JP2006514531A - Drug metabolizing enzymes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a purified polypeptide which is a drug metabolizing enzyme.
An isolated polypeptide selected from the group consisting of the following (a) to (d). (A) a polypeptide comprising an amino acid sequence selected from the group having SEQ ID NO: 1-12, (b) at least 90% identical to an amino acid sequence selected from the group having SEQ ID NO: 1-12 A polypeptide having a native amino acid sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-12, and (d) SEQ ID NO: 1-12 An immunogenic fragment of a polypeptide having an amino acid sequence selected from the group comprising.

Description

  The present invention relates to nucleic acid sequences and amino acid sequences of drug metabolizing enzymes. The present invention also provides diagnosis and treatment of gastrointestinal diseases, including autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine disorders, ophthalmic diseases, metabolic disorders, and liver diseases using these sequences. Regarding prevention. The invention further relates to the evaluation of the effect of exogenous compounds on the expression of nucleic acid and amino acid sequences of drug metabolizing enzymes.

  Drug metabolism and drug movement in the body (pharmacokinetics) are important in determining its effectiveness, toxicity, and interaction with other drugs. Three processes dominate pharmacokinetics: absorption of drugs, distribution of drugs to various tissues, and elimination of drug metabolites. These processes are closely related to drug metabolism because various metabolic modifications transform most of the physicochemical and pharmacological properties of drugs, including solubility, receptor binding, and excretion rates. Involved. Metabolic pathways that modify drugs also accept various natural substrates such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins. Thus, enzymes in these pathways are important sites of biochemical and pharmacological interactions between natural compounds, drugs, carcinogens, mutagens, and xenobiotics.

  It has long been known that genetic differences in drug metabolism cause significant differences in drug effects and levels of toxicity between individuals. For drugs that have a narrow therapeutic index or drugs that require physiological activity, such as codeine, such genetic polymorphisms can be extremely important. In addition, promising new drugs are often excluded in clinical trials because of toxicity that can cause side effects in only a subset of patients. Advances in pharmacogenomic research, where drug-metabolizing enzymes are an important part, will develop tools that can withstand the question of drug efficacy and toxicity and expand such information (Evans, WE and RV Relling (1999) Science 286: 487-491).

Drug metabolic reactions are divided into Phase I, which functionalizes drug molecules and prepares them for further metabolism, followed by Phase II. In general, Phase I reaction products are partially or completely inert, and Phase II reaction products are the major excretory species. However, phase I reaction products may be more active than the original drug administered, and this metabolic activation principle is utilized as a prodrug (eg, L-dopa). In addition, some non-toxic compounds (eg, aflatoxin, benzo-α-pyrene) are metabolized to toxic intermediates via these pathways. Phase I reactions are usually the rate limiting step in drug metabolism. However, pre-exposure to the compound or other compounds can induce the expression of Phase I enzymes, thereby increasing substrate flux through metabolic pathways (Klaassen, CD, et al. (1996) Casarett and Doull's Toxicology: The Basic Science of Poisons , McGraw-Hill, New York, NY, 113-186; Katzung, BG (1995) Basic and Clinical Pharmacology , Appleton and Lange, Norwalk, CT, 48-59; Gibson, GG and P. Skett (1994) Introduction to Drug Metabolism , Blackie Academic and Professional, London).

  Drug metabolizing enzymes (DME) have a wide range of substrate specificities. This is in contrast to the immune system, where a wide variety of antibodies have a high specificity for their antigen. The ability of DME to metabolize diverse molecules creates potential drug interactions at the level of metabolism. For example, the induction of one DME by one compound can affect the metabolism of other compounds by that enzyme.

  DMEs are classified according to the type of reaction they catalyze and the associated cofactors. The main classes of Phase I enzymes include, but are not limited to, cytochrome P450 and flavin-containing monooxygenases. Other enzyme classes involved in Phase I catalytic cycles and reactions include, but are not limited to, NADPH cytochrome P450 reductase (CPR), microsomal cytochrome b5 / NADH cytochrome b5 reductase system, ferredoxin / ferredoxin reductase redox couple, Aldo / keto reductase and alcohol dehydrogenase are included. The main classes of Phase II enzymes include, but are not limited to, UDP glucuronyl transferase, sulfotransferase, glutathione S transferase, N acyl transferase, and N acetyl transferase.

Cytochrome P450 and members of the P450 catalytic cycle-related enzyme cytochrome P450 superfamily enzymes catalyze the oxidative metabolism of various substrates. Such substrates include natural compounds such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins, drugs, carcinogens, mutagens, and xenobiotics. Cytochrome P450, also known as P450 heme-thiolate protein, usually acts as a terminal oxidase in a multicomponent electron transport chain called the P450-containing monooxygenase system. Specific reactions catalyzed include hydroxylation, epoxidation, N-oxidation, sulfoxidation, N-dealkylation, S-dealkylation, and O-dealkylation, desulfation, deamination, and azo, nitro, And reduction of the N-oxide group. These reactions are related to glucocorticoid, cortisol, estrogen and androgen steroid production in animals, insecticide resistance in insects, herbicide resistance in plants and flower color development, and environmental purification bioremediation by microorganisms. Cytochrome P450 can act on drugs, carcinogens, mutagens, and xenobiotics to cause detoxification or conversion of the substance to a more toxic product. Cytochrome P450 is abundant in liver but also appears in other tissues, and these enzymes are located in microsomes (ExPASYENZYME EC 1.14.14.1; Prosite PDOC00081 Cytochrome P450 cysteine heme-iron ligand signature; PRINTS EP450I E-Class P450 Group I signature; see Graham-Lorence, S. and Peterson, JA (1996) FASEB J. 10: 206-214).

  400 cytochrome P450s have been identified in a variety of organisms including bacteria, fungi, plants, and animals (Graham-Lorence, supra). Class B is found in prokaryotes and fungi, and class E is found in bacteria, plants, insects, vertebrates, and mammals. Five subclasses or groups are included in the large family of E-class cytochrome P450s (PRINTS EP450I E-Class P450 Group I signature).

  All cytochrome P450s use heme cofactors and share structural properties. Most cytochrome P450s are 400-530 amino acids long. The secondary structure of this enzyme is about 70% alpha helix and about 22% beta sheet. The region surrounding the heme binding site at the C-terminus of this protein is conserved in all cytochrome P450s. A signature sequence of 10 amino acids in this heme-iron binding region is identified, which contains a conserved cysteine involved in heme iron binding at the fifth coordination site. In eukaryotic cytochrome P450, the transmembrane region is usually found in the first 15-20 amino acids of this protein and generally consists of about 15 hydrophobic residues followed by a positively charged residue (Prosite PDOC00081). Supra; see Graham-Lorence supra).

  Cytochrome P450 enzymes are involved in cell proliferation and development. This enzyme has a role in chemical mutagenesis and carcinogenesis that occurs by metabolizing chemicals into reactive intermediates that form adducts with DNA (Nebert, DW and Gonzalez, FJ (1987) Ann. Rev. Biochem. 56: 945-993). These adducts can cause nucleotide changes and DNA rearrangements that lead to carcinogenesis. Cytochrome P450 expression in the liver and other tissues is induced by xenobiotics such as polycyclic aromatic hydrocarbons, peroxisome proliferators, phenobarbital, and glucocorticoid dexamethasone (Dogra, SC et al. (1998) Clin. Exp. Pharmacol. Physiol. 25: 1-9). Certain cytochrome P450 proteins may be involved in eye development such that mutations in the P450 gene CYP1B1 cause primary congenital glaucoma (Online Mendelian Inheritance in Man (OMIM) * 601771 Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYP1B1).

Cytochrome P450 is involved in inflammation and infection. Hepatic cytochrome P450 activity is greatly affected by various infections and inflammatory stimuli, with suppressed and induced activity (Morgan, ET (1997) Drug Metab. Rev. 29: 1129-1188). The effects observed in vivo can be mimicked by pro-inflammatory cytokines and interferons. Autoantibodies against two cytochrome P450 proteins were found in patients with autoimmune polyadenoendocrine type I (APECED, one type of multigland autoimmune syndrome) (OMIM * 240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy).

Mutations in cytochrome P450s are the most common congenital adrenal hyperplasia in infantile adrenal disorder, pseudovitamin D deficiency rickets, cerebral tendon xanthomas (progressive neurological dysfunction, early-onset atherosclerosis) And one type of lipid storage disease characterized by cataracts), linked to a variety of metabolic disorders, including heritable resistance to the anticoagulants coumarin and warfarin (Isselbacher, KJ et al. (1994) Harrison's Principles of Internal Medicine , McGraw-Hill, Inc. New York, NY, 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). An extremely high level of expression of cytochrome P450 protein aromatase was found in a juvenile fibrolamellar hepatocellular carcinoma with severe gynecomastia, feminization (Agarwal, VR (1998 ) J. Clin. Endocrinol. Metab. 83: 1797-1800).

The cytochrome P450 catalytic cycle is completed by the reduction of cytochrome P450 by NADPH cytochrome P450 reductase (CPR). It is generally believed that another microsomal electron transport system consisting of cytochrome b5 and NADPH cytochrome b5 reductase is not the primary donor of electrons to the cytochrome P450 catalytic cycle. However, recent research reports by Lamb, DC et al. (1999; FEBS Lett. 462: 283-288) have shown that Candida albicans can be effectively reduced and supported by the microsomal cytochrome b5 / NADPH cytochrome b5 reductase system. ) Identify cytochrome P450 (CYP51). Thus, there appear to be many cytochrome P450s supported by this alternative electron donor system.

  Cytochrome b5 reductase also reduces oxidized hemoglobin (which cannot transport oxygen, methemoglobin or ferrihemoglobin) to active hemoglobin in red blood cells. The presence of high levels of oxidizers or abnormal hemoglobin that is not fully reduced (hemoglobin M) causes methemoglobinemia. Methemoglobinemia can also arise from a genetic deficiency of erythrocyte cytochrome b5 reductase (for review see Mansour, A. and Lurie, A.A. (1993) Am. J. Hematol. 42: 7-12).

  Members of the cytochrome P450 family are also closely involved in vitamin D synthesis and catabolism. Vitamin D exists as two biologically equivalent prohormones, ergocalciferol (vitamin D2) produced in plant tissues and cholecalciferol (vitamin D3) produced in animal tissues. The latter cholecalciferol is formed when 7-dehydrocholesterol is exposed to near ultraviolet (ie 290-310 nm). It is usually produced when the skin is exposed to sunlight for a short time (for review see Miller, W.L. and Portale, A.A. (2000) Trends Endocrinol. Metab. 11: 315-319).

Both prohormone types are further metabolized in the liver by the enzyme 25-hydroxylase to 25-hydroxyvitamin D (25 (OH) D). 25 (OH) D is the most abundant precursor of vitamin D and is further metabolized in the kidney by the enzyme 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase) to form the active 1α, 25 -Dihydroxyvitamin D (1α, 25 (OH) ₂ D). The regulation of 1α, 25 (OH) ₂ D production is mainly performed at this final step in the synthetic pathway. 1α-Hydroxylase activity includes several levels of circulating enzyme products (1α, 25 (OH) ₂ D), as well as levels of parathyroid hormone (PTH), calcitonin, insulin, calcium, phosphorus, growth factors, and prolactin. It depends on the physiological factors. Furthermore, 1α-hydroxylase activity outside the kidney has been reported, and tissue-specific and local regulation of 1α, 25 (OH) ₂ D production is also considered biologically important. Catalysis of 1α, 25 (OH) ₂ D to 24,25-dihydroxyvitamin D (24,25 (OH) ₂ D) involves the enzyme 25-hydroxyvitamin D 24-hydroxylase (24-hydroxylase) It also happens in the kidneys. 24-Hydroxylase can also utilize 25 (OH) D as a substrate (Shinki, T. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 12920-12925; Miller, WL and Portale, AA, supra. , As well as its references).

  Vitamin D25-hydroxylase, 1α-hydroxylase, and 24-hydroxylase are all NADPH-dependent type I (mitochondrial) cytochrome P450 enzymes that show high homology with other members of the family. Vitamin D25-hydroxylase also exhibits broad substrate specificity and may also perform 26-hydroxylation of bile acid intermediates and 25-hydroxylation, 26-hydroxylation, and 27-hydroxylation of cholesterol (Dilworth, FJ et al. (1995) J. Biol. Chem. 270: 16766-16774; Miller, WL and Portale, AA, supra, and references thereof).

The active form of vitamin D (1α, 25 (OH) ₂ D) is involved in calcium and phosphate homeostasis and promotes myeloid and skin cell differentiation. Vitamin D deficiency caused by a deficiency in enzymes involved in vitamin D metabolism (eg, 1α-hydroxylase) is hypocalcemia, hypophosphatemia, vitamin D-dependent (susceptibility) rickets (bone density Causes a decrease, as well as a varus knee with a duck walk, or a disease that exhibits the unique symptoms of O-legs). Vitamin D25-hydroxylase deficiency causes cerebral tendon xanthomatosis (a lipid storage disease characterized by cholesterol and cholestanol deposition in the Achilles tendon, brain, lung, and many other tissues). The disease exhibits progressive neurological dysfunction (including cerebellar ataxia after puberty), atherosclerosis, and cataract. The absence of rickets due to vitamin D25-hydroxylase deficiency suggests that there are alternative pathways for 25 (OH) D synthesis (Griffin, JE and Zerwekh, JE (1983) J. Clin. Invest 72: 1190-1199; Gamblin, GT et al. (1985) J. Clin. Invest. 75: 954-960; and Miller, WL and Portal, AA, supra).

  Ferredoxin and ferredoxin reductase are electron transfer accessory proteins that support at least one human cytochrome P450 species (cytochrome P450c27 encoded by the CYP27 gene) (Dilworth, FJ et al. (1996) Biochem. J. 320: 267-71. ). CYP104D1, a Streptomyces-Griseus cytochrome P450, is heterologously expressed in E. coli and reduced by endogenous ferredoxin and ferredoxin reductase enzymes (Taylor, M. et al. (1999) Biochem. Biophys. Res. Commun. 263: 838-42). This suggests that many cytochrome P450 species are supported by the ferredoxin / ferredoxin reductase pair. Ferredoxin reductase is also found in certain model drug metabolism systems and is known to reduce the antitumor antibiotic actinomycin D to reactive free radical species (Flitter, WD and Mason, RP (1988)). ) Arch. Biochem. Biophys. 267: 632-639).

Dimethylaminohydrolase
NG, NG-dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes the endogenous nitric oxide synthase (NOS) inhibitors NG-monomethyl-arginine and NG, NG-dimethyl-L-arginine to produce L-citrulline It is an enzyme that makes it. Inhibition of DDAH increases the intracellular concentration of NOS inhibitor to a concentration sufficient to inhibit NOS. Thus, DDAH inhibition provides a method for NOS inhibition, and changes in DDAH activity may be involved in pathophysiological changes in nitric oxide production (MacAllister, RJ, et al. (1996) Br. J. Pharmacol 119: 1533-1540). DDAH has been found in neurons exhibiting cytoskeletal abnormalities and oxidative stress in Alzheimer's disease. No DDAH has been found in control neurons of the same age. This suggests that oxidative stress and nitric oxide-mediated phenomena are involved in the pathogenesis of Alzheimer's disease (Smith, MA et al. (1998) Free Radic. Biol. Med. 25: 898-902).

Flavin-containing monooxygenase (FMO)
Flavin-containing monooxygenases oxidize nucleophilic nitrogen, sulfur, and phosphorus heteroatoms of an unusual range of substrates. Like cytochrome P450, FMO is a microsomal enzyme that uses NADPH and O ₂ and its substrate overlaps extensively with that of cytochrome P450. FMO is distributed in tissues such as liver, kidney, and lung.

  Five different known FMO isoforms (FMO1, FMO2, FMO3, FM04, and FMO5) expressed in a tissue-specific manner are found in mammals. These isoforms differ in substrate specificity, and also differ in other properties such as inhibition by various compounds and the stereospecificity of the reaction. FMO has a 13 amino acid signature sequence and its components span the N-terminal two-thirds sequence. These components also include the FATGY motif found in many N hydroxylases, and the FAD binding region (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, oxidation of secondary amines to hydroxylamines and nitrones, oxidation of primary amines to hydroxylamines and oximes, and sulfur-containing compounds and phosphines. Oxidation to S-oxide and P-oxide is included. Hydrazine, iodide, selenide, and boron-containing compounds are also substrates. Although FMO is chemically similar to cytochrome P450, FMO is usually distinguishable from cytochrome P450 in vitro , for example based on its high thermal instability and nonionic surfactant sensitivity of cytochrome P450. However, these properties are difficult to use for identification due to differences in thermal stability and surfactant sensitivity among FMO isoforms.

FMO plays an important role in the metabolism of several drugs and xenobiotics. FMO (liver FMO3) is primarily responsible for the metabolism of (S) nicotine to (S) nicotine N-1′-oxide (excreted in the urine). FMO is also involved in S-oxygenation of cimetidine, an H ₂ -antagonist that is widely used in the treatment of gastric ulcers. The liver phenotype of FMO is not under the same regulatory control as cytochrome P450. For example, in rats, cytochrome P450 is induced by phenobarbital treatment, but FMO1 is suppressed.

  Endogenous substrates of FMO include cysteamine (oxidized to the disulfide cystamine) and trimethylamine (TMA) metabolized to trimethylamine N-oxide. TMA smells like rotten fish, and mutations in the FMO3 isoform cause large amounts of malodorous free amines to be excreted from sweat, urine, and exhaled breath. These symptoms gave rise to the name of fish odor syndrome (OMIM 602079 Trimethylaminuria).

Lysyl oxidase lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidase involved in the formation of a connective tissue matrix by cross-linking between collagen and elastin. LO is secreted as an N-glycosylated precursor protein of approximately 50 kDa and is cleaved by a metalloprotease to become a mature enzyme, which is also active. Copper atoms in LO are involved in the transfer of electrons to and from oxygen and promote oxidative deamination of lysine residues in these extracellular matrix proteins. Although copper coordination is essential for LO activity, the expression of this apoenzyme is not affected by insufficient dietary copper intake. However, functional LO deficiency is linked to skeletal and vascular tissue damage associated with dietary copper deficiency. LO is also inhibited by various semicarbazides, hydrazine, and amino nitrites, and heparin. β-aminopropionitrile is one of the commonly used inhibitors. LO activity increases in response to ozone and cadmium and increases in response to increased levels of hormones released in response to local tissue trauma. Examples of such hormones include transforming growth factor-β, platelet-derived growth factor, angiotensin II, and fibroblast growth factor. Abnormalities in LO activity are linked to Menkes syndrome and occipital horn syndrome. Cytosolic LO enzymes are thought to be involved in abnormal cell growth (reviewed by Rucker, RB et al. (1998) Am. J. Clin. Nutr. 67: 996S-1002S and Smith-Mungo, LI and Kagan, HM (1998) Matrix Biol. 16: 387-398).

Dihydrofolate reductase Dihydrofolate reductase (DHFR) is a ubiquitous enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, which is a de novo synthesis of glycine and purines, In addition, it is an essential process in the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). This basic reaction is
7,8-dihydrofolic acid + NADPH → 5,6,7,8-tetrahydrofolic acid + NADP ⁺ .

The DHFR enzyme can be inhibited by various dihydrofolate analogs such as trimethroprim and methotrexate. Because abundant dTMP is required for DNA synthesis, rapid cell division requires DHFR activity. Replication of DNA viruses (eg, herpes virus) also requires high levels of DHFR activity. Therefore, drugs targeting DHFR are used for cancer chemotherapy and suppression of DNA virus replication (for similar reasons, thymidylate synthases are also target enzymes). Drugs that suppress DHFR have preferential cytotoxicity against rapidly dividing cells (ie, DNA virus-infected cells), but have no specificity, and indiscriminately destroy dividing cells. In addition, cancer cells can become resistant to drugs such as methotrexate as a result of acquired transport defects or duplication of one or more DHFR genes (Stryer, L. (1988) Biochemistry . WH Freeman and Co., Inc. New York. 511-5619).

Aldo / keto reductases Aldo / keto reductases are monomeric NADPH-dependent oxidoreductases with broad substrate specificity (Bohren, KM et al. (1989) J. Biol. Chem. 264: 9547-9551). . These enzymes catalyze the reduction of carbonyl-containing compounds, such as carbonyl-containing sugars and aromatic compounds, to the corresponding alcohols. Thus, various carbonyl-containing drugs and xenobiotics are likely metabolized by this class of enzymes.

  One known reaction catalyzed by the family member aldose reductase is that glucose is reduced to sorbitol and further metabolized to fructose by sorbitol dehydrogenase. Under normal conditions, the reduction of glucose to sorbitol is not the main route. However, in hyperglycemic conditions, sorbitol accumulation appears to be associated with the development of diabetic complications (OMIM * 103880 Aldo-keto reductase family 1, member B1). Members of this enzyme family are also highly expressed in several liver cancers (Cao, D. et al. (1998) J. Biol. Chem. 273: 11429-11435).

Alcohol dehydrogenase Alcohol dehydrogenase (ADH) oxidizes simple alcohols to the corresponding aldehydes. ADH is a cytosolic enzyme that likes the cofactor NAD + and binds to zinc ions. ADH levels are highest in the liver and low in the kidney, lung, and gastric mucosa.

  The known ADH isoform is a dimeric protein composed of 40 kDa subunits. There are five known loci (a, b, g, p, c) that encode these subunits, some of which are characteristic allelic variants (b1, b2, b3, g1, g2). The subunits can form homodimers and heterodimers, and the composition of the subunits determines the specific properties of the active enzyme. Therefore, holoenzymes are classified as class I (subunit configuration, aa, ab, ag, bg, gg), class II (pp), and class III (cc). Class IADH isoenzymes oxidize ethanol and other small fatty alcohols and are inhibited by pyrazole. Class II isoenzymes prefer long chain fatty and aromatic alcohols and cannot oxidize methanol. It is not inhibited by pyrazole. Class III isoenzymes prefer longer long chain fatty alcohols (more than 5 carbons) and aromatic alcohols and are not inhibited by pyrazole.

  Short chain alcohol dehydrogenases include a large number of closely related enzymes with various substrate specificities. Included in this group are the mammalian enzymes D-β-hydroxybutyrate dehydrogenase, (R) -3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin dehydrogenase, NADPH-dependent carbonyl reductase, corticosteroid 11- β-dehydrogenase, estradiol-17-β-dehydrogenase, bacterial enzymes acetoacetyl-CoA reductase, glucose 1-dehydrogenase, 3-β-hydroxysteroid dehydrogenase, 20-β-hydroxysteroid dehydrogenase, ribitol dehydrogenase, 3-oxo Acyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, sorbitol-6-phosphate 2-dehydrogenase, 7-α-hydroxysteroid dehydrogenase, cis-1,2-dihi Doxy-3,4-cyclohexadiene-1-carboxylate dehydrogenase, cis-toluene dihydrodiol dehydrogenase, cis-benzene glycol dehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase, N-acyl mannosamine 1 -Dehydrogenase (N-acylmannosamine 1-dehydrogenase) and 2-deoxy-D-gluconate 3-dehydrogenase (Krozowski, Z. (1994) J. Steroid Biochem. Mol. Biol. 51: 125-130; Krozowski, Z. (1992) Mol. Cell Endocrinol. 84: C25-31; and Marks, AR et al. (1992) J. Biol. Chem. 267: 15459-15463).

UDP glucuronyltransferase
Members of the UDP glucuronyltransferase family (UGT) catalyze the transfer of the glucuronic acid group from the cofactor uridine diphosphate-glucuronic acid (UDP-glucuronic acid) to the substrate. This transition is usually performed on a nucleophilic heteroatom (O, N, or S). Examples of substrates include xenobiotics rendered functional by the Phase I reaction and endogenous compounds such as bilirubin, steroid hormones, and thyroid hormones. The product of glucuronidation is excreted in urine if the molecular weight of the substrate is less than about 250 g / mol, but is excreted in bile if the glucuronidated substrate is larger than that.

  UGT is present in microsomes of the liver, kidney, intestine, skin, brain, spleen, and nasal mucosa. In these positions, UGT is ideally located to reach the product of phase I drug metabolism because it is on the same side of the endoplasmic reticulum membrane as the cytochrome P450 enzyme and the flavin-containing monooxygenase. UGT has a C-terminal transmembrane domain that anchors its UGT to the endoplasmic reticulum membrane, as well as a conserved signature domain of approximately 50 amino acid residues in its C-terminal part (Prosite PDOC00359 UDP-glycosyltransferase signature).

  UGT involved in drug metabolism is encoded by two gene families, UGT1 and UGT2. Members of the UGT1 family arise by alternative splicing of a single locus with constant regions involved in cofactor binding and membrane insertion and a variable substrate binding domain. Members of the UGT2 family are encoded by different loci and are classified into two families, UGT2A and UGT2B. The 2A subfamily is expressed in the olfactory epithelium, and the 2B subfamily is expressed in liver microsomes. Mutations in the UGT gene are called hyperbilirubinemia (OMIM # 143500 Hyperbilirubinemia I), Crigler-Najjar syndrome, characterized by marked hyperbilirubinemia from birth, and Gilbert's disease Related to mild hyperbilirubinemia (OMIM * 191740 UGT1).

Sulfotransferase sulfate conjugation occurs on many of the same substrates that undergo O-glucuronic acid conjugation, producing highly water-soluble sulfate esters. Sulfotransferase (ST) catalyzes this reaction by transferring SO ₃ ⁻ from the cofactor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the substrate. ST substrates are mainly phenols and aliphatic alcohols, but also include aromatic amines and aliphatic amines that are conjugated to produce the corresponding sulfamic acid. Products from these reactions are mainly excreted in urine.

  ST is found in a variety of tissues including the liver, kidney, intestine, lung, platelets, and brain. ST enzymes are generally cytosolic enzymes, and many types are often expressed simultaneously. For example, more than 12 types of ST are present in rat liver cytosol. These biochemically characterized STs fall into five classes based on their substrate selectivity: aryl sulfotransferase, alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester sulfotransferase, and bile salt sulfotransferase. being classified.

  ST enzyme activity varies significantly with sex and age in rats. It is believed that the effects of developmental cues and sex-related hormones combine to produce these differences in the ST expression profile as well as in other DME profiles such as cytochrome P450. Of note, high expression of ST in cats partially compensates for the reduced level of UDP glucuronyltransferase activity.

  Several types of ST have been purified and cloned from human liver cytosol. There are two phenol sulfotransferases that differ in thermostability and substrate selectivity. Thermostable enzymes catalyze the sulfation of phenols such as paranitrophenol, minoxidil, and acetaminophen, and thermolabile enzymes prefer monoamine substrates such as dopamine, epinephrine and levadopa. Other cloned STs include estrogen sulfotransferase and N-acetylglucosamine-6-O-sulfotransferase. This last enzyme illustrates another major role of ST in cell biochemistry. That is, modifications of the sugar structure that can be important for cell differentiation and proteoglycan maturation. Indeed, an inherited deficiency of certain sulfotransferases appears to be associated with patchy corneal degeneration, a disorder characterized by poor synthesis of mature keratan sulfate proteoglycans (Nakazawa, K. et al. (1984) J. Biol. Chem. 259: 13751-13757; OMIM * 217800 Macular dystrophy, corneal).

Galactosyltransferases are a subset of glycosyltransferases (glycosyltransferases), and galactose (Gal) in N-terminal acetylglucosamine (GlcNAc) oligosaccharide chains that are part of glycolipids or glycoproteins released into solution (Kolbinger, F. et al. (1998) J. Biol. Chem. 273: 433-440; Amado, M. et al. (1999) Biochim. Biophys. Acta 1473: 35-53). Galactosyltransferase is a soluble extracellular protein detected on the cell surface and is also present in the Golgi apparatus. β1,3-galactosyltransferase forms a type I sugar chain having a Gal (β1-3) GlcNAc bond. Known human and mouse β1,3-galactosyltransferases are thought to have a short cytosolic domain, one transmembrane domain, and one catalytic domain with eight conserved regions (Kolbinger, F. supra and Hennet, T. et al. (1998) J. Biol. Chem. 273: 58-65). In mouse UDP-galactose: β-N-acetylglucosamine β1,3-galactosyltransferase-I, region 1 is amino acid residue 78-83, region 2 is amino acid residue 93-102, and region 3 is amino acid residue 116. -119, region 4 is amino acid residues 147-158, region 5 is amino acid residues 172-183, region 6 is amino acid residues 203-206, region 7 is amino acid residues 236-246, and region 8 is amino acids Located at residues 264-275. Since one variant of a sequence present in region 8 of mouse UDP-galactose: β-N-acetylglucosamine β1,3-galactosyltransferase-I is also present in bacterial galactosyltransferases, this sequence is Galactosyltransferase sequence motif (Hennet, T. supra). Recent studies suggest that brainiac protein is a β1,3-galactosyltransferase (Yuan, Y. et al. (1997) Cell 88: 9-11; and Hennet, T. supra).

  UDP-Gal: GlcNAc-1,4-galactosyltransferase (-1,4-GalT) (Sato, T. et al. (1997) EMBO J. 16: 1850-1857) has Gal (β1-4) GlcNAc binding Catalyses the formation of type II sugar chains. As in the case of β1,3-galactosyltransferase, a soluble form of the enzyme is formed by membrane bound cleavage. Amino acids conserved in β1,4-galactosyltransferases include two cysteines linked by disulfide bonds and a putative UDP-galactose binding site in the catalytic domain (Yadav, S. and Brew, K (1990) J. Biol. Chem. 265: 14163-14169; Yadav, SP and Brew, K. (1991) J. Biol. Chem. 266: 698-703, and Shaper, NL et al. (1997) J. Biol Chem. 272: 31389-31399). In addition to the synthesis of sugar chains on glycoproteins or glycolipids, β1,4-galactosyltransferase has several specialized roles. In mammals, as part of a heterodimer with α-lactalbumin, certain β1,4-galactosyltransferases function in mammary lactose production during lactation. Β1,4-galactosyltransferase on the surface of sperm acts as a receptor that specifically recognizes eggs. Cell surface β1,4-galactosyltransferases also act in cell adhesion, cell / basement membrane interactions, normal cell migration, and metastatic cell migration (Shur, B. (1993) Curr. Opin. Cell Biol. 5: 854-863; and Shaper, J. (1995) Adv. Exp. Med. Biol. 376: 95-104).

Glutathione S transferase The basic reaction catalyzed by glutathione S transferase (GST) is electrophile conjugation with reduced glutathione (GSH). GST is a homodimeric or heterodimeric protein and is mainly localized in the cytosol, but some activity is also found in microsomes. The main isoenzymes have common structural and catalytic properties, and in humans they are classified into four major classes: α, μ, π, and θ. The two largest classes, α and μ, are identified by their respective protein isoelectric points (α is about 7.5 to 9.0 for pI and μ is about 6.6 for pI). Each GST has a common binding site for GSH and a variable hydrophobic binding site. The hydrophobic binding site in each isoenzyme is specific for a particular electrophilic substrate. Certain amino acid residues within GST have been identified as important for their binding site and catalytic activity. Residues Q67, T68, D101, E104, and R131 are important for GSH binding (Lee, HC 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).

In most cases, GST performs useful functions such as inactivating and detoxifying potential mutagenic and carcinogenic chemicals. However, in some cases their effects can be detrimental and can activate chemicals to make them mutagenic and carcinogenic. Several types of rat and human GST are reliable pre-neoplastic markers that help detect carcinogenesis. The expression of human GST in bacterial strains such as Salmonella typhimurium used in the well-known Ames test to investigate mutagenicity helps determine the role of these enzymes in mutagenesis. Dihalomethanes that cause liver tumors in mice are thought to be activated by GST. This idea is supported by the fact that dihalomethane is more mutagenic in bacterial cells expressing human GST than in non-transduced cells (Thier, R. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8567-8580). Mutagenicity of ethylene dibromide and ethylene dichloride is increased in bacterial cells expressing the human αGST A1-1, and aflatoxin B1 mutagenesis is substantially reduced by enhanced GST expression ( Simula, TP et al. (1993) Carcinogenesis 14: 1371-1376). Thus, control of GST activity would be useful in controlling mutagenesis and carcinogenesis.

  GST appears to be related to the acquired resistance of many cancers to drug treatment, a phenomenon known as multidrug resistance (MDR). MDR occurs in cancer patients treated with a cytotoxin such as cyclophosphamide, becomes resistant to the drug, and then becomes resistant to various other cytotoxics . Elevated GST levels are associated with some of these drug resistant cancers. In addition, after the GST level rises in response to the drug, it is considered that the drug is inactivated by the GSH conjugation reaction catalyzed by GST. Second, elevated GST levels protect cancer cells from other GST-binding cytotoxins. Increased levels of A1-1 in tumors have been linked to drug resistance caused by cyclophosphamide treatment (Dirven H. A. et al. (1994) Cancer Res. 54: 6215-6220). Therefore, modulation of GST activity in cancer tissue would be useful for MDR treatment of cancer patients.

γ-Glutamyltranspeptidase γ-glutamyltranspeptidase is a widely expressed enzyme that cleaves γ-glutamylamide bonds and initiates degradation of extracellular glutathione (GSH). The degradation of GSH provides cells with a region of cysteine gathering for the biosynthetic pathway. γ-glutamyl transpeptidase also contributes to the antioxidant of cells and its expression is caused by oxidative stress. This cell surface localized glycoprotein is expressed at high levels in cancer cells. Some studies suggest that the high activity level of γ-glutamyl transpeptidase at the surface of cancer cells is used to activate precursors, resulting in high concentrations of anticancer therapeutics locally. (Hanigan, MH (1998) Chem. Biol. Interact. 111-112: 333-42; Taniguchi, N. and Ikeda, Y. (1998) Adv. Enzymol. Relat. Areas Mol. Biol. 72: 239-78 Chikhi, N. et al. (1999) Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 122: 367-380).

Acyltransferase
N-acyltransferase enzymes catalyze the transfer of amino acid conjugates to activated carboxyl groups. Endogenous compounds and xenobiotics are activated by acyl-CoA synthetase in the cytosol, microsomes and mitochondria. The acyl-CoA intermediate is then amino acid (usually glycine, glutamine, or taurine by N-acyltransferase in the cytosol or mitochondria, but ornithine, arginine, histidine, serine, aspartic acid, and some dipeptides To form a metabolite having an amide bond. Although this reaction is complementary to O-glucuronidation, amino acid conjugation does not produce reactive and toxic metabolites, often caused by glucuronidation.

  Bile acid-CoA: amino acid N-acyltransferase (BAT) is one of this class of well-characterized enzymes. BAT is responsible for the production of bile acid conjugates that act as surfactants in the gastrointestinal tract (Falany, CN et al. (1994) J. Biol. Chem. 269: 19375-19379; Johnson, MR et al. (1991) J. Biol. Chem. 266: 10227-10233). BAT is also useful as a prognostic predictor of liver cancer 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 relaxes the eukaryotic chromatin structure, thereby allowing transcription factors to reach the promoter element of the DNA template in the affected region (or the entire genome) of the genome. In contrast, histone deacetylation closes the chromatin structure and restricts the arrival of transcription factors, reducing transcription. Finally, the use of chemicals that inhibit histone deacetylation (eg, sodium butyrate) as a general means of stimulating cellular transcription results in an overall increase in gene expression, which is an artifact. Modulation of gene expression by acetylation is also not limited to acetylation of other proteins such as p53, GATA-1, MyoD, ACTR, TFIIE, TFIIF, and high mobility group proteins (HMG). It can also be caused by conversion. In the case of p53, acetylation increases binding to DNA and stimulates transcription of genes regulated by p53. Prototype histone acetylase (HAT) is Gcn5 derived from Saccharomyces cerevisiae . Gcn5 is a member of one family of acetylases, including Tetrahymena p55, human Gcn5, and human p300 / CBP. Histone acetylation is reviewed in Cheung, WL et al. (2000) Curr. Opin. Cell Biol. 12: 326-333 and Berger, S. L (1999) Curr. Opin. Cell Biol. 11: 336-341. Yes. Several acetyltransferase enzymes are commonly used in several major classes of other enzymes such as, but not limited to, acetylcholinesterase and carboxylesterase, alpha / beta hydrolase fold (Center of Applied Molecular Engineering Inst. of Chemistry and Biochemistry-University of Salzburg, http://predict.sanger.ac.uk/irbm-course97/Docs/ms/ ) (Structural Classification of Proteins, http: // scop. mrc-lmb.cam.ac.uk/scop/index.html).

N-acetyltransferase aromatic amine and hydrazine-containing compounds are N-acetylated by N-acetyltransferase enzymes in liver and other tissues. Several xenobiotics can be O-acetylated to some extent by the same enzymes. N-acetyltransferase is a cytosolic enzyme that transfers an acetyl group in two processes using cofactor acetyl-coenzyme A (acetyl-CoA). In the first process, the acetyl group is transferred from acetyl-CoA to the active site cysteine residue, and in the second process, the acetyl group is transferred to the substrate amino group and the enzyme is regenerated.

  In contrast to most other DME classes, the number of known N-acetyltransferases is small. In humans, there are two highly similar enzymes, NAT1 and NAT2, and mice appear to have a third type enzyme, NAT3. The human form of N-acetyltransferase has independent regulation (NAT1 is widely expressed, but NAT2 is expressed only in the liver and intestine) and overlapping substrate selectivity. Both enzymes appear to accept some substrate to some extent, but NAT1 prefers several substrates (paraaminobenzoic acid, paraaminosalicylic acid, sulfamethoxazole, and sulfanilamide), while NAT2 has other substrates (Isoniazid, hydralazine, procainamide, dapsone, aminoglutethimide, and sulfamethazine) are preferred.

  Clinical observations of patients taking isoniazid, an antituberculosis drug, in the 1950s gave rise to descriptions of fast and slow acetylators of this compound. These phenotypes were later shown to be due to mutations in the NAT2 gene that affect the activity or stability of the enzyme. The slow acetylation of isoniazid is very prevalent in the Middle Eastern population (about 70%), slightly inferior in whites (about 50%), and less than 25% in Asian populations. Recently, a functional polymorphism in NAT1 has been detected and about 8% of the tested population shows a slow acetylation phenotype (Butcher, NJ et al. (1998) Pharmacogenetics 8: 67-72). . Because NAT1 can activate several known aromatic amine carcinogens, polymorphisms in the widely expressed NAT1 enzyme appear to be important in determining cancer risk (OMIM * 108345 N-acetyltransferase 1 ).

  Allylamine N-acetyltransferases catalyze N-acetylation of arylamine and heterocyclic amine substrates, often converting relatively inert compounds into chemically active, oncogenic electrophiles (Reviewed in Evans, DAP (1993) Genetic Factors in Drug Therapy: Clinical and Molecular Pharmacogenetics. Cambridge: Cambridge Univ. Press 211-305). Epidemiological studies show that allelic variation that affects cytosolic arylamine N-acetyltransferase activity in peripheral blood mononuclear cells is a specific form involving cancer resulting from exposure to aromatic amine carcinogens present in tobacco May correlate with a predisposition to cancer. Eight percent of the population with a relatively low form of arylamine N-acetyltransferase activity appears less likely to develop these cancers (Doll, MA et al. (1997) Biochem. Biophys. Res. Commun. 233: 584- 591; Butcher, NJ et al. (1998) Pharmacogenetics 8: 67-72).

  Microsomal arylacetamide deacetylase competes with the cellular substrate arylamine N-acetyltransferase for arylamines and heterocyclic substrates, and catalyzes the biotransformation of carcinogens into an active form. In particular, arylacetamido deacetylases convert 4-acetylaminobiphenyl, 2-acetylaminofluorene, and 2-acetylaminonaphthalene in various tissues. Cofactors are not necessary. The activity of this enzyme is highest in liver tissue (Lower, GM and Bryan, GT (1976) J. Toxicol. Environ. Health 1: 421-32; Probst, MR et al. (1994) J. Biol. Chem. 269: 21650 -6).

Covalent modification of methyltransferase cell substrates with methyl groups has been implicated in the pathology of cancer and other diseases (Gloria, L. et al. (1996) Cancer 78: 2300-2306.). Cytosine hypermethylation of eukaryotic DNA interferes with transcriptional activity (Turker, MS and Bestor, TH (1997) Mutat. Res. 386: 119-130). Within the mRNA of higher eukaryotes is N ⁶ -methyladenosine (Bokar, JA et al. (1994) J. Biol. Chem. 269: 17697-17704). Hypermethylated viral DNA is transcribed at a higher rate in infected cells than hypo- or semi-methylated DNA (Willis, DB et al. (1989) Cell. Biophys. 15: 97-111).

  Neurotransmitter metabolism, modulation of cell proliferation and differentiation, induction of immune response and tissue homeostasis may involve neurotransmitter metabolism (Weiss, B. (1991) Neurotoxicology 12: 379-386; Collins, SM et al. (1992) Ann. NY Acad. Sci. 664: 415-424; and Brown, JK and Imam, H. (1991) J. Inherit. Metab. Dis. 14: 436-458). In tissues, the rate of synthesis and degradation that regulates neurotransmitter activity depends on the level of enzymes and cofactors (Brown, J.K. and Imam, H. supra). Many pathways of small molecule degradation, such as neurotransmitter molecules, require methyltransferase activity (Kagan, R.M. and Clarke, S. (1994) Arch. Biochem. Biophys. 310: 417-427). For example, catechol-O-methyltransferase is required for degradation of catecholamines epinephrine or norepinephrine, and in the pineal gland, N-acetyl-5-hydroxytryptamine is converted to melatonin by hydroxyindole-O-methyltransferase. The catechol-O-methyltransferase and hydroxyindole methyltransferase genes have selective initiation codons (Rodriguez, IR et al. (1994) J. Biol. Chem. 269: 31969-31977; and Tenhunen, J. et al. (1994) Eur. J. Biochem. 223: 1049-1059).

  S-adenosylmethionine (AdoMet) is an important methyl group source for intracellular methylation (Bottiglieri, T. and Hyland, K. (1994) Acta Neurol. Scand. Suppl. 154: 19-26 ). Methyltransferase activity catalyzes the transfer of methyl groups from AdoMet to receptor molecules such as phosphotidylethanolamine or polynucleotide 5 'caps of viral mRNA (Montgomery, JA et al. (1982) J Med. Chem. 25: 626-629).

Members of the protein and small molecule S-adenosylmethionine methyltransferase family (AdoMet-MT) utilize AdoMet as a substrate or product and harbor three common consensus sequence motifs (Kagan and Clarke, supra). Motifs I and II are arranged with a characteristic interval of 34 amino acid residues to 90 amino acid residues (mode 52, average 57 ± 13), and motifs II and III are 12 residues to 38 residues (Mode 25, average 22 ± 5). Motif I consists of a portion of the AdoMet binding pocket, and Motif II can also be involved in AdoMet binding, but the role of Motif III is uncertain. The main exceptions to the spacing rules are RNA methyltransferase and many porphyrin precursor methyltransferases. These heterologous genotype motifs have been suggested to be useful in predicting related enzymes from methyltransferases and open reading frames generated by genomic sequencing projects (Kagan and Clarke, supra).

Messenger RNAN ⁶ -adenosine methyltransferase holoenzyme is partially purified from HeLa cell nuclear extract, 875 kDa ssDNA-agarose binding protein, 70 kDa AdoMet binding protein, and about 30 kDa component with unknown function Three subunits are obtained. These three components are absolutely necessary for the m ⁶ A methylation activity of RNA (Bokar, JA, supra).

In many tissues, including brain, intestine, bone marrow, liver and kidney, serine hydroxymethyltransferase converts serine to glycine by transferring the hydroxymethyl side chain group of serine to tetrahydrofolate (methyl receptor). The products of this reaction are N ⁵ , N ¹⁰ -methylenetetrahydrofolic acid and water. N ⁵ , N ¹⁰ -methylenetetrahydrofolic acid is a substrate for novel purine and pyrimidine nucleotide synthesis, homocysteine to methionine conversion, and tRNA methylation during tissue growth and cell proliferation.

  The genes encoding many of the growth-related methyltransferases have not yet been identified and isolated. In their role as rate-limiting steps in the methyltransferase reaction, AdoMet-MTs have been identified as targets for the design of psychiatric, antiviral, anticancer and anti-inflammatory drugs (Bottiglieri, T. and Hyland, K., supra; Gloria, L. et al., Supra) Sequence-specific methylation inhibits the activity of the LMP1 and BCR2 enhancer promoter regions of Epstein-Barr virus (Minarovits, J. et al. (1994) Virology 200: 661-667) 2'-5'-linked oligo (adenylic acid) nucleoside analogues synthesized by interferon-treated mouse L cells act as antiviral agents (Goswami, BB et al. (1982) J. Biol. Chem. 257: 6867-6870). Adenine analog inhibitors of AdoMet-MT reduced nucleic acid methylation and proliferation of leukemic L1210 cells (Kramer, D.L. et al. (1990) Cancer Res. 50: 3838-3842).

The use of experimental neurostimulators has shown that inactivation of neurotransmitters is absolutely essential for the correct functioning of the nervous system (Avery, L. and Horvitz, HR (1990)). J. Ex. Zool. 253: 263-270). Epigenetic or genetic defects in neurotransmitter metabolic pathways can result in a wide range of pathologies in different tissues, including Parkinson's disease and hereditary myoclonus (McCance, KL and Huether, SE (1994) Pathophysiology , Mosby- Year Book, Inc., St. Louis, MO 402-404, and Gundlach, AL (1990) FASEB J. 4: 2761-2766.).

Aminotransferases Aminotransferases constitute a family of pyridoxal 5'-phosphate (PLP) dependent enzymes. PLP-dependent enzymes catalyze component substitution of amino acids. Aspartate aminotransferase (AspAT) is the most widely studied PLP-containing enzyme. AspAT catalyzes the reversible transamination reaction of dicarboxyl L-amino acids (aspartic acid and glutamic acid) and the corresponding 2-oxo acids (oxaloacetic acid and 2-oxoglutaric acid). Other members of this family include pyruvate aminotransferase, branched chain amino acid aminotransferase, tyrosine aminotransferase, aromatic aminotransferase, alanine: glyoxylate aminotransferase (AGT) and kynurenine aminotransferase (Vacca, RA (1997) J. Biol. Chem. 272: 21932-21937).

  Primary hyperoxaluria type I is an autosomal recessive disease that results in a deficiency in the liver-specific peroxisomal enzyme alanine: glyoxylate aminotransferase-1. The phenotype of this disease is a deficiency in glyoxylate metabolism. In the absence of AGT, glyoxylic acid is not transaminated to glycine and is oxidized to oxalic acid. As a result, insoluble calcium oxalate is deposited in the kidney and ureter, eventually resulting in renal failure (Lumb, M. J. et al. (1999) J. Biol. Chem. 274: 20587-20596).

  Kynurenin aminotransferase catalyzes the irreversible transamination of L-tryptophan metabolite L-kynurenine to form kynurenic acid. This enzyme can also catalyze a reversible transamination reaction from L-2-aminoadipic acid to 2-oxoglutarate and vice versa to produce 2-oxoadipate and L-glutamate. Since kynurenic acid is a putative modulator of glutamatergic neurotransmission, deficiency of kynurenine aminotransferase appears to be related to pleotrophic effects (Buchli, R. et al. (1995) J. Biol. Chem. 270: 29330-29335).

Catechol-O-methyltransferase Catechol-O-methyltransferase (COMT) provides S-adenosyl-L-methionine (AdoMet; SAM) to one hydroxyl group of a catechol substrate (eg, levodopa, dopamine, or DBA) Catalyze the transfer of methyl groups in the body. Methylation of the 3′-hydroxyl group takes precedence over methylation of the 4′-hydroxyl group, and the membrane-bound isoform of COMT is more site specific than the soluble form. Translation of the soluble form of this enzyme is either by using the internal start codon of the full-length mRNA (1.5 kb) or by translation of a short mRNA (1.3 kb) transcribed from an internal promoter. The proposed S _N 2 -like methylation reaction requires Mg ^{2+ and} is suppressed by Ca ²⁺ . Binding of donor and substrate to COMT occurs sequentially. First, AdoMet is bonded to Mg ²⁺ independent manner COMT, binding and the binding of catechol substrate Mg ²⁺ is followed.

Inhibition is difficult because the amount of COMT in the tissue is greater than the amount normally required for activity. However, the inhibitor group has in vitro use (eg, gallic acid, tropolone, U-0521, and 3 ′, 4′-dihydroxy-2-methyl-propiofetropolone) and clinical use (eg, nitrocatechol compounds and tolcapone). Administration of these inhibitors increases the half-life of levodopa, followed by production of dopamine. In addition, inhibition of COMT may increase the half-life of various other catechol-structured compounds such as, but not limited to, epinephrine / norepinephrine, isoprenaline, limiterol, dobutamine, fenoldopam, apomorphine, and α-methyldopa. Since deficiency of norepinephrine leads to clinical depression, the use of COMT inhibitors may be useful in the treatment of depression. COMT inhibitors are usually well tolerated and have minimal side effects, eventually being metabolized in the liver, leaving only a few metabolites in the body (Mannisto, PT and Kaakkola, S. (1999) Pharmacological Reviews. 51: 593-628).

Copper-zinc superoxide dismutase Copper-zinc superoxide dismutase is a compact homodimeric metalloenzyme that is involved in cellular defense against oxidative damage. This enzyme has one zinc atom and one copper atom in each subunit and catalyzes the disproportionation reaction of superoxide anion to O ₂ and H ₂ O ₂ . The rate of this disproportionation reaction is diffusion limited and is increased by the presence of favorable electrostatic interactions between the substrate and the enzyme active site. Several examples of this class of enzymes have all been identified in the cytoplasm of eukaryotic cells and have also been identified in the periplasm of several bacterial species. Copper-zinc superoxide dismutases are robust enzymes and are highly resistant to proteolytic digestion and denaturation by urea and SDS. In addition to the compact structure of these enzymes, metal ions and intrasubunit disulfide bonds are believed to contribute to the enzyme's stability. These enzymes are reversibly denatured even at temperatures as high as 70 ° C. (Battistoni, A. et al. (1998) J. Biol. Chem. 273: 5655-5661).

  Overexpression of superoxide dismutase is involved in increasing the freezing tolerance of genetically engineered alfalfa and may also confer resistance to environmental toxins such as the diphenyl ether herbicide acifluorfen (McKersie, BD et al. ( 1993) Plant Physiol. 103: 1155-1163). In addition, yeast cells become more resistant to freeze-thaw damage after exposure to hydrogen peroxide. This is because up-regulation of superoxide dismutase expression upon exposure allows the yeast cells to adapt to further peroxidative stress. In this study, mutations in the yeast superoxide dismutase gene cluster affect the regulation of glutathione metabolism that has long been thought to be important in determining whether an organism survives through a cryopreservation process. More adversely affected freeze-thaw resistance than mutation (Jong-In Park, J.-I. et al. (1998) J. Biol. Chem. 273: 22921-22928).

Superoxide dismutase expression is also associated with Mycobacterium tuberculosis, the organism that causes tuberculosis. Superoxide dismutase is one of 10 major proteins secreted by Mycobacterium tuberculosis, and its expression is upregulated about 5-fold in response to oxidative stress. M. tuberculosis expresses superoxide dismutase almost two orders of magnitude more than the non-pathogenic mycobacteria M. smegmatis and secretes the expressed enzyme at a much higher rate. As a result, M. tuberculosis secretes up to 350 times more enzyme than S. aureus, conferring substantial resistance to oxidative stress (Harth, G. and Horwitz, MA (1999) J. Biol. Chem. 274: 4281). -4292).

  Reduced expression of copper-zinc superoxide dismutase, as well as decreased expression of other enzymes with antioxidant capacity, may be associated with early cancers. The expression level of copper-zinc superoxide dismutase is lower in prostate intraepithelial neoplasia and prostate cancer compared to normal prostate tissue (Bostwick, D. G. (2000) Cancer 89: 123-134).

Phosphodiesterases Phosphodiesterases constitute a class of enzymes that catalyze the hydrolysis of one of the two ester bonds of a phosphodiester compound. Thus, phosphodiesterases are important for various cellular processes. Phosphodiesterases include DNA and RNA endonucleases and exonucleases that are essential for cell growth and replication, and topoisomerases that degrade and reform nucleic acid strands during DNA topology rearrangement. Certain Tyr-DNA phosphodiesterases act on DNA repair by hydrolyzing a dead-end covalent intermediate formed between topoisomerase type I and DNA (Pouliot, JJ et al. (1999) Science 286: 552). Yang, S.-W. (1996) Proc. Natl. Acad. Sci. USA 93: 11534-11539).

  Acidic sphingomyelinase is a phosphodiesterase that hydrolyzes the membrane phospholipid sphingomyelin to produce ceramide and phosphorylcholine. Phosphorylcholine is used for the synthesis of phosphatidylcholine involved in various intracellular signal transduction pathways, while ceramide is an essential precursor for the production of ganglioside, a membrane lipid found in high concentrations in nerve tissue. The deficiency of acid sphingomyelinase accumulates sphingomyelin molecules in lysosomes, thereby causing Niemann-Pick disease (Schuchman, E. H. and S. R. Miranda (1997) Genet. Test. 1: 13-19).

  Glycerophosphoryl diester phosphodiesterase (also called glycerophosphodiester phosphodiesterase) hydrolyzes deacetylated phospholipid glycerophosphodiesters to produce sn-glycerol-3-phosphate and alcohol It is a phosphodiesterase. Glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, and glycerophosphoinositol are examples of substrates for glycerophosphoryl diester phosphodiesterase. Certain glycerophosphoryl diester phosphodiesterases from E. coli have a wide range of specificities for glycerophosphodiester substrates (Larson, T. J. et al. (1983) J. Biol. Chem. 248: 5428-5432).

  Cyclic nucleotide phosphodiesterase (PDE) is an extremely important enzyme for the regulation of cyclic nucleotides cAMP and cGMP. cAMP and cGMP function as intracellular second messengers that transmit various extracellular signals such as hormones, light and neurotransmitters. PDE breaks down cyclic nucleotides into their corresponding monophosphates, thereby regulating their intracellular concentrations and their effects on signaling. Because their role is a regulator of signaling, PDE has been extensively studied as a target for chemotherapy (Perry, MJ and GA Higgs (1998) Curr. Opin. Chem. Biol. 2: 472-481). Torphy, JT (1998) Am. J. Resp. Crit. Care Med. 157: 351-370).

  The mammalian PDE family is classified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitors (Beavo, JA (1995) Physiol. Rev. 75: 725-748; Conti, M (1995) Endocrine Rev. 16: 370-389). Some of these families have unique genes, many of which are expressed as splice variants in various tissues. Within the PDE family there are numerous isoenzymes and numerous splice variants of these isozymes (Conti, M. and S.-LC Jin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63 : 1-38). The presence of numerous PDE families, isoenzymes, and splice variants indicates the diversity and complexity of regulatory pathways involving cyclic nucleotides (Houslay, MD and G. Milligan (1997) Trends Biochem. Sci. 22: 217-224).

PDE1 type (PDE1) is Ca ²⁺ / calmodulin dependent and appears to be encoded by at least three different genes, each with at least two different splice variants (Kakkar, R. et al. 1999) Cell Mol. Life Sci. 55: 1164-1186). PDE1 was found in the lung, heart, and brain. Several PDE1 isoenzymes are regulated by phosphorylation / dephosphorylation in vitro . Phosphorylation of these PDE1 isoenzymes reduces the affinity of this enzyme for calmodulin, reduces PDE activity, and increases the steady state level of cAMP (Kakkar, supra). Since PDE1 is involved in both cyclic nucleotide and calcium signaling, it may provide a useful therapeutic target for disorders of the central nervous system and cardiovascular, immune system (Perry, MJ and GA Higgs (1998) Curr. Opin. Chem. Biol. 2: 472-481).

  PDE2 is a cGMP-stimulated PDE present in the cerebellum, neocortex, heart, kidney, lung, pulmonary artery and skeletal muscle (Sadhu, K. et al. (1999) J. Histochem. Cytochem. 47: 895-906). PDE2 mediates the action of cAMP in catecholamine secretion, is involved in the regulation of aldosterone (Beavo, supra), and is thought to play a role in olfactory signaling (Juilfs, DM et al. (1997) Proc. Natl. Acad. Sci. USA 94: 3388-3395).

  Because PDE3 has a high affinity for both cGMP and cAMP, these cyclic nucleotides act as competitive substrates for PDE3. PDE3 acts to stimulate myocardial contractility, inhibit platelet aggregation, relax vascular and airway smooth muscle, inhibit proliferation of cultured T lymphocytes and vascular smooth muscle cells, and regulate catecholamine-induced free fatty acid release from adipose tissue To do. The PDE3 family of phosphodiesterases is sensitive to specific inhibitors such as cilostamide, enoximone, and lixazinone. PDE3 isoenzymes can be inhibited by cAMP-dependent protein kinases or insulin-dependent kinases (Degerman, E. et al. (1997) J. Biol. Chem. 272: 6823-6826).

  PDE4 is specific for cAMP, is localized to airway smooth muscle, vascular epithelium, and all inflammatory cells and can be activated by cAMP-dependent phosphorylation. PDE4 is widely used as a potential target for new anti-inflammatory agents with an emphasis on the discovery of asthma treatment, as elevated levels of cAMP can suppress inflammatory cell activation and relax bronchial smooth muscle Researched. PDE4 inhibitors are currently undergoing clinical trials for the treatment of asthma, chronic obstructive pulmonary disease, and atopic eczema. All four known isoenzymes of PDE4 are sensitive to the inhibitor rolipram, a compound that 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 potential therapeutic agents for acute lung injury, endotoxemia, rheumatoid arthritis, multiple sclerosis, and various neurological and gastrointestinal diseases (Doherty, AM (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 allosteric cGMP specific binding sites (McAllister-Lucas, LM et al. (1995) J. Biol. Chem. 270: 30671-30679). The binding of cGMP to these allosteric binding sites appears to be more important for phosphorylation of PDE5 by cGMP-dependent protein kinase than direct regulation of catalytic activity. High levels of PDE5 are found in vascular smooth muscle, platelets, lungs, and kidneys. The inhibitor zaprinast is effective against PDE5 and PDE1. Sildenafil was obtained by modifying zaprinast to obtain specificity for PDE5 (VIAGRA; Pfizer, Inc., New York NY). This sildenafil is a treatment for male erectile dysfunction (Terrett, N. et al. (1996) Bioorg. Med. Chem. Lett. 6: 1819-1824). Inhibitors of PDE5 are currently being studied as cardiovascular therapeutics (Perry, M. J. and G. A. Higgs (1998) Curr. Opin. Chem. Biol. 2: 472-481).

  PDEs, which are photoreceptor cyclic nucleotide phosphodiesterases, are important elements of the phototransduction cascade. PDE6 binds to the G protein transducin and hydrolyzes cGMP to regulate cGMP-gated cation channels in the photoreceptor membrane. In addition to the cGMP binding active site, PDE6 also has two high affinity cGMP binding sites that are thought to play a regulatory role in the function of PDE6 (Artemyev, NO et al. (1998) Methods 14: 93-104). . PDE6 deficiency is associated with retinal disease. rd mouse retinal degeneration (Yan, W. et al. (1998) Invest. Opthalmol. Vis. Sci. 39: 2529-2536), human autosomal recessive retinitis pigmentosa (pigment retinitis) (Danciger, M (1995) Genomics 30: 1-7) and Irish setter dog rod / cone dysplasia type 1 (Suber, ML et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3968 -3972) is due to a mutation in the PDE6B gene.

  The PDE7 family of PDEs consists of only one known member with multiple splice variants (Bloom, T. J. and J. A. Beavo (1996) Proc. Natl. Acad. Sci. USA 93: 14188-14192). PDE7 is cAMP-specific, but little is known about other physiological functions. Although mRNA encoding PDE7 is found in skeletal muscle, heart, brain, lung, kidney, and pancreas, expression of PDE7 protein is restricted to specific tissue types (Han, P. et al. (1997) J. Biol. Chem. 272: 16152-16157; Perry, MJ and GA Higgs (1998) Curr. Opin. Chem. Biol. 2: 472-481). PDE7 is closely related to the PDE4 family but is not inhibited by rolipram, a specific inhibitor of PDE4 (Beavo, supra).

  PDE8s are cAMP specific and closely related to the PDE4 family. PDE8 is expressed in the thyroid, testis, eye, liver, skeletal muscle, heart, kidney, ovary, and brain. PDE8 cAMP hydrolysis activity is not inhibited by PDE inhibitors rolipram, vinpocetine, milrinone, IBMX (3-isobutyl-1-methylxanthine), or zaprinast, but PDE8 is inhibited by dipyridamole (Fisher, DA et al. (1998) Biochem. Biophys. Res. Commun. 246: 570-577; Hayashi, M. et al. (1998) Biochem. Biophys. Res. Commun. 250: 751-756; Soderling, SH et al. (1998) Proc. Natl. Acad. Sci. USA 95: 8991-8996).

  PDE9s are cGMP specific and most similar to the PDE8 family of PDEs. PDE9 is expressed in the kidney, liver, lung, brain, spleen, and small intestine. PDE9 is not inhibited by sildenafil (VIAGRA; Pfizer, Inc., New York NY), rolipram, vinpocetine, dipyridamole, or IBMX (3-isobutyl-1-methylxanthine) but is sensitive to the PDE5 inhibitor zaprinast (Fisher, DA et al. (1998) J. Biol. Chem. 273: 15559-15564; Soderling, SH et al. (1998) J. Biol. Chem. 273: 15553-15558).

  PDEs are dual substrate PDEs that hydrolyze both cAMP and cGMP. PDE10 is expressed in the brain, thyroid and testis (Soderling, SH 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).

  PDE contains a catalytic domain of about 270-300 amino acids and an N-terminal regulatory domain responsible for binding cofactors, and in some cases, a hydrophilic C-terminal domain of unknown function (Conti, M. and S.- LC Jin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63: 1-38). A putative zinc binding motif conserved in the catalytic domain of all PDEs was identified. N-terminal regulatory domains include non-catalytic cGMP binding domains in PDE2, PDE5, and PDE6, calmodulin binding domains in PDE1, and domains with phosphorylation sites in PDE3 and PDE4. In PDE5, the N-terminal cGMP-binding domain spans about 380 amino acid residues and contains a tandem repeat of a conserved sequence motif (McAllister-Lucas, LM et al. (1993) J. Biol. Chem. 268 : 22863-22873). This NKXnD motif has been shown to be important for cGMP binding by mutagenesis (Turko, I. V. et al. (1996) J. Biol. Chem. 271: 22240-22244). The PDE family has about 30% amino acid identity within the catalytic domain, but isoenzymes within the same family usually show about 85 to 95% identity in this region (eg, PDE4A and PDE4B). Furthermore, while the similarity outside the catalytic domain within a family is high (greater than 60%), there is little sequence similarity outside this domain between families.

Many of the functions that make up immune and inflammatory responses are inhibited by agents that increase the level of intracellular cAMP (Verghese, MW et al. (1995) Mol. Pharmacol. 47: 1164-1171). Various diseases are caused by increased PDE activity and are associated with decreased levels of cyclic nucleotides. For example, the certain type of diabetes insipidus in the mouse related to the increase in PDE4 activity, elevation of low K _m cAMP PDE activity was observed in leukocytes atopic patients, PDE3 is related to heart disease.

Many inhibitors of PDEs have been identified and clinically tested (Perry, MJ and GA Higgs (1998) Curr. Opin. Chem. Biol. 2: 472-481; Torphy, TJ (1998) Am. J Respir. Crit. Care Med. 157: 351-370). PDE3 inhibitors are under development as antithrombotics, antihypertensives and cardiotonic drugs useful for the treatment of congestive heart failure. Rolipram, a PDE4 inhibitor, is used to treat depression, and other inhibitors of PDE4 are being evaluated as anti-inflammatory drugs. Rolipram was also found to inhibit lipopolysaccharide (LPS) -induced TNF-α, which was shown to enhance HIV-1 replication in vitro . Thus, rolipram appears to inhibit HIV-1 replication (Angel, JB et al. (1995) AIDS 9: 1137-1144). Furthermore, rolipram has been shown to be effective in the treatment of encephalomyelitis based on its ability to suppress the production of cytokines such as TNF-α, TNF-β, and interferon γ. Rolipram was also considered effective against tardive dyskinesia and was effective in the treatment of multiple sclerosis in experimental animal models (Sommer, N. et al. (1995) Nat. Med. 1: 244-248; Sasaki, H. et al. (1995) Eur. J. Pharmacol. 282: 71-76).

  Theophylline is a non-specific PDE inhibitor used to treat bronchial asthma and other respiratory diseases. Theophylline acts on airway smooth muscle function and is thought to have anti-inflammatory or immunomodulatory capacity in the treatment of respiratory diseases (Banner, KH and CP Page (1995) Eur. Respir. J. 8: 996- 1000). Pentoxifylline is another non-specific PDE inhibitor used to treat intermittent claudication and diabetic peripheral vascular disease. Pentoxifylline is also known to block the production of TNF-α and can inhibit HIV-1 replication (Angel et al., Supra).

  PDEs have been reported to affect cell proliferation of various cell types (Conti et al. (1995) Endocrine Rev. 16: 370-389) and are thought to be involved in various cancers. Growth of prostate cancer cell lines DU145 and LNCaP was suppressed by delivery of cAMP derivatives and PDE inhibitors (Bang, Y. J. et al. (1994) Proc. Natl. Acad. Sci. USA 91: 5330-5334). These cells also showed a permanent transformation in phenotype from epithelium to neuronal morphology. PDE inhibitors may also regulate mesangial cell proliferation (Matousovic, K. et al. (1995) J. Clin. Invest. 96: 401-410) and may also regulate lymphocyte proliferation (Joulain, C. et al. (1995) J. Lipid Mediat. Cell Signal. 11: 63-79). Certain cancer treatments have been described as involving processes that deliver PDE to specific cellular compartments of the tumor and lead to cell death (Deonarain, MP and AA Epenetos (1994) Br. J. Cancer 70: 786-794). ).

Phosphotriesterase phosphotriesterase (PTE, paraoxonase) enzymes such hydrolyze toxic organophosphorus compounds, PTE have been isolated from various tissues. PTE enzymes appear to be abundant in mammals but not in birds and insects, accounting for the low resistance of birds and insects to organophosphorus compounds (Vilanova, E. and Sogorb, MA ( 1999) Crit. Rev. Toxicol. 29: 21-57). Phosphotriesterase plays a central role in the detoxification of insecticides by mammals. Phosphotriesterase activity varies from individual to individual and is lower in infants than in adults. PTE knockout mice have significant susceptibility to the organophosphorus toxins diazoxon and chlorpyrifos oxon (Furlong, CE, et al. (2000) Neurotoxicology 21: 91-100). PTE is attracting attention as an enzyme with the ability to detoxify organophosphorus-containing chemical wastes and chemical weapons (eg, parathion) and pesticides and pesticides. Several studies have shown that phosphotriesterase is involved in atherosclerosis and multiple diseases related to lipoprotein metabolism.

Two soluble thioesterases involved in thioesterase fatty acid biosynthesis were isolated from mammalian tissue. One is active only for long chain fatty acyl thioesters and the other is active for thioesters having various lengths of fatty acyl chains. These thioesterases catalyze the termination step of the fatty acid chain in the novel biosynthesis of fatty acids. The termination step of the fatty acid chain involves hydrolysis of the thioester bond that binds the fatty acid acyl chain to the 4'-phosphopantethein prosthetic group of the acyl carrier protein (ACP) subunit of fatty acid synthase (Smith S, (1981a) Methods Enzymol. 71: 181-188; Smith, S. (1981b) Methods Enzymol. 71: 188-200).

  E. coli has two soluble thioesterases, thioesterase type I, which is active only for long chain acylthioesters, and thioesterase type II (TEII), which has specificity for various length chains (Naggert J. Biol. Chem. 266: 11044-11050). E. coli TEII has no sequence similarity with either of the two types of mammalian thioesterases that function as chain-terminating enzymes in novel fatty acid biosynthesis. Unlike mammalian thioesterase, E. coli TEII lacks the characteristic serine active site gly-X-ser-X-gly sequence motif and is not inactivated by the serine denaturing agent diisopropylfluorophosphate. However, modification of histidine 58 with iodoacetamide and diethyl pyrocarbonate results in loss of TEII activity. Overexpression of TEII does not change the fatty acids contained in E. coli. This indicates that it does not function as a fatty acid chain terminating enzyme in fatty acid biosynthesis (Naggert et al., Supra). For this reason, according to Naggert et al. (Supra), the physiological substrate of E. coli TEII is not ACP-phosphopantethein fatty acid ester but coenzyme A (CoA) fatty acid ester.

Carboxylesterases Mammalian carboxylesterases constitute a multigene family that is expressed in various tissues and cell types. Isozymes have significant sequence homology and are classified primarily based on amino acid sequence. Acetylcholinesterase, butyrylcholinesterase, and carboxylesterase are grouped into the serine superfamily of esterases (B-esterases). Other carboxylesterases include thyroglobulin, thrombin, factor IX, gliotactin, and plasminogen. Carboxylesterases catalyze the hydrolysis of the ester and amide groups of the molecule and are involved in the detoxification of drugs, environmental toxins, and carcinogens. As the substrate of carboxylesterase, short chain and long chain acylglycerol, acylcarnitine, carbonic acid, dipivefrin hydrochloride, cocaine, salicylic acid, capsaicin, palmitoyl-CoA, imidapril, haloperidol, pyrrolizidine alkaloid, steroid, p-nitro Phenylacetic acid, malathion, butanilicaine, and isocarboxazide are included. This enzyme often exhibits low substrate specificity. Carboxyl esterases are also important for converting prodrugs to the corresponding free acids. The corresponding free acid may be an active form of its prodrug, such as lovastatin used to lower blood cholesterol (reviewed in Satoh, T. and Hosokawa, M. (1998) Annu. Rev. Pharmacol. Toxicol. 38: 257-288).

  Neuroligins (i) have an N-terminal signal sequence, (ii) are similar to cell surface receptors, (iii) have a carboxylesterase domain, (iv) are highly expressed in the brain, (v) A class of molecules that binds to neulexins in a calcium-dependent manner. Although homologous to carboxylesterases, neuroligins lack active site serine residues and these are thought to play 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 initial oxygenation step in the sterol biosynthesis pathway of eukaryotic cells. Cholesterol is an essential component of the cytoplasmic membrane acquired by the LDL receptor-mediated pathway or the biosynthetic pathway. In biosynthesis, all 27 carbon atoms in the cholesterol molecule are derived from acetyl-CoA (Stryer, L., supra). SE converts squalene to 2,3 (S) -oxide squalene, which is then converted to lanosterol and further to cholesterol. The steps involved in cholesterol biosynthesis are summarized below (Stryer, L (1988) Biochemistry . WH Freeman and Co., Inc. New York. 554-560, and Sakakibara, J. et al. (1995) 270: 17-20. ). Acetate (derived from acetyl-CoA) → 3hydroxy-3-methyl-glutaryl CoA → mevalonic acid → 5-phosphomevalonic acid → 5-pyrophosphomevalonic acid → isopentenyl pyrophosphate → dimethylallyl pyrophosphate → geranyl pyrophosphate → farnesyl pyrophosphate → Squalene → Squalene epoxide → Lanosterol → Cholesterol.

  Cholesterol is essential for eukaryotic cell viability, but excessive levels of serum cholesterol result in the formation of atheroma plaques in higher organism arteries. For example, the deposition of highly insoluble lipids on the walls of essential blood vessels such as coronary arteries can reduce blood flow and prevent sufficient blood from flowing into the tissue, leading to tissue necrosis. HMG-CoA reductase is responsible for the conversion of 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) to mevalonic acid, which is the first step in cholesterol biosynthesis. HMG-CoA is the target of various pharmaceutical compounds designed to reduce plasma cholesterol levels. However, inhibition of HMG-CoA also reduces the synthesis of non-sterol intermediates (eg, mevalonic acid) required for other biochemical pathways. SE catalyzes the rate-limiting reaction that occurs late in the sterol synthesis pathway, and cholesterol is the only end product of this pathway following the catalytic step by SE. Thus, SE is an ideal target for designing antihyperlipidemic drugs that do not reduce other required intermediates (Nakamura, Y. et al. (1996) 271: 8053-8056).

Epoxide hydrolase Epoxide hydrolase (hydrolase) catalyzes the addition of water to an epoxide-containing compound, whereby the epoxide is hydrolyzed to its corresponding 1,2-diol. These are closely related to bacterial haloalkane dehalogenases, and other members of the α / β hydrolase fold family of enzymes (eg, Streptomyces aureofaciens bromoperoxidase A2), Pseudomonas putida hydroxymucone Sequence similarity is shown to hydroxymuconic semialdehyde hydrolases and haloalkane dehalogenase from Xanthobacter autotrophicus . Epoxide hydrolases are ubiquitous in nature and are present in mammals, invertebrates, plants, fungi and bacteria. This family of enzymes is important for detoxification of xenobiotic epoxide compounds, which are often electrophilic and often destructive when introduced into living organisms. Examples of epoxide hydrolase reactions include cis-9,10-epoxyoctadec-9 (Z) -enoic acid (leukotoxin), its corresponding diol, threo-9,10-dihydroxyoctadec-12 (Z) -enoic acid Hydrolysis to form (leucotoxin diol) and its corresponding diol from cis-12,13-epoxyoctadec-9 (Z) -enoic acid (isoleukotoxin), threo-12,13-dihydroxyoctadec-9 ) -enoic acid (isoleukotoxindiol) hydrolysis. Leukotoxin alters membrane permeability and ion transport, causing an inflammatory response. In addition, epoxide carcinogens are known to be produced by cytochrome P450 as an intermediate in the detoxification of drugs and environmental toxins.

  These enzymes have a catalytic triad consisting of Asp (nucleophilic), Asp (acid that supports histidine) and His (water-activated histidine). The reaction mechanism of an epoxide hydrolase proceeds through a covalent ester intermediate that is initiated by one nucleophilic attack of the Asp residue on the first carbon atom of the target molecule's epoxide ring to form a covalent ester intermediate. (Arand, M. et al. (1996) J. Biol. Chem. 271: 4223-4229; Rink, R. et al. (1997) J. Biol. Chem. 272: 14650-14657; Argiriadi, MA et al. (2000) J. Biol. Chem. 275: 15265-15270).

Degradation of tyrosine, an amino acid, into the enzymes succinic acid and pyruvic acid or fumaric acid and acetoacetic acid involved in tyrosine catalysis requires a large number of enzymes, and a large number of intermediate compounds are produced. In addition, many xenobiotic compounds can be metabolized using one or more reactions that are part of the tyrosine catabolism pathway. Although this pathway has been studied primarily in bacteria, tyrosine degradation is known to occur in a variety of organisms and appears to involve a number of similar biological responses.

Enzymes involved in the degradation of tyrosine to succinate and pyruvate (for example Arthrobacter species) include 4-hydroxyphenylpyruvate oxidase, 4-hydroxyphenylacetate 3-hydroxylase (4-hydroxyphenylacetate 3 -hydroxylase), 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase (5-carboxymethyl-2- hydroxymuconic semialdehyde dehydrogenase), trans, cis-5-carboxymethyl-2-hydroxymuconate isomerase, homoprotocatechuate isomerase / decarboxylase , Cis-2-oxohept-3-ene-1,7-dioate hydratase, 2,4-dihydroxyhept-trans-2-ene-1,7- dioate aldolase, and succinic semialdehyde dehydrogenase.

  Enzymes involved in the degradation of tyrosine into fumaric acid and acetoacetate (eg, Pseudomonas) include 4-hydroxyphenylpyruvate dioxygenase, homogentisic acid-1,2-dioxygenase, maleyl acetoacetate isomerase, and fumaryl aceto Acetase is included. If an intermediate from the succinate / pyruvate pathway is accepted, 4-hydroxyphenylacetate 1-hydroxylase may be involved.

  Examples of further enzymes involved in tyrosine metabolism in various organisms include 4-chlorophenylacetate-3,4-dioxygenase, aromatic aminotransferase, 5-oxopent-3- ene-1,2,5-tricarboxylate decarboxylase, 2-oxo-hept-3-ene-1,7-dioate hydratase, and 5-carboxymethyl-2-hydroxymuconate isomerase (Ellis, LBM et al. (1999) Nucleic Acids Res. 27: 373-376; Wackett, LP and Ellis, LBM (1996) J. Microbiol. Meth. 25: 91-93; and Schmidt, M. (1996 ) Amer. Soc. Microbiol. News 62: 102).

  In humans, an acquired or congenital genetic defect in an enzyme in the tyrosine degradation pathway can cause hereditary tyrosineemia. One type of this disease, hereditary tyrosinemia type I (HT1), is caused by a deficiency in fumaryl acetoacetate hydrolase, the final enzyme in the pathway in organisms that metabolize tyrosine to fumarate and acetoacetate. HT1 is characterized by progressive liver injury beginning in early childhood and increases the risk of liver cancer (Endo, F. et al. (1997) J. Biol. Chem. 272: 24426-24432).

Use of microarrays to detect and diagnose diseases associated with drug-metabolizing enzymes Microarray technology provides a simple way to explore the expression of a single polymorphic gene or the expression profile of many related or unrelated genes. obtain. When testing the expression of a single gene, the expression of a particular gene or variant thereof is detected using a microarray. When testing expression profiles, microarrays provide a platform for testing: That is, which genes are tissue-specific, perform housekeeping functions, are part of a signaling cascade, or genes that are specifically associated with a particular genetic predisposition, condition, disease, or disorder It is a test of whether there is.
Potential applications of gene expression profile generation are particularly relevant for disease diagnosis, prognosis, and treatment improvement. For example, both levels and sequences expressed in tissues from colon cancer patients can be compared to levels and sequences expressed in normal tissues.

  For example, colorectal cancer is the fourth most common cancer in the United States and the second most common cause of cancer deaths, with approximately 130,000 new cases and 55,000 deaths each year To do. Colon cancer and rectal cancer share many environmental risk factors, both found in people with unique genetic syndromes (Potter, JD (1999) J Natl Cancer Institute 91 : 916-932). Colon cancer is the only cancer with approximately equal frequency in men and women. In the United States, the survival rate after diagnosis of colon cancer is about 55% (Ries et al. (1990) National Institutes of Health, DHHS Publ No. (NIH) 90-2789).

  Colon cancer is causally related to both genes and the environment. Several molecular pathways are associated with the development of colon cancer, and the expression of key genes in either of these pathways can be lost by congenital or acquired mutations or hypermethylation. There is a particular need to identify genes whose changes in expression can be an early indicator of colon cancer or a predisposition to developing colon cancer.

  It is well known that an unusual pattern of DNA methylation occurs consistently in human tumors and includes a wide range of hypomethylated portions of the genome and portions of locally increased methylation at the same time. Particularly in colon cancer, it is known that these changes occur early in tumor progression, such as precancerous polyps that precede colon cancer. Indeed, DNA methyltransferase, an enzyme that performs DNA methylation, is markedly increased in histologically normal mucosa of patients with benign polyps that precede colon cancer or cancer. This increase then continues during the progression of colon tumors (Wafik et al. (1991) Proc Natl Acad Sci USA 88: 3470-3474). Increased DNA methylation occurs in G + C-rich regions of genomic DNA called “CpG islands”, which are important for maintaining the “open” transcriptional structure around the gene, these This region results in a “closed” structure that stops gene transcription. It has been suggested that differentiation of immortalized cells can be prevented by quiescence or down-regulation of differentiation genes by such abnormal methylation of CpG islands (Anteguera, F. et al. (1990) Cell 62: 503-514).

  Familial colorectal adenomatosis (FAP) is a rare autosomal dominant syndrome that precedes colorectal cancer and is caused by a congenital mutation in the colorectal adenomatous polyposis (APC) gene. FAP is characterized by early onset of multiple colorectal adenomas that progress to cancer at an average age of 44 years. The APC gene is part of the APC-β-catenin-Tcf (T-cell factor) pathway. Failure of this pathway results in loss of regular replication, attachment, and migration of colonic epithelial cells, leading to polyp growth. After the APC-β-catenin-Tcf pathway is activated, a series of other genetic changes follow, with the transition from normal colonic mucosa to metastatic cancer. These changes include mutations in the K-Ras proto-oncogene, changes in methylation patterns, and mutations or loss of the tumor suppressor genes p53 and Smad4 / DPC4. Inheritance of a mutated APC gene is a rare phenomenon, but loss or mutation of APC and the resulting effects on the APC-β-catenin-Tcf pathway are central to most colorectal cancer patients in the general population. It is considered to be the target.

  Hereditary nonpolyposis colorectal cancer (HNPCC) is another congenital autosomal dominant syndrome with a phenotype that is not as well defined as FAP. HNPCC, which accounts for about 2% of colorectal cancer cases, is distinguished by the tendency of early onset of cancer and the onset of other cancers, particularly those related to the endometrium, urinary tract, stomach and biliary system. HNPCC arises from mutations in one or more genes in the DNA mismatch repair (MMR) pathway. Mutations of the two human MMR genes MSH2 and MLH1 are found in most of the HNPCC families identified so far. The DNA MMR pathway identifies and repairs errors that result from the action of replicating DNA polymerases. In addition, loss of MMR activity prevents cancer progression by accumulating mutations and deletions in other genes such as the loss of the BAX gene that regulates apoptosis and the TGFβ receptor II gene that controls cell proliferation. Arise. In patients with DNA MMR deficiency, progression to cancer is more rapid than usual because of the irreparable damage to DNA.

  Although ulcerative enteritis is a minor cause for colorectal cancer, affected patients are about 20 times more likely to develop cancer. Progression is characterized by loss of the p53 gene that can appear early in histologically normal tissues. The progression of disease from ulcerative colitis to dysplasia / cancer without intermediate polyp status suggests that mutagenic activity resulting from exposure of proliferating cells to colon contents in the colonic mucosa is high. To do.

  Almost all colorectal cancers arise from cells in which the estrogen receptor (ER) gene is silenced. Inactivation of ER gene transcription is age related and associated with hypermethylation of the ER gene (Issa, J-P J et al. (1994) Nature Genetics 7: 536-540). By introducing an exogenous ER gene into cultured colon cancer cells, growth is markedly suppressed. A link between the loss of ER protein in colonic epithelial cells and the resulting cancer development has not been established.

It is clear that there are a number of genetic alterations associated with colorectal cancer and associated with the development and progression of the disease, particularly with respect to gene down-regulation or deletion, and these alterations provide an early indicator of cancer development. Can be used to monitor disease progression or to provide promising therapeutic targets. The specific group of genes that has affected certain cases of colorectal cancer depends on the molecular progression of the disease. By identifying additional genes associated with colorectal cancer and precancerous conditions, a more reliable diagnostic pattern associated with the onset and progression of the disease will be obtained.
The discovery of novel drug metabolizing enzymes and polynucleotides encoding them can meet the needs of the art by providing new compositions. This novel composition is useful in the diagnosis, treatment and prevention of gastrointestinal diseases including autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine disorders, ophthalmic disorders, metabolic disorders, and liver disorders. They are also useful for evaluating the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of drug metabolizing enzymes.

  The present invention is generically referred to as “DME” and individually “DME-1”, “DME-2”, “DME-3”, “DME-4”, “DME-5”, “DME-6”. Purified polypeptides that are drug metabolizing enzymes, referred to as "DME-7", "DME-8", "DME-9", "DME-10", "DME-11", and "DME-12" I will provide a. In certain embodiments, the invention provides: (a) a polypeptide consisting of an amino acid sequence selected from the group having SEQ ID NO: 1-12; (b) an amino acid sequence selected from the group having SEQ ID NO: 1-12 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-12, and (d) ) Provided is an isolated polypeptide selected from the group comprising an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-12. In one embodiment, an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-12 is provided.

  The present invention also relates to (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 Or (d) an immunogenic fragment of a polynucleotide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, which encodes a certain polypeptide selected from the group consisting of A polynucleotide is provided. In one embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-12. In another embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO: 13-24.

  The invention further comprises (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, (b) at least 90% of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (d) SEQ ID NO: A recombinant polynucleotide having a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of immunogenic fragments of a polypeptide having an amino acid sequence selected from the group consisting of 1-12 provide. In one embodiment, the present invention provides a cell transformed with a recombinant polynucleotide. In another embodiment, the present invention provides a genetic transformant comprising a recombinant polynucleotide.

  Furthermore, the present invention provides (a) a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12; A polypeptide comprising a natural amino acid sequence having 90% or more identity, and (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, and a method for producing a polypeptide selected from the group consisting of: The production method comprises the steps of (a) culturing cells transformed with the recombinant polynucleotide under conditions suitable for polypeptide expression, and (b) recovering the polypeptide so expressed. And the recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide.

  The invention further comprises (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, (b) at least 90% of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 A polypeptide comprising a native amino acid sequence that is identical, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (d) SEQ ID NO: 1 An isolated antibody is provided that specifically binds to a polypeptide selected from the group consisting of immunogenic fragments of a polypeptide having an amino acid sequence selected from the group consisting of -12.

  The invention further comprises (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24, (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24 and at least A polynucleotide comprising a naturally occurring polynucleotide sequence having 90% identity, (c) a polynucleotide sequence complementary to (a), (d) a polynucleotide sequence complementary to (b), and (e) ( Provided is an isolated polynucleotide selected from the group consisting of the RNA equivalents of a) to (d). In one embodiment, the polynucleotide has at least 60 contiguous nucleotides.

  The invention further provides a method of detecting a target polynucleotide in a sample. Wherein the target polynucleotide is (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24, (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24 A polynucleotide having a natural polynucleotide sequence with at least 90% identity to (c) a polynucleotide complementary to (a), (d) a polynucleotide complementary to (b), and (e) ( a) having a polynucleotide sequence selected from the group consisting of RNA equivalents of a) to (d). The detection method comprises: (a) hybridizing the sample with a probe consisting of at least 20 consecutive nucleotide groups consisting of a sequence complementary to the target polynucleotide in the sample; and (b) The process comprises detecting the presence or absence of the hybridization complex, and optionally detecting the amount of the complex if present. Under conditions such that a hybridization complex is formed between the probe and the target polynucleotide or fragment thereof, the probe specifically hybridizes to the target polynucleotide. In one embodiment, the probe comprises at least 60 contiguous nucleotides.

  The present invention also provides a method for detecting a target polynucleotide in a sample. Wherein the target polynucleotide is (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24, (b) a polynucleotide selected from the group consisting of SEQ ID NO: 13-24 A polynucleotide having a natural polynucleotide sequence having at least 90% identity to the sequence, (c) a polynucleotide complementary to the polynucleotide of (a), (d) a polynucleotide complementary to the polynucleotide of (b) It has the sequence of a polynucleotide selected from the group consisting of nucleotides and RNA equivalents of (e) (a)-(d). The detection method comprises: (a) a process of amplifying a target polynucleotide or fragment thereof using polymerase chain reaction amplification; and (b) detecting the presence / absence of the amplified target polynucleotide or fragment thereof, Optionally including detecting the amount of the polynucleotide or fragment thereof, if present.

  The present invention further provides a composition comprising an effective amount of a polypeptide and a pharmaceutically acceptable excipient. An effective amount of the polypeptide comprises (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 and at least A polypeptide comprising a natural amino acid sequence having 90% identity, (c) a biologically active fragment of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (d) SEQ Selected from the group consisting of immunogenic fragments of amino acid sequences selected from the group consisting of ID NO: 1-12. In one example, a composition comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 is provided. Furthermore, the present invention provides a method for treating diseases and symptoms associated with decreased expression of functional DME, comprising administering the composition to a patient.

  The invention also includes (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (d) In order to confirm the effectiveness as an agonist of a polypeptide selected from the group consisting of an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 Methods for screening compounds are provided. The screening method comprises (a) a process of exposing a sample having the polypeptide to a certain compound, and (b) a process of detecting agonist activity in the sample. Alternatively, the present invention provides a composition comprising an agonist compound identified by this method and a suitable pharmaceutical excipient. In yet another alternative, the present invention provides a method of administering the composition to a patient in need of treatment for a disease or condition associated with reduced expression of functional DME.

  The invention further includes (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, and (d) ) An immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, a method of screening a compound for the effectiveness as an antagonist of a polypeptide selected from the group consisting of provide. The screening method consists of (a) exposing a sample containing the polypeptide to a certain compound, and (b) detecting antagonistic activity in the sample. In one embodiment, the present invention provides a composition comprising an antagonist compound identified by this method and a pharmaceutically acceptable excipient. In yet another alternative, the present invention provides a method of administering the composition to a patient in need of treatment for a disease or condition associated with overexpression of functional DME.

  The invention further comprises (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, (b) at least 90% (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, or (d) SEQ ID NO A method is provided for screening for compounds that specifically bind to a polypeptide selected from the group comprising an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of: 1-12. The screening method comprises: (a) a process of mixing a polypeptide with at least one test compound under appropriate conditions; and (b) a compound that detects binding of the polypeptide to the test compound and thereby specifically binds to the polypeptide. Identifying the process.

  The invention further comprises (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, (b) at least 90% of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12, or (d) SEQ ID NO A method of screening for a compound that modulates the activity of a polypeptide selected from the group comprising an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of: 1-12. The screening method includes: (a) a step of mixing a polypeptide with at least one test compound under conditions where the activity of the polypeptide is allowed; and (b) a step of calculating the activity of the polypeptide in the presence of the test compound. And (c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, the change in the activity of the polypeptide in the presence of the test compound. Means a compound that modulates the activity of a polypeptide.

  The present invention further provides a method of screening for a compound effective to alter the expression of a target polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24, comprising: Exposing a sample containing the target polynucleotide to a compound; (b) detecting a change in expression of the target polynucleotide; and (c) the target polynucleotide in the presence of a variable amount of the compound. The screening method is provided comprising the step of comparing expression with expression in the absence of the compound.

  The invention further includes (a) treating a biological sample containing nucleic acid with a test compound, and (b) (i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24 , (Ii) a polynucleotide comprising a natural polynucleotide sequence having at least 90% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24, (iii) a sequence complementary to (i) (Iv) a polynucleotide complementary to the polynucleotide of (ii), (v) at least 20 consecutive nucleotides of a polynucleotide selected from the group consisting of RNA equivalents of (i) to (iv) A method for calculating the toxicity of a test compound comprising the step of hybridizing a nucleic acid of a treated biological sample with a probe comprising Hybridization occurs under conditions such that a specific hybridization complex is formed between the probe and a target polynucleotide in a biological sample, the target polynucleotide comprising: (i) SEQ ID NO: A polynucleotide comprising a polynucleotide sequence selected from the group consisting of 13-24, (ii) a natural polynucleotide sequence having at least 90% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24 (Iii) a polynucleotide having a sequence complementary to the polynucleotide of (i), (iv) a polynucleotide complementary to the polynucleotide of (ii), and (v) (i) to (iv) ) Selected from the group consisting of RNA equivalents. Alternatively, the target polynucleotide has a fragment of a polynucleotide sequence selected from the group consisting of (i) to (v) above. The toxicity calculation method further includes (c) the process of quantifying the amount of hybridization complex, and (d) the amount of hybridization complex of the treated biological sample, compared to the amount of hybridization of the untreated biological sample. A difference in the amount of hybridization complex in the treated biological sample, including the process of comparing to the amount of hybridization complex, indicates the toxicity of the test compound.

(Description of the present invention)
Before describing the proteins, nucleotide sequences and methods of the present invention, it is to be understood that the present invention is not limited to the particular devices, materials and methods described and may be modified. Also, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention which is limited only by the claims. Please also understand.

  As used in the claims and in the specification, the singular forms “a” and “its” may refer to the plural, unless the context clearly indicates otherwise. You have to be careful. Thus, for example, when “a certain host cell” is described, there may be a plurality of such host cells, and when “a certain antibody” is described, one or more antibodies, and References are also made to antibody equivalents known to those skilled in the art.

  All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined. Although any devices, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, suitable devices, materials, and methods are now described. All publications referred to in this invention are cited for purposes of describing and disclosing cell lines, protocols, reagents and vectors that have been reported in the publications and may be used in connection with the present invention. is there. No disclosure in this specification is an admission that the invention is not entitled to antedate such disclosure by virtue of prior art.

(Definition)
The term “DME” is substantially derived from all species (especially mammals including cattle, sheep, pigs, mice, horses and humans) from any source such as natural, synthetic, semi-synthetic or recombinant. Refers to the amino acid sequence of purified DME.

  The term “agonist” refers to a molecule that enhances or mimics the biological activity of DME. These agonists include proteins, nucleic acids, carbohydrates, small molecules, any other molecules that interact directly with DME or interact with components of biological pathways involving DME to regulate DME activity. It may contain compounds and compositions.

  The term “allelic variant (or variant sequence)” refers to another form of the gene encoding DME. Allelic variants can result from at least one mutation in the nucleic acid sequence. It can also result in altered mRNA or polypeptide. The structure or function of the polypeptide may or may not change. A gene may not have any of its native allelic variant sequences, or may have more than one. The usual mutational changes that give rise to allelic variants are generally attributed to natural deletions, additions or substitutions of nucleotides. These various changes can occur one or more times within a sequence, either alone or together with other changes.

  “Transformed / modified” nucleic acid sequences encoding DME include polypeptides having at least one of the same polypeptides or functional characteristics of DME as a result of various nucleotide deletions, insertions or substitutions Including sequences that yield This definition includes improper or unexpected hybridization to a group of allelic variants at a position that is not a normal chromosomal locus for a polynucleotide sequence encoding DME, and also includes a polynucleotide encoding DME. Polymorphisms that are easily detectable or difficult to detect using certain oligonucleotide probes. The encoded protein may also be “transformed / modified” and may have deletions, insertions or substitutions of amino acid residues that result in a silent change resulting in a functionally equivalent DME. . Intentional amino acid substitutions are in terms of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residue as long as the activity of DME is preserved biologically or immunologically. It can be made on the basis of similarity. For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine. Amino acids having uncharged polar side chains with similar hydrophilicity values include asparagine and glutamine, serine and threonine. Amino acids having uncharged side chains with similar hydrophilicity values include leucine, isoleucine and valine, glycine and alanine, phenylalanine and tyrosine.

  The term “amino acid” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide or protein sequence or fragments thereof, and refers to a natural or synthetic molecule. Where “amino acid sequence” refers to the sequence of a native protein molecule, “amino acid sequence” and similar terms are not intended to limit the amino acid sequence to the complete and intact amino acid sequence associated with the described protein molecule.

“Amplification” refers to the additional replication of a nucleic acid sequence. Amplification is typically performed using polymerase chain reaction (PCR) techniques well known to those skilled in the art.
The term “antagonist” is a molecule that inhibits or attenuates the biological activity of DME. Antagonists include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, any molecule that interacts directly with DME or acts on components of biological pathways involving DME to regulate DME activity. Other compounds and compositions can be included.

The term “antibody” refers to intact immunoglobulin molecules and fragments thereof, such as Fab, F (ab ′) ₂ , and Fv fragments, that are capable of binding epitope determinants. An antibody that binds to a DME polypeptide can be produced using an intact polypeptide group or a fragment group having the small peptide group as an immunizing antigen. The origin of the polypeptide or oligopeptide used to immunize an animal (mouse, rat, rabbit, etc.) may be in the case of RNA translation or chemical synthesis. They can also be conjugated to a carrier protein if desired. Commonly used carriers that chemically bond with peptides include bovine serum albumin, thyroglobulin, and mussel hemocyanin (KLH). This bound peptide is used to immunize the animal.

  The term “antigenic determinant” refers to a region of a molecule (ie, an epitope) that makes contact with a particular antibody. When immunizing a host animal with a protein or protein fragment, multiple regions of the protein can induce the production of antibodies that specifically bind to an antigenic determinant (a specific region or three-dimensional structure of the protein). Certain antigenic determinants can compete with intact antigens (ie, immunogens used to elicit an immune response) in binding to certain antibodies.

The term “aptamer” refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from in vitro evolution processes (for example, SELEX (abbreviation of Systematic Evolution of Ligands by EXponential Enrichment) described in US Pat. No. 5,270,163). Select target-specific aptamer sequences from the library. Aptamer compositions may be double-stranded or single-stranded and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide component of the aptamer can have modified sugar groups (e.g., the 2′-OH group of the ribonucleotide is replaced with 2′-F or 2′-NH ₂ ). Can improve desired properties, for example, resistance to nucleases or longer life in blood. In order to slow down the rate at which aptamers are removed from the circulatory system, aptamers can be conjugated to other molecules (eg, high molecular weight carriers). Aptamers can be specifically cross-linked with their homologous ligands, for example by photoactivation of certain cross-linking agents (see, eg, Brody, EN and L. Gold (2000) J. Biotechnol. 74: 5-13). ).

The term “intramer” means an aptamer expressed in vivo. For example, certain RNA expression systems based on vaccinia virus are used to highly express a specific RNA aptamer in the cytoplasm of leukocytes. (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96: 3606-3610).

  The term “spiegelmer” refers to aptamers comprising L-DNA, L-RNA or other levorotary nucleotide derivatives or nucleotide-like molecules. Aptamers containing levorotatory nucleotides are resistant to degradation by natural enzymes that normally act on substrates containing dextrorotatory nucleotides.

  The term “antisense” refers to any composition capable of base pairing with the “sense” (coding) strand of a particular nucleic acid sequence. Examples of antisense compositions include DNA, RNA, peptide nucleic acids (PNA), oligonucleotides having modified backbone linkages such as phosphorothioic acid, methylphosphonic acid or benzylphosphonic acid, modified sugar groups such as Oligonucleotide having 2'-methoxyethyl sugar or 2'-methoxyethoxy sugar, or oligonucleotide having modified base such as 5-methylcytosine, 2-deoxyuracil or 7-deaza-2'-deoxyguanosine There can be. Antisense molecules can be produced by any method including chemical synthesis or transcription. Complementary antisense molecules, once introduced into a cell, form base pairs with the natural nucleic acid sequence produced by the cell, forming a duplex that prevents transcription or translation. The expression “negative” or “minus” may refer to the antisense strand of a reference DNA molecule, and the expression “positive” or “plus” may refer to the sense strand of a reference DNA molecule.

  The term “biologically active” refers to a protein having the structural, regulatory or biochemical functions of a natural molecule. Similarly, the term “immunologically active” or “immunogenic” means that natural or recombinant DME, synthetic DME, or any oligopeptide thereof, is capable of producing a specific immune response in an appropriate animal or cell. Refers to the ability to induce and bind to a specific antibody.

  The term “complementary” or “complementarity” refers to the relationship between two single stranded nucleic acids that anneal by base pairing. For example, the sequence “5′-AGT-3 ′” forms a pair with its complementary sequence “3′-TCA-5 ′”.

  “Composition comprising (having) a polynucleotide sequence of” or “composition comprising (having) an amino acid sequence of” in a broad sense refers to any composition having a specified polynucleotide sequence or amino acid sequence. Point to. The composition can include a dry formulation or an aqueous solution. A composition comprising a polynucleotide sequence encoding DME or a fragment of DME can be used as a hybridization probe. This probe can be stored in a lyophilized state and can be bound to a stabilizer such as a carbohydrate. In hybridization, the probe is dispersed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, sodium dodecyl sulfate; SDS) and other components (eg, Denhart's solution, milk powder, salmon sperm DNA, etc.) be able to.

  The “consensus sequence” is obtained by repeatedly analyzing the DNA sequence in order to separate unnecessary bases, extended in the 5 ′ and / or 3 ′ direction using an XL-PCR kit (Applied Biosystems, Foster City CA), Resequenced nucleic acid sequences, or one or more overruns using a GELVIEW fragment construction system (GCG, Madison, WI), or a fragment construction computer program such as Phrap (University of Washington, Seattle WA) It refers to nucleic acid sequences constructed from wrapped cDNA or EST, or genomic DNA fragments. Some sequences have undergone both extension and construction to create consensus sequences.

A “conservative amino acid substitution” is a substitution that is predicted to cause little loss of the properties of the original protein when the substitution is made, that is, the structure and particularly the function of the protein are preserved, and a large change due to such substitution occurs. Refers to no substitution. The table below shows the amino acids that can be substituted for the original amino acids in the protein and are recognized as conservative amino acid substitutions.

Conservative amino acid substitutions usually include (a) the backbone structure of the polypeptide in the substitution region, eg, a β sheet or α helix structure, (b) the charge or hydrophobicity of the molecule at the substitution site, and / or (c) the majority of the side chain Is retained.

  “Deletion” refers to a change in an amino acid or nucleotide sequence such that one or more amino acid residues or nucleotides are not lost.

  The term “derivative” refers to a chemically modified polynucleotide or polypeptide. For example, replacement of hydrogen with an alkyl group, acyl group, hydroxyl group or amino group can be included in chemical modification of the polynucleotide sequence. A polynucleotide derivative encodes a polypeptide that retains at least one biological or immunological function of the natural molecule. Polypeptide derivatives are modified by glycosylation, polyethylene glycolation, or any similar process that retains at least one biological or immunological function of the polypeptide of derived origin. Polypeptide.

  “Detectable label” refers to a reporter molecule or enzyme that can generate a measurable signal and is covalently or non-covalently attached to a polynucleotide or polypeptide.

  “Differential expression” refers to the expression of a gene or protein that is increased (upregulated) or decreased (downregulated) or deleted, as determined by comparing at least two different samples. Such a comparison can be performed, for example, between a treated sample and an untreated sample, or a diseased sample and a normal sample.

  “Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon can represent one structural or functional domain of the encoded protein, a new reassortment of stable substructures allows new proteins to be assembled and new Can promote the evolution of protein function.

  A “fragment” refers to a unique portion of DME or a polynucleotide encoding DME that is identical to the parent sequence but shorter in length than the parent sequence. Fragments can have a length that is shorter than the total length of the defined sequence minus 1 nucleotide / amino acid residue. For example, a fragment may have 5 to 1000 consecutive nucleotide or amino acid residues. Fragments used as probes, primers, antigens, therapeutic molecules or for other purposes are at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, It can be 250 or 500 contiguous nucleotides or amino acid residue lengths. Fragments can be preferentially selected from specific regions of a molecule. For example, a polypeptide fragment is a length of contiguous amino acids selected from the first 250 or 500 amino acids (or the first 25% or 50%) of a polypeptide as found within a defined sequence. Can have. These lengths are clearly given by way of example, and in the examples of the present invention may be any length supported by the specification including the sequence listing, tables and drawings.

  A fragment of SEQ ID NO: 13-24 contains a region of a unique polynucleotide sequence that specifically identifies SEQ ID NO: 13-24, eg, different from other sequences in the genome from which the fragment was obtained. . Certain fragments of SEQ ID NO: 13-24 are useful, for example, in hybridization and amplification techniques, or similar methods that distinguish SEQ ID NO: 13-24 from related polynucleotide sequences. The exact length of the fragment of SEQ ID NO: 13-24 and the region of SEQ ID NO: 13-24 that the fragment corresponds to can be routinely determined by one skilled in the art based on the intended purpose for the fragment. is there.

  Certain fragments of SEQ ID NO: 1-12 are encoded by certain fragments of SEQ ID NO: 13-24. The fragment of SEQ ID NO: 1-12 contains a unique amino acid sequence region that specifically identifies SEQ ID NO: 1-12. For example, the fragment of SEQ ID NO: 1-12 is useful as an immunogenic peptide for producing an antibody that specifically recognizes SEQ ID NO: 1-12. The exact length of a fragment of SEQ ID NO: 1-12 and the region of SEQ ID NO: 1-12 is determined routinely by one skilled in the art based on the intended purpose for the fragment. Is possible.

  A “full length” polynucleotide sequence is a sequence having at least one translation start codon (eg, methionine) followed by an open reading frame and a translation stop codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

  “Homology” means sequence similarity, interchangeability, or sequence identity of two or more polynucleotide sequences or two or more polypeptide sequences.

  The term “percent match” or “˜% identical” as applied to a polynucleotide sequence means the percentage of residues that match between at least two or more polynucleotide sequences aligned using a standardized algorithm . Such an algorithm can insert a gap in a standardized and reproducible way in the sequences to be compared in order to optimize the alignment between the two sequences, so that the two sequences can be compared more significantly.

  The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm such as those incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package (a set of molecular biological analysis programs) (DNASTAR, Madison WI). This CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5: 151-153, Higgins, D.G. et al. (1992) CABIOS 8: 189-191. Default parameters for pairwise alignment of polynucleotide sequences are set as Ktuple = 2, gap penalty = 5, window = 4, “diagonals saved” = 4. A “weighted” residue weight table is selected as the default. CLUSTAL V reports the percent identity as a “percent similarity” between aligned polynucleotide sequence pairs.

  Alternatively, the National Local Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) is commonly used and provides a set of free sequence comparison algorithms (Altschul, SF et al. (1990) ) J. Mol. Biol. 215: 403-410). This algorithm is available from several sources, and is also available from NCBI in Bethesda, Maryland and the Internet (http://www.ncbi.nlm.nih.gov/BLAST/). The BLAST software suite includes a variety of sequence analysis programs, including “blastn”, which aligns a known polynucleotide sequence with another polynucleotide sequence obtained from various databases. In addition, a tool called “BLAST 2 Sequences”, which is used to directly compare two nucleotide sequences in a pair-wise manner, can be used.

“BLAST 2 Sequences” can be used interactively by accessing http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (described below). The BLAST program is generally used with parameters such as gaps set to default settings. For example, to compare two nucleotide sequences, blastn may be performed using the “BLAST 2 Sequences” tool Version 2.0.12 (April 21, 2000) set to default parameters. An example of setting default parameters is shown below.
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

  The percent identity can be measured by the full length of a defined sequence (eg, a sequence defined by a particular SEQ ID number). Alternatively, compared to a shorter length, eg, the length of a fragment derived from a defined larger sequence (eg, a fragment of at least 20, 30, 40, 50, 70, 100 or 200 consecutive nucleotides). The coincidence rate may be measured. The lengths listed here are merely exemplary, and using the fragments of any sequence length supported by the sequences described herein, including tables, figures and sequence listings, the percent identity It should be understood that the length that can be measured can be accounted for.

  Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It should be understood that this degeneracy can be used to cause changes in a nucleic acid sequence to produce a large number of nucleic acid sequences in which all nucleic acid sequences encode substantially the same protein.

  The term “percent match” or “˜% identity” as used for a polypeptide sequence refers to the percentage of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. Methods for polypeptide sequence alignment are known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions as detailed above usually preserve the charge and hydrophobicity of the substitution site, thus preserving the structure of the polypeptide (and thus also the function).

  The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (above with reference). Default parameters for pairwise alignment of polypeptide sequences using CLUSTAL V are set as Ktuple = 1, gap penalty = 3, window = 5, “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. Similar to polynucleotide alignment, CLUSTAL V reports the percent identity as the “percent similarity” between aligned polypeptide sequence pairs.

Alternatively, the NCBI BLAST software suite may be used. For example, when making pairwise comparisons of two polypeptide sequences, one may use blastp with the “BLAST 2 Sequences” tool Version 2.0.12 (Apr-21-2000) set with default parameters. An example of setting default parameters is shown below.
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter: on

  The percent identity can be measured for the entire length of a defined polypeptide sequence (eg, a sequence defined by a particular SEQ ID number). Alternatively, compared to a shorter length, eg, the length of a fragment derived from a defined larger polypeptide sequence (eg, a fragment of at least 15, 20, 30, 40, 50, 70 or 150 consecutive residues). Then, the coincidence rate may be measured. The lengths listed here are merely exemplary and may be used at any length using any fragment length supported by the sequences described herein, including tables, figures and sequence listings. It should be understood that the length over which the match rate can be measured can be described.

  “Human Artificial Chromosome (HAC)” is a linear microchromosome containing all the elements necessary for chromosomal replication, segregation and maintenance, which may contain a DNA sequence of about 6 kb (kilobase) to 10 Mb in size. .

  The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been modified to resemble a more human antibody while retaining its original binding ability.

  “Hybridization” is an annealing process in which a single-stranded polynucleotide forms a base pair with a complementary single-stranded polynucleotide under predetermined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share high complementarity. Specific hybridization complexes are formed under permissive annealing conditions and remain hybridized after the “wash” step. The washing step is particularly important in determining the stringency of the hybridization process, and more stringent conditions reduce non-specific binding (ie, binding of pairs between nucleic acid strands that are not perfectly matched). The permissive conditions for nucleic acid sequence annealing can be routinely determined by those skilled in the art. While annealing conditions can be constant for any hybridization experiment, wash conditions can be varied from experiment to experiment to obtain the desired stringency and thus also hybridization specificity. Permissive annealing conditions are, for example, at a temperature of 68 ° C., in the presence of about 6 × SSC, about 1% (w / v) SDS, and about 100 μg / ml shear-denatured salmon sperm DNA.

In general, the stringency of hybridization can be expressed to some extent relative to the temperature at which the wash step is performed. Such washing temperatures are usually selected to be about 5-20 ° C. below the melting point (Tm) of the specific sequence at a given ionic strength and pH. This Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe under a given ionic strength and pH. The formula for calculating Tm and hybridization conditions for nucleic acids are well known and are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, volumes 1-3, Cold Spring Harbor Press, Plainview NY. Refer to Chapter 9 of Volume 2 in particular.

  The high stringency conditions for polynucleotide-polynucleotide hybridization of the present invention include wash conditions for 1 hour at 68 ° C. in the presence of about 0.2 × SSC and about 0.1% SDS. Or you may carry out at the temperature of 65 degreeC, 60 degreeC, 55 degreeC, or 42 degreeC. The SSC concentration can be varied in the range of about 0.1-2 × SSC in the presence of about 0.1% SDS. Usually, a blocking agent is used to prevent nonspecific hybridization. Such blocking agents include, for example, sheared denatured salmon sperm DNA at about 100-200 μg / ml. For example, organic solvents such as formamide at a concentration of about 35-50% v / v can be used for hybridization of RNA and DNA under specific conditions. Useful variations of cleaning conditions will be apparent to those skilled in the art. Hybridization can suggest evolutionary similarity between nucleotides, especially under high stringency conditions. Such similarity strongly suggests a similar role for nucleotide groups and nucleotide-encoded polypeptides.

The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by the formation of hydrogen bonds between complementary bases. Hybridization complexes can be formed in solution (such as C ₀ t or R ₀ t analysis). Alternatively, one nucleic acid sequence is present in a dissolved state and the other nucleic acid sequence is a solid support (eg, paper, membrane, filter, chip, pin or glass slide, or other suitable substrate that is a cell or nucleic acid thereof. Can be formed between two nucleic acid sequences that are immobilized on a substrate to which is immobilized.

  The term “insertion” or “addition” refers to a change in amino acid sequence or nucleic acid sequence to which one or more amino acid residues or nucleotides are added, respectively.

  “Immune response” may refer to symptoms associated with inflammation, trauma, immune disorders, infectious diseases or genetic diseases. These symptoms can be characterized by the expression of various factors that can affect the cellular and systemic defense systems, such as cytokines, chemokines, and other signaling molecules.

  An “immunogenic fragment” is a polypeptide or oligopeptide fragment of DME that can elicit an immune response when introduced into an organism such as a mammal. The term “immunogenic fragment” also includes a polypeptide fragment or oligopeptide fragment of DME useful in any antibody production method disclosed herein or known in the art.

  The term “microarray” refers to the organization of a plurality of polynucleotides, polypeptides or other compounds on a substrate.

  The term “element” or “array element” refers to a polynucleotide, polypeptide or other compound having a unique defined position on the microarray.

  The term “modulate” or “modulate activity” refers to altering the activity of DME. For example, modulation can cause changes in DME protein activity, or binding properties, or other biological, functional, or immunological properties.

  The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides or fragments thereof. The terms “nucleic acid” and “nucleic acid sequence” refer to DNA or RNA of genomic or synthetic origin that is single-stranded or double-stranded, or may represent the sense or antisense strand. Peptide nucleic acid (PNA) or any DNA-like or RNA-like substance.

  “Functionally linked” refers to a state in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Functionally linked DNA sequences can be very close or contiguous. Alternatively, if necessary to join two protein coding regions, they can be in the same reading frame.

  “Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent comprising an oligonucleotide of at least about 5 nucleotides in length, attached to the peptide backbone of amino acid residues ending in lysine. The terminal lysine imparts solubility to the composition. PNA binds preferentially to complementary single-stranded DNA or RNA to stop the elongation of transcription, and can be extended to the lifetime of PNA in cells by polyethylene glycolation.

  “Post-translational modifications” of DME may include lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage and other modifications known in the art. These processes can occur synthetically or biochemically. Biochemical modifications depend on the enzyme environment of DME and will vary depending on the cell type.

  The “probe” is a nucleic acid sequence encoding DME, a complementary sequence thereof, or a fragment thereof used for detecting the same sequence or allelic nucleic acid sequence, related nucleic acid sequence. A probe is an isolated oligonucleotide or polynucleotide that is bound to a detectable label or reporter molecule. Typical labels include radioisotopes, ligands, chemiluminescent reagents and enzymes. A “primer” is a short nucleic acid, usually a DNA oligonucleotide, that can be annealed to a target polynucleotide by forming complementary base pairs. The primer can then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used, for example, for amplification (and identification) of nucleic acid sequences by polymerase chain reaction (PCR).

  Probes and primers as used in the present invention typically contain at least 15 contiguous nucleotides of known sequence. Longer probes and primers to increase specificity, such as probes consisting of at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or 150 consecutive nucleotides of the disclosed nucleic acid sequences; Primers may be used. Some probes and primers are much longer than this. It should be understood that any length of nucleotide as supported herein, including tables, drawings and sequence listings, can be used.

For the preparation and use of probes and primers, see Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, 1-3, Cold Spring Harbor Press, Plainview NY, Ausubel, FM et al., (1987 ) Current Protocols in Molecular Biology , Greene Publ. Assoc. & Wiley-Intersciences, New York NY, Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications Academic Press, San Diego CA, etc. PCR primer pairs can be obtained from known sequences using a computer program for that purpose, such as using Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).

  The selection of oligonucleotides to be used as primers is performed using software known in the art for such purposes. For example, OLIGO 4.06 software is useful for selecting PCR primer pairs up to 100 nucleotides each, derived from oligonucleotides and up to 5,000 larger polynucleotides, up to 32 kilobases of input polynucleotide sequences. It is also useful for analyzing. Similar primer selection programs incorporate additional functionality for extended capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center of the University of Texas Southwestern Medical Center in Dallas, Texas) can select specific primers from the megabase sequence, and thus the entire genome range. This is useful for designing primers. The Primer3 primer selection program (available from the Whitehead Institute / MIT Center for Genome Research, Cambridge, Mass.) Allows the user to enter a “mispriming library” that allows the user to specify sequences to avoid as primer binding sites. Primer3 is particularly useful for selection of oligonucleotides for microarrays (the source code for the latter two primer selection programs can be obtained from the respective sources and modified to meet user-specific needs). The PrimerGen program (publicly available from the UK Human Genome Mapping Project in Cambridge, UK-Resource Center) designs primers based on multiple sequence alignments, thereby maximally conserving or minimally conserving aligned nucleic acid sequences Allows selection of primers that hybridize to any of the regions. This program is therefore useful for the identification of both unique and conserved oligonucleotides and polynucleotide fragments. Oligonucleotide and polynucleotide fragments identified by any of the above selection methods identify fully or partially complementary polynucleotides in hybridization techniques, eg, as PCR or sequencing primers, as microarray elements, or in nucleic acid samples. It is useful as a specific probe. The method for selecting the oligonucleotide is not limited to the above method.

  A “recombinant nucleic acid” is not a natural sequence, but a sequence that artificially combines two or more separated segments of a sequence. This artificial combination is often accomplished by chemical synthesis, but more commonly by artificial manipulation of isolated segments of nucleic acids, eg, genetic engineering as described in Sambrook et al. (Supra). Achieve by technique. The term recombinant nucleic acid also includes nucleic acids in which a portion of the nucleic acid has been modified by addition, substitution or deletion. Often recombinant nucleic acids include nucleic acid sequences operably linked to promoter sequences. Such a recombinant nucleic acid can be part of a vector that is used, for example, to transform a cell.

  Alternatively, such a recombinant nucleic acid can be part of a viral vector, which can be based, for example, on vaccinia virus. Such vectors can be used to inoculate a mammal and the recombinant nucleic acid is expressed to induce a protective immune response in the mammal.

  “Regulatory elements” are nucleic acid sequences that are usually derived from untranslated regions of a gene and include enhancers, promoters, introns, and 5 ′ and 3 ′ untranslated regions (UTRs). Regulatory elements interact with host or viral proteins that regulate transcription, translation or RNA stability.

A “reporter molecule” is a chemical or biochemical moiety used to label a nucleic acid, amino acid or antibody. Reporter molecules include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, color formers, substrates, cofactors, inhibitors, magnetic particles and other components known in the art.
The “RNA equivalent” for a DNA sequence is composed of the same linear nucleic acid sequence as the reference DNA sequence, but all the nitrogenous base thymines are replaced with uracil, and the backbone of the sugar chain is deoxyribose. Rather than ribose.

  The term “sample” is used in its broadest sense. Samples presumed to contain DME, nucleic acids encoding DME, or fragments thereof include body fluids, extracts from chromosomes or organelles or membranes isolated from cells or cells, cells, It may include genomic DNA, RNA, cDNA, tissue, tissue print, etc. present in solution or fixed to a substrate.

  The terms “specific binding” and “specifically bind” refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. This interaction depends on whether there is a specific structure of the protein (eg, an antigenic determinant or epitope) that the binding molecule recognizes. For example, when an antibody is specific for epitope “A”, there is a labeled A and a polypeptide having the epitope A in the reaction containing the antibody or an unlabeled “A” that is not bound. This reduces the amount of labeled A that binds to the antibody.

  The term “substantially purified” refers to a nucleic acid or amino acid sequence that has been removed from the natural environment or isolated or separated, preferably at least about 60% of other naturally associated components, preferably At least about 75%, most preferred refers to at least about 90% absent.

  “Substitution” is the replacement of one or more amino acids or nucleotides with another amino acid or nucleotide, respectively.

  The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. Is included. The substrate can have various surface forms such as wells, grooves, pins, channels, holes, and the like, and polynucleotides and polypeptides bind to the substrate surface.

  A “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue at a given time under given conditions.

  “Transformation” is the process by which exogenous DNA is introduced into a recipient cell. Transformation can occur under natural or artificial conditions according to various methods known in the art and can be any known sequence that inserts an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. Can be based on method. The method of transformation is selected according to the type of host cell to be transformed. Non-limiting transformation methods include bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cell” includes stably transformed cells in which the introduced DNA is replicable as an autonomously replicating plasmid or as part of a host chromosome. Furthermore, cells that transiently express introduced DNA or introduced RNA for a limited time are also included.

  As used herein, a “transgenic organism” is any organism, including but not limited to animals and plants, in which one or more cells of the organism are, for example, used in the art by human involvement. Have heterologous nucleic acid introduced by well-known transgenic technology. Nucleic acids are introduced into cells either directly or indirectly by introduction into cell precursors, by planned genetic manipulation, for example by microinjection or by infection with recombinant viruses. Alternatively, the nucleic acid can be introduced by infection with a recombinant viral vector such as a lentiviral vector (Lois, C. et al. (2002) Science 295: 868-872). The term genetic manipulation does not refer to classical crossbreeding or in vitro fertilization but to the introduction of recombinant DNA molecules. Among the genetic transformants expected in accordance with the present invention are bacteria, cyanobacteria, fungi and animals and plants. The isolated DNA of the present invention can be introduced into a host by methods known in the art, such as infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are well known and are described in references such as Sambrook et al. (1989) supra.

  A “variant / mutant sequence” of a specific nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity with the specific nucleic acid sequence over the length of the entire nucleic acid sequence. For definition, blastn is executed using the “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set to the default parameters. Such nucleic acid pairs are, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% for a given length, It can show 96%, 97%, 98%, 99% or more sequence identity. Certain variants may be described, for example, as “allelic” variants (described above), “splice” variants, “species” variants, or “polymorphic” variants. A splice variant may have significant identity to a reference molecule, but will usually have more or fewer nucleotides due to alternative splicing of exons during mRNA processing. Corresponding polypeptides may have additional functional domains or lack domains present in the reference molecule. Species variants are polynucleotide sequences that vary from species to species. The resulting polypeptides usually have significant amino acid identity with each other. Polymorphic variants differ in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variant sequences can also include “single nucleotide polymorphisms” (SNPs) that differ in one nucleotide of the polynucleotide sequence. The presence of SNPs can indicate, for example, a particular population, condition or disease propensity.

  A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence that has at least 40% sequence identity to that particular polypeptide sequence over the entire length of one polypeptide sequence. For the definition, blastp is executed by using “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as default parameters. Such a polypeptide pair is, for example, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94 for a given length of one polypeptide. %, 95%, 96%, 97%, 98%, 99% or more sequence identity.

(invention)
The present invention is based on the discovery of novel human drug-metabolizing enzymes (DME) and polynucleotides encoding DME, and autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine using these compositions. Based on the discovery of diagnosis, treatment, and prevention of gastrointestinal diseases, including disorders, eye diseases, metabolic disorders, and liver diseases.

  Table 1 summarizes the nomenclature for the full-length polynucleotide and polypeptide sequences of the present invention. Each polynucleotide and its corresponding polypeptide correlates with one Incyte project identification number (Incyte project ID). Each polypeptide sequence was represented by a polypeptide sequence identification number (polypeptide SEQ ID NO) and an Incyte polypeptide sequence number (Incyte polypeptide ID). Each polynucleotide sequence was represented by a polynucleotide sequence identification number (polynucleotide SEQ ID NO) and an Incyte polynucleotide consensus sequence number (Incyte polynucleotide ID). Column 6 shows the Incyte ID numbers of physical full-length clones corresponding to the polypeptide and polynucleotide sequences of the present invention. A full-length clone encodes a polypeptide having at least 95% sequence identity to the polypeptide sequence shown in row 3.

  Table 2 shows the sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) 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 each polypeptide of the present invention. Column 3 shows the GenBank identification number (GenBank ID NO :) of the closest GenBank homolog. Column 4 shows the probability score for the match between each polypeptide and its homologue. Column 5 shows appropriate citations where applicable and one or more annotations of GenBank homologues, all of which are specifically incorporated herein by reference.

  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) of each polypeptide of the present invention. Column 3 shows the number of amino acid residues of each polypeptide. Columns 4 and 5 show potential phosphorylation and glycosylation sites, respectively, as determined by the GCG sequence analysis software package MOTIFS program (Genetics Computer Group, Madison WI). Column 6 shows the amino acid residues that include the signature sequence, domain, and motif. Column 7 shows the analysis method for the analysis of the structure / function of the protein, and the applicable part further shows a searchable database used for the analysis method.

  Both Table 2 and Table 3 summarize the properties of the polypeptides of the present invention, and these properties demonstrate that the claimed polynucleotides are drug metabolizing enzymes. For example, SEQ ID NO: 1 has 40% identity with rabbit UDP glucuronosyltransferase (GenBank ID g165801) from residue C87 to residue K471 by the Basic Local Alignment Search Tool (BLAST). (See Table 2). The BLAST probability score is 1.1e-70, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 1 also has a UDP-glucuronosyltransferase domain, which is a statistically significant match in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM). Determined by searching (see Table 3). Data obtained from BLIMPS, MOTIFS, and PROFILESCAN analyzes further demonstrate that SEQ ID NO: 1 is a member of the UDP-glycosyltransferase superfamily.

  In another example, SEQ ID NO: 3 was determined by the Basic Local Alignment Search Tool (BLAST) to have 49% identity with chicken sulfotransferase (GenBank ID g2687360) from residues K4 to Q302 (Table 2). The BLAST probability score is 2.3e-81, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 3 also has a sulfotransferase domain, which is determined by searching for statistically significant matches in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM) (See Table 3). Data from BLIMPS analysis provides further empirical evidence that SEQ ID NO: 3 is a sulfotransferase.

In another example, SEQ ID NO: 4 has 37% identity to Mycobacterium bovis C-5 sterol desaturase (GenBank ID g9965825) from residue D38 to residue P306. Basic Local Alignment Search Tool (BLAST) (See Table 2). The BLAST probability score is 5.8e-44, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 5 has 44% identity to mouse arylacetamide deacetylase (GenBank ID g12597322) from residue V38 to residue L440, determined by BLAST analysis with a probability score of 2.6e-87 It was. Data from BLIMPS and MOTIFS analyzes provide evidence to further confirm that SEQ ID NO: 5 is a lipolytic enzyme.

  In another example, SEQ ID NO: 6 has 93% identity to human UDP-glucuronosyltransferase (GenBank IDs g3135025 and g8650278) from residue M1 to residue D529. Determined by Tool (BLAST) (see Table 2). The BLAST probability score is 1.2e-274, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 6 also has a UDP-glucuronosyltransferase domain, which is a statistically significant match in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM). Determined by searching (see Table 3). Data from BLIMPS, MOTIFS and PROFILESCAN analyzes provide further confirmatory evidence that SEQ ID NO: 6 is a UDP-glucuronosyltransferase. SEQ ID NO: 7 has 97% identity from residue A9 to residue S615 to the mouse protein arginine methyltransferase (GenBank ID g5257221) determined by BLAST analysis with a probability score of 0.0.

  In another example, SEQ ID NO: 10 may have 100% identity to human NG, NG-dimethylarginine dimethylaminohydrolase (GenBank ID g4160666) from residue M57 to residue S341. Determined by Search Tool (BLAST) (see Table 2). The BLAST probability score is 2.5e-148, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. Data from BLAST analysis using the Prodom database provides further confirmatory evidence that SEQ ID NO: 10 is NG, NG-dimethylarginine dimethylaminohydrolase.

  As another example, SEQ ID NO: 11 has been shown by the Basic Local Alignment Search Tool (BLAST) to have 54% identity with human arylsulfatase (GenBank ID g825628) from residue P76 to residue W554. (See Table 2). The BLAST probability score is 2.2e-146, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 11 also has one sulfatase domain, which searches for statistically significant matches in the PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM). (See Table 3). Data from BLIMPS, MOTIFS, and PROFILESCAN analyzes provide further empirical evidence that SEQ ID NO: 11 is an arylsulfatase. Algorithms and parameters for the analysis of SEQ ID NO: 1-12 are described in Table 7.

  SEQ ID NO: 2, SEQ ID NO: 8-9 and SEQ ID NO: 12 were analyzed and annotated in a similar manner. Table 7 describes the algorithms and parameters for the analysis of SEQ ID NO: 1-12.

  As shown in Table 4, the full-length polynucleotide sequences of the present invention were assembled using cDNA sequences, coding (exon) sequences derived from genomic DNA, or any combination of these two sequences. Column 1 shows the polynucleotide sequence identification number (polynucleotide SEQ ID NO), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in base pairs. ing. Column 2 shows the nucleotide start position (5 ′) and stop position (3 ′) of the cDNA sequence and / or genomic sequence used in the construction (assembly) of the full-length polynucleotide sequence of the present invention, and SEQ ID NO: 13 -24 or the nucleotide start position of a fragment of a polynucleotide sequence useful for techniques (e.g., hybridization or amplification techniques) that distinguish SEQ ID NO: 13-24 from the associated polynucleotide sequence. 5 ') and end position (3').

The polynucleotide fragment described in column 2 of Table 4 may particularly refer to Incyte cDNA from, for example, a tissue-specific cDNA library or a pooled cDNA library. Alternatively, the polynucleotide fragment in column 2 may refer to a GenBank cDNA or EST that contributed to the construction of the full-length polynucleotide sequence. In addition, the polynucleotide fragment in column 2 can identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (ie, sequences that include the “ENST” designation). Alternatively, the polynucleotide fragment described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records database (ie, sequences containing the designation “NM” or “NT”) and from the NCBI RefSeq Protein Sequence Records. In some cases (ie, a sequence containing the designation “NP”). Alternatively, the polynucleotide fragment described in column 2 may refer to a construct consisting of both cDNA and Genscan predicted exons combined by an “exon-stitching” algorithm. For example, a polynucleotide sequence identified as FL_XXXXXX_N ₁ _N ₂ _YYYYY_N ₃ _N ₄ has a cluster identification number of the sequence to which the algorithm is applied is XXXXXX, and a prediction number generated by the algorithm is YYYYY (if present N) _{1,2,3 ..} is a “stitched” sequence that is a specific exon that may have been manually edited during the analysis (see Example 5 ). Alternatively, the polynucleotide fragments in row 2 may refer to exon constructs joined together by an “exon-stretching” algorithm. For example, the polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_1_N is a “stretched” sequence. Where XXXXXX is the Incyte project identification number, gAAAAA is the GenBank identification number of the human genome sequence to which the `` exon stretching '' algorithm is applied, gBBBBB is the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N is a specific number Refers to an exon (see Example 5 ). When a RefSeq sequence is used as a protein homologue of the “exon stretching” algorithm, the RefSeq identifier (labeled “NM”, “NP”, or “NT”) is used instead of the GenBank identifier (ie, gBBBBB). Sometimes used.

Alternatively, prefix codes identify constituent sequences that have been manually edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following table lists the constituent sequence prefix codes and examples of how to analyze the same sequences corresponding to the prefix codes (see Examples 4 and 5 ).

  In some cases, to confirm the final consensus polynucleotide sequence, an Incyte cDNA coverage that overlapped the sequence coverage as shown in Table 4 was obtained, but the associated Incyte cDNA identification number was not shown. .

  Table 5 shows a representative cDNA library for full-length polynucleotide sequences constructed using Incyte cDNA sequences. A representative cDNA library is the Incyte cDNA library that is most frequently represented by the Incyte cDNA sequences used to construct and confirm the polynucleotide sequences described above. The tissues and vectors used to construct the cDNA library are shown in Table 5 and described in Table 6.

  The present invention also includes mutants of DME. Preferred DME variants have at least one functional or structural feature of DME and have at least about 80% amino acid sequence identity to the DME amino acid sequence, or at least about 90% amino acid sequence identity. Or even variants having at least about 95% amino acid sequence identity.

  The present invention also provides a group of polynucleotides encoding DME. In certain embodiments, the present invention provides a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 13-24, encoding DME. The polynucleotide sequence of SEQ ID NO: 13-24 also includes an equivalent RNA sequence as shown in the sequence listing, where the occurrence of the nitrogen base thymine is replaced with uracil and the sugar backbone is substituted for deoxyribose. It consists of ribose.

  The invention also includes variant sequences of polynucleotide sequences encoding DME. In particular, such variant polynucleotide sequences have at least about 70% polynucleotide sequence identity to the polynucleotide sequence encoding DME, or at least about 85% polynucleotide sequence identity, or even at least There will be as much as about 95% polynucleotide sequence identity. Particular embodiments of the present invention may comprise at least 70% polynucleotide sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13-24, or at least 85% polynucleotide sequence identity, and at least A variant sequence of the polynucleotide sequence is provided comprising a sequence selected from the group consisting of SEQ ID NO: 13-24 with as much as 95% polynucleotide sequence identity. Any of the polynucleotide variants described above can encode an amino acid sequence having at least one functional or structural characteristic of DME.

  In yet another example, a polynucleotide variant sequence of the invention is a splice variant sequence of a polynucleotide sequence encoding DME. Some splice variant sequences may have portions with significant sequence identity to the polynucleotide sequence encoding DME, but the addition or absence of a few blocks of sequences caused by alternative splicing of exons during mRNA processing. Loss usually results in having more or fewer polynucleotides. For some splice variant sequences, less than about 70%, or less than about 60%, or less than about 50% polynucleotide sequence identity is found over the entire length with the polynucleotide sequence encoding DME. Some portions of the splice variant sequence may comprise at least about 70%, or at least about 85%, or at least about 95%, or even 100% of the polynucleotide with each portion of the polynucleotide sequence encoding DME. It will have sequence identity. Any of the splice variants described above can encode an amino acid sequence having at least one functional or structural characteristic of DME.

  Those skilled in the art will appreciate that the degeneracy of the genetic code creates various polynucleotide sequences that encode DME, some of which have minimal similarity to any known native gene polynucleotide sequences. Will be understood. Thus, the present invention can cover all possible variations of polynucleotide sequences that can be produced by selection of combinations based on possible codon selection. These combinations are made on the basis of the standard triplet genetic code applied to the polynucleotide sequence of native DME. Also consider that all such mutations are clearly disclosed.

  Nucleotide sequences encoding DME and its mutant sequences are generally capable of hybridizing to the nucleotide sequence of native DME under suitably selected stringency conditions, but include substantially different codons, such as including non-natural codon groups. It may be beneficial to create a nucleotide sequence that encodes a DME having use or a derivative thereof. Codons can be selected to increase the expression rate of peptides generated in a particular eukaryotic or prokaryotic host based on the frequency with which the host utilizes a particular codon. Another reason for substantially altering the nucleotide sequence encoding DME and its derivatives without altering the encoded amino acid sequence is RNA with favorable properties such as longer half-life than transcripts made from the native sequence Sometimes a transcript is made.

  The present invention also includes a process in which DNA sequences encoding DME and a group of derivatives thereof or a group of fragments thereof are completely generated by synthetic chemistry. After production, this synthetic sequence can be inserted into any of a variety of available expression vectors and cell lines using reagents well known in the art. Furthermore, synthetic chemistry can be used to introduce mutations into sequences encoding DME, or any fragment thereof.

  The invention further includes polynucleotide sequences that are capable of hybridizing under the various stringency conditions to the claimed polynucleotide sequences, particularly SEQ ID NO: 13-24 and fragments thereof (e.g. Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399-407, Kimmel, AR (1987) Methods Enzymol. 152: 507-511, etc.). Hybridization conditions, including annealing and washing conditions, are described in “Definitions”.

Methods for DNA sequencing are known in the art, and any embodiment of the present invention can be performed using DNA sequencing methods. Enzymes can be used for the DNA sequencing method, such as Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ). ) Can be used. Alternatively, a polymerase and proofreading exonuclease can be used in combination, as seen, for example, in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated using equipment such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno, NV), the PTC200 thermal cycler (MJ Research, Watertown MA), and the ABI CATALYST 800 thermal cycler (Applied Biosystems). The sequencing is then performed using the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA) or other methods well known in the art. The resulting sequence is analyzed using various algorithms well known in the art. (See Ausubel, FM (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York NY, unit 7.7, Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New York NY, 856-853, etc.) .

  Using various PCR-based methods well known in the art and partial nucleotide sequences, the nucleic acid sequence encoding DME is extended to detect upstream sequences such as promoters and regulatory elements. obtain. For example, restriction site PCR, one of the methods that can be used, is a method of amplifying an unknown sequence from genomic DNA in a cloning vector using universal primers and nested primers (see, for example, Sarkar, G. (1993 ) See PCR Methods Applic. 2: 318--322). Another method is reverse PCR, which amplifies an unknown sequence from a circularized template using primers extended in a wide range of directions. The template is obtained from a restriction enzyme fragment containing a known genomic locus and surrounding sequences (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res. 16: 8186). A third method is a capture PCR method, which is involved in a method for PCR amplification of DNA fragments adjacent to known sequences of human and yeast artificial chromosome DNA. (See Lagerstrom, M. et al. (1991) PCR Methods Applic 1: 111-119). In this method, it is possible to insert a recombinant double-stranded sequence into an unknown sequence region using a plurality of restriction enzyme digestion and ligation reactions before PCR. Other methods that can be used to search for unknown sequences are known in the art (see Parker, J.D. et al. (1991) Nucleic Acids Res. 19: 3055-3060, etc.). In addition, genomic DNA can be walked using PCR, nested primers and PromoterFinder library (Clontech, Palo Alto CA). This procedure is useful for finding intron / exon junctions without having to screen the library. For all PCR-based methods, using commercially available software such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another suitable program, approximately 22-30 nucleotides in length, containing GC Primers can be designed to anneal to the template at a rate of about 50% or more and at a temperature of about 68 ° C to 72 ° C.

  When screening for full-length cDNAs, it is preferable to use a library that is size-selected to contain larger cDNAs. In addition, random primer libraries often contain sequences having the 5 'region of the gene and are suitable for situations in which an oligo d (T) library cannot produce a full-length cDNA. Genomic libraries may be useful for extending sequences into the 5 ′ non-transcribed regulatory region.

  A commercially available capillary electrophoresis system can be used to analyze the size of the sequencing or PCR product or to confirm its nucleotide sequence. Specifically, capillary sequencing is a flowable polymer for electrophoretic separation, a laser activated fluorophore that is specific for four different nucleotides, and detection of the emitted wavelength. And a CCD camera to be used. The output / light intensity can be converted to an electrical signal using suitable software (Applied Biosystems GENOTYPER, SEQUENCE NAVIGATOR, etc.). The entire process from sample loading to computer analysis and electronic data display is computer controllable. Capillary electrophoresis is particularly suitable for sequencing small DNA fragments that are present in small amounts in a particular sample.

  In another embodiment of the invention, the polynucleotide sequences encoding DME or fragments thereof are cloned in a recombinant DNA molecule expressing DME, fragments or functional equivalents thereof in a suitable host cell. Can do. Due to the degeneracy inherent in the genetic code, another group of DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can be created, and these sequences can be used for the expression of DME.

  For various purposes, the nucleotide sequences of the present invention can be recombined using methods generally known in the art to modify the sequences encoding DME. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. It is possible to recombine nucleotide sequences using random fragmentation of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly. For example, site-directed mutagenesis via oligonucleotides can be used to introduce mutations that create new restriction sites, change glycosylation patterns, change codon preference, generate splice variants, and the like.

  Nucleotides of the present invention can be synthesized using MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; U.S. Pat.No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793-797; Christians, FC et al. (1999). Nat. Biotechnol. 17: 259-264; Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319). PKIN biological properties such as activity or ability to bind to other molecules and compounds can be altered or improved. DNA shuffling is a process of creating a library of gene variants using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening to identify gene variant sequences with the desired characteristics. These suitable variants may then be pooled and further repeated for DNA shuffling and selection / screening. Thus, genetic diversity is created by “artificial” breeding and rapid molecular evolution. For example, single gene fragments with random point mutations can be recombined and screened and then shuffled until the desired properties are optimized. Alternatively, recombining a given gene with homologous genes from the same gene family, either from the same species or from different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner It can be made.

In another embodiment, sequences encoding DME can be synthesized in whole or in part using chemical methods well known in the art (eg, Caruthers. MH et al. (1980) Nucleic Acids Symp. Ser). 7: 215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7: 225-232). Alternatively, DME itself or fragments thereof can be synthesized using chemical methods. For example, peptide synthesis can be performed using various liquid phase or solid phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties , WH Freeman, New York NY, 55-60, Roberge, JY et al. (1995) Science 269: 202-204 etc.). Automated synthesis can be achieved using an ABI 431A peptide synthesizer (Applied Biosystems). Furthermore, the variant polypeptide by modifying the amino acid sequence of DME, or any part thereof, directly during synthesis and / or in combination with a sequence group from other proteins or any part thereof Or a polypeptide having one sequence of a natural polypeptide.

  This peptide can be substantially purified using preparative high performance liquid chromatography (see, for example, Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182: 392-421). The composition of this synthetic peptide can be confirmed by amino acid analysis or sequencing (see Creighton, 28-53, etc. above).

  In order to express biologically active DME, a group of nucleotide sequences encoding DME or a derivative thereof can be inserted into a suitable expression vector. This expression vector is a vector having elements necessary for controlling transcription and translation of a coding sequence inserted into a suitable host. Necessary elements include regulatory sequences in the vector and polynucleotide sequences encoding DME (enhancers, constitutive and expression-inducible promoters, 5 'and 3' untranslated regions, etc.). Such elements vary in strength and specificity. More specific translation of sequences encoding DME can be achieved using specific initiation signals. Such signals include the ATG initiation codon and nearby sequences such as the Kozak sequence. In cases where sequences encoding DME, its initiation codon, and upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational control signals may be required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal including an in-frame ATG start codon should be included in the expression vector. Exogenous translation elements and initiation codons can originate from a variety of natural and synthetic products. Inclusion of an enhancer suitable for the particular host cell system used can enhance expression efficiency (see, eg, Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162) .

Methods well known to those skilled in the art can be used to produce expression vectors having sequences encoding DME and suitable transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination techniques (eg Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, Chapters 4 and 8, and 16-17; and Ausubel, FM et al. (1995) Current Protocols in Molecular Biology , John Wiley & Sons, New York NY, ch. Chapters 9, 13 and 16 reference).

  A variety of expression vector / host systems can be utilized to retain and express sequences encoding DME. Such expression vectors / host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, and yeast transformed with yeast expression vectors. Or transformed with an insect cell line infected with a viral expression vector (eg baculovirus), a viral expression vector (eg cauliflower mosaic virus: CaMV or tobacco mosaic virus: TMV) or a bacterial expression vector (eg Ti plasmid or pBR322 plasmid) Plant cell lines and animal cell lines. (Sambrook, supra, Ausubel, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509, Engelhard, EK 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) EMBOJ. 6: 307-311, The McGraw Hill Yearbook of Science and Technology) (1992) McGraw Hill New York NY, 191-196, Logan, J. et al. T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81: 3655-3659, Harrington, JJ et al. ( 1997) See Nat. Genet. 15: 345-355, etc.) Using an expression vector derived from a retrovirus, adenovirus, herpes virus or vaccinia virus, or an expression vector derived from various bacterial plasmids, the nucleotide sequence is determined as a target organ. (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5 (6): 350-356, Yu, M. et al. (1993) Proc. Natl. Acad. Sci USA 90 (13): 6340-6344, Buller, RM (1985) Nature 317 (6040): 813-815; McGregor, DP et al. (1994) Mol.Immunol. 31 (3): 219-226, Verma, IM and N. Somia (1997) Nature 389: 239-242 Etc.). The present invention is not limited by the host cell used.

In bacterial systems, a number of cloning and expression vectors can be selected depending upon the use intended for polynucleotide sequences encoding DME. For example, a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid (Life Technologies) can be used for routine cloning, subcloning and propagation of polynucleotide sequences encoding DME. Ligation of the DME-encoding sequence into the multicloning site of the vector destroys the lacZ gene, allowing a colorimetric screening method for identification of transformed bacteria with recombinant molecules. In addition, these vectors may be useful for in vitro transcription in cloned sequences, dideoxy sequencing, single-stranded rescue with helper phage, and generation of nested deletions (e.g., Van Heeke, G. And SM Schuster (1989) J. Biol. Chem. 264: 5503-5509). For example, when a large amount of DME is required for the production of antibodies, vectors that direct the expression of DME at a high level can be used. For example, vectors containing the strong inducible SP6 bacteriophage promoter or inducible T7 bacteriophage promoter can be used.

For the production of DME, a yeast expression system can be used. A large number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, PGH promoter, etc. can be used for Saccharomyces cerevisiae or Pichia pastoris . In addition, such vectors direct either secretion of the expressed protein or retention within the cell. It also makes it possible to incorporate foreign sequences into the host genome for stable growth (e.g. Ausubel, 1995, supra, Bitter, GA et al. (1987) Methods Enzymol. 153: 516-544, and Scorer CA et al. (1994) Bio / Technology 12: 181-184).

Plant systems can also be used to express DME. Transcription of sequences encoding DME can be achieved by viral promoters such as 35S and 19S from CaMV as used alone or in combination with omega leader sequences from TMV (Takamatsu, N. (1987) EMBO J 6: 307-311). Promoted by a promoter. Alternatively, a plant promoter such as a small subunit of RUBISCO or a heat shock promoter may be used (eg, Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224 : 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. (See The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill New York NY, 191-196, etc.).

  In mammalian cells, a number of virus-based expression systems can be utilized. When an adenovirus is used as an expression vector, a sequence group encoding DME can be ligated to an adenovirus transcription / translation complex composed of a late promoter and a triple leader sequence. With the insertion of the adenovirus genome into the essential E1 or E3 region, infectious viruses that express DME in host cells can be obtained (see, for example, Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81: 3655-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. Proteins can also be expressed at high levels using vectors based on SV40 or EBV.

  Human artificial chromosomes (HACs) can also be used to deliver fragments of DNA that are larger than those contained in and expressed from a plasmid. Approximately 6 kb to 10 Mb of HAC is made for therapy and delivered by conventional delivery methods (liposomes, polycation amino polymers, or vesicles) (eg, Harrington. JJ et al. (1997) Nat Genet. 15: 345). -355.)

  For long-term production of recombinant proteins in mammalian systems, stable expression of DME in cell lines is desirable. For example, expression vectors and selectable marker genes on the same vector or on different vectors can be used to transform a sequence encoding DME into a cell line. The expression vector used can have a replication element of viral origin and / or an endogenous expression element. After the introduction of the vector, the cells can be grown for about 1-2 days in enhanced medium before being transferred to the selective medium. The purpose of the selectable marker is to provide resistance to the selective medium, and the presence of the selectable marker allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems can be used to recover transformed cell lines. Such a selection system includes, but is not limited to, the herpes simplex virus thymidine kinase gene used for tk ^- cells and the herpes simplex virus adenine phosphoribosyltransferase gene used for apr ^- cells. (See, eg, Wigler, M. et al. (1977) Cell 11: 223-232, Lowy, I. et al. (1980) Cell 22: 817-823). Moreover, tolerance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr provides resistance to methotrexate, neo provides resistance to aminoglycoside neomycin and G-418, als provides resistance to chlorsulfuron, and pat provides resistance to phosphinothricin acetyltransferase (Wigler, M. (1980) Proc. Natl. Acad. Sci. USA 77: 3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14 etc.). Other selectable genes, such as trpB and hisD that alter cellular requirements for metabolism, have been described in the literature (eg Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci. USA 85: 8047-8051). Visible markers such as anthocyanins, green fluorescent protein (GFP; 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 transient or stable protein expression resulting from specific vector systems (Rhodes, CA (1995) Methods Mol. Biol 55: 121-131 etc.).

  Even if the presence / absence of marker gene expression suggests the presence of the target gene, the presence and expression of the gene may need to be confirmed. For example, when a sequence encoding DME is inserted into a certain marker gene sequence, a transformed cell group having a sequence group encoding DME can be identified by lack of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding DME under the control of a single promoter. Expression of a marker gene in response to induction or selection usually means that a tandem gene is also expressed.

  In general, a host cell having a nucleic acid sequence encoding DME and expressing DME can be identified using various methods well known to those skilled in the art. These methods include protein-biological, including DNA-DNA or DNA-RNA hybridization, PCR methods, membrane systems for detection and / or quantification of nucleic acids or proteins, solution-based, or chip-based techniques. Examples include, but are not limited to, test methods or immunological assays.

Immunological methods for detection and measurement of DME expression using either specific polyclonal or monoclonal antibodies are known in the art. Such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS) and the like. A two-site, monoclonal-based immunoassay using a group of monoclonal antibodies that react with two non-interfering epitopes on DME is preferred, but competitive binding studies can also be used. These and other assays are known in the art (Hampton. R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press. St Paul. MN, Sect. IV, Coligan, JE et al. (1997). ) Current Protocols in Immunology , Greene Pub. Associates and Wiley-Interscience, New York NY, Pound, JD (1998) Immunochemical Protocols , Humans Press, Totowa NJ, etc.).

A wide variety of labeling and conjugation methods are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding DME include oligo labeling, nick translation, end labeling, or labeling. PCR amplification using the synthesized nucleotides is included. Alternatively, DME-encoding sequences, or any fragment thereof, can be cloned into a vector for generating mRNA probes. Such vectors are known in the art and are commercially available and should be used for in vitro RNA probe synthesis with the addition of a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. Can do. Such methods can be performed using various kits commercially available from, for example, Amersham Pharmacia Biotech, Promega (Madison WI), US Biochemical, and the like. Suitable reporter molecules or labels that can be used to facilitate detection include substrates, cofactors, inhibitors, magnetic particles, radionuclides, enzymes, fluorescent agents, chemiluminescent agents, color formers, and the like.

  Host cells transformed with a nucleotide sequence encoding DME are cultured under conditions suitable for expression and recovery of this protein in cell culture media. Whether a protein produced from a transformed cell is secreted or remains intracellular depends on the sequence used, the vector, or both. One skilled in the art will appreciate that expression vectors having a polynucleotide group encoding DME can be designed to have a signal sequence group that directs secretion of DME across a prokaryotic or eukaryotic cell membrane.

  Furthermore, selection of host cell lines may be made by their ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired form. Such polypeptide modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” or “pro” form of the protein can be used to identify induction, folding and / or activity of the protein to the target. Various host cells (eg CHO, HeLa, MDCK, MEK293, WI38, etc.) with specific cellular equipment and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATCC, Manassas, VA). Available and can be selected to ensure correct modification and processing of the foreign protein.

  In another embodiment of the invention, a natural nucleic acid sequence encoding DME, a modified nucleic acid sequence, or a recombinant nucleic acid sequence is ligated to a heterologous sequence in any of the host systems described above. Can result in translation of certain fusion proteins. For example, a chimeric DME protein containing a heterologous moiety that can be recognized by a commercially available antibody can facilitate the screening of peptide libraries for inhibitors of DME activity. Also, heterologous protein portions and heterologous peptide portions can facilitate the purification of fusion proteins using commercially available affinity substrates. Such moieties include, but are not limited to, glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, There is hemagglutinin (HA). GST on immobilized glutathione, MBP on maltose, Trx on phenylarsine oxide, CBP on calmodulin, and 6-His on metal chelate resins allow for purification of cognate fusion proteins. FLAG, c-myc and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. In addition, if a genetic manipulation is performed so that the fusion protein has a proteolytic cleavage site between the sequence encoding DME and the heterologous protein sequence, DME can be cleaved from the heterologous portion after purification. Fusion protein expression and purification methods are described in Ausubel (1995) chapter 10 above. Various commercially available kits can also be used to facilitate the expression and purification of the fusion protein.

In a further embodiment of the present invention, radiolabeled DME can be synthesized in vitro using TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple the transcription and translation of protein coding sequences operably linked to a T7, T3 or SP6 promoter. Translation takes place in the presence of a radiolabeled amino acid precursor such as ³⁵ S methionine.

  A compound that specifically binds to DME can be screened using the DME of the present invention or a fragment thereof. At least one or more test compounds can be used to screen for specific binding to DME. Examples of test compounds include antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

In certain embodiments, the compound thus identified is closely related to the natural ligand of DME, such as a ligand or a fragment thereof, or a natural substrate, structural or functional mimetic, Alternatively, it is a natural binding partner (see Chapter 5 of Coligan, JE et al. (1991) Current Protocols in Immunology 1 (2)). Similarly, the compound may be closely related to the natural receptor to which DME binds, or at least some fragment of this receptor, such as the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one example, screening for these compounds includes the production of suitable cells that express DME as either secreted proteins or proteins on the cell membrane. Suitable cells include cells from mammals, yeast, Drosophila, or E. coli. Next, cells expressing DME or a cell membrane fraction containing DME are contacted with a test compound and analyzed for stimulation or inhibition of binding or activity of either DME or test compound.

  Some assays simply allow the test compound to be conjugated experimentally to the polypeptide and the binding detected by a fluorophore, radioisotope, enzyme conjugate or other detectable label. For example, the assay can include mixing at least one test compound with DME in solution or immobilized on a solid support, and detecting binding of the DME to the compound. Alternatively, this assay can detect and measure the binding of a test compound in the presence of a labeled competitor. In addition, this assay can be performed using cell-free reconstituted specimens, chemical libraries or natural product mixtures where the test compound is released in solution or immobilized on a solid support.

A compound that modulates the activity of DME can be screened using the DME of the present invention or a fragment thereof. Such compounds can include agonists, antagonists, partial agonists or inverse agonists and the like. In one embodiment, an assay is performed under conditions where DME activity is permissible, wherein DME is mixed with at least one test compound, and the activity of DME in the presence of the test compound is determined in the absence of the test compound. Compare with DME activity. A change in the activity of DME in the presence of a test compound is indicative of the presence of a compound that modulates the activity of DME. Alternatively, the assay is performed by mixing the test compound with an in vitro or cell-free system having DME under conditions suitable for the activity of DME. In any of these assays, the test compound that modulates the activity of DME may indirectly modulate, and does not need to be in direct contact with the test compound. At least one or more test compounds can be screened.

  In another example, homologous recombination in embryonic stem cells (ES cells) is used to “knock out” a polynucleotide encoding DME or a mammalian homolog thereof in an animal model system. Such techniques are well known in the art and are useful for creating animal models of human disease (see US Pat. Nos. 5,175,383 and 5,767,337, etc.). For example, mouse ES cells such as the 129 / SvJ cell line are derived from early mouse embryos and can be grown in culture. This ES cell is transformed with a vector containing a gene of interest disrupted with a marker gene such as the neomycin phosphotransferase gene (neo: Capecchi, M.R. (1989) Science 244: 1288-1292). This vector is integrated into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination is performed using the Cre-loxP system, and the target gene is knocked out in a tissue-specific or developmental stage-specific manner (Marth, JD (1996) Clin. Invest. 97: 1999-2002; Wagner, KU et al. (1997) Nucleic Acids Res. 25: 4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts collected from, for example, the C57BL / 6 mouse strain. These blastocysts are surgically introduced into pseudopregnant females, the genetic traits of the resulting chimeric progeny are identified, and they are crossed to create heterozygous or homozygous systems. The genetically modified animals thus prepared can be tested with potential therapeutic or toxic drugs.

Polynucleotides encoding DME can be engineered in vitro in human blastocyst-derived ES cells. Human ES cells have the potential to differentiate into at least eight separate cell lineages, including endoderm, mesoderm and ectoderm cell types. These cell lines differentiate into, for example, neural cells, hematopoietic lineages and cardiomyocytes (Thomson, JA et al. (1998) Science 282: 1145-1147).

  Polynucleotides encoding DME can be used to produce “knock-in” humanized animals (pigs) or transgenic animals (mouse or rat) modeled on human disease. Using knock-in technology, a region of the polynucleotide encoding DME is injected into animal ES cells and the injected sequence is integrated into the animal cell genome. Transformed cells are injected into blastula and transplanted as described above. Test for genetically modified progeny or inbred lines and treat with potential pharmaceuticals to obtain information on the treatment of human disease. Alternatively, mammalian inbred lines that overexpress DME, such as secreting DME into milk, can be a convenient protein source (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4: 55- 74).

(Treatment)
There are chemical and structural similarities between multiple regions of DME and drug metabolizing enzymes, for example, in the context of sequences and motifs. Furthermore, examples of tissues that express DME include liver tissues, renal cortical tissues, islet cells, and cancer tissues including bone marrow neuroblastoma tumors, which are seen in Table 6. Thus, DME is thought to play a role in gastrointestinal diseases including autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine disorders, ophthalmic disorders, metabolic disorders, and liver disorders. In the treatment of diseases associated with increased DME expression or activity, it is desirable to reduce DME expression or activity. In the treatment of a disease associated with a decrease in DME expression or activity, it is desirable to enhance DME expression or activity.

Thus, in one embodiment, DME or a fragment or derivative thereof can be administered to a subject for the treatment or prevention of a disease associated with decreased expression or activity of DME. Examples of such diseases include, but are not limited to, autoimmune / inflammatory diseases such as acquired immune deficiency syndrome (AIDS) and Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloid Disease, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune multi-endocrine candidiasis ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis , Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, lymphocytic factor-induced incident lymphopenia, neonatal hemolytic disease (fetal erythroblastosis), erythema nodosum, atrophic gastritis, glomerulus Nephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis Dementia, pancreatitis, polymyositis, psoriasis, Reiter syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis (scleroderma), thrombocytopenic purpura Ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infection, bacterial infection, fungal infection, parasitic infection, protozoal infection, helminth infection, trauma Included in cell proliferation abnormalities are actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria , Psoriasis, primary thrombocythemia, and cancers such as adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, Neck, gallbladder, ganglion, digestive tract Includes heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterine cancer, and tubular acidosis and anemia in developmental disorders , Cushing's syndrome, achondroplasia dwarfism, Duchenne / Becker muscular dystrophy, sputum, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, mental retardation), Smith-Magenis syndrome (Smith- Magenis syndrome), spinal dysplasia syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoses, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, Syndenham chorea Includes endocrine disorders, including seizure disorders such as Syndenham's chorea and cerebral palsy, spinal cord fission, anencephalopathy, cranio-vertebral rupture, congenital glaucoma, cataract, sensorineural hearing loss Hypothalamic and pituitary disorders and sexual function resulting from lesions such as primary brain tumors and adenomas, gestational infarction, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immune abnormalities, and head trauma complications Disorders related to hypopituitarism and benign lines, including depression and Sheehan syndrome, diabetes insipidus, Kalman disease, hand-Schuller Christian disease, letterer Zyve disease, sarcoidosis, empty sella syndrome, dwarfism Anti-diuretic hormone (ADH) incompatible secretion syndrome (SIADH) and acromegaly, disorders associated with hypopituitarism, including giantism, and goiter and myxedema, bacterial infectious acute thyroiditis, viral infection Disorders associated with hypothyroidism, including primary subacute thyroiditis, autoimmune thyroiditis (Hashimoto's disease), cretinism, and thyroid poisoning and its various types, Reeve's disease, anterior tibial myxedema, toxic multinodular goiter, thyroid cancer, hyperthyroidism including Plummer's disease, hyperparathyroidism including Conn's disease (chronic hypercalemia), type I and type II diabetes And pancreatic diseases such as hyperplasia and adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing disease, Riddle syndrome, Arnold-Healy-Gordon syndrome, brown cell tumor, Addison Disorders related to adrenal glands such as illness and adrenal insufficiency, and abnormal prolactin production and infertility in women, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, gonadotropin-only deficiency (isolated) gonadotropin deficiency), amenorrhea, milk leakage, hemi-yang, hirsutism and masculinization, breast cancer, postmenopausal osteoporosis, male Leydig cell hyperplasia, male menopause Includes gonad steroid hormone-related diseases such as germ cell dysplasia, hypersexuality associated with Leydig cell tumor, androgen resistance associated with lack of androgen receptor, 5α-reductase syndrome, gynecomastia, etc. , Eye disease, conjunctivitis, dry keratoconjunctivitis, keratitis, episclerosis, iritis, posterior uveitis, glaucoma, transient cataract, ischemic optic neuropathy, optic neuritis, Leber hereditary optic neuropathy, vitreous Exfoliation, retinal detachment, cataract, macular degeneration, central serous chorioretinopathy, retinitis pigmentosa (retinal pigment degeneration), choroidal melanoma, retrobulbar tumor, chiasmal tumor, metabolism Among the disorders are Addison's disease, cerebral tendon xanthoma, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty cirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, Instep Adenoma, glucagonoma, glycogenosis, hereditary fructose intolerance, hyperadrenal medulla, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia , Hypothyroidism, hyperlipidemia, hyperlipidemia, lipid myopathies, lipodystrophy, lysosomal storage disease, Menkes syndrome, occipital horn syndrome, mannosidosis, neuraminidase deficiency Include obesity, obesity, pentose-phenylketonuria, pseudovitamin D deficiency rickets, hypocalcemia, hypophosphatemia, postadolescent cerebellar ataxia, tyrosineemia, and gastrointestinal Diseases include dysphagia, peptic esophagitis, esophageal spasm, esophageal stenosis, esophageal cancer, dyspepsia, digestive disorder, gastritis, gastric cancer, anorexia, nausea, vomiting, gastric failure hemp Sinus or pyloric edema, abdominal angina, heartburn, gastroenteritis, ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary tract disease, hepatitis, hyperbilirubinemia, Hereditary hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colon cancer, colon obstruction , Irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal bleeding, acquired immune deficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatitis, hemochromatosis, Wilson disease, alpha ₁ antitrypsin deficiency Disease, Reye syndrome, primary sclerosing cholangitis, liver infarction, portal vein occlusion and thrombus, central lobular necrosis, liver purpura, hepatic vein thrombosis, hepatic vein obstruction, pre-eclampsia, eclampsia, gestational acute liver fat In the gestational liver And juice stasis, nodular regenerative and adenomas include liver cancer, including carcinomas.

  In another embodiment, a vector capable of expressing DME or a fragment or derivative thereof for the treatment or prevention of diseases associated with decreased expression or activity of DME, including but not limited to the diseases listed above. Can be administered to a subject.

  In yet another embodiment, a composition having substantially purified DME for the treatment or prevention of diseases associated with decreased expression or activity of DME, including but not limited to the diseases listed above. The product can be administered to a subject together with a suitable pharmaceutical carrier.

  In yet another embodiment, an agonist that modulates the activity of DME is provided to a subject for the treatment or prevention of a disease associated with a decrease in DME expression or activity, including but not limited to the diseases listed above. Can be administered.

  In a further embodiment, an antagonist of DME may be administered to a subject for the treatment or prevention of a disease associated with increased DME expression or activity. Examples of such diseases include, but are not limited to, the aforementioned autoimmune / inflammatory diseases, abnormal cell proliferation, developmental or developmental disorders, endocrine disorders, ophthalmic disorders, metabolic disorders, and liver disorders. Gastrointestinal diseases are included. In one embodiment, an antibody that specifically binds to DME can be used directly as an antagonist or indirectly as a targeting or delivery mechanism that delivers the drug to cells or tissues that express DME.

  In another embodiment, a complementary sequence of a polynucleotide encoding DME is expressed for the treatment or prevention of diseases associated with increased DME expression or activity, including but not limited to the diseases listed above. The vector can be administered to a subject.

  In another embodiment, any protein, antagonist, antibody, agonist, complementary sequence, or vector of the invention can be administered in combination with another suitable therapeutic agent. Suitable therapeutic agents for use in combination therapy can be selected by one skilled in the art according to conventional pharmaceutical principles. When combined with a therapeutic agent, it can provide a synergistic effect in the treatment or prevention of the various diseases described above. This method makes it possible to achieve a pharmaceutical effect with a small amount of each drug, thereby reducing the possibility of side effects.

  DME antagonists can be produced using methods common in the art. Specifically, a drug that specifically binds to DME can be identified by making an antibody using purified DME or screening a library of therapeutic agents. DME antibodies can also be produced using methods common in the art. Such antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chains, Fab fragments, and fragments produced by Fab expression libraries. However, it is not limited to these. Neutralizing antibodies (ie, antibodies that inhibit dimer formation) are usually suitable for therapy. Single chain antibodies (eg, camels or llamas) can be potent enzyme inhibitors. There will also be advantages in the design of peptide mimetics and the development of immunosorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74: 277-302).

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans and others, DME or any fragment or oligo with its immunogenic properties. It can be immunized by injection of peptides. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyol, polyanion, peptide, oil emulsion, mussel hemocyanin (KLH), dinitrophenol, etc. There is a surfactant. Among adjuvants used in humans, BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are particularly preferred.

  The oligopeptide, peptide or fragment used for inducing an antibody against DME preferably has an amino acid sequence consisting of at least about 5 amino acids, and generally has a sequence consisting of about 10 or more amino acids. Become. These oligopeptides, peptides or fragments are also preferably identical to part of the amino acid sequence of the natural protein. Short stretches of DME amino acids can be fused with sequences of other proteins such as KLH to produce antibodies against this chimeric molecule.

  Monoclonal antibodies against DME can be produced using any technique that produces groups of antibody molecules by continuous cell lines in the medium. Such technologies include, but are not limited to, hybridoma technology, human B cell hybridoma technology and EBV-hybridoma technology (Kohler, G. et al. (1975) Nature 256: 495-497, Kozbor, D. et al. (1985) .J. Immunol. Methods 81: 31-42, Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2026-2030, Cole, SP et al. (1984) Mol. Cell Biol. 62 : 109-120 etc.).

  In addition, techniques developed for the production of “chimeric antibodies” such as splicing mouse antibody genes into human antibody genes can be used to obtain molecules with suitable antigen specificity and biological activity (eg, Morrison (1984) Proc. Natl. Acad. Sci. 81: 68516855; Neuberger, MS et al. (1984) Nature 312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454. reference). Alternatively, the techniques described for the production of single chain antibodies can be applied using methods known in the art to produce DME specific single chain antibodies. Antibodies with related specificity but different idiotype composition can also be generated by chain shuffling from random combinations of immunoglobulin libraries (Burton DR (1991) Proc. Natl. Acad. Sci. USA 88 : 10134-10137 etc.).

Antibody production can also be accomplished by induction of in vivo production in a lymphocyte population or by screening an immunoglobulin library or panel of highly specific binding reagents as disclosed in the literature. (See Orlando, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3833-3837, Winter, G. et al. (1991) Nature 349: 293-299).

Antibody fragments with specific binding sites for DME can also be produced. For example, but not by way of limitation, such fragments are produced by reducing the F (ab ′) ₂ fragment produced by pepsin digestion of the antibody molecule and the disulfide bridge of the F (ab ′) ₂ fragment. There is a Fab fragment to be. Alternatively, it is possible to quickly and easily identify a monoclonal Fab fragment having a desired specificity by preparing a Fab expression library (see Huse, WD et al. (1989) Science 246: 1275-1281). .

  A variety of immunoassays can be screened to identify antibodies with the desired specificity. Numerous protocols for competitive binding studies using either polyclonal or monoclonal antibodies with established specificity, or immunoradiometric assays are well known in the art. Usually such immunoassays involve the measurement of complex formation between DME and its specific antibody. Two-site monoclonal-based immunoassays are commonly used, using a group of monoclonal antibodies reactive against two non-interfering DME epitopes, but competitive binding studies are also available (Pound, supra).

Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques are used to assess the affinity of antibodies for DME. The affinity is expressed by the binding constant Ka, which is a value obtained by dividing the molar concentration of the DME antibody complex by the molar concentration of free antibody and free antigen under equilibrium conditions. Polyclonal antibodies have heterogeneous affinities for a number of DME epitopes, and Ka determined for a given polyclonal antibody formulation represents the average affinity or binding activity of the group of antibodies to DME. The Ka of a monoclonal antibody preparation monospecific for a particular DME epitope represents a true measure of affinity. A high-affinity antibody preparation having a Ka value of 10 ⁹ to 10 ¹² L / mol is preferably used in an immunoassay in which the DME antibody complex must withstand severe manipulation. A low-affinity antibody preparation with a Ka value of 10 ⁶ to 10 ⁷ L / mol is preferably used for immunopurification and similar treatments in which DME must eventually dissociate from the antibody in an activated state. (Catty, D. (1988) Antibodies, Volume I: A Practical Approach . IRL Press, Washington, DC; Liddell, JE and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies , John Wiley & Sons, New York NY ).

  The antibody titer and binding activity of the polyclonal antibody formulation can be further evaluated to determine the quality and suitability of such reagents for certain applications used later. For example, polyclonal antibody formulations containing at least 1-2 mg / ml specific antibody, preferably 5-10 mg / ml specific antibody, are generally used in processes where the DME antibody complex must be precipitated. Antibody specificity, antibody titer, binding activity, and guidelines for antibody quality and use in various applications are generally available (see, for example, Catty literature, Coligan et al. Above).

In another embodiment of the invention, a polynucleotide encoding DME, or any fragment or complementary sequence thereof, is used for therapeutic purposes. In some embodiments, gene expression can be altered by designing sequences complementary to the coding and regulatory regions of genes encoding DME and antisense molecules (DNA, RNA, PNA, or modified oligonucleotides). . Such techniques are well known in the art, and antisense oligonucleotides or larger fragments can be designed from various positions along the control or coding region of the sequence encoding DME. (See Agrawal, S., ed. (1996) Antisense Therapeutics , Humana Press Inc., Totawa NJ).

  For use in therapy, any gene delivery system suitable for introducing an antisense sequence into a suitable target cell can be used. Antisense sequences can be delivered into cells in the form of expression plasmids that produce sequences complementary to at least a portion of the cellular sequence encoding the target protein during transcription (Slater, JE et al. (1998) J. Allergy Clin Immunol. 102 (3): 469-475 and Scanlon, KJ et al. (1995) 9 (13): 1288-1296, etc.). Antisense sequences can also be introduced into cells using viral vectors such as retroviruses and adeno-associated virus vectors (Miller, AD (1990) Blood 76: 271, supra, Ausubel, Uckert, W. et al. and W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347). Other genetic delivery mechanisms include liposome systems, artificial viral envelopes and other systems known in the art (Rossi, JJ (1995) Br. Med. Bull. 51 (1): 217-225; Boado, RJ et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315, Morris, MC et al. (1997) Nucleic Acids Res. 25 (14): 2730-2736.

In another embodiment of the invention, a polynucleotide encoding DME may be used for somatic or germ cell gene therapy. By performing gene therapy, (i) Severe combined immune deficiency (SCID) − characterized by gene deficiency (eg, X chromosome chain inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288: 669-672)) X1), severe combined immune deficiency associated with congenital adenosine deaminase (ADA) deficiency (Blaese, RM et al. (1995) Science 270: 475-480, Bordignon, C. et al. (1995) Science 270: 470- 475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75: 207-216: Crystal, RG et al. (1995) Hum. Gene Therapy 6: 643-666, Crystal, RG et al. (1995) Hum. Gene Therapy 6: 667-703), thalassamia, familial hypercholesterolemia, hemophilia due to factor VIII or factor IX deficiency (Crystal, 35 RG (1995) Science 270: 404-410, Verma , IM and N. Somia (1997) Nature 389: 239-242), (ii) expressing a conditional lethal gene product (eg, in the case of cancer resulting from uncontrolled cell growth), (iii) Intracellular parasites such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335: 395-396, Poescbla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93: 11395 -11399) and other proteins that have protective functions against human retroviruses, 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 If a deficiency in a gene required for DME expression or regulation causes disease, DME can be expressed from a suitable population of introduced cells to mitigate symptoms caused by the gene deficiency. It is.

In a further embodiment of the present invention, diseases and abnormalities due to DME deficiency are treated by preparing mammalian expression vectors encoding DME and introducing these vectors into DME deficient cells by mechanical means. . Mechanical introduction techniques for in vivo or ex vitro cells include (i) direct microinjection of DNA into individual cells, (ii) gene guns, (iii) transfection via liposomes, ( iv) receptor-mediated gene transfer, and (v) use of DNA transposons (Morgan, RA and WF Anderson (1993) Annu. Rev. Biochem. 62: 191-217, Ivics, Z. (1997) Cell 91 : 501-510; Boulay, JL. And H. Recipon (1998) Curr. Opin. Biotechnol. 9: 445-450).

  Expression vectors that may be effective for DME expression include, but are not limited to, PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA) and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). To express DME, (i) a constitutively active promoter (eg, cytomegalovirus (CMV), rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin gene) (Ii) inducible promoters (eg, tetracycline-regulated promoters (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci, contained in the commercially available T-REX plasmid (Invitrogen)). USA 89: 5547-5551; Gossen, M. et al. (1995) Science 268: 1766-1769; Rossi, FMV and HM Blau (1998) Curr. Opin. Biotechnol. 9: 451-456)), ecdysone inducible promoter. (Included in commercially available plasmids PVGRXR and PIND: Invitrogen), FK506 / rapamycin-inducible promoter, or RU486 / mifepristone-inducible promoter (Rossi, FMV and HM Blau, supra), or (iii ) Derived from normal individuals , It is possible to use natural promoters or tissue-specific promoter of the endogenous gene encoding DME.

  Commercially available liposome transformation kits (eg, Invitrogen's PerFect Lipid Transfection Kit) allow those skilled in the art to deliver polynucleotides to target cells in culture. Also, the effort required to optimize the experimental parameters is minimized. Alternatively, transformation using the calcium phosphate method (Graham. FL and AJ Eb (1973) Virology 52: 456-467) or electroporation (Neumann, B. et al. (1982) EMBO J. 1: 841-845) I do. In order to introduce DNA into primary culture cells, a standardized mammalian transfection protocol modification is required.

In another embodiment of the invention, a disease or disorder caused by a genetic defect associated with DME expression encodes DME under the control of (i) a retroviral terminal repeat (LTR) promoter or an independent promoter. A polynucleotide, (ii) a suitable RNA packaging signal, and (iii) a Rev responsive element (RRE) with additional retroviral cis-acting RNA sequences and coding sequences necessary for efficient vector propagation A retroviral vector consisting of can be made and treated. Retroviral vectors (eg PFB and PFBNEO) are commercially available from Stratagene and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6733-6737). The above data is incorporated herein by reference. The vector is propagated in a suitable vector producing cell line (VPCL), which expresses an envelope gene with affinity for a receptor on the target cell or a pan-affinity envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61: 1647-1650, Bender, MA et al. (1987) J. Virol. 61: 1639-1646, Adam, MA and AD Miller (1988) J. Virol. 62: 3802-3806, Dull , T. et al. (1998) J. Virol. 72: 8463-8471, Zufferey, R. et al. (1998) J. Virol. 72: 9873-9880). In US Pat. No. 5,910,434 to Rigg (“Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant”), a method for obtaining a retroviral packaging cell line is disclosed, with reference to It is a part of this specification. The propagation of retroviral vectors, transduction of cell populations (eg CD4 ⁺ T cells), and return of transduced cells to patients are methods well known to those skilled in the field of gene therapy and are described in numerous references. (Ranga, U. et al. (1997) J. Virol. 71: 7020-7029, Bauer, G. et al. (1997) Blood 89: 2259-2267, Bonyhadi, ML (1997) J. Virol. 71: 4707 -4716, Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 1201-1206, Su, L. (1997) Blood 89: 2283-2290).

  Alternatively, an adenoviral gene therapy delivery system is used to deliver a polynucleotide encoding DME to cells having one or more genetic abnormalities associated with DME expression. Production and packaging of adenoviral vectors are known to those skilled in the art. Replication-deficient adenovirus vectors have proved versatile in introducing genes encoding immunoregulatory proteins into intact pancreatic islets (Csete, ME et al. (1995) Transplantation 27: 263- 268). Potentially useful adenoviral vectors are described in US Pat. No. 5,707,618 (“Adenovirus vectors for gene therapy”) to Armentano, which is incorporated herein by reference. See also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19: 511-544 and Verma, I.M. and N. Somia (1997) Nature 18: 389: 239-242 for adenoviral vectors. Both documents are hereby incorporated by reference.

  Alternatively, a herpes gene therapy delivery system is used to deliver polynucleotides encoding DME to target cells having one or more genetic abnormalities with respect to DME expression. The use of herpes simplex virus (HSV) -based vectors may be particularly useful when introducing DME into central neurons where HSV is tropic. The production and packaging of herpes vectors is known to those skilled in the art. Replication competent herpes simplex virus (HSV) type I vectors have been used to deliver reporter genes to primate eyes (Liu, X. et al. (1999) Exp. Eye Res. 169: 385- 395). The production of HSV-1 viral vectors is also disclosed in US Pat. No. 5,804,413 (“Herpes simplex virus swains for gene transfer”) granted to DeLuca, which is incorporated herein by reference. . US Pat. No. 5,804,413 describes a recombinant HSV d92 comprising a genome with at least one exogenous gene introduced into a cell under the control of a promoter suitable for purposes including human gene therapy. The patent also discloses the generation and use of recombinant HSV lines lacking ICP4, ICP27, and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73: 519-532 and Xu, H. et al. (1994) Dev. Biol. 163: 152-161. Both documents are hereby incorporated by reference. Manipulation of cloned herpesvirus sequences, production of recombinant virus after transfection with multiple plasmids with various segments of large herpesvirus genomes, herpesvirus growth and proliferation, and cell populations Infection with herpesviruses is a technique known to those skilled in the art.

  Alternatively, alphavirus (positive single stranded RNA virus) vectors are used to deliver a group of polynucleotides encoding DME to a group of target cells. Biological research on the prototype alphavirus Semliki Forest Virus (SFV) has been extensive and gene transfer vectors are based on the SFV genome (Garoff, H. and K.-J Li (1998) Cun. Opin. Biotech. 9: 464-469). During the replication of alpha viral RNA, subgenomic RNA is produced that normally encodes the viral capsid proteins. Since this subgenomic RNA replicates at a higher level than the full-length genomic RNA, the capsid protein group is overproduced compared to viral proteins with enzymatic activity (eg, protease and polymerase). Similarly, by introducing a sequence encoding DME into a region encoding the capsid of the α virus genome, RNA encoding a large number of DMEs is produced in the vector-introduced cell, and DME is synthesized at a high level. Usually, infection with alpha virus is accompanied by cell lysis within a few days. On the other hand, the ability of a group of normal hamster kidney cells (BHK-21) having a certain variant of Sindbis virus (SIN) to establish a persistent infection can apply lytic replication of α viruses to gene therapy (Dryga, SA et al. (1997) Virology 228: 74-83). Since the α virus has a wide host range, DME can be introduced into various types of cells. Specific transduction of a subset of cells in a population may require cell sorting prior to transduction. Methods for treating alphavirus infectious cDNA clones, alphavirus cDNA and RNA transfection methods, and alphavirus infection methods are known to those skilled in the art.

  It is also possible to inhibit gene expression using an oligonucleotide derived from the transcription start site. The transcription initiation site is, for example, between about −10 and about +10 counting from the start site. Similarly, inhibition can be achieved using triple helix base pairing methods. Triple helix base pairing is useful because it prevents the double helix from opening and binding to a polymerase, transcription factor, or regulatory molecule. Recent therapeutic advances using triple helix DNA are described in the literature (Gee, JE et al. (1994) in: Huber, BE and BI Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY, (See pages 163-177 etc.). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by preventing transcripts from binding to ribosomes.

  Ribozymes are enzymatic RNA molecules, and ribozymes can 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 and subsequent internal nucleotide strand breaks. For example, hammerhead ribozyme molecules made by genetic engineering may specifically and effectively catalyze the cleavage of nucleotides in sequences encoding DME.

  Specific ribozyme cleavage sites within any RNA target are first identified by scanning the target molecule for ribozyme cleavage sites including GUA, GUU, GUC sequences. Once identified, assess whether a short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site has secondary structural features that render the oligonucleotide dysfunctional. Is possible. Assessment of the suitability of candidate targets can also be performed by testing accessibility to hybridization with complementary oligonucleotide groups using a ribonuclease protection assay.

The complementary ribonucleic acid molecules and ribozymes of the present invention can be made using any method well known in the art for nucleic acid molecule synthesis. Production methods include methods of chemically synthesizing oligonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding DME. Such DNA sequences can be incorporated into a variety of vectors using a suitable RNA polymerase promoter such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.

  RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'end, 3' end, or both of the molecule, or phosphorothioate or 2 'O instead of phosphodiesterase linkages in the main chain of the molecule. -Use of methyl is included. This concept is unique to PNA production but can be extended to all these molecules. This includes adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio- and similar modifications that are not readily recognized by endogenous endonucleases, as well as non-conventional bases such as inosine, Include queosine, wybutosine, etc.

  A further embodiment of the invention includes a method of screening for compounds effective in altering the expression of a polynucleotide encoding DME. Non-limiting compounds that are effective in altering the expression of specific polynucleotides include oligonucleotides, antisense oligonucleotides, triple helix forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and specific polynucleotides There are non-polymeric chemical entities that can interact with sequences. Effective compounds can alter polynucleotide expression by acting as either inhibitors or enhancers of polynucleotide expression. Therefore, in the treatment of diseases associated with increased DME expression or activity, compounds that specifically inhibit the expression of polynucleotides encoding DME are therapeutically useful and are associated with decreased DME expression or activity. In the treatment of disease, compounds that specifically promote the expression of polynucleotides encoding DME may be therapeutically useful.

At least one to a plurality of test compounds can be screened for efficacy in altering the expression of a specific polynucleotide. The test compound is obtained by any method commonly known in the art. Such methods include modifying the expression of the polynucleotide, selecting from an existing, commercial or dedicated, natural or non-natural compound library, and the chemical and / or structural properties of the target polynucleotide. There are chemical modifications of compounds that are known to be effective when rationally designing properties-based compounds and when selecting from libraries of combinatorial or randomly generated compounds. A sample containing a polynucleotide encoding DME is exposed to at least one of the test compounds thus obtained. Samples can be, for example, intact cells, permeabilized cells, or in vitro cell-free or reconstituted biochemical systems. Alterations in the expression of a polynucleotide encoding DME are assayed by any method well known in the art. Usually, the expression of a specific nucleotide is detected by hybridization using a probe having a nucleotide sequence complementary to the sequence of a polynucleotide encoding DME. The amount of hybridization can be quantified, thereby forming the basis for comparison of expression of polynucleotides exposed and not exposed to one or more test compounds. Detection of a change in the expression of the polynucleotide exposed to the test compound indicates that the test compound is effective in altering the expression of the polynucleotide. For compounds effective in modifying the expression of specific polynucleotides, for example, Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) US Pat. No. 5,932,435, Arndt, GM et al. (2000) Nucleic Acids Res. 28: E15) or human cell lines such as HeLa cells (Clarke, ML et al. (2000) Biochem. Biophys. Res. Commun. 268: 8-13). Particular embodiments of the present invention are involved in screening combinatorial libraries of oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, modified oligonucleotides) for examining antisense activity against specific polynucleotide sequences. (Bruice, TW et al. (1997) US Pat. No. 5,686,242, Bruice, TW et al. (2000) US Pat. No. 6,022,691).

Numerous methods for introducing vectors into cells or tissues are available and are equally suitable for in vivo , in vitro and ex vivo use. For ex vivo treatment, the vector can be introduced into stem cells taken from the patient, cloned and expanded back to the patient by autologous transplantation. Delivery by transfection, ribosome injection or polycation amino polymer can be performed using methods well known in the art (Goldman, CK et al. (1997) Nat. Biotechnol. 15: 462-466. Etc. See).

  Any of the above treatment methods can be applied to all subjects requiring treatment, including mammals such as humans, dogs, cats, cows, horses, rabbits, monkeys, and the like.

A further embodiment of the invention relates to the administration of compositions generally having certain active ingredients formulated with certain pharmaceutically acceptable excipients. Excipients include, for example, sugar, starch, cellulose, gum and protein. Various dosage forms are widely known, and details are described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions comprise DME, DME antibodies, mimetics, agonists, antagonists, inhibitors, and the like.

  The compositions used in the present invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal. , Intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal.

  Compositions administered from the lung may be prepared in liquid or dry powder form. Such compositions are usually aerosolized just before the patient inhales. In the case of small molecules (eg, conventional low molecular weight organic drugs), aerosol delivery of fast acting formulations is known in the art. In the case of macromolecules (eg, larger peptides and proteins), recent improvements in pulmonary delivery through the alveolar region of the lung in the art can substantially transport drugs such as insulin into the blood circulation. (See Patton, JS et al., US Pat. No. 5,997,848, etc.). Pulmonary delivery is superior in administration without needle injection and eliminates the need for penetration enhancers that may be toxic.

  Compositions suitable for use in the present invention include compositions containing as much active ingredient as necessary to achieve a given purpose. The determination of an effective dose is within the ability of those skilled in the art.

  In order to deliver a macromolecular group having DME or a group of fragments thereof directly into cells, a special variety of composition groups can be prepared. For example, a liposomal formulation comprising a cell-impermeable polymer can facilitate cell fusion and intracellular delivery of the polymer. Alternatively, DME or a fragment thereof can be attached to the short cationic N-terminal part of the HIV Tat-1 protein. The fusion protein thus generated has been shown to transduce cells of all tissues including the brain in a mouse model system (Schwarze, S.R. et al. (1999) Science 285: 1569-1572).

  For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, such as neoplastic cell culture assays, or in animal models such as mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine beneficial doses and routes for administration to humans.

A therapeutically effective amount refers to the amount of an active ingredient that ameliorates symptoms and conditions, such as DME or a fragment thereof, an antibody of DME, an agonist or antagonist of DME, an inhibitor and the like. Therapeutic efficacy and toxicity can be determined by standard drug techniques in cell culture or animal experiments, for example by measuring ED ₅₀ (50% therapeutically effective dose of a population) or LD ₅₀ (50% lethal dose of a population). Can be determined. The dose ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compositions that exhibit high therapeutic indices are desirable. Data obtained from cell culture assays and animal experiments is used to formulate a range of dosage for use in humans. The dosage contained in such compositions is preferably within a range of blood concentrations that includes ED ₅₀ with little or no toxicity. Depending on the dosage form used, patient sensitivity and route of administration, the dosage will vary within this range.

  The exact dosage will be determined by the practitioner in light of factors related to the subject that requires treatment. Dosage forms and dosages are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors related to the subject include the severity of the disease, the patient's general health, the subject's age, weight and sex (gender), time and frequency of administration, drug combination, response sensitivity and response to treatment. Long-acting compositions may be administered once every 3-4 days, once a week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

  The usual dose depends on the route of administration, but is about 0.1 to 100,000 μg, up to about 1 g in total. Guidance on specific dosages and delivery methods is described in the literature and is usually available to the practitioner. Those skilled in the art will utilize different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, symptoms, sites, etc.

(Diagnosis)
In another embodiment, antibodies that specifically bind to DME are used to diagnose diseases characterized by the expression of DME, or to monitor patients being treated with an agonist or antagonist or inhibitor of DME or DME. Used for. Antibodies useful for diagnostic purposes are formulated in the same manner as described above for the treatment site. Diagnostic assays for DME include methods for detecting DME from human body fluids or from cell or tissue extracts using antibodies and labels. The antibody can be modified or not, and can be labeled covalently or non-covalently with a reporter molecule. A wide variety of reporter molecules are known in the art and can be used, some of which have been described above.

  Various protocols for measuring DME, such as ELISA, RIA, and FACS, are well known in the art and provide a basis for diagnosing altered or abnormal levels of DME expression. Normal or standard DME expression values are determined by binding body fluids or cells collected from normal mammals, eg, human subjects, to antibodies against DME under conditions suitable for complex formation. . The amount of standard complex formation can be quantified by various methods such as photometry. The amount of DME expressed in disease samples from subjects, controls and biopsy tissues is compared to standard values. The deviation between the standard value and the subject establishes the parameter for diagnosing the disease.

  In another embodiment of the invention, a group of polynucleotides encoding DME may be used for diagnostic purposes. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. These polynucleotides can be used to detect and quantify gene expression in biopsy tissue groups where DME expression can correlate with disease. This diagnostic assay can be used to check for the presence or overexpression of DME and to monitor the regulation of DME levels during treatment.

  In one embodiment, nucleic acid sequences encoding DME are identified using hybridization with PCR probes capable of detecting polynucleotide sequences, such as genomic sequences encoding DME or closely related molecules, for example. it can. Probe specificity and hybridization, where the probe is made from a highly specific region (eg, 5 'regulatory region) or from a slightly less specific region (eg, a conserved motif) Alternatively, the stringency of amplification will determine whether the probe will identify only the native sequence encoding DME, or whether it will identify allelic variants or related sequences.

  Probes can also be used to detect related sequences, and can have at least 50% sequence identity with any sequence encoding DME. The hybridization probe of the present invention can be DNA or RNA, and can be derived from the sequence of SEQ ID NO: 13-24, or a genomic sequence including a DME gene promoter, enhancer, and intron.

As a method for preparing a hybridization probe group specific to a DNA group encoding DME, a method of cloning a polynucleotide sequence group encoding a DME or DME derivative group into vectors for preparing an mRNA probe group including. Vectors for making mRNA probes are known to those skilled in the art and are commercially available and used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. obtain. Hybridization probes can be labeled with various reporter populations. Examples of reporter populations include radionuclides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase bound to a probe via an avidin / biotin binding system.

Polynucleotide sequences encoding DME can be used to diagnose diseases associated with DME expression. Examples of such diseases include, but are not limited to, autoimmune / inflammatory diseases such as acquired immune deficiency syndrome (AIDS) and Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloid Disease, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune multi-endocrine candidiasis ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis , Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, pulmonary emphysema, lymphocytic factor-induced incident lymphopenia, neonatal hemolytic disease (fetal erythroblastosis), erythema nodosum, atrophic gastritis, glomerulus Nephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis Scabies, pancreatitis, polymyositis, psoriasis, Reiter syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis , Include uveitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infection, bacterial infection, fungal infection, parasitic infection, protozoal infection, helminth infection, trauma, cell proliferation Abnormalities include actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, Primary thrombocythemia and cancers such as adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, nerve Node, digestive tract, heart, Includes kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterine cancer, and tubular acidosis, anemia, cushing in developmental disorders Syndrome, achondroplasia dwarfism, Duchenne / Becker muscular dystrophy, sputum, gonadal malformation, WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, mental retardation), Smith- Magenis syndrome ), Myelodysplastic syndromes, hereditary mucosal epithelial dysplasia, hereditary keratoses, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, Syndenham chorea ( Syndenham's chorea) and seizure disorders such as cerebral palsy, spinal cord fission, anencephalopathy, craniotomy, congenital glaucoma, cataract, sensorineural hearing loss, and endocrine disorders include primary brain Hypothalamic and pituitary disorders resulting from lesions such as tumors and adenomas, gestational infarctions, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immune abnormalities, complications due to head trauma, and decreased sexual function And disorders related to hypopituitarism and benign linetypes, including Sheehan syndrome, diabetes insipidus, Kalman disease, hand-Schuller Christian disease, letterer Zyve disease, sarcoidosis, empty sella syndrome, dwarfism Anti-diuretic hormone (ADH) incompatible secretion syndrome (SIADH) and acromegaly, disorders associated with hypopituitarism, including giantism, and goiter and myxedema, bacterial infectious thyroiditis, viral infectivity Disorders related to hypothyroidism, including subacute thyroiditis, autoimmune thyroiditis (Hashimoto's disease), cretinism, and thyroid poisoning and its various forms, grave Disease, anterior tibial myxedema, toxic multinodular goiter, thyroid cancer, hyperthyroidism including Plummer disease, hyperparathyroidism including Conn's disease (chronic hypercalemia), type I and type II diabetes and Pancreatic diseases such as complications, hyperplasia and adrenocortical carcinoma and adenoma, alkalosis-related hypertension, amyloidosis, hypokalemia, Cushing's disease, Riddle syndrome, Arnold-Healy-Gordon syndrome, brown cell tumor, Addison's disease Adrenal-related disorders such as adrenal insufficiency and abnormal prolactin production and infertility in women, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency (isolated gonadotropin) deficiency), amenorrhea, milk leakage, semi-middle yang, hirsutism and masculinization, breast cancer, postmenopausal osteoporosis, male Leydig cell hyperplasia, male menopause, reproductive details These include gonad steroid hormone related diseases such as cystic dysplasia, hypersexuality associated with Leydig cell tumor, androgen resistance associated with the absence of androgen receptor, 5α-reductase syndrome, gynecomastia and the like. For eye diseases, conjunctivitis, dry keratoconjunctivitis, keratitis, episclerosis, iritis, posterior uveitis, glaucoma, transient cataract, ischemic optic neuropathy, optic neuritis, Leber hereditary optic neuropathy, vitreous detachment , Retinal detachment, cataract, macular degeneration, central serous chorioretinopathy, retinitis pigmentosa (retinal pigment degeneration), choroidal melanoma, retrobulbar tumor, chiasmal tumor, metabolic disorders Among them are adrenal insufficiency, cerebral tendon xanthoma, adrenal cortical hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty cirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, thyroid Tumor, glucagonoma, glycogenosis, hereditary fructose intolerance, adrenal medulla hyperactivity, hypoadrenosis, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, thyroid Hypolipidemia, hyperlipidemia, lipid myopa -(Lipid myopathies), lipodystrophy, lysosomal storage disease, Menkes syndrome, occipital horn syndrome, mannosidosis, neuraminidase deficiency, obesity, pentose-phenylketonuria, pseudovitamin D deficiency , Hypocalcemia, hypophosphatemia, post-pubertal cerebellar ataxia, tyrosineemia, and gastrointestinal disorders, including dysphagia, peptic esophagitis, esophagus Convulsions, esophageal stricture, esophageal cancer, indigestion, digestive disorder, gastritis, stomach cancer, anorexia, nausea, vomiting, gastric paralysis, sinus or pyloric edema, abdominal angina, heartburn, gastroenteritis, ileus, intestinal infection, digestive Ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary tract disease, hepatitis, hyperbilirubinemia, hereditary hyperbilirubinemia, cirrhosis, hepatic passive congestion, hepatoma, infectious Enteritis, Ulcerative colitis, Ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, Colon cancer, Colon obstruction, Irritable bowel syndrome, Short bowel syndrome, Diarrhea, Constipation, Gastrointestinal bleeding, Acquired immune deficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatitis, hemochromatosis, Wilson's disease, alpha ₁ antitrypsin deficiency, Reye syndrome, primary sclerosing cholangitis, liver infarction, portal circulation obstruction and thrombus, Includes leaflet necrosis, liver purpura, hepatic venous thrombosis, hepatic vein occlusion, preeclampsia, eclampsia, gestational acute liver fat, gestational intrahepatic cholestasis, nodular regeneration and liver cancer including adenoma, carcinoma It is. The polynucleotide sequence encoding DME is a Southern method, Northern method, dot blot method, or other membrane technology, PCR method, or the like that uses body fluids or tissues collected from patients to detect altered DME expression. Can be used for dipstick, dipstick, pin, and multi-format ELISA assays, and microarrays. Such qualitative or quantitative methods are known in the art.

  In certain embodiments, nucleotide sequences encoding DME may be useful in assays that detect related disorders, particularly those mentioned above. Nucleotide sequences encoding DME can be labeled and added to body fluids or tissue samples taken from patients under conditions suitable for the formation of hybridization complexes by a variety of standard methods. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient sample is significantly changed compared to the control sample, the presence of a level change in the nucleotide sequence group encoding DME in the sample is indicative of the presence of the associated disorder. Such assays can also be used to estimate specific therapeutic effects in animal experiments, clinical trials, or to monitor individual patient treatment.

  In order to provide a basis for the diagnosis of disorders associated with the expression of DME, a normal or standard profile of expression is established. This is accomplished by mixing body fluids or cell extracts collected from either normal animals or human subjects with sequences encoding DME or fragments thereof under conditions suitable for hybridization or amplification. Can be achieved. Standard hybridization can be quantified by comparing values obtained from experiments conducted with known amounts of substantially purified polynucleotides to values obtained from normal subjects. The standard value thus obtained can be compared with the value obtained from a sample obtained from a patient showing signs of disease. Deviation from standard values is used to determine the presence of the disease.

  Once the presence of the disease is established and the treatment protocol is initiated, the hybridization assay can be repeated periodically to determine whether the patient's expression level has begun to approach levels observed in normal subjects. The results obtained from continuous assays can be used to show the effect of treatment over a period of days to months.

  For cancer, the presence of an abnormal amount of transcript (underexpressed or overexpressed) in living tissue from an individual indicates the predisposition to the disease or detects the disease before actual clinical symptoms appear A method may be provided. This type of clearer diagnosis allows medical professionals to take advantage of preventive or aggressive treatment early on, thereby preventing the development or further progression of cancer.

Use of oligonucleotides designed from sequences encoding DME for further diagnosis may include the use of PCR. These oligomers can be synthesized chemically, produced enzymatically, or produced in vitro . Oligomers preferably contain a fragment of a polynucleotide encoding DME, or a fragment of a polynucleotide complementary to a polynucleotide encoding DME, and are used to identify specific genes or conditions under optimized conditions Is done. Oligomers can also be used for the detection, quantification, or both of closely related DNA or RNA sequences under moderately stringent conditions.

  In certain embodiments, oligonucleotide primers derived from polynucleotide sequences encoding DME can be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that often cause human congenital or acquired genetic diseases. Although not limited, SNP detection methods include single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP). In SSCP, DNA is amplified by polymerase chain reaction (PCR) using oligonucleotide primers derived from polynucleotide sequences encoding DME. DNA can be derived from, for example, diseased or normal tissue, biopsy samples, body fluids, and the like. SNPs in DNA cause differences in the secondary and tertiary structure of single-stranded PCR products. Differences can be detected using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primer is fluorescently labeled. As a result, the amplimer can be detected by a high-throughput device such as a DNA sequencing machine. Furthermore, a sequence database analysis method called in silico SNP (in silico SNP, isSNP) is used to compare polymorphisms by comparing the sequences of individual overlapping DNA fragments that are arranged in a general consensus sequence. Can be identified. These computer-based methods filter out sequence variations due to sequencing errors using the laboratory preparation of DNA and automated analysis of statistical models and DNA sequence chromatograms. In another embodiment, SNPs are detected and characterized, for example, by mass spectrometry using a high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).

  SNPs can be beneficial for studying the genetic basis of human disease. For example, at least 16 common SNPs are associated with non-insulin dependent diabetes mellitus. SNPs are also useful for studying the differences in outcomes of single gene diseases such as cystic fibrosis, sickle cell anemia, and chronic granulomatous disease. For example, a variant on mannose-binding lectin (MBL2) has been found to correlate with adverse outcomes in cystic fibrosis lungs. SNPs also have utility in pharmacogenomics. Pharmacogenomics identifies genetic variants that affect a patient's response to certain drugs, such as life-threatening toxicities. For example, some mutations in N-acetyltransferase increase the incidence of peripheral neuropathy in response to the antituberculosis agent isoniazid, whereas mutations in the core promoter of the ALOX5 gene are anti-asthma drugs that target the 5-lipoxygenase pathway. Reduces clinical response to treatment. Analyzing the distribution of SNPs in different populations is useful for studying genetic drift, mutation, recombination and selection, as well as investigating the origin and migration of populations (Taylor, JG 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 that can be used to quantify DME expression include radiolabeling or biotin labeling of nucleotides, coamplification of control nucleic acids, and interpolation of results obtained from standard curves (eg, Melby, PC et al. (1993) J. Immunol. Methods 159: 235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236). Accelerate the quantification rate of multiple samples by performing high-throughput assays where the oligomer of interest is present in various dilutions and the quantification is rapid by spectrophotometric or colorimetric reactions .

  In yet another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein can be used as elements in the microarray. Microarrays can be used in transcriptional imaging techniques that simultaneously monitor the associated expression levels of multiple genes. This is described below. Microarrays can also be used to identify genetic variants, mutations and polymorphisms. Use this information to determine gene function, understand the genetic basis of the disease, diagnose the disease, monitor disease progression / regression as a function of gene expression, and develop drug activity in disease treatment And can be monitored. In particular, this information can be used to develop a pharmacogenomic profile of the patient in order to select the most appropriate and effective treatment for the patient. For example, based on a patient's pharmacogenomic profile, a therapeutic agent that is highly effective for the patient and that exhibits few side effects can be selected.

  In another embodiment, DME, DME fragments or antibodies specific for DME may be used as elements on a microarray. Microarrays can be used to monitor or measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.

  Certain embodiments relate to the use of the polynucleotides of the invention to generate a transcription image of a tissue or cell type. A transcript image represents a global pattern of gene expression by a particular tissue or cell type. Comprehensive gene expression patterns are analyzed by quantifying the number and relative abundance of genes expressed at a given time under a given condition (see “Comparative Gene Transcript Analysis” of Seilhamer et al., US Pat. No. 5,840,484). Reference is hereby incorporated by reference). Thus, a transcription image can be generated by hybridizing a polynucleotide of the present invention or a complement thereof to the entire transcript or reverse transcript of a particular tissue or cell type. In certain embodiments, hybridization is generated in a high throughput format such that a polynucleotide of the invention or its complement has a subset of multiple elements on a microarray. The resulting transcript image can provide a profile of gene activity.

Transcript images can be generated using transcripts isolated from tissues, cell lines, biopsies or biological samples thereof. The transcript image thus reflects gene expression in vivo for tissue or biopsy samples and in vitro for cell lines.

  Transcript images that produce expression profiles of polynucleotides of the invention can also be used in conjunction with in vitro model systems and preclinical evaluation of drugs as well as industrial or natural environmental compound toxicity tests. All compounds elicit characteristic gene expression patterns, often referred to as molecular fingerprints or toxicant signatures, that indicate mechanisms of action and toxicity (Nuwaysir, EF et al. (1999) Mol. Carcinog. 24 : 15 3-159, Steiner, S. and NL Anderson (2000) Toxicol. Lett. 112-113: 467-471, which is hereby specifically incorporated by reference). If a test compound has the same signature as that of a compound with known toxicity, it may share toxic properties. A fingerprint or signature is most useful and accurate when it contains expression information from multiple genes and gene families. Ideally, measurement of expression across the genome provides the highest quality signature. Even if there are genes whose expression is not altered by any tested compound, those genes are important because their expression levels can be used to normalize the remaining expression data. The normalize procedure is useful for comparing expression data after treatment with different compounds. Assigning gene function to elements of a toxicity signature helps to interpret toxicity mechanisms, but knowledge of gene function is not required for statistical matching of signature groups leading to toxicity prediction (eg, February 29, 2000) See Press Release 00-02 issued by the National Institute of Environmental Health Sciences at http://www.niehs.nih.gov/oc/news/toxchip. available at .htm). Thus, it is important and desirable to include all expressed gene sequences in toxicological screening using toxicity signatures.

  In one embodiment, the toxicity of a test compound is calculated by treating a biological sample containing nucleic acid with the test compound. Nucleic acid expressed in the treated biological sample can be hybridized with one or more probes specific for the polynucleotide of the invention, thereby quantifying the transcription level corresponding to the polynucleotide of the invention. The transcript level in the treated biological sample is compared to the level in the untreated biological sample. The difference in transcript levels between the two samples is indicative of the toxic response caused by the test compound in the treated sample.

  Another embodiment relates to analyzing the proteome of a tissue or cell type using the polypeptide sequences of the present invention. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of the proteome can be individually subject to further analysis. Proteome expression patterns or profiles can be analyzed by quantifying the number and relative abundance of proteins expressed at a given time under a given condition. Thus, a proteomic profile of a cell can be generated by separating and analyzing a polypeptide of a particular tissue or cell type. In some embodiments, the separation is achieved by two-dimensional gel electrophoresis in which the protein is separated from the sample by one-dimensional isoelectric focusing and separated according to molecular weight by two-dimensional sodium dodecyl sulfate slab gel electrophoresis. (Steiner and Anderson, above). Proteins are visualized in the gel as dispersed, unique points by staining the gel with a substance such as Coomassie Blue, or a silver stain or fluorescent stain. The optical density of each protein spot is usually proportional to the protein level in the sample. Compare the optical density of equally located protein spots from different samples, such as biological samples treated or untreated with test compounds or therapeutic agents, and identify changes in protein spot density associated with treatment To do. The proteins in the spot are partially sequenced using standard methods, for example using chemical or enzymatic cleavage followed by mass spectrometry. The identity of a protein within a spot can be determined by comparing its subsequence, preferably at least 5 consecutive amino acid residues, to the polypeptide sequence of the invention. In some cases, additional sequence data is obtained for definitive protein identification.

  Proteomic profiles can also be generated by quantifying DME expression levels using antibodies specific for DME. In one embodiment, protein expression levels are quantified by using antibodies as elements on the microarray and exposing the microarray to the sample to detect the level of protein binding to each array element (Lueking, A. et al. (1999) Anal Biochern. 270: 103-111, Mendoze, LG et al. (1999) Biotechniques 27: 778-788). Detection can be performed in a variety of ways known in the art, for example, a thiol or amino-reactive fluorescent compound can be reacted with a protein in a sample to detect the amount of fluorescent binding in each array element.

  Toxicity signatures at the proteome level are also useful for toxicological screening and should be analyzed in parallel with the toxicity signature at the transcriptional level. For some proteins in some tissues, the transcript and protein abundance may be poorly correlated (Anderson, NL and J. Seilhamer (1997) Electrophoresis 18: 533-537) Proteome toxicity signatures may be useful in the analysis of compounds that do not significantly affect the proteome but alter the proteome profile. Furthermore, since analysis of transcripts in body fluids is difficult due to rapid degradation of mRNA, proteome profiling can be more reliable and informative in such cases.

  In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. Proteins 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 the untreated biological sample. The difference in the amount of protein in both samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these subsequences to the polypeptides of the invention.

  In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. The protein obtained from the biological sample is incubated with an antibody specific for the polypeptide of the present invention. The amount of protein recognized by the antibody is quantified. The amount of protein in the treated biological sample is compared to the amount of protein in the untreated biological sample. The difference in the amount of protein in both samples is indicative of a toxic response to the test compound in the treated sample.

Microarrays are prepared, used and analyzed using methods known in the art (Brennan, TM et al. (1995), US Pat. No. 5,474,796, Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619, Baldeschweiler et al. (1995) PCT application WO95 / 251116, Shalon, D. et al. (1995) PCT application WO95 / 35505, Heller, RA et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155, Heller, MJ et al. (1997) US Pat. No. 5,605,662 etc.). Various types of microarrays are known and are described in detail in DNA Microarrays: A Practical Approach , M. Schena, edited. (1999) Oxford University Press, London. This document is incorporated herein by reference in particular.

  In another embodiment of the invention, a group of nucleic acid sequences encoding DME can be used to generate a group of hybridization probes useful for mapping native genomic sequences. Either a coding sequence or a non-coding sequence can be used, and in some cases a non-coding sequence is preferred over a coding sequence. For example, conservation of coding sequences within members of a multigene family can cause unwanted cross-hybridization during chromosomal mapping. Nucleic acid sequences can be specific chromosomes, specific regions of chromosomes or artificially formed chromosomes, eg, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacterial P1 products, or single chromosome cDNA (E.g. Harrington, JJ et al. (1997) Nat. Genet. 15: 345-355; Price, CM (1993) Blood Rev. 7: 127-134; and Trask, BJ (1991) Trends) Genet. 7: 149--154). Once mapped, a genetic linkage map can be generated using the nucleic acid sequences of the present invention to correlate, for example, the inheritance of a disease state with the inheritance of a particular chromosomal region or restriction fragment length polymorphism (RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83: 7353-7357).

Fluorescence in situ hybridization (FISH) can be correlated with other physical and genetic map data (see, for example, Heinz-Ulrich, et al. (1995) in Meyers, 965-968, supra). Examples of genetic map data can be found in various scientific journals or the Online Mendelian Inheritance in Man (OMIM) website. Because the correlation between the location of the gene encoding DME on a physical chromosomal map and a specific disease or a predisposition to a specific disease can help determine the region of DNA associated with this disease , Can facilitate the cloning work to determine the location.

  The genetic map can be expanded using physical mapping techniques such as linkage analysis using established chromosomal markers and chromosomal specimen in situ hybridization. By placing a gene on the chromosome of another mammal, such as a mouse, the associated markers can often be revealed even if the exact chromosomal locus is unknown. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. When a gene involved in a disease or syndrome is roughly positioned by genetic linkage to a particular gene region, such as the 11q22-23 region of vasodilatory ataxia, any sequence that maps to that region is: It may indicate relevant or regulatory genes for further investigation (see Gatti, RA et al. (1988) Nature 336: 577-580). The nucleotide sequence of the present invention may be used to find a difference in the chromosomal position among the healthy person, the owner, and the infected person due to translocation, inversion and the like.

  In another embodiment of the invention, DME, its catalytic fragments, or immunogen fragments, or oligopeptides thereof may be used for screening libraries of compounds in any of a variety of drug screening techniques. Fragments used for drug screening can be free in solution, fixed to a solid support, retained on the cell surface, or located intracellularly. Complex formation due to binding of DME to the drug being tested can be measured.

  Another drug screening method is used to screen compounds with suitable binding affinity for the protein of interest with high throughput (see, eg, Geysen, et al. (1984) PCT application WO84 / 03564). In this method, a large number of different small molecule test compounds are synthesized on a solid substrate. The test compound reacts with DME or a fragment thereof and then washed. The bound DME is then detected by various methods well known in the art. Purified DME can also be coated directly onto plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize the peptide on a solid support.

  In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of specifically binding DME compete with the test compound for binding to DME. In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with DME.

  In another embodiment, nucleotide sequences encoding DME are molecular biology techniques that will be developed in the future and are characterized by, but not limited to, the properties of currently known nucleotide sequences (including but not limited to triplet genetic code and specific It can be used for new technologies that depend on base-pairing interactions).

  Without further details, those skilled in the art will be able to make full use of the present invention with the above description. Accordingly, the examples described below are for illustrative purposes only and do not limit the invention in any way.

  All patents, patent applications and publications disclosed herein are U.S. Patent Application Nos. 60 / 269.643, 60 / 271.332, 60 / 276.767, 60 / 282.077, 60 / 285.447, References, including 60 / 287.060 and 60 / 288.543, are specifically incorporated herein by reference.

1 Preparation of cDNA library
The Incyte cDNA group is derived from the cDNA library group described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and dissolved in guanidinium isothiocyanate solution, and other tissues were homogenized and dissolved in phenol or in a suitable mixture of modifiers. TRIZOL (Life Technologies), which is an example of a mixed solution, is a single-phase solution of phenol and guanidine isothiocyanate. The resulting lysate was layered over a cesium chloride cushion solution and centrifuged or extracted with chloroform. RNA was precipitated from the lysate using either isopropanol, sodium acetate and ethanol, or another method.

  In order to increase the purity of RNA, extraction and precipitation of RNA with phenol was repeated as many times as necessary. In some cases, RNA was treated with DNase. In most libraries, poly (A +) RNA was isolated using oligo d (T) -linked paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA) or OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit, such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

  In some cases, RNA was provided to Stratagene and Stratagene produced the corresponding cDNA library. If not, cDNA was synthesized using the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies) using the recommended or similar method known in the art, and a cDNA library was prepared (Ausubel, supra). , 1997, 5.1-6.6 units, etc.). Reverse transcription was initiated using oligo d (T) or random primers. A synthetic oligonucleotide adapter was ligated to a double stranded cDNA and the cDNA was digested with a suitable restriction enzyme. For most libraries, cDNA size selection (300-1000 bp) was performed using SEPHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. The cDNA was ligated into compatible restriction enzyme sites of a suitable plasmid polylinker. Suitable plasmids include, for example, PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics) or pINCY (Incyte Genomics), or derivatives thereof. The recombinant plasmid was transformed into a suitable E. coli cell such as Stratagene XL1-Blue, XL1-BIueMRF or SOLR, or Life Technologies DH5α, DH10B or ELECTROMAX DH10B.

2 Isolation of cDNA clones
The plasmid obtained as in Example 1 was recovered from the host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. At least one of the following was used for purification of the plasmid. That is, Magic or WIZARD Minipreps DNA purification system (Promega), AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD), QIAWELL QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid and QIAWELL 8 Ultra Plasmid purification system, REAL Prep 96 plasmid purification kit Either. The plasmid was precipitated and then resuspended in 0.1 ml distilled water and stored at 4 ° C. either lyophilized or not lyophilized.

  Alternatively, plasmid DNA was amplified from host cell lysates using direct binding PCR in a high throughput format (Rao, V.B. (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples are processed and stored in 384-well plates and the concentration of amplified plasmid DNA is determined fluorimetrically using PICOGREEN dye (Molecular Probes, Eugene OR) and FLUOROSKAN2 fluorescence scanner (Labsystems Oy, Helsinki, Finland) Quantified.

3 Sequencing and analysis
Incyte cDNA recovered from the plasmid as described in Example 2 was sequenced as shown below. Sequencing of cDNA can be performed using standard methods or high-throughput equipment such as the ABI CATALYST 800 thermal cycler (Applied Biosystems) or the PTC-200 thermal cycler (MJ Research) or the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) liquid transfer. Processed in conjunction with the system. The cDNA sequencing reaction was prepared using a reagent provided by Amersham Pharmacia Biotech, or an ABI sequencing kit such as ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). For electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides, use the MEGABACE 1000 DNA sequencing system (Molecular Dynamics) or an ABI PRISM 373 or 377 sequence using standard ABI protocol and base calling software. Thing systems (Applied Biosystems) or other sequence analysis systems known in the art were used. Reading frames within the cDNA sequences were determined using standard methods (reviewed in Ausubel, 1997, 7.7 units supra). Several of the cDNA sequences were selected and the sequences were extended by the method described in Example 8 .

Polynucleotide sequences derived from Incyte cDNA sequences were validated by removing vector, linker and poly (A) sequences and masking ambiguous base pairs. In doing so, algorithms and programs based on BLAST, dynamic programming and adjacent dinucleotide frequency analysis were used. Secondly, Incyte cDNA sequences, or translations thereof, into public databases (eg GenBank primates and rodents, mammals, vertebrates, eukaryotes, BLOCKS, PRINTS, DOMO, PRODOM) and humans PROTEOME database (Incyte Genomics, Palo Alto CA) containing sequences from rat, mouse, nematode, budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), and Candida albicans, and hidden marks such as PFAM Protein family database based on model (HMM) and SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95: 5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30: 242- We queried against a database selected from HMM-based protein domain databases such as 244). (HMM is a probabilistic approach to analyzing the consensus primary structure of gene families. See Eddy, SR (1996) Cuff. Opin. Struct. Biol. 6: 361-365, etc.). Inquiries were made using programs based on BLAST, FASTA, BLIMPS and HMMER. The Incyte cDNA sequence was constructed to yield the full length polynucleotide sequence. Alternatively, the population of Incyte cDNA was extended to full length using GenBank cDNA, GenBank EST, stitched sequences, stretched sequences or Genscan predicted coding sequences (see Examples 4 and 5 ). It was constructed using programs based on Phred, Phrap and Consed, and a population of cDNA was screened for open reading frames using programs based on GenMark, BLAST and FASTA. The full length polynucleotide sequence was translated to derive the corresponding full length polypeptide sequence. Alternatively, the polypeptides of the present invention can begin at any methionine residue of the full-length translated polypeptide. Subsequently, GenBank protein database (genpept), SwissProt, PROTEOME database, BLOCKS, PRINTS, DOMO, PRODOM and Prosite database, protein family database based on hidden Markov model (HMM) such as PFAM, and HMM like SMART The full-length polypeptide sequence was analyzed by querying a protein domain database based on. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm, such as that incorporated into the MEGALIGN multi-sequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.

  Table 7 gives an overview of the tools, programs and algorithms used for analysis and assembly (construction) of Incyte cDNA and full-length sequences, as well as applicable descriptions, references and threshold parameters. The tools, programs and algorithms used are shown in column 1 of Table 7 and a brief description thereof is shown in column 2. Column 3 is a preferred reference, all of which are incorporated herein by reference in their entirety. Where applicable, column 4 indicates the score, probability value, and other parameters used to evaluate the strength with which the two sequences match (the higher the score or the lower the probability value, the 2 High identity between sequences).

  The above programs used for the assembly and analysis of full-length polynucleotide and polypeptide sequences can also be used to identify polynucleotide sequence fragments of SEQ ID NO: 13-24. A fragment of about 20 to about 4000 nucleotides useful for hybridization and amplification techniques is shown in column 4 of Table 2.

4 Identification and editing of coding sequences from genomic DNA Putative drug metabolizing enzymes were first identified by running the Genscan gene identification program in public genomic sequence databases (eg gbpri and gbhtg). Genscan is a universal gene identification program that analyzes genomic DNA sequences from various organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268: 78-94 and Burge, C. and S Karlin (1998) Cuff. Opin. Struct. Biol. 8: 346-354). The program links predicted exons to form a constructed cDNA sequence that spans from methionine to the stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequences that Genscan analyzes at one time was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode a drug metabolizing enzyme, the encoded polypeptide was interrogated and analyzed for drug metabolizing enzymes in a PFAM model. Potential drug metabolizing enzymes were also identified based on their homology to the Incyte cDNA sequence already annotated as drug metabolizing enzymes. The Genscan predicted sequences thus selected were then compared to the public databases genpept and gbpri by BLAST analysis. If necessary, Genscan predicted sequences were edited by comparison with the top BLAST hits from genpept to correct sequence errors predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find public cDNA coverage of any Incyte cDNA or Genscan predicted sequence, thus providing evidence of transcription. . This information was used to correct or confirm the Genscan predicted sequence when the Incyte cDNA coverage was available. Full-length polynucleotide sequences were obtained by constructing Genscan predicted coding sequences with Incyte cDNA sequences and / or public cDNA sequences using the construction process described in Example 3 . Alternatively, the full-length polynucleotide sequence is completely derived from an edited or unedited Genscan predicted coding sequence.

5 Integration of genomic sequence data with cDNA sequence data (assembly)
Stitched sequence
The partial cDNA sequence was extended using exons predicted by the Genscan gene identification program described in Example 4 . Partial cDNAs constructed as described in Example 3 were mapped to genomic DNA and resolved into clusters containing the relevant cDNA and Genscan exons predicted from one or more genomic sequences. Analyze each cluster using graph theory and dynamic programming based algorithms to integrate cDNA and genomic information, and then generate potential splice variants that can be confirmed, edited or extended to yield full-length sequences. did. Sequence sections were identified such that the entire length of the section was present in two or more sequences in the cluster, and the sections so identified were considered equal due to transitivity. For example, if a section exists in one cDNA and two genomic sequences, all three sections were considered equal. This process can link unrelated but contiguous genomic sequences together by cDNA sequences. The sections thus identified are stitched together by a stitch algorithm in the order in which they appear along the parent sequence to produce the longest possible sequence and variant sequence. The linkage (cDNA-cDNA or genomic sequence-genomic sequence) between the intervals generated along one type of parent sequence prevailed over the linkage (cDNA-genomic sequence) that changes the type of parent. The resulting stitch sequences were translated and compared with the public databases genpept and gbpri by BLAST analysis. The incorrect exon predicted by Genscan was corrected by comparing it to the top BLAST hit from genpept. If necessary, the sequences were further extended using additional cDNA sequences or by examination of genomic DNA.

Stretched sequence
The partial DNA sequence was extended to full length by an algorithm based on BLAST analysis. First, using the BLAST program, we query the public databases such as GenBank primate, rodent, mammal, vertebrate and eukaryote databases for the partial cDNA constructed as described in Example 3. It was. The nearest GenBank protein homologue was then compared to either the Incyte cDNA sequence or the GenScan exon predicted sequence described in Example 4 by BLAST analysis. The resulting high scoring segment pair (HSP) was used to produce a chimeric protein and the translated sequence was mapped onto the GenBank protein homologue. Insertions or deletions can occur in the chimeric protein compared to the original GenBank protein homologue. Homologous genomic sequences were searched from public human genome databases using GenBank protein homologues, chimeric proteins or both as probes. In this way, the partial DNA sequence was stretched or extended by the addition of homologous genomic sequences. The resulting stretch sequence was examined to determine whether it contained the complete gene.

6 Chromosome mapping of polynucleotides encoding DME
The sequences used to construct SEQ ID NO: 13-24 were compared to sequences in the Incyte LIFESEQ database and the public domain database using BLAST and other Smith-Waterman algorithm implementations. Sequences in these databases that matched SEQ ID NO: 13-24 were incorporated into clusters of contiguous and overlapping sequences using a construction algorithm such as Phrap (Table 7). Clustered sequences have already been mapped using radiation hybrids and genetic map data available from public sources such as the Stanford Human Genome Center (SHGC), Whitehead Genome Research Institute (WIGR), and Genethon. I confirmed. When a mapped sequence was included in a cluster, the entire sequence of that cluster was assigned to a map location along with the individual sequence numbers.

  The position on the map is expressed as a range or section of a human chromosome. The location on the map of a section in centimorgan units is measured with respect to the end of the p-arm of the chromosome (centiorgan (cM) is a unit of measure based on the frequency of recombination between chromosomal markers. 1 cM is approximately equal to one megabase (Mb) of DNA in humans, although this value varies widely with recombination hot and cold spots). The cM distance is based on genetic markers mapped by Genethon such that the sequence is a boundary for radiation hybrid markers such that they are contained within each cluster.

  Disease genes that have already been identified using publicly available human gene maps and other sources such as NCBI “GeneMap'99” (http: //www.ncbi.nlm.nih.gpv/genemap/) It can be determined whether a class is mapped within or near the above interval.

7 Analysis of Polynucleotide Expression Northern analysis is an experimental technique used to detect the presence of transcribed genetic information, in which the labeled nucleotide sequence to the membrane to which RNA from a particular cell type or tissue is bound. Involved in hybridization. (See Sambrook, Chapter 7; Ausubel. FM et al., Chapters 4 and 16).

  Using similar computer techniques applying BLAST, the same or related molecules were searched in cDNA databases such as GenBank and LifeSeq (Incyte Genomics). Northern analysis is much faster than many membrane-based hybridizations. Furthermore, the sensitivity of the computer search can be changed to determine whether a particular match is classified as an exact match or a similar match. The search criterion is a product score, which is defined by the following equation.

  The product score takes into account both the similarity between the two sequences and the length with which the sequences match. The product score is a normalized value of 0 to 100, and is obtained as follows. Multiply the BLAST score by the nucleotide sequence identity and divide the product by 5 times the shorter length of the two sequences. A BLAST score is calculated by assigning a score of +5 to each base that matches a high scoring segment pair (HSP) and assigning -4 to each mismatch base pair. Two sequences can share two or more HSPs (separated by gaps). If there are two or more HSPs, the product score is calculated using the segment pair with the highest BLAST score. The product score represents the balance between the fractional overlap and the quality of the BLAST alignment. For example, a product score of 100 is obtained only if there is a 100% match over the shorter length of the two sequences compared. The product score 70 is obtained when one end is 100% coincident and 70% overlap, or the other end is 88% coincident and 100% overlap. The product score 50 is obtained when either one end is 100% coincident and 50% overlap, or the other end is 79% coincident and 100% overlap.

Alternatively, the polynucleotide sequence encoding DME is analyzed against the tissue from which it was derived. For example, certain full-length sequences are constructed to at least partially overlap with the Incyte cDNA sequence (see Example 3 ). Each cDNA sequence is derived from a cDNA library made from human tissue. Each human tissue consists of cardiovascular system, connective tissue, digestive system, embryonic structure, endocrine system, exocrine gland, female genital organ, male genital organ, germ cell, blood and immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory Classify into one organ / tissue category such as organ system, sensory organ, skin, stomatognathic system, unclassifiable / mixed, or urinary tract. Count the number of libraries in each category and divide by the total number of libraries in all categories. Similarly, each human tissue is classified into one of the following disease / condition categories: cancer, cell line, development, inflammation, nervousness, trauma, cardiovascular, pool, etc. Count the number of libraries in each category and divide by the total number of libraries in all categories. The resulting percentage reflects tissue-specific and disease-specific expression of cDNA encoding DME. cDNA sequences and cDNA library / tissue information can be obtained from the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).

8 Extended full-length polynucleotide sequence of a polynucleotide encoding DME was also generated by extending the fragment using multiple oligonucleotide primers designed from the appropriate fragment of the full-length molecule. One primer was synthesized to initiate 5 ′ extension of a known fragment and the other primer was synthesized to initiate 3 ′ extension of a known fragment. The starting primer is about 22-30 nucleotides in length, has a GC content of about 50% or more, and is annealed to the target sequence at a temperature of about 68-72 ° C or OLIGO 4.06 software (National Biosciences) or another suitable Designed using a simple program. All nucleotide extensions that resulted in hairpin structures and primer-primer dimers were avoided.

  A selected human cDNA library was used to extend the sequence. Where more than one extension was necessary or desirable, additional primers or nested sets of primers were designed.

High fidelity amplification was obtained by PCR using methods well known to those skilled in the art. PCR was performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture has template DNA and 200 nmol of each primer. It also contains a buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), Pfu DNA polymerase (Stratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. Step 1: 94 ° C, 3 minutes, Step 2: 94 ° C, 15 seconds, Step 3: 60 ° C, 1 minute, Step 4: 68 ° C, 2 minutes, Step 5: Repeat steps 2, 3, and 4 20 times . Step 6: Store at 68 ℃, 5 minutes, Step 7: Store at 4 ℃. Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters: Step 1: 94 ° C, 3 minutes, Step 2: 94 ° C, 15 seconds, Step 3: 57 ° C, 1 minute, Step 4: 68 ° C, 2 minutes, Step 5: Repeat steps 2, 3, and 4 20 times . Step 6: Store at 68 ° C for 5 minutes, Step 7: Store at 4 ° C.

  The DNA concentration in each well is 1 × TE and 100 μl PICOGREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 0.5 μl undiluted PCR product. (Coming Costar, Acton MA) was distributed to each well so that DNA could bind to the reagent. Plates were scanned with Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure sample fluorescence and quantify DNA concentration. An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a 1% agarose minigel to determine which reaction was successful in extending the sequence.

  The extended nucleotide is desalted and concentrated, transferred to a 384-well plate, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and before religation reaction to pUC 18 vector (Amersham Pharmacia Biotech). Sonicated or sheared. For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments were excised, and the agar was digested with Agar ACE (Promega). The extended clone was religated to the pUC 18 vector (Amersham Pharmacia Biotech) using T4 ligase (New England Biolabs, Beverly MA), treated with Pfu DNA polymerase (Stratagene) to fill the restriction site overhang, Cells were transfected. The transformed cells were selected and transferred to a medium containing antibiotics, and each colony was excised and cultured overnight at 37 ° C. in a 384 well plate of LB / 2X carbenicillin culture solution.

  Cells were lysed and DNA was PCR amplified using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) as follows. Step 1: 94 ° C, 3 minutes, Step 2: 94 ° C, 15 seconds, Step 3: 60 ° C, 1 minute, Step 4: 72 ° C, 2 minutes, Step 5: Repeat steps 2, 3, and 4 29 times . Step 6: Store at 72 ° C for 5 minutes, Step 7: Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recovery were reamplified using the same conditions as above. Samples are diluted with 20% dimethyl sulfoxide (1: 2, v / v), and the DYENAMIC energy transfer sequencing primer and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE terminator cycle sequencing reaction kit (Terminator cycle sequencing ready) reaction kit) (Applied Biosystems).

  Similarly, full-length nucleotide sequences are verified using the above procedure, or 5 ′ regulatory sequences are obtained using oligonucleotides designed for such extensions and appropriate genomic libraries.

9 Identification of SNP (Single Nucleotide Polymorphism) in Polynucleotides Encoding DME A common DNA sequence variant known as single nucleotide polymorphism (SNP) is SEQ ID NO: 13- using the LIFESEQ database (Incyte Genomics). Identified in 24. As described in Example 3 , sequences from the same gene were constructed together in clusters, which allowed identification of all sequence variants within the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. The pre-filter eliminates the majority of base call errors by requiring a minimum Phred quality score of 15, and also introduces sequence alignment errors and errors caused by improper trimming of vector sequences, chimeras and splice variants Removed. The original chromatogram file in the vicinity of the putative SNP was analyzed by an automated procedure for advanced chromosome analysis. Clone error filters used statistically generated algorithms to identify errors introduced during the experimental process, such as those caused by reverse transcriptase, polymerase, or somatic mutations. The cluster error filter used statistically generated algorithms to identify errors due to clustering of related homologs or pseudogenes, or errors caused by contamination with non-human sequences. The last group of filters removed duplicates and SNPs present in immunoglobulins or T cell receptors.

  Several SNPs were selected for further characterization by mass spectrometry using the high-speed MASSARRAY system (Sequenom, Inc.) and analyzed for allelic frequency at SNP sites in four different human populations. The white population consisted of 92 people (46 men and 46 women), including 83 Utah, 4 French, 3 Venezuela and 2 Amish. The African population consists of 194 African-Americans (97 men and 97 women). The Hispanic population is comprised of 324 Mexican Hispanics (162 men and 162 women). The Asian population is composed of 126 people (64 men and 62 women), with parent breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asians. Has been. The frequency of alleles was first analyzed in the white population, and in some cases, SNPs that did not show allelic variance in this population were not further examined in the other three populations.

10 Labeling and use of individual hybridization probes
CDNA, mRNA, or genomic DNA is screened using a hybridization probe derived from SEQ ID NO: 13-24. Although specifically described for labeling oligonucleotides of about 20 base pairs, virtually the same procedure is used for larger nucleotide fragments. Oligonucleotides were designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences), each oligomer 50 pmol, [γ- ³² P] adenosine triphosphate (Amersham Pharmacia Biotech) 250 μCi, T4 polynucleotide kinase Label by mixing (DuPont NEN, Boston MA). The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia Biotech). In a typical membrane-based hybridization analysis of human genomic DNA digested with any one of Ase I, Bgl II, Eco RI, Pst I, Xba1 or Pvu II (DuPont NEN), 10 ⁷ counts per minute An aliquot containing a labeled probe is used.

  DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are washed sequentially at room temperature, for example under conditions consistent with 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. Visualize and compare hybridization patterns using autoradiography or alternative imaging means.

11 Microarray Binding or synthesis of array elements on a microarray can be accomplished using photolithography, piezoelectric printing (see inkjet printing, Baldeschweiler, etc.), mechanical microspotting techniques and derivatives thereof. It is. In each of the above technologies, the substrate should be a solid with a uniform, non-porous surface (Schena (1999) supra). Recommended substrates include silicon, silica, glass slides, glass chips and silicon wafers. Alternatively, elements similar to dot blot or slot blot methods may be utilized to place and bond elements to the surface of the substrate using thermal, ultraviolet, chemical or mechanical bonding procedures. Conventional arrays can be made using available methods and machines well known to those skilled in the art and can have any suitable number of elements (eg, Schena, M. et al. (1995) Science 270: 467-470, Shalon D. et al. (1996) Genome Res. 6: 639-645, Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16: 27-31).

  Full-length cDNAs, expressed sequence tags (ESTs), or fragments or oligomers thereof can be microarray elements. Fragments or oligomers suitable for hybridization can be selected using software known in the art such as Laser GENE software (DNASTAR). The array element is hybridized with the polynucleotide in the biological sample. The polynucleotide in the biological sample is conjugated to a fluorescent label or other molecular tag to facilitate detection. Following hybridization, unhybridized nucleotides from the biological sample are removed and hybridization at each array element is detected using a fluorescent scanner. Alternatively, hybridization can be detected using laser desorption and mass spectrometry. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to elements on the microarray can be calculated. The preparation and use of the microarray in one example is detailed 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 consists of MMLV reverse transcriptase, 0.05 pg / μl oligo (dT) primer (21mer), 1 × first strand buffer, 0.03 unit / μl RNase inhibitor, 500 μM Reverse transcription using dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed using a GEMBRIGHT kit (Incyte) in a volume of 25 ml containing 200 ng poly (A) ⁺ RNA. Specific control poly (A) ⁺ RNA is synthesized from non-coding yeast genomic DNA by in vitro transcription. After incubation at 37 ° C for 2 hours, each reaction sample (one Cy3 and the other Cy5 labeled) was treated with 2.5 ml of 0.5M sodium hydroxide and incubated at 85 ° C for 20 minutes to react. To break down RNA. Samples are purified using two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA). After mixing, the two reaction samples are ethanol precipitated using 1 ml glycogen (1 mg / ml), 60 ml sodium acetate and 300 ml 100% ethanol. The sample is then completely dried using SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl of 5 × SSC / 0.2% SDS.

Microarray Preparation Array elements are made using the sequences of the present invention. Each array element is amplified from bacterial cells containing a vector with a cloned cDNA insert. PCR amplification uses primers complementary to the vector sequence located on the side of the cDNA insert. Array elements are amplified from an initial amount of 1-2 ng to a final amount greater than 5 μg in 30 cycles of PCR. Amplified array elements are purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).

  The purified array element is fixed on a polymer-coated slide glass. Microscope slides (Corning) are sonicated in 0.1% SDS and acetone during and after treatment and thoroughly washed with distilled water. The slides were etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed thoroughly in distilled water, and 0.05% aminopropylsilane (Sigma) in 95% ethanol. ) To coat. The coated glass slide is cured in an oven at 110 ° C.

  Array elements are added to the coated glass substrate using the method described in US Pat. No. 5,807,522. This patent is hereby incorporated by reference. 1 μl of array element DNA having an average concentration of 100 ng / μl is filled into an open capillary printing element by a high-speed robot apparatus. The instrument now adds approximately 5 nl of array element samples per slide.

  The microarray is UV crosslinked using a STRATALINKER UV crosslinking agent (Stratagene). The microarray is washed once with 0.2% SDS at room temperature and three times with distilled water. After incubating the microarray for 30 minutes at 60 ° C. in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA), 0.2% SDS and Nonspecific binding sites are blocked by washing with distilled water.

The hybridization hybridization reaction solution contained 9 μl of a sample mixture containing 0.2 μg of each of the Cy3 and Cy5 labeled cDNA synthesis products in 5 × SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65 ° C. for 5 minutes, aliquoted onto the microarray surface and covered with a 1.8 cm ² cover glass. The array is transferred to a waterproof chamber with a cavity slightly larger than the microscope slide. Keep the chamber interior at 100 humidity by adding 140 μl of 5 × SSC to the corner of the chamber. The chamber containing the array is incubated at 60 ° C. for about 6.5 hours. The array was washed in the first wash buffer (1 × SSC, 0.1% SDS) for 10 minutes at 45 ° C. and in the second wash buffer (0.1 × SSC) for 10 minutes each at 45 ° C. 3 Wash once and dry.

The detection reporter-labeled hybridization complex is an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara CA) capable of generating spectral lines at 488 nm for Cy3 excitation and 632 nm for Cy5 excitation. Detect with the microscope provided. The excitation laser light is focused on the array using a 20 × microscope objective (Nikon, Inc., Melville NY). A slide containing the array is placed on a computer-controlled XY stage of a microscope and passed through an objective lens for raster scanning. The 1.8 cm × 1.8 cm array used in this example scans with a resolution of 20 μm.

  In two different scans, the mixed gas multiline laser sequentially excites the two fluorophores. The emitted light is separated based on wavelength and sent to two photomultiplier detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. The signal is filtered using a suitable filter placed between the array and the photomultiplier tube. The maximum emission wavelength of the fluorophore used is 565 nm for Cy3 and 650 nm for Cy5. The instrument can record spectra from both fluorophores simultaneously, but with a suitable filter in the laser source, it scans once per fluorophore and typically scans each array twice.

  The sensitivity of the scan is usually calibrated using the signal intensity generated by the cDNA control species added to the sample mixture at a known concentration. A specific location on the array contains a complementary DNA sequence, and the intensity of the signal at that location is correlated with the hybridization species at a weight ratio of 1: 100,000. When two samples from different sources (such as cells to be tested and control cells) are each labeled with a different fluorophore and hybridized to a single array to identify genes expressed differently from the others. Performs calibration by labeling a sample of cDNA to be calibrated with two fluorophores and adding equal amounts of each to the hybridization mixture.

  The photomultiplier tube output 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 is displayed as an image in which the signal intensity is mapped using a linear 20 color conversion from a blue (low signal) to red (high signal) pseudo color range. Data is also analyzed quantitatively. When exciting and measuring two different fluorophores simultaneously, using the emission spectra of each fluorophore, the data first corrects for optical crosstalk between the fluorophores (due to overlapping emission spectra).

  A grid is overlaid on the fluorescent signal image, whereby the signal from each spot is collected on each element of the grid. The fluorescence signals within each element are integrated to give a numerical value depending on the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).

In an example of disease diagnosis using microarrays, the component polynucleotide sequence group of SEQ ID NO: 18 (Clone Ids below) normalizes the expression of SEQ ID NO: 18 in colon polyps or colon cancer patient tissues. Used to compare with expression in colon tissue. Expression of SEQ ID NO: 18 in tissue samples from patients with colorectal polyp (polyp) and colorectal cancer (tumor) was shown to be down-regulated by about 3 to about 8 times compared to normal colon tissue.

  In this way, DME expression of SEQ ID NO: 18 was compared in matched normal colon tissue and cancerous colon tissue or colon polyp tissue samples. In one example, matched normal colon and cancer colon tissue samples were obtained from 3 individuals and were provided by Huntsman Cancer Institute, (Salt Lake City, UT). Donor 3583 is a 59-year-old male diagnosed with a tubulovillous adenoma hyperplastic polyp. Donor 3647 was diagnosed with moderately differentiated adenocarcinoma at age 83 (sex unknown). Donor 3649 (sex, age unknown) was diagnosed with well-differentiated adenocarcinoma.

  In another example, matched normal and cancerous colon or polyp tissue samples were provided by Huntsman Cancer Institute, (Salt Lake City, UT). Donor 3754 is an age-gendered donor diagnosed with a pendunculated colorectal polyp. Donor 3755 is an unidentified age-gender donor with a family history of colorectal cancer diagnosed as a colorectal polyp. Donor 3583 is a 58-year-old man diagnosed with ductal choriodenoma hyperplastic polyp. Donor 3311 is an 85-year-old man diagnosed with invasive poorly differentiated adenocarcinoma and metastasis to lymph nodes. Donor 3756 is a 78-year-old woman diagnosed with invasive moderately differentiated adenocarcinoma. Donor 3757 is a 75-year-old woman diagnosed with invasive moderately to poorly differentiated adenocarcinoma and metastasis to lymph nodes. Donor 3649 is an 86-year-old individual diagnosed with invasive well-differentiated adenocarcinoma and gender unknown. Donor 3647 is an invasive, moderately well-differentiated adenocarcinoma, an 83-year-old individual diagnosed with metastasis to lymph nodes and unidentified. Donor 3839 is a 60-year-old individual whose gender is unknown and has been diagnosed with colorectal cancer. Donor 3581 is a male (age unknown) diagnosed with a colorectal tumor. Donors 3754, 3755, 3311, 3756 and 3757 matched a common control sample consisting of a pool of normal colon tissue from three additional donors. All other comparisons were made against matched normal and tumor or polyp tissue from the same donor.

12 Complementary polynucleotides
A sequence complementary to a sequence encoding DME or any portion thereof is used to detect, reduce or inhibit the expression of native DME. Although the use of oligonucleotides comprising about 15-30 base pairs is described, essentially the same method is used for fragments of smaller or larger sequences. Appropriate oligonucleotides are designed using Oligo 4.06 software (National Biosciences) and DME coding sequences. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to inhibit the promoter from binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to inhibit the ribosome from binding to the transcript encoding DME.

13 Expression of DME
DME expression and purification can be performed using bacterial or viral based expression systems. In order to express DME in bacteria, the cDNA is subcloned into a suitable vector containing an antibiotic resistance gene and an inducible promoter that increases the level of cDNA transcription. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter in combination with the lac operator regulator and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host such as BL21 (DE3). Antibiotic-resistant bacteria express DME when induced with isopropyl β-D thiogalactopyranoside (IPTG). Expression of DME in eukaryotic cells is carried out by infecting insect cell lines or mammalian cell lines with a recombinant form of Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The baculovirus non-essential polyhedrin gene is replaced with the cDNA encoding DME by either homologous recombination or bacterial-mediated gene transfer with transfer plasmid mediation. The infectivity of the virus is maintained and a high level of cDNA is transcribed by a strong polyhedrin promoter. Recombinant baculoviruses are often used to infect Spodoptera frugiperda (Sf9) insect cells, but are also used to infect human hepatocytes. In the latter case, further genetic modification of the baculovirus is required. (See Engelhard. EK 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, DME becomes a fusion protein synthesized with, for example, glutathione S-transferase (GST) or a peptide epitope tag such as FLAG or 6-His, so that recombinant fusion from unpurified cell lysates Protein affinity-based purification can be performed quickly in one step. GST is a 26 kDa enzyme from Schistosoma japonicum that enables purification of the fusion protein on immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharmacia Biotech). After purification, the GST moiety can be proteolytically cleaved from DME at specifically engineered sites. FLAG is an 8-amino acid peptide that allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, in which 6 histidine residues are continuously extended, allows purification on a metal chelate resin (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995) chapters 10 and 16 above. The purified DME obtained by these methods can be used directly to perform the applicable assays of Examples 17, 18, and 19 below.

14 Functional assays
DME function is assessed by expression of sequences encoding DME at physiologically enhanced levels in mammalian cell culture systems. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses cDNA at high levels. Vectors selected include the pCMV SPORT plasmid (Life Technologies) and the pCR 3.1 plasmid (Invitrogen, Carlsbad CA), both having a cytomegalovirus promoter. Using a liposomal formulation or electroporation, 5-10 μg of the recombinant vector is transiently transfected into a human cell line, eg, an endothelial or hematopoietic cell line. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is simultaneously transfected. The expression of the labeled protein provides a means to distinguish between transfected and non-transfected cells. Moreover, expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. The labeled protein can be selected from, for example, green fluorescent protein (GFP; Clontech), CD64 or CD64-GFP fusion protein. Identify transfected cells that express GFP or CD64-GFP using flow cytometry (FCM), an automated, laser-optical technique, and evaluate the apoptotic status and other cellular properties of the cells To do. FCM detects and measures the uptake of fluorescent molecules that diagnose phenomena that precede or occur simultaneously with cell death. These phenomena include changes in nuclear DNA content as measured by DNA staining with propidium iodide, changes in cell size and particle size as measured by forward and 90 ° side scatter, bromodeoxyuridine. Down-regulation of DNA synthesis measured by a decrease in the uptake of protein, changes in cell surface and protein expression measured by reactivity with specific antibodies, and fluorescein-conjugated annexin V protein on the cell surface There is a change in the plasma membrane composition measured by binding. The flow cytometry method is described in Ormerod, MG (1994) Flow Cytometry Oxford, New York, NY.

  The effect of DME on gene expression can be assessed using highly purified cell populations transfected with either the DME encoding sequence and either CD64 or CD64-GFP. CD64 or CD64-GFP is expressed on the transfected cell surface and binds to a conserved region of human immunoglobulin G (IgG). Transfected and non-transfected cells can be efficiently separated using magnetic beads coated with either human IgG or antibodies to CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods well known in the art. Expression of mRNA encoding DME and other genes of interest can be analyzed by Northern analysis or microarray technology.

15 Production of DME-specific antibodies Using polyacrylamide gel electrophoresis (PAGE; see, for example, Harrington, MG (1990) Methods Enzymol. 182: 488-495) or DME substantially purified by other purification techniques And immunize animals (eg, rabbits, mice, etc.) using standard protocols to produce antibodies.

  Alternatively, the DME amino acid sequence can be analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions, the corresponding oligopeptides synthesized and used to generate antibodies in a manner well known to those skilled in the art. To produce. The selection of appropriate epitopes, such as those near the C-terminus or in adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, chapter 11 above).

  Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chemistry and by reaction using N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Bind to KLH (Sigma-Aldrich, St. Louis MO) to enhance immunogenicity (see Ausubel, 1995 et al., Supra). Rabbits are immunized with oligopeptide-KLH conjugate in complete Freund's adjuvant. In order to test the anti-peptide activity and anti-DME activity of the obtained antiserum, the peptide or DME is bound to a substrate, blocked with 1% BSA, washed with a rabbit antiserum, washed, and further radioactive. React with iodine labeled goat anti-rabbit IgG.

16 Purification of natural DME using specific antibodies Natural or recombinant DME is substantially purified by immunoaffinity chromatography using antibodies specific for DME. The immunoaffinity column is formed by covalently binding an activated chromatography resin such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) and an anti-DME antibody. After binding, the resin is blocked and washed according to the manufacturer's instructions.

  The culture medium containing DME is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of DME (for example, with a high ionic strength buffer in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and DME (eg, with a pH 2-3 buffer, or a chaotrope such as high concentrations of urea or thiocyanate ions), and DME is collected.

17 Identification of molecules interacting with DME
DME or a biologically active fragment thereof is labeled with ¹²⁵ I Bolton Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529-539). Candidate molecules pre-sequenced in multiwell plates are incubated with labeled DME, washed and all wells with labeled DME complex are assayed. Data obtained at various DME concentrations are used to calculate the quantity, affinity and association value of DME bound to the candidate molecule.

  Alternatively, molecules that interact with DME may be selected from the yeast two-hybrid system described in Fields, S. and O. Song (1989, Nature 340: 245-246) or the MATCHMAKER system ( Analysis is performed using a commercial kit based on a two-hybrid system such as Clontech).

  DME can also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) using an i-throughput yeast two-hybrid system to determine all interactions between proteins encoded by two large libraries of genes (Nandabalan K. et al. (2000) US Pat. No. 6,057,101).

18 Demonstration of DME activity
The cytochrome P450 activity of DME is measured using 4-hydroxylation of aniline. Aniline is converted to 4-aminophenol by this enzyme, and the maximum absorption wavelength of aniline is 630 nm (Gibson and Skett, supra). Although this assay is a convenient measure, it underestimates the total hydroxylation that also occurs at the 2- and 3-positions. The assay is performed at 37 ° C., and the reaction buffer contains an aliquot of enzyme and an appropriate amount of aniline (about 2 mM). In this reaction, the buffer contains NADPH or a cofactor system that produces NADPH. Some preparations of this reaction buffer include 85 mM Tris (pH 7.4), 15 mM magnesium chloride, 50 mM nicotinamide, 40 mg trisodium isocitrate and 2 units of isocitrate dehydrogenase. Add 8 mg NADP ⁺ to 10 ml reaction buffer stock solution just before. The reaction is performed with an optical cuvette and the absorbance at 630 nm is measured. In this assay, the rate of increase in absorbance is proportional to the enzyme activity. A standard curve can be generated using known concentrations of 4-aminophenol.

DME's 1α, 25-dihydroxyvitamin D24-hydroxylase activity is determined from ³ H-labeled 1α, 25-dihydroxyvitamin D (1α, 25 (OH) ₂ D) to 24, in transgenic rats expressing DME. The conversion to 25-dihydroxyvitamin D (24,25 (OH) ₂ D) is monitored and determined. 1 μg of 1α, 25 (OH) ₂ D dissolved in ethanol (or ethanol alone as a control) is used to express a DME-expressing male transgenic rat group or a DME-deficient mutant or DME Is administered intravenously to the same group of control rats, except that is not expressed. Rats were sacrificed by decapitation after 8 hours, kidneys were quickly removed, washed, and 9 volumes of ice-cold buffer (15 mM Tris-acetate (pH 7.4), 0.19 M sucrose, 2 mM magnesium acetate, and 5 mM Homogenize in sodium succinate). Next, a predetermined amount (eg 3 ml) of each homogenate is added to 0.25 nM 1α, 25 (OH) ₂ [ ^1-3 H] D (specific activity is about 3.5 GBq / mmol), with constant oxygen presence. Incubate for 15 minutes at 37 ° C. Total lipids were extracted as described (Bligh, EG and WJ Dyer (1959) Can. J. Biochem. Physiol. 37: 911-917) and n-hexane / chloroform / methanol (10:10) at a flow rate of 1 ml / min. 2.5: 1.5) Analyze the chloroform phase with a FINEPAK SIL column (JASCO, Tokyo, Japan) using a solvent system. Alternatively, the chloroform phase can be reversed using a J SPHERE ODS-AM column (YMC Co. Ltd., Kyoto, Japan) in an acetonitrile buffer system (40-100%, in water, 30 minutes) at a flow rate of 1 ml / min. Analyze by HPLC. This eluate is collected in 30 second (or less) fractions and the amount of ³ H present in each fraction is measured using a scintillation counter. By comparing the chromatogram of the control sample (ie, the sample containing 1α, 25-dihydroxyvitamin D or 24,25-dihydroxyvitamin D (24,25 (OH) ₂ D)) with the chromatogram of the reaction product Determine the relative mobilities of the substrate (1α, 25 (OH) ₂ [1- ³ H] D) and the product (24,25 (OH) ₂ [1- ³ H] D) Correlate with each collected fraction. The amount of 24,25 (OH) ₂ [1- ³ H] D produced in control rats is calculated from the amount of 24,25 (OH) ₂ [1- ³ H] D in transgenic rats expressing DME. Subtract. The difference in production of 24,25 (OH) ₂ [1- ³ H] D between the transgenic and control rats is proportional to the amount of DME 25-hydrolase activity present in the sample. Confirmation of identity between substrate and product (s) is performed by mass spectroscopy (Miyamoto, Y. et al. (1997) J. Biol. Chem. 272: 14115-14119).

  To measure the flavin-containing monooxygenase (FMO) activity of DME, chromatographic analysis of metabolite groups is performed. For example, Ring, BJ et al. (1999; Drug Metab. Dis. 27: 1099-1103) incubated FMO in 0.1 M sodium phosphate buffer (pH 7.4 or 8.3) and 1 mM NADPH at 37 ° C. Was stopped with an organic solvent and product formation was determined by HPLC. Alternatively, the activity is measured by monitoring oxygen uptake using a Clark-type electrode. For example, Ziegler, DM, and Poulsen, LL (1978; Methods Enzymol. 52: 142-151) describe FMO in a NADPH-producing cofactor system (similar to that described above) containing the substrate methimazole. Incubated at 37 ° C. The oxygen uptake rate is proportional to the enzyme activity.

The UDP glucuronyltransferase activity of DME is measured using colorimetric determination of free amino groups (Gibson and Skett, supra). Incubation of amine-containing substrate (eg 2-aminophenol) at 37 ° C, reaction buffer containing necessary cofactors (40 mM Tris (pH 8.0), 7.5 mM MgCl ₂ , 0.025% Triton X-100, 1 mM) Ascorbic acid, 0.75 mM UDP-glucuronic acid) was performed with an aliquot of this enzyme. Allow sufficient time to stop the reaction by adding ice-cold 20% trichloroacetic acid (in 0.1 M phosphate buffer (pH 2.7)), incubate on ice, and centrifuge to clarify the supernatant. All unreacted 2-aminophenol is destroyed in this step. Sufficient freshly prepared sodium nitrite is then added, and this step forms the diazonium salt of the glucuronidation product. Sufficient ammonium sulfamate is added to remove excess nitrous acid and the diazonium salt is reacted with an aromatic amine (eg, N-naphthylethylenediamine) to produce a colored azo compound. This compound can be assayed spectrophotometrically (such as 540 nm). A standard curve can be generated using known concentrations of aniline. This aniline will form a chromophore with properties similar to 2-aminophenol glucuronide.

  For measurement of DME glutathione S-transferase (GST) activity, a model substrate such as 2,4-dinitro-1-chlorobenzene is used. This substrate reacts with glutathione to form the product 2,4-dinitrophenyl-glutathione, which has a maximum absorption wavelength of 340 nm. An important note is that GSTs have various substrate specificities, and the choice of model substrate should be based on the substrate preference of the target GST. The assay is performed at room temperature and a certain amount of enzyme is placed in an appropriate reaction buffer (eg, 1 mM glutathione, 1 mM dinitrochlorobenzene, 90 mM potassium phosphate buffer, pH 6.5). The reaction is performed with an optical cuvette and the absorbance at 340 nm is measured. In this assay, the rate of increase in absorbance is proportional to the enzyme activity.

For measuring the N-acyltransferase activity of DME, a radiolabeled amino acid substrate is used, and incorporation of the radiolabel into the conjugated product group is measured. The reaction buffer in which the enzyme is incubated contains an unlabeled acyl-CoA compound and a radiolabeled amino acid to separate the radiolabeled acyl conjugate from the unreacted amino acid, so that n-butanol or other suitable organic Extract into solvent. For example, Johnson, M. R et al. (1990; J. Biol. Chem. 266: 10227-10233) measured the bile acid-CoA: amino acid N-acyltransferase activity and used this enzyme for cholyl-CoA and ³ H-glycine. Alternatively, incubation was performed with ³ H-taurine, and tritiated cholate conjugates were separated by extraction into n-butanol, and the radioactivity in the extract was measured by scintillation. Alternatively, for the measurement of N-acyltransferase activity, the following reduced CoA (CoASH) spectrophotometric method is used.

N-acetyltransferase activity of DME is measured using the radiolabel transfer of [ ¹⁴ C] acetyl-CoA to a substrate molecule (eg Deguchi, T. (1975) J. Neurochem. 24: 1083-5 See). Alternatively, a spectrophotometric assay based on the DTNB (5,5'-dithio-bis (2-nitrobenzoic acid; Ellman reagent) reaction with CoASH can be used.Free thiol-containing CoASH is a substrate catalyzed by N-acetyltransferase Absorption (412 nm) of DTNB conjugate is used to detect CoASH (De Angelis, J. et al. (1997) J. Biol. Chem. 273: 3045-3050) Enzyme activity is proportional to the rate of radioactivity incorporation into the substrate or the rate of increase in absorbance in this spectrophotometric assay.

The protein arginine methyltransferase activity of DME is measured at 37 ° C. for various times. S- adenosyl-L-[methyl - ³ H] methionine ^([3 H] AdoMet; specific activity = 75 Ci / mmol; NEN Life Science Products) is used as a methyl donor substrate. Useful methyl acceptor substrates include glutathione S-transferase fibrillar lysine-arginine domain fusion protein (GST-GAR), heterogeneous nuclear ribonucleoprotein (hnRNP) or in lysates from cells treated with adenosine dialdehyde. Hypomethylated proteins present in The methylation reaction is stopped by adding SDS-PAGE sample buffer. The products of the reaction are separated by SDS-PAGE and visualized by fluorometric analysis. The presence of ³ H-labeled methyl donor substrate is indicative of the protein arginine methyltransferase activity of DME (Tang, J. et al. (2000) J. Biol. Chem. 275: 7723-7730 and Tang, J. et al. (2000 ) J. Biol. Chem. 275: 19866-19876).

Catechol-O-methyltransferase activity of DME is 50 mM Tris-HCl (pH 7.4), 1.2 mM MgCI2 _, 200 μM SAM (S-adenosyl-L-methionine) iodide (0.5 μCi of methyl- [H ³ ] SAM ), 1 mM dithiothreitol, and various concentrations of catechol substrate (eg, L-dopa, dopamine, or DBA) in a final volume of 1.0 ml reaction mixture. The reaction is started by adding a sample containing 250-500 μg of purified or unpurified DME and is carried out at 37 ° C. for 30 minutes. To stop the reaction, cool quickly on ice and immediately extract with 7 ml ice-cold n-heptane. After centrifugation at 1000 xg for 10 minutes, a 3 ml aliquot group of this organic extract is analyzed for radioactive content in a liquid scintillation counter. The level of catechol-related radioactivity in the organic phase is proportional to the catechol-O-methyltransferase activity of DME (Zhu, BT and JG Liehr (1996) 271: 1357-1363).

To determine the DHFR activity of DME, the disappearance of NADPH is examined at ₃₄₀ nm (ε ₃₄₀ = 11,800 M ⁻¹ cm ⁻¹ ) by spectrophotometry at 15 ° C. The standard assay mix contains 100 μM NADPH, 14 mM 2-mercaptoethanol, MTEN buffer (50 mM 2-morpholinoethanesulfonic acid, 25 mM tris (hydroxymethyl) aminomethane, 25 mM ethanolamine, and 100 mM NaCl, pH 7.0). To 2.0 ml. The reaction is started by adding 50 μm dihydrofolic acid (as substrate). The oxidation of NADPH to NADP ⁺ in the reaction is consistent with the reduction of dihydrofolate and is proportional to the amount of DHFR activity in the sample (Nakamura, T. and Iwakura, M. (1999) J. Biol. Chem. 274). : 19041-19047).

  The aldo / keto reductase activity of DME is measured by the decrease in absorbance at 340 nm when NADPH is consumed. A standard reaction mixture contains 135 mM sodium phosphate buffer (pH 6.2-7.2 depending on the enzyme), 0.2 mM NADPH, 0.3 M lithium sulfate, 0.5-2.5 mg enzyme, and a suitable level of substrate. The mixture is incubated at 30 ° C. and the reaction is measured continuously with a spectrophotometer. Enzyme activity is calculated as the number of moles of NADPH consumed per mg of enzyme.

The alcohol dehydrogenase activity of DME is measured using the increase in absorbance at 340 nm when NAD ⁺ is reduced to NADH. The standard reaction mixture is 50 mM sodium phosphate (pH 7.5) and 0.25 mM EDTA. The reaction is incubated at 25 ° C. and monitored with a spectrophotometer. Enzyme activity is calculated as the number of moles of NADH produced per mg of enzyme.

The carboxylesterase activity of DME is determined using 4-methylumbelliferyl acetate as a substrate. The enzymatic reaction is initiated by adding approximately 10 μl of DME-containing sample with 0.5 mM _4- methylumbelliferyl acetic acid to 1 ml of reaction buffer (90 mM KH ₂ PO ₄ , 40 mM KCl, pH 7.3) at 37 ° C. . The production of 4-methylumbelliferone is monitored for 1.5 minutes with a spectrophotometer (ε ₃₅₀ = 12.2 mM ⁻¹ cm ⁻¹ ). Specific activity is expressed as micromolar of product formed per milligram of protein per minute and corresponds to the activity of DME in the sample (Evgenia, V. et al. (1997) J. Biol. Chem. 272: 14769-14775 ).

Alternatively, the cocaine benzoyl ester hydrolase activity of DME is measured using a reaction buffer (50 mM NaH ₂ PO ₄ , pH 7.4) containing about 0.1 ml DME and 3.3 mM cocaine, 1 mM benzamidine and 1 mM EDTA and 1 mM dithiothreitol. ), Incubated at 37 ° C. and measured. A total volume of 0.4 ml reaction is incubated for 1 hour and terminated with an equal volume of 5% trichloroacetic acid. Add 0.1 ml of internal standard 3,4-dimethylbenzoic acid (10 μg / ml). The precipitated protein is separated by centrifugation at 12,000 xg for 10 minutes. The supernatant is transferred to a clean test tube and extracted twice with 0.4 ml of methylene chloride. The two extracts are combined and dried under a stream of nitrogen. The residue is resuspended in 14% acetonitrile, 250 mM KH ₂ PO ₄ (pH 4.0) containing 8 μl diethylamine per 100 ml, and then injected onto a C18 reverse phase HPLC column for separation. The column eluate is monitored at 235 nm. To quantify DME activity, compare the peak area ratio of the analyte to the internal standard. A standard curve is generated with a benzoic acid standard prepared in a trichloroacetic acid treated protein matrix (Evgenia, V. et al. (1997) J. Biol. Chem. 272: 14769-14775).

  Alternatively, DME carboxylesterase activity on the water soluble substrate paranitrophenylbutyric acid is determined by spectrophotometry well known to those skilled in the art. In this method, DME-containing samples are diluted with 0.5 M Tris-HCl (pH 7.4 or 8.0) or sodium acetate (pH 5.0) in the presence of 6 mM taurocholic acid. The assay is initiated by adding freshly prepared para-nitrophenylbutyric acid solution (100 μg / ml in sodium acetate at pH 5.0). The carboxylesterase activity is then monitored and compared to a controlled autohydrolysis of the substrate with a spectrophotometer set at 405 nm (Wan, L. et al. (2000) J. Biol. Chem. 275: 10041-10046).

The sulfotransferase activity of DME is measured using ³⁵ S incorporation from [ ³⁵ S] PAPS into model substrates (such as phenol) (Folds, A. and JL Meek (1973) Biochim. Biophys. Acta 327: 365 -374). A certain amount of enzyme was added at 37 ° C in 1 mL of 10 mM phosphate buffer (pH 6.4), 50 mM phenol, and 0.4-4.0 mM [ ³⁵ S] adenosine 3'-phosphate 5'-phosphosulfate (PAPS ). Allow sufficient time to transfer 5-20% of the radiolabel to the substrate and add 0.2 ml of 0.1 M barium acetate to precipitate the protein and phosphate buffer. Next, 0.2 ml of 0.1 M barium hydroxide (Ba (OH) ₂ ) is added followed by 0.2 ml zinc sulfate (ZnSO ₄ ). Centrifugation to clarify the supernatant, thereby removing protein and unreacted [ ³⁵ S] PAPS. The radioactivity of the supernatant is measured by scintillation. Enzyme activity is determined from the number of moles of radioactivity in the reaction product.

Samples containing DME were prepared with 2.5 μmol imidazole HC1 (pH 6.8), 3.75 μg protamine chloride, 25 nmol fully desulfated N-resulfated heparin (as hexosamine), and 50 pmol (approximately A final reaction volume of 50 μl with 5 × 10 ⁵ cpm) of [ ³⁵ S] PAPS is incubated at 37 ° C. for 20 minutes, and the heparan sulfate 6-sulfotransferase activity of DME is measured in vitro . This reaction is stopped by immersing the reaction tube in hot water for 1 minute. 0.1 μmol of chondroitin sulfate A (as glucuronic acid) is added to the reaction mixture as a carrier. ³⁵ S-labeled polysaccharides were precipitated from chilled triple volume ethanol with 1.3% potassium acetate and completely removed from unincorporated [ ³⁵ S] PAPS and its degradation products by gel chromatography using a desalting column. To separate. One unit of enzyme activity is defined as the amount required to transfer 1 pmol of sulfuric acid per minute and is determined by the amount of [ ³⁵ S] PAPS incorporated into the precipitated polysaccharide (Habuchi, H. et al. ( 1995) J. Biol. Chem. 270: 4172-4179).

  Alternatively, DME heparan sulfate 6-sulfotransferase activity is separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, regenerated and measured. After separation, the gel is washed with buffer (0.05 M Tris-HCl, pH 8.0), cut into 3-5 mm segments and stirred with 100 μl at 4 ° C. for 48 hours with the same buffer with 0.15 M NaCl. The eluted enzyme is collected by centrifugation and assayed for sulfotransferase activity as described above (Habuchi, H. et al. (1995) J. Biol. Chem. 270: 4172-4179).

Alternatively, the sulfotransferase activity of DME is determined by measuring the transfer of [ ³⁵ S] sulfate from [ ³⁵ S] PAPS to the immobilized peptide. This immobilized peptide is the N-terminal 15 residues of the mature P-selectin glycoprotein ligand-1 polypeptide with an added C-terminal cysteine residue. This peptide spans three potential tyrosine sulfation sites. This peptide is linked to the iodoacetamide activated resin by this cysteine residue (density of 1.5-3.0 μmol peptide per ml resin). This enzyme assay consists of 10 μl peptide derivatized beads in a final volume of 130 μl containing 40 mM Pipes (pH 6.8), 0.3 M NaCl, 20 mM MnCl ₂ , 50 mM NaF, 1% Triton X-100, and 1 mM 5′-AMP. And 2-20 μl of DME-containing sample. The assay is initiated with the addition of 0.5 μCi [ ³⁵ S] PAPS (1.7 μM; 1Ci = 37 GBq). After 30 minutes at 37 ° C., the reaction beads are washed with 65 ° C., 6M guanidine, and the radioactivity incorporated into the beads is measured with a liquid scintillation counter. [ ³⁵ S] sulfuric acid transferred to the bead-bound peptide is measured to determine DME activity in the sample. One unit of activity is defined as 1 pmol of product formed per minute (Ouyang, YB. Et al. (1998) Biochemistry 95: 2896-2901).

Alternatively, DME sulfotransferase assay was performed using 50 mM Hepes-NaOH (pH 7.0), 250 mM sucrose, 1 mM dithiothreitol, 14 μM [ ³⁵ S] PAPS (15 Ci / mmol) and dopamine (25 μM), p-nitro Perform with [ ³⁵ S] PAPS as the sulfate donor in a final volume of 30 μl containing phenol (5 μM) or other candidate substrate. The assay reaction is initiated by the addition of purified DME enzyme reagent, or a sample with DME activity, and the reaction is continued for 15 minutes at 37 ° C. and heated at 100 ° C. for 3 minutes to complete. The formed sediment is removed by centrifugation. The supernatant is then analyzed by either thin layer chromatography or two-dimensional thin layer separation to examine ³⁵ S sulfate. ^A suitable standard is analyzed in parallel with the supernatant to allow identification of ³⁵ S sulfate, and the enzyme specificity of the DME-containing sample is determined based on the relative rate of transfer of the reaction product (Sakakibara, Y. et al. (1998) ) J. Biol. Chem. 273: 6242-6247).

The squalene epoxidase activity of DME is as follows: purified DME (or unpurified mixture with DME), 20 mM Tris-HCl (pH 7.5), 0.01 mM FAD, 0.2 units NADPH-cytochrome C (P-450) reductase , 0.01 mM [ ^l4 C] squalene (diffused with 20 μl Tween 80), and assay in a mixture with 0.2% Triton X-100. The reaction is started by adding 1 mM NADPH and incubated at 37 ° C. for 30 minutes. Non-saponified lipids are analyzed by silica gel TLC developed with ethyl acetate / benzene (0.5: 99.5, v / v). The reaction product is compared with the reaction product from the reaction mixture without DME. The presence of 2,3 (S) -oxide squalene is confirmed using a suitable lipid standard (Sakakibara, J. et al. (1995) 270: 17-20).

  The epoxide hydrolase activity of DME is measured by disappearance of the substrate using gas chromatographic analysis (GC) of the ether extract or by disappearance of the substrate and diol formation by GC analysis of the reaction mixture quenched with acetone. Samples containing DME or epoxide hydrolase control samples were prepared with 10 mM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid (EDTA), and 5 mM epoxide substrate (e.g., ethylene oxide, styrene oxide, propylene oxide, isoprene monoxide, Epichlorohydrin, epibromohydrin, epifluorohydrin, glycidol, 1,2-epoxybutane, 1,2-epoxyhexane, 1,2-epoxyoctane). At various times, a portion of the sample is removed from the reaction mixture and added to 1 ml ice-cold acetone with an internal standard for GC analysis (eg 1-nonanol). Proteins and salts were removed by centrifugation (15 min, 4000 × g) and the extract was purified by GC using a 0.2 mm × 25 m CP-Wax57-CB column (CHROMPACK, Middelburg, The Netherlands) and a flame ionization detector. analyse. The identification of GC products is performed using suitable standards and controls well known to those skilled in the art. One unit of DME activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of diol per minute (Rink, R. et al. (1997) J. Biol. Chem. 272: 14650-14657).

  The aminotransferase activity of DME was determined by measuring samples containing DME at 37 ° C. with 150 mM Tris acetate buffer (pH 8.0) containing 70 μM PLP in a final volume of 200 μl in the presence of 1 mM L-kynurenine and 1 mM 2-oxoglutaric acid. Incubate for time and assay. The formation of kynurenic acid is quantified by HPLC with spectrophotometric detection at 330 nm using suitable standards and controls well known to those skilled in the art. Alternatively, L-3-hydroxykynurenine is used as a substrate and xanthurenic acid production is determined by HPLC analysis of the product with UV detection at 340 nm. Production of kynurenic acid and xanthurenic acid each show aminotransferase activity (Buchli, R. et al. (1995) J. Biol. Chem. 270: 29330-29335).

  Alternatively, the measurement of DME aminotransferase activity can be accomplished by monitoring changes in the UV / VIS absorption spectrum of the enzyme-binding cofactor pyridoxal 5'-phosphate (PLP) under single turnover conditions. This is done by determining the activity of purified DME or an unpurified sample containing DME on various amino acids and oxo acid substrates. This reaction is performed at 25 ° C. in 50 mM 4-methylmorpholine (pH 7.5) with 9 μM purified DME or DME-containing sample and the substrate to be tested (amino acid and oxoacid substrate). To examine the half-reaction of amino acids to oxoacids, the decrease in absorbance at 360 nm and the increase in absorbance at 330 nm resulting from the conversion of enzyme-bound PLP to pyridoxamine 5 ′ phosphate (PMP) are measured. The specificity and relative activity of DME is determined by the activity of enzyme reagents for specific substrates (Vacca, R. A. et al. (1997) J. Biol. Chem. 272: 21932-21937).

  The superoxide dismutase activity of DME is assayed from cell pellets, cultured supernatants, or purified protein reagents. Samples or lysates are separated by electrophoresis on a 15% non-denaturing polyacrylamide gel. The gel is incubated for 30 minutes in 2.5 mM nitroblue tetrazolium followed by 20 minutes in 30 mM potassium phosphate, 30 mM TEMED, and 30 μM riboflavin (pH 7.8). Superoxide dismutase activity is visualized as a white band against the background blue, for which the gel is illuminated on a light box. Quantification of superoxide dismutase activity is performed by densitometric scanning of active gels using suitable superoxide dismutase positive and negative controls (eg, various amounts of commercially available E. coli superoxide dismutase, etc.). (Harth, G. and Horwitz, MA (1999) J. Biol. Chem. 274: 4281-4292).

19 Identification of DME inhibitors
As described in the assay of Example 17 , the compound to be tested is dispensed into the wells of a multiwell plate at various concentrations, along with a suitable buffer and substrate. DME activity can be measured for each well to determine the ability of each compound to inhibit DME activity and dose-response profiles. This assay can also be used to identify molecules that enhance DME activity.

  Those skilled in the art may make various modifications to the methods and systems described in this invention without departing from the scope and spirit of the invention. While the invention has been described with reference to several embodiments, it should be understood that the scope of the invention should not be unduly limited to such specific embodiments. . Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

(Short description of the table)
Table 1 outlines the nomenclature for the full-length polynucleotide and polypeptide sequences of the present invention.
Table 2 shows GenBank identification numbers and annotations of GenBank homologs closest to the polypeptides of the invention. The probability score for each polypeptide and its GenBank homologue is also shown.

  Table 3 shows the structural features of the polynucleotide sequences of the present invention, including predicted motifs and domains, along with methods, algorithms and searchable databases for use in polypeptide analysis.

  Table 4 shows the cDNA and genomic DNA fragments used to construct the polynucleotide sequences of the present invention, along with selected fragments of the polynucleotide sequences.

  Table 5 shows a representative cDNA library of the polynucleotides of the present invention.

  Table 6 is an appendix explaining the tissues and vectors used for preparing the cDNA library shown in Table 5.

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

Claims (79)

An isolated polypeptide selected from the group consisting of the following (a) to (f):
(A) Polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 (SEQ ID NOs: 1 to 12) (b) From SEQ ID NO: 1-5 and SEQ ID NO: 8-12 A polypeptide having a natural amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: (c) a natural such that at least 93% is identical to the amino acid sequence of SEQ ID NO: 6 A polypeptide having an amino acid sequence (d) a polypeptide having a natural amino acid sequence that is at least 97% identical to the amino acid sequence of SEQ ID NO: 7 (e) selected from the group consisting of SEQ ID NO: 1-12 A biologically active fragment of a polypeptide having a selected amino acid sequence (f) selected from the group having SEQ ID NO: 1-12 Immunogenic fragments of polypeptides having different amino acid sequences 2. The isolated polypeptide of claim 1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12. An isolated polynucleotide encoding the polypeptide of claim 1. An isolated polynucleotide encoding the polypeptide of claim 2. 5. The isolated polynucleotide of claim 4 having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. A cell transformed with the recombinant polynucleotide according to claim 6. A genetic transformant comprising the recombinant polynucleotide according to claim 6. A method for producing the polypeptide of claim 1, comprising:
(A) culturing cells transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. Process,
(B) recovering the polypeptide so expressed. 10. The method of claim 9, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12. 2. An isolated antibody that specifically binds to the polypeptide of claim 1. An isolated polynucleotide selected from the group consisting of the following (a) to (i):
(A) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 13-24; (b) selected from the group consisting of SEQ ID NO: 13-17 and SEQ ID NO: 20-24; A polynucleotide having a natural polynucleotide sequence such that it is at least 90% identical to the polynucleotide sequence (c) a natural poly at least 93% identical to that of SEQ ID NO: 18 A polynucleotide having a nucleotide sequence (d) a polynucleotide having a natural polynucleotide sequence which is at least 97% identical to the polynucleotide sequence of SEQ ID NO: 19 (e) complementary to the polynucleotide of (a) Polynucleotide (f) (b) Plastid complementary to the polynucleotide (g) a polynucleotide complementary to the polynucleotide of (c)
(H) a polynucleotide complementary to the polynucleotide of (d) (i) an RNA equivalent of (a) to (h) An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 12. A method for detecting a target polynucleotide having the sequence of the polynucleotide of claim 12 in a sample comprising:
(A) hybridizing the sample with a probe having a group of at least 20 consecutive nucleotides having a sequence complementary to the target polynucleotide in the sample, the probe comprising a hybridization complex Specifically hybridizing to the target polynucleotide under conditions formed by the body between the probe and the target polynucleotide or fragments thereof;
(B) detecting the presence / absence of the hybridization complex, and optionally detecting the amount of the complex if present.
A method wherein the probe specifically hybridizes to the target polynucleotide under conditions such that a hybridization complex is formed between the probe and the target polynucleotide or fragment. 15. The method of claim 14, wherein the probe comprises at least 60 consecutive nucleotides. A method for detecting a target polynucleotide having the sequence of the polynucleotide of claim 12 in a sample comprising:
(A) amplifying the target polynucleotide or fragment thereof using polymerase chain reaction amplification;
(B) detecting the presence or absence of the amplified target polynucleotide or a fragment thereof, and optionally detecting the amount of the target polynucleotide or a fragment thereof when present. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. The composition of claim 17, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12. A method for treating diseases and pathologies associated with decreased expression of functional DME (a novel drug metabolizing enzyme),
18. A method of treatment comprising the step of administering the composition of claim 17 to a patient in need of such treatment. A method of screening a compound to confirm its effectiveness as an agonist of the polypeptide of claim 1 comprising:
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) A screening method comprising the step of detecting agonist activity in the sample. 21. A composition comprising an agonist compound identified by the method of claim 20 and a pharmaceutically acceptable excipient. A method of treating a disease or condition associated with decreased expression of functional DME, comprising:
22. A method of treatment comprising the step of administering the composition of claim 21 to a patient in need of such treatment. A method of screening a compound to confirm its effectiveness as an antagonist of the polypeptide of claim 1 comprising:
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) a screening method comprising the step of detecting antagonist activity in the sample. 24. A composition comprising an antagonist compound identified by the method of claim 23 and a pharmaceutically acceptable excipient. A method of treating a disease or condition associated with overexpression of functional DME, comprising:
25. A method of treatment comprising the step of administering the composition of claim 24 to a patient in need of such treatment. A method for screening for a compound that specifically binds to the polypeptide of claim 1, comprising:
(A) mixing the polypeptide of claim 1 with at least one test compound under suitable conditions;
(B) detecting the binding of the polypeptide of claim 1 to a test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. A method for screening for a compound that modulates the activity of the polypeptide of claim 1, comprising:
(A) mixing the polypeptide of claim 1 with at least one test compound under conditions that permit the activity of the polypeptide of claim 1;
(B) calculating the activity of the polypeptide of claim 1 in the presence of a test compound;
(C) comparing the activity of the polypeptide of claim 1 in the presence of a test compound with the activity of the polypeptide of claim 1 in the absence of the test compound;
2. A method wherein the change in activity of the polypeptide of claim 1 in the presence of a test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1. A method of screening for compounds effective to alter the expression of a target polynucleotide comprising the sequence of claim 5 comprising:
(A) exposing a sample containing the target polynucleotide to a compound under conditions suitable for expression of the target polynucleotide;
(B) detecting altered expression of the target polynucleotide;
(C) comparing the expression of the target polynucleotide in the presence of a variable amount of the compound and in the absence of the compound. A method for calculating the toxicity of a test compound comprising:
(A) treating a biological sample having a nucleic acid group with the test compound;
(B) a process of hybridizing a nucleic acid group of the treated biological sample and a probe having at least 20 consecutive nucleotide groups of the polynucleotide of claim 12, wherein the hybridization comprises the probe and the probe 13. A polynucleotide sequence of the polynucleotide of claim 12 or a sequence thereof, wherein the target polynucleotide is formed under conditions that form a specific hybridization complex with a target polynucleotide of a biological sample. A process that is a polynucleotide having fragments;
(C) quantifying the yield of the hybridization complex;
(D) comparing the amount of the hybridization complex in the treated biological sample with the amount of the hybridization complex in an untreated biological sample;
A difference in the amount of the hybridization complex in the treated biological sample is indicative of the toxicity of the test compound. A diagnostic test for a condition or disease associated with the expression of DME in a biological sample, comprising:
(A) mixing the antibody with the polypeptide and binding the biological sample with the antibody of claim 11 under conditions suitable to form a complex of the antibody and the polypeptide. When,
(B) detecting the complex, and
The method wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample. The antibody is
The antibody according to claim 11, which is any one of (a) a chimeric antibody, (b) a single chain antibody, (c) a Fab fragment, (d) a F (ab ′) ₂ fragment, and (e) a humanized antibody. A composition comprising the antibody of claim 11 and an acceptable excipient. A method for diagnosing a medical condition or disease associated with the expression of DME in a subject comprising:
35. A method comprising administering to said subject an effective amount of the composition of claim 32. The composition of claim 32, wherein the antibody is labeled. 35. A method of diagnosing a medical condition or disease associated with the expression of DME in a subject comprising the step of administering to the subject an effective amount of the composition of claim 34. A method for preparing a polyclonal antibody having the specificity of the antibody of claim 11, comprising:
(A) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 or an immunogenic fragment thereof under conditions that elicit an antibody response The process of becoming
(B) isolating the antibody from the animal;
(C) a polyclonal so that said isolated antibody is screened with said polypeptide, thereby specifically binding to a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 And a step of identifying an antibody. 37. A polyclonal antibody produced by the method of claim 36. 38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier. A method for producing a monoclonal antibody having the specificity of the antibody according to claim 11, comprising:
(A) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 or an immunogenic fragment thereof under conditions that elicit an antibody response The process of becoming
(B) isolating cells producing antibodies from said animal;
(C) fusing the antibody-producing cells with immortalized cells to form hybridoma cells producing monoclonal antibodies;
(D) culturing the hybridoma cells;
(E) isolating a monoclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 from the culture. 40. A monoclonal antibody produced by the method of claim 39. 41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier. 12. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library. 12. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library. A method for detecting in a sample a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-12, comprising:
(A) incubating the antibody of claim 11 with a sample under conditions permitting specific binding between the antibody and the polypeptide;
(B) detecting specific binding, and
A method wherein the specific binding indicates that a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12 is present in the sample. A method for purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-12,
(A) incubating the antibody of claim 11 with a sample under conditions permitting specific binding between the antibody and the polypeptide;
(B) separating the antibody from the sample and obtaining a purified polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12. A microarray, wherein at least one element of the microarray is a polynucleotide according to claim 13. A method for creating an expression profile of a sample comprising a polynucleotide comprising:
(A) labeling a sample polynucleotide;
(B) contacting the microarray element of claim 46 with a labeled polynucleotide of the sample under conditions suitable for formation of a hybridization complex; and (c) quantifying the expression of the polynucleotide in the sample. A method comprising the steps of: An array comprising various nucleotide molecules attached to unique physical locations on a solid substrate,
At least one said nucleotide molecule has an oligonucleotide or polynucleotide that is capable of specifically hybridizing with at least 30 contiguous nucleotides of the target polynucleotide;
13. An array wherein the target polynucleotide is the polynucleotide of claim 12. 49. The array of claim 48, wherein the sequence of the first oligonucleotide or polynucleotide is completely complementary to at least 30 contiguous nucleotides of the target polynucleotide. 49. The array of claim 48, wherein the sequence of the first oligonucleotide or polynucleotide is completely complementary to at least 60 contiguous nucleotides of the target polynucleotide. 49. The array of claim 48, wherein the sequence of the first oligonucleotide or polynucleotide is completely complementary to the target polynucleotide. 49. The array of claim 48, wherein the array is a microarray. 49. The array of claim 48, comprising the target polynucleotide hybridized to a nucleotide molecule comprising a first sequence of the oligonucleotide or polynucleotide. 49. The array of claim 48, wherein a linker connects at least one of the nucleotide molecules and the solid substrate. 49. The array of claim 48, wherein each unique physical location on the substrate comprises a plurality of nucleotide molecules, wherein the plurality of nucleotide molecules at any single unique physical location have the same sequence. And each of the unique physical locations on the substrate comprises nucleotide molecules having a sequence that is different from the sequence of the nucleotide molecules at another unique physical location on the substrate. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 1. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 2. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 3. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 4. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 5. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 6. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 7. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 8. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 9. The polypeptide according to claim 1, which has the amino acid sequence of SEQ ID NO: 10. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 11. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 12. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 13. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 14. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 15. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 16. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 17. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 18. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 19. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 20. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 21. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 22. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 23. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 24.