CA2394160A1 - Human oxidoreductase proteins - Google Patents

Abstract

The invention provides human oxidoreductase proteins (ORP) and polynucleotid es which identify and encode ORP. The invention also provides expression vector s, host cells, antibodies, agonists, and antagonists. The invention also provid es methods for diagnosing, treating, or preventing disorders associated with expression of ORP.

Description

HUMAN OXIDOREDUCTASE PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of oxidoreductase proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative disorders including cancer; endocrine, metabolic, reproductive, neurological, viral, and autoimmune/inflammatory disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of oxidoreductase proteins.
BACKGROUND OF THE INVENTION
Many pathways of biogenesis and biodegradation require oxidoreductase (dehydrogenase or reductase) activity, coupled to the reduction or oxidation of a donor or acceptor cofactor. Potential cofactors include cytochromes, oxygen, disulfide, iron-sulfur proteins, flavin adenine dinucleotide (FAD), and the nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E.A. and Leech, A.R. (1983) Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U.K. pp. 779-793).
Reductase activity catalyzes the transfer of electrons between substrates) and cofactors) with concurrent oxidation of the cofactor. The reverse dehydrogenase reaction catalyzes the reduction of a cofactor and consequent oxidation of the substrate. Oxidoreductase enzymes are a broad superfamily of proteins that catalyze numerous reactions in all cells of organisms ranging from bacteria to plants to humans. These reactions include metabolism of sugar, certain detoxification reactions in the liver, and the synthesis or degradation of fatty acids, amino acids, glucocorticoids, estrogens, androgens, and prostaglandins. Different family members are named according to the direction in which their reactions are typically catalyzed; thus they may be referred to as oxidoreductases, oxidases, reductases, or dehydrogenases. In addition, family members often have distinct cellular localizations, including the cytosol, the plasma membrane, mitochondria) inner or outer membrane, and peroxisomes.
Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier, providing activated one-carbon units to a wide variety of biosynthetic reactions, including synthesis of purines, pyrimidines, and the amino acid methionine. Tetrahydrofolate is generated by the activity of a holoenzyme complex called tetrahydrofolate synthase, which includes three enzyme activities:
tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and tetrahydrofolate synthetase. Thus, tetrahydrofolate dehydrogenase plays an important role in generating building blocks for nucleic and amino acids, crucial to proliferating cells.
Intracellular redox status plays a critical role in the assembly of proteins.
A major rate limiting step in protein folding is the thiol:disulfide exchange necessary for correct protein assembly. Although incubation of reduced, unfolded proteins in buffers containing defined ratios of oxidized and reduced thiols can lead to folding into native conformation, the rate of folding is slow, and the attainment of the native conformation decreases proportionately with protein size and the number of cysteine residues.
Certain cellular compartments such as the endoplasmic reticulum of eukaryotes and the periplasmic space of prokaryotes are maintained in a more oxidized state than the surrounding cytosol. Correct disulfide formation can occur in these compartments, but it occurs at a rate that is insufficient for normal cell processes and inadequate for synthesizing secreted proteins.
Protein disulfide isomerases (PDIs), thioredoxins, and glutaredoxins are able to catalyze the formation of disulfide bonds and regulate the redox environment in cells to enable the necessary thiol:disulfide exchanges. Each of these classes of molecules has a somewhat different function, but all belong to a group of disulfide-containing redox proteins that contain a conserved active-site sequence and are ubiquitously distributed in eukaryotes and prokaryotes. PDIs are found in the endoplasmic reticulum of eukaryotes and in the periplasmic space of prokaryotes. PDIs function by exchanging their own disulfide for thiols in a folding peptide chain. In contrast, reduced thioredoxins and glutaredoxins are generally found in the cytoplasm and function by directly reducing disulfides in the substrate proteins. Thioredoxin (Trx), a heat-stable, redox-active protein, contains an active site cysteine disulfide/dithiol. Oxidized thioredoxin, Trx-S, can be reduced to the dithiol form by NADPH and a specific flavoprotein enzyme, thioredoxin reductase. Reduced thioredoxin, Trx-(SH), participates in a number of redox reactions mostly linked to reduction of protein disulfides.
Trx and thioredoxin reductase (TR), together with NADPH, form a redox complex in which TR
catalyzes the electron transport from NADPH to Trx. The reduced thioredoxin then functions as an electron donor in a wide variety of different metabolic processes.
Disulfide-containing redox proteins not only facilitate disulfide formation, but also regulate and participate in a wide variety of physiological processes. The thioredoxin system serves, for example, as a hydrogen donor for ribonucleotide reductase and controls the activity of enzymes by redox reactions.
Mammalian thioredoxin (MT) acts as a hydrogen donor for ribonucleotide reductase and methionine sulfoxide reductase, facilitates refolding of disulfide-containing proteins, and activates the glucocorticoid and interleukin-2 receptors. MT also modulates the DNA binding activity of some transcription factors either directly (TFIIIC, BZLF1, and NF-kB) or indirectly (AP-1) through the nuclear factor Ref-1. The importance of the redox regulation of transcription factors is exemplified by the v-fos oncogene where a point mutation of the thioredoxin-modulated cysteine residue results in constitutive activation of the AP-1 complex. Thioredoxin, secreted by cells using a leaderless pathway, stimulates the proliferation of lymphoid cells, fibroblasts, and a variety of human solid tumor cell lines.
Furthermore, thioredoxin is an essential component of early pregnancy factor, inhibits human immunodeficiency virus expression in macrophages, reduces H202, scavenges free radicals, and protects cells against oxidative stress (Abate, C. et al. (1990) Science 249: 1157-1161; Rosen, A. et al. (1995) Int. Immunol. 7: 625-633; Tagaya, Y. et al. (1989) EMBO J. 8: 757-764; Newman, G. W. (1994) J.
Expt. Med. 180: 359-363; and Makino, Y. (1996) J. Clin. Invest. 98: 2469-2477).
Short-chain alcohol dehydrogenases (SCADs) are a family of dehydrogenases that share only 15°1o to 30% sequence identity, with similarity predominantly in the coenzyme binding domain and the substrate binding domain. In addition to the well-known role in detoxification of ethanol, SCADS are also involved in synthesis and degradation of fatty acids, steroids, and some prostaglandins, and are therefore implicated in a variety of disorders such as lipid storage disease, myopathy, SCAD deficiency, and certain genetic disorders. For example, retinol dehydrogenase is a SCAD-family member (Simon, A. et al. (1995) J. Biol. Chem. 270:1107-1112) that converts retinol to retinal, the precursor of retinoic acid. Retinoic acid, a regulator of differentiation and apoptosis, has been shown to down-regulate genes involved in cell proliferation and inflammation (Chaff, X. et al. (1995) J.
Biol. Chem. 270:3900-3904).
In addition, retinol dehydrogenase has been linked to hereditary eye diseases such as autosomal recessive childhood-onset severe retinal dystrophy (Simon, A. et al. (1996) Genomics 36:424-430).
Propagation of nerve impulses, modulation of cell proliferation and differentiation, induction of the immune response, and tissue homeostasis involve neurotransmitter metabolism (Weiss, B. (1991) Neurotoxicology 12:379-386; Collies, S.M. et al. (1992) Ann. N.Y. Acad. Sci.
664:415-424; Brown, J.K. and Imam, H. (1991) J. Inherit. Metab. Dis. 14:436-458). Many pathways of neurotransmitter metabolism require oxidoreductase activity, coupled to reduction or oxidation of a cofactor, such as NAD+/NADH (Newsholme, E.A. and Leech, A.R. (1983) Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U.K. pp. 779-793). Degradation of catecholamines (epinephrine or norepinephrine) requires alcohol dehydrogenase (in the brain) or aldehyde dehydrogenase (in peripheral tissue). NAD+-dependent aldehyde dehydrogenase oxidizes 5-hydroxyindole-3-acetate (the product of 5-hydroxytryptamine (serotonin) metabolism) in the brain, blood platelets, liver and pulmonary endothelium (Newsholme, E.A. and Leech, A.R. supra p. 786). Other neurotransmitter degradation pathways that utilize NAD+/NADH-dependent oxidoreductase activity include those of L-DOPA
(precursor of dopamine, a neuronal excitatory compound), glycine (an inhibitory neurotransmitter in the brain and spinal cord), histamine (liberated from mast cells during the inflammatory response), and taurine (an inhibitory neurotransmitter of the brain stem, spinal cord and retina) (Newsholme, E.A. and Leech, A.R. supra, pp. 790, 792). Epigenetic or genetic defects in neurotransmitter metabolic pathways can result in a spectrum of disease states in different tissues including Parkinson's disease and inherited myoclonus (McCance, K.L. and Huether, S.E. (1994) Pathoph siy ology, Mosby-Year Book, Inc., St.
Louis, MO pp. 402-404; Gundlach, A.L. (1990) FASEB J. 4:2761-2766).
3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabolism.
It catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with concomitant oxidation of NAD

to NADH, in the mitochondria and peroxisomes of eukaryotic cells. In peroxisomes, 3HACD and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme, defects in which are associated with peroxisomal bifunctional enzyme deficiency. This interruption in fatty acid metabolism produces accumulation of very-long chain fatty acids, disrupting development of the brain, bone, and adrenal glands. Infants born with this deficiency typically die within 6 months (Watkins, P. et al.
(1989) J. Clin. Invest. 83:771-777; Online Mendelian Inheritance in Man (OMIM), #261515). The neurodegeneration that is characteristic of Alzheimer's disease involves development of extracellular plaques in certain brain regions. A major protein component of these plaques is the peptide amyloid-~i (A(3), which is one of several cleavage products of amyloid precursor protein (APP). 3HACD, which has been shown to bind the A~3 peptide, is overexpressed in neurons affected in Alzheimer's disease. In addition, an antibody against 3HACD can block the toxic effects of A(3 in a cell culture model of Alzheimer's disease (Yan, S. et al. (1997) Nature 389:689-695; OMIM, #602057).
17(3-hydroxysteroid dehydrogenase (17(3HSD6) plays an important role in the regulation of the male reproductive hormone, dihydrotestosterone (DHTT). l7~iHSD6 acts to reduce levels of DHTT by oxidizing a precursor of DHTT, 3a-diol, to androsterone which is readily glucuronidated and removed from tissues. 17/3HSD6 is active with both androgen and estrogen substrates when expressed in embryonic kidney 293 cells. At least five other isozymes of 17(3HSD have been identified that catalyze oxidation and/or reduction reactions in various tissues with preferences for different steroid substrates (Biswas, M.G. and Russell, D.W. (1997) J. Biol. Chem. 272:15959-15966). For example, l7~iHSD1 preferentially reduces estradiol and is abundant in the ovary and placenta.
17(3HSD2 catalyzes oxidation of androgens and is present in the endometrium and placenta.
17(3HSD3 is exclusively a reductive enzyme in the testis (Geissler, W.M. et al. (1994) Nature Genet.
7:34-39). An excess of androgens such as DHTT can contribute to certain disease states such as benign prostatic hyperplasia and prostate cancer.
Steroids, such as estrogen, testosterone, corticosterone, and others, are generated from a common precursor, cholesterol, and are interconverted into one another. A wide variety of enzymes act upon cholesterol, including a number of dehydrogenases. One such dehydrogenase is 3-oxo-5-a-steroid dehydrogenase (OASD), a microsomal membrane protein highly expressed in prostate and other androgen-responsive tissues. OASD catalyzes the conversion of testosterone into dihydrotestosterone, which is the most potent androgen. Dihydrotestosterone is essential for the formation of the male phenotype during embryogenesis, as well as for proper androgen-mediated growth of tissues such as the prostate and male genitalia. A defect in OASD that prevents the conversion of testosterone into dihydrotestosterone leads to a rare form of male pseudohermaphroditis, characterized by defective formation of the external genitalia (Andersson, S., et al. (1991) Nature 354:159-161; Labrie, F., et al.
(1992) Endocrinology 131:1571-1573; OMIM #264600). Thus, DASD plays a central role in sexual differentiation and androgen physiology.
The discovery of new oxidoreductase proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative disorders including cancer; endocrine, metabolic, reproductive, neurological, viral, and autoimmunelinflammatory disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of oxidoreductase proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, oxidoreductase proteins, referred to collectively as "ORP" and individually as "ORP-1," "ORP-2," "ORP-3," "ORP-4," "ORP-5," "ORP-6," "ORP-7,"
"ORP-8," "ORP-9," "ORP-10," "ORP-11," "ORP-12," "ORP-13," "ORP-14," "ORP-15,"
"ORP-16," "ORP-17," "ORP-18," "ORP-19," "ORP-20," "ORP-21," "ORP-22," "ORP-23,"
"ORP-24,"
"ORP-25," "ORP-26," "ORP-27." In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: l-27. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-27.
The invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID N0:1-27. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:28-54.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID N0:1-27.
The invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected fiom the group consisting of SEQ ID N0:28-54, c) a polynucleodde sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-27. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional ORP, comprising administering to a patient in need of such treatment the composition.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional ORP, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional ORP, comprising administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected liom the group consisting of SEQ ID N0:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID N0:1-27. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that modulates the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-27. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID N0:28-54, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the. treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID
N0:28-54, ii) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, ii) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:28-54, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above;
c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding ORP.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of ORP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA
was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding ORP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described.
All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"ORP" refers to the amino acid sequences of substantially purified ORP
obtained from any SpeCles, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.

The term "agonist" refers to a molecule which intensifies or mimics the biological activity of ORP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of ORP either by directly interacting with ORP
or by acting on components of the biological pathway in which ORP
participates.
An "allelic variant" is an alternative form of the gene encoding ORP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding ORP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as ORP or a polypeptide with at least one functional characteristic of ORP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding ORP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding ORP. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent ORP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of ORP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.

The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of ORP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of ORP either by directly interacting with ORP or by acting on components of the biological pathway in which ORP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind ORP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic ORP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding ORP or fragments of ORP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp , Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
A "fragment" is a unique portion of ORP or the polynucleotide encoding ORP
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25 % or 50% of a polypeptide) as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ 1D N0:28-54 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:28-54, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:28-54 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish ' SEQ ID N0:28-54 from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:28-54 and the region of SEQ ID N0:28-54 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.

A fragment of SEQ ID NO:1-27 is encoded by a fragment of SEQ ID N0:28-54. A
fragment of SEQ ID NO: l-27 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-27. For example, a fragment of SEQ ID NO:1-27 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-27.
The precise length of a fragment of SEQ ID NO:1-27 and the region of SEQ ID NO:1-27 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full-length" polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A "full-length" polynucleotide sequence encodes a "full-length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off.- 50 Expect: l0 Word Size: ll Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight fable. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity"

between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (Apr-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 ~ g/ml sheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T"~ for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al., 1989, Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 p g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of ORP
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of ORP which is useful in any of the antibody production methods disclosed herein or known in the art.

The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of ORP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of ORP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an ORP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of ORP.
"Probe" refers to nucleic acid sequences encoding ORP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA
polymerise enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerise chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo~v, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU
primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead InstitutelMIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, su ra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA
stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding ORP, or fragments thereof, or ORP itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed liom their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed" cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants, and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook, J. et al. (1989), su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic"
(as defined above), "splice," "species," or "polymorphic" variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%
or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human oxidoreductase proteins (ORP), the polynucleotides encoding ORP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative disorders including cancer; endocrine, metabolic, reproductive, neurological, viral, and autoimmunelinflammatory disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding ORP.
Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each ORP were identified, and column 4 shows the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA
libraries. In some cases, GenBank sequence identifiers are also shown in column 5. The Incyte clones and GenBank cDNA
sequences, where indicated, in column 5 were used to assemble the consensus nucleotide sequence of each ORP and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding ORP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID N0:28-54 and to distinguish between SEQ ID N0:28-54 and related polynucleotide sequences.
The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
Column 3 lists tissue categories which express ORP as a fraction of total tissues expressing ORP.
Column 4 lists diseases, disorders, or conditions associated with those tissues expressing ORP as a fraction of total tissues expressing ORP. Column 5 lists the vectors used to subclone each cDNA library.
The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which eDNA clones encoding ORP were isolated. Column 1 references the nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
SEQ ID N0:31 maps to chromosome 3 within the interval from 23.2 to 31.4 centiMorgans.
SEQ ID N0:42 maps to chromosome 22 within the interval from 0 to 40.2 centiMorgans. SEQ ID
N0:48 maps to chromosome 7 within the interval from 100.5 to 114.5 centiMorgans, to chromosome 17 within the interval from 67.6 to 69.3 centiMorgans, and to chromosome 17 within the interval from 83.8 centiMorgans to the q terminus. SEQ ID N0:53 maps to chromosome 11 within the interval from 64.9 to 70.9 centiMorgans.
The invention also encompasses ORP variants. A preferred ORP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the ORP amino acid sequence, and which contains at least one functional or structural characteristic of ORP.
The invention also encompasses polynucleotides which encode ORP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:28-54, which encodes ORP. The polynucleotide sequences of SEQ
ID N0:28-54, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding ORP. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding ORP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID
N0:28-54 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:28-54.
Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of ORP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding ORP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring ORP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode ORP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring ORP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding ORP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding ORP
and its derivatives without altering the encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode ORP
and ORP
derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding ORP or any fragment thereof.
Also encompassed by the invention are polynucleodde sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:28-54 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI
CATALYST
800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding ORP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.

2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al.
(1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode ORP may be cloned in recombinant DNA molecules that direct expression of ORP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express ORP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter ORP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of ORP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding ORP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.) Alternatively, ORP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of ORP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing.
(See, e.g., Creighton, su ra, pp. 28-53.) In order to express a biologically active ORP, the nucleotide sequences encoding ORP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding ORP. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding ORP. Such signals include the ATG initiation colon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding ORP and its initiation colon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation colon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding ORP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding ORP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Broglie, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105; The MeGraw Hill Yearbook of Science and Technology (1992) MeGraw Hill, New York NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding ORP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding ORP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding ORP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem.
264:5503-5509.) When large quantities of ORP are needed, e.g. for the production of antibodies, vectors which direct high level expression of ORP may be used. For example, vectors containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of ORP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra;
Bitter, supra; and Scorer, supra.) Plant systems may also be used for expression of ORP. Transcription of sequences encoding ORP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, supra; Brogue, supra; and Winter, supra.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfec;tion. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp.
191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding ORP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses ORP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of ORP in cell lines is preferred. For example, sequences encoding ORP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk~ and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate Q-glucuronide, or luciferase and its substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.) Although the presencelabsence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding ORP is inserted within a marker gene sequence, transformed cells containing sequences encoding ORP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding ORP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding ORP and that express ORP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of ORP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on ORP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al.
(1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect.
IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding ORP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding ORP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US
Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding ORP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode ORP may be designed to contain signal sequences which direct secretion of ORP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Mantissas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding ORP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric ORP
protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of ORP activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the ORP encoding sequence and the heterologous protein sequence, so that ORP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, su ra, ch. 10). A
variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled ORP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
ORP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to ORP. At least one and up to a plurality of test compounds may be screened for specific binding to ORP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of ORP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which ORP
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express ORP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.

coli. Cells expressing ORP or cell membrane fractions which contain ORP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either ORP or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with ORP, either in solution or affixed to a solid support, and detecting the binding of ORP to the compound.
Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
ORP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of ORP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for ORP
activity, wherein ORP is combined with at least one test compound, and the activity of ORP in the presence of a test compound is compared with the activity of ORP in the absence of the test compound. A change in the activity of ORP in the presence of the test compound is indicative of a compound that modulates the activity of ORP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising ORP under conditions suitable for ORP
activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of ORP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding ORP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding ORP may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding ORP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding ORP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress ORP, e.g., by secreting ORP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of ORP and oxidoreductase proteins. In addition, the expression of ORP is closely associated with cancerous, inflamed, reproductive, and gastrointestinal tissues. Therefore, ORP
appears to play a role in cell proliferative disorders including cancer;
endocrine, metabolic, reproductive, neurological, viral, and autoimmune/inflammatory disorders. In the treatment of disorders associated with increased ORP expression or activity, it is desirable to decrease the expression or activity of ORP. In the treatment of disorders associated with decreased ORP
expression or activity, it is desirable to increase the expression or activity of ORP.
Therefore, in one embodiment, ORP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ORP.
Examples of such disorders include, but are not limited to, a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease, myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an endocrine disorder, such as disorders of the hypothalamus and pituitary resulting from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma, disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism, disorders associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, disorders associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease, disorders associated with hyperparathyroidism including Conn disease (chronic hypercalemia), pancreatic disorders such as Type I or Type II diabetes mellitus and associated complications, disorders associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, disorders associated with gonadal steroid hormones such as:
in women, abnormal prolactin production, infertility, endometriosis, perturbations of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, hypergonadal disorders associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia; a metabolic disorder, such as Addison's disease, cystic fibrosis, diabetes, fatty hepatocirrhosis, galactosemia, goiter, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypothyroidism hyperlipidemia, hyperlipemia, lipid myopathies, obesity, lipodystrophies, and phenylketonuria, congenital adrenal hyperplasia, pseudovitamin D-deficiency rickets, cerebrotendinous xanthomatosis, and coumarin resistance; a reproductive disorder, such as disorders of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, disruptions of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea, disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia; a neurological disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; an autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and a viral disorder, such as viral infections, e.g., those caused by adenoviruses (acute respiratory disease, pneumonia), arenaviruses (lymphocytic choriomeningitis), bunyaviruses (Hantavirus), coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses (hepatitis), herpesviruses (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus), llaviviruses (yellow fever), orthomyxoviruses (influenza), papillomaviruses (cancer), paramyxoviruses (measles, mumps), picornoviruses (rhinovirus, poliovirus, coxsackie-virus), polyomaviruses (BK virus, JC virus), poxviruses (smallpox), reovirus (Colorado tick fever), retroviruses (human immunodeficiency virus, human T lymphotropic virus), rhabdoviruses (rabies), rotaviruses (gastroenteritis), and togaviruses (encephalitis, rubella).
In another embodiment, a vector capable of expressing ORP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ORP including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified ORP
in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ORP including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of ORP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ORP including, but not limited to, those listed above.
In a further embodiment, an antagonist of ORP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ORP.
Examples of such disorders include, but are not limited to, those cell proliferative disorders including cancer; endocrine, metabolic, reproductive, neurological, viral, and autoimmune/inflammatory disorders described above.
In one aspect, an antibody which specifically binds ORP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues .
which express ORP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding ORP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ORP including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of ORP may be produced using methods which are generally known in the art.
In particular, purified ORP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind ORP. Antibodies to ORP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with ORP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to ORP
have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein.
Short stretches of ORP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to ORP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the fiybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce ORP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R.
(1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for ORP may also be generated. For example, such fragments include, but are not limited to, F(ab~2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab~2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between ORP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering ORP epitopes is generally used, but a competitive binding assay may also be employed (Pound, su ra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for ORP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of ORP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple ORP
epitopes, represents the average affinity, or avidity, of the antibodies for ORP. The I~ determined for a preparation of monoclonal antibodies, which are monospecific for a particular ORP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in which the ORP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with I~ ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of ORP, preferably in active form, from the antibody (Catty, D.
(1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A.
Cryer (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of ORP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al., supra.) In another embodiment of the invention, the polynucleotides encoding ORP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding ORP.
Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding ORP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding ORP may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in ORP expression or regulation causes disease, the expression of ORP
from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in ORP
are treated by constructing mammalian expression vectors encoding ORP and introducing these vectors by mechanical means into ORP-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem.
62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin.
Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of ORP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). ORP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or j3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M. V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding ORP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to ORP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding ORP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus ci~-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.

Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al.
(1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding ORP to cells which have one or more genetic abnormalities with respect to the expression of ORP. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E.
et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S.
Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr.
19:511-544; and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding ORP to target cells which have one or more genetic abnormalities with respect to the expression of ORP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing ORP to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res.169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV
vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol.
163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding ORP to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV
genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469).
During alphavirus RNA
replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for ORP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of ORP-coding RNAs and the synthesis of high levels of ORP in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al.
(1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of ORP
into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A
complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecples may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding ORP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding ORP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerise promoters such as T7 or SP6. Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding ORP. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased ORP
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding ORP may be therapeutically useful, and in the treament of disorders associated with decreased ORP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding ORP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding ORP is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding ORP are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding ORP. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem.
Biophys. Res. Commun.
268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechnol. 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of ORP, antibodies to ORP, and mimetics, agonists, antagonists, or inhibitors of ORP.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g.
larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S.
Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising ORP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, ORP or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example ORP or fragments thereof, antibodies of ORP, and agonists, antagonists or inhibitors of ORP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDSO (the dose therapeutically effective in 50% of the population) or LDSO (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDSO/EDso ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy.
Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 g to 100,000 g, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind ORP may be used for the diagnosis of disorders characterized by expression of ORP, or in assays to monitor patients being treated with ORP or agonists, antagonists, or inhibitors of ORP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for ORP include methods which utilize the antibody and a label to detect ORP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring ORP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of ORP
expression. Normal or standard values for ORP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to ORP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of ORP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding ORP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of ORP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of ORP, and to monitor regulation of ORP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding ORP or closely related molecules may be used to identify nucleic acid sequences which encode ORP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identities only naturally occurring sequences encoding ORP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the ORP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:28-54 or from genomic sequences including promoters, enhancers, and introns of the ORP gene.
Means for producing specific hybridization probes for DNAs encoding ORP
include the cloning of polynucleotide sequences encoding ORP or ORP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerises and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidinlbiotin coupling systems, and the like.
Polynucleotide sequences encoding ORP may be used for the diagnosis of disorders associated with expression of ORP. Examples of such disorders include, but are not limited to, a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease, myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an endocrine disorder, such as disorders of the hypothalamus and pituitary resulting from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma, disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism, disorders associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, disorders associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease, disorders associated with hyperparathyroidism including Conn disease (chronic hypercalemia), pancreatic disorders such as Type I or Type II diabetes mellitus and associated complications, disorders associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, disorders associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbations of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, hypergonadal disorders associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia; a metabolic disorder, such as Addison's disease, cystic fibrosis, diabetes, fatty hepatocirrhosis, galactosemia, goiter, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypothyroidism hyperlipidemia, hyperlipemia, lipid myopathies, obesity, lipodystrophies, and phenylketonuria, congenital adrenal hyperplasia, pseudovitamin D-deficiency rickets, cerebrotendinous xanthomatosis, and coumarin resistance; a reproductive disorder, such as disorders of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, disruptions of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea, disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
a neurological disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; an autoimmune/inllammatory disorder, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystids, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and a viral disorder, such as viral infections, e.g., those caused by adenoviruses (acute respiratory disease, pneumonia), arenaviruses (lymphocytic choriomeningitis), bunyaviruses (Hantavirus), coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses (hepatitis), herpesviruses (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus), ilaviviruses (yellow fever), orthomyxoviruses (influenza), papillomaviruses (cancer), paramyxoviruses (measles, mumps), picornoviruses (rhinovirus, poliovirus, coxsackie-virus), polyomaviruses (BK virus, JC virus), poxviruses (smallpox), reovirus (Colorado tick fever), retroviruses (human immunodeficiency virus, human T
lymphotropic virus), rhabdoviruses (rabies), rotaviruses (gastroenteritis), and togaviruses (encephalitis, rubella). The polynucleotide sequences encoding ORP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered ORP expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding ORP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding ORP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding ORP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of ORP, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding ORP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) 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.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding ORP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding ORP, or a fragment of a polynucleotide complementary to the polynucleotide encoding ORP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA
or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding ORP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding ORP are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR
products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In f~CCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence database analysis methods, termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of ORP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described in Seilhamer, J.J. et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, antibodies specific for ORP, or ORP or fragments thereof may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number 5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under. given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, su ra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for ORP to quantify the levels of ORP expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal.
Biochem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J.
Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.

USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding ORP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP). (See, e.g., Larder, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding ORP
on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA
associated with that disorder and thus may further positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known.
This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or. affected individuals.
In another embodiment of the invention, ORP, its catalytic or immunogenic liagments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be liee in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between ORP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with ORP, or fragments thereof, and washed. Bound ORP is then detected by methods well known in the art. Purified ORP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding ORP specifically compete with a test compound for binding ORP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with ORP.
In additional embodiments, the nucleotide sequences which encode ORP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, in particular U.S. Ser. No. 60/172,367, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI
cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, su ra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), pcDNA2.l plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4 C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fiuorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN TI fluorescence scanner (Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, su ra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table S summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
'The polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM
is a probabilistic approach which analyzes consensus primary structures of gene families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:28-54. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and.l6.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIF'ESEQ (Incyte Genomics).
This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity S x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps).
If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding ORP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
V. Chromosomal Mapping of ORP Encoding Polynucleotides The cDNA sequences which were used to assemble SEQ ID N0:28-54 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:28-54 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and G~nethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
The genetic map locations of SEQ ID N0:31, SEQ ID N0:42, SEQ ID N0:48, and SEQ
ID
N0:53 are described in The Invention as ranges, or intervals, of human chromosomes. More than one map location is reported for SEQ ID N0:48, indicating that previously mapped sequences having similarity, but not complete identity, to SEQ ID N0:48 were assembled into their respective clusters.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (eM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Gen~thon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI
"GeneMap'99" World Wide Web site (http://www.ncbi.nlm.nih.gov/genemap~, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VI. Extension of ORP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:28-54 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO

4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 C to about 72 C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)ZS04, and (3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94 C, 3 min; Step 2: 94 C, 15 sec; Step 3: 60 C, 1 min; Step 4: 68 C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 C, 5 min; Step 7: storage at 4 C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94 C, 3 min; Step 2:
94 C, 15 sec; Step 3: 57 C, 1 min; Step 4: 68 C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68 C, 5 min; Step 7: storage at 4 C.
The concentration of DNA in each well was determined by dispensing 100 p1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 1 to 10 1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1:
94 C, 3 min; Step 2: 94 C, 15 sec; Step 3: 60 C, 1 min; Step 4: 72 C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72 C, 5 min; Step 7: storage at 4 C. DNA was quantified by PICOGREEN
reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC
DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:28-54 are used to obtain 5' regulatory sequences using the procedure above, along with oligonucleotides designed for such extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:28-54 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments.
Oligonucleotides are designed using state-of-the-art software such as OLIGO
4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 Ci of [-y-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst 1, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
VIII. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing, See, e.g., Baldeschweiler, s-upra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol.
16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation Total RNA is isolated liom tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/fil oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/~.~1 RNase inhibitor, 500 EiM dATP, 500 ~M dGTP, 500 NM dTTP, 40 EiM
dCTP, 40 NM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37 C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium hydroxide and incubated for 20 minutes at 85 C to the stop the reaction and degrade the RNA.
Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ~.~1 SX SSC/0.2% SDS.

Microarra,~paration Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure described in US
Patent No. 5,807,522, incorporated herein by reference. 1 E.il of the array element DNA, at an average concentration of 100 ng/Eil, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 C
followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 fil of sample mixture consisting of 0.2 ~g each of Cy3 and Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65 C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 Eil of SX SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60 C. The arrays are washed for 10 min at 45 C in a first wash buffer (1X SSC, 0.1 % SDS), three times for 10 minutes each at 45 C in a second wash buffer (0.1X SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Ine., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, Hamamatsu Photonics Systems, Bridgewater N.>7 corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices, Ine., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the lluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
IX. Complementary Polynucleotides Sequences complementary to the ORP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring ORP. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OL1G0 4.06 software (National Biosciences) and the coding sequence of ORP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the ORP-encoding transcript.
X. Expression of ORP
Expression and purification of ORP is achieved using bacterial or virus-based expression systems. For expression of ORP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter, and the TS or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express ORP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
Expression of ORP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Auto~raphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding ORP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc.
Natl. Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.) In most expression systems, ORP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from ORP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, su ra, ch. 10 and 16). Purified ORP obtained by these methods can be used directly in the assays shown in Examples XI and XV.
XI. Demonstration of ORP Activity For purposes of example, an assay demonstrating the activity of a short-chain alcohol dehydrogenase is described. Essentially the same method is used for other types of oxidoreductases, with suitable substitution of the substrate and electron acceptor. ORP
activity is demonstrated by the oxidation of NADPH to NADP in the presence of substrate (Kunau and Dommes (1978) Eur. J.
Biochem. 91:533-544). Substrates include, but are not limited to, all-trans-retinaldehyde and cis-4-dienoyl-CoA. ORP is preincubated for 10 minutes at 37 C in 60 pM potassium phosphate (pH 7.4), 125 nM NADPH, and 0.2 pM CoA (coenzyme A). The reaction is initiated by addition of the appropriate substrate (12.5 to 150 ~.~M final concentration). The change in absorbance of the reaction at 340 nm, due to the oxidation of NADPH to NADP, is measured using a spectrophotometer at 23 °C. Units of ORP activity are expressed as Nmoles of NADP formed per minute. A reaction lacking ORP is used as a negative control.
Alternatively, ORP activity is assayed by measuring the reduction of insulin.
Aliquots of ORP are preincubated at 37 °C for 20 min with 2 E~l of 50 mM Hepes, pH
7.6, 100 ~g/ml bovine serum albumin, and 2 mM DTT in a total volume of 70 iil. Then, 40 iM of a reaction mixture composed of 200 Eil of Hepes (1 M), pH 7.6, 40 p1 of EDTA (0.2 M), 40 E~l of NADPH (40 mg/ml), and 500 Nl of insulin (10 mg/ml) is added. The reaction is initiated with the addition of 10 Eil of thioredoxin reductase from calf thymus (3.0 A412 unit), and incubation is continued for 20 min at 37 °C. The reaction rate is followed by monitoring the oxidation of NADPH
at 412 nM. The oxidation of NADPH is proportional to the amount of insulin reduction.
XII. Functional Assays ORP function is assessed by expressing the sequences encoding ORP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G.
( 1994) Flow C~rtometry, Oxford, New York NY.
The influence of ORP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding ORP and either CD64 or CD64-GFP.
CD64 and CD64-GFP
are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding ORP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XIII. Production of ORP Specific Antibodies ORP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the ORP amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. I 1.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KhH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-ORP
activity by, for example, binding the peptide or ORP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIV. Purification of Naturally Occurring ORP Using Specific Antibodies Naturally occurring or recombinant ORP is substantially purified by immunoaffinity chromatography using antibodies specific for ORP. An immunoaffinity column is constructed by covalently coupling anti-ORP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing ORP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of ORP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/ORP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and ORP is collected.
XV. Identification of Molecules Which Interact with ORP
ORP, or biologically active fragments thereof, are labeled with'zsI Bolton-Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a mufti-well plate are incubated with the labeled ORP, washed, and any wells with labeled ORP complex are assayed. Data obtained using different concentrations of ORP
are used to calculate values for the number, affinity, and association of ORP
with the candidate molecules.
Alternatively, molecules interacting with ORP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989, Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
ORP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent No. 6,057,101 ).
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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P ., SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
YUE, Henry LAL, Preeti TANG, Y. Tom HILLMAN, Jennifer BAUGHN, Mariah R.
AZIMZAI, Yalda LU, Dyung Aina M.
<120> HUMAN OXIDOREDUCTASE PROTEINS
<130> PF-0754 PCT
<140> To Be Assigned <141> Herewith <150> 60/172,367 <151> 1999-12-16 <160> 54 <170> PERL Program <210> 1 <211> 468 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 543496CD1 <400> 1 Met Ala Ala Arg Leu Val Ser Arg Cys Gly Ala Val Arg Ala Ala Pro His Ser Gly Pro Leu Val Ser Trp Arg Arg Trp Ser Gly Ala Ser Thr Asp Thr Val Tyr Asp Val Val Val Ser Gly Gly Gly Leu Val Gly Ala Ala Met Ala Cys Ala Leu Gly Tyr Asp Ile His Phe His Asp Lys Lys Ile Leu Leu Leu Glu Ala Gly Pro Lys Lys Val Leu Glu Lys Leu Ser Glu Thr Tyr Ser Asn Arg Val Ser Ser Ile Ser Pro Gly Ser Ala Thr Leu Leu Ser Ser Phe Gly Ala Trp Asp His Ile Cys Asn Met Arg Tyr Arg Ala Phe Arg Arg Met Gln Val Trp Asp Ala Cys Ser Glu Ala Leu Ile Met Phe Asp Lys Asp Asn Leu Asp Asp Met Gly Tyr Ile Val Glu Asn Asp Val Ile Met His Ala Leu Thr Lys Gln Leu Glu Ala Val Ser Asp Arg Val Thr Val Leu Tyr Arg Ser Lys Ala Ile Arg Tyr Thr Trp Pro Cys Pro Phe Pro Met Ala Asp Ser Ser Pro Trp Val His Ile Thr Leu Gly Asp Gly Ser Thr Phe Gln Thr Lys Leu Leu Ile Gly Ala Asp Gly His Asn Ser Gly Val Arg Gln Ala Val Gly Ile Gln Asn Val Ser Trp Asn Tyr Asp Gln Ser Ala Val Val Ala Thr Leu His Leu Ser Glu Ala Thr Glu Asn Asn Val Ala Trp Gln Arg Phe Leu Pro Ser Gly Pro Ile Ala Leu Leu Pro Leu 5er Asp Thr Leu Ser Ser Leu Val Trp Ser Thr Ser His Glu His Ala Ala Glu Leu Val Ser Met Asp Glu Glu Lys Phe Val Asp Ala Val Asn Ser Ala Phe Trp Ser Asp Ala Asp His Thr Asp Phe Ile Asp Thr Ala Gly Ala Met Leu Gln Tyr Ala Val Ser Leu Leu Lys Pro Thr Lys Val Ser Ala Arg Gln Leu Pro Pro Ser Val Ala Arg Val Asp Ala Lys Ser Arg Val Leu Phe Pro Leu Gly Leu Gly His Ala Ala Glu Tyr Val Arg Pro Arg Val Ala Leu Ile Gly Asp Ala Ala His Arg Val His Pro Leu Ala Gly Gln Gly Val Asn Met Gly Phe Gly Asp Ile Ser Ser Leu Ala His His Leu Ser Thr Ala Ala Phe Asn Gly Lys Asp Leu Gly Ser Val Ser His Leu Thr Gly Tyr Glu Thr Glu Arg Gln Arg His Asn Thr Ala Leu Leu Ala Ala Thr Asp Leu Leu Lys Arg Leu Tyr Ser Thr Ser Ala Ser Pro Leu Val Leu Leu Arg Thr Trp Gly Leu Gln Ala Thr Asn Ala Val Ser Pro Leu Lys Glu Gln Ile Met Ala Phe Ala Ser Lys <210> 2 <211> 254 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 907607CD1 <400> 2 Met Ala Ala Thr Ala Arg Arg Gly Trp Gly Ala Ala Ala Val Ala Ala Gly Leu Arg Arg Arg Phe Cys His Met Leu Lys Asn Pro Tyr Thr Ile Lys Lys Gln Pro Leu His Gln Phe Val Gln Arg Pro Leu Phe Pro Leu Pro Ala Ala Phe Tyr His Pro Val Arg Tyr Met Phe Ile Gln Thr Gln Asp Thr Pro Asn Pro Asn Ser Leu Lys Phe Ile Pro Gly Lys Pro Val Leu Glu Thr Arg Thr Met Asp Phe Pro Thr Pro Ala Ala Ala Phe Arg Ser Pro Leu Ala Arg Gln Leu Phe Arg Ile Glu Gly Val Lys Ser Val Phe Phe Gly Pro Asp Phe Ile Thr Val Thr Lys Glu Asn Glu Glu Leu Asp Trp Asn Leu Leu Lys Pro Asp Ile Tyr Ala Thr Ile Met Asp Phe Phe Ala Ser Gly Leu Pro Leu Val Thr Glu Glu Thr Pro Ser Gly Glu Ala Gly Ser Glu Glu Asp Asp Glu Val Val Ala Met Ile Lys Glu Leu Leu Asp Thr Arg Ile Arg Pro Thr Val Gln Glu Asp Gly Gly Asp Val Ile Tyr Lys Gly Phe Glu Asp Gly Ile Val Gln Leu Lys Leu Gln Gly Ser Cys Thr Ser Cys Pro Ser Ser Ile Ile Thr Leu Lys Asn Gly Ile Gln Asn Met Leu Gln Phe Tyr Ile Pro Glu Val Glu Gly Val Glu Gln Val Met Asp Asp Glu Ser Asp Glu Lys Glu Ala Asn Ser Pro <210> 3 <211> 555 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1290078CD1 <400> 3 Met Gln Ser Cys Glu Ser Ser Gly Asp Ser Ala Asp Asp Pro Leu Ser Arg Gly Leu Arg Arg Arg Gly Gln Pro Arg Val Val Val Ile Gly Ala Gly Leu Ala Gly Leu Ala Ala Ala Lys Ala Leu Leu Glu Gln Gly Phe Thr Asp Val Thr Val Leu Glu Ala Ser Ser His Ile Gly Gly Arg Val Gln Ser Val Lys Leu Gly His Ala Thr Phe Glu Leu Gly Ala Thr Trp Ile His Gly Ser His Gly Asn Pro Ile Tyr His Leu Ala Glu Ala Asn Gly Leu Leu Glu Glu Thr Thr Asp Gly Glu Arg Ser Val Gly Arg Ile Ser Leu Tyr Ser Lys Asn Gly Val Ala Cys Tyr Leu Thr Asn His Gly Arg Arg Ile Pro Lys Asp Val Val Glu Glu Phe Ser Asp Leu Tyr Asn Glu Val Tyr Asn Leu Thr Gln Glu Phe Phe Arg His Asp Lys Pro Val Asn Ala Glu Ser Gln Asn Ser Val Gly Val Phe Thr Arg Glu Glu Val Arg Asn Arg Ile Arg Asn Asp Pro Asp Asp Pro Glu Ala Thr Lys Arg Leu Lys Leu Ala Met Ile Gln Gln Tyr Leu Lys Val Glu Ser Cys Glu Ser Ser Ser His Ser Met Asp Glu Val Ser Leu Ser Ala Phe Gly Glu Trp Thr Glu Ile Pro Gly Ala His His Ile Ile Pro Ser Gly Phe Met Arg Val Val Glu Leu Leu Ala Glu Gly Ile Pro Ala His Val Ile Gln Leu Gly Lys Pro Val Arg Cys Ile His Trp Asp Gln Ala Ser Ala Arg Pro Arg Gly Pro Glu Ile Glu Pro Arg Gly Glu Gly Asp His Asn His Asp Thr Gly Glu Gly Gly Gln Gly Gly Glu Glu Pro Arg Gly Gly Arg Trp Asp Glu Asp Glu Gln Trp Ser Val Val Val Glu Cys Glu Asp Cys Glu Leu Ile Pro Ala Asp His Val Ile Val Thr Val Ser Leu Gly Val Leu Lys Arg Gln Tyr Thr Ser Phe Phe Arg Pro Gly Leu Pro Thr Glu Lys Val Ala Ala Ile His Arg Leu Gly Ile Gly Thr Thr Asp Lys Ile Phe Leu Glu Phe Glu Glu Pro Phe Trp Gly Pro Glu Cys Asn Ser Leu Gln Phe Val Trp Glu Asp Glu Ala Glu Ser His Thr Leu Thr Tyr Pro Pro Glu Leu Trp Tyr Arg Lys Ile Cys Gly Phe Asp Val Leu Tyr Pro Pro Glu Arg Tyr Gly His Val Leu Ser Gly Trp Ile Cys Gly Glu Glu Ala Leu Val Met Glu Lys Cys Asp Asp Glu Ala Val Ala Glu Ile Cys Thr Glu Met Leu Arg Gln Phe Thr Gly Asn Pro Asn Ile Pro Lys Pro Arg Arg Ile Leu Arg Ser Ala Trp Gly Ser Asn Pro Tyr Phe Arg Gly Ser Tyr Ser Tyr Thr Gln Val Gly Ser Ser Gly Ala Asp Val Glu Lys Leu Ala Lys Pro Leu Pro Tyr Thr Glu Ser Ser Lys Thr Ala Pro Met Gln Val Leu Phe Ser Gly Glu Ala Thr His Arg Lys Tyr Tyr Ser Thr Thr His Gly Ala Leu Leu Ser Gly Gln Arg Glu Ala Ala Arg Leu Ile Glu Met Tyr Arg Asp Leu Phe Gln Gln Gly Thr <210> 4 <211> 337 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1302741CD1 <400> 4 Met Ala Leu Gln Thr Leu Gln Ser Ser Trp Val Thr Phe Arg Lys Ile Leu Ser His Phe Pro Glu Glu Leu Ser Leu Ala Phe Val Tyr Gly Ser Gly Val Tyr Arg Gln Ala Gly Pro Ser Ser Asp Gln Lys Asn Ala Met Leu Asp Phe Val Phe Thr Val Asp Asp Pro Val Ala Trp His Ser Lys Asn Leu Lys Lys Asn Trp Ser His Tyr Ser Phe Leu Lys Val Leu Gly Pro Lys Ile Ile Thr Ser Ile Gln Asn Asn Tyr Gly Ala Gly Val Tyr Tyr Asn Ser Leu Ile Met Cys Asn Gly Arg Leu Ile Lys Tyr Gly Val Ile Ser Thr Asn Val Leu Ile Glu Asp Leu Leu Asn Trp Asn Asn Leu Tyr Ile Ala Gly Arg Leu Gln Lys Pro Val Lys Ile Ile Ser Val Asn Glu Asp Val Thr Leu Arg Ser Ala Leu Asp Arg Asn Leu Lys Ser Ala Val Thr Ala Ala Phe Leu Met Leu Pro Glu Ser Phe Ser Glu Glu Asp Leu Phe Ile Glu Ile Ala Gly Leu Ser.Tyr Ser Gly Asp Phe Arg Met Val Val Gly Glu Asp Lys Thr Lys Val Leu Asn Ile Val Lys Pro Asn Ile Ala His Phe Arg Glu Leu Tyr Gly Ser Ile Leu Gln Glu Asn Pro Gln Val Val Tyr Lys Ser Gln Gln Gly Trp Leu Glu Ile Asp Lys Ser Pro Glu Gly Gln Phe Thr Gln Leu Met Thr Leu Pro Lys Thr Leu Gln Gln Gln Ile Asn His Ile Met Asp Pro Pro Gly Lys Asn Arg Asp Val Glu Glu Thr Leu Phe Gln Val Ala His Asp Pro Asp Cys Gly Asp Val Val Arg Leu Gly Leu Ser Ala Ile Val Arg Pro Ser Ser Ile Arg Gln Ser Thr Lys Gly Ile Phe Thr Ala Gly Leu Lys Lys Ser Val Ile Tyr Ser Ser Leu Lys Leu His Lys Met Trp Lys Gly Trp Leu Arg Lys Thr Ser <210> 5 <211> 109 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1541028CD1 <400> 5 Met Ser Ala Asn Thr Phe Gly Asn Ala Gly Phe Ser Val Leu Leu Pro Gly Ala Arg Leu Glu Gly Arg Cys Gly Pro Thr Asn Ala Arg Val Arg Cys His Leu Gly Leu Lys Ile Pro Pro Gly Cys Glu Leu Val Val Gly Gly Glu Pro Gln Cys Trp Ala Glu Gly His Cys Leu Leu Val Asp Asp Ser Phe Leu His Thr Val Ala His Asn Gly Ser Pro Glu Asp Gly Pro Arg Val Val Phe Ile Val Asp Leu Trp His Pro Asn Val Ala Gly Ala Glu Arg Gln Ala Leu Asp Phe Val Phe Ala Pro Asp Pro <210> 6 <211> 385 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 1597687CD1 <400> 6 Met Lys Met Leu Pro Gly Val Gly Val Phe Gly Thr Gly Ser Ser Ala Arg Val Leu Val Pro Leu Leu Arg Ala Glu Gly Phe Thr Val Glu Ala Leu Trp Gly Lys Thr_Glu Glu Glu Ala Lys Gln Leu Ala Glu Glu Met Asn Ile Ala Phe Tyr Thr Ser Arg Thr Asp Asp Ile Leu Leu His Gln Asp Val Asp Leu Val Cys Ile Ser Ile Pro Pro Pro Leu Thr Arg Gln Ile Ser Val Lys Ala Leu Gly Ile Gly Lys Asn Val Val Cys Glu Lys Ala Ala Thr Ser Val Asp Ala Phe Arg Met Val Thr Ala Ser Arg Tyr Tyr Pro Gln Leu Met Ser Leu Val Gly Asn Val Leu Arg Phe Leu Pro Ala Phe Val Arg Met Lys Gln Leu Ile Ser Glu His Tyr Val Gly Ala Val Met Ile Cys Asp Ala Arg Ile Tyr Ser Gly Ser Leu Leu Ser Pro Ser Tyr Gly Trp Ile Cys Asp Glu Leu Met Gly Gly Gly Gly Leu His Thr Met Gly Thr Tyr Ile Val Asp Leu Leu Thr His Leu Thr Gly Arg Arg Ala Glu Lys Val His Gly Leu Leu Lys Thr Phe Val Arg Gln Asn Ala Ala Ile Arg Gly Ile Arg His Val Thr Ser Asp Asp Phe Cys Phe Phe Gln Met Leu Met Gly Gly Gly Val Cys Ser Thr Val Thr Leu Asn Phe Asn Met Pro Gly Ala Phe Val His Glu Val Met Val Val Gly Ser Ala Gly Arg Leu Val Ala Arg Gly Ala Asp Leu Tyr Gly Gln Lys Asn Ser Ala Thr Gln Glu Glu Leu Leu Leu Arg Asp Ser Leu Ala Val Gly Ala Gly Leu Pro Glu Gln Gly Pro Gln Asp Val Pro Leu Leu Tyr Leu Lys Gly Met Val Tyr Met Val Gln Ala Leu Arg Gln Ser Phe Gln Gly Gln Gly Asp Arg Arg Thr Trp Asp Arg Thr Pro Val Ser Met Ala Ala Ser Phe Glu Asp Gly Leu Tyr Met Gln Ser Val Val Asp Ala Ile Lys Arg Ser Ser Arg Ser Gly Glu Trp Glu Ala Val Glu Val Leu Thr Glu Glu Pro Asp Thr Asn Gln Asn Leu Cys Glu Ala Leu Gln Arg Asn Asn Leu <210> 7 <211> 312 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1690348CD1 <400> 7 Met Ala Cys Ala Ala Val Met Ile Pro Gly Leu Leu Arg Cys Ser 1 5 ~ 10 15 Val Gly Ala Ile Arg Ile Glu Ala Ala Ser Leu Arg Leu Thr Leu Ser Thr Leu Arg His Leu Thr Leu Thr Ser Ile Met Lys Ser Lys Arg Lys Thr Asp His Met Glu Arg Thr Ala Ser Val Leu Arg Arg Glu Ile Val Ala Ala Ala Lys Val Cys Gly Ala Ala Ser Glu Ser Pro Ser Val Lys Ser Leu Arg Leu Leu Val Ala Asp Gln Asp Phe Ser Phe Lys Ala Gly Gln Trp Val Asp Phe Phe Ile Pro Gly Val Ser Val Val Gly Gly Phe Ser Ile Cys Ser Ser Pro Arg Leu Leu Glu Gln Glu Arg Val Ile Glu Leu Ala Val Lys Tyr Thr Asn His Pro Pro Ala Leu Trp Val His Asn Thr Cys Thr Leu Asp Cys Glu Val Ala Val Arg Val Gly Gly Glu Phe Phe Phe Asp Pro Gln Pro Ala Asp Ala Ser Arg Asn Leu Val Leu Ile Ala Gly Gly Val Gly Ile Asn Pro Leu Leu Ser Ile Leu Arg His Ala Ala Asp Leu Leu Arg Glu Gln Ala Asn Lys Arg Asn Gly Tyr Glu Ile Gly Thr Ile Lys Leu Phe Tyr Ser Ala Lys Asn Thr Ser Glu Leu Leu Phe Lys Lys Asn Ile Leu Asp Leu Val Asn Glu Phe Pro Glu Lys Ile Ala Cys Ser Leu His Val Thr Lys Gln Thr Thr Gln Ile Asn Ala Glu Leu Lys Pro Tyr Ile Thr Glu Gly Arg Ile Thr Glu Lys Glu Ile Arg Asp His Ile Ser Lys Glu Thr Leu Phe Tyr Ile Cys Gly Pro Pro Pro Met Thr Asp Phe Phe Ser Lys Gln Leu Glu Asn Asn His Val Pro Lys Glu His Ile Cys Phe Glu Lys Trp Trp <210> 8 <211> 160 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1865603CD1 <400> 8 Met Leu Arg His Leu Pro Ser Arg Leu Pro Val Lys Met Trp Gly Arg Thr Leu Glu Lys Gln Ser Trp Arg Asp Ser Ser Gln Thr Pro Pro Pro Cys Leu Ile Arg Arg Leu Asp His Ile Val Met Thr Val Lys Ser Ile Lys Asp Thr Thr Met Phe Tyr Ser Lys Ile Leu Gly Met Glu Val Met Thr Phe Lys Glu Asp Arg Lys Ala Leu Cys Phe Gly Asp Gln Lys Phe Asn Leu His Glu Val Gly Lys Glu Phe Glu Pro Lys Ala Ala His Pro Val Pro Gly Ser Leu Asp Ile Cys Leu Ile Thr Glu Val Pro Leu Glu Glu Met Ile Gln His Leu Lys Ala Cys Asp Val Pro Ile Glu Glu Gly Pro Val Pro Arg Thr Gly Ala Lys Gly Pro Ile Met Ser Ile Tyr Phe Arg Asp Pro Asp Arg Asn Leu Ile Glu Val Ser Asn Tyr Ile Ser Ser <210> 9 <211> 487 <212> PRT -<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1976472CD1 <400> 9 Met Asp Trp Ile Arg Leu Thr Lys Ser Gly Lys Asp Leu Thr Gly Leu Lys Gly Arg Leu Ile Glu Val Thr Glu Glu Glu Leu Lys Lys His Asn Lys Lys Asp Asp Cys Trp Ile Cys Ile Arg Gly Phe Val Tyr Asn Val Ser Pro Tyr Met Glu Tyr His Pro Gly Gly Glu Asp Glu Leu Met Arg Ala Ala Gly Ser Asp Gly Thr Glu Leu Phe Asp Gln Val His Arg Trp Val Asn Tyr Glu Ser Met Leu Lys Glu Cys Leu Val Gly Arg Met Ala Ile Lys Pro Ala Val Leu Lys Asp Tyr Arg Glu Glu Glu Lys Lys Val Leu Asn Gly Met Leu Pro Lys Ser Gln Val Thr Asp Thr Leu Ala Lys Glu Gly Pro Ser Tyr Pro Ser Tyr Asp Trp Phe Gln Thr Asp Ser Leu Val Thr Ile Ala Ile Tyr Thr Lys Gln Lys Asp Ile Asn Leu Asp Ser Ile Ile Val Asp His Gln Asn Asp Ser Phe Arg Ala Glu Thr Ile Ile Lys Asp Cys Leu Tyr Leu Ile His Ile Gly Leu Ser His Glu Val Gln Glu Asp Phe Ser Val Arg Val Val Glu Ser Val Gly Lys Ile Glu Ile Val Leu Gln Lys Lys Glu Asn Thr Ser Trp Asp Phe Leu Gly His Pro Leu Lys Asn His Asn Ser Leu Ile Pro Arg Lys Asp Thr Gly Leu Tyr Tyr Arg Lys Cys Gln Leu Ile Ser Lys Glu Asp Val Thr His Asp Thr Arg Leu Phe Cys Leu Met Leu Pro Pro Ser Thr His Leu Gln Val Pro Ile Gly Gln His Val Tyr Leu Lys Leu Pro Ile Thr Gly Thr Glu Ile Val Lys Pro Tyr Thr Pro Val Ser Gly Ser Leu Leu Ser Glu Phe Lys Glu Pro Val Leu Pro Asn Asn Lys Tyr Ile Tyr Phe Leu Ile Lys Ile Tyr Pro Thr Gly Leu Phe Thr Pro Glu Leu Asp Arg Leu Gln Ile Gly Asp Phe Val Ser Val Ser Ser Pro Glu Gly Asn Phe Lys Ile Ser Lys Phe Gln Glu Leu Glu Asp Leu Phe Leu Leu Ala Ala Gly Thr Gly Phe Thr Pro Met Val Lys Ile Leu Asn Tyr Ala Leu Thr Asp Ile Pro Ser Leu Arg Lys Val Lys Leu Met Phe Phe Asn Lys Thr Glu Asp Asp Ile Ile Trp Arg Ser Gln Leu Glu Lys Leu Ala Phe Lys Asp Lys Arg Leu Asp Val Glu Phe Val Leu Ser Ala Pro Ile Ser Glu Trp Asn Gly Lys Gln Gly His Ile Ser Pro Ala Leu Leu Ser Glu Phe Leu Lys Arg Asn Leu Asp Lys Ser Lys Val Leu Val Cys Ile Cys Gly Pro Val Pro Phe Thr Glu Gln Gly Val Arg Leu Leu His Asp Leu Asn Phe Ser Lys Asn Glu Ile His Ser Phe Thr Ala <210> 10 <211> 524 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2050821CD1 <400> 10 Met Ser Leu Leu Ser Leu Pro Trp Leu Gly Leu Arg Pro Val Ala Met Ser Pro Trp Leu Leu Leu Leu Leu Val Val Gly Ser Trp Leu Leu Ala Arg Ile Leu Ala Trp Thr Tyr Ala Phe Tyr Asn Asn Cys Arg Arg Leu Gln Cys Phe Pro Gln Pro Pro Lys Arg Asn Trp Phe Trp Gly His Leu Gly Leu Ile Thr Pro Thr Glu Glu Gly Leu Lys Asp Ser Thr Gln Met Ser Ala Thr Tyr Ser Gln Gly Phe Thr Val Trp Leu Gly Pro Ile Ile Pro Phe Ile Val Leu Cys His Pro Asp Thr Ile Arg Ser Ile Thr Asn Ala Ser Ala Ala Ile Ala Pro Lys Asp Asn Leu Phe Ile Arg Phe Leu Lys Pro Trp Leu Gly Glu Gly Ile Leu Leu Ser Gly Gly Asp Lys Trp Ser Arg His Arg Arg Met Leu Thr Pro Ala Phe His Phe Asn Ile Leu Lys Ser Tyr Ile Thr Ile Phe Asn Lys Ser Ala Asn Ile Met Leu Asp Lys Trp Gln His Leu Ala Ser Glu Gly Ser Ser Arg Leu Asp Met Phe Glu His Ile Ser Leu Met Thr Leu Asp Ser Leu Gln Lys Cys Ile Phe Ser Phe Asp Ser His Cys Gln Glu Arg Pro Ser Glu Tyr Ile Ala Thr Ile Leu Glu Leu Ser Ala Leu Val Glu Lys Arg Ser Gln His Ile Leu Gln His Met Asp Phe Leu Tyr Tyr Leu Ser His Asp Gly Arg Arg Phe His Arg Ala Cys Arg Leu Val His Asp Phe Thr Asp Ala Val Ile Arg Glu Arg Arg Arg Thr Leu Pro Thr Gln Gly Ile Asp Asp Phe Phe Lys Asp Lys Ala Lys Ser Lys Thr Leu Asp Phe Ile Asp Val Leu Leu Leu Ser Lys Asp Glu Asp Gly Lys Ala Leu Ser Asp Glu Asp Ile Arg Ala Glu Ala Asp Thr Phe Met Phe Gly Gly His Asp Thr Thr Ala Ser Gly Leu Ser Trp Val Leu Tyr Asn Leu Ala Arg His Pro Glu Tyr Gln Glu Arg Cys Arg Gln Glu Val Gln Glu Leu Leu Lys Asp Arg Asp Pro Lys Glu Ile Glu Trp Asp Asp Leu Ala Gln Leu Pro Phe Leu Thr Met Cys Val Lys Glu Ser Leu Arg Leu His Pro Pro Ala Pro Phe Ile Ser Arg Cys Cys Thr Gln Asp Ile Val Leu Pro Asp Gly Arg Val Ile Pro Lys Gly Ile Thr Cys Leu Ile Asp Ile Ile Gly Val His His Asn Pro Thr Val Trp Pro 425 . 430 435 Asp Pro Glu Val Tyr Asp Pro Phe Arg Phe Asp Pro Glu Asn Ser Lys Gly Arg Ser Pro Leu Ala Phe Ile Pro Phe Ser Ala Gly Pro Arg Asn Cys Ile Gly Gln Ala Phe Ala Met Ala Glu Met Lys Val Val Leu Ala Leu Met Leu Leu His Phe Arg Phe Leu Pro Asp His Thr Glu Pro Arg Arg Lys Leu Glu Leu Ile Met Arg Ala Glu Gly Gly Leu Trp Leu Arg Val Glu Pro Leu Asn Val Gly Leu Gln <210> 11 <211> 144 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2408443CD1 <400> 11 Met Val Thr Leu Tyr Ser Ser Pro Ser Cys Thr Ser Cys Arg Lys Ala Lys Gln Trp Leu Val Asp His Asn Leu Pro Phe Ile Glu Arg Asn Leu Asn Lys Glu Pro Leu Arg Ala Glu Asp Val Lys Ala Met Leu Arg Leu Thr Glu Asp Gly Thr Glu Glu Leu Ile Ser Thr Arg Ser Lys Ile Phe Ser Glu Leu Thr Ile Asp Leu Asp Asp Met Ser Ile Asn Lys Leu Ile Asp Leu Ile Val Met Tyr Pro Ser Leu Leu Lys Arg Pro Ile Ile Leu Asp Asp Gln Arg Met Gln Ile Gly Tyr Asn Asp Asp Glu Ile Arg Arg Phe Leu Pro Arg Glu Val Arg Gln Arg Glu Leu Ile Arg Ala Thr Phe Lys Ala Asp Phe Ala Glu Glu Ala Lys Asp Leu Val Val Glu Glu Gly <210> 12 <211> 373 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2508668CD1 <400> 12 Met Trp Val Cys Ser Thr Leu Trp Arg Val Arg Thr Pro Ala Arg Gln Trp Arg Gly Leu Leu Pro Ala Ser Gly Cys His Gly Pro Ala Ala Ser Ser Tyr Ser Ala Ser Ala Glu Pro Ala Arg Val Arg Ala Leu Val Tyr Gly His His Gly Asp Pro Ala Lys Val Val Glu Leu Lys Asn Leu Glu Leu Ala Ala Val Arg Gly Ser Asp Val Arg Val Lys Met Leu Ala Ala Pro Ile Asn Pro Ser Asp Ile Asn Met Ile Gln Gly Asn Tyr Gly Leu Leu Pro Glu Leu Pro Ala Val Gly Gly Asn Glu Gly Val Ala Gln Val Val Ala Val Gly Ser Asn Val Thr Gly Leu Lys Pro Gly Asp Trp Val Ile Pro Ala Asn Ala Gly Leu Gly Thr Trp Arg Thr Glu Ala Val Phe Ser Glu Glu Ala Leu Ile Gln Val Pro Ser Asp Ile Pro Leu Gln Ser Ala Ala Thr Leu Gly Val Asn Pro Cys Thr Ala Tyr Arg Met Leu Met Asp Phe Glu Gln Leu Gln Pro Gly Asp Ser Val Ile Gln Asn Ala Ser Asn Ser Gly Val Gly Gln Ala Val Ile Gln Ile Ala Ala Ala Leu Gly Leu Arg Thr Ile Asn Val Val Arg Asp Arg Pro Asp Ile Gln Lys Leu Ser Asp Arg Leu Lys Ser Leu Gly Ala Glu His Val Ile Thr Glu Glu Glu Leu Arg Arg Pro Glu Met Lys Asn Phe Phe Lys Asp Met Pro Gln Pro Arg Leu Ala Leu Asn Cys Val Gly Gly Lys Ser Ser Thr Glu Leu Leu Arg Gln Leu Ala Arg Gly Gly Thr Met Val Thr Tyr Gly Gly Met Ala Lys Gln Pro Val Val Ala Ser Val Ser Leu Leu Ile Phe Lys Asp Leu Lys Leu Arg Gly Phe Trp Leu Ser Gln Trp Lys Lys Asp His Ser Pro Asp Gln Phe Lys Glu Leu Ile Leu Thr Leu Cys Asp Leu Ile Arg Arg Gly Gln Leu Thr Ala Pro Ala Cys Ser Gln Val Pro Leu Gln Asp Tyr Gln Ser Ala Leu Glu Ala Ser Met Lys Pro Phe Ile Ser Ser Lys Gln Ile Leu Thr Met <210> 13 <211> 305 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2536830CD1 <400> 13 Met Ala Lys Phe Val Ile Ala Gly Arg Ala Asp Cys Pro Tyr Tyr Ala Lys Thr Glu Leu Val Ala Asp Tyr Leu Gln Lys Asn Leu Pro Asp Phe Arg Ile His Lys Ile Thr Gln Arg Pro Glu Val Trp Glu Asp Trp Leu Lys Asp Val Cys Glu Lys Asn Lys Trp Ser His Lys Asn Ser Pro Ile Ile Trp Arg Glu Leu Leu Asp Arg Gly Gly Lys Gly Leu Leu Leu Gly Gly Tyr Asn Glu Phe Leu Glu His Ala Gln Leu Tyr Tyr Asp Val Thr Ser Ser Met Thr Thr Glu Leu Met Met Val Ile Ala Gln Glu Asn Leu Gly Ala His Ile Glu Lys Glu Gln Glu Glu Glu Ala Leu Lys Thr Cys Ile Asn Pro Leu Gln Val Trp Ile Thr Ser Ala Ser Ala Pro Ala Cys Tyr Asn Leu Ile Pro Ile Leu Thr Ser Gly Glu Val Phe Gly Met His Thr Glu Ile Ser Ile Thr Leu Phe Asp Asn Lys Gln Ala Glu Glu His Leu Lys Ser Leu Val Val Glu Thr Gln Asp Leu Ala Ser Pro Val Leu Arg Ser Val Ser Ile Cys Thr Lys Val Glu Glu Ala Phe Arg Gln Ala His Val Ile Val Val Leu Asp Asp Ser Thr Asn Lys Glu Val Phe Thr Leu Glu Asp Cys Leu Arg Ser Arg Val Pro Leu Cys Arg Leu Tyr Gly Tyr Leu Ile Glu Lys Asn Ala His Glu Ser Val Arg Val Ile Val Gly Gly Arg Thr Phe Val Asn Leu Lys Thr Val Leu Leu Met Arg Tyr Ala Pro Arg Ile Ala His Asn Ile Ile Ala Val Ala Leu Gly Val Glu Gly Glu Ala Lys Ala Ile Leu Ala Arg Lys Leu Lys Thr Ala Pro Ser Cys Glu <210> 14 <211> 500 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2645179CD1 <400> 14 Met Glu Ala Ala Arg Pro Pro Pro Thr Ala Gly Lys Phe Val Val Val Gly Gly Gly Ile Ala Gly Val Thr Cys Ala Glu Gln Leu Ala Thr His Phe Pro Ser Glu Asp Ile Leu Leu Val Thr Ala Ser Pro Val Ile Lys Ala Val Thr Asn Phe Lys Gln Ile Ser Lys Ile Leu Glu Glu Phe Asp Val Glu Glu Gln Ser Ser Thr Met Leu Gly Lys Arg Phe Pro Asn Ile Lys Val Ile Glu Ser Gly Val Lys Gln Leu Lys Ser Glu Glu His Cys Ile Val Thr Glu Asp Gly Asn Gln His Val Tyr Lys Lys Leu Cys Leu Cys Ala Gly Ala Lys Pro Lys Leu Ile Cys Glu Gly Asn Pro Tyr Val Leu Gly Ile Arg Asp Thr Asp Ser Ala Gln Glu Phe GTn Lys Gln Leu Thr Lys Ala Lys Arg Ile Met Ile Ile Gly Asn Gly Gly Ile Ala Leu Glu Leu Val Tyr Glu Ile Glu Gly Cys Glu Val Ile Trp Ala Ile Lys Asp Lys Ala Ile Gly Asn Thr Phe Phe Asp Ala Gly Ala Ala Glu Phe Leu Thr Ser Lys Leu Ile Ala Glu Lys Ser Glu Ala Lys Ile Ala His Lys Arg Thr Arg Tyr Thr Thr Glu Gly Arg Lys Lys Glu Ala Arg Ser Lys Ser Lys Ala Asp Asn Val Gly Ser Ala Leu Gly Pro Asp Trp His Glu Gly Leu Asn Leu Lys Gly Thr Lys Glu Phe Ser His Lys Ile His Leu Glu Thr Met Cys Glu Val Lys Lys Ile Tyr Leu Gln Asp Glu Phe Arg Ile Leu Lys Lys Lys Ser Phe Thr Phe Pro Arg Asp His Lys Ser Val Thr Ala Asp Thr Glu Met Trp Pro Val Tyr Val Glu Leu Thr Asn Glu Lys Ile Tyr Gly Cys Asp Phe Ile Val Ser Ala Thr Gly Val Thr Pro Asn Val Glu Pro Phe Leu His Gly Asn Ser Phe Asp Leu Gly Glu Asp Gly Gly Leu Lys Val Asp Asp His Met His Thr Ser Leu Pro Asp Ile Tyr Ala Ala Gly Asp Ile Cys Thr Thr Ser Trp Gln Leu Ser Pro Val Trp Gln Gln Met Arg Leu Trp Thr Gln Ala Arg Gln Met Gly Trp Tyr Ala Ala Lys Cys Met Ala Ala Ala Ser Ser Gly Asp Ser Ile Asp Met Asp Phe Ser Phe Glu Leu Phe Ala His Val Thr Lys Phe Phe Asn Tyr Lys Val Val Leu Leu Gly Lys Tyr Asn Ala Gln Gly Leu Gly Ser Asp His Glu Leu Met Leu Arg Cys Thr Lys Gly Arg Glu Tyr Ile Lys Val Val Met Gln Asn Gly Arg Met Met Gly Ala Val Leu Ile Gly Glu Thr Asp Leu Glu Glu Thr Phe Glu Asn Leu Ile Leu Asn Gln Met Asn Leu Ser Ser Tyr Gly Glu Asp Leu Leu Asp Pro Asn Ile Asp Ile Glu Asp Tyr Phe Asp <210> 15 <211> 369 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2754425CD1 <400> 15 Met Val Trp Ala Pro Leu Gly Pro Pro Arg Thr Asp Cys Leu Thr Leu Leu His Thr Pro Ser Lys Asp Ser Pro Lys Met Ser Leu Glu Trp Leu Val Ala Trp Ser Trp Ser Leu Asp Gly Leu Arg Asp Cys Ile Ala Thr Gly Ile Gln Ser Val Arg Asp Cys Asp Thr Thr Ala Val Ile Thr Val Ala Cys Leu Leu Val Leu Phe Val Trp Tyr Cys Tyr His Val Gly Arg Glu Gln Pro Arg Pro Tyr Val Ser Val Asn Ser Leu Met Gln Ala Ala Asp Ala Asn Gly Leu Gln Asn Gly Tyr Val Tyr Cys Gln Ser Pro Glu Cys Val Arg Cys Thr His Asn Glu Gly Leu Asn Gln Lys Leu Tyr His Asn Leu Gln Glu Tyr Ala Lys Arg Tyr Ser Trp Ser Gly Met Gly Arg Ile His Lys Gly Ile Arg Glu Gln Gly Arg Tyr Leu Asn Ser Arg Pro Ser Ile Gln Lys Pro Glu Val Phe Phe Leu Pro Asp Leu Pro Thr Thr Pro Tyr Phe Ser Arg Asp Ala Gln Lys His Asp Val Glu Val Leu Glu Arg Asn Phe Gln Thr Ile Leu Cys Glu Phe Glu Thr Leu Tyr Lys Ala Phe Ser Asn Cys Ser Leu Pro Gln Gly Trp Lys Met Asn Ser Thr Pro Ser Gly Glu Trp Phe Thr Phe Tyr Leu Val Asn Gln Gly Val Cys Val Pro Arg Asn Cys Arg Lys Cys Pro Arg Thr Tyr Arg Leu Leu Gly Ser Leu Arg Thr Cys Ile Gly Asn Asn Val Phe Gly Asn Ala Cys Ile Ser Val Leu Ser Pro Gly Thr Val Ile Thr Glu His Tyr Gly Pro Thr Asn Ile Arg Ile Arg Cys His Leu Gly Leu Lys Thr Pro Asn Gly Cys Glu Leu Val Val Gly Gly Glu Pro Gln Cys Trp Ala Glu Gly Arg Cys Leu Leu Phe Asp Asp Ser Phe Leu His Ala Ala Phe His Glu Gly Ser Ala Glu Asp Gly Pro Arg Val Val Phe Met Val Asp Leu Trp His Pro Asn Val Ala Ala Ala Glu Arg Gln Ala Leu Asp Phe Ile Phe Ala Pro Gly Arg <210> 16 <211> 145 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2821526CD1 <400> 16 Met Glu Leu Val Gln Val Leu Lys Arg Gly Leu Gln Gln Ile Thr Gly His Gly Gly Leu Arg Gly Tyr Leu Arg Val Phe Phe Arg Thr Asn Asp Ala Lys Val Gly Thr Leu Val Gly Glu Asp Lys Tyr Gly Asn Lys Tyr Tyr Glu Asp Asn Lys Gln Phe Phe Gly Arg His Arg Trp Val Val Tyr Thr Thr Glu Met Asn Gly Lys Asn Thr Phe Trp Asp Val Asp Gly Ser Met Val Pro Pro Glu Trp His Arg Trp Leu His Ser Met Thr Asp Asp Pro Pro Thr Thr Lys Pro Leu Thr Ala Arg Lys Phe Ile Trp Thr Asn His Lys Phe Asn Val Thr Gly Thr Pro Glu Gln Tyr Val Pro Tyr Ser Thr Thr Arg Lys Lys Ile Gln Glu Trp Ile Pro Pro Ser Thr Pro Tyr Lys <210> 17 <211> 255 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2876494CD1 <400> 17 Met Lys Val Leu Ala Thr Ser Phe Val Leu Gly Ser Leu Gly Leu Ala Phe Tyr Leu Pro Leu Val Val Thr Thr Pro Lys Thr Leu Ala Ile Pro Glu Lys Leu Gln Glu Ala Val Gly Lys Val Ile Ile Asn Ala Thr Thr Cys Thr Val Thr Cys Gly Leu Gly Tyr Lys Glu Glu Thr Val Cys Glu Val Gly Pro Asp Gly Val Arg Arg Lys Cys Gln Thr Gln Arg Leu Glu Cys Leu Thr Asn Trp Ile Cys Gly Met Leu His Phe Thr Ile Leu Ile Gly Lys Glu Phe Glu Leu Ser Cys Leu Ser Leu Arg Thr Cys Ile Gly Asn Asn Val Phe Gly Ser Ser Asp Ile Leu Glu Phe Gly Gln Glu Ala Phe Arg Phe Thr Trp Arg Leu Ala Arg Gly Val Ile Ser Thr Asp Asp Glu Val Phe Lys Pro Phe Gln Ala Asn Ser His Phe Val Lys Phe Lys Tyr Ala Gln Glu Tyr Asp Ser Gly Thr Tyr Arg Cys Asp Val Gln Leu Val Lys Asn Leu Arg Leu Val Lys Arg Leu Tyr Phe Gly Leu Arg Val Leu Pro Pro Asn Leu Val Asn Leu Asn Phe His Gln Ser Leu Thr Glu Asp Gln Lys Leu Ile Asp Glu Gly Leu Glu Val Asn Leu Asp Ser Tyr Ser Lys Pro His His Pro Lys Trp Lys Lys Lys Val Ala Ser Ala Leu Gly Ile Gly Ile Ala Ile Gly Val Val Gly Gly Val Leu Val Arg Ile Val Leu Cys Ala Leu Arg Gly Gly Leu Gln Gln <210> 18 <211> 246 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3403225CD1 <400> 18 Met Leu Val Thr Leu Gly Leu Leu Thr Ser Phe Phe Ser Phe Leu Tyr Met Val Ala Pro Ser Ile Arg Lys Phe Phe Ala Gly Gly Val Cys Arg Thr Asn Val Gln Leu Pro Gly Lys Val Val Val Ile Thr Gly Ala Asn Thr Gly Ile Gly Lys Glu Thr Ala Arg Glu Leu Ala Ser Arg Gly Ala Arg Val Tyr Ile Ala Cys Arg Asp Val Leu Lys Gly Glu Ser Ala Ala Ser Glu Ile Arg Val Asp Thr Lys Asn Ser Gln Val Leu Val Arg Lys Leu Asp Leu Ser Asp Thr Lys Ser Ile Arg Ala Phe Ala Glu Gly Phe Leu Ala Glu Glu Lys Gln Leu His Ile Leu Ile Asn Asn Ala Gly Val Met Met Cys Pro Tyr Ser Lys Thr Ala Asp Gly Phe Glu Thr His Leu Gly Val Asn His Leu Gly Thr Gly Val Thr Thr Tyr Ala Val His Pro Gly Val Val Arg Ser Glu Leu Val Arg His Ser Ser Leu Leu Cys Leu Leu Trp Arg Leu Phe Ser Pro Phe Val Lys Thr Ala Arg Glu Gly Ala Gln Thr Ser Leu His Cys Ala Leu Ala Glu Gly Leu Glu Pro Leu Ser Gly Lys Tyr Phe Ser Asp Cys Lys Arg Thr Trp Val Ser Pro Arg Ala Arg Asn Asn Lys Thr Ala Glu Arg Leu Trp Asn Val Ser Cys Glu Leu Leu Gly Ile Arg Trp Glu <210> 19 <211> 467 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4163943CD1 <400> 19 Met Ala Ala Ala Ala Arg Ala Arg Val Ala Tyr Leu Leu Arg Gln Leu Gln Arg Ala Ala Cys Gln Cys Pro Thr His Ser His Thr Tyr Ser Gln Ala Pro Gly Leu Ser Pro Ser Gly Lys Thr Thr Asp Tyr Ala Phe Glu Met Ala Val Ser Asn Ile Arg Tyr Gly Ala Ala Val Thr Lys Glu Val Gly Met Asp Leu Lys Asn Met Gly Ala Lys Asn Val Cys Leu Met Thr Asp Lys Asn Leu Ser Lys Leu Pro Pro Val Gln Val Ala Met Asp Ser Leu Val Lys Asn Gly Ile Pro Phe Thr Val Tyr Asp Asn Val Arg Val Glu Pro Thr Asp Ser Ser Phe Met Glu Ala Ile Glu Phe Ala Gln Lys Gly Ala Phe Asp Ala Tyr Val Ala Val Gly Gly Gly Ser Thr Met Asp Thr Cys Lys Ala Ala Asn Leu Tyr Ala Ser Ser Pro His Ser Asp Phe Leu Asp Tyr Val Ser Ala Pro Ile Gly Lys Gly Lys Pro Val Ser Val Pro Leu Lys Pro Leu Ile Ala Val Pro Thr Thr Ser Gly Thr Gly Ser Glu Thr Thr Gly Val Ala Ile Phe Asp Tyr Glu His Leu Lys Val Lys Ile Gly Ile Thr Ser Arg Ala Ile Lys Pro Thr Leu Gly Leu Ile Asp Pro Leu His Thr Leu His Met Pro Ala Arg Val Val Ala Asn Ser Gly Phe Asp Val Leu Cys His Ala Leu Glu Ser Tyr Thr Thr Leu Pro Tyr His Leu Arg Ser Pro Cys Pro Ser Asn Pro Ile Thr Arg Pro Ala Tyr Gln Gly Ser Asn Pro Ile Ser Asp Ile Trp Ala Ile His Ala Leu Arg Ile Val Ala Lys Tyr Leu Lys Arg Ala Val Arg Asn Pro Asp Asp Leu Glu Ala Arg Ser His Met His Leu Ala Ser Ala Phe Ala Gly Ile Gly Phe Gly Asn Ala Gly Val His Leu Cys His Gly Met Ser Tyr Pro Ile Ser Gly Leu Val Lys Met Tyr Lys Ala Lys Asp Tyr Asn Val Asp His Pro Leu Val Pro His Gly Leu Ser Val Val Leu Thr Ser Pro Ala Val Phe Thr Phe Thr Ala Gln Met Phe Pro Glu Arg His Leu Glu Met Ala Glu Ile Leu Gly Ala Asp Thr Arg Thr Ala Arg Ile Gln Asp Ala Gly Leu Val Leu Ala Asp Thr Leu Arg Lys Phe Leu Phe Asp Leu Asp Val Asp Asp Gly Leu Ala Ala Val Gly Tyr Ser Lys Ala Asp Ile Pro Ala Leu Val Lys Gly Thr Leu Pro Gln Glu Arg Val Thr Lys Leu Ala Pro Arg Pro Gln Ser Glu Glu Asp Leu Ala Ala Leu Phe Glu Ala Ser Met Lys Leu Tyr <210> 20 <211> 317 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4293484CD1 <400> 20 Met Ala Asp Ser Ala Gln Ala Gln Lys Leu Val Tyr Leu Val Thr Gly Gly Cys Gly Phe Leu Gly Glu His Val Val Arg Met Leu Leu Gln Arg Glu Pro Arg Leu Gly Glu Leu Arg Val Phe Asp Gln His Leu Gly Pro Trp Leu Glu Glu Leu Lys Thr Gly Thr Arg Asn Val Ile Glu Ala Cys Val Gln Thr Gly Thr Arg Phe Leu Val Tyr Thr Ser Ser Met Glu Val Val Gly Pro Asn Thr Lys Gly His Pro Phe Tyr Arg Gly Asn Glu Asp Thr Pro Tyr Glu Ala Val His Arg His Pro Tyr Pro Cys Ser Lys Ala Leu Ala Glu Trp Leu Val Leu Glu Ala Asn Gly Arg Lys Val Arg Gly Gly Leu Pro Leu Val Thr Cys Ala Leu Arg Pro Thr Gly Ile Tyr Gly Glu Gly His Gln Ile Met Arg Asp Phe Tyr Arg Gln Gly Leu Arg Leu Gly Gly Trp Leu Phe Arg Ala Ile Pro Ala Ser Val Glu His Gly Arg Val Tyr Val Gly Asn Val Ala Trp Met His Val Leu Ala Ala Arg Glu Leu Glu Gln Arg Ala Thr Leu Met Gly Gly Gln Val Tyr Phe Cys Tyr Asp Gly Ser Pro Tyr Arg Ser Tyr Glu Asp Phe Asn Met Glu Phe Leu Gly Pro Cys Gly Leu Arg Leu Val Gly Ala Arg Pro Leu Leu Pro Tyr Trp Leu Leu Val Phe Leu Ala Ala Leu Asn Ala Leu Leu Gln Trp Leu Leu Arg,Pro Leu Val Leu Tyr Ala Pro Leu Leu Asn Pro Tyr Thr Leu Ala Val Ala Asn Thr Thr Phe Thr Val Ser Thr Asp Lys Ala Gln Arg His Phe Gly Tyr Glu Pro Leu Phe Ser Trp Glu Asp Ser Arg Thr Arg Thr Ile Leu Trp Val Gln Ala Ala Thr Gly Ser Ala Gln <210> 21 <211> 181 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4440080CD1 <400> 21 Met Phe Ser Ala Ile Arg Ser Gln His Ser Gly Val Asp Ile Cys Ile Asn Asn Ala Gly Leu Ala Arg Pro Asp Thr Leu Leu Ser Gly Ser Thr Ser Gly Trp Lys Asp Met Phe Asn Val Asn Val Leu Ala Leu Ser Ile Cys Thr Arg Glu Ala Tyr Gln Ser Met Lys Glu Arg Asn Val Asp Asp Gly His Ile Ile Asn Ile Asn Ser Met Ser Gly His Arg Val Leu Pro Leu Ser Val Thr His Phe Tyr Ser Ala Thr Lys Tyr Ala Val Thr Ala Leu Thr Glu Gly Leu Arg Gln Glu Leu Arg Glu Ala Gln Thr His Ile Arg Ala Thr Cys Ile Ser Pro Gly Val Val Glu Thr Gln Phe Ala Phe Lys Leu His Asp Lys Asp Pro Glu Lys Ala Ala Ala Thr Tyr Glu Gln Met Lys Cys Leu Lys Pro Glu Asp Val Ala Glu Ala Val Ile Tyr Val Leu Ser Thr Pro Ala His Ile Gln Ile Gly Asp Ile Gln Met Arg Pro Thr Glu Gln Val Thr <210> 22 <211> 360 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5495687CD1 <400> 22 Met Val Pro Ala Ala Gly Arg Arg Pro Pro Arg Val Met Arg Leu Leu Gly Trp Trp Gln Val Leu Leu Trp Val Leu Gly Leu Pro Val Arg Gly Val Glu Val Ala Glu Glu Ser Gly Arg Leu Trp Ser Glu Glu Gln Pro Ala His Pro Leu Gln Val Gly Ala Val Tyr Leu Gly Glu Glu Glu Leu Leu His Asp Pro Met Gly Gln Asp Arg Ala Ala Glu Glu Ala Asn Ala Val Leu Gly Leu Asp Thr Gln Gly Asp His Met Val Met Leu Ser Val Ile Pro Gly Glu Ala Glu Asp Lys Val Ser Ser Glu Pro Ser Gly Val Thr Cys Gly Ala Gly Gly Ala Glu Asp Ser Arg Cys Asn Val Arg Glu Ser Leu Phe Ser Leu Asp Gly Ala Gly Ala His Phe Pro Asp Arg Glu Glu Glu Tyr Tyr Thr Glu Pro Glu Val Ala Glu Ser Asp Ala Ala Pro Thr Glu Asp Ser Asn Asn Thr Glu Ser Leu Lys Ser Pro Lys Val Asn Cys Glu Glu Arg Asn Ile Thr Gly Leu Glu Asn Phe Thr Leu Lys Ile Leu Asn Met Ser Gln Asp Leu Met Asp Phe Leu Asn Pro Asn Gly Ser Asp Cys Thr Leu Val Leu Phe Tyr Thr Pro Trp Cys Arg Phe Ser Ala Ser Leu Ala Pro His Phe Asn Ser Leu Pro Arg Ala Phe Pro Ala Leu His Phe Leu Ala Leu Asp Ala Ser Gln His Ser Ser Leu Ser Thr Arg Phe Gly Thr Val Ala Val Pro Asn Ile Leu Leu Phe Gln Gly Ala Lys Pro Met Ala Arg Phe Asn His Thr Asp Arg Thr Leu Glu Thr Leu Lys Ile Phe Ile Phe Asn Gln Thr Gly Ile Glu Ala Lys Lys Asn Val Val Val Thr Gln Ala Asp Gln Ile Gly Pro Leu Pro Ser Thr Leu Ile Lys Ser Val Asp Trp Leu Leu Val Phe Ser Leu Phe Phe Leu Ile Ser Phe Ile Met Tyr Ala Thr Ile Arg Thr Glu Ser Ile Arg Trp Leu Ile Pro Gly Gln Glu Gln Glu His Val Glu <210> 23 <211> 476 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5527735CD1 <400> 23 Met Ala Ser Pro Phe Ser Gly Ala Leu Gln Leu Thr Asp Leu Asp Asp Phe Ile Gly Pro Ser Gln Glu Cys Ile Lys Pro Val Lys Val Glu Lys Arg Ala Gly Ser Gly Val Ala Lys Ile Arg Ile Glu Asp Asp Gly Ser Tyr Phe Gln Ile Asn Gln Asp Gly Gly Thr Arg Arg Leu Glu Lys Ala Lys Val Ser Leu Asn Asp Cys Leu Ala Cys Ser Gly Cys Ile Thr Ser Ala Glu Thr Val Leu Ile Thr Gln Gln Ser His Glu Glu Leu Lys Lys Val Leu Asp Ala Asn Lys Met Ala Ala Pro Ser Gln Gln Arg Leu Val Val Val Ser Val Ser Pro Gln Ser Arg Ala Ser Leu Ala Ala Arg Phe Gln Leu Asn Pro Thr Asp Thr Ala Arg Lys Leu Thr Ser Phe Phe Lys Lys Ile Gly Val His Phe Val Phe Asp Thr Ala Phe Ser Arg His Phe Ser Leu Leu Glu Ser Gln Arg Glu Phe Val Arg Arg Phe Arg Gly Gln Ala Asp Cys Arg Gln Ala Leu Pro Leu Leu Ala Ser Ala Cys Pro Gly Trp Ile Cys Tyr Ala Glu Lys Thr His Gly Ser Phe Ile Leu Pro His Ile Ser Thr Ala Arg Ser Pro Gln Gln Val Met Gly Ser Leu Val Lys Asp Phe Phe Ala Gln Gln Gln His Leu Thr Pro Asp Lys Ile Tyr His Val Thr Val Met Pro Cys Tyr Asp Lys Lys Leu Glu Ala Ser Arg Pro Asp Phe Phe Asn Gln Glu His Gln Thr Arg Asp Val Asp Cys Val Leu Thr Thr Gly Glu Val Phe Arg Leu Leu Glu Glu Glu Gly Val Ser Leu Pro Asp Leu Glu Pro Ala Pro Leu Asp Ser Leu Cys Ser Gly Ala Ser Ala Glu Glu Pro Thr Ser His Arg Gly Gly Gly Ser Gly Gly Tyr Leu Glu His Val Phe Arg His Ala Ala Arg Glu Leu Phe Gly Ile His Val Ala Glu Val Thr Tyr Lys Pro Leu Arg Asn Lys Asp Phe Gln Glu Val Thr Leu Glu Lys Glu Gly Gln Val Leu Leu His Phe Ala Met Ala Tyr Gly Phe Arg Asn Ile Gln Asn Leu Val Gln Arg Leu Lys Arg Gly Arg Cys Pro Tyr His Tyr Val Glu Val Met Ala Cys Pro Ser Gly Cys Leu Asn Gly Gly Gly Gln Leu Gln Ala Pro Asp Arg Pro Ser Arg Glu Leu Leu Gln His Val Glu Arg Leu Tyr Gly Met Val Arg Ala Glu Ala Pro Glu Asp Ala Pro Gly Val Gln Glu Leu Tyr Thr His Trp Leu Gln Gly Thr Asp Ser Glu Cys Ala Gly Arg Leu Leu His Thr Gln Tyr His Ala Val Glu Lys Ala Ser Thr Gly Leu Gly Ile Arg Trp <210> 24 <211> 621 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5540437CD1 <400> 24 Met Ser Gly Cys Gly Leu Phe Leu Arg Thr Thr Ala Ala Ala Arg Ala Cys Arg Gly Leu Val Val Ser Thr Ala Asn Arg Arg Leu Leu Arg Thr Ser Pro Pro Val Arg Ala Phe Ala Lys Glu Leu Phe Leu Gly Lys Ile Lys Lys Lys Glu Val Phe Pro Phe Pro Glu Val Ser Gln Asp Glu Leu Asn Glu Ile Asn Gln Phe Leu Gly Pro Val Glu Lys Phe Phe Thr Glu Glu Val Asp Ser Arg Lys Ile Asp Gln Glu Gly Lys Ile Pro Asp Glu Thr Leu Glu Lys Leu Lys Ser Leu Gly Leu Phe Gly Leu Gln Val Pro Glu Glu Tyr Gly Gly Leu Gly Phe Ser Asn Thr Met Tyr Ser Arg Leu Gly Glu Ile Ile Ser Met Asp Gly Ser Ile Thr Val Thr Leu Ala Ala His Gln Ala Ile Gly Leu Lys Gly Ile Ile Leu Ala Gly Thr Glu Glu Gln Lys Ala Lys Tyr Leu Pro Lys Leu Ala Ser Gly Glu His Ile Ala Ala Phe Cys Leu Thr Glu Pro Ala Ser Gly Ser Asp Ala Ala Ser Ile Arg Ser Arg Ala Thr Leu Ser Glu Asp Lys Lys His Tyr Ile Leu Asn Gly Ser Lys Val Trp Ile Thr Asn Gly Gly Leu Ala Asn Ile Phe Thr Val Phe Ala Lys Thr Glu Val Val Asp Ser Asp Gly Ser Val Lys Asp Lys Ile Thr Ala Phe Ile Val Glu Arg Asp Phe Gly Gly Val Thr Asn Gly Lys Pro Glu Asp Lys Leu Gly Ile Arg Gly Ser Asn Thr Cys Glu Val His Phe Glu Asn Thr Lys Ile Pro Val Glu Asn Ile Leu Gly Glu Val Gly Asp Gly Phe Lys Val Ala Met Asn Ile Leu Asn Ser Gly Arg Phe Ser Met Gly Ser Val Val Ala Gly Leu Leu Lys Arg Leu Ile Glu Met Thr Ala Glu Tyr Ala Cys Thr Arg Lys Gln Phe Asn Lys Arg Leu Ser Glu Phe Gly Leu Ile Gln Glu Lys Phe Ala Leu Met Ala Gln Lys Ala Tyr Val Met Glu Ser Met Thr Tyr Leu Thr Ala Gly Met Leu Asp Gln Pro Gly Phe Pro Asp Cys Ser Ile Glu Ala Ala Met Val Lys Val Phe Ser Ser Glu Ala Ala Trp Gln Cys Val Ser Glu Ala Leu Gln Ile Leu Gly Gly Leu Gly Tyr Thr Arg Asp Tyr Pro Tyr Glu Arg Ile Leu Arg Asp Thr Arg Ile Leu Leu Ile Phe Glu Gly Thr Asn Glu Ile Leu Arg Met Tyr Ile Ala Leu Thr Gly Leu Gln His Ala Gly Arg Ile Leu Thr Thr Arg Ile His Glu Leu Lys Gln Ala Lys Val Ser Thr Val Met Asp Thr Val Gly Arg Arg Leu Arg Asp Ser Leu Gly Arg Thr Val Asp Leu Gly Leu Thr Gly Asn His Gly Val Val His Pro Ser Leu Ala Asp Ser Ala Asn Lys Phe Glu Glu Asn Thr Tyr Cys Phe Gly Arg Thr Val Glu Thr Leu Leu Leu Arg Phe Gly Lys Thr Ile Met Glu Glu Gln Leu Val Leu Lys Arg Val Ala Asn Ile Leu Ile Asn Leu Tyr Gly Met Thr Ala Val Leu Ser Arg Ala Ser Arg Ser Ile Arg Ile Gly Leu Arg Asn His Asp His Glu Val Leu Leu Ala Asn Thr Phe Cys Val Glu Ala Tyr Leu Gln Asn Leu Phe Ser Leu Ser Gln Leu Asp Lys Tyr Ala Pro Glu Asn Leu Asp Glu Gln Ile Lys Lys Val Ser Gln Gln Ile Leu Glu Lys Arg Ala Tyr Ile Cys Ala His Pro Leu Asp Arg Thr Cys <210> 25 <211> 245 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5596281CD1 <400> 25 Met Gly Arg Leu Asp Gly Lys Val Ile Ile Leu Thr Ala Ala Ala Gln Gly Ile Gly Gln Ala Ala Ala Leu Ala Phe Ala Arg Glu Gly Ala Lys Val Ile Ala Thr Asp Ile Asn Glu Ser Lys Leu Gln Glu Leu Glu Lys Tyr Pro Gly Ile Gln Thr Arg Val Leu Asp Val Thr Lys Lys Lys Gln Ile Asp Gln Phe Ala Ser Glu Val Glu Arg Leu Asp Val Leu Phe Asn Val Ala Gly Phe Val His His Gly Thr Val Leu Asp Cys Glu Glu Lys Asp Trp Asp Phe Ser Met Asn Leu Asn Val Arg Ser Met Tyr Leu Met Ile Lys Ala Phe Leu Pro Lys Met Leu Ala Gln Lys Ser Gly Asn Ile Ile Asn Met Ser Ser Val Ala Ser Ser Val Lys Gly Val Val Asn Arg Cys Val Tyr Ser Thr Thr Lys Ala Ala Val Ile Gly Leu Thr Lys Ser Val Ala Ala Asp Phe Ile Gln Gln Gly Ile Arg Cys Asn Cys Val Cys Pro Gly Thr Val Asp Thr Pro Ser Leu Gln Glu Arg Ile Gln Ala Arg Gly Asn Pro Glu Glu Ala Arg Asn Asp Phe Leu Lys Arg Gln Lys Thr Gly Arg Phe Ala Thr Ala Glu Glu Ile Ala Met Leu Cys Val Tyr Leu Ala Ser Asp Glu Ser Ala Tyr Val Thr Gly Asn Pro Val Ile Ile Asp Gly Gly Trp Ser Leu <210> 26 <211> 159 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5731013 <400> 26 Met Thr Ala Arg Gly Thr Pro Ser Arg Phe Leu Ala Ser Val Leu His.Asn Gly Leu Gly Arg Tyr Val Gln Gln Leu Gln.Arg Leu Ser Phe Ser Val Ser Arg Asp Gly Ala Ser Ser Arg Gly Ala Arg Glu Phe Val Glu Arg Glu Val Ile Asp Phe Ala Arg Arg Asn Pro Gly Val Val Ile Tyr Val Asn Ser Arg Pro Cys Cys Val Pro Arg Val Val Ala Glu Tyr Leu Asn Gly Ala Val Arg Glu Glu Ser Ile His Cys Lys Ser Val Glu Glu Ile Ser Thr Leu Val Gln Lys Leu Ala Asp Gln Ser Gly Leu Asp Val Ile Arg Ile Arg Lys Pro Phe His Thr Asp Asn Pro Ser Ile Gln Gly Gln Trp His Pro Phe Thr Asn Lys Pro Thr Thr Phe Arg Gly Leu Arg Pro Arg Glu Val Gln Asp Pro Ala Pro Ala Gln Val Gln Ala Gln <210> 27 <211> 291 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5731162CD1 <400> 27 Met Ala Cys Leu Ser Pro Ser Gln Leu Gln Lys Phe Gln Gln Asp Gly Phe Leu Val Leu Glu Gly Phe Leu Ser Ala Glu Glu Cys Val Ala Met Gln Gln Arg Ile Gly Glu Ile Val Ala Glu Met Asp Val Pro Leu His Cys Arg Thr Glu Phe Ser Thr Gln Glu Glu Glu Gln Leu Arg Ala Gln Gly Ser Thr Asp Tyr Phe Leu Ser Ser Gly Asp Lys Ile Arg Phe Phe Phe Glu Lys Gly Val Phe Asp Glu Lys Gly Asn Phe Leu Val Pro Pro Glu Lys Ser Ile Asn Lys Ile Gly His Ala Leu His Ala His Asp Pro Val Phe Lys Ser Ile Thr His Ser Phe Lys Val Gln Thr Leu Ala Arg Ser Leu Gly Leu Gln Met Pro Val Val Val Gln Ser Met Tyr Ile Phe Lys Gln Pro His Phe Gly Gly Glu Val Ser Pro His Gln Asp Ala Ser Phe Leu Tyr Thr Glu Pro Leu Gly Arg Val Leu Gly Val Trp Ile Ala Val Glu Asp Ala Thr Leu Glu Asn Gly Cys Leu Trp Phe Ile Pro Gly Ser His Thr 24/4() Ser Gly Val Ser Arg Arg Met Val Arg Ala Pro Val Gly Ser Ala Pro Gly Thr Ser Phe Leu Gly Ser Glu Pro Ala Arg Asp Asn Ser Leu Phe Val Pro Thr Pro Val Gln Arg Gly Ala Leu Val Leu Ile His Gly Glu Val Val His Lys Ser Lys Gln Asn Leu Ser Asp Arg Ser Arg Gln Ala Tyr Thr Phe His Leu Met Glu Ala Ser Gly Thr Thr Trp Ser Pro Glu Asn Trp Leu Gln Pro Thr Ala Glu Leu Pro Phe Pro Gln Leu Tyr Thr <210> 28 <211> 1557 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 543496CB1 <400> 28 ctgagtgcga cggcgcaggt ctgcaccatg gcggcccggc ttgtcagccg atgcggggct 60 gtgcgtgcag ctccccacag cggcccgctg gtgtcctggc gcaggtggtc cggcgcctca 120 acagacaccg tgtatgacgt ggtggtgtcg ggtggaggcc tggtgggcgc tgccatggcc 180 tgtgccttgg gatatgatat tcactttcat gacaagaaaa tcctgttgct cgaagcaggt 240 ccaaagaaag tactggagaa attgtcagaa acttacagca acagggtcag ctccatttcc 300 cctggctctg caacgcttct cagtagtttt ggtgcctggg accatatctg caacatgaga 360 tacagagcct ttcggcgaat gcaggtgtgg gacgcctgct cagaggccct gataatgttt 420 gataaggata atttagatga catgggctat atcgtggaga atgatgtcat catgcatgct 480 ctcactaagc agttggaggc tgtgtctgac cgagtgacgg ttctctacag gagcaaagcc 540 attcgctata cctggccttg tccatttcct atggccgact ccagcccttg ggttcatatt 600 accctaggtg atggcagcac cttccagacc aaattgttga taggtgcaga tggtcacaac 660 tccggagtac ggcaggctgt tggaatccag aatgtgagct ggaactatga ccagtctgct 720 gttgtggcta ctctgcattt atcagaggcc acagaaaaca acgtagcctg gcagagattt 780 cttccctctg ggcctattgc tctgctcccg ctctcagaca ccttgagttc cttggtttgg 840 tccacgtccc atgaacatgc agcagagcta gttagcatgg atgaggaaaa atttgtggat 900 gccgttaact ctgccttttg gagtgatgct gaccacacgg acttcatcga cacagctggt 960 gccatgctgc agtatgctgt cagccttctg aagcccacta aggtctcggc tcgccagctg 1020 cccccaagcg tagccagggt ggatgccaaa agccgagttc tgtttcctct tgggttggga 1080 catgctgctg agtacgtcag gcctcgggtg gcgctcattg gggatgcagc ccacagagtc 1140 catccgcttg caggacaggg tgtcaacatg ggctttgggg atatctccag cttggcccat 1200 cacctcagta cggcagcctt caatgggaag gacttaggtt ccgtgagcca cctcacaggt 1260 tatgaaacag aaagacagcg tcacaacact gctcttctgg ctgctacaga cttactaaaa 1320 aggctctatt ctaccagtgc ctccccgctt gtgttgctca ggacgtgggg cttgcaggcc 1380 acaaatgcag tgtctccact caaagaacag attatggcct ttgcaagcaa atgagtactc 1440 ctctcctaaa gaaagattac gttgatgaaa aagaacatcc tgcccaggac ccatcataca 1500 tattttcaag atcttattta atttaataaa cttactttac attaaaaaaa aaaaaaa 1557 <210> 29 <211> 1106 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 907607CB1 <400> 29 cccacgcgtc cgggttcccc ggcctctctt ggtcagggtg acgcagtagc ctgcaaacct 60 cggcgcgtag gccaccgcac ttatccgcag caggaccgcc cgcagccggt agggtgggct 120 cttcccagtg cccgcccagc taccggccag cctgcggctg cgcagatctt tcgtggttct 180 gtcagggaga cccttaggca ctccggacta agatggcggc gacggccagg cggggctggg 240 gagctgcggc tgttgccgcc gggctgcgca ggcggttctg tcatatgttg aagaatccat 300 acaccattaa gaaacagcct ctgcatcagt ttgtacaaag accacttttc ccactacctg 360 cagcctttta tcacccagtg agatacatgt ttattcaaac acaagatacc ccaaatccaa 420 acagcttaaa gtttatacca ggaaaaccag ttcttgagac aaggaccatg gattttccca 480 ccccagctgc agcatttcgc tcccctctgg ctaggcagtt atttaggatt gaaggagtaa 540 aaagtgtctt ctttggacca gatttcatca ctgtcacaaa ggaaaatgaa gaattagact 600 ggaatttact gaaaccagat atttatgcaa caatcatgga cttctttgca tctggcttac 660 ccctggttac tgaggaaaca ccttcaggag aagcaggatc tgaagaagat gatgaagttg 720 tggcaatgat taaggaattg ttagatacta gaatacggcc aactgtgcag gaagatggag 780 gggatgtaat ctacaaaggc tttgaagatg gcattgtaca gctgaaactc cagggttctt 840 gtaccagctg ccctagttca atcattactc tgaaaaatgg aattcagaac atgctgcagt 900 tttatattcc ggaggtagaa ggcgtagaac aggttatgga tgatgaatca gatgaaaaag 960 aagcaaactc accttaaaat aatctggatt ttctttgggc ataacagtca gacttgttga 1020 taatatatat caagttttta ttattaatat gctgaggaac ttgaagatta ataaaatatg 1080 ctcttcagag aatgatatat aaaaaa 1106 <210> 30 <211> 2180 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1290078CB1 <400> 30 gccccacaca ggccgcggcg gctggctcgg gcccctacgg tcccggcggc ggctggagga 60 ggaagccagg cggctggcgg aggaggagag acggaggagg ccgagaccgg agcgccgctc 120 gccgcagact tacttccccg gctcagcagg gaaaggttcc tagaaggtga gcgcggacgg 180 tatgcaaagt tgtgaatcca gtggtgacag tgcggatgac cctctcagtc gcggcctacg 240 gagaagggga cagcctcgtg tggtggtgat cggcgccggc ttggctggcc tggctgcagc 300 caaagcactt cttgagcagg gtttcacgga tgtcactgtg cttgaggctt ccagccacat 360 cggaggccgt gtgcagagtg tgaaacttgg acacgccacc tttgagctgg gagccacctg 420 gatccatggc tcccatggga accctatcta tcatctagca gaagccaacg gcctcctgga 480 agagacaacc gatggggaac gcagcgtggg ccgcatcagc ctctattcca agaatggcgt 540 ggcctgctac cttaccaacc acggccgcag gatccccaag gacgtggttg aggaattcag 600 cgatttatac aacgaggtct ataacttgac ccaggagttc ttccggcacg ataaaccagt 660 caatgctgaa agtcaaaata gcgtgggggt gttcacccga gaggaggtgc gtaaccgcat 720 caggaatgac cctgacgacc cagaggctac caagcgcctg aagctcgcca tgatccagca 780 gtacctgaag gtggagagct gtgagagcag ctcacacagc atggacgagg tgtccctgag 840 cgccttcggg gagtggaccg agatccccgg cgctcaccac atcatcccct cgggcttcat 900 gcgggttgtg gagcGgctgg cggagggcat ccctgcccac gtcatccagc tagggaaacc 960 tgtccgctgc attcactggg accaggcctc agcccgcccc agaggccctg agattgagcc 1020 ccggggtgag ggcgaccaca atcacgacac tggggagggt ggccagggtg gagaggagcc 1080 ccgggggggc aggtgggatg aggatgagca gtggtcggtg gtggtggagt gcgaggactg 1140 tgagctgatc ccggcggacc atgtgattgt gaccgtgtcg ctaggtgtgc taaagaggca 1200 gtacaccagt ttcttccggc caggcctgcc cacagagaag gtggctgcca tccaccgcct 1260 gggcattggc accaccgaca agatctttct ggaattcgag gagcccttct ggggccctga 1320 gtgcaacagc ctacagtttg tgtgggagga cgaagcggag agccacaccc tcacctaccc 1380 acctgagctc tggtaccgca agatctgcgg ctttgatgtc ctctacccgc ctgagcgcta 1440 cggccatgtg ctgagcggct ggatctgcgg ggaggaggcc ctcgtcatgg agaagtgtga 1500 tgacgaggca gtggccgaga tctgcacgga gatgctgcgt cagttcacag ggaaccccaa 1560 cattccaaaa cctcggcgaa tcttgcgctc ggcctggggc agcaaccctt acttccgcgg 1620 ctcctattca tacacgcagg tgggctccag cggggcggat gtggagaagc tggccaagcc 1680 cctgccgtac acggagagct caaagacagc gcccatgcag gtgctgtttt ccggtgaggc 1740 cacccaccgc aagtactatt ccaccaccca cggtgctctg ctgtccggcc agcgtgaggc 1800 tgcccgcctc attgagatgt accgagacct cttccagcag gggacctgag ggctgtcctc 1860 gctgctgaga agagccacta actcgtgacc tccagcctgc cccttgctgc cgtgtgctcc 1920 tgccttcctg atcctctgta gaaaggattt ttatcttctg tagagctagc cgccctgact 1980 gccttcagac ctggccctgt agcttttctt tttctccagg ctgggccgtg agcaggtggg 2040 ccgttgagtt acctctgtgc tggatcccgt gcccccactt gcctaccctc tgtcctgcct 2100 tgttattgta agtgccttca atactttgca ttttgggata ataaaaaagg ctccctcccc 2160 tgcccctcaa aaaaaaaaaa 2180 <210> 31 <211> 1311 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1302741CB1 <400> 31 ccgccgccca cccgagccta tctgggctgc gtcttctcgc cgctgctctt cgtggcccaa 60 cgccccaatc cttgcgtgtg cttgcagtcc caccccacac tcagccttgt gtccctcgat 120 ccagtctccg acttccattt cccaccctaa accgcctacc cggtgtctgt tccccgcccg 180 gttgtcctcg ccctgctgcg ctgagtgtcc cctgttagcc tcgaccccat ggcgctgcag 240 acgctgcaga gctcgtgggt gaccttccgc aagatcctgt ctcacttccc cgaggagctg 300 agtctggctt tcgtctacgg ctccggggtg taccgccagg cagggccgag ttcagaccag 360 aagaatgcta tgctggactt tgtgttcaca gtagatgacc ctgtcgcatg gcattcaaag 420 aacctgaaga aaaattggag tcactactct ttcctaaaag ttttagggcc caagattatc 480 acgtccatcc agaataacta tggcgctgga gtttactaca attcattgat catgtgtaat 540 ggtaggctta tcaaatatgg agttattagc actaacgttc tgattgaaga tctcctcaac 600 tggaataact tatacattgc tggacgactc caaaaaccgg tgaaaattat ctcagtgaac 660 gaggatgtca ctcttagatc agccctcgat agaaatctga agagtgctgt gaccgctgct 720 ttcctcatgc tccccgaaag cttttctgaa gaagacctct tcatagagat tgccggtctc 780 tcctattcag gtgactttcg gatggtggtt ggagaagata aaacaaaagt gttgaatatt 840 gtgaagccca atatagccca ctttcgagag ctctatggca gcatactaca ggaaaatcct 900 caagtggtgt ataaaagcca gcaaggctgg ctggagatag ataaaagccc agaaggacag 960 ttcactcagc tgatgacatt gcccaaaacc ttacagcaac agataaatca tattatggac 1020 cctcctggaa aaaacagaga tgtggaagaa actttattcc aagtggctca tgatcccgac 1080 tgtggagatg tggtgcgact agggctttca gcaatcgtga gaccgtctag tataagacag 1140 agcacgaaag gcatttttac tgctggcctg aagaagtcag tgatttatag ttcactaaaa 1200 ctgcacaaaa tgtggaaagg gtggctgagg aaaacatcct gattttgctt gcttttatat 1260 atgttatatg tagatgaata aagtgtttga tcctttttga caaaaaaaaa a 1311 <210> 32 <211> 921 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1541028CB1 <400> 32 ggcccctgcg ccccacgtgg cgccctttcc gggcgggggg ggtggggagg cacctccagc 60 gacaactctc ccccctcccc cggcacccgg ctgatttgct gtgccactgg gagggttcgg 120 gggtggctga gctgagaggg ctccgggaag gagtgacgtc aggacctgcc ttcagccccc 180 tttgtgccgc gggacgccca gcggcacgac gtggagctcc tggagagcag cttccctgcc 240 attttgcggg acttcggggc tgtgagctgg gacttctcag ggactacccc tccgcctcgg 300 ggctggtccc cacctctggc ccccgggtgc taccagctcc tgctgtacca agcaggccgg 360 tgccaaccca gcaactgccg ccggtgcccg ggggcctatc gggcactgag ggggcttcga 420 agctttatga gtgccaacac cttcggcaat gccggctttt ccgttctcct gcctggggcc 480 cggctcgagg gccgctgtgg gcccaccaat gcccgggtca gatgccatct gggcctaaag 540 atccctcctg gctgtgagct ggtggtcggc ggtgagcccc agtgctgggc tgaggggcac 600 tgtctactgg tggacgactc ttttctacac acagtggctc acaatggctc ccccgaagat 660 gggcctcgag tggtcttcat cgtggacctc tggcacccca acgtggcagg ggctgagcgc 720 caggccctcg actttgtctt cgccccagac ccttgaagga aggtgctccc ttcacacacc 780 caggctggag agacactgcg ctcagggacg gcttgatggt agccaggacc tcctctctac 840 tgcgggggtg ggcgggggcg gaggatggga actggctagt gagcactgaa atataaattc 900 tgaatcctca aaaaaaaaaa a 921 <210> 33 <211> 2032 <212> DNA
<213> Homo Sapiens 27/4() <220>
<221> misc_feature <223> Incyte ID No: 1597687CB1 <400> 33 agaaaccagg cggcgcgttc ccggtggcgg cgccctggac tcccgggccc gcgcatcccc 60 gccagccttc cttaaggcgg atgggtggcc cccgagaccc cgtcggaccc atggtttcca 120 gtgcagcgcg gagtgggcga tgccagcgtg ccaggagcca tgtctgacca ggacgtttgg 180 aagatcatat ccatgccaga ggctcttgtg aggagatgag ttggtaaaga gagaggctgg 240 gatgaagatg ctgccaggag tgggcgtgtt tgggactggc agctccgccc gagttctggt 300 cccactgctg agggcagaag ggttcactgt tgaggccctg tgggggaaga ctgaggagga 360 ggcgaagcag cttgctgagg agatgaacat cgccttctac accagccgga ctgatgacat 420 cttgctgcat caagatgtgg atctggtgtg catcagcatc ccccctccac tcacccggca 480 gatatccgtg aaggctctag gtattgggaa gaatgtggtt tgcgagaagg cagcaacatc 540 ggtggatgcc ttccggatgg tgacagcctc gcgctactac ccgcagctca tgagcctggt 600 agggaacgtg ctgcgcttcc tgcctgcctt cgtgcgcatg aaacagctga tttcggaaca 660 ctatgtggga gcggtgatga tctgtgatgc ccgcatctac tcaggcagcc tgctgagccc 720 cagctatggc tggatctgtg atgagctcat gggcggcggg ggcttgcaca ccatggggac 780 ctacattgtg gacctgctga cccacctgac cggccggaga gccgagaagg tgcacgggct 840 gctcaagaca ttcgtgaggc agaacgctgc catccgtggc atccggcacg tcactagcga 900 tgacttctgt ttcttccaga tgctcatggg tgggggtgtg tgtagcacag tgacactcaa 960 cttcaacatg ccaggcgcct ttgtgcatga agtcatggtg gtaggctctg caggacgcct 1020 cgtcgcccgg ggagccgacc tctatgggca gaagaactct gccacgcaag aggagctgct 1080 cttgagggac tcgctggcag tgggcgcagg actgcctgag caggggcccc aggatgtccc 1140 gctgctgtac ctgaagggca tggtctacat ggtgcaggcc ttgcgccagt ccttccaggg 1200 gcagggcgac cgccgcacct gggaccgcac ccctgtctcc atggccgcct ccttcgagga 1260 tgggctgtac atgcagagcg tggtggatgc catcaagagg tcgagccgat ccggggagtg 1320 ggaggctgtg gaggtgctga cggaggagcc cgacaccaac cagaacctgt gtgaggcact 1380 tcagcggaac aacctatgag cctgcacctg ggctccttgc cacagggcag agggaccagg 1440 gaggggaaca ggagccagac atgacaggga cttggcccta gcagagacag tgtctttcat 1500 ttaatgagtc tgggtgaagc cagaggtggc tctgggactt cgccccctcc agtggctcat 1560 ctgctcctca gacttgcagg gtggcaaatg ttcctgttgc caggttacca cggtgtgcac 1620 agggcagagc tgccgccagg cctcctcatg gtgatgccag tgagtcggac caggaaaggc 1680 tcaccctggg gagcccttca gcctgatccg cagtcctaac agggtgagaa agcacaagag 1740' gggccagctc ggctgaggcc tgaggagaaa tgatgagaag ttttgtttcg ttcttgggct 1800 ggggcatctc aggaacagtg agggcaacaa gctgtccctc agggaccctc acctgccctc 1860 ccatgtgacc aggcccttcc caggtgggga ctggggcttg ctttattaag tgagctcttg 1920 ggcttttgaa gtaggacaga gggccctggt ttggcagaga ggaggcaagg gagggcttta 1980 gaacatctga gaaatgttaa taaataattc agaaaataaa aaaaaaaaaa as 2032 <210> 34 <211> 1134 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1690348CB1 <400> 34 cgtggacttc taagaaagcg ccatggcctg tgctgctgtt atgattcctg ggttgttgcg 60 gtgctctgtt ggagccatcc gtattgaggc tgcgtcactg agattgacac tcagcacttt 120 gcgccatctt actctaacca gcataatgaa atccaaaagg aaaactgatc acatggagag 180 aactgcaagt gtccttcgac gggagattgt ggcagcagct aaggtgtgtg gagctgccag 240 tgagtcaccg tcagtgaaga gcctccgctt gcttgttgct gatcaagact tttcctttaa 300 agctggccag tgggttgatt tctttattcc aggagtctct gtggttggtg ggttttcaat 360 atgctccagt cccagactgc tagaacaaga gagagtgata gaattggcag tgaaatatac 420 gaaccaccct cctgccctct gggttcacaa tacgtgtaca cttgactgtg aagtggctgt 480 gagagtgggt ggagagttct tctttgaccc tcagcctgcg gatgcctcta gaaacctcgt 540 gttgattgca ggaggagtcg gaattaaccc tctgctttcc atcctgcggc acgcagcaga 600 tctcctcaga gagcaggcaa acaaaagaaa tggatatgag ataggaacaa taaaactatt 660 ctacagtgca aaaaatacca gcgaactcct gtttaagaaa aatatccttg atttagtaaa 720 tgaatttcct gagaagattg catgcagttt gcatgttaca aaacagacta cacaaatcaa 780 tgcggaactc aagccataca tcacggaagg aagaataacg gagaaggaga taagagatca 840 tatttcaaaa gagactttgt tctatatttg tggcccacct ccaatgacag actttttctc 900 caagcaactg gaaaacaacc atgtacccaa agaacacatt tgctttgaga agtggtggta 960 ggaggcagac aaaggcagaa aaaataaaga ggtgagatct actcaggaga gctcctgtcc 1020 tttgtggcat gattaatttt ttttatctct acttgagttg tcttattttt taaggctata 1080 aacttagtga ccagctggat aataaaagcc agctggcaga cttaaatgat aaac 1134 <210> 35 <211> 734 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 661 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte ID No: 1865603CB1 <400> 3S
gggaggaccc tgggcaaaga cacctaccct gccatgctgc gccatctgcc ctccaggctg 60 ccagtcaaga tgtggggcag gactttggag aaacagtcat ggagggacag cagtcagacc 120 cctcccccat gtcttatccg tagacttgac cacatcgtga tgacggtgaa gagcatcaaa 180 gacaccacca tgttttattc caagatcctg ggcatggagg tcatgacttt taaggaagac 240 cggaaagcac tgtgttttgg agaccagaaa tttaacctcc acgaggtggg aaaggaattt 300 gaacccaaag ccgctcaccc agttcctggc tccctggaca tatgtctgat cacagaggtg 360 cctttggagg aaatgatcca gcacctcaag gcttgtgatg tccctattga ggaggggcca 420 gtccccagaa caggggcaaa agggcctatc atgtccatct acttccgaga ccccgacaga 480 aatctgattg aggtgtccaa ctacatctcc tcgtgatgga ggctggacct cctccattct 540 gtcccccttg atgtcgccct ctcctttcct tcctgcaaac ccccacccag gcccagagat 600 ctccaaagac tgaggactta ggcacttcac acatcctgct gaggggggac ccaagacagg 660 nttgggacca aacctacatg tctgtatgtc atcaaagttg gcctaataaa tgcataacct 720 ctcaatgaaa aaaa 734 <210> 36 <211> 2221 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1976472CB1 <400> 36 gtcgggggct tggcctctgc ccggccacag agccggagct ggaggtgctg tcccgtctgg 60 cggcgatccc cgggcagggc ccggggccgg ggtttgaaga tgctgaacgt cccttcccag 120 tctttcccgg cccccaggtc gcagcagcgt gtcgcctccg gggggcgtac aaggtacctt 180 taaaacaggg cagaagcctt atggattgga ttcgactgac caaaagtgga aaggatctaa 240 cgggattaaa aggcaggtta attgaagtaa ctgaagaaga acttaagaaa cacaacaaaa 300 aagatgattg ttggatatgc ataagaggtt tcgtttataa tgtcagccct tatatggagt 360 atcatcctgg tggagaagat gaactaatga gagcagcagg atcagatggt actgaacttt 420 ttgatcaggt tcatcgttgg gtcaattatg aatccatgct gaaagaatgc ctggttggca 480 gaatggccat taaacctgct gttctgaaag actatcgtga ggaggaaaag aaagtcttaa 540 atggcatgct tcccaagagc caagtgacag atacacttgc caaagaaggt cctagttatc 600 caagctatga ttggttccaa acagactctt tagtcaccat tgccatatat actaaacaga 660 aggatatcaa tttagactca ataatagttg atcatcagaa tgattccttt agagcagaaa 720 caattattaa ggattgttta tatcttatac atattgggct aagccatgag gttcaggaag 780 atttttctgt gcgggttgtt gagagtgtgg gaaaaataga gattgttcta caaaaaaaag 840 agaatacttc ttgggacttt cttggccatc ccctgaagaa tcataattca cttattccaa 900 ggaaagatac aggtttgtac tacagaaagt gccagttaat ttccaaggaa gatgttactc 960 atgatacgag gcttttctgt ttgatgctgc caccaagcac tcatcttcaa gtgcccattg 1020 ggcaacatgt ttacctcaag ctacctatta caggtacaga aatagtaaag ccatatacac 1080 ctgtatctgg ttccttactc tcagagttca aggaaccagt tcttcccaac aataaataca 1140 tctacttttt gataaaaatc tatcccactg gactcttcac accagagctt gatcgtcttc 1200 agattggaga ttttgtttct gtaagcagtc ctgagggcaa ttttaaaata tccaagttcc 1260 aagaattaga agatctcttt ttgttggcag ctggaacagg cttcacacca atggttaaaa 1320 tactgaatta tgctttgact gatataccca gtctcaggaa agtgaagctg atgttcttca 1380 ataaaacaga agatgatata atttggagaa gccaattgga gaaattagca tttaaagata 1440 aaagactgga tgttgaattt gttctctcag cacctatttc tgaatggaat ggcaaacagg 1500 gacatatttc accagctctt ctttctgaat ttttgaaaag aaatttggac aaatccaaag 1560 ttctcgtctg catttgtgga ccagtgccat ttacagaaca aggagtaagg ttgctgcatg 1620 atctcaactt ttccaaaaat gagatccata gttttacagc ataatgaaga gctgtcattg 1680 tcctttattc aactagttta tctaaatttg tgattgctta gggtttttta agagaacatt 1740 tttgtacata acaaaaggtt aactagaatc cagccttcag tttcttaaat gaaatcaaat 1800 gttccttcag tacaggtaac ttcttggctt tcttttgtac cacaacttat tttactactg 1860 atatttgacc tggaaagtta atcatggcaa caaatacata caggattctt tgttatgaat 1920 cacaaatttc cttgccattt aaattatatc actgttctac aataagcact tgtgttttat 1980 gacatgataa agtaaatgat cacttcgatc atgtttctat gctgtaaaat gtcttattag 2040 caatacagat taaattttac cttgcactgt taactcagga aatgatcatt tatgtccttg 2100 ttattaatgt taaaacatag aatgttataa cttattaagt gatccaaaca tttttttgtg 2160 tgtgtatggc attgatgcag aatagaataa aattatactt aagttctttt taaaaaaaaa 2220 a 2221 <210> 37 <211> 1706 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2050821CB1 <400> 37 agaagaggtt gtgtgggaca agctgctccc gacagaagga tgtcgctgct gagcctgccc 60 tggctgggcc tcagaccggt ggcaatgtcc ccatggctac tcctgctgct ggttgtgggc 120 tcctggctac tcgcccgcat cctggcttgg acctatgcct tctataacaa ctgccgccgg 180 ctccagtgtt tcccacagcc cccaaaacgg aactggtttt ggggtcacct gggcctgatc 240 actcctacag aggagggctt gaaggactcg acccagatgt cggccaccta ttcccagggc 300 tttacggtat ggctgggtcc catcatcccc ttcatcgttt tatgccaccc tgacaccatc 360 cggtctatca ccaatgcctc agctgccatt gcacccaagg ataatctctt catcaggttc 420 ctgaagccct ggctgggaga agggatactg ctgagtggcg gtgacaagtg gagccgccac 480 cgtcggatgc tgacgcccgc cttccatttc aacatcctga agtcctatat aacgatcttc 540 aacaagagtg caaacatcat gcttgacaag tggcagcacc tggcctcaga gggcagcagt 600 cgtctggaca tgtttgagca catcagcctc atgaccttgg acagtctaca gaaatgcatc 660 ttcagctttg acagccattg tcaggagagg cccagtgaat atattgccac catcttggag 720 ctcagtgccc ttgtagagaa aagaagccag catatcctcc agcacatgga ctttctgtat 780 tacctctccc atgacgggcg gcgcttccac agggcctgcc gcctggtgca tgacttcaca 840 gacgctgtca tccgggagcg gcgtcgcacc ctccccactc agggtattga tgattttttc 900 aaagacaaag ccaagtccaa gactttggat ttcattgatg tgcttctgct gagcaaggat 960 gaagatggga aggcattgtc agatgaggat ataagagcag aggctgacac cttcatgttt 1020 ggaggccatg acaccacggc cagtggcctc tcctgggtcc tgtacaacct tgcgaggcac 1080 ccagaatacc aggagcgctg ccgacaggag gtgcaagagc ttctgaagga ccgcgatcct 1140 aaagagattg aatgggacga cctggcccag ctgcccttcc tgaccatgtg cgtgaaggag 1200 agcctgaggt tacatccccc agctcccttc atctcccgat gctgcaccca ggacattgtt 1260 ctcccagatg gccgagtcat ccccaaaggc attacctgcc tcatcgatat tataggggtc 1320 catcacaacc caactgtgtg gccggatcct gaggtctacg accccttccg ctttgaccca 1380 gagaacagca aggggaggtc acctctggct tttattcctt tctccgcagg gcccaggaac 1440 tgcatcgggc aggcgttcgc catggcggag atgaaagtgg tcctggcgtt gatgctgctg 1500 cacttccggt tcctgccaga ccacactgag ccccgcagga agctggaatt gatcatgcgc 1560 gccgagggcg ggctttggct gcgggtggag cccctgaatg taggcttgca gtgactttct 1620 gacccatcca cctgtttttt tgcagattgt catgaataaa acggtgctgt caaaaaaaaa 1680 aaaaaaaaac taaaaaaaaa aaaaaa 1706 <210> 38 <211> 549 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2408443CB1 <400> 38 cccacgcgtc cgtgtcatgt acttttgtta atatttttgc tataataaac ttagtatatt 60 tcaaggaggg caacatttta tggttacatt atactcgtct ccgagttgca cctcttgtcg 120 taaagcaaaa caatggctgg ttgaccataa tctcccattt attgaacgta atttaaataa 180 agaaccattg cgtgcggaag atgtcaaagc aatgttacga ttgactgaag atgggacgga 240 agaattaatt tcaacacgct caaaaatttt ttctgagttg acgattgact tagatgatat 300 gtcaattaat aaattgattg acctcatcgt catgtatccg tctttactga agcggccaat 360 tattcttgac gatcagcgca tgcaaattgg gtacaatgat gatgaaattc gtcgcttttt 420 accacgtgaa gttcgtcagc gagagttaat tcgcgcaaca tttaaagctg acttcgcaga 480 agaggcaaaa gatttagtgg ttgaagaagg ctgagcacgt cagtcttttt ttctttacca 540 tgacaaaag 549 <210> 39 <211> 1363 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2508668CB1 <400> 39 cgtcaggctc tgtgttggtt ggagcgagca tgtgggtctg cagtaccctg tggcgggtgc 60 gaacccccgc ccggcagtgg cgggggctgc tcccagcttc tggctgtcac ggacctgccg 120 cctcctccta ctccgcatcc gccgagcctg cccgggtccg ggcgcttgtc tatgggcacc 180 acggggatcc agccaaggtc gtcgaactca agaacctgga gctagctgct gtgagaggat 240 cagatgtccg tgtgaagatg ctggcggccc ctatcaatcc atctgacata aatatgatcc 300 aaggaaacta cggactcctt cctgaactgc ctgctgttgg agggaacgaa ggtgttgcac 360 aggtggtagc ggtgggcagc aatgtgaccg ggctgaagcc aggagactgg gtgattccag 420 caaatgctgg tttaggaacc tggcggaccg aggctgtgtt cagcgaggaa gcactgatcc 480 aagttccgag tgacatccct cttcagagcg ctgccaccct gggtgtcaat ccctgcacag 540 cctacaggat gttgatggac ttcgagcaac tgcagccagg ggattctgtc atccagaatg 600 catccaacag cggagtgggg caagcggtca tccagatcgc cgcagccctg ggcctaagaa 660 ccatcaatgt ggtccgagac agacctgata tccagaagct gagtgacaga ctgaagagtc 720 tgggggctga gcatgtcatc acagaagagg agctaagaag gcccgaaatg aaaaacttct 780 ttaaggacat gccccagcca cggcttgctc tcaactgtgt tggtgggaaa agctccacag 840 agctgctgcg gcagttagcg cgtggaggaa ccatggtaac ctatgggggg atggccaagc 900 agcccgtcgt agcctctgtg agcctgctca tttttaagga tctcaaactt cgaggctttt 960 ggttgtccca gtggaagaag gatcacagtc cagaccagtt caaggagctg atcctcacac 1020-tgtgcgatct catccgccga ggccagctca cagcccctgc ctgctcccag gtcccgctgc 1080 aggactacca gtctgccttg gaagcctcca tgaagccctt catatcttca aagcagattc 1140 tcaccatgtg atcatcccaa aagagctgga gtgacatggg aggggaggcg gatctgaggg 1200 gctgggtgca ggcccctcag ttggggctcc caccttcccc agactactgt tctcctcact 1260 gcctcttcct attaggagga tggtgaagcc agccacggtt ttccccaggg ccagccttaa 1320 ggtatctaat aaagtctgaa ctctcccttc caaaaaaaaa aaa 1363 <210> 40 <211> 1196 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2536830CB1 <400> 40 ggttgccagg caaccgcgaa ccgcggtccc tgcctctgag tctctctcga ccatggccaa 60 attcgtcatc gcgggtagag cagattgtcc atattatgct aaaacagaac ttgtggcaga 120 ctatttacaa aagaatcttc ctgattttcg gatacataaa atcacacaac gtcctgaggt 180 ttgggaggat tggctaaaag atgtgtgtga aaagaataag tggagtcaca agaattcccc 240 tatcatctgg agagagctgt tggatcgtgg aggaaagggt ttgcttttgg gaggatataa 300 tgagttcctg gagcatgctc agctttacta tgatgtcacc tctagcatga cgactgaact 360 gatgatggta attgctcaag agaacctggg ggcacatata gaaaaagagc aggaggaaga 420 agccctgaaa acttgcatca accccttgca ggtctggatc accagtgcct ctgctcctgc 480 ctgctacaac ctaattccca tattgacgag tggcgaagtg tttgggatgc atacagaaat 540 tagcataact ctatttgaca acaagcaggc ggaagaacat ctcaaaagcc ttgtggtgga 600 gacccaagac ctggcatctc ccgtcctgcg cagtgtctcc atctgcacga aggtggagga 660 ggccttccgc caggcccacg tcattgtggt gctggatgac agcaccaaca aggaggtgtt 720 cactctggag gactgcctcc gaagcagggt gcctctctgc aggctctatg ggtacctgat 780 agagaaaaat gctcatgagt ctgtcagagt catcgtggga gggagaacct ttgtaaacct 840 gaagacagtt ttactcatga gatatgcccc acgcattgca cacaacatta ttgctgtggc 900 gctgggggtg gaaggtgaag cgaaagccat actggccaga aaactgaaga cagctccttc 960 atgtgagtga atgtctcttt atgtttttgt ttttattttg aaacaacatg atttcaaagc 1020 agctaggaag aacactgtcc cctctaggaa cattctgagt atctggaata ggaagtataa 1080 aacagacata aagaacttat tgtgtgattt gactactgtg caaacatttt tttttactgg 1140 cttttgtgtc tggctttttc catcattata aaaatcaata aatatttaat aagaaa 1196 <210> 41 <211> 1926 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2645179CB1 <400> 41 gcttcctctc ctggagtcca gagtcccgct gctccgccgc gatattcagt aaaccactgg 60 gagtccggca gcatggaggc agcgcgccct cccccgacgg cagggaagtt cgtggtggtc 120 ggcggcggca tcgcgggcgt cacttgtgcg gagcagttgg ctactcactt tccatcggaa 180 gatattctct tggtaacagc ttctcctgtt attaaagcag ttacaaattt caagcagatt 240.
tctaaaatat tggaagaatt cgatgttgaa gaacaatcaa gtaccatgtt aggaaaacgc 300 tttcccaaca ttaaggttat agaatctggc gtaaagcaac tgaagagtga agaacactgc 360 attgtaacag aagatggcaa tcagcacgta tataagaaac tctgtctgtg tgctggagct 420 aaaccaaagt tgatatgtga aggaaatcct tatgtattag gaatccgtga tacagacagt 480 gctcaggaat ttcagaaaca gcttactaaa gctaaaagaa taatgatcat agggaacggt 540 ggtattgcac ttgagttagt gtatgaaatt gaaggctgtg aagtgatttg ggccattaaa 600 gataaagcta tagggaatac tttcttcgat gcaggagcag ctgaattctt gacttcaaag 660 ctcattgctg aaaaatcaga ggctaaaatt gcacataaaa gaaccagata tacaactgaa 720 ggaaggaaaa aggaagctag aagcaaatct aaagcagata atgtaggaag tgcattggga 780 ccagattggc atgaaggctt gaatcttaaa ggaacaaaag agttttctca taagattcac 840 cttgaaacta tgtgtgaagt aaagaaaatc taccttcagg atgagtttag aattttgaag 900 aaaaagtcct tcacttttcc aagagaccat aagtcagtta cagctgatac agagatgtgg 960 cctgtctatg tggaattgac caatgaaaag atatatggct gcgatttcat tgtcagtgct 1020 acaggagtta caccaaatgt agaacctttt ctccatggta acagttttga tctaggagaa 1080 gatggtggcc tgaaagtgga tgatcatatg cacacatccc ttcctgatat ctatgctgcc 1140 ggtgacatct gtactacatc ctggcagctg agcccagtct ggcagcagat gaggctgtgg 1200 acccaggcta gacagatggg atggtatgca gcaaagtgca tggctgcagc gagttcagga 1260 gactctattg acatggattt cagctttgaa ctgtttgctc atgtgacaaa attttttaac 1320 tataaggttg tactgctggg aaaatacaat gcacagggct taggttcaga tcatgaatta 1380 atgctgagat gtaccaaagg acgagaatac atcaaagtcg tcatgcaaaa tggacgaatg 1440 atgggagctg tcttaattgg tgaaaccgat ttagaagaaa catttgaaaa cctaatctta 1500 aaccaaatga atctttcatc atatggagaa gatctgctag atccaaatat tgatatagaa 1560 gattattttg actaaaaatg gaatttcttc aggaatcata taaagttcca aatgacacca 1620 gaaaaatcac aagtcaataa aatgaatgac tgtattgagt taatgatgac cacactgaaa 1680 attacagaag tgataatgat attagtggaa aaatataaaa acataaattc taagtttgaa 1740 atcagttcaa agtttattta tagatatatc tttccaatac aacactgacc gcttagataa 1800 aaatcttaag ttatttattt ctgtgtttta aacataaata tgtttacttg tgatttagct 1860 ttggagcaaa tttaggtaag ttatctactt agccaaatgt actctagtag actagaacca 1920 ttcttt 1926 <210> 42 <211> 1727 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2754425CB1 <400> 42 agggaccgac ggcacttgat aatgtgacaa aaaccagcct cttccacccc actgcagtga 60 cttttgccgg aaagtggagc agtgggcacg gccaaccttg tcgttcatca atgccgcccc 120 tggcagtatc tgaggatcgg tggcagccat gcctccccct gccccagccg ctccttcccc 180 cccacgctaa tctgcatggt gtgggcgccc ttgggacccc cgaggactga ttgtctgacc 240 ttgcttcaca cgcccagtaa ggactccccc aagatgtcgc tcgagtggct ggtggcctgg 300 agctggtcgc tggatggcct gagggactgc atcgccaccg gcatccagtc cgtgcgggac 360 tgcgacacca ccgctgtcat cactgtggcc tgcctcctgg tcctcttcgt gtggtactgt 420 tatcacgtgg gcagggagca gccccggccc tacgtctccg tcaactccct catgcaggct 480 gccgatgcca acgggctgca gaatggctac gtgtactgcc agtcccctga gtgcgtgcgc 540 tgcacccaca acgagggcct caaccagaag ctgtaccaca acctgcagga gtacgccaag 600 cgctactcct ggtccggcat gggccgcatc cacaagggca tccgcgagca gggccggtac 660 ctcaacagcc ggccctccat ccagaagccc gaggtcttct tcctgcccga cctgcccacc 720 acgccctatt tctcccggga cgcacagaaa catgatgtgg aagtgctgga acggaacttc 780 cagaccatcc tgtgtgagtt tgagaccctc tacaaagctt tctcaaactg cagcctcccg 840 caaggatgga aaatgaacag cacccccagc ggggagtggt tcacctttta cttggtcaat 900 cagggggttt gtgttcccag gaactgtagg aagtgcccac ggacgtaccg cttgctcgga 960 agccttcgga cctgtattgg gaacaatgtt tttgggaacg cgtgcatctc tgtgctgagc 1020 cctgggactg tgataacgga gcactatgga cccaccaaca tccgcatccg atgccattta 1080 ggtctgaaaa ctccaaatgg ctgtgagctg gtggtggggg gagagcccca gtgctgggca 1140 gaagggcgct gccttctctt tgatgactct ttcctgcatg ctgcgttcca tgaaggttca 1200 gcagaggatg gcccacgggt ggttttcatg gtggatttgt ggcatccaaa cgtcgcagcg 1260 gccgaacggc aggctcttga tttcatcttt gctccgggac gatgagagta tttcccatgc 1320 tggagtcggc gagaagggcc gaggcggggc ctgggcagac tgtggtccgg tccagtccct 1380 accggtgttg tttccatgct cagaaacctg cctcagcgga aagctcttat ttgggatttt 1440 atatcatgtc gggtccctct ttcccttggt tattgtaaat ggaaactttt cggcttgtat 1500 ttccttagat tttttttttt tccttccaat catttgcttc agagactcct ttctggccta 1560 acagcgcatt cctttgattg gtccttgagt gaccagagac ttagtgccct tgtaagtctg 1620 tcttctgttg ctacttgttt ttttcagtgc tctgaaatag agtaactaaa tggttatttg 1680 tctgaatata ataatgtaaa acttcttgtg gtcatcttaa aaaaaaa 1727 <210> 43 <211> 611 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2821526CB1 <400> 43 cccatccggt gggttttgct cggcgcaccc gggaggcggg gccagcgagg caagatggag 60 ttagtgcagg tcctgaaacg cgggctgcag cagatcaccg gccacggcgg tctccgaggc 120 tatctacggg tttttttcag gacaaatgat gcgaaggttg gtacattagt gggggaagac 180 aaatatggaa acaaatacta tgaagacaac aagcaatttt ttggccgtca ccgatgggtt 240 gtatatacta ctgaaatgaa tggcaaaaac acattctggg atgtggatgg aagcatggtg 300 cctcctgaat ggcatcgttg gcttcacagt atgactgatg atcctccaac aacaaaacca 360 cttactgctc gtaaattcat ttggacgaac cataaattca acgtgactgg caccccagaa 420 caatatgtac cttattctac cactagaaag aagattcagg agtggatccc accttcaaca 480 ccttacaagt aaagacaatg aagaacagtt gaaacatgca aaatatggag cttttcatgt 540 aattactctt ttactgttta ccattcacta taattcacaa ttaaaattgt gtgactaaac 600 aaaaaaaaaa a 611 <210> 44 <211> 1352 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2876494CB1 33/4() <400> 44 gttttattga caatacatgc atcatatctt ttgactttga aggatatctc atgtcaaagg 60 aatcaagtta tgatttatag aggattcagc tggaatacct tgtgggtgct ggctgagggt 120 ggcaaaacgc ctaccgagac atgaaggttt tagccactag ttttgtcctt gggagcctgg 180 ggttggcctt ctacctgcct ttggtggtga ctacacctaa aacactggcc atccctgaga 240 agctgcaaga agctgtgggg aaagttatca tcaatgccac aacctgtact gtcacctgtg 300 gccttggcta taaggaggag accgtctgtg aggtgggccc tgatggagtg agaaggaaat 360 gtcagactca gcgcttagaa tgtctgacca actggatctg tgggatgctc,catttcacca 420 ttctcattgg caaggaattt gagcttagct gtctgagttc agacatcttg gagtttggac 480 aggaagcttt ccggttcacc tggagacttg ctcgaggtgt catctccact gacgatgagg 540 tcttcaaacc ctttcaagcc aactcccact ttgtgaagtt taaatatgct caggagtatg 600 actctgggac atatcgctgt gatgtgcagc tggtaaaaaa cttgagactt gtcaagaggc 660 tctattttgg gttgagggtc cttcctccta acttggtgaa tctgaatttc catcagtcac 720 ttactgagga tcagaagtta atagatgagg gattggaagt taatctggac agctactcca 780 agcctcacca cccaaagtgg aaaaagaagg tggcgtcagc cttgggaata ggaattgcca 840 ttggagtggt tggtggcgtg ttggtgagga ttgtcctctg tgcgctaagg gggggcctgc 900 agcagtgaca gcttcaagaa cttaacagcc ttgctcctga agaactggct gcccaggaag 960 ccaagctagc tttttagggg agtgttccag ctgctggtag tggatcagct tagagggaac 1020 actcccacag ccaaaagaat gagtgggaga aatggagggg acaatctcct gggagctatg 1080 cgcagtaacc taacttcctt atgtcccatg gatctcttcc tgatcttccc tgcccattgg 1140 gtacccagga aactgcaagc attgcctgtg ttcctgggaa gagttctaag aagcttgcat 1200 tcattttcta ccctttatga cttggatgcc tccccacctc catttcccct cttctgagct 1260 gtgtattcat gtagagggat gtattcagcc tttttagtga acattttttt tcaataaaag 1320 taattcacag tagctgtatg gagacctttt tc 1352 <210> 45 <211> 1458 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3403225CB1 <400> 45 ggacggagag gagcagagaa gcagcagaag cagccaagag ctggagccag accaggaacc 60 tgagccagag ctggggttga agctggagca gcagcaaaag caacagcagc tacagaagtt 120 ggaacgatgc tggtcacctt gggactgctc acctccttct tctcgttcct gtatatggta 180 gctccatcca tcaggaagtt ctttgctggt ggagtgtgta gaacaaatgt gcagcttcct 240 ggcaaggtag tggtgatcac tggcgccaac acgggcattg gcaaggagac ggccagagag 300 ctcgctagcc gaggagcccg agtctatatt gcctgcagag atgtactgaa gggggagtct 360 gctgccagtg aaatccgagt ggatacaaag aactcccagg tgctggtgcg gaaattggac 420 ctatccgaca ccaaatctat ccgagccttt gctgagggct ttctggcaga ggaaaagcag 480 ctccatattc tgatcaacaa tgcgggagta atgatgtgtc catattccaa gacagctgat 540 ggctttgaaa cccacctggg agtcaaccac ctgggcaccg gggtcaccac ctacgcagtg 600 cacccaggcg tcgtccgctc tgagctggtc cggcactcct ccctgctctg cctgctctgg 660 cggctcttct ccccctttgt caagacggca cgggaggggg cgcagaccag cctgcactgc 720 gccctggctg agggcctgga gcccctgagt ggcaagtact tcagtgactg caagaggacc 780 tgggtgtctc caagggcccg aaataacaaa acagctgagc gcctatggaa tgtcagctgt 840 gagcttctag gaatccggtg ggagtagctg gtggaagagc tgcagcttta tcaggcccaa 900 tccatgccat aatgaacagg gaccaaggag aaggccaacc ctaaaggatt gtcctcttgg 960 ccagctggtg ctgcgaatcc tgcctgctct gatcctcttg acccttctgg gaatgtttgc 1020 acacctgaca ctcttgtgag actggcttat ggcatgagtt gtggacacct atagagtgtt 1080 cttctctaag acctggaaag tcagcaaccc tctgggggca gcaggactgg gcagatccca 1140 ggctgggcat gggggtggca gaagagcccg agaaattggg tcagttccct catcagcacc 1200 agaggctcag ctgaggcaag aagagcacca tcactgccta tttctagggg ctatacactc 1260 caactcttgg ttgatctctt tctttttaaa aatatttgcc accaccctgg agtctagacc 1320 aacacacaaa gatcctggct aaccctggcc tatttagatt ccttcctctc acctggacct 1380 tcccatttca atcatgcaga tggtttcttt ttgtaaagag ttccgtttgc ctttcaattt 1440 ttagagaaaa taaagact 1458 <210> 46 <211> 1884 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4163943CB1 <400> 46 ggactccaag cgccatggcc gctgccgccc gagcccgggt cgcgtacttg ctgaggcaac 60 tgcaacgcgc agcgtgccag tgcccaactc attctcatac ttactcccaa gcccctggac 120 tttcaccttc tgggaaaaca acagattatg cctttgagat ggctgtttca aatattagat 180 atggagcagc agttacaaag gaagtaggaa tggacctaaa aaacatgggt gctaaaaatg 240 tgtgcttgat gacagacaag aacctctcca agctccctcc tgtgcaagta gctatggatt 300 ccctagtgaa gaatggcatc ccctttacgg tttatgataa tgtgagagtg gaaccaacgg 360 attcaagctt catggaagct attgagtttg cccaaaaggg agcttttgat gcctatgttg 420 ctgtcggtgg tggctctacc atggacacct gtaaggctgc taatctgtat gcatccagcc 480 ctcattctga tttcctagat tatgtcagtg cccccattgg caagggaaag cctgtgtctg 540 tgcctcttaa gcctctgatt gcagtgccaa ctacctcagg aaccgggagt gaaactactg 600 gggttgccat ttttgactat gaacacttga aagtaaaaat tggcatcact tcgagagcca 660 tcaaacccac actgggactg attgatcctc tgcacaccct ccacatgcct gcccgagtgg 720 tcgccaacag tggctttgat gtgctttgcc atgccctgga gtcatacacc accctgccct 780 accacctgcg gagcccctgc ccttcaaatc ccatcacacg gcctgcgtac cagggcagca 840 acccaatcag tgacatttgg gctatccacg cgctgcggat cgtggctaag tatctgaaga 900 gggctgtcag aaatcccgat gatcttgaag caaggtctca tatgcacttg gcaagtgctt 960 ttgctggcat cggctttgga aatgctggtg ttcatctgtg ccatggaatg tcttacccaa 1020 tttcaggttt agtgaagatg tataaagcaa aggattacaa tgtggatcac ccactggtgc 1080 cccatggcct ttctgtggtg ctcacgtccc cagcggtgtt cactttcacg gcccagatgt 1140 ttccagagcg acacctggag atggcagaaa tactgggagc cgacacccgc actgccagga 1200 tccaagatgc agggctggtg ttggcagaca cgctccggaa attcttattc gatctggatg 1260 ttgatgatgg cctagcagct gttggttact ccaaagctga tatccccgca ctagtgaaag 1320 gaacgctgcc ccaggaaagg gtcaccaagc ttgcaccccg tccccagtca gaagaggatc 1380 tggctgctct gtttgaagct tcaatgaaac tgtattaatt gtcattttaa ctgaaagaat 1440 taccgctggc cattgtagtg ctgagagcaa gagctgatct agctagggct ttgtcttttc 1500 atctttgtgc ataacttacc tgttaccagt ataggtggga tatacattta tcttgcagga 1560 aattccccaa agctcagagt ccagttcctt ccataaaaca ggctggacaa atgaccacta 1620 tgttagaccc ccaggctcga cttcaggggt cagtgttcct gtcccaaacc ccacacagaa 1680 tactctgcct ctgcttcatg tagcaaatga gcaaaaactc agtatctatc aaaagtgtaa 1740 attatatttc ctatgcctag taattcactt catgtctaaa aatttatctg atagaaacac 1800 tagcaccagt acatacagaa gcatggcaag gatgtttctg gcagcacttt tctaataata 1860 aaagatttga aacaaaaaaa aaaa 1884 <210> 47 <211> 1400 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4293484CB1 <400> 47 ctccaggcca gtctgggcac cctgggatag cggctgcagc tcccaggatc agctctgccc 60 tcccgccaaa cgccagcctc gtcaccgctc cagggcacct ccagcagtaa caggtggttg 120 cagcaggtgg cagccagccc ctggatgagc caaggtctct tccccagcca ggcatggccg 180 actctgcaca ggcccagaag ctggtgtacc tggtcacagg gggctgtggc ttcctgggag 240 agcacgtggt gcgaatgctg ctgcagcggg agccccggct cggggagctg cgggtctttg 300 accaacacct gggtccctgg ctggaggagc tgaagacagg tacccggaac gtgatcgagg 360 cttgtgtgca gaccggaaca cggttcctgg tctacaccag cagcatggaa gttgtggggc 420 ctaacaccaa aggtcacccc ttctacaggg gcaacgaaga caccccatac gaagcagtgc 480 acaggcaccc ctatccttgc agcaaggccc tggccgagtg gctggtcctg gaggccaacg 540 ggaggaaggt ccgtgggggg ctgcccctgg tgacgtgtgc ccttcgtccc acgggcatct 600 acggtgaagg ccaccagatc atgagggact tctaccgcca gggcctgcgc ctgggaggtt 660 ggctcttccg ggccatcccg gcctctgtgg agcatggccg ggtctatgtg ggcaatgttg 720 cctggatgca cgtgctggca gcccgggagc tggagcagcg ggcaaccctg atgggcggcc 780 aggtatactt ctgctacgat ggatcaccct acaggagcta cgaggatttc aacatggagt 840 tcctgggccc ctgcggactg cggctggtgg gcgcccgccc attgctgccc tactggctgc 900 tggtgttcct ggctgccctc aatgccctgc tgcagtggct gctgcggcca ctggtgctct 960 acgcacccct gctgaacccc tacacgctgg ccgtggccaa caccaccttc accgtcagca 1020 ccgacaaggc tcagcgccat ttcggctatg agcccctgtt ctcgtgggag gatagccgga 1080 cccgcaccat tctctgggta caggccgcta cgggttcagc ccagtgacgg tggggctggg 1140 gcctggaggc ccagatacag cacatccacc caggtcccga gccctcacac cctggacggg 1200 aagggacagc tgcattccag agcaggaggc agggctctgg ggccagaatg gctgtccttg 1260 tcgtagagcc ctccacattt tctttttctt ttttgagaca gggtcttgct ctgtcaccca 1320 gactggagtg cagtggtgtg atcatagctc actgcaccct caacctcctg ggttcaagca 1380 atcctcctgc ctcagcctcc 1400 <210> 48 <211> 1313 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4440080CB1 <400> 48 gaggctggct agggagccgt gggagtgagc tgctcacccc agtcagaccc ctgttgggtt 60 tcataggagc tggctgctga atgtaagagt gcaggctacc ccgggacttt gatcccctac 120 agatgtgacc tatcaaatga agaggacatc ctctccatgt tctcagctat ccgttctcag 180 cacagcggtg tagacatctg catcaacaat gctggcttgg cccggcctga caccctgctc 240 tcaggcagca ccagtggttg gaaggacatg ttcaatgtga acgtgctggc cctcagcatc 300 tgcacacggg aagcctacca gtccatgaag gagcggaatg tggacgatgg gcacatcatt 360 aacatcaata gcatgtctgg ccaccgagtg ttacccctgt ctgtgaccca cttctatagt 420 gccaccaagt atgccgtcac tgcgctgaca gagggactga ggcaagagct tcgggaggcc 480 cagacccaca tccgagccac gtgcatctct ccaggtgtgg tggagacaca attcgccttc 540 aaactccacg acaaggaccc tgagaaggca gctgccacct atgagcaaat gaagtgtctc 600 aaacccgagg atgtggccga ggctgttatc tacgtcctca gcacccccgc acacatccag 660 attggagaca tccagatgag gcccacggag caggtgacct agtgactgtg ggagctcctc 720 cttccctccc cacccttcat ggcttgcctc ctgcctctgg attttaggtg ttgatttctg 780 gatcacggga taccacttcc tgtccacacc ccgaccaggg gctagaaaat ttgtttgaga 840 tttttatatc atcttgtcaa attgcttcag ttgtaaatgt gaaaaatggg ctggggaaag 900 gaggtggtgt ccctaattgt tttacttgtt aacttgttct tgtgcccctg ggcacttggc 960 ctttgtctgc tctcagtgtc ttccctttga catgggaaag gagttgtggc caaaatcccc 1020 atcttcttgc acctcaacgt ctgtggctca gggctggggt ggcagaggga ggccttcacc 1080 ttatatctgt gttgttatcc agggctccag acttcctcct ctgcctgccc cactgcaccc 1140 tctccccctt atctatctcc ttctcggctc cccagcccag tcttggcttc ttgtcccctc 1200 ctggggtcat ccctccactc tgactctgac tatggcagca gaacaccagg gcctggccca 1260 gtggatttca tggtgatcat taaaaaagaa aaatcgcaac caaaaaaaaa aaa 1313' <210> 49 <211> 1459 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5495687CB1 <400> 49 gggccgcgcc cgcgctccca ggctctcctc ccccagcctt cctccggctg gcagcacgac 60 tcgcgtagcc gtgcgccgat tgcctctcgg cctgggcaat ggtcccggct gccggtcgac 120 gaccgccccg cgtcatgcgg ctcctcggct ggtggcaagt attgctgtgg gtgctgggac 180 ttcccgtccg cggcgtggag gttgcagagg aaagtggtcg cttatggtca gaggagcagc 240 ctgctcaccc tctccaggtg ggggctgtgt acctgggtga ggaggagctc ctgcatgacc 300 cgatgggcca ggacagggca gcagaagagg ccaatgcggt gctggggctg gacacccaag 360 gcgatcacat ggtgatgctg tctgtgattc ctggggaagc tgaggacaaa gtgagttcag 420 agcctagcgg cgtcacctgt ggtgctggag gagcggagga ctcaaggtgc aacgtccgag 480 agagcctttt ctctctggat ggcgctggag cacacttccc tgacagagaa gaggagtatt 540 acacagagcc agaagtggcg gaatctgacg cagccccgac agaggactcc aataacactg 600 aaagtctgaa atccccaaag gtgaactgtg aggagagaaa cattacagga ttagaaaatt 660 tcactctgaa aattttaaat atgtcacagg accttatgga ttttctgaac ccaaacggta 720 gtgactgtac tctagtcctg ttttacaccc cgtggtgccg cttttctgcc agtttggccc 780 ctcactttaa ctctctgccc cgggcatttc cagctcttca ctttttggca ctggatgcat 840 ctcagcacag cagcctttct accaggtttg gcaccgtagc tgttcctaat attttattat 900 ttcaaggagc taaaccaatg gccagattta atcatacaga tcgaacactg gaaacactga 960 aaatcttcat ttttaatcag acaggtatag aagccaagaa gaatgtggtg gtaactcaag 1020 ccgaccaaat aggccctctt cccagcactt tgataaaaag tgtggactgg ttgcttgtat 1080 tttccttatt ctttttaatt agttttatta tgtatgctac cattcgaact gagagtattc 1140 ggtggctaat tccaggacaa gagcaggaac atgtggagta gtgatggtct gaaagaagtt 1200 ggaaagagga acttcaatcc ttcgtttcag aaattagtgc tacagtttca tacattttct 1260 ccagtgacgt gttgacttga aacttcaggc agattaaaag aatcatttgt tgaacaactg 1320 aatgtataaa aaaattataa actggtgttt taactagtat tgcaataagc aaatgcaaaa 1380 atattcaata gatgcactat tcttgttttt actgcatgaa cgtaattcag tatttggaaa 1440 gtaatccagt ttgaaatgg ~ 1459 <210> 50 <211> 2101 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5527735CB1 <400> 50 cggctcgagg cggccgtcat ggcgtcgccc ttcagcgggg cgctgcagct gacggacctg 60 gatgacttca tcgggccgtc tcaggagtgc atcaagcctg tcaaagtgga aaaaagggcg 120 ggaagtggcg tggccaagat tcgcattgaa gatgacggga gctacttcca aattaaccaa 180 gacggcggga cccggaggct ggagaaggcc aaggtctcgc taaacgactg cctggcgtgc 240 agcggctgca tcacctccgc agagaccgtg cttatcaccc agcagagcca cgaggagctg 300 aagaaggttc tagatgctaa caagatggcg gcacccagtc agcagaggct ggttgtagtt 360 tcggtctcac cacagtctag agcatcgctg gctgcacggt ttcagctgaa tcctacagat 420 actgccagga aattaacctc attctttaaa aaaatagggg tgcacttcgt cttcgacacc 480 gccttctcaa ggcacttcag cctcctggag agccagcgag agtttgtgcg gcgattccga 540 ggacaggccg actgcagaca ggcactgccc ctgctggcct ctgcctgccc aggctggatc 600 tgctatgccg agaagactca cggcagcttc atcctccccc acatcagcac cgcccggtcc 660 ccgcagcagg tcatgggctc cctggtcaag gacttcttcg cccagcagca gcacttgacc 720 cctgacaaga tctaccacgt cacagtgatg ccctgctatg acaaaaagct ggaagcctcc 780 agacccgact ttttcaacca ggagcaccag acacgggatg tggactgtgt cctcacaaca 840 ggagaagttt tcaggttgct ggaggaagag ggcgtctccc tccccgacct ggaaccagcc 900 cctctggaca gcctgtgcag cggtgcctct gcagaggagc ccaccagcca tcggggaggg 960 ggctcggggg gctacctgga gcatgtgttc cggcacgcgg cccgagagct ctttggaatc 1020 catgtggctg aggttaccta caaacccctg aggaacaaag acttccagga ggtgacactg 1080.' gagaaggagg gccaggtgct gctgcacttc gcaatggcgt acggcttccg caacatccag 1140 aacctggtgc agaggctcaa acgagggcgc tgcccctacc actacgtgga ggtcatggcc 1200 tgcccctcag gctgcctgaa cggcgggggc cagctccagg ccccagacag gcccagcaga 1260 gagctcctcc agcacgtgga gagactgtac ggcatggtcc gggctgaggc gcccgaggac 1320 gcgcctgggg ttcaggagct gtacacacac tggctgcagg gcacggactc ggagtgtgca 1380 ggtcgcttgc tgcatacgca gtaccacgcc gtggagaagg ccagcactgg cctgggcatc 1440 cggtggtagg ggctgcagga ccaggactcc caggaggccg tgtccatgtg tgacagcaga 1500 accacatgcc ccaagacccc agggcttccc ccaaaattct gagtgagctg cagggtgtgc 1560 tgggacccga gtaggagcta ggactagcca ggacccgcag ccgcctcgtc acctccagtt 1620 gggtgcctct gggttcccac tggctctgcc caggtggggt ggggtggccc aggcagcaga 1680 aggttccctg aggtcccaga gcctgttccg ttggccctgg gccgaggccc acaggtgctg 1740 cccttgctgc tgctggtcgg gcacccaagt gcgtgagggg cttcagcctg tcccggggtt 1800 gcctgaggca gagcaagacg ggttctcacc cctgacttct ggaggcttcc cttgaagctc 1860 tgtgcaaaag gtgggagaca gagctggacc tgcaggggtg gtcccgccac aaccctgcgt 1920 gtggaccctg gcaggggggg ggtgccaggc ccctggaaag caggggttac cgttacgagg 1980 ctgtggtccg gggcaagcca agtacgaagc agcagccatc gcgggctgca tcatccccca 2040 gccaggtccc caccaggcct gtctcccagc gtttgtctaa taaacgcacc cctcgtaaaa 2100 g 2101 <210> 51 <211> 2440 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 5540437CB1 <400> 51 gtgtccctgc ggcgctaaga aggggagact gaggctgagg ctggggaaca tcgggcagca 60 tgagcggctg cgggctcttc ctgcgcacca cggctgcggc tcgtgcctgc cggggtctgg 120 tggtctctac cgcgaaccgg cggctactgc gcaccagccc gcctgtacga gctttcgcca 180 aagagctttt cctaggcaaa atcaagaaga aagaagtttt cccatttcca gaagttagcc 240 aagatgaact taatgaaatc aatcagttct tgggacccgt ggaaaaattc ttcactgaag 300 aggtggactc ccgaaaaatt gaccaggaag ggaaaatccc agatgaaact ttggagaaat 360 tgaagagcct agggcttttt gggctgcaag tcccagaaga atatggtggc ctgggcttct 420 ccaacaccat gtactcaaga ctaggggaga tcatcagcat ggatgggtcc atcactgtga 480 ccctggcagc gcaccaggct attggcctca aggggatcat cttggctggc actgaggagc 540 agaaagccaa atacttgcct aaactggcgt ccggggagca cattgcagcc ttctgcctca 600 cggagccagc cagtgggagc gatgcagcct caatccggag cagagccaca ctaagtgaag 660 acaagaagca ctacatcctc aatggctcca aggtctggat tactaatgga ggactggcca 720 atatttttac tgtgtttgca aagactgagg tcgttgattc tgatggatca gtgaaagaca 780 aaatcacagc attcatagta gaaagagact ttggtggagt cactaatggg aaacccgaag 840 ataaattagg cattcggggc tccaacactt gtgaagtcca ttttgaaaac accaagatac 900 ctgtggaaaa catccttgga gaggtcggag atgggtttaa ggtggccatg aacatcctca 960 acagcggccg gttcagcatg ggcagcgtcg tggctgggct gctcaagaga ttgattgaaa 1020 tgactgctga gtacgcctgc acaaggaaac agtttaacaa gaggctcagt gaatttggat 1080 tgattcagga gaaatttgca ctgatggctc agaaggctta cgtcatggag agtatgacct 1140 acctcacagc agggatgctg gaccaacctg gctttcccga ctgctccatc gaggcagcca 1200 tggtgaaggt gttcagctcc gaggccgcct ggcagtgtgt gagtgaggcg ctgcagatcc 1260 tcgggggctt gggctacaca agggactatc cgtacgagcg catactgcgt gacacccgca 1320 tcctcctcat cttcgaggga accaatgaga ttctccggat gtacatcgcc ctgacgggtc 1380 tgcagcatgc cggccgcatc ctgactacca ggatccatga gcttaaacag gccaaagtga 1440 gcacagtcat ggataccgtt ggccggaggc ttcgggactc cctgggccga actgtggacc 1500 tggggctgac aggcaaccat ggagttgtgc accctagtct tgcggacagt gccaacaagt 1560 ttgaggagaa cacctactgc ttcggccgga ccgtggagac actgctgctc cgctttggca 1620 agaccatcat ggaggagcag ctggtactga agcgggtggc caacatcctc atcaacctgt 1680 atggcatgac ggccgtgctg tcgcgggcca gccgctccat ccgcattggg ctccgcaacc 1740 acgaccacga ggttctcttg gccaacacct tctgcgtgga agcttacttg cagaatctct 1800 tcagcctctc tcagctggac aagtatgctc cagaaaacct agatgagcag attaagaaag 1860 tgtcccagca gatccttgag aagcgagcct atatctgtgc ccaccctctg gacaggacat 1920 gctgaggcag gggacagtgt cccctgctac cgcccgcccc tacccatggc ccgttgctgg 1980 atgactgtta ctcttttttc agaaggtgtt gggattatca caggttaagc cttttgttcc 2040 ccgtctgcac ctgaagggtt gtcgcctggc ctgggagagc ctcttccagg ttttgacctg 2100.
caggcagtgc tctctaacag gaccatcaca gcttctgaac tgagccggag agagagaatg 2160' gaattgctga cccctggaac tggcgggtat tctggtcatt gaggagacac catagtggaa 2220 actggggctt atgctgctgc ctccagggtg tgaggtgggt ggggacctgt gtcaggtgtg 2280 gatagccatt tctgctcaac cacacattct ctaagaaaca gcttgaaagc tctgtctggg 2340 tcattcattt aaactagaag cagaggcact taaaacatgt accaggaacc atttaacaaa 2400 gaatataaaa tgtcacaatc tgtgtactgt taaaaaaaaa 2440 <210> 52 <211> 1072 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5596281CB1 <400> 52 cggacggtgg gtccagacaa aggttgtctc aagtttgttg ctcaaaccga gttctggaga 60 acgccatcag ctcgctgctt aaaattaaac cacaggttcc attatgggtc gacttgatgg 120 gaaagtcatc atcctgacgg ccgctgctca ggggattggc caagcagctg ccttagcttt 180 tgcaagagaa ggtgccaaag tcatagccac agacattaat gagtccaaac ttcaggaact 240 ggaaaagtac ccgggtattc aaactcgtgt ccttgatgtc acaaagaaga aacaaattga 300 tcagtttgcc agtgaagttg agagacttga tgttctcttt aatgttgctg gttttgtcca 360 tcatggaact gtcctggatt gtgaggagaa agactgggac ttctcgatga atctcaatgt 420 gcgcagcatg tacctgatga tcaaggcatt ccttcctaaa atgcttgctc agaaatctgg 480 caatattatc aacatgtctt ctgtggcttc cagcgtcaaa ggagttgtga acagatgtgt 540 gtacagcaca accaaggcag ccgtgattgg cctcacaaaa tctgtggctg cagatttcat 600 ccagcagggc atcaggtgca actgtgtgtg cccaggaaca gttgatacgc catctctaca 660 agaaagaata caagccagag gaaatcctga agaggcacgg aatgatttcc tgaagagaca 720 aaagacggga agattcgcaa ctgcagaaga aatagccatg ctctgcgtgt atttggcttc 780 tgatgaatct gcttatgtaa ctggtaaccc tgtcatcatt gatggaggct ggagcttgtg 840 attttaggat ctccatggtg ggaaggaagg caggcccttc ctatccacag tgaacctggt 900 tacgaagaaa actcaccaat catctccttc ctgttaatca catgttaatg aaaataagct 960 ctttttaatg atgtcactgt ttgcaagagt ctgattcttt aagtatatta atctctttgt 1020 aatctcttct gaaatcattg taaagaaata aaaatattga actcaaaaaa as 1072 <210> 53 <211> 1040 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5731013CB1 <400> 53 ccttcggctc gagcggcaac cggaagcctc gaggtttagt cccgcccccc tctcctcgct 60 gcttaggctc cgcggcctcc aagctgtagc tatgacggcg cgcgggactc cgagccgctt 120 cttggccagc gttctccaca acggactggg tcgctatgtg cagcagctgc agcgtctgag 180 cttcagcgtc agccgcgacg gcgcctcgtc tcgcggcgcc agggagttcg tggagcggga 240 ggtgatcgac ttcgcccgac ggaatccagg ggtcgtaata tatgtaaact cgcgtccgtg 300 ctgcgtgccc agagtagtgg ccgaatacct taacggggct gtgcgcgagg agagcatcca 360 ctgcaagtcg gtcgaggaga tctcgacgct ggtgcagaag ctggccgacc agtcgggctt 420 ggacgtgatc cgcatccgca agcccttcca caccgacaac cctagcatcc agggccagtg 480 gcaccccttc accaacaagc cgaccacgtt ccgcgggcta cgcccccgag aggttcagga 540 tcctgcccca gcccaggtgc aagcacagtg aagagttgcc ccaccaactg cagccccagg 600 ctttggactg ttactccggt aaaggtggtt cttccccttt gggattccaa gcccaggcaa 660 atggaaccca tcaatgggca agttgacaga ggttctgctt gggataatga agagctgcct 720 gtttctttcc agtgcctgct tctgggggca gtgaccttgt gaaccactca tttttatgca 780 agtggcatcc ctaaaacctg agatgaggaa gacttcaagg gttttacagg acccttgttt 840 tttaaatcca aattgataat aatgatctca aaacacagtg agaggtctga aggctggctt 900 ctgaagaatc cctgatgtct tattggaaca accactgagc tacggagagc tctgctgtga 960 tgggctaggc actttatatc tgtgtgaata cagatttata aaacaggtta ataaacttat 1020 ccaaggtcaa aaaaaaaaaa 1040 <210> 54 <211> 1624 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5731162CB1 <400> 54 ctcagcctcg cgagcagctg ggactacagg tgcccgccac cacgcccggc taattttttg 60 tatttttagt agagacgggg tttcaccgtg ttagccagga tggtctcgat ttcctgacct 120 cgtgatccgc gcgcctctgc ctccaaagtg ctgggattac aggcccgagc cgccgcaccc 180 ggcctgcagg tgttgttctg accccagctc cactgtgcca gcccgacttg agatgccaag 240 tatcttgggc ctgaggattg aggcctagag agaggcaggc gttcctcaga gtcacaggga 300 atggcggcgc ctggactggg actcagccca gctgcttggc ctgaccctct cacagcataa 360 tttcccggca cctggaggcc gcgcttagaa gccgcccagt gccctgagcg tctccatggc 420 ctgcctgagc ccctcgcagc tccagaagtt ccaacaggat ggattcctgg tgctggaagg 480 attcttgtct gcggaagagt gtgtggccat gcaacaaagg attggcgaga tagtggctga 540 aatggatgtt cctctccact gccgcacaga attctccacc caggaagagg agcagcttcg 600 agcccagggc agcacagact atttcttgag cagtggtgac aagattcgat tcttctttga 660 gaaaggcgtt tttgatgaga aaggaaattt cctggtccct ccggagaaat ccatcaacaa 720 aattggccac gctctgcacg cccacgaccc cgtcttcaag agcatcacac actccttcaa 780 ggtgcagacc ttggccagaa gtctgggcct ccagatgccc gtggtggtgc agagcatgta 840 catctttaag caacctcact ttggcggtga agtctcccct catcaggacg cctccttcct 900 gtacacggag cccctgggcc gggtgctggg cgtgtggatc gcagtggagg atgccacgct 960 ggagaacggc tgtctctggt tcatccctgg ctcccacacc agtggtgtgt caagaaggat 1020 ggtccgggcc cctgttggct cagcgcctgg taccagcttc cttgggtcag agccagcccg 1080 ggataacagc ctctttgtgc ccaccccagt gcagagaggg gccctggtcc tcatccatgg 1140 agaagtggta cacaagagca agcagaacct ctctgaccgc tcgcgccagg cctacacttt 1200 ccacctcatg gaggcctctg gcaccacctg gagcccggag aactggctcc agccaacagc 1260 tgaactgccc tttccccaac tgtacaccta aaggctctcg cagggcagga gccctcgccc 1320 ctcccgggtg aagctgtggg ctgtaaacac cagtgccttg ctcagcctcc tggttgcaac 1380 agggaggtct tgtctcccct cctgggcttt cctcctgccc tgtgggcagc agcctaggct 1440 gggtcagggg cttccctaag atcttcacct ctctgcctcc ctactgcccc aacatagcct 1500 tgaggaggct tctcagccac caaagggttc tggccccttc tcactctcct ctcctctcag 1560 atggaactct ggttattatg gtgttagtta tcgaataaaa acgacttcag aatgaaaaaa 1620 aaaa 1624

Claims (28)

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-27.

2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO:1-27.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 selected from the group consisting of SEQ ID
NO:28-54.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method for producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. An isolated antibody which specifically binds to a polypeptide of claim 1.

11. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
a) a polynucleotide sequence selected from the group consisting of SEQ ID
NO:28-54, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:28-54, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).

12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 11.

13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising:
a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.

15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising:
a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

16. A composition comprising an effective amount of a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

17. A composition of claim 16, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-27.

18. A method for treating a disease or condition associated with decreased expression of functional ORP, comprising administering to a patient in need of such treatment the composition of claim 16.

19. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

20. A composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.

21. A method for treating a disease or condition associated with decreased expression of functional ORP, comprising administering to a patient in need of such treatment a composition of claim 20.

22. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

23. A composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.

24. A method for treating a disease or condition associated with overexpression of functional ORP, comprising administering to a patient in need of such treatment a composition of claim 23.

25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of:

a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

26. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

27. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

28. A method for assessing toxicity of a test compound, said method comprising:
a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 11 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 11 or fragment thereof;
c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.