CA2422530A1 - Human drug metabolizing enzymes - Google Patents

Abstract

The invention provides human drug metabolizing enzymes (DME) and polynucleotides which identify and encode DME. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of DME.

Description

DRUG METABOLIZING ENZXMES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of drug metabolizing enzymes and to the use of these sequences in the diagnosis, treatment, and prevention of autoimrntme/inflammatory, cell proliferative, developmental, endocrine, eye, metabolic, and gastrointestinal disorders, including liver disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of drug metabolizing enzymes.
BACKGROUND OF THE INVENTION
The metabolism of a drug and its movenxent through the body (pharmacolcinetics) are inxportant in determining its effects, toxicity, and interactions with other drugs. The three processes governing pharmacoltinetics are the absorption of the drug, distribution to various tissues, and elimination of drug metabolites. These processes are intimately coupled to drug metabolism, since a variety of metabolic modifications alter most of the physicochemical and pharmacological properties of drugs, including solubility, binding to receptors, and excretion rates. The metabolic pathways which modify drugs also accept a variety of naturally occurring substrates such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins. The enzymes in these pathways are therefore important sites of biochemical and pharmacological interaction between natural compounds, drugs, carcinogens, mutagens, and xenobiotics.
It has long been appreciated that inherited differences in drug metabolism lead to drastically different levels of drug efficacy and toxicity among individuals. For drugs with narrow therapeutic indices, or drugs which require bioactivation (such as codeine), these polymorplusms can be critical.
Moreover, promising new drugs are frequently eliminated in clinical trials based on toxicities which may only affect a segment of the patient group. Advances in pharmacogenomics research, of which drug metabolizing enzymes constitute an hnportant part, are promising to expand the tools and information that can be brought to bear on questions of drug efficacy and toxicity (See Evans, ~UV.E.
and R.V. Relling (1999) Science 286:487-491).
Drug metabolic reactions are categorized as Phase I, which functionalize the drug molecule and prepare it for further metabolism, and Phase II, which are conjugative. In general, Phase I
reaction products are partially or fully inactive, and Phase II reaction products are the chief excreted species. However, Phase I reaction products are sometimes more active than the original administered drugs; this metabolic activation principle is exploited by pro-drugs (e.g. L-dopa).
Additionally, some nontoxic compounds (e.g. aflatoxin, benzo[a]pyrene) are metabolized to toxic intermediates through these pathways. Phase I reactions are usually rate-limiting in drug metabolism.
Prior exposure to the compound, or other compounds, can induce the expression of Phase I enzymes however, and thereby increase substrate flux thxough the metabolic pathways.
(See Klaassen, C.D. et al. (1996) Casarett and Doull's Toxicology: The Basic Science of Poisons, MeGraw-Hill, New York, NY, pp. 113-186; Katzung, B.G. (1995) Basic and Clinical Pharmacolo~y, Appleton and Lange, Norwalk, CT, pp. 48-59; Gibson, G.G. and P. Skett (1994) Introduction to Drug Metabolism, Blackie Academic and Professional, London.) Drug metabolizing enzymes (DMEs) have broad substrate specificities. This can be contrasted to the immune system, where a large and diverse population of antibodies are highly specific for theix antigens. The ability of DMEs to metabolize a wide variety of molecules creates the potential for drug interactions at the level of metabolism. For example, the induction of a DME by one compound may affect the metabolism of another compound by the enzyme.
DMEs have been classified according to the type of reaction they catalyze and the cofactors involved. The major classes of Phase I enzymes include, but are not limited to, cytochrome P450 and flavin-containing monooxygenase. Other enzyme classes involved in Phase I-type catalytic cycles and reactions include, but are not limited to, NADPH cytochrome P450 reductase (CPR), the rndcrosomal cytochrome b5/NADH cytochrome b5 reductase system, the ferredoxin/ferredoxin reductase redox pair, aldo/keto reductases, and alcohol dehydrogenases. The major classes of Phase II enzymes include, but axe not limited to, UDP glucuronyltransferase, sulfotransferase, glutathione S-transferase, N-acyltransferase, and N-acetyl transferase.
Cytochrome P450 and P450 cata~ic cycle-associated enzymes Members of the cytochrome P450 superfamily of enzymes catalyze the oxidative metabolism of a variety of substrates, including natural compounds such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins, as well as drugs, carcinogens, mutagens, and xenobiotics. Cytochromes P450, also known as P450 heme-thiolate proteins, usually act as terminal oxidases in mufti-component electron transfer chains, called P450-containing monooxygenase systems. Specific reactions catalyzed include hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, and O-dealkylations, desulfation, deamination, and reduction of azo, vitro, and N-oxide groups. These reactions are involved in steroidogenesis of glucocorticoids, cortisols, estxogens, and androgens in animals; insecticide resistance in insects; herbicide resistance and flower coloring in plants; and environmental bioremediation by microorganisms. Cytochrome P450 actions on drugs, carcinogens, mutagens, and xenobiotics can result in detoxification or in conversion of the substance to a moxe toxic product. Cytochromes P450 are abundant in the liver, but also occur in other tissues; the enzymes are located in microsomes. (See ExPASY ENZYME EC 1.14.14.1; Prosite Cytochrome.P450 cysteine heme-iron ligand signature; PRINTS EP450I E-Class P450 Group I

signature; Graham Lorence, S. and Peterson, J.A. (1996) FASEB J. 10:206-214.) Four hundred cytochromes P450 have been identified in diverse organisms including bacteria, fungi, plants, and animals (Graham Lorence, supxa). The B-class is found in prokaryotes and fungi, while the E-class is found in bacteria, plants, insects, vertebrates, and mammals. Five subclasses or groups are found within the larger family of E-class cytochromes P450 (PRINTS
EP450I E-Class P450 Group I signature).
All cytochromes P450 use a heme cofactor and share structural attributes. Most cytochromes P450 are 400 to 530 amino acids in length. The secondary structure of the enzyme is about 70%
alpha-helical and about 22% beta-sheet. The region around the heme-binding site in the C-terminal part of the protein is conserved among cytochromes P450. A ten amino acid signature sequence in this heme-iron ligand region has been identified which includes a conserved cysteine involved in binding the heme iron in the fifth coordination site. In eukaryotic cytochromes P450, a membrane-spanning region is usually found in the first 15-20 amino acids of the protein, generally consisting of approximately 15 hydrophobic residues followed by a positively charged residue. (See Prosite PDOC00081, suura; Graham Lorence, supra.) Cytochrome P450 enzymes are involved in cell proliferation and development.
The enzymes have roles in chemical mutagenesis and carcinogenesis by metabolizing cliemicals to xeactive intermediates that form adducts with DNA (Nebert, D.W. and Gonzalez, F.J.
(1987) Ann. Rev.
Biochem. 56:945-9J3): These adducts can cause nucleotide changes and DNA
rearrangements that lead to oncogenesis. Cytochrome P450 expression in liver and other tissues is induced by xenobiotics such as polycyclic aromatic hydrocarbons, peroxisomal proliferators, phenobarbital, and the glucocorticoid dexamethasone (Dogra, S.C. et al. (1998) Clip. Exp. Pharmacol.
Physiol. 25:1-9). A
cytochrome P450 protein may participate in eye development as 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).
Cytochromes P450 are associated with inflammation and infection. Hepatic cytochrome P450 activities are profoundly affected by various infections and inflammatory stimuli, some of which axe suppressed and some induced (Morgan, E:T. (1997) Drug Metab. Rev.
29:1129-1188).
Effects observed in vivo can be mimicked by proinflammatory cytokines and interferons.
Autoantibodies to two cytochrome P450 proteins were found in patients with autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), a polyglandular autoimmune syndrome (OMIM *240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy).
Mutations in cytochromes P450 have been linked to metabolic disorders, including congenital adrenal hyperplasia, the most common adrenal disorder of infancy and childhood; pseudovitamin D-deficiency rickets; cerebrotendinous xanthomatosis, a lipid storage disease characterized by progressive neuxologic dysfunction, premature atherosclerosis, and cataracts;
and an inherited resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J.
et al. (1994) Harrison's Principles of Internal Medicine, McGraw-HiII, Inc. New York, NY, pp. 1968-1970; Takeyama, K. et al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998) N. Engl. J. Med.
338:653-661; OMIM
'213700 Cerebrotendinous xanthomatosis; and OMIM #122700 Coumarin resistance).
Extremely high levels of expression of the cytochrome P450 protein aromatase were found in a Pxbrolamellar hepatocelluIar carcinoma from a boy with severe gynecomastia (feminization) (Agarwal, V.R. (1998) T. Clin. Endocrinol. Metab. 83:1797-1800).
The cytochrome P450 catalytic cycle is completed through reduction of cytochrome P450 by NADPH cytochrome P450 reductase (CPR). Another microsomal electron transport system consisting of cytochrome b5 and NADPH cytochrome b5 reductase has been widely viewed as a minor contributor of electrons to the cytochrome P450 catalytic cycle.
However, a recent report by Lamb, D.C. et al. (1999; FEBS Lett. 462:283-288) identifies a Candida albicans cytochrome P450 (CYP51) which can be efficiently reduced and supported by the xnicrosomal cytochrome b5/NADPH
cytochrome b5 reductase system. Therefore, there are likely many cytochromes P450 which are supported by this alternative electron donor system.
Cytochrome b5 reductase is also responsible for the reduction of oxidized hemoglobin (methemoglobin, or ferrihemoglobin, which is unable to carry oxygen) to the active hemoglobin (ferrohemoglobin) in red blood cells. Methemoglobinemia results when there is a high Ievel of oxidant drugs ox an abnormal hemoglobin (hemoglobin M) which is not efficiently reduced.
Methemoglobinemia can also result from a hereditary def ciency in red cell cytochrome b5 reductase (Reviewed in Mansour, A. and Lurie, A.A. (1993) Am. J. Hematol. 42:7-12).
Members of the eytochrome P450 family are also closely associated with 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 form, cholecalciferol, is formed upon the exposure of 7-dehydrocholesterol to near ultraviolet light (i.e., 290-310 mn), normally resulting from even minimal periods of skin exposure to sunlight (reviewed in Miller, W.L. and Portale, A.A. (2000) Trends Endocrinol.
Metab. 11:315-319).
Both prohormone forms are further metabolized in the liver to 25-hydroxyvitaxnin D -(25(OH)D) by the enzyme 25 hydroxylase. 25(OH)D is the most abundant precursor form of vitamin D which must be further metabolized in the kidney to the active form, 1a,25-dihydroxyvitamin D
(Ia,25(OH)ZD), by the enzyme 25-hydroxyvitamin D Ia-hydroxylase (1a-hydroxylase). Regulation of 1a,25(OH)ZD production is primarily at this final step in the synthetic pathway. The activity of la-hydroxylase depends upon several physiological factors including the circulating level of the enzyme product (1a,25(OH)ZD) and the levels of parathyroid hormone (PTH), calcitonin, insulin, calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal la-hydroxylase activity has been reported, suggesting that tissue-specific, local regulation of 2a,25(OI3)2D
production may also be biologically important. The catalysis of 1a,25(OI~zD to 24,25-dihydroxyvitamin D (24,25(OH)ZD), involving the enzyme 25-hydroxyvitamin D
24-hydroxylase (24-hydroxylase), also occurs in the kidney. 24 hydroxylase can also use 25(OI~D as a substrate (Shinki, T. et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12920-12925; Miller, W.L. and Portals, A.A. supra; and references within).
Vitamin D 25-hydroxylase, la-hydroxylase, and 24-hydroxylase are all NADPPI
dependent, type I (mitochondrial) cytochrome P450 enzymes that show a high degree of homology with other members of the family. Vitamin D 25-hydroxylase also shows a broad substrate specificity and may also perform 26-hydroxylation of bile acid intermediates and 25, 26, and 27-hydroxylation of cholesterol (Dilworth, F.J. et al. (1995) J. Biol. Chem. 270:16766-16774;
Miller, W.L. and Portals, A.A. supra; and references within).
The active form of vitamin D (1a,25(OIT)~D) is involved in calcium and phosphate homeostasis and promotes the differentiation of myeloid and skin cells.
Vitamin D deficiency resulting from deficiencies in the enzymes involved in vitamin D metabolism (e.g., la-hydroxylase) causes hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive) rickets, a disease characterized by loss of bone density and distinctive clinical features, including bandy or bow leggedness accompanied by a waddling gait. Deficiencies in vitamin D 25-hydroxylase cause cerebrotendinous xanthomatosis, a lipid-storage disease characterized by the deposition of cholesterol and cholestanol in the Achilles' tendons, brain, lungs, and many other tissues. The disease presents with progressive neurologic dysfunction, including postpubescent cerebellar ataxia, atherosclerosis, and cataracts. Vitamin D 25-hydroxylase deficiency does not result in rickets, suggesting the existence of alternative pathways for the synthesis of 25(OIT)D (Griffin, J.E.
and Zerwelch, J.E.
(1983) 3. Clin. Invest. 72:1190-1199; Gamblin, G.T, et al. (1985) J. Clin.
Invest. 75:954-960; and Miller, W.L. and Portals, A.A. supra).
Ferredoxin and ferredoxin reductase are electron transport accessory proteins which support at least one human cytochrome P450 species, cytochrome P450c27 encoded by the CYP27 gene (Dilworth, F.J. et al. (1996) Biochem. J. 320:267-71). A Streptom~ces~riseus cytochrome P450, CYP104D1, was heterologously expressed in E. coIi and found to be reduced by the endogenous ferredoxin and ferredoxin reductase enzymes (Taylor, M. et al. (1999) Biochem.
Biophys. Res.
Commun. 263:838-42), suggesting that many cytochrome P450 species may be supported by the ferredoxin/ferredoxin xeductase pair. Ferredoxin reductase has also been found in a model drug metabolism system to reduce actinomycin D, an antitumor antibiotic, to a reactive free radical species (Flitter, W.D. and Mason, R.P. (1988) Arch. Bioehem. Biophys. 267:632-639).

Flavin-containing monooxy,~enase (FMO) Flavin-containing monooxygenases oxidize the nucleophilic nitrogen, sulfur, and phosphorus heteroatom of an exceptional range of substrates. Like cytochromes PA~50, FMOs are microsomal and use NADPI and O2; there is also a great deal of substrate overlap with cytochromes P450. The tissue distribution of FMOs includes liver, kidney, and lung.
There are five different known isoforms of FMO in mammals (FMOI, FM02, FM03, FMO~, and FM05), which are expressed in a tissue-specific manner. The isoforms differ in their substrate specificities and other properties such as inhibition by various compounds and stereospecificity of reaction. FMOs have a 13 amino acid signature sequence, the components of which span the N-terminal two-thirds of the sequences and include the FAD binding region and the FATGY motif which has been found in nnany N-hydroxylating enzymes (Stehr, M. et al. (1998) Trends Biochem.
Sci. 23:56-57; PRINTS FMOXYGENASE Flavin-containing monooxygenase signature).
Specific reactions include oxidation of nucleophilic tertiary amines to N-oxides, secondary amines to hydroxylammes and nitrones, primary amines to hydroxylamines and oximes, and sulfur-containing compounds and phosphines to S- and P-oxides. Hydrazmes, iodides, selenides, and boxon-containing compounds are also substrates. Although FMOs appear similar to cytochromes P450 in their chemistry, they can generally be distinguished front cytochromes P450 in vitro based on, for example, the higher heat lability of FMOs and the nonionic detergent sensitivity of cytochromes P450; however, use of these properties in identification is complicated by further variation among FMO isoforms with respect to thermal stability and detergent sensitivity.
FMOs play important roles in the metabolism of several drugs and xenobiotics.
FMO (FMO3 in liver) is predominantly responsible for metabolizing (S)-nicotine to (S) nicotine N-1=oxide, which is excreted in urine. FMO is also involved in S-oxygenation of cimetidine, an Ha-antagonist widely used for the treahnent of gastric ulcers. Liver-expressed forms of FMO are not under the same regulatory control as cytochrome P450. Xn rats, for example, Phenobarbital treatment leads to the induction of cytochrome P450, but the repression of FMOl.
Endogenous substrates of FMO include cysteamine, which is oxidized to the disulfide, cystamine, and trimethylauiine (TMA), which is metabolized to trimethylamiue N-oxide. TMA
smells Iike rotting fish, and mutations in the FM03 isoform lead to large amounts of the malodorous free amine being excreted in sweat, urine, and breath. These symptoms have led to the designation 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 connective tissue matrices by crosslinking collagen and elastin.
LO is secreted as an N-glycosylated precursor protein of approximately 50 kDa and cleaved to the mature form of the enzyme by a metalloprotease, although the precursor form is also active. The copper atom in LO is involved in the transport of electrons to and from oxygen to facilitate the oxidative deamination of lysine residues in these extracellular matrix proteins. While the coordination of copper is essential to LO activity, insufficient dietary intake of copper does not influence the expression of the apoenzyme.
However, the absence of the functional LO is linked to the skeletal and vascular tissue disorders that are associated with dietary copper deficiency. LO is also inhibited by a variety of sernicarbazides, hydrazines, and amino nitrites, as well as heparin. Beta-aminopropionitrile is a commonly used inhibitor. LO activity is increased in response to ozone, cadmium, and elevated levels of hormones released in response to local tissue trauma, such as transforming growth factor-beta, platelet-derived growth factor, angiotensin II, and f broblast growth factor. Abnormalities in LO activity have been linked to Menkes syndrome and occipital horn syndrome. Cytosolic forms of the enzyme have been implicated in abnormal cell proliferation (reviewed in Rucker, R.B. et al.
(1998) Am. J. Clin. Nutr.
67:996S-10025 and Smith-Mungo, L.I. and Kagan, H.M. (1998) Matrix Biol. 16:387-398).
Dihydrofolate reductases Dihydrofolate reductases (DHFR) are ubiquitous enzymes that catalyze the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential step in the de novo synthesis of glycine and purines as well as the conversion of deoxyuridine monophosphate (dIJMP) to deoxythymidine monophosphate (dTMP). The basic reaction is as follows:
7,8-dihydrofolate + NADPH --i 5,6,7,8 tetrahydrofolate + NADP*
The enzymes can be inhibited by a number of dihydrofolate analogs, including.
trimethroprim and methotrexate. Since an abundance of dTMP is required fox DNA synthesis, rapidly dividing cells require the activity of DHFR. The replication of DNA viruses (i.e., herpesvirus) also requires high levels of DHFR activity. As a result, drugs that target DHFR have been used for cancer chemotherapy and to inhibit DNA virus replication. (For similar reasons, thymidylate synthetases are also target enzymes.) Drugs that inhibit DHFR are preferentially cytotoxic for rapidly dividing cells (or DNA virus-infected cells) but have no specificity, resulting in the indiscriminate destruction of dividing cells. Furthermore, cancex cells may become resistant to drugs such as methotrexate as a result of acquired transport defects or the duplication of one or more DHFR
genes (Stryer, L. (1988) Biochenustrv. W.H Freeman and Co., lnc. New York. pp. 511-5619).
Aldo/keto reductases Aldo/keto reductases are monomeric NADPH-dependent oxidoreductases with broad substrate specificities (Bohren, K.M. et al. (1989) J. Biol. Chem. 264:9547-9551). These enzymes catalyze the reduction of carbonyl-containing compounds, including carbonyl-containing sugars and aromatic compounds, to the corresponding alcohols. Therefore, a variety of carbonyl-containing drugs and xenobiotics are likely metabolized by enzymes of this class.
One known reaction catalyzed by a family member, aldose reductase, is the reduction of glucose to sorbitol, which is then further metabolized to fructose by sorbitol dehydrogenase. Under normal conditions, the reduction of glucose to sorbitol is a minor pathway. In hyperglycemic states, however, the accumulation of sorbitol is implicated in the development of diabetic complications (OM1M X203880 Aldo-keto reductase family I, member BI). Members of this enzyme family are also highly expressed in some liver cancers (Cao, D. et al. (1998) J. Biol.
Chem. 273:11429-11435).
Alcohol deh~ enases Alcohol dehydrogenases (ADHs) oxidize simple alcohols to the corresponding aldehydes.
ADH is a cytosolic enzyme, prefers the cofactor NAD+, and also binds zinc ion.
Liver contains the highest levels of ADH, with lower levels in kidney, lung, and the gastric mucosa.
Known ADH isoforms are dimeric proteins composed of 40 kDa subunits. There are five known gene loci which encode these subunits (a, b, g, p, c), and some of the loci have characterized allelic variants (b1, b2, b3, gl, g2). The subunits can form homodimers and heterodimers; the subunit composition determines the specific properties of the active enzyme. The holoenzymes have therefore been categorized as Class I (subunit compositions aa, ab, ag, bg, gg), Class II (pp), and Class T1I (cc). Class I ADH isozymes oxidize ethanol and other small aliphatic alcohols, and are inhibited by pyrazole. Class II isozymes prefer longer chain aliphatic and aromatic alcohols, axe unable to oxidize methanol, and are not inhibited by pyrazole. Class DI
isozymes prefer even longer chain aliphatic alcohols (five carbons and longer) and aromatic alcohols, and are not inhibited by pyrazole.
The short-chain alcohol dehydrogenases include a number of related enzymes with a variety of substrate specificities. Included in this group are the mammalian enzymes D-beta-hydroxybutyrate dehydrogenase, (R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin dehydrogenase, NADPH-dependent carbonyl reductase, corticosteroid I I-beta-dehydrogenase, and estradiol I7-beta-dehydrogenase, as well as the bacterial enzymes acetoacetyl-CoA reductase, glucose 1-dehydrogenase, 3-beta-hydroxystexoid dehydrogenase, 20-beta-hydroxysteroid dehydrogenase, ribitol dehydrogenase, 3-oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, sorbitol-6-phosphate 2-dehydrogenase, 7-alpha-hydroxysteroid dehydrogenase, cis-1,2-dihydroxy-3,4-cyclohexadiene-1-carboxylate dehydrogenase, cis-toluene dihydrodiol dehydrogenase, cis-benzene glycol dehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase, N-acyhnannosamine 1-dehydxogenase, and 2-deoxy D-gluconate 3-dehydrogenase (Kxozowski, 2. (1994) 3. Steroid Biochem. Mol. Biol. 51:125-130; Krozowski, Z. (1992) Mol. Cell Endocrinol.
84:C25-31; and Marks, A.R. et al. (1992) J. Biol. Chem. 267:154.59-15463).

UDP ~lucuronyltransferase Members of the UDP glucuronyltransferase family (TJGTs) catalyze the transfer of a glucuronic acid group from the cofactor uridine diphosphate-glucuronic acid (UDP-glucuronic acid) to a substrate. The transfer is generally to a nucleophilic heteroatom (O, N, or S). Substrates include xenobiotics which have been functionalized by Phase I reactions, as well as endogenous compounds such as bilirubin, steroid hormones, and thyroid hormones. Products of glucuronidation are excreted in urine if the molecular weight of the substrate is less than about 250 glmol, whereas larger glucuronidated substrates are excreted in bile.
UGTs are located in the microsomes of liver, kidney, intestine, skin, brain, spleen, and nasal mucosa, where they are on the same side of the endoplasmic reticulum membrane as cytochrome P450 enzymes and flavin-containing monooxygenases, and therefore are ideally located to access products of Phase I drug metabolism. UGTs have a C-terminal membrane-spanning domain which anchors them in the endoplasmic reticulum membrane, and a conserved signature domain of about 50 amino acid residues in their C terminal section (Prosite PDOC00359 UDP-glycosyltransferase signature).
UGTs involved in drug metabolism are encoded by two gene families, UGT1 and UGT2.
Members of the UGTl family result from alternative splicing of a single gene locus, which has a variable substrate binding domain and constant region involved in cofactor binding and membrane insertion. Members of the UGT2 family are encoded by separate gene loci, and are divided into two families, UGT2A and UGT2B. The 2A subfamily is expressed iu olfactory epithelium, and the 2B
subfamily is expressed in liver microsomes. Mutations in UGT genes are associated with hyperbilirubinemia (OMIM #143500 Hyperbilirubinemia n; Crigler-Najjar syndrome, characterized by intense hyperbilirubinemia from birth (OMIM #218800 Crigler-Najjar syndrome); and a milder form of hyperbilirubinemia termed Gilbert's disease (OM1M *191740 UGT1).
Sulfotransferase Sulfate conjugation occurs on many of the same substrates which undergo O-glucuronidation to produce a highly water-soluble sulfuric acid ester. Sulfotrausferases (ST) catalyze this reaction by transferring SO 3 from the cofactor 3'-phosphoadenosine-5'-phosphosulfate (PADS) to the substrate.
ST substrates are predominantly phenols and aliphatic alcohols, but also include aromatic amines and aliphatic amines, which are conjugated to produce the corresponding sulfamates. The products of these reactions are excreted mainly in urine.
STs are found in a wide range of tissues, including liver, kidney, intestinal tract, lung, platelets, and brain. The enzymes are generally cytosolic, and multiple forms are often co-expressed.
For example, there are more than a dozen forms of ST in rat liver cytosol.
These biochemically characterized STs fall into five classes based on their substrate preference:
arylsulfotransferase, alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester sulfotransferase, and bile salt sulfotransferase.
ST enzyme activity varies greatly with sex and age in xats. The combined effects of developmental cues and sex-related hormones are thought to lead to these differences in ST
expression profiles, as well as the profiles of other DMEs such as cytochromes P450. Notably, the high expression of STs in cats partially compensates for their low level of UDP glucuronyltransferase activity.
Several forms of ST have been purified from human liver cytosol and cloned.
There are two phenol sulfotransferases with different thermal stabilities and substrate preferences. The thermostable enzyme catalyzes the sulfation of phenols such as pare-nitrophenol, minoxidil, and acetaminophen; the thermolabile enzyme prefers monoamine substrates such as dopamine, epinephrine, and levadopa. Other cloned STs include an estrogen sulfotransferase and an N-acetylglucosamine-6-O-sulfotransferase. This last enzyme is illustrative of the other major role of STs in cellular biochemistry, the modification of carbohydrate structures that may be important in cellular differentiation and maturation of proteoglycans. Tndeed, an inherited defect in a sulfotransferase has been implicated in macular corneal dystrophy, a disorder characterized by a failure to synthesize mature kexatan sulfate proteoglycans (Nakazawa, K. et al. (1984) J. Biol. Chem.
259:13751-13757; OMIM ~=2I7800 Macular dystrophy, corneal).
Galactosyltransferases Galactosyltransferases are a subset of glycosyltransferases that transfer galactose (Gal) to the terminal N-acetylglucosamine (GlcNAc) oligosaccharide chains that are part of glycoproteins or glycolipids that are free in solution (Kolbinger, F. et al. (1998} J. Biol.
Chem. 273:433-440; Amado, M. et al. (1999) Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases have been detected on the cell surface and as soluble extracellular proteins, in addition to being present in the Golgi. (31,3-. galactosyltransferases form Type I carbohydrate chains with Gal (j31-3}GIcNAc linkages. Known human and mouse X31,3-galactosyltransferases appear to have a short cytosolic domain, a single transmembrane domain, and a catalytic domain with eight conserved regions.
(Kolbinger, supxa and Hennet, T. et al. (1998) J. Biol. Chem. 273:58-65}. In mouse UDP-galactose:(3-N-acetylglucosamine (31,3-galactosyltransferase I region 1 is located at amino acid residues 78-83, region 2 is located at amino acid residues 93-102, region 3 is located at anuno acid residues 116-I
19, region 4 is located at amino acid residues I47-158; region 5 is located at amino acid residues 172-183, region 6 is located at amino acid residues 203-206, region 7 is located at amino acid residues 236-246, and region 8 is located at amino acid residues 264-275. A variant of a sequence found within mouse UDP-galactose:(3-N-acetylglucosamine j31,3-galactosyltransferase-Ixegion 8 is also found in bacterial galactosyltransferases, suggesting that this sequence defines a galactosyltransferase sequence motif (Hennet, su ra . Recent work suggests that brainiac protein is a (31,3-galactosyltransferase (Yuan, Y.
et al. (1997} Cell 88:9-11; and Hennet, supra).
UDP-Gal:GlcNAc-1,4-galactosyltransferase (-1,4-GaIT) (Sato, T. et al., (1997) EMBO J.
16:1850-1857) catalyzes the formation of Type If carbohydrate chains with Gal ((i1-4)GlcNAc linkages. As is the case with the /31,3-galactosyltransferase, a soluble form of the enzyme is formed by cleavage of the membrane-bound form. Amino acids conserved among (31,4-galactosyltransferases include two cysteines linked through a disulfide-bond and a putative UDP-galactose-binding site in the catalytic domain (Yadav, S. and Brew, K. (1990) J. Biol. Chem. 265:14163-14169; Yadav, S.P.
and Brew, K. (1991) J. Biol. Chem. 266:698-703; and Shaper, N.L. et al. (1997) J. Biol. Chetn.
272:31389-31399). (31,4-galactosyltransferases have several specialized roles in addition to synthesizing carbohydrate chains on glycoproteins or glycolipids. In mammals a (31,4-galactosyltransferase, as part of a heterodimer with a-Iactalbumin, functions in lactating mammary gland lactose production. A (31,4-galactosyltransferase on the surface of sperm functions as a receptor that specifically recognizes the egg. Cell surface J31,4-galactosyltransferases also function in cell adhesion, cell/basal lamina interaction, and normal and metastatic cell migration. (Shun B.
(1993) Curr. Qpin. Cell Biol. 5:854-863; and Shaper, J. (1995) Adv. Exp. Med.
Biol. 376:95-104).
Glutathione S-transfexase The basic reaction catalyzed by glutathione S-transferases (GST) is the conjugation of an electrophile with reduced glutathione (GSH). GSTs are homodimeric or heterodimeric proteins Localized mainly in the cytosol, but some level of activity is present in microsomes as well. The major isozymes share common structural and catalytic properties; in humans they have been classified into four major classes, Alpha, Mu, Pi, and Theta. The two largest classes, Alpha and Mu, are identified by their respective pxotein isoelectric points; pI ~ 7.5-9.0 (Alpha), and pI -- 6.6 (Mu).
Each GST possesses a common binding site for GSH and a variable hydrophobic binding site. The hydrophobic binding site iu each isozyme is specific for particular electrophilic substrates. Specific amino acid residues within GSTs have been identified as important for these binding sites and for catalytic activity. Residues Q67, T68, D101, E104, and 8131 are important for the binding of GSH
(Lee, H.-C. et aI. (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, GSTs perform the beneficial function of deactivation and detoxification of potentially mutagenic and carcinogenic chemicals. However, in some cases their action is detrimental and results in activation of chemicals with consequent mutagenic and carcinogenic effects. Some forms of rat and human GSTs are reliable preneoplastic markers that aid in the detection of carcinogenesis. Expression of human GSTs in bacterial strains, such as Salmonella ~hiznurium used in the well-Irnown Ames test for mutagenicity, has helped to establish the role of these enzymes in mutagenesis. Dihalomethanes, which produce liver tumors in mice, are believed to be activated by GST. This view is supported by the finding that dihalomethanes are more mutagenic in bacterial cells expressing human GST than in untransfected cells (Thier, R. et al. (1993) Proc. Natl. Acad. Sci. USA
90:8567-8580). The mutagenicity of ethylene dibromide and ethylene dichloride is increased in bacterial cells expressing the human Alpha GST, A1-1, while the mutagenicity of aflatoxin B 1 is substantially reduced by enhancing the expression of GST (Simula, T.P. et al.
(1993) Carcinogenesis 14:1371-1376). Thus, control of GST activity may be useful in the control of mutagenesis and carcinogenesis.
GST has been implicated in the acquired resistance of many cancers to drug treatment, the phenomenon Irnown as mufti-drug resistance (MDR). MDR occurs when a cancer patient is treated with a cytotoxic drug such as cyclophosphamide and subsequently becomes resistant to this drug and to a variety of other cytotoxic agents as well. Increased GST levels are associated with some of these drug resistant cancers, and it is believed that this increase occurs in response to the drug agent which is then deactivated by the GST catalyzed GSH conjugation reaction. The increased GST levels then protect the cancer cells from other cytotoxic agents which bind to GST.
Increased levels of Al-1 in tumors has been linked to drug resistance induced by cyclophosphamide treatment (Dirven H.A. et al.
(1994) Cancer Res. 54: 6215-6220). Thus control of GST activity in cancerous tissues may be useful in treating MDR in cancer patients.
Gamma-~lutam 1y transpeptidase Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes that initiate extracellular glutathione (GSH) breakdown by cleaving gamma-glutamyl amide bonds. The breakdown of GSH provides cells with a regional cysteine pool for biosynthetic pathways.
Gamma-glutamyl transpeptidases also contribute to cellular antioxidant defenses and expression is induced by oxidative stress. The cell surface-localized glycoproteins.are expressed at high levels in cancer cells. Studies have suggested that the high level of gamma-glutamyl transpeptidase activity present on the surface of cancer cells could be exploited to activate precursor drugs, resulting in high local concentrations of anti-cancer therapeutic agents (Hanigan, M.H. (1998) Chew. Biol. Interact.
111-112:333-42; Taniguchi, N, and lReda, 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).
Ac~transfarase N-acyltransfexase enzymes catalyze the transfer of an amino acid conjugate to an activated carboxylic group. Endogenous compounds and xenobiotics axe activated by acyl-CoA synthetases in the cytosol, microsomes, and mitochondria. The acyl-CoA intermediates are then conjugated with an amino acid (typically glycine, glutamine, or taurine, but also ornithine, arginine, histidine, serine, aspartic acid, and several dipeptides) by N-acyltransferases in the cytosol or mitochondria to form a metabolite with an amide bond. This reaction is complementary to O-glucuronidation, but amino acid conjugation does not produce the reactive and toxic metabolites which often result from glucuronidation.
One well-characterized enzyme of this class is the bile acid-CoA:amino acid N-acylbransferase (BAT) responsible for generating the bile acid conjugates which serve as detergents in the gastrointestinal tract (Falany, C.N. et al. (I994) J. Biol. Chem.
269:1.9375.-19379; Johnson, M.R, et al. (1991) J. Biol. Chem. 266:10227-10233). BAT is also useful as a predictive indicator far prognosis of hepatocellular carcinoma patients after partial hepatectomy (Furutani, M. et al. (1996) Hepatology 24:1441-1445).
Acetyltransferases Acetyltransferases have been extensively studied for their role in histone acetylation. Histone acetylation results in the relaxing of the chromatin structure in eukaryotic cells, allowing transcription factors to gain access to promoter elements of the DNA templates in the affected region of the genome (or the genome in general). In contrast, histone deacetylation results in a reduction in transcription by closing the chromatin structure and limiting access of transcription factors. To this end, a common means of stimulating cell transcription is the use of chemical agents that inhibit the deacetylation of histories (e.g., sodium butyrate), resulting in a global (albeit artifactual) increase in gene expression. The modulation of gene expression by acetylation also results from the acetylation of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR, TFIlE, TFIIF and the high mobility group proteins (I-BVIG). In the case of p53, acetylation results in increased DNA
binding, leading to the stimulation of transcription of genes regulated by p53. The prototypic histone acetylase (HAT) is Gcn5 from Saccharomyces cerevisiae. GcnS is a member of a family of acetylases that includes Tetrahymena p55, human GcnS, and human p300lCBP. Histone acetylation is reviewed in (Cheung, W.L. et al. (2000) Curr. Opin. Cell Biol. 12:326-333 and Bergen, S.L (1999) Curr. Opin.
Cell Biol. 11:336-341). Some acetyltransferase enzymes possess the alphalbeta hydrolase fold (Center of Applied Molecular Engineering lest. of Chemistry and Biochemistry -University of Salzburg, http://predict.sanger.ac.uk/irbm-course97/Docshnsl) common to several other major classes of enzymes, including but not limited to, acetylcholinesterases and carboxylesterases (Structural Classification of Proteins, http:/lstop.mre-lmb.cam.ac.ulclscoplindex.html).
N-acetyltransfexase Aromatic amines and hydrazine-containing compounds are subject to N-acetylation by the N-acetyltransferase enzymes of liver and other tissues, Some xenobiotics can be O-acetylated to some extent by the same enzymes. N-acetyltransferases are cytosolic enzymes which utilize the cofactor acetyl-coenzyme A (acetyl-CoA) to transfer the acetyl group in a two step process. Tn the first step, the acetyl group is transferred from acetyl-CoA to an active site cysteine residue; in the second step, the acetyl group is transferred to the substrate amino group and the enzyme is regenerated.
In contrast to most other DME classes, there axe a limited number of known N-acetyltransferases. In humans, there are two highly similar enzymes, NAT1 and NAT2; mice appear to have a third form of the enzyme, NAT3. The human forms of N-acetyltransferase have independent regulation (NAT1 is widely-expressed, whereas NAT2 is in liver and gut only) and overlapping substrate preferences. Both enzymes appear to accept most substrates to some extent, but NATl does prefer some substrates (para-aminobenzoic acid, para-aminosalicylic acid, sulfamethoxazole, and sulfanilamide), while NAT2 prefers others (isoniazid, hydralazine, procainamide, dapsone, aminoglutethimide, and sulfamethazine).
Clinical obsexvations of patients taking the antituberculosis drug isoniazid in the 1950s led to the description of fast and slow acetylators of the compound. These phenotypes were shown subsequently to be due to mutations in the NAT2 gene which affected enzyme activity or stability.
The slow isoniazid acetylator phenotype is very prevalent in Middle Eastern populations (approx.
70%), and is less prevalent in Caucasian (approx. 50%) and Asian (<25%) populations. More recently, functional polymorphism in NAT1 has been detected, with approximately 8% of the population tested showing a slow acetylator phenotype (Butcher, N. J. et al.
(1998) Pharmacogenetics 8:67-72). Since NAT1 can activate some known aromatic amine carcinogens, polymorphism in the widely-expressed NAT1 enzyme may be important in determining cancer risk (OMIM
=108345 N-acetyltransferase 1).
Aminotransferases Aminotransferases comprise a family of pyridoxal 5'-phosphate (PLP) -dependent enzymes that catalyze transformations of amino acids. Aspartate aminotransferase (AspAT) is the most extensively studied PLP-containing enzyme. It catalyzes the reversible transamination of dicarboxylic L-amino acids, aspartate and glutamate, and the corresponding 2-oxo acids, oxalacetate and 2-oxoglutarate. Other members of the family include pyruvate aminotransferase, branched-chain amino acid aminotransferase, tyrosine aminotransferase, aromatic aminotransferase, alanine:glyoxylate aminotransferase (AGT), and kynurenine aminotransferase (Vacca, R.A. et al.
(1997) J. Biol. Chem. 272:21932 21937).
Primary hyperoxaluria type-1 is an autosomal recessive disorder resulting in a deficiency in the liver-specific peroxisomal enzyme, alanine:glyoxylate aminotransferase-1.
The phenotype of the disorder is a deficiency in glyoxylate metabolism. In the absence of AGT, glyoxylate is oxidized to oxalate rather than being transaminated to glycine. The result is the deposition of insoluble calcium.
oxalate in the kidneys and urinary tract, ultimately causing renal failure (Lumb, M.J, et al. (1999) J.
Biol. Chem. 274:20587-20596).
Kynurenine amii~otransferase catalyzes the irreversible transamination of the L-tryptophan metabolite L-kynurenine to form kynurenic acid. The enzyme may also catalyze the reversible transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate. Kynurenic acid is a putative modulator of glutamatergic neurotransmission; thus a deficiency in kynurenine aminotransferase may be associated with pleotrophic effects (Buchli, R. et al. (1995) J. Biol. Chem. 270:29330-29335).
Catechol-O-methyltransferase Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the catechol substrate (e.g., L-dope, dopamine, or DBA). Methylation of the 3'-hydroxyl group is favored over methylation of the 4 =hydroxyl group and the membrane bound isoform of COMT is more regiospecific than the soluble form. Translation of the soluble form of the enzyme results from utilization of an internal start codon in a full length mRNA (1.5 kb) or from the translation of a shorter mRNA (1.3 kb), transcribed from an internal promoter. The proposed SN2-like methylation reaction xequires Mgr" and is inhibited by Ca'~'~. The binding of the donor and substrate to COMT occurs sequentially. AdoMet first binds COMT in a Mgr-independent manner, followed by the binding of Mg*'' and the binding of the catechol substrate.
The amount of COMT in tissues is relatively high compared to the amount of activity normally required, thus inhibition is problematic. Nonetheless, inhibitors have been developed for in vitro use (e.g., gallates, tropolone, U-0521, and 3',4.'-dihydroxy-2-methyl-pxopiophetropolone) and for clinical use (e.g., nitrocatechol-based compounds and tolcapone).
Administration of these inhibitors results in the increased half life of L-dope and the consequent formation of dopamine. Inhibition of COMT is also likely to increase the half life of various other catechol-structure compounds, including but not limited to epinephrine/norepinephrine, isoprenaline, rimiterol, dobutamine, fenoldopam, apomorphine, and a methyldopa. A deficiency in norepinephrine has been linked to clinical depression, hence the use of COMT inhibitors could be usefull in the treatment of depression.
COMT inhibitors are genexally well tolerated with minimal side effects and are ultimately metabolized in the liver with only minor accumulation of metabolites in the body (Mannisto, P.T. and Kaakkola, S. (1999) Pharmacol. Rev. 51:593-628).
Copier-zinc superoxide dismutases Copper-zinc superoxide dismutases are compact homodimeric metalloenzymes involved in cellular defenses against oxidative damage. The enzymes contain one atom of zinc and one atom of copper per subunit and catalyze the dismutation of superoxide anions into OZ
and H~Oz. The rate of dismutation is diffusion-limited and consequently enhanced by the presence of favorable electrostatic interactions between the substrate and enzyme active site. Examples of this class of enzyme have been identified in the cytoplasm of all the eukaryotic cells as well as in the periplasm of several bacterial species. Copper-zinc superoxide dismutases are robust enzymes that are highly resistant to proteolytic digestion and denaturing by urea and. SDS. In addition to the compact structure of the enzymes, the presence of the metal ions and intrasubunit disulfide bonds is believed to be responsible for enzyme stability. The enzymes undergo reversible denaturation at temperatwres as high as 70°C
(Battistoni, A. et al. (1998) J. Biol. Chem. 273:5655-5661).
Overexpression of superoxide dismutase has been implicated in enhancing freezing tolerance of transgenic alfalfa as well as providing resistance to environmental toxins such as the diphenyl ether herbicide, acifluorfen (McKersie, B.D. et al. (1993) Plant Physiol. 103:1155-1163). In addtion, yeast cells become more resistant to freeze-thaw damage following exposure to hydrogen peroxide which causes the yeast cells to adapt to further peroxide stress by upregulating expression of supexoxide dismutases. In this study, mutations to yeast superoxide dismutase genes had a more detrimental effect on freeze-thaw resistance than mutations which affected the regulation of glutathione metabolism, long suspected of being important in determining an organism's survival through the process of cryopreservation (long-In Park, J: I. et al. (1998) J. Biol. Chem.
273:22921-22928).
Expression of superoxide dismutase is also associated with Mycobacteriurn tuberculosis, the organism that causes tuberculosis. Superoxide dismutase is one of the ten maj or proteins secreted by M. tuberculosis and its expression is upregulated approximately 5-fold in response to oxidative stress.
M. tuberculosis expresses almost two orders of magnitude more superoxide dismutase than the nonpathogenic mycobacterium M, sme~matis, and secretes a much higher proportion of the expressed enzyme. The result is the secretion of 350-fold more enzyme by M. tubexculosis than M. sme matis, providing substantial xesistance to oxidative stress (Harth, G. and Hoxwitz, M.A. (1999) J. Biol.
Chem. 274:4281-4292).
The xeduced expression of copper-zinc superoxide dismutases, as well as other enzymes with anti-oxidant capabilities, has been implicated in the early stages of cancer.
The expression of copper-zinc superoxide dismutases has been shown to be lower in prostatic intraepithelial neoplasia and prostate carcinomas, compaxed to normal prostate tissue (Bostwick, D.G. (2000) Cancer 89:123-134).
Phosnhodiestexases Phosphodiesterases make up a class of enzymes which catalyze the hydrolysis of one of the two estex bonds in a phosphodiester compound. Phosphodiesterases are therefore crucial to a variety of cellular processes. Phosphodiestexases include DNA and RNA endonucleases and exonucleases, s which are essential for cell growth and replication, and topoisomerases, which break and rejoin nucleic acid strands during topological rearrangement of DNA. A Tyr-DNA
phosphodiesterase functions in. DNA repair by hydrolyzing dead-end covalent intermediates formed between topoisomerase I and DNA (Pouliot, J.J. et al. (1999) Science 286:552-555;
Yang, S; W. (1996) Proc.
Natl. Acad. Sci. USA 93:11534-11539).
x6 Acid sphingomyelinase is a phosphodiesterase which hydrolyzes the membrane phospholipid sphingomyelin to produce ceramide and phosphorylcholine. Phosphorylcholine is used in the synthesis of phosphatidylcholine, which is involved in numerous intracellular signaling pathways, while ceramide is an essential precursor for the generation of gangliosides, membrane lipids found in high concentration in neural tissue. Defective acid sphingomyelinase leads to a build-up of sphingomyelin molecules in lysosomes, resulting in Niemann-Pict' disease (Schuchman, E.H. and S.R. Miranda (1997) Genet. Test. 1:13-19).
Glycerophosphoryl diester phosphodiesterase (also lrnown as glycerophosphodiester phosphodiesterase) is a phosphodiesterase which hydrolyzes deacetylated phospholipid glycerophosphodiesters to produce sn-glycerol-3-phosphate and an alcohol.
Glycerophosphocholine, glycerophosphoethanolamine, glycerophosphoglycerol, and glycerophosphoinositol are examples of substrates fox glycerophosphoryl diester phosphodiesterases. A
glycerophosphoryl diester phosphodiesterase from E. coli has broad specificity fox glycerophosphodiester substrates (Larson, T.J, et al. (1983) J. Biol. Chem. 248:5428-5432).
1S Cyclic nucleotide phosphodiesterases (PDEs) are crucial enzymes in the regulation of the cyclic nucleotides cAMP and cGMP. cAMP and cGMP function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters. PDEs degrade cyclic nucleotides to their corresponding monophosphates, thereby regulating the intracellular concen~ations of cyclic nucleotides and their effects on signal transduction. Due to their roles as regulators of signal transduction, PDEs have been extensively studied as chemotherapeutic targets (Ferry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-4.81;
Tozphy, J.T. (1998) Am. J. Resp. Crit. Care Med. 157:351-370).
Families of mammalian PDEs have been classified based on their substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory agents (Beavo, 3.A. (1995) Physiol. Rev.
75:725-748; Coati, M. et al. (1995) Endocrine Rev. 16:370-389). Several of these families contain distinct genes, many of which are expressed in different tissues as splice variants. Within PDE
families, there are multiple isozymes and multiple splice variants of these isozymes (Coati, M. and S.-L.C. Tin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63:1-38). The existence of multiple PDE
families, isozymes, and splice variants is an indication of the variety and complexity of the regulatory pathways involving cyclic nucleotides (Houslay, M.D. and G. Milligan (X997) Trends Biochem. Sci.
22:217-224).
Type 1 PDEs (PDEIs) are Ca'+lcalmodulin-dependent and appear to be encoded by at least three different genes, each having at least two different splice variants (Kakkar, R. et al. (1999) Cell Mol. Life Sci. 55:1164-1186). PDEls have been found in the lung, heart, and brain. Some PDE1 isozymes are regulated in vitro by phosphorylation/dephosphorylation.
Phosphorylation of these PDE1 isozymes decreases the affinity of the enzyme for calmodulin, decreases PDE activity, axed increases steady state levels of cAMP (Kakkar, s_upra). PDEls may provide useful therapeutic targets for disorders of the central nervous system and the cardiovascular and immune systems, due to the involvement of PDEIs in both cyclic nucleotide and calcium signaling (Perry, M.J. and G.A. I3iggs (1998) Curx. Opin. Chem. Biol. 2:472-481).
PDE2s are cGMP-stimulated PDEs that have been found in the cerebellum, neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle (Sadhu, K. et al. (1999) J. Histochem. Cytochem.
47:895-906). PDE2s are thought to mediate the effects of cAMP on catecholamine secretion, participate in the regulation of aldosterone (Beavo, supra), and play a role in olfactory signal transduction (Juilfs, D.M. et al. (1997) Proc. Natl. Aced. Sci. USA 94:3388-3395).
PDE3s have high affnuty fox both cGMP and CAMP, and so these cyclic nucleotides act as competitive substrates for PDE3s. PDE3s play roles in stimulating myocardial contractility, inhibiting platelet aggregation, relaxing vascular and airway smooth muscle, inhibiting proliferation of T-lymphocytes and cultured vascular smooth muscle cells, and regulating catecholamine-induced ~ release of free fatty acids from adipose tissue. The PDE3 family of phosphodiesterases are sensitive to specific inhibitors such as cilostamide, enoximone, and lixazinone.
Isozymes of PDE3 can be regulated by CAMP-dependent protein kinase, or by insulin-dependent kinases (Degerman, E. et al.
(1997) J. Biol. Chem. 272:6823-6826).
PDE4s are specific for CAMP; are localized to airway smooth muscle, the vascular endothelium, and all inflammatory cells; and can be activated by cAMP-dependent phosphorylation.
Since elevation of cAMP levels can lead to suppression of inflammatory cell activation and to relaxation of bronchial smooth muscle, PDE4s have been studied extensively as possible targets for novel anti-inflammatory agents, with special emphasis placed on the discovery of asthma treatments.
PDE4 inhibitors are currently undergoing clinical trials as treatments for asthma, chronic obstructive pulmonary disease, and atopic eczema. All four known isozymes of PDE4 are susceptible to the inlubitor rolipram, a compound which has been shown to improve behavioral memory in mice (Bared, M. et al. (1998) Proc. Natl. Aced. Sci. USA 95:15020-15025). PDE4 inhibitors have also been studied as possible therapeutic agents against acute lung injury, endotoxemia, rheumatoid arthritis, multiple sclerosis, and various neurological and gastrointestinal indications (Doherty, A.M. (1999) Curr. Opin. Chem. Biol. 3:466-473).
PDE5 is highly selective for cGMP as a substrate (Turko, LV. et al. (1998) Biochemistry 37:4200-4205), and has two allosteric cGMP-specific binding sites (McAllister-Lucas, L.M. et al.
(1995) J. Biol. Chem. 270:30671-30679). Binding of cGMP to these allosteric binding sites seems to be important for phosphorylation of PDE5 by cGMP-dependent protein kinase rather than for direct regulation of catalytic activity. I3igh levels of PDE5 are found in vascular smooth muscle, platelets, lung, and kidney. The inhibitor zaprinast is effective against PDE5 and PDEls.
Modification of zaprinast to provide specificity against PDES has resulted in sildenafil (VIAGRA; Pfizer, Inc., New York NY), a treatment for male erectile dysfunction (Terrett, N. et al. (1996) Bioorg. Med. Chem.
Lett. 6:1819-1824). Inhibitors of PDES are currently being studied as agents for cardiovascular therapy (Penny, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE6s, the photoreceptor cyclic nucleotide phosphodiesterases, are crucial components of the phototransduction cascade. In association with the G-protein transducin, PDE6s hydrolyze cGMP
to regulate cGMP-gated canon channels in photoreceptor membranes. In addition to the cGMP-binding active site, PDE6s also have two high-affinity cGMP-binding sites which are thought to play a regulatory role in PDE6 function (Artemyev, N.O. et al. (1998) Methods 14:93-104). Defects in PDE6s have been associated with retinal disease. Retinal degeneration in the rd mouse (Yan, W. et al. (I998) Invest. Opthalmol. Vis. Sci. 39:2529-2536), autosomal recessive retinitis pigmentosa in humans (Danciger, M. et al. (1995) Genomics 30:1-7), and rod/cone dysplasia 1 in Irish Setter dogs (Sober, M.L. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3968-3972) have been attributed to mutations in the PDE6B gene.
The PDE7 family of PDEs consists of only one known member having multiple splice variants (Bloom, T.J. and J.A. Beavo (1996) Proc. Natl. Acad. Sci. USA
93:14188-14192). PDE7s are cAMP specific, but little else is known about their physiological function. Although mRNAs encoding PDE7s are found in skeletal muscle, heart, brain, lung, kidney, and pancreas, expression of PDE7 proteins is restricted to specific tissue types (Han, P. et al. (1997) J.
Biol. Chem. 272:16I52-16157; Perxy, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE7s are very closely related to the PDE4 family; however, PDE7s are not inhibited by rolipram, a specific inhibitor of PDE4s (Beavo, supra).
PDEBs are cAMP specific, and are closely related to the PDE4 family. PDE8s are expressed in thyroid gland, testis, eye, liver, skeletal muscle, heart, kidney, ovary, and brain. The cAMP-hydrolyzing activity of PDEBs is not inhibited by the PDE inhibitors rolipram, vinpocetine, milrinone, 1BMX (3-isobutyl-1-methylxanthine), or zaprinast, but PDEBs are inhibited by dipyridamole (Fisher, D.A. et al. (I998) Biochem. Biophys. Res. Common. 246:570-577; Hayashi, M. et al. (1998) Biochem. Biophys. Res. Common. 250:751-756; Soderling, S.H. et al. (1998) Proc. Natl. Acad. Sci.
USA 95:8991-8996).
PDE9s are cGMP speck and most closely resemble the PDEB family of PDEs. PDE9s are expressed in kidney, liver, lung, brain, spleen, and small intestine. PDE9s are not inhibited by sildenafil (VIAGRA; Pfizer, Inc., New York NY), rolipram, vinpocetine, dipyridamole, or IBMX (3-isobutyl-1-methylxanthine), but they are sensitive to the PDE5 inhibitor zaprinast (Fisher, D.A. et al.
(1998) J. Biol. Chem. 273:15559-15564; Soderling, S.H. et al. (1998) J. Biol.
Chem. 273:I5553-15558).
PDElOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDElOs are expressed in brain, thyroid, and testis. (Soderling, S.H. et al. (1999) Proc. Natl.
Acad. Sci. USA 96:7071-7076;
Fujishige, K. et al. (1999) J. Biol. Chem. 274:18438-18445; Louglmey, K. et al (1.999) Gene 234:109-117).
PDEs are composed of a catalytic domain of about 270-300 amino acids, an N-terminal regulatory domain responsible for binding cofactors, and, in some cases, a hydrophilic C-terminal domain of unlmown function (Conti, M. and S.-L.C. Jin (I999) Prog. Nucleic Acid Res. Mol. T3iol.
63:1-38). A conserved, putative zinc binding motif, HDX~HXGXXN, has been identified in the catalytic domain of all PDEs. N-terminal regulatory. domains include non-catalytic cGMP-binding domains in PDE2s, PDESs, and PDE6s; calmodulin-binding domains in PDEIs; and domains containing phosphorylation sites in PDE3s and PDE4s. In PDES, the N-terminal cGMP-binding domain spans about 380 amino acid residues and comprises tandem repeats of the conserved sequence motif N(R/K)XnFX3DE (McAllister-Lucas, L.M. et al. (1993) J. Biol. Chem.
268:22863-22873). The NKXnD motif has been shown by mutagenesis to be important for cGMP binding (Turko, LV. et al.
(1996) J. Biol. Chem. 271:22240-22244). PDE families display approximately 30%
amino acid identity within the catalytic domain; however, isozymes within the same family typically display about 85-95% identity in this region (e.g. PDE4A vs PDE4B). Furthermore, within a family there is extensive similarity (>60%) outside the catalytic domain; while across families, there is little or no sequence similarity outside this domain.
Many of the constituent functions of immune and inflammatory responses are inhibited by agents that increase intracellular levels of CAMP (Verghese, M.W. et al.
(1995) Mol. Pharmacol.
47:1164-1171). A variety of diseases have been attributed to increased PDE
activity and associated with decreased levels of cyclic nucleotides. Fox example, a form of diabetes insipidus in mice has been associated with increased PDE4 activity, an increase in low-Km cAMP PDE
activity has been reported in leukocytes of atopic patients, and PDE3 has been associated with cardiac disease.
Many inhibitors of PDEs have been identified and have undergone clinical evaluation (Perry, M.J. and G.A. Iiiggs (1998) Curr. Opin. Chem. Biol. 2:472-481; Torphy, T.J.
(1998) Am. J. Respir.
Crit. Care Med. 157:351-370). PDE3 inhibitors are being developed as antithrombotic agents, antihypertensive agents, and as cardiotonic agents useful in the treatment of congestive heart failure.
Rolipram, a PDE4 inhibitor, has been used in the treatment of depression, and other inhibitors of PDE4 are undergoing evaluation as anti-inflammatory agents. Rolipram has also been shown to inhibit lipopolysacchari.de (LPS) induced TNF-a which has been shown to enhance HIV-1 replication in vitro. Therefore, rolipram may inhibit HIV-1 replication (Angel, J.B. et al. (1995) AIDS
9:1137-1144). Additionally, rolipram, based on its ability to suppress the production of cytokines such as TNF-oc and (3 and interferon ~y, has been shown to be effective in the treatment of encephalomyelitis. Rolipram may also be effective in treating tardive dyskinesia and was effective in treating multiple°sclerosis in an experimental animal model (Sommer, N.
et al. (1995) Nat. Med.
1:244-248; Sasaki, H. et al. (1995) Eur. J. Pharmacol. 282:71-76).
Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases. Theophylline is believed to act on airway smooth muscle function and in an anti-inflammatory or immunomodulatory capacity in the treatment of respiratory diseases (Banner, K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000). Pentoxifylline is another nonspecific PDE
inhibitor used in the treatment of intermittent claudication and diabetes-induced peripheral vascular disease. Pentoxifylline is also known to block TNF-ec production and may inhibit HIV-1 replication (Angel et al., supra).
PDEs have been reported to affect cellular proliferation of a variety of cell types (Coati et al.
(1995) Endocrine Rev. 16:370-389) and have been implicated in various cancers.
Growth of prostate carcinoma cell lines DUi45 and LNCaP was inhibited by delivery of cAMP
derivatives and PDE
inhibitors (Bang, Y.J. et aI. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334).
These cells also showed a permanent conversion in phenotype from epithelial to neuronal morphology. It has also been suggested that PDE inhibitors have the potential to regulate mesangial cell proliferation (Matousovic, K. et al. (1995) J. Clin. Invest. 96:401-410) and lymphocyte proliferation (Joulain, C. et al. (1995) J. Lipid Mediat. Cell Signal. 11:63-79). A cancer treatment has been described that involves intracellular delivery of PDEs to particular cellular compartments of tumors, resulting in cell death (Deonarain, M.P. and A.A. Epenetos (1994) Bx. J. Cancer 70:786-794).
Phosphotriesterases Phosphotriesterases (PTE, paraoxonases) are enzymes that hydrolyze toxic organophosphorus compounds and have been isolated from a variety of tissues. The enzymes appear to be lacking in birds and insects and abundant in mammals, explaining the reduced tolerance of birds and insects to oxganophosphorus compound (Vilanova, E. and Sogoxb, M.A. (I999) Crit. Rev.
Toxicol. 29:21-57).
Phosphotriesterases play a central role in the detoxification of insecticides by mammals.
Phosphotriesterase activity varies among individuals and is lower in infants than adults. PTE
knockout mice are markedly more sensitive to the organophosphate-based toxins diazoxon and chlorpyrifos oxon (Furlong, C.E., et al. (2000) Neurotoxicology 21:91-100).
PTEs have attracted interest as enzymes capable of the detox~cation of organophosphate-containing chemical waste and warfare reagents (e.g., parathion), in addition to pesticides and insecticides. Some studies have also implicated phosphotriesterase in atherosclerosis and diseases involving lipoprotein metabolism.
Thioesterases Two soluble thioesterases involved in fatty acid biosynthesis have been isolated from mammalian tissues, one wluch is active only toward long-chain fatty-acyl thioesters and one which is active toward thioesters with a wide range of fatty-acyl chain-lengths. These thioesterases catalyze the chain-terminating step in the de fzovo biosynthesis of fatty acids. Chain termination involves the hydrolysis of the thioester bond which limes the fatty acyl chain to the 4 =phosphopantetheine prosthetic group of the acyl carrier protein (ACP) subunit of the fatty acid synthase (Smith, S. (1981x) Methods Enzymol. 71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-200).
E. coli contains two soluble thioesterases, thioesterase I which is active only toward long-chain acyl thioesters, and thioesterase II (TEII) which has a broad chain-length specificity (Naggext, J.
et aI. (1991) J. Biol. Chem. 266:11044-11050). E. coli TEII does not exhibit sequence similarity with either of the two types of mammalian thioesterases which function as chain-terminating enzymes in de rzovo fatty acid biosynthesis. Unlike the mammalian thioesterases, E_. coli TEII Lacks the characteristic serine active site gly-X-ser-X-gly sequence motif and is not inactivated by the serine modifying agent diisopropyl fluorophosphate. However, modification of histidine 58 by iodoacetamide and diethylpyrocarbonate abolished TEII activity. Overexpression of TEII did not alter fatty acid content in E. coli, which suggests that it does not function as a chain-terminating enzyme in fatty acid biosynthesis (Naggert et al., su ra . For that reason, Naggert et al. su ra) proposed that the physiological substrates for E. coli TEII may be coenzyme A
(CoA)-fatty acid.esters instead of ACP-phosphopanthetheine-fatty acid esters.
Carboxylesterases Mammalian carboxylesterases constitute a multigene family expressed in a variety of tissues and cell types. Isozymes have significant sequence homology and are classified primarily on the basis of 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 ester- and amide- groups from molecules and are involved in detoxification of drugs, environmental toxins, and carcinogens. Substrates for carboxylesterases include short- and long-chain acyl-glycerols, acylcaxnitine, carbonates, dipivefrin hydrochloride, cocaine, salicylates, capsaicin, palinitoyl-coenzyme A, inaidapril, haloperidol, pyrrolizidine allcaloids, steroids, p-nitrophenyl acetate, malathion, butanilicaine, and isocarboxazide. The enzymes often demonstrate low substrate specificity. Carboxylesterases are also important for the conversion of prodrugs to their respective free acids, which may be the active form of the drug (e.g., lovastatin, used to lower blood cholesterol) (reviewed in Satoh, T. and Hosokawa, M. (1998) Annu. Rev.
Pharmacol.
Toxico1.38;257-288).
Neuroligins are a class of molecules that (i) have N-terminal signal sequences, (ii) resemble cell-surface receptors, (iii) contain carboxylestexase domains, (iv) are highly expressed in the brain, and (v) bind to neurexins in a calcium dependent manner. Despite the homology to caxboxylesterases, neuroligins lack the active site serine residue, implying a zole 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 micxosomal membrane-bound, FAD-dependent oxidoreductase that catalyzes the first oxygenation step in the sterol biosynthetic pathway of eukaryotic cells. Cholesterol is an essential structural component of cytoplasmic membranes acquired via the LDL receptor-mediated pathway or the biosynthetic pathway. In the latter case, all 27 carbon atoms in the cholesterol molecule are derived from acetyl-CoA
(Stryer, L., supxa). SE
converts squalene to 2,3(S)-oxidosqualene, which is then converted to lanosterol and then cholesterol.
The steps involved in cholesterol biosynthesis are summarized below (Stryer, L. (1988) Biochemistry.
W.H Freeman and Co., Inc. New York. pp. 554-560 and Sakakibara, J. et al.
(1995) 270:17-20):
acetate (from Acetyl-CoA) ~ 3-hydoxy-3-methyl-glutaryl CoA ~ mevalonate ~ 5-phosphomevalonate -~ 5 pyrophosphomevalonate -» isopentenyl pyrophosphate ~ dimethylallyl pyrophosphate -~ geranyl pyrophosphate -~ farnesyl pyrophosphate -~ squalene -~ squalene epoxide ~
lanosterol -~ cholesterol.
While cholesterol is essential for the viability of eukaryotic cells, inordinately high serum cholesterol levels result in the formation of atherosclerotic plaques in the arteries of higher organisms. This deposition of highly insoluble lipid material onto the walls of essential blood vessels (e.g., coronary arteries) results in decreased blood flow and potential necrosis of the tissues deprived of adequate blood flow. HMG-CoA reductase is responsible for the conversion of 3-hydroxyl-3 methyl-glutaryl CoA (HMG-CoA) to mevalonate, which represents the first comnutted step in cholesterol biosynthesis. HMG-CoA is the target of a number of pharmaceutical compounds designed to lower plasma cholesterol levels. However, inhibition of MHG-CoA
also results in the reduced synthesis of non-sterol intermediates (e.g., mevalonate) required for other biochemical pathways. SE catalyzes a rate-limiting reaction that occurs later in the sterol synthesis pathway and cholesterol is the only end product of the pathway following the step catalyzed by SE. As a result, SE
is the ideal target fox the design of anti-hyperlipidemic drugs that do not cause a reduction in other necessary intermediates (Nakamura, Y. et al. (1996) 271:8053-8056).
Epoxide hydrolases Epoxide hydrolases catalyze the addition of water to epoxide-containing compounds, thereby hydrolyzing epoxides to their corresponding 1,2-diols. They are related to bacterial haloalkane dehalogenases and show sequence similarity to other members of the a!(3 hydrolase fold family of enzymes (e.g., bromoperoxidase A2 from Streptomyces auxeofaciens, hydroxymuconic semialdehyde hydrolases from Pseudomonas~utida, and haloalkane dehalogenase from Xanthobacter autotrophicus). Epoxide hydrolases are ubiquitous in nature and have been found in mammals, invertebrates, plants, fungi, and bacteria. This family of enzymes is important for the detoxification of xenobiotic epoxide compounds which. are often highly electrophilic and destructive when introduced into an organism. Examples of epoxide hydrolase reactions include the hydrolysis of cis-9,10-epoxyoctadec-9(Z)-enoic acid (leukotoxin) to form its corresponding diol, threo-9,10-dihydroxyoctadec-12(Z)-enoic acid (leukotoxin diol), and the hydrolysis of cis-12,13-epoxyoctadec-9(Z)-enoic acid (isoleukotoxin) to form its corresponding diol threo-12,13-dihydroxyoctadec-9(Z)-enoic acid (isoleukotoxin diol). Leukotoxins alter membrane permeability and ion transport and cause inflammatory responses. In addition, epoxide carcinogens are known to be produced by cytochrome P450 as intermediates in the detoxification of drugs and environmental toxins.
The enzymes possess a catalytic triad composed of Asp (the nucleophile), Asp (the histidine-supporting acid), and His (the water-activating histidine). The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate initiated by the nucleophilzc attack of one of the Asp residues on the primary carbon atom of the epoxide ring of the target molecule, leading to a covalently bound ester intermediate (Arand, M. et al.
(1996) J. Biol. Chem.
271:4223-4229; Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657;
Argiriadi, M.A. et al. (2000) J. Biol. Chem. 275:15265-15270).
Enzymes involved in tyrosine catalysis The degradatizrn of the amino acid tyrosine, to either succinate and pyruvate or fumarate and acetoacetate, requires a large number of enzymes and generates a large number of intermediate compounds. In addition, many xenobiotic compounds may be metabolized using one or more reactions that are part of the tyrosine catabolic pathway. While the pathway has been studied primarily in bacteria, tyrosine degradation is losown to occur in a variety of organisms and is likely to involve many of the same biological reactions.
The enzymes involved in the degradation of tyrosine to succinate and pyruvate (e.g., in Artlzrobacter species) include 4-hydroxyphenylpyruvate oxidase, 4-hydroxyphenylacetate 3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase, tra~zs,cis-5-carboxymethyl-2-hydroxymuconate isomerase, homoprotocatechuate isomerase/decarboxylase, cis-2-oxohept-3-ene-1,7-dioate hydratase, 2,4-dihydroxyhept-trarzs-2-ene-1,7-dioate aldolase, and succinic semialdehyde dehydrogenase.
The enzymes involved in the degradation of tyrosine to fumarate and acetoacetate (e.g., in Pseudorrzoraas species) include 4-hydroxyphenylpyruvate dioxygenase, homogentisate 1,2-dioxygenase, maleylacetoacetate isomerase, and fumarylacetoacetase. 4-hydroxyphenylacetate 1-hydroxylase may also be involved if intermediates from the succinate/pyruvate pathway are accepted.

Additional enzymes associated with tyrosine metabolism in different organisms include 4-chlorophenylacetate-3,4-dioxygenase, aromatic aminotransferase, 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase, 2-oxo-kept-3-ene-1,7-dioate hydratase, and 5-carboxymethyl-2-hydroxymuconate isomerase (Elks, L.B.M. et al. (1999) Nucleic Acids Res.
27:373-376; Wackett, L.P. and Ellis, L.B.M. (1996) J. Microbiol. Meth. 25:91-93; and Schmidt, M.
(1996) Amer. Soc. Microbiol. News 62:102).
In humans, acquired or inherited genetic defects in enzymes of the tyrosine degradation pathway may result in hereditary tyrosinemia. One form of this disease, hereditary tyrosinemia 1 (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase, the last enzyme in the pathway in organisms that metabolize tyrosine to fumarate and acetoacetate.
FTTI is characterized by progressive liver damage beginning at infancy, and increased risk for liver cancer (Endo, F. et al.
(1997) J. Biol. Chem. 272:24426-24432).
The discovery of new drug metabolizing enzymes, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of autoimmunelinflammatory, cell proliferative, developmental, endocrine, eye, metabolic, and gastrointestinal disorders, including liver disoxders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of drug metabolizing enzymes.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, drug metabolizing enzymes, referred to collectively as "DME" and individually as "DME-1," "DME-2," "DME-3," "DME-4,"
"DME-5,"
"D~-6~~, "D~-7~~a "D~-8 ~a~ "D~-9~» "DME-10,T~ '~~-11~~a "DME-12,» "DME-13,"
"DME-14," "DME-15," "DME-16 ", "DME-17 ", and "DME-18." In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D N0:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-18. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID N0:1-18.
The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the 3S group consisting of SEQ ID N0:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ
ID N0:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ lD NO:1-18.
In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ lD N0:1-I8. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO: I9-36.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ lD NO:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ?D N0:1-18, and d) an immunogenic fragment of a polypeptide having an amino IS acid sequence selected from the group consisting of SEQ m NO:1-18. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenie organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ~ NO:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ?I? N0:1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, whexein said cell is transformed with a xecombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to au amino acid sequence selected from the group consisting of SEQ ID NO:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD
N0:1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-18.

The invention further pxovides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:19-36, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ll~
N0:19-36, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the polynueleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method fox detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:19-36, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:19-36, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe compxi.ses at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynueleotade comprising a polynucleotide sequence selected from the group consisting of SEQ ll~ N0:19-36, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID
N0:19-36, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method comprises a) amplifying said target polynucleotide ox fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 117 NO:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ZD
N0:1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D N0:1-18, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ 1D NO:1-18. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional DME, comprising administering to a patient in need of such treatment the composition.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D
N0:1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected froxri the group consisting of SEQ >D N0:1-18. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional DME, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ >D N0:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D N0:1-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-18. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Tn another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional DME, comprising administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-18, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:l-18, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~ NO:1-18. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide. , The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino.
acid sequence selected from the group consisting of SEQ ID N0:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-1S, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-18, and d) au immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~ NO: l-18. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ m N0:19-36, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
The invention further provides a method fox assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:19-36, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:19-36, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID
N0:19-36, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:19-36, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for polynucleotides of the invention.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descxiptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present pxoteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as coxmnonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINZTTONS
"DME" refers to the amino acid sequences of substantially purified DME
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, marine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of DME. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, ox any other compound or composition which modulates the activity of DME either by directly interacting with DME or by acting on components of the biological pathway in which DME
participates.
An "allelic variant" is an alternative form of the gene encoding DME. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, ox substitutions of nucleotides.
Each of these types of changes may occur alone, ox in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding DME include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as DME or a polypeptide with at least one functional characteristic of DME. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding DME, and improper ox unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding DIvlE. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a~ silent change and result in a functionally equivalent DME. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, andlor the amphipathic nature of the residues, as long as the biological or immunological activity of D1VIE is retained. For example, negatively charged annino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, ox a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of DME. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of DME either by directly interacting with DME or by acting on components of the biological pathway in which DME
participates. --The terra "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab,~F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind DME polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The palypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albunun, thyxoglobulin, and keyhole limpet hemocyanin (KLT~. The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) fox binding to an antibody.
The term "aptamex" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, I5 e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.) The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegehner" refers to an aptamer which includes L DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars ox 2'-methoxyethoxy sugars; ox oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into, a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" ox "plus" can refer to the sense strand of a reference DNA molecule.

The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic DME, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding DME or fragments of DME may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR ldt (Applied Biosystems, Foster City CA) in the 5' andlor the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly, Sex Arg His, Lys Asn Asp, Gln, His Asp ~ Asn, Glu Cys Ala, Sex Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, lle Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Tle, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge ox hydrophobicity of the molecule at the site of the substitution, andlor (c) the bulls of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refexs to a chemically modified polynucieotide or polypeptide.
Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which xetains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, ox any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule ox enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, fox example, a treated and an untreated sample, or a diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel xeassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of DME or the polynucleotide encoding DME
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, pximer, antigen, therapeutic molecule, or for othex purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from-certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%).of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID N0:19-36 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:19-36, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ 1D N0:19-36 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:19-36 from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:19-36 and the region of SEQ ll7 N0:19-36 to which the fragment cozresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID N0:1-18 is encoded by a fragment of SEQ ID N0:19-36. A
fragment of SEQ ID N0:1-18 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-18. Fox example, a frabarnent of SEQ ID NO:1-18 is useful as an immunogenic peptide fox the development of antibodies that specifically recognize SEQ m NO:1-18.
The precise length of a'fragment of SEQ ID NO:1-18 and the region of SEQ ID NO: l-18 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation codon (e.g., mefihionine) followed by an open reading frame and a translation termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. Tlus program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Iiiggins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS
8:189-191. For gairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Tnformation (NCBl] Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http:llwww.ncbi.nhn.nih.govlBLASTI. The BLAST software suite includes various sequence analysis programs including "blastn,"'that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http:!/www.ncbi.uhn.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs axe commonly used with gap and other parameters set to default settings. Fox example, to compare two nucleotide sequences, one may use blastn with the 'BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for nzatch: 1 Perzalty for rnistzzatclz: -2 Opefz Gap: 5 and Extefzsiozz Gap: 2 pefzalties Gap x drop-off.' S0 Expect: 10 Ward Size: 1.1 Filter: ova Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, ftgures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incozporated into the MEGALIGN
version 3.I2e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, fox a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matr-iac: BLOSUM62 Opetz Gap: 11 arzd Exteizsion Gap: 1 perzalties Gap x drop-off.' SO
Expect: 10 Word Size: 3 FiZter.~ orz Percent identity may be measured over the length of an entire defined polypeptide sequence, fox example, as defined by a particular SE(~ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which captain all of the elements required for chromosome replication, segregation and maintenance.

The term "humanized antibody" xefers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ~,glml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is caxried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring fIaxbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ~,g/m1. Oxganic solvent, such as formamide at a concentration of about 35-50% vlv, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) oz formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an annino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of DME
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide ox oligopeptide fragment of DMB which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of DME. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of DME.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragnnent thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense ox the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA
like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.

PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an DME may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of DME.
"Probe" refers to nucleic acid sequences encoding DME, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. .
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 4.0, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo , Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful fox designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge IJK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved ox least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments ident~ed by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.

An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that alI occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term. "sample" is used in its broadest sense. A sample suspected of containing DIME, nucleic acids encoding DME, or fragments thereof may comprise a bodily fluid;
an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, prefexably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymexs, xnicroparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or axtificial conditions accoxding to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or .RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microiujection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods la~own in the art, fox example infection, transfection, transformation or transconjugation. Techniques fox transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et aI. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95010, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human drug metabolizing enzymes (DME), the polynucleotides encoding DME, and the use of these compositions fox the diagnosis, treatment, or prevention of autoimmune/inflammatory, cell proliferative, developmental, endocrine, eye, metabolic, and gastrointestinal disorders, including liver disorders.
Table I summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ll~). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ll~ NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ll~) as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corxesponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (Genbank 7D NO:) of the nearest GenBank homolog.
Column 4 shows the probability score for the match between each palypeptide and its GenBank homolog. Column 5 shows the annotation of the GenBank homolog along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification numbex (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 xesidues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI). ~ Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and 'these properties establish that the claimed polypeptides are drug metabolizing enzymes. SEQ >l7 NO: I is 64% identical to bovine arylacetyl acyl-CoA N-acetyltransferase (GenBank ID
83004445) as determined by the Basic Local Alignment Search Tool (BLAST, see Table 2). The BLAST
probability score is 7.1e-86, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
In an alternative example, SEQ 1D N0:2 is 51% identical to hamster carboxylesterase precursor (GenBank 1D 82641986) as determined by BLAST analysis with a probability score of 4.3e-128. SEQ 1D N0:2 also contains carboxylesterase domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM
database of conserved protein family domains (see Table 3). Data from BLM'S, MOTIFS, and analyses provide further corroborative evidence that SEQ 177 N0:2 is a carboxylesterase.
Tn an alternative example, SEQ ID N0:3 is 45% identical to a C. el, e~ans acetyltransferase (GenBank ID 81825778) as determined by BLAST analysis with a probability score of 8.1e-67. SEQ
1D N0:3 also contains an alphalbeta hydrolase fold, consistent with the classification of SEQ >D
NO:3 as an acetyltransferase, as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM database of conserved protein family domains (see Table 3). Data from BLnVIPS analysis provides further corroborative evidence that SEQ DJ N0:3 contains an alpha/beta hydrolase fold.
Tn an alternative example, SEQ DJ N0:4 is 68% identical to human sulfotransferase 1C2 (GenBank ID 88117877) as determined by BLAST analysis with a probability score of 9.2e-114.
SEQ ID NO:4 also contains sulfotransferase domains as determined by searching for statistically significant matches in the hidden Markov model (I~VIM)-based PFAM database of conserved protein family domains (see Table 3). Data from BLIMPS analysis provides fiurther corroborative evidence that SEQ ~ N0:4 is a sulfotransferase. BLAST analysis of SEQ ID N0:4 against the DOMO
database also indicates the presence of a 3'-phosphoadenosine 5'-phosphosulfate (PAYS) binding site.
PAPS is a donor group for sulfotransferase reactions (see Table 3). --In an alternative example, SEQ ID N0:5 is 36% identical to human androgen-regulated short-chain dehydrogenase/reductase (GenBank D~ 89622124) as determined by BLAST
analysis with a probability score of 5.3e-37.. SEQ m N0:5 also contains dehydrogenase domains as determined by searching for statistically significant matches in the hidden Markov model (FIMM)-based PFAM
database of conserved protein family domains (see Table 3). Data from BLIMPS
analysis provides further corroborative evidence that SEQ )D N0:5 is a dehydrogenase.
In an alternative example, SEQ >D N0:6 is 83% identical to a rat UDP-glucuronosyltransferase (GenBank ID 8458395) as determined by the Basic Local Alignment Search 3S Tool (BLAST). (See Table 2.) The BLAST probability score is 2.5e-248, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ m N0:6 is also 84% identical to a human UDP-glucuronosyltransferase (GenBank >D g475759) as determined by BLAST analysis with a probability score of 1.1e-245. SEQ ID N0:6 also contains UDP-glucuronosyltransferase domains as determined by searching for statistically significant matches in the hidden Markov model (HN.llVI)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLM'S, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ >D N0:6 is an UDP-glucuronosyltransferase.
In an alternative example, SEQ 1D N0:7 is 87% identical to marine squalene epoxidase (squalene monooxygenase) (GenBank ID g1217593) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.5e-273, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ )D NO:7 is also 86% identical to rat squalene epoxidase (GenBank ID g666019), as determined by the BLAST
analysis, with a probability score of 4.6e-272, and 86% identical to human squalene epoxidase (GenBank ID g4204675) with a probability score of 4.9e-268. SEQ >D N0:7 also contains a monoogygenase signature domain as determined by searching for statistically significant matches in the hidderx Markov model (FEVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLllVIPS analysis pxovides further corroborative evidence that SEQ ID N0:7 is a monooxygenase.
In an alternative example, SEQ m N0:8 is 92% identical to human dihydrofolate reductase (GenBank ID g1617080) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.2e-87, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ 1D N0:8 also contains a dihydrofolate reductase active site domain as determined by searching for statistically significant matches in the hidden Markov model (I-3lV,tM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLllVll'S and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:$ is a dihydrofolate reductase.
In an alternative example, SEQ ~ N0:9 is 66% identical to human heparan sulfate D-glucosaminyl 3-O-sulfotransferase 3B (GenBank ID g4835725) as determined by BLAST analysis with a probability score of 4.8e-114.
Tn an alternative example, SEQ 1D N0:10 is 57% identical to marine flavin-containing monooxygenase 5 (GenBank ID g1899255) as determined by BLAST analysis with a probability score of 1.0e-157. SEQ ID N0:10 also contains a flavin-binding monooxygenase active site domain as determined by searching for statistically significant matches in the hidden Markov model (FIIVIM)-based PFAM database of conserved pxotein family domains. (See Table 3.) Data from BLIMPS
analysis provides further corroborative evidence that SEQ m N0:10 contains flavin-containing monooxygenase domains.
In an alternative example, SEQ ID N0:11 is 48% identical to human 11 beta hydroxysteroid dehydrogenase (GenBank ID 8179475), a member of the short-chain alcohol dehydrogenase family of enzymes, as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.7e-54, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:11 is also 45% identical to ovine 11-beta hydroxysteroid dehydrogenase (GenBank ID 81191) based on BLAST analysis, with a probability score of 5.3e-53; 44% identical to marine 11 beta hydroxysteroid dehydrogenase (GenBank ID
8806928), with a probability score of 2.0e-51; 45% identical to squirrel monkey 11 beta hydroxysteroid dehydrogenase (GenBank ID 8388414), with a probability score of 5.4e-51; and 43%
identical to guinea pig 1 I-beta hydroxysteroid dehydrogenase (GenBank ID
86010775), with a probability score of 4.5e-47. SEQ m.N0:11 also contains a short-chain alcohol dehydrogenase family signature domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.) Data from BUMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:11 is a short-chain alcohol dehydrogenase.
Iu an alternative example, SEQ ID N0:12 is 55 % identical to rabbit flavin-containing dimethylanaline monooxygenase (GenBank 177 8164989), as determined by BLAST
analysis, with a probability score of 1.3e-154. SEQ m N0:12 is also 55% identical to a guinea pig flavin containing monooxygenase (GenBank ID 8559027), with a probablity score of 3.3e-154; 56%
identical to a marine flavin containing monooxygenase (GenBank ll7 81899255), with a probablity score of 1.4e-153; and 53% identical to a human flavin containing monooxygenase (GenBank ff~
8559046), with a probablity score of 1.5e-151. SEQ ID N0:12 also contains a flavin containing monooxygenase domain as determined by searching for statistically significant matches in the hidden Markov model (~llVIM)-based PFAM database of conserved protein family domains. Data from BLM'S analysis provide further corroborative evidence that SEQ ID N0:12 is a flavin containing monooxygenase.
In an alternative example, SEQ ID NO:I3 is 36% identical to rabbit UDP-glucuronosyltransferase (GenBank ID 8165801), a specific type of UDP-glycosyltransferase, as determined by BLAST analysis. The BLAST probability score is 2.8e-70. SEQ m NO:13 is also 33% identical to several human UDP-glucuxonosyltransferases (GenBank ID
81403658, 81407590, and 81923219), all with probability scores of I.2e-69; and 34% identical to a marine UDP-glucuronosyltransferase (GenBank ID 81246787), with a probablity score of 2.0e-69. SEQ ID N0:13 also contains a UDP-glucuronosyltransferase domain as determined by searching for statistically significant matches in the hidden Markov model (I ~llVIM)-based PFAM database of conserved protein family domains. Data from BLM'S and PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:13 is a UDP-glucuronosyltransferase.
In an alternative example, SEQ ll~ NO:I4 is 96% identical to human UDP-glucuronosyltransferases (GenBank ID 83360273 and 8516150), as determined by BLAST analysis, with probability scores of 3.2e-181 and 8.7e-179, respectively. SEQ ID N0:14 is also 92% identical to macaque UDP-glucuronosyltransferase (GenBank ID 84079707), with a probablity score of 7.4e-171. SEQ lD N0:14 also contains a UDP-glucuronosyltransferase domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFA.M
database of conserved protein family domains. Data from BLllVIPS and PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:14 is a UDP-glucuronosyltransferase.
In an alternative example, SEQ ID NO:IS is 43% identical to human 25-hydroxyvitamin D3 Ia-hydroxylase (GenBank 1D 82516244), a member of the NADPH-dependent, type I
(mitochondrial) cytochrorne P450 family of enzymes, as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.7e-95, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:15 also contains a cytochrome P450 active site domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.) Data from BLllVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ m N0:15 is a member of the cytochrome P450 family.
In an alternative example, SEQ ID N0:16 is 40% identical to Bacillus halodurans 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase (aldolase) (GenBank >D 8101746I9), an enzyme involved in the catabolism of tyrosine and xenobiotic compounds that pass through the tyrosine catabolic pathway, as determined by the BLAST analysis. The probability score is 4.7e-45. SEQ. Iv NO: I6 also contains a fumarylacetoacetate hydrolase family active site domain as determined by searching for statistically significant matches in the hidden Markov model (IiIVIM)-based PFAM
database. Fumarylacetoacetate hydrolase is closely related to the Bacillus halodurans aldolase and is also involved with tyrosine catabolism.
In an alternative example, SEQ ID N0:17 shares 44% local identity (i.e., over I67 contiguous amino acid residues) with human glutathione S-transferase theta 1 (GenBank ID
89937245) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 3.2e-58, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:17 is also 44% identical (over 167 contiguous amino acid residues) to human glutathione S-transferase T1 (GenBank ID 85I0905), as determined by BLAST analysis, with a probability score of 1.7e-57 and 41% identical (over 181 contiguous amino acid residues) to marine glutathione S-transferase theta (GenBank ID
81340076), with a probability score of 4.5e-53. SEQ TD NO: I7 also contains a glutathione S-transferase active site domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Iu an alternative example, SEQ ID N0:18 is 96% identical to human (R)-3-hydroxybutyrate dehydrogenase (GenBank ID g177I98), a member of the short-chain alcohol dehydrogenase family of enzymes, as determined by BLAST analysis. The BLAST probability score is 4.2e-170. SEQ ID
N0:18 also contains a short-chain alcohol dehydrogenase active site domain as determined by searching for statistically significant matches in the hidden Markov model (HIVIM)-based PFAM
database of conserved pxotein family domains. Data from BLM'S, MOTIFS, and PROFILESCAN
analyses provide further corroborative evidence that SEQ ID NO:18 is a short-chain alcohol dehydrogenase. The algorithms and parameters for the analysis of SEQ ID NO:1-18 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte polynucleotide consensus 'sequence number (Incyte Polynucleotide ID) for each polynucleotide of the invention.
Colunm 3 shows the length of each polynucleotide sequence in basepairs. Column 4 lists fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID N0:19-36 or that distinguish between SEQ ID
N0:19-36 and related polynucleotide sequences. Column 5 shows identification numbers corresponding to eDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective 2S full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for example, to Incyte cDNAs along with their corresponding eDNA libraries. For example, 7219095H1 is the identification number of an Incyte cDNA sequence, and SPLNDICO1 is the cDNA
library from which it is derived. Incyte cDNAs for which cDNA libraries are not indicated were dexived from pooled cDNA libraries (e.g., 55143913H1). Alternatively, the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., g2787922) which contributed to the assembly of the full length polynucleotide sequences. In addition, the identification numbers in column 5 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the identification numbers in column 5 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq.Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, FL_XXxl~ NI N2 YYYYY_N3 lV4 represents a "stitched"
sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and ~1,2,3...~ if Present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the identification numbers in column 5 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, FLXXXX~fX_gA.AAA.A_gBBBBB_1 1V is the identification number of a "stretched"
sequence, with X~XXXX being the Incyte project identification number, gAAAAA being the GenBanl;. identification number of the human genomic sequence to which the "exon-stretching" algorithm.
was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "NM," "NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were liand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs GNN, GFG,Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES

(Computer Genomics Group, The Sanger Centre, Cambridge, UK).

GBI Hand=edited analysis of genomic sequences.

FL Stitched or stretched genomic sequences (see Example V).

INCY Full length transcript and exon prediction from mapping of EST

sequences to the genome. Genomic location and EST composition data are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirnn the final consensus polynucleotide sequence, but the relevant Tncyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses DME variants. A preferred DME variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the DME amino acid sequence, and which contains at least one functional or structural characteristic of DME.
The invention also encompasses polynucleotides which encode DME. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:19-36, which encodes DME. The polynucleotide sequences of SEQ ID N0:19-36, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thyxnine are replaced with uracil, and the sugax backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding DME. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding DME. A particular aspect of the invention encompasses a variant of a polynucleotide seqnerice comprising a sequence selected from the group consisting of SEQ m N0:19-36 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:19-36. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of DME.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding DME, some bearing minimal similarity to the polynucleotide sequences of any lmown and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring DME, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode DME and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring DME under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding DME or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding DME and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode DME
and DME derivatives, ar fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding DME or any fragment thereof.
Also encompassed by the invention are poiynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:19-36 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions,"
Methods fox DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland O~, Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amexsham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watextown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system {Molecular Dynamics, Sunnyvale CA), or other systems known in the are. The resulting sequences are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel; F.M.
(1997) Short Protocols in Molecular Biolo~y, John~Wiley & Sons, New York- N'Y, unit 7.7; Meyers, R.A. (1995) Molecular Biolog,~and Biotechnolo~y, Whey VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding DME may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify. unlorown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstarom, M. et al. (1991) PCR Methods Applic. 1:111-119.) Iu this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unlaiown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (199x) Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERF1NDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in fording intron/exon junctions. Fox all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
V~hen screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions.of genes, are preferable fox situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into S' non-transcribed regulatory regions. ' Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Outputllight intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode DME may be cloned in recombinant DNA molecules that direct expression of DME, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express DME.
The nucleotide sequences of the present invention can be engineered using methods generally lrnown in the art in order to alter DME-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, andlor expression of the gene product. DNA
shuffling by random frab~nentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECLTLARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of DME, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding DME may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et aI. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Sex.
7:225-232.) Alternatively, DME itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of DME, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-4.21.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.) In order to express a biologically active DME, the nucleotide sequences encoding DME or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding DME. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efFicient translation of sequences encoding DME. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding DME and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and syntheric. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding DME and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding DME. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambxook, supra; Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et aI. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO

J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGxaw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding DME. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding DME can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORTI
plasmid (Life Technologies). Ligation of sequences encoding DME into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509,) When large quantities of DME are needed, e.g. for the production of antibodies, vectors which direct high level expression of DME may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of DME. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, .e.g., Ausubel, 1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of DME. Transcription of sequences encoding DME may be driven by viral promoters, e.g., the 355 and 195 promoters of CaMV
used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J.

6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et aI.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The MeGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding DME
may be ligated into an adenovirus transcriptionltranslation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses DME in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
Natl. Acid. Sci. USA 81:3655-3659.) Tn addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. .HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (Iiposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of DME in cell lines is preferred. For example, sequences encoding DME can be transformed into cell lines using expression vectors which may contain viral origins of replication andlor endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer xesistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection, systems may be used to recovex transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk- and apr' cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 11;223-232; Lowy, I. et al. (1980) Cell 22;817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dlzfr confers resistance to methotrexate; rzeo confexs resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acid. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and lzisD, which altex cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acid. Sci. USA 85; 8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), l3 glucuronidase and its substrate !3-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding DME is inserted within a marker gene sequence, transformed cells containing sequences encoding DME can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding DME under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding DME and that express DME may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not Limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of DME using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linkec>-immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on DME is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and Wiley-Intexscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing Labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding DME
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding DME, or any fragments thereof, may be cloned into a vector fox the producrion of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenzc agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding DME may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode DME may be designed to contain signal sequences which direct secretion of DME through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen fox its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
I5 Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI3$) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encodixig DME may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric DME protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of DME activity.
Heterologous protein and peptide moieties may also facilitate purification of fusion proteius using commercially available afFmity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of theix cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, cahnodulin, and metal-chelate resins, respectively. FLAG, c-n2yc, and hemagglutinin (FiA,) enable inununoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the DME encoding sequence and the heterologous protein sequence, so that DME may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, su ra, ch. 10).
A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled DME may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 3sS-methionine.
DME of the present invention or fragments thereof may be used to screen for compounds that specifically bind to DME. At least one and up to a plurality of test compounds may be screened for specific binding to DME. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of DME, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunoloay 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which DME
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express DME, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Dro~hila, or E. coli. Cells expressing DME or cell membrane fractions which contain DME are then contacted with a test compound and binding, stimulation, or inlubition of activity of either DME or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophoxe, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with DME, either in solution or affixed to a solid support, and detecting the binding of DME to the compound.
Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
DME of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of DME. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for DME
activity, wherein DME is combined with at least one test compound, and the activity of DME in the pxesence of a test compound is compared with the activity of DME in the absence of the test compound. A change in the activity of DME iu the presence of the test compound is indicative of a compound that modulates the activity of DME. Alternatively, a test compound is combined with an in' vitro or cell-free system comprising DME under conditions suitable for DME
activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of DME
may do so indirectly and need not come in direct contact with the test compound. At least one and up S to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding DME or their mammalian homologs rnay be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 1291SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
(1989) Science 244:1288-I292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Tnvest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL16 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding DME may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding DME can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding DME is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress DME, e.g., by secreting DME in its mills, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS

Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of DME and drug metabolizing enzymes. Tn addition, the expression of DME is closely associated with brain tissue, kidney tissue, and rapidly dividing cells, including breast. tumor, brain tumor, other cancers, and dermal fibroblasts. Therefore, DME appears to play a role in autoimmune/inflammatory, cell proliferative, developmental, endocrine, eye, metabolic, and gastrointestinal disorders, including liver disorders. In the treatment of disorders associated with increased DME expression or activity, it is desirable to decrease the expression or activity of DME.
In the treatment of disorders associated with decreased DME expression or activity, it is desirable to increase the expression or activity of DME.
Therefore, in one embodiment, DME or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of DME.
Examples of such disorders include, but are not limited to, an autoimmune/inflarnmatory disorder, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, 1S autoimmune hemolytic anemia, autoirnmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, l3ashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and hehninthic infections, and trauma; a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), anyelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a developmental disorder, such as renal Tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wihns' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus., seizure disorders such as Syndenham's chorea and cerebral palsy, spins bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; an endocrine disorder, such as disorders of the hypothalamus and pituitary resulting from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma; disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sells syndrome, and dwarfism; disorders associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADT~ secretion (SIADH) often caused by benign adenoma; disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism; disorders associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummet's disease; disorders associated with hyperparathyroidism including Conn disease (chronic hypercalemia); pancreatic disorders such as Type I or Type II diabetes mellitus and associated complications; disorders associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease; disorders associated with gonadal steroid hormones such as:
in women, abnormal prolactin production, infertility, endometriosis, perturbations of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis; and, in men, Leydig cell dericiency, male climacteric phase, and germinal cell aplasia, hypergonadal disorders associated with. Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a--reductase, and gynecomastia;
an eye disorder, such as conjunctivitis, keratoconjunctivitis sicca, keratitis, episcleritis, iritis, posterior uveitis, glaucoma, amaurosis fugax, ischemic optic neuropathy, optic neuritis, Leber's hereditary optic neuropathy, toxic optic neuropathy, vitreous detachment, retinal detachment, cataract, macular degeneration, central serous chorioretinopathy, retinitis pigmentosa, melanoma of the choroid, xetrobulbar tumor, and chiasmal tumor; a metabolic disorder, such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, 6~

hypoparathyroidism, hypercholesteroleania, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, Menkes syndxome, occipital horn syndrome, mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria, pseudovitamin D-deficiency rickets; hypocalcemia, hypophosphatemia, postpubescent cerebellar ataxia, and tyrosinemia, and a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, hereditary hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Cxohn's disease, Whipple's disease, Mallory Weiss syndrome, colonic.carcinoma, eolonic obstruction, ixxitable bowel syndrome, shore bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatoxenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphas-antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas.
In another embodiment, a vector capable of expressing DME or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of DME including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified DME
in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of DME including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of DME
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of DME including, but not limited to, those listed above.
In a further embodiment, an antagonist of DME inay be administered to a subject to treat or prevent a disorder associated with increased expression or activity of DME.
Examples of such disorders include, but are not limited to, those autoimmunelinflammatory, cell proliferative, developmental, endocrine, eye, metabolic, and gastrointestinal disorders, including liver disorders, described above. In one aspect, an antibody which specifically binds DME may be used directly as an anfiagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express DME.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding DME may be administered to a subject to treat or pxevent a disorder associated with increased expression or activity of DME including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential fox adverse side effects.
An antagonist of DME may be produced using methods which are generally la~own in the art.
In particular, purified DME may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind DME.
Antibodies to DME may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
Fox the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with DME or with any fragment or oligopeptide thereof wlrich has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, platonic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calinette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to DME
have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein.
Short stretches of DME
amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to DME may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et aI. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.

Immunol. Methods 81:31-4.2; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci.
USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) W addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Mornson, S.L. et aI. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et a1. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techiniques described for the production of single chain antibodies may be adapted, using methods lrnown in the art, to produce DME-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et aI. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites fox DME may also be generated.
Fox example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such . immunoassays typically involve the measurement of comglex formation between DME and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering DME epitopes is generally used, but a competitive binding assay may also be employed (Pound, su ra .
Various methods such as Scatchard analysis in conjunction with radioimmunoassay -techniques may be used to assess the affinity of antibodies fox DME. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of DME-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
The Ka determined for a preparation of pohyclonal antibodies, which are heterogeneous in their affinities for multiple DME epitopes, represents the average affinity, or avidity, of the antibodies fox DME. The Ka determined fox a preparation of monoclonal antibodies, which are monospecific for a particular DME epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in which the DME-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K$ ranging from about 106 to 107 L/mole are preferred fox use in immunopurification and similar procedures which ultimately require dissociation of DME, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Auproach,1RL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2.mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of DME-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, s. upra, and Coligan et al. su era.) In another embodiment of the invention, the polynucleotides encoding DME, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding DME. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding DME. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, I3umana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable fox introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellulaxly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. lmmunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Phannacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Bx. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morns, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding DME may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cel175:207-216; Crystal, R.G. et al.
(1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995} Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in DME expression or regulation causes disease, the expression of DME from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in DME are treated by constructing mammalian expression vectors encoding DME and introducing these vectors by mechanical meaus into DME-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA mieroinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of DME include, but are not linnited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSHIPERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2 LUC, PTK-HYG (Clontech, Palo Alto CA). DME
may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes}, (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (I992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasnud (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486lmifepristone inducible promoter (Rossi, F.M.V. aird Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding DME from a normal individual.
E
Commercially available liposome transformation kits (e.g., the PERFECT L1PID
TRANSF)JCTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (I973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to DME expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding DME under the control of an independent promoter or the retrovirus long terminal repeat (LTR) pxomoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vectox is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of iransduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
~Ranga, U. et al. (1998) Pxoc. Natl. Acad. Sci. USA 95:1201-1206; Su, L.
(1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding DME to cells which have one or more genetic abnormalities with respect to the expression of DME. The construction and packaging of adenovirus-based vectors are well Wown to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev.
Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding DME to target cells which have one or more genetic abnormalities with respect to the expression of DME. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing DME to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy.
Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et a1. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding DME to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for DME into the alphavirus genome in place of the capsid-coding region results in the production of a large number of DME-coding RNAs and the synthesis of lugh levels of DME in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (EHK-21) with a variant of Sindbis virus (SIB in.dicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of DME into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. Sinnilarly, inhitbition can be achieved using triple helix base-pairing methodology.
Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo i~ c Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding DME.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the taxget molecule for xibozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding DME. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding DME. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased DME
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding DME may be therapeutically useful, and in the treatment of disorders associated with decreased DME expression or activity, a compound which specifically promotes expression of the polynucleotide encoding DME may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection firom an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical andlor structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding DME is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding DME are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding DME. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
Commun. 268:$-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are.well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechnol. 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of DME, antibodies to DME, and mimetics, agonists, antagonists, or inhibitors of DME.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form:.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising DME or fragments thereof. Fox example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. ~ Alternatively, DME or a- fragment thereof may be j oined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et I5 aI. (1999) Science 285:1569-I572).
For any.compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Sucl information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example DME
or fragments thereof, antibodies of DME, and agonists, antagonists or inhibitors of DME, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDSO (the dose therapeutically effective in 50% of the population) or LDSO (the dose lethal to 50% of the population) statistics. Tle dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDSalEDso ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are .
used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDjo with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the ~5 active moiety or to maintain the desired effect. Factors which may be taken into account include the identify nucleic acid sequences which encode DME. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif,' and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding DME, allelic variants, ox related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the DME encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ m N0:19-36 or from genomic sequences including promoters, enhancers, and introns of the DME gene.
Means for producing specific hybridization probes for DNAs encoding DME
include the cloning of polynucl~otide sequences encoding DME or DME derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 3sS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding DME may be used for the diagnosis of disorders associated with expression of DME. Examples of such disorders include, but are not limited to, an autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome (ATDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, athexosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systenuc anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and hehninthic infections, and trauma; a cell proliferative disorder, such as actinic kexatosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a developmental disorder, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot .Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; an endocrine disorder, such as disorders of the hypothalamus and pituitary resulting from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma; disorders associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallna:an's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism; disorders associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma; disorders associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism;
disorders associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease;
disorders associated with hyperparathyroidism including Conn disease (chronic hypercalemia);
pancreatic disorders such as Type I or Type lI diabetes mellitus and associated complications;
disorders associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease;
disorders associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbations of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis; and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, hypergonadal disorders associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia; an eye disorder, such as conjunctivitis, 3S keratoconjunctivitis sicca, keratitis, episcleritis, iritis, posterior uveitis, glaucoma, amaurosis fugax, isehemitc optic neuropathy, optic neuritis, Leber's hereditary optic neuropathy, toxic optic neuropathy, vitreous detachment, retinal detachment, cataract, macular degeneration, central serous chorioretinopathy, retinitis pigmentosa, melanoma of the choroid, retrobulbar tumor, and chiasmal tumor; a metabolic disorder, such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, Menkes syndrome, occipital horn syndrome, mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria, pseudovitamin D-deficiency rickets; hypocalcemia, hypophosphatemia, postpubescent cerebellar ataxia, and tyrosinemia, and a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, hereditary hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crvhn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (All~S) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye's syndrome, primary selerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas. The polynucleotide sequences encoding DME may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered DME
expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding DME may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding DME may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding DME in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of DME, a normal or standard profile for expression is established. This may be accomplished by combining body fluids ox cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding DME, under conditions suitable for hybridization or amplification.
Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
IS Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays ri~ay be used to show the efficacy of treatment over a period ranging from several days to months.
'kith respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding DME
may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding DME, or a fragment of a polynucleotide complementary to the polynucleotide encoding DME, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantif catiori of closely related DNA or RNA sequences.
7n a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding DME may be used to detect single nucleotide polymozphisms (SNPs).
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. A11 compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families.
Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by auy tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with.different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, lmowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http:!/www.niehs.nih.govloc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological scxeening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab geI electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver ox fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In. some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for DME to quantify the levels of DME expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the micxoarray to the sample and detecting the levels of protein bound to each array element (Lueling, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods laiown in the art, fox example, by reacting the proteins in the sample with a thiol-or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. Tn addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so pxoteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual piroteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A
difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al.
(1995) PCT application W095/35505; Heller, R.A, et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well laiown and thoroughly described in DNA Microarrays: A Practical Approach, M. Schena, ed.
(1999) Oxford University Pxess, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding DME
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either 3S coding ox noncoding sequences may be used, and in same instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, ox to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.

7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
(See, for example, Landex, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci:
USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, su ra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMLM) World Wide Web site. Correlation between the location of the gene encoding DME on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching fox disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, DME, its catalytic or immunogenic fragments, or oligopeptides thereof can be used fox screening libraries of compounds iu any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between DME and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT

application W084103564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with DME, or fragments thereof, and washed. Bound DME is then detected by methods well known in the art.
Purified DME can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding DME specifically compete with a test compound for binding DME. In this manner, antibodies can be used to detect the presence of any peptide which shares one or moxe antigenic determinants with DME.
I0 In additional embodiments, the nucleotide sequences which encode DME niay be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not !imitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents., applications and publications, mentioned above and below, including U.S. Ser. No.60/236,947, U.S. Sex. No. 60!238,864, U.S. Ser. No.
60/242,323, U.S. Ser. No.
60/247,581, U.S. Ser. No. 60/249,519, U.S. Ser. No. 60/252,834, and U.S. Ser.
No. 601250,567 are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were xepeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly{A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the IJNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods laiown in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL 51000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XLl-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation o~cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 mI of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Bobbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amexsham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled. polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences ox translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (l~-based protein family databases such as PFAM. (I-BVIM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLM'S, and I~~IMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples TV and V) were used to extend lncyte cDNA
assemblages. to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM.
Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analy..Sis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ
ID N0:19-36. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative drug metabolizing enzymes were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA
sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. Tlie maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Gensean predicted cDNA sequences encode drug metabolizing enzymes, the encoded polypeptides were analyzed by querying against PFAM models for drug metabolizing enzymes.
Potential drug metabolizing enzymes were also identified by homology to Incyte cDNA sequences that had been annotated as drug metabolizing enzymes. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan predicted sequences were then edited by comparison to the top BLAST
hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public eDNA coverage of the Genscan-predicted.
sequences, thus providing evidence for transcription. When Ineyte 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 ICI. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Geno~nitc Sequence Data with cDNA Sequence Data "Stitched" Segnences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example TV. Partial cDNAs assembled as described in Example IQ were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one ox more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genorxvc information, generating possible splice variants that were subsequently confined, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional eDNA sequences, or by inspection of genomie DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described iil Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST prograam. The nearest GenBank protein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (hISPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The Analogous computer techniques applying BLAST were used to search fox identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identi~
5 x minimum {length(Seq. I), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compaxed. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by I00% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding DME are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences axe assembled, at least in part, with overlapping Incyte cDNA sequences (see Example 111). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organltissue categories: cardiovascular system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; heroic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following diseaselcondition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding DME. cDNA sequences and eDNA
library/tissue information are found in the L1FESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of DIME Encoding Polynncleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well laiown iun the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NHa)aS04, and 2 mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: stoxage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, I min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing I00 p1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 ~,l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan lI
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence bf the sample and to quantify the concentration of DNA. A 5 ,u1 to 10 ,u1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were xeligated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37°C in 3g4-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Prabes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide (1:2,~v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction lit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:19-36 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the~art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ,uCi of [y-32P] adenosine triphosphate (Amersham Phatxnacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superf'me size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NITj. Hybridization is carried out fox 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtempexature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
X. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), ssupra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, W, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. {See, e.g., Schena, M. et aI. {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 rnay comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyariate method and poly(A)* RNA is purified using the oligo-(dT) cellulose method. Each poly(A)~
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/,ul oligo-(dT) primer (2lmer), 1X
first strand buffer, 0.03 units/,ul RNase inhibitor, 500 ,uM dATP, 500 ~M
dGTP, 500 ~tM dTTP; 40' ~,M dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 {Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)~ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ~tl 5X SSC/0.2% SDS.
Microarra~paration Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified arxay elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VYJR), W est Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
Array elements are applied to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~Cl of the array element DNA, at an average concentration of 100 ng/~.1, is loaded into the open capillary printing element by a high-sgeed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINT~ER UV-crosslinker (Stratagene).
Microarxays are washed at room temperature once in 0.2% SDS and thxee times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~1 of sample mixture consisting of 0.2 ~tg each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65°C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2. coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly laxger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 p1 of 5X SSC in a comer of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45 ° C in a first wash buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a second wash buffer (0.1X
SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Tnnova 70 mixed gas IO W user (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x I.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
Tn two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 82477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophoxes used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluoxophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typicaiiy calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, axe hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (Iow signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluoxophores are excited and measured simultaneously, the data are first corrected for optical crosstaIk (due to overlapping emission spectra) between the fluorophores using each fluoxophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides Sequences complementary to the DME-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring DME. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of DME. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the DME-encoding transcript.
XII. Expression of DME
Expression and purification of DME is achieved using bacterial or virus-based expression systems. For expression of DME in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
I5 transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transforined into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria empress DME upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of DME in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Auto -~raphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding DME by either homologous recombination or ~bactexial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera fruaiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.I~.
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 is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Sclustosoma iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from DME at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffmity purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIA.GEN). Methods for protein expression and purification are discussed in Ausubel (1995, s" upra, ch. 10 and 16). Purified DME obtained by these methods can be used dixectly in the assays shown in Examples XVI, XV1I, and XVJII, where applicable.
.'III. Functional Assays DME function is assessed by expressing the sequences encoding DME at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ,ug of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New Yoxk NY.
The influence of DME on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding DME and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NIA. mRNA can be purified from the cells using methods well known by those of skill in the art.
Expression of mRNA encoding DME and other genes of interest can be analyzed by northern analysis or microarray techniques.

XIV. Production of DME Specific Antibodies DME substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the DME amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appxopriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydxoxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-DME activity by, for example, binding the peptide or DME
to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XV. Purification of Naturally Occurring DME TTsing Specific Antibodies Naturally occurring or recombinant DME is substantially purified by immunoaffmity chromatography using antibodies specific for DME. An immunoaffinity column is constructed by covalently coupling anti-DME antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing DME are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of DME (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt ' antibodylDME binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and DME is collected.
XVI. Identification of Molecules Which Interact with DME
DME, or biologically active fragments thereof, are labeled with lasl Bolton-Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled DME, washed, and any wells with labeled DME complex are assayed. Data obtained using different concentrations of DME are used to calculate values for the numbex, affinity, and association of DME with the candidate molecules.

Alternatively, molecules interacting with DME are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHIVTAKER system (Clontech).
DME may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two Iarge libraries of genes (Nandabalan, K.
et aI. (2000) U.S.
Patent No. 6,057,101).
XVII. Demonstration of DME Activity Cytochrome P450 activity of DME is measured using the 4-hydroxylation of aniline. Aniline is converted to 4-aminophenol by the enzyme, and has an absorption maximum at 630 nm (Gibson and Skett, supra). This assay is a convenient measure, but underestimates the total hydroxylation, which also occurs at the 2- and 3- positions. Assays are performed at 37°C and contain an aliquot of the enzyme and a suitable amount of aniline (approximately 2 mM) in reaction buffer. For this reaction, the buffer must contain NADPH or an NADPH-generating cofactor system. One formulation for this reaction buffer includes 85 mM Tris pH 7.4, 15 mM MgCl2, 50 mM
nicotinamide, 40 mg trisodium isocitrate, and 2 units isocitrate dehydrogenase, with 8 mg NADP*
added to a 10 mL reaction buffer stock just prior to assay. Reactions are carried out in an optical cuvette, and the absorbance at 630 nm is measured. The rate of increase in absorbance is proportional to the enzyme activity in the assay. A standard curve can be constructed using known concentrations of 4-aminophenol.
1a,25-dihydroxyvitamin D 24-hydroxylase activity of DME is determined by monitoring the conversion of 3H-labeled 1x,25-dihydroxyvitamin D (1a,25(OH)zD) to 24,25-dihydroxyvitamin D
(24,25(OH),,D) in transgenic rats expressing DME. 1 ~Cg of 1a,25(OH)ZD
dissolved in ethanol (or ethanol alone as a control) is administered intravenously to approximately 6-week-old male transgenic rats expressing DME or otherwise identical control rats expressing either a defective variant of DME or not expressing DME. The rats are killed by decapitation after 8 hrs, and the kidneys are rapidly removed, rinsed, and homogenized in 9 volumes of ice-cold buffer (15 mM Tris-acetate (pH 7.4), 0.19 M sucrose, 2 mM magnesium acetate, and 5 mM sodium succinate). A portion (e.g., 3 ml) of each homogenate is then incubated with 0.25 nM 1a,25(OH)2[1 3HJD, with a specific activity of approximately 3.5 GBq/mmol, for 15 min at 37 °C under oxygexi with constant shaking.
Total lipids are extracted as described (Bligh, E.G. and W.J. Dyer (I959) Can.
J. Biochem. Physiol.
37: 911-917) and the chloroform phase is analyzed by HPLC using a FINEPAK STL
column (JASCO, Tokyo, Japan) with an h-hexane/chloroform/methanol (10:2.5:1.5) solvent system at a flow rate of 1 ml/min. In the alternative, the chloroform phase is analyzed by reverse phase HPLC using a J
SPHERE ODS-AM column (YMC Co. Ltd., Kyoto, Japan) with an acetonitrile buffer system (40 to 100%, in water, in 30 min) at a flow rate of 1 ml/min. The eluates are collected in fractions of 30 seconds (or less) and the amount of 3H present in each fraction is measured using a scintillation counter. By comparing the chromatograms of control samples (i.e., samples comprising 1a,25-dihydroxyvitamin D or 24,25-dihydroxyvitamin D (24,25(OH)zD), with the chromatograms of the reaction products, the relative mobilities of the substrate (1a,25(OH)2[1 3H]D) and product (24,25(0H)2[1 3H]D) are determined and correlated with the fractions collected. The amount of 24,25(0H)2[1 3H]D produced in control rats is subtracted from that of transgenic rats expressing DME. The difference in the production of 24,25(0H)2[1 3H]D in the transgenic and control animals is proportional to the amount of 25-hydrolase activity of DME present in the sample. Confirmation of the identity of the substrate and products) is confn~ned by means of mass spectroscopy (Miyamoto, Y. et al. (1997) J. Biol. Chem. 272:14115-14119).
Flavin-containing monooxygenase activity of DME is measured by chromatographic analysis of metabolic products. For example, Ring, B.J. 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, stopped the reaction with an organic solvent, and determined product formation by HPLC.
Alternatively, activity is measured by monitoring oxygen uptake using a Clark type electrode. For example, Ziegler, D.M. and Poulsen, L.L. (1978; Methods Enzymol. 52:142-151) incubated the enzyme at 37°C in an NADPH-generating cofactor system (similar to the one described above) containing the substrate methimazole. The rate of oxygen uptake is proportional to enzyme activity.
UDP glucuronyltransferase activity of DME is measured using a colorimetric determination of free amine groups (Gibson and Skett, supra). An amine-containing substrate, such as 2-aminophenol, is incubated at 37 °C with an aliquot of the enzyme in a reaction buffer containing the necessary cofactors (40 mM Tris pH 8.0, 7.5 mM MgCl2, 0.025% Triton X-100, 1 mM ascorbic acid, 0.75 mM UDP-glucuronic acid). After sufficient time, the reaction is stopped by addition of ice-cold 20% trichloroacetic acid in 0.1 M phosphate buffer pH 2.7, incubated on ice, and centrifuged to clarify the supernatant. Any unreacted 2-aminophenol is destroyed in this step. Sufficient freshly-prepared sodium nitrite is then added; this step allows formation of the diazonium salt of the glucuronidated product. Excess nitrite is removed by addition of sufficient ammonium sulfamate, and the diazonium salt is reacted with an aromatic amine (for example, N-naphtlrylethylene diamine)-to produce a colored azo compound which can be assayed spectrophotometrically (at 540 nm, for example). A standard curve can be constructed using known concentrations of aniline, which will form a chromophore with similar properties to 2-aminophenol glucuronide.
Glutathione S-transferase activity of DME is measured using a model substrate, such as 2,4-dinitro-1-chlorobenzene, which reacts with glutathione to form a product, 2,4-dinitrophenyl-glutathione, that has an absorbance maximum at 340 nm. It is important to note that GSTs have differing substrate specificities, and the model substrate should be selected based on the substrate preferences of the GST of interest. Assays are performed at ambient temperature and contain an aliquot of the enzyme in a suitable reaction buffer (for example, 1 mM
glutathione,1 mM
dinitrochlorobenzene, 90 mM potassium phosphate buffer pH 6.5). Reactions are carried out in an optical cuvette, and the absorbance at 340 rim is measured. The rate of incxease in absorbance is proportional to the enzyme activity in the assay.
N-acyltransferase activity of DME is measured using radiolabeled amino acid substrates and measuring radiolabel incorporation into conjugated products. Enzyme is incubated in a reaction buffer containing an unlabeled acyl-CoA compound and radiolabeled amino acid, and the radiolabeled acyl-conjugates are separated from the unreacted amino acid by extraction into n-butanol or other appropriate organic solvent. For example, Johnson, M. R._et al.
(1990; J. Biol. Chem.
266:10227-10233) measured bile acid-CoA:amino acid N-acyltransferase activity by incubating the enzyme with cholyl-CoA and 3H-glycine or 3H taurine, separating the tritiated cholate conjugate by extraction into n-butanol, and measuring the radioactivity in the extracted product by scintillation.
Alternatively, N-acyltransferase activity is measured using the spectrophotometric determination of reduced CoA (CoASH) described below.
N-acetyltransferase activity of DME is measured using the transfer of radiolabel from [14C]acetyl-CoA to a substrate molecule (for example, see Deguchi, T. (1975) J. Neurochem.
24:1083-5). Alternatively, a spectrophotometric assay based on DTNB (5,5'-dithio-bis(2-nitrobenzoic acid; Ellman's reagent) reaction with CoASH may be used. Free thiol-containing CoASH is formed during N-acetyltransferase catalyzed transfer of an acetyl group to a substrate. CoASH is detected using the absorbance of DTNB conjugate at 412 rim (De Angelis, J. et al.
(1997)~J. Biol. Chem.
273:3045-3050). Enzyme activity is pzoportional to the rate of radioactivity incorporation into substrate, or the rate of absorbance increase in the spectrophotometric assay.
In the alternative, histone acetyltransferase activity of ABBR is determined using a mixture of recombinant Xenopus histone H32~H42 tetramers (100 ~uglml), human histone H2A~H2B purified from HeLa cells (100 ~,glml), and 23 pmol [3H]-acetyl coenzyme A (11.2 Ci/mmol) in 12.5 ~,l of buffer (25 mM Tris-HCl, pH 8.5, 1 mM dithiothreitol, 0.5 mM EDTA, 5 mM sodium butyrate, 150 mM NaCI, and 10% glycerol) incubated at 37 °C for 1 h, The histories are resolved in a SDS-18%
polyacrylamide gel and visualized by staining with Coomassie Brilliant Blue dye and by fluorography followed by autoradiography. Radioactive bands corresponding to the positions of histories visualized by staining indicate the incorporation of label into the particular histone substrate by acetylation.
Protein argiizine methyltransferase activity of DME is measured at 37 °C for various periods of time. S-adenosyl-L-[methyl 3H]methionine ([3H]AdoMet; specific activity =
75 Ci/mmol; NEN

Life Science Products) is used as the methyl-donor substrate. Useful methyl-accepting substrates include glutathione S-transferase fibrillarin glycine-arginine domain fusion protein (GST-GAR), heterogeneous nuclear ribonucleoprotein (hnRNP), or hypomethylated proteins present in lysates from adenosine dialdehyde-treated cells. Methylation reactions are stopped by adding SDS-PAGE
sample buffer. The products of the reactions are resolved by SDS-PAGE and visualized by fluorography. The presence of 3H-labeled methyl-donor substrates is indicative of 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 measured in a reaction mixture consisting of 50 mM Tris-HCl (pH 7.4), 1.2 mM MgCl2, 200 ~,M SAM (S-adenosyl-L-methionine) iodide (containing 0.5 p,Ci of methyl-[H3]SAM), 1 mM dithiothreitol, and varying concentrations of catechol substrate (e.g., L-dopa, dopamine, or DBA) in a final volume of 1.0 ml. The reaction is initiated by the addition of 250-500 ~g of purified DME or crude DME-containing sample and performed at 37 °C for 30 min. The reaction is arrested by rapidly cooling on ice and immediately extracting with 7 ml of ice-cold n-heptane. Following centrifugation at 1000 x g for 10 min, 3-ml aliquots of the organic extracts are analyzed for radioactivity content by liquid scintillation counting. The level of catechol-associated radioactivity in the organic phase is proportional to the catechol-O-methyltransferase activity of DME (Zhu, B. T. and J. G. Liehr (1996) 271:1357-1363).
DHFR activity of DME is determined spectrophotometrically at 15 °C by following the disappearance of NADPH at 340 nm (~40 =11,8001VI'~crri'). The standard assay mixture contains 100 ACM NADPH, 14 mM 2-mercaptoethanol, MTEN buffer (50 mM 2-morpholinoethanesulfonic acid, 25 mM tris(hydroxymethyl)aminomethane, 25 mM ethanolamine, and 100 mM
NaCI, pH 7.0), and DME in a final volume of 2.0 ml. The reaction is started by the addition of 50 ~tM dihydrofolate (as substrate). The oxidation of NADPH to NADPf corresponds to the reduction of dihydrofolate in the reaction and is proportional to the amount of DHFR activity in the sample (Nakamura, T. and Iwakura, M. (1999) J. Biol. Chem. 274:19041-19047).
Aldolketo reductase activity of DME is measured using the decrease in absorbance at 340 inn as NADPH is consumed. A standard reaction mixture is 135 mM sodium phosphate buffer (pH 6.2-7.2 depending on enzyme), 0.2 mM NADPH, 0.3 M lithium sulfate, 0.5-2.5 mg enzyme and an appropriate level of substrate. The reaction is incubated at 30° C and the reaction is monitored continuously with a spectrophotometer. Enzyme activity is calculated as mol NADPH consumed / mg of enzyme.
Alcohol dehydrogenase activity of DME is measured using the increase in absorbance at 340 nxn as NAD~ is reduced to NADH. A standard reaction mixture is 50 mM sodium phosphate, pH 7.5, and 0.25 nnM EDTA. The reaction is incubated at 25°C and monitored using a spectrophotometer.

Enzyme activity is calculated as mol NADH produced l mg of enzyme.
Carboxylestexase activity of DME is determined using 4 methylumbelliferyl acetate as a substrate. The enzymatic reaction is initiated by adding approximately 10 ~,l of DME-containing sample to 1 ml of reaction buffer (90 mM KH2P04, 40 mM KCI, pH 7.3) with 0.5 mM
4-methylumbelliferyl acetate at 37°C. The production of 4-methylumbelliferone is monitored with a spectrophotometer (s3so = 12.2 mM-' cmi') for 1.5 min. Specific activity is expressed as micromoles of product formed per minute per milligram of protein and corresponds to the activity of DME in the sample (Evgenia, V. et al. (1997) J. Biol. Chem. 272:14769-14775).
In the alternative, the cocaine benzoyl ester hydrolase activity of DME is measured by incubating approximately 0.1 nil of DME and 3.3 mM cocaine in reaction buffer (50 mM NaH2P04, pH 7.4) with 1 mM benzamidine, 1 mM EDTA, and 1 mM dithiothreitol at 37°C. The reaction is incubated for 1 h in a total volume of 0.4 ml then terminated with an equal volume of 5%
trichloroacetic acid. 0.1 ml of the internal standard 3,4-dimethylbenzoic acid (10 ,uglml) is added.
Precipitated protein is separated by centrifugation at 12,000 x g for 10 min.
The supernatant is transferred to a clean tube and extracted twice with 0.4 ml of methylene chloride. The two extracts axe combined and dried under a stream of nitrogen. The residue is resuspended in 14% acetonitrile, 25O mM KH2PO4, pH 4.0, with 8 ~,1 of diethylamine per 100 ml and injected onto a C18 reverse-phase HPLC column for separation. The column eluate is monitored at 235 nm.
DME activity is quantified by comparing peak area ratios 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) 3. Biol. Chem. 272:14769-14'775).
In another alternative, DME carboxyl esterase activity against the water-soluble substrate para-nitrophenyl butyric acid is deteiwined by spectrophotometric methods well known to those skilled in the art. In this procedure, the 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
taurocholate. The assay is initiated by adding a freshly prepared para-nitrophenyl butyric acid solution (100 ~xg/ml in sodium acetate, pH 5.0). Carboxyl esterase activity is then monitoxed and compared with control autohydrolysis of the substrate using a spectrophotometer set at 405 nm (Wan, L. et al. (2000) J. Biol.
Chem. 275:10041-10046).
Sulfotransferase activity of DME is measured using the incozporation of 35S
from [35S]PAPS
into a model substrate such as phenol (Folds, A. and J. L. Meek (1973) Biochim. Biophys. Acta 327:365-374). An aliquot of enzyme is incubated at 37°C with 1 mL of 10 mM phosphate buffer, pH
6.4, 50 mM phenol, and 0.4-4.0 mM [35S1 adenosine 3'-phosphate 5'-phosphosulfate (DAPS). 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 protein and phosphate buffer. Then 0.2 mL of 0.1 M Ba(OH)2 is added, followed by 0.2 mL ZnS04. The supernatant is cleared by centrifugation, which removes proteins as well as unreacted [35S]PAPS. Radioactivity in the supernatant is measured by scintillation. The enzyme activity is determined from the number of moles of radioactivity in the reaction product.
Heparan sulfate 6-sulfotransferase activity of DME is measured in vitro by incubating a sample containing DME along with 2.5 ,umol imadazole HCl (pH 6.8), 3.75 ~,g of protamine chloride, 25 nmol (as hexosamine) of completely desulfated and N--resulfated heparin, and 50 pmol (about 5 x 105 cpm) of [3sS]PADS in a final reaction volume of 50 p,1 at 37°C for 20 min. The reaction is stopped by immexsing the reaction tubes in a boiling water bath for 1 min. 0.1 ~.mol (as glucuronic acid) of chondroitin sulfate A is added to the reaction mixture as a carrier.
35S-labeled polysaccharides are precipitated with 3 volumes of cold ethanol containing 1.3% potassium acetate and separated completely from unincorporated [35S]PADS and its degradation products by gel chromatography using desalting columns. One unit of enzyme activity is defined as the amount required to transfer 1 pmol of sulfate/min., determined by the amount of [35S]DAPS incorporated into the precipitated polysaccharides (Habuchi, H. et al. (1995) J. Biol. Chem.
270:4172-4179).
In the alternative, heparan sulfate 6-sulfotransferase activity of DME is measured by extraction and renaturation of enzyme from gels following separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Following separation, the gel is washed with buffer (0.05 M Tris-HCI, pH 8.0), cut into 3-5 mm segments and subjected to agitation at 4 °C with 100 ~ti of the same buffer containing 0.15 M NaCI for 48 h. The eluted enzyme is collected by centrifugation and assayed for the sulfotransferase activity as described above (Habuchi, H. et al.
(1995) J. Biol. Chem. 270:4172-4179).
In another alternative, DME sulfotransferase activity is determined by measuring the transfer of [35S]sulfate from [35S]PAPS to an iirunobilized peptide that represents the N-terminal 15 residues of the mature P-selectin glycoprotein ligand-1 polypeptide to which a C-terminal cysteine residue is added. The peptide spans three potential tyrosine sulfation sites. The peptide is linked via the cysteine residue to iodoacetamide-activated xesin at a density of 1.5-3.0 umol peptide/ml of resin.
The enzyme assay is performed by combining 10 ~,l of peptide-derivitized beads with 2-20 ~l of DME-containing sample in 40 mM Pipes (pH 6.8), 0.3 M NaCI, 20 mM MnCl2, 50 mM
NaF, l %
Triton X-100, and 1 mM 5' AMP in a final volume of 130 ~,1. The assay is initiated by addition of 0.5 ~tCi of [35S]PADS (1.7 ACM; 1 Ci = 37 GBq). After 30 min at 37°C, the xeaction beads are washed with 6 M guanidine at 65°C and the radioactivity incorporated into the beads is determined by liquid scintillation counting. Transfer of [35S]sulfate to the bead-associated peptide is measured to determine the DME activity in the sample. One unit of activity is defined as 1 pmol of product formed per min (Ouyang, Y. B. et al. (1998) Biochemistry 95:2896-2901).
In another alternative, DME sulfotransferase assays are performed using [35S]PADS as the sulfate donor in a final volume of 30 ,u1, containing 50 mM Hepes-NaOH (pH
7.0), 250 mM sucrose, 1 mM dithiothxeitol,14 p.M[35S]PADS (I5 Ci/mmol), and dopamine (25 ~C1V~, p.-nitrophenol (5 p,N.n, or other candidate substrates. Assay reactions are started by the addition of a purified DME enzyme preparation or a sample containing DME activity, allowed to proceed for 15 min at 37°C, and terminated by heating at 100°C fox 3 min. The precipitates formed are cleared by centrifugation. The supernatants are then subjected to the analysis of 35S-sulfated product by either thin-layer chromatography or a two-dimensional thin layer separation procedure.
Appropriate standards are run in parallel with the supernatants to allow the identification of the 35S-sulfated products and determine the enzyme specificity of the DME-containing samples based on relative rates of migration of reaction products (Sakakibara, Y. et al. (1998) J. Biol. Chem. 273:6242-6247).
Squalene epoxidase activity of DME is assayed in a mixture comprising purified DME (or a crude mixture comprising DME), 20 mM Tris-HCl (pH 7.5), 0.01 mM FAD, 0.2 unit of NADPH-cytochrome C (P-450) xeductase, 0.01 mM ['øC]squalene (dispersed with the aid of 20 p,1 of Tween 80), and 0.2% Triton X-100. 1 mM NADPH is added to initiate the reaction followed by incubation at 37 °C for 30 min. The nonsaponifiable lipids are analyzed by silica gel TLC developed with ethyl acetate/benzene (0.5:99.5, vlv). The reaction products are compared to those from a reaction mixture without DME. The presence of 2,3(S)-oxidosqualene is confirmed using appropriate lipid standards (Sakalcibara, J, et al. (1995) 270:17-20).
Epoxide hydrolase activity of DME is determined by following substrate depletion using gas chromatographic (GC) analysis of ethereal extracts or by following substrate depletion and diol production by GC analysis of reaction mixtures quenched in acetone. A sample containing DME or an epoxide hydrolase control sample is incubated in 10 mM Tris-HCl (pH 8.0), 1 mM
ethylenediaminetetraacetate (EDTA), and 5 mM epoxide substrate (e.g., ethylene oxide, styrene oxide, propylene oxide, isoprene monoxide, epichloxohydrin, epibromohydrin, epifiuorohydrin, glycidol, 1,2-epoxybutane, 1,2-epoxyhexane, or 1,2-epoxyoctane). A portion of the sample is withdrawn from the reaction mixture at various time points, and added to I ml of ice-cold acetone containing an internal standard for GC analysis (e.gs, 1-nonanol). Protein and salts are removed by centrifugation (15 min, 4000 x g) and the extract is analyzed by GC using a 0.2 mm x 25-m CP-Wax57-CB column (CHROMPACK, Middelburg, The Netherlands) and a flame-ionization detector. The identification of GC products is performed using appropriate standards and controls well known to those skilled in the art. 1 unit of DME activity is defined as the amount of enzyme that catalyzes the production of 1 p,mol of diol/min (Rink, R. et al. (1997) J.
Biol. Chem.
272:14650-14657).
Aminotransferase activity of DME is assayed by incubating samples containing DME for I
hour at 37°C in the presence of 1 mM L-kynurenine and I mM 2-oxoglutarate in a final volume of 200 ~,1 of 150 mM Tris acetate buffer (pH 8.0) containing 70 ACM PLP. The formation of kynurenic acid is quantified by HPLC with spectrophotometric detection at 330 nm using the appropriate standards and controls well known to those skilled in the art. In the alternative, L-3-hydroxykynurenine is used as substrate and the production of xanthurenic acid is determined by HPLC analysis of the pxoducts with W detection at 340 nm. The production of hynurenic acid and xanthurenic acid, respectively, is indicative of aminotransferase activity (Buchli, R. et al. (1995) J.
Biol. Chem. 270:29330-29335).
In another alteniative, aminotransferase activity of DME is measured by determining the activity of purified DME or crude samples containing DME toward various amino and oxo acid substrates under single turnover conditions by monitoring the changes in the 1:JV/VIS.absorption ' spectrum of the enzyme-bound cofactor, pyridoxal 5'-phosphate (PLP). The reactions are performed at 25°C in 50 mM 4-methylmorpholine (pH 7.5) containing 9 ~,M purified DME or DME containing samples and substrate to be tested (amino and oxo acid substrates). The half reaction from amino acid to oxo acid is followed by measuring the decrease in absorbance at 360 nm and the increase in absorbance at 330 nm due to the conversion of enzyme-bound PLP to pyridoxamine 5' phosphate (PMP). The specificity and relative activity of DME is determined by the activity of the enzyme preparation against specific substrates (Vacca, R.A. et al. (1997) J. Biol.
Chem. 272:21932-21937).
Superoxide dismutase activity of DME is assayed from cell pellets, culture supernatants, or purified protein preparations. Samples or lysates are resolved by electrophoresis on 15%
non-denaturing polyacrylamide gels. The gels are incubated for 30 min in 2.S
mM vitro blue tetrazolium, followed by incubation for 20 min in 30 mM potassium phosphate, 30 mM TEMED, and 301tM riboflavin (pH 7.8). Superoxide dismutase activity is visualized as white bands against a blue background, following illumination of the gels on a lightbox. Quantitation of superoxide dismutase activity is performed by densitometric scanning of the activity gels using the appropriate superoxide dismutase positive and negative controls (e.g., various amounts of commercially available E, coli superoxide dismutase (Harth, G. and Horwitz, M. A. (1999) J. Biol. Chem.
274:4281-4292).
XVIII. Identification of DME Inhibitors Compounds to be tested are arrayed in the wells of a mufti-well plate in varying concentrations along with an appropriate buffer and substrate, as described in the assays in Example . XVII. DME activity is measured for each well and the ability of each compound to inlubit DME
activity can be determined, as well as the dose-response profiles. This assay could also be used to identify molecules which enhance DME activity.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the l08 invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the S scope of the following claims.

A

H

N

H H

O v-1t-ir-1W r-1r-1v-1I~r-1a-i~-3~-tc-t~-1i-1 v-1 ~' U W t~1alU~-1a1W WUW alfYlP~lalalPGr-Ipd U

~ U UU ~OPaUU U~U UU UU UU P4U

U N Lf701r-tCJL~01t0~-1l0v-ItW-ir1OO CJIt1 H

~ 01d~L~MOJNN 'd~OM NM d~Li101L Nl~

L~l0~ ON NLnMl~c0tN NN o0d~t~d~

N MO OL~NM c-1t~N 1.nN d~d~Md~c-Id~
r-1C~L~d~l0ODL~00Nc-)L 1.nL~~c0L~CDa000 O ~Nd~M c-3LDl0d~u1r1djd~d~d~d~d~d~r1d~

~ l~L~M L~O Lt1L~tnL~L~L~L~L~L~1~L Mt N

b ., p ..

rS
z U

q ~
Cu O dlOc-1NM 'cHInl0~CO01O ~-iN McNLf11~
W

W ~-INN NN NN NNN NM MM MM MM
CQ

q H

N N

T'~'~N ~-1 'Lf .N c-1r-1r-1A c-Iw)v-I(~r1v-1.-1c-1v-1i-1r1 .--I
~rlf~G7faU~-IC~faAUfaAG-lA~l~lfa~-iA
~r.u U U UU ~OCaUU U~U UU UU UU f~U
(1, U N u101~-1U L~oWor1~oc-I~ .-I~-1OO Utn H O1~HL M00NN d~OM NM d~f.n01L NL~

L l0l~OOJNl(1Ml~07l~a0NN DOd~L~~H

.-1N MCOOOL NM r1L~N ~N d~d~Md~cidi O L~WH lOa0L~00Nr1L~L(1L~0000L~O c0c0 W ~Hd~M c-IlDlfld~LW-1d~'~Hd~d~cNd~CHv-1di L~L~M l~CO~clC~LtdL~C~L~l~L~L~L~N ML~

'~

wz ~H
.~~~y Oi O
W

W

O c-1N Md~I,nlOL'~~

r1NM d~iltl0L~00O1c-)r-1r-1c-1v-1c-Ii-Wv-i W

H

O

~ N Ina1c-1 l~41l0v-1l0c-IL~c--fc-IOO N
U

U d1diL~MO NN ~HOM NM ~N1.t1O1~ NL~
O

~n L~l0l~oO N111ML~OJL~00NN 00d~l~'cN

"~ N Mo0WL'~NM ~-it~N ~N d~diMd~c-id~
O

L~L~d~l0O L~00Nc-IL~Lf1L~a0O L~ODO00 W N d~M c-Il0l0diLnr1'cH'd~d~'d~d~d~'cNr1~H

L~LM L~COt11L~LnL~t~L~L~L~L~l~L~Ml~

~ ' a~ a~ cu I

I o ~ ~ '~
'~ o o ~
u . . ~. ~, .
. u u u I ~-1 ~-I .I-W -1 t51 ''Cf~:,' -rl U7 u t17 I

~ a~ o r1 a~ ~I s14-1 ~o a~
-- rt -o co o ~ .U -. ~ rd ~ Z N at o U o1 Zi -.-1 ~ -~I r-i N N ~,'' cd O
t ~ o, u~ - ~ I o I a, N -W-1 i N U d z i r-I ~-1 U U 01 ~ r ~-I.-I ~I N Zs ~ '' ~ ~ o r U1 I~ ~
z ;
'' >.., , -.-1 u~ ~ U N I ,-I
'at v G' (l3 c-t N ri N N '-t ~-, b7 -.-i -.-I
N O ~ ~., ~' ~ U1 of ,'~ W pa O
.!-1 W
r-I

U N ~,' ,-t f-I v U1 (I$ 4-1 -rl N ~-. -rI N
-.-I N ,.C,' ,"~ tii ~, td O -5 '' '' ., O N Y~ U1 F.., 1~i111 r-I ~-a- U1 i c-t lV N N
, tn W ~
~ D ~ cti U

S 1-s ~ d ~ ttf 1 I a1 N N -- U to ~ N
.-t r-1 vo --1 N -ri O ~I ~ rn o~ ~tf -~I ~ P~ N o~ .t~
O O 2i f.~ rn r1 o ~-I ~i N N
l-I ~i UI ,.Q ,.~', .-, ~ ~ 01 U !-1 b1 O tW-I rtS ~-I
O -.-I ~ ~ o H N !_,'u1 O
~ u1 .>_,'' ~ U1 U of r1 .h O o t~ ~
~' 1~7 U rtf N .!J O N .
N (d N ~ U7 O --, b U ' c6 v ~-i ,.Q
- ~ v '' ~ cIi .1-~
. U U
l I

. ( l O .1-> O ~I -.-I .~I
~ U r- d N t O O
r U U7 -rl 37 U ~-l ~: FC .f-I (U -rl 4-1 4-1 ~1 -. N ctS
O .1.i iw ~I u7 U~ v u7 O U N ~; -.-I
o~ eo ctS O
~

(tS .J-~ O 1I~ R~ r1 U7 r1 cfj ~ Ul 4-I r1 ~-I r1 cCj t57 .1-1 ~-i ~., ~I '~ 4-1 O I~
t'7 I~ N N O

r1 'ZS U 4-I ~1 ~,' U N r1 ~ p'., ~''.,, N fn t11 O 4-t tn cti 't~~ UW d 4-i I O ~,' 'i~ u1 i~

~,-' 4-I I ~ N (fS Ul (fi aW -i c~ U Z.,'' O
'L3 r.~ I 4S N N O N ,s.,' t'-, S=,' ~ tI1 S-1 N

b1 ~ (CS O t-I Id Ul U1 ((j nt U7 .~i-.-I (~ .!-1 N S~-, O N ',~ r1 U ,.C~' r1 N .~I
111 Zf Ul (J ~-, lD

O td Ul ,Sy U1 ((S -rl -rl f.1 (Lt U .-.~1 ,~-1 N
tI$ U U1 L~ It3 .7~ U N ~', I ~I
N H O U

r1 l1$ ~' U ILS U1 ,~-I .h N N Ul P: tdiN .l~ O r-1 O I If3 N ''(j '~ H !-1 ~-1 Zj d~ ~ ~
~-I ~ ~t -I 4 FI,' '' 0 (IS ~i (IS ~ UJ --I ~ ~ N H ' 1 "
' ' .-.
, : r G C, r-1 -rI rtf r ~ . -1 >_, ,T N r-1 - M "Li m , N N ~ ~
u1 N -~I O .I-~ d ~, ~ ~
r1 ~, N N .t~ N P; 4-I ~ ?
.1.-~ ~1 >~ 1 O A u7 f ., O ~ 4-t Ca .1.W N U1 ,~ 'y.,' -.-1 -r-I..
-1 4-I f-I ri U7 f1,' N U ciS -.-I., ao U 4-t r-1 tf~ N -I .
~. i O U7 tit -rl u2 .1--1 x S-I a1 cri U N cd cti ~, s~ f~ ~ O ~'., N
~, u~ ''C3 cd N ~ ~ ao U ~ .1~ rti.R O -r1 tti m d~

>=i ~' .!.-t -ri r1 .C,' ~I ciS .!..JQi F'r Fi f~' H .~., O O M rtS ,i;~,' ~t ~.I
~-1 cd f-i U U1 O I

t~ cd ~r ~ ~I O ~I .1J m ~ H u1 U1 O ttf O
~S-''N cLS O r-I ~ ~-I ~' ~ ~1 N U -~I r1 o ctt r-I U U7 U1 I l~ N ' IJ r-1 N O O 1 ' ~
U ~ -I O t '' t ~'' O
~I O

., . r .~, .I- I , t0 ~ O O N 31 rl ~ S-I f, - O O N U ~ ,it 3-1 4.1 ~ , U ~ -1 O O to .1-i U1 (p ' ' -1 i "
.t~ U U7 - O i !~ U 1 ~ l ~
M ' . , r O .!-al U1 r1 tn N U1 .~ Jy r .h 3 -rt ,i ,-I r1 O r1 >_, r -r3 r .~.,~
~-i -r! ~-I ., ~, N rC3 ~1 N N O ,.
R -a-i N .t-i 4-i H , d~
r1 .U ~i cti .1-i U N f~'' O U U O ..

O 5y N .I~ O ~1 td N -rl O ~-i O tIi ~ ~1 ~Ci N ~ ~., -h cd O U >~ ~-I O ri O N ~ N U N
~., 5C ~.1 a0 O U ' Y
fYl U 'a N ?5 1= ' ' ' U -rl ~I U ' (If ' l0 r ' O tI1 U U S O ~ ~
-rl O Cr c~ O t ~

, , ,. , ( C7 '-' (LS fa , ,. , ,i ., r-I C7 CIi (I$ jr., " U '~' '- dj ~- , ~
(tf ITj H ~i U '~ U a , , ,L; ,-t H U M O 2 "-i N
- ~-'',S ~ ~ b1 ~ 1~
U1 U1 u1 S2i t'~ U7 U r1 trj M

,N

N -~

r1 l0 N L~ c-1 c-Id~

M N

I-I O O O O O O

U H M <-I N M t1~

W Lr d~ ~ O~ IS1N
W

LC7 l0 OJ L~
cN OJ
'' , t L~ N Ln , cf' 01 L~ a0 -i a ~', ~ 1 of cy -I 11~ L~ N M
O

al o d~ N -I N o0 '~, o ~o y -1 ~o q CJ b1 as C3~ b~ b1 ~

N

A A A

~ A

A

N ~ ~ oy -I U

N

~ l~ t0 L~ O N N

~ N L~ N
U c~ ~ dW o r-I

CO L
H ~ '~ M r-I ~o to W
~

H l~ M ( pp r~
.

r1 O

l~
"~

!~

N

~H

O
W

W e'1 N M ~ Ll1lp Lfj ~

O cd U1 U oo ~ ,.-i 0 ~

1 O In (~ U1 r-i >~
f-t 'Z5 ~ u7 Id N ~ r6 ~ N >~' ~,-'N Fq O I

'Cj r1 (C1 ,~i b1 N 1-I Ul -r-1.ii NJ 4-I ct$
F; O

-ri U ~ U ~i .L.1 N U1 ~''H O
'R N l~

'~S '~1 N N -rl O (C3 4-1 ~ -rlN N -r-I
N ~, );'', ~', t7 ~ j," tA.-1 0 ~ m .N x ~ ?-I 4-m1 ~ rd ~ u1 .-, m -r-I Sa ~ cti ~ aS .I-~
tn G~ '~ ~If 0 I .1.~ r-1 ~; a1 .1-)(d ri ctf .~'' O (l2 f-1 S-I ."j ~ O !->

N -~ r1 ~ ~ U Al ,'~ (i3 01 ~' ~'' 01 ~,' O U 1-I N IfS .L.) fit d~ -rl U1 U
d!

Ir7 ~ O ~H N 4-I Uf S~ c-4 O .
O M ~ Q~ O .1.~ N a1 N -ri Fc,' ~,' ~,' t~

N o~ 4-I o0 N D J-> ~- N ~ I W-I I t3~
-.-I -1~ 'Lf U 4-i rt3 U ~-I O
I '~-~ -ri N

f~'' O 01 U1 -r3 S-n-' O .f''-,I td O ~ (IS
O1 ~-I I=,' N 137 ri N ~1 O .U r1 P~l .4~
Ln N ~-I ~-I-I td U7 di 4-I ~ F; .!-I ~-1 O ~H C7 of U -.-I U u1 .i-7 ~1 U1 -1..1 a0 r-i v ~ ~ .I-.7 ~i ~I r1 r1 r1 (IJ 'ZS
~f-1 ~i f=i ~I-1 Ul ~ 'Z$ -r-I
M ,~-I -r1 (a LCI
.~, .. ~ U 0 N N N t>3 ~ cd p ,-i~ ~, r-I
ccf O 1 N F; .R 5r''d ''C3 (r) N ~o d, N ,.c; ~ .t~ ~, m O .!~ ai I ,C, cii o .~' U YA-rl rti .~' ,.c'., -ri t ,-~ to 4 ' ' ' o -a-i r-I N 1 .U u1 r-1.-i N r-1 r- Zf I U ~ 4-1 N U3 r-1 ~ u1 ~C .L.1 ~, U ~-I
y-I ,-1 c~

U7 1IS 'i~ td N W ,.C," O 0 4-1e-I 'L1 J.~
La td N N !~ 00 M (CS O .-t ''Z~
N ~ td 1 '' -I

U ~ ~ ~ U
~ ~ ~ x ~
~ Ii ~ ~ ~ ~ M ~ .~ ~[f O r-I (tS 6-1 'Zj UI
i r >'', r1 O Lr7 l r1 Gl N Ul N N N 'L~ U1 5~ 4-1 Ul U7 r-1 O
1'0 (I3 01 ',~y,t,' ~ r1 -rl ~
,u, 4-i O O to CCI

O ~ O .h >:.,-' .1~ Fn-! O J~' ~ f..,-' O
F'~. !a rf~ U O -.-I UJ ~,-' 4-l ~ to -.-1 O .f~ to i-1 4-I N ctS O O 0 N ,5y Z 01 r-IN ~I 1-I
7 U .-I -r1 .i.1 ctSr1 c-1 (Y1 O '~' r''h--i n'-l, r-I .,..I ~ .,..I,'~-.-I N CJ
O N r1 -rl ~1 ~'' cIS ~,'N ~ ~ tCf ~(,' N fn .. ~ ..

x ti !z, a~ o .u ~,-~I U ~,.u ~ .u .u ~ .~I yo s~ O U N a~ yo U7 N c~f rR h 4-I cB ',~,' rn cCf u7 N
x 0 cti ~S -.-I N u~ O -ri b7 u~ !-I O
m U1 O ~ -rl I-7 ~o ~ t ~ ~
~

U s~ m rd o N z us ~ ~n F. a zs ~-I ~ ~-I o -~1 ~ 07 ~ u~ N
b1 .~', .h S-1 -rl 1n a-I ~ ~' N ~ O F: O > N - l ~ -I ~' U7 ~1 f~
U
!

, . , , -r td ,'~ N O U ~'~ ~; . O , r-~ N U O a-1 - .1-7 O
,.C', O O O -r-I O O f.a-' "~' td 0 O U ~
!-7 , al U1 tIJ ~, ~ Fi ~ (a .)'., U $ U1 ~, ~ liS
iLS u1 I ?-I O ~ r1 r-1 O ?-I ~ O
S~ r1 S-I >-.,' '~ ~., O ~ O ''d rtS ,~' -.-) ~' O ~ (IS
O ~ f.~'' ~i c,' r1 1'37 'z3 ~1 S
.U ~, O O 1 N
O ' f' N U

v ~ x a~ ~ -~1 , , ~
~ x a~ ~I o o t~ ~ ~ --~I o -~I ~ o x ~ E-W, x ~-mn ~ .N .~
1 ~n zi ~
o U' ~-' ~-' 'J S-1 " TCf '--' ~7 ~ ~ .
n U1 u1 O U ~'., -r~ ,~i Td H 1',~ ,C', v Pa ,i,'' W N -.-1 r-1 ..C~' ,i.~'' UJ U U

O

r~ N L' ~-1 1.C1 N o0 ~ Ltd N OD O I~

W N c-1 CH ~-It--t x '-~y-I r1 M o1 01 p O ~ ~ t31 C51137 N

A A ~

U A

U

N N d~ O
N N

~ ~ M L~ ~

~r ~ N 117 U 00 N c-1 [~ 1 ~
r1 H ~ ~
W
~

H t r7 N

..

-~I
O

A

H

OI

O ~-1 W t~ o '~ ~ q ~
M ~ ~ ~

O F r O O r-I
1 j=,' r 'n I N 1 1 U Tl7 )_, N
1 ' l9 ~ ~ 7. ~
~ H ~
~

r-I 1. -r ~ w-I -Li O , -1 I
~

W
~ ~

O W d >3~ rt -.-! cIS N -~ N U In 1 N O U 6-1 , N
-~

m .4 -ri t U ,.~ ,~ ~ ~ ~ ~ r-I N ~ t<1 b7-rl oo ~ ~i ~ o al ~ -r1 U

p ''O .~ ~ W ~-I r .
~,' ca ~.1 ~, N , ~
~ rd b1 -l N

O .I-) -rl ~ M ~-I rtf ~ ~ ~ r L N
O 1-I ,.Q O I

b7 .I-~ N a1 ~1 -.-I o~ aS N .-I .1-Io O
N ~' -r1 ~I T7~ ~L U1 17 .!-~

N r1 N O ~ N o1 4-I N O r1 I N ~,' U -rl ,ice, O U ''CS .~i -r1 ' td ~ ~., UI ~
' tn u~ m v-I O u1 ~I v , f~1.s~
2i .~ ~ .z, 4-I ~ ?~ >_t,-~-I, ~I
N ~ N ctS
~-I W

O U ~ (ij '-' fIt'L~ r1 U1 ~,-' '' .
Ul 'T3 f,' U7 (iS N .!-~
,.C,' r-1 O W tll r-1 f-I ~' ,1.I'Jy ~'., N 4-I(iS
b) ai tn Sue'' '>..,' ca N ',2,' ~ I U r-t ",.~' !~ b1 W ~ ~ ~ O ctt 1 ,-W-1 rU -ri tn .u .t~ a1 -.-1 N N ~-1 td ~-I N
,.~

U O ~ >-: U 4-I r-1 W I N cd ~-I Ci ~
-ri ~-I -.-1 S-I 4-t-I-~ U ai n >~' -.-1 eo -r1 ~, r1 -r1 Ul ffS U1 L!7 Ul ~ I -rI UI -I
-r1 Fi Gi .1-1 .1J ~ M r-l N cW (~S
O C~ l0 '~ N r) '' ~ ' ' ~' O U -rl C
-ri 4-1 l Q a1 ~
' O
cd ,. r ~ N (d d <N .~., ~-1b).!~
>s ~ N -n ~ ,. ., .U ~1 07 4-1 G
~ cd I , ~ -i r-1 f ~ ~
1~ N ~
df !~ -l . 1> r O ~ ~, N N
O U , N U~ O 1 .1.l ' . ' ''d r ' ~

.! U ~-1 N -rl r-,,--, r-I N ",-4'-ri,i", ~ r!1 (Y1 ~ ~, ,i; U I
, ~
~, ~
d N ~-1 r-I
' ' -I -r1 A l~ I~-I O U1 U7 cd I tCi ~ W
, O ~ r1 4-I .!-~-rt f~ Ra N il~.U
, U ~ o ~

O r~ ~-I I m ~-I N s~ ~ ~ O ~ .~ rti rtf -~I O ~ W ai O ~-I~ M H ~
o ~ rti O N ~ 5., :~ J.~ N N S-1 J-I ~- UI N
O tn U O -.-I O .h E7 ~y di ~,' Zi O , N N
a~ '' oo ' 4 ' U7 I '~
b ' ~., ~., -r1-.-I U7 ~ N ~' r1 7 U1 U ~i ~, U7 '~ .!-f I~, U7 U7 - r1 r-1 l ' x cd ctf ~ c0 O R; cd Ra ~ ~y ~ N 43 O ~
N ~, O O O O rtf ,'~ N O di -.-I ~, eo r1 ~
' I

.-I r1 U! r-i ~,-' ~-1 l(itd .~i ~-1 ,.ti' .~,(If '' ~-1 -.-1 U ~., -ri (-icIi U1 O ~ -.-I~-I
a r-I to t~ U7 ~
' ' ~
~
' . O O O (6 U7 u1 I r1 ~ cd O tQ
>_.,'',?~ .~ ,01 Ul O ~ .1-1 ~I ",~ .'1~'O
, ~ M ~ N 00 ' a N -ri N - ~ ' .U t l 4 1 >~ 1 1-1 5 ~ rtS ~ W-1 O U1 --I

, . ~y r -rl ~-I ~, 4-I
t13 . . O c0 ~ N r1 J-1O
U .1~ (d . ,~ O ,t; N ~ IA
ri N ri - UJ O U O N ~ !(S
N ~ 'a c-I i,'' r1 i . I
U ,.~5 N U
l;71 r-1 N P4 ~

~ ~ ~ ~ 'RS O O ~ ., , O
.I-1 O 'T3 W 3 ' p -r1 .t-~
~ ~-I 5 ~ H N ' '' Q N

,. , ~ Id U N r1 . ~ Lt C~ O ~
., Z Pa ~
~

~ ~E ~~ a x x~H~ = W~ x o ~ o~ ~~ ~ ~c~.~ ~-~~ om~r ~
-I m~

, .
u V -~I l .

r c o w w w w w o N M ~ o M

W r-i N M ~-I d~ l~ h ill H

x ~ ~ r P4 <N L ~ ~ L~ M L~
~Z

, n Ol M
, M lf1 O M 01 ~
U

7 ~ b1 b~
H
C

U

-1 r-1 .--1 A

A q A A

N M ~ Ln ~ U U
N

N r , cH M d~ ,-I
U L~ ~ '-I
r1 ~

H L~ L~ L~ L~ L~ M M
RI
H

~j ..

-r-1 O

RI

N
q ~H

H
CX

W c-I M-1 >n v ~ -I ~-1 .-I ~-1r1 u ~
o .
i ~ ~

~r U

J~ N O

r-1 N-1 Ul -R O O
rtS

~

O ~ N

~-I N -r-1 O , c;

'ti o~
.N ~I
U

~
~ ~

~
r1 M ~-1 N
O

I (U zi -~i O
H
~

d c fx t d O

f71 .1-~ (d RS ~ M

O N ,-~ ~-1 l0 U

O ~,' U1 .
.N . in ~, N (fS P
i T~ '.3' .1-W -I

~

x PiNa', c~
cap ~ O
~

~i Ln U1 ~i ~i e~Y
~, ~'I
~

x ~

CD Zi ~1 U M ,r'., N

r1 O

y N N

-I ~I di r U
W
N

X N

ctS v-I
O

N ~-1 A

U' b1 H

N

'~ c-1 -r-1A

.t-'U

d5 t~
N

r1 .1-',di O
(~

H t~
Pa H

N

r1 O

N
Q

~H
r~iy O<

O co W

Pa ~-I
Ul H ~ H

O (a O H ~ O H

, f~ O ~1 tjt O ~ O a fx ~ U ~ O f~~ ~' p cti tn U Gx O l~ f.~ W f~, Cry O I~ W f .
N ~ ., O

-r-I W ~1 W I I at W Q W I . (a W

_1-) I 1 I U1 if~ I tl~ a 1 I CIAI 1 I
''CS ~
fd ~ H H H w w ax rz, H H H w IxE-~ H
O
,ct ~-I Lla tJ~U1 ~., z~', H f=1UU1 U~ ~",W U1 v7 ,.C', ~Lf ct3 a,' a',Ft', H H H O ~~' a',H a', ry' .h a a a a a o x wa a ~
.1-~
a~
~s r a a a a~ a~ w m a~ a~ ~ w cnw vo m x cn oa ~
A

I .
N 01 I 1 op r-I I 1 r~ O

O M 00 dt O d~ h 41 O
I N

~ d W N r-I ~ W W fxO Z
oo n ~ ~-I

H o1 (~ c-t N I U7 Ul W
N O L~ O

I ~ I ~., ~ ~, . ~ . ~ ~ A
x -1 O I~ O d~ ~ x ~? N o ~

, t l UI N r1r-1~, H
, W ~ f~ v-1 L(7 ,h~ W c-IO H W
N N c0 H f~
ri O
W

W~~ - z N ~ ~a AIM x~r r~ ~,~.I rxa O ~1 W~r I t~ 1 ~N C7 N
I 'Z L!7 'z ' >

, L , ~-iM~m W~ .7-~~~I 1~,'t ~~ O ~yH H',T'~U' 'Z,HUo v-I,. O O O1 Id f~ v-I0.i,tia1 Ul U H N 01 O - L~ O
1 W cH '~-~

-<s7 0 ~ a~~ ~ ea w N ~~ ~ ~ ~ I UH
~7~0 CH-mr N b 1- ~ O Pa Pa tiiC ~ o H ~-~ W
t11 '',~, ~ ~ 7 rI 01 . 01 wN ua C7 N t 1 ''~, U7 tn.H ~, U U1 10 I U7 M H ~, fa H >'o o H H
~ lfl I
'-$

W c-i N N W W 'Z$'~N ~
W P~1I M N ~ ~ r1 O l0 C/~
~ ~ l1 l N
~ ~ ' H

t- C 7 .t N W U1 U7 r1r1~ W
~ Pa c-I ~ f 01 ~-i N H
N 1 ~1 f~,H~a I UO~lao I ~1 Tn fi,w I 0~- y-IO~O -~IC75 wOH
~ W~ 4-l ~

' O ~ 0 O InN N 4-t4-IUl U1 W 1~1 1 ~ lx H O
H

H W P, ~o . p i aW~-1 Wo to OH wp;
~ ~ w . c>3 U .-I ~ IH f~ N N H
H ~ ~ N M fa Ln ~ W
W o d~
~ o ~

u1 p N o .u p -- I .r~~-Iv~ u~ ' cnN v~
N U W N b I ,~ O W u~ O H
u1 H W N ~-i W ~ W
C

F G4 M ~ 0.1 I ~W ~ ~fc>iZi H
U a Cl~~ U1 r1 m ~.1 Ul o u1r1H rn 4-1 a U1 Ch I N Z rI m O
H I ~i (!~
m P4 t ~rl ~I W r.~0.,'Wa N tn v N N H(~ ~, O O O C~O~, ~ ~ N N - M
~~ I

O ~ ~ a W uW om u~ m NW H ymn~ R, ~
O twr~ rn o ~n aS H O q ~ I ,-I ~ W
~ ~

,~i 00 (Y., .~I NI!7~-1~-1 ~-4f~SQi ~ '?I5 iR Cl1 d~ ~-1 ~-1 N W H c-! .1.1 H
M M

N ~ ~W Z', N c~f~N N . PH H , , O ~
~ ~-1W W N N U1 O .~y rtSO
.-1 a .1-~ ~ .c,' N

to W W NH ~ ~ ~II.h .t~ .t~~dr-C W , , ~ W
zf W ~H H I .N u;! P4 I 4-I
~ H O
W

u~C=a r~t!~x u7 Oou~ u1 u1 N U cdc1,.p, cry f~ W W to m N Pa N ~-1~
H U1 . Ln W

rti U7 IW H Lr7N .1-I~N N N 1-IW r~ .L~.1.~I u1 U1 -I1 W o ao m~M N .-I t cft ~
U a w Lr ~ - W
~ c0 o --I
a r a , r1 dr r-Ir-IUl~ N N O m H
v~ w ~~~ ~nm 1 ~ a~xi -1 ~' C7 ~I H oo MO~lW
a ~ o~ 1 N ~' ~co ~ ,~a n ~~ WN ~n ~' ,.~M waw~
~' ~' ~' .u r.~ ~ca,C,-sr~, ~ ~ ~ e o wax a ~ w M~-.~~o wo ~nN
>~ t7 o w I
~ tn I
H ~

~ H . 0 o -~I.o o 0 cdo a rtiasU W Ox !a3 ~1 a1 o ~ ,-10~I 0 ,R ~'f.~~ <-I. ~ R, ~ ~
W l~ R Q ~ f~' (' ~ ' o U cN L C! ~1 H ' I
o a1 n r o~ UI
00 u!
o v ~ ~ ' . . ,. -. b1A ~ . . .. ~
O ~ ~ V ~ , , , ~ H ~ ~

C? ~-IN Ul (~S ,C,N(IIH of , N c-1 O1 w a c-1 C (If l ~, Cl~
Gl N (L3 .Li m N ~i t4 H~C~~H UM WU' UWCh U~ U~ UU U~-rx~la t~U~tr~cdfx W H~W~
la UWC7 p,f~

-_.

.,1 -i m ,-I
cd fLl Ln u) r-1 N

O ' .h ~
'~y l0 .1.3 W

>~

O In l0 OJ

r1 O M O 00 ~

~j H t~ . . ~
. N
.
.
.
, r1 N O L~
O L~ H
Lf1 l0 N

r1 .N1.OO1~N M M01 M

f~ 'd~ M N N L~
N c-1N t~
M
M
<N

uW H H
n H
u1 v1 H

H ~

N H
~
N

. vo . o o co ~-1 W .-W
r-t o~
m o~

J.~ lD O1 01 !f7 l0 O M d~ N 01 d~
.1, Il1 L~
M
l0 N

O v-I d~ r1 v-1 ~-I
.4'' c-I ~-I c-IN u-I
r! N
M
d~
~
LO

Wwul rnvl cnuaHtr~u2Hr~ HHu~H v~H

m N

O

s~
2i ~1 r1 l0 o N
r1 N

d~ 01 ~ N t0 M N
P; ' N ri A A a r N Lf1 01 c-1 N a1 d~ t N ' O

''., d~ d~ M ~.-I
O
(~

H t L M
~
H

W
f-a O

U1 r1 N M
H
''~_', N

~ x t~ ~, m ~ F~ f~,'~ z O ~ U O
~n U W f=.iO W W f~ O f~ W U1 O
N

-rl 1 W t=1I 1 W f~ W I W W
u1 W
u1 .l-1 U1 I I td1ClZ1 I I Cl~ I U1 a I
'Z3 tCf ~ w W H ~ ~ ~ H ~ w P;G4 P', G4 H ~ H
~
~

r ~ t ,'~,~ C u H WW w H O TUl7 d, l ', ~ l7 ' ", II 1 . H ~t,'H H FC FC
td a a a a a a J-~
.>, a~
ru a o rx n. a ~za m r~ m w s w ~~ ~

a an ~ w m w M ,~ I
~

z~ ~~n I o ~ H ' ~ w ~ M.. ~ ~ w -I n w a H o'~

r~ M~ ~~ M N wN
~

r~~, p .u .. oorxN
I ~ z N

N Infsl O f-IN ~' N v-I(d 00 -. W
r-I r12 ' W

CxaOJ O O lD W W ~i~ N - I
1.C1 IY., O
M

in N W ,5yh,'r4 ~-l "~ Ll~ O r1 ~ r-1 d~
N d~ U I17 4-I
'J

CO ';l,U1 ,-1~ U! ,h.f W t~ -'Tf'ar .1-1'Jy ~ Cit 1 U . ",.~ ~I
,'~'~

~ I r.~-.-1 l U7 u2 M I at t~ U1 OI ~-t ~o m W M 1 p, a ~

o M z ~ . ,~ p (x o N~ O , O in-d~ N t~ (If. ''[j~ C9 co ' d~ .
O W 00 N to N 00 O ~

U ., O ~N U 'C7U O1 ' w-1 W N N .I-1 N

f~ ~ C7 4-!U2(d l~ W a -r1~, ~ ",O~ w-1 W N N ~H ~ H U CJ t3t M

w O N ~i-- a r.~ w .uu~ ,-I -- ,-I ~n O
I x ~-tN u~ W I f~, ~

o~ - ~ N s~ >;7 try ~ R~s~ b~ b~'z3 z N
I U ~ In u1 I
I

.. U1A ~ N N I W m Ncd t U1 I H m p 1=llD I 1 UI e0 d~ ., ~
Gi '~ ~ t~ W ~
N M L~ M N

, ~ N U1 $-tW N W W W
-~ -NW ~ f-IFi ~ W N ~l~ C7 ~ ~
~ ~ r1 N G U O ~ ~
'~

t L N v-I O 01 i~ ~

-uA wx ~ww - a~n .. rd ~s ~I ~I U xo, o o - u~ 5.,~, ~ ~, o . ~
rn o, rzi ~ s . --a d ~ c a~ zs I mn m ~t S-1a rt .~o Lo u~ o, , ~-~I~ 4.1~ ~-1 o~ ,n .. ~ ~ N >~
' ~-.I .u oo N RW-I~ O ~ N ~-IM O fl 4-I r1(ISf~S f1!Y~ '~, ~., O
U1 ' N , CO' ~ N ~,' f[( ' O ' d~
' U , , N ' ' O U o Cl1 Ul~.,-r1 ~i , H ,'~' 4-1 l.,N I, 0~ [Si~ a H ~', -- ~
~, ~ I tT N ~ o ~-~ "TI
-rl I o o -I a '' ~-I a ~ , , ~ o .-I ~ ~-1 A
N ~ 1 I c~
J ~ 0 1 ~

- n m U o v-t,~ ~ a rc3 'zi5r ~1 5r-~i d o U ~ ~ .-I ~
~ o o o m.nrt~o ~.,o v vi r;~ a o <n .h m t~, o .
~ ? ~ ~ o I ' A ~n a, o u~
' I N '~ 1 j w In ' -I ~ ~ as ' !

, . -1 f l Z O O .4W 'LfN O O ~d a W
, w , - ~ ,~i -1 ' I
~ . W <H d ' N N N M ,5y-r1 ,. ~-t ,-1 ri>=iS..i r! ~i ,'~ Ul r~ f 4-Iz W v-1t~ T~ Cf~ P1 hs U7 zf i M .~ ~ Cl~ r~ 'J"r 4-I .-I o 0 5.
w , , , .~ o ~, o c~ c~ r~
~,'' U1 U1H N -rl-.-1p'., C!a U1 ,~ a~ U1 a~ N ~
o x t11 ~ . ~ S-1 ' z ~ ~ U1 tn N >w >~OI zf S-Irt O ~ O Nm O O O ,-I , ,~irtf ~H rtf N w ,.t~~ ~ m ,r-I10 U rtf U H W
~I cd ;-I U t~ in ~ U U
d~ U U ~I ~I
~Ei ~o M o~

-P~
t>) ~I ~IU r-IN U ',~ w ',~., ~n ~ ~ ~
.-1 d' ~ a1 W ',~ ~-I N N
O M
d~

.l~ .U J-~1 O U1 a w ~ r1~.,-i r-1~-1 rI H ,'x', Q f'', f.~O M 1J InJJ w a w f=m4-1 4-1 rt3 O H L' O 7 <n a~
r1 I o t W w i ~

, a rr1 b1 ((fu1 i~ t31~ ~tf ~1 O
f: 4 4 1' , C C7 1 I U1 u1 f ' 1 I O t O I
4i d~ O I
' ' , - - ., !_,U ~i I ~ I ~; 1 I I ~j t'it -1 rl( U t 00 ~ I 00 1.C1>:,' o ~', b1 z N
W o O b1 ~ O M ,Z, ~-1 cd W W o O N M W p; W a~ r~ O W
~o L N ~
W ~n N
th U
a1 -.-I W ~ x r3 r1,>_,'d~ H t-I -rl$-1~1 d~ ~1 r1~, O M
O -I rn~' r1 d~N ~Hf~ H M tnd~ ~.u ~01d~ tnf~z~
rra(arrlw ul~lFCtnt7~rnP~ N ~U~ .I~H ~.1-~H
H~trW~

Q] O

,_.I

M

~
m >~
O
~n U

r a-I
~
'~

~~

w >~

O M

-r1 L~
a1 ~O

NHH

N cN 01 r1 ~ lD ~.O ~N O
,5y a-I d~ N
OD M

~ <H N
~ ~ ti O 01 OO
N d0 1 O .-) N N M
d~

.~u rNr~~EM-~ H~ r Hs'ncn~nr~tn ~ n . .
>~ .
f~, ~

rU O ~ 1D . L!7 N
W 01 M 01 cH O M
U I,n N d~
.N 01 N O1 O L~ M
.!-~ ~-i t~

N l~ O
O W -1 N N ~ Lf~ M
.s-i N c-I N di lfl N ~-f H a1 c-1 c-1 N
M d~

W rr~ v v 1 u1 v~
W r~ u1 H u1 try u~ H tn rn E-a u1 O

-.~ 0 0 ~ .. .
-~r u7 U N M
N
' ., M In 1. n FC .
R

O

H

A
U

f~ U L~

N N N
U N

L N
U OD L~
r1 ~,' W o ~O
O
f H co Ln !~ n H

OI O
..

W U
~1 O

U7 ~H u7 to H
'Z

O O R O H ~ ~ O
U ~ N f~ W ~ O f~ a1 W f~ CUJ1 O fx -~I m u~ W I W fa W I I W W A W
aJ 'O (a 1 U~ 1 1 I Lf~ CI~ 1 a I I
~, O ,5~ E-~ W ~ f.~ ~ L-~ C-~ W W (x H ~ H C-~
.-1 ..~ ni ~7 ~ W w U cl~ u1 ~ ~ W (x, U u.2 Ca U
'~a ~a a~ ~wa a a a ~ ~° wa a w a~'~aw x mas m a~ m a~ gas m I
t-t N c~ ..
N ~ M FCU '~1~ ~ N W I
wq~ W O ~' a~W H ~ .u1 m Ha O ~
W d~ ~ ~ W H H In o c~ ?-I U
~C !~ ~ o I o tx rn r$ t~ ~ ~-I ~ ~ ~ p W ~ ..
H M ~ N owaNO o o .1.~ ono acnHw UCH ~-t N o p~~Ino 0 4-1 rti faN Ch W
,"~ H r~; o ~ o H ~1 r-! o o ~; ~, M ~ U
Fl W E-~ a u1 ~ ~ H H s-l N t~ a~ -,-! ~ W Z, ~ W r.~
WHO W la fa O~~ 1 WNfi,Mf-~ W I G~,Wpq O R: w .. ~ M .. H H .. ~i .. ~j v N pip tJ~ p ~' A w w a~ .~w ~ N w r~ a H a~ 1 a~ ~I a~ a~ ~n al x w ~ ~ ~ In H O ,~ d7 v cH cN U~ U1 O W ~~ U7 c"1 U1 M U7 Ul ~-I ~i W U1 Cl~ d~
x ~ >:~ ~s of r~ r~ ~c m N m ~ ro W rt ~a ~ W ~~ x H tn o o ~C o - ~ s~ I I H H ~ FC a, .u r1 .u .N .N I ~ r1 o x ~ I
ui a a cu -r1 01 .-1 U U H H ~ U H U ~ U U ~-i CQ o0 W (x~ I v-1 r-1 tv u1 W t~ O o is tt1 ~ ~ ~ i~ W U o ~ ~ ~ ~ ~ d~ ~ co to Z H ~ ~
U 4-1 Ul ,~i L~ ~ ~., N (1 (~ ,~'' '',.J o ~ ~ 'Cl N 'T3 ~ d~ I c-I
.~I r~ w o ~n o x ~~ w w A o a~ oo ur w rv m ~ ~ ,~ u~ W w ~ ~
a~ +-~ a m x w o zs v rx x ~ A ~.I c~ ~.I 1 ~.I ~ I o H - w I ra ~n I ~i O H I O ~~ t7~ O ~ W G, ~ N b~ co f~ '',~, d~ b~
~i "r4' W o t=i ~ N cd W W N I N ~-I N N f.~ cd R; cN U1 w cIf N O va t~ O ~ u~ P ~ E~ tn E-~ ~ .1.~ to .u U .U .1.~ D O oo W W W a ø
u7 zf ~ r.~ ~ e-1 ~ ctf cd cd a,' o~ a,' ~ H W rtf ~o rtS ctS ed ~ ~ cd x N ~
U1 f~ f=mti ~i w 'Z O~ S-I '~' N a a-I a U f~i U2 i-1 t~' ,-I ~ .-I r1 ~ N U Lr) r~, u1 U7 ~ N
,~,i S-I ~ C7 W cN OI ~ O U W x W ~ O ~ 40-I ~ ~t0-1 N 40-I 40-I ~I U ~ ~ H ~
~ yD U
~i .~u ~ w~~~° ~~ ~11°~a°xoHw ~°I~ ~~ s-°I° ~°I .-1 I o ~~wHHa~-I
p ~a-~I omo~ ~n~ o ~a wcnrdw~ 1 ~u~-1~ ~ m~~~~UOO I ~a s ; cd ~ o o x ~ I o ~ ~ ~ ~ w ~ ?W, N ~ cn ~, ~s-I ~ ~ 1 w FC u., c~ N
rs~ ~z~o-,L~~I ~s~ ~ ~,xM ~qH~.~~-1,.~~1,~ I ~~ ~,cno~r~w~~N
r~A ~~NPqn Ha ~ChA.o-I AZNHA~i-~l~A~fa~-~rHnAP~,uytratr~W u'~1 m ,_, .~I
>~ o ~n .ou ~, ~ . ~
w° t~h ran ' z -~I M
rn ~a .-1 o d~ ,n r1 ~Ln C~ M
.~u .~ ~ ~ CN-~ H
N u~2 N ~ .-I r~ o~
W W U'~1 C~~1 C-~ UN1 ~
m N
O
p '-~ w ~r1 ~r1 Ul L~ N
r'1 M
q A
s~ ~ H
a~ N d~ o r1 5, H W WH m h p1 ~ ~ O
W(~O U
tn H ~ ~ oo 1~7 H '.~H H

y a d ~ ~ ~ ~ a r a rn c U O W fm O W W G4 UlO P.t fstO
(U

r-I ~1I W (~ 1 I W W A I W

-a 1 u~ 1 1 ~nv~ I rn a m n W I
~d H w w ~
~i O
~
~

r H w at ~ r~ r=,H H w ~ E-~
. ' ' r1 U~,~, U t4 ,~,"~', W H fitc!~,~'",W c4 ,L, W H Ul ry'H H ~ En O FC H Ft,' (IS C
td .1.-1 .t-a~~ a a - w a a a o x a a a ~

~ w on s:nr~ m r~ x ~ w m al w w OO I O ,9 N

Ln W ~ ~

I Ch 1 a <-Id~ l I t~ ! U1 ',~
N r M

O H C~x,"2' " r1WM M O I
, AM~O , ~, 1 .1~~ (~0~H O
H . ~' O N p1pM.~O
fitO N (s~ u-I
l0 ',X',~,' fil~-!O x-IU1 4-I',~ei'p; H i-1 1 ,1i W M O 1 N v d~

O fit o O O cd N O W W
d~I N W p', ' N H
I M Y
'l'-' L(7 ( ~

,.. O ( ,ti-I-17 I I N
, l ~ , t N Ri -~ a , '-m - ~ o ' n U u~~ .. ,x .
N ~I Ln ~ 1 ~ I ~- , -~ W ,-t n it.. r~
o W
m o r_~n .-1 ~ a o, ,.t~~ ~ u, u1 ~-Io ~o . d~ tn ~-t N o, W rd I W pC~N 'Z-5N N W d, I CJ d~
M O N .-1 .-1M ,~l, M cH

U1S~' N C7 U1 ca ~I b1U7 '~-tN ,."7 I
M O ~f,' R ChM Ix a ',T ' ' O In /-~ N O to ~- \ O a, W U7 a L~
,~t71 fp I UlI M I I . ~
1 R; Ri M =i Ul ~Z I
y-I~ dr 00 0 -I ~
~

t 1 -I.y O1 (Q a0 W I d~ '1..,' CO N N N QW '(j, C .~,U
~ N M l~ -I ...W J I i-!
d ,5-~
VI
l0 ChO1 N W int37 U1 (!?',~tC7 ',~c',N W -O1O N r1 Ix 4.101 r1 d~

,h-iO L b1 QO (~SO (titf$,fa.,'J-~O 1;71N
O N ,-1w (h,' o,~ I ~1O W y 5C0 ~r ~ ~I L a~ ',~
~-1a 1 ~

t a s ~ o o O ~ ~ ~ r.~A N ~d r-IN n , .--1I ~' ~o ~ 1 o ctt. N ~ O ''~
io ,T co .

O O to O I ,r7t~~ q bW T .!-~O O O ~o d~ N oo .-I d~ M
~-i Ul 'z.~., O v-ia O ..CiI O O r1'Z ,~',O y--1 W N N l0 Cl~~IW .-I
00 o~

O O O r-I ~'.,,'~',O ~-1N c-I,~(a O O fit S=iW
U7 1 U ~-I O M

U ,~;b1 O ',1,'''ZS'Z5~i''i~'Zl.fi,~iC7 O -4-I t l 01 Ch ,.4''L~ C7 ",1., ~,' 0 ~,' ~ O ~,I 'fir~ o 0 ~., .~.,a L~
-~-1 M N N U1 .
I

U W -~I N U ,.~t-I ,s~..~U d~ H ~-t to .I~ ~Ho tnN ~I ~ tn . N

O '~,~', ~1 LJtLC1N O N N N --I~ .~j..ii:31U1 M
'~i N O O d 01In r W ~ N d~ N
H l~ ' ' ' L~ ' O 1 d~ S
-; Ln , r ~.,( ~H Z -1-Wd ~ Lf IdH H ~ O O
R w-1-I .1-~ O W , M r1 a~ a ~a ~1 ~ ~ w ~1 -r.l ~ ~ a ~ ~1 z o ~ w In >n ~ 1 Lr~
~I 0 U1 H .l~ TCfcIi-~-tF'.,.~1.I-~S~'~,'~,'H H '~ O ~i '~ laW fit c0 I ~,'''~l t~ fst Li f''.,N H -r-1a-Ia a-i-rl-r-I
N 1 l N to N f b1 ~ ~ q (C) I -ri W ttit~
O N M LtW
t--1 i ~I U ~-1 R r ~ -I-I ~ ft (d Itf O -r O
d~ C7 U ~ W Cj -riN W -I
-I Li t'' l0 N
k ' .~ r c , ,. , -,a U .~ U
-1 " ~ I ~ I U . N U N , ',atM fit',~ CIA
C7t-1 ' d~ U .-1 U l C7 I I
' ' Ch U
" ~ M
~

-, ,7.,~, fitr-11 1 U7 ri 1 I ,'I,'Z ~-,t~ W
O -1J , .il " d M ~ Ch .UZt p~ ..
F Z,ifi1 W
td H --~ H i ~

r -~1t H . o rti-U -W -NH H -riCh U7 -~ o 1.I -1 c~H .-1 ~
I

~ W ~ s~ ~ ~ !~ ~1U ~ yo Sa N W ~ ~ I
caj ~ r-i N o toio -~I~ ~,.~N
~ eo -~ H r1 r-1r1 FfLr-Ir' ~ L ~ 5 H a I ~
O -~ -r-I '' ~ I ' ' ''~ d~ -<N ~i w-I N ~ -~ -1 dt U1 a tn -I In . ..' , , . t- , ~
A fi~ f , a ~ t ., M r-U' r c f m t ~ ~ n a U U7U~ u1 u~Gl fs, f=tf~
r A H C7 r.~4-Im frv"~ r-1 Z

- _ -E

z 1J N OD w-1 N
'fit -V

W ~

tl1 O M
-r1 LW
-I

O1 c-I O
.t-~ d~ ~H c0 r1 r1 ~
~

r1 d~ N
~ N C~
t-i d~
cH
N

O

. . M c-I
r-I . IW 01 lD Ln ,5y . -1 OO
pp ~-1 u-I lO
O
c-I
N
ltd N
v-1 rtj O r1 O d~ OW -1 ~..1 M N v-1 N M d~
v-I N
N M
M ~N
M
M
M
cH

~ ~o c~ cn Cn cn try M rr1 H L-t y H
tl~ ~
u1 H
H
H
H
H

. - ~ ~
C-~
[

~

N 01 l0 . . . l0 U l0 c-I O\ M d~ cH
l O v-I M

M ~1 M lD
lD
l0 .!~ r-1 cN M 01 Ln N
O 01 01 00 t-1 N
.V l~ u-I L~ M

01 v-I

N
d!
N

O N a-I c-1 LC7 6W
,Li rN c-I c-1 -1 N N
-r1 r-1 N M ~N
c-I N
N eN
M d~
M
M
d~
d~

w cn Ewo cn v~ ua va w own u~ u7 H rrs cry ua cm ~
H >n u? H
u7 rr1 ~, H

N

o ~d >~
~
-~1 r1 a1 l0 L!1 M
n-I
Ill N N

FC In N tn LCD
W

N

H

A A

A a N H

N M N M di ~ O N

,~ N Lf1 N ~N

H ~
0.
W

t H

a<

q ~

~ H H ~ ~
~ -1 O U
V m ~ ~ .. WI w w q ~ P~l f.~ L 1 W W
1~ Z3 cCf 1 tr1 I
r''~ O ~~ H W hi P', P; H ~ H H ~ Ri Ri PL'n W H
r-I .~ oS tI~ ~ f~ U v1 try ~ W W W H C~
'~~aaa °xwaaa o°r~
~~ram m wnr~ x w ~~~ ~ w I o I ~o tn N I 1 f31 1 00 ~-1 N I lD N a u-1 ~ O N
~, N l!W -I N L~ U1 CO "~', ~ U] ( M lO CO O
H H C7 ~N W In M N H H 1 d~ ,5-I 01 ~-I N
WfY,W I M '.~ OI fl.IWR',WO M I Ri ,'~ "~'a HOH~N Z ~ I I HOHN t~M I
O U1 O N v-i M ~ ~ H I O U7 O ~-I t~ cH ~ ~ ~
(~' 1~' t~; ''~ ~ ~ l0 N 01 Ri R'i r~' ~ ~ N N N
W 'H7 QI Lf1 ~-1 r1 ~' r-I 4-I a ,'Z.~ W ~.~.' W ~c7 OI r-I CJ ~-1 r-1 4-I
UO ~ L~ W ~ ~~t ~ UO ~ ~ ~ ~ ~~I
W W U t M I N U1 ~ ~ U7 ø~ U7 ~ ~ W W U N M ~ OD N ~ ~ .I-1 UJ Ra Ul (Y., '~-~ M O ~-I ~-i O ,-. p . O O U1 R', ,~ lI7 O OD M O -~ tIS O
W ~..~ d~ o N Ln U H U .1-I U ~ W a M o r-I M U H >_; U .1-1 L7 r.~ a .-1 ~ ~ C7 ~ l'37 p cft ~ C7 (x a N f~ ~ Ch is ~ b1 w w I as U 1 ,-I w r1 o, a 1 w w I ~I w ml w .~I ~-I ~, T~ to H vv In N b! b~ zi U' ~o ~, tn H In I m is tn is ~
C7 d~ ~ ~ ~ M O1 1 I ~ I 00 U1 ~ i;J d~ ~ ~ d~ ~-[ I 1 W N to l0 M d~ W v f~i v W c-1 W N U2 t~ M W V U1 W v ~w~a a~l~x a~ ~ ~,~ W~~n a~~-I H~
w u~ a I N a~ a~ ~-I ~ w H ~n .~ ~ I ~ N ~~ ~., ~.1 a M H ~ .. ~a N . ~l ~ ~I
a W o ~i c<' oo r.~ ~ 'd ~ Zi ~ 1 .n a o ~-1 ~ i:7 ~ Z3 ~ ~1 rd ~
Ul ,ht 01 N M N 1 ~r1 ~., .N ~,' .1-1 c-I l0 ,hi 01 O ~ I -rl ~., .t-~ N ~' .l~
W Ul v-I 4-I Ul fi.1 c--I (Ii (d c~ c>j (ti ,~i ~ M Ul ~ ~-I w1 4-I lD e-I (If (fWa 4-I (I~ (d U4-I OH~o u~ ~ ~ ~ W OH~o>'. zn~-1 ~ ~ >~ u~ ~
~.~1 Uawo ~ . . o,-.I ~~, Uawor-1 ~w o.-1a3, ~ .-l~, m .t~ ~ ~ ~ o m M M .. ~ 5r-~1 ~,.~I N ~ ~ ~ ~ ~ o a c~ I .. ~ ?,-~I as ~,-~1 o arnwa ~I~o~ a~ m ~n m ~ >~,rn~a~wa 1 >'I~, a~ ~n cn ~
~'., U' O ~,' W .!-~ d~ N Zi N O O c>3 O M C7 O ~,' W o0 J.~ ,-I 'Z3 N O J-~ O
a~ Z rn ~-I of w -~I ~ >~ m ~ ~n ~ Z o Z v~ ,-m .-I .~1 ~ ~ u~ ~I ~ w rn zi W o ~., I I .u ~ o ~ o a~ ca o o W o ~ r1 ~, r~ .1y o a~ 5., o a~
s~ u~ m d~ ~ p, ~1 1~ en ~1 ~n ~ .~ ~ rn w m s-~, >~1 ~l ~n ~n ~1 m to ~ ~ ~ ~ o U d~ N C~ ~ U ~.i U 1~-I U ~ a ~ . U ~o ~Q., ~ U ~I .-I U U !.1 ~ U7 W ,'~ O ~ ~, a a a~ ~ a~ ~ N ~ '',~ Ul W p O M ~, o N . ~ N In ~., ~ N
~ >~ wa~~na~l ~1 ~~-I~,~I~1 ~-lawwa~~n~,~l~l~-1 ~~I~-IM~1 ~~u O c~ .~1 u~ c7 o I t~ . . ,~ u~ is m tn ~n as c7 rrz rn ~ o om U ~ u~ b~ tn x tr tn m t57 ~ ~ Pt U' CJ N W N O ~ W cd W td N ~ W ~ ~ W U U d~ P.a 01 is ai W ai L~ W
N W la cf' .r1 O H H 'cN M N .r1 ~1 ~I ~.1 cH .r1 W H H N t~ .r1 ~.! f-1 0~ N S-1 N
rnA H~vi~r~~WZrnH~.l~~.t-~~~rr~~f~H~tn~C7~Hu1H~.1-~Z~01~.1-~H
O
-1 -~I
r~
H
z N U N
O r1 ~r1 l0 W U' t~
O
.r1 di J-1 Op rti H
a-I '~'~, 0.S ~1 N N
H~
N UI N ~ N c-I
.1~ O .L-W -I N 1O
W W t ~ H tall UN2 O ''d >~ 'd -rl -ri .r1 U7 M
m U
H
R
1~ U
H
N N m U r1 ~ o f~ d~
HWH
OI O

W A O M U d~
u~ H ~ ~-I -- ~-1 <n rn o ~ z z ~ o '~

t ~ O fx P; w ~ U ~ O
d ~
u1 U O fx w w w f.~ rn O t>~ f~ Gy fx, N

-~I R w I r r w W A w w w w m u>

.u I I Lr~ t!~ CIa r U7a I I 1 I r Zf cIi 5r H H w w w fxw H H H r~ r~;
O
"ct r-i U2 U1 ,~' ,'~"' ~' W H f~ U1 U~ W W U W
, ' 'L,' cti cai r.~F! H H H H O FC,r~ C!~
.r~ , -LI

~~R ~

.. O 00 00 ~ rCIH FC, rI I
M 01 c-I O

v-I LC7 M '~N d~ ~ ~, d, H W r1 l~
O M Ln tt1 ~x ~C7H ~~ ~ ~~ W

N E-toH Ft,'.. ~ ~ -I
.

rx ~o ~ ~ O .,~.r.rA x a 4.1c~
O ~ ~o ~, I ~

r o O . O ~ o ~-r.-t ~ o di oo co ~o W fs, -r w O u7 d~ f~,' ~N ~ ~JC N d~
tn d~ M M ~, d~ cft o t~ m o p; w f~~ ~-rO f~ m >' d~
f~ U U P; C~ W
d~ U

~-Iw w M O O O I p; (tito N
I ,'N

o P; .. ~ ~ W
0 o, . . ..
. .

0 .. O w ~ N .~I-~IM
. ~ oo ~ ..,~ o ~ W o H U r~ ~ co m O d~ S
w ~

M W H w M d~ a r r oo x w 1 r In LC1 H o N 4.rH I t1~ ''Cia ~ N N H
M ~r d~

N If) N d, -1~ ~,.~.,(~ N F(','fit ,Y, O ,~C, U d, ~ ,~ W
~i I U
N
A;
O
U

1 a .. ~ to N N I [-~ ,.~..
OI ,~Z . I I .,C~'. I ,~, 00 U7 ..
Pi WH ',~ I ,.C,d~ W W
FG' O ~., O l~ H
I o1 01 tn w H ~I .r.mn i3tM,rW n o H U oo N N
a <-r ~ ~
Ln N [h ",~'(IS r1 O N N ,-iF(', U d~ M M d~ F(', w ~H d~ ~, L~

N ~ w .I~ >~ u7 tn~ ~ r~ O O ~ ~ N
O ~ r.~ W ~ H ~ W
w H ~

o O W ~ .~ H ~ ~ ~ W ~ W ~ N N
~ m , , I ~
~ , O O W b1 UI . d~.!-1N .. I U N 4-1 r~t77 LIB O N H O ,r.1W N I
L~ ~0 ~ tP
L~

u1 ~,"'Z, .ri W H W t~ff1U1 x t~ N f4 ~-IO
W d to o 01O a~ FC v-I
L~ W
M

N f~ O '~',fll O N a ,'~i'Jy'?t,'~H v-I .U ~,' ~iS-1 Ul <N F(,'d~ d~ d~I-1c-1' OI
U ' " cH ' ~
4 ~ '' " ' 1 ~

- ,~ N U ~ U U S4 ~, td cd 2i ., ~, U C7,.c,I a, W ~, M
~, ~I x O ,5y cN O~~ I U O H .1.t~ ..~y .r1 O I 1 r M H ',~ .. r 1 I I ' N

1. O GI r-1 w cH ~..IO O O Q ~ N .t~ Q ,C~, O c-r 01 O r-IO ., w W ~
N I

O W n W -.-rN tn b~ m n tn fa H U U t ~ U
O co ~ ao.i~M ~n N
o t~

,~i d~ d~ ~ r1 d~ d~d~d~ ,5-I.O !d Ul (IS~
Cll M N di di L'-~C Ul p'., d~ N
M

N w w r.~ rt3 ca o w w w x R; O ,h U ~ G4 a fryU ~o f~ ttf H fly 'Z, O
~-I

in H H 4-r m .n -- d~ w H .u a~ <a~
zi I w ~-r s~ w W U ~.i ~I d, 4yO U . ,-r N ~ N ..-r ~', co . . .. M W O
~ . .

N ~I ~', N ~ M w ~ ~ ~ w W U U O r-Icd c0 ~n p ~0 d~ tn N ~ W i.a ~o 01 N t~

S-r O O R p., ~ ~ oo O O O u1 Zn rd -.-1 U ,.,~
o W M r1 .-1 ~t~1 ~ 'Tj ' N o~ ~ FC
a~

U1 tf1R; ~ O .-r U1 6-iS-1i-1~o W a r1 .~'' U
~ p, W R; d~ ~-r ~ ~ ~ 11~
x~ d< d~ ,-r d~ ~
xx~a ~n ~~~~ n x n ~~~ ~ x.~~.~w,-rH~d~ ~,,~.u Q (d U U O . . (a U U U W W A I f 3 1 r1 1 I 1 U 1 I tIrftSO i I

rd O O (W O O M ~-r O O O ~',N ~i o 4 .r-v In .n L~ ~o u1 ~,'t_',M P J.~ ~ ~I
~ o~ L~ L~ ~t cN

O

O 'JW-IM 'ct~r1 I~ '~'a' U1 Ul ~ l ~ ' ,'~ M ~-1 ~-I .~~ ~' d. ~
5C cH M M -~ ~ A
v-I

M U1 U .U w ~ r-C W y y , , , G4 r ,.1=, f~ C7 O r.~ w C7 , U I H H N v N U1u1 ~'.., U ~ ~ U ~ U (~ f.~ C7 a U1 w L>i o r-I

~
in !~
O
u7 N M N Lp U ~.
N

J-> d~ M Lf~ 01 ~y cH
.1-1 wChrn~

O p~

M c0 O

H O

~ ~ ~ ~

- ~ l0 M O
-I 00 t~ l0 r1 N O N
r-1 N
cff O
N

N

~ O1 M
of a-r N LCl N
~ N c'~1 c-I C
M N l0 t-1 M c-4 c-r d~ N c~S
M v--1 ~ u~ulcrlHH orn HcntrlCnHH

,. , ~ H

~ H

Q rIODMlO00t~ NO ~O~MN._.1D010d~
U
l N

.1-1N ~ v-1 01 O W 01 d~ v-I
.r.)-1 O1 M <H
M O1 l0 O M
~N

Ww cHr~~~C-MnH~ '~ nc H
i ~ N o N m ~
N
H

t t t r r~ t r~
~lt r C-rr i U

~
rd -~I

r-t u't d~ In M
.~i U~

n c c~ m A

A

U w N L~

h ~H

M d~ c-I ~H

U r~ o~
.-1 R

HW L
H M l~

p1 ..

~qZ ~

H H

rr~ In m ~ H H O

r-1 O , C'., ~ U ~ O

U (Y)L W P;

ri I W 1 I W q t!?

O W H
Q

~, Pa W H En f ..

_ '~ a a "

I rx a a a a o a ~

~q w w w r~ cn w M W

t N

U' ~ O

H

-1 00 ~-1 L~ O

~' ~ ~

N~-tI ~~- UW ~ N
I

~ 0 H H Gv U1 O H q c-1 o ..I ~
0 ., '~ ~ W W

o U r- M p; U U
o - I W-! ~ 7 ~ C) to ' t -t - . - W r.C
o ZS ~ "W U o~ 'ZS
~1 ~ H '~ '~' H ~-I
O

a a~ la a~ cn w a~
q~ ~ N x M

W ?-I~t-Itn W U''~1 ~N tl~ '~.t ~', q N

W W rtf Cn O \
-I-~-1 d~ ~; I M
I ~-1 d~

rn milNH ~t~o,r.~oxMxo ~nN

a~ a~ r.~,a~ H m n~
M o I x x ~ N
U1 U1 ~ N w ry'U7 N ,Ljl0 ~ H (Y, N q !

~sc~~ mM O~a o x as ~n ~~ I

F,"~''a-I ~-1 q W !~
I I uo ~ tn [-~
di N N O 'Z3 W q N
O ,.C,'r'Y.'F4' W O O1 ~ N

as ~, ~a ~, x ~-Io, M z~ ~ H ~I
r~
a U1 O O tti .C, O a O
N (d W M U1 N S-I~-I>~''W-I q O ~I
Ul C7 ~--o N PO I
.-I

U ~ Zi N Zf H x W
4-1 m oo I ,- ..
~ W
o ~ ?~ b~ o y-I o ~, ~ N o I M m U

O O O
H
~

O N N ~I O q L
~i N ~ O O N ~ ' '~ ~ 't3 O
N U y h ' . ( . W d~ ~
QJ W cd ., 1 H .-I.a..1 ~y .x O ai -r! W
W O
y-i O

rn ~ :~ .~ .~ ~ ~ >~
~ I ~ w ~ ~
w q ~ r1 .-IN .,..1'~ H -.-i oo t71.. . ~ N b1 . w Q, N of cd 'd ~I W w ~S
of ao -~IN N H -~1 t~ ~ H

f-I ~.~.i..Cia \ U~ rLi v-1U7 N ~-t U7 Ul ~m Uc~U .-I~Na~~~ oaow .v~rU
' J.) I I ,a~ Ul P', I I
~, ~ O .I O M
H -L~ ~ U
I CJ ' i ~

td O ~ H .N
I>f ;'I~-1~ c~ ~, o ,-1 lD wi O I H ; y ~ U i O
' ~ H
O '' N

, , E- P ~
N r1 v ~' ~' -1 -1 W ' O c-1!a ~ ~ W ' -1 ~

. . r r O ,I,~i cn rn rn N r Ul , rn q c4 4-Ir.~ Ch q i 4-a u~ m rx v ~ a v q H

s~

,., -.-I

,~ .u c~3 a s~
o uz ti) U
O

.I-~
~y J~

O
r1 r1 W

Ua S-;

O

.N

t~S

r-1 r-I

.-i O

s~
q~
m O
~
U

.h O
.i1 O
,~', .-I

W
W

N

N
O

~' '[j r1 r1 r1 ~C
W' v q~

N
N

U
r-I

q H
W

H

!~

p1 O

W ~
q U
O

U1 ~-I
H r ''~', s~

.r-N

O

fat L~ l0 00dl l0 01 CN L~Ol001O l0t~00 l0c--1 OO

01O Ln OM dtlOMOlc-Ir)O OdtODt-Ic-I~OLf7l~LnL~L~I!lMu-I1.ndt01O1~HhcN

00l~00 L~r-tOJdtv-1Lnr1Q1L~c-IL~IS1l0MO d4O O1nLnc-IdtLf1f'1c-11 MN COCO
n M L~O1r1 00v-INv-Ir1L~r-I00~-Ic-Il0Mv-Il0N u-iN Nv-III1c-Iv-I~-Iv-Ir-1.
Ndtv-1r-I
v-i O

.,.I

.,a N

O

M M c-Iw1 d1L~

NM l0NO100M " 01 dl dt01M O LN c-t l0l~

00 MN O ODM L~O s-) ON dt l0MO lDL~O lD OO

IW -IM c-I N.Nc-I00ON !.nr1m-ILIBc-101N riu-Ic-Ir1r-1c-1c-1eNtOdtr-1a-101r!M v-1w-I

I p N U

O .1J .N1J

O r1l>7 r1r1 O '~O d O N O O
O O

O y -Ii-1NN ..~ r1 M -.s-IMw1~-I M

I N N OO OO M M N O O r-1 MO OO O M

U1 tno o UU ~W o o M W C-~ o oW Z~ W o N

r.~ ~ o o H~ HW H o o ~ ~ H Hp pH p o a >a N o o ~H pf=,O o o H Hd~ O N pH H H o a N ~ o o W ChH

~ o o U H~-i o H~ ~~ ~ o a N o o ,~~ ~Z,H o o p ~o ~ o HO O ~-Io a I I wH H tr~ I I H HI I ~H ~1H H o~ I
cd o,InM u1~ ar4x ~ ~ x U1 I

f N ~-w a~ v~ a u1 0~w dt ~-1 t71L~00 v~ v~ (Y.i O c-I~-' v00W'~-nM (.Y1'~.~~'.r O..r (zt I N O w-)c-IInO v-1 a0U Mv-I c-3v--IO r-Ir1t() ~ ~ q I

N 01N M ',~.,r~-i'f4n.'Z,'c-10 0Lnl017O1',T~,M Nw-1M l01'7WPite~
t.f7LE~ dtM ~h U C~0101 Lf7O dl[~,~7,,'',--IdWpdt00N C~l0Nfzlv-Il.f~HN N~ rM L~l0c-IMM
61.
117 , -rl N

~", N O1l0 01N OM 01l9NlflCO~-IM Nf.~NlDMl0Lf1OW-1dla7~Ol0WN C~l~L''~
dt 7 L~231b) OM Nl0c-I00O~ ~7OdlN~ l~N Z31N ~Hl~01M L~01L~Oo-1cHdttytN
dt VI

dt 01Q1r-Il0~lDLc)....M00N l0d~ dtl00101r-Ia7L~~ 01O lc7lf7 dt L~H s-IO Nv-iCOIl7L~H H r-Il0C~ Lf7OD O 01..'ItON dtN G1O[W-Iw-I H
Q) O (~ Nl0Ml~MO s-IfY~(Y1lD~-Il0 aLn O Inl0MLf1l0N -1a IS1Lt7~f~
a O

Cf M LtJC~ ~l0l0~ l~ ~ J ~~ r N
! Fi , t l C DL LCJC LL LC1U CTicHC7L~00C~l0LI1L~l~l~filODLf1111C)CJ

I ~ I 1 M v-f r-I s-S
Lt7 (0' -. M I _~_. ~ N 1 W M -I 1 . pp w I 1 M
CO

dl N 01 a1 O ~-1 t ''(j C1~ ~-1 cN d7 .I-1 cN o0 (U O 01 Ly-I O If1 LI7 ~, I O lp M v-I

.I-1 t~ O w-I v-I O ri w-1 N r1 L~
O

U dl dl O . ~-1 I,17 c-1 I 1 O
~ M ~ .

N I 1 ~ .M 01 I N N v-I M l0 ~ M dt . O
I C~

r1 M N t11 v--) O~ l0 Lf1 c-I l11 N M
I>f 00 O M O l0 I~
~N 111 N CS1 l~ I I 01 i I N M 1 ~) f:~ N v-i M o0 oD
Lf1 dS
N

Tn In a0 v-1 i-1 00 c-I v-I w-1 r1 c-i f~ M t0 ~-1 v-I tI7 r1 N u-i .-1 N

U

~
,~,' N
.!-~

b1 t~ oo dt o7 dW o o .-1 0 ~, O 1.f7 l0 M O c--I I~ C~ 01 L~

N L~ OJ 'cr s-1 c-I l0 O LO 111 OO
~

U3 ~ ~-1 v-! r-I ~-i c--I N c-i r1 c-1 t-l N

U

N Pa al al H !Y7 P4 p U f~l r1 U U U l0 pa U U U E~ U

N In 01 c-I U L'~ 01 lD v-1 ~O

O O1 dt L~ M ~ N N dt O M
~

~ L~ lD L~ O O N Ln M C~ OO

~ N M 00 CO t~ N M c-I i~ N

~ O N c--I

~' dt dt M ,-I ~o O
p H ( t~ M t~ CO Lc7 ~ !f1 l t~
W
H

O

'~'J

., O
~

NO

U

~p ~H
~
f r-OI

O dl O ~-I N M dt Ln ~p L~ OO
W

W r1 N N N N N N N N N

s~

m W v-1 O'd'Ln 00N M lOc-ICHOr-IN COd~M 'd~ O N L~ l~

L~c-iO1d~00LnII1~O L"d~~O1.17l~O1II1MMO I!1NlDNO Or-IOl0l(7NO1LI7 L~LC101NL~L~01O InN N l0L~Nl0O01O u-1NO L~N l000N00d~c-It~N

M i-1t17O1~-iv-1r-1t17CW -1v-1N 1Ov-iv-Ic-Ir1v-Iv-Iv-Ir-Ic-1c-1d~~-ILW-101O1c-!L~c-I

O

-N

N

O

W O100 00 t~Oa0 In L~ c-1 d~ Lf1O1l0<Nd~L~ O l0 N c-IO Mo1 l~lf1ODo~ Lf1l0NC~ N

L~ 01r-IO cpt 01 N o0 d~oN d~d~ tt1NM M c-tl0~ot~ d~

L(1 M ~-1t!7~--Ic-1L~M cH 00w-Iv-I~-Ii-1v-1c-1dWc-1l~L~d Wi-1s-IM L~LI~c-1w-Ic-IL~
-! -1 'i~

N H

N r1-I~
.,_.1 O N~ O O O N

I ~ O .-. O~ O

c-Id1l1~M LI1l0M O ~I I O

OO OO OO O c-IO Nt-I O r-j CHN r-I O O

u1 W ZZ HIxo 0o M P; H MH N o H H
~ p~ O~ ~W o 0 ~ 1 ' . l 0 o .I-H p O,O o o O p .

HH H Hf~0 0o Ho ~I H oH o o Z H

O In ~~ ~H of oIo H N ~ H H

o U o o I H W ~ M NI ~I H 1 I~ I CSIoO
Ix O
N v-I,a~ WZ ~a1N o~~-I t~1M ~ l G~1 oal o cooU

H U
~1 M v V~ vv L~ COM vOD l0~ ~ N" N LnOv f~ l~ 00 01l~c-Iv-I c-Iv-I O NM dW -I~NcHO

E~~-Is-Ir1u-Is-1c-IM M'L~,.~Ci~ t"1O r~ N 10 l0 II I

O xx xH Hx ~ OO M InN ,'l,~-IN~Or1f~L~fii.1JJ-1M',1',OM O NW ',I,' U ~-1i-1d~01M CH01O O.r1v-Id~N d~L~Nd~N N NN r1~-IM~DL~M c-ILW-Id~

L NN L~t!W-1O i-1v-IL~O1lO01a~l~l~'00t~l0d~d1''~''dL~d~d~l~~ c-1d~!n N f71l~11100N 00011'~C71~ M t0L~MWrILC700t31!nNl0U?N t57O1II7~ Z~OJM 1.f1 Lf1l0v-iLf1lDCO d~NCO01 NM v-Id~M II O1O c-Il0O

~nd~Nd~o0o HH .--IIn~ cH In1~H N oo HH d~o H Mo1N
N CNL~r1d~MN ~ PGP7LnON M~ Od~pLlt!Wic-IpC1pq l0lfl,~,LrlHlN L~
.h t!1 C7LL~l0L 00l~C7 C7Ch111lb1MC7lb7C7Inb1cNU'U'C7IntnC)C7f~tnl0 -I

~ 1 1 ~ I ~

Q~ ~ ~ O l~ in N

_ _ '1'S tf~ ,-1 O~ N d .1J O1 N r1 N N Lf1 F-i'' 1.f1 .1~ ' ' r1 L~ lfl N 111 M lD 1 In Ln U . v-1 ~ t~ tn N 01 1 IM
N 00 01 ~
M

r-I M 01 N LO M a0In (tS u) l~ 1~ !~ N

N t t 1 c-1 to a1I
a o tf1 M a~ N
N

U1 W i c-i u-i w1 L~ c-Ic-1 fs C~ ri ~-I N c~1 N

N

U

G' .~i Nh ~i d~ L~~ ~ N N LnNL(7 U7 ~-'I c-I N c-I s-1 01v-Ic-I
I-a N

'~

.,-1 N P4 al fYl Pa al P4c-1Pa ~ _ .

N M ~H In O1 ~ ~

~ L~ a0 N N a0 d~l~d~

~i Ln N <N d~ M d~c-1d~
' f-". d~ d~ d~ cN d~ d~r1d~
O
f~

H t~ r- ~ t~ ~ ~ M~
W
H

N

O
..

NO

~

U t"'-, O

~H U

r~-1r Ot O 0001 O r-I N M doI,y-1~p W

I

i a _r., .N ~-Id~LnM 01M ~ L~N l~MN

O ~-3OO OO O OM r-1NO

N W H HH HZ u1H H HH
u1 H ~ O ~

O O A DCO b ~O
H H

S-IU H aH H ~~ H HW

C-~P:C~-~H ~~ ~ HWHP'~O

Ix p W ,'OuzPa~ W C~"~.4, PaU
W

C~

A v-I~ .-ia-ic-1~ c-I~-1a--Ic-1v-1 H P~lU v-1f~alW U f~lP~f~9n1Pa U ~oP4U UU t~UU U UU

.U ow-IU~ oWO ~-I~-Ir-Iv-ioin N L M 00N Nd~O NCHIn01L~
U

.1.~.r..O p0N Lc'IM l~l~N N 00<H
O

~y-nODo0L~N Mc-IL~t1~cH~HMcH

U d~toOJl~NN o-1Lno0O L~N
O

-~aM c-1lOl0d~L!)c-Id~d~cHd~G~
~-I

H M C~OJL(7lt11t~LL~l~tL~
W

O

'C3 .,i O

NO

r~

U

A

~
Ot O wIN Mdi117l0t~01v-IN Ml0 W

W N N NN NN N NM M MM

~ 4f N
O

' ~1 ~, ~ ~ O ~
~
r j r1 4S ,!~ ~ O ~ -C,' .5=, ~-1 ~' B .!-1 U ~51 r1 U r1 r-I
~-I r1 (CS
O

.1~ -rlO O c6 M O U1 U ~I , , N Ut -a O .1-3 U1 U ~
t11 of N
ai ctf Q
L
-' 5y U1 ,-I Ql N ..~i ''Ci4-I ~ .
U ~-! -a ~', ''d .
~y " ' ~' a ' -a U
ii T' j ~ ~
, (ISN O , G' 4-1U
C N f~ ~t.Li 4-I dP a ~ N ,.C O
, US

O U ~ ,s; I .!~ O o >_,'Z$ ~
O ~ r1 2i U a ' N
r-I O N ~- to ~ N J rti ~-I tn -1J ~ ' N
~1 U ~ ~l .!~ !~ ' tai ' ''Cf '' .N
~iS

. , Q Lf ~
4-1 (IiU1 ILf I ~., , U Ul to ,S.a r1 ~ td Gi tT blrl r-1 ( ' .~, N -' ,3 ~.,'' L~
U UJ

.~ .I
N CJ -a W O M ~ O U -a a a O O ~ O , ~
',-y,' O N
N ~ ~
O O H

zi -a ~.I rti 7u N ~I ~ o .
.u a -a ~ ~-1 O u a ~ .u o ~I ~ ~I v a t~ u~
~, ~
u ~
m ~
u ~
a~
u7 . c a~ .u a~ m a~ ~ I
. .N a r-I ~s o u7 a~
a~ 1 tn a~
a .>, s~

~ O ~ ~ U ~ U N
O ~ O ~ O N
U a ~;
U N
a r1 .!~
c~
O
.h P-t ~, O (if ~1 r1 .ri ' f-t'~
4J o J ~ ,fi -.-I
A~ -~ ~ ' Fa C-7 (J7 -a ~ G 'd a ~ td N U
s~ N
rd -.-1 N

- ~ I o a rW-I aJ zi t ., .I-a ~
x td c~
N ~I
-a U 6-IU1 ~1-I r-I ~ ~ N ni ~-1 ~ -~-i ~-I td -.-f td O ',~, >r,' .U z!
N U N
.fit ~-1 ~ .!~
.h a ~.I ~u .u -a o o 5S o .u u7 ~ ~, ca .u o w ~I ~ v 3 ~1-1 ~ -a U p a~ 2i o U U rtf 23 ,t,' ~I f17 t11.~, .!-~ 'ZicIS U tLS 4-I O -C~
O ~ U1 -.-1 -.-1 .~' .l~
.t~ '' ~C N J ..R
ttf ~ -a ) cd -a tf1 ~
,.tt f~
N Tt'i -a ~
i,' a (ii , , .- -a N O
U1 .U -a ~y U7 '' U cd i ' ~1 Gi ~ ~
TLf O
r1 M N -a N a ctS N ~ N -U -a ~-t 2 h O ~ ,.c, ?a .U ..~
-rl ' N U .t~ cd ' ' rt N
N ai u:
~
U
c~

(f1 , c~ >- O W ri U d~ .~., -t-1t1-t ~ O ., U , fIf ~'.,f U ~1 ~ ~ .f~ ~-i ~., .a Lj c ~ U U CI
Cf1 S '~ -i '' -.-t f-I a O
N Fi ~-I
~
' ''(j , I ~ ~I N t~
a ~ .CI -rl -t- O ' .~
'Z3 O r1 O ciS W .!~
N -ri N ~ O U1 -a !~ ~ >~ ~-t ~ " 4 1 ~-I
H 1 O ~

, . -I- 1i W
.17 ~ ~ ., .1- ~ O
t>3 ~ - c0 -a -a ~ a ~-I 5r ~ .h U tIf ,U 4-1 -a t!? tf1 O
3 'Ti >=i U 4-1 U ' O

N
-.-1 r-i ~
U

-a tt I - O U1 -a ~, N -a G,' f-I ~-t -r1 cd U -1~
b7 U l O
b7 1 ? ~
rti O N ~- 'Zi ~ l .t~
~' '~f N

, r ] .J~ r-1O
, ~ -a r) O O .1~ U U
O ~' O .!-a ~ ~., U
~'-r ~-I 1 ~.'Tj ~2 'ii ~I
.-l ' N ctf O ~' J (l7 N
-a (!j ~ U
-! cd ~'., ' ' ., . O N r-!
-a ~ ~-I - N Ilittf 'Cf a N r .! G .' .fit r J-.1 ' ~
f~'' U1 U da N N (7S 4-I
P.t li;f ~' U
'd R
(IS .1.~
N
ttS
U
?

O
a ~I u7 ai zn u~ a~ ~ ~ ?, ., , -I
N U1 x td -a u~ , ~d -a !~ N a ~ .u -~, u I -a N .u .u ~ u7 '~ -a ~d U ~
! ~1 ) ~-I
L3) z5 U d~

. . ttf -rlFi . -.-I1 .!- U ri .~
. U) b1 4-1 O O r1 r1 -a , ice .-I Ci Tll O
'~ t r-I
td a U7 O U t ~
~' O
S-t -a O In 'z3 . . N ~ O
3-I ~ , - r1 U ~
U 1 O ~ ~ U1 S-1 N S-1 .U
-.-I ' -' ~ U ~
ctf h i v O O
U = iJ7 Li ~ ~1 a1 . . , ta ~-I U -.-i U . . S 0.i -a Cl~
N '' : f..~ Id (ti U -I Fi I
-a tti U1 ~-I ~"., i -a ,ij ~-1 7 f~-a .I-1 ~ ri O ,.Q .tiri ca ~ (., O .!-1 U (S U .~.1 U a U r1 F r1 ~-1 zi , O
-1 ',~y zi a -I

~ rt ~-1 ~n M ~ ~I .u a s~ ,-I
~ -~1s >., O a~ -a a~ ~u ~ ~t .u o t~ o .>, ~ ~d O ~n rt ~ o .u R~ ~ ~, ~I P O
s~ ~I ~n rt td I ~ a~ a r-I rn r.~
,.~ u~ .>~
-i o~
a~ a ~s O
~ ~s -a ~n ~ a~
.u rI

r - ,-I a~ -~1 4-I a~ r-1~
r-I ?-1N -a a~ ~n m ~1 ~s O f ,-1 N U1 ~ -~1 ~a U1 a N ~-I f~''CS IJ ?, ~y in a U U7 (U t~
U1 'Ci S a~
I ' . O U1 t Q l ' - -! ..Q
td l I
i-1 I N N
U , r ,. r ,.Q ~, ti$ r1 U M
c~ r 4-1 N N U O ''C~
a r- ,fit .1 O
N cd ' ~ F4 d ' cd ~-I
5 -a ~, -a v-I

c a f-1 f r-i .!.-a~-f ~ ~ , -, -a ~I ~-1 ~ M
a ~ ~ Ti O ~I U1 TS
O ~ t N O
N ~ ra b7-a -h r1 7 OJ m N ~ o1 ~ of 1 rn ~ ~
a ca d . c ~ r o o o ~ . 0 O 'q m ~ o >~ g a~ U ~1 o a r1 ca r7 ,.,~ ~ ~I
~ -a U
aai -a o~
ca o -a -a ~i ~., 7a_, it ,-I

~1 ~I i1 N ~1 z5 N c~ ~1 ~1 ~
rt 4-1zi N !~ rtf N N ~I
o N ~ N 4-1 H 4-I 4-t U ~ ~ '~ zi bt ~ ?-I u1 O -a 4 ,.q f~ N -a ~
zi ~

U
.>
;
O

, u1 U a 4-I>~
;j G,'N ~ ''~ a O -r-I -1 cd ~
17 4-I r1 ~-I '~ ~, 4.1 -a UI td ~., a N 5., l .1~
,5y 'J -'Ti -1 r .
zs zi zt U zi ~ m ~ r1 y z3 O
ra o ~ -a u~ N ~ t~ ~ ~ r-I
c~ ,~ r1 r1 ~ O
.N -a .~;
o ~I S-I
~I a ~
u7 rt a~ a~ a~ r1 ai a~ rt -a ai a~ ~
U -a U ~n a~ .1~ ~1 o ca w .U .!~ t~ ~ to ~
r1 .i.~tt$ 1 a7 ~
-a U N ) s~, .u u> 1 ~ p a~ '~ U f ' .u -h 1 ~ av c~ f U (A U ~
W ~n ~ z .>, . .t .1~t!1 it ai ~ ~ .. f-, ~-I,.4'' ~ c~ >:2, . J-1 of .L~
- -a 4-t~
3 , ~-I O
>~ f ai N
F~ O ~ ~
' ~3 ~ ~ 3~ 3 b7 rt N ~ ~I f~ H
~1 -a ~-i r-I ,--I~-I r-I N -ri (ii r-1 r1 N
.-I .1.1f31a i-1 rJl N (d ' .U
Jy O IJ7 ..~ ,~1 'Ci f(S m O ~ O U qi O O f N
O ' >_; cU ~ N
lLj ~ N a-I
O
O
~
~

~-I
U

, .1- O O .l~
UI Ui U1 a tt7 r-1 ~ ~1 tI Lf -LW
1 N ~y -a ~I ~-1 -I UI ~1 O ~ ~ U ,--1 N ~ N U
.1..7 U1 Zi U t(f Zi 5r cd U
~ ~ .~
~ 'Ii Zi O
cd -r1 -~-1-.-I a rti -a -a ~
-I t31~-1 r-1 O b1 ~-1N
.! ' ~I PW cd N N N
ZS ~., ~
cd ~ -.-1 f-I N
' Id "~ O f-i (t$ ~,' ~' 'Zf~-I
Ul ~' 4-i .!J U U r1 1.~
Ci ~ r~; FC N R3 .t~ O .L~
4-1 ~ .h -.-1 !-I 'Tj cd ?1 ,.Q O ? ,Li, N
td ' U
a,' - .1-1 U t O
O -1 .!~
rtf U ~
O ~ '' O ' >=i W . FC .l .~ ~y cn m Ul .1.t N U U , O U
~-I ~ ~ N O -r-1 ~.1 r ~ rtS
r-1 Zf ~. .!~
.u ~ ~-1 ,i, O ~ cd I ~ U
~ O 25 ~ dl ~ if7 N
N
tn .N

a m ~ ,~ ~ ~ A ~ H ~ ~ 3 0 O > -a -~' ~ ~ ~ .~
rn .N ~ .u ' o o ~ c~a ~
~ ~
o .u b) ~
In o ( '' i r ~ 237 bt-a U U N k~ 27lU
O N td r-f O ~ '~
~., Ip N >:,'' L -a U U
U O
O

~
O

>~ s~ >~ ~ >~ ~ a o ~ >~ , t37 3 ~ ua ~ ~1 ~1 a~ a~ ~n ~t ~I M ~
7u .-I ~1 ai 1~
s~
m ~I

-a -a -a rI ~1 ~ ~ o ~ -a -a 7a cd ui ~ -a o o ~i rI r1 u7 ~I ,~ U u-1-a U ' ~ o U1 U7 tn ' U1 ~,' N O U1 U7 td ZS M C5 a .!-7 U N tIS ttiU1 N ~ ~1 ~ c~ -a ~ ~U N ~ ~,' U N .4-7 -a a ~; U N ~ a~
cd ~ .-1 r1 .!-7 -.-I ~ ~I O U
~ ~ N
u-I ~ $
~ Zs 3 .u ~ it a .s, c~
~ v 3 ~ rd a~
~
~n o~atas~,a~o o ~s .Nrtsmm~U~ art-ao~ m .

a~~~
u 'd 2i ~ 1 Zs t3? a~ ~ ~d zi ,.
r-I .~ r~, o -a r~ ~-I u-70 o ai ~I ~
m ~I p ~I a r1 c~

N N N f-I N >_,' ',~ N N ~'Z$
r1 (d U2 r-1 U N ~ ~ Ci U 'Lf -1~ U d? O
,5y O O
N U Ul ' ~

~
O

-f-1 -~7.1-.7 .1.~ .-i U7 .1..7 d~
r1 U rti (fS -r-1 .7..7~'', f~ -,~r.1..1 'Ci IiS .~., O .t> U , u1 -a U i-1 UI .1-7 r-I ctiaLi ,i; U N d-7 5S 5r U ~.,"
,-i ttS U1 ?-1 t37 )ti .1~
U N O ~ r1 tIi ~ ~, t~ U H
4-I J ttf pa ~-I
U ~
t ~
!

U

- .
3 ~ ~ ',?a ~ ~ -a U (L3 ~ ~ -O N .~, W -i RS N a -r-I-O if) .~, .t~ U
S-I .!_t U1 .-1 ~ U7 1-i a1 .Q
~
U
?-I
o1 a N
L~

O -h r1 ?-IS-t ~i 'Li .U ~ S-1 ~.1 -a .I-7U (U a I O -I-.7 ',~,' U1 U7 -.-I
3-IO 1.~-a ,-I r1 ~ a .1-1O
U -a 'T~t~ .1-I rCi' U1 zf (a tU
~-i .t.1 ri (6 ~ U O ~
to r1 cai -a a '~ J..7 ..
of r-I
U7 ~
~; S-1 f-I S-I
O N
f;
-!
N
t'Wo .!-7U1N N ~., U1 U1 M U1 U ~I rti U! U1 , N U t37 cd (U O u1 U U r 3 tIS t~

~-I .t~
~
l~

~
O

>~ ~ -a ,~ s~ >~ >~ >~ r1 o .u ~ >~ .
-a ~ c> o a~ ~ 3 a~ o ~ ~ Zs o ~ m s s; ~I
~ u ~ ~ ~' i ~ .~
to .~1 u7 u -~1 u~
~I

. o o o ca a o o a~
U . .~ -~I a~ o ~ ~, ~ ca ca ,.~
~ U ,~ ~ a u7 O 3 ' ~ ~
' ' -a u7 ' g ~s u~
o ~o U .I U .~., U ~ U U .1-a ~ N ~-1 ~, ~, U U -a C d7 ~, -a N
r1 1 ~j ft$ Ul U7 7 <U O ' a ~' ~ ! -' -a 1 O 'd N o1 -' ' ' , . . Lf U
U2 , Ul - - ~., to r1 U1 ., r-I ~, TT3'G,' .i1 U~ O ~., U1 .I3 O ~., G, 'd ''(j -r! ~1 r U )-' ~, 4J 'i-5 .L !y .1-!
~,' U1 4.1 ~', O O
~1 tiS O 01 .t.~ F; td O
i "' ., -t N t~ cd ~ ~.1 tIf ''Ci r1 cti td ~
A ~-1 tr7cd U1 N r--I r-I ri O
.1--7 ~,' cti td rtf ,t ~ 7 ~d ., ~,' ~.i v-t u w-I ~-I
o J..7 ~ r1 it O
3 -I-a ~I .t~
~i .h -t~
--. 3 3 3 ~ v U u~ ~ 3 -a s ~ u ~i ~, cn o U o 0 u~
O !~ ai 7 ~ a U
F <U '1 ~ o ~u iii ' u7 m rd ' ~s a ~.I
~a o . .!- I I .R
~y-a ~ 5y Ci -1 rt N ~ a~f .-1 N .t~ I U pnri 1-IctS
O ~ ~i ~, N
U 5yrl ctS cd ~ of (1J ~ O O N ~,' ~t ~
-1-7 ' ,h ,.c,'' ~ -a W-I
S .h G,' ( N ~-i i 1-I
-' ''d '' r1 i U

-I 4-i<'I ~I tll a U d$ 0.'iU1 Ul N I~ ~ a ,.f~
-I (Ii ., .1~ ~-, r1 O
I cli ''[j (ti U ~
~, ' .l-7 .1-7 i U
U .S;
" N
~-1 tij(d (t$(p rti (I$ U O f~ lLi N ~ U ~-1 >?I 131 N N O
U Fi "~S .-I TIl a r1 O S-I Id ~ S-l O
S-1 ~t '~
-' O
1 ~' (IS

i-I-a r-t!-I !-I U O ~,' ~-1 ~-1Ulrf N , U N O r-1 O ,.C,' N :?y ,5t~-Ix-I -rI .tJ
,i., O ~-I -a r-1 ~-I tti i-I ~I N
~-I ' N
.1- c~
rl N
cti '' ~ ,q O f-1 .~ '.~' U2 ~ ~ -ar3 ~-I U1 U1 r1 O N ~ .t~ U I I ,.L~~ i-1 O . ~ ~ f..,-' U cd 'J ~ ~ rat I-I ' . 4-I
-h O ~ ~
U ~
4.t U
~
~

1-lt-l ~l t-l t-l U rti Iii a-l Fl C
~ ~.,~I -!~ ,3 S-1 ~ uM-I-~
~I ~ .~ -. ~ 4-l $ U U -1 O .~i -' -~ .~, -I ri -~ 4~-I
-1 ',03 'Ti O
f~.' ~1 O

f~

1~
C~ L1 ~

O1 M d~ L ~ N C~
O

O N ~-1 O ~ O

H ~ H
H H H

-a a ~ ~ P O H
' a m ~ -~ U r~
r~

o ~ ~
o zs ~ ~ v rt ai ~a ~ ~ .~ r1 ~a I s~ ~ ~' t ~

r o R, v ,~ u~ ~ -a o g ca la o ~a s~ ~ U v -a u o v ~s l -. r I o .u .u o <n ~ v -a ~I o c~ a ,-,~ v ~ s~ ~
~' ~ .R
-S
-a 2f I

i~ f ~

_ ~l U cd 1-1 trZ ~ U U1 ~ ~
N r N ~ -a 5y .1.~ ca N - d . '' -t , ' r-I Id N N 'i~ U ~t h L~
N N ~

~' , c -ri N ri O
~y'ZJy Ul U O N r1 <.I U1 -rl - (~ ~, i-1 ~-I .I~
, U Q~ ~ a N a r-i ~ 'C3 .!~ U ~.,'' N O .-I .I-~ U1 N O rd ~ F; '!-~' r-I .1~ -rI a a O

I -a r1 cd rt w ~ ~u v o ?m5 ~ ,?,.x .N ,-I v v -a ~I
-a o a p, N '' .u ~ .~ v 01 Ul O
U ~
l ~I
"
N O '~
J

., I a ~., t~ .l-~ U
r r1 N 4-I ~., ri .!-1 (a ~-1 f: Cl1 U7 ~I 't~
, r-1 U N J
.1 ' ~N to I -a N -a G o O -a (I$ .~., ,rS U1 a ~.,-' U ~ UI -a (d 1~ O O r-I td N ',~' r-t W l ai 'Jr-rl n 3 ~I fi l t W

r ~ ~ .u .u -,~ >-~ ~, -a m v IIS o ~ v ~I >o, r N U1 U ~ U
~ ~
o ~ ~I ~I
o (CS r-I
W 'C~ ~
U ''O f~
'~ v ~t ~ ~ _ ., j O ~ ~ '' . cCf a ~ a O ~ N ~ a FC N a ~ -.-I O f~ ~ v 2 ~ ~
u7 ~ .t~ ,.~ b~

. N
.--I ~ ',~y ((f ,~.'' , C
w-1 la O .~' -a N r-I m U1 ZS J~ U ~-I r-I
-a , U ~,'' ~ a !-I ~-i U
v ~-I r-I
~ ~-I

O I -a .l-~ a f-1 f-~ ~ ~ m ~ O r'-r ~'Ci 4-t rti r-1 U 'z3 aS ~I -a >~ ~d !
~ ctf f~ N

~1 ~ N W-I cii ',~ ~, ~ N O N a )~ a 4-1 U .
~ rI ~.,' ''a~ ~ r1 a N N >_,' -4-I O t~ m O (d 4 >=i U t~ ~-1 U I
~

- ~.I i-1 u U O -rt Zf a -a U
u1 O .. . r-1 ~'d f~ (0 O S-1 N N (IS tfS v fa ~
i-1 ~
~
-I
!
~
i a td r 4-t ,SZ t~ ~1 ~ v is f~ -a ~ Ul ccS ~ O O 'Lf ~
-r--.
N U N u1 N
4-1 a r0 ~ "
~ qi ~
S ' Ul N
t77 "' '' , a ~-I N U~ U r1 -!J cd O ~
N fir N ~., r1 r-! ~'., I 'd ? a , -I
C
.1~ N .!-~ N b~ ~ r-I
N O -a ~' P4 ~

, , C O ~ -a N .1~ U t~ ,5 ~I ~.,'' O , ~I ~-1 4-I O '~
Z3 II 4-1 ~ rti U1 v U~ N
r-I b~ ~ m u1 cIf O ctf 'd v r-1 ,.q a O v td -O , ~ -~-I ~ -a w ~ , N c~S G v cti N f~
~-! N I-t rti O ~-1 -a N N

l ~ N
5~ a >_ ' U

, O cd U U ,s~
1S 1-1 O '-' tii O .1~ r cd U ~ tti .1~ i N 1~ ~ . I
1-I , 1 , U1 N -.-I
O 1t-1 t~ .1~ 1if ''t~
(iS ~ rti . .t-1 ~;

v U U7 N .~ ~-I .N -a uy ~ rci -a O -a m .
N ~-1 O ,s~ -a O c>3 ai -J-1 U ~ ' -~-I t7~'zJ
f-I 1 ~ v ~ ~ ~ N tf~
~ ~'' U t~ cti ' ) ~.
U ''d ' - N ~i r1 U7 r-I
v , . Cj ~ f-1 ~-1 ?-I ~ N
. 'C3 -a . ' , f-n -~
f '' ' -1 -a ~ S-1 ~C -a a U!
., ~, ~ U U7 N UI O t~ Jy N .U !~, O 4-I a) ~ Cla W F~ .!~ O .u N 'i~

N .I-~ ~ O O ,5y r-I ,.'3 U~ ,C', -a O 4-i ,L1 ~, -a U U7 F; O U' f.,'' H -a .!-~ N .~.) ~ r-I iv " U7 U1 N ~
.S' LI ' ' fi N U r1 O rd O U ' 'J

. , ~ U U1 S-1 , "~5 U7 . ~ H ~-1 U1 ,L, .!~ U '~$
rd , U7 'Q
>_,' t(i ~1 >0a ~' ~-I ~ O ' -a -a >_; -~ ~ -a ~ O ~ '' ~

.1- Ti O ~-1 r1 -~-i ,R U ',.~,", N N f: -a .I t~ G; N ttS
'',r N O .1~ b1 ~-1 tti is N ?-I ~
~ U1 a td ~ ,'~ ri -a 'ti -i N .!~ ,.t''., (U

-a U .~1 ca v u~ .~ ~ m m -a 4-I s~ v 3 o tn ca ~ m -a v ~ r1 ,~ rt ~ ~I s~ :a ~ is w .I-~a U .U r1 -.-1 ,.q ~n -a r17 -a .l-1 a -ri -! O -rI
N r-! ~I O c~S -a -a cd ~ (ti ,~ a ..R U~ r-1 UI h i ' ~
Ul r1 a ~ ~1 O rtS rt3 f ~
W ~ N J
~ Zi tR ~

I . ifs cd ,.c, ~, - -I N rt ~ N U H
b~ rti I -a .U .1-.~ O
U .1~ O f-I .~ a ~d 5, ~-I
cd ~ .~-~ ~ N
~ ~ as N ~ N -a N

~ , ~ ~ ut .u m 3 v cd ~ ~, ~I ~ s~ U ~ .u ~ o v .u m a -a ~ H
~t ~r v o ~i v o ~ -a ,~ ~v m ~
~.I ~
ua~~
o I ~n ~

. . -a ~~s~zt~m zs~l?~zs~v r-I ~ ~av v , N ' , U1 U Li ''C3 O (If tIl ~' U N ''O ''(j O

., .-I 1I U U a U7 O ri N , -a ra rti b~ O N ~ ~ ~ O O fst v U ?~ tT U7 ~I ~ U N ~
~ ''Ci c o ,--I O -I~ u1 ui V ~ .'~ .~
' > cd u1 ~ ~ f~
O 3 , ' a ~
' x, a a ~,-' 4-1 cCS ~ U cd - a Ul r1 a ctf U 1-~ ~ O
N ~ -~-1 1=; TJ v ,.q -1~
J~ ri td ,~ ~ ri di ' ' "

i-1 .1.1 ,~; N r-1 . N f-1 ~ cd R~ ~1 ~ a N J-~
~ -4 ~ ,.~, '~ U O U1 a v td U 4-I ~ ~
., -a O td v ~
~ .s, z! ~ s~ .-s tai v -- o v ~I
~

. s~, ~ v v 3 o v a m U ca .u ~ > O Zs U ~ ~
U
O

~I r1 ~-I U1 U N ~ ~
4-1 ~ O r1 ,S; O U1 ~,' ~ U ~
r1 .f'., ~ ,h S~ ~
', ,5y ~ O ,-1 ~,' F4 ' v N (Y1 N ' a O 'i~ 4-I a C

l -!
l (d ~
Z

,. 4-i r1 , , ~ .1..) ~
r , -a O -a v -a r ~
r -i ~
~
~
O
~

':-J~ b14 ~Cf ~ ,~ ~,' ,.>_,'U U ~
~ -I c t,' O ~ N
d ~ u1 ~ N ,t~-I ~ '~ N ~
~ J-.~ U >w ~ N .h ~ t I ~ O
~

N U7 ~' ~', d$ , ~'.,v 4-1 r-1 O .-1 'L.,' N O .!.) (a b7 ~ , 1 'L.,' (~ r-t ~-I cd 'Cf 'i~ -of U1 (ff Td O -a rti 'a 'd rCf N .~, I ~ 4 N
' ' . O ~, N (d .1 .1.~ .t-1 1~ "~1' U~ 'Z3 N ~1 - -1 O N U v O '.'S S-I
td LS !I$ U W ~ O ~ N ,.C~-' 11 a ~ N .~., ~,' ,k, U O ~
r1 O -a Td -a v N ''d ?

, .t (iS r-I .1-1 r-l , r1 O ri W !-~
U .!..l ,'~ r-I O N UI ~,' r-1 N ~' ~1 r-I 1~
U '~ ~' W O .N N r) r1 >:'' "

-1 tIS ~

, , $
O .1-~ O ~ a ~-i a N (U . -1 U ~; ctS
crs !~' v N cti a N .~-> O ri -ri ,~ O to I U t=' U1 ' N N 4-.1 UI ~ u1 O ~y -t-W-i .U , f-I O ~ ~ Ul 7y,i; r-i ..L; -U S-I ~ ~,' N ',~ ~-1 W
~ c~ rd d tti f~H ~ ice'' a -,-I f~ a -a O O N ~ ~ ~
.!-~t~ td O N ctf I cti ia , , . - ~-1 cd 'd ~ 4 U ~
td a ~ cti tti > ; ~r O
I O ni ~
'' > - -a O O 'Zi Fi 4-t rd N I"., d, .1.~ UI O cti ,.C', Fi ~-I O ~ ~ ctf -a 1 N .~
.I N Ft ' d fy N o0 U ' ~
r1 J-> 4-1 ,C,' ~, 1 4 G' ~ N -.-I ~

. , . ~1 O .. , Jya r-I .!~
,. - P-t .h t3o-1 v ~ <v J-~ N ~ O
U ~t ~-I E-~ O N 4-! !-~ N tl~ 'Z3 -a r1 ~ 'Lf U -a s~ ~ rti ~ U -,-1 a US cc! ,~i O of tti b~ -~ 01 ,.~
U cn U ~,' a1 -a rti a ZS ~; ''U .l-~ N F,' (S (CS tn U1 ~
U -I $
~
S
S
a I

r ,-.I ~, ,' N U ~-I r-I N
> .1 N rt1 U 1.7 W -a ,7y m ~ a ~.i .. ~ -r-i d -(I
, t ~
t s ~ t~ .e, o o ~ ~.I o ra -a ~I
~ I <n a o ca a -a ~I .u >~ at m -a o o -a r1 v ~a ~ ,-I a~
u o -a ~ a z5 a o ~

. .
a , ~ ~ '~
o -a o >~ m o, -a ~ ~ a w ~ ~' '~
u ~ v 3 o s ~

o . -~ o z .I .
.u o z ., u ~ o v .u o ,.~ u~ N o 3 ~ ,~
m , ~ _ O ~, ~ .~' l~
4~ O rt3 ~ N r1 -a I ! rtf -~ ' ~I ' _ U
' b v ~
' O
~

- - ~ ~I -r-I O 1W
''C3~, r-1 rtS LS F: .~' > ~I ~ Ul , S-7 N i~ N
~ ~, '' f-1 (tf d fl ~-t O U N S~
!~ N (l5 O a Si ' ~ ~

. v t1$ ~., ~-1 ~I (d ''d . Cf ~ N r-I tJ2 -a N .h O
Zi t F.. '~ t >=,' v i~ -a zi a u~ a U7 ~ ' 1 ~ ~
' -I
-!
L
>
t~ (if o1 ., 5t ~, ~4-1 W Ul N ~ U -rl US >~' N r N O v U7 ~., U ICf N
't~ . N G,' lif O l-~ S
.. O >_ 1 - ~ ~
- ' r't5 G' ctf Cp . ' r1 O r-i N f-1 v ''Cj f~ O cd F(', N S-1 r-I
(d ''(j .1.~tfS ~I t57-a N ~-i N ''Ci, ti a ~y U U ~ .R ~-s=i N , !-~ U1 4-I ~' .!-~ a ~ ~,' ~--r-3U a .l-U ~ fi ~ ~ O O ~
'Ti >~ N >_ ' O
rI ' U1 -a -a N
l s ~ j ., U ~I N v U r1 ,?~ N f-1 ~ W
U , r-i I-I 4-I ~
. -I
r f ' ~
' ' . ; - ",.~ '~' N R3 ,~' ~ 'Wrl tIf , 5a (i N ~-I U ~ U (U '-I
o o r1 ltS Q? ,-I .>~
s~ Cj ,.1~ ~
,~S n-i V1 ",3 ~
d o ~n o, o - .u s~ ~I as v v a~ v ~C
u ,. .I ~ o ~a .u -a1~ . s~, ca H s ; as v as ~ ~ u1 ~ s~ ~ ,~ v a ~ tU
' ' . . .I-~ O U a N -N tL3 ~ , N
. ~ p, r1~ ~ .I-~ f-1 ~ cif u1 ZS N
,~-~ 1~ tn .uu~g s~.u3s~v~-I~~-I o ~ ~wvc~u~v~N o .-a~svm ~lvv~~
' T

p W, Cj O O O -a (tf O -a UI ~', r1 ~''., v ~-1 ~1 O 'ZS
v-I !~ O C3 H U $ G; U1 U U ~-' ~

a O N r-I O N -a -!.~ ~j -.-IO ~ ctE O ,'~ N ''(~ U1 ~y N ctf ~ U ~, (U ~; cd t~
I-I i-l !-." ~ ~5 a a ~

S-1U N U I-1 rI UI ciS ri .1->U ~., ''t~ rt ~
r1 4-I .-I N td v ~,' 'CS O .1~ ~i-1 ~

~

U tff -a cd ~ , .!~ U1 ~-I a N di ciS UI ~If U.t W-I O
U1.I-~ t!I r-i O .U a N N Fi O
u7 r-1 1-~ r1 ~ 4-1 U) N -! cd U7 v rt ~ v 5r !-~ N L.,-' ca a U1 4-I ~y f-I ',~ ~
u1 ' U7 UI .I~ 'Zi v ~i O .-1 N cti t~ ~, , y -a td 4-I 0? N b1 W ~ t0 cif W Y51 a U a -r-I W U ~
>_,' N O ~-1 .1~ rtf 1~ -a ,~ N (ti ~
1~ ~-1 q 3 ~ v <n s~ v .~ .u ~ o 3 ~ o .-I u a ,.~ , ,.
~ o w o ~ -a .N o~ ~s p, o uz -a ~ -a o ,.~ r1 -a o v c~ ~I ~ s~ ca E-~ ~ o u U ~s ti v ca u v ~ ~
-a zi -a ~n v t' N ~ ~ m .
.1-1O ~,' O a r1 -rl U tIf ~a UI 'r> U N (~ .u .u ~-.~
' '~C U7 O ,.~1 .~ ,~ l1 .C tl~ N zi .~
'' U~ U7 N (U
U v J.~

U7 ~ N U1 O a Q1 UI .t~ tt1 ~,' ~r (d(~ d cn cct O S.-t O ~r S~ cti N 1.~ N ~,' r1 N
ItS U. cti 4-1 u2 ~ rCi tIf cd BLS O
fIS !~ (S ~-t ~. $-i f-W.-1 U7 U O
N ~ ~ N
U7 O ~.I Ul N to a ' , W a U r-1 r1 ~-1S-I u1 O ~ U ~ N .u N , ~1 ~i ~
R U O , ~-1 cti >~ 1-I o1 r1 r-i ,t; ~1 ,f, UI cd X~1 a ~-I f-t U O rd O i cd (IS -a ~-I
S~ -rl CS O a ~tf , .1.~ N ~
~ u1 ' J-~
.t-1"~ N 1 > U1 rtf ,-1 O 2~ ' I~ fd tti o ~
' l' . , ~ r-t , -rl , , ~ v a I-~ .t. ~ U N ~I U
U . , N
P ,-Q ~
1 .R ~
~ -1 O
~ ~
~ ~
~ ~
U ~
~
O ~

l-l.~ f.~~ !-~ I -. ~ U N 40-I
~.-14 ~1 t-l U U r 'J' R~ ~
-f -r -.-~'i .~
1 r- -I C-f $ -1 ~ ~ ~
C '~ F~' --~ -r -I
-1 ~ U ,~ .~ U . ~ t~
~ ,.C, O ''~-~'T ~ 'J~ FC

H G

f f~ ' -~ W

. >
~

N M M

O

f-1O ~1 ~i H

H

H H

a ~4 1z6 -o ~ ~t s a m ~ o ~d a~ ~
~

~~ ~~
~ a~

N O ((f U r-1 .N

N U

N

d~ W
-r-I
r1 td ~1 ~
~ .s'-, O O O
~ N
N

~

N U ~
N ~-1 ~
U O

( Jl r-1 N N

(11 O
..~i ''C3 ~ (v ~' U .1.) r-1 N
.I-) --l 4-I
U 1-~
~;

N t~f O ~,' N -rI

~,' .t~
-r-1 .R .'y.' -r1 O
U1 (f( ~1 -I~
~ ~

'~ 1~

~!-~
''Ci O O
f..,'' ~
H

F N
~;

N O -~-1 (U C5~

-'~

-t-~
~
N
~, ~-I

O rt J-~
.1-~
cd O U N
N rd ~ N r1 -rl N

4a ~-1 S~ .!~
~ N

N -'-t cd ~
u!

'~ .U
.7.~
W .-i of ~ N

~'~~~

rI
''C~

O r-I
~ U
'Jv y~i m cd v N
?-I
?i r ~ ~ U
~ N

- .
a -O ~ ~, U N
N .!-~

O -~I
,~ w-1 ~I
'Zs '~i -N
~ ~i d , ~
O
~ O

r--.-I
r1 (U
-.-i cd U

~U i~~7 ~ ~
O N
~

.

O fsW1 ,~'., C~ .U
l.~
~

-i .
U
U
.t~ -O
O

~ b~
cd 'b -.-i ~~
~
~

.t-3 -r i O O O
t N
~

G'N -r ! N r~
~i "~1' ~ ~ ~
~

U
U O

-1-~N .1~

N

U1 r1 td U
',~
O

-r-1(Ii ctS
-r-f -r3 r-I
~i v 3 ~ o o ~,~ ~

m ~, v~
N ~
.N ~t A

N

U1 ,i,'' -r1 N
rtf r1 .h ~

~1U
''C~
N

N ~ ~-i cd 'C~
u1 zt ~rI,Ci HIS
O ~-I
~., r1 a E~ U

b~ rt 'z5 ~3 a O

.uU

U

N H

,5rH
~ O

-~-1H

a ~

n i W ~ N °' ,~ 1l o ~l ,p o ~ c. ° c~Gv ro v~ Y ~ i, > N
... ~. ~~ oc~a °'o~~ o .~, o o c"~ ~ ~ ~ ~ ~ oo v ~"'w ,-4 :-: rn .>~
N a w. b o o .a H o ~ ~~Wi Ww ~'.c ~ °~' .fl ~ ;d °ir on ,~ ~ v ~, y U ~ ~ O c~V ~ by ~ > ~ O
," ~ W n II ~ ~ O ..t; W , ~ ran .'O M Q
Cct ~ t~ ~ t. G ~ ~ ~ cd ~ ~, ~ O 'C O , p~~p N
1 w O ~Y ~ ~l C. 0~1 ~ 4~ GL 4~-r 0.! ~l, .-a Cry O O o0 ~ U ~ ~ , .("~. O
'~ O~ 0o ~ ~ c~
. 'r U ~ ~ '~_ ~ _ ~ ~ N .~
'-' ~ 00 ~ M ~.-~ O d: ~ a M ,~' t3~
U U U Pa '-' N ~ 00 0~o cad ~ o ~ ~ ~ o ,.7 o d A"
a U U U ~ °' ~ ~ ° ~ ~ ~ W xt ~ ~ W ~ ~ H
a.~ ao~o~ ;~~~~'.aHt~ ~, a'N ~P~

0 o U o ~ ~ m ''~ ,r; W vi N ~ ~ ~ o ..: ~ ° a w w ~ w ~;~".; ~~ ~~~ x~ ~' 3u, N o b U' v~ ~ ~' ~ o~ ~
..
U U ~ ~ ~ cct ~ "C1 ~. T1 ~ '~ ~ ~ _v ~~
a-a a..~ v) au '~' ~~..tt c~ r~~ r4 U b ~jlU~w ~ cadQ~~ cGd O ~m~U ccf ~w~, O U C/7 ~ H ~ ~'' (~ ~ N ~.U, U
w P. Ca. ~ A. ~? v~ U '~ ~ ~ .d > a :b ~~.-i ~O ~ o ~i °°
P. f~ ~'~' f3. ri ~ U at a 'L7 N U ~ ,.C""
~Nz wz~ ~~ x~~N~ ~N:~Az H
° a ,.d ~ .
U N U b ~ N O ~ ~ ~ by O w a ~ p ~ .-WG ~ '~ N ° m ~ (n '~ ~ ~ hi N ~ ~ ~ 'C V ~ ~Nr .,~ ~ O V s~S w N ~ ~ O" ~ ~ b ~ N c~ b '~~' cad ,~ ~ a N ~ .a ~' b .~ N b p ,~; N U° ~ ~ c~G ~ O" b ~
U 2j "" O r~ rn ~ ~ .a ~, N cn C ~ ~~ v c°d ° ° ~ ,'-~ v ~ '~ ~ v~ c.,~d °°
U r~ ~ ° H ~~ .~ 'o '~ a H ~ y cd r i.. U V1 4"' ~ x TJ O ~ ~ U
O ~ ~ ..~' ~ w c~nv ~ ~ ~ O . t3"
._ p ~ ~~a~ O ~;~ ~~w ~ ~
~y.., N .'-' v Pa .fl b0 ,~"' ~ P-~ ~ .O G _ m m ~ ~ O ~ a c~ N y ~ ".a, by o m ~ ~ ~ ° ~ °' ~ ~ c~ ° '~ ~ o ~ a' .~ ~ i a U
~ ~ ~ ~ .~ U .D f3, N ~ ~ '~ ~ ~ ~i~.~ ~ ° G
H O 'd ° ~ ~'i N N O O .~..~ ~ ~.' (r .r O a a .~ ..., ° .~ ''a ~ ~.a ~. ~ O ~, ° ~ O
Gp ~ ~~ ~ V ~ .~ ~ N ~ ~ ~ 'N Q ~ ~ ~ O ..-'~' TJ 'G ~ ,O O
N cUd ~ O ,.~', N N ,.~ N ~ '~-~ c~
f3~ '.,.., U U ~' ~ ...n U
'tar p vy'''n .i~ p 'v~ ~ v .~ c~ c~ y 4~ ti O ~ ~ ~ ~ b°A a a ~ w ° a. m ~~ ~ w H
U o a H
w >~ ~ ~ ~ ~ w t7 i H N
U t~
°
;" i H
a> '"' H iti ~ ~ N by V'7 N
O O II ~0 H
T.~,' V1 y rN.n 'O ~ ~ N
R ~ a'i"~" ~ ~ N M ...
O
N
a, z ~ .~ c~
(V ~ N p' N
~ vv u7 m N
~D O > ~ t~ ~ ~ sw O ~ ~ ~ vi ...0, O
~~ v~ V7 Pa ° ~ ~ cat ci~ ~ ~ ~ N bD U O b°p U ' ~ f~' TJ
'b ~ O N v w <t' ~ ~ ~ 'd ~ O ~ ~ d' ~ 7~' O v ~ N [-~ r.Wn N ~ ~ ~ ~ ~ ~ ,~ ~ N
N c'~ N ~ ~ ~ '~ ~ O ~ p ~.,.°~, ~ ~ Ti ~ 4.. ~ ~'" .a: ~ C7 ~ r p A'' vD ~ ~ Pa O °~ ~ ~ '"
n r, ~ U? .d oo '-. ~ _, C~ P.r p ~ ~r O p ~ ~ ~ YN', ~ ~ oo ~ av ° '~ o i '~ °~ ~ ,-; '~ >, .~ G ~ o v v V N ~ Ga vi C/1 d' ~ O ~ ~ ~ 01 i~,'. p ~ ~ ~ [1 ~ ~ P-~ O
c~ '~"~' N ~ by ~ ~ N 'p w.r ~'' d' H ~~iy..] y 'C~' ui ~ ~ W
a~ a Pa ~'' .-; '~ ~ b d,~' o~o r-~ '~ '~ '~ m b p°., m f~j ~ °~
~
i ~ ° w ~ ~ a'' .~ °' ° U ~ ~ ~ ~'. ~ ~ .~ a~ ~ G C7 ri a) ,~ ~Y .~ by ~ ~ ~ ~ ~"~ ~ N ~O O \O ~ O ~ .~ p O ~U ti ~'' y .-w it ~ ~ a'0.. .-i N ~ a .d tn t ~ w H N p t*,:, bA ' ~ ~'' a -d i H a~ "' ~ ~ ~ a a G ~ a~ .~ ~, ~w' ate', C~ C7 ,-., Z W oo a cn ~~ ~ c~ ri C~ Z ~ U P-. N W r° U C'J ~ Pa ~r ~
_ ~ b a v ~ ~ ~ ~ ~' ~ ° b CC3 ~ ~ ,b ~ ~ ~ :.~ v ~ ~ ~zy ~ A.
U ~N~ ~~N~ OHf3~
O b0 ~y ~ ~ ~ ~' ~ N N ~_ y,~" N
cct CT p b ~~ ~ P.i a3 . N O T~ p N
N ~ G N N Z . by v~ T1 C ~, CY d"
v ~ A .~ ~ ~ ~ ' p p O
~D ~ ,~ ~ ~ ~ ~ ° ~, O O R~
'o '~r'~ ~ ~ P", ~ 'o ~ en oA o ~ p ~ ~ a U V ~ .Cbp ,~ ~ '~ ~ 'p ~'' U by O b G", N
Vy .
a p ~ '~ ~ '~ ~ .~ a °
p ~ m ~ ~i., c~ tU.m~ ~' O u~ .fl .u U Tf .r, bu 3 ~ ~'n ~', o ~° ~ ~ ~ °~' ~~ a ~
.~ :.. '~ ~ ,b o ~c .~ .-. se ..~ o ~'O ,~ '~ U V ~ ~ ~ U ,~ ~ ~ U ~ ~ .Or., ~ ~ ~ U
a~ ~ ~ ~ y ~ o ~ ~~ o ~ .n o ~?
o ~ .fl ~ f~ o '~ a. ~"o °~,' 3 a, a. ~ °~' a~ ~ -a a, ~
~ b ~ ~ ~ U o °~' ~ a ~ ~ ~ ~ -d U

cG b O
O b pa ~ ti tH

~ Pa P-~ P., U ran -~i E-~

<11.0> INCYTE GENOMICS, INC.
AZIMZAI, Yalda BAUGHN, Mariah R.
BOROWSKY, Mark L.
DING, Li DUGGAN, Brendan ELLIOTT, Vicki GANDHI, Ameena R.
GRIFFIN, Jennifer A.
HAFALIA, April J.A.
ISON, Craig H.
KHAN, Farrah A.
LAL, Preeti LEE, Ernestine A.
LU, Dyung Aina M.
ARVIZU, Chandra POLICKY, Jennifer L.
RAMKUMAR, Jayala~ni RING, Huijun Z.
SANJANWALA, Madhu S.
TANG, Y. Tom TRIBOULEY, Catherine M.
WALIA, Narinder K.
WALSH, Roderick T.
WARREN, Bridget XU, Yuming YANG, Junming YAO, Monique YUE, Henry <120> DRUG METABOLIZING ENZYMES
<130> PI-0233 PCT
<140> To Be Assigned <141> Herewith <150> 60/236,947; 60/238,864; 60/242,323; 60/247,581; 60/249,519; 60/252,834 60/250,567 <151> 2000-09-29; 2000-10-06; 2000-10-20; 2000-11-09; 2000-11-16; 2000-11-22;

<160> 36 <170> PERL Program <210> 1 <211> 256 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472792CD1 <400> 1 Met Phe His Leu Gln Ser Pro His Val Leu Gln Met Leu Glu Lys Ser Met Arg Lys Cys Leu Pro Glu Ser Leu Lys Met Lys Gln Glu Met Thr Asp Asp Phe Asp His Tyr Thr Asn Ser Tyr His Ile Tyr Ser Lys Asp Pro Glu Asn Cys Gln Glu Cys Leu Asp Met Ser Gly Ile Ile Asn Trp Lys Gln His Leu Gln Ile Gln Ser Ser Gln Ser Arg Leu Asn Glu Val I1e G1n Ser Leu Val A1a Ala Lys Leu Val Lys Val Lys Arg Ser Gln Cys Gln Leu Tyr Glu Met Pro Glu Thr Ala Lys Lys Leu Val Pro Phe Leu Leu Glu Thr Lys Asn Leu Cys Tyr Lys Ser Gly Ile Leu Lys A1a Ile Asn Gln Glu Met Phe Lys Leu Ser Ser Leu Lys Thr Thr His Ala Ser Leu Met Asn Lys Phe Trp His Phe Gly Gly Asn Glu Arg Asn Gln Arg Phe Ile Glu Cys Cys Ile Gln Asn Leu Pro Phe Cys Cys Leu Leu Gly Pro Glu Arg Thr Thr Val Ser Trp Phe Val Met Asp His Thr Gly Glu Leu Trp Met Ala A1a Ile Met Pro Glu Ser Arg Gly Gln Gly Leu Met Ser Tyr Leu Ile Trp Ser Gln Phe Gln Ile Leu Asp Lys Leu Gly Phe Pro Leu Tyr Tyr His Ala Asp Arg Ala Asn Lys Cys Val Gln G1y Val Ser His Ala Leu His His Ile Leu Met Pro Cys Asp Gln Asn Gln <210> 2 <211> 618 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473645CD1 <400> 2 Met Val Val Val Pro Phe Thr Ile Pro Phe Asp Ser Ser Val Trp Leu Leu Gln Lys Leu Asn Ser Pro Trp Arg Met Thr Val Asp Tyr His Thr Leu Asn Gln Ala Val Thr Leu Ile Ile Ala Ala Val Leu Gly Val Ala Ser Leu Leu Glu Gln Gly Phe Ser Arg Ala Ile Trp G1u Asn Arg G1u Ser Leu Ser Lys Val Cys Val Ser Gln Gly Pro Ser Arg Pro Leu Ala Cys Ala Thr Asn Gly Asp Ile Lys Val Gln Gly Gly Pro Ser Ala Glu Gly Pro Gln Arg Asn Thr Arg Leu Gly Trp Ile Gln G1y Lys Gln Val Thr Val Leu Gly Ser Pro Val Pro Val Asn Val Phe Leu Gly Va1 Pro Phe Ala Ala Pro Pro Leu Gly Ser Leu Arg Phe Thr Asn Pro Gln Pro Ala Ser Pro Trp Asp Asn Leu Arg Glu A1a Thr Ser Tyr Pro Asn Leu Cys Leu Gln Asn Ser Glu Trp Leu Leu Leu Asp Gln His Met Leu Lys Va1 His Tyr Pro Lys Phe Gly Val Ser Glu Asp Cys Leu Tyr Leu Asn Ile Tyr Ala Pro Ala His Ala Asp Thr Gly Ser Lys Leu Pro Val Leu Val Trp Phe Pro Gly Gly Ala Phe Lys Thr Gly Ser Ala Ser Ile Phe Asp Gly Ser Ala Leu Ala Ala Tyr Glu Asp Val Leu Val Val Val Va1 Gln Tyr Arg Leu Gly Ile Phe Gly Phe Phe Thr Thr Trp Asp Gln His Ala Pro Gly Asn Trp Ala Phe Lys Asp Gln Val Ala Ala Leu Ser Trp Val Gln Lys Asn Ile Glu Phe Phe Gly Gly Asp Pro Ser Ser Val Thr Ile Phe Gly Glu Ser Ala Gly Ala Ile Ser Val Ser Ser Leu Ile Leu Ser Pro Met Ala Lys Gly Leu Phe His Lys Ala I1e Met Glu Ser Gly Val Ala Ile Ile Pro Tyr Leu Glu Ala His Asp Tyr Glu Lys Ser Glu Asp Leu Gln Val Val Ala His Phe Cys Gly Asn Asn Ala Ser Asp Ser Glu Ala Leu Leu Arg Cys Leu Arg Thr Lys Pro Ser Lys Glu Leu Leu Thr Leu Ser Gln Lys Thr Lys Ser Phe Thr Arg Val Val Asp Gly Ala Phe Phe Pro Asn Glu Pro Leu Asp Leu Leu Ser Gln Lys Ala Phe Lys Ala Ile Pro Ser Ile Ile Gly Val Asn Asn His Glu Cys Gly Phe Leu Leu Pro Met His Ile Pro Pro Gln Tyr Leu His Leu Val Ala Asn Glu Tyr Phe His Asp Lys His Ser Leu Thr Glu Ile Arg Asp Ser Leu Leu Asp Leu Leu Gly Asp Val Phe Phe Val Val Pro Ala Leu Ile Thr Ala Arg Tyr His Arg Asp Ala Gly Ala Pro Val Tyr Phe Tyr Glu Phe Arg His Arg Pro Gln Cys Phe Glu Asp Thr Lys Pro Ala Phe Val Lys Ala Asp His Ala Asp Glu Val Arg Phe Val Phe G1y Gly Ala Phe Leu Lys Gly Asp Ile Val Met Phe Glu Gly Ala Thr Glu Glu Glu Lys Leu Leu Ser Arg Lys Met Met Lys Tyr Trp Ala Thr Phe Ala Arg Thr Gly Asn Pro Asn Gly Asn Asp Leu Ser Leu Trp Pro Ala Tyr Asn Leu Thr Glu Gln Tyr Leu G1n Leu Asp Leu Asn Met Ser Leu Gly Gln Arg Leu Lys Glu Pro Arg Arg Asp Val Trp Val Thr Gly Tyr Pro Gln Pro Trp Lys Ala Ala Ile Ile Gln Asn Lys Lys Pro Arg Ser Gln Ile Leu Gly Ile Lys Gly Arg Ile Ser Asn Ala Lys Lys Lys <210> 3 <211> 342 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3348779CD1 <400> 3 Met A1a Asp Asp Leu Glu Gln G1n Ser Gln G1y Trp Leu Ser Ser Trp Leu Pro Thr Trp Arg Pro Thr Ser Met Ser Gln Leu Lys Asn Val Glu Ala Arg Ile Leu Gln Cys Leu Gln Asn Lys Phe Leu Ala Arg Tyr Val Ser Leu Pro Asn Gln Asn Lys Ile Trp Thr Val Thr Val Ser Pro Glu Gln Asn Asp Arg Thr Pro Leu Val Met Val His Gly Phe Gly Gly Gly Val Gly Leu Trp Ile Leu Asn Met Asp Ser Leu Ser Ala Arg Arg Thr Leu His Thr Phe Asp Leu Leu Gly Phe Gly Arg Ser Ser Arg Pro Ala Phe Pro Arg Asp Pra Glu Gly Ala Glu Asp Glu Phe Val Thr Ser Ile Glu Thr Trp Arg G1u Thr Met Gly Ile Pro Ser Met Ile Leu Leu Gly His Ser Leu Gly Gly Phe Leu Ala Thr Ser Tyr Ser Ile Lys Tyr Pro Asp Arg Val Lys His Leu Ile Leu Val Asp Pro Trp G1y Phe Pro Leu Arg Pro Thr Asn Pro Ser Glu Ile Arg Ala Pro Pro Ala Trp Val Lys Ala Val Ala Ser Val Leu Gly Arg Ser Asn Pro Leu Ala Val Leu Arg Val Ala Gly Pro Trp Gly Pro Gly Leu Val G1n Arg Phe Arg Pro Asp Phe Lys Arg Lys Phe Ala Asp Phe Phe Glu Asp Asp Thr Ile Ser Glu Tyr Ile Tyr His Cys Asn Ala G1n Asn Pro Ser Gly Glu Thr Ala Phe Lys A1a Met Met Glu Ser Phe Gly Trp Ala Arg Arg Pro Met Leu Glu Arg Ile His Leu Ile Arg Lys Asp Val Pro Ile Thr Met Ile Tyr Gly Ser Asp Thr Trp Ile Asp Thr Ser Thr Gly Lys Lys Val Lys Met G1n Arg Pro Asp Ser Tyr Val Arg Asp Met Glu Ile Lys Gly Ala Ser His His Val Tyr Ala Asp G1n Pro His Ile Phe Asn Ala Val Val Glu Glu I1e Cys Asp Ser Val Asp <210> 4 <211> 297 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 71680316CD1 <400> 4 Met Ala Leu His Asp Met Glu Asp Phe Thr Phe Asp G1y Thr Lys Arg Leu Ser Val Asn Tyr Val Lys Gly Ile Leu Gln Pro Thr Asp Thr Cys Asp Ile Trp Asp Lys Ile Trp Asn Phe Gln Ala Lys Pro Asp Asp Leu Leu Ile Ser Thr Tyr Pro Lys Ala Gly Thr Thr Trp Met His Glu Ile Leu Asp Met Ile Leu Asn Asp Gly Asp Val Glu Lys Cys Lys Arg Ala Gln Thr Leu Asp Arg His Ala Phe Leu Glu Leu Lys Phe Pro Leu Glu Phe Val Leu Glu Met Ser Ser Pro G1n Leu I1e Lys Thr His Leu Pro Ser His Leu Ile Pro Pro Ser I1e Trp Lys Glu Asn Cys Lys Ile Val Tyr Val A1a Arg Asn Pro Lys Asp Cys Leu Val Ser Tyr Tyr His Phe His Arg Met Ala Ser Phe Met Pro Asp Pro Gln Asn Leu Glu Glu Phe Tyr Glu Lys Phe Met Ser Gly Lys Val Val Gly Arg Ser Trp Phe Asp His Val Lys Gly Trp Trp Ala Ala Lys Asp Thr His Arg Ile Leu Tyr Leu Phe Tyr Glu Asp Ile Lys Lys Asn Pro Lys His Glu Ile His Lys Val Leu Glu Phe Leu Glu Lys Thr Leu Ser Gly Asp Val Ile Asn Lys I1e Val His His Thr Ser Phe Asp Val Met Lys Asp Asn Pro Met Ala Asn His Thr Ala Val Pro A1a His Ile Phe Asn His Ser Ile Ser Lys Phe Met Arg Lys Gly Met Pro Gly Asp Trp Lys Asn His Phe Thr Val Ala Met Asn Glu Asn Phe Asp Lys His Tyr Glu Lys Lys Met Ala Gly Ser Thr Leu Asn Phe Cys Leu Glu Ile <210> 5 <211> 320 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 868788CD1 <400> 5 Met Ser Leu Tyr Arg Ser Val Val Trp Phe Ala Lys Gly Leu Arg Glu Tyr Thr Lys Ser Gly Tyr Glu Ser Ala Cys Lys Asp Phe Val Pro His Asp Leu Glu Val Gln Ile Pro Gly Arg Va1 Phe Leu Val Thr Gly Gly Asn Ser Gly Ile Gly Lys Ala Thr Ala Leu Glu Ile Ala Lys Arg Gly Gly Thr Val His Leu Val Cys Arg Asp Gln Ala Pro A1a Glu Asp A1a Arg Gly Glu Ile Ile Arg Glu Ser Gly Asn G1n Asn Ile Phe Leu His Ile Val Asp Leu Ser Asp Pro Lys Gln Ile Trp Lys Phe Val Glu Asn Phe Lys Gln Glu His Lys Leu His Val Leu Ile Asn Asn Ala Gly Cys Met Val Asn Lys Arg Glu Leu Thr Glu Asp G1y Leu Glu Lys Asn Phe Ala A1a Asn Thr Leu G1y Va1 Tyr Ile Leu Thr Thr Gly Leu Ile Pro Val Leu Glu Lys Glu His Asp Pro Arg Va1 Ile Thr Val Ser Ser Gly Gly Met Leu Val G1n Lys Leu Asn Thr Asn Asp Leu Gln Ser Glu Arg Thr Pro Phe Asp Gly Thr Met Va1 Tyr Ala Gln Asn Lys Val Ser Glu Arg Gln G1n Val Val Leu Thr Glu Arg Trp Ala Gln Gly His Pro Ala Tle His Phe Ser Ser Met His Pro Gly Trp Ala Asp Thr Pro Gly Val Arg Gln Ala Met Pro Gly Phe His Ala Arg Phe G1y Asp Arg Leu Arg Ser Glu Ala Gln Gly Glu Asp Thr Met Leu Trp Leu A1a Leu Ser Ser Ala Ala Ala Ala Gln Pro Ser Gly Arg Phe Phe Gln Asp Arg Lys Pro Val Ser Thr His Leu Pro Leu Ala Thr Ala Ser Ser Ser Pro Ala Glu Glu G1u Lys Leu Ile Glu Ile Leu Glu Gln Leu Ala Gln Thr Phe Lys <210> 6 <211> 530 <212> P12T
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5672227CD1 <400> 6 Met Pro Gly Lys Trp Ile Ser Ala Leu Leu Leu Leu Gln Ile Ser Phe Cys Phe Lys Ser Gly Asn Cys Gly Lys Val Leu Va1 Trp Pro Met G1u Tyr Ser His Trp Met Asn Ile Lys Ile Ile Leu Glu Glu Leu Val Gln Lys G1y His Glu Val Thr Val Leu Arg Pro Ser A1a Phe Val Phe Leu Asp Pro Lys G1u Thr Ser Asp Leu Lys Phe Val Thr Phe Pro Thr Ser Phe Ser Ser His Asp Leu Glu Asn Phe Phe 80 .85 90 Thr Arg Phe Val Asn Val Trp Thr Tyr Glu Leu Pro Arg Asp Thr g5 100 105 Cys Leu Ser Tyr Phe Leu Tyr Leu Gln Asp Thr Ile Asp G1u Tyr Ser Asp Tyr Cys Leu Thr Val Cys Lys Glu Ala Val Ser Asn Lys Gln Phe Met Thr Lys Leu G1n G1u Ser Lys Phe Asp Val Val Phe Ser Asp Ala Ile Gly Pro Cys Gly Glu Leu Ile Ala Glu Leu Leu Gln Ile Pro Phe Leu Tyr Ser Leu Arg Phe Ser Pro Gly Tyr Thr Ile Glu Lys Tyr Ile Gly Gly Val Leu Phe Pro Pro Ser Tyr Val Pro Val Val Met Ser Glu Leu Ser Asp Gln Met Ile Phe Met G1u Arg I1e Lys Asn Met Ile His Met Leu Tyr Phe Asp Phe Trp Phe Gln Ile Tyr Asp Leu Lys Lys Trp Asp Gln Phe Tyr Ser Glu Val Leu Gly Arg Pro Thr Thr Leu Phe Glu Thr Met Gly Lys Ala Glu Met Trp Leu Ile Arg Thr Tyr Trp Asp Phe Glu Phe Pro Arg Pro Phe Leu Pro Asn Val Asp Phe Val Gly Gly Leu His Cys Lys Pro Ala Lys Pro Leu Pro Lys Glu Met Glu Glu Phe Val Gln Ser Ser Gly Glu Asn Gly Ile Val Val Phe Ser Leu Gly Ser Met Ile Ser Asn Met Ser Glu Glu Ser Ala Asn Met Ile A1a Ser Ala Leu Ala Gln Ile Pro Gln Lys Val Leu Trp Arg Phe Asp Gly Lys Lys Pro Asn Thr Leu Gly Ser Asn Thr Arg Leu Tyr Lys Trp Leu Pro Gln Asn Asp Leu Leu Gly His Pro Lys Thr Lys Ala Phe Ile Thr His Gly Gly Thr Asn Gly Ile Tyr Glu Ala Ile Tyr His Gly Ile Pro Met Val Gly Ile Pro Leu Phe Ala Asp Gln His Asp Asn Ile Ala His Met Lys Ala Lys Gly Ala Ala Leu Ser Val Asp Ile Arg Thr Met Ser Ser Arg Asp Leu Leu Asn A1a Leu Lys Ser Val I1e Asn Asp Pro Val Tyr Lys Glu Asn Val Met Lys Leu Ser Arg Ile His His Asp Gln Pro Met Lys Pro Leu Asp Arg Ala Val Phe Trp Ile Glu Phe Val Met Arg His Lys Gly Ala Lys His Leu Arg Val Ala A1a His Asn Leu Thr Trp Ile Gln Tyr His Ser Leu Asp Val I1e A1a Phe Leu Leu Ala Cys Val Ala Thr Val Ile Phe Ile Ile Thr Lys Phe Cys Leu Phe Cys Phe Arg Lys Leu Ala Lys Lys Gly Lys Lys Lys Lys Arg Asp <210> 7 <211> 576 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483529CD1 <400> 7 Met Trp Thr Phe Leu G1y I1e Ala Thr Phe Thr Tyr Phe Tyr Lys Lys Cys G1y Asp Val Thr Leu Ala Asn Lys Glu Leu Leu Leu Cys Val Leu Val Phe Leu Ser Leu Gly Leu Val Leu Ser Tyr Arg Cys Arg His Arg Asn Ala Gly Leu Leu Gly Arg Gln Gln Ser Ala Ala Gly Gly Ser Gln Phe Ala Ala Leu Ser Asp Ile Leu Ser Ala Leu Pro Leu Ile Gly Phe Leu Trp Ala Lys Ser Pro Pro Glu Ser Glu Lys Lys Glu Glu Pro Glu Ser Ser Lys Cys Arg Lys Glu Thr Ser Val Ser Glu Thr Thr Leu Thr Gly Ala Ala Thr Ser Val Thr Ala Ser Ser Leu Asn Asp Pro G1u Val Ile Ile Val Gly Ser Gly Val Leu Gly Ser Ala Leu Ala Thr Val Leu Ser Arg Asp Gly Arg Lys Val Thr Val Ile Glu Arg Asp Leu Lys Glu Pro Asp Arg Ile Val Gly Glu Leu Leu Gln Pro Gly Gly Tyr Arg Val Leu Lys Glu Leu Gly Leu Gly Asp Thr Val Glu Gly Phe Asn Ala His Leu Ile His Gly Tyr Ile Val His Asp Tyr Glu Ser Arg Ser Glu Val Gln Ile Pro Tyr Pro Va1 Ser Glu Asn Asn Gln Val Gln Asn Gly Val Ala Phe His His Gly Lys Phe Ile Met Ser Leu Arg Lys Ala Ala Met A1a Glu Pro Asn Val Arg Val I1e Glu Gly Val Val Leu Gln Leu Leu G1u Asp G1y Asp Thr Val Thr Gly Val Gln Tyr Lys Asp Lys Glu Thr Gly Asp Thr Lys Glu Leu His Ala Pro Leu Thr Val Val Ala Asp Gly Leu Phe Ser Lys Phe Arg Lys Asn Leu Ile Ser Ser Lys Val Ser Va1 Ser Ser His Phe Val Gly Phe Leu Met Lys Asn Ala Pro Gln Phe Lys Ala Asn Phe Ala Glu Leu Val Leu Leu Asn Pro Ser Pro Val Leu Ile Tyr Gln Ile Ser Ala Ser Glu Thr Arg Val Leu Val Asp Ile Arg Gly Glu Met Pro Arg Asn Leu Arg G1u Tyr Met Ala Glu Gln Ile Tyr Pro Gln Ile Pro Asp His Leu Lys Glu Ala Phe Leu Glu Ala Ser G1n Ser Ala Arg Leu Arg Thr Met Pro Ala Ser Phe Leu Pro Pro Ser Pro Val Asn Lys Arg Gly Val Leu Leu Leu Gly Asp Ala Tyr Asn Met Arg His Pro Leu Thr Gly Gly Gly Met Thr Val A1a Phe Lys Asp Ile Lys Leu Trp Arg Lys Leu Leu Lys G1y I1e Pro Asp Leu Tyr Asp Asp Ala Ala Ile Phe Glu Ala Lys Lys Ser Phe Tyr Trp Ala Arg Lys Thr Ser His Ser Phe Val Val Asn Ile Leu Ala Gln A1a Leu Tyr Glu Leu Phe Ser Ala Thr Asp Asp Ser Leu His G1n Leu Arg Lys Ala Cys Phe Leu Tyr Phe Lys Leu Gly Gly Glu Cys Va1 Ala Gly Pro Val Gly Leu Leu Ser Val Leu Ser Pro Asn Pro Leu Val Leu Ile Gly His Phe Phe Ala Val Ala Ile Tyr Ala Val Tyr Phe Cys Phe Lys Ser Glu Pro Trp Ile Thr Lys Pro Arg Ala Leu Leu Ser Ser Gly Ala Val Leu Tyr Lys Ala Cys Ser Va1 Ile Phe Pro Leu Ile Tyr Ser Glu Met Lys Tyr Met Val His <210> 8 <211> 187 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5521346CD1 <400> 8 Met Phe Leu Leu Leu Asn Cys Ile Val Ala Val Ser G1n Asn Met Gly Ile Gly Lys Asn Gly Asp Leu Pro Arg Pro Pro Leu Arg Asn Glu Phe Arg Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val G1u Gly Lys Gln Asn Leu Val Ile Met Gly Arg Lys Thr Trp Phe Ser Ile Pro Glu Lys Asn Arg Pro Leu Lys Asp Arg Ile Asn Leu Val Leu Ser Arg Glu Leu Lys G1u Ala Pro Gln G1y Ala His Phe Leu Ala Arg Ser Leu Asp Asp Ala Leu Lys Leu Thr G1u Arg Pro Glu Leu Ala Asn Lys Val Asp Met Ile Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His Leu G1y His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu Ser Asp Thr Phe Phe Ser Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu Pro Glu Tyr Pro Gly Val Leu Ser Asp Va1 Gln Glu Gly Lys His Ile Lys Tyr Lys Phe Glu Va1 Cys Glu Lys Asp Asp <210> 9 <211> 342 <212> PRT
<213> Homo Sapiens <220> .
<221> misc_feature <223> Incyte ID No: 71177017CD1 <400> 9 Met Ala Gly Ser Gly Gly Leu G1y G1y Gly Ala Gly Gly G1y Gln Gly Ala Gly Ala G1y Gln Gly Ala Ala Leu Arg A1a Ser Arg Ala Pro Met Leu Leu Val Ala Leu Val Leu Gly Ala Tyr Cys Leu Cys Ala Leu Pro Gly Arg Cys Pro Pro Ala Ala Arg Ala Pro Ala Pro Ala Pro Ala Pro Ser Glu Pro Ser Ser Ser Val His Arg Pro Gly Ala Pro Gly Leu Pro Leu Ala Ser Gly Pro Gly Arg Arg Arg Phe Pro Gln Ala Leu Ile Va1 Gly Val Lys Lys Gly Gly Thr Arg Ala Leu Leu Glu Phe Leu Arg Leu His Pro Asp Val Arg Ala Leu Gly Ser Glu Pro His Phe Phe Asp Arg Cys Tyr Glu Arg Gly Leu Ala Trp Tyr Arg Ser Leu Met Pro Arg Thr Leu Asp Gly G1n Ile Thr Met Glu Lys Thr Pro Ser Tyr Phe Val Thr Arg Glu Ala Pro Arg Arg Ile His Ala Met Ser Pro Asp Thr Lys Leu Ile Val Val Val Arg Asn Pro Val Thr Arg Ala Ile Ser Asp Tyr Ala Gln Thr Leu Ser Lys Thr Pro Gly Leu Pro Ser Phe Arg Ala Leu Ala Phe Arg His Gly Leu Gly Pro Val Asp Thr Ala Trp Ser Ala Val Arg Ile Gly Leu Tyr A1a Gln His Leu Asp His Trp Leu Arg Tyr Phe Pro Leu Ser His Phe Leu Phe Val Ser Gly G1u Arg Leu Val Ser Asp Pro Ala Gly Glu Val Gly Arg Val Gln Asp Phe Leu Gly Leu Lys Arg Val Val Thr Asp Lys His Phe Tyr Phe Asn Ala Thr Lys Gly Phe Pro Cys Leu Lys Lys Ala G1n Gly Gly Ser Arg Pro Arg Cys Leu Gly Lys Ser Lys Gly Arg Pro His Pro Arg Val Pro Gln Ala Val Val Arg Arg Leu Gln Glu Phe Tyr Arg Pro Phe Asn Arg Arg Phe Tyr Gln Met Thr Gly Gln Asp Phe Gly Trp Gly <210> 10 <211> 549 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Inoyte ID No: 7472836CD1 <400> 10 Met Pro Gly Lys Arg Ile Ala Val Ile Gly Ala Glu Val Ser Gly Leu Asn Ala Ile Lys Ser Cys Leu G1u Glu Gly Leu Glu Pro Thr Cys Phe Glu Gly Gly Ser Asp Ile Gly Gly Val Trp Arg Tyr Glu Val Ser Thr Ser Glu Lys Met Pro Ser Ile Tyr Lys Ser Va1 Thr Ile Asn Thr Ser Lys Glu Met Met Cys Phe Ser Asp Phe Pro Val Pro Asp His Phe Pro Asn Tyr Met His Asn Ser Lys Leu Met Asp Tyr Phe Gly Met Tyr Ala Thr His Phe Gly Leu Leu Asn Tyr Ile Arg Phe Lys Thr Glu Va1 Gln Ser Val Arg Lys His Pro Asp Phe Ser Ile Asn Gly Gln Trp Asp Val Val Val Glu Thr Glu Glu Lys Gln G1u Thr Leu Val Phe Asp Gly Val Leu Val Cys Ser Gly His His Thr Asp Pro Tyr Leu Pro Leu Gln Ser Phe Pro Gly Ile Glu Lys Phe Glu G1y Cys Tyr Phe His Ser Arg Glu Tyr Lys Ser Pro Glu Asp Phe Ser Gly Lys Arg Ile Ile Val Ile G1y Ile Gly Asn Ser Gly Val Asp Ile Ala Val Glu Leu Ser Arg Val Ala Lys Gln I1e Glu Lys Arg Pro Leu Asn Ser Arg Met Asp Cys Gly His Thr 2l5 220 225 Cys Thr Gln Ile Tyr Pro Phe Thr Tyr Met Tyr His His Val Gly Phe Leu His Cys Pro Tyr Phe Phe Leu Phe Pro Asp Gln Glu Ile Ser Ala Ser Cys Met Phe Ser Leu Ala Gly Leu Asn Pro Gly Leu Gly Leu Glu His Phe Gln Pro Leu Ile Lys Val Phe Arg Ala Leu Ser Gln His Pro Thr Val Ser Asp Asp Leu Pro Asn His Ile Ile Ser Gly Lys Val Gln Val Lys Pro Ser Val Lys G1u Phe Thr Glu Thr Asp Ala Ile Phe Glu Asp Ser Thr Val Glu Glu Asn Ile Asp Val Val I1e Phe Ala Thr Gly Tyr Ser Phe Ser Phe Ser Phe Leu Asp Gly Leu Ile Lys Val Thr Asn Asn Glu Val Ser Leu Tyr Lys Leu Met Phe Pro Pro Asp Leu Glu Lys Pro Thr Leu Ala Val Ile Gly Leu Ile Gln Pro Leu Gly Ile Ile Leu Pro Ile Ala Glu Leu Gln Ser Arg Trp Ala Thr Arg Va1 Phe Lys G1y Leu Ile Lys Leu Pro Ser Ala Glu Asn Met Met Ala Asp Ile Ala Gln Arg Lys Arg Ala Met Glu Lys Arg Tyr Val Lys Thr Pro Arg His Thr Ile Gln Val Asp His Ile Glu Tyr Met Asp Glu Ile Ala Met Pro Ala Gly Val Lys Pro Asn Leu Leu Phe Leu Phe Leu Ser Asp Pro Lys Leu Ala Met Glu Va1 Phe Phe Gly Pro Cys Thr Pro Tyr Gln Tyr His Leu His Gly Pro Glu Lys Trp Asp Gly Ala Arg Arg Ala Asn Leu Thr Gln Arg Glu Arg Ile Ile Lys Pro Leu Arg Thr Arg Ile Thr Ser Glu Asp Ser His Pro Ser Ser Gln Leu Ser Trp Ile Lys Met Ala Pro Val Ser Leu Ala Phe Leu Ala Ala Gly Leu Ala Tyr Phe Arg Tyr Thr His Tyr G1y Lys Trp Lys <210> 11 <211> 286 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7455721CD1 <400> 11 Met Lys Val Leu Leu Leu Thr Gly Leu Gly Ala Leu Phe Phe Ala Tyr Tyr Trp Asp Asp Asn Phe Asp Pro Ala Ser Leu Gln G1y Ala Arg Val Leu Leu Thr Gly Ala Asn Ala Gly Val Gly Glu Glu Leu A1a Tyr His Tyr Ala Arg Leu Gly Ser His Leu Val Leu Thr Ala His Thr GIu Ala Leu Leu Gln Lys VaI Val Gly Asn Cys Arg Lys Leu Gly Ala Pro Lys Val Phe Tyr Ile Ala Ala Asp Met Ala Ser Pro Glu Ala Pro Glu Ser Val Val Gln Phe Ala Leu Asp Lys Leu Gly Gly Leu Asp Tyr Leu Val Leu Asn His Ile Gly Gly Ala Pro Ala Gly Thr Arg Ala Arg Ser Pro Gln Ala Thr Arg Trp Leu Met Gln Val Asn Phe Val Ser Tyr Val Gln Leu Thr Ser Arg Ala Leu Pro Ser Leu Thr Asp Ser Lys G1y Ser Leu Val Val Val Ser Ser Leu Leu Gly Arg Val Pro Thr Ser Phe Ser Thr Pro Tyr Ser Ala Ala Lys Phe Ala Leu Asp Gly Phe Phe Gly Ser Leu Arg Arg Glu Leu Asp Val Gln Asp Val Asn Val Ala Ile Thr Met Cys Val Leu Gly Leu Arg Asp Arg Ala Ser Ala A1a Glu Ala Val Arg Gly Val Thr Arg Val Lys Ala Ala Pro Gly Pro Lys Ala Ala Leu Ala Val Ile Arg Gly Gly Ala Thr Arg Ala Ala Gly Val Phe Tyr Pro Trp Arg Phe Arg Leu Leu Cys Leu Leu Arg Arg Trp Leu Pro Arg Pro Arg Ala Trp Phe Ile Arg Gln Glu Leu Z~sn Val Thr Ala Ala Ala 275 280 , 285 Ala <210> 12 <211> 525 <212> PRT

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472837CD1 <400> 12 Met Arg Lys Lys Asn Ala Val Ile Gly Ala Gly Val Ser Gly Leu Asp Ala Ile Lys Ser Cys Leu Glu Glu G1y Leu Glu Pro Val Cys Phe Glu Lys Ser Asn Glu Ile Gly Gly Leu Trp Arg Tyr Gln Glu Thr Pro Glu Ser Gly Arg Pro Gly Ile Tyr Lys Ser Met Ile Phe Asn Ala Ser Lys Glu Thr Thr A1a Phe Ser Asp Tyr Pro Phe Pro Asp His Tyr Pro Asn Tyr Leu His Asn Ser Lys Met Met Glu Tyr Leu Arg Met Tyr Thr Arg His Phe His Leu Met Lys His Ile Gln Phe Lys Va1 Cys Arg Va1 Arg Lys His Pro Asp Phe Ser Ser Ser 110 115 , 120 Gly Gln Trp Asp Val Val Val Glu Thr Asp Gly Lys G1n Lys Ser Tyr Val Phe Asp G1y Ile Met Thr Cys Ser Gly Tyr Tyr Asn Ser Lys Cys Leu Pro Leu Lys Asp Phe Pro Gly Ile Glu Asn Phe Gln Gly Pro Tyr Leu His Thr Ser Ala Tyr Lys His Leu Asp Tyr Phe Val Gly Lys Arg Val Val Val Val Ser Ile G1u Asp Ser Gly Ala Asp Val Ala Gly Glu Ile Ser His Val Ala G1u Gln Val Phe Leu Ser Thr Arg Gln Gly Ala Trp Ile Trp Asn Arg Val Trp Asp Asn Gly Asn Pro Leu Asp I1e Thr Leu Phe Thr His Tyr Asn Arg Ile Met Glu Lys Ile Phe Pro Thr Phe Met Ile Asn Arg Trp Val Glu Asn Lys Ser Asn Ala Arg Leu Asn His Asp Asn Tyr Gly Leu Gln Pro Gln His Arg Phe Leu Ser His G1n Ala Thr Val Asn Asp Asp Leu Pro Asn His Ile Ile Ser Gly Arg Val Leu Met Lys Pro Asp Met Arg Glu Phe Thr Thr Thr Ser A1a Phe Phe Glu Asp Gly Thr Glu Lys Asn Ile Asp Ala Val Ile Phe Ala Thr Gly Tyr Thr Leu Ser Phe Pro Phe Leu Glu Asp Asp Ser Ala Ile Leu Asp Ser Gln Tyr Ser Val Phe Lys Phe Met Phe Pro Pro Gln Leu Glu Lys Pro Thr Leu Ser Phe Ile Gly Ile Leu G1n Pro Val Gly Ala Ile Ile Pro I1e Ser Glu Leu Gln Ser Arg Cys Ala Val Gln Val Phe Glu Gly Leu Lys Lys Leu Leu Ser Thr Ser Ala Met Ile Ala Asp Ile Asn Arg Arg Lys Lys Lys Met Ala Lys Arg Phe Ile Lys Ser Pro Arg Asp Thr His Gln Val Pro Cys Ile Asp Tyr Met Asp Glu Ile Ala Leu Asn I1e Arg Val Lys Pro Asn Leu Phe Ser Leu Leu Leu Trp Asp Thr Arg Leu Ala Lys Glu Ile Phe Tyr Gly Pro Cys Thr Ala Cys Gln Tyr Cys Leu Gln Gly Pro G1y Lys Trp Thr Arg A1a G1n Lys Ala Ile Leu Thr Glu Arg Asp Arg Ile Leu Lys Pro Leu Arg Thr Arg Val Val Lys His Thr Arg Pro Ser Ser Ser Ser Leu Cys Cys Pro Phe Ser Leu Cys Phe His Val Arg His Pro Cys Gln <210> 13 <211> 523 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484241CD1 <400> 13 Met Val Gly Gln Arg Val Leu Leu Leu Val Ala Phe Leu Leu Ser Gly Val Leu Leu Ser Glu Ala Ala Lys Ile Leu Thr Ile Ser Thr Leu Gly Gly Ser His Tyr Leu Leu Leu Asp Arg Val Ser Gln Ile Leu Gln Glu His Gly His Asn Val Thr Met Leu His Gln Ser Gly Lys Phe Leu Ile Pro Asp Ile Lys G1u Glu Glu Lys Ser Tyr Gln Val Ile Arg Trp Phe Ser Pro Glu Asp His Gln Lys Arg Ile Lys Lys His Phe Asp Ser Tyr Ile Glu Thr Ala Leu Asp Gly Arg Lys Glu Ser Glu Ala Leu Val Lys Leu Met Glu Ile Phe Gly Thr Gln 110 11.5 120 Cys Ser Tyr Leu Leu Ser Arg Lys Asp Ile Met Asp Ser Leu Lys Asn Glu Asn Tyr Asp Leu Val Phe Val Glu Ala Phe Asp Phe Cys Ser Phe Leu Ile Ala Glu Lys Leu Val Lys Pro Phe Val Ala Ile Leu Pro Thr Thr Phe Gly Ser Leu Asp Phe Gly Leu Pro Ser Pro Leu Ser Tyr Val Pro Val Phe Pro Ser Leu Leu Thr Asp His Met Asp Phe Trp Gly Arg Val Lys Asn Phe Leu Met Phe Phe Ser Phe Ser Arg Ser Gln Trp Asp Met Gln Ser Thr Phe Asp Asn Thr Ile Lys G1u His Phe Pro Glu Gly Ser Arg Pro Val Leu Ser His Leu Leu Leu Lys Ala Glu Leu Trp Phe Va1 Asn Ser Asp Phe Ala Phe Asp Phe Ala Arg Pro Leu Leu Pro Asn Thr Val Tyr Ile Gly Gly Leu Met Glu Lys Pro Ile Lys Pro Val Pro Gln Asp Leu Asp Asn Phe Ile Ala Asn Phe Gly Asp Ala Gly Phe Val Leu Val Ala Phe G1y Ser Met Val Asn Thr Cys Gln Asn Pro G1u Ile Phe Lys Glu Met Asn Asn Ala Phe Ala His Leu Pro Gln Gly Val Ile Trp Lys Cys Gln Cys Ser His Trp Pro Lys Asp Val His Leu Ala Ala Asn Val Lys Ile Val Asp Trp Leu Pro Gln Ser Asp Leu Leu Ala His Pro Ser Ile Arg Leu Phe Val Thr His G1y Gly Gln Asn Ser Ile Met Glu Ala I1e G1n His Gly Val Pro Met Val Gly Ile Pro Leu Phe Gly Asp Gln Pro Glu Asn Met Val Arg Val Glu Ala Lys Lys Phe Gly Val Ser Ile Gln Leu Lys Lys Leu Lys Ala Glu Thr Leu Ala Leu Lys Met Lys Gln Ile Met Glu Asp Lys Arg Tyr Lys Ser Ala Ala Val Ala Ala Ser Val Ile Leu Arg Ser His Pro Leu Ser Pro Thr Gln Arg Leu Val Gly Trp Ile Asp His Val Leu Gln Thr Gly Gly Ala Thr His Leu Lys Pro Tyr Val Phe Gln Gln Pro Trp His Glu Gln Tyr Leu Leu Asp Val Phe Val Phe Leu Leu Gly Leu Thr Leu Gly Thr Leu Trp Leu Cys Gly Lys Leu Leu Gly Met Ala Val Trp Trp Leu Arg Gly Ala Arg Lys Val Lys Glu Thr <210> 14 <211> 353 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484251CD1 <400> 14 Met Va1 Leu Lys Trp Ala Ser Val Leu Leu Leu Ile His Leu Ser Cys Tyr Phe Ser Ser Gly Ser Cys Gly Lys Val Leu Val Trp Ala Thr Glu Tyr Ser Leu Trp Met Asn Met Lys Thr Ile Leu Lys Glu Leu Val G1n Arg Gly His Glu Val Thr Val Leu Ala Ser Ser Ala Ser Ile Leu Phe Asp Pro Asn Asp Ser Ser Thr Leu Lys Leu Glu Val Tyr Pro Thr Ser Leu Thr Lys Thr Glu Phe G1u Asn Ile Val Met Gln Gln Val Lys Arg Asn Val Asp Phe Val Gly Gly Phe His Cys Lys Pro Ala Lys Pro Leu Pro Lys Glu Met G1u G1u Phe Val Gln Ser Ser Gly Glu Asn Gly Val Val Val Phe Ser Leu Gly Ser Met Val Ser Asn Met Thr Ala Glu Arg Ala Asn Va1 Ile Ala Thr Ala Leu Ala Lys I1e Pro Gln Lys Val Leu Trp Arg Phe Asp G1y Asn Lys Pro Asp Ala Leu Gly Leu Asn Thr Arg Leu Tyr Lys Trp Ile Pro Gln Asn Asp Leu Leu Gly His Pro Lys Thr Arg A1a Phe Ile Thr His Gly Gly Ala Asn Gly Ile Tyr Glu Ala Ile Tyr His Gly Ile Pro Met Val Gly I1e Pro Leu Phe Trp Asp Gln Pro Asp Asn Ile A1a His Met Lys Ala Lys Gly Ala Ala Val Arg Leu Asp Phe His Thr Met Ser Ser Thr Asp Leu Leu Asn Ala Leu Lys Thr Val Ile Asn Asp Pro Ser Tyr Lys Glu Asn Val Met Lys Leu Ser Arg Ile Gln His Asp Gln Pro Val Lys Pro Leu Asp Arg Ala Val Phe Trp I1e Glu Phe Val Met Cys His Lys Gly Ala Lys His Leu Arg Val Ala Ala Arg Asp Leu Thr Trp Phe Gln Tyr His Ser Leu Asp Val Ile G1y Phe Leu Leu Ala Cys Val Ala Thr Val Leu Phe Ile Ile Thr Lys Cys~Cys Leu Phe Cys Phe Trp Lys Phe Ala Arg Lys Gly Lys Lys Gly Lys Arg Asp <210> 15 <211> 525 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473890CD1 <400> 15 Met Gln Thr Ser Ala Met Ala Leu Leu Ala Arg Ile Leu Arg Ala Gly Leu Arg Pro Ala Pro Glu Arg Gly Gly Leu Leu Gly Gly Gly Ala Pra Arg Arg Pro Gln Pro Ala Gly A1a Arg Leu Pro Ala Gly Ala Arg Ala Glu Asp Lys Gly Ala Gly Arg Pro Gly Ser Pro Pro Gly Gly Gly Arg Ala Glu Gly Pro Arg Ser Leu Ala Ala Met Pro Gly Pro Arg Thr Leu Ala Asn Leu Ala Glu Phe Phe Cys Arg Asp Gly Phe Ser Arg Ile His Glu Ile Gln Gln Lys His Thr Arg Glu Tyr Gly Lys Ile Phe Lys Ser His Phe Gly Pro Gln Phe Val Val Ser Ile Ala Asp Arg Asp Met Val Ala Gln Val Leu Arg A1a Glu 125 ~ 130 135 Gly Ala Ala Pro G1u Arg Ala Asn Met Glu Ser Trp Arg G1u Tyr Arg Asp Leu Arg Gly Arg Ala Thr Gly Leu Ile Ser Ala Glu Gly Glu Gln Trp Leu Lys Met Arg Ser Val Leu Arg Gln Arg Ile Leu Lys Pro Lys Asp Val Ala Ile Tyr Ser Gly Glu Val Asn Gln Val Ile Ala Asp Leu Ile Lys Arg Ile Tyr Leu Leu Arg Ser Gln A1a Glu Asp Gly Glu Thr Val Thr Asn Val Asn Asp Leu Phe Phe Lys Tyr Ser Met Glu Gly Val Ala Thr Ile Leu Tyr Glu Ser Arg Leu 230 . 235 240 Gly Cys Leu Glu Asn Ser Ile Pro Gln Leu Thr Val Glu Tyr I1e Glu Ala Leu Glu Leu Met Phe Ser Met Phe Lys Thr Ser Met Tyr Ala Gly Ala Ile Pro Arg Trp Leu Arg Pro Phe Ile Pro Lys Pro Trp Arg Glu Phe Cys Arg Ser Trp Asp Gly Leu Phe Lys Phe Ser Gln Ile His Val Asp Asn Lys Leu Arg Asp Ile Gln Tyr Gln Met Asp Arg Gly Arg Arg Val Ser Gly Gly Leu Leu Thr Tyr Leu Phe Leu Ser Gln Ala Leu Thr Leu Gln Glu Ile Tyr Ala Asn Val Thr Glu Met Leu Leu Ala Gly Va1 Asp Thr Thr Ser Phe Thr Leu Ser Trp Thr Val Tyr Leu Leu Ala Arg His Pro G1u Val Gln Gln Thr Val Tyr Arg Glu I1e Val Lys Asn Leu G1y Glu Arg His Val Pro Thr Ala Ala Asp Val Pro Lys Val Pro Leu Va1 Arg A1a Leu Leu Lys Glu Thr Leu Arg Leu Phe Pro Val Leu Pro Gly Asn Gly Arg Val Thr Gln Glu Asp Leu Val Ile Gly Gly Tyr Leu Ile Pro Lys Gly Thr Gln Leu Ala Leu Cys His Tyr Ala Thr Ser Tyr Gln Asp G1u Asn Phe Pro Arg Ala Lys Glu Phe Arg Pro Glu Arg Trp Leu Arg Lys Gly Asp Leu Asp Arg Val Asp Asn Phe Gly Ser Ile Pro Phe Gly His Gly Val Arg Ser Cys Ile Gly Arg Arg Ile Ala Glu Leu Glu Ile His Leu Va1 Val Ile Gln Val Gly Arg Gly Gly Gln Thr Leu Ala Phe Gly Phe Gly Leu Phe Leu Leu G1y Thr Thr Gly <210> 16 <211> 314 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484470CD1 <400> 16 Met Leu Val Ser G1y Arg Arg Arg Leu Leu Thr Ala Leu Leu Gln Ala Gln Lys Trp Pro Phe Gln Pro Ser Arg Asp Met Arg Leu Val Gln Phe Gln Ala Pro His Leu Va1 Gly Pro His Leu Gly Pro Glu Met G1u Asn Gly Gly Arg Val Ile Asn Leu Asn Ala Phe Asp Ser Met Leu Pro Lys Met Met Thr Gln Phe Leu Glu Gln Gly Glu Ala I1e Leu Ser Val Ala Arg Arg Ala Leu Gly A1a G1n Leu Pro Val Leu Pro Trp Leu Glu Val Thr Ser Leu Ala Pro Ala Thr Trp Pro Asp Lys Val Va1 Cys Val Gly Met Asn Tyr Val Asp His Cys Lys Glu Gln Asn Val Pro Val Pro Thr Glu Pro Ile Asn Phe Ser Lys Phe Ala Asn Ser Ile Val Glu Pro Tyr Asp Glu Met Val Leu Pro Ser Glu Ser Gln Glu Val Asp Trp Glu Val Glu Gln Ala Val Val T1e Gly Lys Lys Gly Lys His Ile Lys Ala Thr Asp Ala Met Ala His Val Ala Gly Phe Thr Ala Ala His Asn Val Ser Ala His Asp Trp Gln Met Arg His Asp Gly Lys Gln Trp Leu Leu Gly Lys Thr Phe Asn Thr Phe Tyr Pro Leu Gly Leu A1a Leu Val Thr Lys Asp Ser Val Ala Asp Ala His Ile Leu Lys Thr Cys Cys Gln Val Asn Gly Glu Val Val Gln Ser Asn Asn Thr Asn Gln Met Val Val Lys Thr Glu Glu Arg Ile Ala Trp Val Ser Gln Phe Val Thr Phe Tyr Pro Gly Asp Val Ile Leu Thr Gly Thr Ser Pro Gly Val G1y Val Phe Arg Lys Pro Ser Val Phe Leu Lys Lys Gly Asp G1u Val Gln Cys Glu Ile Glu Glu Leu Gly Val Ile Ile Ser Lys Val Val <210> 17 <211> 315 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 318172CD1 <400> 17 Met A1a Leu Glu Leu Tyr Met Asp Leu Leu Ser Ala Pro Cys Arg Ala Val Tyr Ile Phe Ser Lys Lys His Asp Ile Gln Phe Asn Phe Gln Phe Val Asp Leu Leu Lys Gly His His His Ser Lys G1u Tyr Ile Asp Ile Asn Pro Leu Arg Lys Leu Pro Ser Leu Lys Asp Gly Lys Phe Ile Leu Ser Glu Ser Pro Gln Leu Leu Leu Thr Pro Gln Gly Ser Phe Pro Ala Leu Thr Asp Cys Gln Thr Asp Ser Ser Gly Glu Gly Leu Ser Arg Gly Arg Leu Gly Ser Ser Leu Leu Trp Lys Pro Gln Gln Leu Gly Pro Gly His Thr Gly Val Val Asp Val Val Ala Leu Arg Lys Ala Arg Ala Val Pro Leu Thr Gly Pro Phe Cys Ser Ala A1a Ile Leu Tyr Tyr Leu Cys Arg Lys Tyr Ser Ala Pro Ser His Trp Cys Pro Pro Asp Pro His A1a Arg A1a Arg Val Asp Glu Phe Val A1a Trp Gln His Thr A1a Phe Gln Leu Pro Met Lys Lys Ile Val Trp Leu Lys Leu Leu Ile Pro Lys Ile Thr Gly Glu G1u Val Ser Ala Glu Lys Met Glu His Ala Val Glu Glu Val Lys Asn Ser Leu Gln Leu Phe Glu Glu Tyr Phe Leu Gln Asp Lys Met Phe Ile Thr Gly Asn Gln Ile Ser Leu Ala Asp Leu Val Ala Val Val Glu Met Met Gln Pro Met Ala Ala Asn Tyr Asn Val Phe Leu Asn Ser Ser Lys Leu Ala Glu Trp Arg Met Gln Val Glu Leu Asn Ile Gly Ser Gly Leu Phe Arg Glu Ala His Asp Arg Leu Met G1n Leu Ala Asp Trp Asp Phe Ser Thr Leu Asp Ser Met Val Lys Glu Asn Ile Ser Glu Leu Leu Lys Lys Ser Ser Trp Leu Trp Pro Leu <210> 18 <211> 343 <212> PRT
<213> Homo Sapiens <220>
<22l> misc_feature <223> Incyte ID No: 7484475CD1 <400> 18 Met Leu Ala Thr Arg Leu Ser Arg Pro Leu Ser Arg Leu Pro Gly Lys Thr Leu Ser Ala Cys Asp Arg Glu Asn Gly Ala Arg Arg Pro Leu Leu Leu Gly Ser Thr Ser Phe Ile Pro Ile Gly Arg Arg Thr Tyr Ala Ser A1a Ala Gly Pro Val Gly Ser Lys Ala Val Leu Val Thr Gly Cys Asp Ser Gly Phe Gly Phe Ser Leu Ala Lys His Leu His Ser Lys Gly Phe Leu Val Phe A1a Gly Cys Leu Met Lys Asp Lys Gly His Asp Gly Val Lys Glu Leu Asp Ser Leu Asn Ser Asp Arg Leu Arg Thr Val Gln Leu Asn Val Cys Ser Ser ~Glu Glu Val Glu Lys Val Va1 Glu Ile Val Arg Ser Ser Leu Lys Asp Pro Glu Lys Gly Met Trp Gly Leu Val Asn Asn A1a Gly Ile Ser Thr Phe Gly Glu Val Glu Phe Thr Ser Leu Glu Thr Tyr Lys Gln Val Ala Glu Val Asn Leu Trp Gly Thr Val Arg Met Thr Lys Ser Phe Leu Pro Leu Ile Arg Arg Ala Lys Gly Arg Va1 Val Asn Ile Ser Ser Met Leu Gly Arg Met Ala Asn Pro Ala Arg Ser Pro Tyr Cys I1e Thr Lys Phe Gly Val G1u Ala Phe Ser Asp Cys Leu Arg Tyr Glu Met Tyr Pro Leu Gly Val Lys Val Ser Va1 Val Glu Pro Gly Asn Phe Ile A1a Ala Thr Ser Leu Tyr Ser Pro Glu Ser Ile Gln Ala 245 250 ~ 255 I1e Ala Lys Lys Met Trp Glu Glu Leu Pro Glu Val Val Arg Lys Asp Tyr Gly Lys Lys Tyr Phe Asp Glu Lys Ile Ala Lys Met Glu Thr Tyr Cys Ser Ser Gly Ser Thr Asp Thr Ser Pro Val Ile Asp Ala Val Thr His Ala Leu Thr Ala Thr Thr Pro Tyr Thr Arg Tyr His Pro Met Asp Tyr Tyr Trp Trp Leu Arg Met Gln Ile Met Thr His Leu Pro Gly Ala Ile Ser Asp Met Ile Tyr Ile Arg <210> 19 <211> 970 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472792CB1 <400> 19 atgttccatt tgcagagtcc ccatgtactg cagatgctag agaaatccat gaggaagtgc 60 ctccctgaat ccctaaagat gaaacaggag atgacagatg actttgatca ctacaccaac 120 agctaccata tctattctaa agatcccgag aactgtcaag aatgccttga catgtcaggt 180 atcatcaact ggaaacaaca tttgcagatc caaagctcac agtccagact gaatgaggta 240 atacaaagtc ttgtagctgc taaattggtc aaagtcaaaa gatcacaatg ccagctttat 300 gaaatgcctg agacagcaaa gaaactggtt ccctttctgc tagagacaaa gaacttatgt 360 tataaatctg gaatacttaa ggccattaac caagagatgt ttaaactctc atctctgaaa 420 accacccatg cttccttgat gaataaattc tggcattttg gtggcaatga gaggaaccag 480 agattcattg agtgctgtat tcagaacctc ccattctgct gtctgctggg gcctgaaagg 540 accacggtgt cctggtttgt aatggaccat actggagagc tgtggatggc agccatcatg 600 cctgagtccc ggggccaggg cctcatgtcc tatcttatct ggtcccagtt ccagattctg 660 gacaaacttg gcttccccct atattaccat gcagacagag ccaacaaatg tgtacagggt 720 gtaagtcatg ctctgcatca tattctcatg ccctgtgacc agaaccaatg aaactgtgtt 780 tctctgtgaa gctagccctg aacataagac agtgttgtgt ggaatgtgca tgcgagtgga 840 ggagtgtatg gtggacagag agaaaggatg aattttaata aacaggaaag gagtggatga 900 tatttgggct ctggggaagc agtgtgaatg gtcaataggt cttccttggt ccctgcactt 960 gaattctcct <210> 20 <211> 1857 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473645CB1 <400> 20 atggtggtgg tgcctttcac aattccattc gattcatcag tctggctgct gcagaagttg 60 aacagcccct ggagaatgac tgtagactac cacacactca accaagcggt aactctgatc 120 atagctgctg tgctaggtgt ggcatccctg cttgagcagg gtttcagcag agcaatctgg 180 gagaacaggg agagcctatc aaaggtctgt gtctcccagg ggccatccag gccactggcc 240 tgtgctacta atggggacat caaggtccaa ggagggcctt ctgctgaagg gccacagagg 300 aacaccaggc tgggatggat tcagggcaag caagtcactg tgctgggaag ccctgtgcct 360 gtgaacgtgt tcctcggagt CCCCtttgCt gCtCCCCCgC tgggatccct gcgatttacg 420 aacccgcagc ctgcatcgcc ctgggataac ttgcgagaag ccacctccta ccctaatttg 480 tgcctccaga actcagagtg gctgctctta gatcaacata tgctcaaggt gcattacccg 540 aaattcggag tgtcagaaga ctgcctctac ctgaacatct atgcgcctgc ccacgccgat 600 acaggctcca agctccccgt cttggtgtgg ttcccaggag gtgccttcaa gactggctca 660 gcctccatct ttgatgggtc cgccctggct gcctatgagg acgtgctggt tgtggtcgtc 720 cagtaccggc taggaatatt tggtttcttc accacatggg atcagcatgc tccggggaac 780 tgggccttca aggaccaggt ggctgctctg tcctgggtcc agaagaacat cgagttcttc 840 ggtggggacc ccagctctgt gaccatcttt ggcgagtccg cgggagccat aagtgtttct 900 agtcttatac tgtctcccat ggccaaaggc ttattccaca aagccatcat ggagagtggg 960 gtggccatca tcccttacct ggaggcccat gattatgaga agagtgagga cctgcaggtg 1020 gttgcacatt tctgtggtaa caatgcgtca gactctgagg ccctgctgag gtgcctgagg 1080 acaaaaccct ccaaggagct gctgaccctc agccagaaaa caaagtcttt cactcgagtg 1140 gttgatggtg ctttctttcc taatgagcct ctagatctat tgtctcagaa agcatttaaa 1200 gcaattcctt ccatcatcgg agtcaataac cacgagtgtg gcttcctgct gcctatgcac 1260 atcccgcctc agtatttgca ccttgtggct aatgaatact tccatgacaa gcactccctg 1320 actgaaatcc gagacagtct tctggacttg cttggagatg tgttctttgt ggtccctgca 1380 ctgatcacag ctcgatatca cagagatgct ggtgcacctg tctacttcta tgagtttcgg 1440 caccggcctc agtgctttga agacacgaag ccagcttttg tcaaagccga ccacgctgat 1500 gaagtccgct ttgtgttcgg tggtgccttc ctgaaggggg acattgttat gttcgaagga 1560 gccacggagg aggagaagtt actgagccgg aagatgatga aatactgggc tacctttgct 1620 cgaacCggga atcctaatgg gaacgacctg tctctgtggc cagcttataa tctgactgag 1680 cagtacctcc agctggactt gaacatgagc ctcggacaga gactcaaaga accgcggaga 1740 gatgtgtggg tgacggggta tcctcagcca tggaaagctg ccatcatcca gaataaaaaa 1800 cctagaagtc aaattctagg catcaagggt cggatcagca atgccaagaa gaaatga 1857 <210> 21 <211> 1468 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3348779CB1 <400> 21 acatcctttc ctcccacccc atacacactg ttaattagga atcagtgggc tcaatgtagc 60 aggctctggg aagatcctga atccttcttg ggctccatga gggaggtggg acaagctgtc 120 cattctactt acactggaag atcctggctt ccacattctt cagctgagac atggaagtga 180 ggcgctgggc gggctcgcca ggacctggca aggcttgttt actatggccg atgatctgga 240 gcagcagtct caaggctggc tgagtagctg gctgcccacg tggcgcccca cttccatgtc 300 tcagctgaag aatgtggaag ccaggatcct ccagtgtctc cagaataagt tcctggccag 360 atatgtatcc ctcccaaacc agaataagat ctggacggtg actgtgagcc ccgagcaaaa 420 cgaccgcacc cccttggtga tggtgcatgg ttttgggggc ggcgtgggtc tctggatcct 480 caacatggac tcactgagtg cccgccgcac actgcacacc ttcgatctgc ttggcttcgg 540 gcgaagctca aggccagcat tcccaaggga cccggagggg gctgaggatg agtttgtgac 600 atcgatagag acatggcggg agaccatggg gatccccagc atgatcctcc tggggcacag 660 tttgggagga ttcctggcca cttcttactc aatcaagtac cctgatagag ttaaacacct 720 catcctggtg gacccatggg gctttcccct ccgaccaact aaccccagtg agatccgtgc 780 acccccagcc tgggtcaaag ccgtggcatc tgtcctagga cgttccaatc cattggctgt 840 tcttcgagta gctgggccct gggggcctgg tctggtgcag cgattccggc cggacttcaa 900 acgcaagttt gcagacttct ttgaagatga taccatatca gagtatattt accactgcaa 960 cgcacagaat cccagtggtg agacagcatt caaagccatg atggagtcct ttggctgggc 1020 ccggcgccct atgctggagc gaattcactt gattcgaaaa gatgtgccta tcactatgat 1080 ctacgggtcc gacacctgga tagataccag tacgggaaaa aaggtgaaga tgcagcggcc 1140 ggattcctat gtccgagaca tggagattaa gggtgcctcc caccatgtct atgctgacca 1200 gccacacatc ttcaatgctg tggtggagga gatctgcgac tcagttgatt gagctgctct 1260 ctgaagagga agaggagaaa gccagagagt cactcttacc tccctgtctg cttactcacc 1320 cactctgtcc tttcctcacc aactaacatg tgccagccag gcagagtctt gtgctgttcc 1380 cagaacagga cgacagtgaa aagaacactc ttgaccctac actgaaggct gaaggcagaa 1440 gccacaagag gccttgagtg ccaccccc 1468 <210> 22 <211> 1134 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 71680316CB1 <400> 22 atggccttac acgacatgga ggattttaca tttgatggaa caaagcgctt aagtgtcaac 60 tacgtgaagg gaattcttca accgacagac acctgtgaca tctgggataa gatctggaac 120 ttccaagcca agcctgatga cctgcttatt tctacctatc ctaaagcagg aacaacatgg 180 atgcatgaaa ttttagacat gattctaaat gatggtgatg tggagaaatg caaaagagcc 240 cagactctag atagacacgc tttccttgaa ctgaaatttc ccttggagtt cgttcttgaa 300 atgtcctcac cacaactgat aaaaacacat ctcccttcac atctgattcc accatctatc 360 tggaaagaaa actgcaagat tgtctatgtg gccagaaatc ccaaggattg cctggtgtcc 420 tactaccact ttcacaggat ggcttccttt atgcctgatc ctcagaactt agaggaattt 480 tatgagaaat tcatgtccgg aaaagttgtt ggcaggtcct ggtttgacca tgtgaaagga 540 tggtgggctg caaaagacac gcaccggatc ctctacctct tctacgagga tattaaaaaa 600 aatccaaaac atgagatcca caaggtgttg gaattcttgg agaaaacttt gtcaggtgat 660 gttataaaca agattgtcca ccatacctca tttgatgtaa tgaaggataa tcccatggcc 720 aaccatactg cggtacctgc tcacatattc aatcactcca tctcaaaatt tatgaggaaa 780 gggatgcctg gagactggaa gaaccacttt actgtggcta tgaatgagaa ctttgataag 840 cattatgaaa agaagatggc agggtccaca ctgaacttct gcctggagat ctgagaggaa 900 caacaacaaa ctaggtgaca gagactatgc caactatttc gccttttatt ctgttgagca 960 aggaactgtg actgaatgtg gagcttatga gcttcagtcc atctcctata gtgtggctag 1020 tttgctataa tattaaaaca.tgatttaaaa tatcaacaaa ccagttactc cagcaaataa 1080 aataagagaa ttagagagca gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1134 <210> 23 <211> 1109 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 868788CB1 <400> 23 ctcgagccgc gcgccgggag cgcggtgggg ctaggcgtgg ggcgctcccg gcatgtccct 60 gtaccgcagc gtcgtgtggt tcgccaaggg gctgcgcgag tacaccaaga gtggctatga 120 atctgcatgt aaagactttg tccctcatga cttggaggtc cagattcctg gaagagtctt 180 tttggtcact ggaggaaaca gcggcattgg caaagcaact gcccttgaaa tcgccaagcg 240 aggtggcaca gttcacctgg tttgtcgaga tcaagcccca gcagaagatg ccaggggtga 300 gatcatccgg gagagcggta accagaacat ttttctgcac attgtggact tgtctgatcc 360 caagcaaatc tggaaatttg ttgaaaattt caagcaggaa cataaactcc atgttctgat 420 caataatgca ggttgcatgg tcaataaaag agagctcaca gaagatggac ttgaaaaaaa 480 ctttgctgcc aatactctgg gtgtgtacat tctcacgacc ggcctgatcc ctgtgctgga 540 gaaagaacac gacccccgag tgataaccgt ctcctcagga ggaatgttgg ttcagaaact 600 gaacaccaat gatctccagt ccgaaagaac accatttgat ggaactatgg tctatgcaca 660 aaacaaggtc agtgagaggc agcaagtggt tctgacggag cggtgggccc aagggcaccc 720 ggccatccat ttttcttcca tgcatcctgg ctgggccgac accccaggtg tgaggcaggc 780 gatgccgggg ttccacgcca ggttcgggga ccgcctgcgc tccgaggccc agggcgagga 840 caccatgctg tggctggccc tctcctctgc cgcagccgca cagcccagcg gccgcttctt 900 tcaagatcgg aagccagttt ctacacactt gcctctcgct acagcgtcct cctcaccggc 960 cgaagaggag aaactcattg aaatcctgga acagctggct cagacattta aataggccaa 1020 cccagacaca gtgcggtacc agaattgctt agaagatacc agaaggtgcg gtctagggac 1080 cagtgaataa gaagacccat ttcaatttc 1109 <210> 24 <211> 1614 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5672227CB1 <400> 24 aaaaggactt tgattttcag gatgcctggg aagtggattt ctgctctgct cctgctgcaa 60 ataagtttct gctttaaatc tgggaactgc ggaaaggtgt tggtatggcc gatggaatat 120 agtcactgga tgaatataaa gataatactg gaggaacttg tacagaaggg ccatgaagtc 180 actgttctga gaccttcagc ttttgtcttt cttgatccca aggaaacttc tgaccttaaa 240 tttgtaactt ttcctacatc tttcagtagt catgacctgg aaaatttttt tacaaggttt 300 gtgaatgtat ggacttatga gttgccaaga gacacatgtt tgtcatattt tctttatcta 360 caagatacga ttgatgaata ttctgactac tgtctgactg tttgtaaaga agcagtttca 420 aacaaacagt tcatgacaaa actacaggaa tccaaatttg atgtcgtttt ctcagatgcc 480 attggtccct gtggagagct gatagctgaa ctgctccaga ttccttttct gtacagtctt 540 cgcttctctc ctggctacac aattgaaaag tacattggag gagttctatt CCCtCCCtCC 600 tatgtacctg ttgttatgtc agaattaagt gatcaaatga ttttcatgga gaggataaaa 660 aatatgatac atatgcttta ttttgacttt tggtttcaaa tttatgatct gaagaagtgg 720 gaccagtttt atagtgaagt tctaggaaga cccactacat tatttgagac aatggggaaa 780 gctgaaatgt ggctcattcg aacctattgg gattttgaat ttcctcgccc attcttacca 840 aatgttgatt ttgttggagg acttcactgt aaaccagcca aacccctgcc taaggaaatg 900 gaagagtttg tgcagagctc tggagaaaat ggtattgtgg tgttttctct ggggtcgatg 960 atcagtaaca tgtcagaaga aagtgccaac atgattgcat cagcccttgc ccagatccca 1020 caaaaggttc tatggagatt tgatggcaag aagccaaata ctttaggttc caatactcga 1080 ctgtacaagt ggttacccca gaatgacctt cttggtcatc ccaaaaccaa agcttttata 17.40 actcatggtg gaaccaatgg catctatgag gcgatctacc atgggatccc tatggtgggc 1200 attcccttgt ttgcggatca acatgataac attgctcaca tgaaagccaa gggagcagcc 1260 ctcagtgtgg acatcaggac catgtcaagt agagatttgc tcaatgcatt gaagtcagtc 1320 attaatgacc ctgtctataa agagaatgtc atgaaattat caagaattca tcatgaccaa 1380 ccaatgaagc ccctggatcg agcagtcttc tggattgagt ttgtcatgcg ccacaaagga 1440 gccaagcacc ttcgagtcgc agctcacaac ctcacctgga tccagtacca ctctttggat 1500 gtgatagcat tcctgctggc ctgcgtggca actgtgatat ttatcatcac aaaattttgc 1560 ctgttttgtt tccgaaagct tgccaaaaaa ggaaagaaga agaaaagaga ttag 1614 <210> 25 <211> 2076 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483529CB1 <400> 25 atgtggactt tccttggcat cgccaccttc acctatttct ataagaaatg cggggacgtc 60 accctggcca acaaggagct cctgctgtgc gtgctggtgt tcctgtccct gggcctggtg 120 ctctcctacc gctgtcgcca ccgcaacgca ggcctcctgg ggcgccagca gagcgccgcc 180 ggcggctccc agttcgccgc cctctccgac atcctctcgg ctttgcctct catcggcttc 240 ctctgggcca agtcaccccc tgagtcagag aagaaagagg agccggagtc cagtaagtgc 300 agaaaagaaa ccagcgtgtc agaaacaaca ctgacggggg ccgctacctc agtaacagca 360 tcttctctga atgatccgga agttatcatc gtggggtctg gtgtccttgg gtcggctttg 420 gcgacggttc tttccagaga tggaagaaag gtgacggtaa tcgagagaga cttaaaagag 480 ccagacagaa tcgttggcga gcttctgcag ccaggtggtt accgcgtcct taaagagctg 540 ggcctcggag atacagtgga gggttttaat gcccatctga tacacggcta catagttcat 600 gattatgaaa gcagatccga agtgcagatt ccctacccgg tgtcggaaaa caaccaagtg 660 cagaatggag ttgctttcca ccatggcaag ttcatcatga gtctccgaaa agcagctatg 720 gcagagccca atgtaagggt tatagaaggt gttgtacttc agttattaga ggacggtgac 780 actgtgacgg gcgttcagta caaggacaag gagactggag acaccaagga gctccacgcc 840 ccgttgactg ttgttgcaga tgggctcttc tccaagttca ggaagaacct catctccagt 900 aaagtttctg tttcatctca ctttgtgggt ttccttatga agaatgcacc acagtttaaa 960 gccaattttg cagagcttgt tctgctcaac ccaagtccag ttctcatcta ccagatctca 1020 gccagcgaga ctcgggtact tgttgacatc cgtggggaaa tgccaaggaa cttaagagag 1080 tacatggctg agcagattta cccccaaata cctgaccacc tgaaggaagc gtttctagag 1140 gcatctcaga gcgctcgtct gcggaccatg ccagcaagct tcctgcctcc ttcaccagtg 1200 aacaaacgag gtgttcttct tttgggagac gcatataata tgaggcatcc acttactggt 1260 ggaggaatga ctgttgcttt taaagatata aaactatgga gaaaactgct aaagggtatc 1320 cctgaccttt atgatgatgc agctattttc gaggccaaaa aatcatttta ctgggcaaga 1380 aaaacatctc attcctttgt cgtgaatatc cttgctcagg ctctttatga attattttct 1440 gccacagatg attccctgca tcaactaaga aaagcctgtt ttctttattt caaacttggt 1500 ggcgaatgtg ttgcgggtcc tgttgggctg ctttctgtat tgtctcctaa ccctctagtt 1560 ttaattggac acttctttgc tgttgcaatc tatgccgtgt atttttgctt taagtcagaa 1620 ccttggatta caaaacctcg agcccttctc agtagtggtg ctgtattgta caaagcgtgt 1680 tctgtaatat ttcctctaat ttactcagaa atgaagtata tggttcatta agcttaaagg 1740 ggaaccattt gtgaatgaat atttggaact taccaagtcc taagagactt ttggaagagg 1800 atatatatag catagtacca taccacttat aaagtggaaa ctcttggacc aagatttgga 1860 ttaatttgtt tttgaagttt tttgtatata aatatgtaaa tacatgcttt aatttgcaat 1920 ttaaaatgaa ggggttaaat aagttagaca tttaaaagaa atgattgtta ccataaatta 1980 gtgctaatgc tgaggagaac tacagttttt cttttgaatt tagtatttga gatgagttgt 2040 tgggacatgc aaataaaatg aagaatgaaa aaaaaa 2076 <210> 26 <211> 1570 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5521346CB1 <400> 26 ggccagggcc aaagcgactt tcgctacttg gattggtcgg cgtagctttg ggcggccgga 60 ccttagaaag tcacacatct gcgcgcctgt gaggcccctg cttctgcgga tgctgaggca 120 cgtaaaaaaa tttgaagaag ggaatttcgc ggcattcttg gcctggcttc ctggcgtagc 180 cagcaagttc ggaggtgtta accgctgctg tcatgtttct tttgctaaac tgcatcgtcg 240 ctgtgtccca aaacatgggc atcggcaaga acggggacct gcccaggccg ccgctcagga 300 atgaattcag gtatttccag agaatgacca caacttcttc agtagagggt aaacagaatc 360 tggtgattat gggtaggaag acctggttct ccattcctga gaagaatcga cctttaaagg 420 atagaattaa tttagttctc agcagagaac tcaaggaagc tccacaagga gctcattttc 480 ttgccagaag tttggatgat gccttaaaac ttactgaacg accagaatta gcaaataaag 540 tagacatgat ttggatagtt ggtggcagtt ctgtttataa ggaagccatg aatcacctag 600 gccatcttaa actatttgtg acaaggatca tgcaggactt tgaaagtgac acgttttttt 660 cagaaattga cttggagaaa tataaacttc tgcctgaata cccaggtgtt ctctctgatg 720 tccaggaggg gaaacacatc aagtacaaat ttgaagtatg tgagaaggat gattaatatg 780 aaggtgtttt ctggtttaag ttgttccccc tccctctgag aaaagtatgc atttttacat 840 tagaaaaggg acttttgttg acttcagatc tatggataat tatttctaag caacgtgttt 900 ttattcctca ctaatcttgg ctatatcaga taccatttat gaaacattct tgctataact 960 gtctctccaa gaccccgact gagtccccag cacctgctac agtgagctgc cattccacac 1020 ccatcacatg tggcactctt gccactcctt gacattgtca ggcttttcta atgttggtag 1080 tat tattaa agatgaagat gcacataccc ttcagctgag cagtttcact agtaggaaat 1140 accaaaagct tcgtacatgt atatccagag gtttgtagac aaatgttgca gccttttttg 1200 taacagtgaa aaactgaaaa caacctggaa gtccagtgat gggaaaatga atatatttct 1260 gtcttagatt ggggaaccca aagcagattc caagactgaa atttaagtga aagcagttta 1320 tttgctaggt cataccagaa gtcatcaatc gaagtatgga gaaatggaac tgagaaggta 1380 aaaaccagtt caaaatcgct cttgtggccc aggctggagt gcggtggtgt gacctcggct 1440 cactgcttct ccagatgggc gcacagccat gacagtgcag atcctgctga agctggaccg 1500 cctggacctc gcccggaagg agctgaagag aatgcaggac ctggacgagg atgccaccct 1560 cacccagctc 1570 <210> 27 <211> 1591 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 71177017CB1 <400> 27 ggggagggcg tccagacccc gcccccggtc ccgccccgcg CCCgCtCCCa ggctgcggga 60 ccgccgggcg cagagcacaa gccgggcacc cacggactga gcggcgcgcg ggccgagatg 120 CgCJCCCgCC tgctgccccc gctcccgggc ctgggagcgg cgcgacccca tggcaggtag 180 cggcggcctg ggcggcgggg ccgggggcgg ccagggcgca ggggccgggc aaggggccgc 240 tctgcgggcg tcccgcgcgc cgatgctgct cgtggccctg gtgctcggcg cctactgcct 300 ctgcgccctc cccggccgct gcccgccggc cgcccgcgcc cccgcgccgg cccccgcgcc 360 CtCCgagCCg tCCagCtCCg tCCdCCgCCC gggagcaccc ggcctgcctt tggccagcgg 420 tcccggccgc cggcgcttcc cgcaagcgct catcgttggc gtgaagaagg gcggcacgcg 480 cgccctgctg gagtttctgc ggctgcaccc cgacgtccgc gcgctgggct ctgagcccca 540 cttcttcgac aggtgctacg agcgcggcct cgcctggtac cggagtctga tgccccgaac 600 cctggatggg cagatcacca tggagaagac ccccagctac ttcgtgacgc gagaggcccc 660 ccgccgcatc cacgccatgt ccccggacac gaagctgatc gtggtggtgc ggaaccccgt 720 gacccgggcc atctccgact acgcccagac gctctccaag accccgggcc tgcccagctt 780 CCgCgCCCtg gCCttCCgCC aCggCCtggg ccccgtggac acagcctgga gcgccgtccg 840 catcggcctg tacgcccagc acctggacca ctggctgcgc tacttccccc tgtcccactt 900 cctgttcgtc agcggggagc gtctggtcag cgacccggcc ggagaggtcg gccgcgtgca 960 ggacttcctg ggcctgaaac gggtcgtcac ggacaagcac ttctacttca acgccaccaa 1020 gggCttCCCC tgcctcaaga aggcccaggg cggcagccgt ccccgctgcc tgggcaagtc 1080 caagggccgg ccacacccac gcgtgcccca ggccgtggtc cggcgcctgc aggagttcta 1140 ccggcccttc aaccgcaggt tctaccagat gacgggccag gacttcggct ggggctgagc 1200 ggcaccctgg ggatgctcag caccttgatt gacacccgct cgcctggcca gagcgggctg 1260 cgtgcacatg ctgggcagag aggaatattt aagaaataaa gcttggaccc agatttttcc 1320 acaaaaaaaa aaaaatcaaa aggatagaaa taaggttcta Cgcatcagaa tagaaaaaaa 1380 aagggggggc ccccggtcta agtgagcccc ttttggcccc ggggaatttt aatttcccgg 1440 gagccggggt aacccttgca gggggggttt accaaggttt ttttccccct aataggtggg 1500 gggttccgta tttttaaaga agctttgtgg gcggtgtaaa ttcacatggg gggtcaacat 1560 ataggccgtg gttttctccc cccggggggg g 1591 <210> 28 <211> 1870 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472836CB1 <400> 28 tgagctactt tgtacttgaa aatgagaaat agttgtgtga ggtaggcgtt ttccgtcact 60 ctaagagcct gtgaaaggca taaaaatgtt gagtaagaca ttccttaaat gcactaacca 120 gcaattttag gaaaaaaaaa gaatgctctt ggatgataga gaaagataca tggcaataca 180 aatctgattt tatattaaga agtttcctga tgtcagaaac atgccaggaa aaagaattgc 240 agtgattggc gctgaagtca gtggattaaa cgctattaag agctgtctgg aagagggact 300 ggagcccacc tgttttgaag gaggcagtga cattggagga gtatggagat atgaggtaag 360 tacttcagag aaaatgccta gtatctacaa atctgtgacc atcaatactt ccaaggagat 420 gatgtgcttc agtgacttcc ctgtccctga tcattttccc aactacatgc acaactccaa 480 actcatggac tacttcggga tgtatgccac acactttggc ctcctgaatt acattcgttt 540 taagactgaa gtgcaaagtg tgaggaagca cccagatttt tctatcaatg gacaatggga 600 tgttgttgtg gagactgaag agaaacaaga gactttggtc tttgatgggg tcttagtttg 660 cagtggacac cacacagatc cctacttacc acttcagtcc ttcccaggca ttgagaaatt 720 tgaaggctgt tatttccata gtcgggaata caaaagtccc gaggactttt cagggaaaag 780 aatcatagtg atcggcattg gaaattctgg agtggatatt gcggtggagc tcagtcgtgt 840 agcaaaacag attgaaaaga gaccactgaa ttccaggatg gattgtggac acacatgcac 900 acaaatatac ccgttcacat acatgtacca tcatgtagga ttcctccatt gcccctactt 960 ttttctcttt cctgatcaag agatatcagc cagctgcatg ttttcccttg caggcctaaa 1020 cccaggcctg ggccttgaac attttcagcc actgataaag gtatttaggg ctttaagtca 1080 gcatccaact gtcagtgatg acctgccaaa tcacataatt tctggaaaag tccaagtaaa 1140 gcccagcgtg aaggagttca cagaaacaga tgccattttt gaagacagca ctgtagagga 1200 gaatattgat gttgtcatct ttgctacagg atacagtttt tctttttctt tccttgatgg 1260 tctgatcaag gttactaaca atgaagtatc tctgtataag cttatgttcc ctcctgacct 1320 ggagaagcca accttggctg tcatcggtct tatccaacca ctgggcatca tcttacctat 1380 tgcagagctc caatctcgtt gggctacacg agtgttcaaa gggctgatca aattaccctc 1440 agcggagaac atgatggcag atattgccca gaggaaaagg gctatggaaa aacgatatgt 1500 aaagacaccc cgccacacaa tccaagtgga tcacattgag tacatggatg agattgccat 1560 gccagcaggg gtgaaaccca acctgctctt cctctttctc tcagatccaa agctggccat 1620 ggaggttttc tttggcccct gcaccccata ccagtaccac ctccatgggc ccgagaaatg 1680 ggatggggcc cggagagcta acctgaccca gagagagagg atcatcaagc ccctgaggac 1740 tcgcattact agtgaggaca gccacccatc ctcacagctc tcttggataa agatggcccc 1800 agtgagcctg gcatttctgg ctgctggctt ggcatacttt cgatatactc attacggtaa 1860 atggaaataa 1870 <210> 29 <211> 1840 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7455721CB1 <400> 29 gctggtttca aggaacagcg aagggtctgt tttagtggag cggagtgagc aaggaggaca 60 ggtccacaca gggcgtttgt agccaccaag gagtctggtt tttattctaa ctggaacagg 120 aaggattttc agtccaggag aggcaaggtg tgcagatgga gagggctgag aagggtggag 180 ggggagggtg caggtgcggt ggttcatgga gccgcggtgg ttcatggagc caccgtggtg 240 gcctggacag gtggtggctg tggaggtgga taggaaggcg tggatccaag gcggtggcat 300 caccaaacac ccaccaaaca caggtggctg tgggccaggc ctgtgccggc cacctctgtc 360 ccctgtccct ctgcaggccc acacaggacc atgaaggtgc ttctcctcac agggctgggg 420 gccctgttct tcgcctatta ttgggatgac aacttcgacc cagccagcct ccagggagcg 480 cgagtgctgc tgacaggggc caacgctggt gttggtgagg agctggccta tcactacgcg 540 cgtctgggct cccacctggt gctcactgcc cacactgagg ctctcctgca gaaggtggta 600 gggaactgcc ggaagctggg cgcccccaag gtcttctaca tcgcggcgga catggcctcc 660 cctgaggcgc ccgagagcgt ggtgcagttt gcgctggaca agctgggcgg gctggactac 720 ctcgtgctga accacatcgg cggcgccccg gccggcacgc gagcccgcag cccccaggca 780 actcgctggc tcatgcaggt aaactttgtg agctacgtgc aactgacgtc gcgggcgctg 840 cccagcctga cggacagcaa gggctccctg gtggtggtgt cctcgctgct cggccgcgtg 900 cccacgtcgt tctccactcc ctactcggcg gccaagtttg cgctggacgg cttcttcggc 960 tccctgcggc gggagctgga cgtgcaggac gtgaacgtgg ccatcaccat gtgcgtcctg 1020 ggcctccgag atcgcgcctc cgccgccgag gcagtcaggg gagtcacgag ggtcaaggcg 1080 gccccggggc ccaaggcagc cctggccgtg atccgcggcg gcgccacgcg cgcggccggc 1140 gtcttctacc cgtggcgttt ccgcctgctg tgcttgctcc ggcgctggct accgcgcccg 1200 cgggcctggt ttatccgcca ggagctcaac gtcacggccg cggcagcctg agcaccgggg 1260 ggtgcccctc cagtcccaga cggcaatgtt cctccctcca actgtccctg gagccagaac 1320 actcacagag acacccctga gagggtggcc acagcccaag atgaagtcat caagacagaa 1380 aagcaaaacc gagaaaaacg acgggcacct ggaaccagtc acggcttggg aggtgcaggt 1440 gccccgtgtt aggcgccttt gtcggggact tgcaaggcct cacctgtttg gccatgattg 1500 atgacgtgac tgcttccatt ttgcagatga ggaaactaag gctcagagag gccacgccac 1560 CCttgagCCa cccatggacc cctctccatc tCCtgCCtgC gCCtttaagt ccctgattta 1620 ttctttccat tcattccatc tgggaggaac ccccccaact cctgccagct tcccctagct 1680 ggggtctctg gtactcttca cacctgcagg ggcgtctaca ctgttcgtct acctggtggc 1740 agggtctgag cgggaggagg agggaaagag tgtgttctga gctggaccca gcctcttgtt 1800 cgagaataaa aactcttctt ctcttgcaaa aaaaaaaaaa 1840 <210> 30 <211> 1578 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472837CB1 <400> 30 atgagaaaaa aaaatgcagt gattggtgct ggggtcagcg gattagatgc cattaagagc 60 tgcttggagg agggactcga gcctgtatgt tttgaaaaaa gcaatgagat tggaggactg 120 tggagatacc aggaaacacc tgaaagtgga aggcctggga tatacaaatc tatgatcttc 180 aacgcttcaa aggagacgac agccttcagc gactaccctt tccctgatca ttatcccaac 240 tatttgcaca attccaaaat gatggagtat ctcaggatgt acaccaggca ctttcacctc 300 atgaagcaca tccagtttaa ggtgtgcaga gtgaggaagc acccagattt ttcatcctct 360 ggccagtggg atgttgtggt agaaaccgat gggaagcaga aatcctatgt ctttgatggg 420 atcatgactt gcagtggcta ttacaattca aagtgtttgc ccttaaagga tttccccggg 480 atcgagaact tccaaggtcc atatctccac acctcggcat acaagcatct cgactatttt 540 gtggggaaga gagtggttgt ggtcagcatt gaggattctg gagcagatgt ggctggcgag 600 atcagtcatg ttgctgaaca ggttttcctc agtacaagac aaggtgcatg gatatggaac 660 cgggtttggg ataatgggaa tccccttgat atcacactct tcactcatta taatagaata 720 atggaaaaaa ttttccccac attcatgatc aacagatggg tagaaaataa atcaaatgca 780 aggcttaacc atgacaatta tgggctacag cctcaacaca gatttctcag ccatcaggct 840 actgtgaatg atgatctgcc taaccacata atttctggaa gagtcctgat gaaaccagac 900 atgagagagt tcaccacaac atcagccttc tttgaggatg gcactgaaaa gaacattgat 960 gctgtcatct ttgctacagg ctacaccttg tctttccctt tcttggagga tgactcagca 1020 atcctggaca gccagtactc cgtgtttaaa tttatgttcc ctccccagct ggagaagcca 1080 acactaagct tcattggcat tctccagcca gtgggagcca tcattcccat ttcagaactc 1140 cagagccgat gcgctgtgca agtattcgag ggtctgaaaa aattactctc cacaagtgcc 1200 atgatagctg acatcaacag gaggaaaaag aaaatggcaa aaaggtttat aaaaagccca 1260 agagatacac atcaagtgcc atgtattgac tacatggatg aaattgcact gaatattaga 1320 gtcaaaccca acctgttctc tctcctcctc tgggatacaa ggttagccaa agagattttc 1380 tatgggcctt gcaccgcatg tcaatattgc ctacaggggc cagggaaatg gaccagggcc 1440 cagaaggcca ttcttactga aagagaccgg atcctcaaac ccctgagaac acgtgtggtc 1500 aaacacacta gaccttcttc ctctagtctg tgctgccctt tttctctttg tttccatgtg 1560 aggcatccat gccagtga 1578 <210> 31 <211> 2263 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484241CB1 <400> 31 gtactgaggc gcgcacagag tccttagccc ggcgcagggc gcgcagccca ggctgagatc 60 cgctgcttct gtggaagtga gcatggttgg gcagcgggtg ctgcttctag tggccttcct 120 tctttctggg gtcctgctct cagaggctgc caaaatcctg acaatatcta cactgggtgg 180 aagccattac ctactgttgg accgggtgtc tcagattctt caagagcatg gtcataatgt 240 gactatgctt catcagagtg gaaagttttt gatcccagat attaaagagg aggaaaaatc 300 ataccaagtt atcaggtggt tttcacctga agatcatcaa aaaagaatta agaagcattt 360 tgatagctac atagaaacag cattggatgg cagaaaagaa tctgaagccc ttgtaaagct 420 aatggaaata tttgggactc aatgtagtta tttgctaagc agaaaggata taatggattc 480 cttaaagaat gagaactatg atctggtatt tgttgaagca tttgatttct gttctttcct 540 gattgctgag aagcttgtga aaccatttgt ggccattctt eccaccacat tcggctcttt 600 ggattttggg ctaccaagcc ccttgtctta tgttccagta ttcccttcct tgctgactga 660 tcacatggac ttctggggcc gagtgaagaa ttttctgatg ttctttagtt tctccaggag 720 ccaatgggac atgcagtcta catttgacaa caccatcaag gagcatttcc cagaaggctc 780 taggccagtt ttgtctcatc ttctactgaa agcagagttg tggtttgtta actctgattt 840 tgcctttgat tttgcccggc ccctgcttcc caacactgtt tatattggag gcttgatgga 900 aaaacctatt aaaccagtac cacaagactt ggacaacttc attgccaact ttggggatgc 960 agggtttgtc cttgtggcct ttggctccat ggtgaacacc tgtcagaatc cggaaatctt 1020 caaggagatg aacaatgcct ttgctcacct accccaaggg gtgatatgga agtgtcagtg 1080 ttctcattgg cccaaagatg tccacctggc tgcaaatgtg aaaattgtgg actggcttcc 1140 tcagagtgac ctcctggctc acccaagcat ccgtctgttt gtcacccacg gcgggcagaa 1200 tagcataatg gaggccatcc agcatggtgt gcccatggtg gggatccctc tctttggaga 1260 ccagcctgaa aacatggtcc gagtagaagc caaaaagttt ggtgtttcta ttcagttaaa 1320 gaagctcaag gcagagacat tggctcttaa gatgaaacaa atcatggaag acaagagata 1380 caagtccgcg gcagtggctg ccagtgtcat cctgcgctcc cacccgctca gccccacaca 1440 gcggctggtg ggctggattg accacgtcct ccagacaggg ggcgcgacgc acctcaagcc 1500 ctatgtctt~t cagcagccct ggcatgagca gtacctgctc gacgtttttg tgtttctgct 1560 ggggctcact ctggggactc tatggctttg tgggaagctg ctgggcatgg ctgtctggtg 1620 gctgcgtggg gccagaaagg tgaaggagac ataaggccag gtgcagcctt ggcggggtct 1680 gttt'ggtggg cgatgtcacc atttctaggg agcttcccac tagttctggc agccccattc 1740 tctagtcctt ctagttatct cctgttttct tgaagaacag gaaaaatggc caaaaatcat 1800 cctttccaca ttgggtccct gtttttggtg cccacagtga gctccttttt ggctgagcag 1860 gcatggagac tgtaggtttc cagatttcct gaaaaataaa agtttacagc gttatctctc 1920 cccaacctca ctaaatgatt ggccaagaga tttctgtcct aattgcccag aattctgtca 1980 tctggctact caaggctatc ggggaatggg gcaagtttgc actggcagct ggccaggatg 2040 aaggcagcgg aaagtgggtg gagggttagc taacctgtgg ggtctgaaga gagagaaaag 2100 tggccaaaaa tcctccagaa tgcttgacct gttgaaccag aatgttttgc tatttagtct 2160 tggcctatat tcatgcaacc taagcagcaa gctatcatgg gcatgctgat aaagaaacac 2220 ttctgtttcc tggttacagt ctctggctgg aaccctgaag gaa 2263 <210> 32 <211> 1224 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484251CB1 <400> 32 atggttctga aatgggcttc agttcttctg ctgatacatc tcagttgtta ctttagctct 60 gggagttgtg gaaaggtgct ggtttgggcc acagaataca gcctttggat gaatatgaag 120 acaatcctga aagagcttgt tcagagaggt catgaggtga ctgtactggc atcttcagct 180 tccattcttt ttgatcccaa cgactcatcc actcttaaac tcgaagttta tcctacatct 240 ttaactaaaa ctgaatttga gaatatcgtc atgcaacagg ttaagagaaa cgttgatttt 300 gttggaggat ttcactgcaa acctgccaaa cccctaccta aggaaatgga ggagtttgta 360 cagagctctg gagaaaatgg tgttgtggtg ttttctctgg ggtcaatggt cagtaacatg 420 acagcagaaa gggccaacgt aattgcaaca gcccttgcca agatcccaca aaaggttctg 480 tggagatttg atgggaataa accagatgcc ttaggtctca atactcggct gtataagtgg 540 ataccccaga atgaccttct aggtcatcca aaaaccagag cttttataac tcatggtgga 600 gccaatggca tctatgaggc aatctaccat gggatcccta tggtgggcat tccattgttt 660 tgggatcaac ctgataacat tgctcacatg aaggccaagg gagcagctgt tagactggac 720 ttccacacaa tgtcgagtac agacctgctg aatgcactga agacagtaat taatgatcct 780 tcatataaag agaatgttat gaaattatca agaattcaac atgatcaacc agtaaagccc 840 ctggatcgag cagtcttctg gattgaattt gtgatgtgcc acaaaggagc caaacacctt 900 cgagttgcag cccgtgacct cacctggttc cagtaccact ctttggatgt gattgggttt 960 ctgctggcct gtgtggcaac tgtgctattt atcatcacaa agtgttgtct gttttgtttc 1020 tggaagttcg ctagaaaagg aaagaaagga aaaagggatt agttaaatct gagatttgaa 1080 gctggaaaac ctgatagata gggatacttc agttgattcc agcaataaat attgtgatgc 1140 aagatttctt tcttcctgat aatgatcgta caagaaaaag tatttaatta aggagtctta 1200 gattatttac tgctaatact gtta 1224 <210> 33 <211> 1724 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473890CB1 <400> 33 gagaagcact gctctgacaa gttaagacgc aggaaacagc aacccaaaga gcagcccccg 60 aaacaaagtt gctcagacaa acaagccgat gcagacgagc gccatggccc tgctggcgcg 120 gatcctgaga gccgggctgc ggccggcgcc cgagcggggt gggctcctgg gcggcggggc 180 cccgcggcgg cctcaacccg cgggcgcacg gctcccggcg ggggcgcggg ccgaggacaa 240 aggcgccggg cggccggggt cgccgccggg agggggccga gccgagggtc cccggagcct 300 cgccgccatg ccggggccga ggaccctcgc caacctggcg gagttcttct gcagggacgg 360 cttcagccgc atccacgaga tccagcagaa gcacacacgg gaatatggaa aaatcttcaa 420 gtctcacttt ggtcctcagt ttgtagtatc tattgcagac cgcgatatgg tggctcaggt 480 gctccgggcg gagggcgctg cgcccgagag agccaacatg gagtcctggc gggagtaccg 540 agacttgcgg gggagagcca ccgggctcat ctcggcggag ggtgaacagt ggctcaagat 600 gagaagcgta ttgagacaaa gaattctgaa accgaaagat gtggccattt attctggaga 660 agtcaaccaa gttattgctg acttaattaa aagaatctac ctcctcagga gccaggcaga 720 agatggagaa accgtgacca atgtcaatga tcttttcttc aaatattcaa tggaaggagt 780 ggccaccatc ctttatgaga gtcgtttggg ctgcctggaa aacagcatcc cacagctgac 840 tgtggaatac atcgaggccc tggagctcat gtttagcatg ttcaagacct ccatgtatgc 900 aggcgccatc cccagatggc ttcgcccctt catcccaaag ccctggcggg aattctgcag 960 gtcctgggat ggactcttca aattcagcca aattcatgtt gacaacaagt tgagggacat 1020 acagtaccaa atggaccgag gccggagggt gagcggggga cttctcacat acctcttcct 1080 tagccaggct ctgacgctgc aggagatcta cgccaacgtg actgagatgc tgctggccgg 1140 cgtcgacacg acgtccttca ccttgtcttg gactgtgtac ctcctggcaa ggcacccaga 1200 agtgcagcag acggtgtacc gggagattgt gaagaattta ggggaaaggc atgttccaac 1260 tgcagctgat gtccccaagg tcccgctggt cagagctctc cttaaggaaa ccctgaggct 1320 gtttccagtg ctgccaggga acggccgggt cacccaggaa gacctggtta ttggcgggta 1380 tctgattccg aaaggcaccc agctggccct ttgccactat gccacatcgt accaggatga 1440 gaacttccct cgggccaagg agttccggcc tgagcgctgg ctgcggaaag gagacttaga 1500 tagagttgac aattttggat ccatcccctt tggtcatggg gttcgcagct gcatagggcg 1560 gagaattgca gaactggaga ttcacctcgt cgtgatccag gtagggcgtg gaggccaaac 1620 gttggccttt gggttcggcc tctttctact gggcacaaca ggataaagac ttccaggttg 1680 aaaagcaaag acggttttgg aaacttaaga tcaagagccg tcag 1724 <210> 34 <211> 945 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484470CB1 <400> 34 atgctggtct ctggtagaag aaggttgctc acagctctgc tgcaggctca gaagtggccc 60 tttcaaccct ccagagacat gagactagtc cagttccagg caccccacct ggtggggcct 120 cacttgggcc cagagatgga gaatggtgga agggttatca acctcaatgc ctttgactct 180 atgctcccca aaatgatgac acagttccta gagcagggag aggccatcct ctcagtggca 240 agaagagcct tgggtgccca gttgccagtc ctaccatggt tggaagtgac ctccctggct 300 ccagccacat ggccagataa ggtggtgtgt gtgggcatga attatgtgga ccactgcaaa 360 gaacagaatg tgcccgtgcc cacggaaccg atcaacttca gcaagtttgc caactccatt 420 gtggagccct atgatgagat ggtcctccca tcagagagcc aggaggtaga ctgggaagtg 480 gagcaggctg tggtcattgg aaagaaaggc aagcacatca aggccacaga tgccatggcc 540 catgtggctg gcttcactgc ggctcataac gtgagtgctc atgactggca aatgagacat 600 gatgggaaac agtggctgct gggaaaaacc ttcaacacct tctaccctct gggccttgcc 660 ttggtgacca aggacagtgt agcagatgca cacatcttaa agacctgctg ccaagtgaat 720 ggggaagtgg tccagagcaa caacaccaac cagatggtgg tcaagacaga ggagcggata 780 gcgtgggtct cccagtttgt caccttttac ccaggggatg tcatcctgac tgggacctcc 840 ccaggtgttg gtgtattcag gaaaccttct gtctttctca agaagggaga tgaagtccag 900 tgtgaaattg aagaactagg tgtcatcatc agcaaggtgg tgtga 945 <210> 35 <211> 1127 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 318172CB1 <400> 35 atggccctgg agctctacat ggacctgctg tcagcaccct gccgtgccgt ctacatcttc 60 tcgaagaagc atgacatcca gttcaacttt cagtttgtgg atctgctgaa aggtcaccac 120 cacagcaaag aatacattga catcaacccc ctcaggaagc tgcccagcct caaagatggg 180 aaatttatct taagtgaaag CCCCCaaCtC CtaCtCaCCC CgCagggCtC CttCCCagCC 240 cttactgact gtcagacaga cagctctggt gaaggtcttt ctcggggccg tttgggctcc 300 tctttgctct ggaagcccca gcagttgggc ccaggccaca ccggggtggt tgacgtagtg 360 gcactcagga aggcaagggc agtgcccctg accggcccct tctgcagcgc ggccatcctt 420 tactacctgt gccgcaagta cagcgcacca tcgcactggt gcccgccaga cccgcacgca 480 cgtgcccgtg tggatgagtt cgtggcttgg caacacacgg cctttcagct gcccatgaag 540 aagatagtct ggctcaagtt gctgatccca aagataacag gggaggaagt ttcagctgag 600 aagatggagc atgcagtgga agaggtgaag aacagcctgc agctctttga ggagtatttt 660 ctgcaggata agatgttcat caccgggaac caaatctcac tggctgacct ggtggccgtg 720 gtggagatga tgcagcccat ggcagccaac tataatgtct tcctcaacag ctccaagcta 780 gctgagtggc gtatgcaggt ggagctgaat attggctctg gcctctttag ggaggcccat 840 gatcgactaa tgcagttggc cgactgggac ttttcaacat tggattcaat ggtcaaggag 900 aatatttctg agttgctgaa gaagagcagt tggctctggc ctctttaggg aggcccatga 960 tcgactaatg cagttggccg actgggactt ttcaacattg gattcaatgg tcaaggagaa 1020 tatttctgag ttgctgaaga agagcaggtg accctaggcg cagcctgtcc cgcagggcct 1080 ggctggctta gcaatctgag ccaccttcct taaaggaaat gttaaaa 1127 <210> 36 <211> 1257 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte TD No: 7484475CB1 <400> 36 gccgcctgca ggcctcgcgc tgacaggatc ttttgtgacg ctgcaaatgt cctgaaccac 60 cgggaggaac tgggccattc taacacccgt tgctaccatg ctggccaccc gcctctccag 120 acecctgtca cggctcccag gaaaaaccct aagtgcctgt gatagagaaa atggagcaag 180 acgcccacta ttgcttggtt ctacttcctt tatcccgatt ggccgtcgga cttatgccag 240 tgcggcgggg ccggttggca gcaaagctgt cctggtcaca ggctgtgact ctggatttgg 300 gttctcattg gccaagcatc tgcattcgaa aggcttcctt gtgtttgctg gctgcttgat 360 gaaggacaaa ggccatgatg gggtcaagga gctggacagc ctaaacagtg accgattgag 420 aaccgtccag ctcaatgtct gcagcagcga agaggtggag aaagtggtgg agattgtccg 480 ctcgagcctg aaggaccctg agaaaggcat gtggggcctc gttaacaatg ccggcatctc 540 aacgttcggg gaggtggagt tcaccagcct ggagacctac aagcaggtgg cagaagtgaa 600 cctttggggc acagtgcgga tgacgaaatc ctttctcccc ctcatccgaa gggccaaagg 660 ccgcgtcgtc aatatcagca gcatgetggg ccgcatggcc aacccggccc gctccccgta 720 ctgcatcacc aagttcgggg tagaggcttt ctcggactgc ctgcgctatg agatgtaccc 780 cctgggcgtg aaggtcagcg tggtggagcc cggcaacttc atcgctgcca ccagccttta 840 cagccctgag agcattcagg ccatcgccaa gaagatgtgg gaggagctgc ctgaggtcgt 900 gcgcaaggac tacggcaaga agtactttga tgaaaagatc gccaagatgg agacctactg 960 cagcagtggc tccacagaca cgtcccctgt catcgatgct gtcacacacg ccctgaccgc 1020 caccaccccc tacacccgct accaccccat ggactactac tggtggctgc gaatgcagat 1080 catgacccac ttgcctggag ccatctccga catgatctac atccgctgaa gagtctcgct 1140 gtggcctctg tcagggatcc ctggtggaag gggaggggag ggaggaaccc atatagtcaa 1200 ctcttgatta tccacgtgtg gattatccac catgccagga agacccataa ctggttt 1257

Claims

What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:1-16, c) a polypeptide comprising a naturally occurring amino acid sequence at least 97%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:17-18, d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, and e) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18.
2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO:1-18.
3. An isolated polynucleotide encoding a polypeptide of claim 1.
4. An isolated polynucleotide encoding a polypeptide of claim 2.
5. An isolated polynucleotide of claim 4 selected from the group consisting of SEQ ID
NO:19-36.
6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim 6.
8. A transgenic organism comprising a recombinant polynucleotide of claim 6.
9. A method of producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:1-18.
11. An isolated antibody which specifically binds to a polypeptide of claim 1.
12. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:19-36, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:19-36, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.
15. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:1-18.
19. A method for treating a disease or condition associated with decreased expression of functional DME, comprising administering to a patient in need of such treatment the composition of claim 17.
20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with decreased expression of functional DME, comprising administering to a patient in need of such treatment a composition of claim 21.
23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.

25. A method for treating a disease or condition associated with overexpression of functional DME, comprising administering to a patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions; and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method comprising:
a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with the expression of DME in a biological sample, the method comprising:
a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
31. The antibody of claim 11, wherein the antibody is:
a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an acceptable excipient.
33. A method of diagnosing a condition or disease associated with the expression of DME in a subject, comprising administering to said subject an effective amount of the composition of claim 32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with the expression of DME in a subject, comprising administering to said subject an effective amount of the composition of claim 34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising:

a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18.

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

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

39. A method of snaking a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:

a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18.

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

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

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

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

44. A method of detecting a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18 in a sample, the method comprising:

a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-18 in the sample.

15. A method of purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-18 from a sample, the method comprising:

a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-18.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim 13.

47. A method of generating a transcript image of a sample which contains polynucleotides, the method comprising:

a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

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

49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:1.
57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:2.
58. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:3.
59. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:4.
60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:5.
61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:7.
63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:8.
64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:9.
65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:10.
66. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:11.
67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:12.
68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:13.
69. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:14.
70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:15.
71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:16.
72. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:17.
73. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:18.
74. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:19.
75. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:20.
76. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
No:21.
77. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:22.

78. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:23.
79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:24.
80. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:25.
81. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:26.
82. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:27.
83. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:28.
84. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:29.
85. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:30.
86. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:31.
87. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:32.
88. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:33.
89. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:34.

90. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:35.
91. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:36.