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DRUG METABOLIZING ENZYMES
TECHNICAL FIELD
The invention relates to novel nucleic acids, drug metabolizing enzymes
encoded by these
nucleic acids, and to the use of these nucleic acids and proteins in the
diagnosis, treatment, and
prevention of autoimmune/inflammatory, cell proliferative, developmental,
endocrine, eye, metabolic,
and gastrointestinal disorders, including liver disorders. The invention also
relates to the assessment of
the effects of exogenous compounds on the expression of nucleic acids and drug
metabolizing
enzymes.
BACKGROUND OF THE INVENTION
The metabolism of a drug and its movement through the body (pharmacokinetics)
are
important in determining its effects, toxicity, and interactions with other
drugs. The three processes
governing pharmacokinetics are the absorption 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
polymorphisms can be critical.
Moreover, promising new drugs are frequently eliminated in clinical trials
based on toxicities which
may only affect a segment of the patient group. Advances in pharmacogenomics
research, of which
drug metabolizing enzymes constitute an important part, are promising to
expand the tools and
information that can be brought to bear on questions of drug efficacy and
toxicity (See Evans, W.E.
and R.V. 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
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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 through the metabolic pathways.
(See Klaassen, C.D.
et al. (1996) Casarett and Doull's Toxicology: The Basic Science of Poisons,
McGraw-Hill, New
York, NY, pp. 113-186; Katzung, B.G. (1995) Basic and Clinical PharmacoloQV,
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 their 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 microsomal
cytochrome b5/NADH cytochrome b5 reductase system, the ferredoxin/ferredoxin
reductase redox
pair, aldo/keto reductases, and alcohol dehydrogenases. The major classes of
Phase II enzymes
include, but are not limited to, UDP glucuronyltransferase, sulfotransferase,
glutathione S-transferase,
N-acyltransferase, and N-acetyl transferase.
Cytochrome P450 and P450 catalytic cycle-associated enz~
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 multi-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, estrogens, and
androgens in animals;
insecticide resistance in insects; herbicide resistance and flower coloring in
plants; and environmental
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bioremediation by microorganisms. Cytochrome P450 actions on drugs,
carcinogens, mutagens, and
xenobiotics can result in detoxification or in conversion of the substance to
a more toxic product.
Cytochromes P450 are abundant in the liver, but also occur in other tissues;
the enzymes are located
in microsomes. (See ExPASY ENZYME EC 1.14.14.1; Prosite PDOC00081 Cytochrome
P450
cysteine heme-iron ligand signature; PRINTS EP450I E-Class P450 Group I
signature; Graham-
Lorence, S. and 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, supra). The B-class is found in
prokaryotes and fungi,
while the E-class is found in bacteria, plants, insects, vertebrates, and
mammals. Five subclasses or
groups are found within the larger 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, supra; Graham-Lorence, supra.)
Cytochrome P450 enzymes are involved in cell proliferation and development.
The enzymes
have roles in chemical mutagenesis and carcinogenesis by metabolizing
chemicals to reactive
intermediates that form adducts with DNA (Nebert, D.W. and Gonzalez, F.J.
(1987) Ann. Rev.
Biochem. 56:945-993). 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
3o 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 are
suppressed and some induced (Morgan, E.T. (1997) Drug Metab. Rev. 29:1129-
1188). Effects
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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 neurologic dysfunction, premature atherosclerosis, and cataracts;
and an inherited
resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J.
et al. (1994) Harnson's
Principles of Internal Medicine, McGraw-Hill, Inc. New York, NY, pp. 1968-
1970; Takeyama, K. et
al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998) N. Engl. J. Med.
338:653-661; OMIM
*213700 Cerebrotendinous xanthomatosis; and OMIM #122700 Coumarin resistance).
Extremely
high levels of expression of the cytochrome P450 protein aromatase were found
in a fibrolamellar
hepatocellular carcinoma from a boy with severe gynecomastia (feminization)
(Agarwal, V.R. (1998)
J. Clip. 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 ~ .
2o Lamb, D.C. et al. (1999; FEBS Lett. 462:283-288) identifies a Candida
adbicans cytochrome P450
(CYP51) which can be efficiently reduced and supported by the microsomal
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 level of oxidant
drugs or an abnormal hemoglobin (hemoglobin M) which is not efficiently
reduced.
Methemoglobinemia can also result from a hereditary deficiency in red cell
cytochrome b5 reductase
(Reviewed in Mansour, A. and Lurie, A.A. (1993) Am. J. Hematol. 42:7-12).
Members of the cytochrome P450 family are also closely associated with vitamin
D synthesis
and catabolism. Vitamin D exists as two biologically equivalent prohormones,
ergocalciferol (vitamin
DZ), 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.,
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290-310 nm), 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-
hydroxyvitamin D
(25(OH)D) by the enzyme 25-hydroxylase. 25(OITjD is the most abundant
precursor form of vitamin
D which must be further metabolized in the kidney to the active forth, 1a,25-
dihydroxyvitamin D
(1a,25(OH)ZD), by the enzyme 25-hydroxyvitamin D la-hydroxylase (la-
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,
l0 calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal
la-hydroxylase activity
has been reported, suggesting that tissue-specific, local regulation of
1a,25(OH)2D production may
also be biologically important. The catalysis of 1a,25(OH)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(OH)D as a substrate
(Shinki, T. et al. (1997)
Proc. Natl. Acad. Sci. U.S.A. 94:12920-12925; Miller, W.L. and Portale, A.A.
supra; and references
within). ,.' ~.
Vitamin D 25-hydroxylase, la-hydroxylase, and 24-hydroxylase are all NADPH-
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 Portale,
A.A. supra; and references within).
The active form of vitamin D (1a,25(OH)ZD) 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(OI~D (Griffin, J.E. and Zerwekh, J.E. (1983)
J. Clin. Invest.
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72:1190-1199; Gamblin, G.T. et al. (1985) J. Clin. Invest. 75:954-960; and
Miller, W.L. and Portale,
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 Streptomyces griseus
cytochrome P450,
CYP104D1, was heterologously expressed in E. coli and found to be reduced by
the endogenous
ferredoxin and ferredoxin reductase enzymes (Taylor, M. et al. (1999) Biochem.
Biophys. Res.
Commun. 263:838-42), suggesting that many cytochrome P450 species may be
supported by the
ferredoxin/ferredoxin reductase 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. Biochem. Biophys. 267:632-639).
Flavin-containing monooxyQenase (FMO)
Flavin-containing monooxygenases oxidize the nucleophilic nitrogen, sulfur,
and phosphorus
heteroatom of an exceptional range of substrates. Like cytochromes P450, FMOs
are microsomal
and use NADPH and 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 (FMO1, FM02, FM03,
FM04; .:'
and FMOS), 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 many N-hydroxylating enzymes (Stehr, M. et al. (1998) Trends
Biochem. Sci.
23:56-57; PRINTS FMOXYGENASE Flavin-containing monooxygenase signature).
Specific reactions include oxidation of nucleophilic tertiary amines to N-
oxides, secondary
amines to hydroxylamines and nitrones, primary amines to hydroxylamines and
oximes, and sulfur-
containing compounds and phosphines to S- and P-oxides. Hydrazines, iodides,
selenides, and boron-
containing compounds are also substrates. Although FMOs appear similar to
cytochromes P450 in
their chemistry, they can generally be distinguished from 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 (FM03
in liver) is predominantly responsible for metabolizing (S)-nicotine to (S)-
nicotine N-1=oxide, which is
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excreted in urine. FMO is also involved in S-oxygenation of cimetidine, an H2-
antagonist widely used
for the treatment of gastric ulcers. Liver-expressed forms of FMO are not
under the same regulatory
control as cytochrome P450. In rats, for example, phenobarbital treatment
leads to the induction of
cytochrome P450, but the repression of FMO1.
Endogenous substrates of FMO include cysteamine, which is oxidized to the
disulfide,
cystamine, and trimethylamine (TMA), which is metabolized to trimethylamine N-
oxide. TMA smells
like 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,~-~: -
insufticient dietary intake of copper does not influence the expression of the
apoenzyme. .However, . ..
the absence of the functional LO is linked to the skeletal and vascular tissue
disorders that.are . : . _
associated with dietary copper deficiency. LO is also inhibited by a variety
of semicarbazides,. w.. . . .
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 fibroblast 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 (dUIVlP) to
deoxythymidine monophosphate (dTMP). The basic reaction is as follows:
7,8-dihydrofolate + NADPH 1 5,6,7,8-tetrahydrofolate + NADP+
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The enzymes can be inhibited by a number of dihydrofolate analogs, including
trimethroprim and
methotrexate. Since an abundance of dTMP is required for DNA synthesis,
rapidly dividing 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, cancer 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)
l0 Biochemistry. W.H Freeman and Co., Inc. 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
(OMIM *103880 Aldo-keto reductase family 1, member B1). 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 dehydro~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, gv gx). 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 I(I (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, are
unable to oxidize methanol,
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and are not inhibited by pyrazole. Class III isozymes prefer even longer chain
aliphatic alcohols (five
carbons and longer) and aromatic alcohols, and are not inhibited by pyrazole.
The short-chain alcohol dehydrogenases include a number of related enzymes
with a variety
of substrate specificities. Included in this group are the mammalian enzymes D-
beta-hydroxybutyrate
dehydrogenase, (R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin
dehydrogenase,
NADPH-dependent carbonyl reductase, corticosteroid 11-beta-dehydrogenase, and
estradiol 17-beta-
dehydrogenase, as well as the bacterial enzymes acetoacetyl-CoA reductase,
glucose 1-
dehydrogenase, 3-beta-hydroxysteroid dehydrogenase, 20-beta-hydroxysteroid
dehydrogenase, ribitol
dehydrogenase, 3-oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate
dehydrogenase, sorbitol-6-
l0 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-
acylinannosamine 1-
dehydrogenase, and 2-deoxy-D-gluconate 3-dehydrogenase (Krozowski, Z. (1994)
J. Steroid
Biochem. Mol. Biol. 51:125-130; Krozowski, Z. (1992) Mol. Cell Endocrinol.
84:C25-31; and Marks,
A.R. et al. (1992) J. Biol. Chem. 267:15459-15463).
UDP glucuron~ltransferase
Members of the UDP glucuronyltransferase family (UGTs) catalyze the transfer
of a
glucuronic acid group from the cofactor uridine diphosphate-glucuronic acid
(UDP-glucuronic acid) to
a substrate. The transfer is 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 g/mol,
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
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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 in olfactory
epithelium, and the 2B
subfamily is expressed in liver microsomes. Mutations in UGT genes are
associated with
hyperbilirubinemia (OMIM #143500 Hyperbilirubinemia I); Crigler-Najjar
syndrome, characterized by
intense hyperbilirubinemia from birth (OMIM #218800 Crigler-Najjar syndrome);
and a milder form of
hyperbilirubinemia termed Gilbert's disease (OMIM *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. Sulfotransferases (ST)
catalyze this reaction by
transferring S03 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 rive classes based on their substrate preference:
arylsulfotransferase,
alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester
sulfotransferase, and bile salt
sulfotransferase.
ST enzyme activity varies greatly with sex and age in rats. The combined
effects of
developmental cues and sex-related hormones are thought to lead to these
differences in ST
expression profiles, as 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 para-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. Indeed, an inherited defect in a sulfotransferase
has been implicated in
macular corneal dystrophy, a disorder characterized by a failure to synthesize
mature keratan sulfate
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proteoglycans (Nakazawa, K. et al. (1984) J. Biol. Chem. 259:13751-13757; OM1M
*217800 Macular
dystrophy, corneal).
Galactosyltransferases
Galactosyltransferases are a subset of glycosyltransferases that transfer
galactose (Gal) to
the terminal N-acetylglucosamine (GlcNAc) oligosaccharide chains that are part
of glycoproteins or
glycolipids that are free in solution (Kolbinger, F. et al. (1998) J. Biol.
Chem. 273:433-440; Amado, M.
et al. (1999) Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases have
been detected on the
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 ((31-3)GlcNAc
linkages. Known
human and mouse (31,3-galactosyltransferases appear to have a short cytosolic
domain, a single
transmembrane domain, and a catalytic domain with eight conserved regions.
(Kolbinger, supra 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 amino acid residues 116-
119, region 4 is located at
amino acid residues 147-158, region 5 is located at amino acid residues 172-
183, region 6 is located at
amino acid residues 203-206, region 7 is located at amino acid residues 236-
246, and region 8 is
located at amino acid residues 264-275. ~A variant of a sequence found within
mouse UDP-
galactose:(3-N-acetylglucosamine ~i1,3-galactosyltransferase-I region 8 is
also found in bacterial
galactosyltransferases, suggesting that this sequence defines a
galactosyltransferase sequence motif
(Hennet, supra). Recent work suggests that brainiac protein is a ~i1,3-
galactosyltransferase (Yuan,
Y. et al. (1997) Cell 88:9-11; and Hennet, supra).
UDP-Gal:GlcNAc-1,4-galactosyltransferase (-1,4-Gall) (Sato, T. et al., (1997)
EMBO J.
16:1850-1857) catalyzes the formation of Type II carbohydrate chains with Gal
((31-4)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. Chem. 272:31389-31399). ~i1,4-galactosyltransferases have
several specialized
3o roles in addition to synthesizing carbohydrate chains on glycoproteins or
glycolipids. In mammals a
(31,4-galactosyltransferase, as part of a heterodimer with a-lactalbumin,
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 (31,4-
galactosyltransferases also
11
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function in cell adhesion, cell/basal lamina interaction, and normal and
metastatic cell migration. (Shun
B. (1993) Curr. Opin. Cell Biol. 5:854-863; and Shaper, J. (1995) Adv. Exp.
Med. Biol. 376:95-104).
Glutathione S-transferase
The basic reaction catalyzed by glutathione S-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 protein 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 in 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 al. (1995) J. Biol. Chem. 270:99-109). Residues R13, R20, and R69 are
important for the catalytic
activity of GST (Stenberg, G. et al. (1991) Biochem. J. 274:549-555).
In most cases, 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 typhimurium
used in the well-known 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 B1 is
substantially reduced by enhancing the expression of GST (Simula, T.P. et al.
(1993) Carcinogenesis
14:1371-1376). Thus, control of GST activity may be useful in the control of
mutagenesis and
carcinogenesis.
GST has been implicated in the acquired resistance of many cancers to drug
treatment, the
phenomenon known as multi-drug resistance (MDR). MDR occurs when a cancer
patient is treated
with a cytotoxic drug such as cyclophosphamide and subsequently becomes
resistant to this drug and
to a variety of other cytotoxic agents as well. Increased GST levels are
associated with some of
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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
A1-1 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- 1~ utamyl transpeptidase
Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes that
initiate extracellular
glutathione (GSH) breakdown by cleaving gamma-glutamyl amide bonds. The
breakdown of GSH
l0 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) Chem. Biol. Interact. 111-
112:333-42;
Taniguchi, N. and Ikeda, Y. (1998) Adv.:Enzymol. Relat. Areas Mol. Biol.
72:239-78; Chikhi, N. et al.
(1999) Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 122:367-380).
Acyltransferase
N-acyltransferase enzymes catalyze the transfer of an amino acid conjugate to
an activated
carboxylic group. Endogenous compounds and xenobiotics are activated by acyl-
CoA synthetases in
the cytosol, microsomes, and mitochondria. The acyl-CoA intermediates are then
conjugated with an
amino acid (typically glycine, glutamine, or taurine, but also ornithine,
arginine, histidine, serine, aspartic
acid, and several dipeptides) by N-acyltransferases in the cytosol or
mitochondria to form a metabolite
with an amide bond. 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-
acyltransferase (BAT) responsible for generating the bile acid conjugates
which serve as detergents in
the gastrointestinal tract (Falany, C.N. et al. (1994) J. Biol. Chem.
269:19375-19379; Johnson, M.R. et
al. (1991) J. Biol. Chem. 266:10227-10233). BAT is also useful as a predictive
indicator for prognosis
of hepatocellular carcinoma patients after partial hepatectomy (Furutani, M.
et al. (1996) Hepatology
24:1441-1445).
Acetyltransferases
Acetyltransferases have been extensively studied for their role in histone
acetylation. Histone
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acetylation results in the relaxing of the chromatin structure in eukaryotic
cells, allowing transcription
factors to gain access to promoter elements of the DNA templates in the
affected region of the
genome (or the genome in general). In contrast, histone deacetylation results
in a reduction in
transcription by closing the chromatin structure and limiting access of
transcription factors. To this
end, a common means of stimulating cell transcription is the use of chemical
agents that inhibit the
deacetylation of histones (e.g., sodium butyrate), resulting in a global
(albeit artifactual) increase in
gene expression. The modulation of gene expression by acetylation also results
from the acetylation
of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR,
TFII)E, TFI1F and the high
mobility group proteins (HMG). In the case of p53, acetylation results in
increased DNA binding,
leading to the stimulation of transcription of genes regulated by p53. The
prototypic histone acetylase
(HAT) is GcnS from Saccharomyces cerevisiae. GcnS is a member of a family of
acetylases that
includes Tetrahymena p55, human GcnS, and human p300/CBP. 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 alpha/beta
hydrolase fold (Center of
Applied Molecular Engineering Inst. of Chemistry and Biochemistry - University
of Salzburg,
http://predict.Banger.ac.uk/irbm-course97/Docs/ms~ common to several other
major classes of
enzymes, including but not limited to, acetylcholinesterases
and:carboxylesterases (Structural
Classification of Proteins, http://scop.mrc-lmb.cam.ac.uk/scop/index.html).
N-ace~ltransferase
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. In 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 are a limited number of known N-
acetyltransferases. In humans, there are two highly similar enzymes, NATl 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).
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Clinical observations 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 NATl enzyme maybe 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 aminotransferase 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-
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adenosyl-L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the
catechol substrate
(e.g., L-dopa, dopamine, or DBA). Methylation of the 3'-hydroxyl group is
favored over methylation
of the 4 =hydroxyl group and the membrane bound isoform of COMT is more
regiospecific than the
soluble form. Translation of the soluble form of the enzyme results from
utilization of an internal start
codon in a full-length mRNA (1.5 kb) or from the translation of a shorter mRNA
(1.3 kb), transcribed
from an internal promoter. The proposed SN2-like methylation reaction requires
Mg++ and is inhibited
by Ca++. The binding of the donor and substrate to COMT occurs sequentially.
AdoMet first binds
COMT in a Mg++-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-
propiophetropolone) and for clinical use
(e.g., nitrocatechol-based compounds and tolcapone). Administration of these
inhibitors results in the
increased half-life of L-dopa 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 generally 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).
Copper-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 Hz02. 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
3o 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
temperatures as high as 70°C
(Battistoni, A. et al. (1998) J. Biol. Chem. 273:5655-5661).
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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 superoxide
dismutases. In this study, mutations to yeast superoxide dismutase genes had a
more detrimental
effect on freeze-thaw resistance than mutations which affected the regulation
of glutathione
metabolism, long suspected of being important in determining an organism's
survival through the
process of cryopreservation (Jung-In Park, J.-I. et al. (1998) J. Biol. Chem.
273:22921-22928).
Expression of superoxide dismutase is also associated with Mycobacterium
tuberculosis, the
organism that causes tuberculosis. Superoxide dismutase is one of the ten
major proteins secreted by
M. tuberculosis and its expression is upregulated approximately 5-fold in
response to oxidative stress.
M. tuberculosis expresses almost two orders of magnitude more superoxide
dismutase than the
nonpathogenic mycobacterium M smegmatis, and secretes a much higher proportion
of the expressed
enzyme. The result is the secretion of 350-fold more enzyme by M. tuberculosis
than M.
smegmatis, providing substantial resistance to oxidative stress (Harth, G. and
Horwitz, M.A. (1999) J.
Biol. Chem. 274:4281-4292).
The reduced 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, compared to normal prostate tissue (Bostwick, D.G. (2000)
Cancer 89:123-134).
Phosphodiesterases
Phosphodiesterases make up a class of enzymes which catalyze the hydrolysis of
one of the
two ester bonds in a phosphodiester compound. Phosphodiesterases are therefore
crucial to a variety
of cellular processes. Phosphodiesterases include DNA and RNA endonucleases
and exonucleases,
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.
3o USA 93:11534-11539).
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
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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-Pick disease (Schuchman, E.H. and
S.R. Miranda
(1997) Genet. Test. 1:13-19).
Glycerophosphoryl diester phosphodiesterase (also known 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 for glycerophosphoryl diester phosphodiesterases. A
glycerophosphoryl diester
phosphodiesterase from E. coli has broad specificity for glycerophosphodiester
substrates (Larson,
T.J. et al. (1983) J. Biol. Chem. 248:5428-5432).
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
concentrations 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
(Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481; Torphy,
J.T. (1998) Am. J.
Resp. Crit. Care Med. 157:351-370).
Families of mammalian PDEs have been classified based on their substrate
specificity and
affinity, sensitivity to cofactors, and sensitivity to inhibitory agents
(Beavo, J.A. (1995) Physiol. Rev.
75:725-748; Conti, M. et al. (1995) Endocrine Rev. 16:370-389). Several of
these families contain
distinct genes, many of which are expressed in different tissues as splice
variants. Within PDE
families, there are multiple isozymes and multiple splice variants of these
isozymes (Conti, M. and S.-
L.C. Jin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63:1-38). The existence of
multiple PDE families,
isozymes, and splice variants is an indication of the variety and complexity
of the regulatory pathways
involving cyclic nucleotides (Houslay, M.D. and G. Milligan (1997) Trends
Biochem. Sci. 22:217-224).
Type 1 PDEs (PDEls) are Caz+/calinodulin-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, and
increases steady state levels of cAMP (Kakkar, supra). PDEls may provide
useful therapeutic
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targets for disorders of the central nervous system and the cardiovascular and
immune systems, due to
the involvement of PDEls in both cyclic nucleotide and calcium signaling
(Perry, M.J. and G.A. Higgs
(1998) Curr. 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. Acad. Sci. USA 94:3388-
3395).
PDE3s have high affinity for both cGMP and cAMP, and so these cyclic
nucleotides act as
l0 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 iniMammatory 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
inhibitor rolipram, a compound which has been shown to improve behavioral
memory in mice (Barad,
M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:15020-15025). PDE4 inhibitors
have also been
studied as possible therapeutic agents against acute lung injury, endotoxemia,
rheumatoid arthritis,
multiple sclerosis, and various neurological and gastrointestinal indications
(Doherty, A.M. (1999)
C~rr. Opin. Chem. Biol. 3:466-473).
PDES is highly selective for cGMP as a substrate (T~rko, 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 PDES by cGMP-dependent protein kinase
rather than for direct
regulation of catalytic activity. High levels of PDES are found in vascular
smooth muscle, platelets,
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lung, and kidney. The inhibitor zaprinast is effective against PDES 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 (Petty, M.J. and G.A. Higgs (1998) Curt. 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
l0 regulatory role in PDE6 function (Artemyev, N.O. et al. (1998) Methods
14:93-104). Defects in
PDE6s have been associated with retinal disease. Retinal degeneration in the
rd mouse (Yan, W. et
al. (1998) Invest. Opthalmol. Vis. Sci. 39:2529-2536), autosomal recessive
retinitis pigmentosa in
humans (Danciger, M. et al. (1995) Genomics 30:1-7), and rod/cone dysplasia 1
in Irish Setter dogs
(Suber, M.L. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3968-3972) have been
attributed to
mutations in the PDE6B gene.
The PDE7 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. 272:16152-16157;
Perry, M.J. and G.A. Higgs (1998) C~rr. 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. PDEBs 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, IBMX (3-isobutyl-1-methylxanthine), or zaprinast, but PDEBs are
inhibited by dipyridamole
(Fisher, D.A. et al. (1998) Biochem. Biophys. Res. Commun. 246:570-577;
Hayashi, M. et al. (1998)
Biochem. Biophys. Res. Commun. 250:751-756; Soderling, S.H. et al. (1998)
Proc. Natl. Acad. Sci.
3o USA 95:8991-8996).
PDE9s are cGMP specific and most closely resemble the PDE8 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-
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methylxanthine), but they are sensitive to the PDES inhibitor zaprinast
(Fisher, D.A. et al. (1998) J.
Biol. Chem. 273:15559-15564; Soderling, S.H. et al. (1998) J. Biol. Chem.
273:15553-15558).
PDElOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDElOs are
expressed in brain, thyroid, and testis. (Soderling, S.H. et al. (1999) Proc.
Natl. Acad. Sci. USA
96:7071-7076; Fujishige, K. et al. (1999) J. Biol. Chem. 274:18438-18445;
Loughney, K. et al (1999)
Gene 234:109-117).
PDEs are composed of a catalytic domain of about 270-300 amino acids, an N-
terminal
regulatory domain responsible for binding cofactors, and, in some cases, a
hydrophilic C-terminal
domain of unknown function (Conti, M. and S.-L.C. Jin (1999) Prog. Nucleic
Acid Res. Mol. Biol.
63:1-38). A conserved, putative zinc-binding motif 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 PDEls; 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 a conserved sequence motif
(McAllister-Lucas, L.M.
et al. (1993) J. Biol. Chem. 268:22863-22873). The NKXnD motif has been shown
by mutagenesis to
be important for cGMP binding (Turko, LV. et al. (1996) J. Biol: Chem.
271:22240-22244). PDE
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. For 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. Higgs (1998) C~rr. 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
lipopolysaccharide (LPS) induced TNF-a which has been shown to enhance HIV-1
replication in
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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-a 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,
to 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-a 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 DU145 and LNCaP was inhibited by delivery of CAMP
derivatives and PDE
inhibitors (Bang, Y.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334).
These cells also
showed a permanent 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,
2o K. et al. (1995) J. Clip. 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) Br. 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
organophosphorus compound (Vilanova, E. and Sogorb, M.A. (1999) 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 detoxification of organophosphate-containing chemical waste and
warfare reagents
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(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 which is active only toward long-chain fatty-acyl
thioesters and one which is
active toward thioesters with a wide range of fatty-acyl chain-lengths. These
thioesterases catalyze
the chain-terminating step in the de novo biosynthesis of fatty acids. Chain
termination involves the
hydrolysis of the thioester bond which links the fatty acyl chain to the 4'-
phosphopantetheine prosthetic
group of the acyl carrier protein (ACP) subunit of the fatty acid synthase
(Smith, S. (1981a) Methods
l0 Enzymol. 71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-200).
E. coli contains two soluble thioesterases, thioesterase I which is active
only toward long-
chain acyl thioesters, and thioesterase II (TEII) which has a broad chain-
length specificity (Naggert, J.
et al. (1991) J. Biol. Chem. 266:11044-11050). E. coli TEII does not exhibit
sequence similarity with
either of the two types of mammalian thioesterases which function as chain-
terminating enzymes in de
novo 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., supra). For that reason, Naggert et al. (supra)
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, acylcarnitine, carbonates, dipivefrin hydrochloride, cocaine,
salicylates, capsaicin,
palinitoyl-coenzyme A, imidapril, haloperidol, pyrrolizidine alkaloids,
steroids, p-nitrophenyl acetate,
malathion, butanilicaine, and isocarboxazide. The enzymes often demonstrate
low substrate
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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 carboxylesterase domains, (iv) are
highly expressed in the brain,
and (v) bind to neurexins in a calcium-dependent manner. Despite the homology
to carboxylesterases,
neuroligins lack the active site serine residue, implying a role in substrate
binding rather than catalysis
(Ichtchenko, K. et al. (1996) J. Biol. Chem. 271:2676-2682).
Sdualene epoxidase
Squalene epoxidase (squalene monooxygenase, SE) is a microsomal membrane-
bound, FAD-
dependent oxidoreductase that catalyzes the first oxygenation step in the
sterol biosynthetic pathway
of eukaryotic 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., supra). 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 committed 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
3o 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 for the
design of anti-hyperlipidemic drugs that do not cause a reduction in other
necessary intermediates
(Nakamura, Y. et al. (1996) 271:8053-8056).
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oxide 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/~i
hydrolase fold family of
enzymes (e.g., bromoperoxidase A2 from Streptomyces aureofaciens,
hydroxymuconic
semialdehyde hydrolases from Pseudomonas putida, 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
l0 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
nucleophilic 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 degradation of the amino acid tyrosine, to either succinate and pyruvate
or fumarate and
acetoacetate, requires a large number of enzymes and generates a large number
of intermediate
compounds. In addition, many xenobiotic compounds may be 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 known 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
Arthrobacter species) include 4-hydroxyphenylpyruvate oxidase, 4-
hydroxyphenylacetate
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3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxymethyl-2-
hydroxymuconic
semialdehyde dehydrogenase, traps,cis-5-carboxymethyl-2-hydroxymuconate
isomerase,
homoprotocatechuate isomerase/decarboxylase, cis-2-oxohept-3-ene-1,7-dioate
hydratase,
2,4-dihydroxyhept-traps-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
Pseudomonas 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.
l0 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-hept-3-ene-1,7-
dioate hydratase, and
5-carboxymethyl-2-hydroxymuconate isomerase (Ellis, L.B.M. et al. (1999)
Nucleic Acids Res.
27:373-376; Wackett, L.P. and Ellis, L.B.M. (1996) J. Microbiol. Meth. 25:91-
93; and Schmidt, M.
15 (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 enzyrrie in the
pathway in organisms that metabolize tyrosine to fumarate and acetoacetate.
HT1 is characterized
20 by progressive liver damage beginning at infancy, and increased risk for
liver cancer (Endo, F. et al.
(1997) J. Biol. Chem. 272:24426-24432).
Cytosine Deaminase
The bacterial cytosine deaminase catalyzes the hydrolysis of cytidine into
uridine and
ammonia. It also can convert 5-fluorocytosine (SFC), an antifungal agent into
5-fluorouracil (SFII), an
25 anticancer drug (Senter, P.D. et al. (1991) Bioconjug. Chem. 2:447-451).
This ability has been
recently utilized in cancer therapy research where it has been shown to act as
a suicide gene. A
suicide gene codes for enzymes that convert nontoxic compounds or prodrugs,
such as SFC, into toxic
products, such as SFU. Cytosine deaminase also kills cells through the
bystander effect, where non-
adjacent tumor cells are killed by the release of SFU (Gnant, M.F. et al.
(1999) Cancer Res.
30 59:3396-3403). Many suicide gene therapy approaches have been successfully
used in animal models
of cancer, and are currently being tested in clinical trials. Among more than
30 suicide genes
described, the most potent and widely used are the HSVltk and the cytosine
deaminase genes
(Singhal S. and Kaiser L.R. (1998) Surg. Oncol. Clip. N. Am. 7:505-536;
(Sandalon, Z. et al. (2001)
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Gene Ther. 8:232-238).
Acetyl-CoA Carboxylase
Acetyl-CoA carboxylase, a biotin-dependent enzyme, is found in all animals,
plants, and
bacteria. It catalyzes the carboxylation of Acetyl-CoA from COZ and H20 using
the energy of ATP
hydrolysis. Acetyl-CoA carboxylation is the rate limiting step in the
biogenesis of long-chain fatty
acids. Two isoforms of Acetyl-CoA carboxylase, types I and types II, are
expressed in humans in a
tissue-specific manner (Ha et al. (1994) Eur. J. Biochem. 219:297-306). Acetyl-
CoA carboxylase is a
multicomponent enzyme containing carbamoyl-phosphate synthase activity, a
biotin carboxyl carrier
protein, and a carboxyl transferase functional unit. Carbamoyl-phosphate
synthase catalyzes the
ATP-dependent synthesis of carbamyl-phosphate from gultamine or ammonia. It
initiates both the
urea cycle and the biosynthesis of arginine and pyrimidines. The carboxyl
transferase domain
functions in the transcarboxylation from biotin to an acceptor molecule. There
are two recognized
types of carboxyl transferase: one uses acyl-CoA and the other, 2-oxo acid as
the acceptor molecule
of carbon dioxide. All of the members in this family utilize acyl-CoA as the
acceptor molecule.
Tryptophan decarboxylase
Tryptophan decarboxylase (Tdc), also known as dopa decarboxylase, is a
pyridoxal-phosphate
protein which acts on three different substrates: L-tryptophan, 5-hydroxy-L-
tryptophan and
dihydroxy-L-phenylalanine. Tdc is an important enzyme in the biosynthesis of
terpenoid indole
alkaloids in Catharanthus roseus from which it was cloned. Its amino acid
sequence shows a strong
similarity with the alpha-methyldopa-hypersensitive protein of Drosophila
melanogaster. It also
shows significant similarity to feline glutamate decarboxylase and murine
ornithine decarboxylase.
Tdc, along with tyrosine hydroxylase, regulates cerebral dopamine synthesis.
(See Ouwerkerk, P. B.
et al. (1999) Plant Mol. Biol. 41:491-503; De Luca, V. et al. (1989) Proc.
Natl. Acad. Sci. USA
86:2582-2586.)
Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydro e~ nase
Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme is one
of the
four enzymes of the peroxisomal beta-oxidation pathway. The full-length human
cDNA has been
sequenced and mapped to chromosome 3q26.3-3q28. This enzyme, found mainly in
liver and kidney,
contains the tripeptide SKL at the carboxy terminus, which serves as a
peroxisomal targeting signal.
3o The human and rat bifunctional enzyme cDNAs are 80% homologous (Hoefler, G.
et al. (1994)
Genomics 19:60-67). Deficiency of enoyl-CoA hydratase: 3-hydroxyacyl-CoA
dehydrogenase leads
to clinical manifestations resembling Zellweger syndrome and accumulation of
very long-chain fatty
acids (Fukuda, S. et al. (1998) J. Inherit. Metab. Dis. 21:23-28).
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C7, a novel nucleolar protein, is the mouse homologue of the Drosophila late
puff product L82
and an isoform of human OXRl (oxidation resistance). Antibodies to recombinant
C7 protein
localized to nucleoli in a variety of cell types, suggest its involvement in
the formation or function of the
nucleole (Fischer, H. et al. (2001) Biochem. Biophys. Res. Commun. 281:795-
803).
Mammalian glucosamine-6-phosphate deaminase (GNPDA) was first detected in
hamster
spermatozoa. A human testis homolog was described by Shevchenko, V. et al.
(1998; Gene
216:31-38) and was mapped to chromosome Sq3l. In mouse spermatids, GNPDA
localized close to
the developing acrosome vesicle and in mouse spermatozoa close to the
acrosomal region. Following
the induction of the acrosome reaction, GNPDA fluorescence in spermatozoa was
either reduced or
l0 absent thus suggesting that GNPDA plays a role in the acrosome reaction
(Montag, M. et al. (1999)
FEBS Lett. 458:141-144).
Expressionprofiling
Microarrays are analytical tools used in bioanalysis. A microarray has a
plurality of molecules
spatially distributed over, and stably associated with, the surface of a solid
support. Microarrays of
polypeptides, polynucleotides, and/or antibodies have been developed and find
use in a variety of
applications, such as gene sequencing, monitoring gene expression, gene
mapping, bacterial
identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays find use is in gene expression
analysis. Array
technology can provide a simple way to explore the expression of a single
polymorphic gene or the
expression profile of a large number of related or unrelated genes. When the
expression of a single
gene is examined, arrays are employed to detect the expression of a specific
gene or its variants.
When an expression profile is examined, arrays provide a platform for
identifying genes that are tissue
specific, are affected by a substance being tested in a toxicology assay, are
part of a signaling
cascade, carry out housekeeping functions, or are specifically related to a
particular genetic
predisposition, condition, disease, or disorder.
Lung cancer is the leading cause of cancer death for men and the second
leading cause of
cancer death for women in the U.S. The vast majority of lung cancer cases are
attributed to smoking
tobacco, and increased use of tobacco products in third world countries is
projected to lead to an
epidemic of lung cancer in these countries. Exposure of the bronchial
epithelium to tobacco smoke
appears to result in changes in tissue morphology, which are thought to be
precursors of cancer.
Lung cancers are divided into four histopathologically distinct groups. Three
groups (squamous cell
carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-
small cell lung cancers
(NSCLCs). The fourth group of cancers is referred to as small cell lung cancer
(SCLC). Collectively,
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NSCLCs account for ~70% of cases while SCLCs account for -18% of cases. The
molecular and
cellular biology underlying the development and progression of lung cancer are
incompletely
understood. Deletions on chromosome 3 are common in this disease and are
thought to indicate the
presence of a tumor suppressor gene in this region. Activating mutations in K-
ras are commonly
found in lung cancer and are the basis of one of the mouse models for the
disease.
There is a need in the art for new compositions, including nucleic acids and
proteins, for the
diagnosis, prevention, and treatment of autoimmune/inflammatory, cell
proliferative, developmental,
endocrine, eye, metabolic, and gastrointestinal disorders, including liver
disorders.
1o SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides, drug
metabolizing
enzymes, referred to collectively as "DME" and individually as "DME-1," "DME-
2," "DME-3,"
"DME-4," "DME-5," "DME-6," "DME-7," "DME-8," "DME-9," "DME-10," "DME-11," "DME-
12," and "DME-13," and methods for using these proteins and their encoding
polynucleotides for the
detection, diagnosis, and treatment of diseases and medical conditions.
Embodiments also provide
methods for utilizing the purified drug metabolizing enzymes and/or their
encoding polynucleotides for
facilitating the drug discovery process, including determination of efficacy,
dosage, toxicity, and
pharmacology. Related embodiments provide methods for utilizing the purified
drug metabolizing
enzymes and/or their encoding polynucleotides for investigating the
pathogenesis of diseases and
medical conditions.
An embodiment provides an isolated polypeptide selected from the group
consisting of a) a
polypeptide comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
13, b) a polypeptide comprising a naturally occurring amino acid sequence at
least 90% identical or at
least about 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-13, c) a biologically active fragment of a polypeptide having an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of
a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-13. Another
embodiment provides an isolated polypeptide comprising an amino acid sequence
of SEQ >D NO:1-13.
Still another embodiment provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ B7 N0:1-13, b) a polypeptide comprising a naturally
occurnng amino acid
sequence at least 90% identical or at least about 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment
of a polypeptide
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having an amino acid sequence selected from the group consisting of SEQ >D
N0:1-13, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-13. In another embodiment, the polynucleotide
encodes a polypeptide
selected from the group consisting of SEQ >D NO:1-13. In an alternative
embodiment, the
polynucleotide is selected from the group consisting of SEQ ll~ N0:14-26.
Still another embodiment provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ >D N0:1-13, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-13, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-13, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:l-13.
Another embodiment provides a cell transformed with the recombinant
polynucleotide. Yet another
embodiment provides a transgenic organism comprising~the recombinant
polynucleotide.
Another embodiment provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ )D NO:1-13, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical or at least about 90% identical to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-13, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ >D NO:l-13, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ll~ N0:1-13.
The method comprises a) culturing a cell under conditions suitable for
expression of the polypeptide,
wherein said cell is transformed with a recombinant polynucleotide comprising
a promoter sequence
operably linked to a polynucleotide encoding the polypeptide, and b)
recovering the polypeptide so
expressed.
Yet another embodiment provides an isolated antibody which specifically binds
to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ )D NO:1-13, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:1-13, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-13,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
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group consisting of SEQ ~ NO:1-13.
Still yet another embodiment provides an isolated polynucleotide selected from
the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ >D N0:14-26, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical or at least about 90% identical to a
polynucleotide sequence selected
from the group consisting of SEQ >D N0:14-26, c) a polynucleotide
complementary to the
polynucleotide of a), d) a polynucleotide complementary to the polynucleotide
of b), and e) an RNA
equivalent of a)-d). In other embodiments, the polynucleotide can comprise at
least about 20, 30, 40,
60, 80, or 100 contiguous nucleotides.
l0 Yet another embodiment provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide being selected from the group consisting of a) a
polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ >D N0:14-26,
b) a polynucleotide
comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ >D N0:14-26, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex. In a related embodiment, the method can include
detecting the amount of
the hybridization complex. In still other embodiments, the probe can comprise
at least about 20, 30,
40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide being selected from the group consisting of
a) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
>l7 N0:14-26, b) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:14-26, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target polynucleotide
or fragment thereof. In a related embodiment, the method can include detecting
the amount of the
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amplified target polynucleotide or fragment thereof.
Another embodiment provides a composition comprising an effective amount of a
polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-13, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical or at least about 90% identical to
an amino acid sequence
selected from the group consisting of SEQ )D N0:1-13, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ >D NO:1-13,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-13, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition can comprise an amino acid sequence selected from
the group consisting
of SEQ )Z7 NO:1-13. Other embodiments provide a method of treating a disease
or condition
associated with decreased or abnormal expression of functional DME, comprising
administering to a
patient in need of such treatment the composition.
Yet another embodiment provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-13, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ )D N0:1-13, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D NO:1-13, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ )D NO:1-13. The method comprises a)
exposing a sample
comprising the polypeptide to a compound, and b) detecting agonist activity in
the sample. Another
embodiment provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. Yet another embodiment provides a
method of treating a
disease or condition associated with decreased expression of functional DME,
comprising
administering to a patient in need of such treatment the composition.
Still yet another embodiment provides a method for screening a compound for
effectiveness
as an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ >D NO:1-13, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical or
at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
>D NO:1-13, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ >D N0:1-13, and d) an immunogenic fragment of a polypeptide
having an amino
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acid sequence selected from the group consisting of SEQ )D NO:1-13. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in
the sample. Another embodiment provides a composition comprising an antagonist
compound
identified by the method and a pharmaceutically acceptable excipient. Yet
another embodiment
provides a method of treating a disease or condition associated with
overexpression of functional
DME, comprising administering to a patient in need of such treatment the
composition.
Another embodiment provides a method of screening for a compound that
specifically binds to
a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-13, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ ll~ N0:1-13, c)
a biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
>D N0:1-13, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ D7 N0:1-13. The method comprises a)
combining the
polypeptide with at least one test compound under suitable conditions, and b)
detecting binding of the
polypeptide to the test compound, thereby identifying a compound that
specifically binds to the
polypeptide.
Yet another embodiment provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ )D NO:1-13, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical or at least
about 90% identical to an
amino acid sequence selected from the group consisting of SEQ ll~ NO:1-13, c)
a biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D N0:1-13, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ >l7 N0:1-13. The method comprises a)
combining the
polypeptide with at least one test compound under conditions permissive for
the activity of the
polypeptide, b) assessing the activity of the polypeptide in the presence of
the test compound, and c)
comparing the activity of the polypeptide in the presence of the test compound
with the activity of the
polypeptide in the absence of the test compound, wherein a change in the
activity of the polypeptide in
the presence of the test compound is indicative of a compound that modulates
the activity of the
polypeptide.
Still yet another embodiment provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
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polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
Another embodiment provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound; b)
hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:14-26, ii) a
polynucleotide comprising a naturally occurnng polynucleotide sequence at
least 90% identical or at
least about 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:14-26, 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 N0:14-26, ii) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical or at least about 90% identical to a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:14-26, iii) a polynucleotide complementary to the
polynucleotide of i), iv) a
polynucleotide complementary to the polynucleotide of ii), and v) an RNA
equivalent of i)-iv).
Alternatively, the target polynucleotide can comprise a fragment of a
polynucleotide selected from the
group consisting of i)-v) above; c) quantifying the amount of hybridization
complex; and d) comparing
the amount of hybridization complex in the treated biological sample with the
amount of hybridization
complex in an untreated biological sample, wherein a difference in the amount
of hybridization
complex in the treated biological sample is indicative of toxicity of the test
compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and
polypeptide
embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptide embodiments of the invention. The probability scores
for the matches
between each polypeptide and its homolog(s) are also shown.
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Table 3 shows structural features of polypeptide embodiments, including
predicted motifs and
domains, along with the methods, algorithms, and searchable databases used for
analysis of the
polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide embodiments, along with selected fragments of the
polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze
polynucleotides and
l0 polypeptides, along with applicable descriptions, references, and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is
understood that
embodiments of the invention are not limited to the particular machines,
instruments, materials, and
methods described, as these may vary. It is also to be understood that the
terminology used herein is
for the purpose of describing particular embodiments only, and is not intended
to limit the scope of the
invention.
As used herein and in the appended claims, the singular forms "a," "an," and
"the" include
plural reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a
host cell" includes a plurality of such host cells, and a reference to "an
antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled in the art,
and so forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with various embodiments of the invention. Nothing herein
is to be construed as an
admission that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
3o DEFINITIONS
"DME" refers to the amino acid sequences of substantially purified DME
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and human,
and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
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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, 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.
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, or
substitutions of nucleotides.
l0 Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding 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 or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide encoding
DME. 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 one or more similarities in
polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of DME is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and 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" can refer to an oligopeptide,
a peptide, a
polypeptide, or a protein sequence, or a fragment of any of these, and to
naturally occurnng or
3o synthetic molecules. Where "amino acid sequence" is recited to refer to a
sequence of a naturally
occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid. Amplification
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may be carried out using polymerase chain reaction (PCR) technologies or other
nucleic acid
amplification technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of DME. Antagonists may include proteins such as antibodies, anticalins,
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 term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
l0 Antibodies that bind DME polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
, 5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have modified sugar groups (e.g., the
2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
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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 polynucleotide having a specific nucleic acid sequence.
Antisense compositions
may include, DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having
modified backbone
linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates;
oligonucleotides
having modified sugar groups such as 2'-methoxyethyl sugars or 2'-
methoxyethoxy sugars; or
oligonucleotides having modified bases such as 5-methyl cytosine, 2'-
deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense molecules may be produced by any method including
chemical synthesis
or transcription. Once introduced into a cell, the complementary antisense
molecule base-pairs with a
naturally occurring nucleic acid sequence produced by the cell to form
duplexes which block either
transcription or translation. The designation "negative" or "minus" can refer
to the antisense strand,
and the designation "positive" or "plus" can refer to the sense strand of a
reference DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic 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" and a "composition
comprising a given
polypeptide" can refer to any composition containing the given polynucleotide
or polypeptide. The
composition may comprise a dry formulation or an aqueous solution.
Compositions comprising
polynucleotides encoding 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
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(e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other
components (e.g., Denhardt's
solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(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, Ser
Arg ~ His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
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absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
l0 "Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of DME or a polynucleotide encoding DME which
can be
identical in sequence to, but shorter in length than, the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from about S to about 1000 contiguous nucleotides or
amino acid residues. A
fragment used as a probe, primer, antigen, therapeutic molecule, or for other
purposes, may be at least
5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500
contiguous nucleotides or amino
acid residues in length. Fragments may be preferentially selected from certain
regions of a molecule.
For example, a polypeptide fragment may comprise a certain length of
contiguous amino acids
selected from the first 250 or 500 amino acids (or first 25% or 50%) of a
polypeptide as shown in a
certain defined sequence. Clearly these lengths are exemplary, and any length
that is supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ )D N0:14-26 can comprise a region of unique polynucleotide
sequence
that specifically identifies SEQ )D N0:14-26, for example, as distinct from
any other sequence in the
genome from which the fragment was obtained. A fragment of SEQ >D N0:14-26 can
be employed
in one or more embodiments of methods of the invention, for example, in
hybridization and
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amplification technologies and in analogous methods that distinguish SEQ ID
N0:14-26 from related
polynucleotides. The precise length of a fragment of SEQ >D N0:14-26 and the
region of SEQ ID
N0:14-26 to which the fragment corresponds are routinely determinable by one
of ordinary skill in the
art based on the intended purpose for the fragment.
A fragment of SEQ D7 NO:l-13 is encoded by a fragment of SEQ ID N0:14-26. A
fragment of SEQ ID N0:1-13 can comprise a region of unique amino acid sequence
that specifically
identifies SEQ ID N0:1-13. For example, a fragment of SEQ 117 N0:1-13 can be
used as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID N0:1-13.
The precise length of a fragment of SEQ ID N0:1-13 and the region of SEQ 117
NO:1-13 to which
the fragment corresponds can be determined based on the intended purpose for
the fragment using
one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide is one containing at least a translation
initiation codon (e.g.,
methionine) followed by an open reading frame and a translation temlination
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 one
or more
computer algorithms or programs known in the art or described herein. For
example, percent identity
can be determined using the default parameters of the CLUSTAL V algorithm as
incorporated into
the MEGALIGN version 3.12e sequence alignment program. This program is part of
the
LASERGENB software package, a suite of molecular biological analysis programs
(DNASTAR,
Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989;
CABIOS 5:151-
153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191). For pairwise
alignments of
polynucleotide sequences, the default parameters are set as follows: Ktuple=2,
gap penalty=S,
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
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which can be used is provided by the National Center for Biotechnology
Information (NCBI) Basic
Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol.
Biol. 215:403-410), which
is available from several sources, including the NCBI, Bethesda, MD, and on
the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
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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 incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=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, for 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:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ B7 number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
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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 ~.g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carned out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor
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/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
3o apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acids by
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virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex
may be formed in solution (e.g., Cot or Rot analysis) or formed between one
nucleic acid present in
solution and another nucleic acid immobilized on a solid support (e.g., paper,
membranes, filters, chips,
pins or glass slides, or any other appropriate substrate to which cells or
their nucleic acids have been
fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
polynucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
to 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 or
oligopeptide fragment
of DME which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray'' refers to an arrangement of a plurality of
polynucleotides,
polypeptides, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, antibody, or
other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of 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 fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
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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 acids encoding DME, their complements, or fragments
thereof,
which are used to detect identical, allelic or related nucleic acids. Probes
are isolated oligonucleotides
or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include
radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers"
are short nucleic
acids, usually DNA oligonucleotides, which may be annealed to a target
polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a
DNA polymerase enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic
acid, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in 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. (1999) Short
Protocols in Molecular
Bi_ oloQV, 4~' ed., John Wiley & Sons, New York NY), and Innis, M. et al.
(1990; PCR Protocols, A
Guide to Methods and Applications, Academic Press, San Diego CA). PCR primer
pairs can be
derived from a known sequence, for example, by using computer programs
intended for that purpose
such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge MA).
3o 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
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programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a nucleic acid that is not naturally occurnng
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,
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translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA molecule, is composed of the same
linear
sequence of nucleotides as the reference DNA molecule with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing DME,
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 about 60%
free, preferably at least about 75% free, and most preferably at least about
90% free from other
components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene expression
by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
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cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
cells" includes stably transformed cells in which the inserted DNA is capable
of replication either as
an autonomously replicating plasmid or as part of the host chromosome, as well
as transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
l0 and plants, in which one or more of the cells of the organism contains
heterologous nucleic acid
introduced by way of human intervention, such as by transgenic techniques well
known in the art. The
nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor of the cell,
by way of deliberate genetic manipulation, such as by microinjection or by
infection with a
recombinant virus. In another embodiment, the nucleic acid can be introduced
by infection with a
recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002)
Science 295:868-872). The
term genetic manipulation does not include classical cross-breeding, or in
vitro fertilization, but rather
is directed to the introduction of a recombinant DNA molecule. The transgenic
organisms
contemplated in accordance with the present invention include bacteria,
cyanobacteria, fungi, plants
and animals. The isolated DNA of the present invention can be introduced into
the host by methods
known in the art, for example infection, transfection, transformation or
transconjugation. Techniques
for transferring the DNA of the present invention into such organisms are
widely known and provided
in references such as Sambrook et al. (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
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reference molecule. Species variants are polynucleotides that vary from one
species to another. The
resulting polypeptides will generally have significant amino acid identity
relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a
particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity 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 95%, 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.
TIIE INVENTION
Various embodiments of the invention include new human drug metabolizing
enzymes (DME),
the polynucleotides encoding DME, and the use of these compositions for the
diagnosis, treatment, or
prevention of autoimmune/inflammatory, cell proliferative, developmental,
endocrine, eye, metabolic, .
and gastrointestinal disorders, including liver disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
embodiments of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to
a single Incyte project identification number (Incyte Project >D). Each
polypeptide sequence is
denoted by both a polypeptide sequence identification number (Polypeptide SEQ
)D NO:) and an
Incyte polypeptide sequence number (Incyte Polypeptide 117) 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 >D) 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
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide >D) for polypeptides of the
invention. Column 3
shows the GenBank identification number (GenBank B7 NO:) of the nearest
GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
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Column S shows the annotation of the GenBank homolog(s).
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ >D NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and 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. For example, SEQ
)D N0:2 is 84% identical, from residue M1 to residue S2457, to a rat acetyl-
CoA carboxylase
(GenBank ID g3080546) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 0.0, which indicates the probability
of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID N0:2 also contains a
carboxyl
transferase domain and a biotin-requiring enzyme domain as determined by
searching fur statistically
significant matches in the hidden Markov model (HIVIM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIIVVIPS, MOTIFS, PROFILESCAN, and
other BLAST
analyses provide further corroborative evidence that SEQ ID N0:2 is an acetyl-
CoA carboxylase.
In an alternative example, SEQ ID NO:S is 87% identical, from residue M1 to
residue D274,
to human glucosamine-6-phosphate deaminase (GenBank ID g2632113) as determined
by BLAST.
(See Table 2.) The BLAST probability score is 8.2e-133. SEQ ID NO:S also
contains a
glucosamine-6-phosphate isomerases/6-phosphogluconolactonases domain as
determined by searching
for statistically significant matches in the HIvIM-based PFAM database of
conserved protein family
domains. (See Table 3.) Data from BLM'S, MOTIFS, and other BLAST analyses
provide further
corroborative evidence that SEQ >D NO:S is a glucosamine-6-phosphate
deaminase.
In an alternative example, SEQ B7 N0:8 is 97% identical, from residue M1 to
residue L723,
to human enoyl-CoA: hydratase/3-hydroxyacyl-CoA dehydrogenase (GenBank )D
g452045) as
determined by BLAST. (See Table 2.) The BLAST probability score is 0Ø SEQ ID
N0:8 also
contains a 3-hydroxyacyl-CoA dehydrogenase, C-terminal domain, a 3-hydroxyacyl-
CoA
dehydrogenase, NAD binding domain, and an enoyl-CoA hydratase/3-hydroxyacyl-
CoA
dehydrogenase/isomerase family signature as determined by searching for
statistically significant
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matches in IEVIM-based PFAM database of conserved protein family domains. (See
Table 3.) Data
from BLIMPS, MOTIFS, PROFILESCAN, and other BLAST analyses provide further
corroborative
evidence that SEQ ll~ N0:8 is an enoyl-CoA:3-hydroxyacyl-CoA dehydrogenase
bifunctional enzyme.
In an alternative example, SEQ D7 N0:13 is 97% identical, from residue M1 to
residue L614,
to rat acetylcholinesterase T subunit (GenBank ID g262093) as determined by
BLAST. (See Table
2.) The BLAST probability score is 0Ø SEQ )17 N0:13 also contains a
carboxylesterase domain as
determined by searching for statistically significant matches in HIVIM-based
PFAM database of
conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:13
is an
to acetylcholinesterase. SEQ ID NO:1, SEQ ID N0:3-4, SEQ D7 N0:6-7, and SEQ ID
N0:9-12 were
analyzed and annotated in a similar manner. The algorithms and parameters for
the analysis of SEQ
ID NO:1-13 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled
using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any
combination of these two
types of sequences. Column 1 lists the polynucleotide sequence identification
number (Polynucleotide
SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number
(Incyte ID) for
each polynucleotide of the invention, and the length of each polynucleotide
sequence in basepairs.
Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA
and/or genomic
sequences used to assemble the full length polynucleotide embodiments, and of
fragments of the
polynucleotides which are useful, for example, in hybridization or
amplification technologies that
identify SEQ ID N0:14-26 or that distinguish between SEQ D7 N0:14-26 and
related polynucleotides.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotides. In addition, the
polynucleotide fragments described in column 2 may identify sequences derived
from the ENSEMBL
(The Sanger Centre, Cambridge, UK) database (i.e., those sequences including
the designation
"ENST"). Alternatively, the polynucleotide fragments described in column 2 may
be derived from the
NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences
including the
designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e.,
those sequences
including the designation "NP"). Alternatively, the polynucleotide fragments
described in column 2
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, a polynucleotide sequence identified as
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FL_XXXXXX_NI NZ YYYYY N3 NQ 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 NI,2,3..., if
present, represent specific exons
that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLX~,'XXXX_gAAAAA~BBBBB_1 N is a "stretched" sequence, with XXXXXX being the
Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the GenBank
l0 identification number or NCBI RefSeq identification number of the nearest
GenBank protein homolog,
and N referring to specific exons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier
(denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
2o ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
Table 4 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotides which
were assembled using Incyte cDNA sequences. The representative cDNA library is
the Incyte
cDNA library which is most frequently represented by the Incyte cDNA sequences
which were used
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to assemble and confum the above polynucleotides. 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.
Various embodiments also encompass polynucleotides which encode DME. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ )D N0:14-26, which encodes DME. The
polynucleotide sequences
l0 of SEQ >D N0:14-26, as presented in the Sequence Listing, embrace the
equivalent RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar
backbone is composed of ribose instead of deoxyribose.
The invention also encompasses variants of a polynucleotide encoding DME. In
particular,
such a variant polynucleotide will have at least about 70%, or alternatively
at least about 85%, or even
at least about 95% polynucleotide sequence identity to a polynucleotide
encoding DME. A particular
aspect of the invention encompasses a variant of a polynucleotide comprising a
sequence selected
from the group consisting of SEQ ID N0:14-26 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 >D N0:14-26. Any one of the
polynucleotide variants
described above can encode a polypeptide which contains at least one
functional or structural
characteristic of DME.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide encoding DME. A splice variant may have portions which have
significant sequence
identity to a polynucleotide encoding DME, but will generally have a greater
or lesser number of
polynucleotides due to additions or deletions of blocks of sequence arising
from alternate splicing of
exons during mRNA processing. A splice variant may have less than about 70%,
or alternatively less
than about 60%, or alternatively less than about 50% polynucleotide sequence
identity to a
polynucleotide encoding DME over its entire length; however, portions of the
splice variant will have
at least about 70%, or alternatively at least about 85%, or alternatively at
least about 95%, or
alternatively 100% polynucleotide sequence identity to portions of the
polynucleotide encoding DME.
For example, a polynucleotide comprising a sequence of SEQ )D N0:24 and a
polynucleotide
comprising a sequence of SEQ >D N0:25 are splice variants of each other. Any
one of the splice
variants described above can encode a polypeptide which contains at least one
functional or structural
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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 known and naturally occurring gene, may
be produced. Thus,
the invention contemplates each and every possible variation of polynucleotide
sequence that could be
made by selecting combinations based on possible codon choices. These
combinations are made in
accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of
naturally occurring DME, and all such variations are to be considered as being
specifically disclosed.
Although polynucleotides which encode DME and its variants are generally
capable of
l0 hybridizing to polynucleotides encoding naturally occurring DME under
appropriately selected
conditions of stringency, it may be advantageous to produce polynucleotides
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 polynucleotides which encode DME
and DME
derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the synthetic
polynucleotide may be inserted into any of the many available expression
vectors and cell systems
using reagents well known in the art. Moreover, synthetic chemistry may be
used to introduce
mutations into a polynucleotide encoding DME or any fragment thereof.
Embodiments of the invention can also include polynucleotides that are capable
of hybridizing
to the claimed polynucleotides, and, in particular, to those having the
sequences shown in SEQ 117
N0:14-26 and fragments thereof, under various conditions of stringency (Wahl,
G.M. and S.L. Berger
(1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511).
Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Biosciences, Piscataway NJ),
or combinations
of polymerises and proofreading exonucleases such as those found in the
ELONGASE amplification
CA 02453075 2004-O1-05
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system (Invitrogen, Carlsbad CA). Preferably, sequence preparation is
automated with machines
such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200
thermal cycler
(MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied
Biosystems).
Sequencing is then carned out using either the ABI 373 or 377 DNA sequencing
system (Applied
Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences),
or other
systems known in the art. The resulting sequences are analyzed using a variety
of algorithms which
are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995)
Molecular Biology and
Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding 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 unknown sequence from
genomic DNA within a
cloning vector (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 (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
A third method, capture
PCR, involves PCR amplification of.DNA fragments adjacent to known sequences
in human and
yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods
Applic. 1:111-119). In
this method, multiple restriction enzyme digestions and ligations may be used
to insert an engineered
double-stranded sequence into a region of unknown sequence before performing
PCR. Other
methods which may be used to retrieve unknown sequences are known in the art
(Parker, J.D. et al.
(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested
primers, and
PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This
procedure
avoids the need to screen libraries and is useful in fording intron/exon
junctions. For all PCR-based
methods, primers may be designed using commercially available software, such
as OLIGO 4.06
primer analysis software (National Biosciences, Plymouth MN) or another
appropriate program, to be
about 22 to 30 nucleotides in length, to have a GC content of about SO% or
more, and to anneal to the
template at temperatures of about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
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Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotides or fragments thereof
which encode
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 polynucleotides which encode substantially the same or a
functionally equivalent
polypeptides may be produced and used to express DME.
The polynucleotides of the invention can be engineered using methods generally
known 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, and/or expression of the gene
product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be
used to engineer the nucleotide sequences. For example, oligonucleotide-
mediated site-directed
mutagenesis may be used to introduce mutations that create new restriction
sites, alter glycosylation
patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of 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,
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fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
In another embodiment, polynucleotides encoding DME may be synthesized, in
whole or in
part, using one or more chemical methods well known in the art (Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232).
Alternatively, DME itself or a fragment thereof may be synthesized using
chemical methods known in
the art. For example, peptide synthesis can be performed using various
solution-phase or solid-phase
techniques (Creighton, T. (1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204).
Automated synthesis may be
achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid
sequence of 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 (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-
421). The
composition of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing.
(Creighton, supra, pp. 28-53).
In order to express a biologically active DME, the polynucleotides 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
polynucleotides encoding
DME. Such elements may vary in their strength and specificity. Specific
initiation signals may also be
used to achieve more efficient translation of polynucleotides encoding DME.
Such signals include the
ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases
where a
polynucleotide sequence 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 synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used (Scharf, D. et
al. (1994) Results Probl.
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Cell Differ. 20:125-162).
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing polynucleotides encoding DME and appropriate
transcriptional and translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination (Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel
et al., supra, ch. 1, 3,
and 15).
A variety of expression vector/host systems may be utilized to contain and
express
polynucleotides encoding DME. These include, but are not limited to,
microorganisms such as bacteria
l0 transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast
transformed with yeast expression vectors; insect cell systems infected with
viral expression vectors
(e.g., baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower
mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression
vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems (Sambrook, supra; Ausubel et al.,
supra; Van Heeke, G.
and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu, N.
(1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y
(1992) McGraw
Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. USA
81:3655-3659; Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
Expression vectors derived from
retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various
bacterial plasmids, may be
used for delivery of polynucleotides to the targeted organ, tissue, or cell
population (Di Nicola, M. et
al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad.
Sci. USA 90:6340-
6344; Buller, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al.
(1994) Mol. Immunol.
31:219-226; Verma, 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 polynucleotides encoding DME. For example, routine
cloning, subcloning,
and propagation of polynucleotides encoding DME can be achieved using a
multifunctional E. coli
vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid
(Invitrogen).
Ligation of polynucleotides 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
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sequence (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 polynucleotide sequences into the host genome for stable
propagation (Ausubel et al.,
l0 supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer,
C.A. et al. (1994)
Bio/Technology 12:181-184).
Plant systems may also be used for expression of DME. Transcription of
polynucleotides
encoding DME may be driven by viral promoters, e.g., the 35S and 19S promoters
of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMEO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue,
R. et al: (1984)
Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ.
17:85-105). These constructs
can be introduced into plant cells by direct DNA transformation or pathogen-
mediated transfection
(The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New
York NY, pp.
191-196).
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, polynucleotides encoding
DME may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses DME in host cells (Logan, J. and T. Shenk
(1984) Proc. Natl. Acad.
Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous
sarcoma virus (RSV)
enhancer, may be used to increase expression in mammalian host cells. 5V40 or
EBV-based vectors
may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes (Harnngton, J.J. et al. (1997) Nat.
Genet. 15:345-355).
For long term production of recombinant proteins in mammalian systems, stable
expression of
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DME in cell lines is preferred. For example, polynucleotides encoding DME can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively
(Wigler, M. et al. (1977)
Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also,
antimetabolite, antibiotic, or herbicide
resistance can be used as the basis for selection. For example, dhfr confers
resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively (Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al.
(1981) J. Mol. Biol.
150:1-14). Additional selectable genes have been described, e.g., trpB and
hisD, which alter cellular
requirements for metabolites (Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech), (3-
2o 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 (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
polynucleotides 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 polynucleotide 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
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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.
T_mmunological 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-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on DME is
preferred, but a competitive
binding assay may be employed. These and other assays are well known in the
art (Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. N; Coligan,
l0 J.E. et al. (1997) Current Protocols in ImmunoloQV, Greene Pub. Associates
and Wiley-Interscience,
New York NY; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa
NJ).
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding DME
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively,
polynucleotides encoding DME, or any fragments thereof, may be cloned into a
vector for the
production of an mRNA probe. Such vectors are known in the art, are
commercially available, and
may be used to synthesize RNA probes in vitro by addition of an appropriate
RNA polymerase such
as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of
commercially available kits, such as those provided by Amersham Biosciences,
Promega (Madison
WI), and US Biochemical. Suitable reporter molecules or labels which may be
used for ease of
detection include radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents, as
well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with polynucleotides encoding 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 for its ability to modulate
expression of the
inserted polynucleotides or to process the expressed protein in the desired
fashion. Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
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"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
polynucleotides
encoding 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 proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MEP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MEP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the 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 et al. (supra, ch. 10 and
16). A variety of
commercially available kits may also be used to facilitate expression and
purification of fusion proteins.
In another embodiment, synthesis of radiolabeled DME may be achieved an vitro
using the
TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These
systems couple
transcription and translation of protein-coding sequences operably associated
with the T7, T3, or SP6
promoters. Translation takes place in the presence of a radiolabeled amino
acid precursor, for
example, 35S-methionine.
DME, fragments of DME, or variants of DME may be used to screen for compounds
that
specifically bind to DME. One or more test compounds may be screened for
specific binding to
DME. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test
compounds can be screened
for specific binding to DME. Examples of test compounds can include
antibodies, anticalins,
oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of DME can be used to screen for binding of
test
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compounds, such as antibodies, to DME, a variant of DME, or a combination of
DME and/or one or
more variants DME. In an embodiment, a variant of DME can be used to screen
for compounds that
bind to a variant of DME, but not to DME having the exact sequence of a
sequence of SEQ ID NO:1-
13. DME variants used to perform such screening can have a range of about 50%
to about 99%
sequence identity to DME, with various embodiments having 60%, 70%, 75%, 80%,
85%, 90%, and
95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to
DME can be
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 (Coligan, J.E.
et al. (1991) Current
Protocols in Immunology 1(2):Chapter 5). In another embodiment, the compound
thus identified can
be a natural ligand of a receptor DME (Howard, A.D. et al. (2001) Trends
Pharmacol. Sci.22:132-
140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding
to DME can be
closely related to the natural receptor to which DME binds, at least a
fragment of the receptor, or a
fragment of the receptor including all or a portion of the ligand binding site
or binding pocket. For
example, the compound may be a receptor for DME which is capable of
propagating a signal, or a
decoy receptor for DME which is not capable of propagating a signal
(Ashkenazi, A. and V.M. Divit
(1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends
Tmmunol. 22:328-336).
The compound can be rationally designed using known techniques. Examples of
such techniques
include those used to construct the compound etanercept (ENBREL; Immunex
Corp., Seattle WA),
which is efficacious for treating rheumatoid arthritis in humans. Etanercept
is an engineered p75
tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human
IgGI (Taylor, P.C. et al.
(2001) Curr. Opin. Irmnunol. 13:611-616).
In one embodiment, two or more antibodies having similar or, alternatively,
different
specificities can be screened for specific binding to DME, fragments of DME,
or variants of DME.
The binding specificity of the antibodies thus screened can thereby be
selected to identify particular
fragments or variants of DME. In one embodiment, an antibody can be selected
such that its binding
specificity allows for preferential identification of specific fragments or
variants of DME. In another
embodiment, an antibody can be selected such that its binding specificity
allows for preferential
diagnosis of a specific disease or condition having increased, decreased, or
otherwise abnormal
production of DME.
In an embodiment, anticalins can be screened for specific binding to DME,
fragments of
DME, or variants of DME. Anticalins are ligand-binding proteins that have been
constructed based on
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a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:8177-
8184; Skerra, A.
(2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can
include a beta-barrel
having eight antiparallel beta-strands, which supports four loops at its open
end. These loops form the
natural ligand-binding site of the lipocalins, a site which can be re-
engineered in vitro by amino acid
substitutions to impart novel binding specificities. The amino acid
substitutions can be made using
methods known in the art or described herein, and can include conservative
substitutions (e.g.,
substitutions that do not alter binding specificity) or substitutions that
modestly, moderately, or
significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to,
stimulate, or inhibit
DME involves producing appropriate cells which express DME, either as a
secreted protein or on the
cell membrane. Preferred cells include cells from mammals, yeast, Drosophila,
or E. coli. Cells
expressing DME or cell membrane fractions which contain DME are then contacted
with a test
compound and binding, stimulation, or inhibition 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 fluorophore, 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.
An assay can be used to assess the ability of a compound to bind to its
natural ligand and/or to
inhibit the binding of its natural ligand to its natural receptors. Examples
of such assays include radio-
labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be
introduced into a polypeptide
compound (such as a receptor) to improve or alter its ability to bind to its
natural ligands (Matthews,
D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related
embodiment, one or more amino
acid substitutions can be introduced into a polypeptide compound (such as a
ligand) to improve or alter
its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells
(1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-
10988).
DME, fragments of DME, or variants of DME 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
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presence 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 in 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 to a
plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding DME or their mammalian
homologs may be
"knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337). For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,
M.R. (1989) Science
244:1288-1292). The vector integrates into the corresponding region of the
host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Cfin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from
the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and
the resulting chimeric progeny are genotyped and bred to produce heterozygous
or homozygous
strains. Transgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
Polynucleotides encoding 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,
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a mammal inbred to overexpress DME, e.g., by secreting DME in its milk, may
also serve as a
convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists between
regions of DME and drug metabolizing enzymes. The expression of DME is closely
associated with
kidney, liver, and brain tissues, and with adrenal tumor, bladder tumor, and
lung tumor tissues. In
addition, examples of tissues expressing DME can be found in Table 6 and can
also be found in
Example Xi. Therefore, DME appears to play a role in autoimmune/inflammatory,
cell proliferative,
developmental, endocrine, eye, metabolic, and gastrointestinal disorders,
including liver disorders. In
l0 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/inflatnmatory disorder, such
as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune
hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-
candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic
infections, and trauma; a cell proliferative disorder, such as actinic
keratosis, 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,
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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 Syndenharri 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, Kallman'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 Cone 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
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,
keratoconjunctivitis sicca,
keratitis, episcleritis, iritis, posterior uveitis, glaucoma, amaurosis fugax,
ischemic optic neuropathy,
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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, Crohn's disease, Whipple's disease, Mallory-
Weiss syndrome, colonic
carcinoma, colonic obstruction, irntable bowel syndrome, short bowel syndrome,
diarrhea, constipation,
2o gastrointestinal hemorrhage, acquired immunodeficiency syndrome (A)DS)
enteropathy, jaundice,
hepatic encephalopathy, hepatorenal 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
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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 may 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 autoimmune/inflammatory, 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
antagonist or indirectly as a targeting or delivery mechanism for bringing a
pharmaceutical agent to
cells or tissues which express DME.
to In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding DME may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of DME including, but not limited to, those
described above.
In other embodiments, any protein, agonist, antagonist, antibody,
complementary sequence, or
vector embodiments may be administered in combination with other appropriate
therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made
by one of ordinary
skill in the art, according to conventional pharmaceutical principles. The
combination of therapeutic
agents may act synergistically to effect the treatment or prevention of the
various disorders described
above. Using this approach, one may be able to achieve therapeutic efficacy
with lower dosages of
each agent, thus reducing the potential for adverse side effects.
An antagonist of DME may be produced using methods which are generally known
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. Single chain antibodies (e.g., from
camels or llamas) may be
potent enzyme inhibitors and may have advantages in the design of peptide
mimetics, and in the
development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J.
Biotechnol. 74:277-
302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
dromedaries, llamas, humans, and others may be immunized by injection with DME
or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvants may be used to increase immunological response. Such
adjuvants include, but are
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not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to 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
l0 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 (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Immunol. Methods
81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
Cole, S.P. et al. (1984)
Mol. Cell Biol. 62:109-120).
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used (Mornson, S.L. et al.
(1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
Takeda, S. et al. (1985)
Nature 314:452-454). Alternatively, techniques described for the production of
single chain antibodies
may be adapted, using methods known in the art, to produce 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 (Burton, D.R.
(1991) Proc. Natl. Acad.
Sci. USA 88:10134-10137).
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-
3837; Winter, G. et al.
(1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for DME may also be
generated. For
example, such fragments include, but are not limited to, F(ab')z 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
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easy identification of monoclonal Fab fragments with the desired specificity
(Huse, W.D. et al. (1989)
Science 246:1275-1281).
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
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, supra).
l0 Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for 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 polyclonal antibodies, which are heterogeneous
in their affinities for
multiple DME epitopes, represents the average affinity, or avidity, of the
antibodies for DME. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular DME
epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ke ranging
from about 109 to 1012 L/mole are preferred for use in immunoassays in which
the DME-antibody
complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka ranging
from about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures
which ultimately require dissociation of DME, preferably in active form, from
the antibody (Catty, D.
(1988) Antibodies, Volume I: A Practical Approach, IRI, 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 S-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
(Catty, supra; Coligan et al.,
supra).
In another embodiment of the invention, 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,
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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 (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein (Slater, J.E. et
al. (1998) J. Allergy Clin. Immunol. 102:469-475; Scanlon, K.J. et al. (1995)
9:1288-1296). Antisense
l0 sequences can also be introduced intracellularly through the use of viral
vectors, such as retrovirus and
adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et
al., supra; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery
mechanisms include
liposome-derived systems, artificial viral envelopes, and other systems known
in the art (Rossi, J.J.
(1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Morris,
M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
In another embodiment of the invention, polynucleotides encoding 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)-Xl disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor 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
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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 means into DME-deficient cells. Mechanical transfer
technologies for use with
cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii) ballistic gold
particle delivery, (iii) liposome-mediated transfection, (iv) receptor-
mediated gene transfer, and (v) the
use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-
217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J.-L. and H. Recipon (1998)
Curr. Opin. Biotechnol.
9:445-450).
l0 Expression vectors that may be effective for the expression of DME include,
but are not
limited to, the PCDNA 3.1, EPTTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). 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 (1992)
Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) C~rr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding DME from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMEO 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) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
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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
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.5. Patent No. 5,910,434 to Rigg ("Method
for obtaining
l0 retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998)
Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, an adenovirus-based gene therapy delivery system is used to
deliver
polynucleotides encoding 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 known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy'),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999; Annu.
Rev. Nutr. 19:511-544) and Verma, LM. and N. Somia (1997; Nature 18:389:239-
242).
In another embodiment, a herpes-based, gene therapy delivery system is used to
deliver
polynucleotides encoding 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.
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Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
this patent are the construction and use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999; J. Virol. 73:519-
532) and Xu, H. et al.
(1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesvirus
sequences, the generation of
recombinant virus following the transfection of multiple plasmids containing
different segments of the
large herpesvirus genomes, the growth and propagation of herpesvirus, and the
infection of cells with
herpesvirus are techniques well known to those of ordinary skill in the art.
In another embodiment, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding 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) C~rr. 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 high 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 (BHK-21) with a variant of Sindbis virus (SIN)
indicates that the lytic
replication of alphaviruses can be altered to suit the needs of the gene
therapy application (Dryga,
S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses
will allow the introduction
of 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
3o and +10 from the start site, may also be employed to inhibit gene
expression. Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
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been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and
B.I. Carr, Molecular and
Immunolo ig 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 RNA molecules encoding DME.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
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 may be prepared by any
method
known in the art for the synthesis of nucleic acid molecules. These include
techniques for chemically
synthesizing oligonucleotides such as solid phase phosphoramidite chemical
synthesis. Alternatively,
RNA molecules may be generated by in vitro and in vivo transcription of DNA
molecules encoding
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
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limited to, oiigonucleotides, 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.
l0 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 from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding 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. 268:8-13).
A particular embodiment of the present invention involves screening a
combinatorial library of
oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide
nucleic acids, and modified
oligonucleotides) for antisense activity against a specific polynucleotide
sequence (Bruice, T.W. et al.
(1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No.
6,022,691).
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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 (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.
l0 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 tg on'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, intra-
arterial, intramedullary, intrathecal.
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means. ,
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery 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. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
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macromolecule. Alternatively, DME or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example 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 SO% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDso 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 EDso
with little or no toxicity.
2o The dosage varies within this range depending upon the dosage form
employed, the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 /,cg to 100,000 /.cg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
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conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind DME may be used for
the diagnosis
of disorders characterized by expression of DME, or in assays to monitor
patients being treated with
DME or agonists, antagonists, or inhibitors of DME. Antibodies useful for
diagnostic purposes may be
prepared in the same manner as described above for therapeutics. Diagnostic
assays for DME
include methods which utilize the antibody and a label to detect DME in human
body fluids or in
extracts of cells or tissues. The antibodies may be used with or without
modification, and may be
labeled by covalent or non-covalent attachment of a reporter molecule. A wide
variety of reporter
molecules, several of which are described above, are known in the art and may
be used.
A variety of protocols for measuring DME, including ELISAs, RIAs, and FACS,
are known in
the art and provide a basis for diagnosing altered or abnormal levels of DME
expression. Normal or
standard values for DME expression are established by combining body fluids or
cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibodies
to DME under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of DME
expressed in subject,
control, and disease samples from biopsied tissues are compared with the
standard values. Deviation
between standard and subject values establishes the parameters for diagnosing
disease.
In another embodiment of the invention, polynucleotides encoding DME may be
used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotides, complementary
RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and
quantify gene
expression in biopsied tissues in which expression of DME may be correlated
with disease. The
diagnostic assay may be used to determine absence, presence, and excess
expression of DME, and to
monitor regulation of DME levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotides,
including genomic sequences, encoding DME or closely related molecules may be
used to 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,
or related sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the DME encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:14-26 or from
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genomic sequences including promoters, enhancers, and introns of the DME gene.
Means for producing specific hybridization probes for polynucleotides encoding
DME include
the cloning of polynucleotides 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 35S, or by enzymatic
labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling systems, and the
like.
Polynucleotides 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
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections, and trauma; a cell
proliferative disorder, such as
actinic keratosis, 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, C~shing's
syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,
epilepsy, gonadal
dysgenesis, WAGR syndrome (Wilins' tumor, aniridia, genitourinary
abnormalities, and mental
retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary
rnucoepithelial dysplasia,
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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, Kallman's disease, Hand-
Schuller-Christian
disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwa~sm;
disorders associated
l0 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 Plummet's
disease; disorders associated with hyperparathyroidism including Coon 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, C~shing'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, 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,
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,
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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, lipodystroplues, 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,
Crohn's disease, Whipple's disease,
Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irntable bowel
syndrome, short
bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired
immunodeficiency
syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal
syndrome, hepatic
steatosis, hemochromatosis, Wilson's disease, alpha,-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. Polynucleotides 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, polynucleotides encoding DME may be used in assays
that detect the
presence of associated disorders, particularly those mentioned above.
Polynucleotides complementary
to 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
3o standard value. If the amount of signal in the patient sample is
significantly altered in comparison to a
control sample then the presence of altered levels of polynucleotides encoding
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
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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 or 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
l0 presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier, thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding 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
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from polynucleotides
encoding DME
may be used to detect single nucleotide polymorphisms (SNPs). SNPs are
substitutions, insertions and
deletions that are a frequent cause of inherited or acquired genetic disease
in humans. Methods of
SNP detection include, but are not limited to, single-stranded conformation
polymorphism (SSCP) and
fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived
from polynucleotides
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encoding DME are used to amplify DNA using the polymerise chain reaction
(PCR). The DNA may
be derived, for example, from diseased or normal tissue, biopsy samples,
bodily fluids, and the like.
SNPs in the DNA cause differences in the secondary and tertiary structures of
PCR products in
single-stranded form, and these differences are detectable using gel
electrophoresis in non-denaturing
gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which
allows detection of the
amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence
database analysis methods, termed in silico SNP (isSNP), are capable of
identifying polymorphisms by
comparing the sequence of individual overlapping DNA fragments which assemble
into a common
consensus sequence. These computer-based methods filter out sequence
variations due to laboratory
l0 preparation of DNA and sequencing errors using statistical models and
automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and
characterized by mass
spectrometry using, for example, the high throughput MASSARRAY system
(Sequenom, Inc., San
Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) C~rr. Opin.
Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of DME include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244;
Duplaa, C. et al. (1993)
Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples may
be accelerated by
running the assay in a high-throughput format where the oligomer or
polynucleotide of interest is
presented in various dilutions and a spectrophotometric or colorimetric
response gives rapid
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quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotides described herein may be used as elements on a microarray. The
microarray can be
used in transcript imaging techniques which monitor the relative expression
levels of large numbers of
genes simultaneously as described below. The microarray may also be used to
identify genetic
variants, mutations, and polymorphisms. This information may be used to
determine gene function, to
understand the genetic basis of a disorder, to diagnose a disorder, to monitor
progression/regression of
disease as a function of gene expression, and to develop and monitor the
activities of therapeutic
agents in the treatment of disease. In particular, this information may be
used to develop a
l0 pharmacogenomic profile of a patient in order to select the most
appropriate and effective treatment
regimen for that patient. For example, therapeutic agents which are highly
effective and display the
fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, DME, fragments of DME, or antibodies specific for DME
may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;
hereby expressly incorporated by reference herein). Thus a transcript image
may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
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molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to
that of a compound
with known toxicity, it is likely to share those toxic properties. These
fingerprints or signatures are
most useful and refined when they contain expression information from a large
number of genes and
gene families. Ideally, a genome-wide measurement of expression provides the
highest quality
signature. Even genes whose expression is not altered by any tested compounds
are important as
well, as the levels of expression of these genes are used to normalize the
rest of the expression data.
The normalization procedure is useful for comparison of expression data after
treatment with different
to compounds. While the assignment of gene function to elements of a toxicant
signature aids in
interpretation of toxicity mechanisms, knowledge of gene function is not
necessary for the statistical
matching of signatures which leads to prediction of toxicity (see, for
example, Press Release 00-02
from the National Institute of Environmental Health Sciences, released
February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In an embodiment, the toxicity of a test compound can be assessed by treating
a biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of the
present invention, so that transcript levels corresponding to the
polynucleotides of the present invention
may be quantified. The transcript levels in the treated biological sample are
compared with levels in
an untreated biological sample. Differences in the transcript levels between
the two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another embodiment relates to the use of the polypeptides disclosed herein to
analyze the
proteome of a tissue or cell type. The term proteome refers to the global
pattern of protein expression
in a particular tissue or cell type. Each protein component of a proteome can
be subjected individually
to further analysis. Proteome expression patterns, or profiles, are analyzed
by quantifying the number
of expressed proteins and their relative abundance under given conditions and
at a given time. A
profile of a cell's proteome may thus be generated by separating and analyzing
the polypeptides of a
particular tissue or cell type. In one embodiment, the separation is achieved
using two-dimensional gel
electrophoresis, in which proteins from a sample are separated by isoelectric
focusing in the first
dimension, and then according to molecular weight by sodium dodecyl sulfate
slab gel electrophoresis
in the second dimension (Steiner and Anderson, supra). The proteins are
visualized in the gel as
discrete and uniquely positioned spots, typically by staining the gel with an
agent such as Coomassie
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Blue or silver or fluorescent stains. The optical density of each protein spot
is generally proportional to
the level of the protein in the sample. The optical densities of equivalently
positioned protein spots
from different samples, for example, from biological samples either treated or
untreated with a test
compound or therapeutic agent, are compared to identify any changes in protein
spot density related to
the treatment. The proteins in the spots are partially sequenced using, for
example, standard methods
employing chemical or enzymatic cleavage followed by mass spectrometry. The
identity of the protein
in a spot may be determined by comparing its partial sequence, preferably of
at least 5 contiguous
amino acid residues, to the polypeptide sequences of interest. In some cases,
further sequence data
may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for 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 microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol- or
amino-reactive fluorescent compound and detecting the amount of fluorescence
bound at each array
element.
Toxicant signatures at the proteome level are also useful for toxicological
screening; and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the amino acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
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sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art
(Brennan,
T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al. (1995)
PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci.
USA 94:2150-2155;
Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of
microarrays are well known
and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A
Practical Approach, Oxford
University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding DME
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a mufti-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (PACs), bacterial artificial chromosomes (BACs),
bacterial Pl
constructions, or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once
mapped, the nucleic acid sequences may be used to develop genetic linkage
maps, for example, which
correlate the inheritance of a disease state with the inheritance of a
particular chromosome region or
restriction fragment length polymorphism (RFLP) (Larder, E.S. and D. Botstein
(1986) Proc. Natl.
Acad. Sci. USA 83:7353-7357).
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples
of genetic map data
can be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM)
World Wide Web site. Correlation between the location of the gene encoding 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
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linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation
(Gatti, R.A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, DME, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between 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 (Geysers, et al.
(1984) PCT application
W084/03564). In this method, large numbers of different small test compounds
are synthesized on a
solid substrate. The test compounds are reacted with 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 more
antigenic determinants with DME.
In additional embodiments, the nucleotide sequences which encode DME may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
3o properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
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whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/303,745, U.S. Ser. No. 60/305,402, U.S. Ser. No.
60/308,158, and U.S. Ser.
No. 60/322,127, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
lncyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and
lysed in guanidinium
isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and
guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or
extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the
UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen),
using the
recommended procedures or similar methods known in the art (Ausubel et al.,
supra, ch. 5). Reverse
transcription was initiated using oligo d(T) or random primers. Synthetic
oligonucleotide adapters were
ligated to double stranded cDNA, and the cDNA was digested with the
appropriate restriction enzyme
or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using
SEPHACRYL
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham
Biosciences) or preparative agarose gel electrophoresis. cDNAs were ligated
into compatible
restriction enzyme sites of the polylinker of a suitable plasmid, e.g.,
PBLUESCRIPT plasmid
(Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1 plasmid (Invitrogen,
Carlsbad CA), PBK-
CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid
(Stratagene),
pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Iucyte Genomics), or pINCY
(Incyte Genomics),
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or derivatives thereof. Recombinant plasmids were transformed into competent
E. coli cells including
XL1-Blue, XLl-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX
DH10B
from Invitrogen.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
Q1AWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without 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
cycfing steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Biosciences or supplied in ABI sequencing
kits such as the
ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Amersham
Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard
ABI protocols and base calling software; or other sequence analysis systems
known in the art.
Reading frames within the cDNA sequences were identified using standard
methods (Ausubel et al.,
supra, ch. 7). Some of the cDNA sequences were selected for extension using
the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
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vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo
Sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans,
Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); hidden
Markov model (HMM)-based protein family databases such as PFAM, INCY, and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain
databases such as
l0 SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002)
Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which
analyzes consensus
primary structures of gene families; see, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.
6:361-365.) The queries were performed using programs based on BLAST, FASTA,
BL>IVVIPS, and
HHIVIMER. The Incyte cDNA sequences were assembled to produce full length
polynucleotide
sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,
stretched
sequences, or Genscan-predicted coding sequences (see Examples IV and V) were
used to extend
Incyte cDNA assemblages to full length. Assembly was performed using programs
based on Phred,
Phrap, and Consed, and cDNA assemblages were screened for open reading frames
using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences
were translated
. to derive the corresponding full length polypeptide sequences.
Alternatively, a polypeptide may begin
at any of the 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, the PROTEOME databases, BLOCKS, PRINTS, DOMO,
PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such
as PFAM,
INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full
length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(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
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column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:14-26. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
1o 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 (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94;
Burge, C. and S. Karfin
(1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted exons to form an
assembled cDNA sequence extending from a methionine to a stop codon. The
output of Genscan is a
FASTA database of polynucleotide and polypeptide sequences. The maximum range
of sequence for
Genscan to analyze at once was set to 30 kb. To determine which of these
Genscan predicted cDNA
sequences encode 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 cDNA coverage of the Genscan-predicted sequences, thus
providing evidence
for transcription. When Incyte cDNA coverage was available, this information
was used to correct or
confirm the Genscan predicted sequence. Full length polynucleotide sequences
were obtained by
assembling Genscan-predicted coding sequences with Incyte cDNA sequences
and/or public cDNA
sequences using the assembly process described in Example III. Alternatively,
full length
polynucleotide sequences were derived entirely from edited or unedited Genscan-
predicted coding
sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
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"Stitched" Seguences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
2o with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Se4uences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
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VI. Chromosomal Mapping of DME Encoding Polynucleotides
The sequences which were used to assemble SEQ 1D N0:14-26 were compared with
sequences from the Incyte L1FESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:14-26 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
l0 of all sequences of that cluster, including its particular SEQ 117 NO:, to
that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site
(http://www.ncbi.nlm.nih.gov/genemapn, can be employed to determine if
previously identified disease
genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound (Sambrook, supra, ch. 7;
Ausubel et al., supra,
ch.4).
Analogous computer techniques applying BLAST were used to search for 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:
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BLAST Score x Percent Identity
x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
5 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
l0 gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotides encoding DME are analyzed with respect to the
tissue sources
from which they were derived. For example, some full length sequences are
assembled, at least in
part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA
sequence is derived
from a cDNA library constructed from a human tissue. Each human tissue is
classified into one of the
following organ/tissue categories: cardiovascular system; connective tissue;
digestive system;
embryonic structures; endocrine system; exocrine glands; genitalia, female;
genitalia, male; germ cells;
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 disease/condition
categories: cancer, cell line, developmental, inflammation, neurological,
trauma, cardiovascular, pooled,
and other, and the number of libraries in each category is counted and divided
by the total number of
libraries across all categories. The resulting percentages reflect the tissue-
and disease-specific
expression of cDNA encoding DME. cDNA sequences and cDNA library/tissue
information are
found in the LIFESEQ GOLD database (Iucyte Genomics, Palo Alto CA).
VIII. Extension of DME Encoding Polynucleotides
Full length polynucleotides are produced by extension of an appropriate
fragment of the full
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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.
l0 High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)2S04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme
(Invitrogen), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer pair
PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C> 15 sec; Step
3: 60°C, 1 min; Step 4: 68°C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min;
Step 7: storage at 4 °C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~,1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~cl 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 relegation into pUC 18 vector (Amersham
Biosciences). For shotgun
sequencing, the digested nucleotides were separated on low concentration (0.6
to 0.8%) agarose gels,
fragments were excised, and agar digested with Agar ACE (Promega). Extended
clones were
religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
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Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site overhangs, and
transfected into competent E. coli cells. Transformed cells were selected on
antibiotic-containing
media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the following
parameters: Step
1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1
min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and
4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at
4°C. DNA was quantified by
PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries
were reamplified using the same conditions as described above. Samples were
diluted with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing primers
and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above
procedure or are used
to obtain 5' regulatory sequences using the above procedure along with
oligonucleotides designed for
such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in DME Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ID N0:14-26 using the LIFESEQ database (Incyte Genomics).
Sequences from the
same gene were clustered together and assembled as described in Example III,
allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minimum Phred quality score of 15, and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original
chromatogram files
in the vicinity of the putative SNP. Clone error filters used statistically
generated algorithms to identify
errors introduced during laboratory processing, such as those caused by
reverse transcriptase,
polymerase, or somatic mutation. Clustering error filters used statistically
generated algorithms to
identify errors resulting from clustering of close homologs or pseudogenes, or
due to contamination by
non-human sequences. A final set of filters removed duplicates and SNPs found
in immunoglobulins
or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
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four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental breakdown
of 43 % Chinese, 31 % Japanese, 13 % Korean, 5 % Vietnamese, and 8 % other
Asian. Allele
frequencies were first analyzed in the Caucasian population; in some cases
those SNPs which showed
no allelic variance in this population were not further tested in the other
three populations.
X. Labeling and Use of Individual Hybridization Probes
l0 Hybridization probes derived from SEQ ID N0:14-26 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 /.cCi of
[y 32P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide
kinase (DuPont NEN,
Boston MA). The labeled oligonucleotides are substantially purified using a
SEPHADEX G-25
superfine size exclusion dextran bead column (Amersham Biosciences). An
aliquot containing 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
1I, Eco RI, Pst I,
Xba I, or Pvu 1I (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing; see, e.g.,
Baldeschweiler et al., supra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena, M., ed.
(1999) DNA Microarrays: A Practical Approach, Oxford University Press,
London). Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
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analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and
may contain any appropriate number of elements (Schena, M. et al. (1995)
Science 270:467-470;
Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31).
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/p,l oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/pl RNase inhibitor, 500 pM dATP, 500 p,M dGTP, 500
~,M dTTP, 40 pM
dCTP, 40 pM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse
transcription
reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with
GEMBRIGHT kits
(Incyte). 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 O.SM 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.
(CLONTECI-1], 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 p1 SX SSC/0.2% SDS.
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Microarrav Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than S p,g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
l0 Scientific Products Corporation (VWR), West Chester PA), washed extensively
in distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/p,l, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°C
followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
p.1 of SX SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60°C. The arrays are washed for 10 min at 45°C in a
first wash buffer (1X SSC, 0.1%
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
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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 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
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
1o emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
Array elements
that exhibited at least about a two-fold change in expression, a signal-to-
background ratio of at least
2.5, and an element spot size of at least 40% were identified as
differentially expressed using the
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GEMTOOLS program (Incyte Genomics).
Expression
SEQ )D N0:24 showed differential expression, as determined by microarray
analysis. For
example, when the expression pattern of genes in normal lung tissue was
compared to that in
tumorous lung tissue from the same donor, SEQ 1D N0:24 was found to be
upregulated by at least
two-fold in three out of five donors. In each case, the experiment was done in
duplicate and the
results confirmed the original analysis. Analysis of gene expression patterns
associated with the
development and progression of the disease can yield tremendous insight into
the biology underlying
this disease, and can lead to the development of improved diagnostics and
therapeutics. Therefore,
l0 SEQ >D N0:24 and SEQ ID NO:11 (which is encoded by SEQ )D N0:24) can be
used as diagnostic
tools and as therapeutics for lung cancer.
XII. 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.
XIII. 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
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express 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 Autographica 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 bacterial-mediated
transposition involving
transfer plasmid intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter
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drives high levels of cDNA transcription. Recombinant baculovirus is used to
infect Spodoptera
frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the
latter requires additional genetic modifications to baculovirus (Engelhard,
E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945).
In most expression systems, 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 Schistosoma japonacum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from DME
at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity
purification using
commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a
stretch of six consecutive histidine residues, enables purification on metal-
chelate resins (QIAGEN).
Methods for protein expression and purification are discussed in Ausubel et
al. (supra, ch. 10 and 16).
Purified DME obtained by these methods can be used directly in the assays
shown in Examples XVII,
XVIII, and XIX, where applicable.
XIV. 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 plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid
(Invitrogen), both of
which contain the cytomegalovirus promoter. 5-10 p.g 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 /.cg 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
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light scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine
uptake; alterations in expression of cell surface and intracellular proteins
as measured by reactivity
with specific antibodies; and alterations in plasma membrane composition as
measured by the binding
of fluorescein-conjugated Annexin V protein to the cell surface. Methods in
flow cytometry are
discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
The influence of 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
NY). 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.
XV. 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 animals (e.g., rabbits, mice, etc.) 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
2o synthesized and used to raise antibodies by means known to those of skill
in the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art (Ausubel et al., supra, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MES) to
increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-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.
3o XVI. Purification of Naturally Occurring DME Using Specific Antibodies
Naturally occurring or recombinant DME is substantially purified by
imrnunoaffinity
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
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SEPHAROSE (Amersham Biosciences). 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
antibody/DME binding
(e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such
as urea or tluocyanate
ion), and DME is collected.
XVII. Identification of Molecules Which Interact with DME
DME, or biologically active fragments thereof, are labeled with lasl Bolton-
Hunter reagent
l0 (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 number, 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 MATCHMAKER 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 large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVIII. 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 MgClz, 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 earned 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.
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1x,25-dihydroxyvitamin D 24-hydroxylase activity of DME is determined by
monitoring the
conversion of 3H-labeled 1a,25-dihydroxyvitamin D (1a,25(OH)ZD) to 24,25-
dihydroxyvitamin D
(24,25(OH)ZD) in transgenic rats expressing DME. 1 p.g 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 S mM sodium succinate). A portion
(e.g., 3 ml) of each
homogenate is then incubated with 0.25 nM 1a,25(OH)2[1 3H]D, with a specific
activity of
approximately 3.5 GBq/mmol, for 15 min at 37 °C under oxygen with
constant shaking. Total lipids
are extracted as described (Bligh, E.G. and W.J. Dyer (1959) Can. J. Biochem.
Physiol. 37: 911-917)
and the chloroform phase is analyzed by HPLC using a FINEPAK SIL column
(JASCO, Tokyo,
Japan) with an n-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 confirmed 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)
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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
l0 product. Excess nitrite is removed by addition of sufficient ammonium
sulfamate, and the diazonium
salt is reacted with an aromatic amine (for example, N-naphthylethylene
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 nm is measured. The rate of increase 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.
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CA 02453075 2004-O1-05
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N-acetyltransferase activity of DME is measured using the transfer of
radiolabel from
[iaC]acetyl-CoA to a substrate molecule (for example, see Deguchi, T. (1975)
J. Neurochem.
24:1083-S). 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 nm (De Angelis, J. et al. (1997)
J. Biol. Chem.
273:3045-3050). Enzyme activity is proportional to the rate of radioactivity
incorporation into
substrate, or the rate of absorbance increase in the spectrophotometric assay.
Protein arginine 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
SO mM Tris-HCl (pH 7.4), 1.2 mM MgCl2, 200 p.M SAM (S-adenosyl-L-methionine)
iodide
(containing 0.5 pCi 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 p.g of purified DME or crude DME-containing sample and
performed at 37 °C for
min. The reaction is arrested by rapidly cooling on ice and immediately
extracting with 7 ml of
25 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
30 disappearance of NADPH at 340 nm (ego = 11,800 M-'~cmn). The standard assay
mixture contains
100 ~M 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
~.M dihydrofolate (as
111
CA 02453075 2004-O1-05
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substrate). The oxidation of NADPH to NADP+ 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).
Aldo/keto reductase activity of DME is measured using the decrease in
absorbance at 340 nm
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
nm as NAD+ is reduced to NADH. A standard reaction mixture is 50 mM sodium
phosphate, pH 7.5,
and 0.25 mM EDTA. The reaction is incubated at 25 °C and monitored
using a spectrophotometer.
Enzyme activity is calculated as mol NADH produced / mg of enzyme.
Carboxylesterase activity of DME is determined using 4-methylumbelliferyl
acetate as a
substrate. The enzymatic reaction is initiated by adding approximately 10 p1
of DME-containing
sample to 1 ml of reaction buffer (90 mM KHZPO4, 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 mMu cmu) 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 ml 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 p,g/ml) 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
are combined and dried under a stream of nitrogen. The residue is resuspended
in 14% acetonitrile,
250 mM KIIzP04, 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) J. Biol. Chem. 272:14769-14775).
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In another alternative, DME carboxyl esterase activity against the water-
soluble substrate
para-nitrophenyl butyric acid is determined 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 ~.g/ml
in sodium acetate, pH 5.0).
Carboxyl esterase activity is then monitored 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 incorporation of 355
from [355]PAPS
into a model substrate such as phenol (Folds, A. and J. L. Meek (1973)
Biochim. Biophys. Acta
l0 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 [35S] adenosine 3'-phosphate S'-
phosphosulfate (PAPS). 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 [355]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 pmol imidazole 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 lOs
cpm) of [355]PAPS in a final reaction volume of 50 p.1 at 37°C for 20
min. The reaction is stopped by
immersing 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 [355]PAPS 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]PAPS 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 ~1 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.
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Chem. 270:4172-4179).
In another alternative, DME sulfotransferase activity is determined by
measuring the transfer
of [355]sulfate from [3SS]PAPS to an immobilized 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 resin at a density of 1.5-3.0 p,mol
peptide/ml of resin. The enzyme
assay is performed by combining 10 p.1 of peptide-derivitized beads with 2-20
p1 of DME-containing
sample in 40 mM Pipes (pH 6.8), 0.3 M NaCI, 20 mM MnCl2, 50 mM NaF, 1% Triton
X-100, and 1
mM 5'-AMP in a final volume of 130 p1. The assay is initiated by addition of
0.5 p,Ci of [35S]PAPS
to (1.7 pM; 1 Ci = 37 GBq). After 30 min at 37°C, the reaction 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]PAPS as the
sulfate donor in a final volume of 30 p,1, containing 50 mM Hepes-NaOH (pH
7.0), 250 mM sucrose, 1
mM dithiothreitol, 14 pM[35S]PAPS (15 Ci/mmol), and dopamine (25 ~M), p-
nitrophenol (5 p.M), 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
2o terminated by heating at 100°C for 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 355-
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) reductase, 0.01 mM ['4C]squalene (dispersed with
the aid of 20 p1 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, v/v). The reaction products are compared
to those from a
reaction mixture without DME. The presence of 2,3(5)-oxidosqualene is
confirmed using appropriate
lipid standards (Sakakibara, J. et al. (1995) 270:17-20). ,
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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, epichlorohydrin, epibromohydrin,
epifluorohydrin, 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 1 ml of ice-cold
acetone containing an internal
standard for GC analysis (e.g., 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 1
hour at 37°C in the presence of 1 mM L-kynurenine and 1 mM 2-
oxoglutarate in a final volume of 200
p1 of 150 mM Tris acetate buffer (pH 8.0) containing 70 p,M 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 products with
UV detection at 340 nm. The production of kynurenic acid and xanthurenic acid,
respectively, is
indicative of aminotransferase activity (Buchli, R. et al. (1995) J. Biol.
Chem. 270:29330-29335).
In another alternative, 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
UV/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 pM 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
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CA 02453075 2004-O1-05
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purified protein preparations. Samples or lysates are resolved by
electrophoresis on 15%
non-denaturing polyacrylamide gels. The gels are incubated for 30 min in 2.5
mM vitro blue
tetrazolium, followed by incubation for 20 min in 30 mM potassium phosphate,
30 mM TEMED, and
30 pM 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).
XIX. Identification of DME Inhibitors
Compounds to be tested are arrayed in the wells of a multi-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 inhibit 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 compositions, methods,
and systems of
the invention will be apparent to .those skilled in the art without departing
from the scope and spirit of
the invention. It will be appreciated that the invention provides novel and
useful proteins, and their
encoding polynucleotides, which can be used in the drug discovery process, as
well as methods for
2o using these compositions for the detection, diagnosis, and treatment of
diseases and conditions.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Nor should the description of such embodiments be considered exhaustive or
limit the invention to the
precise forms disclosed. Furthermore, elements from one embodiment can be
readily recombined with
elements from one or more other embodiments. Such combinations can form a
number of
embodiments within the scope of the invention. It is intended that the scope
of the invention be
defined by the following claims and their equivalents.
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CA 02453075 2004-O1-05
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CA 02453075 2004-O1-05
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<110> INCYTE GENOMICS, INC.
GRIFFIN, Jennifer A.
RAMKUMAR, Jayala~ani
EMERLING, Brooke M.
RICHARDSON, Thomas W.
LI, Joana X.
WARREN, Bridget A.
HONCHELL, Cynthia D.
BAUGHN, Mariah R.
TANG, Y. Tom
LEE, Ernestine A.
ELLIOTT, Vicki S.
YUE, Henry
LEE, Sally
SWARNAKAR, Anita
FORSYTHE, Ian J.
SANJANWALA, Madhusudan M.
YAO, Monique G.
ZEBARJADIAN, Yeganeh
GORVAD, Ann E.
BECHA, Shanya D.
BURFORD, Neil
<120> DRUG'METABOLIZING ENZYMES
<130> PF-1057 PCT
<140> To Be Assigned
<141> Herewith,. .
<150> US .60/303,745
<151> 2001-07-06
<150> US 60/305,402
<151> 2001-07-13
<150> US 60/308,158
<151> 2001-07-27
<150> US 60/322,127
<151> 2001-09-14
<160> 26
<170> PERL Program
<210> 1
<211> 191
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7484676CD1
<400> 1
Met Glu Ala Lys Ala Ala Pro Lys Pro Ala Ala Ser Gly Ala Cys
1/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
1 5 10 15
Ser Val Ser Ala Glu Glu Thr Glu Lys Trp Met Glu Glu Ala Met
20 25 30
His Met Ala Lys Glu Ala Leu Glu Asn Thr Glu Val Pro Val Gly
35 40 45
Cys Leu Met Val Tyr Asn Asn Glu Val Val Gly Lys Gly Arg Asn
50 55 60
Glu Val Asn Gln Thr Lys Asn Ala Thr Arg His Ala Glu Met Val
65 70 75
Ala Ile Asp Gln Val Leu Asp Trp Cys Arg Gln Ser Gly Lys Ser
80 85 90
Pro Ser Glu Val Phe Glu His Thr Val Leu Tyr Val Thr Val Glu
95 100 105
Pro Cys Ile Met Cys Ala Ala Ala Leu Arg Leu Met Lys Ile Pro
110 115 120
Leu Val Val Tyr Gly Cys Gln Asn Glu Arg Phe Gly Gly Cys Gly
125 130 135
Ser Val Leu Asn Ile Ala Ser Ala Asp Leu Pro Asn Thr Gly Arg
140 145 150
Pro Phe Gln Cys Ile Pro Gly Tyr Arg Ala Glu Glu Ala Val Glu
155 160 165
Met Leu Lys Thr Phe Tyr Lys Gln Glu Asn Pro Asn Ala Pro Lys
170 175 180
Ser Lys Val Arg Lys Lys Glu Cys Gln Lys Ser
185 190
<210> 2 .. ..
<211> 2458
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7620224CD1
<400> 2
Met Val Leu Leu Leu Cys Leu Ser Cys Leu Ile Phe Ser Cys Leu
1 5 10 15
Thr Phe Ser Trp Leu Lys Ile Trp Gly Lys Met Thr Asp Ser Lys
20 25 30
Pro Ile Thr Lys Ser Lys Ser Glu Ala Asn Leu Ile Pro Ser Gln
35 40 45
Glu Pro Phe Pro Ala Ser Asp Asn Ser Gly Glu Thr Pro Gln Arg
50 55 60
Asn Gly Glu Gly His Thr Leu Pro Lys Thr Pro Ser Gln Ala Glu
65 70 75
Pro Ala Ser His Lys Gly Pro Lys Asp Ala Gly Arg Arg Arg Asn
80 85 90
Ser Leu Pro Pro Ser His Gln Lys Pro Pro Arg Asn Pro Leu Ser
95 100 105
Ser Ser Asp Ala Ala Pro Ser Pro Glu Leu Gln Ala Asn Gly Thr
110 115 120
Gly Thr Gln Gly Leu Glu Ala Thr Asp Thr Asn Gly Leu Ser Ser
125 130 135
Ser Ala Arg Pro Gln Gly Gln Gln Ala Gly Ser Pro Ser Lys Glu
140 145 150
2/34
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Asp Lys Lys Gln Ala Asn Ile Lys Arg Gln Leu Met Thr Asn Phe
155 160 165
Ile Leu Gly Ser Phe Asp Asp Tyr Ser Ser Asp Glu Asp Ser Val
170 175 180
Ala Gly Ser Ser Arg Glu Ser Thr Arg Lys Gly Ser Arg Ala Ser
185 190 195
Leu Gly Ala Leu Ser Leu Glu Ala Tyr Leu Thr Thr Gly Glu Ala
200 205 210
Glu Thr Arg Val Pro Thr Met Arg Pro Ser Met Ser Gly Leu His
215 220 225
Leu Val Lys Arg Gly Arg Glu His Lys Lys Leu Asp Leu His Arg
230 235 240
Asp Phe Thr Val Ala Ser Pro Ala Glu Phe Val Thr Arg Phe Gly
245 250 255
Gly Asp Arg Val Ile Glu Lys Val Leu Ile Ala Asn Asn Gly Ile
260 265 270
Ala Ala Val Lys Cys Met Arg Ser Ile Arg Arg Trp Ala Tyr Glu
275 280 285
Met Phe Arg Asn Glu Arg Ala Ile Arg Phe Val Val Met Val Thr
290 295 300
Pro Glu Asp Leu Lys Ala Asn Ala Glu Tyr Ile Lys Met Ala Asp
305 310 315
His Tyr Val Pro Val Pro Gly Gly Pro Asn Asn Asn Asn Tyr Ala
320 325 330
Asn Val Glu Leu Ile Val Asp Ile Ala Lys Arg Ile Pro Val Gln
335 340 345
Ala Val Trp Ala Gly Trp Gly His.Ala.Ser Glu Asn Pro Lys Leu
350 355 360
Pro Glu Leu Leu Cys Lys Asn Gly Val Ala Phe Leu Gly Pro Pro
365 370 375
Ser Glu Ala Met Trp Ala Leu Gly Asp.Lys Ile Ala Ser Thr Val
380 385 390
Val Ala Gln Thr Leu Gln Val Pro Thr Leu Pro Trp Ser Gly Ser
395 400 405
Gly Leu Thr Val Glu Trp Thr Glu Asp Asp Leu Gln Gln Gly Lys
410 415 420
Arg Ile Ser Val Pro Glu Asp Val Tyr Asp Lys Gly Cys Val Lys
425 430 435
Asp Val Asp Glu Gly Leu Glu Ala Ala Glu Arg Ile Gly Phe Pro
440 445 450
Leu Met Ile Lys Ala Ser Glu Gly Gly Gly Gly Lys Gly Ile Arg
455 460 465
Lys Ala Glu Ser Ala Glu Asp Phe Pro Ile Leu Phe Arg Gln Val
470 475 480
Gln Ser Glu Ile Pro Gly Ser Pro Ile Phe Leu Met Lys Leu Ala
485 490 495
Gln His Ala Arg His Leu Glu Val Gln Ile Leu Ala Asp Gln Tyr
500 505 510
Gly Asn Ala Val Ser Leu Phe Gly Arg Asp Cys Ser Ile Gln Arg
515 520 525
Arg His Gln Lys Ile Val Glu Glu Ala Pro Ala Thr Ile Ala Pro
530 535 540
Leu Ala Ile Phe Glu Phe Met Glu Gln Cys Ala Ile Arg Leu Ala
545 550 555
Lys Thr Val Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr
560 565 570
3/34
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Ser Gln Asp Gly Ser Phe His Phe Leu Glu Leu Asn Pro Arg Leu
575 580 585
Gln Val Glu His Pro Cys Thr Glu Met Ile Ala Asp Val Asn Leu
590 595 600
Pro Ala Ala Gln Leu Gln Ile Ala Met Gly Val Pro Leu His Arg
605 610 615
Leu Lys Asp Ile Arg Leu Leu Tyr Gly Glu Ser Pro Trp Gly Val
620 625 630
Thr Pro Ile Ser Phe Glu Thr Pro Ser Asn Pro Pro Leu Ala Arg
635 640 645
Gly His Val Ile Ala Ala Arg Ile Thr Ser Glu Asn Pro Asp Glu
650 655 660
Gly Phe Lys Pro Ser Ser Gly Thr Val Gln Glu Leu Asn Phe Arg
665 670 675
Ser Ser Lys Asn Val Trp Gly Tyr Phe Ser Val Ala Ala Thr Gly
680 685 690
Gly Leu His Glu Phe Ala Asp Ser Gln Phe Gly His Cys Phe Ser
695 700 705
Trp Gly Glu Asn Arg Glu Glu Ala Ile Ser Asn Met Val Val Ala
710 715 720
Leu Lys Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr Val Glu
725 730 735
Tyr Leu Ile Asn Leu Leu Glu Thr Glu Ser Phe Gln Asn Asn Asp
740 745 750
Ile Asp Thr Gly Trp Leu Asp Tyr Leu Ile Ala Glu Lys Val Gln
755 760 765
Ala Glu Lys Pro Asp Ile Met Leu Gly.Va1 Val Cys Gly Ala Leu
770 775 780
Asn Val Ala Asp Ala Met Phe Arg Thr Cys Met Thr Asp Phe Leu
785 790 795
His Ser Leu Glu Arg Gly Gln Val Leu Pro Ala Asp Ser Leu Leu
800 805 810
Asn Leu Val Asp Val Glu Leu Ile Tyr Gly Gly Val Lys Tyr Ile
815 820 825
Leu Lys Val Ala Arg Gln Ser Leu Thr Met Phe Val Leu Ile Met
830 835 840
Asn Gly Cys His Ile Glu Ile Asp Ala His Arg Leu Asn Asp Gly
845 850 855
Gly Leu Leu Leu Ser Tyr Asn Gly Asn Ser Tyr Thr Thr Tyr Met
860 865 870
Lys Glu Glu Val Asp Ser Tyr Arg Ile Thr Ile Gly Asn Lys Thr
875 880 885
Cys Val Phe Glu Lys Glu Asn Asp Pro Thr Val Leu Arg Ser Pro
890 895 900
Ser Ala Gly Lys Leu Thr Gln Tyr Thr Val Glu Asp Gly Gly His
905 910 915
Val Glu Ala Gly Ser Ser Tyr Ala Glu Met Glu Val Met Lys Met
920 925 930
Ile Met Thr Leu Asn Val Gln Glu Arg Gly Arg Val Lys Tyr Ile
935 940 945
Lys Arg Pro Gly Ala Val Leu Glu Ala Gly Cys Val Val Ala Arg
950 955 960
Leu Glu Leu Asp Asp Pro Ser Lys Val His Pro Ala Glu Pro Phe
965 970 975
Thr Gly Glu Leu Pro Ala Gln Gln Thr Leu Pro Ile Leu Gly Glu
980 985 990
4/34
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Lys Leu His Gln Val Phe His Ser Val Leu Glu Asn Leu Thr Asn
995 1000 1005
Val Met Ser Gly Phe Cys Leu Pro Glu Pro Val Phe Ser Ile Lys
1010 1015 1020
Leu Lys Glu Trp Val Gln Lys Leu Met Met Thr Leu Arg His Pro
1025 1030 1035
Ser Leu Pro Leu Leu Glu Leu Gln Glu Ile Met Thr Ser Val Ala
1040 1045 1050
Gly Arg Ile Pro Ala Pro Val Glu Lys Ser Val Arg Arg Val Met
1055 1060 1065
Ala Gln Tyr Ala Ser Asn Ile Thr Ser Val Leu Cys Gln Phe Pro
1070 1075 1080
Ser Gln Gln Ile Ala Thr Ile Leu Asp Cys His Ala Ala Thr Leu
1085 1090 1095
Gln Arg Lys Ala Asp Arg Glu Val Phe Phe Ile Asn Thr Gln Ser
1100 1105 1110
Ile Val Gln Leu Val Gln Arg Tyr Arg Ser Gly Ile Arg Gly Tyr
1115 1120 1125
Met Lys Thr Val Val Leu Asp Leu Leu Arg Arg Tyr Leu Arg Val
1130 1135 1140
Glu His His Phe Gln Gln Ala His Tyr Asp Lys Cys Val Ile Asn
1145 1150 1155
Leu Arg Glu Gln Phe Lys Pro Asp Met Ser Gln Val Leu Asp Cys
1160 1165 1170
Ile Phe Ser His Ala Gln Val Ala Lys Lys Asn Gln Leu Val Ile
1175 1180 1185
Met Leu Ile Asp Glu Leu Cys Gly Pro Asp Pro Ser Leu Ser Asp
1190 1195 1200
Glu Leu Ile Ser Ile Leu Asn Glu Leu Thr Gln Leu Ser Lys Ser
1205 1210 1215
Glu His Cys Lys Val Ala Leu Arg Ala Arg Gln Ile Leu Ile Ala
1220 1225 1230
Ser Leu Leu Pro Ser Tyr Glu Leu Arg His Asn Gln Val Glu Ser
1235 1240 1245
Ile Phe Leu Ser Ala Ile Asp Met Tyr Gly His Gln Phe Cys Pro
1250 1255 1260
Glu Asn Leu Lys Lys Leu Ile Leu Ser Glu Thr Thr Ile Phe Asp
1265 1270 1275
Val Leu Pro Thr Phe Phe Tyr His Ala Asn Lys Val Val Cys Met
1280 1285 1290
Ala Ser Leu Glu Val Tyr Val Arg Arg Gly Tyr Ile Ala Tyr Glu
1295 1300 1305
Leu Asn Ser Leu Gln His Arg Gln Leu Pro Asp Gly Thr Cys Val
1310 1315 1320
Val Glu Phe Gln Phe Met Leu Pro Ser Ser His Pro Asn Arg Met
1325 1330 1335
Thr Val Pro Ile Ser Ile Thr Asn Pro Asp Leu Leu Arg His Ser
1340 1345 1350
Thr Glu Leu Phe Met Asp Ser Gly Phe Ser Pro Leu Cys Gln Arg
1355 1360 1365
Met Gly Ala Met Val Ala Phe Arg Arg Phe Glu Asp Phe Thr Arg
1370 1375 1380
Asn Phe Gly Glu Val Ile Ser Cys Phe Ala Asn Val Pro Lys Asp
1385 1390 1395
Thr Pro Leu Phe Ser Glu Ala Arg Thr Ser Leu Tyr Ser Glu Asp
1400 1405 1410
5/34
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Asp Cys Lys Ser Leu Arg Glu Glu Pro Ile His Ile Leu Asn Met
1415 1420 1425
Ser Ile Gln Cys Ala Asp His Leu Glu Asp Glu Ala Leu Val Pro
1430 1435 1440
Ile Leu Arg Thr Phe Val Gln Ser Lys Lys Asn Ile Leu Val Asp
1445 1450 1455
Tyr Gly Leu Arg Arg Ile Thr Phe Leu Ile Ala Gln Glu Lys Glu
1460 1465 1470
Phe Pro Lys Phe Phe Thr Phe Arg Ala Arg Asp Glu Phe Ala Glu
1475 1480 1485
Asp Arg Ile Tyr Arg His Leu Glu Pro Ala Leu Ala Phe Gln Leu
1490 1495 1500
Glu Leu Asn Arg Met Arg Asn Phe Asp Leu Thr Ala Val Pro Cys
1505 1510 1515
Ala Asn His Lys Met His Leu Tyr Leu Gly Ala Ala Lys Val Lys
1520 1525 1530
Glu Gly Val Glu Val Thr Asp His Arg Phe Phe Ile Arg Ala Ile
1535 1540 1545
Ile Arg His Ser Asp Leu Ile Thr Lys Glu Ala Ser Phe Glu Tyr
1550 1555 1560
Leu Gln Asn Glu Gly Glu Arg Leu Leu Leu Glu Ala Met Asp Glu
1565 1570 1575
Leu Glu Val Ala Phe Asn Asn Thr Ser Val Arg Thr Asp Cys Asn
1580 1585 1590
His Ile Phe Leu Asn Phe Val Pro Thr Val Ile Met Asp Pro Phe
1595 1600 1605
Lys Ile Glu Glu Ser Val Arg Tyr Met Val.Met Arg Tyr Gly Ser
1610 1615 1620
Arg Leu Trp Lys Leu Arg Val Leu Gln Ala:.Glu Val Lys Ile Asn
1625 1630 1635
Ile Arg Gln.Thr Thr Thr Gly Ser Ala Val Pro Ile Arg Leu Phe
1640 1645 1650
Ile Thr Asn Glu Ser Gly Tyr Tyr Leu Asp Ile Ser Leu Tyr Lys
1655 1660 1665
Glu Val Thr Asp Ser Arg Ser Gly Asn Ile Met Phe His Ser Phe
1670 1675 1680
Gly Asn Lys Gln Gly Pro Gln His Gly Met Leu Ile Asn Thr Pro
1685 1690 1695
Tyr Val Thr Lys Asp Leu Leu Gln Ala Lys Arg Phe Gln Ala Gln
1700 1705 1710
Thr Leu Gly Thr Thr Tyr Ile Tyr Asp Phe Pro Glu Met Phe Arg
1715 1720 1725
Gln Ala Leu Phe Lys Leu Trp Gly Ser Pro Asp Lys Tyr Pro Lys
1730 1735 1740
Asp Ile Leu Thr Tyr Thr Glu Leu Val Leu Asp Ser Gln Gly Gln
1745 1750 1755
Leu Val Glu Met Asn Arg Leu Pro Gly Gly Asn Glu Val Gly Met
1760 1765 1770
Val Ala Phe Lys Met Arg Phe Lys Thr Gln Glu Tyr Pro Glu Gly
1775 1780 1785
Arg Asp Val Ile Val Ile Gly Asn Asp Ile Thr Phe Arg Ile Gly
1790 1795 1800
Ser Phe Gly Pro Gly Glu Asp Leu Leu Tyr Leu Arg Ala Ser Glu
1805 1810 1815
Met Ala Arg Pro Glu Ala Ile Pro Lys Ile Tyr Val Ala Ala Asn
1820 1825 1830
6/34
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Ser Gly Ala Arg Ile Gly Met Ala Glu Glu Ile Lys His Met Phe
1835 1840 1845
His Val Ala Trp Val Asp Pro Glu Asp Pro His Lys Gly Phe Lys
1850 1855 1860
Tyr Leu Tyr Leu Thr Pro Gln Asp Tyr Thr Arg Ile Ser Ser Leu
1865 1870 1875
Asn Ser Val His Cys Lys His Ile Glu Glu Gly Gly Glu Ser Arg
1880 1885 1890
Tyr Ile Met Thr Asp Ile Ile Gly Lys Asp Asp Gly Leu Gly Val
1895 1900 1905
Glu Asn Leu Arg Gly Ser Gly Met Ile Ala Gly Glu Ser Ser Leu
1910 1915 1920
Ala Tyr Glu Glu Ile Val Thr Ile Ser Leu Val Thr Cys Arg Ala
1925 1930 1935
Ile Gly Ile Gly Ala Tyr Leu Val Arg Leu Gly Gln Arg Val Ile
1940 1945 1950
Gln Val Glu Asn Ser His Ile Ile Leu Thr Gly Ala Ser Ala Leu
1955 1960 1965
Asn Lys Val Leu Gly Arg Glu Val Tyr Thr Ser Asn Asn Gln Leu
1970 1975 1980
Gly Gly Val Gln Ile Met His Tyr Asn Gly Val Ser His Ile Thr
1985 1990 1995
Val Pro Asp Asp Phe Glu Gly Val Tyr Thr Ile Leu Glu Trp Leu
2000 2005 2010
Ser Tyr Met Pro Lys Asp Asn His Ser Pro Val Pro Ile Ile Thr
2015 2020 2025
Pro Thr Asp Pro Ile Asp Arg Glu Ile Glu Phe Leu Pro Ser Arg
2030 2035 2040
Ala Pro Tyr Asp Pro Arg Trp Met Leu Ala Gly Arg Pro His Pro
2045 2050 2055
Thr Leu Lys Gly Thr Trp Gln Ser Gly Phe Phe Asp His Gly Ser
2060 2065 2070
Phe Lys Glu Ile Met Ala Pro Trp Ala Gln Thr Val Val Thr Gly
2075 2080 2085
Arg Ala Arg Leu Gly Gly Ile Pro Val Gly Val Ile Ala Val Glu
2090 2095 2100
Thr Arg Thr Val Glu Val Ala Val Pro Ala Asp Pro Ala Asn Leu
2105 2110 2115
Asp Ser Glu Ala Lys Ile Ile Gln Gln Ala Gly Gln Val Trp Phe
2120 2125 2130
Pro Asp Ser Ala Tyr Lys Thr Ala Gln Ala Ile Lys Asp Phe Asn
2135 2140 2145
Arg Glu'Lys Leu Pro Leu Met Ile Phe Ala Asn Trp Arg Gly Phe
2150 2155 2160
Ser Gly Gly Met Lys Asp Met Tyr Asp Gln Val Leu Lys Phe Gly
2165 2170 2175
Ala Tyr Ile Val Asp Gly Leu Arg Gln Tyr Lys Gln Pro Ile Leu
2180 2185 2190
Ile Tyr Ile Pro Pro Tyr Ala Glu Leu Arg Gly Gly Ser Trp Val
2195 2200 2205
Val Ile Asp Ala Thr Ile Asn Pro Leu Cys Ile Glu Met Tyr Ala
2210 2215 2220
Asp Lys Glu Ser Arg Gly Gly Val Leu Glu Pro Glu Gly Thr Val
2225 2230 2235
Glu Ile Lys Phe Arg Lys Lys Asp Leu Ile Lys Ser Met Arg Arg
2240 2245 2250
7/34
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Ile Asp Pro Ala Tyr Lys Lys Leu Met Glu Gln Leu Gly Glu Pro
2255 2260 2265
Asp Leu Ser Asp Lys Asp Arg Lys Asp Leu Glu Gly Arg Leu Lys
2270 2275 2280
Ala Arg Glu Asp Leu Leu Leu Pro Ile Tyr His Gln Val Ala Val
2285 2290 2295
Gln Phe Ala Asp Phe His Asp Thr Pro Gly Arg Met Leu Glu Lys
2300 2305 2310
Gly Val Ile Ser Asp Ile Leu Glu Trp Lys Thr Ala Arg Thr Phe
2315 2320 2325
Leu Tyr Trp Arg Leu Arg Arg Leu Leu Leu Glu Asp Gln Val Lys
2330 2335 2340
Gln Glu Ile Leu Gln Ala Ser Gly Glu Leu Ser His Val His Ile
2345 2350 2355
Gln Ser Met Leu Arg Arg Trp Phe Val Glu Thr Glu Gly Ala Val
2360 2365 2370
Lys Ala Tyr Leu Trp Asp Asn Asn Gln Val Val Val Gln Trp Leu
2375 2380 2385
Glu Gln His Trp Gln Ala Gly Asp Gly Pro Arg Ser Thr Ile Arg
2390 2395 2400
Glu Asn Ile Thr Tyr Leu Lys His Asp Ser Val Leu Lys Thr Ile
2405 2410 2415
Arg Gly Leu Val Glu Glu Asn Pro Glu Val Ala Val Asp Cys Val
2420 2425 2430
Ile Tyr Leu Ser Gln His Ile Ser Pro Ala Glu Arg Ala Gln Val
2435 2440 2445
Val His Leu Leu Ser Thr Met Asp Ser-Pro Ala Ser Thr
2450 2455 .
<210> 3
<211> 431
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7487081CD1
<400> 3
Met Pro Pro Ile Leu Gln Arg Leu Gln Gln Ala Thr Lys Met Met
1 5 10 15
Ser Arg Arg Lys Ile Leu Leu Leu Val Leu Gly Cys Ser Thr Val
20 25 30
Ser Leu Leu Ile His Gln Gly Ala Gln Leu Ser Trp Tyr Pro Lys
35 40 45
Leu Phe Pro Leu Ser Cys Pro Pro Leu Arg Asn Ser Pro Pro Arg
50 55 60
Pro Lys His Met Thr Val Ala Phe Leu Lys Thr His Lys Thr Ala
65 70 75
Gly Thr Thr Val Gln Asn Ile Leu Phe Arg Phe Ala Glu Arg His
80 85 90
Asn Leu Thr Val Ala Leu Pro His Pro Ser Cys Glu His Gln Phe
95 100 105
Cys Tyr Pro Arg Asn Phe Ser Ala His Phe Val His Pro Ala Thr
110 115 120
Arg Pro Pro His Val Leu Ala Ser His Leu Arg Phe Asp Arg Ala
8/34
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125 130 135
Glu Leu Glu Arg Leu Met Pro Pro Ser Thr Val Tyr Val Thr Ile
140 145 150
Leu Arg Glu Pro Ala Ala Met Phe Glu Ser Leu Phe Ser Tyr Tyr
155 160 165
Asn Gln Tyr Cys Pro Ala Phe Arg Arg Val Pro Asn Ala Ser Leu
170 175 180
Glu Ala Phe Leu Arg Ala Pro Glu Ala Tyr Tyr Arg Ala Gly Glu
185 190 195
His Phe Ala Met Phe Ala His Asn Thr Leu Ala Tyr Asp Leu Gly
200 205 210
Gly Asp Asn Glu Arg Ser Pro Arg Asp Asp Ala Ala Tyr Leu Ala
215 220 225
Gly Leu Ile Arg Gln Val Glu Glu Val Phe Ser Leu Val Met Ile
230 235 240
Ala Glu Tyr Phe Asp Glu Ser Leu Val Leu Leu Arg Arg Leu Leu
245 250 255
Ala Trp Asp Leu Asp Asp Val Leu Tyr Ala Lys Leu Asn Ala Arg
260 265 270
Ala Ala Ser Ser Arg Leu Ala Ala Ile Pro Ala Ala Leu Ala Arg
275 280 285
Ala Ala Arg Thr Trp Asn Ala Leu Asp Ala Gly Leu Tyr Asp His
290 295 300
Phe Asn Ala Thr Phe Trp Arg His Val Ala Arg Ala Gly Arg Ala
305 310 315
Cys Val Glu Arg Glu Ala Arg Glu Leu Arg Glu Ala Arg Gln Arg
320 325 330
Leu Leu Arg Arg Cys Phe Gly Asp Glu Pro Leu Leu Arg Pro Ala
335 340 . 345
Ala Gln Ile Arg Thr Lys Gln Leu Gln Pro Trp Gln Pro Ser Arg
350 355 360
Lys Val Asp Ile Met Gly Tyr Asp Leu Pro Gly Gly Gly Ala Gly
365 370 375
Pro Ala Thr Glu Ala Cys Leu Lys Leu Ala Met Pro Glu Val Gln
380 385 390
Tyr Ser Asn Tyr Leu Leu Arg Lys Gln Lys Arg Arg Gly Gly Ala
395 400 405
Arg Ala Arg Pro Glu Pro Val Leu Asp Asn Pro Pro Pro Arg Pro
410 415 420
Ile Arg Val Leu Pro Arg Gly Pro Gln Gly Pro
425 430
<210> 4
<211> 570
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2176536CD1
<400> 4
Met Ser Val Ser Asn Leu Ser Trp Leu Lys Lys Lys Ser Gln Ser
1 5 10 15
Val Asp Ile Asn Ala Pro Gly Phe Asn Pro Leu Ala Gly Ala Gly
20 25 30
9/34
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Lys Gln Thr Pro Gln Ala Ser Lys Pro Pro Ala Pro Lys Thr Pro
35 40 45
Ile Ile Glu Glu Glu Gln Asn Asn Ala Ala Asn Thr Gln Lys His
50 55 60
Pro Ser Arg Arg Ser Glu Leu Lys Arg Phe Tyr Thr Ile Asp Thr
65 70 75
Gly Gln Lys Lys Thr Leu Asp Lys Lys Asp Gly Arg Arg Met Ser
80 85 90
Phe Gln Lys Pro Lys Gly Thr Ile Glu Tyr Thr Val Glu Ser Arg
95 100 105
Asp Ser Leu Asn Ser Ile Ala Leu Lys Phe Asp Thr Thr Pro Asn
110 115 120
Glu Leu Val Gln Leu Asn Lys Leu Phe Ser Arg Ala Val Val Thr
125 130 135
Gly Gln Val Leu Tyr Val Pro Asp Pro Glu Tyr Val Ser Ser Val
140 145 150
Glu Ser Ser Pro Ser Leu Ser Pro Val Ser Pro Leu Ser Pro Thr
155 160 165
Ser Ser Glu Ala Glu Phe Asp Lys Thr Thr Asn Pro Asp Val His
170 175 180
Pro Thr Glu Ala Thr Pro Ser Ser Thr Phe Thr Gly Ile Arg Pro
185 190 195
Ala Arg Val Val Ser Ser Thr Ser Glu Glu Glu Glu Ala Phe Thr
200 205 210
Glu Lys Phe Leu Lys Ile Asn Cys Lys Tyr Ile Thr Ser Gly Lys
215 220 225
Gly Thr Val Ser Gly Val Leu Leu Val Thr Pro Asn Asn Ile Met
230 235 240
Phe Asp Pro His Lys Asn Asp Pro Leu Val Gln Glu Asn Gly Cys
245 250 255
Glu Glu Tyr Gly Ile Met Cys Pro Met Glu Glu Val Met Ser Ala
260 265 270
Ala Met Tyr Lys Glu Ile Leu Asp Ser Lys Ile Lys Glu Ser Leu
275 280 285
Pro Ile Asp Ile Asp Gln Leu Ser Gly Arg Asp Phe Cys His Ser
290 295 300
Lys Lys Met Thr Gly Ser Asn Thr Glu Glu Ile Asp Ser Arg Ile
305 310 315
Arg Asp Ala Gly Asn Asp Ser Ala Ser Thr Ala Pro Arg Ser Thr
320 325 330
Glu Glu Ser Leu Ser Glu Asp Val Phe Thr Glu Ser Glu Leu Ser
335 340 345
Pro Ile Arg Glu Glu Leu Val Ser Ser Asp Glu Leu Arg Gln Asp
350 355 360
Lys Ser Ser Gly Ala Ser Ser Glu Ser Val Gln Thr Val Asn Gln
365 370 375
Ala Glu Val Glu Ser Leu Thr Val Lys Ser Glu Ser Thr Gly Thr
380 385 390
Pro Gly His Leu Arg Ser Asp Thr Glu His Ser Thr Asn Glu Val
395 400 405
Gly Thr Leu Cys His Lys Thr Asp Leu Asn Asn Leu Glu Met Ala
410 415 420
Ile Lys Glu Asp Gln Ile Ala Asp Asn Phe Gln Gly Ile Ser Gly
425 430 435
Pro Lys Glu Asp Ser Thr Ser Ile Lys Gly Asn Ser Asp Gln Asp
440 445 450
10/34
CA 02453075 2004-O1-05
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Ser Phe Leu His Glu Asn Ser Leu His Gln Glu Glu Ser Gln Lys
455 460 465
Glu Asn Met Pro Cys Gly Glu Thr Ala Glu Phe Lys Gln Lys Gln
470 475 480
Ser Val Asn Lys Gly Lys Gln Gly Lys Glu Gln Asn Gln Asp Ser
485 490 495
Gln Thr Glu Ala Glu Glu Leu Arg Lys Leu Trp Lys Thr His Thr
500 505 510
Met Gln Gln Thr Lys Gln Gln Arg Glu Asn Ile Gln Gln Val Ser
515 520 525
Gln Lys Glu Ala Lys His Lys Ile Thr Ser Ala Asp Gly His Ile
530 535 540
Glu Ser Lys Cys Tyr Arg Val Asn Glu Val Ser Ser Ser Asn Cys
545 550 555
Met Val Ser Pro Ser Phe Leu Thr Asp Ser Lys Ala Ala Val Pro
560 565 570
<210> 5
<211> 276
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7498076CD1
<400> 5
Met Arg Leu Val Ile Leu Asp Asn Tyr Asp Leu Ala Ser Glu Trp
1 5 10 15
Ala Ala Lys Tyr Ile Cys Asn Arg Ile Ile Gln Phe Lys Pro Gly
20 25 30
Gln Asp Arg Tyr Phe Thr Leu Gly Leu Pro Thr Gly Ser Thr Pro
35 40 45
Leu Gly Cys Tyr Lys Lys Leu Ile Glu Tyr His Lys Asn Gly His
50 55 60
Leu Ser Phe Lys Tyr Val Lys Thr Phe Asn Met Asp Glu Tyr Val
65 70 75
Gly Leu Pro Arg Asn His Pro Glu Ser Tyr His Ser Tyr Met Trp
80 85 90
Asn Asn Phe Phe Lys His Ile Asp Ile Asp Pro Asn Asn Ala His
95 100 105
Ile Leu Asp Gly Asn Ala Ala Asp Leu Gln Ala Glu Cys Asp Ala
110 115 120
Phe Glu Asn Lys Ile Lys Glu Ala Gly Gly Ile Asp Leu Phe Val
125 130 135
Gly Gly Ile Gly Pro Asp Gly His Ile Ala Phe Asn Glu Pro Gly
140 145 150
Ser Ser Leu Val Ser Arg Thr Arg Leu Lys Thr Leu Ala Met Asp
155 160 165
Thr Ile Leu Ala Asn Ala Lys Tyr Phe Asp Gly Asp Leu Ser Lys
170 175 180
Val Pro Thr Met Ala Leu Thr Val Gly Val Gly Thr Val Met Asp
185 190 195
Ala Arg Glu Val Met Ile Leu Ile Thr Gly Ala His Lys Ala Phe
200 205 210
11/34
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Ala Leu Tyr Lys Ala Ile Glu Glu Gly Val Asn His Met Trp Thr
215 220 225
Val Ser Ala Phe Gln Gln His Pro Arg Thr Ile Phe Val Cys Asp
230 235 240
Glu Asp Ala Thr Leu Glu Leu Arg Val Lys Thr Val Lys Tyr Phe
245 250 255
Lys Gly Leu Met His Val His Asn Lys Leu Val Asp Pro Leu Phe
260 265 270
Ser Met Lys Asp Gly Asn
275
<210> 6
<211> 867
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1388154CD1
<400> 6
Met Arg Lys Val Ala His Ser Leu Thr Ala Ser Leu Ala Asn Gly
1 5 10 15
Leu Phe His Ser Arg Gly Met Pro Ala Gly Ala Phe Gly Ser Arg
20 25 30
Arg Arg Arg Leu Ala Ala Pro Pro Leu Leu Arg Pro Ala Gly Arg
35 40 45
Gly Arg Gly Gly Arg Gln Arg Cys Gln Arg Gly Arg Ser Cys Gly
50 55 60
Ala Arg Glu Glu Glu Val Glu Pro Gly Thr Ala Arg Pro Pro Pro
65 70 75
Ala Ala Ser Ala Met Asp Ala Ser Leu Glu Lys Ile Ala Asp Pro
80 85 90
Thr Leu Ala Glu Met Gly Lys Asn Leu Lys Glu Ala Val Lys Met
95 100 105
Leu Glu Asp Ser Gln Arg Arg Thr Glu Glu Glu Asn Gly Lys Lys
110 115 120
Leu Ile Ser Gly Asp Ile Pro Gly Pro Leu Gln Gly Ser Gly Gln
125 130 135
Asp Met Val Ser Ile Leu Gln Leu Val Gln Asn Leu Met His Gly
140 145 150
Asp Glu,Asp Glu Glu Pro Gln Ser Pro Arg Ile Gln Asn Ile Gly
155 160 165
Glu Gln Gly His Met Ala Leu Leu Gly His Ser Leu Gly Ala Tyr
170 175 180
Ile Ser Thr Leu Asp Lys Glu Lys Leu Arg Lys Leu Thr Thr Arg
185 190 195
Ile Leu Ser Asp Thr Thr Leu Trp Leu Cys Arg Ile Phe Arg Tyr
200 205 210
Glu Asn Gly Cys Ala Tyr Phe His Glu Glu Glu Arg Glu Gly Leu
215 220 225
Ala Lys Ile Cys Arg Leu Ala Ile His Ser Arg Tyr Glu Asp Phe
230 235 240
Val Val Asp Gly Phe Asn Val Ser Tyr Asn Lys Lys Pro Val Ile
245 250 255
Tyr Leu Ser Ala Ala Ala Arg Pro Gly Leu Gly Gln Tyr Leu Cys
12/34
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260 265 270
Asn Gln Leu Gly Leu Pro Phe Pro Cys Leu Cys Arg Val Pro Cys
275 280 285
Asn Thr Val Phe Gly Ser Gln His Gln Met Asp Val Ala Phe Leu
290 295 300
Glu Lys Leu Ile Lys Asp Asp Ile Glu Arg Gly Arg Leu Pro Leu
305 310 315
Leu Leu Val Ala Asn Ala Gly Thr Ala Ala Val Gly His Thr Asp
320 325 330
Lys Ile Gly Arg Leu Lys Glu Leu Cys Glu Gln Tyr Gly Ile Trp
335 340 345
Leu His Val Glu Gly Val Asn Leu Ala Thr Leu Ala Leu Gly Tyr
350 355 360
Val Ser Ser Ser Val Leu Ala Ala Ala Lys Cys Asp Ser Met Thr
365 370 375
Met Thr Pro Gly Pro Trp Leu Gly Leu Pro Ala Val Pro Ala Val
380 385 390
Thr Leu Tyr Lys His Asp Asp Pro Ala Leu Thr Leu Val Ala Gly
395 400 405
Leu Thr Ser Asn Lys Pro Thr Asp Lys Leu Arg Ala Leu Pro Leu
410 415 420
Trp Leu Ser Leu Gln Tyr Leu Gly Leu Asp Gly Phe Val Glu Arg
425 430 435
Ile Lys His Ala Cys Gln Leu Ser Gln Arg Leu Gln Glu Ser Leu
440 445 450
Lys Lys Val Asn Tyr Ile Lys Ile Leu Val Glu Asp Glu Leu Ser
455 460 465
Ser Pro Val Val Val Phe Arg Phe Phe Gln Glu Leu Pro Gly Ser
470 475 480
Asp Pro Val Phe Lys Ala Val Pro Val Pro Asn Met Thr Pro Ser
485 490 495
Gly Val Gly Arg Glu Arg His Ser Cys Asp Ala Val Asn Arg Trp
500 505 510
Leu Gly Glu Gln Leu Lys Gln Leu Val Pro Ala Ser Gly Leu Thr
515 520 525
Val Met Asp Leu Glu Ala Glu Gly Thr Cys Leu Arg Phe Ser Pro
530 535 540
Leu Met Thr Ala Ala Val Leu Gly Thr Arg Gly Glu Asp Val Asp
545 550 555
Gln Leu Val Ala Cys Ile Glu Ser Lys Leu Pro Val Leu Cys Cys
560 565 570
Thr Leu Gln Leu Arg Glu Glu Phe Lys Gln Glu Val Glu Ala Thr
575 580 585
Ala Gly Leu Leu Tyr Val Asp Asp Pro Asn Trp Ser Gly Ile Gly
590 595 600
Val Val Arg Tyr Glu His Ala Asn Asp Asp Lys Ser Ser Leu Lys
605 610 615
Ser Asp Pro Glu Gly Glu Asn Ile His Ala Gly Leu Leu Lys Lys
620 625 630
Leu Asn Glu Leu Glu Ser Asp Leu Thr Phe Lys Ile Gly Pro Glu
635 640 645
Tyr Lys Ser Met Lys Ser Cys Leu Tyr Val Gly Met Ala Ser Asp
650 655 660
Asn Val Asp Ala Ala Glu Leu Val Glu Thr Ile Ala Ala Thr Ala
665 670 675
Arg Glu Ile Glu Glu Asn Ser Arg Leu Leu Glu Asn Met Thr Glu
13/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
680 685 690
Val Val Arg Lys Gly Ile Gln Glu Ala Gln Val Glu Leu Gln Lys
695 700 705
Ala Ser Glu Glu Arg Leu Leu Glu Glu Gly Val Leu Arg Gln Ile
710 715 720
Pro Val Val Gly Ser Val Leu Asn Trp Phe Ser Pro Val Gln Ala
725 730 735
Leu Gln Lys Gly Arg Thr Phe Asn Leu Thr Ala Gly Ser Leu Glu
740 745 750
Ser Thr Glu Pro Ile Tyr Val Tyr Lys Ala Gln Gly Ala Gly Val
755 760 765
Thr Leu Pro Pro Thr Pro Ser Gly Ser Arg Thr Lys Gln Arg Leu
770 775 780
Pro Gly Gln Lys Pro Phe Lys Arg Ser Leu Arg Gly Ser Asp Ala
785 790 795
Leu Ser Glu Thr Ser Ser Val Ser His Ile Glu Asp Leu Glu Lys
800 805 810
Val Glu Arg Leu Ser Ser Gly Pro Glu Gln Ile Thr Leu Glu Ala
815 820 825
Ser Ser Thr Glu Gly His Pro Gly Ala Pro Ser Pro Gln His Thr
830 835 840
Asp Gln Thr Glu Ala Phe Gln Lys Gly Val Pro His Pro Glu Asp
845 850 855
Asp His Ser Gln Val Glu Gly Pro Glu Ser Leu Arg
860 865
<210> 7
<211> 422
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7481664CD1
<400> 7
Met Asp Ser Ile Ser Thr Ala Ile Leu Leu Leu Leu Leu Ala Leu
1 5 10 15
Val Cys Leu Leu Leu Thr Leu Ser Ser Arg Asp Lys Gly Lys Leu
20 25 30
Pro Pro Gly Pro Arg Pro Leu Ser Ile Leu Gly Asn Leu Leu Leu
35 40 45
Leu Cys Ser Gln Asp Met Leu Thr Ser Leu Thr Lys Leu Ser Lys
50 55 60
Glu Tyr Gly Ser Met Tyr Thr Val His Leu Gly Pro Arg Arg Val
65 70 75
Val Val Leu Ser Gly Tyr Gln Ala Val Lys Glu Ala Leu Val Asp
80 85 90
Gln Gly Glu Glu Phe Ser Gly Arg Gly Asp Tyr Pro Ala Phe Phe
95 100 105
Asn Phe Thr Lys Gly Asn Gly Ile Ala Phe Ser Ser Gly Asp Arg
110 115 120
Trp Lys Val Leu Arg Gln Phe Ser Ile Gln Ile Leu Arg Asn Phe
125 130 135
Gly Met Gly Lys Arg Ser Ile Glu Glu Arg Ile Leu Glu Glu Gly
140 145 150
14/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
Ser Phe Leu Leu Ala Glu Leu Arg Lys Thr Glu Gly Glu Pro Phe
155 160 165
Asp Pro Thr Phe Val Leu Ser Arg Ser Val Ser Asn Ile Ile Cys
170 175 180
Ser Val Leu Phe Gly Ser Arg Phe Asp Tyr Asp Asp Glu Arg Leu
185 190 195
Leu Thr Ile Ile Arg Leu Ile Asn Asp Asn Phe Gln Ile Met Ser
200 205 210
Ser Pro Trp Gly Glu Leu Tyr Asn Ile Phe Pro Ser Leu Leu Asp
215 220 225
Trp Val Pro Gly Pro His Gln Arg Ile Phe Gln Asn Phe Lys Cys
230 235 240
Leu Arg Asp Leu Ile Ala His Ser Val His Asp His Gln Ala Ser
245 250 255
Leu Asp Pro Arg Ser Pro Arg Asp Phe Ile His Cys Phe Leu Thr
260 265 270
Lys Met Ala Glu Glu Lys Glu Asp Pro Leu Ser His Phe His Met
275 280 285
Asp Thr Leu Leu Met Thr Thr His Asn Leu Leu Phe Gly Gly Thr
290 295 300
Lys Thr Val Ser Thr Thr Leu His His Ala Phe Leu Ala Leu Met
305 310 315
Lys Tyr Pro Lys Val Gln Ala Arg Val Gln Glu Glu Ile Asp Leu
320 325 330
Val Val Gly Arg Ala Arg Leu Pro Ala Leu Lys Asp Arg Ala Ala
335 340 345
Met Pro Tyr Thr Asp Ala Val Ile His Glu Val Gln Arg Phe Ala
350 355 360
Asp Ile Ile Pro Met Asn Leu Pro His Arg Val Thr Arg Asp Thr
365 370 375
Ala Phe Arg Gly Phe Leu Ile Pro Lys Gly Ala Val Cys Ala Trp
380 385 390
Glu Ser Arg Trp Arg Ala Trp Ser Ser Phe Cys Thr Ser Pro Pro
395 400 405
Ser Cys Arg Ala Phe Arg Cys Ser Arg Trp Val Arg Pro Arg Thr
410 415 420
Ser Thr
<210> 8
<211> 723
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7497661CD1
<400> 8
Met Ala Glu Tyr Thr Arg Leu His Asn Ala Leu Ala Leu Ile Arg
1 5 10 15
Leu Arg Asn Pro Pro Val Asn Ala Ile Ser Thr Thr Leu Leu Arg
20 25 30
Asp Ile Lys Glu Gly Leu Gln Lys Ala Val Ile Asp His Thr Ile
35 40 45
Lys Ala Ile Val Ile Cys Gly Ala Glu Gly Lys Phe Ser Ala Gly
15/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
50 55 60
Ala Asp Ile Arg Gly Phe Ser Ala Pro Arg Thr Phe Gly Leu Thr
65 70 75
Leu Gly His Val Val Asp Glu Ile Gln Arg Asn Glu Lys Pro Val
80 85 90
Val Ala Ala Ile Gln Gly Met Ala Phe Gly Gly Gly Leu Glu Leu
95 100 105
Ala Leu Gly Cys His Tyr Arg Ile Ala His Ala Glu Ala Gln Val
110 115 120
Gly Leu Pro Glu Val Thr Leu Gly Leu Leu Pro Gly Ala Arg Gly
125 130 135
Thr Gln Leu Leu Pro Arg Leu Thr Gly Val Pro Ala Ala Leu Asp
140 145 150
Leu Ile Thr Ser Gly Arg Arg Ile Leu Ala Asp Glu Ala Leu Lys
155 160 165
Leu Gly Ile Leu Asp Lys Val Val Asn Ser Asp Pro Val Glu Glu
170 175 180
Ala Ile Arg Phe Ala Gln Arg Val Ser Asp Gln Pro Leu Glu Ser
185 190 195
Arg Arg Leu Cys Asn Lys Pro Ile Gln Ser Leu Pro Asn Met Asp
200 205 210
Ser Ile Phe Ser Glu Ala Leu Leu Lys Met Arg Arg Gln His Pro
215 220 225
Gly Cys Leu Ala Gln Glu Ala Cys Val Arg Ala Val Gln Ala Ala
230 235 240
Val Gln Tyr Pro Tyr Glu Val Gly Ile Lys Lys Glu Glu Glu Leu
245 250 255
Phe Leu Tyr Leu Leu Gln Ser Gly Gln Ala Arg Ala Leu Gln Tyr
260 265 270
Ala Phe Phe Ala Glu Arg Lys Ala Asn Lys Trp Ser Thr Pro Ser
275 280 285
Gly Ala Ser Trp Lys Thr Ala Ser Ala Arg Pro Val Ser Ser Val
290 295 300
Gly Val Val Gly Leu Gly Thr Met Gly Arg Gly Ile Val Ile Ser
305 310 315
Phe Ala Arg Ala Arg Ile Pro Val Ile Ala Val Asp Ser Asp Lys
320 325 330
Asn Gln Leu Ala Thr Ala Asn Lys Met Ile Thr Ser Val Leu Glu
335 340 345
Lys Glu Ala Ser Lys Met Gln Gln Ser Gly His Pro Trp Ser Gly
350 355 360
Pro Lys Pro Arg Leu Thr Ser Ser Val Lys Glu Leu Gly Gly Val
365 370 375
Asp Leu Val Ile Glu Ala Val Phe Glu Glu Met Ser Leu Lys Lys
380 385 390
Gln Val Phe Ala Glu Leu Ser Ala Val Cys Lys Pro Glu Ala Phe
395 400 405
Leu Cys Thr Asn Thr Ser Ala Leu Asp Val Asp Glu Ile Ala Ser
410 415 420
Ser Thr Asp Arg Pro His Leu Val Ile Gly Thr His Phe Phe Ser
425 430 435
Pro Ala His Val Met Lys Leu Leu Glu Val Ile Pro Ser Gln Tyr
440 445 450
Ser Ser Pro Thr Thr Ile Ala Thr Val Met Asn Leu Ser Lys Lys
455 460 465
Ile Lys Lys Ile Gly Val Val Val Gly Asn Cys Phe Gly Phe Val
16/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
470 475 480
Gly Asn Arg Met Leu Asn Pro Tyr Tyr Asn Gln Ala Tyr Phe Leu
485 490 495
Leu Glu Glu Gly Ser Lys Pro Glu Glu Val Asp Gln Val Leu Glu
500 505 510
Glu Phe Gly Phe Lys Met Gly Pro Phe Arg Val Ser Asp Leu Ala
515 520 525
Gly Leu Asp Val Gly Trp Lys Ser Arg Lys Gly Gln Gly Leu Thr
530 535 540
Gly Pro Thr Leu Leu Pro Gly Thr Pro Ala Arg Lys Arg Gly Asn
545 550 555
Arg Arg Tyr Cys Pro Ile Pro Asp Val Leu Cys Glu Leu Gly Arg
560 565 570
Phe Gly Gln Lys Thr Gly Lys Gly Trp Tyr Gln Tyr Asp Lys Pro
575 580 585
Leu Gly Arg Ile His Lys Pro Asp Pro Trp Leu Ser Lys Phe Leu
590 595 600
Ser Arg Tyr Arg Lys Thr His His Ile Glu Pro Arg Thr Ile Ser
605 610 615
Gln Asp Glu Ile Leu Glu Arg Cys Leu Tyr Ser Leu Ile Asn Glu
620 625 630
Ala Phe Arg Ile Leu Gly Glu Gly Ile Ala Ala Ser Pro Glu His
635 640 645
Ile Asp Val Ile Tyr Leu His GIy Tyr Gly Trp Pro Arg His Val
650 655 660
Gly Gly Pro Met Tyr Tyr Ala Ser Thr Val Gly Leu Pro Thr Val
665 670 675
Leu Glu Lys Leu Gln Lys Tyr Tyr Arg Gln Asn Pro Asp Ile Pro
680 685 690
Gln Leu Glu Pro Ser Asp Tyr Leu Arg Arg Leu Val Ala Gln Gly
695 700 705
Ser Pro Pro Leu Lys Glu Trp Gln Ser Leu Ala Gly Pro His Ser
710 715 720
Ser Lys Leu
<210> 9
<211> 362
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7495116CD1
<400> 9
Met Ala Ala Gly Ala Gly Ala Gly Ser Ala Pro Arg Trp Leu Arg
1 5 10 15
Ala Leu Ser Glu Pro Leu Ser Ala Ala Gln Leu Arg Arg Leu Glu
20 25 30
Glu His Arg Tyr Ser Ala Ala Gly Val Ser Leu Leu Glu Pro Pro
35 40 45
Leu Gln Leu Tyr Trp Thr Trp Leu Leu Gln Trp Ile Pro Leu Trp
50 55 60
Met Ala Pro Asn Ser Ile Thr Leu Leu Gly Leu Ala Val Asn Val
65 70 75
17/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
Val Thr Thr Leu Val Leu Ile Ser Tyr Cys Pro Thr Ala Thr Glu
80 85 90
Glu Ala Pro Tyr Trp Thr Tyr Leu Leu Cys Ala Leu Gly Leu Phe
95 100 105
Ile Tyr Gln Ser Leu Asp Ala Ile Asp Gly Lys Gln Ala Arg Arg
110 115 120
Thr Asn Ser Cys Ser Pro Leu Gly Glu Leu Phe Asp His Gly Cys
125 130 135
Asp Ser Leu Ser Thr Val Phe Met Ala Val Gly Ala Ser Ile Ala
140 145 150
Ala Arg Leu Gly Thr Tyr Pro Asp Trp Phe Phe Phe Cys Ser Phe
155 160 165
Ile Gly Met Phe Val Phe Tyr Cys Ala His Trp Gln Thr Tyr Val
170 175 180
Ser Gly Met Leu Arg Phe Gly Lys Val Asp Val Thr Glu Ile Gln
185 190 195
Met Ala Leu Val Ile Val Phe Val Leu Ser Ala Phe Gly Gly Ala
200 205 210
Thr Met Trp Asp Tyr Thr Gly Thr Ser Val Leu Ser Pro Gly Leu
215 220 225
His Ile Gly Leu Ile Ile Ile Leu Ala Ile Met Ile Tyr Lys Lys
230 235 240
Ser Ala Thr Asp Val Phe Glu Lys His Pro Cys Leu Tyr Ile Leu
245 250 255
Met Phe Gly Cys Val Phe Ala Lys Val Ser Gln Lys Leu Val Val
260 265 270
Ala His Met Thr Lys Ser Glu Leu Tyr Leu Gln Asp Thr Val Phe
275 280 285
Leu Gly Pro Gly Leu Leu Phe Leu Asp Gln Tyr Phe Asn Asn Phe
290 295 300
Ile Asp Glu Tyr Val Val Leu Trp Met Ala Met Val Ile Ser Ser
305 310 315
Phe Asp Met Val Ile Tyr Phe Ser Ala Leu Cys Leu Gln Ile Ser
320 325 330
Arg His Leu His Leu Asn Ile Phe Lys Thr Ala Cys His Gln Ala
335 340 345
Pro Glu Gln Val Gln Val Leu Ser Ser Lys Ser His Gln Asn Asn
350 355 360
Met Asp
<210> 10
<211> 350
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7498400CD1
<400> 10
Met Thr Asn Glu Gly His Pro Trp Val Ser Leu Val Val Gln Lys
1 5 10 15
Thr Arg Leu Gln Ile Ser Gln Asp Pro Ser Leu Asn Tyr Glu Tyr
20 25 30
Leu Pro Thr Met Gly Leu Lys Ser Phe Ile Gln Ala Ser Leu Ala
18/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
35 40 45
Leu Leu Phe Gly Lys His Ser Gln Ala Ile Val Glu Asn Arg Val
50 55 60
Gly Gly Val His Thr Val Gly Asp Ser Gly Ala Phe Gln Leu Gly
65 70 75
Val Gln Phe Leu Arg Ala Trp His Lys Asp Ala Arg Ile Val Tyr
80 85 90
Ile Ile Ser Ser Gln Lys Glu Leu His Gly Leu Val Phe Gln Asp
95 100 105
Met Gly Phe Thr Val Tyr Glu Tyr Ser Val Trp Asp Pro Lys Lys
110 115 120
Leu Cys Met Asp Pro Asp Ile Leu Leu Asn Val Val Glu Ser Lys
125 130 135
Gln Ile Phe Pro Phe Phe Asp Ile Pro Cys Gln Gly Leu Tyr Thr
140 145 150
Ser Asp Leu Glu Glu Asp Thr Arg Ile Leu Gln Tyr Phe Val Ser
155 160 165
Gln Gly Phe Glu Phe Phe Cys Ser Gln Ser Leu Ser Lys Asn Phe
170 175 180
Gly Ile Tyr Asp Glu Gly Val Gly Met Leu Val Val Val Ala Val
185 190 195
Asn Asn Gln Gln Leu Leu Cys Val Leu Ser Gln Leu Glu Gly Leu
200 205 210
Ala Gln Ala Leu Trp Leu Asn Pro Pro Asn Thr Gly Ala Arg Val
215 220 225
Ile Thr Ser Ile Leu Cys Asn Pro Ala Leu Leu Gly Glu Trp Lys
230 235 240
Gln Ser Leu Lys Glu Val Val Glu Asn Ile Met Leu Thr Lys Glu
245 250 255
Lys Val Lys Glu Lys Leu Gln Leu Leu Gly Thr Pro Gly Ser Trp
260 265 270
Gly His Ile Thr Glu Gln Ser Gly Thr His Gly Tyr Leu Gly Leu
275 280 285
Asn Ser Gln Gln Val Glu Tyr Leu Val Arg Lys Lys His Ile Tyr
290 295 300
Ile Pro Lys Asn Gly Gln Ile Asn Phe Ser Cys Ile Asn Ala Asn
305 310 315
Asn Ile Asn Tyr Ile Thr Glu Gly Ile Asn Glu Ala Val Leu Leu
320 325 330
Thr Glu Ser Ser Glu Met Cys Leu Pro Lys Glu Lys Lys Thr Leu
335 340 345
Ile Gly Ile Lys Leu
350
<210> 11
<211> 204
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1709240CD1
<400> 11
Met Ala Glu Lys Pro Lys Leu His Tyr Phe Asn Ala Arg Gly Arg
1 5 10 15
19/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
Met Glu Ser Thr Arg Trp Leu Leu Ala Ala Ala Gly Val Glu Phe
20 25 30
Glu Glu Lys Phe Ile Lys Ser Ala Glu Asp Leu Asp Lys Leu Arg
35 40 45
Asn Asp Gly Tyr Leu Met Phe Gln Gln Val Pro Met Val Glu Ile
50 55 60
Asp Gly Met Lys Leu Val Gln Thr Arg Ala Ile Leu Asn Tyr Ile
65 70 75
Ala Ser Lys Tyr Asn Leu Tyr Gly Lys Asp Ile Lys Glu Arg Ala
80 85 90
Leu Ile Asp Met Tyr Ile Glu Gly Ile Ala Asp Leu Gly Glu Met
95 100 105
Ile Leu Leu Leu Pro Val Cys Pro Pro Glu Glu Lys Asp Ala Lys
110 115 120
Leu Ala Leu Ile Lys Glu Lys Ile Lys Asn Arg Tyr Phe Pro Ala
125 130 135
Phe Glu Ala Asp Ile His Leu Val Glu Leu Leu Tyr Tyr Val Glu
140 ' 145 150
Glu Leu Asp Ser Ser Leu Ile Ser Ser Phe Pro Leu Leu Lys Ala
155 160 165
Leu Glu Thr Arg Ile Ser Asn Leu Pro Thr Val Lys Lys Phe Leu
170 175 180
Gln Pro Gly Ser Pro Arg Lys Pro Pro Met Asp Glu Lys Ser Leu
185 190 195
Glu Glu Ala Arg Lys Ile Phe Arg Phe
200
<210> 12
<211> 204
<212> PRT
<213> Homo sapiens
<220>
<221> mist feature
<223> Incyte ID No: 4739684CD1
<400> 12
Met Ala Glu Lys Pro Lys Leu His Tyr Phe Asn Ala Arg Gly Arg
1 5 10 15
Met Glu Ser Thr Arg Trp Leu Leu Ala Ala Ala Gly Val Glu Phe
20 25 30
Glu Glu Lys Phe Met Gln Val Pro Met Val Glu Ile Asp Gly Met
35 40 45
Lys Leu Val Gln Thr Arg Ala Ile Leu Asn Tyr Ile Ala Ser Lys
50 55 60
Tyr Asn Leu Tyr Gly Lys Asp Ile Lys Glu Arg Ala Leu Ile Asp
65 70 75
Met Tyr Ile Glu Gly Ile Ala Asp Leu Gly Glu Met Ile Leu Leu
80 85 90
Leu Pro Val Cys Pro Pro Glu Glu Lys Asp Ala Lys Leu Ala Leu
95 100 105
Ile Lys Glu Lys Ile Lys Asn Arg Tyr Phe Pro Ala Phe Glu Lys
110 115 120
Val Leu Lys Ser His Gly Gln Asp Tyr Leu Val Gly Asn Lys Leu
125 130 135
Ser Arg Ala Asp Ile His Leu Val Glu Leu Leu Tyr Tyr Val Glu
20/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
140 145 150
Glu Leu Asp Ser Ser Leu Ile Ser Ser Phe Pro Leu Leu Lys Ala
155 160 165
Leu Lys Thr Arg Ile Ser Asn Leu Pro Thr Val Lys Lys Phe Leu
170 175 180
Gln Pro Gly Ser Pro Arg Lys Pro Pro Met Asp Glu Lys Ser Leu
185 190 195
Glu Glu Ala Arg Lys Ile Phe Arg Phe
200
<210> 13
<211> 614
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 72461345CD1
<400> 13
Met Arg Pro Pro Gln Cys Leu Leu His Thr Pro Ser Leu Ala Ser
1 5 10 15
Pro Leu Leu Leu Leu Leu Leu Trp Leu Leu Gly Gly Gly Val Gly
20 25 30
Ala Glu Gly Arg Glu Asp Ala Glu Leu Leu Val Thr Val Arg Gly
35 40 45
Gly Arg Leu Arg Gly Ile Arg Leu Lys Thr Pro Gly Gly Pro Val
50 55 60
Ser Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val Gly Ser
65 70 75
Arg Arg Phe Met Pro Pro Glu Pro Lys Arg Pro Trp Ser Gly Ile
80 85 90
Leu Asp Ala Thr Thr Phe Gln Asn Val Cys Tyr Gln Tyr Val Asp
95 100 105
Thr Leu Tyr Pro Gly Phe Glu Gly Thr Glu Met Trp Asn Pro Asn
110 115 120
Arg Glu Leu Ser Glu Asp Cys Leu Tyr Leu Asn Val Trp Thr Pro
125 130 135
Tyr Pro Arg Pro Thr Ser Pro Thr Pro Val Leu Ile Trp Ile Tyr
140 145 150
Gly Gly Gly Phe Tyr Ser Gly Ala Ser Ser Leu Asp Val Tyr Asp
155 160 165
Gly Arg Phe Leu Ala Gln Val Glu Gly Thr Val Leu Val Ser Met
170 175 180
Asn Tyr Arg Val Gly Thr Phe Gly Phe Leu Ala Leu Pro Gly Ser
185 190 195
Arg Asp Ala Pro Gly Asn Val Gly Leu Leu Asp Gln Arg Leu Ala
200 205 210
Leu Gln Trp Val Gln Glu Asn Ile Ala Ala Phe Gly Gly Asp Pro
215 220 225
Met Ser Val Thr Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val
230 235 240
Gly Met His Ile Leu Ser Leu Pro Ser Arg Ser Leu Phe His Arg
245 250 255
Ala Val Leu Gln Ser Gly Thr Pro Asn Gly Pro Trp Ala Thr Val
260 265 270
21/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
Ser Ala Gly Glu Ala Arg Arg Arg Ala Thr Leu Leu Ala Arg Leu
275 280 285
Val Gly Cys Pro Pro Gly Gly Ala Gly Gly Asn Asp Thr Glu Leu
290 295 300
Ile Ser Cys Leu Arg Thr Arg Pro Ala Gln Asp Leu Val Asp His
305 310 315
Glu Trp His Val Leu Pro Gln Glu Ser Ile Phe Arg Phe Ser Phe
320 325 330
Val Pro Val Val Asp Gly Asp Phe Leu Ser Asp Thr Pro Glu Ala
335 340 345
Leu Ile Asn Thr Gly Asp Phe Gln Asp Leu Gln Val Leu Val Gly
350 355 360
Val Val Lys Asp Glu Gly Ser Tyr Phe Leu Val Tyr Gly Val Pro
365 370 375
Gly Phe Ser Lys Asp Asn Glu Ser Leu Ile Ser Arg Ala Gln Phe
380 385 390
Leu Ala Gly Val Arg Ile Gly Val Pro Gln Ala Ser Asp Leu Ala
395 400 405
Ala Glu Ala Val Val Leu His Tyr Thr Asp Trp Leu His Pro Glu
410 415 420
Asp Pro Ala His Leu Arg Asp Ala Met Ser Ala Val Val Gly Asp
425 430 435
His Asn Val Val Cys Pro Val Ala Gln Leu Ala Gly Arg Leu Ala
440 445 450
Ala Gln Gly Ala Arg Val Tyr Ala Tyr Ile Phe Glu His Arg Ala
455 460 465
Ser Thr Leu Thr Trp Pro Leu Trp Met Gly Val Pro His Gly Tyr
470 475 480
Glu Ile Glu Phe Ile Phe Gly Leu Pro Leu Asp Pro Ser Leu Asn
485 490 495
Tyr Thr Val Glu Glu Arg Ile Phe Ala Gln Arg Leu Met Lys Tyr
500 505 510
Trp Thr Asn Phe Ala Arg Thr Gly Asp Pro Asn Asp Pro Arg Asp
515 520 525
Ser Lys Ser Pro Arg Trp Pro Pro Tyr Thr Thr Ala Ala Gln Gln
530 535 540
Tyr Val Ser Leu Asn Leu Lys Pro Leu Glu Val Arg Arg Gly Leu
545 550 555
Arg Ala Gln Thr Cys Ala Phe Trp Asn Arg Phe Leu Pro Lys Leu
560 565 570
Leu Ser Ala Thr Asp Thr Leu Asp Glu Ala Glu Arg Gln Trp Lys
575 580 585
Ala Glu Phe His Arg Trp Ser Ser Tyr Met Val His Trp Lys Asn
590 595 600
Gln Phe Asp His Tyr Ser Lys Gln Glu Arg Cys Ser Asp Leu
605 610
<210> 14
<211> 788
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7484676CB1
22/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
<400> 14
ggctgggtat ggaggcgaag gcggcaccca agccagctgc aagcggcgcg tgctcggtgt 60
cggcagagga gaccgaaaag tggatggagg aggcgatgca catggccaaa gaagccctcg 120
aaaatactga agttcctgtt ggctgtctta tggtctacaa caatgaagtt gtagggaagg 180
ggagaaatga agttaaccaa accaaaaatg ctactcgaca tgcagaaatg gtggccatcg 240
atcaggtcct cgattggtgt cgtcaaagtg gcaagagtcc ctctgaagta tttgaacaca 300
ctgtgttgta tgtcactgtg gagccgtgca ttatgtgtgc agctgctctc cgcctgatga 360
aaatcccgct ggttgtatat ggctgtcaga atgaacgatt tggtggttgt ggctctgttc 420
taaatattgc ctctgctgac ctaccaaaca ctgggagacc atttcagtgt atccctggat 480
atcgggctga ggaagcagtg gaaatgttaa agaccttcta caaacaagaa aatccaaatg 540
caccaaaatc gaaagttcgg aaaaaggaat gtcagaaatc ttgaacatgt tctgatgaaa 600
gaaccaagtg acccaaagtg acctggacaa gattcataga ctgaaagctg ttgacatcgt 660
tgaatcatat gtttatatat tgtttttaat ctgcaggaaa atggtgtctc tcatcatttg 720
ctctgattaa ggggacaaat tagcactttt tagaagtaag accttgcagc acatgacagc 780
gctcgttt 788
<210> 15
<211> 7446
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7620224CB1
<400> 15
cctgtgggcg cctgtcagcc tcactcaaga atggagctgg tggctgcatc cccagtgacg 60
attttctgaa tggtcttgct tctttgtcta tcttgtctga ttttctcctg tctgaccttt 120
tcctggttaa aaatctgggg gaaaatgacg gactccaagc cgatcaccaa gagtaaatca 180
gaagcaaacc tcatcccgag ccaggagccc tttccagcct ctgataactc aggggagaca 240
ccgcagagaa atggggaggg ccacactctg cccaagacac ccagccaggc cgagccagcc 300
tcccacaaag gccccaaaga tgccggtcgg cggagaaact ccctaccacc ctcccaccag 360
aagcccccaa gaaaccccct ttcttccagt gacgcagcac cctccccaga gcttcaagcc 420
aacgggactg ggacacaagg tctggaggcc acagatacca atggcctgtc ctcctcagcc 480
aggccccagg gccagcaagc tggctccccc tccaaagaag acaagaagca ggcaaacatc 540
aagaggcagc tgatgaccaa cttcatcctg ggctcttttg atgactactc ctccgacgag 600
gactctgttg ctggctcatc tcgtgagtct acccggaagg gcagccgggc cagcttgggg 660
gccctgtccc tggaggctta tctgaccaca ggtgaagctg agacccgcgt ccccactatg 720
aggccgagca tgtcgggact ccacctggtg aagaggggac gggaacacaa gaagctggac 780
ctgcacagag actttaccgt ggcttctccc gctgagtttg tcacacgctt tgggggggat 840
cgggtcatcg agaaggtgct tattgccaac aacgggattg ccgccgtgaa gtgcatgcgc 900
tccatccgca ggtgggccta tgagatgttc cgcaacgagc gggccatccg gtttgttgtg 960
atggtgaccc ccgaggacct taaggccaac gcagagtaca tcaagatggc ggatcattac 1020
gtccccgtcc caggagggcc caataacaac aactatgcca acgtggagct gattgtggac 1080
attgccaaga gaatccccgt gcaggcggtg tgggctggct ggggccatgc ttcagaaaac 1140
cctaaacttc cggagctgct gtgcaagaat ggagttgctt tcttaggccc tcccagtgag 1200
gccatgtggg ccttaggaga taagatcgcc tccaccgttg tcgcccagac gctacaggtc 1260
ccaaccctgc cctggagtgg aagcggcctg acagtggagt ggacagaaga tgatctgcag 1320
cagggaaaaa gaatcagtgt cccagaagat gtttatgaca agggttgcgt gaaagacgta 1380
gatgagggct tggaggcagc agaaagaatt ggttttccat tgatgatcaa agcttctgaa 1440
ggtggcggag ggaagggaat ccggaaggct gagagtgcgg aggacttccc gatccttttc 1500
agacaagtac agagtgagat cccaggctcg cccatctttc tcatgaagct ggcccagcac 1560
gcccgtcacc tggaagttca gatcctcgct gaccagtatg ggaatgctgt gtctctgttt 1620
ggtcgcgact gctccatcca gcggcggcat cagaagatcg ttgaggaagc accggccacc 1680
atcgccccgc tggccatatt cgagttcatg gagcagtgtg ccatccgcct ggccaagacc 1740
gtgggctatg tgagtgcagg gacagtggaa tacctctata gtcaggatgg cagcttccac 1800.
23/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
ttcttggagc tgaatcctcg cttgcaggtg gaacatccct gcacagaaat gattgctgac 1860
gttaatctgc cggccgccca gctacagatc gccatgggcg tgccactgca ccggctgaag 1920
gatatccggc ttctgtatgg agagtcacca tggggagtga ctcccatttc ttttgaaacc 1980
ccctcaaacc ctcccctcgc ccgaggccac gtcattgccg ccagaatcac cagcgaaaac 2040
ccagacgagg gttttaagcc gagctccggg actgtccagg aactgaattt ccggagcagc 2100
aagaacgtgt ggggttactt cagcgtggcc gctactggag gcctgcacga gtttgcggat 2160
tcccaatttg ggcactgctt ctcctgggga gagaaccggg aagaggccat ttcgaacatg 2220
gtggtggctt tgaaggaact gtccatccga ggcgacttta ggactaccgt ggaatacctc 2280
attaacctcc tggagaccga gagcttccag aacaacgaca tcgacaccgg gtggttggac 2340
tacctcattg ctgagaaagt gcaggcggag aaaccggata tcatgcttgg ggtggtatgc 2400
ggggccttga acgtggccga tgcgatgttc agaacgtgca tgacagattt cttacactcc 2460
ctggaaaggg gccaggtcct cccagcggat tcactactga acctcgtaga tgtggaatta 2520
atttacggag gtgttaagta cattctcaag gtggcccggc agtctctgac catgttcgtt 2580
ctcatcatga atggctgcca catcgagatt gatgcccacc ggctgaatga tggggggctc 2640
ctgctctcct acaatgggaa cagctacacc acctacatga aggaagaggt tgacagttac 2700
cgaattacca tcggcaataa gacgtgtgtg tttgagaagg agaacgatcc tacagtcctg 2760
agatccccct cggctgggaa gctgacacag tacacagtgg aggatggggg ccacgttgag 2820
gctgggagca gctacgctga gatggaggtg atgaagatga tcatgaccct gaacgttcag 2880
gaaagaggcc gggtgaagta catcaagcgt ccaggtgccg tgctggaagc aggctgcgtg 2940
gtggccaggc tggagctcga tgacccttct aaagtccacc cggctgaacc gttcacagga 3000
gaactccctg cccagcagac actgcccatc ctcggagaga aactgcacca ggtcttccac 3060
agcgtcctgg aaaacctcac caacgtcatg agtggctttt gtctgccaga gcccgttttt 3120
agcataaagc tgaaggagtg ggtgcagaag ctcatgatga ccctccggca cccgtcactg 3180
ccgctgctgg agctgcagga gatcatgacc agcgtggcag gccgcatccc cgcccctgtg 3240
gagaagtctg tccgcagggt gatggcccag tatgccagca acatcacctc ggtgctgtgc 3300
cagttcccca gccagcagat agccaccatc ctggactgcc atgcagccac cctgcagcgg 3360
aaggctgatc gagaggtctt cttcatcaac acccagagca tcgtgcagtt ggtccagaga 3420
taccgcagcg ggatccgcgg ctatatgaaa acagtggtgt tggatctcct gagaagatac 3480
ttgcgtgttg agcaccattt tcagcaagcc cactacgaca agtgtgtgat aaacctcagg 3540
gagcagttca agccagacat gtcccaggtg ctggactgca tcttctccca cgcacaggtg 3600
gccaagaaga accagctggt gatcatgttg atcgatgagc tgtgtggccc agacccttcc 3660
ctgtcggacg agctgatctc catcctcaac gagctcactc agctgagcaa aagcgagcac 3720
tgcaaagtgg ccctcagagc ccggcagatc ctgattgcct ccctcctccc ctcctacgag 3780
ctgcggcata accaggtgga gtccattttc ctgtctgcca ttgacatgta cggccaccag 3840
ttctgccccg agaacctcaa gaaattaata ctttcggaaa caaccatctt cgacgtcctg 3900
cctactttct tctatcacgc aaacaaagtc gtgtgcatgg cgtccttgga ggtttacgtg 3960
cggaggggct acatcgccta tgagttaaac agcctgcagc accggcagct cccggacggc 4020
acctgcgtgg tagaattcca gttcatgctg ccgtcctccc acccaaaccg gatgaccgtg 4080
cccatcagca tcaccaaccc tgacctgctg aggcacagca cagagctctt catggacagc 4140
ggcttctccc cactgtgcca gcgcatggga gccatggtag ccttcaggag attcgaggac 4200
ttcaccagaa attttggtga agtcatctct tgcttcgcca acgtgcccaa agacaccccc 4260
ctcttcagcg aggcccgcac ctccctatac tccgaggatg actgcaa,gag cctcagagaa 4320
gagcccatcc acattctgaa tatgtccatc cagtgtgcag accacctgga ggatgaggca 4380
ctggtgccga ttttacggac attcgtacag tccaagaaaa atatccttgt ggattatgga 4440
ctccgacgaa tcacattctt gattgcccaa gagaaagaat ttcccaagtt tttcacattc 4500
agagcaagag atgagtttgc agaagatcgc atttaccgtc acttggaacc tgccctggcc 4560
ttccagctgg aactcaaccg gatgcgtaac ttcgatctga ccgccgtgcc ctgtgccaac 4620
cacaagatgc acctttacct gggtgctgcc aaggtgaagg aaggtgtgga agtgacggac 4680
cataggttct tcatccgcgc catcatcagg cactctgacc tgatcacaaa ggaagcctcc 4740
ttcgaatacc tgcagaacga gggtgagcgg ctgctcctgg aggccatgga cgagctggag 4800
gtggcgttca ataacaccag cgtgcgcacc gactgcaacc acatcttcct caacttcgtg 4860
cccactgtca tcatggaccc cttcaagatc gaggagtccg tgcgctacat ggttatgcgc 4920
tacggcagcc ggctgtggaa actccgtgtg ctacaggctg aggtcaagat caacatccgc 4980
cagaccacca ccggcagtgc cgttcccatc cgcctgttca tcaccaatga gtcgggctac 5040
tacctggaca tcagcctcta caaagaagtg actgactcca gatctggaaa tatcatgttt 5100
cactccttcg gcaacaagca agggccccag cacgggatgc tgatcaatac tccctacgtc 5160
24/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
accaaggatc tgctccaggc caagcgattc caggcccaga ccctgggaac cacctacatc 5220
tatgacttcc cggaaatgtt caggcaggct ctctttaaac tgtggggctc cccagacaag 5280
tatcccaaag acatcctgac atacactgaa ttagtgttgg actctcaggg ccagctggtg 5340
gagatgaacc gacttcctgg tggaaatgag gtgggcatgg tggccttcaa aatgaggttt 5400
aagacccagg agtacccgga aggacgggat gtgatcgtca tcggcaatga catcaccttt 5460
cgcattggat cctttgggcc tggagaggac cttctctacc tccgggcatc cgagatggcg 5520
cggccagagg cgattcccaa aatttacgtg gcagccaaca gtggcgcccg tattggcatg 5580
gcagaggaga tcaaacacat gttccacgtg gcttgggtgg acccagaaga cccccacaaa 5640
ggatttaaat acctgtacct gactccccaa gactacacca gaatcagctc cctgaactcc 5700
gtccactgta aacacatcga ggaaggagga gagtccagat acatcatgac cgatatcatc 5760
gggaaggatg atggcttggg cgtggagaat ctgaggggct caggcatgat tgctggggag 5820
tcctctctcg cttacgaaga gatcgtcacc attagcttgg tgacctgccg agccattggg 5880
attggggcct acttggtgag gctgggccag cgagtgatcc aggtggagaa ttcccacatc 5940
atcctcacag gagcaagtgc tctcaacaag gtcctgggaa gagaggtcta cacatccaac 6000
aaccagctgg gtggcgttca gatcatgcat tacaatggtg tctcccacat caccgtgcca 6060
gatgactttg agggggttta taccatcctg gagtggctgt cctatatgcc aaaggataat 6120
cacagccctg tccctatcat cacacccact gaccccattg acagagaaat tgaattcctc 6180
ccatccagag ctccctacga cccccggtgg atgcttgcag gaaggcctca cccaactctg 6240
aagggaacgt ggcagagcgg attctttgac cacggcagtt tcaaggaaat catggcaccc 6300
tgggcgcaga ccgtggtgac aggacgagca aggcttgggg ggattcccgt gggagtgatt 6360
gctgtggaga cacggactgt ggaggtggca gtccctgcag accctgccaa cctggattct 6420
gaggccaaga taattcagca ggcaggacag gtgtggttcc cagactcagc ctacaaaacc 6480
gcccaggcca tcaaggactt caaccgggag aagttgcccc tgatgatctt tgccaactgg 6540
agggggttct ccggtggcat gaaagacatg tatgaccagg tgctgaagtt tggagcctac 6600
atcgtggacg gccttagaca atacaaacag cccatcctga tctatatccc gccctatgcg 6660
gagctccggg gaggctcctg ggtggtcata gatgccacca tcaacccgct gtgcatagaa 6720
atgtatgcag acaaagagag caggggtggt gttctggaac cagaggggac agtggagatt 6780
aagttccgaa agaaagatct gataaagtcc atgagaagga tcgatccagc ttacaagaag 6840
ctcatggaac agctagggga acctgatctc tccgacaagg accgaaagga cctggagggc 6900
cggctaaagg ctcgcgagga cctgctgctc cccatctacc accaggtggc ggtgcagttc 6960
gccgacttcc atgacacacc cggccggatg ctggagaagg gcgtcatatc tgacatcctg 7020
gagtggaaga ccgcacgcac cttcctgtat tggcgtctgc gccgcctcct cctggaggac 7080
caggtcaagc aggagatcct gcaggccagc ggggagctga gtcacgtgca tatccagtcc 7140
atgctgcgtc gctggttcgt ggagacggag ggggctgtca aggcctactt gtgggacaac 7200
aaccaggtgg ttgtgcagtg gctggaacag cactggcagg caggggatgg cccgcgctcc 7260
accatccgtg agaacatcac gtacctgaag cacgactctg tcctcaagac catccgaggc 7320
ctggttgaag aaaaccccga ggtggccgtg gactgtgtga tatacctgag ccagcacatc 7380
agcccagctg agcgggcgca ggtcgttcac ctgctgtcta ccatggacag cccggcctcc 7440
acctga 7446
<210> 16
<211> 1444
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7487081CB1
<400> 16
ccaacccggg ccagaggcag agcccaggcg tgcggtgctt ctcgggaaca ctggccctgc 60
taccagagcc gcaggatccc ccatgagtgc ctgtgaaggg agtcctggat agtgaggccc 120
tggctggtcc aggggtgggt actccgccat gccacccatc ctccagcgcc tgcagcaggc 180
caccaagatg atgagccgcc ggaaaatcct gctgctggtg ctagggtgca gcaccgtaag 240
ccttctcatc caccaggggg cgcagctcag ctggtacccc aagctgttcc ccttgagctg 300
ccctcctctg cggaactcgc cgccgcgccc caagcacatg actgtggcct tcctgaagac 360
25/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
tcacaagacg gcaggcacga cggtgcagaa catcctgttt cgctttgccg agcgccacaa 420
cctgacggtg gccctgccgc acccgagctg cgagcaccag ttctgctacc cccgcaactt 480
ctcggcgcac ttcgtgcacc cggccacgcg gccgccgcac gtgctggcca gccacctgcg 540
cttcgaccgt gcggagctgg agcgcctcat gccgcccagc accgtctatg tcaccatcct 600
gcgcgagccg gccgccatgt tcgagtcgct cttcagctac tacaaccagt actgcccggc 660
cttccggcgc gtgcccaacg cctcgctcga ggccttcctg cgcgcgcccg aggcatacta 720
ccgcgctggc gagcacttcg ccatgttcgc acacaacacg ctggcctacg acctgggcgg 780
cgacaatgag cgcagcccgc gcgacgacgc cgcctacctg gcgggcctca tccgccaggt 840
ggaggaggtt ttctcgctcg tcatgatcgc cgagtacttc gacgagtcgc tagtgctgct 900
gcggcgccta ctggcctggg acctggacga cgtgctctac gccaagctca acgcgcgcgc 960
cgccagctcg cgcctggccg ccatccccgc ggcgctggcg cgggcggcgc gcacctggaa 1020
cgccctggac gccggcctct acgaccactt caacgccacc ttctggcgcc acgtggcgcg 1080
cgcgggccgc gcgtgcgtgg agcgcgaggc gcgcgagctg cgcgaggccc gccagcgcct 1140
actgcggcgc tgcttcgggg acgagccact gctgcggcct gccgcgcaga tccgcaccaa 1200
gcagctgcag ccgtggcagc ccagccgcaa ggtggacatt atgggctatg acctgcccgg 1260
cggcggcgcc ggcccggcca ccgaggcctg cctcaagctg gccatgcccg aggtccagta 1320
ctcgaactac ctgttgcgca agcagaagcg ccggggcggt gcgcgggctc ggcccgagcc 1380
cgtcctggac aatcccccgc ctcggcccat ccgagtgctg cctcgcggcc ctcaaggtcc 1440
ctga 1444
<210> 17
<211> 2508
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2176536CB1
<400> 17
ggagctcagc gccggcgccg cgccgcccag ccccgccgag aggggcgcac tcgccgccgc 60
ggggcccgcc gccgctcacc gcagccccct cctggcgacc cgcaagtcct ctcaaactgt 120
gagtaactaa gtggtttgtg catcattcca gaagcaaagc taaaattttt agcggtgttg 180
tcgacttgac ctgctaattt cctgttctgg aatcgagaga agactcctca acaagttgct 240
gcaatgtctg tgtctaatct atcatggctg aagaaaaagt cccagtcggt ggatattaat 300
gctccagggt tcaacccttt ggctggtgca ggaaagcaaa caccacaagc cagtaagccc 360
ccggcaccca agacccccat cattgaagaa gagcagaaca atgcagcaaa tactcagaaa 420
catccttcca gaaggagcga actgaagagg ttctacacaa ttgacactgg ccaaaagaag 480
accctagaca agaaagatgg aagacgaatg tcttttcaga aacctaaagg gactattgag 540
tatactgttg aatcaaggga ttctttgaat agcatagccc tgaagtttga tacaacacct 600
aacgaacttg ttcaattaaa taagttattc tcccgagcag ttgttactgg acaggttctg 660
tatgttcctg atcctgaata tgtctccagt gttgagagct ctccatctct aagccccgta 720
agtcctctgt caccaacatc atctgaggct gaatttgata agaccactaa tcctgatgtc 780
catccaacag aagcaactcc ctcatctact ttcactggta ttcgacctgc acgagttgta 840
tcttcaactt ctgaggagga ggaagcattt actgagaaat ttcttaaaat taattgcaaa 900
tatattacca gtggcaaggg cacagtcagt ggtgtgctgc tagttacacc aaataatata 960
atgtttgatc cacataaaaa tgaccctttg gttcaagaga atggctgtga ggaatatggc 1020
atcatgtgtc caatggaaga ggtgatgtca gctgcaatgt acaaagaaat tttggatagc 1080
aaaataaagg aatctttacc catagatata gatcagctat caggaaggga cttctgccat 1140
tcaaagaaaa tgacaggaag taacactgag gaaatagact caagaatccg agatgcaggt 1200
aatgatagtg ccagcactgc tcctaggagc actgaggagt ctctttctga agatgtgttc 1260
acagaatcag aactttcccc tatacgagag gagcttgtat cttcagatga actgcgacaa 1320
gataaatctt ctggtgcgtc atcagaatct gtgcaaactg tcaatcaggc tgaagtagaa 1380
agtctgacag tcaaatcaga atctactggt actcctggtc acttaagatc tgatactgaa 1440
cattctacaa atgaagttgg gactttatgt cataaaactg atttaaataa tcttgaaatg 1500
gccattaagg aagatcagat tgcagataac tttcaaggaa tatcaggtcc taaagaagac 1560
26/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
agcacaagta taaaaggtaa ttcagaccag gattcttttc ttcatgagaa ttcgttacac 1620
caagaagaga gtcaaaaaga aaatatgcct tgtggggaaa cagcagaatt taaacaaaag 1680
caaagtgtta acaaaggaaa acaaggaaag gagcaaaatc aggactcaca gacagaggca 1740
gaagagctac gcaaactttg gaaaacccat actatgcaac aaactaaaca gcaaagggaa 1800
aatattcaac aagtgtcaca aaaagaagct aagcataaaa ttacatctgc tgatggacac 1860
atagaaagta agtgttatag agtaaatgaa gttagttcat ccaattgtat ggtgtctccg 1920
tctttcctca ctgactcaaa agccgctgta ccttagggca acctgaagga taagtgagtg 1980
acccttgacc ggtgattctc tggggacaag tatatacagt ctccagtgtc tactgtcttt 2040
tcttatccca caggtggatt gtacacataa tgagcatcat tgctacccac catgtttaaa 2100
cattcttggc tcactacatc gtatcattaa acaatttatt tcttgagaaa ttgcacaaaa 2160
gctgtataat acctttgatc actccatttt ctcttaaaaa gtttttggtg tgttaccatt 2220
aaaaatgtct tttttcatat ataacatttt aattgccaaa gttgtaatcc catcttttat 2280
aaaatgtaca gttaaaccta aaagtgtact tacagaattc ttccacgtat cgagaagttt 2340
catcatcatc atctaattat tttcttttaa aaacctgccc tcaggacaaa taaaaacact 2400
attttatctg tcagttttct ggtactgttt acttctgaaa attacactat tttcagtctt 2460
ttatttctaa tatactgtgc ttctgttaat aaattatggg ttaaatgg 2508
<210> 18
<211> 2162
<212> DNA
<213> Homo sapiens
<220>
<221> mist feature
<223> Incyte ID No: 7498076CB1
<400> 18
tccgggtccg tgggattcgc gctccactgg tcagctgggg tcgctctcgg gtggttgggt 60
gttgcttgtt cccgctgttc cagcgtcgaa gaaccattgg gtctgccggt ttgaacttgt 120
tctggaagct gtgcgtcacc gtaatgaggc ttgtaattct tgataactat gacttggcta 180
gtgaatgggc agccaaatac atctgtaatc gcatcattca gttcaaacct ggacaggaca 240
gatattttac actgggttta ccaacaggga gtacaccttt aggatgctat aaaaaactaa 300
tagaatatca taagaatgga cacctttctt ttaaatatgt gaagaccttt aatatggatg 360
aatatgtagg acttccaaga aatcatcctg aaagctacca ttcttatatg tggaataatt 420
tttttaagca tatcgatata gatcctaata atgcacatat ccttgacggg aatgctgcag 480
atttacaagc agaatgtgat gcttttgaaa acaaaataaa agaagctgga ggaatagatc 540
tttttgttgg aggaattggt ccagatggtc atatcgcttt caatgagcct ggatccagtt 600
tagtgtcaag gacaagatta aagactctag caatggatac catcttggca aatgccaaat 660
attttgatgg agatttatca aaagtgccaa ctatggctct aactgttggt gtggggacag 720
tgatggatgc tagagaagta atgatcctta taacaggggc acacaaggca tttgccctgt 780
acaaagcaat agaagaagga gtcaatcaca tgtggactgt ttccgctttc cagcagcatc 840
cccggactat ttttgtatgc gatgaagatg ctactttaga attaagagtt aaaactgtga 900
aatactttaa aggtctaatg catgtgcaca ataaacttgt ggatccacta ttcagtatga 960
aagatggaaa ctgaaggaga ctggagcaaa attcagcttg aatgaacaga gcacttttta 1020
ctaagtagta gatgaatttt cagctatgca atatgacaaa acatggggaa ttttgaagat 1080
tgtcattttt tcattcgagt ctctatgtta aacattccat attttgaata tttatatctt 1140
gtacttgggt ttaagagaag tagctggctc tcaagattga ctggctattt attataaagt 1200
actgaagtca catagccacc tataaaacag catagaaatg tctgcctgtt taaaaagtca 1260
ttttaaaggt agagtgtcca ccatcaggca ccatttgtga tatgactcca gtggcatata 1320
tttcattttt taatgacaag acactccaaa cctttcagat aacaaactat cattgcagac 1380
cttcactttt ggaatgcaat ctttatattt tctgtgcatc acacacatgc ttttctgcac 1440
gtggttgcct tagtcatctt cctacagcac catctagaca tcaaaaattg tgctatatat 1500
cattggtaaa ggaaatttga agagatgaca gtgcctaaaa gtacagttta catccttttg 1560
gaaagtatgt gtaagtgcat gttttttgtg caccttcttc tatagcactt ttttacaaat 1620
atcttatttt tatttaacga cttgggttca tgtccctaat ataagtatct tgacaattat 1680
gagctttata cctagcaagc cacttcagga aattcttttg gagaatattt tctgattatt 1740
27/34
gcc
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
gttaaactta atatacaatt agctttattc cttataaaat gtctaaaaga ataatacgaa 1800
gtatatataa aaggaattac tgtaaactac attgccatag caatttacat aaaagtatat 1860
tgttttctat ctttaactca aataaagcgt gtaataaata agttatctaa atttccagaa 1920
gtgaacctga agaatacatg cattcgactc ccaacaatta tagtacatgt gaacctgcat 1980
tcattttggg tttgttacca tcatgcatta ggagagttag gttaaaagat gtctttgcct 2040
ctgtggggag caaaaaaatg ctccttttgc ttattttgca tttcataagc ccacctggta 2100
gaagaagcct tacttatctg cttgtctatg tgattaaaat gtagtttctt aaaatattaa 2160
as 2162
<210> 19
<211> 4333
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1388154CB1
<400> 19
agatcggcac tgcgaaatgg ggataatgat ctcctagagc tgctgaaata ggaaaatgga 60
agaacagcag ctgatgtatt aatattatca ttattactac tacccattac ctgacaagtg 120
ataattgtga catgaaagag gctggaccgg ttgttacaac aattttttcc gtctagaatt 180
cggcggtaag gtctgagaca acacctcagc ttagatcagt gccttctctg aacagcgttc 240
actaagcagc ccccaacccc aaaaccccca agtccccggg cgccgaggac gctgcgagtc 300
ctgcgcatgc gcaaggttgc ccactcgctc accgcctcct tggcgaatgg cctgttccat 360
tctcgaggga tgccggcggg agcgttcggc agccggcggc gccgcctggc agctcctcct 420
cttctccgcc ccgctggccg cgggcgcggg ggacgtcagc gctgccagcg tggaaggagc 480
tgcggggcgc gggaggagga agtagagccc gggaccgcca ggccaccacc ggccgcctca 540
gccatggacg cgtccctgga gaagatagca gaccccacgt tagctgaaat gggaaaaaac 600
ttgaaggagg cagtgaagat gctggaggac agtcagagaa gaacagaaga ggaaaatgga 660
aagaagctca tatccggaga tattccaggc ccactccagg gcagtgggca agatatggtg 720
agcatcctcc agttagttca gaatctcatg catggagatg aagatgagga gccccagagc 780
cccagaatcc aaaatattgg agaacaaggt catatggctt tgttgggaca tagtctggga 840
gcttatattt caactctgga caaagagaag ctgagaaaac ttacaactag gatactttca 900
gataccacct tatggctatg cagaattttc agatatgaaa atgggtgtgc ttatttccac 960
gaagaggaaa gagaaggact tgcaaagata tgtaggcttg ccattcattc tcgatatgaa 1020
gacttcgtag tggatggctt caatgtgtca tataacaaga agcctgtcat atatcttagt 1080
gctgctgcta gacctggcct gggccaatac ctttgtaatc agctcggctt gcccttcccc 1140
tgcttgtgcc gtgtaccctg taacactgtg tttggatccc agcatcagat ggatgttgcc 1200
ttcctggaga aactgattaa agatgatata gagcgaggaa gactgcccct gttgcttgtc 1260
gcaaatgcag gaacggcagc agtaggacac acagacaaga ttgggagatt gaaagaactc 1320
tgtgagcagt atggcatatg gcttcatgtg gagggtgtga atctggcaac attggctctg 1380
ggttatgtct cctcatcagt gctggctgca gccaaatgtg atagcatgac gatgactcct 1440
ggcccgtggc tgggtttgcc agctgttcct gcggtgacac tgtataaaca cgatgaccct 1500
gccttgactt tagttgctgg tcttacatca aataagccca cagacaaact ccgtgccctg 1560
cctctgtggt tatctttaca atacttggga cttgatgggt ttgtggagag gatcaagcat 1620
gcctgtcaac tgagtcaacg gttgcaggaa agtttgaaga aagtgaatta catcaaaatc 1680
ttggtggaag atgagctcag ctccccagtg gtggtgttca gatttttcca ggaattacca 1740
ggctcagatc cggtgtttaa agccgtccca gtgcccaaca tgacaccttc aggagtcggc 1800
cgggagaggc actcgtgtga cgcggtgaat cgctggctgg gagaacagct gaagcagctg 1860
gtgcctgcaa gcggcctcac agtcatggat ctggaagctg agggcacgtg tttgcggttc 1920
agccctttga tgaccgcagc agttttagga actcggggag aggatgtgga tcagctcgta 1980
gcctgcatag aaagcaaact gccagtgctg tgctgtacgc tccagttgcg tgaagagttc 2040
aagcaggaag tggaagcaac agcaggtctc ctatatgttg atgaccctaa ctggtctgga 2100
ataggggttg tcaggtatga acatgctaat gatgataaga gcagtttgaa atcagatccc 2160
gaaggggaaa acatccatgc tggactcctg aagaagttaa atgaactgga atctgaccta 2220
28/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
acctttaaaa taggccctga gtataagagc atgaagagct gcctttatgt cggcatggcg 2280
agcgacaacg tcgatgctgc tgagctcgtg gagaccattg cggccacagc ccgggagata 2340
gaggagaact cgaggcttct ggaaaacatg acagaagtgg ttcggaaagg cattcaggaa 2400
gctcaagtgg agctgcagaa ggcaagtgaa gaacggcttc tggaagaggg ggtgttgcgg 2460
cagatccctg tagtgggctc cgtgctgaat tggttttctc cggtccaggc tttacagaag 2520
ggaagaactt ttaacttgac agcaggctct ctggagtcca cagaacccat atatgtctac 2580
aaagcacaag gtgcaggagt cacgctgcct ccaacgccct cgggcagtcg caccaagcag 2640
aggcttccag gccagaagcc ttttaaaagg tccctgcgag gttcagatgc tttgagtgag 2700
accagctcag tcagtcacat tgaagactta gaaaaggtgg agcgcctgtc cagtgggccg 2760
gagcagatca ccctcgaggc cagcagcact gagggacacc caggggctcc cagccctcag 2820
cacaccgacc agaccgaggc cttccagaaa ggggtcccac acccagaaga tgaccactca 2880
caggtagaag gaccggagag cttaagatga gactcattgt gtggtttgag actgtactga 2940
gtattgtttc agggaagatg aagttctatt ggaaatgtga actgtgccac atactaatat 3000
aaattactgt tgtttgtgct tcactgggat tttggcacaa atatgtgcct gaaaggtagg 3060
ctttctagga ggggagtcag cttgtctaac ttcatgtaca tgtagaacca catgtttgct 3120
gtcctactac cacttttccc taagttacca taaacacatt ttattcacaa aaaacacttc 3180
gaatttcaag tgtctaccag tagcaccctt gctctttcta aacataagcc taagtatatg 3240
aggttgcccg tggcaacttt ttggtaaaac agcttttcat tagcactctc caggttctct 3300
gcaacacttc acagaggcga gactggctgt atcctttgct gtcggtcttt agtacgatca 3360
agttgcaata tacggtggga ctgctagact tgaaggagag cagtgattgt gggattgtaa 3420
ataagagcat cagaagccct ccccagctac tgctcttcgt ggagacttag taaggactgt 3480
gtctacttga gctgtggcaa ggctgctgtc tgggactgtc ctctgccaca aggccatttc 3540
tcccattata taccgtttgt aaagagaaac tgtaaagtct cctcctgacc atatattttt 3600
aaatactggc aaagctttta aaattggcac acaagtacag actgtgctca tttctgttta 3660
gtatctgaaa acctgataga tgctaccctt aagagcttgc tcttccgtgt gctacgtagc 3720
acccacctgg ttaaaatctg aaaacaagta cccctttgac ctgtctccca ctgaagcttc 3780
tactgccctg gcagctcgcc tgggcccaac tcagaaacag gagccagcag agcactctct 3840
cacgctgatc cagccgggca ccctgcttaa gtcagtagaa gctcgctggc actgcccgtt 3900
cctacttttc cgaagtactg cgtcactttg tcgtaagtaa tggcccctgt gccttcttaa 3960
tccagcagtc aagcttttgg gagacctgaa aatgggaaaa ttcacactgg gtttctggac 4020
tgtagtattg gaagccttag ttatagtata ttaagcctat aattatactc tgatttgatg 4080
ggatttttga catttacact tgtcaaaatg cagggggttt tttttggtgc agatgattaa 4140
acagtcttcc ctatttggtg caatgaagta tagcagataa aatgggggag gggtaaatta 4200
tcaccttcaa gaaaattaca tgtttttata tatatttgga attgttaaat tggttttgct 4260
gaaacatttc acccttgaga tattatttga atgttggttt caataaaggt tcttgaaatt 4320
gttaaaaaaa aaa 4333
<210> 20
<211> 1727
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7481664CB1
<400> 20
tacggccgga atcccgggtc gacccacgcg tccgagagct gcaggctcta gcgcatccca 60
gccagtgtct cctgcagctc agcagctgcc ttcaccatgg acagcataag cacagccatc 120
ttactcctgc tcctggctct cgtctgtctg ctcctgaccc taagctcaag agataaggga 180
aagctgcctc cgggacccag acccctctca atcctgggaa acctgctgct gctttgctcc 240
caagacatgc tgacttctct cactaagctg agcaaggagt atggctccat gtacacagtg 300
cacctgggac ccaggcgggt ggtggtcctc agcgggtacc aagctgtgaa ggaggccctg 360
gtggaccagg gagaggagtt tagtggccgc ggtgactacc ctgccttttt caactttacc 420
aagggcaatg gcatcgcctt ctccagtggg gatcgatgga aggtcctgag acagttctct 480
atccagattc tacggaattt cgggatgggg aagagaagca ttgaggagcg aatcctagag 540
29/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
gagggcagct tcctgctggc ggagctgcgg aaaactgaag gcgagccctt tgaccccacg 600
tttgtgctga gtcgctcagt gtccaacatt atctgttccg tgctcttcgg cagccgcttc 660
gactatgatg atgagcgtct gctcaccatt atccgcctta tcaatgacaa cttccaaatc 720
atgagcagcc cctggggcga gttgtacaac atcttcccga gcctcctgga ctgggtgcct 780
gggccgcacc aacgcatctt ccagaacttc aagtgcctga gagacctcat cgcccacagc 840
gtccacgacc accaggcctc gctagacccc agatctcccc gggacttcat ccactgcttc 900
ctcaccaaga tggcagagga gaaggaggac ccactgagcc acttccacat ggataccctg 960
ctgatgacca cacataacct gctctttggc ggcaccaaga cggtgagcac cacgctgcac 1020
cacgccttcc tggcactcat gaagtaccca aaagttcaag cccgcgtgca ggaggagatc 1080
gacctcgtgg tgggacgcgc gcggctgccg gcgctgaagg accgcgcggc catgccttac 1140
acagacgcgg tgatccacga ggtgcagcgc tttgcagaca tcatccccat gaacttgccg 1200
caccgcgtca ctagggacac ggcctttcgc ggcttcctga tacccaaggg cgccgtctgt 1260
gcctgggaga gtcgctggcg cgcatggagc tctttctgta cctcaccgcc atcctgcaga 1320
gcttttcgct gcagccgctg ggtgcgcccg aggacatcga cctgacccca ctcagctcag 1380
gtcttggcaa tttgccgcgg cctttccagc tgtgcctgcg cccgcgctaa cgccccggcc 1440
cttccagatt cgcctgtgag cgatgaggcc cgcccatgcg ggttgctacg tccccttctt 1500
ggtccacagt ctgccctcat ccctctggca gtcacgctgt cttccctgca tgctgtgcct 1560
gccgcgtgcc cttcccccat ccctccaatc tgtgccccgt ctgcagggca gaggcagatg 1620
tggcatgtct ttttgtaccc acagagcttg ttctatggca cgcccttttc taggcttttt 1680
gtatcatttc ttagtacatt gtaatagatt caaaccagtc ttggctg 1727
<210> 21
<211> 2400
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7497661CB1
<400> 21
atggccgagt atacgcggct gcacaacgcc ttggcgctaa tccgcctccg aaacccgccg 60
gtcaacgcga tcagtacgac tttactccgt gacataaaag aaggactaca gaaagctgta 120
atagaccata caataaaagc cattgtgatt tgtggagcag agggcaaatt ttctgcaggt 180
gctgatattc gtggcttcag tgctcctagg acatttggcc ttacactggg acatgtagta 240
gatgaaatac agagaaatga gaagcccgtg gtggcagcaa tccaaggcat ggctttcgga 300
gggggactag agctggccct gggctgtcac tataggattg cccacgcaga ggctcaagtt 360
ggcttaccag aagttacact gggacttctc cctggtgcaa gaggaaccca gcttctcccc 420
agactcactg gagttcctgc tgcacttgac ttaattacct caggaagacg tattttagca 480
gatgaagcac tcaagctggg cattctagat aaagttgtaa actcagaccc ggttgaagaa 540
gcaatcagat ttgctcagag agtttcagat caacctctag aatcccgtag actctgcaac 600
aagccaattc agagcttgcc caacatggac agcattttta gtgaggccct cttgaagatg 660
cggaggcagc accctgggtg tcttgcacag gaggcttgtg tccgtgcagt ccaggctgct 720
gtgcagtatc cctatgaagt gggcatcaag aaggaggagg agctgtttct atatcttttg 780
caatcagggc aggctagagc cctgcaatat gctttcttcg ctgaaaggaa agcaaataag 840
tggtcaactc cctccggagc atcgtggaaa acagcatcag cgcggcctgt ctcctcagtt 900
ggtgttgttg gcttgggaac aatgggccga ggcattgtca tttcttttgc aagggccagg 960
attcctgtga ttgctgtaga ctcggacaaa aaccagctag caactgcaaa caagatgata 1020
acctctgtct tggaaaaaga agcctccaaa atgcaacaga gcggccaccc ttggtcagga 1080
ccaaaaccca ggttaacttc atctgtgaag gagcttggtg gtgtagattt agtcattgaa 1140
gcagtatttg aggaaatgag cctgaagaag caggtctttg ctgaactctc agctgtgtgc 1200
aaaccagaag catttttgtg cactaatact tcagccctgg atgttgatga gattgcttct 1260
tccactgatc gtcctcactt ggtcattggc acccacttct tttcgccagc tcatgtcatg 1320
aagttgttag aggttattcc cagccaatac tcttccccca ctaccattgc cactgttatg 1380
aacttatcaa aaaagattaa aaagattgga gtcgttgtag gcaactgttt tggatttgtg 1440
gggaatcgaa tgttgaatcc ttactacaat caggcatatt tcttgttaga agaaggcagc 1500
30/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
aaaccagagg aggtagatca ggtgctggaa gagtttggtt ttaaaatggg accttttaga 1560
gtgtctgatc ttgctgggtt ggatgtgggc tggaaatcta gaaaggggca aggtcttact 1620
ggacctacat tgcttccagg aactcctgcc cgaaaaaggg gtaataggag gtactgccca 1680
attcctgatg tgctctgtga attaggacga tttggccaga agacaggtaa gggttggtat 1740
caatatgaca agccattggg taggattcac aaacctgatc cctggctttc caaattccta 1800
tcacggtata gaaaaaccca tcacattgaa ccacgtacca ttagccagga tgagatcctt 1860
gaacgctgct tatattcact tatcaatgaa gcattccgta tcttgggaga agggatagct 1920
gctagcccag agcacattga tgtcatctac ttgcatgggt atgggtggcc aaggcacgtg 1980
ggtgggccca tgtactatgc ttccacagtt gggttgccta cagttctaga gaaattgcag 2040
aaatattaca gacagaatcc tgacatcccc cagctggagc ccagtgacta cctgaggagg 2100
ctggttgccc agggaagccc tcctctgaaa gaatggcaaa gcttggcagg accccatagc 2160
agtaaactgt gattcagcct tctgcatttt gccatgtatg gtggcatcta atttcagtgt 2220
gattcattcc taaaatccaa acagatttct ctgaagtaca aatgatgatt gctgctgttg 2280
ctgctgctgc tggtggtggt ggtgctaaat ggtcagcaaa agcccctctc tgtatcttcc 2340
tacctcataa ttctaatggc cagatttcag gaatgtgttt tctatacctc tgacggtact 2400
<210> 22
<211> 1929
<212> DNA
<213> Homo sapiens
<220>
<221> mist feature
<223> Incyte ID No: 7495116CB1
<400> 22
ggagcccccc acgagggaag ggcgttccag ggacaagggc cgtacccctt gacggggctg 60
caaaaggggt ccccaccgaa acccccaggt ttcggcagcg tggtcctcca gtggtaacac 120
tgggcgaccg gagtcgtgtt ccgagcccta aggagagaac cggctcggtt cgtgggtgca 180
gcccgcgagg agacgacctc agggcgtaaa tccgacgccc gttgggttta agacatcaga 240
cctcaggcgc agtgcagccg gcacctgggg agtcaaggac cttgatcgcc gataacaagt 300
tgccgttggc gggcgccgag ggtgacccgc gagcgcggca cacgcctccc gcccctagca 360
ggaccccctc cgcccgcccc cagccggacc cctcccgagg ccccgcccca cccgcgagcc 420
gcagccgcgg cccacacagc ttctggggct ggggccccgg cagccgggca ggccggcctg 480
acctcgacct ccgccgtgcg ggcccgaccg gtgagtccag cccggcagtc gcaggacccg 540
gccgccagcc tctccctcca cctctccctg cccccagcgc caggcgcggg ctgcgctcgg 600
tggcggcggc ggggccctca ggcggccatg gcggcaggcg ccggggccgg gtccgcgccg 660
cgctggctga gggcgctgag cgagccgctg agcgcggcgc agctgcggcg actggaggag 720
caccgctaca gcgcggcggg cgtctcgctg ctcgagccgc cgctgcagct ctactggacc 780
tggctgctcc agtggatccc gctctggatg gcccccaact ccatcaccct gctggggctc 840
gccgtcaacg tggtcaccac gctcgtgctc atctcctact gtcccacggc caccgaagag 900
gcaccatact ggacatacct tttatgtgca ctgggacttt ttatttacca gtcactggat 960
gctattgatg ggaaacaagc cagaagaaca aactcttgtt cccctttagg ggagctcttt 1020
gaccatggct gtgactctct ttccacagta tttatggcag tgggagcttc aattgccgct 1080
cgcttaggaa cttatcctga ctggtttttt ttctgctctt ttattgggat gtttgtgttt 1140
tattgcgctc attggcagac ttatgtttca ggcatgttga gatttggaaa agtggatgta 1200
actgaaattc agatggcttt agtgattgtc tttgtgttgt ctgcatttgg aggagcaaca 1260
atgtgggact atacgggcac cagtgtcttg tcacctggac tccacatagg actaattatt 1320
atactggcaa taatgatcta taaaaagtca gcaactgatg tgtttgaaaa gcatccttgt 1380
ctttatatcc taatgtttgg atgtgtcttt gctaaagtct cacaaaaatt agtggtagct 1440
cacatgacca aaagtgaact atatcttcaa gacactgtct ttttggggcc aggtcttttg 1500
tttttagacc agtactttaa taactttata gacgaatatg ttgttctatg gatggcaatg 1560
gtgatttctt catttgatat ggtgatatac tttagtgctt tgtgcctgca aatttcaaga 1620
caccttcatc taaatatatt caagactgca tgtcatcaag cacctgaaca ggttcaagtt 1680
ctttcttcaa agagtcatca gaataacatg gattgaagag acttccgaac acttgctatc 1740
tcttgctgct gctgtttcat ggaaggagat attaaacatt tgtttaattt ttatttaagt 1800
31/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
gttataccta tttcagcaaa taaaatattt cattgcttat attaaaaaaa aaaaaaaagg 1860
gcggccgcac agattaggtg aactcgtcga cccgggaaat taataccgga ccggtacctg 1920
caaggcggg
1929
<210> 23
<211> 1367
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7498400CB1
<400> 23
agcgggggga agacttctgg gcagaagcgg aacacaggag cagagacaca tagtcttggc 60
tccagtttcg tttcagttat gcccaccctt tcagtgttca tggatgtgcc cctcgcccac 120
aagctagagg gcagcttgtt aaagacctac aaacaagatg attacccgaa caagatattc 180
ttagcctata gaggcacctt cccacagccc catggagtcc aggagagatt gtttgcaggc 240
tgtctgcaga gctcagccct gggggcccaa accaggcatc tggagctccc tctgtggttt 300
tcctcacagt ctgcatgaca aatgaaggcc atccctgggt ttctctcgtg gtgcagaaga 360
ctcgactaca gatttcacag gatccctccc tgaattatga gtacttgccc accatgggcc 420
tgaaatcatt catccaggcc tctctagcac tcctctttgg aaagcacagc caagccattg 480
tggagaacag ggtagggggt gtacacactg ttggtgacag tggtgccttc cagcttggcg 540
tccagtttct cagagcttgg cataaggatg ctcgtatagt ttacatcatc tcttctcaaa 600
aagaactgca tggactcgtc ttccaggaca tgggctttac agtttatgaa tactctgtct 660
gggaccccaa gaagctatgc atggaccccg acatactcct caatgtggtg gagagcaagc 720
agatattccc attttttgat attccctgtc aaggtttata caccagtgac ttggaagaag 780
atactagaat cttacaatac tttgtgtctc aaggctttga gttcttctgc agccagtctc 840
tgtccaagaa ttttggcatt tatgatgaag gagtggggat gctagtggtg gtggcagtca 900
acaaccagca gctgctgtgt gtcctctccc agctggaagg attagcccag gccctgtggc 960
taaacccccc caacacgggt gcacgtgtca tcacctccat cctctgcaac cctgctctgc 1020
tgggagaatg gaagcagagt ctaaaagaag ttgtagagaa catcatgcta accaaggaaa 1080
aagtgaagga gaaactccag ctcctgggaa cccctgggtc ctggggtcac atcaccgagc 1140
agagtgggac ccacggctat cttggactca actcccagca ggtggaatac ctggtcagga 1200
agaagcacat ctatatcccc aagaacggtc agattaactt cagctgtatc aatgccaaca 1260
acataaatta catcactgag ggcatcaatg aggctgtcct cctcacagag agctcagaga 1320
tgtgtcttcc aaaggaaaaa aaaacactga ttggaataaa actttag 1367
<210> 24
<211> 724
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1709240CB1
<400> 24
caaagagctt ataaatacat taggacctgg aattcagttg tcgagccagg acggtgacag 60
cgtttaacaa agcttagaga aacctccagg agactgctat catggcagag aagcccaagc 120
tccactactt caatgcacgg ggcagaatgg agtccacccg gtggctcctg gctgcagctg 180
gagtagagtt tgaagagaaa tttataaaat ctgcagaaga tttggacaag ttaagaaatg 240
atggatattt gatgttccag caagtgccaa tggttgagat tgatgggatg aagctggtgc 300
agaccagagc cattctcaac tacattgcca gcaaatacaa cctctatggg aaagacataa 360
aggagagagc cctgattgat atgtatatag aaggtatagc agatttgggt gaaatgatcc 420
tccttctgcc cgtatgtcca cctgaggaaa aagatgccaa gcttgccttg atcaaagaga 480
32/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
aaataaaaaa tcgctacttc cctgcctttg aggctgacat tcatctggtg gaacttctct 540
actacgtcga ggagcttgac tccagtctta tctccagctt ccctctgctg aaggcccttg 600
aaaccagaat cagcaacctg cccacagtga agaagtttct acagcctggc agcccaagga 660
agcctcccat ggatgagaaa tctttagaag aagcaaggaa gattttcagg ttttaataac 720
gcag 724
<210> 25
<211> 791
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4739684CB1
<400> 25
gctgcaggaa ttcggtacca aagagcttat aaatacatta ggacctggaa ttcagttgtc 60
gagccaggac ggtgacagcg tttaacaaag cttagagaaa cctccaggag actgctatca 120
tggcagagaa gcccaagctc cactacttca atgcacgggg cagaatggag tccacccggt 180
ggctcctggc tgcagctgga gtagagtttg aagagaaatt tatgcaagtg ccaatggttg 240
agattgatgg gatgaagctg gtgcagacca gagccattct caactacatt gccagcaaat 300
acaacctcta tgggaaagac ataaaggaga gagccctgat tgatatgtat atagaaggta 360
tagcagattt gggtgaaatg atcctccttc tgcccgtatg tccacctgag gaaaaagatg 420
ccaagcttgc cttgatcaaa gagaaaataa aaaatcgcta cttccctgcc tttgaaaaag 480
tcttaaagag ccatggacaa gactaccttg ttggcaacaa gctgagccgg gctgacattc 540
atctggtgga acttctctac tacgtcgagg agcttgactc cagtcttatc tccagcttcc 600
ctctgctgaa ggccctgaaa accagaatca gcaacctgcc cacagtgaag aagtttctac 660
agcctggcag cccaaggaag cctcccatgg atgagaaatc tttagaagaa gcaaggaaga 720
ttttcaggtt ttaataacgc agtcatggag gccaagaact tgcaatacca atgttctaaa 780
gtttgcaaca a 791
<210> 26
<211> 2544
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 72461345CB1
<400> 26
ttgagttaca tgccgcgtac gtagctccgg aattctgtgc tcgaggcggg gtctcggggg 60
ctcctgagcc ggccgcctcc aggaagcagg cgccgcgcgg tattgccgca tgcacctcgg 120
tcgtggggac cccgctgcag cagccctgta cacacgggtg aactcctgga gggcggggcg 180
gggggcaatc tgcgcgggtc aggcccctcg gagcaggctg ggggcgcccg agccaggccg 240
ctcccacctg ccagccgcgt ggccaatgaa tgctaggcct ggtgatgtca tgccccgacc 300
ggaccctggt gacgaaagtc cgaagtcacc cgtcagggaa ccagcacaga cccacccgcg 360
ggggttccag aagtttccac tgccgccgcg gagtgcgggc tcgcccagca gccttgcgcg 420
tgctaccacg ctgtccgtgc cttctcagac gccgcctgcc ctgcagccat gaggcccccg 480
cagtgtctgc tgcacacgcc ttccctggct tccccactcc ttctcctcct cctctggctc 540
ctgggtggag gagtgggggc tgagggccgg gaggatgcag agctgctggt gacggtgcgt 600
gggggccggc tgcggggcat tcgcctgaag acccccgggg gccctgtctc tgcttttctg 660
ggcatcccct ttgcagagcc acctgtgggc tcacgtagat ttatgccacc agagcccaag 720
cggccctggt caggaatatt ggatgctacc accttccaaa atgtctgcta ccaatacgtg 780
gacaccctgt accctgggtt tgagggtacc gagatgtgga accccaatcg agagctgagt 840
gaagactgcc tttatcttaa tgtgtggaca ccatacccca ggcctacttc tcccacacct 900
33/34
CA 02453075 2004-O1-05
WO 03/004608 PCT/US02/21105
gtcctcatct ggatctatgg gggtggtttc tacagtggag catcctcctt ggacgtgtat 960
gacggccgtt tcctggccca ggttgaggga accgtgttgg tatctatgaa ctaccgagtg 1020
ggaacctttg gcttcttggc tctaccagga agcagagacg cccctggcaa tgtaggcctg 1080
ctggatcaac ggcttgcctt gcaatgggta caagaaaata tcgcagcctt tgggggagac 1140
ccaatgtcag tgactctgtt tggggagagt gcaggtgcag cctcagtggg catgcacatt 1200
ctgtccctgc ccagcaggag cctcttccac agggctgtcc tgcagagtgg cacacccaac 1260
gggccctggg ccactgtgag tgcgggagag gccaggcgca gggccacact gctggcccgc 1320
cttgtgggct gtcccccagg tggcgctggt ggcaatgaca ccgagctgat atcctgcttg 1380
aggacaaggc ccgctcagga cctggtggac cacgagtggc atgtgctgcc tcaagaaagt 1440
atcttccggt tttccttcgt gcctgtggtg gacggggatt tcctcagtga cacgccggag 1500
gccctcatca atactggaga ttttcaagac ctgcaggtgc tggtgggtgt ggtgaaggac 1560
gagggctcct actttctggt ttacggggtc ccaggcttca gcaaagacaa tgaatctctc 1620
atcagccggg cccagttcct ggctggggtg cggatcggtg taccccaagc gagtgacctg 1680
gcggccgagg ctgtggtcct gcattataca gactggctgc accctgagga ccctgcccac 1740
ctgagagatg ccatgagtgc ggtggtaggc gaccacaacg ttgtgtgccc tgtggcccag 1800
ctggctgggc gactggctgc ccaaggggct cgggtctatg cctacatctt tgaacaccgt 1860
gcctccacat tgacttggcc cctctggatg ggggtgcccc atggctatga aatcgagttc 1920
atctttgggc tccccctgga tccctcactg aactacaccg tggaggagag aatctttgct 1980
cagcgactta tgaaatactg gaccaatttt gcccgcacag gagaccccaa tgaccctcga 2040
gactctaagt ctccacggtg gccaccgtac accactgccg cgcagcaata cgtgagcctg 2100
aacctgaagc ctttggaggt gcggcgggga ctgcgcgccc agacctgcgc cttctggaat 2160
cgttttctcc ccaaattgct cagcgccaca gacacgctgg acgaggcgga gcgccagtgg 2220
aaggccgagt tccaccgctg gagctcctac atggtgcact ggaagaacca gttcgaccac 2280
tatagcaagc aggaacgctg ctcagacctg tgaccccttg ggggacccca ggtcctgccg 2340
tcctgcccga gcccctgatt gtatatacta tttatttaag ggctgggata taatacaacc 2400
gagcccccag gccctgtcca ccccgccccg acttcctccc actaggggat cctcatcttc 2460
tgcatgtttt gggctgagct cccctccccg cggtgccttc gcccctctgg gccgccaata 2520
aactgttaca gctaaaaaaa aaaa 2544
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