BACKGROUND OF THE INVENTION Metalloproteins, including metallothioneins, ferritins, globins, and selenium-binding proteins, function in many aspects of metabolism, ranging from detoxification to storage. Mutation of the genes encoding metal-binding proteins can lead to life-threatening disorders, and, where examined, expression of these genes is often tightly regulated in the cell (Aisen, P. et al. (2001) Int. J. Bioch. Cell Bio. 33: 940-959).

Metallothioneins (MTs) are a group of small (61 amino acids), cysteine rich proteins that bind heavy metals such as cadmium, zinc, mercury, lead, and copper and are thought to play a role in metal detoxification or the metabolism and homeostasis of metals. They are present in a wide variety of eukaryotes including invertebrates, vertebrates, plants, and fungi. The primary structure of the vertebrate MTs is strongly conserved between species, particularly the positions of the cysteine residues which serve to chelate heavy metal ions via thiolate complexes.

At least two closely related but chromatographically distinct isoforms of MT, MT-I and MT-II, have been observed in every vertebrate species examined. All isoforms of MT sequenced thus far share 20 cysteine residues found in the same positions and lack aromatic amino acids or leucine (Schmidt, C. J. and D. H. Hamer (1983) Gene 24: 137-146; Schmidt, C. J. et al. (1985) J. Biol Chem.

260: 7731-7737). MT-II isoforms differ from MT-I only in the presence of an aspartate residue at position 10 or 11 while MT-I contains either glycine or valine at these positions. Recently, a third class of MT, MT-0, has been discovered in human liver that is characterized by a negatively charged amino acid at position 8 (glutamate) and a lysine substitution at the highly conserved Glu23 (Soumillion, A. et al. (1992) Eur. J. Biochem. 209: 999-1004) In humans, only a single sequence of MT-0 and Mut-n have been found, while at least six isoforms of MT-I have been identified. These various isoforms of

MT-I differ only by a few residues distributed throughout the molecule. The localization and identification of 12 functional MT genes on human chromosome 16 indicates that several additional MT isoforms exist.

The reason for multiple human MT genes is unclear. It is possible that different isoforms are responsible for binding different metals, or that different control sequences for the transcription of these various isoforms respond to different stimuli, such as heavy metals, glucocorticoids, or enterotoxin (Schmidt, et al. supra). A third possibility is that multiple isoforms could play different roles in metal homeostasis during development or in different tissues.

Acute or chronic exposure to heavy metals such as lead, arsenic, mercury or cadmium leads to a variety of diseases and disorders involving neuromuscular, CNS, cardiovascular, and gastrointestinal effects. MTs may play a role in the prevention or alleviation of these conditions. In addition, MTs are transcriptionally regulated by glucocorticoids which suggests that MTs have a direct role in the effects of glucocorticoids to treat inflammatory disease, immune disorders, and cancer.

Metal ions such as iron, zinc, copper, cobalt, manganese, molybdenum, selenium, nickel, and chromium are important as cofactors for a number of enzymes. For example, copper is involved in hemoglobin synthesis, connective tissue metabolism, and bone development, by acting as a cofactor in oxidoreductases such as superoxide dismutase, ferroxidase (ceruloplasmin), and lysyl oxidase. Copper and other metal ions must be provided in the diet, and are absorbed by transporters in the gastrointestinal tract. Plasma proteins transport the metal ions to the liver and other target organs, where specific transporters move the ions into cells and cellular organelles as needed. Imbalances in metal ion metabolism have been associated with a number of disease states (Danks, D. M. (1986) J.

Med. Genet. 23: 99-106).

Iron plays an essential role in oxygen transport and redox reactions, particularly cell respiration; however, iron is also toxic when present in excess. In humans, unregulated iron absorption leads to cirrhosis, endocrine failure, arthritis and cardiomyopathy, as well as hepatocellular carcinoma (Griffiths, W. J. H. et al. (1999) Mol. Med. Today 5: 431-438). Iron-overloading is also the cause of hereditary hemochromatosis, a disorder that leads to death if left untreated. Iron metabolism requires functioning iron-transporter proteins, such as transferrins, as well as iron storage proteins, such as ferritin.

Transferrins are eukaryotic iron-binding glycoproteins that control the level of free iron in biological fluids (Crichton, R. R. and M. Charloteaux-Wauters (1987) Eur. J. Biochem. 164: 485-506).

Transferrin family members are proteins of 650 to 700 residues which have evolved from the duplication of a domain of around 340 residues. Each of the duplicated domains binds one atom of

iron. Each iron atom is bound by four conserved residues: an aspartic acid, two tyrosines, and a histidine (Anderson, B. F. et al. (1989) J. Mol. Biol. 209: 711-734). The importance of the function of the transferrins in maintaining homeostasis of the mechanisms regulating iron absorption and transport is appropriately illustrated in conditions which result from iron overload. Briefly described, iron overload occurs when transferrin fails to bind all of the nonheme iron in the circulation. In this state, the unbound circulating iron may accumulate in specific organs, including the liver, heart, pancreas and gonads. Excess iron in these tissues catalyzes oxidative damage, resulting in cirrhosis, hepatoma, cardiomyopathy, diabetes, hypogonadism and arthritis (Aisen, (2001), supra). Atransferrinemia (also called hypotransferrinemia) is a condition caused by a genetic mutation that causes severe deficiency in plasma transferrin, resulting in massive deposition of iron in non-hematopoietic tissues. The consequence of this condition is severe iron deficiency anemia leading to death from liver failure (for a review of genetics of iron storage, see Beutler, E. (2001) Drug Metab. Dispos. 29: 495-499). Aberrant expression of transferrin which results in the disruption of iron homeostasis is associated with neurodegenerative disorders such as Alzheimer's disease and Restless legs syndrome (Thompson, K. J. et al. (2001) Brain Res. Bull. 55: 155-164).

Ferritin is a ubiquitous iron-binding protein that is involved in iron storage and detoxification in microbes, plants, and animals. Mammalian ferritin consists of 24 subunits of two types, H (for heart, or heavy) and L (for light or liver). These subunits assemble into a spherical structure which can accommodate up to 4,000 iron atoms as ferrihydrite, FeOOH (Aisen, P. et al. (1999) Curr. Opin.

Chem. Biol. 3: 200-206). Ferritin expression is regulated at the post-transcriptional level in response to intracellular iron levels. At low intracellular iron concentrations, iron regulatory proteins (IRPs) bind to iron responsive elements of ferritin mRNA, preventing translation of the protein. At high intracellular iron concentrations, when ferritin is needed to sequester iron in order to prevent oxidative damage, IRPs are prevented from binding to the iron responsive elements (Aisen, (2001), supra).

Globins are heme-binding proteins found in all living systems, from bacteria to plants to animals.

These proteins carry out crucial oxygen transport and storage functions. Hemoglobin and myoglobin, globins that transport and store oxygen respectively, are composed of a protein moiety, the globin part, and a heme group, a complex of iron with a porphyrin ring. It is the iron ion, in the 2+ oxidation state, that allows for the reversible binding of oxygen to the heme. Mutation of globin genes gives rise to serious, and often lethal, disorders such as sickle-cell anemia, which is characterized by chronic hemolytic anemia, severe pain and tissue damage that can lead to stroke, kidney failure, heart disease, infection and other complications (Bunn, H. F. and Forget, B. G. (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects (Saunders, Philadelphia) ). Mutations that affect gene expression levels

are equally serious, and give rise to thalassemias.

The importance of oxygen supply in higher organisms is reflected in the specialization and regulation of the globins. Myoglobin, a 17.8 KDa monomeric protein, is found in red muscle and is used to store oxygen. Hemoglobin, the carrier protein that delivers oxygen from the lungs to respiring tissues, is a heterotetramer composed of pairs of globin subunits. The four subunits work together leading to cooperative binding of oxygen, conferring on hemoglobin its transport capacity. Hemoglobin is highly expressed in red blood cells, yielding a concentration of 15 g per 100 ml of normal blood (Hardison, R. C. (1996) 93: 5675-5679).

The individual globin polypeptides, or globin chains, in hemoglobin are developmentally regulated. a-and (3-globin make up adult human hemoglobin, are about 50% identical and arose through duplication and mutation of an ancestral globin gene. The (3-globin gene subsequently underwent further duplication events to give rise to e-globin, 8-globin and y-globin genes. Embryonic hemoglobin is composed of a and E-globin, fetal hemoglobin is composed of a-and y-globin, and ß- globin is expressed starting shortly before birth and combines with a-globin to form the adult form of hemoglobin. e-and y-globin are adapted to the needs of the fetus: they have higher affinities for oxygen, facilitating the transfer of oxygen from the mother to the fetus. 8-globin evolved in and is found only in adult primate hemoglobin, at very low levels.

Hemoglobin production is also tightly coordinated to ensure proper oxygen transport and delivery. Globin gene expression is carefully regulated to avoid producing excess unmatched globin chains, which can affect the material properties of red blood cell membranes and the state of red blood cellhydration, inducing thalassemia (Schrier, S. L. (1994) Annu. Rev. Med. 45: 211-218). Thus, in humans, the (3-and ß-like globins on chromosome 11 are coordinately regulated along with the (X- globin genes located on chromosome 16. This regulation involves sequential activation and silencing of globin genes in erythroid cells, and is carried out by multiple cis-and trans-acting factors (Kollia, P. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 5693-5698).

Selenium is a micronutrient that has been implicated in cancer prevention by reducing the number of DNA adducts by carcinogens (Harrison, P. R. et al. (1997) Biomed. Environ. Sci. 10: 235- 245). In mammals, selenium-containing proteins can be divided into three classes: proteins that incorporate selenium in a non-specific manner, selenocysteine-containing selenoproteins, and specific selenium-binding proteins, such as glutathione peroxidase (Behne, D. and A. Kyriakopoulos (2001) Annu. Rev. Nutr. 21: 453-473). In the genetic code, UGA serves as either a signal for termination or as a codon for selenocysteine (Sec). Sec differs from other amino acids in much of the biosynthetic machinery governing its incorporation into protein. Sec-containing proteins have diverse functions.

The more recently evolved selenoproteins appear to take advantage of unique redox properties of Sec that are superior to those of Cys for specific biological functions (Gladyshev V. N. and Kryukov G. V.

(2001) Biofactors 14: 87-92). Selenium-binding proteins are thought to mediate the inhibitory effects of selenium on growth in mammalian cells and tumorigenesis in rodents. Several selenium-binding proteins have been identified in mouse, rat and human, and are expressed in tissues involved in detoxification, such as the liver (Bansal, M. P. et al. (1989) Carcinogenesis 10: 541-546; Bansal, M. P. et al. (1989) J. Biol. Chem. 264: 13780-13784). In mouse and rat, selenium-binding proteins were also found in blood, duodenum, kidney, pancreas testis, ovary and in mammary tumors (Morrison, D. G. et al. (1989) In Vivo 3: 167-172).

In humans, a 56 kDa selenium-binding protein is expressed in liver, kidney and lung tissue.

Using a cell-free transport assay, this protein has been shown to participate in late stages of intra- Golgi protein transport (Porat, A. et al. (2000) J. Biol. Chem. 275: 14457-14465). A 56 kDa protein that differs from the 56 kDa selenium-binding protein by only 14 residues has been found to associate with an acetamenophen metabolite correlated with hepatotoxicity (Lanfear, J. et al. (1993) 14: 335-340; Pumford, N. R. et al. (1992) Biochem. Biophys. Res. Commun. 182: 1348-55). Both the selenium- binding and the acetamenophen-binding proteins may mediate detoxification mechanisms and anti- carcinogenic functions of selenium (Lanfear et al., supra).

Expression profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.

One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants.

When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.

For example, both the levels and sequences expressed in tissues from subjects with obesity or type II diabetes may be compared with the levels and sequences expressed in normal tissue. The

primary function of adipose tissue is the ability to store and release fat during periods of feeding and fasting. White adipose tissue is the major energy reserve in periods of fasting, and its reserve is mobilized during energy deprivation. Adipose tissue is one of the primary target tissues for insulin, and adipogenesis and insulin resistance are linked in type II diabetes, non-insulin dependent diabetes mellitus (NIDDM). Cytologically the conversion of a preadipocytes into mature adipocytes is characterized by deposition of fat droplets around the nuclei. The conversion process in vivo can be induced by thiazolidinediones and other PPARY agonists (Adams et al. (1997) J Clin Invest 100: 3149- 3153) which also lead to increased sensitivity to insulin and reduced plasma glucose and blood pressure.

Preadipocyte cell lines are used to study adipose cells. The most important function of adipose tissue is its ability to store and release fat during periods of feeding and fasting. White adipose tissue is the major energy reserve in periods of excess energy use. Its primary purpose is mobilization during energy deprivation. Understanding how various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity. Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked and present intriguing relations. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance. The majority of research in adipocyte biology to date has been done using transformed mouse preadipocyte cell lines. The culture condition which stimulates mouse preadipocyte differentiation is different from that for inducing human primary preadipocyte differentiation. In addition, primary cells are diploid and may therefore reflect the in vivo context better than aneuploid cell lines. Understanding the gene expression profile during adipogenesis in humans will lead to understanding the fundamental mechanism of adiposity regulation.

Furthermore, through comparing the gene expression profiles of adipogenesis between donor with normal weight and donor with obesity, identification of crucial genes, potential drug targets for obesity and type It diabetes, will be possible.

Pickup and Crook (1998; Diabetologia 41: 1241-8) have suggested that NIDDM may result from the inability of an individual with hypersensitive acute-phase immune response to carry out normal cell signaling and repair. Steps in this process are highly correlated with long-term lifestyle and environment and include: 1) high glucose stimulation of insulin and cytokine production, 2) influence of various cytokines on tissue remodeling during adipocyte differentiation and their affect on signaling pathways, and 3) occurrence of tissue damage when cytokines continue to be produced, extracellular matrix components (ECM) are not recycled, and homeostasis is not timely restored. Understanding how human adipocytes differentiate and how they contribute to the regulation of energy balance in

physiological and pathophysiological situations may lead to development of novel therapeutics to treat obesity and type 1 : 1 diabetes.

The potential application of gene expression profiling is also relevant to improving diagnosis, prognosis, and treatment of cancer, such as breast, colon, ovarian and lung cancer. Breast cancer is the most frequently diagnosed type of cancer in American women and the second most frequent cause of cancer death. The lifetime risk of an American woman developing breast cancer is 1 in 8, and one-third of women diagnosed with breast cancer die of the disease. A number of risk factors have been identified, including hormonal and genetic factors. One genetic defect associated with breast cancer results in a loss of heterozygosity (LOH) at multiple loci such as p53, Rb, BRCA1, and BRCA2. Another genetic defect is gene amplification involving genes such as c-myc and c-erbB2 (Her2-neu gene). Steroid and growth factor pathways are also altered in breast cancer, notably the estrogen, progesterone, and epidermal growth factor (EGF) pathways. Breast cancer evolves through a multi-step process whereby premalignant mammary epithelial cells undergo a relatively defined sequence of events leading to tumor formation. An early event in tumor development is ductal hyperplasia. Cells undergoing rapid neoplastic growth gradually progress to invasive carcinoma and become metastatic to the lung, bone, and potentially other organs. Variables that may influence the process of tumor progression and malignant transformation include genetic factors, environmental factors, growth factors, and hormones.

Colorectal cancer is the fourth most common cancer and the second most common cause of cancer death in the United States with approximately 130,000 new cases and 55,000 deaths per year.

Colon and rectal cancers share many environmental risk factors and both are found in individuals with specific genetic syndromes. (See Potter (1999) J Natl Cancer Institute 91: 916-932 for a review of colorectal cancer. ) Colon cancer is the only cancer that occurs with approximately equal frequency in men and women, and the five-year survival rate following diagnosis of colon cancer is around 55% in the United States (Ries et al. (1990) National Institutes of Health, DHHS Publ No. (NIH) 90-2789).

Colon cancer is causally related to both genes and the environment. Several molecular pathways have been linked to the development of colon cancer, and the expression of key genes in any of these pathways may be lost by inherited or acquired mutation or by hypermethylation. There is a particular need to identify genes for which changes in expression may provide an early indicator of colon cancer or a predisposition for the development of colon cancer.

There are a number of genetic alterations associated with colon cancer and with the development and progression of the disease, particularly the downregulation or deletion of genes, that potentially provide early indicators of cancer development, and which may also be used to monitor

disease progression or provide possible therapeutic targets. The specific genes affected in a given case of colon cancer depend on the molecular progression of the disease. Identification of additional genes associated with colon cancer and the precancerous state would provide more reliable diagnostic patterns associated with the development and progression of the disease.

Ovarian cancer is the leading cause of death from a gynecologic cancer. The majority of ovarian cancers are derived from epithelial cells, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result, the long-term survival rates for this disease is very low.

Identification of early-stage markers for ovarian cancer would significantly increase the survival rate.

Genetic variations involved in ovarian cancer development include mutation of p53 and microsatellite instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors.

Lung cancer is the leading cause of cancer death in the United States, affecting more than 100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains thousands of noxious substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in the exposed bronchial epithelium. In nearly 80% of patients diagnosed with lung cancer, metastasis has already occurred.

Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and liver. The decision to treat with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to identification, staging, and treatment of lung cancer could positively affect patient outcome.

Lung cancers progress through a series of morphologically distinct stages from hyperplasia to invasive carcinoma. Malignant lung cancers are divided into two groups comprising four histopathological classes. The Non Small Cell Lung Carcinoma (NSCLC) group includes squamous cell carcinomas, adenocarcinomas, and large cell carcinomas and accounts for about 70% of all lung cancer cases. Adenocarcinomas typically arise in the peripheral airways and often form mucin secreting glands. Squamous cell carcinomas typically arise in proximal airways. The histogenesis of squamous cell carcinomas may be related to chronic inflammation and injury to the bronchial epithelium, leading to squamous metaplasia. The Small Cell Lung Carcinoma (SCLC) group accounts for about 20% of lung cancer cases. SCLCs typically arise in proximal airways and exhibit a number of paraneoplastic syndromes including inappropriate production of adrenocorticotropin and anti-diuretic hormone.

Osteosarcoma is the most common malignant bone tumor in children. Approximately 80% of

patients present with non-metastatic disease. After the diagnosis is made by an initial biopsy, treatment involves the use of 3-4 courses of neoadjuvant chemotherapy before definitive surgery, followed by post-operative chemotherapy. With currently available treatment regimens, approximately 30-40% of patients with non-metastatic disease relapse after therapy. Currently, there is no prognostic factor that can be used at the time of initial diagnosis to predict which patients will have a high risk of relapse. The only significant prognostic factor predicting the outcome in a patient with non-metastatic osteosarcoma is the histopathologic response of the primary tumor resected at the time of definitive surgery. The degree of necrosis in the primary tumor is a reflection of the tumor response to neoadjuvant chemotherapy. A higher degree of necrosis (good or favorable response) is associated with a lower risk of relapse and a better outcome. Patients with a lower degree of necrosis (poor or unfavorable response) have a much higher risk of relapse and poor outcome even after complete resection of the primary tumor. Unfortunately, poor outcome cannot be altered despite modification of post-operative chemotherapy to account for the resistance of the primary tumor to neoadjuvant chemotherapy. Thus, there is an urgent need to identify prognostic factors that can be used at the time of diagnosis to recognize the subtypes of osteosarcomas that have various risks of relapse, so that more appropriate chemotherapy can be used at the outset to improve the outcome.

Prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells. Androgen responsive cells become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent cells evolve from the hyperplastic population.

These cells represent a more advanced form of prostate tumor that may become invasive and potentially become metastatic to the bone, brain, or lung. A variety of genes may be differentially expressed during tumor progression. For example, loss of heterozygosity (LOH) is frequently observed on chromosome 8p in prostate cancer. Fluorescence in situ hybridization (FISH) revealed a deletion for at least 1 locus on 8p in 29 (69%) tumors, with a significantly higher frequency of the deletion on 8p21. 2-p21. 1 in advanced prostate cancer than in localized prostate cancer, implying that deletions on 8p22-p21. 3 play an important role in tumor differentiation, while 8p21. 2-p21. 1 deletion plays a role in progression of prostate cancer (Oba, K. et al. (2001) Cancer Genet. Cytogenet. 124: 20-26).

Tangier disease (TD) is a genetic disorder characterized by near absence of circulating high density lipoprotein (HDL) and the accumulation of cholesterol esters in many tissues, including tonsils, lymph nodes, liver, spleen, thymus, and intestine. Low levels of HDL represent a clear predictor of premature coronary artery disease and homozygous TD correlates with a four-to six-fold increase in

cardiovascular disease compared to controls. HDL plays a cardio-protective role in reverse cholesterol transport, the flux of cholesterol from peripheral cells such as tissue macrophages through plasma lipoproteins to the liver. The HDL protein, apolipoprotein A-1, plays a major role in this process, interacting with the cell surface to remove excess cholesterol and phospholipids. This pathway is severely impaired in TD and the defect lies in a specific gene, the ABC1 transporter. This gene is a member of the family of ATP-binding cassette transporters, which utilize ATP hydrolysis to transport a variety of substrates across membranes.

Cell lines Human umbilical vein endothelial cells (HUVECs) are a primary cell line derived from the endothelium of the human umbilical vein. Activation of vascular endothelium is a central event in a wide range of physiological and disease processes such as vascular tone regulation, coagulation and thrombosis, atherosclerosis, inflammation and some infectious diseases. Blood vessel walls are composed of two tissue layers: an endothelial cell (EC) layer which comprises the lumenal surface of the vessel, and an underlying vascular smooth muscle cell (VSMC) layer. Through dynamic interactions with each other and with surrounding tissues, the vascular endothelium and smooth muscle tissues maintain vascular tone, control selective permeability of the vascular wall, direct vessel remodeling and angiogenesis, and modulate inflammatory and immune responses. The inflammatory response is a complex vascular reaction mediated by numerous cytokines, chemokines, growth factors, and other signaling molecules expressed by activated ECs, VSMCs and leukocytes. Inflammation protects the organism during trauma and infection, but can also lead to pathological conditions such as atherosclerosis.

Human peripheral blood mononuclear cells (PBMCs) represent the major cellular components of the immune system. PBMCs contain about 12% B lymphocytes, 25% CD4+ and 15% CD8+ lymphocytes, 20% NK cells, 25% monocytes, and 3% various cells that include dendritic cells and progenitor cells. The proportions, as well as the biology of these cellular components tend to vary slightly between healthy individuals, depending on factors such as age, gender, past medical history, and genetic background.

The human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells. C3A cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin- like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with a- fetoprotein iii) conversion of ammonia to urea and glutamine; iv) metabolism of aromatic amino

acids; and v) proliferation in glucose-free and insulin-free medium. The C3A cell line is now well established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22: 866- 875; Nagendra et al. (1997) Am J Physiol 272: G408-G416).

Primary prostate epithelial cells (PrEC) is a line derived from primary prostate epithelial cells isolated from a normal donor Drug treatment TNF-a, also called cachectin, is produced by neutrophils, activated lymphocytes, macrophages, NK cells, LAK cells, astrocytes, endothelial cells, smooth muscle cells, and some transformed cells. TNF-a occurs as a secreted, soluble form and as a membrane-anchored form, both of which are biologically active. Two types of receptors for TNF-a have been described and virtually all cell types studied show the presence of one or both of these receptor types. TNF-a and TNF-ß are extremely pleiotropic fac-tors due to the ubiquity of their receptors, to their ability to activate multiple signal transduction path-ways and to their ability to induce or suppress the expression of a wide number of genes. TNF-a and TNF-ß play a critical role in mediation of the inflammatory response and in mediation of resistance to infections and tumor growth. It is likely that the serum TNF-a concentration can serve as a biomarker for staging the invasiveness of breast cancer.

Clinically, TNF-a treatment in combination with Interferon-gamma (IFN-y) may provide a successful approach to overcome the cellular heterogeneity of advanced breast tumors. In in vitro studies, TNF- a has been demonstrated to be antitumorigenic in MCF-7 cells by inducing apoptosis and inhibiting proliferation.

The growth factors Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGFa) play a critical role in tumor development, growth, and progression. They are important in the growth of normal as well as hyperproliferative prostate epithelial cells, particularly at early stages of tumor development and progression, and affect signaling pathways in these cells in various ways (Lin J et al. (1999) Cancer Res. 59: 2891-2897; Putz T et al.

(1999) Cancer Res 59: 227-233). The relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied.

(See Khazaie, K. et al. (1993) Cancer and Metastasis Rev. 12: 255-274, and references cited therein for a review of this area. ) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility,

chemotaxis, secretion and differentiation.

Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema. Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone.

Gemfibrozil is a fibric acid antilipemic agent that lowers serum triglycerides and produces favorable changes in lipoproteins. Gemfibrozil is effective in reducing the risk of coronary heart disease in men (Frick, M. H. , et al. (1987) New Engl. J. Med; 317: 1237-1245). The compound can inhibit peripheral lipolysis and decrease hepatic extraction of free fatty acids, which, decreases hepatic triglyceride production. Gemfibrozil also inhibits the synthesis and increases the clearance of apolipoprotein B, a carrier molecule for VLDL. Gemfibrozil has variable effects on LDL cholesterol.

Although it causes moderate reductions in patients with type IIa hyperlipoproteinemia, changes in patients with either type Db or type IV hyperlipoproteinemia are unpredictable. In general, the HMG- CoA reductase inhibitors are more effective than gemfibrozil in reducing LDL cholesterol. At the molecular level gemfibozil may function as a peroxisome proliferator-activated receptor (PPAR) agonist. Gemfibrozil is rapidly and completely absorbed from the GI tract and undergoes enterohepatic recirculation. Gemfibrozil is metabolized by the liver and excreted by the kidneys, mainly as metabolites, one of which possesses pharmacologic activity. Gemfibozil causes peroxisome proliferation and hepatocarcinogenesis in rats, which is a cause for concern generally for fibric acid derivative drugs. In humans, fibric acid derivatives are known to increase the risk of gall bladder disease although gemfibrozil is better tolerated than other fibrates. The relative safety of gemfibrozil in humans compared to rodent species including rats may be attributed to differences in metabolism and clearance of the compound in different species (Dix, K. J. , et al. , (1999) Drug Metab. Distrib. 27 (1) 138-146 ; Thomas, B. F. , et al. , (1999) Drug Metab. Distrib. 27 (1) 147-157).

There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of blood disorders, heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflammatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders.

SUMMARY OF THE INVENTION Various embodiments of the invention provide purified polypeptides, metalloproteins, referred

to collectively as'MEPR'and individually as'MEPR-1,''MEPR-2,''MEPR-3,''MEPR-4,''MEPR- 5,''MEPR-6,''MEPR-7,'and'MEPR-8'and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions.

Embodiments also provide methods for utilizing the purified metalloproteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified metalloproteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.

An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO : 1-8.

Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO : 1-8. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO : 9-16.

Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8.

Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.

Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8.

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.

Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0 : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0 : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0 : 1-8.

Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0 : 9-16, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0 : 9-16, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a) -d). In other embodiments, the polynucleotide can comprise at least about 20,30, 40, 60,80, or 100 contiguous nucleotides.

Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0 : 9-16, b) a polynucleotide

comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of 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. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20,30, 40,60, 80, or 100 contiguous nucleotides.

Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of 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. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional MEPR, comprising administering to a patient in need

of such treatment the composition.

Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional MEPR, comprising administering to a patient in need of such treatment the composition.

Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.

Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional MEPR, comprising administering to a patient in need of such treatment the composition.

Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ

ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8. 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.

Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1-8. 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.

Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, 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.

Another embodiment 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO : 9-16, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).

Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide 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 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.

Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog (s) are also shown.

Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.

Table 5 shows representative cDNA libraries for polynucleotide embodiments.

Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.

DESCRIPTION OF THE INVENTION Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, 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 invention.

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 various embodiments of 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 "MEPR"refers to the amino acid sequences of substantially purified MEPR obtained from any species, 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 MEPR. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of MEPR either by directly interacting with MEPR or by acting on components of the biological pathway in which MEPR participates.

An"allelic variant"is an alternative form of the gene encoding MEPR. 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 MEPR include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as MEPR or a polypeptide with at least one functional characteristic of MEPR. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding MEPR, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding MEPR. 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 MEPR. Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of MEPR 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 glutamin ; 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"can refer to an oligopeptide, a peptide, a polypeptide, or a 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. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.

The term"antagonist"refers to a molecule which inhibits or attenuates the biological activity of MEPR. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of MEPR either by directly interacting with MEPR or by acting on components of the biological pathway in which MEPR 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 MEPR 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"aptamer"refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e. g. , SELEX (Systematic Evolution of Ligands by EXponential Enrichment), described in U. S. Patent No.

5,270, 163), which selects for target-specific aptamer sequences from large combinatorial libraries.

Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e. g. , the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH2), which may improve a desired property, e. g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e. g. , a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.

Aptamers may be specifically cross-linked to their cognate ligands, e. g. , by photo-activation of a cross-linker (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74: 5-13).

The term"intramer"refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96: 3606-3610).

The term"spiegelmer"refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.

The term"antisense"refers to any composition capable of base-pairing with the"sense" (coding) strand of a polynucleotide having 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 MEPR, 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"and a"composition comprising a given polypeptide"can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding MEPR or fragments of MEPR 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 (Accelrys, Burlington MA) 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, Gin, 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 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.

"Differential expression"refers to increased or upregulated; or decreased, downregulated, or

absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.

"Exon shuffling"refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.

A"fragment"is a unique portion of MEPR or a polynucleotide encoding MEPR which can be 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 about 5 to about 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 ID NO : 9-16 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO : 9-16, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO : 9-16 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO : 9-16 from related polynucleotides.

The precise length of a fragment of SEQ ID NO : 9-16 and the region of SEQ ID NO : 9-16 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 : 1-8 is encoded by a fragment of SEQ ID NO : 9-16. A fragment of SEQ ID NO : 1-8 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO : 1-8. For example, a fragment of SEQ ID NO : 1-8 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO : 1-8. The precise length of a fragment of SEQ ID NO : 1-8 and the region of SEQ ID NO : 1-8 to which the fragment

corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.

A"full length"polynucleotide 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, alternatively, 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 identical nucleotide 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 one or more computer algorithms or programs known in the art or described herein. For example, percent identity can 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.

Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used 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 2.0. 12 (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 : 10 Word Size : 11 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 identical 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. The phrases"percent similarity'and"% similarity, "as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.

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=l, gap penalty=3, window=5, and"diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table.

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 2.0. 12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example: Matrix : BLOSUM62 Open Gap : Il 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 step (s) 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 yg/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. and D. W.

Russell (2001 ; Molecular Cloning : A Laboratory Manual, 3rd ed. , vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 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 Itg/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 acids 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 present in solution and another nucleic acid 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 polynucleotide 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 MEPR 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 MEPR 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, antibodies, or other chemical compounds on a substrate.

The terms"element"and"array element"refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.

The term"modulate"refers to a change in the activity of MEPR. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of MEPR.

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 MEPR 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 MEPR.

"Probe"refers to nucleic acids encoding MEPR, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. 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 polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e. g. , by the polymerase 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, for example, Sambrook, J. and D. W. Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed. , vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY), Ausubel, F. M. et al. (1999; Short Protocols in Molecular Biology, 4'ed., John Wiley & Sons, New York NY), and 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 Institute/MIT 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 nucleic acid 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 and Russell (supra). 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 molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule 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 MEPR, nucleic acids encoding MEPR, or fragments thereof 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 from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 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"or"expression profile"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. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295: 868-872). 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 and Russell (supra).

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 2.0. 9 (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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 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 alternate splicing during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.

Species variants are polynucleotides that vary from one species to another. The resulting polypeptides will generally 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 or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the"BLAST 2 Sequences"tool Version 2.0. 9 (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 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.

THE INVENTION Various embodiments of the invention include new human metalloproteins (MEPR), the polynucleotides encoding MEPR, and the use of these compositions for the diagnosis, treatment, or prevention of blood disorders, heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflammatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders.

Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project Nid). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide m) as shown.

Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have

at least 95% sequence identity to the polypeptide sequences shown in column 3.

Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog (s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog (s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites and potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Accelrys, Burlington MA), in addition to amino acid residues comprising signature sequences, domains, and motifs. Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are metalloproteins. For example, SEQ ID NO : 1 is 91% identical, from residue S6 to residue A39, to human metallothionein-Ie (GenBank ID g386865) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2. ) The BLAST probability score is 3. 0e-15, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO : 1 also has homology to proteins that are localized to the cytoplasm, have small-molecule binding properties, and are members of a family of cysteine-rich heavy metal binding proteins, as determined by BLAST analysis using the PROTEOME database.

SEQ ID NO : 1 also contains a metallothionein domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3. ) Data from BLIMPS and BLAST analyses provide further corroborative evidence that SEQ ID NO : 1 is a metallothionein.

For example, SEQ ID NO : 4 is 90% identical, from residue A54 to residue H130, to Otolemur crassicaudatus delta-globin (GenBank ID gl418276) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2. ) The BLAST probability score is l. Oe-58, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO : 4 also has homology to proteins that are hemoglobins, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO : 4 also contains a globin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3. ) Data from BLIMPS and additional BLAST analyses provide further corroborative evidence that SEQ ID NO : 4 is a globin.

For example, SEQ ID NO : 6 is 100% identical, from residue MI to residue H138 and from residue L262 to residue P698,, to human transferrin (GenBank ID g248648) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2. ) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.

SEQ ID NO : 6 also has homology to proteins that are found in the endosome/endosomal vesicles, the cytoplasm and extracellular spaces (excluding the cell wall), have iron binding activity, are involved in iron transport and cell proliferation, and may have roles in the immune response and phagocytosis, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO : 6 also contains a transferrin domain as determined by searching for statistically significant matches in the hidden Markov model (fEqM)-based PFAM and SMART databases of conserved protein families/domains.

(See Table 3. ) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO : 6 is a transferrin protein.

For example, SEQ ID NO : 8 is 100% identical, from residue M7 to residue G93, to human selenoprotein W (GenBank ID g2326175) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2. ) The BLAST probability score is 4.6E-39, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO : 8 also has homology to selenoprotein W, as determined by BLAST analysis using the PROTEOME database. Data from BLAST analyses against the DOMO database provide further corroborative evidence that SEQ ID NO : 8 is a selenoprotein.

SEQ ID NO : 2-3, SEQ ED NO : 5, and SEQ ID NO : 7 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO : 1-8 are described in Table 7.

As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two

types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.

Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO : 9-16 or that distinguish between SEQ ID NO : 9-16 and related polynucleotides.

The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i. e., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i. e. , those sequences including the designation"NM"or"NT") or the NCBI RefSeq Protein Sequence Records (i. e., those sequences including the designation"NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an"exon stitching"algorithm. For example, a polynucleotide sequence identified as FL_XXXXXX_N1_N2_YYYYY_N3_N4 represents a"stitched"sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and Nl 2 3, if present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching"algorithm. For example, a polynucleotide sequence identified as FLXXXXXXgAAAAAgBlJVis a"stretched"sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the"exon-stretching"algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the"exon-stretching"algorithm, a RefSeq identifier (denoted by"NM," "NP,"or"NT") may be used in place of the GenBank identifier (i. e., gBBBBB).

Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V). Prefix Type of analysis and/or examples of programs GNN, GFG, Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES (Computer Genomics Group, The Sanger Centre, Cambridge, UK) GBI Hand-edited analysis of genomic sequences. FL Stitched or stretched genomic sequences (see Example V). INCY Full length transcript and exon prediction from mapping of EST sequences to the genome. Genomic location and EST composition data are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.

Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.

Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with allele frequencies in different human populations. Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PID) for polynucleotides of the invention. Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID). Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full- length polynucleotide sequence (CB 1 SNP). Column 7 shows the allele found in the EST sequence.

Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST. Columns 11-14

show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of allele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.

The invention also encompasses MEPR variants. Various embodiments of MEPR variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the MEPR amino acid sequence, and can contain at least one functional or structural characteristic of MEPR.

Various embodiments also encompass polynucleotides which encode MEPR. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO : 9-16, which encodes MEPR. The polynucleotide sequences of SEQ ID NO : 9-16, 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 variants of a polynucleotide encoding MEPR. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding MEPR. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO : 9-16 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 NO : 9-16. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of MEPR.

In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding MEPR. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding MEPR, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding MEPR over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding MEPR. For example, a polynucleotide comprising a sequence of SEQ ID NO : 13 is a splice variant of a polynucleotide

comprising a sequence of SEQ ID NO : 14. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of MEPR.

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 MEPR, 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 MEPR, and all such variations are to be considered as being specifically disclosed.

Although polynucleotides which encode MEPR and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring MEPR under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding MEPR 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 MEPR 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 polynucleotides which encode MEPR and MEPR derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide 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 polynucleotide encoding MEPR or any fragment thereof.

Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO : 9-16 and fragments thereof, under various conditions of stringency (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), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA). 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 (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).

The nucleic acids encoding MEPR 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 (Sarkar, G. (1993) PCR Methods Applic. 2318-322). 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 (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 (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 (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, polynucleotides or fragments thereof which encode MEPR may be cloned in recombinant DNA molecules that direct expression of MEPR, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express MEPR.

The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter MEPR-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 No.

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 MEPR, 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"artificiar'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, polynucleotides encoding MEPR may be synthesized, in whole or in part, using one or more chemical methods well known in the art (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, MEPR itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; 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 MEPR, 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 (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 (Creighton, supra, pp. 28-53).

In order to express a biologically active MEPR, the polynucleotides encoding MEPR 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 polynucleotides encoding MEPR. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding MEPR. Such signals include the ATG initiation codon and adjacent sequences, e. g. the Kozak sequence. In cases where a polynucleotide sequence encoding MEPR and its initiation codon 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 codon 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 (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 polynucleotides encoding MEPR and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and Russell, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1,3, and 15).

A variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding MEPR. 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 (Sambrook and Russell, supra ; Ausubel et al., supra ; Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264: 5503-5509; 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 ; The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc.

Natl. Acad. Sci. USA 81: 3655-3659; 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 polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5: 350-356; Yu, M. et al. (1993) Proc.

Natl. Acad. Sci. USA 90: 6340-6344; Buller, R. M. et al. (1985) Nature 317: 813-815; McGregor, D. P. et al. (1994) Mol. Immunol. 31: 219-226; Verma, I. M. 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 polynucleotides encoding MEPR. For example, routine cloning, subcloning,

and propagation of polynucleotides encoding MEPR can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).

Ligation of polynucleotides encoding MEPR 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 (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264: 5503-5509). When large quantities of MEPR are needed, e. g. for the production of antibodies, vectors which direct high level expression of MEPR may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of MEPR. 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 pastors. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra ; Bitter, G. A. et al. (1987) Methods Enzymol. 153: 516-544; Scorer, C. A. et al. (1994) Bio/Technology 12: 181-184).

Plant systems may also be used for expression of MEPR. Transcription of polynucleotides encoding MEPR may be driven by 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 maybe used (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). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technolog (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, polynucleotides encoding MEPR 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 MEPR in host cells (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 (Harrington, J. J. et al. (1997) Nat. Genet. 15 : 345-355).

For long term production of recombinant proteins in mammalian systems, stable expression of MEPR in cell lines is preferred. For example, polynucleotides encoding MEPR 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 thymine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively (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 (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 (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), p- glucuronidase and its substrate p-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 (Rhodes, C. A. (1995) Methods Mol. Biol. 55: 121-131).

Although the presence/absence 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 MEPR is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding MEPR can be identified by the absence of marker gene function.

Alternatively, a marker gene can be placed in tandem with a sequence encoding MEPR 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 polynucleotide encoding MEPR and that express MEPR 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 MEPR 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 MEPR is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art (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 Immunology. Greene Pub. Associates and Wiley- Interscience, New York NY; 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 MEPR include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Alternatively, polynucleotides encoding MEPR, 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 Biosciences, 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 polynucleotides encoding MEPR 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 MEPR may be designed to contain signal sequences which direct secretion of MEPR 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 polynucleotides 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, Manassas 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 polynucleotides encoding MEPR 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 MEPR protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of MEPR 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 (MEPR), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MEPR, 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 MEPR encoding sequence and the heterologous protein sequence, so that MEPR may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16).

A variety of commercially available kits may also be used to facilitate expression and purification of

fusion proteins.

In another embodiment, synthesis of radiolabeled MEPR 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.

MEPR, fragments of MEPR, or variants of MEPR may be used to screen for compounds that specifically bind to MEPR. One or more test compounds may be screened for specific binding to MEPR. In various embodiments, 1, 2,3, 4,5, 10,20, 50, 100, or 200 test compounds can be screened for specific binding to MEPR. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e. g. , ligands or receptors), or small molecules.

In related embodiments, variants of MEPR can be used to screen for binding of test compounds, such as antibodies, to MEPR, a variant of MEPR, or a combination of MEPR and/or one or more variants MEPR. In an embodiment, a variant of MEPR can be used to screen for compounds that bind to a variant of MEPR, but not to MEPR having the exact sequence of a sequence of SEQ ID NO : 1-8. MEPR variants used to perform such screening can have a range of about 50% to about 99% sequence identity to MEPR, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.

In an embodiment, a compound identified in a screen for specific binding to MEPR can be closely related to the natural ligand of MEPR, e. g. , a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J. E. et al. (1991) Current Protocols in Immunology 1 (2): Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor MEPR (Howard, A. D. et al. (2001) Trends Pharmacol. Sci. 22: 132- 140; Wise, A. et al. (2002) Drug Discovery Today 7: 235-246).

In other embodiments, a compound identified in a screen for specific binding to MEPR can be closely related to the natural receptor to which MEPR binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket. For example, the compound may be a receptor for MEPR which is capable of propagating a signal, or a decoy receptor for MEPR which is not capable of propagating a signal (Ashkenazi, A. and V. M. Divit (1999) Curr. Opin. Cell Biol. 11: 255-260; Mantovani, A. et al. (2001) Trends Immunol. 22 : 328-336).

The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; Amgen Inc. , Thousand Oaks

CA), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG, (Taylor, P. C. et al.

(2001) Curr. Opin. Immunol. 13: 611-616).

In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to MEPR, fragments of MEPR, or variants of MEPR. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of MEPR. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of MEPR.

In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of MEPR.

In an embodiment, anticalins can be screened for specific binding to MEPR, fragments of MEPR, or variants of MEPR. Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7: R177-R184 ; Skerra, A.

(2001) J. Biotechnol. 74: 257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e. g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.

In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit MEPR involves producing appropriate cells which express MEPR, either as a secreted protein or on the cell membrane. Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli.

Cells expressing MEPR or cell membrane fractions which contain MEPR are then contacted with a test compound and binding, stimulation, or inhibition of activity of either MEPR 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 MEPR, either in solution or affixed to a solid support, and detecting the binding of MEPR 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 compound (s) may be free in solution or affixed to a solid support.

An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio- labeling assays such as those described in U. S. Patent No. 5,914, 236 and U. S. Patent No. 6,372, 724.

In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1: 25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B. C. and J. A. Wells (1991) Proc. Natl. Acad.

Sci. USA 88: 3407-3411; Lowman, H. B. et al. (1991) J. Biol. Chem. 266: 10982-10988).

MEPR, fragments of MEPR, or variants of MEPR may be used to screen for compounds that modulate the activity of MEPR. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for MEPR activity, wherein MEPR is combined with at least one test compound, and the activity of MEPR in the presence of a test compound is compared with the activity of MEPR in the absence of the test compound. A change in the activity of MEPR in the presence of the test compound is indicative of a compound that modulates the activity of MEPR. Alternatively, a test compound is combined with an in vitro or cell-free system comprising MEPR under conditions suitable for MEPR activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of MEPR 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 MEPR 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 MEPR 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 MEPR 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 MEPR 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 MEPR, e. g. , by secreting MEPR 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 MEPR and metalloproteins. In addition, examples of tissues expressing MEPR are tumorous tissues, preadipocytes, and cancerous lung tissue, and further examples can be found in Table 6 and in Example XI. Therefore, MEPR appears to play a role in blood disorders, heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflammatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders. In the treatment of disorders associated with increased MEPR expression or activity, it is desirable to decrease the expression or activity of MEPR. In the treatment of disorders associated with decreased MEPR expression or activity, it is desirable to increase the expression or activity of MEPR.

Therefore, in one embodiment, MEPR 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 MEPR. Examples of such disorders include, but are not limited to, heavy metal toxicity, such as is caused by lead, arsenic, mercury, cadmium and copper; a blood disorder such as anemia

including (3-thalassemia, hemorrhage, thrombosis, embolism, lymphadenopathy, splenomegaly, phagocytic disorders, hematopoietic disorders, hemoglobin disorders including sickle cell anemia, bone marrow disorders, leukemia including chronic myelogenous leukemia and other myeloproliferative disorders, lymphoma including non-Hodgkin's lymphoma, Hodgkin's disease, complications related to blood transfusion, complications related to bone marrow transplantation, and clotting disorders including von Willebrand's disease and hemophilia; 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, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation, congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation- induced lung disease, and complications of lung transplantation; 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, Sjögren'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; 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 including Down syndrome, 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, hemiplegic migraine, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as diabetes, GRACILE syndrome, obesity, and osteoporosis; an immune system disorder, such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with

thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, 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; a lipid disorder such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2 gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin : cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay- Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; and a vesicle trafficking disorder, such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS), allergies including hay fever, asthma, and urticaria ives), autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage,

and viral, bacterial, fungal, heltninthic, and protozoal infections.

In another embodiment, a vector capable of expressing MEPR 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 MEPR including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantially purified MEPR 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 MEPR including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity of MEPR may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of MEPR including, but not limited to, those listed above.

In a further embodiment, an antagonist of MEPR may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of MEPR. Examples of such disorders include, but are not limited to, those blood disorders, heavy metal toxicity, cancer, and cardiovascular, autoimmune/inflammatory, neurological, metabolic, immune system, lipid and vesicle trafficking disorders described above. In one aspect, an antibody which specifically binds MEPR 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 MEPR.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding MEPR may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of MEPR including, but not limited to, those described above.

In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments 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 MEPR may be produced using methods which are generally known in the art. In particular, purified MEPR may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind MEPR. Antibodies to MEPR 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. In an embodiment, neutralizing antibodies (i. e. , those which inhibit dimer formation) can be used therapeutically. Single chain antibodies (e. g. , from camels or llamas) may be potent enzyme inhibitors and may have application in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol.

74: 277-302).

For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with MEPR 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 MEPR 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 substantially identical to a portion of the amino acid sequence of the natural protein.

Short stretches of MEPR 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 MEPR 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 hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (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; 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 (Morrison, S. L. et al. (1984) Proc. Natl. Acad.

Sci. USA 81: 6851-6855; Neuberger, M. S. et al. (1984) Nature 312: 604-608; 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 MEPR-specific single chain antibodies.

Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (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 (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 MEPR may also be generated.

For example, such fragments include, but are not limited to, F (abt) 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 (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 MEPR and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering MEPR epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for MEPR. Affinity is expressed as an association constant, Ka which is defined as the molar concentration of MEPR-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 MEPR epitopes, represents the average affinity, or avidity, of the antibodies for MEPR. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular MEPR epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10"L/mole are preferred for use in immunoassays in which the MEPR- antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10'L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of MEPR, 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 MEPR-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra ; Coligan et al., supra).

In another embodiment of the invention, polynucleotides encoding MEPR, 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 MEPR. 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 MEPR (Agrawal, S. , ed. (1996) Antisense Therapeutics, Humana Press, 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 (Slater, J. E. et al. (1998) J. Allergy Clin. Immunol. 102: 469-475; Scanlon, K. J. et al. (1995) 9: 1288-1296). Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A. D. (1990) Blood 76: 271; Ausubel et al., supra ; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63: 323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J. J.

(1995) Br. Med. Bull. 51: 217-225; Boado, R. J. et al. (1998) J. Pharm. Sci. 87: 1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res. 25: 2730-2736).

In another embodiment of the invention, polynucleotides encoding MEPR 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)-Xl 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 VDI or Factor IX deficiencies (Crystal, R. G. (1995) Science 270: 404-410; Verma, I. M. 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 MEPR expression or regulation causes disease, the expression of MEPR 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 MEPR are treated by constructing mammalian expression vectors encoding MEPR and introducing these vectors by mechanical means into MEPR-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. Récipon (1998) Curr. Opin.

Biotechnol. 9: 445-450).

Expression vectors that may be effective for the expression of MEPR include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA 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). MEPR may be expressed using (i) a constitutively active promoter, (e. g. , from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or p-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 MEPR from a normal individual.

Commercially available liposom 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 MEPR expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding MEPR 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 cis-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, 1. 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 No. 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 an embodiment, an adenovirus-based gene therapy delivery system is used to deliver

polynucleotides encoding MEPR to cells which have one or more genetic abnormalities with respect to the expression of MEPR. 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 No. 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, I. M. and N. Somia (1997; Nature 18: 389: 239-242).

In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding MEPR to target cells which have one or more genetic abnormalities with respect to the expression of MEPR. The use of herpes simplex virus (HSV) -based vectors may be especially valuable for introducing MEPR 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 No. 5,804, 413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U. S. Patent No. 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). 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 embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding MEPR 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 MEPR into the alphavirus genome in place of the capsid-coding region results in the production of a large number of MEPR-coding RNAs and the synthesis of high levels of MEPR 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 MEPR 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 (Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunolosic 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 molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding MEPR.

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, GW, 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 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 molecules encoding MEPR. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase 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 2'0-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.

In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art. RNAi is a post- transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i. e. , the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene. PTGS can also be accomplished by use of DNA or DNA fragments as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391: 806-811) and Gura, T.

(2000; Nature 404: 804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.

RNAi can be induced in mammalian cells by the use of small interfering RNA also known as siRNA. SiRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.

SiRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3'overhangs. The use of siRNA for inducing

RNAi in mammalian cells is described by Elbashir, S. M. et al. (2001; Nature 411: 494-498).

SiRNA can either be generated indirectly by introduction of dsRNA into the targeted cell, or directly by mammalian transfection methods and agents described herein or known in the art (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/introductory methods). Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e. g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3'adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred. Regions to be avoided for target siRNA sites include the 5'and 3'untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. The selected target sites for siRNA can then be compared to the appropriate genome database (e. g., human, etc. ) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration.

The selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commercially available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).

In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e. g. , Brummelkamp, T. R. et al. (2002) Science 296: 550-553; and Paddison, P. J. et al. (2002) Genes Dev. 16: 948-958). In these and related embodiments, shRNAs can be delivered to target cells using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PSILENCER1. 0-U6 (circular) plasmid (Ambion). Once delivered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene- specific silencing.

In various embodiments, the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis. Expression levels of the mRNA of a targeted gene, can be determined by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein. Expression levels of the protein encoded by the targeted gene can be determined by Western analysis using

standard techniques known in the art.

An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding MEPR. 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 MEPR expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding MEPR may be therapeutically useful, and in the treatment of disorders associated with decreased MEPR expression or activity, a compound which specifically promotes expression of the polynucleotide encoding MEPR may be therapeutically useful.

In various embodiments, one or more 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 MEPR 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 MEPR 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 MEPR. 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 liposom injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (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 Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of MEPR, antibodies to MEPR, and mimetics, agonists, antagonists, or inhibitors of MEPR.

In various embodiments, the compositions described herein, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-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 allows 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 MEPR or fragments thereof. For example, liposom preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, MEPR 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 MEPR or fragments thereof, antibodies of MEPR, and agonists, antagonists or inhibitors of MEPR, 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 ED^o (the dose therapeutically effective in 50% of the population) or LD50 (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 LD50/ED50 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 ED50 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/, tg 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 MEPR may be used for the diagnosis of disorders characterized by expression of MEPR, or in assays to monitor patients being treated with MEPR or agonists, antagonists, or inhibitors of MEPR. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for MEPR include methods which utilize the antibody and a label to detect MEPR 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 MEPR, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of MEPR expression. Normal or standard values for MEPR expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to MEPR under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of MEPR 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, polynucleotides encoding MEPR may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, 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 MEPR may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of MEPR, and to monitor regulation of MEPR levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding MEPR or closely related molecules may be used to identify nucleic acid sequences which encode MEPR. 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 identifies only naturally occurring sequences encoding MEPR, 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 MEPR encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO : 9-16 or from genomic sequences including promoters, enhancers, and introns of the MEPR gene.

Means for producing specific hybridization probes for polynucleotides encoding MEPR include the cloning of polynucleotides encoding MEPR or MEPR 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 polymerases 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 avidin/biotin coupling systems, and the like.

Polynucleotides encoding MEPR may be used for the diagnosis of disorders associated with expression of MEPR. Examples of such disorders include, but are not limited to, heavy metal toxicity, such as is caused by lead, arsenic, mercury, cadmium and copper; a blood disorder such as anemia including-thalassemia, hemorrhage, thrombosis, embolism, lymphadenopathy, splenomegaly, phagocytic disorders, hematopoietic disorders, hemoglobin disorders including sickle cell anemia, bone marrow disorders, leukemia including chronic myelogenous leukemia and other myeloproliferative disorders, lymphoma including non-Hodgkin's lymphoma, Hodgkin's disease, complications related to blood transfusion, complications related to bone marrow transplantation, and clotting disorders including von Willebrand's disease and hemophilia; 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, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a cardiovascular disease such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis,

balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation, congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation- induced lung disease, and complications of lung transplantation; 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, Sjögren'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; 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 including Down syndrome, 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, hemiplegic migraine, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as diabetes, GRACILE syndrome, obesity, and osteoporosis; an immune system disorder, such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCUM), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, 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, Sjögren'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; a lipid disorder such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2 gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin : cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay- Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; and a vesicle trafficking disorder, such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS), allergies including hay fever, asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage, and viral, bacterial, fungal, helminthic, and protozoal infections. Polynucleotides encoding MEPR 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 MEPR expression. Such qualitative or quantitative methods are well known in the art.

In a particular embodiment, polynucleotides encoding MEPR may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding MEPR 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 polynucleotides encoding MEPR

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 MEPR, 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 MEPR, 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 MEPR 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 MEPR, or a fragment of a polynucleotide complementary to the polynucleotide encoding MEPR, 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 polynucleotides encoding MEPR 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 polynucleotides encoding MEPR 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 fSCCP, 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).

SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity. For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations (Taylor, J. G. et al. (2001) Trends Mol. Med. 7: 507-512; Kwok, P. -Y. and Z. Gu (1999) Mol. Med. Today 5: 538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11: 637-641).

Methods which may also be used to quantify the expression of MEPR include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from

standard curves (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 polynucleotides 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 below. 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, MEPR, fragments of MEPR, or antibodies specific for MEPR 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 (Seilhamer et al. ,"Comparative Gene Transcript Analysis, "U. S. Patent No. 5,840, 484; hereby 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). 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 an embodiment, the toxicity of a test compound can be 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 embodiment relates to the use of the polypeptides disclosed herein 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, supra). 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 interest. In some cases, further sequence data may be obtained for definitive protein identification.

A proteomic profile may also be generated using antibodies specific for MEPR to quantify the levels of MEPR 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 maybe 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 (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 ; Shalon, D. et al. (1995) PCT application W095/35505 ; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155; Heller, M. J. et al. (1997) U. S. Patent No. 5,605, 662). Various types of microarrays are well known and thoroughly described in Schena, M. , ed. (1999; DNA Microarrays : A Practical Approach, Oxford University Press, London).

In another embodiment of the invention, nucleic acid sequences encoding MEPR 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 PI constructions, or single chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat. Genet. 15 : 345- 355; Price, C. M. (1993) Blood Rev. 7: 127-134; Trask, B. J. (1991) Trends Genet. 7: 149-154). Once mapped, the nucleic acid sequences 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) (Lander, 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 (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 MEPR 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 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (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, MEPR, its catalytic or immunogenic fragments, 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 free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between MEPR 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 (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 MEPR, or fragments thereof, and washed.

Bound MEPR is then detected by methods well known in the art. Purified MEPR 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 MEPR specifically compete with a test compound for binding MEPR. In

this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with MEPR.

In additional embodiments, the nucleotide sequences which encode MEPR 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 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, including U. S. Ser. No. 60/350,701, U. S. Ser. No. 60/348,769, U. S. Ser. No. 60/366,059, and U. S. Ser.

No. 60/379,907, are hereby expressly incorporated by reference.

EXAMPLES I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the L1FESEQ GOLD database (Incyte Genomics, Palo Alto CA). 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 (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl 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 (Invitrogen), using the

recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). 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 S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) 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 (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK- CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5a, DH10B, or ElectroMAX DH10B from Invitrogen.

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 lyophibzation, 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 fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II 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 Biosciences 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 (Amersham Biosciences); 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 (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VID.

The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly (A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof 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; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic Acids Res. 29: 41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30: 242-244). (HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct. Biol.

6 : 361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages 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 polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionin residues of the full length translated polypeptide. Full length polypeptide

sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using 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.

Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 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 or the lower the probability value, the greater the identity between two sequences).

The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID NO : 9-16. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.

IV. Identification and Editing of Coding Sequences from Genomic DNA Putative metalloproteins were initially identified by running the Genscan gene identification program against public genomic sequence databases (e. g. , gbpri and gbhtg). Genscan is a general- purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268: 78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8: 346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode metalloproteins, the encoded polypeptides were analyzed by querying against PFAM models for metalloproteins. Potential metalloproteins were also identified by homology to

Incyte cDNA sequences that had been annotated as metalloproteins. These selected Genscan- predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example m. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched"Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example m were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified were then"stitched"together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.

Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.

"Stretched"Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example m were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore"stretched"or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.

VI. Chromosomal Mapping of MEPR Encoding Polynucleotides The sequences which were used to assemble SEQ ID NO : 9-16 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 NO : 9-16 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). 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 Généthon 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.

Map locations are represented by ranges, or intervals, of human chromosomes. The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm. (The centiMorgan (cM) 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 Généthon 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.

VII. 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 (Sambrook and Russell, supra, ch. 7; Ausubel et al. , supra, ch. 4).

Analogous computer techniques applying BLAST were used to search for identical or related molecules in databases such as GenBank or LIFESEQ (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 5 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.

Alternatively, polynucleotides encoding MEPR are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the

following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells ; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue-and disease-specific expression of cDNA encoding MEPR. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).

VIII. Extension of MEPR Encoding Polynucleotides Full length polynucleotides are 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 was synthesized 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) 2SO4, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), 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 lil PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE and 0.5 iLl 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 81 to 10 1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose 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 Biosciences). 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 Biosciences), 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 Biosciences) 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 DYNAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).

In like manner, full length polynucleotides are verified using the above procedure or are used to obtain 5'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in MEPR Encoding Polynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ID NO : 9-16 using the LIFESEQ database (Incyte Genomics). Sequences from the same gene were clustered together and assembled as described in Example m, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants.

An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.

Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc. ) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.

X. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID NO : 9-16 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 tici of [Y_32p] adenosine triphosphate (Amersham Biosciences), 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 Biosciences). An aliquot containing 107 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 I, 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.

XI. 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 et al., supra), 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, M. , ed.

(1999) DNA Microarrays : A Practical Approach, Oxford University Press, London). 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 (Schena, M. et al. (1995) Science 270: 467-470; Shalon, 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 from 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/1 oligo- (dT) primer (21mer), 1X first strand buffer, 0.03 units/lll RNase inhibitor, 500 yM dATP, 500 ßM dGTP, 500 JiM dTTP, 40 AM dCTP, 40 uM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly (A) + RNA with GEMBRIGHT kits (Incyte Genomics). 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 0. 5M 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, 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 ul 5X SSC/0.2% SDS.

Microarray Preparation 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 jig.

Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).

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 U. S.

Patent No. 5,807, 522, incorporated herein by reference. 1 Al of the array element DNA, at an average concentration of 100 ng/yl, is loaded into the open capillary printing element by a high-speed robotic

apparatus. The apparatus then deposits about 5 nl 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 vil of sample mixture consisting of 0.2 yg each of Cy3 and Cy5 labeled cDNA synthesis products in 5X 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 1 of 5X 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, Inc. , Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. 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 R1477, Hamamatsu Photonics Systems, Bridgewater NJ) 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 Cy5. 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, Inc., 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 fluorophores 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 Genomics) Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentially expressed.

Expression For example, SEQ ID NO : 11 shows differential expression associated with tumor tissues, as determined by microarray analysis. Pair comparisons of matched tumorous and microscopically normal tissue from five donors with colon cancer, six donors with lung cancer, and one donor each with breast cancer and ovarian cancer were carried out. The expression of SEQ ID NO : 11 was decreased by at least two-fold in colon, lung and breast cancer tissues, and upregulated by at least two-fold in the ovarian cancer tissue. Therefore, SEQ ID NO : 11 is useful in diagnostic assays for cancer and as a potential biological marker and therapeutic agent in the treatment of cancer, particularly cancers of the colon, lung and breast.

In another example, SEQ ID NO : 9 shows differential expression associated with colon cancer, as determined by microarray analysis. Gene expression in normal colon tissue (mRNA pooled from 3 different donors) was compared to colon polyps from 2 different donors and colon tumors from

3 different donors. The expression of SEQ ID NO : 9 was decreased by at least two-fold in all five diseased tissue samples, as compared to the normal colon tissue. Therefore, SEQ ID NO : 9 is useful in diagnostic assays for colon cancer and as a potential biological marker and therapeutic agent in the treatment of colon cancer.

In another example, SEQ ID NO : 9 shows differential expression associated with Tangier disease, as determined by microarray analysis. The expression of SEQ ID NO : 9 was increased at least two-fold in Tangier disease-derived fibroblasts compared to normal fibroblasts. In addition, both types of cells were cultured in the presence of cholesterol and compared with the same cell type cultured in the absence of cholesterol. Human fibroblasts were obtained from skin explants from both normal subjects and two patients with homozygous Tangier disease. Cell lines were immortalized by transfection with human papillomavirus 16 genes E6 and E7 and a neomycin resistance selectable marker. TD-derived cells are deficient in an assay of apoA-I mediated tritiated cholesterol efflux.

Therefore, SEQ ID NO : 9 is useful in diagnostic assays for Tangier disease.

In a further example, SEQ ID NO : 11 shows differential expression associated with obesity, as determined by microarray analysis. Human primary subcutaneous preadipocytes were isolated from adipose tissue of a 28-year-old healthy female with body mass index (BMI) of 23.59 and a 40-year-old healthy female with a body mass index (BMI) of 32.47. The preadipocytes were cultured and induced to differentiate into adipocytes treated with human insulin and PPAR-y agonist for 3 days and subsequently to medium containing insulin alone for 1,2, 4 days, 1.1 or 2.1 weeks. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. The expression of SEQ ID NO : 11 was increased by at least two-fold the tissues treated for at least one week. Therefore, SEQ ID NO : 11 is useful as a potential biological marker and therapeutic agent in the treatment of obesity.

For example, SEQ ID NO : 12 shows differential expression associated with lung cancer, as determined by microarray analysis. Pair comparison of normal and cancerous lung tissue from individual donors was carried. Expression of SEQ ID NO : 12 was decreased by at least 5.5 fold in 5 lung squamous cell carcinomas and one lung adenocarcinoma, as compared to normal lung tissue.

Therefore, SEQ ID NO : 12 is useful in diagnostic and disease staging assays for lung cancer and as a potential biological marker and therapeutic agent in the treatment of lung cancer.

For example, SEQ ID NO : 14 showed decreased expression in breast ductal carcinoma cells versus a nonmalignant mammary epithelial cell line. The gene expression profile of a nonmalignant mammary epithelial cell line was compared to the gene expression profiles of breast carcinoma lines at

different stages of tumor progression. Cell lines compared included: a) BT-20, a breast carcinoma cell line derived in vitro from the cells emigrating out of thin slices of tumor mass isolated from a 74-year- old female, b) BT-474, a breast ductal carcinoma cell line that was isolated from a solid, invasive ductal carcinoma of the breast obtained from a 60-year-old woman, c) BT-483, a breast ductal carcinoma cell line that was isolated from a papillary invasive ductal tumor obtained from a 23-year- old normal, menstruating, parous female with a family history of breast cancer, d) Hs 57 8T, a breast ductal carcinoma cell line isolated from a 74-year-old female with breast carcinoma, e) MCF7, a nonmalignant breast adenocarcinoma cell line isolated from the pleural effusion of a 69-year-old female, f) MCF-10A, a breast mammary gland (luminal ductal characteristics) cell line isolated. from a 36-year-old woman with fibrocystic breast disease, g) MDA-MB-468, a breast adenocarcinoma cell line isolated from the pleural effusion of a 51-year-old female with metastatic adenocarcinoma of the breast, and h) HMEC, a primary breast epithelial cell line isolated from a normal donor. Therefore, SEQ ID NO : 14 is useful in monitoring treatment of, and diagnostic assays for, breast carcinoma and other cell proliferative disorders.

For example, SEQ ID NO : 16 showed differential expression in tumorous or diseased tissue versus non-tumorous or healthy tissues, as determined by microarray analysis. Array elements that exhibited about at least a two-fold change in expression and a signal intensity over 250 units, a signal- to-background ratio of a least 2.5, and an element spot size of at least 40% were identified as differentially expressed using the GEMTOOLS program (Incyte Genomics).

For example, SEQ ID NO : 16 showed decreased expression in human umbilical vascular endothelial cell line (HUVEC) cells treated with tumor necrosis factor alpha (TNF-a) versus untreated HUVEC cells, as determined by microarray analysis. Expression of SEQ ID NO : 16 decreased by at least twofold in HUVEC cells treated with 0. lng/ml TNF-a. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, cardiovascular and inflammatory disorders.

As another example, SEQ ID NO : 16 showed decreased expression in preadipocyte cells treated with differentiation medium versus untreated preadipocyte cells, as determined by microarray analysis. The preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in the differentiation medium containing active components PPAR-y agonist and human insulin (Zen-Bio). Thiazolidinediones or PPAR-y agonists can bind and activate an orphan nuclear receptor, PPAR-y, and some of them have been proven to be able to induce human adipocyte differentiation.

Human preadipocytes were treated with human insulin and PPAR-y agonist for 3 days and subsequently

were switched to medium containing insulin for a variety of durations before the cells were collected for analysis. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. Between 80% and 90% of the preadipocytes finally differentiated to adipocytes observed under phase contrast microscope. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, metabolic disorders such as obesity and diabetes.

As another example, SEQ ID NO : 16 showed decreased expression in breast cancer cells treated with mammary epithelium growth medium (MEGM) versus untreated breast cancer cells, as determined by microarray analysis. SEQ ID NO : 16 also showed decreased expression in breast cancer cells treated with epidermal growth factor (EGF) versus untreated breast cancer cells. BT-20 is a breast carcinoma cell line derived in vitro from the cells emigrating out of thin slices of the tumor mass isolated from a 74-year-old female. In one experiment, Individual breast tumor cell lines grown under optimal conditions (MEGM) were compared to the same cells maintained in culture under suboptimal conditions. In another experiment, EGF-treated cells were compared to control cells maintained in culture for the same duration in the absence of EGF. Expression of SEQ ID NO : 16 decreased by at least twofold in treated BT-20 cells. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, breast cancer.

As another example, SEQ ID NO : 16 showed decreased expression in tissue associated with osteosarcoma versus normal osteoblasts, as determined by microarray analysis. Messenger RNA from normal human osteoblasts was compared with mRNA from biopsy specimens, osteosarcoma tissues, or primary cultures or metastasized tissues. Expression of SEQ ID NO : 16 decreased by at least twofold in tissue associated with osteosarcoma. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, osteosarcoma.

As another example, SEQ ID NO : 16 showed decreased expression in peripheral blood mononuclear cells (PBMCs) treated with dexamethasone versus untreated PBMCs, as determined by microarray analysis. PBMCs from the blood of 4 healthy volunteer donors were stimulated with LPS in the presence or absence of dexamethasone, for 2,4, 24, and 72 hours. In addition, matching PBMCs were treated for the same duration with dexamethasone alone to monitor the effects of this factor in the absence of stimulation. The treated PBMCs were compared to matching PBMCs kept in culture for 24 hours in the presence of medium alone. Expression of SEQ ID NO : 16 decreased by at least twofold in peripheral blood mononuclear cells (PBMCs) treated with dexamethasone. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for,

autoimmune/inflammatory disorders.

As another example, SEQ ID NO : 16 showed decreased expression in tissue associated with colon cancer versus normal colon tissue, as determined by microarray analysis. Matched normal and colon-cancer associated tissues from a single donor were compared. Expression of SEQ ID NO : 16 decreased by at least twofold in tissue associated with colon cancer. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, colon cancer.

As another example, SEQ ID NO : 16 showed decreased expression in prostate carcinoma cell lines versus nontumorigenic primary prostate epithelial (PrEC) cells, as determined by microarray analysis. Gene expression profiles of the prostate carcinoma lines CA-HPV-10, LNCaP, PC-3, and DU 145, MDAPCa2b were compared to the gene expression profile of nontumorigenic primary prostate epithelial PrEC cells. RNA was harvested when the cells grown in the defined serum-free TCH medium reached 70-80% confluence. Expression of SEQ ID NO : 16 decreased by at least twofold in cell lines associated with prostate cancer. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, prostate cancer.

As another example, SEQ ID NO : 16 showed decreased expression in brain tissue associated with Alzheimer's Disease (AD) versus normal brain tissue, as determined by microarray analysis.

Specific dissected brain regions from a normal 61-year-old female were compared to dissected regions from the brain of a female with severe AD and two normal male brains. The diagnosis of normal or severe AD was established by a certified neuropathologist based on microscopic examination of multiple sections throughout the brain. Expression of SEQ ID NO : 16 decreased by at least twofold in tissue associated with AD. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, Alzheimer's disease.

As another example, SEQ ID NO : 16 showed decreased expression in C3A (hepatocyte) cells treated with gemfibrozil versus untreated C3A cells, as determined by microarray analysis. Early confluent C3A cells were treated with various amounts of Gemfibrozil (120,600, 800, and 1200 ug/ml) dissolved CMC, for 1,3, and 6 hours. Parallel samples of C3A cells were treated with 1% CMC only, as a control. Expression of SEQ ID NO : 16 decreased by at least twofold in cells treated with gemfibrozil. Therefore, SEQ ID NO : 16 is useful in monitoring treatment of, and diagnostic assays for, metabolic, cardiovascular, and liver disorders.

XII. Complementary Polynucleotides Sequences complementary to the MEPR-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring MEPR. 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 OLIGO 4.06 software (National Biosciences) and the coding sequence of MEPR. 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 MEPR-encoding transcript.

XIII. Expression of MEPR Expression and purification of MEPR is achieved using bacterial or virus-based expression systems. For expression of MEPR 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 T5 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 MEPR upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG). Expression of MEPR in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding MEPR 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 Spodopterafrugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.

Infection of the latter requires additional genetic modifications to baculovirus (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, MEPR 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 Biosciences).

Following purification, the GST moiety can be proteolytically cleaved from MEPR 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 et al. (supra, ch. 10 and 16).

Purified MEPR obtained by these methods can be used directly in the assays shown in Examples XVII and XVIB, where applicable.

XIV. Functional Assays MEPR function is assessed by expressing the sequences encoding MEPR 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 (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 mg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposom formulations or electroporation. 1-2 Hg 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 Cytometry, Oxford, New York NY).

The influence of MEPR on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding MEPR 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 MEPR and other genes of interest can be analyzed by northern analysis or microarray techniques.

XV. Production of MEPR Specific Antibodies MEPR 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 animals (e. g. , rabbits, mice, etc. ) and to produce antibodies using standard protocols.

Alternatively, the MEPR 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 (Ausubel et al., supra, ch. 11).

Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-MEPR activity by, for example, binding the peptide or MEPR to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

XVI. Purification of Naturally Occurring MEPR Using Specific Antibodies Naturally occurring or recombinant MEPR is substantially purified by immunoaffinity chromatography using antibodies specific for MEPR. An immunoaffinity column is constructed by covalently coupling anti-MEPR antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing MEPR are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of MEPR (e. g. , high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/MEPR 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 MEPR is collected.

XVII. Identification of Molecules Which Interact with MEPR MEPR, or biologically active fragments thereof, are labeled with 125I Bolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J. 133: 529-539). Candidate molecules previously

arrayed in the wells of a multi-well plate are incubated with the labeled MEPR, washed, and any wells with labeled MEPR complex are assayed. Data obtained using different concentrations of MEPR are used to calculate values for the number, affinity, and association of MEPR with the candidate molecules.

Alternatively, molecules interacting with MEPR 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).

MEPR 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).

XVIII. Demonstration of MEPR Activity MEPR activity can be measured using a modified double antibody radioimmunoassay for metallothionein (Hogstrand, C. and C. Haux (1990) Toxicol. Appl. Pharmacol. 103: 56-65, with modifications as described in Hogstrand, C. et al. ( (1994) J. Exp. Biol. 186: 55-73). Alternatively, a radioimmunoassay for ferritin can be carried out (Spectria, Orion Diagnostics; Punnonen, K. et al.

(1997) Blood 89: 1052-1057). MEPR activity can also be assayed for binding of radioactive metal isotopes, such as 75Se (Bansal, M. P. et al. (1989) Carcinogenesis 10: 541-546; Bansal, M. P. et al.

(1990) Carcinogenesis 11: 2071-2073). Metal content of cells or tissues can be determined using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) in a Thermo Jarrel Ash, Polyscan 61E to assess metal-to-protein ratios (Bongers, J. et al. (1988) Anal. Chem. 60: 2683-2686; Valls, M. et al. (2001) J. Biol. Chem. 276: 32835-32843).

MEPR activity can be measured using a cell-free intra-Golgi transport assay (Porat, A. et al.

(2000) J. Biol. Chem. 275: 14457-14465). MEPR activity can also be measured by its inclusion in coated vesicles. MEPR can be expressed by transforming a mammalian cell line such as COS7, HeLa, or CHO with an eukaryotic expression vector encoding MEPR. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A small amount of a second plasmid, which expresses any one of a number of marker genes, such as-galactosidase, is co-transformed into the cells in order to allow rapid identification of those cells which have taken up and expressed the foreign DNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of MEPR and p-galactosidase.

Transformed cells are collected and cell lysates are assayed for vesicle formation. A non- hydrolyzable form of GTP, GTPyS, and an ATP regenerating system are added to the lysate and the mixture is incubated at 37° C for 10 minutes. Under these conditions, over 90% of the vesicles remain coated (Orci, L. et al (1989) Cell 56 : 357-368). Transport vesicles are salt-released from the Golgi membranes, loaded under a sucrose gradient, centrifuged, and fractions are collected and analyzed by SDS-PAGE. Co-localization of MEPR with clathrin or COP coatamer is indicative of MEPR activity in vesicle formation. The contribution of MEPR to vesicle formation can be confirmed by incubating lysates with antibodies specific for MEPR prior to GTPrS addition. The antibody will bind to MEPR and interfere with its activity, thus preventing vesicle formation.

In the alternative, MEPR activity is measured by its ability to alter vesicle trafficking pathways. Vesicle trafficking in cells transformed with MEPR is examined using fluorescence microscopy. Antibodies specific for vesicle coat proteins or typical vesicle trafficking substrates such as transferrin or the mannose-6-phosphate receptor are commercially available. Various cellular components such as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and the plasmalemma are examined. Alterations in the numbers and locations of vesicles in cells transformed with MEPR as compared to control cells are characteristic of MEPR activity.

The oxygen-binding ability of MEPR can be determined on a modified Hem-O-Scan (Aminco) at 37°C (Li, X. et al. (2002) J. Biol. Chem. 277 : 13479-13487). Oxygen-dependent activity of MEPR can be determined using a ring bioassay (Stamler, J. S. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 444-448). Alternatively, oxygen-dependent effects of MEPR on blood flow can be determined (Stamler, J. S. et al. (1997) Science 276: 2034-2037).

Various modifications and variations of the described compositions, 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. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions.

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.

Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Table 1 Incyte Project ID Polypeptide Incyte Polynucleotide Incyte SEQ ID NO: Polypeptide ID SEQ ID NO: Polynucleotide ID Incyte Full Length Clones 7505815 1 7505815CD1 9 7505815CB1 1213932CA2 7497777 2 7497777CD1 10 7497777CB1 7506267 3 7506267CD1 11 7506267CB1 7505842 4 7505842CD1 12 7505842CB1 60204109CA2, 60206384CA2 7509316 5 7509316CD1 13 7509316CB1 90134781CA2 7509328 6 7509328CD1 14 7509326CB1 90134648CA2 7504913 7 7504913CD1 15 7504913CB1 7511011 8 711011CD1 16 7511011CB1 1852816CA2 Table 2 Polypeptide SEQ Incyte GenBank ID NO: Probability Annotation ID NO: Polypeptide ID or PROTEOME Score ID NO: 9 7505815CD1 g386865 3.0E-15 [Homo sapiens] human metallothionein-Ie Schmidt, C.J., et al. J. Biol. Chem. 260:7731-7737 (1985) 9 7505815CD1 347614#MT1E 2.6E-16 [Homo sapiens][Transporter; Small molecule-binding protein][Cytoplasmic] Metallothionein 1E, a member of a family of cysteine-rich heavy metal-binding proteins that functions in metal homeostasis, controls hepatic zinc levels, and protects against heavy- metal toxicity Barnes, N. L., et al. Int. J. Biochem. Cell Biol. 32:895-903 (2000) Garrett, S. H., et al. Prostate 43:125-35 (2000) 9 7505815CD1 422378#Mt1 6.9E-16 [Mus musculus][Active transporter, secondary; Transporter; Small molecule-binding protein][CytopIasmic] Metallothionein 1, a member of a family of cysteine-rich heavy metal binding proteins that functions in metal ion homeostasis, controls hepatic zinc levels, and protects against heavy-metal toxicity van Lookeren Campagne, M., et al. Proc. Natl. Acad. Sci. U S A 96:12870-5 (1999) 10 7497777CD1 g13603867 2.3E-65 [Homo sapiens] ferritin heavy polypeptide-like 17 Wang, P.J., et al. Nat. Genet. 27:422-426 (2001) 10 7497777CD1 709627#FTHL17 2.0E-66 [Homo sapiens] Protein with high similarity to ferritin heavy polypeptide (human FTH1), which is an iron-storage protein involved in c-myc (MYC) control of cell transformation and proliferation, member of the ferritin family of non-heme iron storage proteins 10 7497777CD1 590115#Fth1 5.8E-60 [Rattus norvegicus][Small molecule-binding protein][Cytoplasmic] Ferritin heavy subunit H, iron-storage protein that is upregulated by cAMP-mediated thyrotropin stimulation; elevated expression is associated with absence epilepsy Vet, J. A., et al. Biochim. Biophys. Acta 1360:39-44 (1997) 11 7506267CD1 g14290607 7.7E-177 [Homo sapiens] Similar to selenium binding protein 1 11 7506267CD1 752259#SELENB 6.6E-178 [Homo sapiens] Selenium binding protein 1 P1 Chang, P. W., et al. J. Cell. Biochem. 64:217-24 (1997) Table 2 Polypeptide SEQ Incyte GenBank ID NO: Probability Annotation ID NO: Polypeptide ID or PROTEOME Score ID NO: 11 7506267CD1 318898#Selenbp1 3.0E-160 [Mus musculus][Small molecule-binding protein] Protein with strong similarity to human SELENBP1, a selenium-binding protein Lanfear, J., et al. Carcinogenesis 14:335-40 (1993) 12 7505842CD1 g1418276 1.0E-58 [Otolemur crassicaudatus] delta-globin Tagle D. A. et al. (1992) Genomics 13:741-60. 12 7505842CD1 721689#1qi8_B 5.4E-66 [Protein Data Bank] Hemoglobin 12 7505842CD1 729719#1fhj_B 6.1E-61 [Protein Data Bank] Hemoglobin (Beta Chain) 13 7509316CD1 g248648 3.3E-38 [Homo sapiens] transferrin Hershberger, C. L. et al. (1991) A cloned gene for human transferrin. Ann. N. Y. Acad. Sci. 646: 140-154 13 7509316CD1 339610#TF 2.8E-39 [Homo sapiens][Ligand; Small molecule-binding protein][Endosome/Endosomal vesicles; Cytoplasmic; Extracellular (excluding cell wall)] Transferrin, an iron-binding protein involved in iron transport, has a role in cell proliferation, may have roles in the immune response and phagocytosis Adrian, G. S. et al. (1992) Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans. Biochim Biophys Acta 1132: 168-176 13 7509316CD1 591483#Tf 1.5E-28 [Rattus norvegicus][Small molecule-binding protein][Extracellular (excluding cell wall)] Transferrin, an iron-binding protein involved in iron transport, has a role in cell proliferation Huggenvik, J. I. et al. (1987) Transferrin messenger ribonucleic acid: molecular cloning and hormonal regulation in rat Sertoli cells. Endocrinology 120: 332-340 14 7509328CD1 g248648 0.0 [Homo sapiens] transferrin Hershberger, C. L. et al. (supra) 15 7504913CD1 g13528828 9.9E-53 [Homo sapiens] grancalcin Table 2 Polypeptide SEQ Incyte GenBank ID NO; Probability Annotation ID NO: Polypeptide ID or PROTEOME Score ID NO: 15 7504913CD1 428784#GCA 8.4E-54 [Homo sapiens][Small molecule-binding protein][Cytoplasmic; Plasma membrane] Grancalcin, member of the penta-EF-hand calcium-binding protein family, may be involved in leukocyte adhesion and migration, possibly functions in granule-membrane fusion and degranulation Lollike, K. et al. Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner. J Biol Chem 276, 17762-9. (2001). 15 7504913CD1 338260#SRI 3.8E-19 [Homo sapiens][Regulatory subunit; Ligand][Cytolasmic; Unspecified membrane] Sorcin, an EF-hand calcium-binding protein that may be involved in multidrug resistance and is a potential modulator of intracellular calcium levels through interaction with the ryanodine receptor Pack-Chung, E. et al. Presenilin 2 interacts with sorcin, a modulator of the ryanodine receptor. J Biol Chem 275, 14440-5 (2000). 16 7511011CD1 g2326175 4.60E-39 [Homo sapiens] selenoprotein W Vendeland, S. C. et al. Rat skeletal muscle selenoprotein W: cDNA clone and mRNA modulation by dietary selenium Proc. Natl. Acad. Sci. U.S.A. 92, 8749-8753 (1995) 16 7511011CD1 337958#SEPW1 6.2E-42 [Homo sapiens] Selenoprotein W, mRNA contains a selenocysteine insertion sequence (SECIS) element which directs selenocysteine incorporation at UGA codons Gu, Q. P. et al. Selenoprotein W accumulates primarily in primate skeletal muscle, heart, brain and tongue. Mol Cell Biochem 204, 49-56. (2000). 16 7511011CD1 747967#Sepw1 3.3E-36 [Rattus norvegicus][Small molecule-binding protein] Selenoprotein W, mRNA contains a selenocysteine insertion sequence (SECIS) element which directs selenocysteine incorporatino at UGA codons, may have antioxidant function and play a role in glutathione metabolism Table 3 SEQ Incyte Amino Acid Signature Sequences, Domains and Motifs Analytical Methods ID Polypeptide Residues and Databases NO: ID 1 7505815CD1 39 signal_cleavage: M1-A20 SPSCAN Signal Peptide: M1-K21, M1-C22, M1-G25 HMMER Metallothionein: M1-A39 HMMER_PFAM Vertebrate metallothioneins proteins BL00203: M1-C38 BLIMPS_BLOCKS Crustacean metallothionein signature PR00858: A8-C26 BLIMPS_PRINTS Vertebrate metallothionein signature PR00860: C5-G18, C19-C28 BLIMPS_PRINTS Mollusc metallothionein signature PR00875: C22-E33, C19-K29 BLIMPS_PRINTS METALLOTHIONEIN BLAST_DOMO DM00214#P04356#16-60: S6-A39 DM00214#S54333#16-61: S6-A39 DM00214#B53640#17-62: C5-C38 DM00214#S24596#16-53: N4-A39 Potential Phosphorylation Sites: S32 MOTIFS Potential Glycosylation Sites: N4 MOTIFS 2 7497777CD1 183 Ferritin: C17-K173 HMMER_PFAM Ferritin iron-binding regions proteins BL00540: V9-L49, A100-S154, G165-L176 BLIMPS_BLOCKS FERRITIN IRON STORAGE MULTIGENE FAMILY CHAIN SUBUNIT HEAVY BLAST_PRODOM PRECURSOR LIVER PD000971: C17-K173 FERRITIN IRON-BINDING REGIONS BLAST_DOMO DM00494#P49946#8-171: N12-L176 DM00494#P18685#11-169: N12-S161 DM00494#P17663#8-171: N12-L176 DM00494#P49947#8-171: N12-L176 3 7506267CD1 325 PROTEIN SELENIUM-BINDING LIVER SELENIUM R11G10.2 PD014082: M1- BLAST_PRODOM R173 PROTEIN SELENIUM-BINDING LIVER SELENIUM F42G8.8 R11G10.2 BLAST_PRODOM PD011335: G161-R272 SELENIUM-BINDING PROTEIN LIVER SELENIUM PD017101: E273-I325 BLAST_PRODOM Table 3 SEQ Incyte Amino Acid Signature Sequences, Domains and Motifs Analytical Methods ID Polypeptide Residues and Databases NO: ID Potential Phosphorylation Sites: S87 S101 S151 S169 S224 S260 T14 T36 T284 Y28 MOTIFS 4 7505842CD1 130 Globin: A54-H130, H3-D53 HMMER_PFAM Globins profile BL01033: G25-F46, H76-F87 BLIMPS-BLOCKS Alpha haemoglobin signature PR00612: L32-F42, D63-H76, Q111-H127 BLIMPS_PRINTS Beta haemoglobin signature PR00814: G30-F46, D48-D63, Q111-H127 BLIMPS_PRINTS PROTEIN HEME TRANSPORT RESPIRATORY OXYGEN HEMOGLOBIN BLAST_PRODOM ERYTHROCYTE CHAIN BETA ALPHA PD000028: D53-Y129, M1-A54 HEMOGLOBIN BETA CHAIN HEME OXYGEN TRANSPORT RESPIRATORY BLAST_PRODOM PROTEIN ERYTHROCYTE CNA PD000054: D83-H130 GLOBINS BLAST_DOMO DM00015#B90227#1-139: D53-A124, V2-A54 DM00015#B25880#1-139: D53-A124, V2-A54 DM00015#A39733#2-140: A54-A124, V2-A54 DM00015#P04245#1-138: D53-A124, L4-A54 Potential Phosphorylation Sites: S45 S50 T5 T39 T71 MOTIFS 5 7509316CD1 82 signal_cleavage: M1-A19 SPSCAN Signal Peptide: M1-C17, M1-A19, M1-P21, M1-D22, M1-V25, M1-C28, M1-T24 HMMER Transferrin: V25-A76 HMMER_PFAM Transferrins proteins BL00205: V25-C38, C58-P83 BLIMPS_BLOCKS TRANSFERRINS DM00330# BLAST_DOMO P02787#20-349: V20-A76 P19134#19-348: V20-A76 P27425#18-351: D22-A76 P09571#1-334: V20-A76 Potential Phosphorylation Sites: S31 S40 S63 T24 T78 MOTIFS Table 3 SEQ Incyte Amino Acid Signature Sequences, Domains and Motifs Analytical Methods ID Polypeptide Residues and Databases NO: ID 6 7509328CD1 575 signal_cleavage: M1-A19 SPSCAN Signal Peptide:L M1-C17, MI-A19, M1-P21, M1-D22, M1-V25, M1-C28, M1-T24 HMMER Transferrin: V238-K560, V25-H138, L139-E224 HMMER_PFAM Transferrin: V238-S564, V25-P237 HMMER_SMART Transferrins proteins BL00205: V25-C38, C264-Y293, V328-L363, E408-T433, BLIMPS_BLOCKS T433-R486, T522-L534 Transferring signatures (transferrin_1.prf): G296_V350, N95-A140 PROFILESCAN Transferrins signatures (transferrin_2.prf): C391-N442 PROFILESCAN Transferrins signatures (transferrin_3.prf): M437-L503 PROFILESCAN Transferrin signature PR00422: Y410-H431, A282-V301, L340-M360, W446-R464, BLIMPS_PRINTS C473-K489 PRECURSOR SIGNAL GLYCOPROTEIN IRON RANSPORT METAOLBINDING BLALST_PRODOM REPEAT SEROTRANSFERIN SIDEROPHILIN BETAIMETAL PD000899: V25- H138, E374-K560, V238-E403, K135-E224 TRANSFERRINS DM00330# BLAST_DOMO P02787#351-680: K23-L558 P19134#350-676: K23-L558 P27425#353-688: K23-N557 P09571#336-669: K23-N557 Transferrins sgnature 1: Y114-D123, Y115-D123, Y322-S331 MOTIFS Transferrins signature 2: Y413-F428 MOTIFS Transferrins signature 3: D454-V484 MOTIFS Potent4ial Phosphorylation Sites: S31 S40 S63 S106 S124 S244 S331 S384 S388 S562 MOTIFS S565 T24 T226 T269 T463 T485 T571 Y455Y543 Potential Glycosylation Sites: N12 N83N309 N507 MOTIFS 7 7504913CD1 106 GRANCALCIN CLACIUM-BINDING REPEAT PD060233: M1-G51 BLALST_PRODOM CALPAIN CATALYTIC DOMAIN DM01246#P28676#32-70: A32-A70 BLAST_DOMO CALMODULIN REPEAT DM00011#P28676#71-114:E71-T106 BLAST_DOMO Table 3 SEQ Incyte Amino Acid Signature Sequences, Domains and Motifs Analyltical Methods ID Polypeptide Residues and Databases NO: ID Potential Photential Phosphorylation Sites: S49 T93 T106 MOTIFS Potential Glycosylation Sites: N12 N83 MOTIFS 8 7511011CD1 93 SELNOPROTEIN W SELENIUM SELENOCYSTEINE PD0129878: A8-Q92 BLAST_PRODOM Potential Phosphorylation Sites: S65 MOTIFS Table 4 Polynucleotide SEQ Sequence Fragments ID NO:/ Incyte ID/ Sequence Length 9/ 7505815cb1/ 329 1-321, 1-327, 1-329, 2-245, 4-312, 5-231, 5-308, 5-324, 6-251, 8-284, 12-274, 12-297, 39-87, 86-329, 87-212, 87-243, 87-266, 87-288, 87- 294, 87-310, 87-313, 87-314, 87-316, 87-317, 87-328, 87-329, 89-206, 89-212, 90-249, 91-293, 91-298, 94-275, 103-329, 114-309, 116- 329, 119-313, 122-245, 123-313, 129-207, 133-329 136-319, 137-329, 139-319, 154-313, 164-313, 166-298, 175-313, 187-319, 191-329, 196-313, 197-313, 231-316 10/ 7497777CB1/ 1-1197, 451-1202, 451-1206, 454-1202, 583-1202, 590-1043, 612-12053, 718-937 1206 11/ 7506267CB1/ 1-538, 1-539, 10-108, 10-134, 10-218, 10-228, 10-232, 10-233, 10-238, 10-239, 10-244, 10-248, 10-252, 10-253, 10-254, 10-257, 10-258, 1387 10-260, 10-263, 10-270, 10-279, 10-285, 10-295, 10-297, 10-309, 10-464, 10-537, 10-1270, 11-267, 12-218, 12-245, 12-257, 12-267, 12- 307, 14-241, 14-259, 14-265, 14-457, 14-499, 14-527, 124-540, 15-223, 15-234, 15-258, 15-263, 15-283, 125-287, 15-288, 15-293, 125-480, 16-147, 16-248, 126-264, 16-280, 17-100, 17-168, 17-209, 17-234, 127-240, 17-241, 17-276, 17-279, 17-284, 127-297, 17-298, 17-302, 17- 314, 17-321, 17-477, 20-217, 20-251, 21-295, 21-303, 21-539, 22-212, 22-530, 27-326, 29-289, 29-540, 30-286, 30-289, 30-302, 30-306, 30-333, 31-309, 35-472, 37-295, 39-310, 40-292, 44-460, 45-293, 48-391, 49-508, 50-499, 71-358, 72-300, 82-322, 83-396, 129-426, 157- 394, 159-402, 166-324, 175-420, 231-414, 235-537, 239-488, 239-495, 239-525, 240-496, 250-540, 265-444, 265-446, 280-495, 287-539, 289-540, 294-409, 294-540, 296-395, 335-476, 349-40, 356-540, 372-539, 390- 535, 437-515, 448-713, 448-720, 538-747, 538787, 538-796, 538-844, 538-949, 538-1064, 538-1088, 538-1103, 538-1127, 538-1128, 538- 1133, 538-1134, 538-1135, 538-1196, 538-1204, 538-1213, 538-1218, 539-783, 539-801, 539-812, 540-779, 540-1135, 540-1255, 540- 12712, 541-1105, 542-1096, 542-1236, 543-798, 545-776, 545-796, 547-803, 547-1275, 549-695, 549-767, 549-1164, 553-927, 553-1226, 544-846, 558-999, 560-876, 569-1223, 570-856, 572-869, 574-1203, 575-843, 578-1139, 589-864, 589-921, 593-1249, 596-942, 600-1216, 604-791, 606-884, 606-1044, 606-1285, 607-1228, 611-890, 611-1043, 611-1285, 615-840, 615-910, 615-911, 615-1017, 628-903, 629- 1056, 634-922, 636-1213, 638-892, 651-1200, 654-1217, 657-908, 658-1258, 659-1275, 660-847, 665-1265, 667-1135, 668-922, 668- 1315, 669-951, 671-849, 675-933, 679-1260, 682-1087, 683-934, 683-954, 683-1118, 683-1248, 683-1271, 683-1281, 688-1251, 689- 1247, 691-925, 693-963, 699-1268, 701-1228, 704-1268, 707-1283, 708-1262, 711- Table 4 Polynucleotide SEQ Sequence Fragments ID NO:/ Incyte ID/ Sequence Length 1290, 712-1007, 713-990, 714-1219, 715-967, 75-1251, 717-933, 717-1282, 720-984, 730-1292 733-1284, 734-1288, 737-1009, 739- 1038, 740-1031, 741-1014, 749-1259, 751-1036, 751-1231, 754-1022, 756-1237, 758-1134, 759-1290, 759-1292, 759-1294, 765-1220, 766- 1294, 768-1027, 769-951, 769-1294, 770-1251, 790-1301, 792-1057, 792-1288, 796-1232, 797-1267, 797-1274, 799-1069, 799-1197, 799- 1296, 799-1298, 801-1274, 804-1234, 804-1257, 805-932, 806-1051, 808-1065, 809-1268, 810-1256, 811-1293, 811-1294, 813-989, 816- 1294, 817-1295, 818-1035, 818-1274, 823-1270, 825-1116, 826-1049, 826-1128, 828-1274, 839-1137, 839-1288, 839-1297, 841-1129, 841- 1270, 842-1287, 844-1270, 845-1270, 847-1287, 848-1270, 848-1287, 848-1301, 851-1121, 855-1290, 859-1130, 860-1085, 860-1096, 860- 1274, 860-1308, 861-1114, 864-1268, 864-1274, 866-1271, 866-1300, 867-1281, 868-1128, 870-1314, 871-1073, 871-1142, 871-1294, 873- 1288, 873-1293, 875-1274, 879-1268, 879-1270, 881-1278, 881-1341, 889-1143, 890-1149. 898-1270, 900-1327, 901-1137, 905-1057, 905-1115, 905-11323, 905-1143, 905-1257, 908-1270, 909-1276, 910-1172, 9120-1273, 912- 1274, 913-1144, 914-1270, 915-1274, 917-1163, 917-1266, 917-1270, 9129-1270, 921-1270, 923-1126, 923-1183, 924-114, 928-1204, 938- 1270, 943-1274, 952-1274, 954-1277, 955-1268, 957-1058, 957-1206, 961-1274, 964-1233, 967-1258, 967-1268, 968-1272, 973-1278, 985- 1260, 992-1091, 992-1101, 1001-1274, 1002-1274, 1020-1298, 1027-1256, 1027-1318, 1028-1272, 1029-12257, 1035-1250 1035-1258, 1041-1278, 1045-1274, 1046-1305, 1052-1272, 1052-1289, 1054-1291, 1056-1243, 1060-1284, 1074-1270, 1076-1274, 1078-1175, 1088- 1303, 1093-1287, 1094-1255, 1095-1310, 1095-1348, 1106-1292, 1120-12912, 1132-1274, 1156-1288, 1171-1275, 1173-1275, 1179-1270, 1181-1274, 1183-1298, 1186-1274, 1198-1387, 1205-1268 12/ 7505842CB1/ 1-742, 22-739, 23-139, 23-251, 23-290, 124-346, 128-333, 173-275, 175-320, 176-292, 176-323, 178-316, 179-293, 179-308, 179-325, 179- 742 337, 179-340, 179-353, 179-358, 179-371, 179-373, 179-375, 180-361, 180-366, 180-373, 181-306, 181-315, 181-318, 181-325, 181-373, 182-364, 184-313, 184-373, 187-295, 187-371, 187-373, 188-373, 189-373, 190-326, 196-290, 196-373, 201-305, 205-330, 205-373, 371- 739, 371-742, 373-723, 373-741, 386-741, 395-742, 402-739, 417-690, 436-742, 439-721, 454-726, 496-699 13/ 7509316CB1/ 1-165, 1-168, 1-169, 1-175, 1-178, 1-181, 1-186, 1-192, 1-198, 1-205, 1-214, 1-216, 1-217, 1-219, 1-226, 1-229, 1-231, 1-232, 1-233, 1- 452 241, 1-243, 1-244, 1-246, 1-247, 1-249, 1-250, 1-252, 1-253, 1-254, 1-255, 1-256, 1-32, 1-452, 2-177, 2-146, 2-205, 2-247, 2-248, 2-250, 2-255, 4-152, 4-1284, 4-210, 4-248, 4-255, 5-145, 8-255, 11-132, 24-255, 39-138, 41-255, 78-249, 296-440, 338-445 Table 4 Polynucleotide SEQ Sequence Fragments ID NO:/ Incyte ID/ Sequence Length 14/ 7509328CB1/ 1-179, 1-196, 1-279, 1-297, 1-316, 12-846, 1-1931, 3-243, 4-284, 8-254, 9-268, 9-302, 10-257, 14-292, 49-1248, 92-438, 118-400, 126-410, 2317 163-427, 164-446, 166-432, 443-1052, 446-667, 446-685, 446-688, 446-691, 446-693, 446-700, 446-718, 446-723, 446-733, 446-739, 446- 746, 446-964, 4469-966, 446-970, 446-972, 447-689, 450-566, 450-1021, 451-657, 451-1114, 452-732, 459-753, 459-1030, 463-1212, 465- 728, 465-813, 467-1082, 469-975, 474-643, 475-712, 476-1060, 476-1140, 477-773, 479-952, 484-879, 484-1108, 484-1123, 485-637, 485- 996, 485-1169, 486-969, 488-836, 488-968, 489-785, 490-1070, 496-753, 496-982, 497-949, 498-754, 503-694, 503-734, 503-770, 503- 1128, 505-755, 509-1070, 511-1113, 519-794, 525-729, 526-1151, 529-1114, 531-595, 534-1151, 537-738, 537-789, 539-646, 539-830, 541-790, 544-799, 545-1157, 549-801, 549-822, 555-823, 555-999, 556-822, 58-1231, 563-1153, 564-1335, 565-849, 573-750, 573-1050, 578-699, 578-704, 579-824, 579-846, 581-835, 581-874, 584-1158, 588-864, 592-874, 594-871, 594-877, 594-893, 595-875, 599-812, 599-909, 601-828, 601-866, 601-1210, 602-864, 603-819, 603-1115, 603-1236, 604-879, 605-858, 605-861, 605-862, 607-1273, 608-878, 608-893, 609-1140, 614-893, 615-859, 615-1285, 617-888, 617-900, 619-855, 627-871, 635-907, 635-1228, 639-1270, 642-1262, 650-1238, 650-1320, 652-894, 64-924, 656-903, 657-1249, 658-1154, 667-928, 667- 930, 674-746, 674-1195, 675-1264, 679-1140, 688-947, 688-960, 688-986, 690-9012, 694-1101, 695-974, 696-1018, 699-925, 700-1085, 702-899, 703-1009, 704-1060, 705-892, 705-978, 705-983, 705-988, 705-1048, 706-1077, 707-1091, 708-959, 708-971, 708-1144, 716- 988, 716-992, 717-1303, 719-979, 721-981, 721-1063, 722-870, 722-1034, 722-1317, 733-901, 735-980, 735-1014, 735-1084, 741-999, 745-1270, 746-996, 747-1098, 747-1231, 747-1273, 749-960, 749-993, 758-1025, 760-1013, 760-1361, 762-1026, 762-1169, 762-1212, 762-1272, 763-1009, 763-1043, 764-1059, 765-1054, 766-1008, 766-1214, 770-1233, 770-1357. 776-916, 777-1077, 778-1057, 779-1116, 785-1041, 785-1061, 789-1346, 797-981, 797-1071, 804-1038, 804-1047, 805-1067, 812-1402, 815-1273, 816-1100, 816-1432, 817-1059, 817-1082, 817-1120, 817-1124, 817-1446, 818-1076, 818-1080, 820-1048, 824-969, 824-1082, 824-1120, 828-1152, 833-1018, 833-1061, 833-1093, 833-1507, 835-1094, 835-1108, 836-1108, 836-1109, 837-1076, 837-1133, 837- 1153, 837-1313, 837-1402, 838-1253, 847-978, 847-1112, 847-1118, 847-1119, 851-1094, 851-1135, 8511142, 854-1541, 857-1307, 861- 1120, 865-1537, 8691149, 869-1553, 872-1158, 876-1011, 876-1080, 878-1157, 879-1159, 883-1229, 883-1408, 884-1413, 885-1148, 885- 1158, 889-1559, 890-1571, 891-1134, 891-1156, 896-1150, 896-1185, 898-1464, 902-1177, 902-1811, 903-1812, 906-1147, 907-1120, 907- 1156, 907-1166, 908-1311, 908-1472, 912-1180, 918-1441, 921-1192, 921-1214, 922-1179, 923-1169, 925-1081, 929-1212, 929-1506, 934- 1607, 936-1811, 937-1410, 937-1606, 940-1220, 941-1239, 941-1607,l 941-1897, 942-1094, 945-1225, Table 4 Polynucleotide SEQ Sequence Fragments ID NO:/ Incyte ID/ Sequence Length 946-1215, 959-1184, 949-1539, 950-1385, 955-1134, 961-1251, 964-1284, 964-1559k 964-1622, 969-1264, 972-1240, 972-1262, 972- 1267, 974-1237, 974-1541, 980-1247, 980-1397, 982-1223, 982-1776, 982-1811, 983-1249, 989-1213, 993-1564, 995-1239, 1000-1812, 1001-1551, 1001-1683, 1003-1266, 1003-1811, 1005-1280, 1005-1942, 1008-1221, 1008-1251, 1008-1268, 1011-1541, 1013-1651, 1014- 1310, 1020-1288, 1020-1293, 1021-1683, 1022-1601, 1023-1272, 1023-1653,1023-1682, 1024-1281, 1024-1284, 1025-1202, 1025-1314, 1026-1365, 1031-1811, 1033-1420, 1034-1250, 1036-1297, 1041-1303, 1041-1476, 1045-1317, 1045-1811, 1046-1296, 12046-1320, 1046- 1583, 1048-1485, 1048-1687, 1049-1812, 12049-1933, 1052-1289, 1056-1311, 1056-1341, 1056-1605, 1056-1632, 1062-1314, 1064-1293, 1064-1313, 1064-1373, 1065-1337, 1066-1607, 1067-1319, 1070-1327, 1070-1339, 1074-1325, 1082-1355, 1083-1397, 1098-1723, 1099- 1651, 1103-1389, 1103-1407, 1106-1812, 1110-1390, 1111-1641, 1115- 1627, 1116-1681, 1117-1641, 1119-1674, 1122-1520, 1122-1756, 1125-1265, 1125-1394, 1132-1718, 1142-1411, 1143-1861, 1146-1410, 1146-1434, 1147-1376, 1147-1410, 1147-1726, 1150-1406, 1150-1408, 1151-1400, 1157-1435, 1158-1422, 1158-1425, 1158-1435, 1158- 1601, 1158-1608, 1158-1619, 1158-1903, 1159-1454, 1160-1490, 1161-1641, 1162-1841, 1168-1431, 1169-1879, 1171-1623, 1171-1863, 1173-1426, 1174-1421, 1174-1441, 1174-1446, 1174-1657, 1175-1460, 1179-1449 1181-1695, 1182-1438, 1184-1726, 1186-1429, 1187- 1379, 1187-1652, 1189-1633, 119-1470, 1192-1643, 1193-1326, 1194-1428, 1194-1439, 1198-1416, 1199-1280, 1199-1723, 1207-1931, 1213-1467, 1213-1488, 1213-1543, 1215-1549, 1218-1481, 1224-1476, 1224-1881, 1226-1512, 1226-1560, 1232-1525, 1233-1428, 1233- 1491, 1234-1484, 1236-1697, 1240-1908, 1240-1909, 1240-1913, 1240-1930, 1240-1931, 1241-1931, 1242-1504, 1243-1752, 1243-1812, 1244-1827, 1249-1604, 1250-1441, 1250-1445, 1250- 1498, 1250-1504, 1250-1521, 1253-1503, 1255-1931, 1256-1492, 1256-1509, 1256-1511, 1260-1524, 1261-1565, 1265-1538, 1267-1931, 1270-1884, 1271-1499, 1271-1513, 1271-1558, 1272-1815, 1273-1542, 1273-1572, 1274-1742, 1274-1747, 1274-1778, 1274-1879, 1275- 1544, 1275-1557, 1275-1851, 1277-1543, 1280-1430, 1281-1537, 1281-1555, 1281-1761, 1281-1898, 1293-1481, 1295-1544, 1296-1954, 1298-1561, 1307-1575, 1308-1812, 1309-1540, 1309-1814, 1310-1556, 1311-1647, 1314-1503, 1314-1949, 1318-1916, 1319-1954, 1323- 1648, 1323-1863, 1323-1877, 1324-1598, 1325-1581, 1325-1588, 1331-1636, 1331-1782, 1331-1919, 1333-1687, 1335-1920, 1337-1898, 1338-1601, 1339-1928, 1341-1937, 1344-1612, 1346-1499, 1350-1950, 1351-1879, 1356-1919, 1357-1645, 1358-1655, 1358-1904, 1359- 1925, 1360-1953, 1361-1609, 1364-1804, 1364-1892, 1367-1856, 1367-1881, 1367-1891, 1371-1614, 1375-1919, 1376-1621, 1377-1840, 1378-1842, 1378-1870, 1378-1957, 1379-1625, 1380- Table 4 Polynucleotide SEQ Sequence Fragments ID NO:/ Incyte ID/ Sequence Length 1607, 1380-1627, 1381-1914, 1383-1947, 1384-1893, 1384-1920, 1386-1645, 1388-1931, 1390-1761, 1392-1612, 1392-1644, 1393-1933, 1394-1948, 1395-1918, 1396-1668, 1397-1687, 1399-1889, 1400-1654, 1404-1807, 1404-1919, 1405-1630, 1406-1732, 1410-1641, 1410- 1918, 1410-1948, 1412-1678, 1414-1681, 1415-1699, 1416-1703, 1419-1919, 1422-1643, 1423-1705, 1423-1791, 1427-1929, 1428-1715, 1429-1683, 1429-1716, 1430-1947, 1430-1949, 1434-1559, 1441-1892, 1443-1663, 1444-1945, 1444-1947, 1446-1686, 1446-1918, 1448- 1919, 1448-1953, 1449-1705, 1454-1733, 1455-1677, 1455-1690, 1457-1893, 1458-1918, 1463-1920, 1463-1932, 1463-1947, 1464-1929, 1464-1931, 1467-1731, 1471-1805, 1471-1949, 1472-1715, 1472-1898, 1473-1723, 1473-1931, 1474-1937, 1477-1930, 1478-1930, 1480- 1949, 1482-1721, 1485-1916, 1488-1859, 1489-1934, 1494-1760, 1495-1974, 1496-1929, 1503-1930, 1505-1770, 1505-1817, 1506-1942, 1507-1931, 1508-1787, 1508-1791, 1510-1931, 1517- 1891, 1520-1797, 1522-1931, 1523-1938, 1524-1948, 1525-1744, 1526-1779, 1526-806, 1530-1931, 12534-1961, 1538-1670, 1538-1754, 1538-1801, 1538-1810, 1542-1955, 1543-1948. 1544-1806, 1544-1930, 1547-1730, 1547-1931, 12547-1949, 1548-1930, 1548-1931, 1550- 1941, 1551-12932, 1552-1929, 1553-1931, 1554-1928, 1554-1930, 1556-1929, 12559-1930 1559-1933, 1564-1833, 1564-1851, 1565-1931, 1568-1927, 1569-1803, 1575-1930, 1576-1931, 1577-1937, 1580-1933, 1581-1931, 1593-1930, 1593-1941, 1594-1856, 1597-1874, 1604- 1846, 1605-1930, 1607-1929, 1609-1931, 1613-1951, 1622-1878, 1632-198, 1641-1880, 1641-1893, 1642-1898, 1643-1909, 1653-1906, 1655-1921, 1661-1811, 1664-1933, 1666-1933, 1668-1914, 1680-1972, 1688-1930, 1692-1893, 1694-1931, 16997-1969, 1698-1931, 1699- 1842, 1702-1879, 1711-1929, 1712-1931, 1718-1931, 1720-1930, 1721-1807, 1732-1889, 1744-1842, 1751-1931, 1764-1903, 1764-1906, 1764-2027, 1765-1967, 1768-2317, 1772-1876, 1772-1931, 1772-1952, 1774-1941, 1781-1931, 1785-1929, 1786-1931, 1793-1931, 1795- 1940, 1807-1931, 1808-1931, 1827-1931, 1833-1952, 1842-1930, 1842-1931, 1843-1949, 1851-1931 15/ 7504913CB1/ 1-247, 17-294, 20-286, 20-290, 20-332, 28-287, 30-814, 31-268, 32-437, 36-386, 36-391, 38-308, 38-341, 43-116, 43-442, 46-314, 51-310, 814 52-302, 69-277, 76-324, 76-348, 88-363, 92-364, 93-341, 97-394, 392-611, 392-617, 446-695, 471-1814, 479-814, 543-663, 553-814, 559- 810, 564-811, 565-814, 596-812 16/ 7511011CB1/ 1-235, 90-321, 97-394, 98-405, 98-520, 105-382, 115-407, 115-422, 117-406, 118-346, 118-359, 118-380, 118-382, 118-390, 120-369, 120- 581 373, 121-266, 121-352, 121-382, 121-415, 124-370, 125-340, 126-401, 127-353, 127-372, 127-386, 127-419, 133-422, 135-412, 136-358, 137-388, 137-457, 138-514, 141-332, 141-336, 141-370, 141-377, 141-381, 141-402, 141-4120, 141-418, 141-419, 141-435, 141-440, 141- 5812, 143-310, 143-322, 143-354, 143-372, 143-377, 143-392, 143-424, 143-482, 144-310, 144-319, 144-378, 144-384, 144-387, 144-388, 144-397, 144-413, 144-414, 144-415, 144-417, 144-422, 144-423, 1244-443, 144-445, 144-446, 144-452, 144-490, 145-417, 145-442, 146- 341, 146-347, 146-352, 146-358, 146-362, 146-368, 146-369, 146-371, 146-373, 146-374, 146-375, 146-377, 146-379, 146-381, 146-384, 146-385, 146-386, 146-387, 146-389, 146-391, 146-394, 146-396, 146-397, 146-398, 146-402, 146-404, 146-405, 146-408, 146-410, 146- 413, 146-414, 146-417, 146-421, 146-422, 146-423, 146-426, 146-427, 146-430, Table 4 Polynucleotide SEQ Sequence Fragments ID NO:/ Incyte ID/ Sequence Length 1246-431, 146-432, 146-433, 146-435, 146-437, 146-439, 146-440, 146-442, 146-443, 146-444, 146-445, 146-457, 146-460, 146-462, 146- 463, 146-464, 146-465, 146-467, 146-469, 146-471, 146-484, 147-334, 147-347, 147-351, 147-361, 147-374, 147-380, 147-383, 147-385, 147-391, 147-394, 147-408, 147-410, 147-415, 147-416, 147-421, 147-422, 147-424, 147-426, 147-428, 147-429, 147-437, 147-440, 147- 443, 147-450, 147-454, 147-577, 148-307, 148-378, 148-385, 148-396, 148-404, 148-419, 148-422, 148-425, 148-454, 148-581, 149-376, 149-409, 149-425, 1249-444, 150-264, 150-395, 150-418, 150-421, 150-442, 151-395, 151-424, 151-426, 151-432, 151-444, 153-323, 153- 388, 153-410, 1253-419, 153-435, 154-432, 154-445, 155-452, 155-572, 1257-355, 157-376, 157-390, 157-403, 157-408, 157-413, 157-417, 157-426, 1257-462, 158-412, 158-519, 159-359, 159-401, 159-402, 159-406, 159-416, 160-385, 160-414, 160-417, 160-464, 162-354, 162- 361, 162-395, 163-407, 163-448, 163-450, 163-461, 163-463, 164-402, 164-403, 164-416, 164-417, 164-422, 164-425, 165-368, 165-468, 166-402, 168-381, 168-404, 168-424, 168-440, 168-461, 1469-390, 169-459, 170- 339, 170-380, 170-402, 170-414, 170-461, 170-508, 171-429, 172-401, 172-449, 172-452, 173-387, 173-398, 174-472, 175-458, 176-409, 176-413, 177-448, 178-413, 178-420, 178-425, 181-442, 186-455, 187-495, 189-454, 193-435, 201-446, 204-452, 301-440, 309-539, 347- 496, 367-508 Table 5 Polynucleotide SEQ Incyte Project ID : Representative Library ID NO: 9 7505815CB1 BRSTTUT01 10 7497777CB1 TESTNOF01 11 7506267CB 1 COLTDIT04 12 7505842CB1 UCMCNOT02 13 7509316CB1 LIVRTUT09 14 7509328CB1 LIVRTUT10 15 7504913CB1 EOSINOT03 16 7511011CB1 ENDANOT01 Table 6 Library Vector Library Description BRSTTUT01 PSPORT1 Library was cosntructed using RNA isolated from breast tumor tissue removed from a 55-year-old aucasian female during a unilateral extended simple mastectomy. Pathology indicated in vasive grade 4 mammary adenocarcinoma of mixed lobular and ductal type, extensively involving the left breast. The tumor was identified in the deep dermis near the lactiferous ducts with extracapsular extension. Seven mid and low and five high axiallary lymph nodes were positive for tumor. Proliferative fibrocysytic changes were characterized by apocrine metaplasia, sclerosing adenosis, cyst formation, and ductal hyprplasia without atypia. Patient history included atrial tachycardia, blood in the stool, and a benign breast neoplasm. Family history included benign hypertension, atherosclerotic coronary artery disease, cerebrovascular disease, and depresive disorder. COLTDIT04 pINCY Library was constructed using RNA isolated from diseased transverse colon tissue removed from a 16-year-old Caucaisan male during partial colectomy, temporary ileostomy, and colonoscopy. Pathology indicated innumerable (greater than 100) adenomatous polyps with low-grade dysplasia involing the entire colonic mucosa in the setting of familial polyposis coli. Family history included benign col on neoplasm, benign hypertension, cerebrovascular disease, breast cancer, uterine cancer, and type II diabetes. ENDANOT01 PBLUESCRIPT Library was cosntructed using RNA isolated from aortic enothelial cell tissue from an explanted heart removed from a male during a heart transplant. EOSINOT03 PSPORT1 Library was constructed using RNA isolated from pooled eosinophils obtained from allergic asthmatic individuals. LIVRTUT09 pINCY Library was constructed using RNA isolated from an untreated C3A hepatocyte cell line which is a derivative of Hep G2, a cell line derived from a hepatoblastoma removed from a 15-year-old Caucasian male. LIVRTUT10 pINCY Library was constructed using RNA isolated from a treated C3A hepatocyte cell line, which is a derivative of Hep G2, a cell line derived from a hepatoblastoma removed from a 15-year-old Caucasian male. The cells were treated with acetaminophen, 1 mM for 48 hours. TESTNOF01 PSPORT1 This 5' cap isolated full-length library was constructed using RNA isolated from testis tissue removed from a 26-year-old Caucasian male who died from head trauma due to a motor vehicle accident. Serologies were negative. Patient history inclued a hrnia at birth, tobacco use (1 1/2 ppd), marijuana use, nad daily alcohol use (beer and hard liquor). UCMCNOT02 pINCY Library was constructed using RNA isolated from mononuclear cells obtained from the umbilical cord blood of nine individuals.

Table 7 Program Description Reference Parameter Threshold ABI FATURA A program that removes vector sequences and masks Applied Biosystems, Foster City, CA. ambiguous bases in nucleic acid sequences. ABI/PARACEL PDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch <50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA. ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA. BLAST A Basic Local Alignment Search Tool useful in Altschul, S.F. et al. (1990) J. Mol. Biol. ESTs: Probability value = 1.0E- sequence similarity search for amino acid and nucleic 215:403-410; Altschul, S.F. et al. (1997) 8 or less; Full Length sequences: acid sequences. BLAST includes five funcitons: Nucleic Acids Res. 25:3389-3402. Probability value= 1.0E-10 or bnlastp, blastn, blastx, tblastn, and tblastx. less FASTA A Pearson and Lipman algorithm that searches for Pearson, W.R. and D.J. Lipman 91988) Proc. ESTs: fasta E value = 1.06E-6; similarity between a query sequence and a group of Natl. Acad Sci. USA 85:2444-2448; Pearson, Assembled ESTs: fasta Identity sequences of the same type. FASTA comprises as W.R. (1990) Methods Enzymol. 183:63-98; =905% or greater and Match least five funcitons: fasta, tfasta, fastx, tfastx, and and Smith, T.F. and M.S. Waterman (1981) length = 200 bases or greater; ssearch. Adv. Appl. Math. 2:482-489. fastx E value=1.0E-8 or less; Full Length sequences: fastx score = 100 or greater BLIMPS A BLocks IMProved Searcher that matches a Henikoff, S. and J.G. Henikoff (19910 Probability value=1.0E-3 or sequence against those in BLOCKS, PRINTS, Nucleic Acids Res. 19:6565-6572; Henikoff, less DOMO, PRODOM, and PFAM databases to search J.G. an S. Henikoff (1996) Methods for gene families, sequence homoloogy, and structural Enzymol. 266:88-105; and Attwood, T.K. et fingerprint regions. al. (1997) J. Chem. Inf. Comput. Sci. 37:417- 424.

Table 7 Program Description Reference Parameter Threshold HMMER An algorithm for searching a query sequence against Krogh, A. et al. (1994) J. Mol. Biol. PFAM, INCY, SMART or hidden Markov model (HMM)-based dtabases of 235:1501-1531; Sonnhammer, E.L.L. et al. TIGRFAM hits: Probability protein family consensus sequences, such as PFAM, (1988) Nucleic Acids Res. 26:320-322; value= 1.0E-3 or less; Signal INCY, SMART and TIGRFAM. Durbin, R. et al. (1998) Our World View, in peptide hits: Score = 0 or greater a Nutshell, Cambridge Univ. Press, pp. 1- 350. ProfileScan An algorithm that searches for structural and Gribskov, M. et al. (1988) CABIOS 4:61-66; Normalized quality score # GCG sequence motifs in protein sequences that match Gribskov, M. et al. (1989) Methods specified "HIGH" value for that sequence patterns defined in Prosite. Enzymol. 183:146-159; Bairoch, A. et al. particular Prosite motif. (1997) Nucleic Acids Res. 25:217-221. Generally, score = 1.4-2.1. Phred A base-calling algorithm that examines automated Ewing, B. et al. (998) Genome Res. 8:175- sequencer traces with high sensitivity and probability. 185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194. Phrap A Phils Revised Assembly Program including Smith, T.F. and M.S. Waterman (1981) Adv. Score = 120 or greater; match SWAT and CrossMatch, programs based on efficient Appl. Math. 2:482-489; Smith, T.F. and length = 56 or greater implementation of the Smith-Waterman algorithm, M.S. Waterman (1981) J. Mol. Biol. 147:195- useful in searching sequence homology and 197; and Green, P., University of assembling DNA sequences. Washington, Seattle, WA. Consed A graphical tool for viewing and editing Phrap Gordon, D. et al. (1998) Genome Res. 8:195- assemblies. 202. SPScan A weight matrix analysis program that scans protein Nielson, H. et al. (1997) Protein Engineering Score = 3.5 or greater sequences for the presence of secretory signal 10:1-6; Claverie, J.M. and S. Audic (1997) peptides. CABIOS 12:431-439. TMAP A program that uses weight matrices to delineate Persson, B. and P. Argos (1994) J. Mol. Biol. transmembrane segments on protein sequences and 237:182-192; Persson, B. and P. Argos determine orientation. (1996) Protein Sci. 5:363-371.

Table 7 Program Description Reference Parameter Threshold TMHMMER A program that uses a hidden Markov model (HMM) Sonnhammer, E.L. et al. (1998) Proc. Sixth to delineate t5ransmembrane segments on protein Intl. Conf. On Intelligent Systems for Mol. sequences and determine orientation. Biol., Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence (AAAI) Press, Menlo Park, CA, and MIT Press, Cambridge, MA, pp. 175-182. Motifs A program that searches amino acid sequences for Bairoch, A. et al. (1997) Nucleic Acids Res. patterns that matched those defined in Prosite. 25:217-221; isconsin Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI.

Table 8 SEQ PID EST I SNP ID EST CB1 EST Allele Allele Amino Acid Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele 1 Allele 1 Allele 1 Allele 1 NO: frequency frequency frequency frequency 11 7506267 1356525H1 SNP00131678 158 704 G G A V216 n/a n/a n/a n/a 11 7506267 1356525H1 SNP00131679 220 766 G G C V236 n/a n/a n/a n/a 11 7506267 1611995H1 SNP00131680 18 891 T T G L278 n/a n/a n/a n/a 11 7506267 1782943H1 SNP00029841 125 1170 G G C noncoding n/a n/a n/a n/a 13 7509316 1481535H1 SNP00011450 34 27 A G A noncoding n/a n/a n/a n/a 13 7509316 1481535H1 SNP00042260 148 141 G G C C38 n/a n/a n/a n/a 13 7509316 1481535H1 SNP00042261 199 189 C C T P54 n/a n/a n/a n/a 13 7509316 1484574H1 SNP00042261 189 190 C C T P54 n/a n/a n/a n/a 13 7509316 1484575H1 SNP00042261 188 188 C C T P54 n/a n/a n/a n/a 13 7509316 2513108H1 SNP00011450 28 26 G G A noncoding n/a n/a n/a n/a 13 7509316 2513108H1 SNP00042260 142 140 G G C G38 n/a n/a n/a n/a 13 7509316 2708505H1 SNP00042260 150 139 G G C K37 n/a n/a n/a n/a 13 7509316 2816717H1 SNP00042261 189 187 C C T G53 n/a n/a n/a n/a 13 7509316 3245068H1 SNP00011450 35 25 G G A noncoding n/a n/a n/a n/a 13 7509316 4028005H1 SNP00042260 145 138 G G C R37 n/a n/a n/a n/a 13 7509316 4028241H1 SNP00011450 31 24 G G A noncoding n/a n/a n/a n/a 13 7509316 4191474H1 SNP00011450 17 11 G G A noncoding n/a n/a n/a n/a 13 7509316 4191474H1 SNP00042260 131 125 G G C D33 n/a n/a n/a n/a 13 7509316 4227071H1 SNP00011450 34 23 G G A noncoding n/a n/a n/a n/a 13 7509316 4227071H1 SNP00042260 148 135 G G C S36 n/a n/a n/a n/a 13 7509316 4342713H1 SNP00042260 51 132 G G C G35 n/a n/a n/a n/a 13 7509316 4793988H1 SNP00011450 31 21 G G A noncoding n/a n/a n/a n/a 13 7509316 4793988H1 SNP00042260 145 136 G G C T36 n/a n/a n/a n/a 13 7509316 4794505H1 SNP00011450 25 15 G G A noncoding n/a n/a n/a n/a 13 7509316 4794505H1 SNP00042260 139 128 G G C E34 n/a n/a n/a n/a 13 7509316 4795812H1 SNP00011450 19 20 G G A noncoding n/a n/a n/a n/a 13 7509316 4795812H1 SNP00042260 133 134 G G C A36 n/a n/a n/a n/a 13 7509316 4892776H1 SNP00042260 146 142 G G C W38 n/a n/a n/a n/a Table 8 SEQ PID EST I SNP ID EST CB1 EST Allele Allele Amino Acid Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele 1 Allele 1 Allele 1 Allele 1 NO: frequency frequency frequency frequency 13 7509316 4893331H1 SNP00011450 19 20 G G A noncoding n/a n/a n/a n/a 13 7509316 4893666H1 SNP00042260 145 137 G G C E37 n/a n/a n/a n/a 13 7509316 4984592H1 SNP00042260 136 137 G G C G34 n/a n/a n/a n/a 13 7509316 4987182H1 SNP00011450 19 26 G G A noncoding n/a n/a n/a n/a 13 7509316 4988137H1 SNP00011450 8 8 G G A noncoding n/a n/a n/a n/a 13 7509316 4988137H1 SNP00042260 122 120 G G C W31 n/a n/a n/a n/a 13 7509316 4990739H1 SNP00042260 56 54 G G C R9 n/a n/a n/a n/a 13 7509316 4990855H1 SNP00042260 123 123 G G C G32 n/a n/a n/a n/a 13 7509316 4991206H1 SNP00042260 133 130 G G C E34 n/a n/a n/a n/a 13 7509316 5842311H1 SNP00042260 133 124 G G C E32 n/a n/a n/a n/a 13 7509316 6451018H1 SNP00042260 138 73 G G C G15 n/a n/a n/a n/a 14 7509328 027508H1 SNP00128003 103 479 C C T V147 n/a n/a n/a n/a 14 7509328 087739H1 SNP00011452 91 1241 G G C L401 n/a n/a n/a n/a 14 7509328 087859H1 SNP00053101 47 1272 C C T R412 n/a n/a n/a n/a 14 7509328 087893H1 SNP00053101 8 1271 C C T V411 n/a n/a n/a n/a 14 7509328 087902H1 SNP00042263 157 809 C C T N257 n/a n/a n/a n/a 14 7509328 088616H1 SNP00042265 21 1043 C C T L335 n/a n/a n/a n/a 14 7509328 1309840H1 SNP0011453 185 144 C C T P466 0.86 n/a n/a n/a 14 7509328 138047H1 SNP00042263 104 806 C C T V256 n/a n/a n/a n/a 14 7509328 138047H1 SNP00131332 95 797 G G C E253 n/a n/a n/a n/a 14 7509328 138207H1 SNP00011452 24 1234 G G C R399 n/a n/a n/a n/a 14 7509328 139157H1 SNP00042262 156 538 A A G Q167 n/a n/a n/a n/a 14 7509328 1478266H1 SNP00042264 165 924 G G A G296 n/a n/a n/a n/a 14 7509328 1481535H1 SNP00011450 34 37 A G A noncoding n/a n/a n/a n/a 14 7509328 1481535H1 SNP00042260 148 151 G G C C38 n/a n/a n/a n/a 14 7509328 1481626H1 SNP00042261 199 199 C C T P54 n/a n/a n/a n/a 14 7509328 1481993H1 SNP00042265 95 416 C C T F126 n/a n/a n/a n/a 14 7509328 1484574H1 SNP00042261 189 200 C C T P54 n/a n/a n/a n/a Table 8 SEQ PID EST I SNP ID EST CB1 EST Allele Allele Amino Acid Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele 1 Allele 1 Allele 1 Allele 1 NO: frequency frequency frequency frequency 14 7509328 1484575H1 SNP00042261 188 198 C C T P54 n/a n/a n/a n/a 14 7509328 166252H1 SNP00011452 217 1240 G G C R401 n/a n/a n/a n/a 14 7509328 166252H1 SNP00042265 19 1042 C C T P335 n/a n/a n/a n/a 14 7509328 166450H1 SNP00128003 98 475 C C T T146 n/a n/a n/a n/a 14 7509328 166712H1 SNP00042263 103 804 C C T L256 n/a n/a n/a n/a 14 7509328 166712H1 SNP00131332 94 795 G G C E253 n/a n/a n/a n/a 14 7509328 167403H1 SNP00042262 70 553 A A G K172 n/a n/a n/a n/a 14 7509328 167785H1 SNP00042264 221 920 G G A M294 n/a n/a n/a n/a 14 7509328 1907408H1 SNP00042262 25 541 A A G E168 n/a n/a n/a n/a 14 7509328 1954427H1 SNP00011453 153 1433 C C T N465 0.86 n/a n/a n/a 14 7509328 2049444H1 SNP00042263 31 793 C C T A252 n/a n/a n/a n/a 14 7509328 2049444H1 SNP00131332 22 784 G G C R249 n/a n/a n/a n/a 14 7509328 2293567H1 SNP00131332 134 800 G G C W254 n/a n/a n/a n/a 14 7509328 2513108H1 SNP00011450 28 36 G G A noncoding n/a n/a n/a n/a 14 7509328 2513108H1 SNP00042260 142 150 G G C G38 n/a n/a n/a n/a 14 7509328 2514002H1 SNP00042262 16 556 A A G D173 n/a n/a n/a n/a 14 7509328 2706088H1 SNP00128003 86 477 C C T L147 n/a n/a n/a n/a 14 7509328 2706212H1 SNP00011452 14 1238 G G C K400 n/a n/a n/a n/a 14 7509328 2706212H1 SNP00011453 207 1431 C C T Q465 0.86 n/a n/a n/a 14 7509328 2708505H1 SNP00042260 150 149 G G C K37 n/a n/a n/a n/a 14 7509328 2708594H1 SNP00042262 67 555 A A G N173 n/a n/a n/a n/a 14 7509328 271694H1 SNP00042265 8 1041 C C T L335 n/a n/a n/a n/a 14 7509328 2761563H1 SNP00053100 54 1123 T T C L362 n/a n/a n/a n/a 14 7509328 2816717H1 SNP00042261 189 197 C C T G53 n/a n/a n/a n/a 14 7509328 2963778H1 SNP00011453 37 1426 T C T I463 0.86 n/a n/a n/a 14 7509328 2966284H1 SNP00042262 78 554 A A G K172 n/a n/a n/a n/a 14 7509328 2967829H1 SNP00042263 104 807 C C T H257 n/a n/a n/a n/a 14 7509328 2967829H1 SNP00131332 95 798 G G C G254 n/a n/a n/a n/a Table 8 SEQ PID EST ID SNP ID EST CB1 EST Allele Allele Amino Acid Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele 1 Allele 1 Allele 1 Allele 1 NO: frequency frequency frequency frequency 14 7509328 3093681H1 SNP00011452 194 1239 G G C V401 n/a n/a n/a n/a 14 7509328 3245068H1 SNP00011450 35 35 G G A noncoding n/a n/a n/a n/a 14 7509328 3636093H1 SNP00053101 96 1269 C C T H411 n/a n/a n/a n/a 14 7509328 3664421H1 SNP00042265 73 414 C C T L126 n/a n/a n/a n/a 14 7509328 3784014H1 SNP00011453 11 1432 C C T T465 0.86 n/a n/a n/a 14 7509328 3944996H1 SNP00042265 38 415 C C T S126 n/a n/a n/a n/a 14 7509328 3955420H1 SNP00042265 147 411 C C T R125 n/a n/a n/a n/a 14 7509328 4028005H1 SNP00042260 145 148 G G C R37 n/a n/a n/a n/a 14 7509328 4028241H1 SNP00011450 31 34 G G A noncoding n/a n/a n/a n/a 14 7509328 4138382H1 SNP00042265 265 413 C C T G125 n/a n/a n/a n/a 14 7509328 4191474H1 SNP00011450 17 21 G G A noncoding n/a n/a n/a n/a 14 7509328 4191474H1 SNP00042260 131 135 G G C D33 n/a n/a n/a n/a 14 7509328 4227071H1 SNP00011450 34 33 G G A noncoding n/a n/a n/a n/a 14 7509328 4227071H1 SNP00042260 148 145 G G C S36 n/a n/a n/a n/a 14 7509328 4342713H1 SNP00042260 51 142 G G C G35 n/a n/a n/a n/a 14 7509328 4416770H1 SNP00011453 71 1429 C C T T464 0.86 n/a n/a n/a 14 7509328 4418261H1 SNP00042264 214 921 G G A A295 n/a n/a n/a n/a 14 7509328 4419180H1 SNP00042264 215 922 G G A G295 n/a n/a n/a n/a 14 7509328 4698918H1 SNP00042265 15 1038 C C T H334 n/a n/a n/a n/a 14 7509328 4770684H1 SNP00011453 115 1428 C C T R464 0.86 n/a n/a n/a 14 7509328 4793419H1 SNP00042264 189 923 G G A A295 n/a n/a n/a n/a 14 7509328 4793988H1 SNP00011450 31 31 G G A noncoding n/a n/a n/a n/a 14 7509328 4793988H1 SNP00042260 145 146 G G C T36 n/a n/a n/a n/a 14 7509328 4794505H1 SNP00011450 25 25 G G A noncoding n/a n/a n/a n/a 14 7509328 4794505H1 SNP00042260 139 138 G G C E34 n/a n/a n/a n/a 14 7509328 4794831H1 SNP00042265 216 412 C C T A125 n/a n/a n/a n/a 14 7509328 4795812H1 SNP00011450 19 30 G G A noncoding n/a n/a n/a n/a 14 7509328 4795812H1 SNP00042260 133 144 G G C A36 n/a n/a n/a n/a Table 8 SEQ PID EST ID SNP ID EST CB1 EST Allele Allele Amino Acid Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele 1 Allele 1 Allele 1 Allele 1 NO: frequency frequency frequency frequency 14 7509328 4796329H1 SNP00011452 169 1236 G G C D400 n/a n/a n/a n/a 14 7509328 4796621H1 SNP00042263 137 803 C C T S255 n/a n/a n/a n/a 14 7509328 4796621H1 SNP00131332 128 794 G G C E252 n/a n/a n/a n/a 14 7509328 4797414H1 SNP00042263 42 801 C C T R255 n/a n/a n/a n/a 14 7509328 4797414H1 SNP00131332 33 802 G G C D252 n/a n/a n/a n/a 14 7509328 4892727H1 SNP00042265 193 418 C C T P127 n/a n/a n/a n/a 14 7509328 4892776H1 SNP00042260 146 152 G G C W38 n/a n/a n/a n/a 14 7509328 4893331H1 SNP00011450 19 32 G G A noncoding n/a n/a n/a n/a 14 7509328 4893666H1 SNP00042260 145 147 G G C E37 n/a n/a n/a n/a 14 7509328 4897226H1 SNP00011452 168 1231 G G C G398 n/a n/a n/a n/a 14 7509328 4984592H1 SNP00042260 136 139 G G C G34 n/a n/a n/a n/a 14 7509328 4985310H1 SNP00042262 48 552 A A G K172 n/a n/a n/a n/a 14 7509328 4985527H1 SNP00011452 109 1130 G G C stop364 n/a n/a n/a n/a 14 7509328 4985561H1 SNP00128003 80 478 C C T A147 n/a n/a n/a n/a 14 7509328 4986173H1 SNP00042263 98 802 C C T T255 n/a n/a n/a n/a 14 7509328 4986173H1 SNP00131332 89 793 G G C G252 n/a n/a n/a n/a 14 7509328 4986233H1 SNP00042264 153 918 G G A V294 n/a n/a n/a n/a 14 7509328 4986382H1 SNP00053100 68 1122 T T C L362 n/a n/a n/a n/a 14 7509328 4987182H1 SNP00011450 19 26 G G A noncoding n/a n/a n/a n/a 14 7509328 4988137H1 SNP00011450 8 18 G G A noncoding n/a n/a n/a n/a 14 7509328 4988137H1 SNP00042260 122 130 G G C W31 n/a n/a n/a n/a 14 7509328 4988346H1 SNP00042264 76 300 G G A D88 n/a n/a n/a n/a 14 7509328 4990651H1 SNP00042265 70 417 C C T Q127 n/a n/a n/a n/a 14 7509328 4990739H1 SNP00042260 56 64 G G C R9 n/a n/a n/a n/a 14 7509328 4990855H1 SNP00042260 123 133 G G C G32 n/a n/a n/a n/a 14 7509328 4991206H1 SNP00042260 133 140 G G C E34 n/a n/a n/a n/a 14 7509328 4993563H1 SNP00042265 27 1040 C C T D334 n/a n/a n/a n/a 14 7509328 5035934H1 SNP00053100 81 1119 T T C C361 n/a n/a n/a n/a Table 8 SEQ PID EST ID SNP ID EST CB1 EST Allele Allele Amino Acid Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele 1 Allele 1 Allele 1 Allele 1 NO: frequency frequency frequency frequency 14 7509328 5035934H1 SNP00053101 229 1267 C C T S410 n/a n/a n/a n/a 14 7509328 5575378H1 SNP00011452 85 1242 G G C G402 n/a n/a n/a n/a 14 7509328 5842311H1 SNP00042260 133 134 G G C E32 n/a n/a n/a n/a 14 7509328 5973220H1 SNP00011451 359 1280 G G A T414 n/a n/a n/a n/a 14 7509328 5997189H1 SNP00011451 303 1273 G G A G412 n/a n/a n/a n/a 14 7509328 6449152H1 SNP00011452 118 1204 G G C S389 n/a n/a n/a n/a 14 7509328 6449152H1 SNP00011453 311 1397 C C T N453 0.86 n/a n/a n/a 14 7509328 7025579H1 SNP00042264 165 306 G G A D90 n/a n/a n/a n/a 15 7504913 2439170H1 SNP00012381 135 371 G T G A80 n/a n/a n/a n/a 15 7504913 6438969H1 SNP00012381 286 367 T T G T78 n/a n/a n/a n/a 16 7511011 1231661H1 SNP00064728 157 318 A A G D41 n/a n/a n/a n/a 16 7511011 1251130H1 SNP00130948 231 383 A A G I63 n/a n/a n/a n/a 16 7511011 1286974H1 SNP00073765 164 311 C C T R39 n/a n/a n/a n/a 16 7511011 1463636H1 SNP00073765 37 298 C C T D34 n/a n/a n/a n/a 16 7511011 148830H1 SNP00073765 96 272 C C T L26 n/a n/a n/a n/a 16 7511011 148831H1 SNP00064728 146 315 A A G Q40 n/a n/a n/a n/a 16 7511011 148831H1 SNP00130948 211 380 A A G M62 n/a n/a n/a n/a 16 7511011 1599862H1 SNP00073765 175 310 C C T G38 n/a n/a n/a n/a 16 7511011 1736835H1 SNP00130948 206 379 A A G K61 n/a n/a n/a n/a 16 7511011 1738265H1 SNP00064728 172 319 A A G E41 n/a n/a n/a n/a 16 7511011 1738539H1 SNP00144655 27 403 C C T G69 n/a n/a n/a n/a 16 7511011 1804824H1 SNP00064728 169 314 A A G M40 n/a n/a n/a n/a 16 7511011 2222443H1 SNP00130948 220 384 A A G N63 n/a n/a n/a n/a 16 7511011 2379235H1 SNP00064728 61 313 A A G R39 n/a n/a n/a n/a 16 7511011 2379235H1 SNP00130948 126 378 A A G K61 n/a n/a n/a n/a 16 7511011 2387087H1 SNP00073765 166 321 C C T T42 n/a n/a n/a n/a 16 7511011 2562570H1 SNP00064728 180 316 A A G L40 n/a n/a n/a n/a 16 7511011 2562570H1 SNP00130948 245 381 A A G stop62 n/a n/a n/a n/a Table 8 SEQ PID EST ID SNP ID EST CB1 EST Allele Allele Amino Acid Caucasian African Asian Hispanic ID SNP SNP Allele 1 2 Allele 1 Allele 1 Allele 1 Allele 1 NO: frequency frequency frequency frequency 16 7511011 2785776H1 SNP00064728 145 317 A A G N41 n/a n/a n/a n/a 16 7511011 2785776H1 SNP00130948 210 382 A A G L62 n/a n/a n/a n/a 16 7511011 2823772H1 SNP00073765 146 307 C C T P37 n/a n/a n/a n/a 16 7511011 2964404H1 SNP00073765 129 293 C C T Q33 n/a n/a n/a n/a 16 7511011 2967859H1 SNP00073765 135 309 C C T A38 n/a n/a n/a n/a 16 7511011 3084740H1 SNP00064728 142 312 A A G H39 n/a n/a n/a n/a 16 7511011 3084740H1 SNP00130948 207 377 A A G K61 n/a n/a n/a n/a 16 7511011 3112595H1 SNP00073765 161 306 C C T P37 n/a n/a n/a n/a 16 7511011 3203018H1 SNP00064728 162 310 A A G G38 n/a n/a n/a n/a 16 7511011 3203018H1 SNP00130948 227 375 A A G E60 n/a n/a n/a n/a 16 7511011 3498930H1 SNP00073765 212 308 C C T R38 n/a n/a n/a n/a 16 7511011 359786H1 SNP00130948 88 371 G A G A59 n/a n/a n/a n/a 16 7511011 3716539H1 SNP00064728 161 311 A A G S39 n/a n/a n/a n/a 16 7511011 3824025H1 SNP00144655 102 400 C C T N68 n/a n/a n/a n/a 16 7511011 3967011H1 SNP00064728 190 306 A A G H37 n/a n/a n/a n/a 16 7511011 3967011H1 SNP00130948 255 372 A A G D59 n/a n/a n/a n/a 16 7511011 4061982H1 SNP00073765 128 305 C C T P37 n/a n/a n/a n/a 16 7511011 4904854H2 SNP00073765 122 292 C C T F32 n/a n/a n/a n/a 16 7511011 5016305H1 SNP00144655 174 399 C C T T68 n/a n/a n/a n/a 16 7511011 5054003H1 SNP00064728 173 320 A A G I42 n/a n/a n/a n/a 16 7511011 5056713H1 SNP00073765 134 312 C C T P39 n/a n/a n/a n/a 16 7511011 5215823H1 SNP00144655 62 402 C C T A69 n/a n/a n/a n/a 16 7511011 5709223J1 SNP00064728 136 298 A A G E34 n/a n/a n/a n/a 16 7511011 5698223H1 SNP00130948 201 363 A A G E56 n/a n/a n/a n/a 16 7511011 6000209H1 SNP00130948 225 376 A A G G60 n/a n/a n/a n/a 16 7511011 6064259H1 SNP00130948 207 370 A A G V58 n/a n/a n/a n/a 16 7511011 6168703H1 SNP00073765 148 296 C C T H34 n/a n/a n/a n/a 16 7511011 6404752H1 SNP00073767 171 572 C C A noncoding n/a n/a n/a n/a Table 8