JP2005508614A - Drug metabolizing enzymes - Google Patents

Abstract

  Various embodiments of the present invention provide groups of human drug metabolizing enzymes (DME) and polynucleotides that identify and encode DME. Examples of the present invention also provide expression vectors, host cells, antibodies, agonists and antagonists. Another embodiment provides a method for diagnosing, treating or preventing a disease associated with abnormal expression of DME.

Description

  The present invention relates to novel nucleic acid groups, drug metabolizing enzyme groups encoded by these nucleic acids, and autoimmune / inflammatory diseases, abnormal cell proliferation, developmental / developmental disorders, endocrine disorders, ophthalmic disorders using these nucleic acids and proteins. The present invention relates to the diagnosis, treatment and prevention of gastrointestinal disorders including disorders, metabolic disorders, and liver disorders. The invention also relates to the assessment of the effect of exogenous compounds on the expression of the nucleic acids and drug metabolizing enzymes.

  Drug metabolism and drug movement within the body (pharmacokinetics) are important in determining its effects, toxicity, and interactions with other drugs. Three processes govern pharmacokinetics: drug absorption, distribution to various tissues, and elimination of drug metabolites. These processes are closely related to drug metabolism, which means that various metabolic modifications can account for most of the drug's physicochemical and pharmacological properties, including solubility, receptor binding, and excretion rates. This is to transform. 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 for biochemical and pharmacological interactions between natural compounds, drugs, carcinogens, mutagens, and xenobiotics.

  It has long been recognized that genetic differences in drug metabolism result in 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 harm only a portion of the patient population. Advances in pharmacogenomic research, where drug-metabolizing enzymes are an important part, will develop tools to address the question of drug efficacy and toxicity, and such information could be expanded (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, and phase II of conjugation. 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 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, pages 113-186; Katzung, BG (1995) Basic and Clinical Pharmacology , Appleton and Lange, Norwalk, CT, pages 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 ligand region was 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 one 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 the 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 influenced 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 a 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 erythrocytes. The presence of high levels of oxidizing agents 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 light (ie 290-310 nm). It is usually produced when the skin is exposed to short-term sunlight (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 the active form of 1α, 25-dihydroxyvitamin in the kidney by the enzyme 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase) It is further metabolized to 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 has several levels, including circulating levels of enzyme products (1α, 25 (OH) ₂ D) and levels of parathyroid hormone (PTH), calcitonin, insulin, calcium, phosphorus, growth hormone, and prolactin Depends on the physiological factors. Furthermore, extra-renal 1α-hydroxylase activity has been reported, suggesting that tissue-specific and local regulation of lα, 25 (OH) ₂ D production may also be biologically important. The catalytic action of 1α, 25 (OH) ₂ D on 24,25-dihydroxyvitamin D (24,25 (OH) ₂ D) involves the enzyme 25-hydroxyvitamin D 24-hydroxylase (24-hydroxylase). This 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, lα-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 Portal, 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 of enzymes related to vitamin D metabolism (eg, 1α-hydroxylase), is hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive) rickets (decreased bone density and , Varus knee with a duck walk, or a disease that shows the characteristic 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 adolescence), atherosclerosis, and cataract. The absence of rickets due to vitamin D25-hydroxylase deficiency suggests that there is another pathway 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, was found to be heterologously expressed in E. coli and reduced by endogenous ferredoxin and ferredoxin reductase enzymes (Taylor, M. (1999) Biochem. Biophys. Res. Commun. 263: 838-42). This suggests that many cytochrome P450 species are supported by ferredoxin / ferredoxin reductase pairs. Ferredoxin reductase also reduced actinomycin D, an antitumor antibiotic, to a reactive free radical species in a model drug metabolism system (Flitter, WD and Mason, RP (1988) Arch. Biochem. Biophys. 267: 632-639).

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 the liver, kidneys, and lungs.

  Five different known FMO isoforms (FMO1, FMO2, FMO3, FM04, and FMO5) that are 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 non-ionic detergent 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 also, H ₂ is used widely in the treatment of gastric ulcers - involved in S- oxygenation of cimetidine is an antagonist. The liver expression form 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 for 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 a large amount 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, the absence of functional LO is linked to skeletal and vascular tissue damage associated with dietary copper deficiency. LO is also inhibited by various semicarbazides, hydrazines, 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 ⁺
It is.

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 the same reason, thymidylate synthase is also a target enzyme). 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. Furthermore, 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 reductase Aldo / keto reductase is a monomeric NADPH-dependent oxidoreductase with a 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 standard 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 I ADH isoenzymes oxidize ethanol and other small fatty alcohols and are inhibited by pyrazole. Class II isoenzymes like long chain fatty alcohols 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 number of closely related enzymes with varying 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 and bacterial enzymes acetoacetyl-CoA reductase, glucose 1-dehydrogenase, 3-β-hydroxysteroid dehydrogenase, 20-β-hydroxysteroid dehydrogenase, ribitol dehydrogenase, 3- Oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, sorbitol-6-phosphate 2-dehydrogenase, 7-α-hydroxysteroid dehydrogenase, cis-1,2-dihydroxy-3,4-cyclohexadiene-1 -c arboxylate dehydrogenase, cis-toluene dihydrodiol dehydrogenase, cis-benzene glycol dehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase, N-acylmannosamine 1-dehydrogenase, and There is 2-deoxy-D-gluconate 3-dehydrogenase (Krozowski, Z. (1994) J. Steroid Biochem. Mol. Biol. 51: 125-130; Krozowski, Z. (1992) Mol. Cell Endocrinol. 84: C2531; 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 a 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 products 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. UGT2 family members 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, 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 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, kidneys, intestine, lungs, 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. Based on their substrate selectivity, these biochemically characterized STs fall into five classes: aryl sulfotransferases, alcohol sulfotransferases, estrogen sulfotransferases, tyrosine ester sulfotransferases, and bile salt sulfotransferases. being classified.

  ST enzyme activity varies markedly 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, modification 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 glycosyltransferases that transfer galactose (Gal) to a terminal N-acetylglucosamine (GlcNAc) oligosaccharide chain that is part of a glycolipid or glycoprotein that is free in solution. (Kolbinger, F. et al. (1998) J. Biol. Chem. 273: 433-440; Amado, M. et al. (1999) Biochim. Biophys. Acta 1473: 35-53). 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, 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 found in region 8 of mouse UDP-galactose: β-N-acetylglucosamine β1,3-galactosyltransferase-I is also found in bacterial galactosyltransferases, this sequence is It is suggested to define a galactosyltransferase sequence motif (Hennet, T. supra). Recent studies suggest that brainiac protein is a type of 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, the soluble form of this enzyme is formed by membrane bound cleavage. Amino acids conserved in β1,4-galactosyltransferase include two cysteine residues linked by disulfide bonds and one 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 synthesizing sugar chains on glycoproteins or glycolipids, β1,4-galactosyltransferase has several specialized roles. In mammals, certain β1,4-galactosyltransferases function in lactose production during lactation as part of a heterodimer with α-lactalbumin. Β1,4-galactosyltransferase on the surface of sperm acts as a receptor that specifically recognizes eggs. Cell surface β1,4-galactosyltransferases also act on 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 serves a useful function, such as inactivating and detoxifying potential mutagenic and carcinogenic chemicals. However, in some cases their effects are 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 human αGST, A1-1, and mutagenesis of aflatoxin B1 is substantially reduced by promoting GST expression ( Simula, TP et al. (1993) Carcinogenesis 14: 1371-1376). Thus, control of GST activity would be useful in mutagenesis and control of 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. The elevated GST level then protects the cancer cells from other GST-binding cytotoxins. Increased levels of A1-1 in tumors are related 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 high levels of γ-glutamyltranspeptidase activity on the surface of cancer cells can be used to activate precursors, resulting in high local concentrations of anticancer therapeutics (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 And a metabolite having an amide bond is formed. 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 chromatin structure in eukaryotic cells, 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. For this reason, the use of chemicals that inhibit histone deacetylation (eg, sodium butyrate) as a general means of stimulating cell transcription is an artificial result that generally increases gene expression. Modulation of gene expression by acetylation is also acetylated by other proteins, including but not limited to 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. The 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. Review of histone acetylation (Cheung, WL et al. (2000) Curr. Opin. Cell Biol. 12: 326-333 and Berger, S. L (1999) Curr. Opin. Cell Biol. 11: 336-341) Please refer. 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 the antituberculous drug isoniazid 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 enzyme activity or stability. The slow acetylation of isoniazid is highly prevalent in the Middle Eastern population (about 70%), slightly inferior in whites (about 50%), and less than 25% in the Asian population. 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 ).

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 causes 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 urinary tract, eventually causing 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) It catalyzes the transfer of methyl groups in the body. The methylation of the 3′-hydroxyl group takes precedence over the 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 of binding and catechol substrate Mg ²⁺ is followed.

Inhibition is difficult because the amount of COMT in the tissue is greater than the amount of activity normally required. 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, Developed for nitrocatechol compounds and tolcapone. Administration of these inhibitors increases the half-life of levodopa, followed by dopamine formation. 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. It is. 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, have minimal side effects, are ultimately metabolized in the liver, and only a few metabolites accumulate in the body (Mannisto, PT and Kaakkola, S. (1999) Pharmacol. Rev. 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 enhancing 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. Mycobacterium 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, Mycobacterium tuberculosis secretes up to 350 times more enzyme than shrimp and confers 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 stage cancer. 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 tyrosine DNA phosphodiesterases function in DNA repair by hydrolyzing a dead-end covalent intermediate formed between topoisomerase I and DNA (Pouliot, JJ et al. (1999) Science 286: 552-555; Yang, S.-W. (1996) Proc. Natl. Acad. Sci. USA 93: 11534-11539).

  Acidic sphingomyelinase is a phosphodiesterase that hydrolyzes sphingomyelin, a membrane phospholipid, to produce ceramide and phosphorylcholine. Phosphorylcholine is used to synthesize phosphatidylcholine involved in various intracellular signaling pathways, while ceramide is an essential precursor for the production of ganglioside, a membrane lipid found in high concentrations in neural tissue. 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 broad specificity for glycerophosphodiester substrates (Larson, T. J. et al. (1983) J. Biol. Chem. 248: 5428-5432).

  Cyclic nucleotide phosphodiesterase (PDE) is a critical enzyme for the regulation of cyclic nucleotides cAMP and cGMP. cAMP and cGMP function as intracellular second messengers that transmit a variety of extracellular signals such as hormones, light and neurotransmitters. PDE breaks down cyclic nucleotides into their corresponding monophosphates, thereby regulating their intracellular concentration and their effect on signal transduction. Since 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).

  Families of mammalian PDEs are classified based on their substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitors (Beavo, JA (1995) Physiol. Rev. 75: 725-748; Conti M. et al. (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 is found in the lungs, 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 and immune systems (Perry, MJ and GA Higgs (1998) Curr Opin. Chem. Biol. 2: 472-481).

  PDE2 is a cGMP-stimulated PDE found 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 effect of cAMP on catecholamine secretion and appears to be involved in the regulation of aldosterone (Beavo, supra) and appears 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 T lymphocyte and vascular smooth muscle cell proliferation, 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 suppressed 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, localizes to airway smooth muscle, vascular endothelium, and all inflammatory cells and can be activated by cAMP-dependent phosphorylation. PDE4 has been extensively studied as a potential target for new anti-inflammatory agents with an emphasis on the discovery of asthma treatment, as elevated cAMP levels inhibit inflammatory cell activation and may relax bronchial smooth muscle . PDE4 inhibitors are currently in 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 treatments for acute lung injury, endotoxemia, rheumatoid arthritis, multiple sclerosis, and various neurological and gastrointestinal disorders (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. In order to obtain specificity for PDE5, zaprinast was modified to generate sildenafil (VIAGRA; Pfizer, Inc., New York NY). 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, N. O. 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 (retinitis pigmentosa) (Danciger, M. (1995) Genomics 30: 1-7) and Irish setter dog cadaver / cone dysplasia type 1 (Suber, ML et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3968-3972) Caused by mutations 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. MRNA encoding PDE7 is found in skeletal muscle, heart, brain, lung, kidney and pancreas, but expression of PDE7 protein is limited to certain 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. The cAMP hydrolysis activity of PDE8 is not suppressed by PDE inhibitors rolipram, vinpocetine, milrinone, IBMX (3-isobutyl-1-methylxanthine) or zaprinast, whereas PDE8 is suppressed 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).

  PDE10s 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).

  PDEs have a catalytic domain of about 270-300 amino acids and an N-terminal regulatory domain that serves to bind cofactors, and in some cases have 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, while isoenzymes within the same family usually show about 85-95% identity in this region (eg, PDE4A and PDE4B). Furthermore, while 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, accompanied by decreased levels of cyclic nucleotides. For example certain types of diabetes insipidus in the mouse is related to the increase of 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 in 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). Rolipram has also 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 inhibited 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 leading to cell death (Deonarain, MP and AA Epenetos (1994) Br. J. Cancer 70: 786-794). .

Phosphotriesterase phosphotriesterase enzyme (PTE, paraoxonase (paraoxonase)) such hydrolyze toxic organophosphorus compounds, PTE have been isolated from various tissues. The PTE enzyme appears to be abundant in mammals but not in birds and insects and explains 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 waste 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 for 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 a wide range of fatty acyl chains. These thioesterases catalyze the chain termination step in the novel biosynthesis of fatty acids. Chain termination involves hydrolysis of the thioester bond that joins the fatty 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, a thioesterase type I that is active only on long-chain acyl thioesters and a thioesterase type II (TEII) that has specificity for chains of various lengths (Naggert J. Biol. Chem. 266: 11044-11050). E. coli TEII shows 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 diethylpyrocarbonate abolishes TEII activity. The overexpression of TEII does not change the fatty acid content in E. coli, indicating that it does not function as a chain terminator 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. Carboxylesterase substrates include short and long chain acylglycerols, acylcarnitines, carbonic acid, dipivefrin hydrochloride, cocaine, salicylic acid, capsaicin, palmitoyl-CoA, imidapril, haloperidol, pyrrolizidine alkaloids, steroids, p-nitro Phenylacetic acid, malathion, butanilicaine, and isocarboxazide are included. These enzymes often show low substrate specificity. Carboxyl esterases are also important for converting prodrugs to the corresponding free acids. The corresponding free acid can 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 bind 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. Pp. 554-560, and Sakakibara, J. et al. (1995) 270: 17- 20): Acetate (derived from acetyl-CoA) → 3 hydroxy-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 at elevated serum cholesterol levels, atheromatous plaques form in higher organism arteries. For example, if there is this highly insoluble lipid deposit on the wall of an essential blood vessel such as a coronary artery, the blood flow decreases and sufficient blood does not flow to the tissue, and tissue necrosis may occur. 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 later 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 catalyzes the addition of water to an epoxide-containing compound, which hydrolyzes the epoxide to the corresponding 1,2-diol. These are closely related to the bacterial haloalkane dehalogenase and have sequence similarity to other members of the α / βhydrolase fold family of enzymes (eg, bromoperoxidase A2 from Streptomyces aureofaciens) (Bromoperoxidase A2, Pseudomonas-Putida-derived hydroxymuconic semialdehyde hydrolases, and Xanthobacter autotrophicus- derived haloalkane dehalogenase). Epoxide hydrolases are ubiquitous and are found in mammals, invertebrates, plants, fungi, and bacteria. This enzyme family 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 (Z ) -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 via a covalent ester intermediate, initiated by one nucleophilic attack of the Asp residue group on the first carbon atom of the epoxide ring of the target molecule, where the covalent ester intermediate is (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 the amino acid tyrosine to the enzymes succinate and pyruvate or fumaric acid and acetoacetate for 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 catabolic 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 to fumaric acid and acetoacetate (eg, Pseudomonas) include 4-hydroxyphenylpyruvate dioxygenase, homogentisic acid-1,2-dioxygenase, maleylacetoacetate isomerase, and fumarylacetate 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 of 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 is at increased risk of liver cancer (Endo, F. et al. (1997) J. Biol. Chem. 272: 24426-24432).

The cytosine deaminase of cytosine deaminase catalyzes the hydrolysis of cytidine to uridine and ammonia. It can also convert the antifungal agent 5-fluorocytosine (5FC) to the anticancer agent 5-fluorouracil (5FU) (Senter, PD et al. (1991) Bioconjug. Chem. 2: 447-451). This ability has recently been used in cancer treatment research, indicating that it acts as a suicide gene. The enzyme encoded by the suicide gene converts a non-toxic compound or prodrug such as 5FC into a toxic product such as 5FU. Cytosine deaminase also kills cells by a bystander effect, where non-adjacent tumor cells die by the release of 5FU (Gnant, MF et al. (1999) Cancer Res. 59: 3396-3403). Many suicide gene therapy approaches have been successful in animal models of cancer and are currently being tested in clinical trials. The most powerful and widely used gene described in over 30 suicide genes is the gene for HSV1tk and cytosine deaminase (Singhal S. and Kaiser LR (1998) Surg. Oncol. Clin. N. Am. 7: 505-536; (Sandalon, Z. et al. (2001) Gene Ther. 8: 232-238).

Acetyl CoA carboxylase Acetyl CoA carboxylase is a biotin-dependent enzyme that is found in all animals, plants and bacteria. Acetyl-CoA carboxylase uses the energy of ATP hydrolysis to catalyze the carboxylation of acetyl-CoA from CO ₂ and H ₂ O. Acetyl-CoA carboxylation is the rate-limiting step in the biosynthesis of long chain fatty acids. Two isoforms of acetyl-CoA carboxylase, type I and type II, are tissue-specific expressed in humans (Ha et al. (1994) Eur. J. Biochem. 219: 297-306). Acetyl-CoA carboxylase is a multi-component enzyme having carbamoyl phosphate synthase activity, biotin carboxyl carrier protein, and carboxyl group functional unit. Carbamoyl phosphate synthase catalyzes the ATP-dependent synthesis of carbamoyl phosphate from glutamine or ammonia. This initiates both the urea cycle and the biosynthesis of arginine and pyrimidine. The carboxyltransferase domain functions at the carboxyl group transfer from biotin to the receptor molecule. There are two recognized types of carboxyltransferases. One uses acyl-CoA and the other uses 2-oxoacids as carbon dioxide acceptor molecules. All members of this family use acyl CoA as the acceptor molecule.

Tryptophan decarboxylase Tryptophan decarboxylase (Tdc), also known as dopa decarboxylase, is a pyridoxal phosphate protein that acts on three different substrates. L-tryptophan, 5-hydroxy-L-tryptophan, dihydroxy-L-phenylalanine. Tdc is an important enzyme for biosynthesis of terpenoid indole alkaloids in the clone Catharanthus roseus. Its amino acid sequence shows a strong similarity to the Drosophila α-methyldopa hypersensitive protein. Tdc also shows significant similarity to feline glutamate decarboxylase and murine ornithine decarboxylase. Tdc, together with tyrosine hydroxylase, regulates brain dopamine synthesis (Ouwerkerk, PB et al. (1999) Plant Mol. Biol. 41: 491-503; De Luca, V. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2582-2586).

Enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase The enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase biceps enzyme is one of four enzymes in the peroxisomal β-oxidation pathway. A full-length human cDNA was sequenced and mapped to chromosome 3q26.3-3q28. This enzyme is found mainly in the liver and kidney and has the tripeptide SKL at the carboxy terminus, which serves as a peroxisome targeting signal. The cDNA of this double-headed enzyme in human and rat is 80% homologous (Hoefler, G. et al. (1994) Genomics 19: 60-67). Enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase deficiency results in clinical manifestations resembling Zerweger syndrome and accumulation of very long chain fatty acids (Fukuda, S. et al. (1998) J. Inherit. Metab. Dis. 21: 23-28).

  C7 is a novel nucleolar protein, the mouse homologue of L82, a Drosophila late puff product, and is an isoform of human OXR1 (oxidation resistance). Antibodies to recombinant C7 proteins localized in the nucleolus of various cell types suggest C7 involvement in nucleolus formation or function (Fischer, H. et al. (2001) Biochem. Biophys. Res Commun. 281: 795-803).

  Mammalian glucosamine-6-phosphate deaminase (GNPDA) was first detected in hamster sperm. The human testis homologue was described in Shevchenko, V. et al. (1998; Gene 216: 31-38) and mapped to chromosome 5q31. In mouse sperm cells, GNPDA was located near the developing acrosome vesicle and in mouse sperm near the acrosome region. After induction of the acrosome reaction, GNPDA fluorescence in sperm was reduced or absent, suggesting that GNPDA plays a role in the acrosome reaction (Montag, M. et al. (1999) FEBS Lett. 458: 141 -144).

Expression profiling microarrays are analytical tools used for biological analysis. A microarray has a plurality of molecules that are spatially distributed on the surface of a solid support and are stably associated with that surface. Polyarrays of polypeptides, polynucleotides, and / or antibodies have been developed and their various uses include, for example, gene sequencing, gene expression monitoring, gene mapping, bacterial identification, drug discovery, combinatorial chemistry.

  In particular, a region using a microarray is gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of many related or unrelated genes. When testing the expression of a single gene, the array is used to detect the expression of a particular gene or variant thereof. When testing expression profiles, the array provides a platform to identify genes such as: That is, which genes are tissue specific, affected by the substance being tested in the toxicity assay, are part of a signaling cascade, perform housekeeping functions, or have a specific genetic predisposition or condition Identification of genes that are specifically associated with a disease or disorder.

  Lung cancer is the leading cause of cancer death in US men and the second leading cause of cancer death in women. The majority of lung cancer cases are believed to be caused by smoking, and the spread of lung cancer is expected in third world countries due to increased tobacco consumption. Exposure of bronchial epithelium to tobacco smoke appears to change tissue morphology, which is thought to be a precursor to cancer.

  Lung cancer is divided into four histopathologically distinct groups. Group 3 (squamous cell carcinoma, adenocarcinoma, large cell carcinoma) is classified as non-small cell lung cancer (NSCLC). The fourth group of cancers is called small cell lung cancer (SCLC). Combined NSCLC accounts for about 70% of all cases and SCLC is about 18%. The molecular and cell biology understanding of the development and progression of lung cancer is incomplete. Deletion at chromosome 3 is common in lung cancer and appears to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K-ras are commonly found in lung cancer and are the basis for one mouse model of this disease.

  The art includes nucleic acids for the diagnosis, prevention, and treatment of gastrointestinal disorders, including autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental and developmental disorders, endocrine disorders, eye disorders, metabolic disorders, and liver disorders. There is a need for new compositions containing proteins.

  The various embodiments of the present invention are collectively referred to as “DME”, individually “DME-1”, “DME-2”, “DME-3”, “DME-4”, “DME-5”, Drugs called “DME-6”, “DME-7”, “DME-8”, “DME-9”, “DME-10”, “DME-11”, “DME-12”, “DME-13” Provided are purified polypeptides that are metabolic enzymes, and methods for detecting, diagnosing, and treating diseases and medical conditions using these proteins and the polynucleotides that encode them. Some embodiments also provide methods for promoting drug discovery processes using purified drug metabolizing enzymes and / or polynucleotides encoding them, such as methods for determining efficacy, dose, toxicity, and pharmacology. . Some related embodiments provide methods of investigating the pathogenesis of a disease and a disease state using purified drug-metabolizing enzymes and / or polynucleotides encoding them.

  Some embodiments include: (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13; (b) an amino acid selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a natural amino acid sequence with at least 90% identity to the sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and an isolated polypeptide selected from the group consisting of: Another embodiment provides an isolated polypeptide having the amino acid sequence of SEQ ID NO: 1-13.

  In another embodiment, (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, (b) a certain amino acid selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a certain natural amino acid sequence having at least 90% identity to the sequence, (c) a biological activity of the polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 A fragment, or (d) an immunogenic fragment of a polynucleotide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and an isolated polypeptide encoding 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: 1-13. In another embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO: 14-26.

  Another embodiment is (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, (b) a certain selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a certain natural amino acid sequence having at least 90% identity to the amino acid sequence, (c) the biological of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 An active fragment, and (d) an immunogenic fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and a polynucleotide encoding a certain polypeptide selected from the group consisting of: Recombinant polynucleotides having operably linked promoter sequences are provided. Another embodiment provides cells transformed with the recombinant polynucleotide. Yet another embodiment provides a genetically transformed organism having this recombinant polynucleotide.

  Another embodiment is (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and (b) a certain selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a certain natural amino acid sequence having at least 90% identity with the amino acid sequence, and (c) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 A method of producing a polypeptide selected from the group consisting of a biologically active fragment and (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 To do. The production method comprises the steps of (a) culturing a cell under conditions suitable for expression of the polypeptide, transforming the cell with a recombinant polynucleotide, and (b) a polypeptide so expressed. The process consists of recovering the peptide. This recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide.

  Yet another embodiment is (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, (b) a certain selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a certain natural amino acid sequence having at least 90% identity to the amino acid sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, isolated to specifically bind to a polypeptide selected from the group consisting of Antibodies are provided.

  Yet another embodiment is (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26, (b) one selected from the group consisting of SEQ ID NO: 14-26 A polynucleotide having a natural polynucleotide sequence having at least 90% identity to the polynucleotide sequence, a polynucleotide complementary to (c) (a), a polynucleotide complementary to (d) (b), And (e) an RNA equivalent selected from the group consisting of the RNA equivalents of (a)-(d). In another embodiment, the polynucleotide may consist of at least about 20, 30, 40, 60, 80, or 100 consecutive nucleotide groups.

  Another embodiment is a method for detecting a target polynucleotide in a sample, wherein the target polynucleotide has (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26, b) a polynucleotide having a natural polynucleotide sequence having at least 90% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26, complementary to (c) (a) A polynucleotide complementary to (d) (b), and (e) an RNA equivalent of (a)-(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) It consists of a process of detecting the presence or absence of the hybridization complex. 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 thereof. In certain related embodiments, the method can include detecting the amount of the hybridization complex. In yet another embodiment, the probe may consist of at least about 20, 30, 40, 60, 80, or 100 consecutive nucleotide groups.

  Yet another embodiment is a method for detecting a target polynucleotide in a sample, wherein the target polynucleotide has (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26, (b) a polynucleotide having a natural polynucleotide sequence having at least 90% identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26, complementary to (c) (a) A polynucleotide complementary to (d) (b), and (e) an RNA equivalent of (a)-(d). The detection method includes (a) a process of amplifying a target polynucleotide or a fragment thereof using polymerase chain reaction amplification, and (b) a process of detecting the presence or absence of the amplified target polynucleotide or a fragment thereof. In certain related embodiments, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.

  Another embodiment provides a composition comprising an effective amount of a polypeptide and a pharmaceutically acceptable excipient. An effective amount of the polypeptide is (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, (b) a certain selected from the group consisting of SEQ ID NO: 1-13 A polypeptide comprising a natural amino acid sequence having at least 90% identity to the amino acid sequence, (c) a biological activity of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 Selected from the group consisting of a fragment and (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13. In one embodiment, the composition may have an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13. Another embodiment provides a method of treating a disease or condition associated with reduced or abnormal expression of functional DME and a method comprising administering the composition to a patient in need of such treatment.

  Another embodiment is (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, (b) a certain selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a natural amino acid sequence having at least 90% identity to the amino acid sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and (d) an immunogenic fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, in order to confirm the effectiveness as an agonist of a polypeptide selected from the group consisting of: Methods for screening certain 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. Another embodiment provides a composition having an agonist compound identified by this method and an acceptable pharmaceutical excipient. Still other examples provide methods of treating diseases and conditions associated with reduced expression of functional DME and include administering the composition to patients in need of such treatment.

  Yet another embodiment is (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, (b) selected from the group consisting of SEQ ID NO: 1-13, or A polypeptide having a natural amino acid sequence having at least 90% identity to the amino acid sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, And (d) an immunogenic fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, to confirm the effectiveness as an antagonist of the polypeptide selected from the group consisting of Provides a method for screening certain compounds. The screening method consists of (a) exposing a sample having the polypeptide to a certain compound, and (b) detecting antagonistic activity in the sample. Another embodiment provides a composition having an antagonist compound identified by this method and an acceptable pharmaceutical excipient. Yet another embodiment provides a method of administering this composition to a patient in need of treatment for a disease or condition associated with overexpression of functional DME.

  Another embodiment is: (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13; (b) a certain amino acid selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a natural amino acid sequence with at least 90% identity to the sequence, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and ( d) An immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and a method for screening for a compound that specifically binds to a polypeptide selected from the group consisting of: The screening method comprises: (a) mixing the polypeptide with at least one test compound under appropriate conditions; (b) detecting binding between the polypeptide and the test compound and specifically binding to the polypeptide It consists of the process of identifying the compound to be.

  In another embodiment, (a) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 A polypeptide having a certain natural amino acid sequence having at least 90% identity or at least about 90% identity with, (c) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 An activity of a polypeptide selected from the group consisting of a biologically active fragment, and (d) an immunogenic fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 Methods are provided for screening for certain compounds that modulate. The screening method comprises: (a) mixing the polypeptide with at least one test compound under conditions acceptable for the activity of the polypeptide; and (b) converting 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. A change in the activity of the polypeptide at is indicative of a compound that modulates the activity of the polypeptide.

  Yet another embodiment provides a method of screening a compound for the effect of altering the expression of a target polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26 . The method comprises: (a) exposing a sample having the target polynucleotide to a compound; (b) detecting alterations in expression of the target polynucleotide; and (c) a variable amount of the compound. The process consists of comparing the expression of the target polynucleotide in the presence to the expression in the absence of the compound.

  Another embodiment provides a method for calculating the toxicity of a test compound. This method includes the following processes. (a) A process of treating a biological sample having a nucleic acid group with a test compound. (b) A process of hybridizing the nucleic acid group of the treated biological sample. The following probe is used in this process. (i) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26; (ii) at least a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26; A polynucleotide having a natural polynucleotide sequence with 90% identity, a polynucleotide having a sequence complementary to (iii) (i), a polynucleotide complementary to the polynucleotide of (iv) (ii), (v ) a probe consisting of at least 20 contiguous nucleotide groups of a polynucleotide selected from the group consisting of RNA equivalents of (i) to (iv). Hybridization occurs under conditions such that a specific hybridization complex is formed between the probe and a target polynucleotide in the biological sample. The target polynucleotide is (i) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26, (ii) a certain selected from the group consisting of SEQ ID NO: 14-26 A polynucleotide having a natural polynucleotide sequence having at least 90% or at least about 90% identity to the polynucleotide sequence, (iii) a polynucleotide complementary to the polynucleotide of (i), (iv) of (ii) The polynucleotide is selected from the group consisting of a polynucleotide complementary to the polynucleotide and an RNA equivalent of (v) (i) to (iv). Alternatively, the target polynucleotide may have a fragment of a polynucleotide sequence selected from the group consisting of (i) to (v) above. The toxicity calculation method further includes the following processes. (c) quantifying the amount of hybridization complex, and (d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in the untreated biological sample. is there. Differences in the amount of hybridization complexes in the treated biological sample indicate the toxicity of the test compound.

(Description of the present invention)
While the proteins, nucleic acids, and methods of the present invention will be described, it should be understood that prior to that, embodiments of the present invention are not limited to the specific devices, materials and methods described and may be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

  The singular forms “a” and “its” in the claims and specification may refer to the plural unless the context clearly dictates otherwise. Thus, for example, when “a certain host cell” is described, there may be a plurality of such host cells, when “a certain antibody” is described, one or more antibodies, and It also refers 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 are reported in the publications and may be used in connection with various embodiments of the invention. It is what you are doing. No disclosure in this specification is an admission that the invention is not entitled to antedate such disclosure by virtue of prior art.

(Definition)
“DME” refers to substantially purified DME obtained from all species (particularly mammals including cattle, sheep, pigs, mice, horses and humans), such as natural, synthetic, semi-synthetic or recombinant. Refers to the amino acid sequence.

  The term “agonist” refers to a molecule that enhances or mimics the biological activity of DME. Agonists include proteins, nucleic acids, carbohydrates, small molecules, and any other compound that regulates DME activity by interacting directly with DME or acting on components of biological pathways involving DME. Or a composition.

  The term “allelic variant” refers to another form of the gene encoding DME. A group of allelic variants can result from at least one mutation in the nucleic acid sequence. In addition, transformed mRNA groups can be generated. In addition, the structure or function of the polypeptide from which this variant may occur may or may not be altered. A gene may not have any naturally occurring allelic variants or may have more than one. The usual mutational changes that give rise to allelic variants are generally due 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.

  A “transformed / modified” nucleic acid sequence encoding DME includes a polypeptide having at least one of the same polypeptides or functional characteristics of DME, with various nucleotide deletions, insertions or substitutions. Some sequences lead to peptides. 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 encoding DME, and also includes a polynucleotide encoding DME. It includes polymorphisms that are easily detectable or difficult to detect using certain oligonucleotide probes. The encoded protein can also be “transformed / modified” and can 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 residues to the extent that DME activity is preserved biologically or immunologically. It can be made based on one or more similarities. 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 can include asparagine and glutamine, and serine and threonine. Amino acids having uncharged side chains with similar hydrophilicity values may include leucine, isoleucine and valine, glycine and alanine, and phenylalanine and tyrosine.

  The terms “amino acid” and “amino acid sequence” refer to oligopeptides, peptides, polypeptides, protein sequences, or fragments of any thereof, and refer to natural or synthetic molecules. When an “amino acid sequence” refers to the sequence of a natural protein molecule, the “amino acid sequence” and similar terms are not limited to the complete and intact amino acid sequence associated with that protein molecule described.

  “Amplification” refers to the act of creating additional replicas of a nucleic acid. Amplification uses polymerase chain reaction (PCR) techniques or other nucleic acid amplification techniques known to those skilled in the art.

  The term “antagonist” refers to a molecule that inhibits or attenuates the biological activity of DME. Antagonists include antibodies, proteins, such as antibodies, nucleic acids, carbohydrates, and small molecules that directly interact with DME or modulate DME activity by acting on components of biological pathways involving DME. Or any other compound or composition.

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 animals such as mice, rats, rabbits may be in the case of RNA translation or chemical synthesis. They can also be conjugated to a carrier protein if desired. Examples of commonly used carriers that chemically bond to 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 an intact antigen (ie, an immunogen used to elicit an immune response) for 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 (eg, SELEX (abbreviation of Systematic Evolution of Ligands by EXponential Enrichment), described in US Pat. No. 5,270,163). This is a process of selecting target-specific aptamer sequences from a large group of combinatorial libraries. Aptamer compositions are double-stranded or single-stranded and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. Each nucleotide component of the aptamer may have a modified sugar group (eg, the 2′-OH group of a ribonucleotide can be replaced by 2′-F or 2′-NH ₂ ), which Desired properties such as resistance or longer life in blood can be improved. By conjugating the aptamer with another molecule (eg, a high molecular weight carrier), clearance of the aptamer from the circulatory system can be delayed. Aptamers can be specifically cross-linked with their homologous ligands, for example by photoactivation of certain cross-linking agents (Brody, EN and L. Gold (2000) J. Biotechnol. 74: 5-13).

The term “intramer” refers to an aptamer expressed in vivo . For example, certain RNA aptamers are expressed at high levels in the cytoplasm of leukocytes using an RNA expression system based on vaccinia virus (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96: 3606-3610).

  The term “spiegelmer” refers to aptamers such as left-handed nucleotide derivatives such as L-DNA and L-RNA or left-handed nucleotide-like molecules. Aptamers with levorotatory nucleotides are resistant to degradation by natural enzymes that normally act on substrates with dextrorotatory nucleotides.

  The term “antisense” refers to any composition capable of forming base pairs with the “sense” (coding) strand of a polynucleotide having a specific nucleic acid sequence. Antisense compositions include DNA, RNA, peptide nucleic acids (PNA), modified backbone linkages such as phosphorothioic acid, methylphosphonic acid or benzylphosphonic acid, modified sugar groups such as 2 ' There are oligonucleotides with -methoxyethyl sugar or 2'-methoxyethoxy sugar, or oligonucleotides with modified bases such as 5-methylcytosine, 2-deoxyuracil or 7-deaza-2'-deoxyguanosine obtain. Antisense molecules can be produced by any method, such as chemical synthesis or transcription. When introduced into a cell, a complementary antisense molecule forms a base pair with the natural nucleic acid sequence produced by the cell to form a duplex that interferes with 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.

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

  A “composition comprising (having) a polynucleotide of” or “a composition comprising (having) a polypeptide of” refers to any composition having the designated polynucleotide or polypeptide. The composition can include a dry formulation or an aqueous solution. Compositions having a group of polynucleotides encoding DME or a group of fragments of DME can be used as hybridization probes. These probes can be stored in lyophilized form and can be conjugated with stabilizers such as carbohydrates. In hybridization, the probe can be dispersed in an aqueous solution having a salt (eg, NaCl), a surfactant (eg, sodium dodecyl sulfate; SDS) and other components (eg, Denhart's solution, milk powder, salmon sperm DNA, etc.).

  The “consensus sequence” is subjected to repeated DNA sequence analysis to remove unnecessary bases, extended in the 5 ′ and / or 3 ′ direction using an XL-PCR kit (Applied Biosystems, Foster City CA), and sequenced again. One or more overlapping cDNAs or ESTs or genomic DNA using a synthesized nucleic acid sequence or a fragment binding computer program such as GELVIEW fragment binding system (GCG, Madison, WI) or Phrap (University of Washington, Seattle WA) Refers to a nucleic acid sequence assembled from fragments. Some sequences produce consensus sequences by both extension and assembly.

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 such substitution greatly changes. Refers to no replacement. 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-helical structure, (b) the molecular charge or hydrophobicity at the substitution site, and / or (c) the side chain. Hold the most.

  “Deletion” refers to a change in an amino acid sequence that lacks one or more amino acid residues, or a change in a nucleotide sequence that lacks one or more nucleotides.

  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. A polynucleotide derivative encodes a polypeptide that retains at least one biological or immunological function of the natural molecule. A polypeptide derivative is a polypeptide that has been modified by the following process. That is, glycosylation, polyethylene glycolation, or any similar process that retains at least one biological or immunological function of the originating polypeptide.

  “Detectable label” refers to a reporter molecule or enzyme that is covalently or non-covalently bound to a polynucleotide or polypeptide capable of generating a measurable signal.

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

  “Exon shuffling” refers to the recombination of different coding region (exon) groups. An exon can represent one structural or functional domain of the encoded protein, so it is possible to assemble a new protein group with a new combination of stable substructure groups, and a new protein function Can promote the evolution of

  The term “fragment” refers to a unique portion of DME or a polynucleotide encoding DME that may be identical in sequence to the parent sequence but shorter in length than the parent sequence. A fragment may have a length that is less than the total length of the defined sequence minus one nucleotide / amino acid residue. For example, a fragment can have from about 5 to about 1000 contiguous 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 residues long. Fragments can be preferentially selected from specific regions of a molecule. For example, a polypeptide fragment is a length 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 a continuous amino acid. These lengths are clearly given by way of example, and in embodiments of the present invention may be any length supported by this specification including the sequence listing, tables and drawings.

  A fragment of SEQ ID NO: 14-26 has a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 14-26, for example, different from other sequences in the genome from which the fragment was obtained. sell. Similar methods of distinguishing certain fragments of SEQ ID NO: 14-26 from one or more embodiments of the methods of the invention, eg, hybridization and amplification techniques, or SEQ ID NO: 14-26 from related polynucleotides Can be used. The exact length of a fragment of SEQ ID NO: 14-26 and the region of SEQ ID NO: 14-26 to which the fragment corresponds is routinely determined by those skilled in the art based on the purpose intended for the fragment. Can be determined.

  Certain fragments of SEQ ID NO: 1-13 are encoded by certain fragments of SEQ ID NO: 14-26. Certain fragments of SEQ ID NO: 1-13 may have unique amino acid sequence regions that specifically identify SEQ ID NO: 1-13. For example, a fragment of SEQ ID NO: 1-13 can be used as an immunogenic peptide in the development of an antibody that specifically recognizes SEQ ID NO: 1-13. The exact length of a fragment of SEQ ID NO: 1-13 and the region of SEQ ID NO: 1-13 corresponding to this fragment is described herein based on the intended purpose of the fragment. Or may be determined by one or more analytical methods known in the art.

  A “full length” polynucleotide 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.

  The term “homology” means sequence similarity, interchangeability, or sequence identity between two or more polynucleotide sequences or two or more polypeptide sequences.

  The terms “percent match” and “˜% identical” as applied to a polynucleotide sequence refer to the percentage of residues that match between two or more polynucleotide sequences aligned using a standardized algorithm. . Since the standardization algorithm optimizes the alignment between the two sequences, a gap group can be inserted into the two sequences to be compared in a standardized and reproducible manner, so that the two sequences can be compared more significantly.

  To determine percent identity (identity) between polynucleotide sequences, one or more computer algorithms or programs known in the art or described herein may be used. For example, to determine the match rate, the default parameters of the CLUSTAL V algorithm as incorporated in the MEGALIGN version 3.12e sequence alignment program can be used. This program is part of the LASERGENE software package (a set of molecular biological analysis programs) (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989; CABIOS 5: 151-153) and Higgins, D.G. et al. (1992; CABIOS 8: 189-191). The 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 by 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) provides a set of commonly used and freely available sequence comparison algorithms (Altschul, SF (1990) J. Mol. Biol. 215: 403-410). The BLAST algorithm is available from several sources and is also available from NCBI and the Internet (http://www.ncbi.nlm.nih.gov/BLAST/) in Bethesda, Maryland. The BLAST software suite includes a variety of sequence analysis programs, one of which is “blastn” that aligns a known polynucleotide sequence with another polynucleotide sequence obtained from various databases. A tool called “BLAST 2 Sequences” is also available, which is used to directly compare two nucleotide sequences. “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 generally uses parameters such as gaps set to default settings. For example, to compare two nucleotide sequences, blastn can 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 for the full length of a defined sequence (eg, a sequence defined with a particular SEQ ID number). Alternatively, the percentage match of shorter lengths, eg, lengths of defined, larger fragments (eg, fragments of at least 20, 30, 40, 50, 70, 100 or at least 200 consecutive nucleotides) Can also be measured. The lengths listed here are merely exemplary, and the percent identity can be measured using any fragment length supported by the sequences described herein, including tables, figures, and sequence listings. It should be understood that a certain length can be described.

  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 conserve the structure of the polypeptide (and therefore also the function) since it preserves the charge and hydrophobicity of the substitution site.

  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 (described above with reference). The 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. As with polynucleotide alignment, the percent identity of aligned polypeptide sequence pairs is reported by CLUSTAL V as “percent similarity”.

  Alternatively, the NCBI BLAST software suite can be used. For example, when comparing two polypeptide sequences pairwise, blastp of the “BLAST 2 Sequences” tool Version 2.0.12 (April 21, 2000) can be used with default parameters set. 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 full length of a defined polypeptide sequence (eg, the sequence defined by a particular SEQ ID number). Alternatively, 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 at least 150 consecutive residues). The coincidence rate can also be measured. The lengths listed here are merely exemplary, and the percent identity can be measured using any fragment length supported by the sequences described herein, including tables, figures, and sequence listings. It should be understood that a certain length can be described.

  A “human artificial chromosome (HAC)” is a linear microchromosome and can have a DNA sequence of about 6 kb to 10 Mb in size. It has all the elements necessary for chromosome replication, segregation and maintenance.

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

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

  Hybridization under high stringency conditions between polynucleotides of the invention includes wash conditions at 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 0.1% SDS. Alternatively, the temperature may be about 65 ° C, 60 ° C, 55 ° C, or 42 ° C. 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, in the hybridization of RNA and DNA under specific conditions, an organic solvent such as formamide at a concentration of about 35-50% v / v can also be used. Useful variations of the wash conditions will be apparent to those skilled in the art. Hybridization can indicate evolutionary similarity between nucleotides, particularly under high stringency conditions. Evolutionary 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 is present in a dissolved state and the other nucleic acid is a solid support (eg, paper, membrane, filter, chip, pin or glass slide, or other suitable substrate to which the cell or its nucleic acid is immobilized. Formed between two nucleic acids as fixed to the substrate).

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

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

  “Immunogenic fragment” refers to a polypeptide or oligopeptide fragment of DME that elicits an immune response when introduced into an organism, eg, 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, antibodies, or other compounds on a substrate.

  The term “element” or “array element” refers to a polynucleotide, polypeptide, antibody, or other compound having a specific designated position on a microarray.

  The term “modulate” or “modulate activity” refers to altering the activity of DME. For example, modulation results in an increase or decrease in DME protein activity, or a change in 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” also refer to DNA or RNA of genomic or synthetic origin that can be single-stranded or double-stranded, or can represent the sense or antisense strand It may also refer to 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, and can be in the same reading frame if necessary to join two protein coding regions.

  “Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent having 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 this composition. PNA binds preferentially to complementary single-stranded DNA or RNA and stops transcriptional elongation. Polyethylene glycolation can extend the lifetime of PNA in cells.

  “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 with cell type.

  The “probe” refers to a nucleic acid encoding DME, a complementary sequence thereof, or a fragment thereof, which is used for detecting the same nucleic acid, allelic nucleic acid, or related nucleic acid. A probe is an isolated oligonucleotide or polynucleotide that is a sequence attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, 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 amplification (and identification) of nucleic acids, eg, by polymerase chain reaction (PCR).

  The probes and primers used in the present invention usually consist of at least 15 consecutive nucleotide groups of known sequence. To increase specificity, longer probes and primers, such as probes consisting of at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or at least 150 consecutive nucleotides of the disclosed nucleic acid sequence And primers may also be used. Some probes and primers are much longer than this. It should be understood that nucleotides of any length supported by this specification, including tables, drawings and sequence listings may 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. (1999). ) Short Protocols in Molecular Biology , 4th edition, John Wiley & Sons, New York NY), and Innis, M. et al. (1990; PCR Protocols, A Guide to Methods and Applications , Academic Press, San Diego CA) It is described in the literature. To obtain a PCR primer pair from a known sequence, for example, a computer program therefor, such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA) can be used.

  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 sequences. 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. Using the Primer3 primer selection program (available from the Whitehead Institute / MIT Center for Genome Research, Cambridge, Mass.), You can enter a “mispriming library” that allows you to specify the sequences you want 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. Therefore, this program is useful for identifying oligonucleotides and polynucleotide fragments, whether unique or conserved. Oligonucleotides and polynucleotide fragments identified by any of the above selection methods identify complete or partially complementary polynucleotides in hybridization techniques, eg, as PCR or sequencing primers, as microarray elements, or in nucleic acid samples 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 nucleic acid, but a nucleic acid having a sequence that is an artificial combination of two or more separated segments of sequence. This artificial combination is often achieved by chemical synthesis, but more commonly by artificial manipulation of isolated segments of nucleic acids, for example by genetic engineering techniques such as those described in Sambrook (supra). To do. 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 recombinant nucleic acids 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.

  A “regulatory element” is a nucleic acid sequence usually derived from the untranslated region of a gene, including enhancers, promoters, introns, and 5 ′ and 3 ′ untranslated regions (UTRs). Regulatory elements interact with host or viral proteins that control transcription, translation or RNA stability.

  A “reporter molecule” is a chemical or biochemical component 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.

  An “RNA equivalent” for a DNA molecule consists of the same linear nucleotide sequence as the reference DNA molecule, but all the nitrogenous base thymines are replaced by uracil and the backbone of the sugar chain is deoxyribose. Without 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 cells and chromosomes isolated from cells, organelles, and membranes, There can be cells, genomic DNA, RNA, cDNA, tissue, tissue prints, etc. present in solution or fixed to a substrate.

  The terms “specific binding” and “specifically bind” refer to the interaction of a protein or peptide with an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. This interaction depends on the presence or absence of a specific structure of the protein (eg, an antigenic determinant or epitope) that is recognized by the binding molecule. For example, if an antibody is specific for epitope “A”, a polypeptide with epitope A may bind or bind in a reaction involving unbound labeled “A” and the antibody. If unlabeled “A” is present, the amount of labeled A bound to the antibody is reduced.

  The term “substantially purified” refers to an isolated or separated nucleic acid or amino acid sequence that has been removed from its natural environment and has at least about 60% removal of the naturally bound composition. Preferably about 75% or more, and most preferably about 90% or more.

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

  “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. included. The substrate can have various surface forms such as wells, grooves, pins, channels, holes, and the like, and polynucleotides and polypeptides are bound 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” refers to 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 is any known method that inserts an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. Based on this 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. A “transformed cell” includes a stably transformed cell 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 RNA for a limited period 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, subject to the present technology by human intervention. It has heterologous nucleic acid introduced by transgenic techniques known in the art. Nucleic acids are introduced into cells either directly or indirectly by introduction into cell precursors. This is done by planned genetic manipulation, for example by microinjection or by introduction of recombinant viruses. In another embodiment, the introduction of the nucleic acid can be accomplished by infecting a recombinant viral vector such as a lentiviral vector (Lois, C. et al. (2002) Science 295: 868-872). The term genetic engineering does not refer to classical crossbreeding or in vitro fertilization but to the introduction of recombinant DNA molecules. Among the genetically transformed organisms contemplated 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” of a specific nucleic acid sequence is a sequence determined as a nucleic acid sequence having at least 40% sequence identity to the specific nucleic acid sequence over a certain length of one nucleic acid sequence. For the determination, blastn is executed by using “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as 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. Variants can be described, for example, as “allelic” variants (described above) or “splice” variants, “species” variants, “polymorphic” variants. Splice variants may have significant identity to the 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 domain groups or may lack domain groups present in the reference molecule. Species variants are polynucleotides that vary from species to species. The resulting polypeptides usually have significant amino acid identity with each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between certain individuals. Polymorphic variants can also include “single nucleotide polymorphisms” (SNPs) that differ in one nucleotide base of the polynucleotide sequence. The presence of SNPs can indicate, for example, a particular population, condition or disease propensity.

  A “variant” of a specific polypeptide sequence is a sequence determined as a polypeptide sequence having at least 40% sequence identity to the specific polypeptide sequence over a certain length of one polypeptide sequence. It is. For the determination, blastp is executed by using “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as default parameters. Such polypeptide pairs are, for example, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% for one predetermined length of the polypeptide. 95%, 96%, 97%, 98%, 99% or more sequence identity.

(invention)
Various embodiments of the present invention include a novel group of human drug metabolizing enzymes (DME) and polynucleotides encoding DME, and using these compositions, autoimmune / inflammatory diseases, cell proliferation Includes diagnosis, treatment, or prevention of gastrointestinal disorders, including disorders, developmental or developmental disorders, endocrine disorders, ocular disorders, metabolic disorders, and liver disorders.

  Table 1 summarizes the nomenclature of the full-length polynucleotide embodiments and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide correlates to 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).

  Table 2 shows sequences homologous to the polypeptides of the present invention 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 homologue. Column 4 shows the probability score for a match between each polypeptide and one or more of its homologues. Column 5 shows the annotation of GenBank homolog (s).

  Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 each show the polypeptide sequence identification number (SEQ ID NO :) and the corresponding Incyte polypeptide sequence number (Incyte polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues 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.

  Tables 2 and 3 together 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: 2 was determined by the Basic Local Alignment Search Tool (BLAST) that residues M1 to S2457 were 84% identical to rat acetyl-CoA carboxylase (GenBank ID g3080546) ( (See Table 2). The BLAST probability score is 0.0, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 2 also has one carboxyltransferase domain and one biotin-requiring enzyme domain, which is a conserved PFAM database of protein family domains based on the Hidden Markov Model (HMM) Were determined by searching for statistically significant matches (see Table 3). Data from BLIMPS, MOTIFS, PROFILESCAN, and another BLAST analysis provide further confirmation that SEQ ID NO: 2 is an acetyl CoA carboxylase.

  In another example, SEQ ID NO: 5 was determined by BLAST that residues M1 through D274 were 87% identical to human glucosamine-6-phosphate deaminase (GenBank ID g2632113) (see Table 2). ). The BLAST probability score is 8.2e-133. SEQ ID NO: 5 also has one glucosamine-6-phosphate isomerase / 6-phosphogluconolactonases domain, which is statistically found in the PFAM database of conserved protein family domains based on HMM. Determined by searching for significant matches (see Table 3). Data from BLIMPS, MOTIFS and another BLAST analysis provide further confirmation that SEQ ID NO: 5 is glucosamine-6-phosphate deaminase.

  In another example, SEQ ID NO: 8 indicates that residues M1 through L723 are 97% identical to human enoyl CoA: hydratase / 3-hydroxyacyl CoA dehydrogenase (GenBank ID g452045) Judged by Local Alignment Search Tool (BLAST) (see Table 2). The BLAST probability score is 0.0. SEQ ID NO: 8 also includes one 3-hydroxyacyl CoA dehydrogenase C-terminal domain, one 3-hydroxyacyl CoA dehydrogenase NAD binding domain, and one enoyl CoA hydratase / 3-hydroxyacyl CoA dehydrogenase / isomerase family Although having a signature, this was determined by searching for statistically significant matches in the PFAM database of conserved protein family domains based on HMM (see Table 3). Data from BLIMPS, MOTIFS, PROFILESCAN, and another BLAST analysis provide further confirmation that SEQ ID NO: 8 is an enoyl CoA: 3-hydroxyacyl CoA dehydrogenase biceps enzyme.

  In another example, SEQ ID NO: 13 shows that the Basic Local Alignment Search Tool (BLAST ) (See Table 2). The BLAST probability score is 0.0. SEQ ID NO: 13 also has one carboxylesterase domain, which was determined by searching for statistically significant matches in the PFAM database of conserved protein family domains based on HMM (See Table 3). Data from BLIMPS, MOTIFS, and PROFILESCAN analyzes provide further confirmation that SEQ ID NO: 13 is acetylcholinesterase. SEQ ID NO: 1, SEQ ID NO: 3-4, SEQ ID NO: 6-7, and SEQ ID NO: 9-12 were analyzed and annotated in a similar manner. The algorithm and parameters for analysis of SEQ ID NO: 1-13 are listed in Table 7.

  As shown in Table 4, full-length polynucleotide embodiments were assembled using cDNA sequences or 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) and 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. Yes. Column 2 relates to the cDNA and / or genomic sequence used in the assembly of the full-length polynucleotide embodiment, and also for example to identify SEQ ID NO: 14-26, or in relation to SEQ ID NO: 14-26 The starting nucleotide (5 ′) position and the ending nucleotide (3 ′) position of a fragment of a polynucleotide useful in a hybridization technique or amplification technique for distinguishing from a group of polynucleotides to be performed are indicated.

The polynucleotide fragment described in column 2 of Table 4 may refer to, for example, an Incyte cDNA group derived from, for example, a tissue-specific cDNA library group or a pooled cDNA library group. Alternatively, the polynucleotide fragment described in column 2 may refer to the GenBank cDNA or EST that contributed to the assembly of the full-length polynucleotide. In addition, the polynucleotide fragments listed in column 2 may identify sequences from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (ie, sequences that include the designation “ENST”). Alternatively, the polynucleotide fragments listed 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. (Ie, sequences that include the designation “NP”). Alternatively, the polynucleotide fragment listed in column 2 may refer to an assembly consisting of both cDNA and Genscan predicted exons grouped together by an “exon-stitching” algorithm. For example, among the polynucleotide sequences, the sequence identified as FL_XXXXXX_N ₁ _N ₂ _YYYYY_N ₃ _N ₄ is a “stitched” sequence, of which XXXXXX is the identification number of the cluster of sequences to which the algorithm applies, YYYYY is the number of predictions made by the algorithm, and if there are N _1,2,3... , YYYYY represents a specific group of exons that were manually edited during the analysis (see Example 5 ). Alternatively, the polynucleotide fragments in row 2 may refer to an assembly of exons joined together by an “exon-stretching” algorithm. For example, among the polynucleotide sequences, the sequence identified as FLXXXXXX_gAAAAA_gBBBBB_1_N is the “stretch” sequence identification number. 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 of the nearest GenBank protein homolog, or NCBI RefSeq identification number, N is specified Exon (see Example 5 ). When a RefSeq sequence is used as a protein homolog for the “exon stretching” algorithm, the RefSeq identifier represented by “NM”, “NP”, or “NT” is the GenBank identifier (ie, gBBBBB ) Can be used instead.

Alternatively, the prefix code identifies a component sequence derived from a manually edited component sequence, a component sequence predicted from a genomic DNA sequence, or a combined sequence analysis method. The following table lists the constituent sequence prefix codes and examples of sequence analysis methods corresponding to the prefix codes (see Examples 4 and 5 ).

  In some cases, Incyte cDNA coverage overlapping with the sequence coverage as shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but no corresponding Incyte cDNA identification number was given.

  Table 5 shows a representative cDNA library for full-length polynucleotides assembled using Incyte cDNA sequences. A representative cDNA library is an Incyte cDNA library, which is most often represented by a group of Incyte cDNA sequences, which were used to assemble and confirm the polynucleotide. The tissues and vectors used to create the cDNA library are shown in Table 5 and described in Table 6.

  The present invention also includes DME variants. 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.

  Various embodiments also include a polynucleotide encoding DME. In certain embodiments, the present invention provides a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NO: 14-26, encoding DME. The polynucleotide sequence of SEQ ID NO: 14-26 also includes an equivalent RNA sequence as shown in the sequence listing, where the occurrence of the nitrogen base thymine is replaced by uracil and the sugar backbone is derived from ribose rather than deoxyribose. It is configured.

  The present invention also includes a group of polynucleotide variants encoding DME. Specifically, such variant polynucleotides have at least about 70%, or at least about 85%, or even at least about 95% polynucleotide sequence identity to a given polynucleotide sequence encoding DME. It will be. In certain embodiments of the present invention, SEQ ID NO: 14 having at least about 70%, or at least about 85%, or at least about 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 14-26 A polynucleotide variant having a sequence selected from the group consisting of NO: 14-26. Any of the polynucleotide variants described above may encode a polypeptide having at least one functional or structural characteristic of DME.

  Additionally or alternatively, certain polynucleotide variants of the invention are splice variants of a polynucleotide encoding DME. Some splice variants may have multiple portions with significant sequence identity to the polynucleotide encoding DME, but the addition or absence of a few blocks of sequence resulting from 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% of polynucleotide sequence identity is found over the entire length with the polynucleotide encoding DME, Some portions of this splice variant sequence are at least about 70%, or at least about 85%, or at least about 95%, or even 100% of the polynucleotide sequence identity with each portion of the polynucleotide encoding DME. It will have sex. For example, a polynucleotide having the sequence of SEQ ID NO: 24 and a polynucleotide having the sequence of SEQ ID NO: 25 are mutual splice variants. Any of the splice variants described above can encode a polypeptide 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 may cover all possible polynucleotide sequence variations 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.

  Polynucleotides encoding DME and its variants are generally capable of hybridizing to native DME polynucleotides under suitably selected stringency conditions, but include substantially different codons, such as including non-natural codon groups. It may be beneficial to create a polynucleotide 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 the 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 the production by complete synthetic chemistry of polynucleotide groups or fragments thereof encoding DME and its derivatives. After production, the synthetic polynucleotide can be inserted into any of a variety of available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry can be used to induce mutations in certain polynucleotides encoding DME or any fragment thereof.

  Embodiments of the present invention are capable of hybridizing under various stringency conditions to the claimed polynucleotide, in particular, the polynucleotide having the sequence shown in SEQ ID NO: 14-26, and fragments thereof. A group of polynucleotides (Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399-407; Kimmel, AR (1987) Methods Enzymol. 152: 507-511). Hybridization conditions, including annealing and washing conditions, are described in “Definitions”.

Methods for DNA sequencing are well known in the art, and DNA sequencing methods can be used in any embodiment of the invention. Enzymes can be used for DNA sequencing methods. For example, Klenow fragment of DNA polymerase 1, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ) can be used. Alternatively, a polymerase and a proofreading exonuclease can be used in combination, as seen, for example, in the ELONGASE amplification system (Invitrogen, Carlsbad CA). 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 (Amersham Biosciences) or other systems known in the art. The resulting sequences are analyzed using various algorithms well known in the art (Ausubel et al., Supra, Chapter 7; Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New York NY, 856- Page 853).

  Numerous methods based on the PCR method well known in the art and partial nucleotide sequences can be used to extend DME-encoding nucleic acids and detect upstream sequences such as promoters and regulatory elements . For example, one of the methods that can be used, restriction site PCR, is a method of amplifying an unknown sequence from genomic DNA in a cloning vector using universal primers and nested primers (Sarkar, G. (1993) PCR Methods Applic. 2: 318-322). Another method is inverse PCR, which amplifies an unknown sequence from a circularized template using a group of primers extending in various directions. The template is obtained from a group of restriction enzymes consisting of a certain known genomic locus and its surrounding sequences (Triglia, T. et al. (1988) Nucleic Acids Res. 16: 8186). A third method is capture PCR, which includes PCR amplification of DNA fragments adjacent to known sequences of human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic .1: 111-119). In this method, a recombinant double-stranded sequence can be inserted into an unknown sequence region using multiple restriction enzyme digestion and ligation reactions prior to PCR. Several other methods are known in the art that can be used to search for unknown sequences (eg Parker, J.D. et al. (1991) Nucleic Acids Res. 19: 3055-3060). In addition, genomic DNA can be walked using PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA). This procedure is useful for discovery of intron / exon junctions without the need to screen libraries. All PCR-based methods use commercially available software such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another suitable program, about 22-30 nucleotides in length and about 50% GC content. Thus, the primer group can be designed to anneal to the template at a temperature of about 68 ° C. to 72 ° C.

  When screening for full-length cDNA groups, it is preferable to use library groups that are size-selected to include larger cDNA groups. In addition, random primer libraries often contain sequences having the 5 ′ region of the gene cluster, and are suitable for situations where 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.

  Commercially available capillary electrophoresis systems 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 fluorescent dye 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, a polynucleotide encoding DME or a fragment thereof may be cloned into a recombinant DNA molecule that allows DME, a fragment or a functional equivalent thereof to be expressed in a suitable host cell. Due to the degeneracy inherent in the genetic code, other polynucleotides encoding substantially the same or functionally equivalent polypeptides can be produced and these sequences can be utilized for expression of DME.

  For various purposes, the polynucleotides of the invention can be recombined using a number of methods generally known in the art to modify the sequences encoding DME. The various purposes of this recombination include, but are not limited to, cloning of the gene product or modification of processing and / or expression. Nucleotide sequences can be recombined 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, alter glycosylation patterns, change codon preference, generate splice variants, and the like.

  Nucleotides of the present invention are disclosed in 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. Nat. Biotechnol. 17: 259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319). Modify or improve the biological properties of DME, such as its ability to bind molecules and compounds. DNA shuffling is a process of creating a library of gene variants using gene fragment recombination via PCR. The library is then subjected to a selection or screening procedure that identifies a group of genetic variants with the desired characteristics. These suitable variants can then be pooled and further repeated for DNA shuffling and selection / screening. Thus, “artificial” breeding and rapid molecular evolution create diverse genes. For example, single gene fragments with random point mutations can be recombined, screened, and then shuffled until the desired properties are optimized. Alternatively, a method for controlling the genetic diversity of a plurality of naturally occurring genes by recombining a fragment group of a predetermined gene with a homologous gene fragment of the same gene family obtained from either the same species or different species. Can be maximized.

In another embodiment, the polynucleotide 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; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7: 225-232). Alternatively, chemical methods known in the art can be used to synthesize DME itself or fragments thereof. For example, peptide synthesis can be performed using a variety of liquid or solid phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties , WH Freeman, New York NY, pages 55-60; Roberge, JY et al. (1995) ) Science 269: 202-204). Automated synthesis can be achieved using an ABI 431A peptide synthesizer (Applied Biosystems). Further, variant polypeptides may be obtained by directly modifying the amino acid sequence of DME, or any part thereof, and / or combining it with sequences from other proteins or any part thereof. Or a polypeptide having one sequence of a natural polypeptide.

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

  In order to express biologically active DME, a polynucleotide encoding DME or a derivative thereof may be inserted into a suitable expression vector. A suitable 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 DME-encoding polynucleotides (enhancers, constitutive and expression-inducible promoters, 5 ′ and 3 ′ untranslated regions, etc.). Necessary elements vary in strength and specificity. Specific initiation signals can also be used to achieve more efficient translation of a group of polynucleotides encoding DME. Examples of initiation signals include ATG initiation codons and nearby sequences such as Kozak sequences. If the polynucleotide sequence 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 such as 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 increase the efficiency of expression (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).

Methods which are well known to those skilled in the art can be used to create expression vectors with polynucleotides 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 (Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, 4 , 8, and 16-17; Ausubel et al., Supra, 1, 3 and 15).

A variety of expression vector / host systems may be utilized to maintain and express the polynucleotide encoding DME. Such expression vector / host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, 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, animal cell lines (Sambrook, supra; Ausubel et al., Supra; 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) EMBO J. 6: 307- 311; “McGraw Hill Science and Technology The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81: 3655 -3659; Harrington, JJ et al. (1997) Nat. Genet. 15: 345-355). Polynucleotides can be delivered to target organs, tissues or cell populations using expression vectors from retroviruses, adenoviruses, herpesviruses or vaccinia viruses, or expression vectors from various bacterial plasmids (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5: 350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6340-6344; Buller, RM et al. (1985) Nature 317: 813-815; McGregor, DP et al. (1994) Mol. Immunol. 31: 219-226; Verma, IM and N. Somia (1997) Nature 389: 239-242). The present invention is not limited by the host cell used.

  In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding DME. For example, a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen) can be used for routine cloning, subcloning and propagation of a polynucleotide encoding DME. Ligation of the polynucleotide encoding DME to the multicloning site of the vector disrupts the lacZ gene, allowing a colorimetric screening method for the identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription in cloned sequences, dideoxy sequencing, single-stranded rescue with helper phage, generation of nested deletions (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 with a strong inducible SP6 bacteriophage promoter or an inducible T7 bacteriophage promoter can be used.

  For the production of DME, a yeast expression system can be used. A large number of vectors with constitutive or inducible promoters such as alpha factor, alcohol oxidase, PGH promoter, etc. can be used in Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secreted or intracellular retention of the expressed protein and incorporate foreign polynucleotide sequences into the host genome for stable growth. (Ausubel et al., Supra; Bitter, GA et al. (1987) Methods Enzymol. 153: 516-544; Scorer, CA et al. (1994) Bio / Technology 12: 181-184).

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

  In mammalian cells, a number of virus-based expression systems can be utilized. When an adenovirus is used as an expression vector, a group of polynucleotides encoding DME can be ligated to an adenovirus transcription / translation complex consisting of a late promoter and a triple leader sequence. With the insertion of the adenoviral genome into the essential E1 or E3 region, infectious viruses that express DME in host cells can be obtained (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.

  In addition, human artificial chromosomes (HACs) can be used to deliver fragments of DNA that are larger than fragments that can be contained in a plasmid or that can be expressed from a plasmid. Approximately 6 kb to 10 Mb HACs have been generated for therapy and delivered by conventional delivery methods (liposomes, polycation amino polymers, or vesicles) (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 polynucleotide 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 in enhanced medium for about 1-2 days before being transferred to the selective medium. The purpose of the selectable marker is to confer resistance to the selective agent, and the presence of the selectable marker allows for the growth and recovery of cells that can 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. (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 the aminoglycosides 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). Additional selectable genes, such as trpB and hisD, are described, which transform the cellular requirements for metabolites (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 can be used. These markers can be used not only to identify transformants, but also to quantify transient or stable protein expression resulting from a particular vector system (Rhodes, CA (1995) Methods Mol. Biol. 55: 121-131).

  Even if the presence or 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 marker gene sequence, a transformed cell group having a polynucleotide group encoding DME can be identified by the lack of marker gene function. Alternatively, a marker gene can be placed in tandem with one sequence encoding DME under the control of a single promoter. Expression of a marker gene in response to induction or selection usually also indicates tandem gene expression.

  In general, a host cell having a polynucleotide 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. This includes but is not limited to test methods or immunological assays.

Immunological methods for the 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 well 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 , Humana Press, Totowa NJ).

  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, a polynucleotide encoding DME, 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 the synthesis of RNA probes in vitro with the addition of a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. Can do. These methods can be performed using various commercial kits such as Amersham Biosciences, Promega (Madison WI), U.S. Biochemical. Suitable reporter molecules or labels that can be used to facilitate detection include substrates, cofactors, inhibitors, magnetic particles, as well as radionuclides, enzymes, fluorescent agents, chemiluminescent agents, color formers, and the like.

  Host cells transformed with a polynucleotide encoding DME can be cultured under conditions suitable for expression and recovery of the 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. Those 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 a host cell line may be made by its ability to modulate the expression of the inserted polynucleotide 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, HEK293, WI38, etc.) with specific cellular equipment and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATCC, Manassas, VA). And can be selected to ensure correct modification and processing of the foreign protein.

  In another embodiment of the invention, a natural, modified or recombinant polynucleotide encoding DME is linked to a heterologous sequence and the translation of a fusion protein is performed in any of the host systems described above. Can result. 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 the fusion protein using a commercially available affinity matrix. 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 et al., Supra, chapters 10 and 16. Various commercially available kits can also be used to facilitate the expression and purification of the fusion protein.

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

  DME or a fragment or mutant thereof can be used to screen for a compound that specifically binds to DME. One or more test compounds can be used to screen for specific binding to DME. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to DME. Examples of test compounds include antibodies, anticalins, oligonucleotides, proteins (eg, ligands and receptors), or small molecules.

  In a related embodiment, the DME variant can be used to screen the binding of a test compound, eg, an antibody, to the DME, to the DME variant, or to the combined DME and / or one or more DME variants. In certain embodiments, a DME variant can be used to screen for compounds that bind to the DME variant but do not bind to DME having the exact sequence of SEQ ID NO: 1-13. DME variants used to perform such screening may have a range of sequence identity to about 50% to about 99% DME, with various embodiments being 60%, 70%, 75%, 80%, It can have 85%, 90%, and 95% sequence identity.

In some embodiments, a compound identified by a specific binding screen to DME may be closely related to the natural ligand of DME, for example, a ligand or a fragment thereof, or a natural substrate, structural or functional It is a mimetic or natural binding partner (Coligan, JE et al. (1991) Current Protocols in Immunology 1 (2): Chapter 5). In another embodiment, the compound thus identified can be the natural ligand of the receptor DME (Howard, AD et al. (2001) Trends Pharmacol. Sci. 22: 132-140; Wise, A. et al. (2002) Drug Discovery Today 7: 235-246).

In another embodiment, the compound identified by screening for specific binding to DME is a natural receptor to which DME binds, or at least to some fragment of the receptor, or for example, all or part of a ligand binding site or binding pocket. May be closely related to certain fragments of the receptor. For example, the compound may be a DME receptor capable of propagating a signal, or a DME decoy receptor that cannot propagate a signal (Ashkenazi, A. and VM Divit (1999) Curr. Opin. Cell Biol. 11: 255 -260; Mantovani, A. et al. (2001) Trends Immunol. 22: 328-336). The compound can be rationally designed using known techniques. Examples of such techniques include those used to make the compound etanercept (ENBREL; Immunex Corp., Seattle WA). Etanercept is effective in treating human rheumatoid arthritis. Etanercept is a genetically engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG ₁ (Taylor, PC et al. (2001) Curr. Opin. Immunol. 13: 611-616) .

  In one embodiment, two or more antibodies with similar or different specificities can be screened for specific binding to DME or a fragment or variant thereof. The binding specificity of the antibody thus screened can be selected to identify a particular fragment or variant of DME. In one embodiment, antibodies with binding specificities that can preferentially identify specific fragments or variants of DME can be selected. In another embodiment, antibodies with binding specificities that can preferentially diagnose specific diseases or conditions with increased, decreased, or abnormal DME production can be selected.

  In one embodiment, anticalin can be screened for specific binding to DME or a fragment or variant thereof. Anticarin is a ligand binding protein made based on a lipocalin scaffold (Weiss, GA and HB Lowman (2000) Chem. Biol. 7: R177-R184; Skerra, A. (2001) J. Biotechnol. 74: 257-275). The protein structure of lipocalins may include a β-barrel with an eight-stranded antiparallel β-strand that supports four loops at its open end. These loops form the natural ligand binding site of lipocalin, which can be engineered again by amino acid substitution in vitro to give a novel binding specificity. This amino acid substitution can be made using methods known in the art or as described herein. In addition, conservative substitutions (for example, substitutions that do not change the binding specificity) or substitutions that slightly, moderately or greatly change the binding specificity can be performed.

  In one embodiment, screening for compounds that specifically bind to or stimulate or inhibit DME includes the generation 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 cell membrane fractions containing DME, are contacted with a test compound and analyzed for binding, stimulation, or inhibition of the activity of either DME or the test compound.

  Some assays simply allow the test compound to be conjugated experimentally to the polypeptide and the binding detected with 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, detection and measurement of test compound binding in the presence of labeled competitor can be performed. In addition, this assay can be performed using cell-free reconstituted specimens, chemical libraries, or natural product mixtures, where the test compound (s) are released in solution or immobilized on a solid support.

  The assay can be used to assess the ability of a compound to bind to its natural ligand and / or inhibit its binding to its natural receptor. Examples of such assays include radiolabel assays such as those described in US Pat. Nos. 5,914,236 and 6,372,724. In related embodiments, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or modify its ability to bind to a natural ligand (Matthews, DJ and JA Wells. (1994). ) Chem. Biol. 1: 25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or modify its ability to bind to its natural receptor (Cunningham, BC and JA Wells ( 1991) Proc. Natl. Acad. Sci. USA 88: 3407-3411; Lowman, HB et al. (1991) J. Biol. Chem. 266: 10982-10988).

  DME or fragments or variants thereof can be used to screen for compounds that modulate the activity of DME. 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 embodiment, homologous recombination in embryonic stem cells (ES cells) is used to “knock out” a polynucleotide encoding DME or a mammalian homologue thereof in an animal model system. Such techniques are well known in the art and are useful for generating animal models of human disease (see, eg, US Pat. Nos. 5,175,383 and 5,767,337). For example, mouse ES cells such as the 129 / SvJ cell line are derived from early mouse embryos and can be grown in culture. The ES cells are transformed with a vector having the target gene 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. Genetically modified animals produced in this way can be tested with potential therapeutic and toxic drugs.

  Polynucleotides encoding DME can be manipulated 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, for example, into neural cells, hematopoietic lineages and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282: 1145-1147).

  Polynucleotides encoding DME can be used to create “knock-in” humanized animals (pigs) or transgenic animals (mouse or rat) that model 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 inbreds and treat with potential medicines to obtain information on the treatment of human disease. Alternatively, mammalian inbred lines that overexpress DME, such as secreting DME into milk, can also be a convenient source of this protein (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 with respect to sequences and motifs. DME expression is closely related to kidney tissue, liver tissue, brain tissue, adrenal tumor tissue, bladder tumor tissue, lung tumor tissue. See also Table 6 and Example 11 for several examples of tissues that express DME. Thus, DME is thought to play a role in gastrointestinal diseases including autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine disorders, ocular diseases, metabolic disorders, and liver diseases. 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. Such diseases include, but are not limited to, autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine disorders, ophthalmic diseases, metabolic disorders, and gastrointestinal disorders including liver disorders, and autoimmunity / Inflammatory diseases include acquired immune deficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, Autoimmune thyroiditis, autoimmune multiglandular endocrine candidiasis ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphocyte injury Factorized incident lymphopenia, neonatal hemolytic disease (fetal erythroblastosis), erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout, Graves' disease, Thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter syndrome, Rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, cancer, hemodialysis, extracorporeal circulation Complications, viral infections, bacterial infections, fungal infections, parasitic infections, protozoal infections, helminth infections, trauma, cell proliferation abnormalities include actinic keratosis, arteriosclerosis, atherosclerosis, lubrication Bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma, leukemia, lymphoma , Black Cancers such as myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, mammary gland, neck, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary Includes cancer of the pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterus. Developmental disorders include tubular acidosis, anemia, Cushing syndrome, chondrogenic dystrophy, Duchenne / Becker muscular dystrophy, epilepsy, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, Mental retardation), Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoses, hereditary neuropathies (Charcot-Marie-Tooth disease and neurofibromatosis, etc.) ), Seizure disorders such as hypothyroidism, hydrocephalus, Syndenham's chorea and cerebral palsy, spina bifida, anencephalopathy, cranial spinal vertebrae, congenital glaucoma, cataract, sensorineural hearing loss. Endocrine disorders include hypothalamus and pituitary glands that result from lesions such as primary brain tumors, adenomas, gestational infarctions, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immune abnormalities, head trauma complications, etc. Disability and eg sexual dysfunction and Sheehan syndrome, diabetes insipidus, Kalman disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, Turkish empty sella syndrome, dwarfism Disorders associated with hypopituitarism, including, for example, acromegaly, giantosis, disorders associated with hyperpituitary gland, including antidiuretic hormone (ADH) incompatible secretion syndrome (SIADH), which is likely to occur with benign adenomas, and For hypothyroidism including goiter, myxedema, bacterial infectious acute thyroiditis, viral infectious subacute thyroiditis, autoimmune thyroiditis (Hashimoto's disease), cretin disease Consecutive disorders, including thyroid poisoning and its various forms, Graves' disease, anterior tibial myxedema, toxic multinodular goiter, thyroid cancer, Plumer's hyperthyroidism, and Conn's disease (chronic low potassium Parathyroidism, including type I and type II diabetes and complications, eg, adrenal carcinoma, hyperplasia and adenoma, alkalosis-related hypertension, amyloidosis , Hypokalemia, Cushing's disease, Riddle syndrome, Arnold-Healy-Gordon syndrome, pheochromocytoma, Addison's disease such as Addison's disease and abnormal prolactin production in women, infertility, endometriosis, menstrual cycle perturbation, Polycystic ovary disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, milk leakage, semi-yin yang, hirsutism and masculinization, breast cancer, Post-surgical osteoporosis, male Leydig cell dysfunction, male menopause, germ cell aplasia, hypersexuality associated with Leysch cell tumor, androgen resistance associated with lack of androgen receptor, 5α-reductase syndrome, gynecomastia Including gonadal steroid hormone related diseases. Eye diseases include conjunctivitis, dry keratoconjunctivitis, keratitis, suprasclerosis, iritis, posterior uveitis, glaucoma, transient cataract, ischemic optic neuropathy, optic neuritis, Leber hereditary optic neuropathy, moderate Toxic optic neuropathy, vitreous detachment, retinal detachment, cataract, macular degeneration, central serous chorioretinopathy, retinitis pigmentosa (pigmentary retinitis), choroidal melanoma, retrobulbar tumor, chiasmal tumor There is. Metabolic disorders include Addison's disease, cerebral tendon xanthoma, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty cirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, thyroid Tumor, glucagonoma, glycogenosis, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, Hyperlipidemia, hyperlipidemia, lipid myopathies, lipodystrophy, lysosomal storage disease, Menkes syndrome, occipital horn syndrome, mannosidosis, neuraminidase deficiency, obesity, pentose -Phenylketonuria, pseudovitamin D deficiency rickets, hypocalcemia, hypophosphatemia, postadolescent cerebellar ataxia, tyrosineemia. Gastrointestinal disorders include dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal cancer, dyspepsia, digestive disorders, gastritis, gastric cancer, loss of appetite, nausea, vomiting, gastric insufficiency, sinus or pyloric edema, abdomen Angina, heartburn, gastroenteritis, ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary tract disease, hepatitis, hyperbilirubinemia, hereditary hyperbilirubinemia, hard Metamorphosis, passive hepatic congestion, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colon cancer, colonic obstruction, irritable bowel syndrome, short small intestine Syndrome, diarrhea, constipation, gastrointestinal bleeding, acquired immune deficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, fatty liver, hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, Reye syndrome, Primary sclerosing bile Inflammation, liver infarction, portal vein occlusion and thrombus, central lobular necrosis, liver purpura, hepatic vein thrombus, hepatic vein obstruction, preeclampsia, eclampsia, gestational acute fatty liver, intrahepatic cholestasis, There are liver tumors including nodular hyperplasia, adenoma, and carcinoma.

  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, a subject may be administered an antagonist of DME 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 above mentioned autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine disorders, ophthalmic disorders, metabolic disorders, and gastrointestinal disorders including liver disorders It is. 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, agonist, antagonist, complementary sequence, or vector embodiment 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 increase the 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, purified DME can be used to produce antibodies or screen a library of therapeutic agents to identify drugs that specifically bind to DME. 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) may be potent enzyme inhibitors and appear to have advantages in the design of peptidomimetics and in 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 oligos thereof with 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).

  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 (Morrison, SL (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855; Neuberger, MS et al. (1984) Nature 312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454). 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 combinatorial immunoglobulin libraries (Burton DR (1991) Proc. Natl. Acad. Sci. USA 88: 10134-10137).

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

Antibody fragments 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, by producing a Fab expression library, it is possible to quickly and easily identify a monoclonal Fab fragment having the desired specificity (Huse, WD et al. (1989) Science 246: 1275-1281).

  Screening with various immunoassays (immunoassays) can identify antibodies with the desired specificity. Numerous protocols for competitive binding studies using either polyclonal or monoclonal antibodies with established specificity, or for 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 represented by a 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 the Ka determined for a given polyclonal antibody formulation represents the average affinity or binding activity of the group of antibodies to DME. Monoclonal antibodies are monospecific for certain DME epitopes, and the Ka determined for certain formulations of monoclonal antibodies 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. For immunopurification and similar treatments where DME must eventually dissociate from the antibody (preferably in an activated state), a low affinity antibody formulation with a Ka value of 10 ⁶ to 10 ⁷ liter / mol is used. (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 formulation for certain applications used later. For example, polyclonal antibody formulations containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of 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 (Catty, supra; Coligan et al., Supra).

In another embodiment of the invention, a polynucleotide encoding DME, or any fragment or complementary sequence thereof, is used for therapeutic purposes. In one embodiment, gene expression can be altered by designing sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) complementary to the coding and regulatory regions of the gene encoding DME. . Such techniques are well known in the art, and antisense oligonucleotides or larger fragments can be designed from the control region of the sequence encoding DME or from various positions along the coding region (Agrawal, S , Editing (1996) Antisense Therapeutics , Humana Press, 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 a sequence 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: 469-475; Scanlon, KJ et al. (1995) 9: 1288-1296). Antisense sequences can also be introduced into cells using viral vectors such as retroviral and adeno-associated viral vectors (Miller, AD (1990) Blood 76: 271; Ausubel et al., Supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63: 323-347). Other gene delivery mechanisms include liposome systems, artificial viral envelopes and other systems known in the art (Rossi, JJ (1995) Br. Med. Bull. 51: 217-225; Boado, RJ (1998) J. Pharm. Sci. 87: 1308-1315; Morris, MC et al. (1997) Nucleic Acids Res. 25: 2730-2736).

  In another embodiment of the invention, a polynucleotide encoding DME can be used for somatic or germ cell gene therapy. Gene therapy treats (i) gene deficiency (eg, severe combined immunodeficiency (SCID) − characterized by X chromosome linkage inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288: 669-672) X1 disease), severe combined immunodeficiency syndrome 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, and hemophilia caused by factor 8 or factor 9 deficiency (Crystal, 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) Intravesicular parasites such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335: 395-396, Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93: 11395 -11399) and other proteins such as human retroviruses, hepatitis B or C virus (HBV, HCV), parasitic fungi such as Candida albicans and Paracoccidioides brasiliensis, and parasites such as Plasmodium falciparum and Trypanosoma cruzi It can be expressed. When a deficiency in a gene required for DME expression or regulation causes a disease, it is possible to express DME from a suitable population of introduced cells to alleviate the expression of symptoms caused by the gene deficiency.

  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) with receptor-mediated gene transfer, and (v) with the use of DNA transposons (Morgan, RA and WF Anderson (1993) Annu. Rev. Biochem. 62: 191-217, Ivics, Z. (1997) Cell 91: 501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9: 445-450).

  Expression vectors that can affect the expression of DME 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), PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA) are included. 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 regulatable 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 (ii i) It is possible to use a natural promoter or a tissue-specific promoter of an endogenous gene encoding DME derived from a normal individual.

  By using a commercially available liposome transformation kit (for example, Invitrogen's PerFect Lipid Transfection Kit), those skilled in the art do not need much effort to optimize the parameters of the experiment, and the polynucleotide group can be used as the target cell group in culture. Can be delivered. Alternatively, the calcium phosphate method (Graham. F.L. and A.J. Eb (1973) Virology 52: 456-467) or electroporation (Neumann, E. et al. (1982) EMBO J. 1: 841-845). In order to introduce DNA into primary culture cells, modifications to standardized mammalian transfection protocols are required.

In another embodiment of the present invention, a disease or disorder caused by a genetic defect associated with DME expression comprises (i) a polyvirus encoding DME under the control of a retroviral terminal repeat (LTR) promoter or an independent promoter. Nucleotides; (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 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 cited as a part of this specification. This vector is propagated in a suitable vector producing cell line (VPCL). VPCL expresses an envelope gene with affinity for a receptor on each 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). US Pat. No. 5,910,434 to “Rigg” (“Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant”) discloses a method for obtaining a retroviral packaging cell line. Part. Propagation of retroviral vectors, transduction of cell populations (eg CD4 ⁺ T cell populations), and return of transduced cell populations to patients are techniques well known to those skilled in the field of gene therapy, (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 Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 1201-1206; Su, L. (1997) Blood 89: 2283-2290).

  In one embodiment, an adenoviral gene therapy delivery system is used to deliver a polynucleotide encoding DME to cells having one or more genetic abnormalities associated with expression of DME. The production and packaging of adenoviral vectors is well known to those skilled in the art. Replication-deficient adenovirus vectors have been proved to be able to be used in a variety of ways to introduce genes encoding various immunoregulatory proteins into intact islets (Csete, ME et al. (1995) Transplantation 27: 263-268). Adenoviral vectors that may be used are described in US Pat. No. 5,707,618 (“Adenovirus vectors for gene therapy”) to Armentano, which is incorporated herein by reference. For adenoviral vectors, see Antinozi, PA et al. (1999; Annu. Rev. Nutr. 19: 511-544) and Verma, IM and N. Somia (1997; Nature 18: 389: 239-242). .

  In another embodiment, a herpes gene therapy delivery system is used to deliver a polynucleotide encoding DME to target cells having one or more genetic abnormalities associated with expression of DME. 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. A replication competent herpes simplex virus (HSV) type 1 vector has been used to deliver a reporter gene to the primate eye (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”) to DeLuca, which is hereby incorporated 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 Goins, W.F. et al. (1999; J. Virol. 73: 519-532) and Xu, H. et al. (1994; Dev. Biol. 163: 152-161). 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.

  In another embodiment, alphavirus (positive single stranded RNA virus) vector is used to deliver a polynucleotide encoding DME to 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 to a higher level than the full-length genomic RNA, the capsid proteins are overproduced compared to viral proteins with enzymatic activity (eg, proteases and polymerases). 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 cells, 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) with a 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 host range of α virus is wide, 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 a transcription initiation site. This position 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 inhibits the ability of the double helix to open sufficiently so that it can bind 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, Mt. Kisco NY, pp. 163-177. ). 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 that can 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, artificially engineered hammerhead ribozyme molecules may specifically and effectively catalyze the cleavage of nucleotides of RNA molecules 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, evaluate whether a short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene with 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.

Complementary ribonucleic acid molecules and ribozymes can be made using any method known in the art for nucleic acid molecule synthesis. As a production method, there is a method of chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA molecules 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 adjacent 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 originally in the production of PNA groups, but can be extended to all these molecules. For this purpose, acetyl-, methyl-, thio- and similar modifications of adenine, cytidine, guanine, thymine, and uridine that are not easily recognized by endogenous endonucleases, or non-conventional bases such as inosine , Queosine, wybutosine.

  A further embodiment of the invention includes a method of screening for compounds effective in altering the expression of a polynucleotide encoding DME. Compounds that may be effective in causing, but not limited to, expression modification of specific polynucleotides include oligonucleotides, antisense oligonucleotides, triplex-forming oligonucleotides, polypeptide transcriptional regulators such as transcription factors, and There are non-polymeric chemical entities that can interact with specific polynucleotide 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 expression or activity of DME. In the treatment of disease, compounds that specifically promote the expression of polynucleotides encoding DME may be therapeutically useful.

  At least one or more test compounds can be screened for effectiveness in altering the expression of a particular polynucleotide. Any method known in the art can be used to obtain the test compound. As an acquisition method, there is chemical modification of a known compound effective in the following cases. When modifying the expression of a polynucleotide, when selecting from a library of existing, commercial or private, natural or non-natural compounds, and compounds based on the chemical and / or structural properties of the target polynucleotide. There is a case of rational design and a case of selecting from a library of compounds generated in combination or at random. A sample with a polynucleotide encoding DME is exposed to at least one of the test compounds thus obtained. The sample can be, for example, an intact cell, a permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. 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. Quantifying the amount of hybridization can form the basis for comparison of expression of polynucleotides that are exposed to or 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. Screening for compounds effective for altered expression of certain polynucleotides can be performed, such as the 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). Certain embodiments of the invention relate to the process of screening a combinatorial library of oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, modified oligonucleotides) for antisense activity against a particular polynucleotide sequence (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 or by ribosome injection or polycation amino polymers can be performed using methods well known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15: 462-466).

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

A further embodiment of the invention relates to the administration of certain 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 consist of 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, intestinal, topical, sublingual or rectal.

  Compositions administered from the lungs can 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 traditional 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.

  A variety of specially shaped composition groups can be prepared to deliver macromolecular groups with DME or fragments thereof directly into cells. 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 proteins thus generated have been shown to transduce cell groups of all tissues, including the brain, of a mouse model system (Schwarze, SR 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 the amount of 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 measured 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 develop a range of dosage for human use. The dosage contained in such compositions is preferably in a range of blood concentrations that includes ED ₅₀ with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, patient sensitivity and route of administration.

  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 relating to the subject may include the severity of the disease, the general health of the patient, the patient's age, weight and sex (gender), time and frequency of administration, concomitant medications, 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 0.1-100,000 μg, up to a total of about 1 g. Guidance on specific dosages and delivery methods is described in the literature and is usually available to practitioners. 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 DME expression or to monitor patients treated with DME or DME agonists, antagonists or inhibitors Used for. Antibodies useful for diagnostic purposes are made in the same manner as described above in the treatment section. Diagnostic assays for DME include methods for detecting DME from human body fluids or from cell or tissue extracts using antibodies and labels. This antibody is used with or without modification. It can also 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 diagnosis. Polynucleotides that can be used include oligonucleotides, complementary RNA and DNA molecules, and PNA. 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, hybridization with PCR probes that can detect a group of polynucleotides, such as a group of genomic sequences that encode DME or a group of closely related molecules, can be used to identify nucleic acid sequences that encode DME. . 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 target hybridization probe of the present invention can be DNA or RNA, and can be derived from the sequence of SEQ ID NO: 14-26, or the genomic sequence including the promoter, enhancer, and intron of the DME gene.

As a means for preparing a hybridization probe group specific to a polynucleotide group encoding DME, a method of cloning a polynucleotide group encoding DME or a 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.

  A polynucleotide encoding DME can be used to diagnose a disease associated with DME expression. Such diseases include, but are not limited to, autoimmune / inflammatory diseases, cell proliferation abnormalities, developmental or developmental disorders, endocrine disorders, ophthalmic diseases, metabolic disorders, and gastrointestinal disorders including liver disorders, and autoimmunity / Inflammatory diseases include acquired immune deficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, Autoimmune thyroiditis, autoimmune multiglandular endocrine candidiasis ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphocyte injury Factorized incident lymphopenia, neonatal hemolytic disease (fetal erythroblastosis), erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout, Graves' disease, Thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter syndrome, Rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, cancer, hemodialysis, extracorporeal circulation Complications, viral infections, bacterial infections, fungal infections, parasitic infections, protozoal infections, helminth infections, trauma, cell proliferation abnormalities include actinic keratosis, arteriosclerosis, atherosclerosis, slipping Bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma, leukemia, lymphoma , Black Cancers such as myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, mammary gland, neck, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary Includes cancer of the pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterus. Developmental disorders include tubular acidosis, anemia, Cushing syndrome, chondrogenic dystrophy, Duchenne / Becker muscular dystrophy, epilepsy, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, Mental retardation), Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoses, hereditary neuropathies (Charcot-Marie-Tooth disease and neurofibromatosis, etc.) ), Seizure disorders such as hypothyroidism, hydrocephalus, Syndenham's chorea and cerebral palsy, spina bifida, anencephalopathy, cranial spinal vertebrae, congenital glaucoma, cataract, sensorineural hearing loss. Endocrine disorders include hypothalamus and pituitary glands that result from lesions such as primary brain tumors, adenomas, gestational infarctions, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immune abnormalities, head trauma complications, etc. Disability and eg sexual dysfunction and Sheehan syndrome, diabetes insipidus, Kalman disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, Turkish empty sella syndrome, dwarfism Disorders associated with hypopituitarism, including, for example, acromegaly, giantosis, disorders associated with hyperpituitary gland, including antidiuretic hormone (ADH) incompatible secretion syndrome (SIADH), which is likely to occur with benign adenomas, and For hypothyroidism including goiter, myxedema, bacterial infectious acute thyroiditis, viral infectious subacute thyroiditis, autoimmune thyroiditis (Hashimoto's disease), cretin disease Consecutive disorders, including thyroid poisoning and its various forms, Graves' disease, anterior tibial myxedema, toxic multinodular goiter, thyroid cancer, Plumer's hyperthyroidism, and Conn's disease (chronic low potassium Parathyroidism, including type I and type II diabetes and complications, eg, adrenal carcinoma, hyperplasia and adenoma, alkalosis-related hypertension, amyloidosis , Hypokalemia, Cushing's disease, Riddle syndrome, Arnold-Healy-Gordon syndrome, pheochromocytoma, Addison's disease such as Addison's disease and abnormal prolactin production in women, infertility, endometriosis, menstrual cycle perturbation, Polycystic ovary disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, milk leakage, semi-yin yang, hirsutism and masculinization, breast cancer, Post-surgical osteoporosis, male Leydig cell dysfunction, male menopause, germ cell aplasia, hypersexuality associated with Leysch cell tumor, androgen resistance associated with lack of androgen receptor, 5α-reductase syndrome, gynecomastia Including gonadal steroid hormone related diseases. Eye diseases include conjunctivitis, dry keratoconjunctivitis, keratitis, suprasclerosis, iritis, posterior uveitis, glaucoma, transient cataract, ischemic optic neuropathy, optic neuritis, Leber hereditary optic neuropathy, moderate Toxic optic neuropathy, vitreous detachment, retinal detachment, cataract, macular degeneration, central serous chorioretinopathy, retinitis pigmentosa (pigmentary retinitis), choroidal melanoma, retrobulbar tumor, chiasmal tumor There is. Metabolic disorders include Addison's disease, cerebral tendon xanthoma, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty cirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, thyroid Tumor, glucagonoma, glycogenosis, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, Hyperlipidemia, hyperlipidemia, lipid myopathies, lipodystrophy, lysosomal storage disease, Menkes syndrome, occipital horn syndrome, mannosidosis, neuraminidase deficiency, obesity, pentose -Phenylketonuria, pseudovitamin D deficiency rickets, hypocalcemia, hypophosphatemia, postadolescent cerebellar ataxia, tyrosineemia. Gastrointestinal disorders include dysphagia, peptic esophagitis, esophageal spasm, esophageal stenosis, esophageal cancer, dyspepsia, digestive disorders, gastritis, gastric cancer, loss of appetite, nausea, vomiting, gastric insufficiency, sinus or pyloric edema, abdomen Angina, heartburn, gastroenteritis, ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary tract disease, hepatitis, hyperbilirubinemia, hereditary hyperbilirubinemia, hard Metastasis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colon cancer, colonic obstruction, irritable bowel syndrome, short small intestine Syndrome, diarrhea, constipation, gastrointestinal bleeding, acquired immune deficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, fatty liver, hemochromatosis, Wilson's disease, alpha-1-antitrypsin deficiency, Reye syndrome, Primary sclerosing bile Inflammation, liver infarction, portal circulation occlusion and thrombus, central lobular necrosis, liver purpura, hepatic vein thrombus, hepatic vein occlusion, preeclampsia, eclampsia, gestational acute fatty liver, intrahepatic cholestasis, There are liver tumors including nodular hyperplasia, adenoma, and carcinoma. Polynucleotides encoding DME include Southern, Northern, dot blot, or other membrane-based technologies, PCR methods, dipsticks, pins, and multi-format ELISA assays, and It can be used in microarrays that utilize body fluids or tissues collected from patients to detect altered DME expression. Such qualitative or quantitative methods are known in the art.

  In certain embodiments, nucleotide groups encoding DME may be used in assays that detect related disorders, particularly those mentioned above. A polynucleotide complementary to the sequence encoding DME can be labeled by standard methods and added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of a hybridization complex. 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 a patient sample is significantly changed compared to a control sample, the presence of a level change in the polynucleotide group encoding DME in that sample reveals the presence of the associated disease. Such assays can also be used to evaluate 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 achieved by mixing a body fluid or cell extract collected from either a normal animal or human subject with a sequence encoding DME or a fragment 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 that observed by 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 transcripts (underexpressed or overexpressed) in living tissue from an individual indicates a predisposition to the disease or a method to detect the disease before clinical symptoms appear. Can 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 and 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, single nucleotide polymorphisms (SNPs) can be detected using oligonucleotide primers derived from a group of polynucleotides encoding DME. 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 using oligonucleotide primers derived from a group of polynucleotides encoding DME and polymerase chain reaction (PCR). This DNA can be derived from, for example, diseased or normal tissue, biopsy samples, body fluids, and the like. SNPs in DNA make a difference in the secondary and tertiary structure of single-stranded PCR products. Differences can be detected using gel electrophoresis in non-denaturing gels. In fSSCP, oligonucleotide primers are 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) identifies polymorphisms by comparing the sequences of individual overlapping DNA fragments that are assembled into a common consensus sequence. Can do. These computer-based methods filter out sequence variations that result from sequencing errors in the laboratory preparation of DNA or using statistical models and automated analysis of 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 used to study the genetic basis of human disease. For example, at least 16 common SNPs are associated with non-insulin dependent diabetes. 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, mutants in MBL2, a mannose-binding lectin, have been shown to correlate with adverse lung outcome in cystic fibrosis. SNPs also have utility in pharmacogenomics. Pharmacogenomics identifies genetic variants that affect a patient's response to drugs (eg, life-threatening toxicity). For example, certain mutations in N acetyltransferase are associated with a high incidence of peripheral neuropathy in response to isoniazid, an antituberculosis drug. On the other hand, certain mutations in the core promoter of the ALOX5 gene result in a reduced clinical response to treatment with certain anti-asthma drugs that target the 5-lipoxygenase pathway. Analyzing the distribution of SNPs in different population groups is useful for investigating genetic drift, mutation, genetic recombination, gene selection, and tracking the origin of the population and its migration (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 regulatory nucleic acids, and interpolation of results obtained from standard curves (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 derived from any of the polynucleotides described herein can be used as elements in a microarray. Microarrays can be used in transcriptional imaging techniques that simultaneously monitor the relative 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 has the least side effects can be selected.

  In another embodiment, DME, DME fragments, or antibodies specific for DME may be used as elements on a microarray. The microarray 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 present invention to produce 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. Global gene expression patterns can be analyzed by quantifying the number and relative abundance of genes expressed at a given time under given conditions (Seilhamer et al. US Pat. No. 5,840,484 “Comparative Gene Transcript Analysis”). ; Specifically incorporated herein by reference). Thus, a transcript image can be generated by hybridizing a polynucleotide of the present invention or its complement to a whole tissue or cell type transcript or reverse transcript. In certain embodiments, hybridization is generated in a high throughput format such that the polynucleotides of the invention or their complements have 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 generate the expression profiles of polynucleotides of the invention can also be used in in vitro model systems and preclinical evaluation of drugs, or in connection with toxicity tests of industrial or natural environmental compounds. All compounds display a mechanism of action and toxicity, eliciting characteristic gene expression patterns, often referred to as molecular fingerprints or toxicant signatures (Nuwaysir, EF et al. (1999) Mol. Carcinog 24: 153-159; Steiner, S. and NL Anderson (2000) Toxicol. Lett. 112-113: 467-471). 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 various compounds. Assigning gene function to elements of a toxic signature helps to interpret toxic mechanisms, but knowledge of gene function is not required for statistical matching of signature groups, which leads to prediction of toxicity (e.g. National Institute of (See Environmental Health Sciences Press Release 00-02, February 29, 2000, http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and desirable to include all expressed gene sequences in toxicological screening using toxicity signatures.

  In some embodiments, the toxicity of a test compound can be assessed by treating a biological sample with 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 transcription level in the treated biological sample is compared to the level in the untreated biological sample. Differences in transcript levels between the two samples indicate a toxic response caused by the test compound in the treated sample.

  Another embodiment relates to analysis of a proteome of a tissue or cell type using the polypeptides disclosed in 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 subjected 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 given conditions. Thus, a proteomic profile of a cell can be generated by isolating and analyzing polypeptides 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, supra). Proteins are visualized in the gel as dispersed, unique points by staining the gel with a substance such as Coomassie Blue, or silver stain or fluorescent stain. The optical density of each protein spot is usually proportional to the protein level in the sample. The optical density of equally located protein spots from different samples, eg, biological samples either treated or untreated with the test compound or therapeutic agent, are compared to identify changes in protein spot density associated with the treatment. Proteins within the spot are partially sequenced using standard methods using, for example, 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 interest. 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 some embodiments, these antibodies are used as elements on the microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the level of protein binding to each array element (Lueking, A. et al. (1999). Anal. Biochem. 270: 103-111; Mendoze, LG et al. (1999) Biotechniques 27: 778-788). Detection can be performed in various ways known in the art, for example, a thiol-reactive or amino-reactive fluorescent compound can be reacted with a protein in the 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 transcript level. For some proteins in some tissues, the correlation between transcript abundance and protein abundance is poor (Anderson, NL and J. Seilhamer (1997) Electrophoresis 18: 533-537). Proteome toxicity signatures may be useful in the analysis of compounds that do not affect so much 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 embodiment, 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 embodiment, 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 by 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). Various types of microarrays are well known and are described in detail in Schena, M., Editing (1999; DNA Microarrays: A Practical Approach , Oxford University Press, London).

  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 result in 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 Mapped to library (Harrington, JJ et al. (1997) Nat. Genet. 15: 345-355; Price, CM (1993) Blood Rev. 7: 127-134; Trask, BJ (1991) Trends Genet. 7 : 149-154). Once mapped, nucleic acid sequences can be used to develop genetic linkage maps that correlate inheritance of a disease state with, for example, inheritance of a specific chromosomal region or restriction fragment length polymorphism (RFLP) (Lander, ES 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 (Heinz-Ulrich et al. (1995) in Meyers, supra, pages 965-968). Examples of genetic map data can be found in various scientific journals or the Online Mendelian Inheritance in Man (OMIM) website. The correlation between the location of the gene encoding DME on a physical chromosomal map and a particular disease or a predisposition to a particular disease can help determine the region of DNA associated with this disease Can facilitate the cloning task of determining the position.

  Genetic maps can be expanded using physical mapping techniques such as linkage analysis with established chromosomal markers and in situ hybridization of chromosomal specimens. 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 for researchers searching for disease genes using gene discovery techniques such as positional cloning. Once a gene (s) involved in a disease or syndrome is roughly positioned by genetic linkage to a specific genomic region, such as the 11q22-23 region of vasodilatory ataxia, it is mapped to that region Any sequence may represent a related or regulatory gene for further investigation (Gatti, RA et al. (1988) Nature 336: 577-580). The nucleotide sequence of the present invention can also be used for detecting a difference in chromosomal location among the healthy person, the owner, and the affected person due to translocation, inversion, and the like.

  In another embodiment of the invention, DME, its catalytic fragment group, or immunogen fragment group, or its oligopeptide group may be used for screening libraries of compound groups 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 the binding of DME to the drug being tested can be measured.

  Another drug screening method is used for high throughput screening of compounds with suitable binding affinity for the protein of interest (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 embodiment, a competitive drug screening assay can be used in which neutralizing antibodies capable of specifically binding DME compete with test compounds 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 characteristics 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.

  Includes all patent applications, patents, publications, U.S. Patent Application Nos. 60 / 303,745, 60 / 305,402, 60 / 308,158, and 60 / 322,127 mentioned above and below. And are hereby incorporated 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 a suitable mixture of denaturants. TRIZOL (Invitrogen), which is an example of a mixed solution, is a single-phase solution of phenol and guanidine isothiocyanate. The resulting lysate was layered on 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 conventional method.

  To increase RNA purity, RNA extraction and precipitation 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 POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

  In some cases, RNA was provided to Stratagene, which produced the corresponding set of cDNA libraries. If not, cDNA was synthesized using the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen) in the recommended or similar manner known in the art to create a cDNA library (see Ausubel et al., Supra). Chapter 5). Reverse transcription was initiated using oligo d (T) or random primers. A synthetic oligonucleotide adapter was ligated to double stranded cDNA and the cDNA was digested with a suitable restriction enzyme or group of enzymes. For most library size selection (300-1000 bp), SEPHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis was used. The cDNA was ligated to suitable restriction enzyme sites on the polylinker of the appropriate plasmid. Suitable plasmids include, for example, PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen) 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 plNCY (Incyte Genomics), or derivatives thereof. The recombinant plasmid was transformed into qualified E. coli cells such as Stratagene XL1-Blue, XL1-BIueMRF or SOLR, or Invitrogen 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. Plasmid purification methods include Magic or WIZARD Minipreps DNA purification system (Promega), AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD), QIAGEN QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid and QIAWELL 8 Ultra Plasmid purification systems, REAL At least one of the Prep 96 plasmid purification kits was used. 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 FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland) did.

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 ABI CATALYST 800 thermal cycler (Applied Biosystems) or PTC-200 thermal cycler (MJ Research), HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) liquid. Treated in combination with the transfer system. A cDNA sequencing reaction was prepared using a reagent provided by Amersham Biosciences, or a reagent of an ABI sequencing kit, for example, ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). For electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides, ABI PRISM 373 or 377 sequencing using MEGABACE 1000 DNA sequencing system (Amersham Biosciences) or standard ABI protocol and base calling software Systems (Applied Biosystems) or other sequence analysis systems known in the art were used. Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, chapter 7). Several cDNA sequences were selected and extended with the technique disclosed in Example 8 .

Polynucleotide sequences derived from Incyte cDNA were validated by removing vectors, linkers and poly (A) sequences and masking ambiguous bases. At that time, an algorithm and a program based on BLAST, dynamic programming, and adjacent dinucleotide frequency analysis were used. Next, the following database group was queried for Incyte cDNA sequences or their translations. That is, selected public databases (e.g. GenBank primates, rodents, mammals, vertebrates, eukaryotic databases and BLOCKS, PRINTS, DOMO, PRODOM) and humans, rats, mice, nematodes ( PROTEOME database family (Incyte Genomics, Palo Alto CA) with sequence groups from Caenorhabditis elegans ), budding yeast ( Saccharomyces cerevisiae ), fission yeast ( Schizosaccharomyces pombe ) and Candida albicans , and hidden Markov model (HMM) based protein family Databases such as PFAM, INCY, and TIGRFAM (Haft, DH et al. (2001) Nucleic Acids Res. 29: 41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl Acad. Sci. USA 95: 5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30: 242-244) (HMM is a probabilistic approach to analyzing consensus primary structure of gene families; example Eddy, SR (1996) Curr. Opin. Struct. Biol. 6: 361-365). Queries were made using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequence was assembled to yield the full length polynucleotide sequence. Alternatively, use the GenBank cDNA group, GenBank EST group, stitched sequence group, stretched sequence group, or Genscan predicted coding sequence group (see Examples 4 and 5 ) to complete the Incyte cDNA assembly group to full length. Elongated. Assembly was performed using a program based on Phred, Phrap and Consed, and a cDNA assembly was screened for open reading frames using a program based on GenMark, BLAST and FASTA. The full length polynucleotide sequence was translated to obtain the corresponding full length polypeptide sequence. Alternatively, the polypeptide can start at any methionine residue of the full-length translated polypeptide. For further analysis of full-length polypeptide sequences, query the GenBank protein databases (genpept), SwissProt, PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM and Prosite databases, PFAM, INCY, and TIGRFAM Hidden Markov Model (HMM) based protein family database group, and HMM based protein domain database group such as SMART. Full-length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Sequence alignments between polynucleotides and polypeptides are generated using default parameters specified by the CLUSTAL algorithm incorporated into the MEGALIGN multi-sequence alignment program (DNASTAR). This also calculates the percent identity between aligned sequences.

  Table 7 gives an overview of the tools, programs and algorithms used for analysis and assembly 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 shows the parameters, such as score, probability value, etc., 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 sequences and polypeptide sequences were also used to identify the polynucleotide sequence fragments of SEQ ID NO: 14-26. A group of fragments of about 20 to about 4000 nucleotides useful for hybridization and amplification techniques is shown in column 2 of Table 4.

4. Identification and Editing of Coding Sequences from Genomic DNA First, putative drug-metabolizing enzymes were identified by running the Genscan gene identification program against 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, Burge, C. and S Karlin (1998) Curr. Opin. Struct. Biol. 8: 346-354). This program links predicted exons together to form an assembled cDNA sequence that spans from methionine to a 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. In order to determine which of these Genscan predicted cDNA sequences encode a drug metabolizing enzyme, the encoded polypeptides were queried and analyzed for drug metabolizing enzymes from the PFAM model group. Potential drug-metabolizing enzymes were also identified by their homology to the Incyte cDNA sequences already annotated as drug-metabolizing enzymes. These selected Genscan predicted sequences 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 errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan predicted sequence, thus providing evidence of transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan predicted coding sequences with Incyte cDNA sequences and / or public cDNA sequences using the assembly process described in Example 3 . Alternatively, the full-length polynucleotide sequence is completely derived from the edited or unedited Genscan predicted coding sequence.

5 Assembly of genomic sequence data with cDNA sequence data
Stitched sequence
In order to extend the partial cDNA sequence group, the exon group predicted by the Genscan gene identification program described in Example 4 was used. The assembled partial cDNA groups as described in Example 3 were mapped to genomic DNA and resolved into cluster groups with related cDNA groups and exons predicted from Genscan from one or more genomic sequences. . To integrate the cDNA and genome information, each cluster was analyzed using a certain algorithm based on graph theory and dynamic programming to generate potential splice variants. The sequences were subsequently confirmed, edited or extended to create a full length sequence. A sequence segment group in which the total length of the segment is in two or more sequences was identified in the cluster, and the segment groups identified as such were considered to be equal due to transitivity. For example, if a section is in one cDNA and two genomic sequences, all three sections were considered equal. This process allows unrelated but contiguous genomic sequences to be cross-linked by joining them with cDNA sequences. The sections thus identified were “stitched” by the stitch algorithm in the order they appear along their parent sequences to generate the longest possible sequence and group of sequence variants. Linkage between sections (cDNA-cDNA or genomic sequence-genomic sequence) that continues along one type of parent sequence takes precedence over linkage (cDNA-genomic sequence) that changes the type of parent. The resulting stitch sequences were translated and compared with public databases genpept and gbpri by BLAST analysis. The incorrect exon group predicted by Genscan was corrected by comparison with the top BLAST hits from genpept. If necessary, the sequences were further extended using additional cDNA sequences or by examining genomic DNA.

Stretched sequence
Partial DNA sequences were extended to full length with one algorithm based on BLAST analysis. First, the BLAST program was used to inquire public cDNA databases such as GenBank primate, rodent, mammalian, vertebrate and eukaryotic databases for the assembled partial cDNA as described in Example 3 . The closest 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 relative 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 assemble SEQ ID NO: 14-26 were compared to the sequences of the Incyte LIFESEQ database and public domain databases using the BLAST et al. Implementation of the Smith-Waterman algorithm. Sequences in these databases that matched SEQ ID NO: 14-26 were assembled into clusters of consecutive overlapping sequences using an assembly algorithm such as Phrap (Table 7). Using the radiation hybrid and genetic map data available from public sources such as the Stanford Human Genome Center (SHGC), Whitehead Genome Research Institute (WIGR), Genethon, etc., any clustered sequences have already been Judged whether it was mapped. If the mapped sequence was contained in a cluster, the entire sequence of that cluster was assigned to a map location along with the individual SEQ ID numbers.

  The location on the map is expressed as a range or section of the human chromosome. The location on a map of a section in centimorgan units is measured relative to the end of the chromosome's short arm (p-arm). On average, 1 cM is approximately equal to 1 megabase (Mb) of human DNA, although this value varies widely due to recombination hot and cold spots). The cM distance is based on a set of genetic markers mapped by Genethon that provide boundaries for radiation hybrid markers whose sequences are contained within each cluster. NCBI “GeneMap'99” (http://www.ncbi.nlm.nih.gov/genemap/) and other publicly available resources such as human genome maps are used to identify disease genes already identified above. It can be determined whether the map is within or near the section.

7 Analysis of Polynucleotide Expression Northern analysis is an experimental technique used to detect the presence of transcripts of genes, labeled nucleotide sequences to membranes to which RNA from specific cell types or tissues is bound. (Sambrook supra, Chapter 7; Ausubel et al., Supra, Chapter 4).

  Similar or related computer technology using BLAST was used to search for identical or related molecules in cDNA databases such as GenBank and LIFESEQ (Incyte Genomics). Northern analysis is much faster than some membrane-based hybridizations. In addition, the sensitivity of computer searches can be modified to determine whether any particular match is classified as strict or homologous. The search criterion is the product score, which is defined by the following formula.

  The product score takes into account both the similarity between two sequences and the length with which the sequences match. The product score is a normalized value from 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. To calculate the BLAST score, a score of +5 is assigned to each matching base in a high scoring segment pair (HSP) and -4 is assigned to each mismatched base. Two sequences can share more than one HSP (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 either one end is 100% coincident and 70% overlap, or the other end is 88% coincident and 100% overlap. A product score of 50 is obtained if either end is 100% coincident and 50% overlap, or 79% coincidence and 100% overlap.

Alternatively, the polynucleotide encoding DME is analyzed for the tissue source from which it was derived. For example, some full length sequences are at least partially assembled using overlapping Incyte cDNA sequences (see Example 3 ). Each cDNA sequence is derived from a cDNA library made from human tissue. Each human tissue is classified into one of the following organ / tissue categories. 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 system, Sensory organs, skin, stomatognathic system, non-classified / 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 lines, development, inflammation, neurological, 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 the cDNA encoding DME. cDNA sequences and cDNA library / tissue information can be obtained from the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).

8 Extension of a polynucleotide encoding DME A full-length polynucleotide is produced by extending the fragment using a set of oligonucleotide primers designed from appropriate fragments of the full-length molecule. One primer was synthesized to initiate a 5 ′ extension of a known fragment and another primer was synthesized to initiate a 3 ′ extension of a known fragment. The OLIGO 4.06 software (National Biosciences) or another appropriate program was used to design the starting primer group. Annealing the target sequence at temperature. All elongations of nucleotide groups that resulted in hairpin structures and primer-primer dimers were avoided.

  The sequence was extended using selected human cDNA libraries. 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 with methods well known to those skilled in the art. PCR was performed in 96-well plates on a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture has template DNA and 200 nmol of each primer. Also contains a reaction buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene) To do. For the primer pair PCI A and PCI B, amplification was performed with the following parameters. Step 1: 3 minutes at 94 ° C. Step 2: 15 seconds at 94 ° C. Step 3: 1 minute at 57 ° C. Step 4: 2 minutes at 68 ° C. Step 5: Repeat steps 2, 3 and 4 20 times. Step 6: 5 minutes at 68 ° C. Step 7: Store at 4 ° C.

  The DNA concentration in each well was determined by adding 100 μl of PICOGREEN quantitative reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1 × TE and 0.5 μl of undiluted PCR product to an opaque fluorometer. The solution was distributed to each well of a plate (Corning Costar, Acton MA), and determination was made 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 gel to determine which reaction was successful in extending the sequence.

  Elongated nucleotides are desalted and concentrated, transferred to a 384-well plate, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), sonicated or sheared, and into the pUC 18 vector (Amersham Biosciences) Was reconsolidated. For shotgun sequencing, digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, fragments were excised, and agar was digested with Agar ACE (Promega). Elongated clones were religated to pUC 18 vector (Amersham Biosciences) using T4 ligase (New England Biolabs, Beverly MA), treated with Pfu DNA polymerase (Stratagene) to fill the restriction site overhangs, and E. coli cells Was transfected. Transfected cell groups were selected in a medium containing antibiotics, and each colony was collected and cultured overnight at 37 ° C. in a 384 well plate group in LB / 2X carbenicillin culture solution.

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

  Similarly, full-length polynucleotides were verified using the above procedure. Alternatively, 5 ′ regulatory sequences were obtained using full-length polynucleotides using the oligonucleotides designed for such extension and some appropriate genomic library in the above procedure.

9 Identification of single nucleotide polymorphisms in polynucleotides encoding DME A common DNA sequence variant known as single nucleotide polymorphism (SNP) uses the LIFESEQ database (Incyte Genomics) in SEQ ID NO: 14-26 Identified. Sequences from the same gene were clustered together and assembled as described in Example 3 to allow identification of all sequence variants in that 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 eliminates errors caused by sequence alignment errors and 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 mutation. Cluster error filters used statistically generated algorithms to identify errors due to clustering of related homologues 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. Selection was performed by mass spectrometry using a high-throughput MASSARRAY system (Sequenom, Inc.) and analyzed for allele frequencies at SNP sites in four different human populations. The white population consists of 92 individuals (46 boys, 46 girls), 83 from Utah, 4 from France, 3 from Venezuela, and 2 from Amish. The African population consists of 194 individuals (97 boys and 97 girls), all of whom are African American. The Hispanic group consists of 324 individuals (162 boys and 162 girls), all of whom are Mexican Hispanics. The Asian population consists of 126 individuals (64 boys, 62 girls). The reported parent breakdown is 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, 8 % Are other Asians. Allele frequency analysis was first made in the Caucasian population, and in some cases, SNPs that did not show allelic variation in this population were not tested 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: 14-26. Although specifically described for labeling oligonucleotides of about 20 base pairs, essentially the same procedure is used for larger nucleotide fragments. Oligonucleotides were designed using modern software such as OLIGO 4.06 software (National Biosciences), 50 pmol of each oligomer, [γ- ³² P] adenosine triphosphate (Amersham Biosciences) 250 μCi, and T4 polynucleotide kinase (DuPont NEN , Boston MA). The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Amersham Biosciences). In a typical membrane-based hybridization analysis of human genomic DNA digested with either one of Ase I, Bgl II, Eco RI, Pst I, Xba I or Pvu II (DuPont NEN) ^Use aliquots containing ⁷ counts of labeled probe.

  DNA obtained 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. In order to remove non-specific signal groups, the blot groups are washed sequentially at room temperature, for example under conditions of up to 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. Visualize and compare hybridization patterns using autoradiography or alternative imaging means.

11 Microarray The concatenation or synthesis of array elements on a microarray uses photolithography, piezo printing (see inkjet printing, Baldeschweiler et al., Etc.), mechanical microspotting technology, and technologies derived therefrom. Can be achieved. In each of the above techniques, the substrate should be a solid with a uniform, non-porous surface (Schena, M., Edit (1999) DNA Microarrays: A Practical Approach , Oxford University Press, London). 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 bind elements to the surface of the substrate using thermal, ultraviolet, chemical or mechanical bonding procedures. Conventional arrays can be made using available methods and machinery known to those skilled in the art and can have any suitable number of elements (Schena, M. et al. (1995) Science 270: 467-470; Shalon, D. et al. (1996) Genome Res. 6: 639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16: 27-31).

  Full-length cDNAs, expressed sequence tags (ESTs), or fragments or oligomers thereof can be elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software known in the art such as LASERGENE software (DNASTAR). The array element group is hybridized with the polynucleotide group in the biological sample. The polynucleotide in the biological sample is conjugated to a molecular tag such as a fluorescent label to facilitate detection. After hybridization, unhybridized nucleotides from the biological sample are removed and hybridization at each array element is detected using a fluorescent scanner. Alternatively, hybridization can also be detected using laser desorption and mass spectrometry. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to an element on the microarray can be calculated. The preparation and use of the microarray in one embodiment is detailed below.

Preparation of tissue or cell samples The guanidinium thiocyanate method is used to isolate total RNA from tissue samples, and the oligo (dT) cellulose method is used for poly (A) ⁺ RNA purification. MMLV reverse transcriptase was used to reverse transcribe each poly (A) ⁺ RNA sample, and 0.05 pg / μl oligo (dT) primer (21mer), 1 × first strand buffer, 0.03 unit / μl RN Anase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences) is used. 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 incubating 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 stop the reaction. To break down RNA. Two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) are used for sample group purification. After mixing, ethanol precipitation of the two reaction samples is performed with 1 ml glycogen (1 mg / ml), 60 ml sodium acetate, and 300 ml 100% ethanol. The sample is then thoroughly dried with 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 a group of bacterial cells containing a group of cloned cDNA inserts and a group of vectors. PCR amplification uses primers complementary to the vector sequences adjacent to the cDNA insert. Array elements are amplified by 30 cycles of PCR from an initial amount of 1-2 ng to a final amount in excess of 5 μg. Amplified array elements are purified using SEPHACRYL-400 (Amersham Biosciences).

  The purified array element is fixed on a polymer-coated slide glass. Microscope slides (Corning) are ultrasonically washed in 0.1% SDS and acetone and thoroughly washed with distilled water during and after treatment. 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. Use to coat. Coated slides are cured in an oven at 110 ° C.

  The array element group is added to the coated glass substrate by the procedure described in US Pat. No. 5,807,522. This patent is hereby incorporated by reference. 1 μl of array element DNA with an average concentration of 100 ng / μl is filled into an open capillary printing element by means of a robotic apparatus. The instrument now places approximately 5 nl of array element samples per slide.

  The microarray is UV cross-linked using STRATALINKER UV cross-linker (Stratagene). The microarray is washed once with 0.2% SDS and three times with distilled water at room temperature. In order to block non-specific binding sites, microarray incubation was performed in 60% at 30 ° C. in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) before Wash with 0.2% SDS and distilled water as done.

The 9 μl sample mixture used for the hybridization reaction contains 0.2 μg of each of the cDNA synthesis products labeled with Cy3 or Cy5 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. To keep the chamber at 100% humidity, add 140 μl of 5 × SSC to one corner of the chamber. The chamber with the array is incubated at 60 ° C. for approximately 6.5 hours. The array is washed for 10 minutes at 45 ° C. in the first wash buffer (1 × SSC, 0.1% SDS) and three times for 10 minutes at 45 ° C. in the second wash buffer (0.1 × SSC). dry.

To detect the detection reporter labeled hybridization complex, an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for Cy3 excitation and 632 nm for Cy5 excitation. ) Is used. A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the excitation laser light on the array. The slide containing the array is placed on a microscope, computer controlled XY stage and raster scanned through the objective lens. 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. A suitable filter group is placed between the array and the photomultiplier tube to filter the signal. 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, scan once for each fluorophore and typically scan each array twice.

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

  A 12-bit RTI-835H analog-to-digital (A / D) conversion board (Analog Devices, Inc., Norwood MA) installed on an IBM compatible PC computer is used to digitize the output of the photomultiplier tube. 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 scale. Data is also analyzed quantitatively. When exciting and measuring two different fluorophores simultaneously, using the emission spectra of each fluorophore, the data is first corrected 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). Array elements that showed an expression change of at least about 2-fold, an SB ratio of 2.5 or greater, and an element spot size of at least 40% were identified in the GEMTOOLS program (Incyte Genomics) as showing differential expression.

Expression
SEQ ID NO: 24 showed differential expression, which was determined by microarray analysis. For example, in a comparison of gene expression patterns, SEQ ID NO: 24 was up-regulated by at least 2-fold in normal lung tissue in 3 out of 5 compared to neoplastic lung tissue from the same donor. In all cases, the experiment was performed twice and the results confirmed the original analysis. Analysis of gene expression patterns associated with tumor development and progression can provide much insight into the biological basis of the disease, as well as improved diagnosis and treatment. Thus, SEQ ID NO: 24 and SEQ ID NO: 11 (SEQ ID NO: 24 is encoded) can be used as a diagnostic tool and therapy for lung cancer.

12 Complementary polynucleotides
A sequence group complementary to a sequence group encoding DME or any part thereof is used to detect, reduce or inhibit the expression of native DME. Although the use of oligonucleotides containing about 15-30 base pairs is described, essentially the same procedure is used for smaller or larger sequence fragments. 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 prevent 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. To express DME in bacteria, the cDNA is subcloned into a suitable vector having 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 regulatory element 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 transcription is performed by a strong polyhedrin promoter. Recombinant baculoviruses are often used to infect Spodoptera frugiperda (Sf9) insect cells of the night owl, but may also be used to infect human hepatocytes. In the case of the latter infection, further genetic modification to the baculovirus is required (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 peptide epitope tags such as FLAG and 6-His, so that recombinant from unpurified cell lysates Affinity-based purification of the fusion protein can be performed rapidly in one step. GST is a 26 kDa enzyme from Schistosoma japonicum that allows purification of fusion proteins on immobilized glutathione while maintaining protein activity and antigenicity (Amersham Biosciences). 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 metal chelate resins (QIAGEN). Methods for protein expression and purification are described in Ausubel et al. (Supra, chapters 10 and 16). 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
The function of DME is assessed by the expression of sequences encoding DME at physiologically enhanced levels in mammalian cell culture systems. The cDNA is subcloned into a mammalian expression vector with a strong promoter that expresses the cDNA at high levels. Vectors selected include the PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and the PCR 3.1 plasmid (Invitrogen), both of which have a cytomegalovirus promoter. Using a liposomal formulation or electroporation, 5-10 μg of the recombinant vector is transiently transfected into a human cell line, such as 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, the expression of 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 expressing GFP or CD64-GFP using flow cytometry (FCM), an automated, laser-optical technology, and determine the apoptotic status and other cellular properties of those cells evaluate. 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, alteration of protein expression in the cell surface and in cells measured by reactivity with specific antibodies, and fluorescein-conjugated annexin V protein to 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 calculated using a highly purified cell population transfected with a sequence of sequences encoding DME 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 these cells by methods well known to those skilled in the art. The 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 Substantially purified DME is performed by polyacrylamide gel electrophoresis (PAGE; see, eg, Harrington, MG (1990) Methods Enzymol. 182: 488-495) or other purification techniques. This is used to immunize animals (eg, rabbits, mice, etc.) to produce antibodies using standard protocols.

  Alternatively, the DME amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine a region with high immunogenicity. Then, a corresponding oligopeptide is synthesized, and an antibody is generated using this oligopeptide by a method well known to those skilled in the art. Methods for selecting appropriate epitopes, such as near the C-terminus or hydrophilic regions, are often described in the art (supra Ausubel et al., Chapter 11).

  Typically, an oligopeptide of about 15 residues in length is 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 increase immunogenicity (supra Ausubel et al.). 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 was 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 Biosciences) and an anti-DME antibody. After binding, the resin is blocked and washed according to the manufacturer's instructions.

  The culture solution 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 (Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). 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 can be obtained by using 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 a high-throughput yeast two-hybrid system to determine all interactions between proteins encoded by the 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). While 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 of reaction buffer stock solution immediately 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.

The 1ME, 25-dihydroxyvitamin D24-hydroxylase activity of DME is expressed in ³ H-labeled 1α, 25-dihydroxyvitamin D (1α, 25 (OH) ₂ D) to 24, in transgenic rats expressing DME. 25 conversion is determined by monitoring the on-dihydroxyvitamin _{D (24,25 (OH) 2 D} ). 1 μg of 1α, 25 (OH) ₂ D dissolved in ethanol (or ethanol alone as a control) is used to express about 6 weeks old male transgenic rats expressing DME, or a DME deletion 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 by HPLC on a FINEPAK SIL column (JASCO, Tokyo, Japan) using a solvent system. Alternatively, reverse phase of chloroform phase using J SPHERE ODS-AM column (YMC Co. Ltd., Kyoto, Japan) in 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 fractions of 30 seconds (or less) and the amount of ³ H present in each fraction is measured with 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. Stop with organic solvent and determine product formation by HPLC. Alternatively, activity measurements are monitored for oxygen uptake using a Clark-type electrode. For example, Ziegler, DM, and Poulsen, LL (1978; Methods Enzymol. 52: 142-151), in the NADPH-producing cofactor system (similar to that described above) in F.A. Incubated at 0 ° C. The oxygen uptake rate is proportional to the enzyme activity.

The UDP glucuronyltransferase activity of DME is measured by colorimetric determination of free amine groups (Gibson and Sketch, 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) With an aliquot of this enzyme in ascorbic acid (0.75 mM UDP-glucuronic acid). 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 the 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, it was incubated with ³ H-taurine, and the tritiated cholate conjugate was 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 a 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. The absorbance of DTNB conjugate (412 nm) is used for detection of 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-accepting 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 centrifuging at 1000 xg for 10 minutes, 3 ml aliquots of this organic extract are analyzed for radioactivity content by liquid scintillation counting. 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. 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) and DME The final volume is 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 reaction 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 can be determined 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. ) Incubate at 37 ° C and measure. 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. Transfer the supernatant to a clean test tube and extract 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 separated by injection onto a C18 reverse phase HPLC column. 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 benzoic acid standards 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). Carboxylesterase activity is then monitored and compared to a control autohydrolysis of the substrate with a spectrophotometer set at 405 nm (Wan, L. et al. (2000) J. Biol. Chem. 275: 10041-10046).

DME sulfotransferase activity is measured using ³⁵ S incorporation from [ ³⁵ S] PAPS into a model substrate (such as phenol) (Folds, A. and JL Meek (1973) Biochim. Biophys. Acta 327: 365- 374). A certain amount of enzyme at 37 ° C with 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) Incubate. After sufficient time for 5-20% of the radiolabel to be transferred to the substrate, 0.2 ml of 0.1 M barium acetate is added 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.

To measure DME's heparan sulfate 6-sulfotransferase activity in vitro , a DME-containing sample was prepared with 2.5 μmol imidazole HC1 (pH 6.8), 3.75 μg protamine chloride, 25 nmol (as hexosamine). Incubate for 20 minutes at 37 ° C. with 50 μl final reaction volume with desulfated N-resulfated heparin and 50 pmol (˜5 × 10 ⁵ cpm) [ ³⁵ S] PAPS. 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 polysaccharide was precipitated with cold triple volume ethanol with 1.3% potassium acetate and completely separated from unincorporated [ ³⁵ S] PAPS and its degradation products by gel chromatography using a desalting column To do. 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 for 48 hours at 4 ° C. in 100 μl of 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. The 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 6M guanidine at 65 ° C., and the radioactivity incorporated into the beads is measured by liquid scintillation counting. [ 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, Y.-B. et al. (1998) Biochemistry 95: 2896-2901).

Alternatively, DME sulfotransferase assay can be 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 This is done using [ 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 a purified DME enzyme preparation, or a sample with DME activity, and the reaction is continued at 37 ° C. for 15 minutes and terminated by heating at 100 ° C. for 3 minutes. 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 movement 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 [ ¹⁴ C] squalene (dispersed with 20 μl Tween 80), and 0.2% Triton X-100. The reaction is started by adding 1 mM NADPH and incubated at 37 ° C. for 30 minutes. Unsaponifiable 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 substrate depletion using gas chromatographic analysis (GC) of ether extracts or by substrate depletion and diol formation by GC analysis of reaction mixtures quenched with acetone. Samples containing DME or epoxide hydrolase control samples were run 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 monooxide, epichloro Hydrin, 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 formation 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 with DME in the presence of 1 mM L-kynurenine and 1 mM 2-oxoglutarate in a final volume of 200 μl, 150 mM Tris acetate buffer (pH 8.0) with 70 μM PLP at 37 ° C. Incubate for 1 hour 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 UV detection at 340 nm and HPLC analysis of the product. Production of kynurenic acid and xanthurenic acid each show aminotransferase activity (Buchli, R. et al. (1995) J. Biol. Chem. 270: 29330-29335).

  Alternatively, DME aminotransferase activity can be measured by monitoring changes in the UV / VIS absorption spectrum of the enzyme-linked cofactor pyridoxal 5'-phosphate (PLP) under single turnover conditions. This is done by determining the activity of purified DME or unpurified samples with 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 conversion of enzyme-bound PLP to pyridoxamine 5 ′ phosphate (PMP) are measured. The specificity and relative activity of DME is measured by the activity of enzyme preparations against 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, culture supernatants, or purified protein preparations. 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 with concentration scans of active gels using appropriate superoxide dismutase positive and negative controls (such as various amounts of commercially available E. coli superoxide dismutase) (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 multi-well plate at various concentrations 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.

  It will be apparent to those skilled in the art that various modifications and variations of the described compositions, methods and systems of the invention can be made without departing from the scope and spirit of the invention. The present invention provides a novel and useful protein group and a polynucleotide group encoding them, and these can be used in the drug discovery process, and the present invention uses these compositions to determine the disease and pathological conditions. It will be appreciated that methods of detection, diagnosis, and treatment are provided. While the invention has been described with reference to several embodiments, it is to be understood that the scope of the invention should not be unduly limited to such specific embodiments . The description of such embodiments should not be considered complete and is not intended to limit the invention to the precise form disclosed. Furthermore, elements of one embodiment can be easily recombined with elements of one or more other embodiments. Such a combination may form a number of embodiments within the scope of the present invention. The scope of the present invention is intended to be defined by the appended claims and their equivalents.

(Short description of the table)
Table 1 summarizes the nomenclature for full-length polynucleotide embodiments and polypeptide embodiments of the invention.

  Table 2 shows the GenBank identification number and annotation of the GenBank homolog that is closest to the polypeptide embodiment of the present invention. Also shown is the probability score for each polypeptide and its homologues (one or more) matching.

  Table 3 shows the structural features of the polypeptide embodiments, such as predicted motifs and domains, along with the methods, algorithms and searchable databases used to analyze the polypeptides.

  Table 4 shows the cDNA and genomic DNA fragments used to assemble the polynucleotide embodiment, along with selected fragments of the polynucleotide.

  Table 5 shows a representative cDNA library of the polynucleotide embodiment.

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

Table 7 shows the tools, programs, and algorithms used for polynucleotide and polypeptide analysis, along with applicable descriptions, references, and threshold parameters.

Claims (81)

An isolated polypeptide selected from the group consisting of:
(A) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 (SEQ ID NOs: 1 to 13) (b) SEQ ID NO: 1-5, SEQ ID NO: 7, SEQ ID NO: A polypeptide comprising a natural amino acid sequence which is at least 90% identical to an amino acid sequence selected from the group consisting of 10-11 (c) appears to be at least 95% identical to the amino acid sequence of SEQ ID NO: 6 (D) a polypeptide having a natural amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 8, and (e) an amino acid sequence of SEQ ID NO: 13 A polypeptide comprising a natural amino acid sequence such that at least 99% is identical to (f) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13, and (g) ) SEQ ID NO: 1-13 selected from the group consisting of Immunogenic fragment of a polypeptide having an acid sequence 2. The isolated polypeptide of claim 1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13. 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: 14-26. A recombinant polynucleotide having a promoter sequence operably linked to the polynucleotide of claim 3. A cell transformed with the recombinant polynucleotide according to claim 6. A genetically transformed organism 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. When,
(B) a method comprising recovering the polypeptide so expressed. 10. The method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13. An isolated antibody that specifically binds to the polypeptide of claim 1. An isolated polynucleotide selected from the group consisting of:
(A) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26 (b) at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26 A polynucleotide comprising such a natural polynucleotide sequence (c) a polynucleotide complementary to the polynucleotide of (a) (d) a polynucleotide complementary to the polynucleotide of (b) (e) (a) to (d) ) RNA equivalent 13. An isolated polynucleotide having 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 at least 20 consecutive nucleotides having a sequence complementary to the target polynucleotide in the sample;
(B) detecting the presence or absence of the hybridization complex, and optionally detecting the amount of the complex if present. 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) A method comprising 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 if present. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. The composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13. A method of treating a disease or condition associated with a decrease in the expression of functional DME (a novel drug-metabolizing enzyme) comprising the step of administering the composition of claim 17 to a patient in need of such treatment A method of treatment characterized. A method of screening a compound to confirm the effectiveness of the polypeptide of claim 1 as an agonist, comprising:
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) A 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. 22. A method of treating a disease or condition associated with decreased expression of functional DME, 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 the effectiveness of the polypeptide of claim 1 as an antagonist comprising:
(A) exposing a sample comprising the polypeptide of claim 1 to a compound;
(B) detecting the antagonist activity in the sample. 24. A composition comprising an antagonist compound identified by the method of claim 23 and a pharmaceutically acceptable excipient. 25. A method of treating a disease or condition associated with overexpression of functional DME, 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 of screening for compounds that modulate (modulate) 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 the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, A method wherein the change in activity of the polypeptide of claim 1 in the presence 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 having 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 the expression modification 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 containing nucleic acid with the test compound;
(B) hybridizing a nucleic acid of the treated biological sample with a probe having at least 20 contiguous nucleotides of the polynucleotide of claim 12, wherein the hybridization comprises the probe and the organism. Wherein the target polynucleotide comprises a polynucleotide sequence of a polynucleotide of claim 12 or a fragment thereof. A polynucleotide, said process;
(C) quantifying the yield of the hybridization complex;
(D) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in the untreated biological sample, Such that differences in the amount of hybridization complexes in a biological sample indicate the toxicity of the test compound. A diagnostic test for a condition or disease associated with the expression of DME in a biological sample,
(A) mixing said biological sample with the antibody of claim 11 under conditions suitable for said antibody to bind said polypeptide and form a complex of antibody and polypeptide; ,
(B) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample. The antibody of claim 11, comprising
(A) chimeric antibody (b) single chain antibody (c) Fab fragment (d) F (ab ′) ₂ fragment (e) An antibody that is one of humanized antibodies. 12. A composition comprising the antibody of claim 11 and an acceptable excipient. 33. A method for 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 32. 33. 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 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 or an immunogenic fragment thereof under conditions that elicit an antibody response;
(B) isolating the antibody from the animal;
(C) screening the isolated antibody with the polypeptide, thereby identifying a polyclonal antibody that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13; Including such methods. 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,
(A) immunizing an animal with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 or an immunogenic fragment thereof under conditions that elicit an antibody response;
(B) isolating antibody producing cells from the animal;
(C) fusing the antibody-producing cells and immortalized cells to form hybridoma cells producing monoclonal antibodies;
(D) culturing the hybridoma cells;
(E) isolating a monoclonal antibody from the culture that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13. 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 according to claim 11, wherein the antibody is produced by screening a Fab expression library. 12. The antibody of claim 11, which is produced by screening a recombinant immunoglobulin library. A method of detecting in a sample a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13,
(A) incubating the antibody and one sample under conditions allowing specific binding between the antibody of claim 11 and the polypeptide;
(B) a step of detecting specific binding, wherein the specific binding indicates that a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-13 is present in the sample. A method characterized by: A method of purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13,
(A) incubating the antibody and one sample under conditions allowing specific binding between the antibody of claim 11 and the polypeptide;
(B) separating the antibody from the sample to obtain a purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-13. 14. A microarray, wherein at least one element of the microarray is a polynucleotide according to claim 13. A method for generating an expression profile of a sample having a group of polynucleotides, comprising:
(A) labeling the polynucleotide in the sample;
(B) contacting the microarray element of claim 46 with a labeled polynucleotide in a sample under conditions suitable for formation of a hybridization complex;
(C) A method comprising quantifying the expression of a polynucleotide in a sample. An array having various nucleotide molecules attached to unique physical locations on a solid substrate, wherein at least one said nucleotide molecule specifically hybridizes with at least 30 consecutive nucleotide groups of a target polynucleotide. 13. An array comprising a first oligonucleotide or polynucleotide sequence capable of soybean, wherein the target polynucleotide is the polynucleotide of claim 12. 49. The array of claim 48, wherein the first oligonucleotide or polynucleotide sequence 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 initial sequence of the 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 having 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 comprises the same sequence. Each of the unique physical locations on the substrate includes a group of nucleotide molecules having a sequence that is different from the sequence of the nucleotide molecules at another unique physical location on the substrate. An array. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 1. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 2. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 3. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 4. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 5. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 6. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 7. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 8. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 9. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 10. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 11. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 12. 2. The polypeptide of claim 1 having the amino acid 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. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 25. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 26.