626, AI589993, AI687065, AW265004, AI280670, AI828367, AI677797, AW193203, AI922577, AI874151, AI355849, AW082040, AI375730, AI952360, AI539153, AI866741, AI620093, AI783861, AW243814, AI678762, AI799472, AI446003, AI224992, AI674912, AW 151750, AW129271, AI476077, AW103371, AI863191, H42825, AW 192375, AW 172723, AW 131999, AI249257, AW403717, AI680539, AI583054, AI628292, AW118512, AW198075, AI500039, AI432736, AW004886, AW193872, AI922365, AW 189415, AI608676, AI537837, AI590686, AI885974, AI587606, AI468872, AI561299, AW195957, AW192288, AW029263, . .

AI554484, AW081255, AI6~5094, AW167882, AW131954, AW152550, AI628833, AA761557, AI433976, AW168031, AI952302, AW102785, AI912409, AI866608, AI866887, AI678443, AI680498, AW084219, AW103886, A1521386, AI633419, AW 103195, AI627748, AW 170674, AW151625, AI339435, AW188491, AL036215, AI367210, AW051107, AW192042, AI345677, AL120853, AI499986, AI697324, AI933785, AW 191844, . AW089327, AI873644, AI633306, AW 117907, AI689175, AW410972, AI434242, AI805769, AI587288, AW235035, AI887211, AI569523, AW189268, AW196868, AI689420, AI475947, AL119791, AI917959, AI288116, AW105620, AI811307, AI249946, AI922618, AI431979, AI678411, ' AL041772, AW029271, AI554544, AW090494, AI818578, AW130584, AI568870, AI866786, AI469811, AI500146, AW 14871'6, AI648684, AI249962, AI288050, AW 103893, AI254727, AW 162189, AI679916, AI446124, AI364788, AF047690, AF119337, AF000145, I48978, I42402, AF008439, E03348, U96683, AF113676, . A18777, I09360, A08916, I89947, A08913, A93016, I89931, AR059958, A08910, I49625, A08909, AR038854; AL122098, AF162270, AL137429, AL137527, AL133072, AC002467, AL122118, AF081197, ALI33645, AL137300, A08908, I66342, AL137294, AF081195;

I41145, AR038969, AF003737, AL137556, AF113689, L30117,.AF125949, AF017152, ALI37712, ALI33077, A089I2, I48979, X93495, AL122111, AF1111~12, AB019565, AL080060, AR019470, AJ242859, AL137538, S68736, I26207, AL133104, AL049452, ' X62580, AL117440, AL080I27, AL080137, S79832, AF022363, AL137476, AF012536, ' ' AL080158,.AL137273, AL080086, AF067790, AL133067, AL137526, AL133093, X87582, E05822, AF132676, AF061836, X84990, A45787, AL137705, AF030513, AL050138, ' AL137283, AR000496, U39656, AF113691, AF104032, AL137463, X81464, Y11587, AL049382, I09499, AL133014, AJ006417, AL122123, AL133558, U67958, AL133081, AF210052, AL049465, AL117585, U80742, A1229'7, AL080074, AL050277, Y16645, ' Y08769, E02253, AL110196, AL122050, X92070, AL133075, E15569, AL096744, U72620, AL133568, AF079765, AF106862, 561953, A21103, AL117432, AF028823, ArF100931, AL122049, L19437, AF118064, ' AF118070, E04233, AL137478, AL080159, AL133640, 272491, AL133098, X52128, AL133557, AB007812, S69510, AF061795, Y14314, AF151685, AL133016, AF158248, U49434, AF'06I98I, AF185576, AFI46568, - U78525, AL080148, AL133113, AL133565, E02221, AL122121., U00763, A18788, I89934, -- -w- ~- I89944, A65341, E03349, 282022, -- 237987, AL110221, A08915, AF032666, X53587, Y10080, AF090896, AF139986, X65873, AJ012755, AL137557, I33392, AL137648, AL137459, U68233, I92592, A93350, E07108, E08631, S78214, AF113013, U91329, I00734, X83508, I68732, AL049464, AL117649, AF113019, AF090934, and AL049466.

HE8UT58 132 11320551 - 1343 15 - AL079991, AI808909, AW409774, AA404732, AW409667, AW341107, AI433410, AI341147, AI885266, AI692534, . AA782257, AI027623, AI681129, AA659407, AA533117, AA778251, AA923181, H29006, AA099037, AI955317, AA099036, AA905366, AA677654, AI276678, AI986154, AA405418, AI347168, AW 135263, AA404602, AA503263, AA868124, H09118, AI420387, H11272, AA468344, AA365338, AW137535, AA402925, AI675497, AA371047, T12623, AA430277, T08839, AA813337, H29109, AF116574, AB014534, AF116573, and AW474492.

HDPBI30 I33 9747111 - 2911 15 - AA714520, N78665, W 15172, 2925 AL134531, AA074818, AI251157, AI311635, AA079403, AW 130754, AI935943, AF083955, AC005015, AL034423, AP000030, AC002992, AC004216, AC003013, U91321, AC003684, AC002528, AL117258, AL021155, AP000045, AF053356, AL033521, AC004598, U91326, AL035072, AD000091, U82668, AC012384, L44140, AF006752, AL034350, AC006039, AC005756, AC005072, AL034429, AC002352, AC005682, AC003663, AC005049, AC007298, AC005620, AC004887, AL117694, AC005911, AC007688, AC006014, AC004797, AL031186, AL031283, AC004963, L47234, 284466, AC004125, AC005529, AL031293, AC006276, AL034400, AC004099, AC005089, AL049871, AC004893, AL080243, AC007021, AL049712, AC007993, AC006581, AC005837, AF139813, M13792, AC005086, AL096791, AJ251973, AC002301, AC006139, AC005488, L78810, AC006115, AC004966, AC006538, 293244, AC004834, AL049570, AC004084, AP000113, AP000251, and AC005696.

HFXJI27 134 12190401 - 3094 15 - AL118569, AL118563, AA928678, AW 182646, AA909963, AA206533, -~

AI890750, AA932204, AA909961, AA233080, AA922947, AI336216, AA922946, AI433872, AW081307, AI245931, AI493179, AI817788, AI373318, AI373111, AI049514, AA670204, AI335925, AA947133, AW022288, AA827770, AI476063, AI888471, AI355877, AA187638, AI358468, AI587384, AI888010, N59174, AI619918, AI266210, N75622, AA610540, AA015788, AI625385, AI127888, AW269052, N39062, AA976881, AW339040, ' AI268557, T62565, D79411, H03654, - w - -. AI268071, AA015789, 824006, 835227, D11573, 879709, AI432041, N22381, T36156, H01601, H13080, AA324493, AA044342, 882836, F12891, AW151373, AA948492, 823916, T63607, T74982, H89128, H02993, 833979, 228580, H84296, AI220697, AA361239, AA341449, H13284, AA018661, N75910, AA044223, AA296530, T63317, AI679618, T63187, 833792, 821997, 836431, T06189, AA334502, AA579309, 882837, 843578, F10497, X85701, 826373, D62713, - 833791, 821886, AW023089, N67948, AA089544, N57061, AA018660, H01498, 821841, 821996, 836149, 220886, AA678512, 858745, AA206395, X85702, AW001736, D29213, AI432644, AI623302, AI431337, AI432653, AW081103, AI431254, AI431230, AI432654, AI492519, AI432662, AI432649, AI431243, AI432647, AI432665, AI432677, AI431248, AI431347, AI431328, - AI431354, AI432655, AI431310, AI431312, AI431330, AI432650, AI432651, AI432661, AI432675, AI432658, AI431241, AI431357, AI432664, AI431351, AI431353, AI432672, AI432674, AI431345, AW 128900, AI431346, AI431255, AI432676, AI432673, AW128846, AI4313.07, AI431316, AI431340, AI791349, AI431238, AW128897, AI492520, AI432645, AI432666, AW129223, AI431751, AI492510, AI432643, AI432657, AI492509, AI431247, AI431236, AI431314, AI431308, AI431231, AI431257, AW129514, AB023187, Y17793, AF064854, AF019249, AW472861, AW517286, AW591915, and AW629869.

Code Description Tissue r an Cell DiseaseVector Line .AR022a Heart ~ a Heart AR023a Liver a Liver AR024a mammary Qlanda mammary oland AR025a Prostate a Prostate AR026a small intestinea small intestine AR027a Stomach a Stomach AR028Blood B cells Blood B cells AR029Blood B cells Blood B cells activated activated AR030Blood B cells Blood B cells resting resting AR031Blood T cells Blood T cells activated activated AR032Blood T cells Blood T cells resting restin AR033brain brain AR034breast breast AR035breast cancer breast cancer AR036Cell Line CAOV3Cell Line AR037cell line PA-1 cell line AR038cell line transformedcell line transformed AR039colon colon AR040colon (9808co65R)colon (9808co65R) AR041colon (9809co15)colon (9809co15) AR042colon cancer colon cancer AR043colon cancer colon cancer (9808co64R) (9808co64R) AR044colon cancer colon cancer 9809co14 9809co14 ' AR045.corn clone 5 corn clone AR046corn c[one 6 com clone AR047corn clone2 corn clone2' AR048com clone3 corn clone3 AR049Corn Clone4 Corn Clone4 AR050Donor II B CellsDonor II
24hrs B Cells 24hrs AR051Donor II B CellsDonor II
72hrs B Cells 72hrs AR052Donor II B-CellsDonor II
24 hrs. B-Cells hrs.

AR053Donor II B-CellsDonor II
72hrs B-Cells 72hrs AR054Donor II RestingDonor II ' ' .
B Cells Resting ' B
Cells AR055Heart Heart AR056Human Lung (clonetech)Human Lung (clonetech) AR057Human Mammary. Human Mammary ' (clontech) (clontech) AR058Human Thymus Human Thymus ' (clonetech) (clonetech) AR059-Jurkat (unstimulated)Jurkat (unstimulated) ARO60Kidne Kidne AR061Liver Liver AR062Liver (Clontech)Liver (Clontech) AR063Lymphocytes w Lymphocytes chronic lymphocytic chronic lymphocytic leukaemia ' leukaemia AR064Lymphocytes Lymphocytes diffuse large B cell lymphomadiffuse large B cell lymphoma AR065Lymphocytes Lymphocytes follicular 1 m homa follicular 1 m homy AR066normal breast normal breast AR067Normal Ovarian Normal Ovarian (4004901) (4004901) AR068Normal Ovary Normal Ovary AR069Normal Ovary Normal Ovary AR070Normal Ovary Normal Ovary AR071Ovarian Cancer Ovarian Cancer AR072Ovarian Cancer Ovarian Cancer (97026001) (97026001) AR073Ovarian Cancer Ovarian Cancer -(97076029) (97076029) AR074Ovarian Cancer Ovarian Cancer (98040011 ) (98040011 ) AR075Ovarian Cancer Ovarian Cancer (98066019) (98066019) AR076Ovarian Cancer Ovarian Cancer (98076017) (98076017) AR077Ovarian Cancer Ovarian Cancer (98096001) (98096001) AR078ovarian cancer ovarian cancer AR079Ovarian Cancer Ovarian Cancer AR080Ovarian Cancer Ovarian Cancer.
-AR081Ovarian Cancer Ovarian Cancer AR082ovarian cancer ovarian-cancer 94.127303 .

94127303 , AR083Ovarian Cancer Ovarian Cancer-AR084Ovarian Cancer Ovarian Cancer AR085Ovarian Cancer Ovarian Cancer 98076045 - - -- w--AR086ovarian cancer.9809G001ovarian cancer AR087Ovarian Cancer Ovarian Cancer AR088Ovarian cancer Ovarian cancer 3rd C00 3rd AR089Prostate Prostate AR090Prostate (clonetech)Prostate (clonetech) AR091rostate cancer rostate cancer AR092prostate cancerprostate #15176 cancer #15176 AR093prostate cancerprostate #15509 cancer #15509 AR094prostate cancerprostate #15673 cancer #15673 AR095Small IntestineSmall Intestine (Clontech) (Clontech) -AR096S leen S Teen AR097Thymus T cells Thymus T
activated cells activated AR098Thymus T cells Thymus T
resting cells resting AR099Tonsil Tonsil AR100Tonsil geminal Tonsil geminal center center centroblast centroblast AR101Tonsil germinalTonsil germinal centerB center B
cell cell AR102Tonsil 1 m h Tonsil 1 node m h node AR103Tonsil memory Tonsil memory B cell B
cell AR104Whole Brain Whole Brain AR105Xeno raft ES-2 Xeno raft AR106Xeno~raft SW626Xeno raft H0002Human Adult Human Adult Heart Uni-ZAP
Heart Heart XR

H0004Human Adult Human Adult Spleen Uni-ZAP
Spleen S Teen XR

H0009Human Fetal . Uni-ZAP
Brain XR

H0011Human Fetal Human Fetal Kidne Uni-ZAP
Kidney Kidne XR

H0012Human Fetal Human Fetal Kidney Uni-ZAP
Kidne Kidne XR

H0013Human 8 Week Human 8 WeekEmbryb Uni-ZAP
Whole O~d XR
Emb o Emb o H0014Human Gall BladderHuman Gall Gall Bladder Uni-ZAP
Bladder XR

H0015Human Gall Bladder,Human Gall Gall Bladder Uni-ZAP
fraction II Bladder ~ XR

H0020Human HippocampusHuman Brain Uni-ZAP
Hi ocam us . XR

H0024Human Fetal Human Fetal Lung Uni-ZAP
Lung III Lun XR

H0028Human Old Ovar Human Old Ovar Bluescri Ovar t H0030Human Placenta Uni-ZAP
~ XR

H0031Human Placenta Human PlacentaPlacenta Uni-ZAP
XR

H0032Human Prostate Human ProstateProstate Uni-ZAP
XR

H0033Human PituitaryHuman Pituitary ~ Uni-ZAP
XR

H0036 Human Adult ,Human AdultSmall Uni-ZAP
Small . Small Iht. XR ' Intestine - Intestine H0038 Human Testes Human TestesTestis Uni-ZAP
XR

H0039 Human PancreasHuman PancreasPancreas diseaseUni-ZAP
Tumor XR

Tumor H0040 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor XR

Tumor H0042 Human Adult Human Adult Lung Uni-ZAP
Pulmonary XR

Pulmonar H0046 Human EndometrialHuman EndometrialUterus diseaseUni-ZAP
XR

Tumor Tumor H0050 Human Fetal Human Fetal Heart Uni-ZAP
Heart Heart XR

H0051 Human HippocampusHuman Brain Uni-ZAP
XR

Hi ocam us H0052 Human CerebellumHuman CerebellumBrain Uni-ZAP
XR

H0056 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein, XR

Endo. remake Vein Endothelialvein Cells H0059 Human Uterine Human UterineUterus diseaseLambda Cancer ZAP II

_ Cancer H0063 Human Th mus Human Th Th mus Uni-ZAP
mus XR

H0068 Human Skin Human Skin Sltin diseaseUni-ZAP
Tumor Tumor . XR

H0069 Human ActivatedActivated Blood Cell . Uni-ZAP
T-Cells T-Cells Line XR

H0071 Human Infant Human InfantAdrenal Uni-ZAP
Adrenal XR

Gland Adrenal Glandland H0075 Human ActivatedActivated Blood ~ Cell Uni-ZAP
T-Cells T-Cells Line XR

(II) H0081 Human Fetal Human Fetal Skin Uni-ZAP
Epithelium Skin XR

(Skin) H0083 HUIvIAN JURKATJurkat Cells Uni-ZAP
XR

MEMBRANEBOUND

POLYSOMES

H0085 Human Colon Human Colon Lambda ZAP II

H0086 Human epithelioidEpithelioid Sk Muscle- diseaseUni-ZAP
XR

sarcoma Sarcoma, muscle H0087 Human Th mus Human Th Bluescri mus. t H0090 Human T-Cell T-Cell L T-Cell diseaseUni-ZAP
L m home .m home XR

HO100 Human Whole Human Whole Embryo Uni-ZAP
Six Week Six XR

Old Embryo Week Old Embr o 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
I;ine XR

treated Treated Cem, Jurkat, ' ~ Ra'i, and ~ -Su t H0129 Jurkat cells, Jurkat Cells Uni-ZAP
thiouridine ' XR

activated, Tract II

H0130 LNCAP untreatedLNCAP Cell Prostate Cell Uni-ZAP
Line Line XR

H0132 LNCAP + 30nM LNCAP Cell Prostate Cell Uni-ZAP
81881 Line Line XR

H0134 Raji Cells, CyclohexamideBlood Cell Uni_-ZAP
cyclohexamide Line XR

treated Treated Cem, Jurkat, Raji, and Su t H0135Human Synovial Human SynovialSynovium Uni-ZAP
Sarcoma XR

Sarcoma H0136Supt Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line XR

. treated Treated Cem, Jurkat, Ra'i, and Su t H0144Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early XR

Sta eHuman Sta a Human H0150Human E idid E ididymis Testis Uni-ZAP
mus XR

H0156Human Adrenal Human AdrenalAdrenal diseaseUni-ZAP
Gland XR

Tumor Gland Tumor Gland H0163Human S novium Human S noviumS novium Uni-ZAP
XR

H0164Human Trachea Human TracheaTrachea diseaseUni-ZAP
Tumor XR

Tumor H0169Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer, XR

Stage C fractionCancer, sta a C

H017012 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old XR

Human Earl Sta a Human H017112 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old XR
I

Human, Earl Sta II a Human HOI72Human Fetal Human Fetal Brain Lambda Brain, Brain ZAP II

random primed H0176CAMAlEe Cell CAMAlEe CellBreast Cell Uni-ZAP
Line Line XR

Line H0178Human Fetal Human Fetal Brain Uni-ZAP
Brain Brain XR

H0179Human Neutro Human NeutroBlood Cell Uni-ZAP
hil hil Line XR

HOI81Human 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 H0188Human Normal Human NormalBreast Uni-ZAP
Breast XR

Breast H0191Human ActivatedHuman Blood Cell Uni-ZAP
Line XR

Macrophage (LPS),Macrophage/Monoc ' .
thiour tes H0194Human Cerebellum,Human CerebellumBrain pBluescript ~

subtracted H0196Human Cardiomyopathy,Human Heart Uni-ZAP
XR

subtracted Cardiom o ath H0197Human Fetal Human Fetal Liver Uni-ZAP
Liver, Liver XR

subtracted H0199Human Fetal Human Fetal Liver ~ Uni-ZAP
Liver, Liver XR

subtracted, ne clone H0204Human Colon Human Colon Colon pBluescript Cancer, subtracted Cancer H0208Early Stage Human Fetal Lung pBluescript Human Lung, Lung 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 H0216Supt cells, CyclohexamideBlood Cell . pBluescript cyclohexamide Line treated, subtractedTreated Cem, Jurkat, Raji, and Supt H0222Activated T-Cells,Activated Blood Cell Uni-ZAP
8 hrs, T-Cells Line XR

subtracted H0244Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old XR

Emb o, subtractedEmbr o .

H0250Human ActivatedHuman Monocytes Uni-ZAP
XR

Monoc tes H0251Human ChondrosarcomaHuman Cartilage diseaseUni-ZAP
XR

Chondrosarcoma H0252Human OsteosarcomaHuman Bone diseaseni-ZAP XR
U

Osteosarcorria H0253Human adult Human Adult Testis Uni-ZAP
testis, large Testis XR

inserts H0254Breast Lymph Breast LymphLymph Uni-ZAP
node cDNA Node Node XR

libr -H025~breast lymph Breast LymphLymph Lambda ZAP
node CDNA Node Node II

librar H0261H. cerebellum, Human CerebellumBrain Uni-ZAP
Enzyme XR

subtracted H0263human colon Human Colon Colon diseaseLambda ZAP
cancer II

Cancer H0264human tonsils Human TonsilTonsil Uni-ZAP
XR

H0265Activated T-CellT-Cells Blood Cell ' Uni-ZAP
Line XR

( l2hs)/Thiouridine labelledEco H0266Human MicrovascularHMEC Vein Cell Lambda ZAP
Line II

Endothelial Cells, fract.
A ' H0268Human UmbilicalHUVE Cells UmbilicalCell Lambda ZAP
Vein Line II

Endothelial vein Cells, fract.
A .

H0271Human Neutrophil,Human NeutrophilBlood Cell Uni-ZAP
- Line XR

Activated Activated H0280K562 + PMA (36 K562 Cell cell lineCell ZAP Ex ress hrs) line Line H0282HBGB"s differentialHuman PrimaryBreast Uni-ZAP
XR

consolidation Breast Cancer -H0286Human OB MG63 Human Bone Cell . Uni-ZAP
treated Line XR

( 10 nM E2) Osteoblastoma fraction I

MG63 cell line H0294Amniotic Cells Amniotic -PlacentaCell 'Uni-ZAP
- TNF Cells - Line XR

induced TNF induced H0295Amniotic Cells Amniotic Placenta Cell ni-ZAP XR
- Primary Cells - U Line Culture Primar Culture H0298HCBB"s differentialCAMAlEe CellBreast Cell Uni-ZAP
Line XR

consolidation Line H0305CD34 positive CD34 PositiveCord Blood ZAP Express cells (Cord Cells Blood) ~ ' H0309Human Chronic Synovium, Synovium diseaseni-ZAP XR
Synovitis Chronic ' U

Synovitisl Osteoarthritis H0316HUMAN STOMACH Human StomachStomach Uni-ZAP
XR

H0318HUMAN B CELL Human B CellLymph diseaseUni-ZAP
~ Node XR

LYMPHOMA L m homa H0327 human corpus Human CorpusBrain Uni-ZAP
colosum XR

Callosum H0328 human ovarian Ovarian CancerOvar diseaseUni-ZAP
cancer XR

H0329 DermatofibrosarcomaDermatofibrosarcomSkin diseaseUni-ZAP
~ XR

Protuberance a Protuberans H0331.Hepatocellular HepatocellularLiver diseaseLambda Tumor ZAP II

Tumor H0333 HemangiopericytomaHemangiopericytomBlood diseaseLambda vessel ZAP II

a H0341 Bone Marrow Bone Marrow Bone CeII Uni-ZAP
Cell Line Cell Marrow Line XR

(RS4;11) Line RS4;11 H0342 Lingual Gyrus Lingual G Brain Uni-Za rus XR

H0344 Adipose tissue Adipose - Uni-ZAP
(human) 6825A XR

(human) H0345 SKIN Skin - 4000868HSkin Uni-ZAP
XR

H0351 Glioblastoma GlioblastomaBrain diseaseUni-ZAP
XR

H0352 wilm"s tumor Wilm"s Tumor diseaseUni-ZAP
XR

H0355 Human Liver Human Liver, pCMVSport normal Adult ' H0357 H. Normalized Human Fetal Liver Uni-ZAP
Fetal Liver XR

Liver, II

H0361 Human rejected Human Rejected diseasepBluescript kidney Kidney H0366 L428 cell line L428 ZAP Ex ress H0369 H. Atrophic Atrophic Uni-ZAP
Endometrium XR

Endometrium and m ometrium H0370 H. Lymph node Lymph node diseaseUni-ZAP
breast . with XR

Cancer Met. Breast Cancer H0373 Human Heart Human Adult Heart CMVS ort Heart 1 H0375 Human Lung Human Lung CMVS ort H0381 Bone Cancer Bone Cancer diseaseUni-ZAP
XR

H0390 Human Amygdala Human Amygdala diseasepBluescript De ression, De ression re-excision H0391 H. Meniingima, Human Menin brain S ortl M6 ima H0392 H. Meningima, Human Menin brain S ortl Ml ima H0393 Fetal Liver, Human Fetal Liver ' Bluescri subtraction Liver t II

H0395 Al-CELL LINE Redd-Sternberg ZAP Ex cell ress H0402 CD34 depleted CD34 DepletedCord ZAP Express Buffy Coat Blood (Cord Blood), Buffy Coat re-excision (Cord Blood) H0404 H. Umbilical HUVE Cells UmbilicalCell Uni-ZAP
Vein Line XR

endothelial vein ' cells, uninduced H0409 H. Striatum Human Brain,Brain pBluescript Depression, .

subtracted Striatum De ression H0411 H Female Bladder,Human FemaleBladder . p5portl Adult Adult Bladder H0412 Human umbilicalHUVE Cells UmbilicalCell pSportl vein Line endothelial vein cells, IL-4 induced 1 s6 H04I3Human UmbilicalHUVE Cells UmbilicalCell pSportl Vein Line Endothelial vein Cells, uninduced H0414Ovarian Tumor Ovarian Tumor,Ovary diseasepSportl I, OV5232 H0415H. Ovarian Tumor,Ovarian Tumor,Ovary ,diseasepCMVSport II, 2.0 H0416Human Neutrophils,Human NeutrophilBlood Cell pBluescript - Line Activated, re-excisionActivated H0421Human Bone Marrow,Bone Marrow pBluescript re-excision H0422T-Cell PHA 16 T-Cells Blood Cell pSportl hrs Line H0423T-Cell PHA 24 T-Cells Blood Cell Sportl hrs Line H0424Human Pituita Human Pituitar Bluescri , subt IX t H0427Human Adipose Human Adipose, p5portl left hi 1i oma H0428Human Ovary Human Ovary Ovary pSportl Tumor H0431H. Kidney Medulla,Kidney medullaKidney pBluescript re-excision H0435Ovarian Tumor Ovarian Tumor;Ovary pCMVSport 10-3-95 2.0 H0436Resting T-Cell T-Cells Blood Cell S ortl Library,II Line H0438H. Whole Brain Human Whole ~ ZAP Express #2, re- Brain excision #2 ~H0441H. Kidney Cortex,Kidney cortexKidney pBluescript subtracted ' H04~k1Spleen metasticSpleen, MetasticSpleen diseasepSportl melanoma malignant melanoma H0445Spleen, ChronicHuman Spleen,Spleen diseasepSportl CLL , 1 m hoc tic leukemia H0448Salivary gland,Human SalivarySalivary Lambda subtracted ZAP II

Gland Qland H0455H. Striatum Human Brain,Brain pBluescript Depression, subt Striatum De ression H0457Human Eosino Human Eosino S ortl hils hils H0459CD34+cells, CD34 positive pCMVSport II, cells 2.0 H0478Salivary Gland,Human SalivarySalivary . pSportl Lib 2 Gland land H0483Breast Cancer Breast Cancer pSportl cell line, Cell MDA 36 line, MDA

H0484Breast Cancer Breast Cancer' pSportl Cell line, Cell angiogenic line, Angiogenic, H0485Hodgkin"s LymphomaHodgkin"s diseasepCMVSport I 2.0 L m homa I

H0486Hodgkin"s LymphomaHodgkin"s diseasepCMVSport II 2.0 L m homa II

H0488Human Tonsils, Human Tonsils~ CMVS ort Lib 2 2.0 H0489.Crohn"s DiseaseIleum Intestine diseaseS ortl . Keratinoc to Keratinoc CMVS ort H0494 to 2.0 H0497 HEL cell line HEL cell HEL pSportl line 92.1.7 H0506 Ulcerative ColitisColon ~ Colon S ortl 'H0509Liver, HepatomaHuman Liver,Liver diseasepCMVSport 3.0 He atoma, atient 8 H0510 Human Liver, Human Liver,Liver pCMVSport normal . 3.0' normal, Patient # 8 H0517 Nasal of s Nasal of CMVS ort s 2.0 H0518 pBMC stimulatedpBMC stimulated pCMVSport w/ poly 3.0 I/C with oly 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 Cehs, ~ 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, progeni for cell line H0530 Human Dermal Human Dermal pSportl Endothelial Endothelial ' Cells;

Cells,untreateduntreated H0538 Merkel Cells Merkel cellsL m h 5 ortl node H0539 Pancreas Islet Pancreas Pancreas diseasepSportl.
Cetl Tumor Islet Cell Tumour H0542 T Cell hel er HeI 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 H054S Human endometrialHuman endometrial pCMVSport 3.0 stromal cells-treatedstromal cells-treated with ro esterone with roe H0547 NTERA2 teratocarcinomaNTERA2, ~ pSportl cell line+retinoicTeratocarcinoma acid (14 da s) cell line H0549 H. Epididiymus,Human Uni-ZAP
caput & XR

corpus Epididiymus, .
caput and co us H0550 H. Epididiymus,Human Uni-ZAP
cauda XR

E ididi mus, cauda H0551 Human Thymus Human Thymus pCMVSport Stromal 3.0 Cells Stromal Cells H0553 Human Placenta Human Placenta CMVS ort 3.0 H0555 Rejected Kidney,Human RejectedKidney diseasepCMVSport lib 4 3.0 Kidne H0556 Activated T- - T-Cells Blood Cell Uni-ZAP
Line XR

cell(12h)/Thiouridine-re-excision ' H0559 HL-60, PMA 4H, HL-60 Cells,Blood CeII Uni-ZAP
re- PMA Line XR

excision stimulated H0560 KMH2 KMH2 CMVS ort 3.0 H0561 L428 L428 CMVS ort 3.0 H0567 Human Fetal Human Fetal pCMVSport Brain, Brain 2.0 normalized A5002F

H0570 Human Fetal Human Fetal pCMVSport Brain, Brain 2.0 normalized C500H

H0572 Human Fetal Human Fetal pCMVSport Brain, Brain 2.0 normalized AC5002 H0574 Hepatocellular HepatocellularLiver diseaseLambda Tumor; re- ZAP II

excision Tumor H0575 Human Adult Human Adult Lung Uni-ZAP
XR

Pulmonary;re-excisionPulmonar H0576 Resting T-CeII;T-Cells Blood Cell Lambda re- Line ZAP II

excision H0578 Human Fetal Fetal Th Th mus S ortl Th mus mus H0580 Dendritic cells,Pooled dendritic pCMVSport pooled 3.0 cells H0581 Human Bone Marrow,Human Bone Bone pCMVSport Marrow 3.0 treated Marrow H0583 B Cell 1 m homaB Cell Lym B Cell diseaseCMVS ort homa 3.0 H0584 Activated T-cells,Activated Blood Cell Uni-ZAP
24 T-Cells Line XR

hrs,re-excision H0586 Healing groin healing groingroin diseasepCMVSport wound, 6.5 3.0 hours post incisionwound, 6.5 hours ost incision H0587 Healing groin Groin=2/19/97groin diseasepCMVSport wound; 7.5 3.0 hours ost incision H0589 CD34 positive CD34 PositiveCord ZAP Express cells (cord Cells Blood blood),re-ex H0590 Human adult Human Adult Small Uni-ZAP
. small Small Int. XR

intestine,re-excisionIntestine H0591 Human T-cell T-Cell LymphomaT-Cell diseaseUni-ZAP
~ XR

1 m homa;re-excision H0592 Healing groin HGS wound . diseasepCMV Sport 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 Cancer;re-Human Lung Lung diseaseLambda 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 H0600 Healing AbdomenAbdomen diseasepCMVSport 3.0 wound;70&90 min ost 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 tes CMVS ort control 1 H0611 H. Leukocytes,H.Leukocytes pCMVSport normalized cot 500 B

H0615 Human Ovarian Ovarian CancerOvary diseaseUni-ZAP
Cancer XR

Reexcision H0616 Human Testes, Human Testes Testis 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 Fetal Heart Uni-ZAP
Heart XR

H0620 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney; Kidney XR

Reexcision H0622 Human PancreasHuman 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; ReexcisionEarl Sta a Human H0625 Ku 812F Baso Ku 812F Baso S ortl hils Line hill H0626 Saos2 Cells; Saos2 Cell pSportl Untreated Line;

Untreated H0628 Human Pre-DifferentiatedHuman Pre- ' Uni-ZAP
XR

Adipocytes Differentiated Adi ocytes H0632 HepatocellularHepatocellularLiver . Lambda Tumor;re- ZAP II

excision Tumor H0633 Lung CarcinomaTNFalpha activated diseasepSportl TNFalpha activatedA549--Lung Carcinoma H0634 Human Testes Human Testes Testis diseaseUni-ZAP
Tumor, re- XR

excision Tumor H0635 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells, T-Cells Line XR

re-excision H0637 Dendritic CellsDentritic pSportl From cells from CD34 Cells CD34 cells H0638 CD40 activatedCD40 activated pSportl monocyte dendridic cellsmonocyte dendridic cells H0641 LPS activated LPS activated~ pSportl derived dendritic cellsmonocyte derived.

dendritic cells H0642 Hep G2 Cells, Hep G2 Cells ' Other lambda - ' libra H06~I4Human PlacentaHuman PlacentaPlacenta Uni-ZAP
(re- XR

excision) H0645 Fetal Heart, Human Fetal Heart ~ Uni-ZAP
re-excision Heart XR

H0646 Lung, Cancer Metastatic pSportl (4005313 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,malignantpotentia Low Malignant Pot H0649 Lung; Normal: Normal Lung pSportl (4005313 B1) H0650 B-Cells B-Cells CMVS ort 3.0 H0653 5tromal Cells Stromal Cells 5 ortl H0654 Lung, Cancer: Metastatic Other (4005313 A3) Invasive Squamous Poorly- cell lung differentiated Carcinoma Metastatic poorly lun 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 ( 15395A 1 F): Carcinoma, Grade II Ovary Pa illar Carcinoma H0660 Ovary, Cancer: Poorly differentiated diseasepSportl ( 15799A 1F) carcinoma, Poorly ovary differentiated carcinoma H0661 Breast, Cancer:Breast cancer diseasepSportl (4004943 A5) H0662 Breast, Normal:Normal BreastBreast p5portl -(400552282) #4005522($2)' ~

H0663 Breast, Cancer:Breast CancerBreast diseasep8portl (4005522 -A2) #4005522(A2) H0665 Stromal cells Stromal cells S ortl 3.88 3.88 H0666 Ovary, Cancer: Ovarian Cancer, diseasep8portl (4004332 A2) Sample #4004332A2 H0667 Stromal cells(HBM3.18)Stromal cell(HBM~ pSportl 3.18) H0668 stromal cell stromal cell pSportl clone 2.5 clone 2.5 H0669 Breast, Cancer:Breast CancerBreast p8portl (4005385 A2) (4005385A2) H0670 Ovary, Cancer(4004650Ovarian Cancer-- p5portl A3): Well-Differentiated4004650A3 Micropapillary , Serous Carcinoma ~~1 H0672Ovary, Cancer: Ovarian Ovary pS ortl (4004576 ~ P

A8) Cancer(4004576A8) H0673Human Prostate Human ProstateProstate Uni-ZAP
Cancer, XR

Stage B2; re-excisionCancer, stage H0674Human Prostate . Human ProstateProstate Uni-ZAP
Cancer, XR

Staoe C; re-excissionCancer, sta a C

H0675Colon, Cancer: Colon Cancer pCMVSport ~ 3.0 (9808C064R) 9808C064R -~

H0676Colon, Cancer: Colon Cancer pCMVSport . 3.0 (9808C064R)-total9808C064R
RNA

H0677TNFR de enerateB-Cells PCRII
oliao H0682Serous Papillaryserous papillary pCMVSport , 3.0 Adenocarcinoma adenocarcinoma (9606G304SPA3B) H0684Serous PapillaryOvarian Cancer-Ovaries pCIvIVSport 3.0 Adenocarcinoma 98106606 H0685Adenocarcinoma Adenocarcinoma pCMVSport of of 3.0 Ovary, Human Ovary, Human Cell Line, Cell # OVCAR-3 Line, # OVCAR--H0686Adenocarcinoma Adenocarcinoma pCMVSport of of 3.0 Ovary, Human Ovary, Human Cell Line Cell Line, # S

H0687Human normal Human normalOvary . pCMVSport 3.0 ova (#96106215)ovar (#96106215) H0689Ovarian.Cancer Ovarian Cancer, pCMVSport 3.0 #98066019 H0690Ovarian Cancer,Ovarian Cancer, pCMVSport # 3.0 97026001 #97026001 H0691Normal Ovary, normal ovary, pCMVSport 3.0 #97106208 #97106208 H0694Prostate gland Prostate prostate pCMVSport gland, 3.0 adenocarcinoma adenocarcinoma,gland, mod/diff, leason N0007Human HippocampusHuman Hi ocam us S0001Brain frontal Brain frontalBrain Lambda cortex cortex ZAP II

S0002Monocyte activatedMonocyte-activatedblood Cell Uni-ZAP
Line XR

S0003Human OsteoclastomaOsteoclastomabone diseaseUni-ZAP
XR

50010Human Amy~dala Am dala Uni-ZAP
XR

50011STROMAL - Osteoclastomabone diseaseUni-ZAP
XR

OSTEOCLASTOMA

50013Prostate Prostate rostate Uni-ZAP
XR

S0022Human OsteoclastomaOsteoclastoma Uni-ZAP
XR

5tromal Cells Stromal Cells -unam lified 50026Stromal cell stromal cellBone marrowCell Uni-ZAP
TF274 Line XR

50027Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
serum Line XR

treated arter 50028Smooth muscle,controlSmooth musclePulmanaryCell Uni-ZAP
Line XR

arter S0029brain stem Brain stem brain Uni-ZAP
XR

50031S final cord S final cords final Uni-ZAP
cord XR

S0032Smooth muscle-ILb- Smooth Pulmanar Cell Uni-ZAP
muscle Line XR

induced arter S0036Human SubstantiaHuman Substantia Uni-ZAP
Nigra XR

Ni ra S0037Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
ILlb Line XR

induced arter 50038Human Whole Human Whole ZAP Express Brain #2 - Brain OliQo dT > l.SKb#2 50040Adipocytes Human Adipocytes Uni-ZAP
XR

from Osteoclastoma 50044Prostate BPH rostate BPH Prostate diseaseUni-ZAP
XR

50045Endothelial Endothelial endothelialCell Uni-ZAP
cells-control cell Line XR

cell-lun S0046Endothelial-inducedEndothelial endothelialCell , Uni-ZAP
cell Line XR

cell-lun S0049Human Brain, Human Brain, Uni-ZAP
Striatum XR

Striatum S0050Human Frontal Human Frontal, disease.Uni-ZAP
Cortex, XR

Schizophrenia Cortex, Schizo hrenia SODS Human ' Human ' diseaseUni-ZAP

Hypothalmus,SchizophrenHypothalamus, is Schizo hrenia S0052neutro hils human neutroblood Cell Uni-ZAP
control hils Line XR

S0053Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line XR

induced induced 50106STRIATUM . BRAIN diseaseUni-ZAP
XR

DEPRESSION

S0110Brain Amygdala Brain ~ diseaseUni-ZAP
~ XR

Depression S0114Anergic T-cell Anergic T-cell ~ Cell Uni-ZAP
Line XR

S0116Bone marrow Bone marrow Bone marrow Uni-ZAP
XR

S0126Osteoblasts Osteoblasts Knee Cell Uni-ZAP
Line XR

S0132Epithelial-TNFaAirway Epithelial , Uni-ZAP
and INF XR

induced S0134A o totic T-cella o totic Cell . Uni-ZAP
cells Line XR

S0136PERM TF274 stromal cellBone marrowCell Lambda Line ZAP II

50142Macrophage-oxLDLmacrophage- blood Cell Uni-ZAP
Line XR

oxidized LDL

treated S0144Macrophage (GM-CSFMacrophage Uni-ZAP
1 (GM- XR

treated) CSF treated) 50148Normal ProstateProstate rostate Uni-ZAP
XR

SO150LNCAP rostate LNCAP Cell Prostate Cell Uni-ZAP
cell line Line Line XR

SO152PC3 Prostate PC3 prostate~ Uni-ZAP
cell line cell XR

line 50180Bone Marrow Bone Marrow diseaseUni-ZAP
Stroma, XR

TNF&LPS ind Stroma, TNF
& LPS

induced S0188.Prostate,$PH, Human Prostate diseasep5portl Lib 2 BPH

S0192Synovial FibroblastsSynovial _ pSportl Fibroblasts (control) 501945 novial h oxiaS novial ~ S ortl Fibroblasts 50196 Synovial IL-1/TNFSynovial . p5portl Fibroblasts stimulated S0198 TTM-pbfd PBLS, TTM PCRII
' receptor enriched S0206 Smooth Muscle- Smooth musclePulmanaryCell pBluescript HASTE Line normalized artery S0210 Messanaial cell,Messan ial S ortl frac 2 cell S0212 Bone Marrow Bone Marrow pSportl 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 50222 H. Frontal H. Brain, Brain diseaseUni-ZAP
Frontal XR

cortex,epileptic;re-Cortex, Epileptic excision S0228 PSMIX PBLS, TTM PCRII

rece for enriched 50242 5ynovial FibroblastsSynovial pSportl Fibroblasts (Ill/TNF), subt 50250 Human OsteoblastsHuman OsteoblastsFemur diseaseCMVS ort II 2.0 ' 50252 TTM-PIMIX pBLS, TTM PCRII

rece for enriched S0254 TTM-PAMIX PBLS, TTM PCRII

rece for enriched S0258 TTM-PNMIX PBLS, TTM PCRII

rece for enriched 50260 S final Cord, S final cords final Uni-ZAP
re-excision cord XR

S0264 PPMIX PPMIX (HumanPituitary ~ CRII
P

Pituitar ) S0268 PRMIX PRMIX (Humanprostate PCRII

Prostate) S0270 PTMIX PTMIX (HumanThymus PCRII

Th mus) 50274 PCMIX PCMIX (HumanBrain PCRII

Cerebellum) S0276~Synovial hypoxia-RSFSynovial Synovial p5portl fobroblasts subtracted' (rheumatoid)tissue S0278 H Macrophage Macrophage Uni-ZAP
(GM-CSF (GM- XR

treated), re-excisiowCSF treated) S0280 Human Adipose Human Adipose Uni-ZAP
Tissue, XR

re-excision Tissue 50282 Brain Frontal Brain frontalBrain Lambda ZAP
Cortex, re- cortex II

excision 50298 Bone marrow Borie mairowBone pSportl marrow stroma,treated stroma,treatedSB

50300 Frontal lobe,dementia;re-Frontal LobeBrain Uni-ZAP
XR

excision dementia/Alzheimer' S0308 S leen/normal ' S Teen S ortl normal S0316Human Normal Human Normal pSportl Cartilaae,FractionCartilage ' I

S0318Human Normal Human Normal pSportl Cartilage Cartilage Fraction II

S0328Palate carcinomaPalate carcinomaUvula diseaseS ortl .

S0330Palate normal Palate normalUvula S ortl S0342Adipocytes;re-excisionHuman Adipocytes Uni-ZAP
from OsteocIastoma XR

S0344Macrophage-oxLDL;macrophage- blood Cell Uni-ZAP
re- oxidized Line XR
excision LDL
treated S0346Human Amygdala;re-Amygdala Uni-ZAP
excision XR

50348Cheek CarcinomaCheek Carcinoma ~ diseaseS ortl S0352Lar nx CarcinomaLar nx carcinoma diseaseS ortl 50354Colon Normal Colon NormalColon S ortl II

S0356Colon CarcinomaColon CarcinomaColon diseaseS ortl 50358Colon Normal Colon NormalColon S ortl III

S0360Colon Tumor Colon Tumor Colon diseaseS ortl II

S0362Human GastrocnemiusGastrocnemius p5portl muscle 50364Human Quadrice Quadrice S ortl s s muscle 50_366_Human Soleus Soleus Muscle S ortl' 50374Normal colon Normal colon S ortl ' S0376Colon Tumor Colon Tumor diseaseS ortl .

S0378Pancreas normalPancreas pSportl PCA4 Normal No PCA4 No 50380Pancreas Tumor Pancreas diseasepSportl PCA4 Tu Tumor PCA4 Tu S0384Tongue carcinomaTon ue carcinoma diseaseS ortl S0386Human Whole Whole brain Brain ZAP Express Brain, re-excision 50388Human Human diseaseUni-ZAP
' Hypothalamus,schizophreHypothalamus, . XR
nia, re-excisionSchizophrenia S0390Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
control; arter Line XR
re-excision 50402Adrenal Gland,normalAdrenal gland; p5portl normal 50404Rectum normal Rectum, normal S ortl 50408Colon, normal Colon, normal S ortl 50410Colon, tumour Colon, tumour 5 ortl S0412Temporal cortex-Temporal diseaseOther Alzheizmer; cortex, subtracted alzheimer S0414Hippocampus, Hippocampus, Other Alzheimer Alzheimer Subtracted Subtracted S0418CHME Cell Line;treatedCHME Cell pCMVSport Line; 3.0 hrs ~ treated 50420CHME Cell CHME Cell pSportl Line,untreated line, untreatetd S0422Mo7e Cell Line Mole CeI1 - CMVS ort GM-CSF Line 3.0 2~5 treated.(lng/ml)GM-CSF treated ( 1 nQ/ml) 50424TF-1 Cell Line TF-1 Cell pSportl GM-CSF Line Treated GM-CSF Treated S0426Monocyte activated;Monocyte-activatedblood Cell Uni-ZAP
re- Line XR
excision S0428Neutrophils human neutrophilsblood Cell ~ Uni-ZAP
control; re- Line XR
excision S0432Sinus piniformisSinus piniformis pSportl Tumour Tumour 50434Stomach Normal Stomach Normal diseaseS ortl S0436Stomach Tumour Stomach Tumour diseaseS ortl S0438Liver Normal Liver Normal p5portl MetSNo MetSNo .

S0442Colon Normal Colon Normal S ortl 50444Colon Tumor Colon Tumour diseaseS ortl S0448Larynx Normal La nx Normal S ortl 50450Lar nx Tumour Lar nx Tumour S ortl 50452Th mus Th mus 5 ortl 50458Thyroid Normal Thyroid normal pSportl (SDCA2 No) 53012Smooth Muscle Smooth musclePulmanaryCell pBluescript Serum arter Line Treated, Norm S3014Smooth muscle, Smooth musclePulmanaryCell , pBluescript serum arte Line induced,re-exc S6014H. by othalamus,H othalamus Brain ZAP Ex frac A ress 56016H. Frontal Cortex,H. Brain, Brain diseaseUni-ZAP
E ile tic Frontal XR
Cortex, E
ile tic S6024Alzheimers, Alzheimer"slSpongyBrain diseaseUni-ZAP
spongy chanoe XR
chanoe 56026Frontal Lobe, Frontal LobeBrain Uni-ZAP
Dementia dementia/Alzheimer' XR
's S6028Human Manic Human Manic Brain diseaseUni-ZAP
Depression de ression ~ XR
Tissue tissue T0002Activated T-cellsActivated Blood Cell pBluescript T-Cell, Line SK-PBL fraction T0004Human White Human White Bluescri Fat Fat t SK-T0006Human Pineal Human Pinneal pBluescript Gland Gland SK-T0010Human Infant Human Infant Other Brain Brain T0023Human PancreaticHuman Pancreatic diseasepBluescript Carcinoma Carcinoma SK-T0041Jurkat T-cell Jurkat T-cell Bluescri G1 hase ~ t SK-T0042Jurkat T-Cell, Jurkat T-Cell' Bluescri S hase Line t SK-T0048Human Aortic Human Aortic pBluescript Endothelium Endothilium 5K-T0049Aorta endothelialAorta endothelial pBluescript cells + cells SK-TNF-a T0060Human White Human White Bluescri Adi ose Fat t SK-T0067Human Th roid Human Th Bluescri roid t SK-T0069Human Uterus, Human Uterus, Bluescri normal t SK-normal T0071HumamBone MarrowHuman Bone pBluescript Marrow SK-T0082Human Adult Human Adult Bluescri Retina Retina t SK-TO110Human colon pBluescript carcinoma SK-(HCC) cell line, remake T0114Human (Caco-2) pBluescript cell line, SK-adenocarcinoma, colon, remake TO115Human Colon pBluescript Carcinoma SK-(HCC) cell line -L0002Atrium cDNA
library Human heart L0005Clontech human aorta olyA+ mRNA (#6572) L0015Human L0021Human adult (K.Okubo) L0022Human adult lung 3"
directed MboI
cDNA

L0040Human colon mucosa L0055Human rom elocyte L0065Liver He G2 cell line.

L0097Subtracted human retinal figment epithelium (RPE) L0105Human aorta aorta polyA+
(TFu'iwara) L0142Human placenta placenta cDNA
(TFu'iwara) L0157Human fetal brain brain (TFu'iwara) L0163Human heart heart cDNA
(YNakamura) L0351Infant brain, BA, M13-Bento Soares derived L0352Normalized infant~ BA, M13-brain, derived Bento Soares L0356S, Human foetal Bluescript Adrenals tissue L0361Stratagene ovary ovary Bluescript (#937217) SK

L0362Stratagene ovarian Bluescript cancer SK-(#937219) L0364NCI CGAP_GCS germ cell Bluescri tumor t SK-L0366Stratagene schizoschizophrenic Bluescript brain brain ' SK-S11 S-11 frontal lobe L0367NCI CGAP_Schl Schwannoma Bluescri tumor t 5K-L0368NCI CGAP_SS1 s novial Bluescri sarcoma t SK-L0369NCI CLAP AA1 adrenal adenomaadrenal Bluescri oland t SK-L0371NCI CGAP Br3 breast tumorbreast Bluescri t SK-L0372NCI CGAP_Col2 colon tumor ' colon Bluescri t SK-L0374NCI CGAP_Co2 tumor colon Bluescri t 5K-L0375NCI_CGAP_Kid6 ~ kidne tumorkidne Bluescri ' t SK-L0376 NCI CGAP_Larl lar nx lar nx ~ Bluescri t SK-L0378 NCI CGAP Lul luno tumor lung Bluescri t SK-L0381 NCI_CGAP_HN4 squamous pharynx Bluescript cell SK-carcinoma L0384 NCI CGAP Pr23 .prostate prostate Bluescript tumor SK-L0387 NCI_CGAP_GCBO germinal tonsil Bluescript center B- SK-cells L0389 NCI_CGAP_HN5 normal gingiva ~ Bluescript (cell SK-line from primary keratinoc t L0393 B, Human Liver ~ Qtl l tissue L0438 normalized infanttotal brain brain Iafmid BA
brain cDNA

L0439.Soares infant whole Lafmid BA
brain 1NIB brain L044.364HB3MK Lafmid BK

L0455 Human retina retina eye lambda gtl0 cDNA
randomly primed sublibrary L0456 Human retina retina eye lambda gtl0 cDNA , Tsp509I-cleaved sublibrar L0462 WATM1 lambda tll L0471 Human fetal Lambda ZAP
heart, Ex ress Lambda ZAP Ex ress L0483 Human ancreatic Lambda ZAPII
islet L0485 STRATAGENE Humanskeletal leg muscle Lambda ZAPII
skeletal musclemuscle ' cDNA
library, cat.
#936215.

L0499 NCI CLAP~HSC2 stem cell bone AMPl 34+/38+ marrow L0512 NCI_CGAP_Ov36 borderline ovary pAMPl ovarian carcinoma L0515 NCI_CGAP_Ov32 papillary ovary pAMPl serous carcinoma L0517 NCI CGAP Prl AMP10 L0518 NCI CGAP_Pr2 ~AMP10 L0519 NCI CGAP Pr3 ~ AMP10 L0520 NCI_CGAP_Alvl alveolar pAMPlO
~ rhabdom osarcoma L0521 NCI CGAP_Ewl Ewina"s sarcoma AMP10 L0525 NCI CGAP Li2 liver AMP10 L0526 NCI_CGAP_Prl2 metastatic pAMPlO
prostate bone lesion L0527 NCI_CGAP Ov2 ovary pAMPlO

L0528 NCI CGAP_Pr5 prostate pAMPlO

L0529 NCI CGAP Pr6 rostate AMP10 , L0534 Chromosome 7 brain ' brain pAMPlO
Fetal Brain cDNA Librar L0535 NCI_CGAP_Br5 infiltratingbreast pAMPlO
ductal ' carcinoma L0543 NCI_CGAP_Pr9 normal prostaticprostate , pAMPlO
a ithelial cells L0544.NCI CGAP_Pr4 rostatic rostate AMP10 ~

intraepithelial neoplasia - high grade L0549NCI_CGAP_HN10 carcinoma pAMPlO
in situ from retromolar tri one L0550NCI_CGAP_HN9 normal squamous pAMPlO

epithelium from retromolar tri~one L0551NCl_CGAP_HN7 normal squamous pAMPlO

epithelium, floor of mouth L0564Jia bone marrowbone marrow Bluescri stroma stroma t L0565Normal Human Bone Hip pBluescript Trabecular Bone Cells L0581Strata~ene liver liver Bluescri (#937224) t SK

L0586HTCDLl pBluescript SK(-) L0588Stratagene endothelial pBluescript cell SK-L0589Stratagene fetal' pBluescript retina SK-L0590Stratagene fibroblast pBluescript SK-(#937212) L0591Stratagene HeLa pBluescript cell s3 SK-L0592Stratagene hNT pBluescript.SK-neuron (#937233) L0593Stratagene pBluescript SK-neuroepithelium (#937231) L0595Stratagene NT2 neuroepithelialbrain z pBluescript neuronal cells SK-recursor 937230 L0596Stratagene colon colon pBluescript SK-(#937204) L0598Morton Fetal cochlea ear ~ pBluescri Cochlea t SK-L0599Stratagene lung lung pBluescript (#937210) SK-L0600Weizmann Olfactoryolfactory nose pBluescript epithelium SK-.

E ithelium L0601Stratagene pancreas pancreas pBluescript ~ SK- , (#937208) L0602Pancreatic Isletancreatic ancreas Bluescri islet t SK-L0603Stratagene placenta placenta , ' pBluescript.SK-(#937225) L0604Stratagene musclemuscle skeletal pBluescript muscle L0605Stratagene fetalfetal spleenspleen pBluescript spleen SK-(#937205) L0606NCI_CGAP_L m5 follicular 1 m h Bluescri 1 m homa node t SK-L0607NCI_CGAP_Lym6 mantle cell lymph pBluescript node SK-I m homa L0608Stratagene lunglung carcinomalung NCI-H69 pBluescript . carcinoma SK-L061522 week old pBluescriptII
human fetal liver cDNA librar SK(_) L0619Chromosome 9 pBluescriptIIKS
exon II

L0622HM1 ~ pcDNAII

(Invitro en) L0623HM3 . pectoral - pcDNAII
muscle (after mastectom (InvitroQen) ) L0626NCI_CGAP_GCl bulk germ pCMV-SPORT2 cell seminoma L0629NCI_CGAP_Mel3 metastatic bowel , pCMV-SPORT4 (skin melanoma rimary) to bowel L0632NCI CGAP_Li5 hepatic adenomaliver pCMV-SPORT4 . NCI_CGAP_Pitl four pooled brain , pCMV-SPORT6 L0636 pituitary adenomas L0637NCI_CGAP_Brn53 three pooledbrain pCMV-SPORT6 menin~iomas L0638NCI_CGAP_Brn35 tumor, 5 brain - pCMV-SPORT6 pooled (see descri lion) L0640NCI CLAP-Brl8 four pooled breast pCMV-high- S PORT6 grade tumors, includin two rima L0641NCI-CGAP_Col7 juvenile colon ~ pCMV-SPORT6 granulosa tumor L0642NCI_CGAP_Col8 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0643NCI_CGAP_Col9 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0644NCI_CGAP_Co20 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0645NCI_CGAP_Co21 moderately colon pCMV-SPORT6 differentiated adenocarcinoma L0646NCI_CGAP_Col4 moderately- colon pCMV-SPORT6 differentiated adenocarcinoma L0647NCI_CGAP_Sar4 five pooled connective pCMV-SPORT6 sarcomas, tissue including m xoid 1i osarcoma L0648NCI_CGAP_Eso2 squamous esophagus pCMV-SPORT6 cell carcinoma L0649NCI_CGAP_GU1 2 pooled genitourinary pCMV-SPORT6 high-grade transitionaltract cell' .

tumors ' L0650NCI_CGAP_Kidl3 2 pooled kidney pCMV-SPORT6 . Wilms"

tumors, one primary and one metast L0653NCI_CGAP_Lu28 two pooled lung pCMV-SPORT6 squamous ' cell carcinomas L065SNCI_CGAP_Lyml2 lymphoma, lymph pCMV-SPORT6 node follicular mixed small and large cell L0657NCI_CGAP_Ov23 tumor, 5 ovary pCMV-SPORT6 pooled (see descri lion) L0659NCI CGAP_Panl adenocarcinomaancreas CMV-SPORT6 L0661NCI_CGAP Me115 malignant skin CMV-SPORT6 p melanoma, metastatic to lymph node L0662NCT_CGAP_Gas4 poorly differentiatedstomach pCMV-SPORT6 adenocarcinoma with signet r L0663NCI_CGAP Ut2 moderately- uterus CMV-SPORT6 p differentiated endometrial adenocarcino L0664NCI_CGAP_Ut3 poorly-differentiateduterus ' pCMV-SPORT6 endometrial adenocarcinoma, L0665NCI_CGAP Ut4 serous papillary, uterus CMV-SPORT6 - p carcinoma, v high , grade, 2 ooled t L0666NCI_CGAP Utl well-differentiateduterus CMV-SPORT6 p endometrial adenocarcinoma, L0667NCI_CGAP_CMLI myeloid cells,whole pCMV-SPORT6 18 blood pooled CML
cases, BCR/ABL rearra L0681Stanley Frontal frontal lobebrain pCR2.1 SN (see individual descri lion) (Invitro en) L0717Gessler Wilms SPORT1 tumor L0719human embryo Whole embryo pSPORTl cDNA

libra L0720PN001-Normal prostate pSportl Human Prostate L0731Soares_pregnant_uterus_, uterus pT7T3-Pac ' NbHPU

L0738Human colorectal T7T3D
cancer L0740Soares melanocytemelanocyte pT7T3D

2NbHM (Pharmacia) with a modified olylinker L0741Soares adult brain pT7T3D
brain N2b4HB55Y (Pharmacia) with a modified of linker L0742Soares adult brain. pT7T3D
brain .

N2b5HB55Y , (Pharmacia) with a modified of linker L0743Soares breast breast pT7T3D
2NbHBst (Pharmacia) with a modified olylinker L0744Soares breast breast pT7T3D
3NbHBst (Pharmacia) with a modified olylinker L0745Soares retina retina eye . pT7T3D
N2b4HR

(Pharmacia) with a modified polylinker L0746Soares retina retina eye pT7T3D
N2b5HR

(Pharmacia) with a modified of linker L0747Soares fetal_heart heart TTT3D
NbHH p 19W (Pharmacia) with a modified of linker L0748Soares fetal . . Liver pT7T3D
liver spleen and 1NFLS Spleen (Pharmacia) with a modified of linker L0749Soares fetal_liver_spleen Liver pT7T3D
and 1NFLS S1 ~ Spleen (Pharmacia) with a modified of linker L0750Soares_fetal , lung pT7T3D
lung_NbHLl 9W ' ' (Pharmacia) with a modified polylinker L0751Soares ovary ovarian ovary pT7T3D
tumor tumor NbHOT . (Pharmacia) with a modified of linker L0752Soares_parathyroidparathyroidparathyroid T7T3D
tumor tumor p NbHPA gland (Pharmacia) ' with a modified of linker L0753Soares_pineal_gland pineal T7T3D
N3H gland p PCT ' (Pharmacia) with a modified of linker L0754Soares placenta placenta pTTT3D
Nb2HP

(Pharmacia) with a modified of linker L0755Soares_placenta placenta T7T3D
8to9wee . p ks_2NbHP8to9W (Pharmacia) with a modified polylinker L0756Soares_multiple multiple-sclerosis T7T3D
~ sclerosis p 2NbHMSP lesions (Pharmacia) with a modified olylinker ~ - ----V TYPE

L0757 Snares senescentsenescent p TTT3D
fibrobla fibroblast ~

sts_NbHSF (Pharmacia) with a modified polylinker V_TYPE

L0758 Soares_testis_NHT , pTTT3D-Pac (Pharmacia) with a modified polylinker L0759 Snares total pTTT3D-Pac fetus_Nb2H

F8 9w ' ' _ (Pharmacia) with a modified of linker L0761 NCI_CGAP_CLL1 B-cell, chronic pTTT3D-Pac lymphotic (Pharmacia) leukemia with a modified of linker L0762 NCI, CGAP_Brl.lbreast pTTT3D-Pac (Pharmacia) with a modified of linker L0763 NCI_CGAP_Br2 breast pTTT3D-Pac (Pharmacia) with a modified of linker L0764 NCI_CGAP_Co3 colon pTTT3D-Pac (Pharmacia) with a modified polylinker L0765 NCI CGAP_Co4 colon pTTT3D-Pac (Pharmacia) with a modified of linker L0766 NCI_CGAP_GCBI germinal pTTT3D-Pac centerB

cell (Pharmacia) with a modified of linker L0767 NCI_CGAP_GC3 pooled germ pTTT3D-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 pTTT3D-Pac oligodendroglioma (Pharmacia) with a modified polylinker L0770 NCI_CGAP_Brn23 glioblastomabrain pTTT3D-Pac (pooled) (Pharmacia) with a modified polylinker L0771NCI_CGAP_Co8 adenocarcinomacolon pT7T3D-Pac (Phannacia) with a modified olylinker L0772NCI_CGAP_CoIO colon tumorRER+colon - pT7T3D-Pac (Pharmacia) ''with a modified of linker L0773NCI_CGAP_Co9 colon tumor colon pT7T3D-Pac RER+ .

(Pharmacia) with a modified of linker L0774NCI_CGAP_Kid3 kidney pT7T3D-Pac (Pharmacia) with a modified polylinker L0775NCI_CGAP_KidS 2 pooled kidney pT7T3D-Pac tumors (clear cell a (Pharmacia) type) with a modified of linker L0776NCI_CGAP_Lu5 carcinoid lung , pTTT3D-Pac (Pharmacia) with a modified of linker L0777Soares_NhHMPu Pooled humanmixed pTTT3D-Pac Sl (see melanocyte, below) (Pharmacia) fetal , heart, and with a pregnant modified.

of linker L0779Soares NFL_T_GBC_S1 pooled TTT3D-Pac p (Pharmacia) with a modified of linker L0780Soares NSF_F8_9W pooled TTT3D-Pac ' OT p PA_P_S 1 (Pharmacia) .

with a modified olylinker L0782NCI_CGAP_Pr21 normal prostateprostate pTTT3D-Pac (Phartnacia) with a modified olylinker L0783NCI_CGAP_Pr22 normal prostateprostate pT7T3D-Pac (Pharmacia).

with a modified of linker L0785Barstead spleen spleen pT7T3D-Pac (Pharmacia) with a modified of linker L0786Soares NbHFB p whole T7T3D-Pac brain (Pharmacia) with a modified of linker L0787NCI_CGAP_Sub1 pT7T3D-Pac _..__.'-___. ... _ (Pharmacia) with a modified of linker L0788NCI_CGAP_Sub2 ~ pT7T3D-Pac (Pharmacia) with a modified olylinker L0789NCI_CGAP_Sub3 pT7T3D-Pac .

(Pharmacia) with a modified poIyIinker L0790NCI_CGAP_Sub4 pT7T3D-Pac (Pharmacia) with a modified olylinker L0791NCI_CGAP_SubS ' pT7T3D-Pac (Pharmacia) with a modified of linker L0792NCI_CGAP_Sub6 pTTT3D-Pac (Pharmacia) with a modified of linker L0794NCI_CGAP_GC6 pooled germ pT7T3D-Pac cell tumors (Pharmacia) with a modified olylinker L0796NCI CGAP_Brn50 medulloblastomabrain TTT3D-Pac p (Pharmacia) with a modified polylinker L0800NCI_CGAP_Col6 colon tumor,colon pT7T3D-Pac RER+

(Pharmacia) with a modified of linker L0803NCI_CGAP_Kidll kidney pTTT3D-Pac (Pharmacia) with a modified of linker L0804NCI_CGAP_Kidl2 2 pooled kidney pT7T3D-Pac tumors ~

(clear cell (Pharmacia) type) with a modified of linker L0805NCI_CGAP_Lu24 carcinoid lung pT7T3D-Pac (Pharmacia) with a modified - of linker L0806NCI_CGAP_Lul9 squamous lung pT7T3D-Pac cell carcinoma, (Pharmacia) poorly differentiated - with a (4 modified polylinker L0808Barstead prostate prostate pT7T3D-Pac BPH

HPLRB41 ~ . ('Pharmacia) with.a modified "- ~-polylinker L0809 NCI_CGAP_Pr28 prostate pTTT3D-Pac (Pharmacia) with a modified of linker L2242 subtracted 3" pancreas AsPC- , pUClB
E5T library . i(ATCC:

CRL-1682) .

OMIM Description - _ Reference 106165 H ertension, essential, 145500 107280 Cerebrovascular disease, occlusive 107280__ Al ha-1-antich mot sin deficienc 107400 Em h sema 107400 Em h sema-cirrhosis 117700 H ocerulo lasminemia, heredita 117700 Hernosiderosis, s stemic, due .to acerulo lasrninemia 122500 Transcortin deficienc 150210 Lactoferrin-deficient neutro hils, 245480 154705 Marfan syndrome, a II

169600 Haile -Haile disease .

180380 Ni ht blindness, con enital statione , rhodo sin-related 180380 Retinitis i mentosa, autosomal recessive 180380 Retinitis i mentosa-4, autosomal dominant 186960 Leukemia/1 m homa, T-cell 190000 Atransferrinemia 193300 Renal cell carcinoma 193300 von Hi el-Lindau s ndrome 203500 Alka tonuria 222900 Sucrose intolerance 227646 Fanconi anemia, a D

232050 Pro ionicacidemia, a II or ccB a 245200 I~rabbe disease 253260 Biotinidase deficienc 276902 Usher s ndrome, a 3 278720 Xeroderma i entosum, ou C

600882 Charcot-Marie-Tooth neuro ath -2B

601154 Cardiom o ath , dilated, 1E

601199 Neonatal~h a arath roidism, 239200 601199 H ocalcemia, autosomal dominant, 601198 601199 Hy ocalciuric hypercalcemia, a I, 145980 _ 601253 Muscular d stro h , limb- irdle, a IC

601471 ~ Moebius s ndrome-2 601682 Glaucoma 1C, rima o en an 1e 601841 Protein C inhibitor deficienc 602011 Pancreatic endocrine tumors Polynucleotide and Polypeptide Tlariants (98] 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 defined 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.
(99] 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.
(100] "Variant"~ refers to a po~lynucleotide 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. . ' [101] 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;
.r (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), (fj, (g), (h), or (i) above.
[102] 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 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.lB 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.
[103] . In a preferred embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under Iower 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.
[104] 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.
[105] The present invention is also directed to proteins which compiise, 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 Iower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins. .
[106J By a nucleic acid having a nucleotide sequence at least, for example, 9S%
"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 9S% identical to a reference nucleotide sequence, up to S% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to S% 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.
[107J As a practical matter, whether any particular nucleic acid molecule -or polypeptide is at least 80%, 8S%, 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. 6:237-24S (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, Misxriatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=S, Gap Size Penalty O.OS, Window Size=S00 or the length of the subject nucleotide sequence, whichever is shorter.
[108J , If the subject sequence is shorter than the query sequence because ~of S' 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 S' 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. , [109] 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 ~ 10% 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 matcliedlaligned with the query .sequence are manually corrected for. No other manual corrections are to be -made for the purposes of the present invention.
[110] 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 222 .

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.
[111] 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=l, 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.
[112] 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-terminal of the subject sequence, which are not matchedlaligned 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. .
[113] 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 matehed/aligned with,the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
[114] 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 50, 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).
(115] 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._ _ __ 2.24 (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.
[116] 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 KGF 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).) [117] 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-la. They used random mutagenesis to generate over 3,500 individual IL-la 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.
[118] 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.
[119] 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 known in the art so as have little effect on activity. .
[120) 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, (I) 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) ih situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).
[121] 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.
[122] The functional activity of 'the polypeptides, and fragments, variants and derivatives of the invention, can be assayed by various methods.
[123] 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, immunofluorescence assays; protein A assays, arid 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.
[124] 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 cari be routinely assayed using techniques known in the art.
[125] 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. ' [126] . 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 Clorie 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 art 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.
[127] 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. - ' [128] ~ 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.
[129] 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. ~ , [130] 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. Fox 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, ox (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 secretary 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.
[131] 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 iminunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 . (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
[132] A fuxther 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 equence encoded by SEQ
ID NO:X, an amino acid sequence encoded by the portion-of SEQ ID NO:X as defined in columnns 8 and 9 of Table 2, amamino 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.
[133] 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.
Polynzicleotide ahd Polypeptide Fragments .
[134] 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 polyriucleotide 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 polynucleotide 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 polyriucleotide sequence in 230 .

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.
[135] 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 example, 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.
[136] Moreover, 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, 2201-225.0, 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 laxger or smaller by several (S, 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.
[137] 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, I9SI-2000, 2001-2050, ZOSI-2100, 2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2.700, 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 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.
[138]~ 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 polypeptides are also encompassed by the invention.

[139] 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.
[140] In additional specific embodiments, polynucleotides of the invention comprise, ox 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.
[141] 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 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.
[142] 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.

[143] 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 IB and the S' IO
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 polynucIeotides, nucleic acids, and polypeptides are also encompassed by the invention.
[144] 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 S' 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 andlor 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.
[145] 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 S' 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 S' 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 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.
[146] ~ 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, 1101-1120, 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, 1041=
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 I0, 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.
[147] 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 andlor 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 of a complete polypeptide retains such immunologic activities can readily be determined byroutine 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.
(148] 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 fxom 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.
[149] The present invention fiu-ther 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, andlor 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.
[150] 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 polypaptide 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-1, 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.
[151] 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 an,d 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.
[152] 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. It 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.
[153] 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%, 9'0%, 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.
[154] Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID N.O:X or the complement thereof, (presented, for example, in Tables 1 A and 2), the cDNA contained in Clone ID NO:Z, or the polynucleotide sequence as defined in column 6 of Table 1B, may be 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 cDNA 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/).
(155] 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-Fasmai~ 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., l, 2, 3 or 4) of the features set out above.
[156] Additionally, Kyte-Doolittle hydrophilic regions arid 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 antienicity 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.
[157] 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 fixnctional activities associated with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimeriaation, as described herein. __ [158] ~ 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.
[159] In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more 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.
[160], 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 NO: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 defned 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 supYa.
[161] The term "epitopes," as used herein, refers to portions 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 epitope," as used herein, is defined as a portion of a protein 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 infi~a. (See, for example, Geysen, et al.; Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983)). The term "antigenic epitope," as used herein, is defined 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. .
[162] 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-S13S
(1985) further described in U.S. Patent No. 4,631,211.) °
[163] In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least S, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 1S, at least 20, at least 2S, 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, 1 S, 20, 2S, 30, 3S, 40, 4S, S0, SS, 60, 6S, 70, 7S, 80, 8S, 90, 9S, 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 241°

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)).
[164] Non-limiting examples of epitopes of polypeptides 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 portion; 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.
[165] 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).
[166] 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., supra; Wilson et al.; supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If ira 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 (I~L,H) 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 ~.g 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, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
[167] 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 thereof), 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 riot 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.
[168] Such fusion proteins as those described above may facilitate purification and may increase half life ih 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 immunoglobulins. See, e.g., EP 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 fusionproteins that have a disulfzde-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-(I995). 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 [169j 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. ' .
[170] 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 linker sequences.
[171] In certain preferred embodiments, proteins of the invention are 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.
[172] ~ 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.
[173] As one of skill in the art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, can be combined with heterologous 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, for 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); K. Johanson et al., J. Biol.
Chem. 270:9459-9471 (1995).
(174] 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 the tag provided in a pQE~ vector (QIAGElV, 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)).
(175] Additional fusion proteins of the invention may be generated through the techniques of, gene-shuffling, motif shuffling, exori-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);
Haiayama, 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.
[176] 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 [177] 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.
[178] 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.
(179] The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac,. tip, 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- (LTAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[180] As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, 6418, 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 as 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.
[181] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA, 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 no't limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, 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. .
[182] 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 murme myeloma cell line, NSO) which are glutamine synthase negative.
Glutamine 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;
W086105807;
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 murine 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.
[183] 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 characteristics 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. . ~ _ [184] 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 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.
[185] 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 sequences 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., Pros. 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).

[186] 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 canon 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.
[187] 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 polypeptides 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 eukaryotic 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.
[188] In one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as 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, Pichia pastoris 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 (AD~1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOXl gene comprises up to approximately 30% of the total soluble protein in Pichia pasto~is. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P.J, et al., Yeast 5:167-77 (1989); Tschopp, J.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 AOXI
regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
[189) In one example, the plasmid vector pPIC9I~ is used to express DNA
encoding a polypeptide of the invention,. as set forth herein, in a Pichea 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 AO~YI
promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
[190] Many other yeast vectors could be used in place of pPIC9IC, such. as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, PHIL-S1, 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.
[191] 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:
[I92] In addition to encompassing host ceps 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 material .(e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endt~genous polynucleotides. For example, techniques known in the arf 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 96/29411, published September 26, 1996; International Publication No. WO 94112650, 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).
[193] , 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 amino 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).
[194] The invention encompasses polypeptides of the ~ present invention which are differentially modified during or after translation, e.g., by glycosylation,~
acetylation, phosphorylation; amidation, derivatization by known protectinglblocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligarld, 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.
[195] 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 C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition br 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.
[196] Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galaetosidase, 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 (lalI 1231, lzsl 131I) carbon (14C) sulfur 35S tritium (3H) indium 111In > > > > > ( )> >
llzln~' 113mIn' llsmln), technetium (99Tc,99mTc), thallium (2olTi), gallium (6sGa, 67Ga), palladium (lo3pd), molybdenum (99Mo), xenon (lssxe), fluorine (18F), ls3Sm, 177Lu, ls9Gd, 149P~' d40La' 175Yb' 166Ho' 90Y' 47SC' 186Re' 188R~' 142Pr' 105' and 97Ru.
[197] 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, 177Lu, 9oy~ ls6Ho, and ls3Sm, to polypeptides.
In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is ~°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 ar-t - see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Ghem. 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. .
[198] 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); POSTTR.ANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New Yorlc, 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)).
[199] 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. ' -[200] 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, 2.500; 3000, 3500, 4000, 4500, 5000, SS00, 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, 90,040, 95,000, or 100,000 lcDa.
[201] 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;
Morpurgo et al., Appl. Biochem. Bioteclanol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), J the disclosures of each of which are incorporated herein by reference.
[202] 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 piotein. There are a number 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 alsoMalik 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. Sulfllydryl 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.
[203] 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.
[204] 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 far 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.
[20S] 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. 68:1-1S
(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.
[206] One system fox 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 (C1SOZCH2CF3). Upon reaction of protein with- tres'ylated 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 grqup.
[207] ~ 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-trichloropenylc.arbonate, 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 reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
[208] 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 averages 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 Carrier Sys. 9:249-304 (1992).
[209] 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 canon 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.
[210] The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present 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.
[211] 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 8 and 9 of Table 2, and/ox 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. . .
[212) 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.
[213] 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 incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
[214] 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 multimeiic fusion protein is recovered from the culture supernatant using techniques known in the art.
[215] 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.
[216] 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, pxoteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag~ fusion proteins of the invention and anti-Flag~ antibody.
[217j The multimers of the invention may be generated using chemical techniques r 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 iri 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).
[218] 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 recornbinantly 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 polypeptide 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 [219] 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, a No:Z, and/or an epitope, of 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.
[220] 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, CHl, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable regions) with a hinge region, CHl~; 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:
[221] 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; Kostelny .
et al., J. Immunol. 148:1547-1553 (1992).
[222] 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 in 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.
[223] Antibodies of the present invention may also be described or specified iri terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homology 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 homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 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 combinations) 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, 10-2 M, 5 X 10-3 M, 10-3 M, 5 X 10-4 M, 10-4 M, 5 X 10-s M, 10-s M, 5 X 10-6 M, 10-6M, 5 X 10-' M, 107 M, 5 X l0-g M, 10-$ M, X 10-9 M, 10-9 M, 5 X 10-'° M, 10-1° M, 5 X 10-il M,-10-1' M, 5 X 10-12 M, 10-12 M, S X
10-13 M, 10-is M, 5 X 10-14 M, 10-14 M, 5,X 10-is M, or 10-is M.
[224] 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%.
[225] 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 immunoprecipitation 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.
[226] .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, for 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 agoriists 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); Taryman 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).
[227] Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both ira vitro and in wivo diagnostic and therapeutic,, methods. For example, the antibodies have utility in 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 lVlanual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);' incorporated by reference herein in its entirety.
[228] As discussed in more detail below, the antibodies of the present invention may be 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 91114438; 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.
[229] 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, c3erivatization by known protecting/blocking 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,rvmetabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical amino acids. ' [230] 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 ~J(bacille Calmette-Guerin) and corynebacterium~ parvum. Such adjuvants are also well known in the art.

[231] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridorna, 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. .
[232] 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.
[233] 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 hybridoma 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.
[234] Another well known method for producing both polyclonal and monoclonal human B cell Iines is transformation using Epstein Barr -Virus (EBV).
Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for --example, the protocol_outlined in Chapter 7.22 ~of Current Protocols ix?
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.
[235] 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 beco~.ne 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 KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells. -[236] 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 CHl domain of the heavy chain.
[237] ~ For examplo, 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-SO (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 S7:I91-280 (1994);
PCT application No. PCT/GB91/Ol 134; PCT publications WO 90/02809; WO
91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WQ 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.
[238] 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 fragnients~ 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 (I988) (said refei~ences incorporated by reference in their entireties).
[239] 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;
Hustori et al., Methods in Enzymology 203:6-88 (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 murine monoclonal antibody and a human imrnunoglobulin 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. Immunol. 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 (U.S:
Patent No. 5,565,332).
[240] 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 using antibody libraries derived from human imrriunoglobulin sequences. See also, U, S. Patent Nos. 4,444,887 and 4,716,111;..
and PCT publications WO 98146645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
[241] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous irnmunoglobulins, 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. IVIonoclonal~
antibodies directed against the antigen can be 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 S98 877; LJ.S. Patent Nos. 5,413,923;
S,62S,126;
5,633,425; S,S69,825; 5,661,016; S,54S,806; 5,814,318; S,88S;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 Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
[242J 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., Bioltechnology 12:899-903 (1988)). , [243J Further, antibodies to the polypeptides of the invention can, in turn, 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.
[244j Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen 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.
Polynucleotides Encoding Antibodies [245] 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 polynuc~eotides 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 ~sequerice 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, and/or toga polypeptide encoded by the cDNA contained in Clone ID
NO:Z.
[246 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 Kutmeier et aL, BioTechniques 17:242 (I994)), 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.
[247) Alternatively, a polynucleotide encoding an antibody may be 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.
[248] Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known xn 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, MoIecuIar.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 axe 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.

[249] In a specific embodiment, the amino acid sequence of the heavy and/or 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 (1998) 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 polypepti'de 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.
[250J In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Nath Acad. Sci. 81:851-85S (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 marine , mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
(251J 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 [252] 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. _ [253] 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 ehain 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, a-nd in vivo genetic recombination. The invention, thus, provides replicable vectors compxising~ 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.
[254] 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.
[255] 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, plasrnid 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 recombinant 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)).
(256] ' 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 pIJR278 (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.
[Z57] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodopter°a frugipe~da cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter).
(258] 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 comply, 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 E1 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)). , v [259] 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, BHI~, Hela, COS, MDCI~, 293, 3T3, WI38,, and in .particular, breast cancer cell lines such as, for example, BT483, HsS78T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, fox example, CRL7030 and Hs578Bst.
[260] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the 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. , [261] 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 camb~e 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 I I(5):155-2I5 (I993)); 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 ~z. Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990); and in Chapters I2 arid 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.
t [262] The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA 'cloning, Vol.3. (Academic Press, New York, I987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will ,also increase (Crouse et.al., Mol. Cell. Biol. 3:257 (1983)).
[263] Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or rnethotrexate, respectively. An advantage of glutamine synthase based vectors are the availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative.
Glutamine 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 incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suplliers, including, for example Lonza Biologics, Inc. (Portsmouth, NH): Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbingtori et al., Biolteclanology 10:169(1992) and in Biblia and Robinson Biotechnol.
Prog. 11:1 (1995) which are incorporated in their entirities by reference herein.
[264] The.host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and Iight chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986);
Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. ' ' .
[265] Once an.antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulim molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art; to facilitate purification.
[266] The present invention encompasses antibodies recombinantly fused or chemically r conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably'at least 10, 20, 30, 40, 50, 60, 70; 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may __ also be used in in vitro immunoassays and purification methods using methods known in the art. See ~e.g., Harbor et al.,,supra,_ and _PCT publication WO 93/21232;
EP 439,095;

Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981;
Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
[267] The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHl domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the,polypeptides to portions of IgA and.IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
5,447,851;
5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570;
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA
89:11337-11341 (1992) (said references incorporated by reference in their entireties).
[268] As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further; the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes 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 immunoglobulins. See EP 394,827; and Traunecker et al., Nature 331.:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al., J.
Biochem. 270:3958-3964, (1995). 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. See, for example, EP A 232,262. 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, for 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, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
[269] Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, ' such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as 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. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the~"flag" tag.
[270] The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linleer known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. 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 125I, 131I, 111In or 99Tc.
[271] Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, . ,ethidium bromide, emetine, mitomycin, etoposide, tenopqside, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antirnetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, S-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide,' busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
(272] The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas eXOtoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, 13-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other 'growth factors.

[273] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide,' nylon, polystyrene, polyvinyl chloride or polypropylene.
[274] Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.

(Marcel Deklcer, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:.A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-l 6 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).
[275] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676;980, which is incorporated herein by reference in its entirety. ' [276] An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factors) and/or cytokine(s) can be used as a therapeutic.
Immzcnophenotyping [277] The antibodies.of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as, cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
[278J These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and "non-self' cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow fox the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
Assays For Antibody Bindihg [279] The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots,.radioimmunoassays, ELISA (enzyme linked irnmunosorbent assay), "sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[280] Immunoprecipitation protocols generally compxise Iysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, O.OI M sodium phosphate at pH 7.2, I% Trasylol) supplemented With protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing ' the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley ~ Sons, Inc., New York, section 10.16.1.
(281] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with~3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol.
1,,,John Wiley &
Sons, Inc., New York, section 10.8.1.
[282]' ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a.
second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the , coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, (I994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 11.2.1.

[283] The binding affinity of an antibody to an antigen and the .off rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or .125I) with the antibody of interest in the presence ~of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated.to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
(284] Antibodies of the invention may be characterized using immunocytochemisty methods on cells (e.g., mammalian cells, such as ECHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
Therapeutic Uses (285] The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including' fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions.as known in the art or as described herein.

[286] In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: eneural disorders, immune system disorders, muscular disorders, reproductive disorders, 'gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions., and/or as described elsewhere herein.
Therapeutic compounds of the invention. include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such' as, for example, a predicted linear epitope shown in column 7 of Table 1A; or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
[287] A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g.
as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
[288] The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lyinphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

[289] The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in~ a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
[290] It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof.
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 104 M, 10-4 M, 5 X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6 M, 5 X
10-' M, 10'' M, 5 X 10-$ M, 10-8 M, 5 X 10-9 M, 10'9 M, 5 X 10-1° M, 10'1° M, 5 X 10-11 M, 10-11 M, 5 X 10-12 M, 1012 M, 5 X 10-13 M, 10-13 M, 5 X 10-14 M, 10-14 M, 5 X
10-15 M, and 10-1s M.
Gene Therapy [291] In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
[292] Any of the methods for gene therapy available in the art can be used according to ' the present invention. Exemplary methods are described below.
[293] For general reviews of the methods .of gene therapy, see Goldspiel et al., Clinical.
Pharmacy 12:488-505 (1993); 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, TIBTECH

11(5):155-215 (1993)..Methods commonly known. in the art of recombinant DNA
technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
[294] In a preferred embodiment, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being 'inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
[295] Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
[296] In a specific embodiment, the nucleic acid'sequences are directly administered ira vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No. 4,980,286),' or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, ISupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it inrlinkage to a ligand subject to receptor-mediated endocytosis~ (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. . In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; W092/20316; W093/14188, WO 93/2022I). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by Homologous recombination (Koller and Smithies, Proc. Natl.
Acad. Sci.
USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
[297] In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Erlzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are clbned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene.to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Gxossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
[298] Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. .
Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (I994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al.,.Science 252:431-434 (1991);
Rosenfeld et al., Cell 68:143- 1S5 (1992); Mastrangeli et al., J. Clin.
Invest. 91:225-234 (1993); PCT Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995).
In a preferred embodiment, adenovirus vectors are used.
[299] Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
[300] Another approach to gene therapy involves transferring a gene to cells in tissue culture by.such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. ' Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
[301] In this embodiment, the nucleic acid is introduced into a cell prior to.
administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any ~ method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.
217:599-618 (1993); Cohere et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac.
Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
(302] The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
[303] Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells.
such as T, lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
(304] In a preferred embodiment,.the cell used for gene therapy is autologous to the patient.
[305] In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used.
Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be, used in accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, CeII 71:973-985 (1992);
Rhein-wald, Meth. Cell. Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc.
61:771 (1986)).
[306] In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that.
expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity [307] The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. ~ Fox example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of~a specific compound is indicated, include ,in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
TherapeuticlProphylactic Administration and Composition [308] The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention. In a preferred embodiment, the compound. is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
[309] Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be selected from among those described herein below.
[310] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the, invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir: Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
[311] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved, by, for~example, and not~by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection,' by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb:
[312] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990);
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.) [313] . In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)): In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);
Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
[314] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
[315] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g:, by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun;. Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.
USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

[316] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be J
employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents., These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such_ compositions will contain a therapeutically effective amount of 'the compound, preferably in purified form, togethei with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[317] In. a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a _ _ __ hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[318] The compounds of the invention can be formulated as neutral or salt forms.
- Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[319] . The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[32U] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to mg/kg of the patient's body weight. Generally, human antibodies have a longer half life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies ~of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
[321] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Diagnosis and Imaging [322] Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to. the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
(323] The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby,an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high- amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment . earlier thereby preventing the development or further progression of the cancer.
[324] Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J.
Cell . Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RLA). Suitable antibody assay labels are known in the art and include enzyme labels, such.as, glucose oxidase; radioisotopes, such as iodine DEMANDE OU BREVET VOLUMINEUX
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Claims (24)

What Is Claimed Is:

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.