WO2004072267A2 - Enzymes - Google Patents

Abstract

Various embodiments of the invention provide human enzymes (ENZM) and polynucleotides which identify and encode ENZM. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of ENZM.

Description

ENZYMES

TECHNICAL FIELD

The invention relates to novel nucleic acids, enzymes encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of automimune/iriflaiTimatory disorders, infectious disorders, immune deficiencies, disorders of metabolism, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and cell proliferative disorders, including cancer. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and enzymes.

BACKGROUND OF THE INVENTION

The cellular processes of biogenesis and biodegradation involve a number of key enzyme classes including oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and others. Each class of enzyme comprises many substrate-specific enzymes having precise and well regulated functions. Enzymes facilitate metabolic processes such as glycolysis, the tricarboxylic cycle, and fatty acid metabolism; synthesis or degradation of amino acids, steroids, phospholipids, and alcohols; regulation of cell signaling, proliferation, inflamation, and apoptosis; and through catalyzing critical steps in DNA replication and repair and the process of translation. Oxidoreductases Many pathways of biogenesis and biodegradation require oxidoreductase (dehydrogenase or reductase) activity, coupled to reduction or oxidation of a cofactor. Potential cofactors include cytochromes, oxygen, disulfide, iron-sulfur proteins, flavin adenine dinucleotide (FAD), and the nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E.A. and A.R. Leech (1983) Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U. K. pp. 779-793). Reductase activity catalyzes transfer of electrons between substrate(s) and cofactor(s) with concurrent oxidation of the cofactor. Reverse dehydrogenase activity catalyzes the reduction of a cofactor and consequent oxidation of the substrate. Oxidoreductase enzymes are a broad superfamily that catalyze reactions in all cells of organisms, including metabolism of sugar, certain detoxification reactions, and synthesis or degradation of fatty acids, amino acids, glucocorticoids, estrogens, androgens, and prostaglandins. Different family members may be referred to as oxidoreductases, oxidases, reductases, or dehydrogenases, and they often have distinct cellular locations such as the cytosol, the plasma membrane, mitochondrial inner or outer membrane, and peroxisomes.

Short-chain alcohol dehydrogenases (SCADs) are a family of dehydrogenases that share only 15% to 30% sequence identity, with similarity predominantly in the coenzyme binding domain and the substrate binding domain. In addition to their role in detoxification of ethanol, SCADs are involved in synthesis and degradation of fatty acids, steroids, and some prostaglandins, and are therefore implicated in a variety of disorders such as lipid storage disease, myopathy, SCAD deficiency, and certain genetic disorders. For example, retinol dehydrogenase is a SCAD-family member (Simon, A. et al. (1995) J. Biol. Che 270:1107-1112) that converts retinol to retinal, the precursor of retinoic acid. Retinoic acid, a regulator of differentiation and apoptosis, has been shown to down-regulate genes involved in cell proliferation and inflammation (Chai, X. et al. (1995) J. Biol. Chem. 270:3900-3904). In addition, retinol dehydrogenase has been linked to hereditary eye diseases such as autosomal recessive childhood-onset severe retinal dystrophy (Simon, A. et al. (1996) Genomics 36:424-430). Membrane-bound succinate dehydrogenases (succinate:quinone reductases, SQR) and fumarate reductases (quinoLfumarate reductases, QFR) couple the oxidation of succinate to fumarate with the reduction of quinone to quinol, and also catalyze the reverse reaction. QFR and SQR complexes are collectively known as succinate:quinone oxidoreductases (EC 1.3.5.1) and have similar compositions. The complexes consist of two hydrophilic and one or two hydrophobic, membrane-integrated subunits. The larger hydrophilic subunit A carries covalently bound flavin adenine dinucleotide; subunit B contains three iron-sulphur centers (Lancaster, CR. and A. Kroger (2000) Biochi Biophys. Acta 1459:422-431). The full-length cDNA sequence for the flavoprotein subunit of human heart succinate dehydrogenase (succinate: (acceptor) oxidoreductase; EC 1.3.99.1) is similar to the bovine succinate dehydrogenase in that it contains a cysteine triplet and in that the active site contains an additional cysteine that is not present in yeast or prokaryotic SQRs (Morris, A.A. et al. (1994) Biochim Biophys. Acta 29:125-128).

Propagation of nerve impulses, modulation of cell proliferation and differentiation, induction of the immune response, and tissue homeostasis involve neurotransmitter metabohsm (Weiss, B. (1991) Neurotoxicology 12:379-386; Collins, S.M. et al. (1992) Ann. N.Y. Acad. Sci. 664:415-424; Brown, J.K. and H. Imam (1991) J. Inherit. Metab. Dis. 14:436-458). Many pathways of neurotransmitter metabohsm require oxidoreductase activity, coupled to reduction or oxidation of a cofactor, such as NAD⁺ NADH (Newsholme and Leech, supra, pp. 779-793). Degradation of catecholamines (epinephrine or norepinephrine) requires alcohol dehydrogenase (in the brain) or aldehyde dehydrogenase (in peripheral tissue). NAD⁺ -dependent aldehyde dehydrogenase oxidizes 5-hydroxyindole-3-acetate (the product of 5-hydroxytιyptamine (serotonin) metabohsm) in the brain, blood platelets, liver and pulmonary endothehum (Newsholme and Leech, supra, p. 786). Other neurotransmitter degradation pathways that utilize NAD⁺/NADH-dependent oxidoreductase activity include those of L-DOPA (precursor of dopamine, a neuronal excitatory compound), glycine (an inhibitory neurotransmitter in the brain and spinal cord), stamine (liberated from mast cells during the inflammatory response), and taurine (an inhibitory neurotransmitter of the brain stem, spinal cord and retina) (Newsholme and Leech, supra, pp. 790, 792). Epigenetic or genetic defects in neurotransmitter metabolic pathways can result in diseases including Parkinson disease and inherited myoclonus (McCance, K.L. and S.E. Huether (1994) Pathophysiology, Mosby-Year Book, Inc., St. Louis, MO pp. 402-404; Gundlach, A.L. (1990) FASEB J. 4:2761-2766). Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier, providing activated one-carbon units to a wide variety of biosynthetic reactions, including synthesis of purines, pyrimidines, and the amino acid met onine. Tetrahydrofolate is generated by the activity of a holoenzyme complex called tetrahydrofolate synthase; which includes three enzyme activities: tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and tetrahydrofolate synthetase. Thus, tetrahydrofolate dehydrogenase plays an important role in generating building blocks for nucleic and amino acids, crucial to proliferating cells.

3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabohsm. It catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with concomitant oxidation of NAD to NADH, in the mitochondria and peroxisomes of eukaryotic cells. In peroxisomes, 3HACD and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme, defects in which are associated with peroxisomal bifunctional enzyme deficiency. This interruption in fatty acid metabohsm produces accumulation of very-long chain fatty acids, disrupting development of the brain, bone, and adrenal glands. Infants born with this deficiency typically die within 6 months (Watkins, P. et al. (1989) J. Clin. Invest. 83:771-777; Online Mendelian Inheritance in Man (OMIM), #261515). The neurodegeneration characteristic of Alzheimer's disease involves development of extracellular plaques in certain brain regions. A major protein component of these plaques is the peptide amyloid-β (Aβ), which is one of several cleavage products of amyloid precursor protein (APP). 3HACD has been shown to bind the Aβ peptide, and is overexpressed in neurons affected in Alzheimer's disease. In addition, an antibody against 3HACD can block the toxic effects of Aβ in a cell culture model of Alzheimer's disease (Yan, S. et al. (1997) Nature 389:689-695; OMIM, #602057).

Steroids such as estrogen, testosterone, and corticosterone are generated from a common precursor, cholesterol, and interconverted. Enzymes acting upon cholesterol include dehydrogenases. Steroid dehydrogenases, such as the hydroxysteroid dehydrogenases, are involved in hypertension, fertility, and cancer (Duax, W.L. and D. Ghosh (1997) Steroids 62:95-100). One such dehydrogenase is 3-oxo-5-α-steroid dehydrogenase (OASD), a microsomal membrane protein highly expressed in prostate and other androgen-responsive tissues. OASD catalyzes the conversion of testosterone into dihydrotestosterone, which is the most potent androgen. Dihydrotestosterone is essential for the formation of the male phenotype during embryogenesis, as well as for proper androgen-mediated growth of tissues such as the prostate and male genitalia. A defect in OASD leads to defective formation of the external genitalia (Andersson, S. et al. (1991) Nature 354:159-161; Labrie, F. et al. (1992) Endocrinology 131:1571-1573; OMIM #264600).

17β-hydroxysteroid dehydrogenase (17βHSD6) plays an important role in the regulation of the male reproductive hormone, dihydrotestosterone (DHTT). 17βHSD6 acts to reduce levels of DHTT by oxidizing a precursor of DHTT, 3α-diol, to androsterone which is readily glucuronidated and removed. 17βHSD6 is active with both androgen and estrogen substrates in embryonic kidney 293 cells. Isozymes of 17βHSD catalyze oxidation and/or reduction reactions in various tissues with preferences for different steroid substrates (Biswas, M.G. and D.W. Russell (1997) J. Biol. Chem. 272:15959-15966). For example, 17βHSDl preferentially reduces estradiol and is abundant in the ovary and placenta. 17βHSD2 catalyzes oxidation of androgens and is present in the endometrium and placenta. 17βHSD3 is exclusively a reductive enzyme in the testis (Geissler, W.M. et al. (1994) Nature Genet. 7:34-39). An excess of androgens such as DHTT can contribute to diseases such as benign prostatic hyperplasia and prostate cancer.

The oxidoreductase isocitrate dehydrogenase catalyzes the conversion of isocitrate to a- ketoglutarate, a substrate of the citric acid cycle. Isocitrate dehydrogenase can be either NAD or NADP dependent, and is found in the cytosol, mitochondria, and peroxisomes. Activity of isocitrate dehydrogenase is regulated developmentally, and by hormones, neurotransmitters, and growth factors.

Hydroxypyruvate reductase (HPR), a peroxisomal 2-hydroxyacid dehydrogenase in the glycolate pathway, catalyzes the conversion of hydroxypyruvate to glycerate with the oxidation of both NADH and NADPH. The reverse dehydrogenase reaction reduces NAD⁺ and NADP⁺. HPR recycles nucleotides and bases back into pathways leading to the synthesis of ATP and GTP, which are used to produce DNA and RNA and to control various aspects of signal transduction and energy metabohsm. Purine nucleotide biosynthesis inhibitors are used as antiproliferative agents to treat cancer and viral diseases. HPR also regulates biochemical synthesis of serine and cellular serine levels available for protein synthesis.

The mitochondrial electron transport (or respiratory) chain is the series of oxidoreductase- type enzyme complexes in the mitochondrial membrane that is responsible for the transport of electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP provides energy to drive energy-requiring reactions. The key respiratory chain complexes are NADH:ubiquinone oxidoreductase (complex I), succinate:ubiquinone oxidoreductase (complex II), cytochrome c_rb oxidoreductase (complex III), cytochrome c oxidase (complex IV), and ATP synthase (complex V) (Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Pubhshing, Inc., New York, NY, pp. 677-678). All of these complexes are located on the inner matrix side of the mitochondrial membrane except complex II, which is on the cytosolic side where it transports electrons generated in the citric acid cycle to the respiratory chain. Electrons released in oxidation of succinate to fumarate in the citric acid cycle are transferred through electron carriers in complex II to membrane bound ubiquinone (Q). Transcriptional regulation of these nuclear-encoded genes controls the biogenesis of respiratory enzymes. Defects and altered expression of enzymes in the respiratory chain are associated with a variety of disease conditions. Other dehydrogenase activities using NAD as a cofactor include 3-hydroxyisobutyrate dehydrogenase (3HBD), which catalyzes the NAD-dependent oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde within mitochondria. 3-hydroxyisobutyrate levels are elevated in ketoacidosis, methylmalonic acidemia, and other disorders (Rougraff, P.M. et al. (1989) J. Biol. Chem. 264:5899-5903). Another mitochondrial dehydrogenase important in amino acid metabohsm is the enzyme isovaleryl-CoA-dehydrogenase (IVD). IVD is involved in leucine metabohsm and catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA. Human IVD is a tetrameric flavoprotein synthesized in the cytosol with a mitochondrial import signal sequence. A mutation in the gene encoding IVD results in isovaleric acidemia (Vockley, J. et al. (1992) J. Biol. Che 267:2494-2501). The mammahan pyruvate dehydrogenase multi-enzyme complex (PDC) is responsible for production of CoAS Ac and NADH. PDC includes three constituent enzymes, pyruvate dehydrogenase (El), lipoate acetyltransferase (E2) and hpoa ide dehydrogenase (E3), and a tightly-associated 50,000-Mr polypeptide of unknown function (protein X) (De Marcucci, O. and Lindsay, J.G. (1985) Eur. J. Biochem 149:641-648). Each PDC contains 6 or 12 protein X subunits. Protein X is tightly associated with E2 and binds to E3. Protein X is thought to help pyruvate dehydrogenase kinase bind to the core of the complex (Ling, M. et al. (1998) Hum. Mol. Genet. 7:501-505).

The family of glutathione peroxidases encompass tetrameric glutathione peroxidases (GPxl- 3) and the monomeric phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4). Although the overall homology between the tetrameric enzymes and GPx4 is less than 30%, a pronounced similarity has been detected in clusters involved in the active site and a common catalytic triad has been defined by structural and kinetic data (Epp, O. et al. (1983) Eur. J. Biochem 133:51-69). GPxl is ubiquitously expressed in cells, whereas GPx2 is present in the hver and colon, and GPx3 is present in plasma. GPx4 is found at low levels in all tissues but is expressed at high levels in the testis (Ursini, F. et al (1995) Meth. Enzymol. 252:38-53). GPx4 is the only monomeric glutathione peroxidase found in mammals and the only mammahan glutathione peroxidase to show high affinity for and reactivity with phosphohpid hydroperoxides, and to be membrane associated. A tandem mechanism for the antioxidant activities of GPx4 and vitamin E has been suggested. GPx4 has alternative transcription and translation start sites which determine its subcellular localization (Esworthy, R.S. et al. (1994) Gene 144:317-318; and Maiorino, M. et al. (1990) Meth. Enzymol. 186:448-450).

The glutathione S -transferases (GST) are a ubiquitous family of enzymes with dual substrate specificities that perform important biochemical functions of xenobiotic biotransformation and detoxification, drug metabohsm, and protection of tissues against peroxidative damage. They catalyze the conjugation of an electrophile with reduced glutathione (GSH) which results in either activation or deactivation/detoxification. The absolute requirement for binding reduced GSH to a variety of chemicals necessitates a diversity in GST structures in various organisms and cell types. GSTs are homodimeric or heterodimeric proteins localized in the cytosol. The major isozymes share common structural and catalytic properties and include four major classes, Alpha, Mu, Pi, and Theta. Each GST possesses a common binding site for GSH, and a variable hydrophobic binding site specific for its particular eleclxophilic substrates. Specific amino acid residues within GSTs have been identified as important for these binding sites and for catalytic activity. Residues Q67, T68, D101, El 04, and R131 are important for the binding of GSH (Lee, H.-C. et al. (1995) J. Biol. Chem 270:99-109). Residues R13, R20, and R69 are important for the catalytic activity of GST (Stenberg, G. et al. (1991) Biochem J. 274:549-555).

GSTs normally deactivate and detoxify potentially mutagenic and carcinogenic chemicals. Some forms of rat and human GSTs are rehable preneoplastic markers of carcinogenesis. Dihalomethanes, which produce hver tumors in mice, are believed to be activated by GST (Thier, R. et al. (1993) Proc. Natl. Acad. Sci. USA 90:8567-8580). The mutagenicity of ethylene dibromide and ethylene dichloride is increased in bacterial cells expressing the human Alpha GST, Al - 1 , while the mutagenicity of aflatoxin Bl is substantially reduced by enhancing the expression of GST (Simula, T.P. et al. (1993) Carcinogenesis 14:1371-1376). Thus, control of GST activity may be useful in the control of mutagenesis and carcinogenesis.

GST has been implicated in the acquired resistance of many cancers to drug treatment, the phenomenon known as multi-drug resistance (MDR). MDR occurs when a cancer patient is treated with a cytotoxic drug such as cyclophosphamide and subsequently becomes resistant to this drug and to a variety of other cytotoxic agents as well. Increased GST levels are associated with some drug resistant cancers, and it is believed that this increase occurs in response to the drug agent which is then deactivated by the GST catalyzed GSH conjugation reaction. The increased GST levels then protect the cancer cells from other cytotoxic agents for which GST has affinity. Increased levels of Al-1 in tumors has been linked to drug resistance induced by cyclophosphamide treatment (Dirven, H.A. et al. (1994) Cancer Res. 54:6215-6220). Thus control of GST activity in cancerous tissues may be useful in treating MDR in cancer patients.

The reduction of ribonucleotides to the corresponding deoxyribonucleotides, needed for DNA synthesis during cell proliferation, is catalyzed by the enzyme ribonucleotide diphosphate reductase. Glutaredoxin is a glutathione (GSH)-dependent hydrogen donor for ribonucleotide diphosphate reductase and contains the active site consensus sequence -C-P-Y-C-. This sequence is conserved in glutaredoxins from such different organisms as Escherichia coli, vaccinia virus, yeast, plants, and mammahan cells. Glutaredoxin has inherent GSH-disulfide oxidoreductase (thioltransferase) activity in a coupled system with GSH, NADPH, and GSH-reductase, catalyzing the reduction of low molecular weight disulfides as well as proteins. Glutaredoxin has been proposed to exert a general thiol redox control of protein activity by acting both as an effective protein disulfide reductase, similar to thioredoxin, and as a specific GSH-mixed disulfide reductase (Padilla, CA. et al. (1996) FEBS Lett. 378:69-73). In addition to their important role in DNA synthesis and cell division, glutaredoxin and other thioproteins provide effective antioxidant defense against oxygen radicals and hydrogen peroxide (Schallreuter, K.U. and J.M. Wood (1991) Melanoma Res. 1:159-167). Glutaredoxin is the principal agent responsible for protein dethiolation in vivo and reduces dehydroascorbic acid in normal human neutrophils (Jung, CH. and J.A. Thomas (1996) Arch. Biochem Biophys. 335:61-72; Park, J.B. and M. Levine (1996) Biochem J. 315:931-938).

The thioredoxin system serves as a hydrogen donor for ribonucleotide reductase and as a regulator of enzymes by redox control. It also modulates the activity of transcription factors such as NF-κB, AP-1, and steroid receptors. Several cytokines or secreted cytokine-like factors such as adult T-cell leukemia-derived factor, 3B6-interleukin-l, T-hybridoma-derived (MP-6) B cell stimulatory factor, and early pregnancy factor have been reported to be identical to thioredoxin (Holmgren, A.

(1985) Annu. Rev. Biochem 54:237-271; Abate, C et al. (1990) Science 249:1157-1161; Tagaya, Y. et al. (1989) EMBO J. 8:757-764; Wakasugi, H. (1987) Proc. Natl. Acad. Sci. USA 84:804-808; Rosen, A. et al. (1995) Int. hrttnunol. 7:625-633). Thus thioredoxin secreted by stimulated lymphocytes (Yodoi, J. and T. Tursz (1991) Adv. Cancer Res. 57:381-411; Tagaya, N. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8282-8286) has extracellular activities including a role as a regulator of cell growth and a mediator in the immune system (Miranda- Vizuete, A. et al. (1996) J. Biol. Che 271:19099-19103; Yamauchi, A. et al. (1992) Mol. Immunol. 29:263-270). Thioredoxin and thioredoxin reductase protect against cytotoxicity mediated by reactive oxygen species in disorders such as Alzheimer's disease (Lovell, M.A. (2000) Free Radic. Biol. Med. 28:418-427). The selenoprotein thioredoxin reductase is secreted by both normal and neoplastic cells and has been implicated as both a growth factor and as a polypeptide involved in apoptosis (Soderberg, A. et al. (2000) Cancer Res. 60:2281-2289). An extracellular plasmin reductase secreted by hamster ovary cells (HT-1080) has been shown to participate in the generation of angiostatin from plasmin. In this case, the reduction of the plasmin disulfide bonds triggers the proteolytic cleavage of plasmin which yields the angiogenesis inhibitor, angiostatin (Stathakis, P. et al. (1997) J. Biol. Chem. 272:20641-20645). Low levels of reduced sulfhydryl groups in plasma has been associated with rheumatoid arthritis. The failure of these sulfhydryl groups to scavenge active oxygen species (e.g., hydrogen peroxide produced by activated neutrophils) results in oxidative damage to surrounding tissues and the resulting inflammation (Hall, N.D. et al. (1994) Rheumatol. Int. 4:35-38). Another example of the importance of redox reactions in cell metabohsm is the degradation of saturated and unsaturated fatty acids by mitochondrial and peroxisomal beta-oxidation enzymes which sequentially remove two-carbon units from Coenzyme A (CoA)-activated fatty acids. The main beta-oxidation pathway degrades both saturated and unsaturated fatty acids while the auxiliary pathway performs additional steps required for the degradation of unsaturated fatty acids. The pathways of mitchondrial and peroxisomal beta-oxidation use similar enzymes, but have different substrate specificities and functions. Mitochondria oxidize short-, medium-, and long-chain fatty acids to produce energy for cells. Mitochondrial beta-oxidation is a major energy source for cardiac and skeletal muscle. In hver, it provides ketone bodies to the peripheral circulation when glucose levels are low as in starvation, endurance exercise, and diabetes (Eaton, S. et al. (1996) Biochem. J. 320:345-357). Peroxisomes oxidize medium-, long-, and very-long-chain fatty acids, dicarboxyhc fatty acids, branched fatty acids, prostaglandins, xenobiotics, and bile acid intermediates. The chief roles of peroxisomal beta-oxidation are to shorten toxic hpophihc carboxylic acids to facilitate their excretion and to shorten very-long-chain fatty acids prior to mitochondrial beta-oxidation (Mannaerts, G.P. and P.P. Van Veldhoven (1993) Biochimie 75:147- 158).

The auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA reductase catalyzes the following reaction: trans-2, cis/trans-4-dienoyl-CoA + NADPH + H⁺— > trans-3-enoyl-CoA + NADP⁺

This reaction removes even-numbered double bonds from unsaturated fatty acids prior to their entry into the main beta-oxidation pathway (Koivuranta, K.T.^'et al. (1994) Biochem J. 304:787-792). The enzyme may also remove odd-numbered double bonds from unsaturated fatty acids (Smeland, T.E. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6673-6677).

Rat 2,4-dienoyl-CoA reductase is located in both mitochondria and peroxisomes (Dommes, V. et al. (1981) J. Biol. Chem 256:8259-8262). Two immunologically different forms of rat mitochondrial enzyme exist with molecular masses of 60 kDa and 120 kDa (Hakkola, E.H. and J.K. Hiltunen (1993) Eur. J. Biochem 215:199-204). The 120 kDa mitochondrial rat enzyme is synthesized as a 335 amino acid precursor with a 29 amino acid N-terminal leader peptide which is cleaved to form the mature enzyme (Hirose, A. et al. (1990) Biochim. Biophys. Acta 1049:346-349). A human mitochondrial enzyme 83% similar to rat enzyme is synthesized as a 335 amino acid residue precursor with a 19 amino acid N-terminal leader peptide (Koivuranta et al., supra). These cloned human and rat mitochondrial enzymes function as homotetramers (Koivuranta et al., supra). A Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is 295 amino acids long, contains a C-terminal peroxisomal targeting signal, and functions as a homodimer (Coe, J.G.S. et al. (1994) Mol. Gen. Genet. 244:661-672; and Gurvitz, A. et al. (1997) J. Biol. Chem 272:22140-22147). All 2,4-dienoyl-CoA reductases have a fairly well conserved NADPH binding site motif (Koivuranta et al., supra).

The main pathway beta-oxidation enzyme enoyl-CoA hydratase catalyzes the reaction:

2-trans-enoyl-CoA + H₂0 < — > 3-hydroxyacyl-CoA

This reaction hydrates the double bond between C-2 and C-3 of 2-trans-enoyl-CoA, which is generated from saturated and unsaturated fatty acids (Engel, C.K. et al. (1996) EMBO J. 15:5135- 5145). This step is downstream from the step catalyzed by 2,4-dienoyl-reductase. Different enoyl- CoA hydratases act on short-, medium-, and long-chain fatty acids (Eaton et al., supra).

Mitochondrial and peroxisomal enoyl-CoA hydratases occur as both mono-functional enzymes and as part of multi-functional enzyme complexes. Human hver mitochondrial short-chain enoyl-CoA hydratase is synthesized as a 290 amino acid precursor with a 29 amino acid N-terminal leader peptide (Kanazawa, M. et al. (1993) Enzyme Protein 47:9-13; and Janssen, U. et al. (1997) Genomics 40:470-475). Rat short-chain enoyl-CoA hydratase is 87% identical to the human sequence in the mature region ofthe protein and functions as a homohexamer (Kanazawa et al., supra; and Engel et al., supra). A mitochondrial trifunctional protein exists that has long-chain enoyl-CoA hydratase, 3- hydroxyacyl-CoA dehydrogenase, and long-chain 3-oxothiolase activities (Eaton et al., supra). In human peroxisomes, enoyl-CoA hydratase activity is found in both a 327 amino acid residue mono- functional enzyme and as part of a multi-functional enzyme, also known as bifunctional enzyme, which possesses enoyl-Co A hydratase, enoyl-CoA isomerase, and 3-hydroxyacyl-CoAhydrogenase activities (FitzPatrick, D.R. et al. (1995) Genomics 27:457-466; and Hoefler, G. et al. (1994) Genomics 19:60-67). A 339 amino acid residue human protein with short-chain enoyl-CoA hydratase activity also acts as an AU-specific RNA binding protein (Nakagawa, J. et al. (1995) Proc. Natl. Acad. Sci. USA 92:2051-2055). All enoyl-CoA hydratases share homology near two active site glutamic acid residues, with 17 amino acid residues that are highly conserved (Wu, W.-J. et al. (1997) Biochemistry 36:2211-2220).

Inherited deficiencies in mitochondrial and peroxisomal beta-oxidation enzymes are associated with severe diseases, some of which manifest soon after birth and lead to death within a few years. Mitochondrial beta-oxidation associated deficiencies include, e.g., carnitine palmitoyl transferase and carnitine deficiency, very-long-chain acyl-CoA dehydrogenase deficiency, medium- chain acyl-CoA dehydrogenase deficiency, short-chain acyl-CoA dehydrogenase deficiency, electron transport flavoprotein and electron transport flavoprotein:ubiquinone oxidoreductase deficiency, trifunctional protein deficiency, and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (Eaton et al, supra). Mitochondrial trifunctional protein (including enoyl-CoA hydratase) deficient patients have reduced long-chain enoyl-CoA hydratase activities and suffer fromnon-ketotic hypoglycemia, sudden infant death syndrome, cardiomyopathy, hepatic dysfunction, and muscle weakness, and may die at an early age (Eaton et al., supra).

Defects in mitochondrial beta-oxidation are associated with Reye's syndrome, a disease characterized by hepatic dysfunction and encephalopathy that sometimes follows viral infection in children. Reye's syndrome patients may have elevated serum levels of free fatty acids (Cotran, R.S. et al. (1994) Robbins Pathologic Basis of Disease, W.B. Saunders Co., Philadelphia PA, p.866). Patients with mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and medium- chain 3-hydroxyacyl-CoA dehydrogenase deficiency also exhibit Reye-like illnesses (Eaton et al., supra; and Egidio, R.J. et al. (1989) Am Fa Physician 39:221-226).

Inherited conditions associated with peroxisomal beta-oxidation include Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, acyl-CoA oxidase deficiency, peroxisomal thiolase deficiency, and bifunctional protein deficiency (Suzuki, Y. et al. (1994) Am. J. Hum. Genet. 54:36-43; Hoefler et al, supra). Patients with peroxisomal bifunctional enzyme deficiency, including that of enoyl-CoA hydratase, suffer fromhypotonia, seizures, psychomotor defects, and defective neuronal migration; accumulate very-long-chain fatty acids; and typically die within a few years of birth (Watkins, P.A. et al (1989) J. Clin. Invest. 83:771-777).

Peroxisomal beta-oxidation is impaired in cancerous tissue. Although neoplastic human breast epithelial cells have the same number of peroxisomes as do normal cells, fatty acyl-CoA oxidase activity is lower than in control tissue (el Bouhtoury, F. et al (1992) J. Pathol 166:27-35). Human colon carcinomas have fewer peroxisomes than normal colon tissue and have lower fatty- acyl-CoA oxidase and bifunctional enzyme (including enoyl-CoA hydratase) activities than normal tissue (Cable, S. et al (1992) Virchows Arch. B Cell Pathol. Incl Mol. Pathol. 62:221-226).

6-phosρhogluconate dehydrogenase (6-PGDH) catalyses the NADP⁺-deρendent oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate with the production of NADPH. The absence or inhibition of 6-PGDH results in the accumulation of 6-phosphogluconate to toxic levels in eukaryotic cells. 6-PGDH is the third enzyme of the pentose phosphate pathway (PPP) and is ubiquitous in nature. In some heterofermentatative species, NAD+ is used as a cofactor with the subsequent production of NADH. The reaction proceeds through a 3-keto intermediate which is decarboxylated to give the enol of ribulose 5-phosphate, then converted to the keto product following tautomerization of the enol (Berdis A.J. and P.F. Cook (1993) Biochemistry 32:2041-2046). 6-PGDH activity is regulated by the inhibitory effect of NADPH, and the activating effect of 6-phosphogluconate (Rippa, M. et al. (1998) Biochim. Biophys. Acta 1429:83-92). Deficiencies in 6-PGDH activity have been linked to chronic hemolytic anemia.

The targeting of specific forms of 6-PGDH (e.g., enzymes found in trypanosomes) has been suggested as a means for controlling parasitic infections (Tetaud, E. et al. (1999) Biochem J. 338:55- 60). For example, the Trypanosoma brucei enzyme is markedly more sensitive to inhibition by the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate, compared to the mammahan enzyme (Hanau, S. et al. (1996) Eur. J. Biochem. 240:592-599).

Ribonucleotide diphosphate reductase catalyzes the reduction of ribonucleotide diphosphates (i.e., ADP, GDP, CDP, and UDP) to their corresponding deoxyribonucleotide diphosphates (i.e., dADP, dGDP, dCDP, and dUDP) which are used for the synthesis of DNA. Ribonucleotide diphosphate reductase thereby performs a crucial role in the de novo synthesis of deoxynucleotide precursors. Deoxynucleotides are also produced from deoxynucleosides by nucleoside kinases via the salvage pathway.

Mammahan ribonucleotide diphosphate reductase comprises two components, an effector- binding component (E) and a non-heme iron component (F). Component E binds the nucleoside triphosphate effectors while component F contains the iron radical necessary for catalysis. Molecular weight deteπninations of the E and F components, as well as the holoenzyme, vary according to the methods used in purification of the proteins and the particular laboratory. Component E is approximately 90-100 kDa, component F is approximately 100-120 kDa, and the holoenzyme is 200- 250 kDa. Ribonucleotide diphosphate reductase activity is adversely effected by iron chelators, such as thiosemicarbazones, as well as EDTA. Deoxyribonucleotide diphosphates also appear to be negative allosteric effectors of ribonucleotide diphosphate reductase. Nucleotide triphosphates (both ribo- and deoxyribo-) appear to stimulate the activity of the enzyme. 3-methyl-4-nitrophenol, a metabolite of widely used organophosphate pesticides, is a potent inhibitor of ribonucleotide diphosphate reductase in mammahan cehs. Some evidence suggests that ribonucleotide diphosphate reductase activity in DNA virus (e.g., herpes virus) -infected cells and in cancer cehs is less sensitive to regulation by allosteric regulators and a correlation exists between high ribonucleotide diphosphate reductase activity levels and high rates of cell proliferation (e.g., in epatomas). This observation suggests that virus-encoded ribonucleotide diphosphate reductases, and those present in cancer cells, are capable of ma taining an increased supply deoxyribonucleotide pool for the production of virus genomes or for the increased DNA synthesis which characterizes cancers cells. Ribonucleotide diphosphate reductase is thus a target for therapeutic intervention (Nutter, L.M. and Y.-C. Cheng (1984) Pharmac. Ther. 26:191-207; and Wright, J.A. (1983) Pharmac. Ther. 22:81-102).

Dihydrodiol dehydrogenases (DD) are monomeric, NAD(P)⁺-dependent, 34-37 kDa enzymes responsible for the detoxification of trøπs-dihydrodiol and anti-άiol epoxide metabohtes of polycychc aromatic hydrocarbons (PAH) such as benzo[α]yrene, benz[α]anthracene, 7-methyl- benz[ ]anthracene, 7,12-dimethyl-benz[fl]anthracene, chrysene, and 5-methyl-chrysene. In mammahan cells, an environmental PAH toxin such as benzo[α]yrene is initially epoxidated by a microsomal cytochrome P450 to yield 7 ?,8R-arene-oxide and subsequently (-)-7i?,8R-dihydrodiol ((- )-trαns-7,8-dihydroxy-7,8-dihyckobenzo[α]pyrene or (-)-trans-B[a P-άioϊ) This latter compound is further transformed to the αrati-diol epoxide of benzofαjpyrene (i.e., (±)-anti-75,8 -dihydroxy-9α,10 - epoxy-7,8,9,10-tetrahydrobenzo[ ]pyrene), by the same enzyme or a different enzyme, depending on the species. This resulting anti-άiol epoxide of benzo[ ]yrene, or the corresponding derivative from another PAH compound, is highly mutagenic. DD efficiently oxidizes the precursor of the πti-diol epoxide (i.e., traπ^-dihydrodiol) to transient catechols which auto-oxidize to quinones, also producing hydrogen peroxide and semiquinone radicals. This reaction prevents the formation of the highly carcinogenic anti-άiol. Aπtϊ-diols are not themselves substrates for DD yet the addition of DD to a sample comprising an anti-άiol compound results in a significant decrease in the induced mutation rate observed in the Ames test. In this instance, DD is able to bind to and sequester the anti-άiol, even though it is not oxidized. Whether through oxidation or sequestration, DD plays an important role in the detoxification of metabohtes of xenobiotic polycychc compounds (Penning, T.M. (1993) Chemico- Biological Interactions 89:1-34).

15-oxoprostaglandin 13-reductase (PGR) and 15-hydroxyprostaglandin dehydrogenase (15- PGDH) are enzymes present in the lung that are responsible for degrading circulating prostaglandins. Oxidative catabolism via passage through the pulmonary system is a common means of reducing the concentration of circulating prostaglandins. 15-PGDH oxidizes the 15-hydroxyl group of a variety of prostaglandins to produce the corresponding 15-oxo compounds. The 15-oxo derivatives usually have reduced biological activity compared to the 15-hydroxyl molecule. PGR further reduces the 13,14 double bond of the 15-oxo compound which typically leads to a further decrease in biological activity. PGR is a monomer with a molecular weight of approximately 36 kDa. The enzyme requires NADH or NADPH as a cofactor with a preference for NADH. The 15-oxo derivatives of prostaglandins PGE_1; PGE₂, and PGE^ are ah substrates for PGR; however, the non-derivatized prostaglandins (i.e., PGE_1; PGE₂, and PGE_2α) are not substrates (Ensor, CM. et al. (1998) Biochem J. 330:103-108). 15-PGDH and PGR also catalyze the metabohsm of lipoxin A₄ (LXA . Lipoxins (LX) are autacoids, hpids produced at the sites of localized inflammation, which down-regulate polymorphonuclear leukocyte (PMN) function and promote resolution of locahzed trauma. Lipoxin production is stimulated by the administration of aspirin in that cells displaying cyclooxygenase II (COX II) that has been acetylated by aspirin and cells that possess 5-hpoxygenase (5-LO) interact and produce lipoxin. 15-PGDH generates I5-0X0-LXA₄ with PGR further converting the 15-oxo compound to 13,14-dihydro-15-oxo-LXA₄ (Chsh, C.B. et al. (2000) J. Biol. Chem 275:25372-25380). This finding suggests a broad substrate specificity of the prostaglandin dehydrogenases and has implications for these enzymes in drug metabohsm and as targets for therapeutic intervention to regulate inflammation.

The GMC (glucose-methanol-choline) oxidoreductase family of enzymes was defined based on sequence ahgnments of Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase, and Hansenula polymorpha methanol oxidase. Despite their different sources and substrate specificities, these four flavoproteins are homologous, being characterized by the presence of several distinctive sequence and structural features. Each molecule contains a canonical ADP-binding, beta-alpha-beta mononucleotide-binding motif close to the amino terminus. This fold comprises a four-stranded parallel beta-sheet sandwiched between a three-stranded antiparallel beta-sheet and alpha-helices. Nucleotides bind in similar positions relative to this chain fold (Cavener, D.R. (1992) J. Mol. Biol. 223:811-814; Wierenga, R.K. et al. (1986) J. Mol. Biol. 187:101-107). Members of the GMC oxidoreductase family also share a consensus sequence near the central region of the polypeptide. Additional members of the GMC oxidoreductase family include cholesterol oxidases from Brevibacterium sterolicum and Streptomyces; and an alcohol dehydrogenase from Pseudomonas oleovorans (Cavener, supra; Henikoff, S. and J.G. Henikoff (1994) Genomics 19:97-107; van Beilen, J.B. et al. (1992) Mol. Microbiol. 6:3121-3136). IMP dehydrogenase and GMP reductase are two oxidoreductases which share many regions of sequence similarity. IMP dehydrogenase (EC 1.1.1.205) catalyes the NAD-dependent reduction of IMP (inosine monophosphate) into XMP (xanthine monophosphate) as part of de novo GTP biosynthesis (Collart, F.R. and E. Huberman (1988) J. Biol. Chem 263:15769-15772). GMP reductase catalyzes the NADPH-dependent reductive deamination of GMP into IMP, helping to maintain the intraceUular balance of adenine and guanine nucleotides (Andrews, S.C. and J.R. Guest (1988) Biochem J. 255:35-43).

Pyridine nucleotide-disulphide oxidoreductases are FAD flavoproteins involved in the transfer of reducing equivalents from FAD to a substrate. These flavoproteins contain a pair of redox-active cysteines contained within a consensus sequence which is characteristic of this protein family (Kurlyan, J. et al. (1991) Nature 352:172-174). Members of this family of oxidoreductases include glutathione reductase (EC 1.6.4.2); thioredoxin reductase of higher eukaryotes (EC 1.6.4.5); trypanothione reductase (EC 1.6.4.8); lipoamide dehydrogenase (EC 1.8.1.4), the E3 component of alpha-ketoacid dehydrogenase complexes; and mercuric reductase (EC 1.16.1.1). Transferases Transferases are enzymes that catalyze the transfer of molecular groups. The reaction may involve an oxidation, reduction, or cleavage of covalent bonds, and is often specific to a substrate or to particular sites on a type of substrate. Transferases participate in reactions essential to such functions as synthesis and degradation of cell components, and regulation of cell functions including cell signaling, cell prohferation, inflammation, apoptosis, secretion and excretion. Transferases are involved in key steps in disease processes involving these functions. Transferases are frequently classified according to the type of group transferred. For example, methyl transferases transfer one- carbon methyl groups, amino transferases transfer nitrogenous amino groups, and similarly denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or aryl, isoprenyl, saccharyl, i phosphorous-containing, sulfur-containing, or selenium-containing groups, as well as small enzymatic groups such as Coenzyme A.

Acyl transferases include peroxisomal carnitine octanoyl transferase, which is involved in the fatty acid beta-oxidation pathway, and mitochondrial carnitine palmitoyl transferases, involved in fatty acid metabohsm and transport. Chohne 0-acetyl transferase catalyzes the biosynthesis of the neurotransmitter acetylcholine. N-acyltransferase enzymes catalyze the transfer of an amino acid conjugate to an activated carboxyhc group. Endogenous compounds and xenobiotics are activated by acyl-CoA synthetases in the cytosol, microsomes, and mitochondria. The acyl-CoA intermediates are then conjugated with an amino acid (typically glycine, glutamine, or taurine, but also ornithine, arginine, histidine, serine, aspartic acid, and several dipeptides) by N-acyltransferases in the cytosol or mitochondria to form a metabolite with an amide bond. One well-characterized enzyme of this class is the bile acid-Co A: amino acid N-acyltransferase (BAT) responsible for generating the bile acid conjugates which serve as detergents in the gastrointestinal tract (Falany, CN. et al. (1994) J. Biol. Chem 269:19375-19379; Johnson, M.R. et al. (1991) J. Biol. Chem 266:10227-10233). BAT is also useful as a predictive indicator for prognosis of hepatocellular carcinoma patients after partial hepatectomy (Furutani, M. et al. (1996) Hepatology 24:1441-1445). Acetyltransferases

Acetyltransferases have been extensively studied for their role in histone acetylation. Histone acetylation results in the relaxing of the chromatin structure in eukaryotic cells, allowing transcription factors to gain access to promoter elements of the DNA templates in the affected region of the genome (or the genome in general). In contrast, histone deacetylation results in a reduction in transcription by closing the chromatin structure and limiting access of transcription factors. To this end, a common means of stimulating cell transcription is the use of chemical agents that inhibit the deacetylation of histones (e.g., sodium butyrate), resulting in a global (albeit artifactual) increase in gene expression. The modulation of gene expression by acetylation also results from the acetylation of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR, TFIIE, TFIIF and the high mobility group proteins (HMG). In the case of p53, acetylation results in increased DNA binding, leading to the stimulation of transcription of genes regulated by p53. The prototypic histone acetylase (HAT) is Gcn5 from Saccharomyces cerevisiae. Gcn5 is a member of a family of acetylases that includes Tetrahymena p55, human Gcn5, and human p300/CBP. Histone acetylation is reviewed in (Cheung, W.L. et al. (2000) Curr. Opin. CeU Biol. 12:326-333 and Berger, S.L (1999) Curr. Opin. CeU Biol. 11:336-341). Some acetyltransferase enzymes possess the alpha/beta hydrolase fold (Center of Apphed Molecular Engineering Inst. of Chemistry and Biochemistry - University of Salzburg, http://predict.sanger.ac.uk irbm-course97 Docs/ms ) common to several other major classes of enzymes, including but not limited to, acetylcholinesterases and carboxylesterases (Structural Classification of Proteins, http://scop.ixtrc-lmb.carriac.uk/scop/index.html). N-acetyltransferases are cytosolic enzymes which utilize the cofactor acetyl-coenzyme A

(acetyl-CoA) to transfer the acetyl group to aromatic amines and hydrazine containing compounds. In humans, there are two highly similar N-acetyltransferase enzymes, NATl and NAT2; mice appear to have a third form of the enzyme, NAT3. The human forms of N-acetyltransferase have independent regulation (NATl is widely-expressed, whereas NAT2 is in hver and gut only) and overlapping substrate preferences. Both enzymes appear to accept most substrates to some extent, but NATl does prefer some substrates (para-aminobenzoic acid, para-aminosalicylic acid, sulfamethoxazole, and sulfanilamide), while NAT2 prefers others (isoniazid, hydralazine, procainamide, dapsone, ammoglutethimide, and sulfamethazine). A recently isolated human gene, tubedown-1, is homologous to the yeast NAT-1 N-acetyltransferases and encodes a protein associated with acetyltransferase activity. The expression patterns of tubedown-1 suggest that it may be involved in regulating vascular and hematopoietic development (Gendron, R.L. et al. (2000) Dev. Dyn. 218:300- 315).

Amino transferases comprise a family of pyridoxal 5 -phosphate (PLP) -dependent enzymes that catalyze transformations of amino acids. Amino transferases play key roles in protein synthesis and degradation, and they contribute to other processes as weU. For example, GABA aminotransferase (GABA-T) catalyzes the degradation of GABA, the major inhibitory amino acid neurotransmitter. The activity of GABA-T is correlated to neuropsychiatric disorders such as alcoholism, epilepsy, and Alzheimer's disease (Sherif, F.M. and S.S. Ahmed (1995) Clin. Biochem. 28: 145-154). Other members of the family include pyruvate aminotransferase, branched-chain amino acid aminotransferase, tyrosine aminotransferase, aromatic aminotransferase, alanine:glyoxylate aminotransferase (AGT), and kynurenine aminotransferase (Vacca, R.A. et al. (1997) J. Biol. Chem 272:21932-21937). Kynurenine aminotransferase catalyzes the irreversible fransamination ofthe L-tryptophan metabohte L-lsynurenine to formkynurenic acid. The enzyme may also catalyzes the reversible transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate. Kynurenic acid is a putative modulator of glutamatergic neurotransmission, thus a deficiency in 1-ynurenine aminotransferase maybe associated with pleiotropic effects (Buchli, R. et al. (1995) J. Biol. Chem 270:29330-29335).

Glycosyl transferases include the mammahan UDP-glucouronosyl transferases, a family of membrane-bound microsomal enzymes catalyzing the transfer of glucouronic acid to hpophihc substrates in reactions that play important roles in detoxification and excretion of drugs, carcinogens, and other foreign substances. Another mammahan glycosyl transferase, mammahan UDP-galactose- ceramide galactosyl transferase, catalyzes the transfer of galactose to ceramide in the synthesis of galactocerebrosides in myelin membranes of the nervous syste The UDP-glycosyl transferases share a conserved signature domain of about 50 amino acid residues (PROSITE: PDOC00359, http://expasy.hcuge.ch/sprot/prosite.html).

Methyl transferases are involved in a variety of pharmacologicaUy important processes. Nicotinamide N-methyl transferase catalyzes the N-methylation of nicotinamides and other pyridines, an important step in the ceUular handling of drugs and other foreign compounds. Phenylemanolamine N-methyl transferase catalyzes the conversion of noradrenalin to adrenalin. 6-0-methylguanine-DNA methyl transferase reverses DNA mefhylation, an important step in carcinogenesis. Uroporphyrin-III C-methyl transferase, which catalyzes the transfer of two methyl groups fromS-adenosyl-L- methionine to uroporphyrinogen III, is the first specific enzyme in the biosynthesis of cobalamin, a dietary enzyme whose uptake is deficient in pernicious anemia. Protein-arginine methyl transferases catalyze the posttranslational methylation of arginine residues in proteins, resulting in the mono- and dimethylation of arginine on the guanidino group. Substrates include histones, myelin basic protein, and heterogeneous nuclear ribonucleoproteins involved in mRNA processing, splicing, and transport. Protein-arginine methyl transferase interacts with proteins upregulated by rnitogens, with proteins involved in chronic lymphocytic leukemia, and with interferon, suggesting an important role for methylation in cytokine receptor signaling (Lin, W.-J. et al. (1996) J. Biol. Chem. 271:15034-15044; Abramovich, C. et al. (1997) EMBO J. 16:260-266; and Scott, H. S. et al. (1998) Genomics 48:330- 340).

Phospho transferases catalyze the transfer of high-energy phosphate groups and are important in energy-requiring and -releasing reactions. The metabolic enzyme creatine kinase catalyzes the reversible phosphate transfer between creat ne/creatine phosphate and ATP/ADP. Glycocyamine kinase catalyzes phosphate transfer from ATP to guanidoacetate, and arginine kinase catalyzes phosphate transfer from ATP to arginine. A cysteine-containing active site is conserved in this famfly (PROSITE: PDOC00103).

Prenyl transferases are heterodimers, consisting of an alpha and a beta subunit, that catalyze the transfer of an isoprenyl group. The Ras farnesyltransferase (FTase) enzyme transfers a farnesyl moiety from cytosohc famesylpyrophosphate to a cysteine residue at the carboxyl terminus of the Ras oncogene protein. This modification is required to anchor Ras to the ceU membrane so that it can perform its role in signal transduction. FTase inhibitors block Ras function and demonstrate antitumor activity_(Buolamwini, J.K. (1999) Curr. Opin. Chem Biol. 3:500-509). Ftase, which shares structural similarity with geranylgeranyl transferase, or Rab GG transferase, prenylates Rab proteins, aUowing them to perform their roles in regulating vesicle transport (Seabra, M.C (1996) J. Biol. Chem 271:14398-14404).

Saccharyl transferases are gfycating enzymes involved in a variety of metabolic processes. Ohgosaccharyl transferase-48, for example, is a receptor for advanced glycation endproducts, which accumulate in vascular complications of diabetes, macrovascular disease, renal insufficiency, and Alzheimer's disease (ThornaUey, P. J. (1998) CeU Mol. Biol. (Noisy-Le-Grand) 44:1013-1023).

Coenzyme A (CoA) transferase catalyzes the transfer of CoA between two carboxyhc acids. Succinyl CoA:3-oxoacid CoA transferase, for example, transfers CoA from succinyl-CoA to a recipient such as acetoacetate. Acetoacetate is essential to the metabohsm of ketone bodies, which accumulate in tissues affected by metabohc disorders such as diabetes (PROSITE: PDOC00980). Transglutaminase transferases (Tgases) are Ca²⁺ dependent enzymes capable of forming isopeptide bonds by catalyzing the transfer of the γ-carboxy group from protein-bound glutamine to the ε-amino group of protein-bound lysine residues or other primary amines. Tgases are the enzymes responsible for the cross-linking of cornified envelope (CE), the highly insoluble protein structure on the surface of comeocytes, into a chemicaUy and mechanicaUy resistant protein polymer. Seven known human Tgases have been identified. Individual transglutaminase gene products are specialized in the cross-linking of specific proteins or tissue structures, such as factor XHIa which stabilizes the fibrin clot inhemostasis, prostrate transglutaminase which functions in semen coagulation, and tissue transglutaminase which is involved in GTP-binding in receptor signaling. Four (Tgases 1, 2, 3, and X) are expressed in terminaUy differentiating epithelia such as the epidermis. Tgases are critical for the proper cross-linking of the CE as seen in the pathology of patients suffering from one form of the skin diseases referred to as congenital ichthyosis which has been linked to mutations in the keratinocyte transglutaminase (TG^) gene (Nemes, Z. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:8402-8407, Aeschlimann, D. et al. (1998) J. Biol. Chem 273:3452- 3460.) Hydrolases

Hydrolases are a class of enzymes that catalyze the cleavage of various covalent bonds in a substrate by the introduction of a molecule of water. The reaction involves a nucleophilic attack by the water molecule's oxygen atom on a target bond in the substrate. The water molecule is spht across the target bond, breaking the bond and generating two product molecules. Hydrolases participate in reactions essential to such functions as synthesis and degradation of ceU components, and for regulation of ceU functions including ceU signaling, ceU proliferation, inflamation, apoptosis, secretion and excretion. Hydrolases are involved in key steps in disease processes involving these functions. Hydrolytic enzymes, or hydrolases, may be grouped by substrate specificity into classes including phosphatases, peptidases, lysophospholipases, phosphodiesterases, glycosidases, glyoxalases, aminohydrolases, carboxylesterases, sulfatases, phosphohydrolases, nucleotidases, lysozymes, and many others.

Phosphatases hydrolyticaUy remove phosphate groups from proteins, an energy-providing step that regulates many ceUular processes, including intraceUular signaling pathways that in turn control cell growth and differentiation, ceU-ceU contact, the ceU cycle, and oncogenesis.

Peptidases, also caUed proteases, cleave peptide bonds that form the backbone of peptide or protein chains. Proteolytic processing is essential to ceU growth, differentiation, remodeling, and homeostasis as weU as inflammation and the immune response. Since typical protein half-hves range from hours to a few days, peptidases are continuaUy cleaving precursor proteins to their active form, removing signal sequences from targeted proteins, and degrading aged or defective proteins.

Peptidases function in bacterial, parasitic, and viral invasion and rephcation within a host. Examples of peptidases include trypsin and chymotrypsin (components of the complement cascade and the blood-clotting cascade) lysosomal cathepsins, calpains, pepsin, renin, and chymosin (Beynon, R. J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York, NY, pp. 1-5).

Lysophosphohpases (LPLs) regulate intraceUular lipids by catalyzing the hydrolysis of ester bonds to remove an acyl group, a key step in hpid degradation. SmaU LPL isoforms, approximately 15-30 kD, function as hydrolases; larger isoforms functionboth as hydrolases and transacylases. A particular substrate for LPLs, lysophosphatidylcholine, causes lysis of ceU membranes. LPL activity is regulated by signaling molecules important in numerous pathways, including the inflammatory response.

The phosphodiesterases catalyze the hydrolysis of one of the two ester bonds in a phosphodiester compound. Phosphodiesterases are therefore crucial to a variety of ceUular processes. Phosphodiesterases include DNA and RNA endo- and exo-nucleases, which are essential to ceU growth and rephcation as weU as protein synthesis. Endonuclease V (deoxyinosine 3'-endonuclease) is an example of a type II site-specific deoxyribonuclease, a putative DNA repair enzyme that cleaves DNAs containing hypoxanthine, uracU, or mismatched bases. Escherichia coli endonuclease V has been shown to cleave DNA containing deoxyxanthosine at the second phosphodiester bond 3' to deoxyxanthosine, generating a 3'-hydroxyl and a 5'-phosphoryl group at the nick site (He, B. et al. (2000) Mutat. Res. 459:109-114). It has been suggested that Escherichia coli endonuclease V plays a role in the removal of deaminated guanine, i.e., xanthine, from DNA, thus helping to protect the ceU against the mutagenic effects of nitrosative deamination (Schouten, K.A. and B. Weiss (1999) Mutat. Res. 435:245-254). In eukaryotes, the process of tRNA splicing requires the removal of smaU tRNA introns that interrupt the anticodon loop 1 base 3' to the anticodon. This process requires the stepwise action of an endonuclease, a hgase, and a phosphotransferase (Hong, L. et al. (1998) Science 280:279-284). Ribonuclease P (RNase P) is a ubiquitous RNA processing endonuclease that is required for generating the mature tRNA 5 '-end during the tRNA splicing process. This is accomplished through the catalysis of the cleavage of P-3'0 bonds to produce 5 '-phosphate and 3'- hydroxyl end groups at a specific site on pre-tRNA. Catalysis by RNase P is absolutely dependent on divalent cations such as Mg²⁺ or Mn²⁺(Kurz, J.C. et al. (2000) Curr. Opin. Chem Biol. 4:553-558). Substrate recognition mechanisms of RNase P are weU conserved among eukaryotes and bacteria (Fabbri, S. et al. (1998) Science 280:284-286). In Saccharomyces cerevisiae, POP1 ('processing of precursor RNAs') encodes a protein component of both RNase P and RNase MRP, another RNA processing protein. Mutations in yeast POP1 are lethal (Lygerou, Z. et al. (1994) Genes Dev. 8:1423- 1433). Another phosphodiesterase, acid sphmgomyelinase, hydrolyzes the membrane phosphohpid sphingomyelin to ceramide and phosphorylcholine. Phosphorylcholine functions in synthesis of phosphatidylcholine, which is involved in intraceUular signaling pathways. Ceramide is an essential precursor for the generation of gangliosides, membrane hpids found in high concentration in neural tissue. Defective acid sphmgomyelinase phosphodiesterase leads to Niemann-Pick disease. Glycosidases catalyze the cleavage of hemiacetyl bonds of glycosides, which are compounds that contain one or more sugar. Mammahan lactase-p orizin hydrolase, for example, is an intestinal enzyme that sphts lactose. Mammahan beta-galactosidase removes the terminal galactose from gangliosides, glycoproteins, and glycosaminoglycans, and deficiency of this enzyme is associated with a gangliosidosis known as Morquio disease type B (PROSITE PCDOC00910). Vertebrate lysosomal alpha-glucosidase, which hydrolyzes glycogen, maltose, and isomaltose, and vertebrate intestinal sucrase-isomaltase, which hydrolyzes sucrose, maltose, and isomaltose, are widely distributed members of this family with highly conserved sequences at their active sites.

The glyoxylase system is involved in gluconeogenesis, the production of glucose from storage compounds in the body. It consists of glyoxylase I, which catalyzes the formation of S-D- lactoylglutathione frommethyglyoxal, a side product of triose-phosphate energy metabohsm, and glyoxylase II, which hydrolyzes S-D-lactoylglutathione to D-lactic acid and reduced glutathione. Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes mellitus, the detoxification of bacterial toxins, and in the control of ceU prohferation and microtubule assembly.

NG,NG-ά me ylarginine dimemylaminohydrolase (DDAH) is an enzyme that hydrolyzes the endogenous nitric oxide synthase (NOS) inhibitors, NG-monomemyl-arginine and NG,NG-dimethyl- L-arginine, to L-citrulline. -hhibiting DDAH can cause increased intraceUular concentration of NOS inhibitors to levels sufficient to inhibit NOS. Therefore, DDAH inhibition may provide a method of NOS inhibition, and changes in the activity of DDAH could play a role in pathophysiological alterations in nitric oxide generation (MacAlhster, R.J. et al. (1996) Br. J. Pharmacol. 119:1533- 1540). DDAH was found in neurons displaying cytoskeletal abnormaUties and oxidative stress in Alzheimer's disease. In age-matched control cases, DDAH was not found in neurons. This suggests that oxidative stress- and nitric oxide-mediated events play a role in the pathogenesis of Alzheimer's disease (Smith, M.A. et al. (1998) Free Rad. Biol. Med. 25:898-902).

Acyl-CoA thioesterase is another member of the carboxylesterase family (Alexson, S.E. et al. (1993) Eur. J. Biochem. 214:719-727). Evidence suggests that acyl-CoA thioesterase has a regulatory role in steroidogenic tissues (Finkielstein, C. et al. (1998) Eur. J. Biochem. 256:60-66).

The alpha/beta hydrolase protein fold is common to several hydrolases of diverse phylogenetic origin and catalytic function. Enzymes with the alpha/beta hydrolase fold have a common core structure consisting of eight beta-sheets connected by alpha-hehces. The most conserved structural feature of this fold is the loops of the nucleophUe-histidine-acid catalytic triad. The histidine in the catalytic triad is completely conserved, while the nucleophile and acid loops accommodate more than one type of amino acid (Ollis, D.L. et al. (1992) Protein Eng. 5:197-211).

Sulfatases are members of a highly conserved gene family that share extensive sequence homology and a high degree of structural similarity. Sulfatases catalyze the cleavage of sulfate esters. To perform this function, sulfatases undergo a unique post-translational modification in the endoplasmic reticulum that involves the oxidation of a conserved cysteine residue. A human disorder caUed multiple sulfatase deficiency is due to a defect in this post-translational modification step, leading to inactive sulfatases (Recksiek, M. et al. (1998) J. Biol. Chem 273:6096-6103).

Phosphohydrolases are enzymes that hydrolyze phosphate esters. Some phosphohydrolases contain a mutT domain signature sequence. MutT is a protein involved in the GO system responsible for removing an oxidatively damaged form of guanine from DNA. A region of about 40 amino acid residues, found in the N-terminus of mutT, is also found in other proteins, including some phosphohydrolases (PROSITE PDOC00695).

Serine hydrolases are a large functional class of hydrolytic enzymes that contain a serine residue in their active site. This class of enzymes contains proteinases, esterases, and Upases which hydrolyze a variety of substrates and, therefore, have different biological roles. Proteins in this superfamuy can be further grouped into subfamilies based on substrate specificity or amino acid siix larities (Puente, X.S. and C Lopez-Otin (1995) J. Biol. Chem 270:12926-12932).

Neuropathy target esterase (NTE) is an integral membrane protein present in ah neurons and in some non-neural-ceU types of vertebrates. NTE is involved in a ceU-signaling pathway controlling interactions between neurons and accessory glial cells in the developing nervous system. NTE has serine esterase activity and efficiently catalyses the hydrolysis of phenyl valerate (PV) in vitro, but its physiological substrate is unknown. NTE is not related to either the major serine esterase famuy, which includes acetylcholinesterase, nor to any other known serine hydrolases. NTE contains at least two functional domains: an N-terminal putative regulatory domain and a C-terminal effector domain which contains the esterase activity and is, in part, conserved in proteins found in bacteria, yeast, nematodes and insects. NTE's effector domain contains three predicted transmembrane segments, and the active-site serine residue hes at the center of one of these segments. The isolated recombinant domain shows PV hydrolase activity only when incorporated into phospholipid liposomes. NTE's esterase activity is largely redundant in adult vertebrates, but organophosphates which react with NTE in vivo initiate unknown events which lead to a neuropathy with degeneration of long axons. These neuropathic organophosphates leave a negatively charged group covalently attached to the active-site serine residue, which causes a toxic gain of function in NTE (Glynn, P. (1999) Biochem. J. 344:625-631). Further, the Drosophila neurodegeneration gene swiss-cheese encodes a neuronal protein involved in gha-neuron interaction and is homologous to the above human NTE (Moser, M. et al. (2000) Mech. Dev. 90:279-282).

Chitinases are chitin-degrading enzymes present in a variety of organisms and participate in processes including ceU waU remodeling, defense and catabohsm. Chitinase activity has been found inhuman serum, leukocytes, granulocytes, and in association with fertilized oocytes in mammals (Escott, G.M. (1995) Infect. Immunol. 63:4770-4773; DeSouza, M.M. (1995) Endrocrinology

136:2485-2496). Glycolytic and proteolytic molecules in humans are associated with tissue damage in lung diseases and with increased tumorigenicity and metastatic potential of cancers (Mulligan, M.S. (1993) Proc. Natl Acad. Sci. 90:11523-11527; Matrisian, L.M. (1991) Am J. Med. Sci. 302:157-162; Witty, J.P. (1994) Cancer Res. 54:4805-4812). The discovery of a human enzyme with chitinolytic activity is noteworthy given the lack of endogenous chitin in the human body (Raghavan, N. (1994) Infect. Immun. 62:1901-1908). However, there is a group of mammahan proteins that share homology with chitinases from various non-mammalian organisms, such as bacteria, fungi, plants, and insects. The members of this fam y differ in their abihty to hydrolyze chitin or chitin-like substrates. Some of the mammahan members of the famUy, such as a bovine whey chitotriosidase and human cartilage proteins which do not demonstrate specific chitinolytic activity, are expressed in association with tissue remodeling events (Rejman, J.J. (1988) Biochem. Biophys. Res. Coirimun. 150:329-334, Nyirkos, P. (1990) Biochem J. 268:265-268). Elevated levels of human cartilage proteins have been reported in the synovial fluid and cartilage of patients with rheumatoid arthritis, a disease which produces a severe degradation of the cartilage and a proliferation of the synovial membrane in the affected joints (Hakala, B.E. (1993) J. Biol. Chem 268:25803-25810).

A smaU subclass of hydrolases acting on ether bonds includes the thioether hydrolases. S- adenosyl-L-homocysteine hydrolase, also known as AdoHcyase or SAHH (PROSITE PDOC00603; EC 3.3.1.1), is a thioether hydrolase first described in rat hver extracts as the activity responsible for the reversible hydrolysis of 5-adenosyl-L-homocysteine (AdoHcy) to adenosine and homocysteine (Sganga, M.W. et al. (1992) PNAS 8'9:6328-6332). SAHH is a cytosohc enzyme that has been found in aU ceUs that have been tested, with the exception of Escherichia coli and certain related bacteria (Walker, R.D. et al. (1975) Can. J. Biochem 53:312-319; Shimizu, S. et al. (1988) FEMS Microbiol. Lett. 51:177-180; Shimizu, S. et al. (1984) Eur. J. Biochem 141:385-392). SAHH activity is dependent on NAD⁺ as a cofactor. Deficiency of SAHH is associated with hypermet oninemia (Online Mendelian Inheritance in Man (OMIM) #180960 HypermetMoninemia), a pathologic condition characterized by neonatal cholestasis, faUure to thrive, mental and motor retardation, facial dysmorphism with abnormal hair and teeth, and myocaridopathy (Labrune, P. et al. (1990) J. Pediat. 117:220-226).

Another subclass of hydrolases includes those enzymes which act on carbon-nitrogen (C-N) bonds other than peptide bonds. To this subclass belong those enzymes hydrolyzing amides, amidines, and other C-N bonds. This subclass is further subdivided on the basis of substrate specificity such as linear amides, cyclic amides, linear amidines, cychc amidines, nitriles and other compounds. A hydrolase belonging to the sub-subclass of enzymes acting on the cychc amidines is adenosine deaminase (ADA). ADA catalyzes the breakdown of adenosine to inosine. ADA is present in many mammahan tissues, including placenta, muscle, lung, stomach, digestive diverticulum, spleen, erythrocytes, fhymus, seminal plasma, thyroid, T-ceUs, bone marrow stemceUs, and Uver. A subclass of AD As, ADAR, act on RNA and are classified as RNA editases. An ADAR fromDrosophila, dADAR, expressed in the developing nervous system, may act on para voltage- gated Na+ channel transcripts in the central nervous system (PaUadino, M.J. et al. (2000) RNA 6:1004-1018). ADA deficiency causes profound lymphopenia with severe combined immunodeficiency (SCID). CeUs from patients with ADA deficiency contain low, sometimes undetectable, amounts of ADA catalytic activity and ADA protein. ADA deficiency stems from genetic mutations in the ADA gene (Hershfield, M.S. (1998) Semin. Hematol. 4:291-298). Metabohc consequences of ADA deficiency are associated with defects in alveogenesis, pulmonary inflammation, and airway obstruction (Blackburn, M.R. et al. (2000) J. Exp. Med. 192:159-170). Pancreatic ribonucleases (RNase) are pyrimidine-specific endonucleases found in high quantity in the pancreas of certain mammahan taxa and of some reptiles (Beintema, J. J. et al (1988) Prog. Biophys. Mol. Biol. 51:165-192). Proteins in the mammalian pancreatic RNase superfam y are noncytosolic endonucleases that degrade RNA through a two-step transphosphorolytic-hydrolytic reaction (Beintema, J.J. et al. (1986) Mol. Biol Evol 3:262-275). SpecificaUy, the enzymes are involved in endonucleolytic cleavage of 3'-phosphomononucleotides and 3'-phosphooligonucleotides ending in C-P or U-P with 2 ',3 -cychc phosphate intermediates. Ribonucleases can unwind the DNA hehx by complexing with single-stranded DNA; the complex arises by an extended multi-site cation-anion interaction between lysine and arginine residues of the enzyme and phosphate groups of the nucleotides. Some of the enzymes belonging to this famuy appear to play a purely digestive role, whereas others exhibit potent and unusual biological activities (D'Alessio, G. (1993) Trends CeU Biol. 3:106-109). Proteins belonging to the pancreatic RNase famUy include: bovine seminal vesicle and brain ribonucleases; kidney non-secretory ribonucleases (Beintema, J.J. et al (1986) FEBS Lett. 194:338-343); Uver-type ribonucleases (Rosenberg, H.F. et al. (1989) PNAS U.S.A. 86:4460-4464); angiogenin, which induces vascularisation of normal and malignant tissues; eosinophil cationic protein (Hofsteenge, J. et al. (1989) Biochemistry 28:9806-9813), a cytotoxin and helminthotoxin with ribonuclease activity; and frog hver ribonuclease and frog sialic acid-binding lectin. The sequences of pancreatic RNases contain 4 conserved disulfide bonds and 3 amino acid residues involved in the catalytic activity. ADP-ribosylation is a reversible post-translational protein modification in which an ADP- ribose moiety is transferred from β-NAD to a target amino acid such as arginine or cysteine. ADP- ribosylarginine hydrolases regenerate arginine by removing ADP-ribose from the protein, completing the ADP-ribosylation cycle (Moss, J. et al. (1997) Adv. Exp. Med. Biol. 419:25-33). ADP- ribosylation is a weU-known reaction among bacterial toxins. Cholera toxin, for example, disrupts the adenylyl cyclase system by ADP-ribosylating the α-subunit of the stimulatory G-protein, causing an increase in intraceUular cAMP (Moss, J. and M. Vaughan (Eds) (1990) ADP-ribosylating Toxins and G-Proteins: Insights into Signal Transduction, American Society for Microbiology, Washington, D.C). ADP-ribosylation may also have a regulatory function in eukaryotes, affecting such processes as cytoskeletal assembly (Zhou, H. et al. (1996) Arch. Biochem Biophys. 334:214-222) and ceU proUferation in cytotoxic T-ceUs (Wang, J. et al. (1996) J. Immunol. 156:2819-2827).

Nucleotidases catalyze the formation of free nucleosides from nucleotides. The cytosohc nucleotidase cN-I (5' nucleotidase-I) cloned from pigeon heart catalyzes the formation of adenosine from AMP generated during ATP hydrolysis (Sala-Newby, G.B. et al. (1999) J. Biol. Chem 274:17789-17793). Increased adenosine concentration is thought to be a signal of metabohc stress, and adenosine receptors mediate effects including vasodUation, decreased stimulatory neuron firing and ischemic preconditioning in the heart (Schrader, J. (1990) Circulation 81:389-391; Rubino, A. et al. (1992) Eur. J. Pharmacol. 220:95-98; de Jong, J.W. et al. (2000) Pharmacol Ther. 87:141-149). Deficiency of pyrimidine 5'-nucleotidase can result in hereditary hemolytic anemia (OMIM #266120). The lysozyme c superfamUy consists of conventional lysozymes c, calcium-binding lysozymes c, and α-lactalbumin (Prager, E.M. and P. JoUes (1996) EXS 75:9-31). The proteins in this superfamily have 35-40% sequence homology and share a common three-dimensional fold, but can have different functions. Lysozymes c are ubiquitous in a variety of tissues and secretions and can lyse the ceU waUs of certain bacteria (McKenzie, H.A. (1996) EXS 75:365-409). Alpha-lactalbumin is a metaUo-protein that binds calcium and participates in the synthesis of lactose (Iyer, L.K. and P.K. Qasba (1999) Protein Eng. 12:129-139). Alpha-lactalbumin occurs in mammahan milk and colostrum (McKenzie, supra).

Lysozymes catalyze the hydrolysis of certain mucopolysaccharides of bacterial cell waUs, specificaUy, the beta (1-4) glycosidic linkages between N-acetylmuramic acid and N- acetylglucosamine, and cause bacterial lysis. Lysozymes occur in diverse organisms including viruses, birds, and mammals. In humans, lysozymes are found in spleen, lung, kidney, white blood ceUs, plasma, saliva, milk, tears, and cartilage (OMIM #153450 Lysozyme; Weaver, L.H. et al. (1985) J. Mol. Biol. 184:739-741). Lysozyme c functions in raminants as a digestive enzyme, releasing proteins from ingested bacterial ceUs, and may perform the same function in human newborns (Braun, O.H. et al. (1995) Klin. Pediatr. 207:4-7). The two known forms of lysozymes, chicken-type and goose-type, were originaUy isolated from chicken and goose egg white, respectively. Chicken-type and goose-type lysozymes have simUar tlκee-dimensional structures, but different amino acid sequences (Nakano, T. and T. Graf (1991) Biochim. Biophys. Acta 1090:273-276). In chickens, both forms of lysozyme are found in neutrophU granulocytes (heterophUs), but only chicken-type lysozyme is found in egg white. GeneraUy, chicken-type lysozyme mRNA is found in both adherent monocytes and macrophages and nonadherent promyelocytes and granulocytes as weU as in ceUs of the bone marrow, spleen, bursa, and oviduct. Goose-type lysozyme mRNA is found in non-adherent ceUs of the bone marrow and lung. Several isozymes have been found in rabbits, including leukocytic, gastrointestinal, and possibly lymphoepithelial forms (OMIM #153450, supra; Nakano and Graf, supra; and GenBank Gl 1310929). A human lysozyme gene encoding a protein similar to chicken-type lysozyme has been cloned (Yoshimura, K. et al. (1988) Biochem Biophys. Res. Commun. 150:794-801). A consensus motif featuring regularly spaced cysteine residues has been derived from the lysozyme C enzymes of various species (PROSITE PS00128). Lysozyme C shares about 40% amino acid sequence identity with α-lactalbumin. Lysozymes have several disease associations. Lysozymuria is observed in diabetic nephropathy (Shima, M. et al. (1986) Clin. Chem. 32:1818-1822), endemic nephropathy (Bruckner, I. et al. (1978) Med. Interne. 16:117-125), urinary tract infections (Heidegger, H. (1990) Minerva Ginecol 42:243-250), and acute monocytic leukemia (Shaw, M.T. (1978) Am J. Hematol. 4:97-103). Nakano and Graf (supra) suggested a role for lysozyme in host defense systems. Older rabbits with an inherited lysozyme deficiency show increased susceptibihty to infections, such as subcutaneous abscesses (OMIM #153450, supra). Human lysozyme gene mutations cause hereditary systemic amyloidosis, a rare autosomal dominant disease in which amyloid deposits form in the viscera, including the kidney, adrenal glands, spleen, and hver. This disease is usuaUy fatal by the fifth decade. The amyloid deposits contain variant forms of lysozyme. Renal amyloidosis is the most common and potentiaUy the most serious form of organ involvement (Pepys, M.B. et al. (1993) Nature 362:553-557; OMIM #105200 Familial Visceral Amyloidosis; Cotran, R.S. et al. (1994) Robbins Pathologic Basis of Disease, W.B. Saunders Company, PhUadelphia PA, pp. 231-238). Increased levels of lysozyme and lactate have been observed in the cerebrospinal fluid of patients with bacterial meningitis (Ponka, A. et al. (1983) Infection 11:129-131). Acute monocytic leukemia is characterized by massive lysozymuria (Den Tandt, W.R. (1988) Int. J. Biochem 20:713-719). Lyases

Lyases are a class of enzymes that catalyze the cleavage of C-C, C-O, C-N, C-S, C-(halide), P-O, or other bonds without hydrolysis or oxidation to form two molecules, at least one of which contains a double bond (Stryer, L. (1995) Biochemistry, W.H. Freeman and Co., New York NY, p.620). Under the International Classification of Enzymes (Webb, E. C. (1992) Enzyme

Nomenclature 1992: Recommendations ofthe Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes, Academic Press, San Diego CA), lyases form a distinct class designated by the numeral 4 in the first digit of the enzyme number (i.e., EC 4.x.x.x). Further classification of lyases reflects the type of bond cleaved as weU as the nature of the cleaved group. The group of C-C lyases includes carboxyl-lyases (decarboxylases), aldehyde-lyases (aldolases), oxo-acid-lyases, and other lyases. The C-0 lyase group includes hydro-lyases, lyases acting on polysaccharides, and other lyases. The C-N lyase group includes ammonia-lyases, amidine- lyases, amine-lyases (deaminases), and other lyases. Lyases are critical components of ceUular biochemistry, with roles in metabohc energy production, including fatty acid metabohsm and the tricarboxylic acid cycle, as weU as other diverse enzymatic processes.

One important family of lyases are the carbonic anhydrases (CA), also caUed carbonate dehydratases, which catalyze the hydration of carbon dioxide in the reaction H₂0 + C0₂ * HC0₃ ^" + H⁺ . C A accelerates this reaction by a factor of over 10⁶ by virtue of a zinc ion located in a deep cleft about 15A below the protein's surface and co-ordinated to the imidazole groups of three His residues. Water bound to the zinc ion is rapidly converted to HC0₃ ^".

Fourteen enzymatic and evolutionarily related forms of carbonic anhydrase are currently known to exist in humans: three cytosohc isozymes (CAI, CAII, and CAIII), two membrane-bound forms (CAIV and CAVII), a mitochondrial form (CAV), a secreted salivary form (CAVT) and a yet uncharacterized isozyme (PROSITE PDOC00146 Eukaryotic-type carbonic anhydrases signature). Though the isoenzymes CAI, CAII, and bovine CAIII have simUar secondary structures and polypeptide-chain folds, CAI has 6 tryptophans, CAII has 7 and CAIII has 8 (Boren, K. et al (1996) Protein Sci. 5:2479-2484). CAII is the predominant CA isoenzyme in the brain of mammals.

CAs participate in a variety of physiological processes that involve pH regulation, C0₂ and HC0₃ ^" transport, ion transport, and water and electrolyte balance. For example, CAII contributes to H⁺ secretion by gastric parietal ceUs, by renal tubular ceUs, and by osteoclasts that secrete H⁺ to acidify the bone-resofbing compartment. In addition, CAII promotes HC0₃ ^" secretion by pancreatic duct ceUs, diary body epithehum, choroid plexus, salivary gland acinar ceUs, and distal colonal epithehum, thus playing a role in the production of pancreatic juice, aqueous humor, cerebrospinal fluid, and saliva, and contributing to electrolyte and water balance. CAII also promotes C0₂ exchange in proximal tubules in the kidney, in erythrocytes, and in lung. CAIV has roles in several tissues: it facilitates HC0₃ ^" reabsorption in the kidney; promotes C0₂ flux in tissues including brain, skeletal muscle, and heart muscle; and promotes C0₂ exchange from the blood to the alveoh in the lung. CAVT probably plays a role in pH regulation in sahva, along with CAII, and may have a protective effect in the esophagus and stomach. Mitochondrial CAV appears to play important roles in gluconeogenesis and ureagenesis, based on the effects of CA inhibitors on these pathways. (Sly, W.S. and P.Y. Hu (1995) Ann. Rev. Biochem 64:375-401.)

The CA XII gene was cloned from a renal ceU carcinoma (RCC) and has been mapped to 15q22. The cDNA sequence encodes a deduced 354-amino acid protein with a predicted molecular mass of 39,448 Da and features of a type I membrane protein. The extraceUular CA domain shows 30 to 42% simUarity with known human CAs, contains aU 3 zinc-binding histidine residues found in active CAs, and contains 2 potential sites for asparagine glycosylation. CA XII has been found in normal human kidney, colon, prostate, and activated lymphocytes and to a lesser extent in pancreas, ovary, and testis. CA XII is overexpressed in 10% of clear ceU renal carcinomas, as compared with the corresponding normal renal tissue. Sequencing revealed no differences between the RCC-derived cDNA and a CA XII cDNA isolated from normal kidney (Tureci, O. et al (1998) Proc. Nat. Acad. Sci. U. S. A. 95: 7608-7613; Ivanov, S. V. et al (1998) Proc. Nat. Acad. Sci. U. S. A. 95:12596-12601).

CA XIV is a type I transmembrane enzyme containing an extracellular CA domain. The CA XIV protein is 337 amino acids in length and contains conserved active-site residues simUar to other CAs. It has residues which form an active site hydrogen-bonded network, including zinc-liganded histidine residues which are essential to CA activity. CA XIV is most similar to CA XII. CA XIV mRNA is most strongly expressed in kidney, heart, but also is expressed in skeletal muscle, liver, brain, and lung. In mouse kidney, CA XIV is intensely expressed in the proximal convoluted tubule where bicarbonate reabsorption and urinary acidification occur. CA XIV is also expressed on the plasma membranes of neurons in many brain regions, including hippocampus. This pattern of expression may be involved in modulating synaptic transmission through extraceUular pH regulation. Thus, CA XIV is a transmembrane CA that is implicated in regulating biocarbonate levels in the extraceUular space of many tissues (Kaunisto, K. et al. (2002) Kidney Int. 61:2111-2118; Mori, K. et al. (1999) J. Biol. Chem. 274:15701-15705).

A number of disease states are marked by variations in CA activity. Mutations in CAII which lead to CAII deficiency are the cause of osteopetrosis with renal tubular acidosis (OMIM #259730 Osteopetrosis with Renal Tubular Acidosis). The concentration of CAII in the cerebrospinal fluid (CSF) appears to mark disease activity in patients with brain damage. High CA concentrations have been observed in patients with brain infarction. Patients with transient ischemic attack, multiple sclerosis, or ep epsy usuaUy have CAII concentrations in the normal range, but higher CAII levels have been observed in the CSF of those with central nervous system infection, dementia, or trigeminal neuralgia (Parkldla, A.K. et al. (1997) Eur. J. Clin. Invest. 27:392-397). Colonic adenomas and adenocarcinomas have been observed to faU to stain for CA, whereas non-neoplastic controls showed CAI and CAII in the cytoplasm of the columnar ceUs lining the upper half of colonic crypts. The neoplasms show staining patterns similar to less mature ceUs lining the base of normal crypts (Gramlich T.L. et al. (1990) Arch. Pathol. Lab. Med. 114:415-419).

Therapeutic interventions in a number of diseases involve altering CA activity. CA inhibitors such as acetazolamide are used in the treatment of glaucoma (Stewart, W.C (1999) Curr. Opin. Opthamol. 10:99-108), essential tremor and Parkinson's disease (Uitti, R.J. (1998) Geriatrics 53:46- 48, 53-57), intermittent ataxia (Singhvi, J.P. et al (2000) Neurology India 48:78-80), and altitude related illnesses (Klocke, D.L. et al. (1998) Mayo Clin. Proc. 73:988-992).

C A activity can be particularly useful as an indicator of long-term disease conditions, since the enzyme reacts relatively slowly to physiological changes. CAI and zinc concentrations have been observed to decrease in hyperthyroid Graves' disease (Yoshida, K. (1996) Tohoku J. Exp. Med. 178:345-356) and glycosylated CAI is observed in diabetes mellitus (Kondo, T. et al. (1987) Clin. Chim Acta 166:227-236). A positive correlation has been observed between CAI and CAII reactivity and endometriosis (Brinton, D.A. et al. (1996) Ann. Clin. Lab. Sci. 26:409-420; D'Cruz , O.J. et al. (1996) Fertil. Steril. 66:547-556). Another important member of the lyase family is orrύthine decarboxylase (ODC), the initial rate-limiting enzyme in polyamine biosynthesis. ODC catalyses the transformation of ornithine into putrescine in the reaction L-orrήthine ^ putrescine + C0₂. Polyamines, which include putrescine and the subsequent metabohc pathway products spermidine and spemiine, are ubiquitous ceU components essential for DNA synthesis, ceU differentiation, and prohferation. Thus the polyamines play a key role in tumor prohferation (Medina, M.A. et al. (1999) Biochem Pharmacol. 57:1341-1344).

ODC is a pyridoxal-5 -phosphate (PLP)-dependent enzyme which is active as a homodimer. Conserved residues include those at the PLP binding site and a stretch of glycine residues thought to be part of a substrate binding region (PROSITE PDOC00685 Orn/DAP/Arg decarboxylase famuy 2 signatures). Mammahan ODCs also contain PEST regions, sequence fragments enriched in proline, glutamic acid, serine, and threonine residues that act as signals for intraceUular degradation (Medina et al, supra).

Many chemical carcinogens and tumor promoters increase ODC levels and activity. Several known oncogenes may increase ODC levels by enhancing transcription of the ODC gene, and ODC itself may act as an oncogene when expressed at very high levels. A high level of ODC is found in a number of precancerous conditions, and elevation of ODC levels has been used as part of a screen for tiimor-promoting compounds (Pegg, A.E. et al. (1995) J. CeU. Biochem Suppl 22:132-138).

Inhibitors of ODC have been used to treat tumors in animal models and human clinical trials, and have been shown to reduce development of tumors of the bladder, brain, esophagus, gastrointestinal tract, lung, oral cavity, mammary gland, stomach, skin and trachea (Pegg et al, supra; McCann, P.P. and A.E. Pegg (1992) Pharmac. Ther. 54:195-215). ODC also shows promise as a target for chemoprevention (Pegg et al, supra). ODC inhibitors have also been used to treat infections by African trypanosomes, malaria, and Pneumocystis carinii, and are potentiaUy useful for treatment of autoimmune diseases such as lupus and rheumatoid arthritis (McCann and Pegg, supra). Another family of pyridoxal-dependent decarboxylases are the group II decarboxylases. This famUy includes glutamate decarboxylase (GAD) which catalyzes the decarboxylation of glutamate into the neurotransmitter GABA; histidine decarboxylase (HDC), which catalyzes the decarboxylation of histidine to histamine; aromatic-L-amino-acid decarboxylase (DDC), also known as L-dopa decarboxylase or tryptophan decarboxylase, which catalyzes the decarboxylation of tryptophan to tiyptamine and also acts on 5-hydroxy-tryptophan and dihydroxyphenylalanine (L- dopa); and cysteine sulfinic acid decarboxylase (CSD), the rate-limiting enzyme in the synthesis of taurine from cysteine (PROSITE PDOC00329 DDC/GAD/HDC/TyrDC pyridoxal-phosphate attachment site). Taurine is an abundant sulfonic amino acid in brain and is thought to act as an osmoregulator in brain ceUs (Bitoun, M. and M. Tappaz (2000) J. Neurochem 75:919-924). Isomerases Isomerases are a class of enzymes that catalyze geometric or structural changes within a molecule to form a single product. This class includes racemases and epimerases, cis-trans- isomerases, intramolecular oxidoreductases, intramolecular transferases (mutases) and intramolecular lyases. Isomerases are critical components of ceUular biochemistry with roles in metabohc energy production including glycolysis, as weU as other diverse enzymatic processes (Stryer, supra, pp.483- 507).

Racemases are a subset of isomerases that catalyze inversion of a molecule's configuration around the asymmetric carbon atom in a substrate having a single center of asymmetry, thereby interconverting two racemers. Epimerases are another subset of isomerases that catalyze inversion of configuration around an asymmetric carbon atom in a substrate with more than one center of symmetry, thereby interconverting two epimers. Racemases and epimerases can act on amino acids and derivatives, hydroxy acids and derivatives, and carbohydrates and derivatives. The interconversion of UDP-galactose and UDP-glucose is catalyzed by UDP-galactose-4'-epimerase. Proper regulation and function of this epimerase is essential to the synthesis of glycoproteins and glycohpids. Elevated blood galactose levels have been correlated with UDP-galactose-4'-epimerase deficiency in screening programs of infants (Gitzelmann, R. (1972) Helv. Paediat. Acta 27:125-130). Correct folding of newly synthesized proteins is assisted by molecular chaperones and folding catalysts, two unrelated groups of helper molecules. Chaperones suppress non-productive side reactions by stoichiometric binding to folding intermediates, whereas folding enzymes catalyze some of the multiple folding steps that enable proteins to attain their final functional configurations (Kern, G. et al. (1994) FEBS Lett. 348:145-148). One class of folding enzymes, the peptidyl prolyl cis-trans isomerases (PPIases), isomerizes certain proline imidic bonds in what is considered to be a rate limiting step in protein maturation and export. PPIases catalyze the cis to trans isomerization of certain proline imidic bonds in proteins. There are three evolutionarily unrelated families of PPIases: the cyclophilins, the FK506 binding proteins, and the newly characterized parvulin family (Rahfeld, J.U. et al. (1994) FEBS Lett. 352:180-184).

The cyclophilins (CyP) were originaUy identified as major receptors for the immunosuppressive drug cyclosporin A (CsA), an inhibitor of T-ceU activation (Handschumacher, R.E. et al. (1984) Science 226:544-547; Harding, M.W. et al. (1986) J. Biol. Chem 261:8547-8555). Thus, the peptidyl-prolyl isomerase activity of CyP may be part of the signaling pathway that leads to T-ceU activation. Subsequent work demonstrated that CyP's isomerase activity is essential for correct protein folding and/or protein trafficking, and may also be involved in assembly/disassembly of protein complexes and regulation of protein activity. For example, in Drosophila, the CyP NinaA is required for correct localization of rhodopsins, whUe a mammahan CyP (Cyp40) is part of the Hsp90/Hsp70 complex that binds steroid receptors. The mammahan CyP (CypA) has been shown to bind the gag protein fromhuman immunodeficiency virus 1 (HIV-1), an interaction that can be inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1 activity, CypA may play an essential function in HIV-1 rephcation. FinaUy, Cyp40 has been shown to bind and inactivate the transcription factor c-Myb, an effect that is reversed by cyclosporin. This effect implicates CyP in the regulation of transcription, transformation, and differentiation (Bergsma, DJ. et al (1991) J. Biol. Chem 266:23204-23214; Hunter, T. (1998) CeU 92:141-143; and Leverson, J.D. and S.A. Ness (1998) Mol. CeU. 1:203-211).

One of the major rate limiting steps in protein folding is the thioldisulfide exchange that is necessary for correct protein assembly. Although incubation of reduced, unfolded proteins in buffers with defined ratios of oxidized and reduced thiols can lead to native conformation, the rate of folding is slow and the attainment of native conformation decreases proportionately with the size and number of cysteines in the protein. Certain ceUular compartments such as the endoplasmic reticulum of eukaryotes and the periplasmic space of prokaryotes are maintained in a more oxidized state than the surrounding cytosol. Correct disulfide formation can occur in these compartments, but at a rate that is insufficient for normal ceU processes and inadequate for synthesizing secreted proteins. The protein disulfide isomerases, thioredoxins and glutaredoxins are able to catalyze the formation of disulfide bonds and regulate the redox environment in ceUs to enable the necessary thioldisulfide exchanges (Loferer, H. (1995) J. Biol. Chem 270:26178-26183).

Each of these proteins has somewhat different functions, but aU belong to a group of disulfide-containing redox proteins that contain a conserved active-site sequence and are ubiquitously distributed in eukaryotes and prokaryotes. Protein disulfide isomerases are found in the endoplasmic reticulum of eukaryotes and in the periplasmic space of prokaryotes. They function by exchanging their own disulfide for a fhiol in a folding peptide chain. In contrast, the reduced thioredoxins and glutaredoxins are generaUy found in the cytoplasm and function by directly reducing disulfides in the substrate proteins. Oxidoreductases can be isomerases as weU. Oxidoreductases catalyze the reversible transfer of electrons from a substrate that becomes oxidized to a substrate that becomes reduced. This class of enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, and reductases. Proper maintenance of oxidoreductase levels is physiologicaUy important. For example, geneticatty- linked deficiencies in hpoamide dehydrogenase can result in lactic acidosis (Robinson, B. H. et al. (1977) Pediat. Res. 11:1198-1202).

Another subgroup of isomerases are the transferases (or mutases). Transferases transfer a chemical group from one compound (the donor) to another compound (the acceptor). The types of groups transferred by these enzymes include acyl groups, amino groups, phosphate groups (phosphotransferases or phosphomutases), and others. The transferase carnitine palmitoyltransferase is an important component of fatty acid metabohsm. GeneticaUy-linked deficiencies in this transferase can lead to myopathy (Scriver, C et al. (1995) The Metabohc and Molecular Basis of Inherited Disease, McGraw-Hill, New York NY, pp.1501-1533).

Yet another subgroup of isomerases are the topoisomersases. Topoisomerases are enzymes that affect the topological state of DNA. For example, defects in topoisomerases or their regulation can affect normal physiology. Reduced levels of topoisomerase II have been correlated with some of the DNA processing defects associated with the disorder ataxia-telangiectasia (Singh, S.P. et al. (1988) Nucleic Acids Res. 16:3919-3929). Ligases

Ligases catalyze the formation of a bond between two substrate molecules. The process involves the hydrolysis of a pyrophosphate bond in ATP or a similar energy donor. Ligases are classified based on the nature of the type of bond they form, which can include carbon-oxygen, carbon-sulfur, carbon-nitrogen, carbon-carbon and phosphoric ester bonds.

Ligases forming carbon-oxygen bonds include the aminoacyl-transfer RNA (tRNA) synthetases which are important RNA-associated enzymes with roles in translation. Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA. The 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, and each class is characterized by a distinctive topology of the catalytic domain. Class I enzymes contain a catalytic domain based on the nucleotide-binding "Rossman fold". Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparaUel β-sheet motif, as well as N- and C- terminal regulatory domains. Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the N- and C-terminal regulatory domains (Hartlein, M. and S . Cusack, (1995) J. Mol. Evol 40:519-530). Autoantibodies against aminoacyl-tRNAs are generated by patients with dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals.

Ligases forming carbon-sulfur bonds (acid-thiol hgases) mediate a large number of ceUular biosynthetic intermediary metabohsm processes involving intermolecular transfer of carbon atom-containing substrates (carbon substrates). Examples of such reactions include the tricarboxyhc acid cycle, synthesis of fatty acids and long-chain phosphohpids, synthesis of alcohols and aldehydes, synthesis of intermediary metabohtes, and reactions involved in the amino acid degradation pathways. Some of these reactions require input of energy, usuaUy in the form of conversion of ATP to either ADP or AMP and pyrophosphate. In many cases, a carbon substrate is derived from a s aU molecule containing at least two carbon atoms. The carbon substrate is often covalently bound to a larger molecule which acts as a carbon substrate carrier molecule within the ceU. In the biosynthetic mechanisms described above, the carrier molecule is coenzyme A. Coenzyme A (CoA) is stiucturatty related to derivatives of the nucleotide ADP and consists of 4-phosphopantetheine linked via a phosphodiester bond to the alpha phosphate group of adenosine 3 ',5 -bisphosphate. The teπninal thiol group of 4'-phosphopantetheine acts as the site for carbon substrate bond formation. The predominant carbon substrates which utilize CoA as a carrier molecule during biosynthesis and intermediary metabohsm in the ceU are acetyl, succinyl and propionyl moieties, coUectively referred to as acyl groups. Other carbon substrates include enoyl hpid, which acts as a fatty acid oxidation intermediate, and carnitine, which acts as an acetyl-CoA flux regulator/mitochondrial acyl group transfer protein. Acyl-CoA and acetyl-CoA are synthesized in the ceU by acyl-CoA synthetase and acetyl-CoA synthetase, respectively.

Activation of fatty acids is mediated by at least three forms of acyl-CoA synthetase activity: i) acetyl-CoA synthetase, which activates acetate and several other low molecular weight carboxyhc acids and is found in muscle mitochondria and the cytosol of other tissues; ii) medium-chain acyl-CoA synthetase, which activates fatty acids containing between four and eleven carbon atoms predominantly from dietary sources), and is present only in hver mitochondria; and iii) acyl CoA synthetase, which is specific for long chain fatty acids with between six and twenty carbon atoms, and is found in microsomes and the mitochondria. Proteins associated with acyl-CoA synthetase activity have been identified from many sources including bacteria, yeast, plants, mouse, and man. The activity of acyl-CoA synthetase may be modulated by phosphorylation of the enzyme by cAMP-dependent protein kinase.

Ligases forming carbon-nitrogen bonds include amide synthases such as glutamine synthetase (glutamate-ammonia hgase) that catalyzes the amination of glutamic acid to glutamine by ammonia using the energy of ATP hydrolysis. Glutamine is the primary source for the amino group in various amide transfer reactions involved in de novo pyrimidine nucleotide synthesis and in purine and pyrimidine ribonucleotide interconversions. Overexpression of glutamine synthetase has been observed in primary hver cancer (Christa, L. et al. (1994) Gastroent. 106:1312-1320).

Acid-amino-acid ligases (peptide synthases) are represented by the ubiquitin conjugating enzymes which are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of ceUular proteins in eukaryotic ceUs and some bacteria. The UCS mediates the elimination of abnormal proteins and regulates the half-hves of important regulatory proteins that control ceUular processes such as gene transcription and ceU cycle progression. In the UCS pathway, proteins targeted for degradation are conjugated to ubiquitin (Ub), a smaU heat stable protein. Ub is first activated by a ubiquitin-activating enzyme (El), and then transferred to one of several Ub- conjugating enzymes (E2). E2 then links the Ub molecule through its C-teπninal glycine to an internal lysine (acceptor lysine) of a target protein. The ubiquitinated protein is then recognized and degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease. The UCS is implicated in the degradation of mitotic cychc kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, ceU surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. (1994) CeU 79:13-21).

Cyclo-hgases and other carbon-nitrogen ligases comprise various enzymes and enzyme complexes that participate in the de novo pathways of purine and pyrimidine biosynthesis. Because these pathways are critical to the synthesis of nucleotides for rephcation of both RNA and DNA, many of these enzymes have been the targets of clinical agents for the treatment of ceU prohferative disorders such as cancer and infectious diseases.

Purine biosynthesis occurs de novo from the amino acids glycine and glutamine, and other smaU molecules. Three of the key reactions in this process are catalyzed by a trifunctional enzyme composed of glycinamide-ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide transformylase (GART). Together these three enzymes combine ribosylamine phosphate with glycine to yield phosphoribosyl arriinoimidazole, a precursor to both adenylate and guanylate nucleotides. This trifunctional protein has been implicated in the pathology of Downs syndrome (Aimi, J. et al. (1990) Nucleic Acid Res. 18:6665-6672). Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that converts inosinic acid to adenylosuccinate, a key step on the path to ATP synthesis. This enzyme is also similar to another carbon-nitrogen hgase, argininosuccinate synthetase, that catalyzes a simUar reaction in the urea cycle (PoweU, S.M. et al. (1992) FEBS Lett. 303:4-10).

Adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase maybe considered as a functional unit, the purine nucleotide cycle. This cycle converts AMP to inosine monophosphate (IMP) and reconverts IMP to AMP via adenylosuccinate, thereby producing NH₃ and foπring fumarate from aspartate. In muscle, the purine nucleotide cycle functions, during intense exercise, in the regeneration of ATP by pulling the adenylate kinase reaction in the direction of ATP formation and by providing Krebs cycle intermediates. In kidney, the purine nucleotide cycle accounts for the release of NH₃ under normal acid-base conditions. In brain, the purine nucleotide cycle may contribute to ATP recovery. Adenylosuccinate lyase deficiency provokes psychomotor retardation, often accompanied by autistic features (Van den Berghe, G. et al (1992) Prog Neurobiol: 39:547-561). A marked imbalance in the enzymic pattern of purine metabohsmis linked with transformation and/or progression in cancer ceUs. In rat hepatomas the specific activities of the anabohc enzymes, IMP dehydrogenase, GMP synthetase, adenylosuccinate synthetase, adenylosuccinase, AMP deaminase and amidophosphoribosyltransferase, increased to 13.5-, 3.7-, 3.1-, 1.8-, 5.5- and 2.8-fold, respectively, of those in normal hver (Weber, G. (1983) Clin. Biochem 16:57-63).

Like the de novo biosynthesis of purines, de novo synthesis of the pyrimidine nucleotides uridylate and cytidylate also arises from a common precursor, in this instance the nucleotide orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP). Again a trifunctional enzyme comprising three carbon-nitrogen Ugases plays a key role in the process. In this case the enzymes aspartate transcarbamylase (ATCase), carbamyl phosphate synthetase II, and dihydroorotase (DHOase) are encoded by a single gene caUed CAD. Together these three enzymes combine the initial reactants in pyrimidine biosynthesis, glutamine, C0₂ and ATP to form dihydroorotate, the precursor to orotate and orotidylate (Iwahana, H. et al. (1996) Biochem. Biophys. Res. Commun. 219:249-255). Further steps then lead to the synthesis of uridine nucleotides from orotidylate. Cytidine nucleotides are derived from uridine-5 -triphosphate (UTP) by the amidation of UTP using glutamine as the amino donor and the enzyme CTP synthetase. Regulatory mutations in the human CTP synthetase are believed to confer multi-drug resistance to agents widely used in cancer therapy (Yamauchi, M. et al. (1990) EMBO J. 9:2095-2099).

Ligases forming carbon-carbon bonds include the carboxylases acetyl-CoA carboxylase and pyruvate carboxylase. Acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA from C0₂ and H₂0 using the energy of ATP hydrolysis. Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. Two isoforms of acetyl-CoA carboxylase, types I and types II, are expressed in human in a tissue-specific manner (Ha, J. et al (1994) Eur. J. Bioche 219:297- 306). Pyruvate carboxylase is a nuclear-encoded mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, a key intermediate in the citric acid cycle.

Ligases forming phosphoric ester bonds include the DNA ligases involved in both DNA rephcation and repair. DNA hgases seal phosphodiester bonds between two adjacent nucleotides in a DNA chain using the energy from ATP hydrolysis to first activate the free 5 -phosphate of one nucleotide and then react it with the 3'-OH group of the adjacent nucleotide. This resealing reaction is used in DNA rephcation to join smaU DNA fragments caUed "Okazaki" fragments that are transiently formed in the process of replicating new DNA, and in DNA repair. DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontroUed methylation of DNA, are corrected before rephcation or transcription of the DNA can occur. Bloom's syndrome is an inherited human disease in which individuals are partiaUy deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts et al, supra, p. 247).

Pantothenate synthetase (D-pantoate; beta-alanine hgase (AMP-forming); EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction. The enzyme is dimeric, with two weU-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. The N-terminal domain is structuraUy very simUar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamUy (von Delft, F. et al. (2000) Structure (Camb) 9:439-450).

Farnesyl diphosphate synthase (FPPS) is an essential enzyme that is required both for cholesterol synthesis and protein prenylation. The enzyme catalyzes the formation of farnesyl diphosphate from dimethylaUyl diphosphate and isopentyl diphosphate. FPPS is inhibited by ώtrogen-containing biphosphonates, which can lead to the inhibition of osteoclast-mediated bone resorptionby preventing protein prenylation (Dunford, J.E. et al. (2001) J. Pharmacol. Exp. Ther. 296:235-242).

5-aιninolevulinate synthase (ALAS; delta-aminolevulinate synthase; EC 2.3.1.37) catalyzes the rate-limiting step in heme biosynthesis in both erythroid and non-eiythroid tissues. This enzyme is unique in the heme biosynthetic pathway in being encoded by two genes, the first encoding ALAS 1, the non-erythroid specific enzyme which is ubiquitously expressed, and the second encoding ALAS2, which is expressed exclusively in erythroid ceUs. The genes for ALASl and ALAS 2 are located, respectively, on chromosome 3 and on the X chromosome. Defects in the gene encoding ALAS2 result in X-linked sideroblastic anemia. Elevated levels of ALAS are seen in acute hepatic porphyrias and can be lowered by zinc mesoporphyrin. Drug Metabolizing Enzymes (DMEs)

The metabohsm of a drug and its movement through the body (pharmacokinetics) are important in determining its effects, toxicity, and interactions with other drugs. The three processes governing pharmacokinetics are the absorption ofthe drug, distribution to various tissues, and elimination of drug metabohtes. These processes are intimately coupled to drug metabohsm, since a variety of metabohc modifications alter most of the physicochemical and pharmacological properties of drugs, including solubility, binding to receptors, and excretion rates. The metabohc pathways which modify drugs also accept a variety of naturaUy occurring substrates such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins. The enzymes in these pathways are therefore important sites of biochemical and pharmacological interaction between natural compounds, drugs, carcinogens, mutagens, and xenobiotics.

It has long been appreciated that inherited differences in drug metabohsm lead to drasticaUy different levels of drug efficacy and toxicity among individuals. For drugs with narrow therapeutic indices, or drugs wliich require bioactivation (such as codeine), these polymorphisms can be critical. Moreover, promising new drugs are frequently eliminated in clinical trials based on toxicities which may only affect a segment of the patient group. Advances in pharmacogenomics research, of which DMEs constitute an important part, are promising to expand the tools and information that can be brought to bear on questions of drug efficacy and toxicity (See Evans, W.E. and R.V. ReUing (1999) Science 286:487-491). DMEs have broad substrate specificities, unlike antibodies, for example, wliich are diverse and highly specific. Since DMEs metabohze a wide variety of molecules, drug interactions may occur at the level of metabohsm so that, for example, one compound may induce a DME that affects the metabohsm of another compound.

Drug metabohc reactions are categorized as Phase I, which prepare the drug molecule for functioning and further metabohsm, and Phase II, which are conjugative. In general, Phase I reaction products are partiaUy or fuUy inactive, and Phase II reaction products are the chief excreted species. However, Phase I reaction products are sometimes more active than the original administered drugs; this metabohc activation principle is exploited by pro-drugs (e.g. L-dopa). AdditionaUy, some nontoxic compounds (e.g. aflatoxin, benzo[ ]pyrene) are metabohzed to toxic intermediates through these pathways. Phase I reactions are usuaUy rate-limiting in drug metabohsm. Prior exposure to the compound, or other compounds, can induce the expression of Phase I enzymes however, and thereby increase substrate flux through the metabohc pathways. (See Klaassen, CD. et al. (1996) Casarett and DouU's Toxicology: The Basic Science of Poisons, McGraw-HiU, New York, NY, pp. 113-186; Katzung, B.G. (1995) Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT, pp. 48- 59; Gibson, G.G. and P. Skett (1994) Introduction to Drug Metabohsm, Blackie Academic and Professional, London.). Drug metaboUzing enzymes (DMEs) have broad substrate specificities. This can be contrasted to the immune system, where a large and diverse population of antibodies are highly specific for their antigens. The abihty of DMEs to metabohze a wide variety of molecules creates the potential for drug interactions at the level of metabohsm For example, the induction of a DME by one compound may affect the metabohsm of another compound by the enzyme. DMEs have been classified according to the type of reaction they catalyze and the cofactors involved. The major classes of Phase I enzymes include, but are not limited to, cytochrome P450 and flavin-containing monooxygenase. Other enzyme classes involved in Phase I-type catalytic cycles and reactions include, but are not limited to, NADPH cytochrome P450 reductase (CPR), the microsomal cytochrome b5/NADH cytochrome b5 reductase system, the ferredoxin/ferredoxin reductase redox pair, aldo/keto reductases, and alcohol dehydrogenases. The major classes of Phase II enzymes include, but are not limited to, UDP glucuronyltransferase, sulfotransferase, glutathione S- transferase, N-acyltransferase, and N-acetyl transferase. Cytochrome P450 and P450 catalytic cycle-associated enzymes

Members of the cytochrome P450 superfamUy of enzymes catalyze the oxidative metabohsm of a variety of substrates, including natural compounds such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins, as weU as drugs, carcinogens, mutagens, and xenobiotics. Cytochromes P450, also known as P450 heme-fhiolate proteins, usuaUy act as terminal oxidases in multi-component electron transfer chains, caUed P450-containing monooxygenase systems. Specific reactions catalyzed include hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, and O- dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-oxide groups. These reactions are involved in steroidogenesis of glucocorticoids, cortisols, estrogens, and androgens in animals; insecticide resistance in insects; herbicide resistance and flower coloring in plants; and environmental bioremediation by microorganisms. Cytochrome P450 actions on drugs, carcinogens, mutagens, and xenobiotics can result in detoxification or in conversion of the substance to a more toxic product. Cytochromes P450 are abundant in the hver, but also occur in other tissues; the enzymes are located in microsomes. (See ExPASY ENZYME EC 1.14.14.1; Prosite PDOC00081 Cytochrome P450 cysteine heme-iron ligand signature; PRINTS EP450I E-Class P450 Group I signature; Graham-Lorence, S. and J.A. Peterson (1996) FASEB J. 10:206-214.)

Four hundred cytochromes P450 have been identified in diverse organisms including bacteria, fungi, plants, and animals (Graham-Lorence and Peterson, supra). The B-class is found in prokaryotes and fungi, while the E-class is found in bacteria, plants, insects, vertebrates, and mammals. Five subclasses or groups are found within the larger farnuy of E-class cytochromes P450 (PRINTS EP450I E-Class P450 Group I signature).

AU cytochromes P450 use a heme cofactor and share structural attributes. Most cytochromes P450 are 400 to 530 amino acids in length. The secondary structure of the enzyme is about 70% alpha-hehcal and about 22% beta-sheet. The region around the heme-binding site in the C-terminal part of the protein is conserved among cytochromes P450. A ten amino acid signature sequence in this heme-iron hgand region has been identified which includes a conserved cysteine involved in binding the heme iron in the fifth coordination site. In eukaryotic cytochromes P450, a membrane-spanning region is usuaUy found in the first 15-20 amino acids of the protein, generaUy consisting of approximately 15 hydrophobic residues foUowed by a positively charged residue. (See Prosite PDOC00081, supra; Graham-Lorence and Peterson, supra.)

Cytochrome P450 enzymes are involved in ceU prohferation and development. The enzymes have roles in chemical mutagenesis and carcinogenesis by metabolizing chemicals to reactive intermediates that form adducts with DNA (Nebert, D.W. and F.J. Gonzalez (1987) Ann. Rev.

Biochem. 56:945-993). These adducts can cause nucleotide changes and DNA rearrangements that lead to oncogenesis. Cytochrome P450 expression in hver and other tissues is induced by xenobiotics such as polycychc aromatic hydrocarbons, peroxisomal proliferators, phenobarbital, and the glucocorticoid dexamethasone (Dogra, S.C. et al. (1998) Clin. Exp. Pharmacol. Physiol. 25:1-9). A cytochrome P450 protein may participate in eye development as mutations in the P450 gene CYPIB 1 cause primary congenital glaucoma (OMIM #601771 Cytochrome P450, subfamUy I (dioxin- inducible), polypeptide 1; CYPIB 1).

Cytochromes P450 are associated with inflammation and infection. Hepatic cytochrome P450 activities are profoundly affected by various infections and inflammatory stimuli, some of which are suppressed and some induced (Morgan, E.T. (1997) Drug Metab. Rev. 29:1129-1188). Effects observed in vivo can be mimicked by proiiiflammatory cytokines and interferons. Autoantibodies to two cytochrome P450 proteins were found in patients with autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), a polyglandular autoimmune syndrome (OMIM #240300 Autoimmune polyenodocrmopathy-candidiasis-ectodermal dystrophy). Mutations in cytochromes P450 have been linked to metabohc disorders, including congenital adrenal hyperplasia, the most common adrenal disorder of infancy and childhood; pseudovitamin D- deficiency rickets; cerebrotendinous xanthomatosis, a hpid storage disease characterized by progressive neurologic dysfunction, premature atherosclerosis, and cataracts; and an inherited resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, Inc. New York, NY, pp. 1968-1970; Takeyama, K. et al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998) N. Engl. J. Med. 338:653-661; OMIM #213700 Cerebrotendinous xanthomatosis; and OMIM #122700 Coumarin resistance). Extremely high levels of expression of the cytochrome P450 protein aromatase were found in a fibrolameUar hepatoceUular carcinoma from a boy with severe gynecomastia (feminization) (Agarwal, V.R. (1998) J. Clin. Endocrinol Metab. 83:1797-1800).

The cytochrome P450 catalytic cycle is completed through reduction of cytochrome P450 by NADPH cytochrome P450 reductase (CPR). Another microsomal electron transport system consisting of cytochrome b5 and NADPH cytochrome b5 reductase has been widely viewed as a minor contributor of electrons to the cytochrome P450 catalytic cycle. However, a recent report by Lamb, D.C. et al (1999; FEBS Lett. 462:283-288) identifies a Candida albicans cytochrome P450 (CYP51) which can be efficiently reduced and supported by the microsomal cytochrome b5/NADPH cytochrome b5 reductase syste Therefore, there are likely many cytochromes P450 which are supported by this alternative electron donor system

Cytochrome b5 reductase is also responsible for the reduction of oxidized hemoglobin (methemoglobin, or ferrihemoglobin, which is unable to carry oxygen) to the active hemoglobin (ferrohemoglobin) in red blood ceUs. Methemoglobinemia results when there is a high level of oxidant drugs or an abnormal hemoglobin (hemoglobin M) which is not efficiently reduced. Methemoglobinemia can also result from a hereditary deficiency in red ceU cytochrome b5 reductase (Reviewed in Mansour, A. and A.A. Lurie (1993) Am J. Hematol. 42:7-12). Members of the cytochrome P450 famUy are also closely associated with vitamin D synthesis and catabohsm Vitamin D exists as two biologicaUy equivalent prohormones, ergocalciferol (vitamin D₂), produced in plant tissues, and cholecalciferol (vitamin D₃), produced in animal tissues. The latter form, cholecalciferol, is formed upon the exposure of 7-dehydrocholesterol to near ultraviolet light (i.e., 290-310 nm), normaUy resulting from even minimal periods of skin exposure to sunhght (reviewed in MiUer, W.L. and A. A. Portale (2000) Trends Endocrinol Metab. 11 :315-319). Both prohormone forms are further metabohzed in the hver to 25-hydroxyvitamin D (25(OH)D) by the enzyme 25-hydroxylase. 25(OH)D is the most abundant precursor form of vitamin D which must be further metabohzed in the kidney to the active form, lα,25-dihydroxy vitamin D (lα,25(OH)₂D), by the enzyme 25-hydroxyvitamin D lα-hydroxylase (lα-hydroxylase). Regulation of lα,25(OH)₂D production is primarily at this final step in the synthetic pathway. The activity of lα-hydroxylase depends upon several physiological factors including the circulating level of the enzyme product (lα,25(OH)₂D) and the levels of parathyroid hormone (PTH), calcitonin, insulin, calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal lα-hydroxylase activity has been reported, suggesting that tissue-specific, local regulation of lα,25(OH)2D production may also be biologicaUy important. The catalysis of 1 α,25(OH)₂D to

24,25-dihydroxyvitarnin D (24,25(OH)₂D), involving the enzyme 25-hydroxyvitamin D 24-hydroxylase (24-hydroxylase), also occurs in the kidney. 24-hydroxylase can also use 25(OH)D as a substrate (Shinki, T. et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12920-12925; MiUer and Portale, supra; and references within). Vitamin D 25-hydroxylase, lα-hydroxylase, and 24-hydroxylase are aU NADPH-dependent, type I (mitochondrial) cytochrome P450 enzymes that show a high degree of homology with other members of the famUy. Vitamin D 25-hydroxylase also shows a broad substrate specificity and may also perform 26-hydroxylation of bUe acid intermediates and 25, 26, and 27-hydroxylation of cholesterol (DUworth, F.J. et al. (1995) J. Biol. Chem 270:16766-16774; MiUer and Portale, supra; and references within).

The active form of vitamin D (1 α,25(OH)₂D) is involved in calcium and phosphate homeostasis and promotes the differentiation of myeloid and skin ceUs. Vitamin D deficiency resulting from deficiencies in the enzymes involved in vitamin D metabolism (e.g., lα-hydroxylase) causes hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive) rickets, a disease characterized by loss of bone density and distinctive clinical features, including bandy or bow leggedness accompanied by a waddling gait. Deficiencies in vitamin D 25-hydroxylase cause cerebrotendinous xanthomatosis, a hpid-storage disease characterized by the deposition of cholesterol and cholestanol in the Achilles' tendons, brain, lungs, and many other tissues. The disease presents with progressive neurologic dysfunction, including postpubescent cerebeUar ataxia, atherosclerosis, and cataracts. Vitamin D 25-hydroxylase deficiency does not result in rickets, suggesting the existence of alternative pathways for the synthesis of 25(OH)D (Griffin, J.E. and J.E. Zerwekh (1983) J. Clin. Invest. 72:1190-1199; Gamblin, G.T. et al. (1985) J. Clin. Invest. 75:954-960; and MiUer and Portale, supra).

Ferredoxin and ferredoxin reductase are electron transport accessory proteins which support at least one human cytochrome P450 species, cytochrome P450c27 encoded by the CYP27 gene (DUworth, F.J. et al. (1996) Biochem J. 320:267-71). A Streptomyces griseus cytochrome P450, CYP 104D 1 , was heterologously expressed in Escherichia coli and found to be reduced by the endogenous ferredoxin and ferredoxin reductase enzymes (Taylor, M. et al. (1999) Biochem. Biophys. Res. Commun. 263:838-842), suggesting that many cytochrome P450 species maybe supported by the ferredoxin/ferredoxin reductase pair. Ferredoxin reductase has also been found in a model drug metabohsm system to reduce actinomycin D, an antitumor antibiotic, to a reactive free radical species (Flitter, W.D. and R.P. Mason (1988) Arch. Biochem Biophys. 267:632-639). Flavin-containing monooxygenase (FMO)

Mavin-containing monooxygenases oxidize the nucleophilic nitrogen, sulfur, and phosphorus heteroatom of an exceptional range of substrates. Like cytochromes P450, FMOs are microsomal and use NADPH and 0₂; there is also a great deal of substrate overlap with cytochromes P450. The tissue distribution of FMOs includes hver, kidney, and lung.

Isoforms of FMO in mammals include FMOl, FM02, FM03, FM04, and FM05, which are expressed in a tissue-specific manner. The isoforms differ in their substrate specificities and properties such as inhibition by various compounds and stereospecificity of reaction. FMOs have a 13 amino acid signature sequence, the components of which span the N-terminal two-thirds ofthe sequences and include the FAD binding region and the FATGY motif found in many N-hydroxylating enzymes (Stehr, M. et al. (1998) Trends Biochem Sci. 23:56-57; PRINTS FMOXYGENASE Flavin- containing monooxygenase signature). Specific reactions include oxidation of nucleophilic tertiary amines to N-oxides, secondary amines to hydroxylamines and nitrones, primary amines to hydroxylamines and oximes, and sulfur-containing compounds and phosphines to S- and P-oxides. Hydrazines, iodides, selenides, and boron-containing compounds are also substrates. FMOs are more heat labUe and less detergent-sensitive than cytochromes P450 in vitro though FMO isoforms vary in thermal stability and detergent sensitivity. FMOs play important roles in the metabohsm of several drugs and xenobiotics. FMO (FM03 in hver) is predominantly responsible for metabolizing (S)-nicotine to (S)-nicotine N-1 '-oxide, which is excreted in urine. FMO is also involved in S-oxygenation of cimetidine, an H₂-antagonist widely used for the treatment of gastric ulcers. Liver-expressed forms of FMO are not under the same regulatory control as cytochrome P450. In rats, for example, phenobarbital treatment leads to the induction of cytochrome P450, but the repression of FMOl . Lysyl oxidase

Lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidase involved in the formation of connective tissue matrices by crosslihking coUagen and elastin. LO is secreted as an N- glycosylated precursor protein of approximately 50 kDa and cleaved to the mature form of the enzyme by a metaUoprotease, although the precursor form is also active. The copper atom in LO is involved in the transport of electrons to and from oxygen to facilitate the oxidative deamination of lysine residues in these extraceUular matrix proteins. WhUe the coordination of copper is essential to LO activity, insufficient dietary intake of copper does not influence the expression of the apoenzyme. However, the absence of the functional LO is linked to the skeletal and vascular tissue disorders that are associated with dietary copper deficiency. LO is also inhibited by a variety of semicarbazides, hydrazines, and amino nitrites, as well as heparin. Beta-aminopropionitrUe is a commonly used inhibitor. LO activity is increased in response to ozone, cadmium, and elevated levels of hormones released in response to local tissue trauma, such as transforming growth factor-beta, platelet-derived growth factor, angiotensin II, and fibroblast growth factor. Abnormalities in LO activity have been linked to Menkes syndrome and occipital horn syndrome. Cytosohc forms of the enzyme have been imphcated in abnormal ceU prohferation (reviewed in Rucker, R.B. et al. (1998) Am J. Clin. Nutr. 67:996S-1002S and Smith-Mungo, L.I. and H.M. Kagan (1998) Matrix Biol. 16:387-398). Dihydrofolate reductases

Dihydrofolate reductases (DHFR) are ubiquitous enzymes that catalyze the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential step in the de novo synthesis of glycine and purines as weU as the conversion of deoxyuridine monophosphate (dUMP) to deox mymidine monophosphate (dTMP). The basic reaction is as foUows:

7,8-dihydrofolate + NADPH → 5,6,7,8-tetrahydrofolate + NADP⁺

The enzymes can be inhibited by a number of dihydrofolate analogs, including trimethroprim and methotrexate. Since an abundance of dTMP is required for DNA synthesis, rapidly dividing ceUs require the activity of DHFR. The rephcation of DNA viruses (i.e., herpesvirus) also requires high levels of DHFR activity. As a result, drugs that target DHFR have been used for cancer chemotherapy and to inhibit DNA virus rephcation. (For simUar reasons, thymidylate synthetases are also target enzymes.) Drugs that inhibit DHFR are preferentiaUy cytotoxic for rapidly dividing ceUs (or DNA virus-infected ceUs) but have no specificity, resulting in the inchscriminate destruction of dividing ceUs. Furthermore, cancer ceUs may become resistant to drugs such as methotrexate as a result of acquired transport defects or the duphcation of one or more DHFR genes (Stryer, L. (1988) Biochemistry. W.H Freeman and Co., Inc. New York. pp. 511-519). Aldo/keto reductases

Aldo/keto reductases are monomeric NADPH-dependent oxidoreductases with broad substrate specificities (Bohren, K.M. et al. (1989) J. Biol. Chem 264:9547-9551). These enzymes catalyze the reduction of carbonyl-containing compounds, including carbonyl-containing sugars and aromatic compounds, to the corresponding alcohols. Therefore, a variety of carbonyl-containing drugs and xenobiotics are likely metabohzed by enzymes of this class.

One known reaction catalyzed by a famUy member, aldose reductase, is the reduction of glucose to sorbitol, which is then further metabohzed to fructose by sorbitol dehydrogenase. Under normal conditions, the reduction of glucose to sorbitol is a minor pathway. In hyperglycemic states, however, the accumulation of sorbitol is implicated in the development of diabetic complications (OMIM #103880 Aldo-keto reductase famUy 1, member Bl). Members of this enzyme famUy are also highly expressed in some hver cancers (Cao, D. et al. (1998) J. Biol. Che 273:11429-11435). Alcohol dehydrogenases

Alcohol dehydrogenases (ADHs) oxidize simple alcohols to the corresponding aldehydes. ADH is a cytosohc enzyme, prefers the cofactor NAD⁺, and also binds zinc ion. Liver contains the highest levels of ADH, with lower levels in kidney, lung, and the gastric mucosa.

Known ADH isoforms are dimeric proteins composed of 40 kDa subunits. There are five known gene loci which encode these subunits (a, b, g, p, c), and some of the loci have characterized aUehc variants (b_1; b₂, b₃, g_1; g₂). The subunits can formhomodimers and heterodimers; the subunit composition determines the specific properties of the active enzyme. The holoenzymes have therefore been categorized as Class I (subunit compositions aa, ab, ag, bg, gg), Class II (pp), and Class III (cc). Class I ADH isozymes oxidize ethanol and other smaU aliphatic alcohols, and are inhibited by pyrazole. Class II isozymes prefer longer chain aliphatic and aromatic alcohols, are unable to oxidize methanol, and are not inhibited by pyrazole. Class III isozymes prefer even longer chain aliphatic alcohols (five carbons and longer) and aromatic alcohols, and are not inhibited by pyrazole.

The short-chain alcohol dehydrogenases include a number of related enzymes with a variety of substrate specificities. Included in this group are the mammahan enzymes D-beta-hydroxybutyrate dehydrogenase, (R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin dehydrogenase, NADPH-dependent carbonyl reductase, corticosteroid 11-beta-dehydrogenase, and estradiol 17-beta- dehydrogenase, as weU as the bacterial enzymes acetoacetyl-CoA reductase, glucose 1- dehydrogenase, 3-beta-hydroxysteroid dehydrogenase, 20-beta-hydroxysteroid dehydrogenase, ribitol dehydrogenase, 3-oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, sorbitol-6- phosphate 2-dehydrogenase, 7-alpha-hydroxysteroid dehydrogenase, cis-l,2-dihydroxy-3,4- cyclohexadiene-1 -carboxylate dehydrogenase, s-toluene dihydrodiol dehydrogenase, s-benzene glycol dehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase, N-acylmaimosamine 1- dehydrogenase, and 2-deoxy-D-gluconate 3-dehydrogenase (Krozowski, Z. (1994) J. Steroid

Biochem Mol. Biol. 51:125-130; Krozowski, Z. (1992) Mol. CeU Endocrinol. 84.C25-31; and Marks,

A.R. et al. (1992) J. Biol. Chem 267:15459-15463). Sulfotransferases

Sulfate conjugation occurs on many ofthe same substrates which undergo O-glucuronidation to produce a highly water-soluble sulfuric acid ester. Sulfotransferases (ST) catalyze this reaction by transferring S0₃ ^" from the cofactor 3'-phosphoadenosine-5'-ρhosphosulfate (PAPS) to the substrate.

ST substrates are predominantly phenols and ahphatic alcohols, but also include aromatic amines and ahphatic amines, which are conjugated to produce the corresponding sulfamates. The products of these reactions are excreted mainly in urine.

STs are found in a wide range of tissues, including hver, kidney, intestinal tract, lung, platelets, and brain. The enzymes are generaUy cytosohc, and multiple forms are often co-expressed.

For example, there are more than a dozen forms of ST in rat hver cytosol. These biochemicaUy characterized STs faU into five classes based on their substrate preference: arylsulfotransferase, alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester sulfotransferase, and bile salt sulfotransferase.

ST enzyme activity varies greatly with sex and age in rats. The combined effects of developmental cues and sex-related hormones are thought to lead to these differences in ST expression profiles, as weU as the profiles of other DMEs such as cytochromes P450. Notably, the high expression of STs in cats partiaUy compensates for their low level of UDP glucuronyltransferase activity.

Several forms of ST have been purified fromhuman hver cytosol and cloned. There are two phenol sulfotransferases with different thermal stabihties and substrate preferences. The thermostable enzyme catalyzes the sulfation of phenols such as para-nitrophenol, minoxidU, and acetaminophen; the thermolabUe enzyme prefers monoamine substrates such as dopamine, epinephrine, and levadopa. Other cloned STs include an estrogen sulfotransferase and an N- acetylglucosamine-6-O-sulfotransferase. This last enzyme is Ulustrative of the other major role of

STs in ceUular biochemistry, the modification of carbohydrate structures that may be important in ceUular differentiation and maturation of proteoglycans. Indeed, an inherited defect in a sulfotransferase has been implicated in macular corneal dystrophy, a disorder characterized by a faUure to synthesize mature keratan sulfate proteoglycans (Nakazawa, K. et al. (1984) J. Biol. Chem.

259:13751-13757; OMIM #217800 Macular dystrophy, corneal).

Galactosyltransferases Galactosyltransferases are a subset of glycosyltransferases that transfer galactose (Gal) to the terminal N-acetylglucosamine (GlcNAc) oligosaccharide chains that are part of glycoproteins or glycolipids that are free in solution (Kolbinger, F. et al (1998) J. Biol. Chem 273:433-440; Amado, M. et al. (1999) Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases have been detected on the ceU surface and as soluble extraceUular proteins, in addition to being present in the Golgi. βl,3- galactosyltransferases form Type I carbohydrate chains with Gal (βl-3)GlcNAc linkages. Known human and mouse βl,3-galactosyltransferases appear to have a short cytosohc domain, a single transmembrane domain, and a catalytic domain with eight conserved regions. (Kolbinger et al, supra; and Hennet, T. et al. (1998) J. Biol. Chem 273:58-65). In mouse UDP-galactose:β-N- acetylglucosamine βl,3-galactosyltransferase-I region 1 is located at amino acid residues 78-83, region 2 is located at amino acid residues 93-102, region 3 is located at amino acid residues 116-119, region 4 is located at amino acid residues 147-158, region 5 is located at amino acid residues 172- 183, region 6 is located at amino acid residues 203-206, region 7 is located at amino acid residues 236-246, and region 8 is located at amino acid residues 264-275. A variant of a sequence found within mouse UDP-galactose:β-N-acetylglucosamine βl,3-galactosyltransferase-I region 8 is also found in bacterial galactosyltransferases, suggesting that this sequence defines a galactosyltransferase sequence motif (Hennet et al, supra). Recent work suggests thatbrainiac protein is a βl,3- galactosyltransferase (Yuan, Y. et al. (1997) CeU 88:9-11; and Hennet et al, supra).

UDP-Gal:GlcNAc-l,4-galactosyltransferase (-1,4-GalT) (Sato, T. et al, (1997) EMBO J. 16:1850-1857) catalyzes the formation of Type II carbohydrate chains with Gal (βl-4)GlcNAc linkages. As is the case with the βl,3-galactosyltransferase, a soluble form of the enzyme is formed by cleavage of the membrane-bound form Amino acids conserved among βl,4-galactosyltransferases include two cysteines linked through a disulfide-bond and a putative UDP-galactose-binding site in the catalytic domain (Yadav, S. and K. Brew (1990) J. Biol. Chem 265:14163-14169; Yadav, S.P. and K. Brew (1991) J. Biol Chem 266:698-703; and Shaper, N.L. et al. (1997) J. Biol. Chem 272:31389-31399). βl,4-galactosyltransferases have several specialized roles in addition to synthesizing carbohydrate chains on glycoproteins or glycohpids. In mammals a βl,4- galactosyltransferase, as part of a heterodimer with α-lactalbumin, functions in lactating mammary gland lactose production. A βl ,4-galactosyltransferase on the surface of sperm functions as a receptor that specificaUy recognizes the egg. CeU surface βl ,4-galactosyltransferases also function in ceU adhesion, cell/basal lamina interaction, and normal and metastatic ceU migration. (Shur, B.

(1993) Curr. Opin. CeU Biol. 5:854-863; and Shaper, J. (1995) Adv. Exp. Med. Biol. 376:95-104). Gamma-glutamyl transpeptidase

Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes that initiate extraceUular glutathione (GSH) breakdown by cleaving gamma-glutamyl amide bonds. The breakdown of GSH provides ceUs with a regional cysteine pool for biosynthetic pathways. Gamma-glutamyl transpeptidases also contribute to ceUular antioxidant defenses and expression is induced by oxidative stress. The ceU surface-localized glycoproteins. are expressed at high levels in cancer ceUs. Studies have suggested that the high level of gamma-glutamyl transpeptidase activity present on the surface of cancer ceUs could be exploited to activate precursor drugs, resulting in high local concentrations of anti-cancer therapeutic agents (Hanigan, M.H. (1998) Chem Biol. Interact. 111-112:333-342; Taniguchi, N. and Y. Ikeda (1998) Adv. Enzymol. Relat. Areas Mol. Biol. 72:239-278; Chikhi, N. et al. (1999) Comp. Biochem Physiol. B. Biochem Mol. Biol. 122:367-380). Aminotransferases

Aminotransferases comprise a fam y of pyridoxal 5 -phosphate (PLP) -dependent enzymes that catalyze transformations of amino acids. Aspartate aminotransferase (Asp AT) is the most extensively studied PLP-containing enzyme. It catalyzes the reversible trarisamination of dicafboxyhc L-amino acids, aspartate and glutamate, and the corresponding 2-oxo acids, oxalacetate and 2-oxoglutarate. Other members of the famUy include pyruvate aminotransferase, branched-chain amino acid aminotransferase, tyrosine aminotransferase, aromatic aminotransferase, alanine:glyoxylate aminotransferase (AGT), and kynurenine aminotransferase (Vacca, R.A. et al. (1997) J. Biol. Chem 272:21932-21937).

Primary hyperoxaluria type-1 is an autosomal recessive disorder resulting in a deficiency in the liver-specific peroxisomal enzyme, alanine: glyoxylate aminotransferase- 1. The phenotype of the disorder is a deficiency in glyoxylate metabohs In the absence of AGT, glyoxylate is oxidized to oxalate rather than being transaminated to glycine. The result is the deposition of insoluble calcium oxalate in the kidneys and urinary tract, ultimately causing renal faUure (Lumb, M.J. et al. (1999) J. Biol. Chem 274:20587-20596).

Kynurenine aminotransferase catalyzes the irreversible transamination of the L-tryptophan metabolite L-kynurenine to formkynurenic acid. The enzyme may also catalyze the reversible traiisamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate. Kynurenic acid is a putative modulator of glutamatergic neurotransmission; thus a deficiency in kynurenine aπ-inotransferase may be associated with pleotrophic effects (Buchh, R. et al. (1995) J. Biol Chem 270:29330-29335). Cateehol-0-methyltransferase Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group of S- adenosyl-L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the catechol substrate (e.g., L-dopa, dopamine, or DBA). Methylation of the 3'-hydroxyl group is favored over methylation of the 4'-hydroxyl group and the membrane bound isoform of COMT is more regiospecific than the soluble form Translation of the soluble form of the enzyme results from utilization of an internal start codon in a full-length mRNA (1.5 kb) or from the translation of a shorter mRNA (1.3 kb), transcribed from an internal promoter. The proposed S_N2-like methylation reaction requires Mg⁴* and is inhibited by Ca^"1"1. The binding of the donor and substrate to COMT occurs sequentiaUy. AdoMet first binds COMT in a Mg^-independent manner, foUowed by the binding of Mg** and the binding of the catechol substrate. The amount of COMT in tissues is relatively high compared to the amount of activity normaUy required, thus inhibition is problematic. Nonetheless, inhibitors have been developed for in vitro use (e.g., gaUates, tropolone, U-0521, and 3',4'-dihydroxy-2-methyl-propiophetropolone) and for clinical use (e.g., nitrocatechol-based compounds and tolcapone). Administration of these inhibitors results in the increased half-hfe of L-dopa and the consequent formation of dopamine. Inhibition of COMT is also likely to increase the half-hfe of various other catechol-structure compounds, including but not limited to epinephrine/norepinephrine, isoprenaline, rimiterol, dobutamine, fenoldopam, apomorphine, and α-methyldopa. A deficiency in norepinephrine has been linked to clinical depression, hence the use of COMT inhibitors could be usefuU in the treatment of depression. COMT inhibitors are generaUy weU tolerated with minimal side effects and are ultimately metabohzed in the hver with only minor accumulation of metabohtes in the body (Mannisto, P.T. and S. Kaakkola (1999) Pharmacol. Rev. 51:593-628). Copper-zinc superoxide dismutases

Copper-zinc superoxide dismutases are compact homodimeric metaUoenzymes involved in ceUular defenses against oxidative damage. The enzymes contain one atom of zinc and one atom of copper per subunit and catalyze the dismutation of superoxide anions into 0₂ and H₂0₂. The rate of dismutation is diffusion-limited and consequently enhanced by the presence of favorable electrostatic interactions between the substrate and enzyme active site. Examples of this class of enzyme have been identified in the cytoplasm of ah the eukaryotic ceUs as weU as in the periplasm of several bacterial species. Copper-zinc superoxide dismutases are robust enzymes that are highly resistant to proteolytic digestion and denaturing by urea and SDS. In addition to the compact structure of the enzymes, the presence of the metal ions and intrasubunit disulfide bonds is beheved to be responsible for enzyme stability. The enzymes undergo reversible denaturation at temperatures as high as 70 °C (Battistoni, A. et al. (1998) J. Biol. Chem 273:5655-5661).

Overexpression of superoxide dismutase has been implicated in enhancing freezing tolerance of transgenic alfalfa as weU as providing resistance to environmental toxins such as the diphenyl ether herbicide, acifluorfen (McKersie, B.D. et al (1993) Plant Physiol. 103:1155-1163). In addition, yeast ceUs become more resistant to freeze-fhaw damage foUowing exposure to hydrogen peroxide which causes the yeast ceUs to adapt to further peroxide stress by upregulating expression of superoxide dismutases. In this study, mutations to yeast superoxide dismutase genes had a more detrimental effect on freeze-thaw resistance than mutations which affected the regulation of glutathione metabohsm, long suspected of being important in determining an organism's survival through the process of cryopreservation (Jong-In Park, J.-I. et al. (1998) J. Biol. Chem 273:22921-22928).

Expression of superoxide dismutase is also associated with Mycobacterium tuberculosis, the organism that causes tuberculosis. Superoxide dismutase is one of the ten major proteins secreted by M. tuberculosis and its expression is upregulated approximately 5-fold in response to oxidative stress. M. tuberculosis expresses almost two orders of magnitude more superoxide dismutase than the nonpathogenic mycobacterium M. smegmatis, and secretes a much higher proportion of the expressed enzyme. The result is the secretion of ~350-fold more enzyme by M. tuberculosis thanM smegmatis, providing substantial resistance to oxidative stress (Harfh, G. and M.A. Horwitz (1999) J. Biol. Chem 274:4281-4292).

The reduced expression of copper-zinc superoxide dismutases, as weU as other enzymes with anti-oxidant capabilities, has been implicated in the early stages of cancer. The expression of copper- zinc superoxide dismutases is reduced in prostatic intraepifhehal neoplasia and prostate carcinomas, (Bostwick, D.G. (2000) Cancer 89:123-134). Phosphoesterases

Phosphotriesterases (PTE, paraoxonases) are enzymes that hydrolyze toxic organophosphorus compounds and have been isolated from a variety of tissues. Phosphotriesterases play a central role in the detoxification of insecticides by mammals. Birds and insects lack PTE, and as a result have reduced tolerance for organophosphorus compounds (VUanova, E. and M.A. Sogorb (1999) Crit. Rev. Toxicol. 29:21-57). Phosphotriesterase activity varies among individuals and is lower in infants than adults. PTE knockout mice are markedly more sensitive to the organophosphate-based toxins diazoxon and chlorpyrifos oxon (Furlong, C.E., et al. (2000) Neurotoxicology 21:91-100). Phosphotriesterase is also implicated in atherosclerosis and diseases involving hpoprotein metabohsm

Glycerophosphoryl diester phosphodiesterase (also known as glycerophosphodiester phosphodiesterase) is a phosphodiesterase which hydrolyzes deacetylated phosphohpid glycerophosphodiesters to produce sn-glycerol-3-phosphate and an alcohol. Glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, and glycerophosphoinositol are examples of substrates for glycerophosphoryl diester phosphodiesterases. A glycerophosphoryl diester phosphodiesterase from -5. coli has broad specificity for glycerophosphodiester substrates (Larson, TJ. et al. (1983) J. Biol Chem 248:5428-5432).

Cychc nucleotide phosphodiesterases (PDEs) are crucial enzymes in the regulation of the cychc nucleotides cAMP and cGMP. cAMP and cGMP function as intraceUular second messengers to transduce a variety of extraceUular signals including hormones, hght, and neurotransmitters. PDEs degrade cychc nucleotides to their corresponding monophosphates, thereby regulating the intraceUular concentrations of cycUc nucleotides and their effects on signal transduction. Due to their roles as regulators of signal transduction, PDEs have been extensively studied as chemotherapeutic targets (Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem Biol. 2:472-481; Torphy, J.T. (1998) Am J. Resp. Crit. Care Med. 157:351-370).

Families of mammaUan PDEs have been classified based on their substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory agents (Beavo, J.A. (1995) Physiol. Rev. 75:725-748; Conti, M. et al. (1995) Endocrine Rev. 16:370-389). Several of these families contain distinct genes, many of which are expressed in different tissues as sphce variants. Within PDE families, there are multiple isozymes and multiple sphce variants of these isozymes (Conti, M. and S.- L.C Jin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63:1-38). The existence of multiple PDE families, isozymes, and sphce variants is an indication of the variety and complexity of the regulatory pathways involving cychc nucleotides (Houslay, M.D. and G. Milhgan (1997) Trends Biochem Sci. 22:217-224). Typ^e 1 PDEs (PDEls) are Ca²⁺/calmodulin-dependent and appear to be encoded by at least three different genes, each having at least two different sphce variants (Kakkar, R. et al. (1999) CeU Mol. Life Sci. 55:1164-1186). PDEls have been found in the lung, heart, and brain. Some PDEl isozymes are regulated in vitro by phosphorylation/dephosphorylation. Phosphorylation of these PDE1 isozymes decreases the affinity of the enzyme for cahnodulin, decreases PDE activity, and increases steady state levels of cAMP (Kakkar et al, supra). PDEls may provide useful therapeutic targets for disorders of the central nervous system and the cardiovascular and immune systems, due to the involvement of PDEls in both cychc nucleotide and calcium signaling (Perry and Higgs, supra).

PDE2s are cGMP-strmulated PDEs that have been found in the cerebeUum, neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle (Sadhu, K. et al. (1999) J. Histochem. Cytochem 47:895-906). PDE2s are thought to mediate the effects of cAMP on catecholamine secretion, participate in the regulation of aldosterone (Beavo, supra), and play a role in olfactory signal transduction (JuUfs, D.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3388-3395).

PDE3s have high affinity for both cGMP and cAMP, and so these cychc nucleotides act as competitive substrates for PDE3s. PDE3s play roles in stimulating myocardial contractility, rnhibiting platelet aggregation, relaxing vascular and airway smooth muscle, inhibiting prohferation of T-lymphocytes and cultured vascular smooth muscle ceUs, and regulating catecholamine-induced release of free fatty acids from adipose tissue. The PDE3 famUy of phosphodiesterases are sensitive to specific inhibitors such as cUostamide, enoximone, and hxazinone. Isozymes of PDE3 can be regulated by cAMP-dependent protein kinase, or by insulin-dependent kinases (Degerman, E. et al. (1997) J. Biol. Chem 272:6823-6826). PDE4s are specific for cAMP; are locahzed to airway smooth muscle, the vascular endothehum, and ah iriflammatory ceUs; and can be activated by cAMP-dependent phosphorylation. Since elevation of cAMP levels can lead to suppression of inflammatory ceU activation and to relaxation of bronchial smooth muscle, PDE4s have been studied extensively as possible targets for novel anti-inflammatory agents, with special emphasis placed on the discovery of asthma treatments. PDE4 inhibitors are currently undergoing clinical trials as treatments for asthma, chronic obstructive pulmonary disease, and atopic eczema. AU four known isozymes of PDE4 are susceptible to the inhibitor rohpram, a compound which has been shown to improve behavioral memory in mice (Barad, M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:15020-15025). PDE4 inhibitors have also been studied as possible therapeutic agents against acute lung injury, endotoxemia, rheumatoid arthritis, multiple sclerosis, and various neurological and gastrointestinal indications (Doherty, A.M. (1999) Curr. Opin. Chem Biol. 3:466-473).

PDE5 is highly selective for cGMP as a substrate (Turko, IV. et al. (1998) Biochemistry 37:4200-4205), and has two aUosteric cGMP-specific binding sites (McAllister-Lucas, L.M. et al (1995) J. Biol. Chem 270:30671-30679). Binding of cGMP to these aUosteric binding sites seems to be important for phosphorylation of PDE5 by cGMP-dependent protein kinase rather than for direct regulation of catalytic activity. High levels of PDE5 are found in vascular smooth muscle, platelets, lung, and kidney. The inhibitor zaprinast is effective against PDE5 and PDEls. Modification of zaprinast to provide specificity against PDE5 has resulted in sUdenafil (VIAGRA; Pfizer, Inc., New York NY), a treatment for male erectUe dysfunction (Terrell, N. et al. (1996) Bioorg. Med. Chem Lett. 6:1819-1824). Inhibitors of PDE5 are currently being studied as agents for cardiovascular therapy (Perry and Higgs, supra).

PDE6s, the photoreceptor cychc nucleotide phosphodiesterases, are crucial components of the phototransduction cascade. In association with the G-protein transducin, PDE6s hydrolyze cGMP to regulate cGMP-gated cation channels in photoreceptor membranes. In addition to the cGMP- binding active site, PDE6s also have two Mgh-affinity cGMP-binding sites which are thought to play a regulatory role in PDE6 function (Artemyev, N.O. et al. (1998) Methods 14:93-104). Defects in PDE6s have been associated with retinal disease. Retinal degeneration in the rd mouse (Yan, W. et al. (1998) Invest. Opthalmol Vis. Sci. 39:2529-2536), autosomal recessive retinitis pigmentosa in humans (Danciger, M. et al. (1995) Genomics 30:1-7), and rod/cone dysplasia 1 in Irish Setter dogs (Suber, M.L. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3968-3972) have been attributed to mutations in the PDE6B gene.

The PDE7 famUy of PDEs consists of only one known member having multiple sphce variants (Bloom TJ. and J.A. Beavo (1996) Proc. Natl. Acad. Sci. USA 93:14188-14192). PDE7s are cAMP specific, but little else is known about their physiological function. Although mRNAs encoding PDE7s are found in skeletal muscle, heart, brain, lung, kidney, and pancreas, expression of PDE7 proteins is restricted to specific tissue types (Han, P. et al. (1997) J. Biol. Chem 272:16152- 16157; Perry and Higgs, supra). PDE7s are very closely related to the PDE4 famUy; however, PDE7s are not inhibited by rohpram, a specific inhibitor of PDE4s (Beavo, supra). PDE8s are cAMP specific, and are closely related to the PDE4 famUy. PDE8s are expressed in thyroid gland, testis, eye, hver, skeletal muscle, heart, kidney, ovary, and brain. The cAMP- hydrolyzing activity of PDE8s is not inhibited by the PDE inhibitors rohpram, vinpocetine, mihinone, IBMX (3-isobutyl-l-methylxanthine), or zaprinast, but PDE8s are inhibited by dipyridamole (Fisher, D.A. et al. (1998) Biochem Biophys. Res. Commun. 246:570-577; Hayashi, M. et al. (1998) Biochem Biophys. Res. Commun. 250:751-756; Soderling, S.H. et al. (1998) Proc. Natl. Acad. Sci. USA 95:8991-8996).

PDE9s are cGMP specific and most closely resemble the PDE8 famUy of PDEs. PDE9s are expressed in kidney, hver, lung, brain, spleen, and smaU intestine. PDE9s are not inhibited by sUdenafil (VIAGRA; Pfizer, Inc., New York NY), rohpram, vinpocetine, dipyridamole, or IBMX (3- isobutyl-1-memylxanthine), but they are sensitive to the PDE5 inhibitor zaprinast (Fisher, D.A. et al. (1998) J. Biol. Chem 273:15559-15564; Soderling, S.H. et al. (1998) J. Biol. Chem 273:15553- 15558).

PDElOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDElOs are expressed in brain, thyroid, and testis. (Soderling, S.H. et al. (1999) Proc. Natl. Acad. Sci. USA 96:7071-7076; Fujishige, K. et al. (1999) J. Biol Chem 274:18438-18445; Loughney, K. et al (1999) Gene 234:109- 117).

PDEs are composed of a catalytic domain of about 270-300 amino acids, an N-teπninal regulatory domain responsible for binding cofactors, and, in some cases, a hydrophihc C-terminal domain of unknown function (Conti and Jin, supra). A conserved, putative zinc-binding motif has been identified in the catalytic domain of aU PDEs. N-terminal regulatory domains include non- catalytic cGMP-binding domains in PDE2s, PDE5s, and PDE6s; calmodulin-binding domains in PDEls; and domains containing phosphorylation sites in PDE3s and PDE4s. In PDE5, the N-terminal cGMP-binding domain spans about 380 amino acid residues and comprises tandem repeats of a conserved sequence motif (McAllister-Lucas, L.M. et al. (1993) J. Biol. Chem 268:22863-22873). The NKXnD motif has been shown by mutagenesis to be important for cGMP binding (Turko, LV. et al. (1996) J. Biol. Chem 271:22240-22244). PDE famUies display approximately 30% amino acid identity within the catalytic domain; however, isozymes within the same famUy typicaUy display about 85-95% identity in this region (e.g. PDE4A vs PDE4B). Furthermore, within a famUy there is extensive simUarity (>60%) outside the catalytic domain; whUe across families, there is little or no sequence similarity outside this domain. Many of the constituent functions of immune and inflammatory responses are inhibited by agents that increase intraceUular levels of cAMP (Verghese, M.W. et al. (1995) Mol. Pharmacol. 47:1164-1171). A variety of diseases have been attributed to increased PDE activity and associated with decreased levels of cychc nucleotides. For example, a form of diabetes insipidus in mice has been associated with increased PDE4 activity, an increase in low-K^ cAMP PDE activity has been reported in leukocytes of atopic patients, and PDE3 has been associated with cardiac disease. Many inhibitors of PDEs have undergone clinical evaluation (Perry and Higgs, supra; Torphy, TJ. (1998) Am J. Respir. Crit. Care Med. 157:351-370). PDE3 inhibitors are being developed as antithrombotic agents, antihypertensive agents, and as cardiotonic agents useful in the treatment of congestive heart faUure. Rohpram, a PDE4 inhibitor, has been used in the treatment of depression, and other PDE4 inhibitors have an anti-inflammatory effect. Rohpram may inhibit HIV-1 rephcation (Angel, J.B. et al. (1995) AIDS 9:1137-1144). AdditionaUy, rohpram suppresses the production of cytokines such as TNF-a and b and interferon g, and thus is effective against encephalomyehtis. Rohpram may also be effective in treating tardive dyskinesia and multiple sclerosis (Sommer, N. et al. (1995) Nat. Med. 1:244-248; Sasaki, H. et al. (1995) Eur. J. Pharmacol. 282:71-76). TheophyUine is a nonspecific PDE inhibitor used in treatment of bronchial asthma and other respiratory diseases. TheophyUine is beheved to act on airway smooth muscle function and in an anti-iiiflammatory or immunomodulatory capacity (Banner, K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000). Pento-dfylline is another nonspecific PDE inhibitor used in the treatment of intermittent claudication and diabetes-induced peripheral vascular disease. Pentoxifylhne is also known to block TNF-a production and may inhibit HIV-1 rephcation (Angel et al, supra).

PDEs have been reported to affect ceUular prohferation of a variety of ceU types (Conti et al. (1995) Endocrine Rev. 16:370-389) and have been implicated in various cancers. Growth of prostate carcinoma cell lines DU145 and LNCaP was inhibited by delivery of cAMP derivatives and PDE inhibitors (Bang, Y.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334). These ceUs also showed a permanent conversion in phenotype from epithelial to neuronal morphology. It has also been suggested that PDE inhibitors can regulate mesangial ceU prohferation (Matousovic, K. et al. (1995) J. Clin. Invest. 96:401-410) and lymphocyte prohferation (Joulain, C. et al. (1995) J. Lipid Mediat. CeU Signal. 11 :63-79). One cancer treatment involves intraceUular dehvery of PDEs to particular cellular compartments of tumors, resulting in ceU death (Deonarain, M.P. and A. A. Epenetos (1994) Br. J. Cancer 70:786-794). UDP Glucuronyltransferases

Members of the UDP glucuronyltransferase famUy (UGTs) catalyze the transfer of a glucuronic acid group from the cofactor uridine diphosphate-glucuronic acid (UDP-glucuronic acid) to a substrate. The transfer is generaUy to a nucleophUic heteroatom (O, N, or S). Substrates include xenobiotics which have been functionahzed by Phase I reactions, as weU as endogenous compounds such as bilirubin, steroid hormones, and thyroid hormones. Products of glucuronidation are excreted in urine if the molecular weight of the substrate is less than about 250 g/mol, whereas larger glucuronidated substrates are excreted in bUe. UGTs are located in the microsomes of hver, kidney, intestine, skin, brain, spleen, and nasal mucosa, where they are on the same side of the endoplasmic reticulum membrane as cytochrome P450 enzymes and flavin-containing monooxygenases. UGTs have a C-tenninal membrane-spanning domain which anchors them in the endoplasmic reticulum membrane, and a conserved signature domain of about 50 amino acid residues in their C terminal section (PROSITE PDOC00359 UDP- glycosyltransferase signature).

UGTs involved in drug metabohsm are encoded by two gene families, UGT1 and UGT2. Members of the UGT1 family result from alternative splicing of a single gene locus, which has a variable substrate binding domain and constant region involved in cofactor binding and membrane insertion. Members of the UGT2 famUy are encoded by separate gene loci, and are divided into two families, UGT2A and UGT2B. The 2 A subfamUy is expressed in olfactory epithehum, and the 2B subfamUy is expressed in hver microsomes. Mutations in UGT genes are associated with hyperbilirubinemia (OMIM #143500 Hyperbilirubinemia I); Crigler-Najjar syndrome, characterized by intense hyperbilirubinemia frombirth (OMIM #218800 Crigler-Najjar syndrome); and a milder form of hyperbilirubinemia termed GUbert's disease (OMIM #191740 UGT1). Thioesterases

Two soluble thioesterases involved in fatty acid biosynthesis have been isolated from mammahan tissues, one which is active only toward long-chain fatty-acyl thioesters and one which is active toward thioesters with a wide range of fatty-acyl chain-lengths. These thioesterases catalyze the chain-terminating step in the de novo biosynthesis of fatty acids. Chain teπriination involves the hydrolysis of the thioester bond which links the fatty acyl chain to the 4'-phosphopantefheine prosthetic group of the acyl carrier protein (ACP) subunit of the fatty acid synthase (Smith, S . (1981a) Methods Enzymol. 71:181-188; Smith, S. (1981b) Methods Enzymol 71:188-200).

E. coli contains two soluble thioesterases, thioesterase I which is active only toward long- chain acyl thioesters, and thioesterase II (TEII) which has a broad chain-length specificity (Naggert, J. et al. (1991) J. Biol. Chem 266:11044-11050). E. coli TEII does not exhibit sequence simUarity with either of the two types of mammahan thioesterases which function as chain-terminating enzymes in de novo fatty acid biosynthesis. Unlike the mammahan thioesterases, E. coli TEII lacks the characteristic serine active site gly-X-ser-X-gly sequence motif and is not inactivated by the serine modifying agent diisopropyl fluorophosphate. However, modification of histidine 58 by iodoacetamide and diethylpyrocarbonate abohshed TEII activity. Overexpression of TEII did not alter fatty acid content in E. coli, which suggests that it does not function as a cham-terminating enzyme in fatty acid biosynthesis (Naggert et al, supra). For that reason, Naggert et al. (supra) proposed that the physiological substrates for E. coli TEII maybe coenzyme A (CoA)-fatty acid esters instead of ACP-phosphopanthetheine-fatty acid esters. Carboxylesterases

Mammahan carboxylesterases are a multigene famUy expressed in a variety of tissues and ceU types. Acetylcholinesterase, butyrylcholinesterase, and carboxylesterase are grouped into the serine superfamUy of esterases (B -esterases). Other carboxylesterases include thyroglobulin, thrombin, Factor IX, ghotactin, and plasminogen. Carboxylesterases catalyze the hydrolysis of ester- and amide- groups from molecules and are involved in detoxification of drugs, environmental toxins, and carcinogens. Substrates for carboxylesterases include short- and long-chain acyl-glycerols, acylcarnitine, carbonates, dipivefrin hydrochloride, cocaine, salicylates, capsaicin, palmitoyl-coenzyme A, imidaprU, haloperidol, pyrrohzidine alkaloids, steroids, p-nitrophenyl acetate, malathion, butanihcaine, and isocarboxazide. Carboxylesterases are also important for the conversion of prodrugs to free acids, wliich may be the active form of the drug (e.g. , lovastatin, used to lower blood cholesterol) (reviewed in Satoh, T. and Hosokawa, M. (1998) Annu. Rev. Pharmacol. Toxicol.38:257-288). Neurohgins are a class of molecules that (i) have N-teπuinal signal sequences, (ii) resemble ceU-surface receptors, (hi) contain carboxylesterase domains, (iv) are highly expressed in the brain, and (v) bind to neurexins in a calcium-dependent manner. Despite the homology to carboxylesterases, neurohgins lack the active site serine residue, implying a role in substrate binding rather than catalysis (Ichtchenko, K. et al. (1996) J. Biol. Chem 271:2676-2682). Squalene epoxidase

Squalene epoxidase (squalene monooxygenase, SE) is a microsomal membrane-bound, FAD- dependent oxidoreductase that catalyzes the first oxygenation step in the sterol biosynthetic pathway of eukaryotic ceUs. Cholesterol is an essential structural component of cytoplasmic membranes acquired via the LDL receptor-mediated pathway or the biosynthetic pathway. SE converts squalene to 2,3(S)-oxidosqualene, which is then converted to lanosterol and then cholesterol.

High serum cholesterol levels result in the formation of atherosclerotic plaques in the arteries of higher organisms. This deposition of highly insoluble hpid material onto the waUs of essential blood vessels results in decreased blood flow and potential necrosis. HMG-CoA reductase is responsible for the first committed step in cholesterol biosynthesis, conversion of 3-hydroxyl-3- methyl-glutaryl CoA (HMG-CoA) to mevalonate. HMG-CoA is the target of a number of pharmaceutical compounds designed to lower plasma cholesterol levels, but inhibition of MHG-CoA also results in the reduced synthesis of non-sterol intermediates required for other biochemical pathways. Since SE catalyzes a rate-limiting reaction that occurs later in the sterol synthesis pathway with cholesterol as the only end product, SE is a better ideal target for the design of anti-hyperhpidemic drugs (Nakamura, Y. et al. (1996) 271:8053-8056). Epoxide hydrolases

Epoxide hydrolases catalyze the addition of water to epoxide-containing compounds, thereby hydrolyzing epoxides to their corresponding 1,2-diols. They are related to bacterial haloalkane dehalogenases and show sequence simUarity to other members of the α/β hydrolase fold famUy of enzymes. This family of enzymes is important for the detoxification of xenobiotic epoxide compounds which are often highly electrophilic and destructive when introduced. Examples of epoxide hydrolase reactions include the hydrolysis of some leukotoxin to leukotoxin diol, and isoleukotoxin to isoleukotoxin diol Leukotoxins alter membrane permeabUity and ion transport and cause inflammatory responses. In addition, epoxide carcinogens are produced by cytochrome P450 as intermediates in the detoxification of drugs and envhonmental toxins. Epoxide hydrolases possess a catalytic triad composed of Asp, Asp, and His (Arand, M. et al. (1996) J. Biol. Chem 271:4223-4229; Rink, R. et al. (1997) J. Biol. Chem 272:14650-14657; Argiriadi, M.A. et al. (2000) J. Biol. Chem 275:15265-15270).

Enzymes involved in tyrosine catalysis

The degradation of the amino acid tyrosine, to either succinate and pyruvate or fumarate and acetoacetate, requires a large number of enzymes and generates a large number of intermediate compounds. In addition, many xenobiotic compounds may be metabohzed using one or more reactions that are part of the tyrosine catabohc pathway. Enzymes involved in the degradation of tyrosine to succinate and pyruvate (e.g., in Arthrobacter species) include 4-hydroxyphenylpyruvate oxidase, 4-hydroxyphenylacetate 3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxyιτιethyl-2-hydroxyrnuconic semialdehyde dehydrogenase, trans,cis- 5-carboxymethγl-2-hydroxymuconate isomerase, homoprotocatechuate isomerase/decarboxylase, cw-2-oxohept-3-ene-l,7-dioate hydratase, 2,4-dihydroxyhept-trani^,-2-ene-l,7-dioate aldolase, and succinic semialdehyde dehydrogenase. Enzymes involved in the degradation of tyrosine to fumarate and acetoacetate (e.g., in Pseudomonas species) include 4-hydroxyphenylpyruvate dioxygenase, homogentisate 1 ,2-dioxygenase, maleylacetoacetate isomerase, fumarylacetoacetase and 4-hydroxyphenylacetate. Additional enzymes associated with tyrosine metabohsm in different organisms include 4-chlorophenylacetate-3,4-dioxygenase, aromatic aminotransferase,

5-oxopent-3-ene-l,2,5-tricarboxylate decarboxylase, 2-oxo-hept-3-ene-l,7-dioate hydratase, and 5-carboxymethyl-2-hydroxymuconate isomerase (Ellis, L.B.M. et al. (1999) Nucleic Acids Res. 27:373-376; Wackett, L.P. and Ellis, L.B.M. (1996) J. Microbiol. Meth. 25:91-93; and Schmidt, M. (1996) Amer. Soc. Microbiol. News 62:102). In humans, acquired or inherited genetic defects in enzymes of the tyrosine degradation pathway may result in hereditary tyrosinemia. One form of this disease, hereditary tyrosinemia 1 (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase, the last enzyme in the pathway in organisms that metabohze tyrosine to fumarate and acetoacetate. HT1 is characterized by progressive hver damage beginning at infancy, and increased risk for hver cancer (Endo, F. et al (1997) J. Biol. Chem 272:24426-24432). Hydroxy acid oxidase

Three mammahan hydroxy acid oxidases (HAOl, HA02, and HA03) have been cloned (Jones et al. (2000) J. Biol Chem 275: 12590-12597). HAOl and HA02 (HAOX2) are fromhuman. HA02 protein is found in the peroxisome. It shares 46.7% amino acid sequence identity with spinach GOX and 46.0% identity with HAOl. HA02 transcripts are found at high levels in both hver and kidney, and at a low level in thymus and the HA02 gene is found on chromosome 1. 2-hydroxyacid oxidases oxidize a broad range of 2-hydroxyacids, ranging from glycolate to long-chain 2-hydroxy fatty acids such as 2-hydroxypalmitate. These enzymes utihze a flavin cofactor and convert 2-hydroxyacids to 2-ketoacids with the concomitant reduction of molecular oxygen to hydrogen peroxide.

Urate oxidase

Urate oxidase, or uricase, is a ubiquitous peroxisomal enzyme that catalyzes the oxidation of uric acid to aUantoin in most mammals. In most mammals, it is present in hver, with little or no detectable activity in other tissues. It is associated with the peroxisomes and exists as a tetramer with an apparent subunit size of 32,000 daltons. Humans and certain primates lack this enzyme activity although there are homologous genomic sequences in humans to porcine and rat urate oxidase. The identification of mice with complete hypoxanthine guanine phosphoribosyltransferase 1 (HPRT) deficiency but without any symptoms of the Lesch-Nyhan syndrome (Hooper, M. et al. (1987) Nature 326:292-295; Kuehn, M. R. et al. (1987) Nature 326:295-298) raises the possibility that the absence of urate oxidase activity in the purine metabohsm pathway may contribute to the development of the neurologic symptoms observed in humans (Lee, C. C. et al. (1988) Science 239:1288-1291; Motojima, K. and Goto, S. (1988) Biochem Biophys. Res. Commun. 155:1266-1270). Expression profiling

Microarrays are analytical tools used inbioanalysis. A microarray has a plurality of molecules spatiaUy distributed over, and stably associated with, the surface of a sohd 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 specificaUy related to a particular genetic predisposition, condition, disease, or disorder. Breast Cancer

More than 180,000 new cases of breast cancer are diagnosed each year, and the mortality rate for breast cancer approaches 10% of aU deaths in females between the ages of 45-54 (Gish, K. (1999) AWIS Magazine 28:7-10). However the survival rate based on early diagnosis of locahzed breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou, CM. et al. (2000) Nature 406:747-752).

Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and maybe passed on from parents to children (Gish, supra). However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epithelial cells. The relationship between expression of epidermal growth factor (EGF) and its receptor,

EGFR, to human mammary carcinoma has been particularly weU 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 ceU functions related to metastatic potential, such as ceU motility, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor famUy, of which EGFR is one, have also been implicated in breast cancer. The abundance of erbB receptors, such as HER- 2/neu, HER-3, and HER-4, and their hgands in breast cancer points to their functional importance in the pathogenesis ofthe disease, and may therefore provide targets for therapy ofthe disease (Bacus, S.S. et al. (1994) Am J. Clin. Pathol 102:S13-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix Gla protein which is overexpressed in human breast carcinoma ceUs; Drgl or RTP, a gene whose expression is (-iminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S 100 protein famUy, aU of which are down-regulated in mammary carcinoma ceUs relative to normal mammary epithelial ceUs (Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395; Uhϊx, W. et al (1999) FEBS Left 455:23-26; Sager, R. et al (1996) Curr. Top. Microbiol. Immunol. 213:51- 64; and Lee, S.W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).

Another genetic defect is gene amplification involving genes such as c-myc and c-efbB2 (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 ceUs undergo a relatively defined sequence of events leading to tumor formation. An early event in tumor development is ductal hyperplasia. CeUs undergoing rapid neoplastic growth graduaUy progress to invasive carcinoma and become metastatic to the lung, bone, and potentiaUy other organs. Variables that may influence the process of tumor progression and malignant transformation include genetic factors, environmental factors, growth factors, and hormones. CeU lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has ^"been shown that these ceU lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, I.I. et al. (1998) Clin. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial ceUs at various stages of malignant transformation.

Tumor ceUs stimulate the formation of stroma that secretes various mediators, such as growth factors, cytokines, and proteases, which are critical for tumor growth. For instance, serum tumor necrosis factor alpha (TNF-α) increased in the circulation of patients with malignancy. Thus it is likely that the serum TNF-α concentration can serve as a biomarker for staging the invasiveness of breast cancer. ClinicaUy, TNF-α treatment in combination with interferon gamma (IFN-γ) may provide a successful approach to overcome the ceUular heterogeneity of advanced breast tumors. In in vitro studies, TNF-α has been demonstrated to be antitumorigenic in MCF-7 ceUs by inducing apoptosis and ihhibiting prohferation.

TNF-α , also called cachectin, is produced by neutrophils, activated lymphocytes, macrophages, NK ceUs, LAK ceUs, astrocytes, endothelial ceUs, smooth muscle ceUs, and some transformed ceUs. TNF-α occurs as a secreted, soluble form and as a membrane-anchored form, both of which are biologicaUy active. Two types of receptors for TNF-α have been described and virtuaUy aU ceU types studied show the presence of one or both of these receptor types. TNF-α and TNF-β are extremely pleiotropic factors due to the ubiquity of their receptors, to their abihty to activate multiple signal transduction path- ways and to their abihty to induce or suppress the expression of a wide number of genes. TNF-α and TNF-β play a critical role in mediation of the inflammatory response and in mediation of resistance to infections and tumor growth. Lung Cancer

The potential apphcation of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of cancer, such as lung cancer. 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 epithehum. In nearly 80% of patients diagnosed with lung cancer, metastasis has already occurred. Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and hver. 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. Earher 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 morphologicaUy distinct stages from hyperplasia to invasive carcinoma. Malignant lung cancers are divided into two groups comprising four histopathological classes. The Non Small CeU Lung Carcinoma (NSCLC) group includes squamous ceU carcinomas, adenocarcinomas, and large ceU carcinomas and accounts for about 70% of aU lung cancer cases. Adenocarcinomas typicaUy arise in the peripheral airways and often formmucin secreting glands. Squamous ceU carcinomas typicaUy arise in proximal airways. The histogenesis of squamous ceU carcinomas may be related to chronic inflammation and injury to the bronchial epithehum, leading to squamous metaplasia. The SmaU CeU Lung Carcinoma (SCLC) group accounts for about 20% of lung cancer cases. SCLCs typicaUy arise in proximal airways and exhibit a number of paraneoplastic syndromes mcluding inappropriate production of adrenocorticotropin and anti-diuretic hormone.

Lung cancer ceUs accumulate numerous genetic lesions, many of which are associated with cytologicaUy visible chromosomal aberrations. The high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease. Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one of the earhest genetic lesions leading to lung cancer. Deletions at chromosome arms 9p and 17p are also common. Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as K-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2. Genes differentiaUy regulated in lung cancer have been identified by a variety of methods. Using mRNA differential display technology, Manda et al (1999; .Genomics 51:5-14) identified five genes differentiaUy expressed in lung cancer ceU lines compared to normal bronchial epithelial ceUs. Among the known genes, pulmonary surfactant apoprotein A and alpha 2 macroglobulin were down regulated whereas nm23Hl was upregulated. Petersen et al. (2000; Int J. Cancer, 86:512-517) used suppression subtractive hybridization to identify 552 clones differentiaUy expressed in lung tumor derived ceU lines, 205 of which represented known genes. Among the known genes, thrombospondin-1, fibronectin, interceUular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentiaUy expressed in lung cancers. Wang et al. (2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentiaUy overexpresssed in squamous ceU carcinoma compared with normal lung epithehu Among the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofilhn 1 and cytokeratin 13.

Colon Cancer ,

WhUe soft tissue sarcomas are relatively rare, more than 50% of new patients diagnosed with the disease will die from it. The molecular pathways leading to the development of sarcomas are relatively unknown, due to the rarity of the disease and variation in pathology. Colon cancer evolves through a multi-step process whereby pre-mahgnant colonocytes undergo a relatively defined sequence of events leading to tumor formation. Several factors participate in the process of tumor progression and malignant transformation including genetic factors, mutations, and selection. To understand the nature of gene alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. Familial adenomatous polyposis (FAP), is caused by mutations in the adenomatous polyposis coli gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q. Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in mis-match repair genes. Although hereditary colon cancer syndromes occur in a smaU percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generaUy apphed. For instance, somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in aU of these genes lead to gene expression changes in colon cancer. Ovarian Cancer

Ovarian cancer is the leading cause of death from a gynecologic cancer. The majority of ovarian cancers are derived from epithelial ceUs, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result, the long-term survival rate 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 microsatelhte instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metabohsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU prohferative disorders, including cancer.

SUMMARY OF THE INVENTION

Various embodiments ofthe invention provide purified polypeptides, enzymes, referred to coUectively as 'ENZM' and individuaUy as ΕNZM-1,' ΕNZM-2,' ΕNZM-3,' ΕNZM-4,' ΕNZM- 5,' 'ENZM-6,' ΕNZM-7,' ΕNZM-8,' ΕNZM-9,' ΕNZM-10,' ΕNZM-11,' ΕNZM-12,' ΕNZM- 13,' ΕNZM-14,' ΕNZM-15,' ΕNZM-16,' ΕNZM-17,' ΕNZM- 18,' ΕNZM-19,' ΕNZM-20,' ΕNZM-21,' ΕNZM-22,' ΕNZM-23,' ΕNZM-24,' ΕNZM-25,' ΕNZM-26,' ΕNZM-27,' ΕNZM- 28,' ΕNZM-29,' ΕNZM-30,' ΕNZM-31,' ΕNZM-32,' 'ENZM-33,' ΕNZM-34,' ΕNZM-35,' ΕNZM-36,' ΕNZM-37,' 'ENZM-38,' and ΕNZM-39' 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 enzymes 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 enzymes 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:l- 39, b) a polypeptide comprising a naturaUy 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: 1-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:l-39.

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:l-39, b) a polypeptide comprising a naturaUy 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 -39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-39. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:40-78.

StiU 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: 1-39, b) a polypeptide comprising a naturaUy 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:l-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. Another embodiment provides a ceU 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: 1-39, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least

90% identical or atleast about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. The method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU 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 specificaUy 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:l-39, b) a polypeptide comprising a naturaUy 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: 1-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39. StiU 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 NO:40-78, b) a polynucleotide comprising a naturaUy 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:40-78, 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 NO:40-78, b) a polynucleotide comprising a naturaUy 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:40-78, 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 specificaUy 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.

StiU 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:40-78, 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:40-78, 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:l-39, b) a polypeptide comprising a naturaUy 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:l-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, 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:l-39. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional ENZM, 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:l-39, b) a polypeptide comprising a naturaUy 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:l-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. The method comprises a) contacting a sample comprising the polypeptide with 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 ENZM, comprising administering to a patient in need of such treatment the composition.

StUl 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:l-39, b) a polypeptide comprising a naturaUy 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:l-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. The method comprises a) contacting a sample comprising the polypeptide with 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 pharmaceuticaUy acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional ENZM, comprising administering to a patient in need of such treatment the composition. Another embodiment provides a method of screening for a compound that specificaUy 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: 1-39, b) a polypeptide comprising a naturaUy 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:l-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. 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:l-39, b) a polypeptide comprising a naturaUy 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:l-39, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-39. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity ofthe polypeptide, b) assessing the activity ofthe 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 ofthe polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.

StiU 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:40-78, the method comprising a) contacting a sample comprising the target polynucleotide with 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:40-78, ii) a polynucleotide comprising a naturaUy 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:40-78, hi) 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:40-78, 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:40-78, 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 hsts 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 apphcable descriptions, references, and threshold parameters.

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

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments ofthe 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 usedherein 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 ceU" includes a plurality of such host ceUs, 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, aU technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skUl in the art to which this invention belongs. Although any machines, materials, and methods simUar 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. AU pubhcations mentioned herein are cited for the purpose of describing and disclosing the ceU lines, protocols, reagents and vectors which are reported in the pubhcations 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 "ENZM" refers to the amino acid sequences of substantially purified ENZM obtained from any species, particularly a mammahan 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 ENZM. Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of ENZM either by dkectly interacting with ENZM or by acting on components of the biological pathway in which ENZM participates.

An "aUelic variant" is an alternative form of the gene encoding ENZM. AUeUc 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 aUehc variants of its naturaUy occurring form. Common mutational changes which give rise to aUehc variants are generaUy 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 ENZM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as ENZM or a polypeptide with at least one functional characteristic of ENZM. Included within this definition are polymorphisms which may or may not be readUy detectable using a particular oUgonucleotide probe of the polynucleotide encoding ENZM, and improper or unexpected hybridization to aUehc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding ENZM. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a sUent change and result in a functionaUy equivalent ENZM. Dehberate amino acid substitutions may be made on the basis of one or more simUarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of ENZM 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 simUar hydroplhlicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having simUar 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 naturaUy occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturaUy 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.

"Amphfication" 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 amphfication technologies weU known in the art.

The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of ENZM. Antagonists may include proteins such as antibodies, anticahns, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of ENZM either by dkectly interacting with ENZM or by acting on components of the biological pathway in which ENZM participates.

The term "antibody" refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind ENZM polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen. The polypeptide or ohgopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemicaUy, and can be conjugated to a carrier protein if desked. Commonly used carriers that are chemicaUy 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 specificaUy to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic detemiinant may compete with the intact antigen (i.e., the immunogen used to ehcit the immune response) for binding to an antibody.

The term "aptamer" refers to a nucleic acid or ohgonucleotide 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 hbraries. 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 desked property, e.g., resistance to nucleases or longer hfetime in blood. Aptamers may be conjugated to other molecules, e.g. , a high molecular weight carrier to slow clearance of the aptamer from the ckculatory system. Aptamers may be specificaUy cross-linked to thek cognate hgands, 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 systemhas 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 naturaUy occurring enzymes, which normaUy 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); ohgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; ohgonucleotides having modified sugar groups such as 2 '-methoxyethyl sugars or 2'-methoxyethoxy sugars; or ohgonucleotides having modified bases such as 5-methyl cytosine, 2 -deoxyuracU, or 7-deaza-2 - deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-paks with a naturaUy occurring nucleic acid sequence produced by the ceU 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 "biologicaUy active" refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule. Likewise, "immunologicaUy active" or "immunogenic" refers to the capability of the natural, recombinant, or synthetic ENZM, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.

"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-paking. For example, 5'-AGT-3' paks 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 ENZM or fragments of ENZM 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 uncaUed 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 GELVEEW 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 especiaUy 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, Gin, His

Asp Asn, Glu

Cys Ala, Ser

Gin Asn, Glu, His

Glu Asp, Gin, His

Gly Ala

His Asn, Arg, Gin, Glu

Ue Leu, Val

Leu Ue, Val

Lys Arg, Gin, Glu

Met Leu, lie

Phe His, Met, Leu, Trp, Tyr

Ser Cys, Thr

Thr Ser, Val

Trp Phe, Tyr

Tyr His, Phe, Trp

Val lie, Leu, Thr

Conservative amino acid substitutions generaUy 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 chemicaUy 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 simUar 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 maybe 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 aUowing acceleration of the evolution of new protein functions.

A "fragment" is a unique portion of ENZM or a polynucleotide encoding ENZM 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 preferentiatty 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:40-78 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:40-78, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO:40-78 can be employed in one or more embodiments of methods ofthe invention, for example, in hybridization and amphfication technologies and in analogous methods that distin uish SEQ ID NO:40-78 fromrelated polynucleotides. The precise length of a fragment of SEQ ID NO:40-78 and the region of SEQ ID NO:40-78 to which the fragment corresponds are routinely deteπninable by one of ordinary skill in the art based on the intended purpose for the fragment.

A fragment of SEQ ID NO:l-39 is encoded by a fragment of SEQ ID NO:40-78. A fragment of SEQ ID NO: 1-39 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO:l-39. For example, a fragment of SEQ ID NO:l-39 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO: 1-39. The precise length of a fragment of SEQ ID NO:l-39 and the region of SEQ ID NO:l-39 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) foUowed by an open reading frame and a translation termination codon. A "fuU length" polynucleotide sequence encodes a "fall length" polypeptide sequence.

"Homology" refers to sequence simUarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences. The terms "percent identity" and "% identity," as apphed 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 ahgnment 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 ahgnment 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 pakwise ahgnments of polynucleotide sequences, the default parameters are set as fohows: 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 avaUable sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Ahgnment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is avaUable from several sources, including the NCBI, Bethesda, MD, and on the Internet at 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 avaUable is a tool caUed "BLAST 2 Sequences" that is used for dkect pakwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at ncbi.nlm.nm.gov/goif/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 simUar amino acid sequences due to the degeneracy ofthe 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 aU encode substantiaUy 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 ahgnment are weU-known. Some ahgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases "percent simUarity" and "% simUarity," as apphed to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences ahgned 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 ahgnment program (described and referenced above). For pakwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as foUows: 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 pakwise 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: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on

Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or ma 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 aU of the elements requked for chromosome rephcation, 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 stUl retains its original binding abihty.

"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base paking 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 paks of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skUl in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desked 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 μg/ml sheared, denatured salmon sperm DNA.

GeneraUy, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typicaUy 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 T_m 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 T_m and conditions for nucleic acid hybridization are weU known and can be found in Sambrook, J. and D.W. RusseU (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 %. TypicaUy, 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 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular ckcumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions wUl be readUy apparent to those of ordinary sk l in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary simUarity between the nucleotides. Such simUarity is strongly indicative of a similar role for the nucleotides and thek 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., C₀t or R₀t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a sohd support (e.g., paper, membranes, filters, chips, pins or glass shdes, or any other appropriate substrate to which ceUs or thek 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 ceUular and systemic defense systems. An "immunogenic fragment" is a polypeptide or oUgopeptide fragment of ENZM which is capable of ehciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or ohgopeptide fragment of ENZM 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 ENZM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of ENZM.

The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oUgonucleotide, 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 oUgonucleotide 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 thek hfespan in the ceU.

"Post-translational modification" of an ENZM may involve hpidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wUl vary by ceU type depending on the enzymatic milieu of ENZM. "Probe" refers to nucleic acids encoding ENZM, thek complements, or fragments thereof, which are used to detect identical, aUelic or related nucleic acids. Probes are isolated ohgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, hgands, chemUuminescent agents, and enzymes. "Primers" are short nucleic acids, usuaUy DNA ohgonucleotides, 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 paks can be used for amphfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typicaUy 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. RusseU (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^th ed., John WUey & 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 paks 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).

Ohgonucleotides 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 paks of up to 100 nucleotides each, and for the analysis of ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 Idlobases. SimUar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (avaUable to the pubhc from the Genome Center at University of Texas South West Medical Center, DaUas 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 (avaUable to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows 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 ohgonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from thek respective sources and modified to meet the user's specific needs.) The PrimeGen program (avaUable to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence ahgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this programis useful for identification of both unique and conserved ohgonucleotides and polynucleotide fragments. The ohgonucleotides 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 partiaUy 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 naturaUy 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 RusseU (supra). The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion ofthe 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 ceU.

Alternatively, such recombinant nucleic acids maybe part of a vkal 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 usuaUy 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 vkal 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 radionuchdes; enzymes; fluorescent, chemUuminescent, 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 aU occurrences of the nitrogenous base thymine are replaced with uracU, 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 ENZM, nucleic acids encoding ENZM, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; 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 smaU 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 wUl reduce the amount of labeled A that binds to the antibody.

The term "substantiaUy purified" refers to nucleic acid or amino acid sequences that are removed from their natural envkonment 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 naturaUy 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, shdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capUlaries. The substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.

A "transcript image" or "expression profile" refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.

"Transformation" describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU 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 ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or vkal infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed ceUs" includes stably transformed cells in which the inserted DNA is capable of rephcation either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs 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 lentivkal 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 dkected 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 RusseU (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 pak 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 "ahelic" (as defined above), "sphce," "species," or "polymorphic" variant. A sphce variant may have significant identity to a reference molecule, but wUl generaUy 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 wUl generaUy 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 atleast 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 pak 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 simUarity over a certain defined length of one of the polypeptides.

THE INVENTION

Various embodiments of the invention include new human enzymes (ENZM), the polynucleotides encoding ENZM, and the use of these compositions for the diagnosis, treatment, or prevention of autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metabohsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU prohferative disorders, including cancer.

Table 1 summarizes the nomenclature for the fuU 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 ID). 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 ID) 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:) ofthe 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 apphcable, aU 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 amino acid residues comprising signature sequences, domains, motifs, potential phosphorylation sites, and potential glycosylation sites. Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were apphed. Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are enzymes. For example, SEQ ID NO:l is 99% identical, from residue Ml to residue F415, to human pyruvate dehydrogenase complex protein X subunit precursor (GenBank ID g2979625) as deteπnined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 2.5e-220, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO: 1 also has homology to dihydrohpoamide dehydrogenase-binding protein (protein X), as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:l also contains a 2-oxo acid dehydrogenases acyltransferase catalytic domain, a biotin-requking enzyme domain, an e3-binding domain, and a pyruvate dehydrogenase complex dihydrohpoamide acetyltransferase catalytic domain, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and TIGRFAM databases of conserved protein famihes/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:l is a pyruvate dehydrogenase complex protein. In another example, SEQ ID NO:4 is 99% identical, fromresidue F81 to residue L483, to human N-acetylglucosam e-6-sulphatase (GenBank ID g31867) as determined by BLAST. (See Table 2.) The BLAST probabihty score is 6.8e-267, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:4 also has homology to proteins that are locahzed to the lysosome, hydrolyze sulfate groups from glycosaminoglycans, are involved in the catabohsm of heparan sulfate and keratan sulfate, and are N-acetylglucosamine-6-sulphate sulphatases, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:4 also contains a sulfatase domain as determined by searching for statisticaUy significant matches in the PFAM database. (See Table 3.) Data from BLIMPS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:4 is an N- acetylglucosamine-6-sulfatase.

In an alternative example, SEQ ID NO:9 is 96% identical, fromresidue Ml to residue Y371, to human acetylcholinesterase (GenBank ID gl77975) as determined by BLAST. (See Table 2.) The BLAST probabUity score is 7.9e-295, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:9 also has homology to proteins that are localized to dendrites, axons, and cell bodies, hydrolyze the neurotransmitter, acetylcholine, and are acetylcholinesterases, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:9 also contains a carboxylesterase domain as deteπnined by searching for statisticaUy significant matches in the PFAM database. (See Table 3.) Data fromBLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:9 is a carboxylesterase.

In an alternative example, SEQ ID NO:14 is 99% identical, fromresidue E115 to residue R293, to human cytochrome P450 2S1 (GenBank ID gl3161184) as determined by BLAST. (See Table 2.) The BLAST probabihty score is 1.6e-153, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO: 14 also has homology to proteins that are members of the cytochrome P450 famUy and are predicted to play a role in extrahepatic xenobiotic metabohsm and possibly the immune response and olfaction, as deteπnined by BLAST analysis using the PROTEOME database. SEQ ID NO: 14 also contains a cytochrome P450 domain as detenriined by searching for statisticaUy significant matches in the PFAM database. (See Table 3.) Data fromBLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further coπoborative evidence that SEQ ID NO: 14 is a cytochrome P450.

In a further example, SEQ ID NO:20 is 97% identical, fromresidue Ml to residue E97, 100% identical fromresidue 1163 to residue L256, and 94% identical, fromresidue A95 to residue L166, to human long-chain 2-hydroxy acid oxidase, HAOX2 (GenBank ID g7208438) as determined by BLAST. (See Table 2.) The BLAST probabihty score is 5.5e-131, for aU three alignments above, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:20 also has homology to proteins that are locahzed to the cytoplasm, function as oxidoreductases, and are hydroxy acid oxidases, as deteπnined by BLAST analysis using the PROTEOME database. SEQ ID NO:20 also contains an FMN-dependent dehydrogenase domain as deteπnined by searching for statisticaUy significant matches in the PFAM database. (See Table 3.) Data fromBLIMPS, analysis and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:20 is a hydroxy acid dehydrogenase.

In another example, SEQ ID NO:23 is 98% identical, fromresidue N85 to residue 1301 and 97% identical, fromresidue Ml to residue V87, to human protem simUar to fucosidase, alpha-L-1, tissue (GenBank ID gl3111748) as determined by BLAST. (See Table 2.) The BLAST probabUity score is 2.9e-168 for each ahgnment, which indicates the probabUity of obtaining the observed polypeptide sequence ahgnments by chance. SEQ ID NO:23 also has homology to proteins that are locahzed to the lysosome and vacuole, have hydrolase function, and are alpha-L-fucosidases, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:23 also contains an alpha-L-fucosidase domain as determined by searching for statisticaUy significant matches in the

PFAM database. (See Table 3.) Data fromBLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:23 is simUar to the alpha-L-fucosidase enzyme.

In yet another example, SEQ ID NO:24 is 89% identical, fromresidue R16 to residue R361 and 100% identical, fromresidue Ml to residue Q48, to human isovaleryl-CoA dehydrogenase

(GenBank ID g306897) as determined by BLAST. (See Table 2.) The BLAST probabUity score is 1.2e-l 86 for both ahgnments, which indicates the probabUity of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:24 also has homology to proteins that are localized to the mitochondrial matix, have oxidoreductase function, and are isovaleryl-CoA dehydrogenases, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:24 also contains an acyl-CoA dehydrogenase, C-terminal domain, middle domain, and N-terminal domain as determined by searching for statisticaUy significant matches in the PFAM database. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:24 is an acyl-CoA dehydrogenase.

In a further example, SEQ ID NO:32 is a sphce variant of human cGMP phosphodiesterase (GenBank ID g2366987) as determined by BLAST. (See Table 2.) The BLAST probabihty score is 0.0, which indicates the probabUity of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:32 also has homology to proteins that are hydrolases, that function in phototransduction, and that have been found in mutated forms in retinitis pigmentosa and stationary night blindness, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:32 also contains a GAF (cychc GMP, adenylyl cyclase, FhlA) domain and a 3 ',5 '-cychc nucleotide phosphodiesterase domain, as deteπnined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein famUies/domains. SEQ ID NO:32 also contains a domain present in phytochromes and cGMP-specific phosphodiesterases and a metal-dependent phosphohydrolases with conserved 'HD' motif domain, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based SMART database of conserved protein families/domains. (See Table 3.) Data fromBLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:32 is a cGMP phosphodiesterase.

In yet a further example, SEQ ID NO:38 is 97% identical, fromresidue Ml to residue G872, to human phosphohpase C-beta-2 (GenBank ID gl90040) as determined by BLAST. (See Table 2.) The BLAST probabihty score is 0.0, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:38 also has homology to proteins that are calcium-dependent, are regulated by G-proteins, and that hydrolyze phosphatidylinositol-4,5- bisphosphate to the second messengers inositol-l,4,5-trisphosphate and diacylglycerol, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:38 contains a C2 (calcium binding) domain and phospholipase C X and Y domains, as determined by searching for statisticaUy significant matches in the PFAM and SMART databases. (See Table 3.) Data fromBLIMPS analyses and BLAST analyses against the PRODOM and DOMO databases provide further corroborative evidence that SEQ ID NO:38 is a phosphohpase C famny member. SEQ ID NO:2-3, SEQ ID NO:5-8, SEQ ID NO.10-13, SEQ ID NO.15-19, SEQ ID NO:21-23, SEQ ID NO.25-31, SEQ ID NO:33-37, and SEQ ID NO:39 were analyzed and annotated in a simUar manner. The algorithms and parameters for the analysis of SEQ ID NO:l-39 are described in Table 7. As shown in Table 4, the fuU 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 hsts the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the coπesponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence inbasepaks. 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:40-78 or that distinguish between SEQ ID NO:40-78 and related polynucleotides . The polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, 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 S anger Centre, Cambridge, UK) database (Le. , 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 apphed, and YYYYY is the number of the prediction generated by the algorithm, and N_{1 2,}s..., if present, represent specific exons that may have been manuaUy 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 FLXXXXXX_gAAAAA_gBBBBB_l_N is 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 apphed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N refeπing 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 foUowing Table Usts examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).

INCY FuU 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 hbraries 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 aUele 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 ofthe 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 aUeles 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 aUele 1 in the population was too low to be detected, while n/a (not avaUable) indicates that the allele frequency was not determined for the population.

The invention also encompasses ENZM variants. Various embodiments of ENZM variants can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the ENZM amino acid sequence, and can contain at least one functional or structural characteristic of ENZM. Various embodiments also encompass polynucleotides which encode ENZM. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:40-78, which encodes ENZM. The polynucleotide sequences of SEQ ID NO:40-78, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracU, and the sugar backbone is composed of ribose instead of deoxyribose.

The invention also encompasses variants of a polynucleotide encoding ENZM. In particular, such a variant polynucleotide wiU have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polynucleotide sequence identity to a polynucleotide encoding ENZM. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:40-78 which has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:40-78. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of ENZM.

In addition, or in the alternative, a polynucleotide variant of the invention is a sphce variant of a polynucleotide encoding ENZM. A sphce variant may have portions which have significant sequence identity to a polynucleotide encoding ENZM, but wUl generaUy have a greater or lesser number of nucleotides due to additions or deletions of blocks of sequence arising from alternate spUcing during mRNA processing. A sphce 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 ENZM over its entke length; however, portions of the sphce variant wiU 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 ENZM. For example, a polynucleotide comprising a sequence of SEQ ID NO:42 and a

' polynucleotide comprising a sequence of SEQ ID NO:52 are sphce variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:49; a polynucleotide comprising a sequence of SEQ ID NO:51 are sphce variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:66 and a polynucleotide comprising a sequence of SEQ ID NO:67 are sphce variants of each other. Any one of the sphce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of ENZM.

It wiU be appreciated by those skiUed in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding ENZM, some bearing minimal simUarity to the polynucleotide sequences of any known and naturaUy 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 apphed to the polynucleotide sequence of naturaUy occurring ENZM, and aU such variations are to be considered as being specificaUy disclosed. Although polynucleotides which encode ENZM and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring ENZM under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding ENZM or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy 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 substantiaUy altering the nucleotide sequence encoding ENZM and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more deskable properties, such as a greater half-hfe, than transcripts produced from the naturaUy occurring sequence. The invention also encompasses production of polynucleotides which encode ENZM and

ENZM derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide may be inserted into any of the many avaUable expression vectors and ceU systems using reagents weU known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a polynucleotide encoding ENZM 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:40-78 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 weU 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 (Apphed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amphfication system (Invitrogen, Carlsbad CA). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 hquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Apphed 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 weU known in the art (Ausubel et al, supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. WUey VCH, New York NY, pp. 856-853).

The nucleic acids encoding ENZM 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 Apphc. 2:318-322). Another method, inverse PCR, uses primers that extend in divergent dkections to amplify unknown sequence from a ckcularized 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 thkd method, capture PCR, involves PCR amphfication of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Apphc. 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 perfoπning 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). AdditionaUy, one may use PCR, nested primers, and PROMOTERFINDER libraries (BD Clontech, Palo Alto CA) to walk' genomic DNA. This procedure avoids the need to screen hbraries and is useful in finding intron/exon junctions. For aU PCR-based methods, primers may be designed using commerciaUy avaUable 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 fuU length cDNAs, it is preferable to use hbraries that have been size-selected to include larger cDNAs. In addition, random-primed hbraries, 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 fuU-length cDNA. Genomic hbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.

Capillary electrophoresis systems which are commerciaUy avaUable may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capUlary 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/hght intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Apphed Biosystems), and the entke process from loading of samples to computer analysis and electronic data display may be computer controUed. CapUlary electrophoresis is especiaUy preferable for sequencing smaU 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 ENZM may be cloned in recombinant DNA molecules that dkect expression of ENZM, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express ENZM.

The polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter ENZM-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 ohgonucleotides ma be used to engineer the nucleotide sequences. For example, ohgonucleotide-mediated site-dkected mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce 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, EC 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 ENZM, 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 hbrary of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occuπing genes in a directed and controllable manner.

In another embodiment, polynucleotides encoding ENZM maybe synthesized, in whole or in part, using one or more chemical methods weU 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, ENZM 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 sohd-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 431 A peptide synthesizer (Apphed Biosystems). AdditionaUy, the amino acid sequence of ENZM, or any part thereof, may be altered during dkect 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 naturaUy occurring polypeptide.

The peptide may be substantiaUy 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 biologicaUy active ENZM, the polynucleotides encoding ENZM 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 ENZM. Such elements may vary in thek strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding ENZM. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding ENZM 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 ceU system used (Scharf, D. et al. (1994) Results Probl CeU Differ. 20:125-162).

Methods which are weU known to those skUled in the art may be used to construct expression vectors containing polynucleotides encoding ENZM and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, 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 ENZM. 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 ceU systems infected with vkal expression vectors (e.g., baculovirus); plant ceU systems transformed with vkal 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 arώnal ceU 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 Hnl, 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; BuUer, 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 ceU employed. In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding ENZM. For example, routine cloning, subcloning, and propagation of polynucleotides encoding ENZM can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORTl plasmid (Invitrogen). Ligation of polynucleotides encoding ENZM into the vector's multiple cloning site disrupts the lacZ gene, aUowing 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 ENZM are needed, e.g. for the production of antibodies, vectors which dkect high level expression of ENZM may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter maybe used.

Yeast expression systems may be used for production of ENZM. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors dkect either the secretion or intraceUular 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, CA. et al. (1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of ENZM. Transcription of polynucleotides encoding ENZM may be driven by vkal 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 smaU subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broghe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl CeU Differ. 17:85-105). These constructs can be introduced into plant ceUs by dkect DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196).

In mammaUan ceUs, a number of vkal-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, polynucleotides encoding ENZM may be hgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the vkal genome may be used to obtain infective virus which expresses ENZM in host ceUs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RS V) enhancer, may be used to increase expression in mammahan host ceUs. S V40 or EBV-based vectors may also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to dehver 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 dehvered 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 mammahan systems, stable expression of ENZM in ceU lines is preferred. For example, polynucleotides encoding ENZM can be transformed into cell lines using expression vectors which may contain vkal origins of rephcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs may be aUowed 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 aUows growth and recovery of ceUs which successfuUy express the introduced sequences. Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk^~ and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetaboUte, 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 emάpat 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 ceUular requkements for metabohtes (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; BD Clontech), β-glucuronidase and its substrate β-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, CA. (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 ENZM is inserted within a marker gene sequence, transformed ceUs containing polynucleotides encoding ENZM can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding ENZM under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.

In general, host ceUs that contain the polynucleotide encoding ENZM and that express ENZM may be identified by a variety of procedures known to those of skUl in the art. These procedures include, but are not United to, DNA-DNA or DNA-RNA hybridizations, PCR amphfication, 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 ENZM 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 ceU sorting (FACS). A two-site, monoclonal-based immunoassay utihzing monoclonal antibodies reactive to two non-interfering epitopes on ENZM 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 WUey- 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 skUled 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 ENZM include ohgolabeling, nick translation, end-labeling, or PCR amphfication using a labeled nucleotide. Alternatively, polynucleotides encoding ENZM, 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 commerciaUy avaUable, 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 commerciaUy avaUable 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 radionuchdes, enzymes, fluorescent, chemUuminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host ceUs transformed with polynucleotides encoding ENZM may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture. The protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used. As wiU be understood by those of skill in the art, expression vectors containing polynucleotides which encode ENZM may be designed to contain signal sequences wliich dkect secretion of ENZM through a prokaryotic or eukaryotic ceU membrane.

In addition, a host ceU strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desked fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation, 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 ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are avaUable from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the coπect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding ENZM may be hgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric ENZM protein containing a heterologous moiety that can be recognized by a commerciaUy avaUable antibody may facUitate the screening of peptide hbraries for inhibitors of ENZM activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially avaUable affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), cahnodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of thek cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, cahnodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable miπiunoaffinity purification of fusion proteins using commerciaUy avaUable monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the ENZM encoding sequence and the heterologous protein sequence, so that ENZM may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel et al (supra, ch. 10 and 16). A variety of commerciaUy avaUable kits may also be used to facilitate expression and purification of fusion proteins.

In another embodiment, synthesis of radiolabeled ENZM maybe 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.

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

In related embodiments, variants of ENZM can be used to screen for binding of test compounds, such as antibodies, to ENZM, a variant of ENZM, or a combination of ENZM and/or one or more variants ENZM. In an embodiment, a variant of ENZM can be used to screen for compounds that bind to a variant of ENZM, but not to ENZM having the exact sequence of a sequence of SEQ ID NO: 1-39. ENZM variants used to perform such screening can have a range of about 50% to about 99% sequence identity to ENZM, 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 ENZM can be closely related to the natural ligand of ENZM, 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 l(2):Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor ENZM (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 ENZM can be closely related to the natural receptor to which ENZM binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the ligand binding site or binding pocket. For example, the compound may be a receptor for ENZM which is capable of propagating a signal, or a decoy receptor for ENZM 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 I Gi (Taylor, P.C et al. (2001) Curr. Opin. Immunol. 13:611-616). In one embodiment, two or more antibodies having simUar or, alternatively, different specificities can be screened for specific binding to ENZM, fragments of ENZM, or variants of ENZM. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of ENZM. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of ENZM. 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 ENZM.

In an embodiment, anticalins can be screened for specific binding to ENZM, fragments of ENZM, or variants of ENZM. 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 ENZM involves producing appropriate cells which express ENZM, either as a secreted protein or on the cell membrane. Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing ENZM or ceU membrane fractions wliich contain ENZM are then contacted with a test compound and binding, stimulation, or inhibition of activity of either ENZM 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 ENZM, either in solution or affixed to a solid support, and detecting the binding of ENZM 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 sohd 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 hgands (Matthews, DJ. 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. Nati. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982- 10988).

ENZM, fragments of ENZM, or variants of ENZM may be used to screen for compounds that modulate the activity of ENZM. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for ENZM activity, wherein ENZM is combined with at least one test compound, and the activity of ENZM in the presence of a test compound is compared with the activity of ENZM in the absence of the test compound. A change in the activity of ENZM in the presence of the test compound is indicative of a compound that modulates the activity of ENZM. Alternatively, a test compound is combined with an in vitro or ceU-free system comprising ENZM under conditions suitable for ENZM activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of ENZM may do so indkectly and need not come in dkect contact with the test compound. At least one and up to a plurality of test compounds may be screened.

In another embodiment, polynucleotides encoding ENZM or thek mammahan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs. Such techniques are weU 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 ceUs, such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture. The ES ceUs 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 ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgicaUy 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 ENZM may also be manipulated in vitro in ES ceUs derived from human blastocysts. Human ES ceUs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types. These ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding ENZM 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 ENZM is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome. Transformed ceUs 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 ENZM, e.g., by secreting ENZM 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 ENZM and enzymes. In addition, examples of tissues expressing ENZM can be found in Table 6 and can also be found in Example XL Therefore, ENZM appears to play a role in autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metabohsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU prohferative disorders, including cancer. In the treatment of disorders associated with increased ENZM expression or activity, it is desirable to decrease the expression or activity of ENZM. In the treatment of disorders associated with decreased ENZM expression or activity, it is desirable to increase the expression or activity of ENZM.

Therefore, in one embodiment, ENZM 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 ENZM. Examples of such disorders include, but are not limited to, an autoimmune/inflammatory disorder such as acquked immunodeficiency syndrome (AIDS),

Addison's disease, adult respkatory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrmopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetahs, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophiha, kritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflarnmation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjbgren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal ckculation, and trauma; an infectious disorder such as a vkal infection, e.g., caused by an adenovirus (acute respkatory disease, pneumonia), an arenavkus (lymphocytic choriomeningitis), a bunyavkus (Hantavirus), a coronavkus (pneumonia, chronic bronchitis), a hepadnavkus (hepatitis), a herpesvkus (herpes simplex virus, variceUa-zoster virus, Epstein-Ban virus, cytomegalovirus), a flavivkus

(yeUow fever), an orthomyxovkus (influenza), a papiUomavkus (cancer), a paramyxovkus (measles, mumps), a picornovirus (rhinovkus, poliovkus, coxsackie-vkus), a polyomavkus (BK virus, JC virus), a poxvirus (smaUpox), a reovkus (Colorado tick fever), a retrovirus (human immunodeficiency virus, human T lymphotropic virus), a rhabdovkus (rabies), a rotavkus (gastroenteritis), and a togavkus (encephalitis, rubeUa), and a bacterial infection, a fungal infection, a parasitic infection, a protozoal infection, and a helminthic infection; an immune deficiency, such as acquked irnmunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVT), DiGeorge's syndrome (thymic hypoplasia), fhymic 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, and immunodeficiency associated with Cushing's disease; a disorder of metabohsm such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty hepatockrhosis, fructose- 1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenahsm, hypoadrenahsm, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperhpidemia, hyperhpemia, a hpid myopafhy, a hpodystrophy, a lysosomal storage disease, mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria, pseudovitamin D-deficiency rickets; a reproductive disorder such as a disorder of prolactin production, infertUity, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia; a neurological disorder such as epUepsy, 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 vkal meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radicuhtis, vkal central nervous system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and metaboUc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis; inherited, metabohc, endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis; mental disorders including mood, anxiety, and schizophrenic disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; a cardiovascular disorder, such as arteriovenous fistula, atherosclerosis, hypertension, vascuhtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrornbolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart faUure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy 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, vkal and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophiha bronchiohtis obhterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in coUagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and nonk-flainmatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation; an eye disorder such as ocular hypertension and glaucoma; a disorder of ceU proUferation such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, ckrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; and a cancer, including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gaU bladder, gangUa, gastrointestinal tract, heart, kidney, hver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.

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

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

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

In a further embodiment, an antagonist of ENZM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ENZM. Examples of such disorders include, but are not limited to, those autoimmune/kiflammatory disorders, infectious disorders, immune deficiencies, disorders of metabohsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU prohferative disorders, including cancer described above. In one aspect, an antibody which specificaUy binds ENZM may be used dkectly as an antagonist or indkectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express ENZM.

In an additional embodiment, a vector expressing the complement ofthe polynucleotide encoding ENZM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ENZM 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 skUl in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention ofthe 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 ENZM may be produced using methods wliich are generaUy known in the art. In particular, purified ENZM may be used to produce antibodies or to screen hbraries of pharmaceutical agents to identify those which specificaUy bind ENZM. Antibodies to ENZM may also be generated using methods that are weU 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 hbrary. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeuticaUy. Single chain antibodies (e.g., from camels or Uamas) may be potent enzyme inhibitors and may have apphcation 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, Uamas, humans, and others may be immunized by injection with ENZM 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, oU emulsions, KLH, and dinitrophenol Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium paryum are especiaUy preferable.

It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to ENZM have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these ohgopeptides, peptides, or fragments are substantiaUy identical to a portion of the amino acid sequence of the natural protein. Short stretches of ENZM 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 ENZM may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EB V-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. CeU 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 maybe adapted, using methods known in the art, to produce ENZM-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin hbraries (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 hbraries or panels of highly specific binding reagents as disclosed in the Uterature (Oriandi, 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 ENZM may also be generated. For example, such fragments include, but are not limited to, F(ab^:)₂ fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression hbraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desked 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 typicaUy involve the measurement of complex formation between ENZM and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering ENZM epitopes is generaUy 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 ENZM. Affinity is expressed as an association constant, K_a, which is defined as the molar concentration of ENZM-antibody complex divided by the molar concentrations of free antigen and free antibody under equihbrium conditions. The K_a determined for a preparation of polyclonal antibodies, which are heterogeneous in thek affinities for multiple ENZM epitopes, represents the average affinity, or avidity, of the antibodies for ENZM. The K_a determined for a preparation of monoclonal antibodies, which are monospecific for a particular ENZM epitope, represents a true measure of affinity. High-affinity antibody preparations with K_a ranging from about 10⁹ to 10¹² L/mole are preferred for use in immunoassays in which the ENZM-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_α ranging from about 10^δ to 10⁷ L/mole are preferred for use in immunopurification and simUar procedures wliich ultimately requke dissociation of ENZM, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John WUey & 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 generaUy employed in procedures requking precipitation of ENZM-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various apphcations, are generaUy avaUable (Catty, supra; CoUgan et al, supra).

In another embodiment of the invention, polynucleotides encoding ENZM, 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 ohgonucleotides) to the coding or regulatory regions of the gene encoding ENZM. Such technology is weU known in the art, and antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding ENZM (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).

In therapeutic use, any gene delivery system suitable for introduction ofthe 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 Chn. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) FASEB J. 9:1288- 1296). Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271-278;

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, JJ. (1995) Br. Med. Bull. 51:217-225; Boado, RJ. 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 ENZM may be used for somatic or geπnline 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) CeU 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, famihal hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (U) express a conditionaUy lethal gene product (e.g., in the case of cancers which result from unregulated ceU prohferation), or (hi) express a protein which affords protection against intraceUular 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. USA93:11395-11399), 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 ENZM expression or regulation causes disease, the expression of ENZM from an appropriate population of transduced ceUs may aUeviate the clinical manifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders caused by deficiencies in ENZM are treated by constructing mammahan expression vectors encoding ENZM and introducing these vectors by mechanical means into ENZM-deficient ceUs. Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) dkect DNA microinjection into individual ceUs, (h) ballistic gold particle dehvery, (in) Uposome-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; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of ENZM include, but are not limited to, the PCDN A 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (BD Clontech, Palo Alto CA). ENZM may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (h) 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), commerciaUy avaUable in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (avaUable 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 ENZM from a normal individual. CommerciaUy avaUable hposome transformation kits (e.g. , the PERFECT LIPID TRANSFECTION KIT, avaUable from Invitrogen) aUow one with ordinary skiU in the art to dehver polynucleotides to target ceUs in culture and requke minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and AJ. Eb (1973) Vkology 52:456-467), or by electroporation (Neumann, E. et al. ( 1982) EMB O J. 1:841-845). The introduction of DNA to primary ceUs requkes modification of these standardized mammahan transfection protocols.

In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to ENZM expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding ENZM under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (k) appropriate RNA packaging signals, and (Ui) a Rev-responsive element (RRE) along with additional retrovirus s-acting RNA sequences and coding sequences requked for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commerciaUy avaUable (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs or a promiscuous envelope protein such as VS Vg (Armentano, D. et al. (1987) J. Vkol 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. MiUer (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Vkol. 72:8463-8471; Zufferey, R. et al. (1998) J. Vkol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging ceU hues producing high transducing efficiency retrovkal supernatant") discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4⁺ T- ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Vkol. 71:7020- 7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Vkol. 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 adenovkus-based gene therapy delivery system is used to dehver polynucleotides encoding ENZM to ceUs which have one or more genetic abnormalities with respect to the expression of ENZM. The construction and packaging of adenovkus-based vectors are weU known to those with ordinary skUl in the art. Rephcation 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). PotentiaUy useful adenovkal vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenovkal vectors, see also Antinozzi, P.A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, LM. and N. Somia (1997; Nature 18:389:239-242).

In another embodiment, a herpes-based, gene therapy delivery system is used to dehver polynucleotides encoding ENZM to target ceUs which have one or more genetic abnormahties with respect to the expression of ENZM. The use of herpes simplex virus (HS V)-based vectors may be especiaUy valuable for introducing ENZM to ceUs of the central nervous system, for which HSV has a tropism The construction and packaging of herpes-based vectors are weU known to those with ordinary skUl in the art. A rephcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to dehver 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 detaU 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 ceU 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. Vkol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesvkus sequences, the generation of recombinant virus foUowing the transfection of multiple plasmids containing different segments of the large herpesvkus genomes, the growth and propagation of herpesvkus, and the infection of ceUs with herpesvkus are techniques weU known to those of ordinary skUl in the art. In another embodiment, an alphavkus (positive, single-stranded RNA virus) vector is used to dehver polynucleotides encoding ENZM to target ceUs. The biology of the prototypic alphavkus, 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 alphavkus RNA rephcation, a subgenomic RNA is generated that normaUy encodes the viral capsid proteins. This subgenomic RNA rephcates to higher levels than the fuU length genomic RNA, resulting in the overproduction of capsid proteins relative to the vkal proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for ENZM into the alphavkus genome in place of the capsid-coding region results in the production of a large number of ENZM-coding RNAs and the synthesis of high levels of ENZM in vector transduced ceUs. While alphavkus infection is typicaUy associated with ceU lysis within a few days, the abUity to estabhsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic rephcation of alphavkuses can be altered to suit the needs of the gene therapy apphcation (Dryga, S.A. et al. (1997) Vkology 228:74-83). The wide host range of alphavkuses wiU aUow the introduction of ENZM into a variety of ceU types. The specific transduction of a subset of ceUs in a population may requke the sorting of ceUs prior to transduction. The methods of manipulating infectious cDNA clones of alphavkuses, performing alphavkus cDNA and RNA transfections, and perforating alphavkus infections, are weU known to those with ordinary skUl in the art.

Ohgonucleotides 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. SimUarly, inhibition can be achieved using triple helix base-paking methodology. Triple hehx pairing is useful because it causes inhibition of the abihty of the double hehx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the hterature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Pubhshing, 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, foUowed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding ENZM.

Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides 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 chemicaUy synthesizing ohgonucleotides such as sohd phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding ENZM. 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 ceU lines, ceUs, or tissues. RNA molecules may be modified to increase intraceUular stability and half-hfe. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2 ' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in aU of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and simUarly modified forms of adenine, cytosine, guanine, thymine, and uracU which are not as easUy 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 sUenced using RNA interference (RNAi) or post-transcriptional gene sUencing (PTGS) methods known in the art. RNAi is a post-transcriptional mode of gene sUencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU specificaUy suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantiaUy reduces the expression of the targeted gene. PTGS can also be accomphshed by use of DNA or DNA fragments as weU. RNAi methods are described by Fke, 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 vkal vector delivery methods described herein or known in the art.

RNAi can he induced in mammahan ceUs by the use of smaU interfering RNA also known as siRNA. siRNA are shorter segments of dsRNA (typicaUy 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 mammahan ceUs is described by Elbashk, S.M. et al. (2001 ; Nature 411 :494-498). siRNA can be generated indkectly by introduction of dsRNA into the targeted ceU. Alternatively, siRNA can be synthesized dkectly and introduced into a ceU by transfection methods and agents described herein or known in the art (such as hposome-mediated transfection, vkal vector methods, or other polynucleotide delivery/introductory methods). Suitable siRNAs can be selected by examining a transcript ofthe 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 (wilhin 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

no by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy avaUable methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).

In alternative embodiments, long-term gene sUencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomphshed 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 ernbodiments, shRNAs can be dehvered to target ceUs using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (ckcular) plasmid (Ambion). Once dehvered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene- specific sUencing.

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, for example, by northern analysis methods using the NORTHERNMAX-GLY kit (Ambion) ; by microaπay 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, for example, by microarray methods; by polyacrylamide gel electrophoresis; and 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 ENZM. Compounds which maybe effective in altering expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, 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 polynucleoti.de expression. Thus, in the treatment of disorders associated with increased ENZM expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding ENZM may be therapeutically useful, and in the treatment of disorders associated with decreased ENZM expression or activity, a compound which specifically promotes expression of the polynucleotide encoding ENZM may be therapeuticaUy 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 naturaUy-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 ENZM is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabihzed cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding ENZM 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 ENZM. 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 ceU 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 ohgonucleotides (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 ceUs or tissues are avaUable and equaUy suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dehvery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).

Any of the therapeutic methods described above may be apphed 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 generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient. Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Pubhshing, Easton PA). Such compositions may consist of ENZM, antibodies to ENZM, and mimetics, agonists, antagonists, or inhibitors of ENZM.

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 hquid or dry powder form These compositions are generaUy aerosolized immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol dehvery of fast-acting formulations is weU-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary dehvery via the alveolar region of the lung have enabled the practical dehvery of drugs such as insulin to blood ckculation (see, e.g., Patton, J.S. et al, U.S. Patent No. 5,997,848). Pulmonary dehvery ahows aάjriinistration without needle injection, and obviates the need for potentiaUy 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 weU within the capability of those skiUed in the art.

Speciahzed forms of compositions may be prepared for dkect intraceUular dehvery of macromolecules comprising ENZM or fragments thereof. For example, hposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular dehvery of the macromolecule. Alternatively, ENZM 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 aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al (1999) Science 285:1569-1572).

For any compound, the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, 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 therapeuticaUy effective dose refers to that amount of active ingredient, for example ENZM or fragments thereof, antibodies of ENZM, and agonists, antagonists or inhibitors of ENZM, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity ma be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED₅₀ (the dose therapeuticaUy effective in 50% of the population) or LD₅₀ (the dose lethal to 50% ofthe 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 ceU culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably wilhin a range of ckculating 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 wiU be deteπnined by the practitioner, in hght of factors related to the subject requking treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desked 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-hfe and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of dehvery is provided in the hterature and generaUy avaUable to practitioners in the art. Those skilled in the art wiU employ different formulations for nucleotides than for proteins or thek inhibitors. SimUarly, dehvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS In another embodiment, antibodies which specifically bind ENZM may be used for the diagnosis of disorders characterized by expression of ENZM, or in assays to monitor patients being treated with ENZM or agonists, antagonists, or inhibitors of ENZM. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for ENZM include methods which utilize the antibody and a label to detect ENZM in human body fluids or in extracts of ceUs 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 ENZM, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of ENZM expression. Normal or standard values for ENZM expression are estabhshed by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to ENZM under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of ENZM expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease. In another embodiment of the invention, polynucleotides encoding ENZM may be used for diagnostic purposes. The polynucleotides which may be used include ohgonucleotides, 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 ENZM may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of ENZM, and to monitor regulation of ENZM levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding ENZM or closely related molecules may be used to identify nucleic acid sequences which encode ENZM. 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 amphfication wiU determine whether the probe identifies only naturaUy occuπing sequences encoding ENZM, aUeUc 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 ENZM 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:40-78 or from genomic sequences including promoters, enhancers, and introns ofthe ENZM gene.

Means for producing specific hybridization probes for polynucleotides encoding ENZM include the cloning of polynucleotides encoding ENZM or ENZM derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commerciaUy avaUable, 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 radionuchdes 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 ENZM may be used for the diagnosis of disorders associated with expression of ENZM. Examples of such disorders include, but are not hmited to, an autoki-mune/kiflamirøtory disorder such as acquked immunodeficiency syndrome (AIDS), Addison's disease, adult respkatory distress syndrome, aUergies, ankylosing spondyhtis, 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, eiythroblastosis fetahs, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophUia, kritable 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 cohtis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal ckculation, and trauma; an infectious disorder such as a vkal infection, e.g., caused by an adenovirus (acute respkatory disease, pneumonia), an arenavkus (lymphocytic choriomeningitis), a bunyavkus (Hantavkus), a coronavirus (pneumonia, chronic bronchitis), a hepadnavkus (hepatitis), a herpesvirus (herpes simplex virus, variceUa-zoster virus, Epstein-Barr virus, cytomegalovirus), a flavivkus (yeUow fever), an orthomyxovkus (influenza), a papiUomavkus (cancer), a paramyxovkus (measles, mumps), a picornovirus (rhinovkus, poliovkus, coxsackie-vkus), a polyomavkus (BK virus, JC virus), a poxvirus (smaUpox), a reovkus (Colorado tick fever), a retrovirus (human immunodeficiency virus, human T lymphotropic virus), a rhabdovkus (rabies), a rotavkus (gastroenteritis), and a togavkus (encephahtis, rubeUa), and a bacterial infection, a fungal infection, a parasitic infection, a protozoal infection, and a helminthic infection; an immune deficiency, such as acquked immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVL), 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, and immunodeficiency associated with Cushing's disease; a disorder of metabohsm such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty hepatockrhosis, fructose- 1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism,

hypoparathyroidism- hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperhpidemia, hyperhpemia, a hpid myopafhy, a hpodystrophy, a lysosomal storage disease, mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria, pseudovitamin D-deficiency rickets; a reproductive disorder such as a disorder of prolactin production, infertUity, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia; a neurological disorder such as epUepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyofrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and vkal meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, vkal central nervous system disease; prion diseases including kuru, Creutzfeldt- Jakob disease, and Gerstor-ann-Straussler-Scheinker syndrome; fatal famUial iiisomnia, nutritional and metaboUc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinal hemangioblastomatosis, encephalotiigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis; inherited, metabohc, endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis; mental disorders including mood, anxiety, and schizophrenic disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; a cardiovascular disorder, such as arteriovenous fistula, atherosclerosis, hypertension, vascuhtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of fhrombolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart faUure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy 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 comphcations 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, vkal and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophiUa bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in coUagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and comphcations of lung transplantation; an eye disorder such as ocular hypertension and glaucoma; a disorder of ceU prohferation such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; and a cancer, including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gaU bladder, gangUa, gastrointestinal tract, heart, kidney, hver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Polynucleotides encoding ENZM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELIS A-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered ENZM expression. Such qualitative or quantitative methods are weU known in the art.

In a particular embodiment, polynucleotides encoding ENZM maybe used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding ENZM 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 ENZM 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 ENZM, a normal or standard profile for expression is estabhshed. This may be accomplished by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding ENZM, under conditions suitable for hybridization or amphfication. 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 substantiaUy 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 estabhsh the presence of a disorder. Once the presence of a disorder is estabhshed 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 aUow health professionals to employ preventative measures or aggressive treatment earher, thereby preventing the development or further progression of the cancer.

Additional diagnostic uses for ohgonucleotides designed from the sequences encoding ENZM may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OUgomers wUl preferably contain a fragment of a polynucleotide encoding ENZM, or a fragment of a polynucleotide complementary to the polynucleotide encoding ENZM, and wiU be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.

In a particular aspect, oUgonucleotide primers derived from polynucleotides encoding ENZM may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquked genetic disease in humans. Methods of SNP detection include, but are not hmited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from polynucleotides encoding ENZM 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, bodUy 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 ohgonucleotide primers are fluorescently labeled, which ahows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. AdditionaUy, sequence database analysis methods, termed in sUico 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 eπors 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 inmonogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be coπelated 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 hfe-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, whUe 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-hpoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and thek 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 ENZM include radiolabeling or biotinylating nucleotides, coamphfication 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. Bioche 212:229-236). The speed of quantitation of multiple samples may be accelerated by mnning the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various άilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

In further embodiments, ohgonucleotides or longer fragments derived from any ofthe 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, ENZM, fragments of ENZM, or antibodies specific for ENZM may be used as elements on a microaπay. 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 ceU type. A transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and thek 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 thek complements to the totality of transcripts or reverse transcripts of a particular tissue or ceU type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or thek 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, ceU 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 ceU 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 prechnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring envkonmental compounds. AU compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysk, 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 simUar 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. IdeaUy, 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 weU, as the levels of expression of these genes are used to normahze the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. WhUe 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 wliich leads to prediction of toxicity (see, for example, Press Release 00-02 from the National Institute of Envkonmental Health Sciences, released February 29, 2000, avaUable at niehs.nm.gov/oc/news/toxchip.htm). Therefore, it is important and deskable in toxicological screening using toxicant signatures to include aU 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 coπesponding 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 ceU type. The term proteome refers to the global pattern of protein expression in a particular tissue or ceU type. Each protein component of a proteome can be subjected individuaUy to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and thek relative abundance under given conditions and at a given time. A profile of a ceU'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 visuahzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or sUver or fluorescent stains. The optical density of each protein spot is generaUy 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 partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed 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 ENZM to quantify the levels of ENZM expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by contacting the microarray with 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 paraUel with toxicant signatures at the transcript level. There is a poor coπelation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. SeUhamer (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 rehable 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 ofthe 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; BaldeschweUer et al. (1995) PCT apphcation W095/25116; Shalon, D. et al. (1995) PCT apphcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microarrays are weU 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 ENZM may be used to generate hybridization probes useful in mapping the naturaUy 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 maybe 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 hbraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, BJ. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic hnkage maps, for example, which coπelate 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-Uhich, 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 Mendehan Inheritance in Man (OMIM) World Wide Web site. Coπelation between the location of the gene encoding ENZM 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 estabhshed chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammahan 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 locahzed 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, ENZM, its catalytic or immunogenic fragments, or ohgopeptides 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 sohd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between ENZM 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 apphcation WO84/03564). In this method, large numbers of different smaU test compounds are synthesized on a sohd substrate. The test compounds are reacted with ENZM, or fragments thereof, and washed. Bound ENZM is then detected by methods weU known in the art. Purified ENZM can also be coated dkectly 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 sohd support.

In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding ENZM specificaUy compete with a test compound for binding ENZM. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic deteiminants with ENZM.

In additional embodiments, the nucleotide sequences which encode ENZM 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 cuπently known, including, but not limited to, such properties as the triplet genetic code and specific base pak 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, apphcations, and publications mentioned above and below, including U.S. Ser. No. 60/447,246, U.S. Ser. No. 60/449,087, U.S. Ser. No. 60/450,622, U.S. Ser. No. 60/456,704, U.S. Ser. No. 60/463,194, U.S. Ser. No. 60/469,358, U.S. Ser. No. 60/475,532, U.S. Ser. No. 60/476,278, and U.S. Ser. No. 60/483,395, are hereby expressly incorporated by reference.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs are derived from cDNA hbraries described in the LIFESEQ database (Incyte, Palo Alto CA). Some tissues are homogenized and lysed in guanidinium isothiocyanate, while others are 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 are centrifuged over CsCl cushions or extracted with chloroform RNA is precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA are repeated as necessary to increase RNA purity. In some cases, RNA is treated with DNase. For most libraries, poly(A)+ RNA is isolated using ohgo d(T)-couρled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworfh CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA is isolated dkectly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).

In some cases, Stratagene is provided with RNA and constructs the coπesponding cDNA hbraries. Otherwise, cDNA is synthesized and cDNA hbraries are constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or simUar methods known in the art (Ausubel et al, supra, ch. 5). Reverse transcription is initiated using ohgo d(T) or random primers. Synthetic oligonucleotide adapters are ligated to double stranded cDNA, and the cDNA is digested with the appropriate restriction enzyme or enzymes. For most hbraries, the cDNA is size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs are hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Invitrogen^ Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2- TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte, Palo Alto CA), pRARE (Incyte), or pINCY (Incyte), or derivatives thereof. Recombinant plasmids are transformed into competent E. coli ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Invitrogen.

II. Isolation of cDNA Clones Plasmids obtained as described in Example I are recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids are purified using at least one of the foUowing: a Magic or WIZARD Iv-inipreps 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. FoUowing precipitation, plasmids are resuspended in 0.1 ml of distiUed water and stored, with or without lyophUization, at 4°C

Alternatively, plasmid DNA is amplified fromhost ceU lysates using dkect link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps are carried out in a single reaction mixture. Samples are processed and stored in 384- weU plates, and the concentration of amplified plasmid DNA is quantified fluorometricaUy 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 are sequenced as follows. Sequencing reactions are processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (HamUton) liquid transfer system cDNA sequencing reactions are prepared using reagents provided by Amersham Biosciences or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides are carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Apphed 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 are identified using standard methods (Ausubel et al, supra, ch. 7). Some of the cDNA sequences are selected for extension using the techniques disclosed in Example VEIL

Polynucleotide sequences derived from Incyte cDNAs are 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 are then queried against a selection of pubhc databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus noryegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizo saccharomyces pombe, and Candida albicans (Incyte, Palo Alto CA); hidden Markov model (HMM)-based protein famUy 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 are performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences are assembled to produce f l length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) are used to extend Incyte cDNA assemblages to fuU length. Assembly is performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages are screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The fuU length polynucleotide sequences are translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the fuU length translated polypeptide. FuU length polypeptide sequences are 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 famUy databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MkaiBio, Alameda CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnment program (DNASTAR), which also calculates the percent identity between aligned sequences.

Table 7 summarizes tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides apphcable 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 thkd column presents appropriate references, aU of which are incorporated by reference herein in thek entirety, and the fourth column presents, where apphcable, the scores, probabUity values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabUity value, the greater the identity between two sequences). The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences are also used to identify polynucleotide sequence fragments from SEQ ID NO:40-78. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amphfication technologies are described in Table 4, column 2.

IV. Identification and Editing of Coding Sequences from Genomic DNA Putative enzymes are initiaUy identified by ronning the Genscan gene identification program against pubhc 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 melhionine 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 is set to 30 kb. To deterrnine which of these Genscan predicted cDNA sequences encode enzymes, the encoded polypeptides are analyzed by querying against PFAM models for enzymes. Potential enzymes are also identified by homology to Incyte cDNA sequences that have been annotated as enzymes. These selected Genscan-predicted sequences are then compared by

BLAST analysis to the genpept and gbpri pubhc databases. Where necessary, the Genscan-predicted sequences are then edited by comparison to the top BLAST hit from genpept to correct eπors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis is also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage is avaUable, this information is used to correct or confirm the Genscan predicted sequence. FuU length polynucleotide sequences are obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example III. Alternatively, fuU length polynucleotide sequences are derived entkely from edited or unedited Genscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences

Partial cDNA sequences are extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III are mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster is analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible sphce variants that are subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval is present on more than one sequence in the cluster are identified, and intervals thus identified are considered to be equivalent by transitivity. For example, if an interval is present on a cDNA and two genomic sequences, then aU three intervals are considered to be equivalent. This process aUows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified are then "stitched" together by the stitching algorithm in the order that they appear along thek parent sequences to generate the longest possible sequence, as weU as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) are given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences are translated and compared by BLAST analysis to the genpept and gbpri pubhc databases. Incoπect exons predicted by Genscan are corrected by comparison to the top BLAST hit from genpept. Sequences are further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary. "Stretched" Sequences

Partial DNA sequences are extended to fuU length with an algorithmbased on BLAST analysis. Fkst, partial cDNAs assembled as described in Example III are queried against pubhc databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases using the BLAST program The nearest GenBank protein homolog is then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein is generated by using the resultant high-scoring segment paks (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 are used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences are therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences are examined to determine whether they contain a complete gene. VI. Chromosomal Mapping of ENZM Encoding Polynucleotides

The sequences used to assemble SEQ ID NO:40-78 are 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:40-78 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data avaUable from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any ofthe clustered sequences have been previously mapped. Inclusion of a mapped sequence in a cluster results 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 Genethon 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 (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. VIL 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 RusseU, supra, ch. 7; Ausubel et al, supra, ch. 4). Analogous computer techniques applying BLAST are used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte). 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 simUar. The basis of the search is the product score, which is defined as:

BLAST Score x Percent Identity 5 x rr-inimum {length(Seq. 1), length(Seq. 2)}

The product score takes into account both the degree of simUarity between two sequences and the length of the sequence match. The product score is a normahzed value between 0 and 100, and is calculated as foUows: 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 pak (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 pak 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 ahgnment. 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 ENZM are analyzed with respect to the tissue sources from which they are 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 hbrary constructed from a human tissue. Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaUa, female; genitalia, male; germ ceUs; hemic and immune system; hver; musculoskeletal system; nervous system; pancreas; respkatory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of hbraries in each category is counted and divided by the total number of hbraries across aU categories. SimUarly, each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding ENZM. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ database (Incyte, Palo Alto CA). VIII. Extension of ENZM Encoding Polynucleotides

FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using oligonucleotide primers designed from this fragment. One primer is synthesized to initiate 5' extension of the known fragment, and the other primer is synthesized to initiate 3' extension of the known fragment. The initial primers are 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 hakpin structures and primer-primer dimerizations is avoided.

Selected human cDNA hbraries are used to extend the sequence. If more than one extension is necessary or desked, additional or nested sets of primers are designed.

High fidelity amphfication is obtained by PCR using methods weU known in the art. PCR is performed in 96-weU plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂S0₄, and 2- mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primer pak 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 pak T7 and SK+ are as foUows: 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 weU is determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 μl of undUuted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent. The plate is 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 μl to 10 μl ahquot of the reaction mixture is analyzed by electrophoresis on a 1 % agarose gel to determine which reactions are successful in extending the sequence. The extended nucleotides are desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to rehgation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides are separated on low concentration (0.6 to 0.8%) agarose gels, fragments are excised, and agar digested with Agar ACE (Promega). Extended clones were rehgated using T4 Ugase (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 ceUs. Transformed ceUs are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37 °C in 384-weU plates in LB/2x carb liquid media. The ceUs are lysed, and DNA is amphfied by PCR using Taq DNA polymerase (Amersham

Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing 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. DNAis quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamplified using the same conditions as described above. Samples are dUuted with 20% dknethysulfoxide (1 :2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).

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

IX. Identification of Single Nucleotide Polymorphisms in ENZM Encoding Polynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) are identified in SEQ ID NO:40-78 using the LIFESEQ database (Incyte). Sequences from the same gene are clustered together and assembled as described in Example III, aUowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters is used to distinguish SNPs from other sequence variants. Preliminary filters remove the majority of basecall eπors by requiring a minimum Phred quality score of 15, and remove sequence alignment eπors and eπors resulting from improper trimming of vector sequences, chimeras, and sphce variants. An automated procedure of advanced chromosome analysis is apphed to the original chromatogram files in the vicinity of the putative SNP. Clone eπor filters use statisticaUy generated algorithms to identify eπors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering eπor filters use statistically generated algorithms to identify eπors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removes duplicates and SNPs found in immunoglobulins or T-cell receptors.

Certain SNPs are 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 comprises 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprises 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprises 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprises 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 are first analyzed in the Caucasian population; in some cases those SNPs which show no allelic variance in this population are not further tested in the other three populations.

X. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:40-78 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base paks, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments. Ohgonucleotides 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 μCi of [γ-³²P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An ahquot 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 ofthe foUowing 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 transfeπed to NYTRAN PLUS nylon membranes (Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentiaUy 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 visuahzed using autoradiography or an alternative imaging means and compared. XI. Microarrays

The linkage or synthesis of array elements upon a microaπay can be achieved utilizing photohthography, piezoelectric printing (ink-jet printing; see, e.g., BaldeschweUer et al, supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and sohd 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 avaUable methods and machines weU known to those of ordinary skUl 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).

FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microaπay. Fragments or ohgomers suitable for hybridization can be selected using software weU 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, microaπay preparation and usage is described in detaU 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/μl oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM 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

GEMB RIGHT kits (Incyte). Specific control poly(A)⁺ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labehng) 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 (BD 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 μl 5X SSC/0.2% SDS. Microarray Preparation

Sequences of the present invention are used to generate aπay elements. Each aπay 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 μg. Amplified aπay elements are then purified using SEPHACRYL-400 (Amersham Biosciences). Purified aπay elements are immobilized on polymer-coated glass slides. Glass microscope shdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distUled 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-Aldrich, St. Louis MO) in 95% ethanol. Coated shdes are cured in a 110°C oven.

Array elements are apphed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference. 1 μl of the aπay element DNA, at an average concentration of 100 ng/μl, is loaded into the open capUlary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of aπay element sample per shde.

Microaπays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microaπays are washed at room temperature once in 0.2% SDS and three times in distUled water. Non-specific binding sites are blocked by incubation of microaπays 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 μl of sample mixture consisting of 0.2 μg 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 microaπay surface and covered with an 1.8 cm² coverslip. The aπays are transfeπed 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 μl of 5X SSC in a comer of the chamber. The chamber containing the aπays 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 (IX 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., MelvUle NY). The slide containing the aπay 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 aπay 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 sequentiaUy. Emitted light is split, based on wavelength, into two photomultipher tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipher 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 aπay 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 typicaUy cahbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A specific location on the aπay contains a complementary DNA sequence, allowing the intensity ofthe signal at that location to be coπelated 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 aπay for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples ofthe calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture. The output of the photomultipher 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 coπected 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 ofthe grid. The fluorescence signal within each element is then integrated to obtain a numerical value coπesponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). 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 differentiaUy expressed. Expression For example, expression of SEQ ID NO:57 was downregulated in breast cancer ceUs versus primary breast epithelial ceUs as determined by microaπay analysis. The gene expression profile of a nonmahgnant mammary epithelial ceU line was compared to the gene expression profiles of breast carcinoma lines at different stages of tumor progression. CeU lines compared included: a) BT-20, a breast carcinoma ceU line derived in vitro from the cells emigrating out of thin shces of tumor mass isolated from a 74-year-old female, b) BT-474, a breast ductal carcinoma ceU 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 ceU line that was isolated from, a papillary invasive ductal tumor obtained from a 23-year-old normal, menstruating, parous female with a famUy history of breast cancer, d) Hs 578T, a breast ductal carcinoma ceU hue isolated from a 74-year-old female with breast carcinoma, e) MCF7, a nonmahgnant 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) ceU line isolated from a 36-year-old woman with fibrocystic breast disease, g) MDA-MB-468, a breast adenocarcinoma ceU 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 ceU line isolated from a normal donor. Expression of SEQ ID NO:57 was decreased at least two-fold in six of seven breast carcinoma lines. Further, expression of SEQ ID NO:57 was increased at least two-fold in one of seven breast carcinoma lines. Therefore, in various embodiments, SEQ ID NO:57 can be used for one or more of the foUowing: i) monitoring treatment of breast cancer, U) diagnostic assays for breast cancer, and Ui) developing therapeutics and/or other treatments for breast cancer. In another example, SEQ ID NO:78 showed differential expression in breast cancer ceU lines treated with TNF-α, as determined by microaπay expression analysis. To provide molecular mechanisms underlying TNF-α effects on normal and cancerous breast ceUs, the gene expression patterns in various time courses foUowing TNF-α treatment were evaluated. Several ceU lines were examined, which included: 1) the MDA-mb-231 breast tumor ceU line isolated from the pleural effusion of a 51 -year old female, which forms poorly differentiated adenocarcinoma in nude mice and ALS treated BALB/c mice, and expresses the Wnt3 oncogene, EGF, and TGF-α; 2) the T-47D breast carcinoma ceU line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast; 3) the MCF7 nonmahgnant breast adenocarcinoma ceU line isolated from the pleural effusion of a 69-year-old female, which has retained characteristics of the mammary epithehum such as the abUity to process estradiol via cytoplasmic estrogen receptors and the capacity to form domes in culture; and 4) the MCF-10A breast mammary gland (luminal ductal characteristics) ceU line isolated from a 36-year-old woman with fibrocystic breast disease. AU ceU lines were treated with 10 ng/ml TNF-α for 1, 4, 8, 24, 48, and 72 hours. Treated ceUs were compared to untreated ceUs kept in culture for the same duration. The expression of SEQ ID NO:78 was upregulated at least two-fold in MDA-mb-231 ceUs upon treatment with TNF-α for at least 24 hours. Therefore, in various embodiments, SEQ ID NO:78 can be used for one or more of the foUowing: i) monitoring freatment of breast cancer, U) diagnostic assays for breast cancer, and hi) developing therapeutics and/or other treatments for breast cancer.

In a further example, expression of SEQ ID NO:57 and SEQ ID NO:60 was downregulated in cancerous lung tissue versus normal lung tissue as determined by microaπay analysis. Grossly uninvolved lung tissue from five donors was compared to lung squamous ceU carcinoma tissue from the same donor, respectively. Expression of SEQ ID NO:57 was decreased at least two-fold in tumorous lung tissue from four of five donors. Expression of SEQ ID NO:57 also was upregulated in cancerous lung tissue versus normal lung tissue as determined by microarray analysis. Grossly uninvolved lung tissue from two donors was compared to lung tumor tissue from the same donor when compared to normal lung tissue from the same donor, respectively. Further, expression of SEQ ID NO:57 was downregulated in cancerous lung tissue versus normal lung tissue as determined by microarray analysis. Grossly uiiinvolved lung tissue from two donors was compared to lung tumor tissue from the same donor, respectively. Expression of SEQ ID NO:57 was decreased atleast two- fold in tumorous lung tissue from one of two donors when compared to normal lung tissue from the same donor, respectively. Further, normal lung tissue from a 68 year-old female was compared to lung tumor from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). Expression of SEQ ID NO:60 was decreased at least two-fold in lung squamous ceU carcinoma in one of five donors when compared to normal lung tissue from the same donor. Therefore, in various embodiments, SEQ ID NO:57 and SEQ ID NO:60 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer, k) diagnostic assays for lung cancer, and hi) developing therapeutics and/or other treatments for lung cancer. AU lung tissue samples were received from the Roy Castle International Centre for Lung Cancer Research (Liverpool, UK).

In another example, expression of SEQ ID NO:65-66 was upregulated in diseased lung tissue versus normal lung tissue as detenrined by microaπay analysis. Grossly uninvolved lung tissue from two donors was compared to lung squamous ceU carcinoma tissue from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). Expression of SEQ ID NO:65-66 was increased at least two-fold in lung squamous ceU carcinoma in two of five donors when compared to normal lung tissue from the same donor. Therefore, in various embodiments, SEQ ID NO:65-66 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer, U) diagnostic assays for lung cancer, and Ui) developing therapeutics and or other treatments for lung cancer. '

In yet another example, SEQ ID NO:70 showed differential expression in lung tumor samples, as determined by microaπay analysis. Several lung squamous ceU carcinoma or lung adenocarcinoma tissue samples were examined and compared to donor-matched grossly uninvolved lung tissue (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). In 5 out of 9 lung tumor samples examined, the expression level of SEQ ID NO:70 was decreased at least 2-fold, and as much as 4-fold, when compared to the expression levels detected in the matched normal lung tissue. In 1 out of 9 lung tumor samples, the expression level of SEQ ID NO:70 was increased at least 3-fold, when compared to the expression levels detected in the matched normal lung tissue.

Therefore, in various embodiments, SEQ ID NO:70 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer; U) diagnostic assays for lung cancer; and hi) developing therapeutics and/or other treatments for lung cancer.

In an alternative example, expression of SEQ ID NO:60 was downregulated in cancerous colon tissue versus normal colon tissue from three donors as determined by microarray analysis. Gene expression profiles were obtained by comparing: i) normal pooled colon tissue from an 83- year-old male to moderately weU-differentiated adenocarcinoma tumor tissue from the same donor. The cancer had metastasized to one lymph node; ii) normal rectal tissue from a male donor with a rectal tumor to tumorous rectal tissue from the same donor; and in) pooled normal colon tissue from a 59-year-old male to colon adenocarcinoma tumor tissue originating from a tubuloviUous adenoma hyperplastic polyp ofthe colon from the same donor. AU tissue samples were obtained from Huntsman Cancer Institute (Salt Lake City, UT). Expression of SEQ ID NO:60 was decreased at least two-fold in colon cancer tissue in each of the three donors when compared to normal colon tissue. Further, SEQ ID NO:78 showed differential expression in colon cancer tissues, as determined by microaπay analysis. Matched normal and tumor samples from various donors diagnosed with colon cancer (Huntsman Cancer Institute, Salt Lake City, UT) were compared by competitive hybridization. In 2 out of 11 tumor tissue samples, the expression of SEQ ID NO:78 was decreased at least two-fold, when compared to grossly uninvolved colon tissue from the same donor. Therefore, in various embodiments, SEQ ID NO:60 and SEQ ID NO:78 can be used for one or more of the foUowing: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and Ui) developing therapeutics and/or other treatments for colon cancer.

In yet another example, expression of SEQ ID NO:60 was downregulated in cancerous ovarian tissue versus normal ovarian tissue as determined by microarray analysis. A normal ovary from a 79 year-old female donor was compared to an ovarian tumor from the same donor (Huntsman Cancer Institute, Salt Lake City, UT). Expression of SEQ ID NO:60 was decreased at least two-fold in ovarian adenocarcinoma when compared to normal ovarian tissue from the same donor. Therefore, in various embodiments, SEQ ID NO:60 can be used for one or more of the foUowing: i) monitoring treatment of ovarian cancer, ii) diagnostic assays for ovarian cancer, and hi) developing therapeutics and/or other treatments for ovarian cancer. In a further example, SEQ ID NO:40, SEQ ID NO:41 , SEQ ID NO:45, SEQ ID NO:56, SEQ

ID NO:60, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:78 showed tissue-specific expression as determined by microarray analysis. RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for thek abihty to provide a complete distribution of RNA n the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%). The normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are dkectly comparable from one tissue to another. The expression of SEQ ID NO:40 was increased by at least two-fold in heart ventricle tissue as compared to the reference sample. Therefore, SEQ ID NO:40 can be used as a tissue marker for heart ventricle tissue. The expression of SEQ ID NO:41 was increased by at least two-fold in brain tissue as compared to the reference sample. Therefore, SEQ ID NO:41 can be used as a tissue marker for brain tissue. The expression of SEQ ID NO:45 was increased by at least two-fold in adrenal glands as compared to the reference sample.

Therefore, SEQ ID NO:45 can be used as a tissue marker for adrenal glands. The expression of SEQ ID NO:56 was increased by at least two-fold in brain, hver, and pancreas as compared to the reference sample. Therefore, SEQ ID NO:56 can be used as a tissue marker for brain, hver, and pancreas. The expression of SEQ ID NO:60 was increased by at least two-fold in tissues fromheart and kidney as compared to the reference sample. Therefore, SEQ ID NO:60 can be used as a tissue marker for tissues in the heart and kidney. The expression of SEQ ID NO:64 was increased by at least two-fold in skeletal muscle and tissues from the hver as compared to the reference sample. Therefore, SEQ ID NO:64 can be used as a tissue marker for skeletal muscle and tissues in the hver. The expression of SEQ ID NO:65 was increased by at least two-fold in tissues from the hver as compared to the reference sample. Therefore, SEQ ID NO:65 can be used as a tissue marker for tissues in the hver. The expression of SEQ ID NO:72 was increased by at least 2-fold in smaU intestine tissue samples, by at least 4-fold in seminal vesicle tissue samples, and by at least 5-fold in liver tissue samples, when compared to the reference sample. Therefore, SEQ ID NO:72 can be used as a tissue marker for smaU intestine, seminal vesicle, and Uver tissues. The expression of SEQ ID NO:74 was increased by at least 2-fold in hver tissue samples, when compared to the reference sample. Therefore, SEQ ID NO:74 can be used as a tissue marker for hver tissue. The expression of SEQ ID NO:75 was increased by at least 5-fold in blood leukocyte samples, and by at least 2-fold in spleen tissue samples, when compared to the reference sample. Therefore, SEQ ID NO:75 can be used a s tissue marker for blood leukocytes and spleen tissue. The expression of SEQ ID NO:78 was increased by at least two-fold in smaU and large intestine tissues, as compared to the reference sample. Therefore, SEQ ID NO:78 can be used as a tissue marker for smaU and large intestine.

XII. Complementary Polynucleotides

Sequences complementary to the ENZM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring ENZM. Although use of oligonucleotides comprising from about 15 to 30 base paks is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments. Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of ENZM. 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 franslation, a complementary ohgonucleotide is designed to prevent ribosomal binding to the ENZM-encoding transcript.

XIII. Expression of ENZM

Expression and purification of ENZM is achieved using bacterial or virus-based expression systems. For expression of ENZM in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that dkects 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 ENZM upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG). Expression of ENZM in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding ENZM by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Vkal infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requkes 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, ENZM 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 ceU lysates. GST, a 26- kUodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). FoUowing purification, the GST moiety can be proteolytically cleaved from ENZM at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commerciaUy avaUable 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 ENZM obtained by these methods can be used dkectly in the assays shown in Examples XVII, XVIII, and XIX, where apphcable. XIV. Functional Assays

ENZM function is assessed by expressing the sequences encoding ENZM at physiologicaUy elevated levels in mammahan ceU culture systems. cDNA is subcloned into a mammahan 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 μg of recombinant vector are transiently transfected into a human ceU line, for example, an endotheUal or hematopoietic ceU line, using either hposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected ceUs fromnontransfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g. , Green Fluorescent Protein (GFP; BD Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward hght scatter and 90 degree side hght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular 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 ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).

The influence of ENZM on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding ENZM and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG). Transfected ceUs are efficiently separated fromnontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the ceUs using methods weU known by those of skUl in the art. Expression of mRNA encoding ENZM and other genes of interest can be analyzed by northern analysis or microarray techniques.

XV. Production of ENZM Specific Antibodies

ENZM substantiaUy purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Haπington, 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 ENZM 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 skUl in the art. Methods for selection of appropriate epitopes, such as those near the C-teπi-inus or inhydrophUic regions are weU described in the art (Ausubel et al , supra, ch. 11).

Typically, oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-malemidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al, supra). Rabbits are immunized with the ohgopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-ENZM activity by, for example, binding the peptide or ENZM 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 ENZM Using Specific Antibodies NaturaUy occuπing or recombinant ENZM is substantiaUy purified by immunoaffinity cliromatography using antibodies specific for ENZM. An immunoaffinity column is constructed by covalently coupling anti-ENZM 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 ENZM are passed over the ύiinmnoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of ENZM (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/ENZM 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 ENZM is coUected.

XVII. Identification of Molecules Which Interact with ENZM ENZM, or biologicaUy 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 weUs of a multi-weU plate are incubated with the labeled ENZM, washed, and any weUs with labeled ENZM complex are assayed. Data obtained using different concenfrations of ENZM are used to calculate values for the number, affinity, and association of ENZM with the candidate molecules.

Alternatively, molecules interacting with ENZM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially avaUable kits based on the two-hybrid system, such as the MATCHMAKER system (BD Clontech). ENZM 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 aU interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).

XVIII. Demonstration of ENZM Activity

ENZM activity is demonstrated through a variety of specific enzyme assays; some of which are outlined below.

ENZM oxidoreductase activity is measured by the increase in extinction coefficient of NAD(P)H coenzyme at 340 nmfor the measurement of oxidation activity, or the decrease in extinction coefficient of NAD(P)H coenzyme at 340 nmfor the measurement of reduction activity (Dalziel, K. (1963) J. Biol. Chem 238:2850-2858). One of three substrates may be used: Asn-βGal, biocytidine, or ubiquinone-10. The respective subunits of the enzyme reaction, for example, cytochrome c b oxidoreductase and cytochrome c, are reconstituted. The reaction mixture contains a)l-2 mg/ml ENZM; and b) 15 mM substrate, 2.4 mM NAD(P)⁺ in 0.1 M phosphate buffer, pH 7.1 (oxidation reaction), or 2.0 mM NAD(P)H, in 0.1 M Na₂HP0₄ buffer, pH 7.4 ( reduction reaction); in a total volume of 0.1 ml. Changes in absorbance at 340 nm(A₃₄₀) are measured at 23.5°C using a recording spectiophotometer (Shimadzu Scientific Instruments, Inc., Pleasanton, CA). The amount of NAD(P)H is stoichiometricaUy equivalent to the amount of subsfrate initiaUy present, and the change in A₃₄₀ is a dkect measure of the amount of NAD(P)H produced; ΔA₃₄₀ = 6620[NADH]. ENZM activity is proportional to the amount of NAD(P)H present in the assay.

Cytochrome P450 activity of ENZM is measured using the 4-hydroxylation of aniline. Aniline is converted to 4-aminophenol by the enzyme, and has an absorption maximum at 630 nm (Gibson, G.G. and P. Skett (1994) Introduction to Drug Metabohsm Blackie Academic and Professional, London). This assay is a convenient measure, but underestimates the total hydroxylation, which also occurs at the 2- and 3- positions. Assays are performed at 37 °C and contain an ahquot of the enzyme and a suitable amount of anUine (approximately 2 mM) in reaction buffer. For this reaction, the buffer must contain NADPH or an NADPH-generating cofactor system One formulation for this reaction buff er includes 85 mM Tris pH 7.4, 15 mM MgCl₂, 50 mM nicotinamide, 40 mg trisodium isocitrate, and 2 units isocitrate dehydrogenase, with 8 mg NADP⁺ added to a 10 mL reaction buffer stock just prior to assay. Reactions are carried out in an optical cuvette, and the absorbance at 630 nmis measured. The rate of increase in absorbance is proportional to the enzyme activity in the assay. A standard curve can be constructed using known concentrations of 4-aminophenol.

Aldo/keto reductase activity of ENZM is proportional to the decrease in absorbance at 340 nm as NADPH is consumed (or increased absorbance if NADPH is produced, i.e., if the reverse reaction is monitored). A standard reaction mixture is 135 mM sodium phosphate buffer (pH 6.2-7.2 depending on enzyme), 0.2 mM NADPH, 0.3 M lithium sulfate, 0.5-2.5 mg ENZM and an appropriate level of substrate. The reaction is incubated at 30 °C and the reaction is monitored continuously with a spectrophotometer. ENZM activity is calculated as mol NADPH consumed / mg of ENZM.

Acyl-CoA dehydrogenase activity of ENZM is measured using an anaerobic electron transfeπing flavoprotein (ETF) assay. The reaction mixture comprises 50 mM Tris-HCI (pH 8.0), 0.5% glucose, and 50 μM acyl-CoA substrate (i.e., isovaleryl-CoA) that is pre-warmed to 32 °C. The mixture is depleted of oxygen by repeated exposure to vacuum foUowed by layering with argon. Trace amounts of oxygen are removed by the addition of glucose oxidase and catalase foUowed by the addition of ETF to a final concentration of 1 μM. The reaction is initiated by addition of purified ENZM or a sample containing ENZM and exciting the reaction at 342 nm. Quenching of fluorescence caused by the transfer of electrons from the substrate to ETF is monitored at 496 nm. 1 unit of acyl-CoA dehydrogenase activity is defined as the amount of ENZM required to reduce 1 μmol of ETF per minute (Reinard, T. et al. (2000) J. Biol. Chem. 275:33738-33743).

Alcohol dehydrogenase activity of ENZM is measured by foUowing the conversion of NAD⁺ to NADH at 340 nm (6₃₄₀ = 6.22 mM^"1 cm^"1) at 25°C in 0.1 M potassium phosphate (pH 7.5), 0.1 M glycine (pH 10.0), and 2.4 mM NAD⁺. Substrate (e.g., ethanol) and ENZM are then added to the reaction. The production of NADH results in an increase in absorbance at 340 nm and coπelates with the oxidation of the alcohol substrate and the amount of alcohol dehydrogenase activity in the ENZM sample (Svensson, S. (1999) J. Biol. Chem. 274:29712-29719). Aldehyde dehydrogenase activity of ENZM is measured by determining the total hydrolase + dehydrogenase activity of ENZM and subtracting the hydrolase activity. Hydrolase activity is first determined in a reaction mixture containing 0.05 M Tris-HCI (pH 7.8), 100 mM 2-mercaptoethanol, and 0.5-18 μM substrate, e.g., 10-HCO-HPteGlu (10-foimyltetrahydrofolate; HPteGlu, tetrahydrofolate) or 10-FDDF (10-formyl-5,8-dideazafolate). Approximately lμg of ENZM is added in a final volume of 1.0 ml. The reaction is monitored and read against a blank cuvette, containing all components except enzyme. The appearance of product is measured at either 295 nm for 5,8-dideazafolate or 300 nm for HPteGlu using molar extinction coefficients of 1.89xl0⁴ and 2.17xl0⁴ for 5,8-dideazafolate and HPteGlu, respectively. The addition of NADP⁺ to the reaction mixture allows the measurement of both dehydrogenase and hydrolase activity (assays are performed as before). Based on the production of product in the presence of NADP⁺ and the production of product in the absence of the cofactor, aldehyde dehydrogenase activity is calculated for ENZM. In the alternative, aldehyde dehydrogenase activity is assayed using propanal as substrate. The reaction mixture contains 60 mM sodium pyrophosphate buffer (pH 8.5), 5 mM propanal, 1 mM NADP⁺, and ENZM in a total volume of 1 ml. Activity is determined by the increase in absorbance at 340 nm, resulting from the generation of NADPH, and is proportional to the aldehyde dehydrogenase activity in the sample (Krupenko, S.A. et al. (1995) J. Biol. Chem. 270:519-522).

6-phosphogluconate dehydrogenase activity of ENZM is measured by incubating purified ENZM, or a composition comprising ENZM, in 120 mM triethanolamine (pH 7.5), 0.1 mM EDTA, 0.5 mM NADP⁺, and 10-150 μM 6-phosphogluconate as substrate at 20-25°C. The production of NADPH is measured fluorimetrically (340 nm excitation, 450 nm emission) and is indicative of 6-phosphogluconate dehydrogenase activity. Alternatively, the production of NADPH is measured photometrically, based on absorbance at 340 nm. The molar amount of NADPH produced in the reaction is proportional to the 6-phosphogluconate dehydrogenase activity in the sample (Tetaud et al., supra). Ribonucleotide diphosphate reductase activity of ENZM is determined by incubating purified

ENZM, or a composition comprising ENZM, along with ditmothreitol, Mg**, and ADP, GDP, CDP, or UDP substrate. The product ofthe reaction, the corresponding deoxyribonucleotide, is separated from the substrate by thin-layer chromatography. The reaction products can be distinguished from the reactants based on rates of migration. The use of radiolabeled substrates is an alternative for increasing the sensitivity ofthe assay. The amount of deoxyribonucleotides produced in the reaction is proportional to the amount of ribonucleotide diphosphate reductase activity in the sample (note that this is true only for pre-steady state kinetic analysis of ribonucleotide diphosphate reductase activity, as the enzyme is subject to negative feedback inhibition by products) (Nutter and Cheng, supra).

Dihydrodiol dehydrogenase activity of ENZM is measured by incubating purified ENZM, or a composition comprising ENZM, in a reaction mixture comprising 50 mM glycine (pH 9.0), 2.3 mM NADP⁺, 8% DMSO, and a trans-dihydrodiol substrate, selected from the group including but not limited to, (±)-trans-naph1halene-l,2-dibydrodiol, (±)-frans-phenanthrene-l,2-dihydrodiol, and (±)- trans-chrysene-1 ,2-dihydrodiol The oxidation reaction is monitored at 340 nm to detect the formation of NADPH, which is indicative of the oxidation of the substrate. The reaction mixture can also be analyzed before and after the addition of ENZM by circular dichroism to determine the stereochemistry of the reaction components and determine which enantiomers of a racemic substrate composition are oxidized by the ENZM (Penning, supra).

Glutathione S-transferase (GST) activity of ENZM, for example, of ENZM-11, is determined by measuring the ENZM catalyzed conjugation of GSH with l-chloro-2,4-dinitrobenzene (CDNB), a common substrate for most GSTs. ENZM is incubated with 1 mM CDNB and 2.5 mM GSH together in 0.1M potassium phosphate buffer, pH 6.5, at 25 ° C. The conjugation reaction is measured by the change in absorbance at 340 nm using an ultraviolet spectrophometer. ENZM activity is proportional to the change in absorbance at 340 nm.

ENZM-9 activity can be monitored using the solutions and reagents in the AMPLEX Red Acetylcholine/Acetylcholinesterase assay kit (Molecular Probes, Eugene OR) by adding 100 μL of AMPLEX Red reagent/horseradish peroxidase/choline oxidase/acetylcholine working solution (400 μM AMPLEX Red reagent containing 2U/ml horseradish peroxidase, 0.2 U/mL choline oxidase, and 100 μM acetylcholine) to ENZM-9 in reaction buffer (50mM Tris-HCI, pH 8.0) in a well of a microplate. The microplate is then incubated at room temperature, protected from hght. Fluorescence is then measured in a fluorescence microplate reader using excitation in the range of 530-560 nm and emission detection at approximately 590 nm over time to follow the kinetics of the reaction.

15-oxoprostaglandin 13-reductase (PGR) activity of ENZM is measured following the separation of contaminating 15-hydroxyprostaglandin dehydrogenase (15-PGDH) activity by DEAE chromatography. Following isolation of PGR containing fractions (or using the purified ENZM), activity is assayed in a reaction comprising 0.1 M sodium phosphate (pH 7.4), 1 mM 2- mercaptoethanol, 20 μg substrate (e.g., 15-oxo derivatives of prostaglandins PGE^ PGE₂, and PGE_2α;), and 1 mM NADH (or a higher concentration of NADPH). ENZM is added to the reaction which is then incubated for 10 min at 37 °C before termination by the addition of 0.25 ml 2 N NaOH. The amount of 15-oxo compound remaining in the sample is deteπnined by measuring the maximum absorption at 500 nm of the terminated reaction and comparing this value to that of a terminated control reaction that received no ENZM. 1 unit of enzyme is defined as the amount required to catalyze the oxidation of 1 μmol substrate per minute and is proportional to the amount of PGR activity in the sample. Choline dehydrogenase activity of ENZM is identified by the ability of E. coli, transformed with an ENZM expression vector, to grow on media containing choline as the sole carbon and nitrogen source. The abUity ofthe transformed bacteria to thrive is indicative of choline dehydrogenase activity (Magne østeras, M. (1998) Proc. Natl. Acad. Sci. USA 95:11394-11399). ENZM thioredoxin activity is assayed as described (Luthman, M. (1982) Biochemistry 21 :6628-6633). Thioredoxins catalyze the formation of disulfide bonds and regulate the redox envkonment in ceUs to enable the necessary thioldisulfide exchanges. One way to measure the thioldisulfide exchange is by measuring the reduction of insulin in a mixture containing 0.1 M potassium phosphate, pH 7.0, 2 mM EDTA, 0.16 μM insulin, 0.33 mM DTT, and 0.48 mM NADPH. Different concentrations of ENZM are added to the mixture, and the reaction rate is foUowed by monitoring the oxidation of NADPH at 340 nM.

ENZM transferase activity is measured through assays such as a methyl transferase assay in which the transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate is measured (Bokar, J.A. et al. (1994) J. Biol. Chem. 269:17697-17704). Reaction mixtures (50 μl final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM dithiothreitol, 3% polyvinylalcohol, 1.5 μCi [ er/ιyZ-³H]AdoMet (0.375 μM AdoMet) (DuPont-NEN), 0.6 μg ENZM, and acceptor substrate (0.4 μg [³⁵S]RNA or 6-mercaptopurine (6-MP) to 1 mM final concentration). Reaction mixtures are incubated at 30°C for 30 minutes, then at 65 °C for 5 minutes. The products are separated by chromatography or electrophoresis and the level of methyl transferase activity is determined by quantification of methyl-³H recovery. Aminotransferase activity of ENZM is assayed by incubating samples containing ENZM for 1 hour at 37 °C in the presence of 1 mM L-kynurenine and 1 mM 2-oxoglutarate in a final volume of 200 μl of 150 mM Tris acetate buffer (pH 8.0) containing 70 μM PLP. The formation of kynurenic acid is quantified by HPLC with spectrophotometric detection at 330 nm using the appropriate standards and controls weU known to those skilled in the art. In the alternative, L-3-hydroxykynurenine is used as substrate and the production of xanthurenic acid is determined by HPLC analysis of the products with UV detection at 340 n The production of kynurenic acid and xanthurenic acid, respectively, is indicative of aminotransferase activity (Buchli et al, supra).

In another alternative, aminotransferase activity of ENZM is measured by determining the activity of purified ENZM or crude samples containing ENZM toward various amino and oxo acid substrates under single turnover conditions by monitoring the changes in the UV7VIS absorption spectrum of the enzyme-bound cofactor, pyridoxal 5 -phosphate (PLP). The reactions are performed at 25°C in 50 mM 4-methylmorpholine (pH 7.5) containing 9 μM purified ENZM or ENZM containing samples and substrate to be tested (amino and oxo acid substrates). The half-reaction from amino acid to oxo acid is foUowed by measuring the decrease in absorbance at 360 nm and the increase in absorbance at 330 nm due to the conversion of enzyme-bound PLP to pyridoxamine 5' phosphate (PMP). The specificity and relative activity of ENZM is determined by the activity of the enzyme preparation against specific substrates (Vacca, supra).

ENZM chitinase activity is deteπnined with the fluorogenic substrates 4-methylumbeUiferyl chitotriose, methylumbeUiferyl chitobiose, or methylumbelhferyl N-acetylglucosamine. Purified ENZM is incubated with 0.5uM substrate at pH 4.0 (0. IM citrate buffer), pH 5.0 (0. IM phosphate buffer), or pH 6.0 (0. IM Tris-HCL). After various times of incubation, the reaction is stopped by the addition of 0. IM glycine buffer, pH 10.4, and the concentration of free methylumbeUiferone is determined fluorometricaUy. Chitinase B from Serratia marcescens may be used as a positive control (Hakala, supra). ENZM isomerase activity is determined by measuring 2-hydroxyhepta-2,4-diene, 1 ,7 dioate isomerase (HHDD isomerase) activity, as described by Garrido-Peritierra, A. and R.A. Cooper (1981; Eur. J. Biochem. 17:581-584). The sample is combined with 5-carboxymethyl-2-oxo-hex-3-ene-l,5, dioate (CMHD), which is the substrate for HHDD isomerase. CMHD concentration is monitored by measuring its absorbance at 246 n Decrease in absorbance at 246 nm is proportional to HHDD isomerase activity of ENZM.

ENZM isomerase activity such as peptidyl prolyl cis/trans isomerase activity can be assayed by an enzyme assay described by Rahfeld (supra). The assay is performed at 10 °C in 35 mM HEPES buffer, pH 7.8, containing chymotrypsin (0.5 mg/ml) and ENZM at a variety of concentrations. Under these assay conditions, the substrate, Suc-Ala-Xaa-Pro-Phe-4-NA, is in equihbrium with respect to the prolyl bond, with 80-95% in trans and 5-20% in cis conformation. An aliquot (2 μl) of the substrate dissolved in dimethyl sulfoxide (10 mg/ml) is added to the reaction mixture described above. Only the cis isomer is a substrate for cleavage by chymotrypsin. Thus, as the substrate is isomerized by ENZM, the product is cleaved by chymotrypsin to produce 4-nitroanilide, which is detected by its absorbance at 390 nm. 4-NitroanUide appears in a time-dependent and a ENZM concentration-dependent manner.

Alternatively, peptidyl prolyl cis-trans isomerase activity of ENZM can be assayed using a chromogenic peptide in a coupled assay with chymotrypsin (Fischer, G. et al. (1984) Biomed. Biochim Acta 43:1101-1111).

UDP glucuronyltransferase activity of ENZM is measured using a colorimetric determination of free amine groups (Gibson, G.G. and P. Skett (1994) Introduction to Drug Metabohsm, BlacMe Academic and Professional, London). An amme-containing substrate, such as 2-aminophenol, is incubated at 37 ° C with an ahquot of the enzyme in a reaction buffer containing the necessary cofactors (40 mM Tris pH 8.0, 7.5 mM MgCl₂, 0.025% Triton X-100, 1 mM ascorbic acid, 0.75 mM UDP-glucuronic acid). After sufficient time, the reaction is stopped by addition of ice-cold 20% trichloroacetic acid in 0.1 M phosphate buffer pH 2.7, incubated on ice, and centrifuged to clarify the supernatant. Any unreacted 2-aminophenol is destroyed in this step. Sufficient freshly-prepared sodium nitrite is then added; this step aUows formation of the diazonium salt of the glucuronidated product. Excess nitrite is removed by addition of sufficient ammonium sulfamate, and the diazonium salt is reacted with an aromatic amine (for example, N-naphthylethylene άiamine) to produce a colored azo compound which can be assayed spectrophotometricaUy (at 540 nm, for example). A standard curve can be constructed using known concentrations of aniline, which will form a chromophore with simUar properties to 2-aminophenol glucuronide.

Adenylosuccinate synthetase activity of ENZM is measured by synthesis of AMP from IMP. The sample is combined with AMP. IMP concentration is monitored spectrophotometricaUy at 248 nm at 23°C (Wang, W. et al. (1995) J. Biol. Chem 270:13160-13163). The increase in IMP concentration is proportional to ENZM activity.

Alternatively, AMP binding activity of ENZM is measured by combining the sample with ³ P-labeled AMP. The reaction is incubated at 37°C and terminated by addition of trichloroacetic acid. The acid extract is neutralized and subjected to gel electrophoresis to remove unbound label. The radioactivity retained in the gel is proportional to ENZM activity.

In another alternative, xenobiotic carboxylic acid:CoA ligase activity of ENZM is measured by combining the sample with γ^"33P-ATP and measuring the formation of γ-³³P- pyrophosphate with time (Vessey, D.A. et al. (1998) J. Biochem. Mol. Toxicol. 12:151-155).

Protein phosphatase (PP) activity can be measured by the hydrolysis of P-nitrophenyl phosphate (PNPP). ENZM is incubated together with PNPP in HEPES buffer pH 7.5, in the presence of 0.1 % β-mercaptoethanol at 37 ° C for 60 min. The reaction is stopped by the addition of 6 ml of 10 N NaOH (Diamond, R.H. et al. (1994) Mol. CeU. Biol. 14:3752-62).

Alternatively, acid phosphatase activity of ENZM is demonstrated by incubating ENZM containing extract with 100 μl of 10 mM PNPP in 0.1 M sodium citrate, pH 4.5, and 50 μl of 40 mM NaCl at 37 °C for 20 min. The reaction is stopped by the addition of 0.5 ml of 0.4 M glycine/NaOH, pH 10.4 (Saftig, P. et al. (1997) J. Biol. Chem 272:18628-18635). The increase in hght absorbance at 410 nm resulting from the hydrolysis of PNPP is measured using a spectrophotometer. The increase in hght absorbance is proportional to the activity of ENZM in the assay.

In the alternative, ENZM activity is determined by measuring the amount of phosphate removed from a phosphorylated protein substrate. Reactions are performed with 2 or 4 nM ENZM in a final volume of 30 μl containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1% 2-mercaptoethanol and 10 μM substrate, ³²P-labeled on serine/threonine or tyrosine, as appropriate. Reactions are initiated with substrate and incubated at 30° C for 10-15 min. Reactions are quenched with 450 μl of 4% (w/v) activated charcoal in 0.6 M HCl, 90 mM Na₄P₂0₇, and 2 mM NaH₂P0₄, then centrifuged at 12,000 x g for 5 min. Acid-soluble ³²Pi is quantified by liquid scintillation counting (Sinclak, C et al. (1999) J. Biol. Chem 274:23666-23672).

In the alternative, phosphohpase activity of ENZM is measured by the hydrolysis of a fatty acyl residue at the sn-1 position of phosphatidylserine. ENZM is combined with the tritium [³H] labeled substrate phosphatidylserine at stoichiometric quantities in a suitable buffer. FoUowing an appropriate incubation time, the hydrolyzed reaction products are separated from the substrates by chromatographic methods. The amount of acylglycerophosphoserine produced is measured by counting tritiated product with the help of a scintillation counter. Various control groups are set up to account for background noise and unincorporated substrate. The final counts represent the tritiated enzyme product [³Hj-acylglycerophosphoserine, which is dkectly proportional to the activity of ENZM in biological samples.

In the alternative, phosphohpase activity of ENZM is measured in a number of manners, several of which are described in Gomez-Cambronero, J. et al. (2003), Methods Mol. Biol. 218:155- 176; Litosch, I. (2003), Biochemistry 42:1618-1623; and IUehberger, D. et al. (2000), Methods Enzymol. 325:167-177. The adenosine deaminase activity of ENZM is deteπnined by measuring the rate of deammation that occurs when adenosine substrate is incubated with ENZM. Reactions are performed with a predetermined amount of ENZM in a final volume of 3.0 ml containing 53.3 mM potassium phosphate and 0.045 mM adenosine. Assay reagents excluding ENZM are mixed in a quartz cuvette and equilibrated to 25° C. Reactions are initiated by the addition of ENZM and are mixed immediately by inversion. The decrease in hght absorbance at 265 nm resulting from the hydrolysis of adenosine to inosine is measured using a spectrophotometer. The decrease in the A₂₆₅ ^ is recorded for approximately 5 minutes. The decrease in hght absorbance is proportional to the activity of ENZM in the assay.

ENZM hydrolase activity is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantified by spectrophotometric (or fluorometric) absorption of the released chromophore (Beynon and Bond, supra, pp.25-55). Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases), aminopeptidase (leucine aminopeptidase), or carboxypeptidase (Carboxypeptidase A and B, procollagen C-proteinase). An assay for carbonic anhydrase activity of ENZM uses the fluorescent pH indicator 8- hydroxypyrene-l,3,6-trisulfonate (pyranine) in combination with stopped-flow fluorometry to measure carbonic arihydrase activity (Shingles, et al. 1997, Anal. Biochem 252:190-197). A pH 6.0 solution is mixed with a pH 8.0 solution and the initial rate of bicarbonate dehydration is measured. Addition of carbonic anhydrase to the pH 6.0 solution enables the measurement of the initial rate of activity at physiological temperatures with resolution times of 2 ms. Shingles et al. (supra) used this assay to resolve differences in activity and sensitivity to sulfonamides by comparing mammahan carbonic anhydrase isoforms. The fluorescent technique's sensitivity aUows the determination of initial rates with a protein concentration as little as 65 ng/ml.

Decarboxylase activity of ENZM is measured as the release of C0₂ from labeled substrate. For example, ornithine decarboxylase activity of ENZM is assayed by measuring the release of C0₂ fromL-[l-¹⁴C]-ornithine (Reddy, S.G et al. (1996) J. Biol. Chem 271:24945-24953). Activity is measured in 200 μl assay buffer (50 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 2 mM dithiothreitol, 5 mM NaF, 0.1% Brij35, 1 mM PMSF, 60 μM pyridoxal-5-phosphate) containing 0.5 mM L-oπrithine plus 0.5 μCi L-[l-¹⁴C]ornithine. The reactions are stopped after 15-30 minutes by addition of 1 M citric acid, and the ¹⁴C0₂ evolved is trapped on a paper disk filter saturated with 20 μl of 2 N NaOH. The radioactivity on the disks is determined by hquid scmtiUation spectography. The amount of ¹⁴C0₂ released is proportional to orrrithine decarboxylase activity of ENZM.

AdoHCYase activity of ENZM in the hydrolytic dkection is performed spectroscopicaUy by measuring the rate of the product (homocysteine) formed by reaction with 5,5'-Dithiobis(2- nitrobenzoic acid) (DTNB). To 800 μl of an enzyme solution containing 4.7 μg of ENZM and 4 units of adenosine deaminase in 50 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA (buffer A), is added 200 μl of 5-Adenosyl-L-homocysteine (500 μM) containing 250 μM DTNB in buffer A. The reaction mixture is incubated at 37 °C for 2 minutes. Hydrolytic activity is monitored at 412 nm continuously using a diode array UV spectrophotometer. Enzyme activity is defined as the amount of enzyme that can hydrolyze 1 μmol of 5-Adenosyl-L-homocysteine/minute (Yuan, C-S et al. (1996) J. Biol. Chem 271:28009-28015).

AdoHCYase activity of ENZM can be measured in the synthetic dkection as the production of S-adenosyl homocysteine using 3-deazaadenosine as a substrate (Sganga et al. supra). Briefly, ENZM is incubated in a 100 μl volume containing 0.1 mM 3-deazaadenosine, 5 mM homocysteine, 20 mM HEPES (pH 7.2). The assay mixture is incubated at 37 °C for 15 minutes. The reaction is terminated by the addition of 10 μl of 3 M perchloric acid. After incubation on ice for 15 minutes, the mixture is centrifuged for 5 minutes at 18,000 x g in a microcentrifuge at 4°C The supernatant is removed, neutralized by the addition of 1 M potassium carbonate, and centrifuged again. A 50 μl ahquot of supernatant is then chromatographed on an Altex Ultrasphere ODS column (5 μm particles, 4.6 x 250 mm) by isocratic elution with 0.2 M ammonium dihydrogen phosphate (Aldrich) at a flow rate of 1 ml/min. Protein is determined by the bicinchoninic acid assay (Pierce).

Alternatively, AdoHCYase activity of ENZM can be measured in the synthetic dkection by a TLC method (Hershfield, M.S. et al. (1979) J. Biol. Chem 254:22-25). In a preincubation step, 50 μM [8^"14C]adenosine is incubated with 5 molar equivalents of NAD⁺ for 15 minutes at 22 °C. Assay samples containing ENZM in a 50 μl final volume of 50 mM potassium phosphate buffer, pH 7.4, 1 mM DTT, and 5 mM homocysteine, are mixed with the preincubated [8^"14C]adenosine/NAD⁺ to initiate the reaction. The reaction is incubated at 37 °C, and 1 μl samples are spotted on TLC plates at 5 minute intervals for 30 minutes. The chromatograms are developed in butanol- 1 /glacial acetic acid/water (12:3:5, v/v) and dried. Standards are used to identify substrate and products under ultraviolet hght. The complete spots containing [¹⁴C] adenosine and [¹⁴C]SAH are then detected by exposing x-ray film to the TLC plate. The radiolabeled substrate and product are then cut from the chromatograms and counted by hquid scintUlation spectrometry. Specific activity of the enzyme is determined from the linear least squares slopes of the product vs time plots and the milligrams of protein in the sample (Bethin, K.E. et al. (1995) J. Biol. Chem 270:20698-20702). Asparaginase activity of ENZM can be measured in the hydrolytic dkection by determining the amount of radiolabeled L-aspartate released from 0.6 mM Λ^-β'-N-acet lglucosaminyl-L- asparagine substrate when it is incubated at 25 °C with ENZM in 50 mM phosphate buffer, pH 7.5 (Kaartinen, V. et al (1991) J. Biol. Chem 266:5860-5869).

Acyl CoA acid hydrolase activity of ENZM in the hydrolytic dkection is performed spectroscopicaUy by monitoring the appearance of the product (CoASH) formed by reaction of substrate (acyl-CoA) and ENZM with 5,5'-Difhiobis(2-nitrobenzoic acid) (DTNB). The final reaction volume is 1 ml of 0.05 M potassium phosphate buffer, pH 8, containing 0.1 mM DTNB, 20 μg/ml bovine serum albumin, 10 μM of acyl-CoA of different lengths (C6-C0A, ClO-CoA, C14-CoA and CI 8-C0A, Sigma), and ENZM. The reaction mixture is incubated at 22 °C for 7 minutes. Hydrolytic activity is monitored spectrophotometricaUy by measuring absorbance at 412 nm (Poupon, V. et al. (1999) J. Biol. Chem 274:19188-19194).

Alternatively, ribonuclease activity of ENZM can be measured spectrophotometricaUy by determining the amount of solubUized RNA that is produced as a result of incubation of RNA substrate with ENZM. 5 μl (20 μg) of a 4 mg/ml solution of yeast tRNA (Sigma) is added to 0.8 ml of 40 mM sodium phosphate, pH 7.5, containing ENZM. The reaction is incubated at 25°C for 15 minutes. The reaction is stopped by addition of 0.5 ml of an ice-cold fresh solution of 20 mM lanthanum nitrate plus 3% perchloric acid. The stopped reaction is incubated on ice for at least 15 min, and the insoluble tRNA is removed by centrifugation for 5 min at 10,000 g. SolubUized tRNA is determined as UV absorbance (260 nm) of the remaining supernatant, with A₂₆₀ of 1.0 coπesponding to 40 μg of solubUized RNA (Rosenberg, H.F. et al. (1996) Nucleic Acids Research 24:3507-3513). RNase P or tRNA splicing endonuclease activity of ENZM can be determined as the abUity of ENZM to cleave ³²P internaUy labeled T. theπnophila pre-tRNA^{G l}. ENZM and substrate are added to reaction vessels and reactions are carried out in MBB buffer (50 mM Tris-HCI (pH 7.5), 10 mM MgCl₂) for 1 hour at 37 °C. Reactions are teπninated with the addition of an equal volume of sample loading buffer (SLB : 40 mM EDTA, 8 M urea, 0.2% xylene cyanol, and 0.2% bromophenol blue). The reaction products are separated by electrophoresis on 8 M urea, 6% polyacrylamide gels and analyzed using detection instruments and software capable of quantification of the products. One unit of ENZM activity is defined as the amount of enzyme requked to cleave 10% of 28 fmol of T. thermophila pre-tRNA^Gln to mature products in 1 hour at 37 °C (True, H.L. et al. (1996) J. Biol. Chem 271:16559-16566).

Alternatively, cleavage of ³²P internaUy labeled substrate tRNA by ENZM can be determined in a 20 μl reaction mixture containing 30 mM HEPES-KOH (pH 7.6), 6 mM MgCl₂, 30 mM KCI, 2 mM DTT, 25 g/ml bovine serum albumin, 1 unit/μl rRNasin, and 5,000-50,000 cpm of gel-purified substrate RNA. 3.0 μl of ENZM is added to the reaction mixture, which is then incubated at 37 °C for 30 minutes. The reaction is stopped by guanidinium/phenol extraction, precipitated with ethanol in the presence of glycogen, and subjected to denaturing polyacrylamide gel electrophoresis (6 or 8% polyacrylamide, 7 M urea) and autoradiography (Rossmanith, W. et al. (1995) J. Biol. Chem 270:12885-12891). The ENZM activity is proportional to the amount of cleavage products detected. ENZM activity can be measured by determining the amount of free adenosine produced by the hydrolysis of AMP, as described by Sala-Newby et al, supra. Briefly, ENZM is incubated with AMP in a suitable buffer for 10 minutes at 37 °C Free adenosine is separated from AMP and measured by reverse phase HPLC.

Alternatively, ENZM activity is measured by the hydrolysis of ADP-ribosylarginine (Konczalik, P. and J. Moss (1999) J. Biol. Chem 274:16736-16740). 50 ng of ENZM is incubated with 100 μM ADP-ribosyl-[¹⁴C]arginine (78,000 cpm) in 50 mM potassium phosphate, pH 7.5, 5 mM dithiothreitol, 10 mM MgCl₂ in a final volume of 100 μl. After 1 h at 37° C, 90 μl of the sample is apphed to a column (0.5 x 4 cm) of Affi-Gel 601 (boronate) equUibrated and eluted with five 1-ml portions of 0.1 M glycine, pH 9.0, 0.1 M NaCl, and 10 mM MgCl₂. Free ¹⁴C-Arg in the total eluate is measured by hquid scintiUation counting. Epoxide hydrolase activity of ENZM can be determined with a radiometric assay utilizing

[H³]-labeled trans-stilbeae oxide (TSO) as substrate. Briefly, ENZM is preincubated in Tris-HCI pH 7.4 buffer in a total volume of 100 μl for 1 minute at 37 °C. 1 μl of [H³]-labeled TSO (0.5 μM in EtOH) is added and the reaction mixture is incubated at 37 °C for 10 minutes. The reaction mixture is extracted with 200 μl n-dodecane. 50 μl of the aqueous phase is removed for quantification of diol product in a hquid scintUlation counter (LSC). ENZM activity is calculated as nmol diol product/min/mg protein (GUI, S.S. et al. (1983) Analytical Biochemistry 131:273-282).

Lysophosphatidic acid acyltransferase activity of ENZM is measured by incubating samples containing ENZM with 1 mM ofthe thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 50 μm LPA, and 50 μm acyl-CoA in 100 mM Tris-HCI, pH 7.4. The reaction is initiated by addition of acyl- CoA, and aUowed to reach equUibrium. Transfer of the acyl group from acyl-CoA to LPA releases free CoA, which reacts with DTNB. The product ofthe reaction between DTNB and free CoA absorbs at 413 nm The change in absorbance at 413 nmis measured using a spectrophotometer, and is proportional to the lysophosphatidic acid acyltransferase activity of ENZM in the sample.

N-acyltransferase activity of ENZM is measured using radiolabeled amino acid substrates and measuring radiolabel incorporation into conjugated products. ENZM is incubated in a reaction buffer containing an unlabeled acyl-CoA compound and radiolabeled amino acid, and the radiolabeled acyl-conjugates are separated from the unreacted amino acid by extraction into n- butanol or other appropriate organic solvent. For example, Johnson, M. R. et al. (1990; J. Biol. Chem. 266:10227-10233) measured bile acid-Co A:amino acid N-acyltransferase activity by incubating the enzyme with cholyl-CoA and ³H-glycine or ³H-taurine, separating the tritiated cholate conjugate by extraction into n-butanol, and measuring the radioactivity in the extracted product by scintillation. Alternatively, N-acyltransferase activity is measured using the spectrophotometric determination of reduced CoA (CoASH) described below.

N-acetyltransferase activity of ENZM is measured using the transfer of radiolabel from [¹⁴C]acetyl-CoA to a substrate molecule (for example, see Deguchi, T. (1975) J. Neurochem.

24:1083-5). Alternatively, a newer spectrophotometric assay based on DTNB reaction with CoASH may be used. Free thiol-containing CoASH is formed during N-acetyltransferase catalyzed transfer of an acetyl group to a substrate. CoASH is detected using the absorbance of DTNB conjugate at 412 nm (De Angelis, J. et al (1997) J. Biol. Chem. 273:3045-3050). ENZM activity is proportional to the rate of radioactivity incorporation into substrate, or the rate of absorbance increase in the spectrophotometric assay.

Galactosyltransferase activity of ENZM is determined by measuring the transfer of galactose from UDP-galactose to a GlcNAc-terminated ohgosaccharide chain in a radioactive assay. (Kolbinger, F. et al. (1998) J. Biol. Chem 273:58-65.) The ENZM sample is incubated with 14 μl of assay stock solution (180 mM sodium cacodylate, pH 6.5, 1 mg/ml bovine serum albumin, 0.26 mM UDP-galactose, 2 μl of UDP-[ H]galactose), 1 μl of MnCl₂ (500 mM), and 2.5 μl of GlcNAcβO- (CH₂)₈-C0₂Me (37 mg/ml in dimethyl sulfoxide) for 60 minutes at 37 °C. The reaction is quenched by the addition of 1 ml of water and loaded on a CI 8 Sep-Pak cartridge (Waters), and the column is washed twice with 5 ml of water to remove unreacted UDP-[³H]galactose. The [³H]galactosylated GlcNAcβO-(CH₂)_s-C0₂Me remains bound to the column during the water washes and is eluted with 5 ml of methanol. Radioactivity in the eluted material is measured by hquid scintillation counting and is proportional to galactosyltransferase activity of ENZM in the starting sample.

Phosphoribosyltransferase activity of ENZM is measured as the transfer of a phosphoribosyl group from phosphoribosylpyrophosphate (PRPP) to a purine or pyrimidine base. Assay mixture (20 μl) containing 50 mM Tris acetate, pH 9.0, 20 mM 2-mercaptoethanol, 12.5 mM MgCl₂, and 0.1 mM labeled substrate, for example, [¹⁴C]uracil, is mixed with 20 μl of ENZM dUuted in 0.1 M Tris acetate, pH 9.7, and 1 mg/ml bovine serum albumin. Reactions are preheated for 1 min at 37 °C, initiated with 10 μl of 6 mM PRPP, and incubated for 5 min at 37 °C The reaction is stopped by heating at 100 °C for 1 min. The product [¹⁴C]UMP is separated from [¹⁴C]uracil on DEAE-cellulose paper (Turner, R.J. et al. (1998) J. Biol. Chem. 273:5932-5938). The amount of [ ¹ C]UMP produced is proportional to the phosphoribosyltransferase activity of ENZM.

ADP-ribosyltransferase activity of ENZM is measured as the transfer of radiolabel from adenine-NAD to agmatine (Weng, B. et al. (1999) J. Biol. Chem. 274:31797-31803). Purified ENZM is incubated at 30 °C for 1 hr in a total volume of 300 μl containing 50 mM potassium phosphate (pH. 7.5), 20 mM agmatine, and 0.1 mM [adenine-U-¹⁴C]NAD (0.05 mCi). Samples (100 μl) are applied to Dowex columns and [¹⁴C]ADP-ribosylagmatine eluted with 5 ml of water for liquid scintillation counting. The amount of radioactivity recovered is proportional to ADP-ribosyltransferase activity of ENZM.

An ENZM activity assay measures aminoacylation of tRNA in the presence of a radiolabeled substrate. ENZM is incubated with [¹⁴C]-labeled amino acid and the appropriate cognate tRNA (for example, [¹⁴Cjalanine and tRNA^ala) in a buffered solution. ¹⁴C-labeled product is separated from free [¹⁴C] amino acid by chromatography, and the incorporated ¹⁴C is quantified using a scintillation counter. The amount of ¹⁴C-labeled product detected is proportional to the activity of ENZM in this assay (Ibba, M. et al (1997) Science 278:1119-1122). Alternatively, argininosuccinate synthase activity of ENZM is measured based on the conversion of [³H]aspartate to [³H] argininosuccinate. ENZM is incubated with a mixture of [³H] aspartate, citruUine, Tris-HCI (pH 7.5), ATP, MgCl₂, KCI, phosphoenolpyruvate, pyruvate kinase, myokinase, and pyrophosphatase, and aUowed to proceed for 60 minutes at 37 °C Enzyme activity was terminated with addition of acetic acid and heating for 30 minutes at 90 °C [³H] argininosuccinate is separated fromun-catalyzed [³Hjaspartate by chromatography and quantified by hquid scintillation spectrometry. The amount of [³H] argininosuccinate detected is proportional to the activity of ENZM in this assay (O'Brien, W. E. (1979) Biochemistry 18:5353-5356).

Alternatively, the esterase activity of ENZM is assayed by the hydrolysis of p- nitrophenylacetate (NPA). ENZM is incubated together with 0.1 μM NPA in 0.1 M potassium phosphate buffer (pH 7.25) containing 150 mM NaCl. The hydrolysis of NPA is measured by the increase of absorbance at 400 nm with a spectiophotometer. The increase in light absorbance is proportional to the activity of ENZM (Probst, M.R. et al. (1994) J. Biol. Chem 269:21650-21656). XIX. Identification of ENZM Agonists and Antagonists

Agonists or antagonists of ENZM activation or inhibition may be tested using the assays described in section XVIII. Agonists cause an increase in ENZM activity and antagonists cause a decrease in ENZM activity.

Various modifications and variations of the described compositions, methods, and systems of the invention wiU be apparent to those skiUed in the art without departing from the scope and spkit of the invention. It wUl be appreciated that the invention provides novel and useful proteins, and thek encoding polynucleotides, which can be used in the drug discovery process, as weU 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 readUy 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 foUowing claims and thek equivalents.

Table 1

Table 1

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table.3

Table 3

Table 3

Table 3

Table 3

Table 3

t oo o

Table 3

o Is⁾

Table 3

t t oo

Table 3

o to o

Table 3

o t

Table 4

t ) O H

Table 4

t o σv

Table 4

Table 4

Table 4

Table 4

Table 5

to

Table 6

Table 6

Table 7

Program Description Reference Parameter Threshold

ABI FACTURA A program that removes vector sequences and masks Apphed Biosystems, Foster City, CA. ambiguous bases in nucleic acid sequences.

ABI/PARACEL A Fast Data Finder useful in comparing and annotating Apphed Biosystems, Foster City, CA; Mismatch <50% FDF amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.

ABI A program that assembles nucleic acid sequences. Apphed Biosystems, Foster City, CA.

AutoAssembler

BLAST A Basic Local Ahgnment Search Tool useful in Altschul, S.F. et al. (1990) I. Mol. Biol. ESTs: Probabihty value=1.0E-8 sequence similarity search for amino acid and nucleic 215:403-410; Altschul, S.F. et al. (1997) or less acid sequences. BLAST includes five functions: blastp, Nucleic Acids Res. 25:3389-3402. Full Length sequences: Probabiht blastn, blastx, tblastn, and tblastx. value= l.OE-10 or less

FASTA A Pearson and Lipman algorithm that searches for Pearson, W.R. and DJ. Lipman (1988) 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 least .R. (1990) Methods Enzymol. 183:63-98; 95% or greater and five functions: fasta, tfasta, fastx, tfastx, and ssearch. and Smith, T.F. and M.S. Waterman (1981) Match length=200 bases or greate 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 sequence Henikoff, S. and I.G. Henikoff (1991) Nucleic Probabihty value=1.0E-3 or less against those in BLOCKS, PRINTS, DOMO, Acids Res. 19:6565-6572; Henikoff, I.G. & S. PRODOM, and PFAM databases to search for gene Henikoff (1996) Methods Enzymol. 266:88- families, sequence homology, and structural fingerprint 105; and Attwood, T.K. et al. (1997) I. Chem. regions. Inf. Comput. Sci. 37:417-424.

HMMER An algorithm for searching a query sequence against Krogh, A. et al. (1994) J. Mol. Biol. 235: 1501- PFAM, INCY, SMART, or ΗGRF hidden Markov model (ΗMM)-based databases of 1531; Sonnhammer, E.L.L. et al. (1988) hits: Probabihty value=1.0E-3 or l protein family consensus sequences, such as PFAM, Nucleic Acids Res. 26:320-322; Durbin, R. et Signal peptide hits: Score= 0 or INCY, SMART, and TIGRFAM. al. (1998) Our World View, in a Nutshell, greater Cambridge Univ. Press, p. 1-350

Table 7 (cont.)

Program Description Reference Parameter Threshold

ProfileScan An algorithm that searches for structural and sequence Gribskov, M. et al. (1988) CABIOS 4:61-66; Normahzed quality scores GCG motifs in protein sequences that match sequence patterns Gribskov, M. et al. (1989) Methods Enzymol. specified "HIGH" value for that defined in Prosite. 183: 146-159; Bairoch, A. et al. (1997) particular Prosite motif. Nucleic Acids Res. 25:217-221. GeneraUy, score=1.4-2.1.

Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probabihty. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.

Phrap A Phils Revised Assembly Program including SWAT and Smith, T.F. and M.S. Waterman (1981) Adv. Score=120 or greater; CrossMatch, programs based on efficient implementation Appl. Math. 2:482-489; Smith, T.F. and M.S. Match length=56 or greater of the Smith-Waterman algorithm, useful in searching Waterman (1981) J. Mol. Biol. 147:195-197; sequence homology and assembling DNA sequences. and Green, P., University of Washington, Seattle, WA.

Consed to A graphical tool for viewing and editing Phrap assembhes. Gordon, D. et al. (1998) Genome Res. 8: 195-202. on 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 peptides. 10: 1-6; Claverie, I.M. and S. Audic (1997) 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 (1996) determine orientation. Protein Sci. 5:363-371.

TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.

Motifs A program that searches amino acid sequences for patterns Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221; that matched those defined in Prosite. Wisconsin Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI.

Table 8

Table 8

Table 8

Table 8

Table 8

t too o

Table 8

Table 8

Claims

What is claimed is:

1. 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: 1-10 and SEQ ID NO: 12-39, b) a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 11, c) a polypeptide comprising a naturally occurrmg amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2-3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 13, SEQ ID NO: 18-19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:36, d) a polypeptide comprising a naturally occurring amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO: 1, e) a polypeptide comprismg a naturally occurrmg amino acid sequence at least 95% identical to an amino acid sequence selected from the group consistmg of SEQ ID NO:6, SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO:28, f) a polypeptide comprising a naturally occurring amino acid sequence at least 93% identical to the amino acid sequence of SEQ ID NO: 21, g) a polypeptide comprismg a naturally occurring amino acid sequence at least 91% identical to the amino acid sequence of SEQ ID NO:25, h) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO: 30, i) a polypeptide comprising a naturally occurring amino acid sequence at least 97% identical to the amino acid sequence of SEQ ID NO: 38, j) a polypeptide consisting essentially of a naturally occurrmg amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of

SEQ ID NO:4, SEQ ID NO:8-10, SEQ ID NO: 14-15, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32-35, SEQ ID NO:37, and SEQ ID NO:39, k) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, and

1) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.

2. An isolated polypeptide of claim 1 selected from the group consistmg of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10 and SEQ ID NO: 12-39, and b) a polypeptide consistmg essentially of the amino acid sequence of SEQ ID NO: 11.

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

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

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:40-78.

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

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

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

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

10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.

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

12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprismg a polynucleotide sequence selected from the group consistmg of SEQ ID NO:40-78, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:47 and SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:61, and SEQ ID NO:63, c) a polynucleotide comprising a naturally occurrmg polynucleotide sequence at least

97% identical to a polynucleotide sequence selected from the group consisting of

SEQ ID NO:40-41, SEQ ID NO:45, and SEQ ID NO:64-65, d) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

98% identical to a polynucleotide sequence selected from the group consisting of

SEQ ID NO:48, SEQ ID NO:55, and SEQ ID NO:71, e) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

96% identical to the polynucleotide sequence of SEQ ID NO:67, f) a polynucleotide comprismg a naturally occurrmg polynucleotide sequence at least

91% identical to the polynucleotide sequence of SEQ ID NO:70, g) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

92% identical to a polynucleotide sequence selected from the group consisting of

SEQ ID NO:69 and SEQ ID NO:77, h) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

95% identical to the polynucleotide sequence of SEQ ID NO:75, i) a polynucleotide consisting essentially of a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:43-44, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53-54, SEQ ID NO:56-57, SEQ ID NO:59-60, SEQ ID NO:62, SEQ ID

NO:66, SEQ ID NO:68, SEQ ID NO:72-74, SEQ ID NO:76, and SEQ ID NO:78, j) a polynucleotide complementary to a polynucleotide of a), k) a polynucleotide complementary to a polynucleotide of b),

1) a polynucleotide complementary to a polynucleotide of c), m) a polynucleotide complementary to a polynucleotide of d) n) a polynucleotide complementary to a polynucleotide of e), o) a polynucleotide complementary to a polynucleotide of f), p) a polynucleotide complementary to a polynucleotide of g), q) a polynucleotide complementary to a polynucleotide of h), r) a polynucleotide complementary to a polynucleotide of i), and s) an RNA equivalent of a)-r).

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

14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprismg a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

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

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprismg: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

18. A composition of claim 17, wherein the polypeptide is selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consistmg of SEQ ID NO: 1-10 and SEQ ID NO: 12-39, and b) a polypeptide consisting essentially of the amino acid sequence of SEQ ID NO: 11.

19. A method for treating a disease or condition associated with decreased expression of functional ENZM, comprising administering to a patient in need of such treatment the composition of claim 17.

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

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

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

23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprismg: a) contacting a sample comprising a polypeptide of claim 1 with a compound, and b) detecting antagonist activity in the sample.

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

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

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

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

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

29. A method of screening for potential toxicity of a test compound, the 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 comprismg at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample indicates potential toxicity of the test compound.

30. A method for a diagnostic test for a condition or disease associated with the expression of ENZM in a biological sample, the method comprising: a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.

31. The antibody of claim 11 , wherein the antibody is: a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')₂ fragment, or e) a humanized antibody.

32. A composition comprising an antibody of claim 11 and an acceptable excipient.

33. A method of diagnosing a condition or disease associated with the expression of ENZM in a subject, comprismg administering to said subject an effective amount of the composition of claim 32.

34. A composition of claim 32, further comprising a label.

35. A method of diagnosing a condition or disease associated with the expression of ENZM in a subject, comprising administering to said subject an effective amount of the composition of claim

34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide consistmg of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from the animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.

37. A polyclonal antibody produced by a method of claim 36.

38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.

40. A monoclonal antibody produced by a method of claim 39.

41. A composition comprismg the monoclonal antibody of claim 40 and a suitable carrier.

42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.

43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39 in a sample, the method comprising: a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39 from a sample, the method comprising: a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim

13.

47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising: a) labeling the polynucleotides of the sample, _t) b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.

49. An array of claim 48, wherem said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

50. An aπay of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.

51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.

52. An aπay of claim 48, which is a microaπay.

53. An aπay of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.

54. An aπay of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An aπay of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.

56. A polypeptide of claim 1, comprismg the amino acid sequence of SEQ ID NO: 1.

57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:2.

58. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:3.

59. A polypeptide of claim 1, comprismg the amino acid sequence of SEQ ID NO:4.

60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:5.

61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:7.

63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:8.

64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ED NO:9.

65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 10.

66. A polypeptide of claim 1, consisting essentially of the amino acid sequence of SEQ ID

67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 12.

68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 13.

69. A polypeptide of claim 1, comprismg the amino acid sequence of SEQ ID NO: 14.

70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 15.

71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 16.

72. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 17.

73. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 18.

74. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 19.

75. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:20.

76. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:21.

77. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:22.

78. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:23.

79. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 24.

80. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:25.

81. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:26.

82. A polypeptide of claim 1, comprising the;amino acid sequence of SEQ ID NO:27.

83. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:28.

84. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:29.

85. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:30.

86. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:31.

87. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:32.

88. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:33.

89. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 34.

90. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ED NO:35.

91. A polypeptide of claim 1, comprismg the amino acid sequence of SEQ ID NO:36.

92. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 37.

93. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:38.

94. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ED NO:39.

95. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:40.

96. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED

NO:41.

97. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:42.

98. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:43.

99. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:44.

100. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:45.

101. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:46.

102. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:47.

103. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:48.

104. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:49. no. A polynucleotide of claim 12, comprismg the polynucleotide sequence of SEQ ED NO:50.

106. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:51.

107. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:52.

108. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED

NO:53.

109. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:54.

110. A polynucleotide of claim 12, comprismg the polynucleotide sequence of SEQ ED NO:55.

111. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:56.

112. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:57.

113. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED

NO:58.

114. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:59.

115. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:60.

116. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:61.

117. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:62.

118. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:63.

119. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:64.

120. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED

NO:65.

121. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:66.

122. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:67.

123. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:68.

124. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:69.

125. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:70.

126. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:71.

127. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:72.

128. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:73.

129. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED

NO:74.

130. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:75.

131. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:76.

132. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:77.

133. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:78.