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DRUG METABOLIZING ENZYMES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of drug
metabolizing
enzymes and to the use of these sequences in the diagnosis, treatment, and
prevention of
autoimmune/inflammatory, cell proliferative, developmental, endocrine, eye,
metabolic, and
gastrointestinal disorders, including liver disorders, and in the assessment
of the effects of exogenous
compounds on the expression of nucleic acid and amino acid sequences of drug
metabolizing
enzymes.
BACKGROUND OF THE INVENTION
The metabolism of a drug and its 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
,20 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
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
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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.,
Amdur, M. O. and J. Doull (1996) Casarett and Doull's Toxicology: The Basic
Science of Poisons,
McGraw-Hill, New York, NY, pp. 113-186; B. G. I~atzung (1995) Basic and
Clinical Pharmacolo~y,
Appleton and Lange, Norwalk, CT, pp. 48-59; G. G. Gibson 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
ferredoxinlferredoxin
reductase redox pair, aldo/keto reductases, and alcohol dehydrogenases. The
major classes of Phase
II enzymes include, but are not limited to, UDP glucuronyltransferase,
sulfotransferase, glutathione S-
transferase, N-acyltransferase, and N-acetyl transferase.
Cytochrome P450 and P450 catalytic cycle-associated enzymes
Members of the cytochrome P450 superfamily of enzymes catalyze the oxidative
metabolism
of a variety of substrates, including natural compounds such as steroids,
fatty acids, prostaglandins,
leukotrienes, and vitamins, as well as drugs, carcinogens, mutagens, and
xenobiotics. Cytochromes
P450, also known as P450 heme-thiolate proteins, usually act as terminal
oxidases in
mufti-component electron transfer chains, called P450-containing monooxygenase
systems. Specific
reactions catalyzed include hydroxylation, epoxidation, N-oxidation,
sulfooxidation, N-, S-, and O-
dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-
oxide groups. These
reactions are involved in steroidogenesis of glucocorticoids, cortisols,
estrogens, and androgens in
animals; insecticide resistance in insects; herbicide resistance and flower
coloring in plants; and
environmental bioremediation by microorganisms. Cytochrome P450 actions on
drugs, carcinogens,
mutagens, and xenobiotics can result in detoxification or in conversion of the
substance to a more
toxic product. Cytochromes P450 are abundant in the 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
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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 fannily 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 a
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 P4S0 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) Clin. Exp. Pharmacol.
Physiol. 25:1-9). A
cytochrome P450 protein may participate in eye development as mutations in the
P450 gene CYP1B 1
cause primary congenital glaucoma (Online Mendelian Inheritance in Man
(OMI1VI) *601771
Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYP1B 1).
Cytochromes P450 are associated with inflammation and infection. Hepatic
cytochrome
P450 activities are profoundly affected by various infections and inflammatory
stimuli, some of
which are suppressed and some induced (Morgan, E.T. (1997) Drug Metab. Rev.
29:1129-1188).
Effects observed in vivo can be mimicked by 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
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progressive neurologic dysfunction, premature atherosclerosis, and cataracts;
and an inherited
resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J.
et al. (1994) Harrison's
Principles of Internal Medicine, McGraw-Hill, Inc. New York, NY, pp. 1968-
1970; Takeyama, K. et
al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998) N. Engl. J. Med.
338:653-661; OMIM
*213700 Cerebrotendinous xanthomatosis; and OMIM #122700 Coumarin resistance).
Extremely
high levels of expression of the cytochrome P450 protein aromatase were found
in a fibrolamellar
hepatocellular carcinoma from a boy with severe gynecomastia (feminization)
(Agarwal, V.R. (1998)
J. Clin. Endocrinol. Metab. 83:1797-1800).
The cytochrome P450 catalytic cycle is completed through reduction of
cytochrome P450 by
NADPH cytochrome P450 reductase (CPR). Another microsomal electron transport
system
consisting of cytochrome b5 and NADPH cytochrome b5 reductase has been widely
viewed as a
minor contributor of electrons to the cytochrome P450 catalytic cycle.
However, a recent report by
Lamb, D. C. et al. (1999; FEBS Lett. 462:283-8) identifies a Candida albicans
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., 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 in
Endocrinology and Metabolism
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(OH)D is the most abundant precursor
form of vitamin
D which must be further metabolized in the kidney to the active form, 1a,25-
dihydroxyvitamin D
(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
4
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enzyme product (1a,25(OH)ZD) and the levels of parathyroid hormone (PTH),
calcitonin, insulin,
calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal la-
hydroxylase
activity has been reported, suggesting that tissue-specific, local regulation
of 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. LT.S.A. 94:12920-
12925; Miller, W.L. and
Portale, A.A. sue; 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(OH)D (Griffin, J.E.
and Zerwekh, J.E.
(1983) J. Clin. Invest. 72:1190-1199; Gamblin, G.T. et al. (1985) J. Clin.
Invest. 75:954-960; and
W.L. and Portale, A.A. supra).
Ferredoxin and ferredoxin reductase are electron transport accessory proteins
which support
at least one hunnan cytochrome P450 species, cytochrome P450c27 encoded by the
CYP27 gene
(Dilworth, F. J. et al. (1996) Biochem. J. 320:267-71). A Stre tp omyces
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
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(Flitter, W. D. and Mason, R. P. (1988) Arch. Biochem. Biophys. 267:632-9).
Flavin-containing monooxy~enase (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 excreted in urine. FMO is also involved in S-oxygenation of
cimetidine, an H Z
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
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formation of connective tissue matrices by crosslinking collagen and elastin.
LO is secreted as a N-
glycosylated precuror protein of approximately 50 kDa Levels 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 electron to and from oxygen to facilitate the
oxidative deamination of
lysine residues in these extracellular matrix proteins. While the coordination
of copper is essential to
LO activity, insufficient dietary intake of copper does not influence the
expression of the apoenzyme.
However, the absence of the functional LO is linked to the skeletal and
vascular tissue disorders that
. are associated with dietary copper deficiency. LO is also inhibited by a
variety of semicarbazides,
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 has been
linked to Menkes syndrome and occipital horn syndrome. Cytosolic forms of the
enzyme hae been
implicated in abnormal cell proliferation (reviewed in Rucker, R.B. et al.
(1998) Am. J. Clin. Nutr.
67:9965-10025 and Smith-Mungo. L.I. and I~agan, 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 (BUMP)
to deoxythymidine monophosphate (dTMP). The basic reaction is as follows:
7,8-dihydrofolate + NADPH --~ 5,6,7,8-tetrahydrofolate + NADP+
The enzymes can be inhibited by a number of dihydrofolate analogs, including
trimethroprim and
methotrexate. Since an abundance of TMP 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)
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
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substrate specificities (Bohren, K. M. et al. (1989) J. Biol. Chem. 264:9547-
51). 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. 273:11429-35).
Alcohol dehydrogenases
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 (b" b," b3, gi, ga). 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 III (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, 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-
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-
acylmannosamine 1-
dehydrogenase, and 2-deoxy-D-gluconate 3-dehydrogenase (Krozowski, Z. (1994)
J. Steroid
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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 ~lucuronyltransferase
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, UGTl and
UGT2.
Members of the UGT1 family result from alternative splicing of a single gene
locus, which has a
variable substrate binding domain and constant region involved in cofactor
binding and membrane
insertion. Members of the UGT2 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 UGTl).
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
(PAPS) 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.
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For example, there are more than a dozen forms of ST in rat liver cytosol.
These biochemically
characterized STs fall into five classes based on their substrate preference:
arylsulfotransferase,
alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester
sulfotransferase, and bile salt
sulfotransferase.
ST enzyme activity varies greatly with sex and age in 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 proteoglycans (Nakazawa, K. et
al. (1984) J. Biol. Chem. w
259:13751-7; OMIM *217800 Macular dystrophy, corneal).
Galactosyltransferases
Galactosyltransferases are a subset of glycosyltransferases that transfer
galactose (Gal) to the
terminal N-acetylglucosamine (GlcNAc) oligosaccharide chains that are part of
glycoproteins or
glycolipids that are free in solution (Kolbinger, F. et al. (1998) J. Biol.
Chem. 273:433-440; Amado,
M. et al. (1999),Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases
have been detected on
the 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)GIcNAc
linkages. Known
human and mouse ~i 1,3-galactosyltransferases appear to have a short cytosolic
domain, a single
transmembrane domain, and a catalytic domain with eight conserved regions.
(Kolbinger, F, supra
and Hennet, T. et al. (1998) J. Biol. Chem. 273:58-65). In mouse UDP-
galactose:(3-N-
acetylglucosamine X31,3-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
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within mouse UDP-galactose: (3-N-acetylglucosamine (31,3-galactosyltransferase-
I region 8 is also
found in bacterial galactosyltransferases, suggesting that this sequence
defines a galactosyltransferase
sequence motif (Rennet, T. supra). Recent work suggests that brainiac protein
is a ~i1,3-
galactosyltransferase. (Yuan, Y. et al. (1997) Cell 88:9-11; and Rennet, T.
supra).
UDP-Gal:GlcNAc-1,4-galactosyltransferase (-1,4-GaIT) (Sato, T. et al., (1997)
EMBO J.
16:1850-1857) catalyzes the formation of Type II carbohydrate chains with Gal
( (31-4)GIcNAc
linkages. As is the case with the ~i1,3-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-bonded
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). (31,4-galactosyltransferases have
several specialized
roles in addition to synthesizing carbohydrate chains on glycoproteins or
glycolipids. In mammals a
~i 1,4-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
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
30, 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-
55).
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
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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
~phimurium 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: 8567-80). The mutagenicity of ethylene dibromide and
ethylene dichloride is
increased in bacterial cells expressing the human Alpha GST, Al-l, while the
mutagenicity of
aflatoxin B 1 is substantially reduced by enhancing the expression of GST
(Simula, T.P. et al. (1993)
Carcinogenesis 14: 1371-6). 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 mufti-drug resistance (MDR). MDR occurs when a cancer
patient is treated
with a cytotoxic drug such as cyclophosphamide and subsequently becomes
resistant to this drug and
to a variety of other cytotoxic agents as well. Increased GST levels are
associated with some of these
drug resistant cancers, and it is believed that this increase occurs in
response to the drug agent which
is then deactivated by the GST catalyzed GSH conjugation reaction. The
increased GST levels then
protect the cancer cells from other cytotoxic agents which bind to GST.
Increased levels of A1-1 in
tumors has been linked to drug resistance induced by cyclophosphamide
treatment (Dirven H.A. et al.
(1994) Cancer Res. 54: 6215-20). Thus control of GST activity in cancerous
tissues may be useful in
treating MDR in cancer patients.
Gamma- lug tam~l transpeptidase
Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes that
initiate extracellular
glutathione (GSH) breakdown by cleaving gamma-glutamyl amide bonds. The
breakdown of GSH
provides cells with a regional cysteine pool for biosynthetic pathways. Gamma-
glutamyl
transpeptidases also contribute to cellular antioxidant defenses and
expression is induced by oxidative
steress. The cell surface-localized glycoproteins.are expressed at high levels
in cancer cells. Studies
have suggested that the high level of gamma-glutamyl transpeptidases 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. 122:367-80).
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
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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-
9; Johnson, M. R. et al.
(1991) J. Biol. Chem. 266:10227-33). BAT is also useful as a predictive
indicator for prognosis of
hepatocellular carcinoma patients after partial hepatectomy (Furutani, M. et
al. ( 1996) Hepatology
24:1441-5).
Acetyltransferases
Acetyltransferases have been extensively studied for their role in histone
acetylation. Histone
acetylation results in the relaxing of the chromatin structure in eukaryotic
cells, allowing transcription
factors to gain access to promoter elements of the DNA templates in the
affected region of the
genome (or the genome in general). In contrast, histone deacetylation results
in a reduction in
transcription by closing the chromatin structure and limiting access of
transcription factors. To this
end, a common means of stimulating cell transcription is the use of chemical
agents that inhibit the
deacetylation of histones (e.g., sodium butyrate), resulting in a global
(albeit artifactual) increase in
gene expression. The modulation of gene expression by acetylation also results
from the acetylation
of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR,
TFITE, TFIIF and the
high mobility group proteins (HMG). In the case of p53, acetylation results in
increased DNA
binding, leading to the stimulation of transcription of genes regulated by
p53. The prototypic histone
acetylase (HAT) is GcnS from Saccharomyces cerevisiae. Gcn5 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) Current Opinion in Cell Biology 12:326-333 and
Berger, S.L (1999)
Current Opinion in Cell Biology 11:336-341). Some acetyltransferase enzymes
posses the alphalbeta
hydrolase fold (Center of Applied Molecular Engineering Inst. of Chemistry and
Biochemistry -
University of Salzburg, http://predict.Banger.ac.uklirbm-course97/Docs/msn
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-acetyltransferase
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
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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, NAT1 and
NAT2; mice appear
to have a third form of the enzyme, NAT3. The human forms of N-
acetyltransferase have
independent regulation (NAT1 is widely-expressed, whereas NAT2 is in liver and
gut only) and
overlapping substrate preferences. Both enzymes appear to accept most
substrates to some extent, but
NAT1 does prefer some substrates (para-aminobenzoic acid, para-aminosalicylic
acid,
sulfamethoxazole, and sulfanilamide), while NAT2 prefers others (isoniazid,
hydralazine,
procainamide, dapsone, aminoglutethimide, and sulfamethazine).
Clinical 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 NAT1 enzyme may be important in determining cancer risk
(OMI1VI *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 included 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
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oxalate in the kidneys and urinary tract, ultimately causing renal failure
(Lumb, M.J. et aI. (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 catalyzes
the reversible
transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce
2-oxoadipate and
L-glutamate. Kynurenic acid is a putative modulator of glutamatergic
neurotransmission, thus a
deficiency in kynurenine aminotransferase may be associated with pleotrophic
effects (Buchli, R. et
al. (1995) J. Biol. Chem. 270:29330-29335).
Catechol-O-methyltransferase:
Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group
of S-
adenosyl-L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the
catechol substrate
(e.g., L-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 Mgr
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) Pharmacological Reviews 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 H202. The rate of
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dismutation is diffusion-limited and consequently enhanced by the presence of
favorable electrostatic
interactions between the substrate and enzyme active site. Examples of this
class of enzyme have
been identified in the cytoplasm of all the eukaryotic cells as well as in the
periplasm of several
bacterial species. Copper-zinc superoxide dismutases are robust enzymes that
are highly resistant to
proteolytic digestion and denaturing by urea and SDS. In addition to the
compact structure of the
enzymes, the presence of the metal ions and intrasubunit disulfide bonds is
believed to be responsible
for enzyme stability. The enzymes undergo reversible denaturation at
temperatures as high as 70 °C
(Battistoni, A. et al. (1998) J. Biol. Chem. 273:5655-5661).
Overexpression of superoxide dismutase has been implicated in enhancing
freezing tolerance
of transgenic Alfalfa as 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
organisms survival
through the process of cryopreservation (Jong-In Park, J-I. et al. (1998) J.
Biol. Chem.
273:22921-22928).
Expression of superoxide dismutase is also associated with Mycobacterium
tuberculosis, the
organism that causes tuberculosis. Superoxide dismutase is one of the ten
major proteins secreted by
M. tuberculosis and its expression is upregulated approximately 5-fold in
response to oxidative stress.
M, tuberculosis expresses almost two orders of magnitude more superoxide
dismutase than the
nonpathogenic mycobacterium M. smegmatis, and secretes a much higher
proportion of the expressed
enzyme. The result is the secretion of 350-fold more enzyme by M. tuberculosis
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
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nucleic acid strands during topological rearrangement of DNA. A Tyr-DNA
phosphodiesterase
functions in DNA repair by hydrolyzing dead-end covalent intermediates formed
between
topoisomerase I and DNA (Pouliot, J.J. et al. (1999) Science 286:552-555;
Yang, S.-W. (1996) Proc.
Natl. Acad. Sci. USA 93:11534-11539).
Acid sphingomyelinase is a phosphodiesterase which hydrolyzes the membrane
phospholipid
sphingomyelin to produce ceramide and phosphorylcholine. Phosphorylcholine is
used in the
synthesis of phosphatidylcholine, which is involved in numerous intracellular
signaling pathways,
while ceramide is an essential precursor for the generation of gangliosides,
membrane lipids found in
high concentration in neural tissue. Defective acid sphingomyelinase leads to
a build-up of
sphingomyelin molecules in lysosomes, resulting in Niemann-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).
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Type 1 PDEs (PDEls) are Ca2+lcalmodulin-dependent and appear to be encoded by
at least
three different genes, each having at least two different splice variants
(Kakkar, R. et al. (1999) Cell
Mol. Life Sci. 55:1164-1186). PDEls have been found in the lung, heart, and
brain. Some PDE1
isozymes are regulated in vitro by phosphorylation/dephosphorylation.
Phosphorylation of these
PDE1 isozymes decreases the affinity of the enzyme for calmodulin, decreases
PDE activity, and
increases steady state levels of cAMP (Kakkar, supra). PDEIs may provide
useful therapeutic targets
for disorders of the central nervous system, and the cardiovascular and immune
systems due to the
involvement of PDEls in both 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, su_pra), 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
competitive substrates for PDE3s. PDE3s play roles in stimulating myocardial
contractility,
inhibiting platelet aggregation, relaxing vascular and airway smooth muscle,
inhibiting proliferation
of T-lymphocytes and cultured vascular smooth muscle cells, and regulating
catecholamine-induced
release of free fatty acids from adipose tissue. The PDE3 family of
phosphodiesterases are sensitive
to specific inhibitors such as cilostamide, enoximone, and lixazinone.
Isozymes of PDE3 can be
regulated by cAMP-dependent protein kinase, or by insulin-dependent kinases
(Degerman, E. et al.
(1997) J. Biol. Chem. 272:6823-6826).
PDE4s are specific for cAMP; are localized to airway smooth muscle, the
vascular '
endothelium, and all inflammatory cells; and can be activated by cAMP-
dependent phosphorylation.
Since elevation of cAMP levels can lead to suppression of inflammatory cell
activation and to
relaxation of bronchial smooth muscle, PDE4s have been studied extensively as
possible targets for
novel anti-inflammatory agents, with special emphasis placed on the discovery
of asthma treatments.
PDE4 inhibitors are currently undergoing clinical trials as treatments for
asthma, chronic obstructive
pulmonary disease, and atopic eczema. All four known isozymes of PDE4 are
susceptible to the
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)
Curr. Opin. Chem. Biol. 3:466-473).
PDES is highly selective for cGMP as a substrate (Turko, LV. et al. (1998)
Biochemistry
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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,
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 (Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE6s, the photoreceptor cyclic nucleotide phosphodiesterases, are crucial
components of the
phototransduction cascade. In association with the G-protein transducin, PDE6s
hydrolyze cGMP to
regulate cGMP-gated cation channels in photoreceptor membranes. In addition to
the cGMP-binding
active site, PDE6s also have two high-affinity cGMP-binding sites which are
thought to play a
regulatory role in PDE6 function (Artemyev, N.O. et al. (1998) Methods 14:93-
104). Defects in
PDE6s have been associated with retinal disease. Retinal degeneration in the
rd mouse (Yan, W. et
al. (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) Curr. Opin. Chem. Biol. 2:472-481). PDE7s
are very closely
related to the PDE4 family; however, PDE7s are not inhibited by rolipram, a
specific inhibitor of
PDE4s (Beavo, supra).
PDEBs are cAMP specific, and are closely related to the PDE4 family. 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 PDE8s are inhibited by
dipyridamole (Fisher,
D.A. et al. (1998) Biochem. Biophys. Res. Commun. 246:570-577; Hayashi, M. et
al. (1998)
Biochem. Biophys. Res. Commun. 250:751-756; Soderling, S.H. et al. (1998)
Proc. Natl. Acad. Sci.
USA 95:8991-8996).
PDE9s are cGMP specific and most closely resemble the PDE8 family of PDEs.
PDE9s are
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expressed in kidney, liver, lung, brain, spleen, and small intestine. PDE9s
are not inhibited by
sildenafil (VIAGRA; Pfizer, Inc., New York NY), rolipram, vinpocetine,
dipyridamole, or IBMX (3-
isobutyl-1-methylxanthine), but they are sensitive to the 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. PDEIOs 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, HDXXHXGXXN, 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 the
conserved sequence
motif N(RlK)XnFX3DE (McAllister-Lucas, L.M. et al. (1993) J. Biol. Chem.
268:22863-22873). The
NKXnD motif has been shown by mutagenesis to be important for cGMP binding
(Turko, LV. et al.
(1996) J. Biol. Chem. 271:22240-22244). PDE families display approximately 30%
amino acid
identity within the catalytic domain; however, isozymes within the same family
typically display
about 85-95% identity in this region (e.g. PDE4A vs PDE4B). Furthermore,
within a family there is
extensive similarity (>60%) outside the catalytic domain; while across
families, there is little or no
sequence similarity outside this domain.
Many of the constituent functions of irrunune 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) Curr. Opin. Chem. Biol. 2:472-481; Torphy, T.J.
(1998) Am. J. Respir.
Crit. Care Med. 157:351-370). PDE3 inhibitors are being developed as
antithrombotic agents,
antihypertensive agents, and as cardiotonic agents useful in the treatment of
congestive heart failure.
Rolipram, a PDE4 inhibitor, has been used in the treatment of depression, and
other inhibitors of
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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 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 b and interferon g, 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. The'ophylline is believed to act on airway smooth
muscle function and in
an anti-inflammatory or immunomodulatory capacity in the treatment of
respiratory diseases (Banner,
K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000). Pentoxifylline is
another nonspecific PDE
inhibitor used in the treatment of intermittent claudication and diabetes-
induced peripheral vascular
disease. Pentoxifylline is also known to block TNF-a production and may
inhibit H1V-1 replication
(Angel et al., supra).
PDEs have been reported to affect cellular proliferation of a variety of cell
types (Conti et al.
(1995) Endocrine Rev. 16:370-389) and have been implicated in various cancers.
Growth of prostate
carcinoma cell lines DU145 and LNCaP was inhibited by delivery of cAMP
derivatives and PDE
inhibitors (Bang, Y.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334).
These cells also
showed a permanent conversion in phenotype from epithelial to neuronal
morphology. It has also
been suggested that PDE inhibitors have the potential to regulate mesangial
cell proliferation
(Matousovic, K. et al. (1995) J. Clin. Invest. 96:401-410) and lymphocyte
proliferation (Joulain, C. et
al. (1995) J. Lipid Mediat. Cell Signal. 11:63-79). A cancer treatment has
been described that
involves intracellular delivery of PDEs to particular cellular compartments of
tumors, resulting in cell
death (Deonarain, M.P. and A.A. Epenetos (1994) 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. 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
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reagents (e.g., parathion), in addition to pesticides and insecticides. Some
studies have also
implicated phosphotriesterase in atherosclerosis and diseases involving
lipoprotein metabolism.
Thioesterases
Two soluble thioesterases involved in fatty acid biosynthesis have been
isolated from
mammalian tissues, one 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 faovo 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 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. (su ra)
proposed that the physiological substrates for E. coli TEII may be coenzyme A
(CoA)-fatty acid esters
instead of ACP-phosphopanthetheine-fatty acid esters.
Carboxvlesterases
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 super family of esterases (B-esterases). Other
carboxylesterases included
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, palmitoyl-coenzyme A, imidapril, haloperidol, pyrrolizidine
alkaloids, steroids,
p-nitrophenyl acetate, malathion, butanilicaine, and isocarboxazide. The
enzymes often demonstrate
low substrate specificity. Carboxylesterases are also important for the
conversion of prodrugs to their
respective free acids, which may be the active form of the drug (e.g.,
lovastatin, used to lower blood
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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).
S ~uaq lene ~oxidase
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., su.~~ra). SE
converts squalene to 2,3(,S~-oxidosqualene, which is then converted to
lanosterol and then cholesterol.
IS 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 results 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 rate-limiting reaction that occurs later in the
sterol synthesis pathway and
cholesterol in 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).
Enoxide hydrolases
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Epoxide hydrolases catalyze the addition of water to epoxide-containing
compounds, thereby
hydrolyzing epoxides to their corresponding 1,2-diols. They are related to
bacterial haloalkane
dehalogenases and show sequence similarity to other members of the a/(3
hydrolase fold family of
enzymes (e.g., bromoperoxidase A2 from Streptomyces 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 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 hydroIase 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 (Michael 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
3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxymethyl-2-
hydroxymuconic
semialdehyde dehydrogenase, trarzs,cis-5-carboxymethyl-2-hydroxymuconate
isomerase,
homoprotocatechuate isomerase/decarboxylase, cis-2-oxohept-3-ene-1,7-dioate
hydratase,
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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.
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-I,7-
dioate hydratase, and
5-carboxymethyl-2-hydroxymuconate isomerase (Ellis, L.B.M. et al. (1999)
Nucleic Acids Res.
27:373-376; Wackett, L.P, and Ellis, L.B.M. (1996) J. Microbiol. Meth. 25:91-
93; and Schmidt, M.
(1996) Amer. Soc. Microbiol. News 62:102).
In humans, acquired or inherited genetic defects in enzymes of the tyrosine
degradation
pathway may result in hereditary tyrosinemia. One form of this disease,
hereditary tyrosinemia 1
(HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase,
the last enzyme in the
pathway in organisms that metabolize tyrosine to fumarate and acetoacetate.
HT1 is characterized
by progressive liver damage beginning at infancy, and increased risk for liver
cancer (Endo, F. et al.
(1997) J. Biol. Chem. 272:24426-24432).
The discovery of new drug metabolizing enzymes and the polynucleotides
encoding them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
prevention, and treatment of autoimmune/inflammatory, cell proliferative,
developmental, endocrine,
eye, metabolic, and gastrointestinal disorders, including liver disorders, and
in the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of drug
metabolizing enzymes.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, drug metabolizing enzymes,
referred to
collectively as "DME" and individually as "DME-1," "DME-2," "DME-3," "DME-4,"
"DME-5,"
"DME-6," "DME-7," "DME-8," "DME-9," "DME-10," "DME-11," "DME-12," "DME-13,"
"DME-
14," "DME-15," "DME-16," "DME-17," "DME-18," "DME-19," "DME-20," "DME-21,"
"DME-
22," "DME-23," and "DME-24." In one aspect, the invention provides an isolated
polypeptide
comprising an amino acid sequence selected from the group consisting of a) an
amino acid sequence
selected from the group consisting of SEQ )D NO:1-24, b) a naturally occurring
amino acid sequence
having at least 90% sequence identity to an amino acid sequence selected from
the group consisting
CA 02397340 2002-07-11
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of SEQ ID NO:1-24, cJ a biologically active fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:l-24, and d) an immunogenic fragment of an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-24. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ )D NO:l-24.
The invention further provides an isolated polynucleotide encoding a
polypeptide
comprising an amino acid sequence selected from the group consisting of a) an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-24, b) a naturally occurring
amino acid sequence
having at least 90% sequence identity to an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-24, c) a biologically active fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:l-24, and d) an immunogenic fragment of an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-24. In one alternative, the
polynucleotide
encodes a polypeptide selected from the group consisting of SEQ )D NO:1-24. In
another
alternative, the polynucleotide is selected from the group consisting of SEQ
ID N0:25-4S.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ )D NO:1-24, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-24,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:l-24, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO: l-24. In one alternative, the invention
provides a cell transformed
with the recombinant polynucleotide. In another alternative, the invention
provides a transgenic
organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ 117 NO:1-24, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ )D NO:1-24,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ >D NO: l-24, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ m NO:1-24. 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.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
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acid sequence selected from the group consisting of SEQ 1D NO:1-24, b) a
naturally occurnng amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-24, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:l-24, and d) an immunogenic
fragment of an
amino acid sequence selected from the group consisting of SEQ ID NO:l-24.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:25-48, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:25- 48, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). In one alternative,
the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a
polynucleotide sequence selected from the group consisting of a) a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:25-48, b) a naturally occurring
polynucleotide sequence
having at least 90% sequence identity to a polynucleotide sequence selected
from the group
consisting of SEQ m N0:25-48, c) a polynucleotide sequence complementary to
a), d) a
polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-
d). The method
comprises a) hybridizing the sample with a probe comprising at least 20
contiguous nucleotides
comprising a sequence complementary to said target polynucleotide in the
sample, and which probe
specifically hybridizes to said target polynucleotide, under conditions
whereby a hybridization
complex is formed between said probe and said target polynucleotide or
fragments thereof, and b)
detecting the presence or absence of said hybridization complex, and
optionally, if present, the
amount thereof. In one alternative, the probe comprises at least 60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:25-48, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ 117
N0:25-48, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerise chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
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The invention further provides a composition comprising an effective amount of
a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-24, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ >D NO:1-24, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-24, and d) an immunogenic
fragment of an
amino acid sequence selected from the group consisting of SEQ ID NO:1-24, and
a pharmaceutically .
acceptable excipient. In one embodiment, the composition comprises an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-24. The invention
additionally provides a
method of treating a disease or condition associated with decreased expression
of functional DME,
comprising administering to a patient in need of such treatment the
composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-24,
b) a naturally
occurnng amino acid sequence having at least 90% sequence identity to an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-24, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID N0:1-24, and d) an
innmunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-24. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
agonist activity in the sample. In one alternative, the invention provides a
composition comprising
an agonist compound identified by the method and a pharmaceutically acceptable
excipient. In
another alternative, the invention provides a method of treating a disease or
condition associated
with decreased expression of functional DME, comprising administering to a
patient in need of such
treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group
consisting of a) an amino acid sequence selected from the group consisting of
SEQ ID NO:1-24, b) a
naturally occurring amino acid sequence having at least 90% sequence identity
to an amino acid
sequence selected from the group consisting of SEQ m NO:l-24, c) a
biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ >D NO:l-24,
and d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:I-24. The method comprises a) exposing a sample comprising the polypeptide
to a compound,
and b) detecting antagonist activity in the sample. In one alternative, the
invention provides a
composition comprising an antagonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
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condition associated with overexpression of functional DME, comprising
administering to a patient
in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-24, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-24, c) a biologically active fragment of
an amino acid
sequence selected from the group consisting of SEQ 117 NO:1-24, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:1-
24. The method
comprises a) combining the polypeptide with at least one test compound under
suitable conditions,
and b) detecting binding of the polypeptide to the test compound, thereby
identifying a compound
that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-24,
b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:l-24, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO: l-24, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:l-24. The
method comprises a) combining the polypeptide with at least one test compound
under conditions
permissive for the activity of the polypeptide, b) assessing the activity of
the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of
the test compound with the activity of the polypeptide in the absence of the
test compound, wherein
a change in the activity of the polypeptide in the presence of the test
compound is indicative of a
compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:25-48, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The invention further provides a method 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 comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
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N0:25-48, ii) a naturally occurring polynucleotide sequence having at least
90% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:25-
48, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to 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 comprising a polynucleotide sequence
selected from the group
consisting of i) a polynucleotide sequence selected from the group consisting
of SEQ ID N0:25-48,
ii) a naturally occurring polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:25-48,
iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide
comprises a fragment of
a polynucleotide sequence selected from the group consisting of i)-v) above;
c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the
treated biological sample with the amount of hybridization complex in an
untreated biological
sample, wherein a difference in the amount of hybridization complex in the
treated biological sample
is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for each polypeptide of the invention. The probability score for the
match between each
polypeptide and its GenBank homolog 'is also shown.
Table 3 shows structural features of each polypeptide sequence, including
predicted motifs
and domains, along with the methods, algorithms, and searchable databases used
for analysis of each
polypeptide.
Table 4 lists the cDNA and genomic DNA fragments which were used to assemble
each
polynucleotide sequence, along with selected fragments of the polynucleotide
sequences.
Table 5 shows the representative cDNA library for each polynucleotide of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction
of the cDNA libraries shown in Table 5.
Table 7 shows the~tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
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DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be noted 'that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Ifnless 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 the invention. Nothing herein is to be
construed as an
admission that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
DEFINITIONS
"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.
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 occurnng form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more
times in a given sequence.
"Altered" nucleic acid sequences encoding DME include those sequences with
deletions,
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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
sequence 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 similarity 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" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or
synthetic molecules. Where "amino acid sequence" is recited to refer to a
sequence of a naturally
occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino
acid sequence to the complete native amino acid sequence associated with the
recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological
activity of DME. Antagonists may include proteins such as antibodies, nucleic
acids, carbohydrates,
small molecules, or any other compound or composition which modulates the
activity of DME either
by directly interacting with DME or by acting on components of the biological
pathway in which
DME participates.
The 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. 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.
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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 "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars 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 sequence" and a "composition
comprising a given amino acid sequence" refer broadly to any composition
containing the given
polynucleotide or amino acid sequence. The composition may comprise a dry
formulation or an
aqueous solution. Compositions comprising polynucleotide sequences encoding
DME or fragments
of DME may be employed as hybridization probes. The probes may be stored in
freeze-dried form
and may be associated with a stabilizing agent such as a carbohydrate. In
hybridizations, the probe
may be deployed in an aqueous solution containing salts (e.g., NaCI),
detergents (e.g., sodium
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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 GELV1EW 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 axe
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
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
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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.
A "fragment" is a unique portion of DME or the polynucleotide encoding DME
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotidelamino acid
residue. For example,
a fragment may comprise from 5 to 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 ID N0:25-48 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:25-48, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:25-48 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:25-48 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:25-48 and the region of SEQ ID N0:25-48 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 ID NO:1-24 is encoded by a fragment of SEQ ID N0:25-48. A
fragment of SEQ ID NO:1-24 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID Nb:l-24. For example, a fragment of SEQ ID NO:1-24 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-
24. The precise length of a fragment of SEQ ID NO:1-24 and the region of SEQ
ID NO:1-24 to
which the fragment corresponds are routinely determinable by one of ordinary
skill in the art based
on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
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"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison Wn. 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=5, window=4, and "diagonals
saved"=4. The "weighted"
residue weight table is selected as the default. Percent identity is reported
by CLUSTAL V as the
"percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison
algorithms is provided by the National Center for Biotechnology 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/BLASTI. 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:/lwww.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 Extensiozt Gap: 2 penalties
Gap x drop-off. 50
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Expect: 10
Word Size: Il
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ TD 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
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 Exterzsiorz Gap: 1 penalties
Gap x drop-off.' S0
Expect: l0
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Word Size: 3
Filter: otz
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ m 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
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 carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2°a ed., vol. 1-3, Cold
Spring Harbor Press,
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CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
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
~glml. 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 apparent to those of ordinary skill in the art.
Hybridization, particularly
under high stringency conditions, may be suggestive of evolutionary similarity
between the
nucleotides. Such similarity is strongly indicative of a similar role for the
nucleotides and their
encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of DME
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide 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, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of DME. For example,
modulation
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WO 01/51638 PCT/USO1/01174
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
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an DME may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of DME.
"Probe" refers to nucleic acid sequences encoding DME, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least IS 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.
CA 02397340 2002-07-11
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Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable. of choosing specific
primers from
megabase sequences and is thus useful 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 sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
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CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently,
a recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
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 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
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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" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a
recipient cell. Transformation may occur under natural or 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 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. 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, cyanobactenia, 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 Ieast
90%, at least 95% or at least
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98% 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 alternative splicing of exons during
mRNA processing. The
corresponding polypeptide may possess additional functional domains or lack
domains that are
present in the reference molecule. Species variants are polynucleotide
sequences that vary from one
species to another. The resulting polypeptides will generally have significant
amino acid identity
relative to each other. A polymorphic variant is a variation in the
polynucleotide sequence of a
particular gene between individuals of a given species. Polymorphic variants
also may encompass
"single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence
varies by one
nucleotide base. The presence of SNPs may be indicative of, for example, a
certain population, a
disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human drug metabolizing enzymes
(DME),
the polynucleotides encoding DME, and the use of these compositions 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
sequences 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
ID NO:) and an
Incyte polypeptide sequence number (Incyte Polypeptide )D) as shown. Each
polynucleotide
sequence is denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ
>D 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 )D NO:) and the
corresponding Incyte
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polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention.
Column 3 shows the GenBank identification number (Genbank ID NO:) of the
nearest GenBank
homolog. Column 4 shows the probability score for the match between each
polypeptide and its
GenBank homolog. Column 5 shows the annotation of the GenBank homolog.
Table 3 shows various structural features of each of the polypeptides of the
invention.
Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID
NO:) and the
corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for
each polypeptide of
the invention. Column 3 shows the number of amino acid residues in each
polypeptide. Column 4
shows 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 each polypeptide of the
invention, and
these properties establish that the claimed polypeptides are drug metabolizing
enzymes. The
algorithms and parameters for the analysis of SEQ IL1 NO:1-24 are described in
Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments
of the polynucleotide sequences which are useful, for example, in
hybridization or amplification
technologies that identify SEQ ID N0:25-48 or that distinguish between SEQ ID
N0:25-48 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start
(5') and stop (3') positions of the cDNA and genomic sequences in column 5
relative to their
respective full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
6537030H1 is the
identification number of an Incyte cDNA sequence, and (OVARDIN02) is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.g., 70614021V1). Alternatively, the identification
numbers in column 5
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WO 01/51638 PCT/USO1/01174
may refer to GenBank cDNAs or ESTs (e.g., 8758933 ) which contributed to the
assembly of the full
length polynucleotide sequences. Alternatively, the identification numbers in
column 5 may refer to
coding regions predicted by Genscan analysis of genomic DNA. For example,
g5091644.v113.gs_l.l.nt.edit is the identification number of a Genscan-
predicted coding sequence,
with 85091644 being the GenBank identification number of the sequence to which
Genscan was
applied. The Genscan-predicted coding sequences may have been edited prior to
assembly. (See
Example IV.) Alternatively, the identification numbers in column 5 may refer
to assemblages of
both cDNA and Genscan-predicted exons brought together by an "exon stitching"
algorithm. (See
Example V.) Alternatively, the identification numbers in column 5 may refer to
assemblages of both
cDNA and Genscan-predicted exons brought together by an "exon-stretching"
algorithm. (See
Example V.) In some cases, Incyte cDNA coverage redundant with the sequence
coverage shown in
column 5 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
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses DME variants. A preferred DME variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the DME amino acid sequence, and which contains at least
one functional or
structural characteristic of DME.
The invention also encompasses polynucleotides which encode DME. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:25-48, which encodes DME. The
polynucleotide
sequences of SEQ ID N0:25-48, 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 a variant of a polynucleotide sequence encoding
DME. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding DME. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:25-48 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
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WO 01/51638 PCT/USO1/01174
of SEQ ID N0:25-48. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of DME.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding DME, some
bearing minimal
similarity to the polynucleotide sequences of any 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 nucleotide sequences which encode DME and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring DME under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding DME or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding DME and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode DME
and
DME derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding DME or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:25-48 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods 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 polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
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combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno
NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800
thermal
cycler (Applied Biosystems). Sequencing is then carried out using either the
ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding DME may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.).
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments
comprising a known genomic locus and surrounding sequences. (See, e.g.,
Triglia, T. et al. (1988)
Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR
amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome
DNA. (See, e.g.,
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. (See, e.g., 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 finding intronlexon 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 50% 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
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sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
s 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, polynucleotide sequences or fragments
thereof
which encode DME may be cloned in recombinant DNA molecules that direct
expression of DME,
or fragments or functional equivalents thereof, in appropriate host cells. Due
to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express DME.
The nucleotide sequences of the present invention can be engineered using
methods
generally 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
Number
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
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optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding DME may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic
Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232.)
Alternatively, DME itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular
Properties, WH Freeman, New
York NY, pp.55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of DME, or any part thereof, may be altered during direct
synthesis and/or
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide
or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, su ~~ra, pp. 28-53.)
In order to express a biologically active DME, the nucleotide sequences
encoding DME or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding DME. Such elements may vary in their
strength and specificity.
Specific initiation signals may also be used to achieve more efficient
translation of sequences
encoding DME. Such signals include the ATG initiation codon and adjacent
sequences, e.g. the
Kozak sequence. In cases where sequences encoding DME and its initiation codon
and upstream
regulatory sequences are inserted into the appropriate expression vector, no
additional transcriptional
or translational control signals may be needed. However, in cases where only
coding sequence, or a
fragment thereof, is inserted, exogenous translational control signals
including an in-frame ATG
initiation colon should be provided by the vector. Exogenous translational
elements and initiation
colons 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. (See,
e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
CA 02397340 2002-07-11
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Methods which are well known to those skilled in the art may be used to
construct
expression vectors containing sequences encoding DME and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook,
J. et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y,
John Wiley & Sons,
New York NY, ch. 9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express
sequences encoding DME. These include, but are not limited to, microorganisms
such as bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast
transformed with yeast expression vectors; insect cell systems infected with
viral expression vectors
(e.g., baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower
mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression
vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra;
Ausubel, supra; Van Heeke,
G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et
al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et 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; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.)
Expression vectors
derived from retrovintses, adenoviruses, or herpes or vaccinia viruses, or
from various bacterial
plasmids, may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell
population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-
356; Yu, M. et al.
(1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985)
Nature
317(6040):813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226;
and Verma, LM.
and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the
host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding DME. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding DME can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORTl
plasmid (Life Technologies). Ligation of sequences encoding DME into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of DME are needed, e.g. for the
production of
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antibodies, vectors which direct high level expression of DME may be used. For
example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of DME. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of DME. Transcription of
sequences
encoding DME may be driven by viral promoters, e.g., the 35S and 19S promoters
of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., 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, sequences 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. (See, e.g., 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. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of DME in cell lines is preferred. For example, sequences encoding DME can be
transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
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CA 02397340 2002-07-11
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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 Bell 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.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
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. (See, e.g.,
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. (See, e.g., 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), f3 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presencelabsence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding DME is inserted within a marker gene sequence,
transformed cells containing
sequences encoding DME can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding DME under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding DME and
that express
DME may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution,
or chip based technologies for the detection and/or quantification of nucleic
acid or protein
sequences.
Immunological methods for detecting and measuring the expression of DME using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
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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.
(See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory
Manual, APS Press, St. Paul
MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y,
Greene Pub. Associates
and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding DME
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding DME, or any fragments
thereof, may be cloned
into a vector 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
Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter
molecules or
labels which may be used for ease of detection include radionuclides, enzymes,
fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates, cofactors,
inhibitors, magnetic
particles, and the like.
Host cells transformed with nucleotide sequences encoding DME may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the
sequence andlor 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 sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
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
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CA 02397340 2002-07-11
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modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences 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 (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-nzyc, and hemagglutinin (HA). GST, MBP, 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 (1995, supra, ch.
10). A variety of commercially available kits may also be used to facilitate
expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled DME may be
achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with
the T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
DME of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to DME. At least one and up to a plurality of test
compounds may be screened
for specific binding to DME. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
DME, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which DME
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express DME, either
as a secreted
CA 02397340 2002-07-11
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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.
DME of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of DME. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for DME
activity, wherein DME is combined with at least one test compound, and the
activity of DME in the
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 Number 5,175,383 and U.S.
Patent Number
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 (March, J.D. (1996) Clin. 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
'S6
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blastocysts such as those from the C57BL16 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with
potential therapeutic or toxic agents.
Polynucleotides encoding DME may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding DME can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding DME is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress DME, e.g., by secreting DME in
its 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. In addition, the
expression of DME is
closely associated with brain, breast, prostate, ovary, testicle, bone, blood,
kidney, lung, thyroid, and
gastrointestinal tissues; Crohn's disease; breast, sigmoid mesentery, and
ureter tumors; and cancers
of the lung, prostate, bone, and blood. Therefore, DME appears to play a role
in
autoimmunelinflammatory, Bell proliferative, developmental, endocrine, eye,
metabolic, and
gastrointestinal disorders, including liver disorders. In the treatment of
disorders associated with
increased DME expression or activity, it is desirable to decrease the
expression or activity of DME.
In the treatment of disorders associated with decreased DME expression or
activity, it is desirable to
increase the expression or activity of DME.
Therefore, in one embodiment, DME or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of DME. Examples of such disorders include, but are not limited to,
an
autoimmunelinflammatory 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),
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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, Cushing's
syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation),
Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as
Syndenham's chorea
and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss; an endocrine disorder, such as disorders of the
hypothalamus and
pituitary resulting from lesions such as primary brain tumors, adenomas,
infarction associated with
pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis,
infections,
immunological disorders, and complications due to head trauma; disorders
associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus,
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,
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thyroid carcinoma, and Plummer's disease; disorders associated with
hyperparathyroidism including
Conn disease (chronic hypercalemia); pancreatic disorders such as Type I or
Type II diabetes
mellitus and associated complications; disorders associated with the adrenals
such as hyperplasia,
carcinoma, or adenoma of the adrenal cortex, hypertension associated with
alkalosis, amyloidosis,
hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon
syndrome,
pheochromocytoma tumors, and Addison's disease; disorders associated with
gonadal steroid
hormones such as: in women, abnormal prolactin production, infertility,
endometriosis,
perturbations of the menstrual cycle, polycystic ovarian disease,
hyperprolactinemia, isolated
gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism
and virilization,
breast cancer, and, in post-menopausal women, osteoporosis; and, in men,
Leydig cell 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,
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, and
postpubescent cerebellar ataxia, 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
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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
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 autoimmunelinflammatory, cell
proliferative,
developmental, endocrine, eye, metabolic, and gastrointestinal disorders,
including liver disorders,
described above. In one aspect, an antibody which specifically binds DME may
be used directly as
an antagonist or indirectly as a targeting or delivery mechanism for bringing
a pharmaceutical agent
to cells or tissues which express DME.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding DME may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of DME including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential 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
CA 02397340 2002-07-11
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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.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with DME or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used
to increase immunological response. Such adjuvants include, but are not
limited to, Freund's,
mineral gels such as aluminum hydroxide, and surface active substances such as
lysolecithin,
pluronic polyols, polyanions, peptides, 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 molecule may be produced.
Monoclonal antibodies to DME may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-
hybridoma technique. (See, e.g., Kohler, G. et 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; and 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. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and 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. (See,
e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
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population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., 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~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of 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).
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 K~ ranging from about 109 to 10'2 L/mole are preferred for use in
immunoassays in which the
DME-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with 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, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
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of DME-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding DME, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding DME. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding DME. (See, e.g., Agrawal, S., ed. (1996) Antisense Thera ep utics,
Humana Press Inc.,
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. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Cli. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of
viral vectors, such as retrovirus and adeno-associated virus vectors. (See,
e.g., Miller, A.D. (1990)
Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.
Ther. 63(3):323-347.)
Other gene delivery mechanisms include liposome-derived systems, artificial
viral envelopes, and
other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado,
R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al.
(1997) Nucleic Acids Res.
25(14):2730-2736.) .
In another embodiment of the invention, polynucleotides encoding DME may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-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 1X 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
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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
falci narum and
Tr~panosoma cruzi). In the case where a genetic deficiency in DME expression
or regulation causes
disease, the expression of DME from an appropriate population of transduced
cells may alleviate the
clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
DME are treated by constructing mammalian expression vectors encoding DME and
introducing
these vectors by mechanical 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; Zvics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of DME include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-H'YG (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) Curr. 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 Blau, H.M. 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 KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
b4
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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
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.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of 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 the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding DME to cells which have one or more genetic
abnormalities with respect
to the expression of DME. The construction and packaging of adenovirus-based
vectors are well
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 Number 5,707,618 to Armentano
("Adenovirus vectors for gene
therapy"), hereby incorporated by reference. For adenoviral vectors, see also
Antinozzi, P.A. et al.
(1999) Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242,
both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding DME to target cells which have one or more genetic
abnormalities with
respect to the expression of DME. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing DME to cells of the central nervous
system, for which HSV has a
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tropism. The constriction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector
has been used to deliver a reporter gene to the eyes of primates (Liu, X. et
al. (1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 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, hereby incorporated by reference. The
manipulation of
cloned herpesvirus sequences, the generation of recombinant virus following
the transfection of
multiple plasmids containing different segments of the large herpesviris
genomes, the growth and
propagation of herpesvirus, and the infection of cells with herpesvirus are
techniques well known to
those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding DME to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic
activity (e.g., protease and polymerise). Similarly, inserting the coding
sequence for DME into the
alphaviris 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 (SII~
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
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-10 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 been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo~ic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding DME.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the 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 of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include
techniques for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite
chemical synthesis. Alternatively, RNA molecules may be generated by in vitro
and in vivo
transcription of DNA sequences encoding DME. Such DNA sequences may be
incorporated into a
wide variety of vectors with suitable RNA polymerase promoters such as T7 or
SP6. Alternatively,
these cDNA constructs that synthesize complementary RNA, constitutively or
inducibly, can be
introduced into Bell 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
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endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding DME. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased DME
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding DME may be therapeutically useful, and in the treament of disorders
associated with
decreased DME expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding DME may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective
in altering polynucleotide expression; selection 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 Schizosaccharom~es
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,
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ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for
antisense activity against a
specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No.
5,686,242; Bruice,
T.W. et al. (2000) U.S. Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be
achieved using methods which are well known in the art. (See, e.g., Goldman,
C.K. et al. (1997)
Nat. Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits,
and monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of DME, antibodies to DME, and mimetics, agonists, antagonists, or
inhibitors of DME.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enterah 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
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macromolecules comprising DME or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, DME or a fragment thereof may be 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 50% 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. 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 ,ug 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,
CA 02397340 2002-07-11
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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
i
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, the polynucleotides encoding DME may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
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
polynucleotide
sequences, 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 occurnng 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
N0:25-4S or from
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genomic sequences including promoters, enhancers, and introns of the DME gene.
Means for producing specific hybridization probes for DNAs encoding DME
include the
cloning of polynucleotide sequences encoding DME or DME derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and
may be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding DME may be used for the diagnosis of
disorders
associated with expression of DME. Examples of such disorders include, but are
not limited to, an
autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome
(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, 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
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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
dwarfism; disorders associated with hyperpituitarism including acromegaly,
giantism, and syndrome
of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by
benign adenoma;
disorders associated with hypothyroidism including goiter, myxedema, acute
thyroiditis associated
with bacterial infection, subacute thyroiditis associated with viral
infection, autoimmune thyroiditis
(Hashimoto's disease), and cretinism; disorders associated with
hyperthyroidism including
thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema,
toxic multinodular goiter,
thyroid carcinoma, and Plummer's disease; disorders associated with
hyperparathyroidism including
Conn disease (chronic hypercalemia); pancreatic disorders such as Type I or
Type 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,
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,
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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, and
postpubescent cerebellar ataxia, 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,
irritable 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. The polynucleotide sequences encoding DME may be used in
Southern or northern
analysis, dot blot, or other membrane-based technologies; in PCR technologies;
in dipstick, pin, and
multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues
from patients to detect
altered DME expression. Such qualitative or quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding DME may be useful in
assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding DME may be labeled by standard methods and added to a fluid
or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding DME in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
DME, a normal or standard profile fox expression is established. This may be
accomplished by
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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 fox a disorder.
Deviation from standard
values is used to establish the 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 the
polynucleotide sequences
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 the polynucleotide sequences encoding DME
are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, 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
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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
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 MASSARR.AY system
(Sequenom, Inc., San
Diego CA).
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. (See, e.g., 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 quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The
microarray can be used in transcript imaging techniques which monitor the
relative expression levels
of large numbers of genes simultaneously as described below. The microarray
may also be used to
identify genetic variants, mutations, and polymorphisms. This information may
be used to determine
gene function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be
used to develop a pharmacogenomic profile of a patient in order to select the
most appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, 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
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and at a given time. (See Seilhamer et al., "Comparative Gene Transcript
Analysis," U.S. Patent
Number 5,840,484, expressly incorporated by reference herein.} Thus a
transcript image may be
generated by hybridizing the polynucleotides of the present invention or their
complements to the
totality of transcripts or reverse transcripts of a particular tissue or cell
type. In one embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol, Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they
contain expression information from a large number of genes and gene families.
Ideally, a genome-
wide measurement of expression provides the highest quality signature. Even
genes whose
expression is not altered by 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
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 one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared
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with levels in an untreated biological sample. Differences in the transcript
levels between the two
samples are indicative of a toxic response caused by the test compound in the
treated sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or
profiles, are analyzed by quantifying the number of expressed proteins and
their relative abundance
under given conditions and at a given time. A profile of a cell's proteome may
thus be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample
are separated by isoelectric focusing in the first dimension, and then
according to molecular weight
by sodium dodecyl sulfate slab 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 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 the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for DME to
quantify the
levels of DME expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lucking, 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
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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
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount
of protein between the two samples is indicative of a toxic response to the
test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See,
e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl.
Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116;
Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997)
Proc. Natl. Acad.
Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No.
5,605,662.) Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical A rpp oath,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding DME
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences 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 (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
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Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., 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
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
l 1q22-23, any sequences mapping to that area may represent associated or
regulatory genes for
further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-
580.) The nucleotide
sequence of the instant invention may also be used to detect differences in
the chromosomal location
due to translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, DME, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used 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. (See, e.g.,
Geysen, et aI. (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.
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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
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not !imitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. Nos. 60/176,139, 601177,443, and 60/178,574, axe hereby
expressly incorporated
by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
Iysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were 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 constricted the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra,
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units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with
the appropriate restriction enzyme or enzymes. For most libraries, the cDNA
was size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRII'T plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XLl-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DH10B from Life Technologies.
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 Iysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega);
an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and
QIAWELL 8
Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems
or the
R.E.A.L. PREP 96 plasmid purification kit from QIA.GEN. 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. 2I6:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton} liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such
as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit
(Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
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ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences 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, and hidden Markov model (HMM)-based protein
family databases such as PFAM. (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,
BLIMPS, and
HMMER. 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 of the invention may begin at any of the methionine residues of
the full length translated
polypeptide. Full length polypeptide sequences were subsequently analyzed by
querying against
databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS,
PRINTS, DOMO,
PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases
such as
PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS
PRO software
(Hitachi Software Engineering, South San Francisco CA) and LASERGENE software
(DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default
parameters
specified by the CLUSTAL algorithm as incorporated into the MEGALIGN
multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly
of Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
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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
m N0:25-48. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization
and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative drug metabolizing enzymes were initially identified by running the
Genscan gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan
is a general-purpose gene identification program which analyzes genomic DNA
sequences from a
variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-
94, and Burge, C. and
S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to
form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of
Genscan is a FASTA database of polynucleotide and polypeptide sequences. 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 andlor 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
"Stitched" Seauences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example 1V. Partial cDNAs assembled as
described in Example
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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 with additional cDNA sequences, or by
inspection of
genomic DNA, when necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described 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.
VI. Chromosomal Mapping of DME Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:25-48 were compared with
sequences from the Incyte LIF'ESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
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SEQ 1D N0:25-48 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
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, or 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/genemap/), 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. (See, e.g., Sambrook,
sera, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.) '
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:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
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(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in
a BLAST alignment. For example, a product score of 100 is produced only for
100% identity over
the entire length of the shorter of the two sequences being 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, polynucleotide sequences 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 ~. 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; hemic 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 L1FESEQ GOLD database (Incyte
Genomics, Palo Alto
CA).
VIII. Extension of DME Encoding Polynncleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
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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)ZS04,
and 2-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step l: 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 SI~+
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: 6$°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~1
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 ~cl to 10 ,u1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Phannacia Biotech), treated with Pfu DNA polymerise (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. cola 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 caxb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (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
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primers and the DYENAMIC Dn2ECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure
or are used to obtain 5'regulatory sequences using the above procedure along
with oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ m N0:25-48 are employed to screen cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
~Y 32P, adenosine triphosphate (Amersham Pharmacia Biotech), 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 Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots axe 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.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, su ra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
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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 Preuaration
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/N1 oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/E.~l RNase inhibitor, 500 ~M dATP, 500 ~M
dGTP, 500 ~.~M dTTP, 40
~M dCTP, 40 E.~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A) ~ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro
transcription from non-coding yeast genomic DNA. After incubation at 37
° C for 2 hr, each reaction
sample (one with Cy3 and another with Cy5 labeling) is treated with 2:5 ml of
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. (CLONTECH), Palo Alto CA) and after combining,
both reaction
samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml
sodium acetate, and 300
ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC
(Savant Instruments
Inc., Holbrook NY) and resuspended in 14 ~1 SX SSC/0.2% SDS.
Microarray Preuaration
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
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slides (Corning) are cleaned by. ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (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 US
Patent No. 5,807,522 , incorporated herein by reference. 1 1.i1 of the array
element DNA, at an
average concentration of 100 ng/~ul, 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 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65°C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cmz 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 E.~l of 5X 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 at 4$8 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.
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Appropriate filters positioned between the array and the photomultiplier tubes
are used to filter the
signals. The 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
(AlD) 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).
XI. Complementary Polynucleotides
Sequences complementary to the DME-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring DME. Although
use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of DME. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a
complementary oligonucleotide is designed to prevent ribosomal binding to the
DME-encoding
transcript.
XII. Expression of DME
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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 drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.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 Schistosoina japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
DME at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
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 (1995, supra, ch. 10 and 16). Purified DME obtained by these methods
can be used directly
in the assays shown in Examples XVI, XVII, and XV>II, where applicable.
XIII. 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 nigh levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are
transiently transfected
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into a human cell line, for example, an endothelial or hematopoietic cell
line, using either liposome
formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser
optics-based technique, is used to identify transfected cells expressing GFP
or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the
uptake of fluorescent molecules that diagnose events preceding or coincident
with cell death. These
events include changes in nuclear DNA content as measured by staining of DNA
with propidium
iodide; changes in cell size and granularity as measured by forward light
scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine
uptake; alterations in expression of cell surface and intracellular proteins
as measured by reactivity
with specific antibodies; and alterations in plasma membrane composition as
measured by the
binding of fluorescein-conZugated 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.
XIV. Production of DME Specific Antibodies
DME substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used
to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the DME amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are
well described in the art. (See, e.g., Ausubel, 1995, supra,, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
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Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, 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.
XV. Purification of Naturally Occurring DME Using Specific Antibodies
Naturally occurring or recombinant DME is substantially purified by
immunoaffinity
chromatography using antibodies specific for DME. An immunoaffinity column is
constructed by
covalently coupling anti-DME antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing DME are passed over the immunoaffinity column, and the column
is
washed under conditions that allow the preferential absorbance of DME (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/DME binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such
as urea or thiocyanate ion), and DME is collected.
XVI. Identification of Molecules Which Interact with DME
DME, or biologically active fragments thereof, are labeled with'zsI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mufti-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).
XVII. Demonstration of DME Activity
Cytochrome P450 activity of DME is measured using the 4-hydroxylation of
aniline. Aniline
is converted to 4-aminophenol by the enzyme, and has an absorption maximum at
630 nm (Gibson
and Skett, supra). This assay is a convenient measure, but underestimates the
total hydroxylation,
CA 02397340 2002-07-11
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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 MgCI Z,
50 mM
nicotinamide, 40 mg trisodium isocitrate, and 2 units isocitrate
dehydrogenase, with 8 mg NADP+
added to a 10 mL reaction buffer stock just prior to assay. Reactions are
carried out in an optical
cuvette, and the absorbance at 630 nm is measured. The rate of increase in
absorbance is
proportional to the enzyme activity in the assay. A standard curve can be
constructed using known
concentrations of 4-aminophenol.
1a,25-dihydroxyvitamin D 24-hydroxylase activity of DME is determined by
monitoring the
conversion of 3H-labeled 1a,25-dihydroxyvitamin D (1a,25(OH)ZD) to 24,25-
dihydroxyvitamin D
(24,25(OH)zD) in transgenic rats expressing DME. 1 pg of 1a,25(OH)ZD dissolved
in ethanol (or
ethanol alone as a control) is administered intravenously to approximately 6-
week-old male
transgenic rats expressing DME or otherwise identical control rats expressing
either a defective
variant of DME or not expressing DME. The rats are killed by decapitation
after 8 hrs, and the
kidneys are rapidly removed, rinsed, and homogenized in 9 volumes of ice-cold
buffer (15 mM Tris-
acetate (pH 7.4), 0.19 M sucrose, 2 mM magnesium acetate, and 5 mM sodium
succinate). A portion
(e.g., 3 ml) of each homogenate is then incubated with 0.25 nM 1a,25(OH)z[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 Dyer, W.J. (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 a 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(OH)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(OH)2[I 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).
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Flavin-containing monooxygenase activity of DME is measured by chromatographic
analysis of metabolic products. For example, Ring, B. J. et al. (1999; Drug
Metab. Dis. 27:1099-
1103) incubated FMO in 0.1 M sodium phosphate buffer (pH 7.4 or 8.3) and 1 mM
NADPH at
37°C, stopped the reaction with an organic solvent, and determined
product formation by HPLC.
Alternatively, activity is measured by monitoring oxygen uptake using a Clark-
type electrode. For
example, Ziegler, D. M. and Poulsen, L. L. (1978; Methods Enzymol. 52:142-151)
incubated the
enzyme at 37 °C in an NADPH-generating cofactor system (similar to the
one described above)
containing the substrate methimazole. The rate of oxygen uptake is
proportional to enzyme activity.
UDP glucuronyltransferase activity of DME is measured using a colorimetric
determination
of free amine groups (Gibson and Skett, supra). An amine-containing substrate,
such as 2-
aminophenol, is incubated at 37 °C with an aliquot of the enzyme in a
reaction buffer containing the
necessary cofactors (40 mM Tris pH 8.0, 7.5 mM MgClz, 0.025% Triton X-100, 1
mM ascorbic acid,
0.75 mM UDP-glucuronic acid). After sufficient time, the reaction is stopped
by addition of ice-cold
20% trichloroacetic acid in 0.1 M phosphate buffer pH 2.7, incubated on ice,
and centrifuged to
clarify the supernatant. Any unreacted 2-aminophenol is destroyed in this
step. Sufficient freshly-
prepared sodium nitrite is then added; this step allows formation of the
diazonium salt of the
glucuronidated product. Excess nitrite is removed by addition of sufficient
ammonium sulfamate,
and the diazonium salt is reacted with an aromatic amine (for example, N-
naphthylethylene diamine)
to produce a colored azo compound which can be assayed spectrophotometrically
(at 540 nm for the
example). A standard curve can be constrncted 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.
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Chem. 266:10227-10233) measured bile acid-CoA:amino acid N-acyltransferase
activity by
incubating the enzyme with cholyl-CoA and 3H-glycine or 3H-taurine, separating
the tritiated cholate
conjugate by extraction into n-butanol, and measuring the radioactivity in the
extracted product by
scintillation. Alternatively, N-acyltransferase activity is measured using the
spectrophotometric
determination of reduced CoA (CoASH) described below.
N-acetyltransferase activity of DME is measured using the transfer of
radiolabel from
[14C]acetyl-CoA to a substrate molecule (for example, see Deguchi, T. (1975)
J. Neurochem.
24:1083-5). Alternatively, a spectrophotometric assay based on DTNB (5,5'-
dithio-bis(2-
nitrobenzoic acid; Ellman's reagent) reaction with CoASH may be used. Free
thiol-containing
CoASH is formed during N-acetyltransferase catalyzed transfer of an acetyl
group to a substrate.
CoASH is detected using the absorbance of DTNB conjugate'at 412 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.
Catechol-O-methyltransferase activity of DME is measured in a reaction mixture
consisting
of 50 mM Tris-HCl (pH 7.4), 1.2 mM MgClz, 200 pM SAM (S-adenosyl-L-methionine)
iodide
(containing 0.5 ~Ci of methyl-[H3]SAM), 1 mM dithiothreitol, and varying
concentrations of
catechol substrate (e.g., L-dopa, dopamine, or DBA) in a final volume of 1.0
ml. The reaction is
initiated by the addition of 250-500 ~g of purified DME or crude DME-
containing sample and
performed at 37 °C for 30 min. The reaction is arrested by rapidly
cooling on ice and immediately
extracting with 7 ml of ice-cold n-heptane. Following centrifugation at 1000 x
g for 10 min, 3-ml
aliquots of the organic extracts are analyzed for radioactivity content by
liquid scintillation counting.
The level of catechol-associated radioactivity in the organic phase is
proportional to the catechol-O-
methyltransferase activity of DME (Zhu, B.T. Liehr, J.G. (1996) 271:1357-
1363).
DHFR activity of DME is determined spectrophotometrically at 15 °C by
following the
disappearance of NADPH at 340 nm (E3ao = 11,800 M-'~crri'). The standard assay
mixture contains
100 pM 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 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 ~.g
enzyme and an
appropriate level of substrate. The reaction is incubated at 30 °C and
the reaction is monitored
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continuously with a spectrophotometer. Enzyme activity is calculated as mol
NADPH consumed /
~,g 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 l p,g of
enzyme.
Carboxylesterase activity of DME activity is determined using 4-
methylumbelliferyl acetate
as a substrate. The enzymatic reaction is initiated by adding approximately 10
~l of DME-
containing sample to 1 ml of reaction buffer (90 mM KHZP04, 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 ($3so = 12.2 mM-' cm') 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 enzyme 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 pglml) 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 KHZP04, pH 4.0, with 8 p1 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).
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 pg/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 35S
from [35S]PAPS
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CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
into a model substrate such as phenol (Folds, A. and Meek, J. L. (1973)
Biochim. Biophys. Acta
327:365-374). An aliquot of enzyme is incubated at 37 °C with 1 mL of
10 mM phosphate buffer, pH
6.4, 50 ~M phenol, and 0.4-4.0 lt.M [35S]PADS. After sufficient time for 5-20%
of the radiolabel to
be transferred to the substrate, 0.2 mL of 0.1 M barium acetate is added to
precipitate protein and
phosphate buffer. Then 0.2 mL of 0.1 M Ba(OH)2 is added, followed by 0.2 mL
ZnS04. The
supernatant is cleared by centrifugation, which removes proteins as well as
unreacted [ 35S]PAPS.
Radioactivity in the supernatant is measured by scintillation. The enzyme
activity is determined
from the number of moles of radioactivity in the reaction product.
Heparan sulfate 6-sulfotransferase activity of DME is measured in vitro by
incubating a
sample containing DME along with 2.5 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
105 cpm) of [35S] adenosine 3'-phosphate 5'-phosphosulfate (PAPS) in a final
reaction volume of 50
p1 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 pmol (as glucuronic acid) of chondroitin sulfate A is
added to the reaction
mixture as a carrier. 35S-Labeled polysaccharides are precipitated with 3
volumes of cold ethanol
containing 1.3% potassium acetate and separated completely from unincorporated
[35S]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
[s5S]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-HCl, pH 8.0), cut into 3-5 mm segments and subjected to
agitation at 4 °C with
100 ~I of the same buffer containing 0.15 M NaC1 for 48 h. The eluted enzyme
is collected by
centrifugation and assayed for the sulfotransferase activity as described
above (Habuchi, H.et al.
(1995) J. Biol. Chem. 270:4172-4179).
In another alternative, DME sulfotransferase activity is determined by
measuring the transfer
of [35S]sulfate from [35S]PAPS to an immobilized peptide that represents the N-
terminal 15 residues
of the mature P-selectin glycoprotein Iigand-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 ~mo1
peptide/ml of resin.
The enzyme assay is performed by combining 10 p1 of peptide-derivitized beads
with 2-20 ~l of
DME-containing sample in 40 mM Pipes (pH 6.8), 0.3 M NaCI, 20 mM MnClz, 50 mM
NaF, 1 %
Triton X-100, and 1 mM 5'-AMP in a final volume of 130 ~I. The assay is
initiated by addition of
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0.5 pCi of [35S]PAPS (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 ~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 ~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
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 35S-
sulfated products and
determine the enzyme specificity of the DME-containing samples based on
relative rates of
migration of reaction products (Sakakibara, Y. et al. (1998) J. Biol. Chem.
273:6242-6247).
Squalene epoxidase activity of DME is assayed in a mixture comprising purified
DME (or a
crude mixture comprising DME), 20 mM Tris-HCl (pH 7.5), 0.01 mM FAD, 0.2 unit
of
NADPH-cytochrome C (P-450) reductase, O.OI mM ['4C]squalene (dispersed with
the aid of 20 ~1 of
Tween 80), and 0.2% Triton X-100. 1 mM NADPH is added to initiate the reaction
followed by
incubation at 37 °C for 30 min. The nonsaponifiable lipids are analyzed
by silica gel TLC developed
with ethyl acetate/benzene (0.5:99.5, v/v). The reaction products are compared
to those from a
reaction mixture without DME. The presence of 2,3(S~-oxidosqualene is
confirmed using
appropriate lipid standards (Sakakibara, J. et al. (1995) 270:17-20).
Epoxide hydrolase activity of DME is determined by following substrate
depletion using gas
chromatographic (GC) analysis of ethereal extracts or by following substrate
depletion and diol
production by GC analysis of reaction mixtures quenched in acetone. A sample
containing DME or
an epoxide hydrolase control sample is incubated in 10 mM Tris-HCl (pH 8.0), 1
mM
ethylenediaminetetraacetate (EDTA), and 5 xnM 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
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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 ~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 nnM 2-
oxoglutarate in a final volume of
200 ~l of 150 mM Tris acetate buffer (pH 8.0) containing 70 ~M PLP. The
formation of kynurenic
acid is quantified by HPLC with spectrophotometric detection at 330 nm using
the appropriate
6
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 W 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 ~M purified
DME or DME containing
samples and substrate to be tested (amino and oxo acid substrates). The half
reaction from amino
acid to oxo acid is followed by measuring the decrease in absorbance at 360 nm
and the increase in
absorbance at 330 nm due to the conversion of enzyme-bound PLP to pyridoxamine
5' phosphate
(PMP). The specificity and relative activity of DME is determined by the
activity of the enzyme
preparation against specific substrates (Vacca, R.A. et al. (1997) J. Biol.
Chem. 272:21932-21937).
Superoxide dismutase activity of DME is assayed from cell pellets, culture
supernatants, or
purified protein preparations. Samples or lysates are resolved by
electrophoresis on 15%
non-denaturing polyacrylamide gels. The gels are incubated for 30 min in 2.5
mM nitro 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).
XVIII. Identification of DME Inhibitors
Compounds to be tested are arrayed in the wells of a multi-well plate in
varying
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WO 01/51638 PCT/USO1/01174
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 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. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
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CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
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129
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
YANG, Junming
BAUGHN, Mariah R.
BURFORD, Neil
AU-YOUNG, Janice
LU, Dyung Aina M.
REDDY, Roopa
RING, Huijun Z.
HILLMAN, Jennifer L.
YUE, Henry
AZIMZAI, Yalda
YAO, Monique G.
GANDHI, Ameena R.
NGUYEN, Danniel B.
BAUGHN, Mariah R.
TANG, Y. Tom
LAL, Preeti
YUE, Henry
BANDMAN, Olga
<120> DRUG METABOLIZING ENZYMES
<130> PI-0007 PCT
<140> To Be Assigned
<141> Herewith
<160> 48
<170> PERL Program
<210> 1
<222> 330
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1799250CD1
<400> 1
Met Ser Pro Leu Ser Ala Ala Arg Ala Ala Leu Arg Val Tyr Ala
1 5 10 15
Val Gly Ala Ala Val Ile Leu Ala Gln Leu Leu Arg Arg Cys Arg
20 25 30
Gly Gly Phe Leu Glu Pro Val Leu Pro Pro Arg Pro Asp Arg Val
35 40 45
Ala Ile Val Thr Gly Gly Thr Asp Gly Ile Gly Tyr Ser Thr Ala
50 55 60
Lys His Leu Ala Arg Leu Gly Met His Val Ile Ile Ala Gly Asn
65 70 75
Asn Asp Ser Lys Ala Lys GIn Val Val Ser Lys Ile Lys Glu Glu
80 85 90
Thr Leu Asn Asp Lys Val Glu Phe Leu Tyr Cys Asp Leu Ala Ser
95 100 105
Met Thr Ser Ile Arg Gln Phe Val Gln Lys Phe Lys Met Lys Lys
110 115 120
Ile Pro Leu His Val Leu Ile Asn Asn Ala Gly Val Met Met Val
125 130 135
Pro Gln Arg Lys Thr Arg Asp Gly Phe Glu Glu His Phe Gly Leu
140 145 150
Asn Tyr Leu Gly His Phe Leu Leu Thr Asn Leu Leu Leu Asp Thr
1/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
155 160 165
Leu Lys Glu Ser Gly Ser Pro Gly His Ser Ala Arg Val Val Thr
170 175 180
Val Ser Ser Ala Thr His Tyr Val Ala Glu Leu Asn Met Asp Asp
185 190 195
Leu Gln Ser Ser Ala Cys Tyr Ser Pro His Ala Ala Tyr Ala Gln
200 205 210
Ser Lys Leu Ala Leu Val Leu Phe Thr Tyr His Leu Gln Arg Leu
215 220 225
Leu Ala Ala Glu Gly Ser His Val Thr Ala Asn Val Val Asp Pro
230 235 240
Gly Val Val Asn Thr Asp Val Tyr Lys His Va1 Phe Trp Ala Thr
245 250 255
Arg Leu Ala Lys Lys Leu Leu Gly Trp Leu Leu Phe Lys Thr Pro
260 265 270
Asp Glu Gly Ala Trp Thr Ser Ile Tyr Ala Ala Val Thr Pro Glu
275 280 285
Leu Glu Gly Val Gly Gly Arg Tyr Leu Tyr Asn Lys Lys Glu Thr
290 295 300
Lys Ser Leu His Val Thr Tyr Asn Gln Lys Leu Gln Gln Gln Leu
305 310 315
Trp Ser Lys Ser Cys Glu Met Thr Gly Val Leu Asp Val Thr Leu
320 325 330
<210> 2
<211> 497
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 2242475CD1
<400> 2
Met Gly Leu Glu Ala Leu Val Pro Leu Ala Val Ile Val Ala Ile
1 5 10 25
Phe Leu Leu Leu Val Asp Leu Met His Arg Arg Gln Arg Trp Ala
20 25 30
Ala Arg Tyr Pro Pro Gly Pro Leu Pro Leu Pro Gly Leu Gly Asn
35 40 45
Leu Leu His Val Asp Phe Gln Asn Thr Pro Tyr Cys Phe Asp Gln
50 55 60
Leu Arg Arg Arg Phe Gly Asp Val Phe Ser Leu Gln Leu AIa Trp
65 70 75
Thr Pro Val Val Val Leu Asn Gly Leu Ala Ala Val Arg Glu Ala
80 85 90
Leu Val Thr His Gly Glu Asp Thr Ala Asp Arg Pro Pro Val Pro
95 100 105
Ile Thr Gln Ile Leu Gly Phe Gly Pro Arg Ser Gln Gly Val Phe
110 115 120
Leu Ala Arg Tyr Gly Pro Ala Trp Arg Glu Gln Arg Arg Phe Ser
125 130 135
Val Ser Thr Leu Arg Asn Leu Gly Leu Gly Lys Lys Ser Leu Glu
140 145 150
Gln Trp Val Thr Glu Glu Ala Ala Cys Leu Cys Ala Ala Phe Ala
155 160 165
Asn His Ser Gly Arg Pro Phe Arg Pro Asn Gly Leu Leu Asp Lys
170 175 180
Ala Val Ser Asn Val Ile Ala Ser Leu Thr Cys Gly Arg Arg Phe
185 190 195
Glu Tyr Asp Asp Pro Arg Phe Leu Arg Leu Leu Asp Leu Ala Gln
200 205 210
Glu Gly Leu Lys Glu Glu Ser Gly Phe Leu Arg Glu Val Leu Asn
2139
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
215 220 225
Ala Val Pro Val Leu Pro His Ile Pro Ala Leu Ala Gly Lys Val
230 235 240
Leu Arg Phe Gln Lys Ala Phe Leu Thr Gln Leu Asp Glu Leu Leu
245 250 255
Thr Glu His Arg Met Thr Trp Asp Pro Ala Gln Pro Pro Arg Asp
260 265 270
Leu Thr Glu Ala Phe Leu Ala Lys Lys Glu Lys Ala Lys Gly Ser
275 280 285
Pro Glu Ser Ser Phe Asn Asp Glu Asn Leu Arg Ile Val Val Gly
290 295 300
Asn Leu Phe Leu Ala Gly Met Val Thr Thr Ser Thr Thr Leu Ala
305 310 315
Trp Ala Leu Leu Leu Met Ile Leu His Pro Asp Val Gln Cys Arg
320 325 330
Val Gln Gln Glu Ile Asp Glu Val Ile Gly Gln Val Arg His Pro
335 340 345
Glu Met Ala Asp Gln Ala His Met Pro Phe Thr Asn Ala Val Ile
350 355 360
His Glu Val Gln Arg Phe Ala Asp Ile Val Pro Met Asn Leu Pro
365 370 375
His Lys Thr Ser Arg Asp Ile Glu Val Gln Gly Phe Leu Ile Pro
380 385 390
Lys Gly Thr Thr Leu Ile Pro Asn Leu Ser Ser Val Leu Lys Asp
395 400 405
Glu Thr Val Trp Glu Lys Pro Leu Arg Phe His Pro Glu His Phe
410 415 420
Leu Asp Ala Gln Gly Asn Phe Val Lys His Glu Ala Phe Met Pro
425 430 435
Phe Ser Ala Gly Arg Arg Ala Cys Leu Gly Glu Pro Leu Ala Arg
440 445 450
Met Glu Leu Phe Leu Phe Phe Thr Cys Leu Leu Gln Arg Phe Ser
455 460 465
Phe Ser Val Pro Thr Gly Gln Pro Arg Pro Ser Asp Tyr Gly Val
470 475 480
Phe Ala Phe Leu Leu Ser Pro Ser Pro Tyr Gln Leu Cys Ala Phe
485 490 495
Lys Arg
<210> 3
<211> 286
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2706492CD1
<400> 3
Met Ala Leu Asp Leu Ala Ser Leu Ala Ser Val Arg Ala Phe Ala
1 5 10 15
Thr Ala Phe Leu Ser Ser Glu Pro Arg Leu Asp Ile Leu Ile His
20 25 30
Asn Ala Gly Ile Ser Ser Cys Gly Arg Thr Arg Glu Ala Phe Asn
35 40 45
Leu Leu Leu Arg Val Asn His Ile Gly Pro Phe Leu Leu Thr His
50 55 60
Leu Leu Leu Pro Cys Leu Lys Ala Cys Ala Pro Ser Arg Val Val
65 70 75
Val Val Ala Ser Ala Ala His Cys Arg Gly Arg Leu Asp Phe Lys
80 85 90
Arg Leu Asp Arg Pro Val Val Gly Trp Arg Gln Glu Leu Arg Ala
95 100 105
3/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Tyr Ala Asp Thr Lys Leu Ala Asn Val Leu Phe Ala Arg Glu Leu
110 115 120
Ala Asn Gln Leu Glu Ala Thr Gly Val Thr Cys Tyr Ala Ala His
125 130 135
Pro Gly Pro Val Asn Ser Glu Leu Phe Leu Arg His Val Pro Gly
140 145 150
Trp Leu Arg Pro Leu Leu Arg Pro Leu Ala Trp Leu Val Leu Arg
155 160 165
Ala Pro Arg Gly Gly Ala Gln Thr Pro Leu Tyr Cys Ala Leu Gln
170 175 180
Glu Gly Ile Glu Pro Leu Ser Gly Arg Tyr Phe Ala Asn Cys His
185 290 195
Val Glu Glu Val Pro Pro Ala Ala Arg Asp Asp Arg Ala Ala His
200 205 210
Arg Leu Trp Glu Ala Ser Lys Arg Leu Ala Gly Leu Gly Pro Gly
215 220 225
Glu Asp Ala Glu Pro Asp Glu Asp Pro Gln Ser Glu Asp Ser Glu
230 235 240
Ala Pro Ser Ser Leu Ser Thr Pro His Pro Glu Glu Pro Thr Val
245 250 255
Ser Gln Pro Tyr Pro Ser Pro Gln Ser Ser Pro Asp Leu Ser Lys
260 265 270
Met Thr His Arg Ile Gln Ala Lys Val Glu Pro Glu Ile Gln Leu
275 280 285
Ser
<210> 4
<211> 469
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2766688CD1
<400> 4
Met Glu Leu IIe Ser Pro Thr Val Ile Ile Ile Leu Gly Cys Leu
1 5 10 15
Ala Leu Phe Leu Leu Leu Gln Arg Lys Asn Leu Arg Arg Pro Pro
20 25 30
Cys Ile Lys Gly Trp Ile Pro Trp Ile Gly Val Gly Phe Glu Phe
35 40 45
Gly Lys Ala Pro Leu Glu Phe Ile Glu Lys Ala Arg Ile Lys Tyr
50 55 60
GIy Pro Ile Phe Thr Val Phe Ala Met Gly Asn Arg Met Thr Phe
65 70 75
Val Thr Glu Glu Glu Gly Ile Asn Val Phe Leu Lys Ser Lys Lys
80 85 90
Val Asp Phe Glu Leu Ala Val Gln Asn Ile Val Tyr His Thr Ala
95 100 105
Ser Ile Pro Lys Asn Val Phe Leu Ala Leu His Glu Lys Leu Tyr
110 115 120
Ile Met Leu Lys Gly Lys Met Gly Thr Val Asn Leu His Gln Phe
125 130 135
Thr Gly Gln Leu Thr Glu Glu Leu His Glu Gln Leu Glu Asn Leu
140 145 150
Gly Thr His Gly Thr Met Asp Leu Asn Asn Leu Val Arg His Leu
155 160 165
Leu Tyr Pro Val Thr Val Asn Met Leu Phe Asn Lys Ser Leu Phe
170 175 180
Ser Thr Asn Lys Lys Lys Ile Lys Glu Phe His Gln Tyr Phe Gln
185 190 195
Val Tyr Asp Glu Asp Phe Glu Tyr Gly Ser Gln Leu Pro Glu Cys
4/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
200 205 210
Leu Leu Arg Asn Trp Ser Lys Ser Lys Lys Trp Phe Leu Glu Leu
215 220 225
Phe Glu Lys Asn Ile Pro Asp Ile Lys Ala Cys Lys Ser Ala Lys
230 235 240
Asp Asn Ser Met Thr Leu Leu Gln Ala Thr Leu Asp Ile Val Glu
245 250 255
Thr Glu Thr Ser Lys Glu Asn Ser Pro Asn Tyr Gly Leu Leu Leu
260 265 270
Leu Trp Ala Ser Leu Ser Asn Ala Val Pro Val Ala Phe Trp Thr
275 280 285
Leu Ala Tyr Val Leu Ser His Pro Asp Ile His Lys Ala Ile Met
290 295 300
Glu Gly Ile Ser Ser Val Phe Gly Lys Ala Gly Lys Asp Lys Ile
305 310 315
Lys Val Ser Glu Asp Asp Leu Glu Asn Leu Leu Leu Ile Lys Trp
320 325 330
Cys Val Leu Glu Thr Ile Arg Leu Lys Ala Pro Gly Val Ile Thr
335 340 345
Arg Lys Val Val Lys Pro Val Glu Ile Leu Asn Tyr Ile Ile Pro
350 355 360
Ser Gly Asp Leu Leu Met Leu Ser Pro Phe Trp Leu His Arg Asn
365 370 375
Pro Lys Tyr Phe Pro Glu Pro Glu Leu Phe Lys Pro Glu Arg Trp
380 385 390
Glu Lys Gly Lys Phe Arg Glu Ala Leu Phe Leu Gly Leu Leu His
395 400 405
Gly Ile Gly Ser Gly Lys Phe Gln Cys Pro Ala Arg Trp Phe Ala
410 415 420
Leu Leu Glu Val Gln Met Cys Ile Ile Leu Ile Leu Tyr Lys Tyr
425 430 435
Asp Cys Ser Leu Leu Asp Pro Leu Pro Lys Gln Ser Tyr Leu His
440 445 450
Leu Val Gly Val Pro Gln Pro Glu Gly Gln Cys Arg Ile Glu Tyr
455 460 465
Lys Gln Arg Ile
<210> 5
<211> 331
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2788823CD1
<400> 5
Met Ser Arg Tyr Leu Leu Pro Leu Ser Ala Leu Gly Thr Val Ala
1 5 10 25
Gly Ala Ala Val Leu Leu Lys Asp Tyr Val Thr Gly Gly Ala Cys
20 25 30
Pro Ser Lys Ala Thr Ile Pro Gly Lys Thr Val Ile Val Thr Gly
35 40 45
Ala Asn Thr Gly Ile Gly Lys Gln Thr Ala Leu Glu Leu Ala Arg
50 55 60
Arg Gly Gly Asn Ile Ile Leu Ala Cys Arg Asp Met Glu Lys Cys
65 70 75
Glu Ala Ala Ala Lys Asp Ile Arg Gly Glu Thr Leu Asn His His
80 85 90
Val Asn Ala Arg His Leu Asp Leu Ala Ser Leu Lys Ser Ile Arg
95 100 105
Glu Phe Ala Ala Lys Ile Ile Glu Glu Glu Glu Arg Val Asp Ile
110 115 120
5/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Leu Ile Asn Asn Ala Gly Val Met Arg Cys Pro His Trp Thr Thr
125 130 135
Glu Asp Gly Phe Glu Met Gln Phe Gly Val Asn His Leu Gly His
140 145 150
Phe Leu Leu Thr Asn Leu Leu Leu Asp Lys Leu Lys Ala Ser Ala
155 160 165
Pro Ser Arg Ile Ile Asn Leu Ser Ser Leu Ala His Val Ala Gly
170 175 180
His Ile Asp Phe Asp Asp Leu Asn Trp Gln Thr Arg Lys Tyr Asn
185 190 195
Thr Lys Ala Ala Tyr Cys Gln Ser Lys Leu Ala Ile Val Leu Phe
200 205 210
Thr Lys Glu Leu Ser Arg Arg Leu Gln Gly Ser Gly Val Thr Val
215 220 225
Asn Ala Leu His Pro Gly Val Ala Arg Thr Glu Leu Gly Arg His
230 235 240
Thr Gly Ile His Gly Ser Thr Phe Ser Ser Thr Thr Leu Gly Pro
245 250 255
Ile Phe Trp Leu Leu Val Lys Ser Pro Glu Leu Ala Ala Gln Pro
260 265 270
Ser Thr Tyr Leu Ala Val Ala Glu Glu Leu Ala Asp Val Ser Gly
275 280 285
Lys Tyr Phe Asp Gly Leu Lys Gln Lys Ala Pro Ala Pro Glu Ala
290 295 300
Glu Asp Glu Glu Val Ala Arg Arg Leu Trp Ala Glu Ser Ala Arg
305 310 315
Leu Val Gly Leu Glu Ala Pro Ser Val Arg Glu Gln Pro Leu Pro
320 325 330
Arg
<210> 6
<211> 509
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3348822CD1
<400> 6
Met Glu Phe Ser Trp Leu Glu Thr Arg Trp Ala Arg Pro Phe Tyr
1 5 10 15
Leu Ala Phe Val Phe Cys Leu Ala Leu Gly Leu Leu Gln Ala Ile
20 25 30
Lys Leu Tyr Leu Arg Arg Gln Arg Leu Leu Arg Asp Leu Arg Pro
35 40 45
Phe Pro Ala Pro Pro Thr His Trp Phe Leu Gly His Gln Lys Phe
50 55 60
Ile Gln Asp Asp Asn Met Glu Lys Leu Glu Glu Ile Ile Glu Lys
65 70 75
Tyr Pro Arg Ala Phe Pro Phe Trp Ile Gly Pro Phe GIn Ala Phe
80 85 90
Phe Cys Ile Tyr Asp Pro Asp Tyr Ala Lys Thr Leu Leu Ser Arg
95 100 105
Thr Asp Pro Lys Ser Gln Tyr Leu Gln Lys Phe Ser Pro Pro Leu
110 115 120
Leu Gly Lys Gly Leu Ala Ala Leu Asp Gly Pro Lys Trp Phe Gln
125 130 135
His Arg Arg Leu Leu Thr Pro Gly Phe His Phe Asn Ile Leu Lys
140 145 150
Ala Tyr Ile Glu Val Met Ala His Ser Val Lys Met Met Leu Asp
155 160 165
Lys Trp Glu Lys Ile Cys Ser Thr G1n Asp Thr Ser Val Glu Val
6/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
170 175 180
Tyr Glu His Ile Asn Ser Met Ser Leu Asp Tle Tle Met Lys Cys
185 190 195
Ala Phe Ser Lys Glu Thr Asn Cys Gln Thr Asn Ser Thr His Asp
200 205 210
Pro Tyr Ala Lys Ala Ile Phe Glu Leu Ser Lys Tle Ile Phe His
215 220 225
Arg Leu Tyr Ser Leu Leu Tyr His Ser Asp Ile Ile Phe Lys Leu
230 235 240
Ser Pro Gln Gly Tyr Arg Phe Gln Lys Leu Ser Arg Val Leu Asn
245 250 255
Gln Tyr Thr Asp Thr Ile Tle Gln Glu Arg Lys Lys Ser Leu Gln
260 265 270
Ala Gly Val Lys Gln Asp Asn Thr Pro Lys Arg Lys Tyr Gln Asp
275 280 285
Phe Leu Asp Ile Val Leu Ser Ala Lys Asp Glu Ser Gly Ser Ser
290 295 300
Phe Ser Asp Ile Asp Val His Ser Glu Val Ser Thr Phe Leu Leu
305 310 315
Ala Gly His Asp Thr Leu Ala Ala Ser Ile Ser Trp Ile Leu Tyr
320 325 330
Cys Leu Ala Leu Asn Pro Glu His Gln Glu Arg Cys Arg Glu Glu
335 340 345
Val Arg Gly Ile Leu Gly Asp Gly Ser Ser Ile Thr Trp Asp Gln
350 355 360
Leu Gly Glu Met Ser Tyr Thr Thr Met Cys Ile Lys Glu Thr Cys
365 370 375
Arg Leu Ile Pro Ala Val Pro Ser Ile Ser Arg Asp Leu Ser Lys
380 385 390
Pro Leu Thr Phe Pro Asp Gly Cys Thr Leu Pro Ala Gly Ile Thr
395 400 405
Val Val Leu Ser Ile Trp Gly Leu His His Asn Pro Ala Val Trp
410 415 420
Lys Asn Pro Lys Val Phe Asp Pro Leu Arg Phe Ser Gln Glu Asn
425 430 435
Ser Asp Gln Arg His Pro Tyr Ala Tyr Leu Pro Phe Ser A1a Gly
440 445 450
Ser Arg Asn Cys Ile Gly Gln Glu Phe Ala Met Tle Glu Leu Lys
455 460 465
Val Thr Ile Ala Leu Ile Leu Leu His Phe Arg Val Thr Pro Asp
470 475 480
Pro Thr Arg Pro Leu Thr Phe Pro Asn His Phe Ile Leu Lys Pro
485 490 495
Lys Asn Gly Met Tyr Leu His Leu Lys Lys Leu Ser Glu Cys
500 505
<210> 7
<211> 433
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4290251CD1
<400> 7
Met Ala Gly Thr Asn Ala Leu Leu Met Leu Glu Asn Phe Ile Asp
1 5 10 15
Gly Lys Phe Leu Pro Cys Ser Ser Tyr Ile Asp Ser Tyr Asp Pro
20 25 30
Ser Thr Gly Glu Val Tyr Cys Arg Val Pro Asn Ser Gly Lys Asp
35 40 45
Glu Ile Glu Ala Ala Val Lys Ala Ala Arg Glu Ala Phe Pro Ser
50 55 60
7139
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Trp Ser Ser Arg Ser Pro Gln Glu Arg Ser Arg Val Leu Asn Gln
65 70 75
Val Ala Asp Leu Leu Glu Gln Ser Leu Glu Glu Phe Ala Gln Ala
80 85 90
Glu Ser Lys Asp Gln Gly Lys Thr Leu Ala Leu Ala Arg Thr Met
95 100 105
Asp Ile Pro Arg Ser Val Gln Asn Phe Arg Phe Phe Ala Ser Ser
110 115 120
Ser Leu His His Thr Ser Glu Cys Thr Gln Met Glu His Leu Gly
125 130 l35
Cys Met His Tyr Thr Val Arg Ala Pro Val Gly Val Ala Gly Leu
140 145 150
Ile Ser Pro Trp Asn Leu Pro Leu Tyr Leu Leu Thr Trp Lys Ile
155 160 165
Ala Pro Ala Met Ala Ala Gly Asn Thr Val Ile Ala Lys Pro Ser
170 175 180
Glu Leu Thr Ser Val Thr Ala Trp Met Leu Cys Lys Leu Leu Asp
185 190 195
Lys Ala Gly Val Pro Pro Gly Val Val Asn Ile Val Phe Gly Thr
200 205 210
Gly Pro Arg Val Gly Glu Ala Leu Val Ser His Pro Glu Val Pro
215 220 225
Leu Ile Ser Phe Thr Gly Ser Gln Pro Thr Ala Glu Arg Ile Thr
230 235 240
Gln Leu Ser Ala Pro His Cys Lys Lys Leu Ser Leu Glu Leu Gly
245 250 255
Gly Lys Asn Pro Ala Ile Ile Phe Glu Asp Ala Asn Leu Asp Glu
260 265 270
Cys Ile Pro Ala Thr Val Arg Ser Ser Phe Ala Asn Gln Val Arg
275 280 285
Ser Tyr Val Lys Arg Ala Leu Ala Glu Ser Ala Gln Ile Trp Cys
290 295 300
Gly Glu Gly Val Asp Lys Leu Ser Leu Pro Ala Arg Asn Gln Ala
305 310 315
Gly Tyr Phe Met Leu Pro Thr Val Ile Thr Asp Ile Lys Asp Glu
320 325 330
Ser Cys Cys Met Thr Glu Glu Ile Phe Gly Pro Val Thr Cys Val
335 340 345
Val Pro Phe Asp Ser Glu Glu Glu Val Ile Glu Arg Ala Asn Asn
350 355 360
Val Lys Tyr Gly Leu Ala Ala Thr Val Trp Ser Ser Asn Val Gly
365 370 375
Arg Val His Arg Val Ala Lys Lys Leu Gln Ser Gly Leu Val Trp
380 385 390
Thr Asn Cys Trp Leu Ile Arg Glu Leu Asn Leu Pro Phe Gly Gly
395 400 405
Met Lys Ser Ser Gly Ile Gly Arg Glu Gly Ala Lys Asp Ser Tyr
410 415 420
Asp Phe Phe Thr Glu Ile Lys Thr Ile Thr Val Lys His
425 ~ 430
<210> 8
<211> 186
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4904188CD1
<400> 8
Met Lys Thr Glu Asp Gly Phe Glu Met Gln Phe Gly Val Asn His
l 5 10 15
Leu Gly His Phe Leu Leu Thr Asn Leu Leu Leu Gly Leu Leu Lys
8139
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
20 25 30
Ser Ser Ala Pro Ser Arg I1e Val Val Val Ser Ser Lys Leu Tyr
35 40 45
Lys Tyr Gly Asp Ile Asn Phe Asp Asp Leu Asn Ser Glu Gln Ser
50 55 60
Tyr Asn Lys Ser Phe Cys Tyr Ser Arg Ser Lys Leu Ala Asn Ile
65 70 75
Leu Phe Thr Arg Glu Leu Ala Arg Arg Leu Glu Gly Thr Asn Val
80 85 90
Thr Val Asn Val Leu His Pro Gly Ile Val Arg Thr Asn Leu Gly
95 100 105
Arg His Ile His Ile Pro Leu Leu Val Lys Pro Leu Phe Asn Leu
110 115 120
Val Ser Trp Ala Phe Phe Lys Thr Pro Val Glu Gly Ala Gln Thr
125 130 135
Ser Ile Tyr Leu Ala Ser Ser Pro Glu Val Glu Gly Val Ser Gly
140 145 150
Arg Tyr Phe Gly Asp Cys Lys Glu Glu Glu Leu Leu Pro Lys Ala
155 160 165
Met Asp Glu Ser Val Ala Arg Lys Leu Trp Asp Ile Ser Glu Val
170 175 180
Met Val Gly Leu Leu Lys
185
<210> 9
<211> 304
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 638419CD1
<400> 9
Met Ala Lys Ile Glu Lys Asn Ala Pro Thr Met Glu Lys Lys Pro
1 5 10 15
Glu Leu Phe Asn Ile Met Glu Val Asp Gly Val Pro Thr Leu Ile
20 25 30
Leu Ser Lys Glu Trp Trp Glu Lys Val Cys Asn Phe Gln Ala Lys
35 40 45
Pro Asp Asp Leu Tle Leu Ala Thr Tyr Pro Lys Ser Gly Thr Thr
50 55 60
Trp Met His Glu Tle Leu Asp Met Ile Leu Asn Asp Gly Asp Val
65 70 75
Glu Lys Cys Lys Arg Ala Gln Thr Leu Asp Arg His Ala Phe Leu
80 85 90
Glu Leu Lys Phe Pro His Lys Glu Lys Pro Asp Leu Glu Phe Val
95 100 105
Leu Glu Met Ser Ser Pro Gln Leu Ile Lys Thr His Leu Pro Ser
110 115 120
His Leu Ile Pro Pro Ser Ile Trp Lys Glu Asn Cys Lys Ile Val
125 130 135
Tyr Val Ala Arg Asn Pro Lys Asp Cys Leu Val Ser Tyr Tyr His
140 145 150
Phe His Arg Met Ala Ser Phe Met Pro Asp Pro Gln Asn Leu Glu
155 160 165
Glu Phe Tyr Glu Lys Phe Met Ser Gly Lys Val Val Gly Arg Ser
170 175 180
Trp Phe Asp His Val Lys Gly Trp Trp Ala Ala Lys Asp Thr His
185 190 195
Arg Ile Leu Tyr Leu Phe Tyr Glu Asp Ile Lys Lys Asn Pro Lys
200 205 210
His Glu Ile His Lys Val Leu Glu Phe Leu Glu Lys Thr Leu Ser
215 220 225
9/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Gly Asp Val Tle Asn Lys Ile Val His His Thr Ser Phe Asp Val
230 235 . 240
Met Lys Asp Asn Pro Met Ala Asn His Thr Ala Val Pro Ala His
245 250 255
Ile Phe Asn His Ser Ile Ser Lys Phe Met Arg Lys Gly Met Pro
260 265 270
Gly Asp Trp Lys Asn His Phe Thr Val Ala Met Asn Glu Asn Phe
275 280 285
Asp Lys His Tyr Glu Lys Lys Met Ala Gly Ser Thr Leu Asn Phe
290 295 300
Cys Leu Glu Ile
<210> 10
<211> 629
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1844394CD1
<400> 10
Met Lys Leu Gln Asn Leu Phe Val Asp Asp Ser Gly Arg Tyr Leu
1 5 10 15
Ala Ile Gln Phe His Leu Glu Cys Ala Tyr Val Phe Leu Tyr Tyr
20 25 30
Tyr Glu Tyr Arg Lys Ala Lys Asp Gln Leu Asp Ile Ala Lys Asp
35 40 45
Ile Ser Gln Leu Gln Ile Asp Leu Thr Gly Ala Leu Gly Lys Arg
50 55 60
Thr Arg Phe Gln Glu Asn Tyr Val Ala Gln Leu Ile Leu Asp Val
65 70 75
Arg Arg Glu Gly Asp Val Leu Ser Asn Cys Glu Phe Thr Pro Ala
80 85 90
Pro Thr Pro Gln Glu His Leu Thr Lys Asn Leu Glu Leu Asn Asp
95 100 105
Asp Thr Ile Leu Asn Asp Ile Lys Leu Ala Asp Cys Glu Gln Phe
110 115 120
Gln Met Pro Asp Leu Cys Ala Glu Glu Ile Ala Ile Ile Leu Gly
125 130 135
Ile Cys Thr Asn Phe Gln Lys Asn Asn Pro Val His Thr Leu Thr
140 145 150
Glu Val Glu Leu Leu Ala Phe Thr Ser Cys Leu Leu Ser Gln Pro
155 160 165
Lys Phe Trp Ala Ile Gln Thr Ser Ala Leu Ile Leu Arg Thr Lys
170 175 180
Leu Glu Lys Gly Ser Thr Arg Arg Val Glu Arg Ala Met Arg Gln
185 190 195
Thr Gln Ala Leu Ala Asp Gln Phe Glu Asp Lys Thr Thr Ser Val
200 205 210
Leu Glu Arg Leu Lys Ile Phe Tyr Cys Cys Gln Val Pro Pro His
215 220 225
Trp Ala Ile Gln Arg Gln Leu Ala Ser Leu Leu Phe Glu Leu Gly
230 235 240
Cys Thr Ser Ser Ala Leu Gln Ile Phe Glu Lys Leu Glu Met Trp
245 250 255
Glu Asp Val Val Ile Cys Tyr Glu Arg Ala Gly Gln His Gly Lys
260 265 270
Ala Glu Glu Ile Leu Arg Gln Glu Leu Glu Lys Lys Glu Thr Pro
275 , 280 285
Ser Leu Tyr Cys Leu Leu Gly Asp Val Leu Gly Asp His Ser Cys
290 295 300
Tyr Asp Lys Ala Trp Glu Leu Ser Arg Tyr Arg Ser Ala Arg Ala
10/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
305 310 315
Gln Arg Ser Lys Ala Leu Leu His Leu Arg Asn Lys Glu Phe Gln
320 325 330
Glu Cys Val Glu Cys Phe Glu Arg Ser Val Lys Ile Asn Pro Met
335 340 345
Gln Leu Gly Val Trp Phe Ser Leu Gly Cys Ala Tyr Leu Ala Leu
350 355 360
Glu Asp Tyr Gln Gly Ser Ala Lys Ala Phe Gln Arg Cys Val Thr
365 370 375
Leu Glu Pro Asp Asn Ala Glu Ala Trp Asn Asn Leu Ser Thr Ser
380 385 390
Tyr Ile Arg Leu Lys Gln Lys Val Lys Ala Phe Arg Thr Leu Gln
395 400 405
Glu Ala Leu Lys Cys Asn Tyr Glu His Trp Gln Ile Trp Lys Asn
410 415 420
Tyr Ile Leu Thr Ser Thr Asp Val Gly Glu Phe Ser Glu Ala Ile
425 430 435
Lys Ala Tyr His Arg Leu Leu Asp Leu Arg Asp Lys Tyr Lys Asp
440 445 450
Val Gln Val Leu Lys Ile Leu Val Arg Ala Val Ile Asp Gly Met
455 460 465
Thr Asp Arg Ser Gly Asp Val Ala Thr Gly Leu Lys Gly Lys Leu
470 475 480
Gln Glu Leu Phe Gly Arg Val Thr Ser Arg Val Thr Asn Asp Gly
485 490 495
Glu Ile Trp Arg Leu Tyr Ala His Val Tyr Gly Asn Gly Gln Ser
500 505 510
Glu Lys Pro Asp Glu Asn Glu Lys Ala Phe Gln Cys Leu Ser Lys
515 520 525
Ala Tyr Lys Cys Asp Thr Gln Ser Asn Cys Trp Glu Lys Asp Ile
530 535 540
Thr Ser Phe Lys Glu Val Val Gln Arg Ala Leu Gly Leu Ala His
545 550 555
Val Ala Ile Lys Cys Ser Lys Asn Lys Ser Ser Ser Gln Glu Ala
560 565 570
Val Gln Met Leu Ser Ser Val Arg Leu Asn Leu Arg Gly Leu Leu
575 580 585
Ser Lys Ala Lys Gln Leu Phe Thr Asp Val Ala Thr Gly Glu Met
590 595 600
Ser Arg Glu Leu Ala Asp Asp Ile Thr Ala Met Asp Thr Leu Val
605 610 615
Thr Glu Leu Gln Asp Leu Ser Asn Gln Phe Arg Asn Gln Tyr
620 625
<210> 11
<211> 320
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2613056CD1
<400> 11
Met Thr Leu Asp Ser Ile Met Lys Cys Ala Phe Ser His Gln Gly
1 5 10 15
Ser Ile Gln Leu Asp Ser Thr Leu Asp Ser Tyr Leu Lys Ala Val
20 25 30
Phe Asn Leu Ser Lys Ile Ser Asn Gln Arg Met Asn Asn Phe Leu
35 40 45
His His Asn Asp Leu Val Phe Lys Phe Ser Ser Gln Gly Gln Ile
50 55 60
Phe Ser Lys Phe Asn Gln Glu Leu His Gln Phe Thr Glu Lys Val
65 70 75
11/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Ile Gln Asp Arg Lys Glu Ser Leu Lys Asp Lys Leu Lys Gln Asp
80 85 90
Thr Thr Gln Lys Arg Arg Trp Asp Phe Leu Asp Ile Leu Leu Ser
95 100 105
Ala Lys Ser Glu Asn Thr Lys Asp Phe Ser Glu Ala Asp Leu Gln
110 115 120
Ala Glu Va1 Lys Thr Phe Met Phe Ala Gly His Asp Thr Thr Ser
125 130 135
Ser Ala Ile Ser Trp Ile Leu Tyr Cys Leu Ala Lys Tyr Pro Glu
140 145 150
His Gln Gln Arg Cys Arg Asp Glu Ile Arg Glu Leu Leu Gly Asp
155 160 165
Gly Ser Ser Ile Thr Trp Glu His Leu Ser Gln Met Pro Tyr Thr
170 175 180
Thr Met Cys Ile Lys Glu Cys Leu Arg Leu Tyr Ala Pro Val Val
185 190 295
Asn Ile Ser Arg Leu Leu Asp Lys Pro Ile Thr Phe Pro Asp Gly
200 205 210
Arg Ser Leu Pro Ala Gly Ile Thr Val Phe Ile Asn Ile Trp Ala
215 220 225
Leu His His Asn Pro Tyr Phe Trp Glu Asp Pro Gln Val Phe Asn
230 235 ~ 240
Pro Leu Arg Phe Ser Arg Glu Asn Ser Glu Lys Ile His Pro Tyr
245 250 255
Ala Phe Ile Pro Phe Ser Ala Gly Leu Arg Asn Cys Ile Gly Gln
260 265 270
His Phe Ala Ile Ile Glu Cys Lys Val Ala Val Ala Leu Thr Leu
275 280 285
Leu Arg Phe Lys Leu Ala Pro Asp His Ser Arg Pro Pro Gln Pro
290 295 300
Val Arg Gln Val Val Leu Lys Ser Lys Asn Gly Ile His Val Phe
305 310 315
Ala Lys Lys Val Cys
320
<210> 12
<211> 56
<212> PRT
<213> Homo sapiens i
<220>
<221> misc_feature
<223> Incyte ID No: 5453617CD1
<400> 12
Met Ser Gly Cys Pro Asn Cys Val Trp Val Glu Tyr Ala Asp Arg
1 5 l0 15
Leu Leu Gln His Phe Gln Asp Gly Gly Glu Arg Ala Leu Ala Ala
20 25 30
Leu Glu Glu His Val Ala Asp Glu Asn Leu Lys Ala Phe Leu Arg
35 40 45
Met Glu Ile Arg Leu His Thr Arg Cys Gly Gly
50 55
<210> 13
<221> 377
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5483256CD1
<400> 13
12/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Met Asp Pro Ala Ala Arg Val Val Arg Ala Leu Trp Pro G1y Gly
1 5 10 15
Cys Ala Leu Ala Trp Arg Leu Gly Gly Arg Pro Gln Pro Leu Leu
20 25 30
Pro Thr Gln Ser Arg Ala Gly Phe Ala Gly Ala Ala Gly Gly Pro
35 40 45
Ser Pro Val Ala Ala A1a Arg Lys Gly Ser Pro Arg Leu Leu Gly
50 55 60
Ala Ala Ala Leu Ala Leu Gly Gly Ala Leu Gly Leu Tyr His Thr
65 70 75
Ala Arg Trp His Leu Arg Ala Gln Asp Leu His Ala Glu Arg Ser
80 85 90
Ala Ala Gln Leu Ser Leu Ser Ser Arg Leu Gln Leu Thr Leu Tyr
95 100 105
Gln Tyr Lys Thr Cys Pro Phe Cys Ser Lys Val Arg Ala Phe Leu
110 115 120
Asp Phe His Ala Leu Pro Tyr Gln Val Val Glu Val Asn Pro Val
125 130 135
Arg Arg Ala Glu Ile Lys Phe Ser Ser Tyr Arg Lys Val Pro Ile
140 145 150
Leu Val Ala Gln Glu Gly Glu Ser Ser Gln Gln Leu Asn Asp Ser
155 160 165
Ser Val Ile Ile Ser Ala Leu Lys Thr Tyr Leu Val Ser Gly Gln
170 175 180
Pro Leu Glu Glu Ile Ile Thr Tyr Tyr Pro Ala Met Lys Ala Val
185 190 195
Asn Glu Gln Gly Lys Glu Val Thr Glu Phe Gly Asn Lys Tyr Trp
200 205 210
Leu Met Leu Asn Glu~Lys Glu Ala Gln Gln Val Tyr Gly Gly Lys
215 220 225
Glu Ala Arg Thr Glu Glu Met Lys Trp Arg Gln Trp Ala Asp Asp
230 235 240
Trp Leu Val His Leu Ile Ser Pro Asn Val Tyr Arg Thr Pro Thr
245 250 255
Glu Ala Leu Ala Ser Phe Asp Tyr Ile Val Arg Glu Gly Lys Phe
260 265 270
Gly Ala Val Glu Gly Ala Val Ala Lys Tyr Met Gly Ala Ala Ala
275 280 285
Met Tyr Leu Ile Ser Lys Arg Leu Lys Ser Arg His Arg Leu Gln
290 295 300
Asp Asn Val Arg Glu Asp Leu Tyr Glu Ala Ala Asp Lys Trp Val
305 310 315
Ala Ala Val Gly Lys Asp Arg Pro Phe Met Gly Gly Gln Lys Pro
320 325 330
Asn Leu Ala Asp Leu Ala Val Tyr Gly Val Leu Arg Val Met Glu
335 340 345
Gly Leu Asp Ala Phe Asp Asp Leu Met Gln His Thr His Ile Gln
350 355 360
Pro Trp Tyr Leu Arg Val Glu Arg Ala Ile Thr Glu Ala Ser Pro
365 370 375
Ala His
<210> 14
<211> 501
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5741354CD1
<400> 14
Met Trp Lys Leu Trp Arg Ala Glu Glu Gly Ala Ala Ala Leu Gly
13/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
1 5 10 15
Gly Ala Leu Phe Leu Leu Leu Phe Ala Leu Gly Val Arg Gln Leu
20 25 30
Leu Lys Gln Arg Arg Pro Met Gly Phe Pro Pro Gly Pro Pro Gly
35 40 45
Leu Pro Phe Ile Gly Asn Ile Tyr Ser Leu Ala Ala Ser Ser Glu
50 55 60
Leu Pro His Val Tyr Met Arg Lys Gln Ser Gln Val Tyr Gly Glu
65 70 75
Ile Phe Ser Leu Asp Leu Gly Gly Ile Ser Thr Val Val Leu Asn
80 85 90
Gly Tyr Asp Val Val Lys Glu Cys Leu Val His Gln Ser Glu Ile
95 100 105
Phe Ala Asp Arg Pro Cys Leu Pro Leu Phe Met Lys Met Thr Lys
110 115 120
Met Gly Gly Leu Leu Asn Ser Arg Tyr Gly Arg Gly Trp Val Asp
125 130 135
His Arg Arg Leu Ala Val Asn Ser Phe Arg Tyr Phe Gly Tyr Gly
140 145 150
Gln Lys Ser Phe Glu Ser Lys Ile Leu Glu Glu Thr Lys Phe Phe
155 160 165
Asn Asp Ala Ile Glu Thr Tyr Lys Gly Arg Pro Phe Asp Phe Lys
170 175 180
Gln Leu Ile Thr Asn Ala Val Ser Asn Ile Thr Asn Leu Ile Ile
185 190 195
Phe Gly Glu Arg Phe Thr Tyr Glu Asp Thr Asp Phe Gln His Met
200 205 210
Ile Glu Leu Phe Ser Glu Asn Val Glu Leu Ala Ala Ser Ala Ser
215 220 225
Val Phe Leu Tyr Asn Ala Phe Pro Trp Ile Gly Ile Leu Pro Phe
230 235 240
Gly Lys His Gln Gln Leu Phe Arg Asn Ala Ala Val Val Tyr Asp
245 250 255
Phe Leu Ser Arg Leu Ile Glu Lys Ala Ser Val Asn Arg Lys Pro
260 265 270
Gln Leu Pro Gln His Phe Val Asp Ala Tyr Leu Asp Glu Met Asp
275 280 285
Gln Gly Lys Asn Asp Pro Ser Ser Thr Phe Ser Lys G1u Asn Leu
290 295 300
Ile Phe Ser Val Gly Glu Leu Ile Ile Ala Gly Thr Glu Thr Thr
305 310 315
Thr Asn Val Leu Arg Trp Ala Ile Leu Phe Met Ala Leu Tyr Pro
320 325 330
Asn Ile Gln Gly Gln Val Gln Lys Glu Ile Asp Leu Ile Met Gly
335 340 345
Pro Asn Gly Lys Pro Ser Trp Asp Asp Lys Cys Lys Met Pro Tyr
350 355 360
Thr Glu Ala Val Leu His Glu Val Leu Arg Phe Cys Asn Ile Val
365 370 375
Pro Leu Gly Ile Phe His Ala Thr Ser Glu Asp Ala Val Val Arg
380 385 390
Gly Tyr Ser Ile Pro Lys Gly Thr Thr Val Ile Thr Asn Leu Tyr
395 400 405
Ser Val His Phe Asp Glu Lys Tyr Trp Arg Asp Pro Glu Val Phe
410 415 420
His Pro Glu Arg Phe Leu Asp Ser Ser Gly Tyr Phe Ala Lys Lys
425 430 435
Glu Ala Leu Val Pro Phe Ser Leu Gly Arg Arg His Cys Leu Gly
440 445 450
Glu His Leu Ala Arg Met Glu Met Phe Leu Phe Phe Thr Ala Leu
455 460 465
Leu Gln Arg Phe His Leu His Phe Pro His Glu Leu Val Pro Asp
470 475 480
Leu Lys Pro Arg Leu Gly Met Thr Leu Gln Pro Gln Pro Tyr Leu
14/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
485 490 495
Ile Cys Ala Glu Arg Arg
500
<210> 15
<211> 144
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5872615CD1
<400> 15
Met Arg Lys Ile Asp Leu Cys Leu Ser Ser Glu Gly Ser Glu Val
1 5 10 15
Ile Leu Ala Thr Ser Ser Asp Glu Lys His Pro Pro Glu Asn Ile
20 25 ~30
Ile Asp Gly Asn Pro Glu Thr Phe Trp Thr Thr Thr Gly Met Phe
35 40 45
Pro Gln Glu Phe Ile Ile Cys Phe His Lys His Val Arg Ile Glu
50 55 60
Arg Leu Val Ile Gln Ser Tyr Phe Val Gln Thr Leu Lys Ile Glu
65 70 75
Lys Ser Thr Ser Lys Glu Pro Val Asp Phe Glu Gln Trp Ile Glu
80 85 90
Lys Asp Leu Val His Thr Glu Gly Gln Leu Gln Asn Glu Glu Ile
95 100 105
Val Ala His Asp Gly Ser Ala Thr Tyr Leu Arg Phe Ile Ile Val
110 215 120
Ser Ala Phe Asp His Phe Ala Ser Val His Ser Val Ser Ala Glu
125 130 135
Gly Thr Val Val Ser Asn Leu Ser Ser
140
<210> 16
<211> 218
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2657543CD1
<400> 16
Met Leu Ser Thr Phe Ala Arg Gln Asn Asp Ile Pro Phe Gln Leu
1 5 10 15
Gln Thr Val Glu Leu Ala Trp Gly Glu His Leu Lys Pro Glu Phe
20 25 30
Leu Lys Val Asn Pro Leu Gly Lys Val Pro Ala Leu Arg Asp Gly
35 40 45
Asp Phe Leu Leu Ala Glu Arg Leu Glu Lys Arg Ser Leu Thr Pro
50 55 60
Pro Ala His Ser Met Val Ile Val Leu Tyr Leu Ser Arg Lys Tyr
65 70 75
Gln Ile Arg Gly His Trp Tyr Pro Pro Glu Leu Gln Ala Arg Thr
80 85 90
Cys Val Asp Glu Tyr Leu Ala Trp Lys His Val Thr Ile Gln Leu
95 100 105
Pro Ala Thr Asn Val Tyr Leu Cys Lys Pro Ala Asp Ala Ala Gln
110 115 120
Leu Glu Arg Leu Leu GIy Arg Leu Thr Pro Ala Leu Gln His Leu
125 130 135
Asp Gly Gly Val Leu Val Ala Arg Pro Phe Leu Ala Met Glu Gln
15/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
140 145 150
Ile Ser Leu Glu Asp Leu Val Leu Thr Glu Val Met Gln Val Lys
155 160 165
Leu Ser Tyr Pro Pro Ala Leu Gly Gly Thr Leu Gly Met GIy Leu
170 175 180
Ser Pro Asn Pro Ser Cys Pro Val Phe Pro Ala His Cys Arg Trp
185 190 195
Leu Arg Pro Leu Pro Arg Leu Ala Leu Ala Gly Ser Val Thr Gly
200 205 210
Pro Tyr Glu Gly Cys Pro Trp Tyr
215
<210> 17
<211> 210
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3041639CD1
<400> 17
Met Ala Cys Ile Leu Lys Arg Lys Ser Val Ile Ala Val Ser Phe
l 5 10 15
Ile Ala Ala Phe Leu Phe Leu Leu Val Val Arg Leu Val Asn Glu
20 25 30
VaI Asn Phe Pro Leu Leu Leu Asn Cys Phe Gly GIn Pro Gly Thr
35 40 45
Lys Trp Ile Pro Phe Ser Tyr Thr Tyr Arg Arg Pro Leu Arg Thr
50 55 60
His Tyr Gly Tyr Ile Asn Val Lys Thr Gln Glu Pro Leu Gln Leu
65 70 75
Asp Cys Asp Leu Cys Ala Ile Val Ser Asn Ser Gly Gln Met Val
80 85 90
Gly Gln Lys Val Gly Asn Glu Ile Asp Arg Ser Ser Cys Ile Trp
95 100 105
Arg Met Asn Asn Ala Pro Thr Lys Gly Tyr Glu Glu Asp Val Gly
110 115 120
Arg Met Thr Met Ile Arg Val Val Ser His Thr Ser Val Pro Leu
125 130 135
Leu Leu Lys Asn Pro Asp Tyr Phe Phe Lys Glu Ala Asn Thr Thr
140 145 150
Ile Tyr Val Ile Trp Gly Pro Phe Arg Asn Met Arg Lys Asp Gly
155 160 165
Asn Gly Ile Val Tyr Asn Met Leu Lys Lys Thr Val Gly Ile Tyr
170 175 180
Pro Asn Ala Gln Ile Tyr Val Thr Thr Glu Lys Arg Met Ser Tyr
185 190 195
Cys Asp Gly Val Phe Lys Lys Glu Thr Gly Lys Asp Ser Thr Glu
200 205 220
<210> 18
<211> 613
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3595451CD1
<400> 18
Met Cys Cys Trp Pro Leu Leu Leu Leu Trp Gly Leu Leu Pro Gly
1 5 10 15
16/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Thr Ala Ala Gly Gly Ser Gly Arg Thr Tyr Pro His Arg Thr Leu
20 25 30
Leu Asp Ser Glu Gly Lys Tyr Trp Leu Gly Trp Ser Gln Arg Gly
35 40 45
Ser Gln Ile Ala Phe Arg Leu Gln Val Arg Thr Ala Gly Tyr Val
50 55 60
Gly Phe Gly Phe Ser Pro Thr Gly Ala Met Ala Ser Ala Asp Ile
65 70 75
Val Val Gly Gly Val Ala His Gly Arg Pro Tyr Leu Gln Asp Tyr
80 85 90
Phe Thr Asn Ala Asn Arg Glu Leu Lys Lys Asp Ala Gln Gln Asp
95 100 105
Tyr His Leu Glu Tyr Ala Met Glu Asn Ser Thr His Thr Ile Ile
110 115 120
Glu Phe Thr Arg Glu Leu His Thr Cys Asp Ile Asn Asp Lys Ser
125 130 135
Ile Thr Asp Ser Thr Val Arg Val Ile Trp Ala Tyr His His Glu
140 145 150
Asp Ala Gly Glu Ala Gly Pro Lys Tyr His Asp Ser Asn Arg Gly
155 160 165
Thr Lys Ser Leu Arg Leu Leu Asn Pro Glu Lys Thr Ser Val Leu
170 175 180
Ser Thr Ala Leu Pro Tyr Phe Asp Leu Val Asn Gln Asp Val Pro
185 190 195
Ile Pro Asn Lys Asp Thr Thr Tyr Trp Cys Gln Met Phe Lys Ile
200 205 210
Pro Val Phe Gln Glu Lys His His Val Ile Lys Val Glu Pro Val
215 220 225
Ile Gln Arg Gly His Glu Ser Leu Val His His Ile Leu Leu Tyr
230 235 240
Gln Cys Ser Asn Asn Phe Asn Asp Ser Val Leu Glu Ser Gly His
245 250 255
Glu Cys Tyr His Pro Asn Met Pro Asp Ala Phe Leu Thr Cys Glu
260 265 270
Thr Val Ile Phe Ala Trp Ala Ile Gly Gly Glu Gly Phe Ser Tyr
275 280 285
Pro Pro His Val Gly Leu Ser Leu Gly Thr Pro Leu Asp Pro His
290 295 300
Tyr Val Leu Leu Glu Val His Tyr Asp Asn Pro Thr Tyr Glu GIu
305 310 315
Gly Leu Ile Asp Asn Ser Gly Leu Arg Leu Phe Tyr Thr Met Asp
320 325 330
Ile Arg Lys Tyr Asp Ala Gly Val Ile Glu Ala Gly Leu Trp Val
335 340 345
Ser Leu Phe His Thr Ile Pro Pro Gly Met Pro Glu Phe Gln Ser
350 355 360
Glu Gly His Cys Thr Leu Glu Cys Leu Glu Glu Ala Leu Glu Ala
365 370 375
Glu Lys Pro Ser Gly Ile His Val Phe Ala Val Leu Leu His Ala
380 385 390
His Leu AIa Gly Arg Gly IIe Arg Leu Arg His Phe Arg Lys Gly
395 400 405
Lys Glu Met Lys Leu Leu Ala Tyr Asp Asp Asp Phe Asp Phe Asn
410 415 420
Phe Gln Glu Phe Gln Tyr Leu Lys Glu Glu Gln Thr Ile Leu Pro
425 430 435
Gly Asp Asn Leu Ile Thr Glu Cys Arg Tyr Asn Thr Lys Asp Arg
440 445 450
Ala Glu Met Thr Trp Gly Gly Leu Ser Thr Arg Ser Glu Met Cys
455 460 465
Leu Ser Tyr Leu Leu Tyr Tyr Pro Arg Ile Asn Leu Thr Arg Cys
470 475 480
Ala Ser Ile Pro Asp Ile Met Glu Gln Leu Gln Phe Ile Gly Val
485 490 ,495
17!39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Lys Glu Ile Tyr Arg Pro Val Thr Thr Trp Pro Phe Ile Ile Lys
500 505 510
Ser Pro Lys Gln Tyr Lys Asn Leu Ser Phe Met Asp Ala Met Asn
515 520 525
Lys Phe Lys Trp Thr Lys Lys Glu Gly Leu Ser Phe Asn Lys Leu
530 535 540
Val Leu Sex Leu Pro Val Asn Val Arg Cys Ser Lys Thr Asp Asn
545 550 555
Ala Glu Trp Ser Ile Gln Gly Met Thr Ala Leu Pro Pro Asp Ile
560 565 570
Glu Arg Pro Tyr Lys Ala Glu Pro Leu Val Cys Gly Thr Ser Ser
575 580 585
Ser Ser Ser Leu His Arg Asp Phe Ser Ile Asn Leu Leu Val Cys
590 595 600
Leu Leu Leu Leu Ser Cys Thr Leu Ser Thr Lys Ser Leu
605 610
<210> 19
<211> 741
<212> PRT
<213> Homo'sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4169101CD1
<400> 19
Met Ala Val Leu Asp Thr Asp Leu Asp His Ile Leu Pro Ser Ser
1 5 10 15
Val Leu Pro Pro Phe Trp Ala Lys Leu Val Val Gly Ser Val Ala
20 25 30
Ile Val Cys Phe Ala Arg Ser Tyr Asp Gly Asp Phe Val Phe Asp
35 40 45
Asp Ser Glu Ala Ile Va1 Asn Asn Lys Asp Leu Gln Ala Glu Thr
50 55 60
Pro Leu Gly Asp Leu Trp His His Asp Phe Trp Gly Ser Arg Leu
65 70 75
Ser Ser Asn Thr Ser His Lys Ser Tyr Arg Pro Leu Thr Val Leu
80 85 90
Thr Phe Arg Ile Asn Tyr Tyr Leu Ser Gly Gly Phe His Pro Val
95 100 105
Gly Phe His Val VaI Asn Ile Leu Leu His Ser Gly Ile Ser Val
110 115 120
Leu Met Val Asp Val Phe Ser VaI Leu Phe Gly Gly Leu Gln Tyr
125 130 135
Thr Ser Lys Gly Arg Arg Leu His Leu AIa Pro Arg Ala Ser Leu
140 145 150
Leu Ala Ala Leu Leu Phe Ala Val His Pro Val His Thr Glu Cys
155 260 165
Val Ala Gly Val Val Gly Arg Ala Asp Leu Leu Cys Ala Leu Phe
270 175 180
Phe Leu Leu Ser Phe Leu Gly Tyr Cys Lys Ala Phe Arg Glu Ser
185 190 195
Asn Lys Glu Gly Ala His Ser Ser Thr Phe Trp Val Leu Leu Ser
200 205 210
Ile Phe Leu Gly Ala Val Ala Met Leu Cys Lys Glu Gln Gly Ile
215 220 225
Thr Val Leu Gly Leu Asn Ala Val Phe Asp Ile Leu Val Ile Gly
230 235 240
Lys Phe Asn Val Leu Glu Ile Val Gln Lys Val Leu His Lys Asp
245 250 255
Lys Ser Leu Glu Asn Leu Gly Met Leu Arg Asn Gly Gly Leu Leu
260 265 270
Phe Arg Met Thr Leu Leu Thr Ser Gly Gly A1a Gly Met Leu Tyr
18/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
275 280 285
Val Arg Trp Arg Ile Met Gly Thr Gly Pro Pro Ala Phe Thr Glu
290 295 300
Val Asp Asn Pro Ala Ser Phe Ala Asp Ser Met Leu Val Arg Ala
305 310 315
Val Asn Tyr Asn Tyr Tyr Tyr Ser Leu Asn Ala Trp Leu Leu Leu
320 325 330
Cys Pro Trp Trp Leu Cys Phe Asp Trp Ser Met Gly Cys Ile Pro
335 340 345
Leu Ile Lys Ser Ile Ser Asp Trp Arg Val Ile Ala Leu Ala Ala
350 355 360
Leu Trp Phe Cys Leu Ile Gly Leu Ile Cys Gln Ala Leu Cys Ser
365 370 375
Glu Asp Gly His Lys Arg Arg Ile Leu Thr Leu Gly Leu Gly Phe
380 385 390
Leu Val Ile Pro Phe Leu Pro Ala Ser Asn Leu Phe Phe Arg Val
395 400 405
Gly Phe Val Val Ala Glu Arg Val Leu Tyr Leu Pro Ser Ile Gly
410 415 420
Tyr Cys Val Leu Leu Thr Phe Gly Phe Gly Ala Leu Ser Lys His
425 430 435
Thr Lys Lys Lys Lys Leu Ile Ala Ala Val Val Leu Gly Ile Leu
440 445 450
Phe Ile Asn Thr Leu Arg Cys Val Leu Arg Ser G1y Glu Trp Arg
455 460 465
Ser Glu Glu Gln Leu Phe Arg Ser Ala Leu Ser Val Cys Pro Leu
470 475 480
Asn Ala Lys Val His Tyr Asn Ile Gly Lys Asn Leu Ala Asp Lys
485 490 495
Gly Asn Gln Thr Ala Ala Ile Arg Tyr Tyr Arg Glu Ala Val Arg
500 505 510
Leu Asn Pro Lys Tyr Val His Ala Met Asn Asn Leu Gly Asn Ile
515 520 525
Leu Lys Glu Arg Asn Glu Leu Gln Glu Ala Glu Glu Leu Leu Ser
530 535 540
Leu Ala Val Gln Ile Gln Pro Asp Phe Ala Ala Ala Trp Met Asn
545 550 555
Leu Gly Ile Va1 Gln Asn Ser Leu Lys Arg Phe Glu Ala Ala Glu
560 565 570
Gln Ser Tyr Arg Thr Ala Ile Lys His Arg Arg Lys Tyr Pro Asp
575 580 585
Cys Tyr Tyr Asn Leu Gly Arg Leu Tyr Ala Asp Leu Asn Arg His
590 595 600
Val Asp Ala Leu Asn Ala Trp Arg Asn Ala Thr Val Leu Lys Pro
605 610 615
Glu His Ser Leu Ala Trp Asn Asn Met Ile Ile Leu Leu Asp Asn
620 625 630
Thr Gly Asn Leu Ala Gln Ala Glu Ala Val Gly Arg Glu Ala Leu
635 640 645
Glu Leu Ile Pro Asn Asp His Ser Leu Met Phe Ser Leu Ala Asn
650 655 660
Val Leu Gly Lys Ser Gln Lys Tyr Lys G1u Ser Glu Ala Leu Phe
665 670 675
Leu Lys Ala Ile Lys Ala Asn Pro Asn Ala Ala Ser Tyr His Gly
680 685 690
Asn Leu Ala Val Leu Tyr His Arg Trp Gly His Leu Asp Leu Ala
695 700 705
Lys Lys His Tyr Glu Ile Ser Leu Gln Leu Asp Pro Thr Ala Ser
710 715 720
Gly Thr Lys Glu Asn Tyr Gly Leu Leu Arg Arg Lys Leu Glu Leu
725 730 735
Met Gln Lys Lys Ala Val
740
19/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
<210> 20
<211> 535
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2925182CD1
<400> 20
Met Arg Leu Arg Asn Gly Thr Val Ala Thr Ala Leu Ala Phe Ile
1 5 10 15
Thr Ser Phe Leu Thr Leu Ser Trp Tyr Thr Thr Trp Gln Asn Gly
20 25 30
Lys Glu Lys Leu Ile Ala Tyr Gln Arg Glu Phe Leu Ala Leu Lys
35 40 45
Glu Arg Leu Arg Ile Ala Glu His Arg Ile Ser Gln Arg Ser Ser
50 55 60
Glu Leu Asn Thr Ile Val Gln Gln Phe Lys Arg Val Gly Ala Glu
65 70 75
Thr Asn Gly Ser Lys Asp Ala Leu Asn Lys Phe Ser Asp Asn Thr
80 85 90
Leu Lys Leu Leu Lys Glu Leu Thr Ser Lys Lys Ser Leu Gln Val
95 100 105
Pro Ser Ile Tyr Tyr His Leu Pro His Leu Leu Lys Asn Glu Gly
110 115 120
Ser Leu Gln Pro Ala Val Gln Ile Gly Asn Gly Arg Thr Gly Val
125 130 135
Ser Ile Val Met Gly Ile Pro Thr Val Lys Arg Glu Val Lys Ser
140 145 150
Tyr Leu Ile Glu Thr Leu His Ser Leu Ile Asp Asn Leu Tyr Pro
155 160 165
Glu Glu Lys Leu Asp Cys Val Ile Val Val Phe Ile Gly Glu Thr
170 175 180
Asp Ile Asp Tyr Val His Gly Val Val Ala Asn Leu Glu Lys Glu
185 190 195
Phe Ser Lys Glu Ile Ser Ser Gly Leu Val Glu Val Ile Ser Pro
200 205 210
Pro Glu Ser Tyr Tyr Pro Asp Leu Thr Asn Leu Lys Glu Thr Phe
215 220 225
Gly Asp Ser Lys Glu Arg Val Arg Trp Arg Thr Lys Gln Asn Leu
230 235 240
Asp Tyr Cys Phe Leu Met Met Tyr Ala Gln Glu Lys Gly Ile Tyr
245 250 255
Tyr Ile Gln Leu Glu Asp Asp Ile Ile Val Lys Gln Asn Tyr Phe
260 265 270
Asn Thr Ile Lys Asn Phe Ala Leu Gln Leu Ser Ser Glu Glu Trp
275 280 285
Met Ile Leu Glu Phe Ser Gln Leu Gly Phe Ile Gly Lys Met Phe
290 295 300
Gln Ala Pro Asp Leu Thr Leu Ile Val Glu Phe Ile Phe Met Phe
305 310 315
Tyr Lys Glu Lys Pro Ile Asp Trp Leu Leu Asp His Ile Leu Trp
320 325 330
Val Lys Val Cys Asn Pro Glu Lys Asp Ala Lys His Cys Asp Arg
335 340 345
Gln Lys Ala Asn Leu Arg Ile Arg Phe Arg Pro Ser Leu Phe Gln
350 355 360
His Val Gly Leu His Ser Ser Leu Ser Gly Lys Ile Gln Lys Leu
365 370 375
Thr Asp Lys Asp Tyr Met Lys Pro Leu Leu Leu Lys Ile His Val
380 385 390
Asn Pro Pro Ala Glu Va1 Ser Thr Ser Leu Lys Val Tyr Gln Gly
395 400 405
20139
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
His Thr Leu Glu Lys Thr Tyr Met Gly Glu Asp Phe Phe Trp Ala
410 415 420
Ile Thr Pro Ile Ala Gly Asp Tyr Ile Leu Phe Lys Phe Asp Lys
425 430 435
Pro Val Asn Val Glu Ser Tyr Leu Phe His Ser Gly Asn Gln Glu
440 445 450
His Pro Gly Asp Ile Leu Leu Asn Thr Thr Val G1u Va1 Leu Pro
455 460 465
Phe Lys Ser Glu Gly Leu Glu Ile Ser Lys Glu Thr Lys Asp Lys
470 475 480
Arg Leu Glu Asp Gly Tyr Phe Arg Ile Gly Lys Phe Glu Asn Gly
485 490 495
Val Ala Glu Gly Met Val Asp Pro Ser Leu Asn Pro Ile Ser Ala
500 505 510
Phe Arg Leu Ser Val Ile Gln Asn Ser Ala Val Trp Ala Ile Leu
515 520 525
Asn Glu Ile His Ile Lys Lys Ala Thr Asn
530 535
<210> 21
<211> 522
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3271838CD1
<400> 21
Met Ala Ala Met Ala Val Ala Leu Arg Gly Leu Gly Gly Arg Phe
1 5 10 15
Arg Trp Arg Thr Gln Ala Val Ala Gly Gly Val Arg Gly Ala Ala
20 25 30
Arg Gly Ala Ala Ala Gly Gln Arg Asp Tyr Asp Leu Leu Val Val
35 40 45
Gly Gly Gly Ser Gly Gly Leu Ala Cys Ala Lys Glu Ala Ala Gln
50 55 60
Leu Gly Arg Lys Val Ser Val Val Asp Tyr Val Glu Pro Ser Pro
65 70 75
Gln Gly Thr Arg Trp Gly Leu Gly Gly Thr Cys Val Asn Val Gly
80 85 90
Cys Ile Pro Lys Lys Leu Met His Gln Ala Ala Leu Leu Gly Gly
95 100 105
Leu Ile Gln Asp Ala Pro Asn Tyr Gly Trp Glu Val Ala Gln Pro
110 115 120
Val Pro His Asp Trp Arg Lys Met Ala Glu Ala Val Gln Asn His
125 130 135
Val Lys Ser Leu Asn Trp Gly His Arg Val Gln Leu Gln Asp Arg
140 145 150
Lys Val Lys Tyr Phe Asn Ile Lys Ala Ser Phe Val Asp Glu His
155 160 165
Thr Val Cys Gly Val Ala Lys Gly Gly Lys Glu Ile Leu Leu Ser
170 175 180
Ala Asp His Ile Ile Ile Ala Thr Gly Gly Arg Pro Arg Tyr Pro
185 190 195
Thr His Ile Glu Gly Ala Leu Glu Tyr Gly Ile Thr Ser Asp Asp
200 205 210
Ile Phe Trp Leu Lys Glu Ser Pro Gly Lys Thr Leu Val Val Gly
215 220 225
Ala Ser Tyr Val Ala Leu Glu Cys Ala Gly Phe Leu Thr Gly Ile
230 235 240
Gly Leu Asp Thr Thr Ile Met Met Arg Ser Ile Pro Leu Arg Gly
245 250 255
Phe Asp Gln Gln Met Ser Ser Met Val Ile Glu His Met Ala Ser
21/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
260 265 270
His Gly Thr Arg Phe Leu Arg Gly Cys Ala Pro Ser Arg Val Arg
275 280 285
Arg Leu Pro Asp Gly G1n Leu Gln Val Thr Trp Glu Asp Arg Thr
290 295 300
Thr G1y Lys Glu Asp Thr Gly Thr Phe Asp Thr Val Leu Trp Ala
305 310 315
Ile Gly Arg Val Pro Asp Thr Arg Ser Leu Asn Leu Glu Lys Ala
320 325 330
Gly Val Asp Thr Ser Pro Asp Thr Gln Lys Ile Leu Val Asp Ser
335 340 345
Arg Glu Ala Thr Ser Val Pro His Ile Tyr Ala Ile Gly Asp Val
350 355 360
Va1 Glu Gly Arg Pro Glu Leu Thr Pro Thr Ala Ile Met Ala Gly
365 370 375
Arg Leu Leu Val Gln Arg Leu Phe Gly Gly Ser Ser Asp Leu Met
380 385 390
Asp Tyr Asp Asn Val Pro Thr Thr Val Phe Thr Pro Leu Glu Tyr
395 400 405
Gly Cys Val Gly Leu Ser Glu Glu Glu Ala Val Ala Arg His Gly
410 415 420
Gln Glu His Val Glu Val Tyr His Ala His Tyr Lys Pro Leu Glu
425 430 435
Phe Thr Val Ala Gly Arg Asp Ala Ser Gln Cys Tyr Val Lys Met
440 445 450
Val Cys Leu Arg Glu Pro Pro Gln Leu Val Leu Gly Leu His Phe
455 460 465
Leu Gly Pro Asn Ala Gly Glu Val Thr Gln Gly Phe Ala Leu Gly
470 475 480
Ile Lys Cys Gly Ala Ser Tyr Ala Gln Val Met Arg Thr Val Gly
485 490 495
Ile His Pro Thr Cys Ser Glu Glu Val Val Lys Leu Arg Ile Ser
500 505 510
Lys Arg Ser Gly Leu Asp Pro Thr Val Thr Gly Cys
515 520
<210> 22
<211> 495
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3292871CD1
<400> 22
Met Lys Asn Lys Thr Cys Val Leu Val Cys Val Ser Val Phe Gly
1 5 10 15
Gly Glu Arg Gly Gln Val Thr Val Pro Arg Val Gly Val Arg Arg
20 25 30
Pro Ser Leu Ala Gly Pro Leu Gln Lys Cys Thr Leu Arg Glu Thr
35 40 45
Arg Val Trp Leu Pro Gln Gly Ser Gly Phe Gln Ser Ser Arg Arg
50 55 60
Glu Lys Tyr Gly Asn Val Phe Lys Thr His Leu Leu Gly Arg Pro
65 70 75
Leu Ile Arg Val Thr Gly Ala Glu Asn Val Arg Lys Ile Leu Met
80 85 90
Gly Glu His His Leu Val Ser Thr Glu Trp Pro Arg Ser Thr Arg
95 100 105
Met Leu Leu Gly Pro Asn Thr Val Ser Asn Ser Ile Gly Asp Ile
110 115 120
His Arg Asn Lys Arg Lys Val Phe Ser Lys Ile Phe Ser His Glu
125 130 135
22!39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Ala Leu Glu Ser Tyr Leu Pro Lys Ile Gln Leu Val Ile Gln Asp
140 145 150
Thr Leu Arg Ala Trp Ser Ser His Pro Glu Ala Ile Asn Val Tyr
155 160 165
Gln Glu Ala Gln Lys Leu Thr Phe Arg Met Ala Ile Arg Val Leu
170 175 180
Leu Gly Phe Ser Ile Pro Glu Glu Asp Leu Gly His Leu Phe G1u
185 190 195
Val Tyr Gln Gln Phe Val Asp Asn Val Phe Ser Leu Pro Val Asp
200 205 210
Leu Pro Phe Ser Gly Tyr Arg Arg Gly Ile Gln Ala Arg Gln Ile
215 220 225
Leu Gln Lys Gly Leu Glu Lys Ala Ile Arg Glu Lys Leu Gln Cys
230 235 240
Thr Gln Gly Lys Asp Tyr Leu Asp Ala Leu Asp Leu Leu Ile Glu
245 250 255
Ser Ser Lys Glu His Gly Lys Glu Met Thr Met Gln Glu Leu Lys
260 265 270
Asp Gly Thr Leu Glu Leu Ile Phe Ala Ala Tyr Ala Thr Thr Ala
275 280 285
Ser Ala Ser Thr Ser Leu Ile Met Gln Leu Leu Lys His Pro Thr
290 295 300
Val Leu Glu Lys Leu Arg Asp Glu Leu Arg Ala His Gly Ile Leu
305 310 315
His Ser Gly Gly Cys Pro Cys Glu Gly Thr Leu Arg Leu Asp Thr
320 325 330
Leu Ser Gly Leu Arg Tyr Leu Asp Cys Val Ile Lys Glu Val Met
335 340 345
Arg Leu Phe Thr Pro Ile Ser Gly Gly Tyr Arg Thr Val Leu Gln
350 355 360
Thr Phe Glu Leu Asp Gly Phe Gln Ile Pro Lys Gly Trp Ser Val
365 370 . 375
Met Tyr Ser Ile Arg Asp Thr His Asp Thr Ala Pro Val Phe Lys
380 385 390
Asp Val Asn Val Phe Asp Pro Asp Arg Phe Ser Gln Ala Arg Ser
395 400 405
Glu Asp Lys Asp Gly Arg Phe His Tyr Leu Pro Phe Gly Gly Gly
410 415 420
Val Arg Thr Cys Leu Gly Lys His Leu Ala Lys Leu Phe Leu Lys
425 430 435
Val Leu Ala Val Glu Leu Ala Ser Thr Ser Arg Phe Glu Leu Ala
440 445 450
Thr Arg Thr Phe Pro Arg Ile Thr Leu Val Pro Val Leu His Pro
455 460 465
Val Asp Gly Leu Ser Val Lys Phe Phe Gly Leu Asp Ser Asn Gln
470 475 480
Asn Glu Ile Leu Pro Glu Thr Glu Ala Met Leu Ser Ala Thr Val
485 490 495
<210> 23
<211> 51
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4109179CD1
<400> 23
Met Glu Glu Lys Thr Ile Leu Ser Cys Ile Leu Arg His Phe Trp
1 5 10 15
Ile Glu Ser Asn Gln Lys Arg Glu G1u Leu Gly Leu Glu Gly Gln
20 25 30
23/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
Leu Ile Leu Arg Pro Ser Asn Gly Ile Trp Ile Lys Leu Lys Arg
35 40 45
Arg Asn Ala Asp Glu Arg
<210> 24
<211> 335
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4780365CD1
<400> 24
Met Ile Leu Phe Leu Ile Met Leu Val Leu Val Leu Phe Gly Tyr
1 5 10 15
Gly Val Leu Ser Pro Arg Ser Leu Met Pro Gly Ser Leu Glu Arg
20 25 30
Gly Phe Cys Met Ala Val Arg Glu Pro Asp His Leu Gln Arg Val
35 40 45
Ser Leu Pro Arg Met Val Tyr Pro Gln Pro Lys Val Leu Thr Pro
50 55 60
Cys Arg Lys Asp Val Leu Val Val Thr Pro Trp Leu Ala Pro Ile
65 70 75
Val Trp Glu Gly Thr Phe Asn Ile Asp Ile Leu Asn Glu Gln Phe
80 85 90
Arg Leu Gln Asn Thr Thr Ile Gly Leu Thr Val Phe Ala Ile Lys
95 100 105
Lys Tyr Val Ala Phe Leu Lys Leu Phe Leu Glu Thr Ala Glu Lys
110 115 120
His Phe Met Val Gly His Arg Val His Tyr Tyr Val Phe Thr Asp
125 130 135
Gln Pro Ala Ala Val Pro Arg Val Thr Leu Gly Thr Gly Arg Gln
140 145 150
Leu Ser Val Leu Glu Val Arg Ala Tyr Lys Arg Trp Gln Asp Val
155 160 165
Ser Met Arg Arg Met Glu Met Ile Ser Asp Phe Cys Glu Arg Arg
170 175 180
Phe Leu Ser Glu Val Asp Tyr Leu Val Cys Val Asp Val Asp Met
185 190 195
Glu Phe Arg Asp His Val Gly Val Glu Ile Leu Thr Pro Leu Phe
200 205 210
Gly Thr Leu His Pro Gly Phe Tyr Gly Ser Ser Arg Glu Ala Phe
215 220 225
Thr Tyr Glu Arg Arg Pro Gln Ser Gln Ala Tyr Ile Pro Lys Asp
230 235 240
Glu Gly Asp Phe Tyr Tyr Leu Gly Gly Phe Phe Gly Gly Ser Val
245 250 255
Gln Glu Val Gln Arg Leu Thr Arg Ala Cys His Gln Ala Met Met
260 265 270
Val Asp Gln Ala Asn Gly Ile Glu Ala Val Trp His Asp Glu Ser
275 280 285
His Leu Asn Lys Tyr Leu Leu Arg His Lys Pro Thr Lys Val Leu
290 295 300
Ser Pro Glu Tyr Leu Trp Asp Gln Gln Leu Leu Gly Trp Pro Ala
305 310 315
Val Leu Arg Lys Leu Arg Phe Thr Ala Val Pro Lys Asn His Gln
320 325 330
Ala Val Arg Asn Pro
335
<210> 25
<211> 1269
24/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1799250CB1
<400> 25
cgcggcggcg gcgcggccgg ggcagccatg tcgccattgt ctgcagcgcg ggcggccctg 60
cgggtctacg cggtaggcgc cgcggtgatc ctggcgcagc tgctgcggcg ctgccgcggg 120
ggcttcctgg agccagttct ccccccacga cctgaccgtg tcgctatagt gacgggaggg 180
acagatggca ttggctattc tacagcgaag catctggcga gacttggcat gcatgttatc 240
atagctggaa ataatgacag caaagccaaa caagttgtaa gcaaaataaa agaagaaacc 300
ttgaacgaca aagtggaatt tttatactgt gacttggctt ccatgacttc catccggcag 360
tttgtgcaga agttcaagat gaagaagatt cctctccatg tcctgatcaa caatgctggg 420
gtgatgatgg tccctcagag gaaaaccaga gatggattcg aagaacattt cggcctgaac 480
tacctagggc acttcctgct gaccaacctt ctcttggata cgctgaaaga gtctgggtcc 540
cctggccaca gtgcgagggt ggtcaccgtc tcctctgcca cccattacgt cgctgagctg 600
aacatggatg accttcagag cagtgcctgc tactcacccc acgcagccta cgcccagagc 660
aagctggccc ttgtcctgtt cacctaccac ctccagcggc tgctggcggc tgagggaagc 720
cacgtgaccg ccaacgtggt ggaccccggg gtggtcaaca cggacgtcta caagcacgtg 780
ttctgggcca cccgtctggc gaagaagctt ctcggctggt tgcttttcaa gacccccgat 840
gaaggagcgt ggacttccat ctacgcagca gtcaccccag agctggaagg agttggtggc 900
cgttacctat acaacaagaa agagaccaag tccctccacg tcacctacaa ccagaaactg 960
cagcagcagc tgtggtctaa gagttgtgag atgactgggg tccttgatgt gaccctgtga 1020
tatcctgtct caggatagct gctgccccaa gaaacacatt gcacctgcca atagcttgtg 1080
ggtctgtgaa gactgcggtg tttgagtttc tCaCaCCCaC CtgCCCa.Cag ggCtCtgtCC 1140
tctagttttg agacagctgc ctcaacctct gcagaacttc aagaagccaa ataaacattt 1200
tggaggataa tcaccccaag tggtcttcaa ccataaactt tgtgattcca aagtgcccag 1260
ttgtcacag 1269
<210> 26
<211> 1593
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2242475CB1
<400> 26
cctgcctggt cctctgtgcc tggtggggtg ggggtgccag gtgtgtccag aggagcccat 60
ttggtagtga ggcaggtatg gggctagaag cactggtgcc cctggccgtg atagtggcca 120
tcttcctgct cctggtggac ctgatgcacc ggcgccaacg ctgggctgca cgctacccac 180
CaggCCCCCt gCCaCtgCCC gggctgggca acctgctgca tgtggacttc cagaacacac 240
catactgctt cgaccagttg cggcgccgct tcggggacgt gttcagcctg cagctggcct 300
ggacgccggt ggtcgtgctc aatgggctgg cggccgtgcg cgaggcgctg gtgacccacg 360
gcgaggacac cgccgaccgc ccgcctgtgc ccatcaccca gatcctgggt ttcgggccgc 420
gttcccaagg ggtgttcctg gcgcgctatg ggcccgcgtg gcgcgagcag aggcgcttct 480
ccgtctccac cttgcgcaac ttgggcctgg gcaagaagtc gctggagcag tgggtgaccg 540
aggaggccgc ctgcctttgt gccgccttcg ccaaccactc cggacgcccc tttcgcccca 600
acggtctctt ggacaaagcc gtgagcaacg tgatcgcctc cctcacctgc gggcgccgct 660
tcgagtacga cgaccctcgc ttcctcaggc tgctggacct agctcaggag ggactgaagg 720
aggagtcggg cttcctgcgc gaggtgctga atgctgtccc cgtcctcccg cacatcccag 780
cgctggctgg caaggtccta cgcttccaaa aggctttcct gacccagctg gatgagctgc 840
taactgagca caggatgacc tgggacccag cccagccacc ccgagacctg actgaggcct 900
tcctggcaaa gaaggagaag gccaagggga gccctgagag cagcttcaat gatgagaacc 960
tgcgcatagt ggtgggtaac ctgttccttg ccgggatggt gaccacctcg accacactgg 1020
cctgggccct gctgctcatg atcctgcatc cggatgtgca gtgccgagta caacaggaaa 1080
tcgatgaggt catagggcag gtgcggcatc cagagatggc agaccaggcc cacatgccgt 1140
tcaccaatgc tgtcatccat gaggtgcagc gctttgcaga cattgtccca atgaatttgc 1200
cacacaagac ttctcgtgac attgaagtgc agggcttcct tatccctaag gggacaaccc 1260
tcatccccaa cctgtcctca gtgctgaagg atgagactgt ctgggagaag cccctccgat 1320
tccaccctga acacttcctg gatgcccagg gcaactttgt gaagcatgag gccttcatgc 1380
25!39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
cattctcagc aggccgcaga gcatgcctgg gggagcccct ggcccgcatg gagctcttcc 1440
tcttcttcac ctgcctcctg caacgcttca gcttctccgt gcccactgga cagccccggc 1500
ccagcgacta tggtgtcttt gcctttctcc ttagcccttc cccctaccag ctctgtgcat 1560
tcaaacgtta gaaggaaaga aattctagtc cag 1593
<210> 27
<211> 1779
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2706492CB1
<400> 27
cagtgagaga actgagaccc agagagatta agtatcttgc ccaaggtcac actttagtaa 60
aaggcaaagt caggatttga atccacacac ttatctagta cactctaaga cacaggggca 120
gattttagta aacagtagga gatggactct cagaatttgg tgcctgggga gagggaagag 180
gagagagatg cctggtgaca agcccagccc ttgcctctcc acaggagagt gggaacaatg 240
aggtcatctt catggccttg gacttggcca gtctggcctc ggtgcgggcc tttgccactg 300
cctttctgag ctctgagcca cggttggaca tcctcatcca caatgccggt atcagttcct 360
gtggccggac ccgtgaggcg tttaacctgc tgcttcgggt gaaccatatc ggtccctttc 420
tgctgacaca tctgctgctg ccttgcctga aggcatgtgc ccctagccgc gtggtggtgg 480
tagcctcagc tgcccactgt cggggacgtc ttgacttcaa acgcctggac cgcccagtgg 540
tgggctggcg gcaggagctg cgggcatatg ctgacactaa gctggctaat gtactgtttg 600
cccgggagct cgccaaccag cttgaggcca ctggcgtcac ctgctatgca gcccacccag 660
ggcctgtgaa ctcggagctg ttcctgcgcc atgttcctgg atggctgcgc ccacttttgc 720
gcccattggc ttggctggtg ctccgggcac caagaggggg tgcccagaca cccctgtatt 780
gtgctctaca agagggcatc gagcccctca gtgggagata ttttgccaac tgccatgtgg 840
aagaggtgcc tccagctgcc cgagacgacc gggcagccca tcggctatgg gaggccagca 900
agaggctggc agggcttggg cctggggagg atgctgaacc cgatgaagac ccccagtctg 960
aggactcaga ggccccatct tctctaagca ccccccaccc tgaggagccc acagtttctc 1020
aaccttaccc cagccctcag agctcaccag atttgtctaa gatgacgcac cgaattcagg 1080
ctaaagttga gcctgagatc cagctctcct aaccctcagg ccaggatgct tgccatggca 1140
cttcatggtc cttgaaaacc tcggatgtgt gcgaggccat gccctggaca ctgacgggtt 1200
tgtgatcttg acctccgtgg ttactttctg gggccccaag ctgtgccctg gacatctctt 1260
ttcctggttg aaggaataat gggtgattat ttcttcctga gagtgacagt aaccccagat 1320
ggagagatag gggtatgcta gacactgtgc ttctcggaaa tttggatgta gtattttcag 1380
gccccaccct tattgattct gatcagctct ggagcagagg cagggagttt gcaatgtgat 1440
gcactgccaa cattgagaat tagtgaactg atccctttgc aaccgtctag ctaggtagtt 1500
aaattacccc catgttaatg aagcggaatt aggctcccga gctaagggac tcgcctaggg 1560
tctcacagtg agtaggagga gggcctggga tctgaaccca agggtctgag gccagggccg 1620
actgccgtaa gatgggtgct gagaagtgag tcagggcagg gcagctggta tcgaggtgcc 1680
ccatgggagt aaggggacgc cttccgggcg gatgcagggc tggggtcatc tgtatctgaa 1740
gcccctcgga ataaagcgcg ttgaccgccg aaaaaaaaa 1779
<210> 28
<211> 1931
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2766688CB1
<400> 28
ggcaacgcgg ctggttctcg cccgtcagtc ctagcccggc cctgcccctc gcttgcattt 60
tttccgcgct ggctgagatt caaagagaag tggaggtggg agggagcgac aatggaaaaa 120
tcacctgaaa actgggacag aggaaggaag ctacagttac gaaggagagc tgcaaaagtt 280
gcagcagaaa ggttgggagt cccgacaggt tccgtagccc acagaaaaga agcaagggac 240
ggcaggactg tttcacactt ttctgcttct ggaaggtgct ggacaaaaac atggaactaa 300
tttccccaac agtgattata atcctgggtt gccttgctct gttcttactc cttcagcgga 360
agaatttgcg tagacccccg tgcatcaagg gctggattcc ttggattgga gttggatttg 420
agtttgggaa agcccctcta gaatttatag agaaagcaag aatcaagtat ggaccaatat 480
26/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
ttacagtctt tgctatggga aaccgaatga cctttgttac tgaagaagaa ggaattaatg 540
tgtttctaaa atccaaaaaa gtagattttg aactagcagt gcaaaatatc gtttatcata 600
cagcatcaat tccaaagaat gtctttttag cactgcatga aaaactctat attatgttga 660
aagggaaaat ggggactgtc aatctccatc agtttactgg gcaactgact gaagaattac 720
atgaacaact ggagaattta ggcactcatg ggacaatgga cctgaacaac ttagtaagac 780
atctccttta tccagtcaca gtgaatatgc tctttaataa aagtttgttt tccacaaaca 840
agaaaaaaat caaggagttc catcagtatt ttcaagttta tgatgaagat tttgagtatg 900
ggtcccagtt gccagagtgt cttctaagaa actggtcaaa atccaaaaag tggttcctgg 960
aactgtttga gaaaaacatt ccagatataa aagcatgtaa atctgcaaaa gataattcca 1020
tgacattatt gcaagctacg ctggatattg tagagacgga aacaagtaag gaaaactcac 1080
ccaattatgg gctcttactg ctttgggctt ctctgtctaa tgctgttcct gttgcatttt 1140
ggacacttgc atacgtcctt tctcatcctg atatccacaa ggccattatg gaaggcatat 1200
cttctgtgtt tggcaaagca ggcaaagata agattaaagt gtctgaggat gacctggaga 1260
atctccttct aattaaatgg tgtgttttgg aaaccattcg tttaaaagct cctggtgtca 1320
ttactagaaa agtggtgaag cctgtggaaa ttttgaatta catcattcct tctggtgact 1380
tgttgatgtt gtctccattt tggctgcata gaaatccaaa gtattttcct gagcctgaat 1440
tgttcaaacc tgaacgttgg gaaaaaggca aatttagaga agcactcttt cttggactgc 1500
ttcatggcat tggaagcggg aagttccagt gtcctgcaag gtggtttgct ctgttagagg 1560
ttcagatgtg tattatttta atactttata aatatgactg tagtcttctg gacccattac 1620
ccaaacagag ttatctccat ttggtgggtg tcccccagcc ggaagggcaa tgccgaattg 1680
aatataaaca aagaatatga catctgttgg gcctcacaag gaccagggcc ttctggagga 1740
gtggcactac cccacctggc agcacctaga cctgagctct acaaaaacac actgcttcac 1800
tttgttttag gacttagttc aagaacacat tcaaatggtg catgtgtttg gtatcttcaa 1860
cagtagacca agaatctaac atcactctca gtaatataga gaccggaata catggtttat 1920
aggaaatgat c 1932
<210> 29
<211> 1282
<212> DNA
<223> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2788823CB1
<400> 29
cgcgcctgcg cctccgctcg cctgtggctg cgtcgcgcgc tcttcctcgg agctacccag 60
gcggctggtg tgcagcaagc tccgcgccga ccccggacgc ctgacgcctg acgcctgtcc 120
ccggcccggc atgagccgct acctgctgcc gctgtcggcg ctgggcacgg tagcaggcgc 180
cgccgtgctg ctcaaggact atgtcaccgg tggggcttgc cccagcaagg ccaccatccc 240
tgggaagacg gtcatcgtga cgggcgccaa cacaggcatc gggaagcaga ccgccttgga 300
actggccagg agaggaggca acatcatcct ggcctgccga gacatggaga agtgtgaggc 360
ggcagcaaag gacatccgcg gggagaccct caatcaccat gtcaacgccc ggcacctgga 420
cttggcttcc ctcaagtcta tccgagagtt tgcagcaaag atcattgaag aggaggagcg 480
agtggacatt ctaatcaaca acgcgggtgt gatgcggtgc ccccactgga ccaccgagga 540
cggcttcgag atgcagtttg gcgttaacca cctgggtcac tttctcttga caaacttgct 600
gctggacaag ctgaaagcct cagccccttc gcggatcatc aacctctcgt ccctggccca 660
tgttgctggg cacatagact ttgacgactt gaactggcag acgaggaagt ataacaccaa 720
agccgcctac tgccagagca agctcgccat cgtcctcttc accaaggagc tgagccggcg 780
gctgcaaggc tctggtgtga ctgtcaacgc cctgcacccc ggcgtggcca ggacagagct 840
gggcagacac acgggcatcc atggctccac cttctccagc accacactcg ggcccatctt 900
ctggctgctg gtcaagagcc ccgagctggc cgcccagccc agcacatacc tggccgtggc 960
ggaggaactg gcggatgttt ccggaaagta cttcgatgga ctcaaacaga aggccccggc 1020
ccccgaggct gaggatgagg aggtggcccg gaggctttgg gctgaaagtg cccgcctggt 1080
gggcttagag gctccctctg tgagggagca gcccctcccc agataacctc tggagcagat 1140
ttgaaagcca ggatggcgcc tccagaccga ggacagctgt CCJCCd.tgCC CgCagCttCC 1200
tggcactacc tgagccggga gacccaggac tggcggccgc catgcccgca gtaggttcta 1260
gggggcggtg ctggccgcag tg 1282
<210> 30
<211> 2416
<212> DNA
<213> Homo Sapiens
27/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
<220>
<221> misc_feature
<223> Incyte ID No: 3348822CB1
<400> 30
agcgtgcgcg ctttggtaac cggctagaaa tcccgcacgc gcgcctgcct cctctcccca 60
ggcctgagct gcccctccca ctgcctttcc ttcttcccgc gagtcagaag cttcgcgagg 120
gcccagagag gcggtggggt gggcgaccct acgccagctc cgggcgggag aaagcccacc 180
ctctcccgcg ccccaggaaa ccgccggcgt tcggcgctgc gcagagccat ggaattctcc 240
tggctggaga cgcgctgggc gcggcccttt tacctggcgt tcgtgttctg cctggccctg 300
gggctgctgc aggccattaa gctgtacctg cggaggcagc ggctgctgcg ggacctgcgc 360
cccttcccag cgccccccac ccactggttc cttgggcacc agaagtttat tcaggatgat 420
aacatggaga agcttgagga aattattgaa aaataccctc gtgccttccc tttctggatt 480
gggccctttc aggcattttt ctgtatctat gacccagact atgcaaagac acttctgagc 540
agaacagatc ccaagtccca gtacctgcag aaattctcac ctccacttct tggaaaagga 600
ctagcggctc tagacggacc caagtggttc cagcatcgtc gcctactaac tcctggattc 660
cattttaaca tcctgaaagc atacattgag gtgatggctc attctgtgaa aatgatgctg 720
gataagtggg agaagatttg cagcactcag gacacaagcg tggaggtcta tgagcacatc 780
aactcgatgt ctctggatat aatcatgaaa tgcgctttca gcaaggagac caactgccag 840
acaaacagca cccatgatcc ttatgcaaaa gccatatttg aactcagcaa aatcatattt 900
caccgcttgt acagtttgtt gtatcacagt gacataattt tcaaactcag ccctcagggc 960
taccgcttcc agaagttaag ccgagtgttg aatcagtaca cagatacaat aatccaggaa 1020
agaaagaaat ccctccaggc tggggtaaag caggataaca ctccgaagag gaagtaccag 1080
gattttctgg atattgtcct ttctgccaag gatgaaagtg gtagcagctt ctcagatatt 1140
gatgtacact ctgaagtgag cacattcctg ttggcaggac atgacacctt ggcagcaagc 1200
atctcctgga tcctttactgcctggctctg aaccctgagc atcaagagag atgccgggag 1260
gaggtcaggg gcatcctggg ggatgggtct tctatcactt gggaccagct gggtgagatg 1320
tcgtacacca caatgtgcat caaggagacg tgccgattga ttcctgcagt cccgtccatt 1380
tccagagatc tcagcaagcc acttaccttc ccagatggat gcacattgcc tgcagggatc 1440
accgtggttc ttagtatttg gggtcttcac cacaaccctg ctgtctggaa aaacccaaag 1500
gtctttgacc ccttgaggtt ctctcaggag aattctgatc agagacaccc ctatgcctac 1560
ttaccattct cagctggatc aaggaactgc attgggcagg agtttgccat gattgagtta 1620
aaggtaacca ttgccttgat tctgctccac ttcagagtga ctccagaccc caccaggcct 1680
cttactttcc ccaaccattt tatcctcaag cccaagaatg ggatgtattt gcacctgaag 1740
aaactctctg aatgttagat ctcagggtac aatgattaaa cgtactttgt ttttcgaagt 1800
taaatttaca gctaatgatc caagcagata gaaagggatc aatgtatggt gggaggattg 1860
gaggttggtg ggataggggt ctctgtgaag agatccaaaa tcatttctag gtacacagtg 1920
tgtcagctag atctgtttct atataacttt gggagatttt cagatctttt ctgttaaact 1980
ttcactacta ttaatgctgt atacaccaat agactttcat atattttctg ttgtttttaa 2040
aatagttttc agaattatgc aagtaataag tgcatgtatg ctcactgtca aaaattccca 2100
acactagaaa atcatgtaga ataaaaattt taaatctcac ttcacttagc cgacattcca 2160
tgccctgacc aatcctactg cttttcctaa aaacagaata atttggtgtg cattctttca 2220
gactttttcc tatacatttt atatgtagaa atgtagcaat gtatttgtat agatgtgatc 2280
attcctatat tgttattgat ttttttcact taataaaaat tcaccttatt ccttatcatt 2340
gctttatggt attctgtaat atgaatgtac tataatttat ttaactattt tccttattgg 2400
gcatttaagt gatttc 2416
<210> 31
<211> 1574
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4290251CB1
<400> 31
ggacaacctg agtgctcagt cgtaaagagg aaaggcagaa tttttccttg ctatggctgg 60
aacaaacgca cttttgatgc tggaaaactt catagatgga aaatttttac cttgtagctc 120
atatatagat tcttacgacc catcaacagg ggaagtgtat tgcagagtgc caaatagtgg 180
aaaagacgag atcgaagccg cggtcaaggc cgccagagaa gcctttccca gctggtcatc 240
ccgcagcccc caggagcgct cacgggtcct gaaccaggtg gcggatttgc tggagcagtc 300
cctggaggag tttgcccagg ccgagtctaa agaccaaggg aaaaccttag cactggcaag 360
aaccatggac attccccggt ctgtgcagaa cttcaggttc ttcgcttcct ccagcctgca 420
28/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
ccacacgtca gagtgcacgc agatggaaca cctgggctgc atgcactaca cggtgcgggc 480
cccggtggga gtcgctggtc tgatcagccc ctggaatttg ccactctact tgctgacctg 540
gaagatagct ccagcgatgg ctgcagggaa cactgtgata gccaagccca gtgagctgac 600
ttcagtgact gcgtggatgt tgtgcaaact cctggataaa gcaggtgttc caccaggtgt 660
ggtcaatatt gtgtttggaa ccgggcccag ggtgggtgag gccctggtgt cccacccaga 720
ggtgcccctg atctccttca ccgggagcca gcccaccgct gagcggatca cccagctgag 780
cgctccccac tgcaaaaagc tctccctgga gctggggggc aagaatcctg ccatcatctt 840
tgaggacgcc aacctggatg agtgcattcc ggcaaccgtc aggtccagct ttgccaacca 900
ggtcagaagt tacgtcaaga gagctcttgc tgaaagtgcc caaatttggt gcggtgaagg 960
agtggataag ttgagcctcc ctgccaggaa ccaggcaggc tactttatgc ttcccacggt 1020
gataacagac attaaggatg aatcctgctg catgacggaa gagatatttg gtccagtgac 1080
gtgtgtcgtc ccctttgata gtgaagagga ggtgattgaa agagccaaca acgttaagta 1140
tgggctggcg gctaccgtgt ggtccagcaa tgtggggcgc gtccaccggg tggctaagaa 1200
gctgcagtct ggcttggtct ggaccaactg ctggctcatc agggagctga accttccttt 1260
cggggggatg aagagttctg gaataggtag agagggagcc aaggactctt acgacttctt 1320
cactgagatc aaaaccatca ccgttaaaca ctgatctttg ctaatggtgg agccactatg 1380
gccaatgcct ggctgcaggc atcagttgtt caatgtggta gatgaaaatc atggcatgaa 1440
ttccagctat gccttgactt ggcagaaggt tatctctagc ttatcctcag ttcttagtaa 1500
ctttacccac tagtgaagag atactgtcta ttttcaatgt ggactcggaa aaaaagactt 1560
ataagtagga agat 1574
<210> 32
<211> 2227
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4904188CB1
<400> 32
gcaggaccca gggcgctgaa ctctcacaac caatcaggcg acccccccag agggaaacta 60
caagtcccag catgccccac gcgcaccgtc aggggccgac ccgccgcgcc ccagcgttct 120
ccgcgtacag gtggtctctt gggttccgga agagcctagg ctggatgtct tgatcaataa 180
cgcagggatc ttccagtgcc cttacatgaa gactgaagat gggtttgaga tgcagttcgg 240
agtgaaccat ctggggcact ttctactcac caatcttctc cttggactcc tcaaaagttc 300
agctcccagc aggattgtgg tagtttcttc caaactttat aaatacggag acatcaattt 360
tgatgacttg aacagtgaac aaagctataa taaaagcttt tgttatagcc ggagcaaact 420
ggctaacatt ctttttacca gggaactagc ccgccgctta gaaggcacaa atgtcaccgt 480
caatgtgttg catcctggta ttgtacggac aaatctgggg aggcacatac acattccact 540
gttggtcaaa ccactcttca atttggtgtc atgggctttt ttcaaaactc cagtagaagg 6.00
tgcccagact tccatttatt tggcctcttc acctgaggta gaaggagtgt caggaagata 660
ctttggggat tgtaaagagg aagaactgtt gcccaaagct atggatgaat ctgttgcaag 720
aaaactctgg gatatcagtg aagtgatggt tggcctgcta aaataggaac aaggagtaaa 780
agagctgttt ataaaactgc atatcagtta tatctgtgat caggaatggt gtggattgag 840
aacttgttac ttgaagaaaa agaattttga tattggaata gcctgctaag aggtacatgt 900
gggtattttg gagttactga aaaattattt ttgggataag agaatttcag caaagatgtt 960
ttaaatatat atagtaagta taatgaataa taagtacaat gaaaaataca attatattgt 1020
aaaattataa ctgggcaagc atggatgaca tattaatatt tgtcagaatt aagtgactca 1080
aagtgctatc gagaggtttt tcaagtatct ttgagtttca tggccaaagt gttaactagt 1140
tttactacaa tgtttggtgt ttgtgtggaa attatctgcc tggtgtgtgc acacaagtct 1200
tacttggaat aaatttactg gtacaaa
1227
<210> 33
<211> 1240
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 638419CB1
<400> 33
cttgcttgca cagtgtcctg gagctggacc tggctctggg tttccaggaa gcagtttgac 60
29/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
taaaggcagc aagctgcttc ctctgctgcc tgaaatacca gattcccaat ggcgaagatt 120
gagaaaaacg ctcccacgat ggaaaaaaag ccagaactgt ttaacatcat ggaagtagat 180
ggagtcccta cgttgatatt atcaaaagaa tggtgggaaa aagtctgtaa tttccaagcc 240
aagcctgatg atcttattct ggcaacttac ccaaagtcag gtacaacatg gatgcatgaa 300
attttagaca tgattctaaa tgatggtgat gtggagaaat gcaaaagagc ccagactcta 360
gatagacacg ctttccttga actgaaattt ccccataaag aaaaaccaga tttggagttc 420
gttcttgaaa tgtcctcacc acaactgata aaaacacatc tcccttcaca tctgattcca 480
ccatctatct ggaaagaaaa ctgcaagatt gtctatgtgg ccagaaatcc caaggattgc 540
ctggtgtcct actaccactt tcacaggatg gcttccttta tgcctgatcc tcagaactta 600
gaggaatttt atgagaaatt catgtccgga aaagttgttg gcaggtcctg gtttgaccat 660
gtgaaaggat ggtgggctgc aaaagacacg caccggatcc tctacctctt ctacgaggat 720
attaaaaaaa atccaaaaca tgagatccac aaggtgttgg aattcttgga gaaaactttg 780
tcaggtgatg ttataaacaa gattgtccac catacctcat ttgatgtaat gaaggataat 840
cccatggcca accatactgc ggtacctgct cacatattca atcactccat ctcaaaattt 900
atgaggaaag ggatgcctgg agactggaag aaccacttta ctgtggctat gaatgagaac 960
tttgataagc attatgaaaa gaagatggca gggtccacac tgaacttctg cctggagatc 1020
tgagaggaac aacaacaaac taggtgacag agactatgcc aactatttcg ccttttattc 1080
tgttgagcaa ggaactgtga ctgaatgtgg agcttatgag cttcagtcca tctcctatag 1140
tgtggctagt ttgctataat attaaaacat gatttaaaat atcaacaaac cagttactcc 1200
agcaaataaa ataagagaat tagagagcag aaaaaaaaaa 1240
<210> 34
<211> 2275
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1844394CB1
<400> 34
gcaattcagt gaggttaaag gactggatgc atttgttctg agcctgctca ctctagatgg 60
tgaatcaatc tacagcctga cctcgaagcc tatactactg ttattagcac gcattatcct 120
agtgaatgta agacataaac tgacagctat tcagagcttg ccatggtgga ctttgagatg 180
tgtgaatatt catcagcatt tgcttgagga acgctcacct ctgcttttta ctcttgccga 240
aaactgtatt gatcaagtga tgaaactaca gaatctgttt gtagatgatt caggtcgata 300
tttggctatt caattccatc tggaatgtgc atatgtgttt ttatattatt atgagtacag 360
aaaagcaaaa gatcagttgg atattgctaa ggacatcagc caattacaaa ttgatttgac 420
aggtgctttg ggaaaaagaa cacggttcca ggaaaattat gtggcacaac tgattctaga 480
tgtaagaagg gaaggggatg tcctttcaaa ttgtgaattc actccagcac ccactcctca 540
ggaacattta accaagaatc ttgagctcaa tgatgacacc attctgaatg acataaagtt 600
agcagattgt gaacagttcc ~agatgccgga tctgtgtgct gaagagatcg ctattattct 660
tggaatctgc actaattttc aaaagaataa cccagtgcac acattaactg aagtggagct 720
tctggcattt acatcatgtt tgctttcaca accaaagttc tgggccattc agacatcagc 780
cttgatcctc cggacaaaac ttgagaaagg aagtactcgc cgagtggaac gtgcaatgag 840
gcagacacag gctcttgcag accaatttga agataaaact acatctgtat tggaacgcct 900
gaagattttc tattgctgtc aagtaccacc tcactgggcc attcagcgcc aacttgcaag 960
tttgctcttt gagttgggat gtaccagttc agcccttcag atatttgaaa agctagaaat 1020
gtgggaagat gttgtcattt gttatgaaag agccgggcag cacggaaagg cagaagaaat 1080
ccttagacaa gagctggaga aaaaagaaac gcctagttta tactgcttgc ttggagatgt 1140
cctcggagac cattcttgct atgacaaggc ctgggagttg tcccggtacc gcagtgctcg 1200
tgctcagcgc tccaaagccc tccttcatct tcggaacaag gagtttcaag agtgtgtaga 1260
gtgcttcgaa cgctcggtta agattaatcc catgcagctc ggggtgtggt tttctctcgg 1320
ttgtgcctat ttggccttgg aagactatca aggttcagca aaggcatttc agcgctgtgt 1380
gactctagaa cccgataatg ctgaagcttg gaacaatttg tcaacttcct atatccgatt 1440
aaaacaaaaa gtaaaagctt ttagaacttt acaagaagct ctcaagtgta actatgaaca 1500
ctggcagatt tggaaaaact acatcctcac cagcactgac gttggggaat tttcagaagc 1560
cattaaagct tatcaccggc tcttggactt acgtgacaaa tacaaagatg ttcaggtcct 1620
taaaattcta gtcagggcag tgattgatgg gatgactgat cgaagtggag atgttgcaac 1680
tggcctcaaa ggaaagctgc aggagttatt tggcagagtg acttcaagag tgacaaatga 1740
tggagaaatc tggaggctgt atgcccacgt atatggaaat gggcagagtg aaaagcctga 1800
tgaaaatgaa aaggcattcc agtgcctctc aaaggcatac aagtgtgaca cccagtccaa 1860
ttgttgggag aaagatatta catcatttaa ggaagttgtt caaagagcct taggacttgc 1920
acatgtggcc ataaaatgca gtaaaaacaa atccagttcc caagaagctg tacaaatgct 1980
30/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
ttcttctgtt cgactcaatt tacggggctt gttatctaaa gcaaagcaac tttttacaga 2040
tgtggcaact ggagaaatgt ccagggaatt agctgatgac ataacagcta tggacacctt 2100
agtgacagag ctccaagacc taagcaacca gtttcgaaat cagtattgat tctgctggaa 2160
gcagattctg gaaaaggtgc tttcacctgc tggtaaaaga tacatctgta tatctgaaat 2220
gcaagatatt gatttttaaa ataaatttgt tttatgactt aaaaaaaaaa aaaaa 2275
<210> 35
<211> 1586
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2613056CB1
<400> 35
tctaaggcac agtatcattt tcagtactga caaggtgttt cattttatat ggttgtcata 60
ataaggcaaa ttcattttgt acgctttata ttttcaaacc cagcaagctc taaaagggac 120
ataaaataac ttagaaattg ggaaagacgg gcatgtgtat gatcatgata ttcatcccct 180
gccccagaac aaatgggagg aacacattgc ccaaaactca cgtctggagc tctttcaaca 240
tgtctccctg atgaccctgg acagcatcat gaagtgtgcc ttcagccacc agggcagcat 300
ccagttggac agtaccctgg actcatacct gaaagcagtg ttcaacctta gcaaaatctc 360
caaccagcgc atgaacaatt ttctacatca caacgacctg gttttcaaat tcagctctca 420
aggccaaatc ttttctaaat ttaaccaaga acttcatcag ttcacagaga aagtaatcca 480
ggaccggaag gagtctctta aggataagct aaaacaagat actactcaga aaaggcgctg 540
ggattttctg gacatacttt tgagtgccaa aagcgaaaac accaaagatt tctctgaagc 600
agatctccag gctgaagtga aaacgttcat gtttgcagga catgacacca catccagtgc 660
tatctcctgg atcctttact gcttggcaaa gtaccctgag catcagcaga gatgccgaga 720
tgaaatcagg gaactcctag gggatgggtc ttctattacc tgggaacacc tgagccagat 780
gccttacacc acgatgtgca tcaaggaatg cctccgcctc tacgcaccgg tagtaaacat 840
atcccggtta ctcgacaaac ccatcacctt tccagatgga cgctccttac ctgcaggaat 900
aactgtgttt atcaatattt gggctcttca ccacaacccc tatttctggg aagaccctca 960
ggtctttaac cccttgagat tctccaggga aaattctgaa aaaatacatc cctatgcctt 1020
cataccattc tcagctggat taaggaactg cattgggcag cattttgcca taattgagtg 1080
taaagtggca gtggcattaa ctctgctccg cttcaagctg gctccagacc actcaaggcc 1140
tccccagcct gttcgtcaag ttgtcctcaa gtccaagaat ggaatccatg tgtttgcaaa 1200
aaaagtttgc taattttaag tcctttcgta taagaattaa tgagacaatt ttcctaccaa 1260
aggaagaaca aaaggataaa tataatacaa aatatatgta tatggttgtt tgacaaatta 1320
tataacttag gatacttctg actggttttg acatccatta acagtaattt taatttcttt 1380
gctgtatctg gtgaaaccca caaaaacacc tgaaaaaact caagctgact tccactgcga 1440
agggaaatta ttggtttgtg taactagtgg tagagtggct ttcaagcata gtttgatcaa 1500
aactccactc agtatctgca ttacttttat ctctgcaaat atctgcatga tagctttatt 1560
ctcagttatc tttccccata ataaaa 1586
<210> 36
<211> 859
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5053617CB1
<400> 36
gtcgagtgcc tcccccaccc cccaccatgt gcttgagtgc acacccggcg ccaggccctg 60
atcctggcac ttcttgtgaa tcacaccgtg tcatacccat gacttccatt gcacagtggg 120
gaaactgagt ctagagaggt gaaataacat gtctaaagtc acaggaagtg aaaaagctga 180
ggacatggag ccagttgccc aatgacagga gagctgaaat gtcctcactg ctgggggtag 240
accgggcctc accagcttcc tggagagtca catgtttgtc tgcatcctca gggggctcgc 300
cggttctcca gcccggactg ctgccagagg cttcctggag gtggcagctt tcttcaaagg 360
caccatcccg gagcgcaagc ccctgatggg cgcagaaaat tcgggacaga ccacgtagag 420
gtgggctccc aagcaggtgc ggacggcacc aggccgccca aggcatcgct gccacctgag 480
ctccagccgc ccacaaactg ctgcatgagt ggctgcccca actgcgtgtg ggtggagtac 540
gcggacaggc tgctgcagca cttccaggac ggtggggagc gggccctggc tgccctggag 600
31/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
gagcacgtgg ctgatgagaa cctcaaggcc ttcctcagga tggagatccg gctgcacacc 660
aggtgcggag gctgagccat ccctgctgga ctccctaccg caggacggag tccaggacgc 720
agccgcagcc tccttccttc acaccccctc acagactcct tgtgtccaac gggaatagga 780
agaattagtt actgacttca cctgagaaaa aaataaattc tctatggtgg tttcaaaaaa 840
aaaaaaaaaa aaaaaaaaa 859
<210> 37
<211> 2302
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5483256CB1
<400> 37
gtttccgggg ctttcagtgg ccggaagtcg cggcgcctgt actgactcta ggaagggctg 60
gagtgttttg aatgggcgcc cgtaagagag gtgggcaagt acgtgttaca gacggccacg 120
ccgcecttta ggcggtcaag gtggggcgag cagacgttcg cccccctgca gtcggccggg 180
tcactaccca agagcctttg gaggcggaag catggaacgg tctgcaaacg ttcccgagcg 240
ggcctctgcg gctctggcgg gcgtttcgaa cttgggcgcc gggcacacgc ccagtcccga 300
gagcgctgag ggttccctta gcgtcgccct caccccggcc aacccgcggg gcgccagagt 360
cctggccctt taaacgccgc gcgtgcctcg gcgtcttcgt ttcgcgcgcc cggccgcggc 420
gccggcggag cgaacatgga cccggctgcg cgggtggtgc gggcgctgtg gcctggtggg 480
tgcgccttgg cctggaggct gggaggccgc ccccagccgc tgctacccac gcagagccgg 540
gctggcttcg cgggggcggc gggcggcccg agccccgtgg ctgcagctcg taaggggagc 600
ccgcggctgc tgggagctgc ggcgctggcc ctggggggag ccctggggct gtaccacacg 660
gcgcggtggc acctgcgcgc ccaggacctc cacgcagagc gctcagccgc gcagctctcc 720
ctgtccagcc gcctgcagct gaccctgtac cagtacaaga cgtgtccctt ctgcagcaag 780
gtccgagcct tcctcgactt ccatgccctg ccctaccagg tggtggaggt gaaccctgtg 840
cgcagggctg agatcaagtt ctcctcctac agaaaggtgc ccatcctggt ggcccaggaa 900
ggagaaagct cgcaacaact aaatgactcc tctgtcatca tcagcgccct caagacctac 960
ctggtgtcgg ggcagcccct ggaagagatc atcacctact acccagccat gaaggctgtg 1020
aacgagcagg gcaaggaggt gaccgagttc ggcaataagt actggctcat gctcaacgag 1080
aaggaggccc agcaagtgta tggtgggaag gaggccagga cggaggagat gaagtggcgg 1140
cagtgggcgg acgactggct ggtgcacctg atctccccca atgtgtaccg cacgcccacc 1200
gaggctctgg cgtcctttga ctacattgtc cgcgagggca agttcggagc cgtggagggt 1260
gccgtggcca agtacatggg tgcagcggcc atgtacctca tcagcaagcg actcaagagc 1320
aggcaccgcc tccaggacaa cgtgcgcgag gacctctatg aggctgctga caagtgggtg 1380
gctgctgtgg gcaaggaccg gcccttcatg gggggccaga agccgaatct cgctgatttg 1440
gcggtgtatg gcgtgctgcg tgtgatggag gggctggatg cattcgatga cctgatgcag 1500
cacacgcaca tccagccctg gtacctgcgg gtggagaggg ccatcaccga ggcctcccca 1560
gcgcactgaa tgtccccgcg cagagcagag ggaaggcagc ggaagacgcc agctgccagg 1620
gcctggggcc actgggccag cgcctggcga tactggttgg gggcaggatc attctgcccc 1680
ttgtccacgc acccccacca gccctctcgc ttctaacaca gggcacctgc tggggctcag 1740
ggatgttagg gacgagttcc agccctgcca ctgccctggg gcgacccctc cctgtccctg 1800
cctccctgct ctgccgcccc tcttcctgga ccctcagtgg ctgtcccatg gctacatcct 1860
gtgggtgggg gccctcgaca ggacagcagg acggtttgtt ttcagtggaa tcccatccct 1920
gggttcccct ggttcccact cttcccaagc ctcccgggac tgggacatgt ttgcaataaa 1980
ggaaaggttt gtggcgcctg tcatggcagg catctcatgg agctccgtgt ggctgagtgc 2040
tgcgtggggc tggcggtcaa gggaggcatc aggcttgggc tgtgccagcc cttgtggtaa 2100
ctaaccgctg gcctggggct tcccaggtgt caggcacggt acggctccgc aggctttgtg 2160
tggcatcgtc cccaggatac cactcagggc acacagctgg gccgtggagc ccagcagcca 2220
gagtgcaggt cggggcaccc tacccacggt ggggctctgc agtggggtca ctcatcaagc 2280
ctcagtttct tcgtcatgtc cc 2302
<210> 38
<211> 1653
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5741354CB1
32/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
<400> 38
ctgggtctca gggctgctgt ggagttcgca cctccagctc gggccgatgt ggaagctttg 60
gagagctgaa gagggcgcgg cggcgctcgg cggcgcgctc ttcctgctgc tcttcgcgct 120
aggggtccgc cagctgctga agcagaggcg gccgatgggc ttccccccgg ggccgccggg 180
gctgccattt atcggcaaca tctattccct ggcagcctca tccgagcttc cccatgtcta 240
catgagaaag cagagccagg tgtacggaga gatcttcagt ttagatcttg gaggcatatc 300
aactgtggtt ctaaatggct atgatgtagt aaaggaatgc cttgttcatc aaagcgaaat 360
ttttgcagac agaccatgcc ttcctttatt catgaagatg acaaaaatgg gaggcttact 420
caattccaga tatggccgag gatgggttga tcacagacga ttagctgtaa acagttttcg 480
atattttgga tatggccaaa agtcttttga atctaaaatc ttggaagaaa ccaaattttt 540
caatgatgct attgaaacat acaaaggtag accttttgac tttaaacagt taataacgaa 600
tgctgtttca aacataacca atctgatcat ttttggagaa cgattcactt atgaagacac 660
cgattttcag cacatgattg agttatttag tgaaaatgtg gaactagctg ccagtgcctc 720
agtcttcttg tataatgcct ttccatggat tggcatcctg ccttttggaa aacatcaaca 780
gctgtttaga aatgcagctg tagtctatga ttttctctcc agactcattg aaaaagcttc 840
agtcaacaga aagcctcagc tacctcagca ttttgttgat gcttatttag atgagatgga 900
tcaaggtaaa aatgacccat catctacttt ctccaaagaa aacctaattt tctcagtggg 960
tgaactcatc attgctggaa ctgaaactac aaccaatgtg ctacggtggg cgattctttt 1020
catggccctt tatcctaata ttcaaggaca agttcagaaa gagattgatt taattatggg 1080
ccctaatggg aagccttctt gggacgacaa atgcaaaatg ccttatactg aggcagtttt 1140
gcatgaagtt ttaagattct gtaatatagt tccattaggg attttccatg caacctctga 1200
agatgcagtt gtacgtggtt attccattcc taaaggcaca acagtaatta caaatcttta 1260
ttctgtacac tttgatgaaa agtactggag agacccagaa gtgttccatc ctgagcgatt 1320
tctggacagc agtggatatt ttgccaagaa ggaagctttg gttccttttt ccctaggaag 1380
aagacattgt cttggagaac acttggctcg gatggaaatg ttcttgtttt ttacagcatt 1440
gcttcagagg tttcatttgc attttccaca tgaactagtt ccagatctga agcccaggtt 1500
aggcatgaCa ttgcagcccc aaccctacct catctgtgct gaaagacgct gaaactgcct 1560
gggatgtttt cgggaacaag aatgtatatt tgccttatcc ctgaacttgg tttaatcaaa 1620
tcaatgtgtg tattagaata aaagtcacag cat 1653.
<210> 39
<211> 683
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5872615CB1
<400> 39
cgcgcgaccc cggactccac ggaggccgcg gcgagcaggc ggagctgcgg gtcgggacgc 60
tctgcgtggg gcggggcgca agggaggttt cgagcccgga aggtccggcg cccagagcta 120
acgggagtcc caggttaaac actttaagat gagaaaaatt gatctctgtc tgagctctga 180
agggtccgaa gtgattttag ctacatcaag tgatgaaaaa cacccacctg aaaatatcat 240
tgatgggaat ccagaaacgt tttggaccac cacaggaatg tttccccagg aattcattat 300
ttgtttccac aaacatgtaa ggattgaaag gcttgtaatc caaagttact ttgtacagac 360
cttgaagatt gaaaaaagca cgtctaaaga gccagttgat tttgagcaat ggattgaaaa 420
agatttggta cacacagagg ggcagcttca aaatgaagaa attgtggcac atgatggctc 480
cgctacttac ttgagattca ttattgtatc agcctttgat cattttgcat ctgtgcatag 540
cgtttctgca gaaggaacag tagtctcaaa tctttcctca taatgataac aaaatgctct 600
tgcatgattt tttaacaata tatttaaaca ggaagttgtc actgatatac tttattaaaa 660
ggatttttat caaaaaaaaa aaa 683
<210> 40
<211> 657
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2657543CB1
<400> 40
atgctatcca cttttgccag gcagaatgac atcccttttc agctgcagac agtggagttg 60
33!39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
gcttgggggg agcacctgaa gcctgagttc ctgaaggtga accccctggg gaaggtgcct 120
gccctcagag atggcgactt cctactagca gagaggctgg agaaaagatc tctgacaccc 180
cctgcccaca gcatggtcat cgttttatac ctgagtcgaa agtaccagat acggggacac 240
tggtacccac ctgagctgca agcccgcacc tgcgtggatg agtacttggc gtggaagcat 300
gtcaccatcc agctgcctgc caccaatgtc tacctgtgca agcctgcaga tgctgcacag 360
ctggagcggc tgttggggag gctgacgcca gccctgcagc acctggatgg gggggtcctg 420
gtggccaggc ccttcctggc ~aatggagcag atctccctgg aagacttggt gctgacggag 480
gtgatgcagg tgaagctttc ctacccacct gccctcgggg ggactctggg catggggctg 540
agCCCCaaCC CCagCtgCCC tgtCttCCCa gCCCaCtgCC gttggctgcg acctCttCCa 600
agactggccc tggctggcag tgtgacaggc ccatatgaag gctgcccttg gtactga 657
<210> 41
<211> 1122
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3041639CB1
<400> 41
tggatctgcg ggaatgtggg ctggagaggt CCtgCCgtgg taCCagCCtC CagCCtgCCC 60
ccaggactgc ccctgaccca ggcgcgcccg ctgctcggtg gcaggagggc cggcggagcg 120
ccatggcctg catcctgaag agaaagtctg tgattgctgt gagcttcata gcagcgttcc 180
ttttcctgct ggttgtgcgt cttgtaaatg aagtgaattt cccattgcta ctaaactgct 240
ttggacaacc tggtacaaag tggataccat tctcctacac atacaggcgg ccccttcgaa 300
ctcactatgg atacataaat gtgaagacac aagagccttt gcaactggac tgtgaccttt 360
gtgccatagt gtcaaactca ggtcagatgg ttggccagaa ggtgggaaat gagatagatc 420
gatcctcctg catttggaga atgaacaatg cccccaccaa aggttatgaa gaagatgtcg 480
gccgcatgac catgattcga gttgtgtccc ataccagcgt tcctcttttg ctaaaaaacc 540
ctgattattt tttcaaggaa gcgaatacta ctatttatgt tatttgggga cctttccgca 600
atatgaggaa agatggcaat ggcatcgttt acaacatgtt gaaaaagaca gttggtatct 660
atccgaatgc ccaaatatac gtgaccacag agaagcgcat gagttactgt gatggagttt 720
ttaagaagga aactgggaag gacagtacag agtgaccatg cagtgttgat tgatcgaaca 780
gcaaccacca catacatgtc ctgccccacc acaaaaggaa ggaaggaata aaagaaagaa 840
agaaagaaac aaacaaacaa acaaacaaaa ctaagcaaga caaaacaaat acccatgtca 900
gtggttcaaa gattaagatt gtggctttgt gtaaagttct ttccctttgt agacttgctg 960
cataattatt caggtatgat ggttacagtt tttaaaaagg aagggaaatt gtggtatgtg 1020
gtatgtaaat atttttaaat gttgtctctc tgttttgatc agtttttgtt ttattcaatt 1080
tgtctttatt aaatcttatc caagccaaaa aaaaaaaaaa ag 1122
<210> 42
<211> 2982
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3595451CB1
<400> 42
agccggtacc ggcgggcagg aggcgcccga ggatgtgctg ctggccgctg ctcctgctgt 60
gggggctgct ccccgggacg gcggcggggg gctcgggccg aacctatccg caccggaccc 120
tcctggactc ggagggcaag tactggctgg gctggagcca gcggggcagc cagatcgcct 180
tccgcctcca ggtgcgcact gcaggctacg tgggcttcgg cttctcgccc accggggcca 240
tggcgtccgc cgacatcgtc gtgggcgggg tggcccacgg gcggccctac ctccaggatt 300
attttacaaa tgcaaataga gagttgaaaa aagatgctca gcaagattac catctagaat 360
atgccatgga aaatagcaca cacacaataa ttgaatttac cagagagctg catacatgtg 420
acataaatga caagagtata acggatagca ctgtgagagt gatctgggcc taccaccatg 480
aagatgcagg agaagctggt cccaagtacc atgactccaa taggggcacc aagagtttgc 540
ggttattgaa tcctgagaaa actagtgtgc tatctacagc cttaccatac tttgatctgg 600
taaatcagga cgtccccatc ccaaacaaag atacaacata ttggtgccaa atgtttaaga 660
ttcctgtgtt ccaagaaaag catcatgtaa taaaggttga gccagtgata cagagaggcc 720
atgagagtct ggtgcaccac atcctgctct atcagtgcag caacaacttt aacgacagcg 780
34/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
ttctggagtc cggccacgag tgctatcacc ccaacatgcc cgatgcattc ctcacctgtg 840
aaactgtgat ttttgcctgg gctattggtg gagagggctt ttcttatcca cctcatgttg 900
gattatccct tggcactcca ttagatccgc attatgtgct cctagaagtc cattatgata 960
atcccactta tgaggaaggc ttaatagata attctggact gaggttattt tacacaatgg 1020
atataaggaa atatgatgct ggggtgattg aggctggcct ctgggtgagc ctcttccata 1080
ccatccctcc agggatgcct gagttccagt ctgagggtca ctgcactttg gagtgcctgg 1140
aagaggctct ggaagccgaa aagccaagtg gaattcatgt gtttgctgtt cttctccatg 1200
ctcacctggc tggcagaggc atcaggctgc gtcattttcg aaaagggaag gaaatgaaat 1260
tacttgccta tgatgatgat tttgacttca atttccagga gtttcagtat ctaaaggaag 1320
aacaaacaat cttaccagga gataacctaa ttactgagtg tcgctacaac acgaaagata 1380
gagctgagat gacttgggga ggactaagca cc~ggagtga aatgtgtctc tcataccttc 1440
tttattaccc aagaattaat cttactcgat gtgcaagtat tccagacatt atggaacaac 1500
ttcagttcat tggggttaag gagatctaca gaccagtcac gacctggcct ttcattatca 1560
aaagtcccaa gcaatataaa aacctttctt tcatggatgc tatgaataag tttaaatgga 1620
ctaaaaagga aggtctctcc ttcaacaagc tggtcctcag cctgccagtg aatgtgagat 1680
gttccaagac agacaatgct gagtggtcga ttcaaggaat gacagcatta cctccagata 1740
tagaaagacc ctataaagca gaacctttgg tgtgtggcac gtcttcttcc tcttccctgc 1800
acagagattt ctccatcaac ttgcttgttt gccttctgct actcagctgc acgctgagca 1860
ccaagagctt gtgatcaaaa ttctgttgga cttgacaatg ttttctatga tctgaacctg 1920
tcatttgaag tacaggttaa agactgtgtc cactttgggc atgaagagtg tggagacttt 1980
tcttccccat tttccctccc tcctttttcc tttccatgtt acatgagaga catcaatcag 2040
gttctcttct ctttcttaga aatatctgat gttatatata catggtcaat aaaataaaac 2100
tggcctgact taagataacc attttaaaaa attgggctgt catgtgggaa taaaagaatt 2160
ctttctttcc tactacattc tgttttattt aaatactcat tgttgctatt tcactttttg 2220
acttgacttt tatatttctt taaaaaattc cttcctttta aaaaatataa aagggactac 2280
tgttCattCC agttttCttC ttctttgttg ttcttctagt gtgacttttc aagtgtaaca 2340
gccattcttc ctgactttaa tattgtccag ttctggtctt ttctgtgaat taccactggg 2400
ccccttacct caatgctttt tgttgatgcc cactctggtt cccttgttta tctgagtctg 2460
ttggtacccc aaatgacccc acacccatct taaagtactt tttttcacct tccctgttta 2520
gtactggcca gatgagtttt ttctagagct ctgtcactat ctgaaaagaa agaggctatg 2580
ggaaacatag aaatggtatg tattaataac tgatcatagg ctgaggagaa aaaatgtagc 2640
tggctgcaaa cccagtgctg tgaggtgact tatatgaggt tccagatcaa agacaggccg 2700
tgtgagccag tccaggaggg tgtaagttct gaatggttcc ttgctgactt tgggtgacac 2760
atgtaccaca tactggctca gtttaagtca tggttctatt gtagatttat ttttatatta 2820
gttaataaat gactttaaat tgtcaccaat tgaaaatctt gtcactcttt tggttttctt 2880
tatatagctc agccaaatct tgttttatgt cctgtcctca tctcttaagc taaatctgtt 2940
tggatcatat taataaacta aatgaaatta aaaaaaaaaa as 2982
<210> 43
<211> 3517
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4169101CB1
<400> 43
tggccactat tacggcgcag tgtgctggaa aggcggggct caggctcctt gcagattcct 60
aaccagcata atgctggagc cgggagccac caacctgcag ttttcagaat ggccgtgttg 120
gacactgatt tggatcacat tcttccatct tctgttcttc ctccattctg ggctaagtta 180
gtagtgggat cggttgccat tgtgtgtttt gcacgcagct atgatggaga ctttgtcttt 240
gatgactcag aagctattgt taacaataag gacctccaag cagaaacgcc cctgggggac 300
ctgtggcatc atgacttctg gggcagtaga ctgagcagca acaccagcca caagtcctac 360
cggcctctca ccgtcctgac tttcaggatt aactactacc tctcgggagg cttccacccc 420
gtgggctttc acgtggtcaa catcctcctg cacagtggca tctctgtcct catggtggac 480
gtcttctcgg ttctgtttgg cggcctgcag tacaccagta aaggccggag gctgcacctc 540
gcccccaggg cgtccctgct ggccgcgctg ctgtttgctg tccatcctgt gcacaccgag 600
tgtgttgctg gtgttgtcgg ccgtgcagac ctcctgtgtg ccctgttctt cttgttatct 660
ttccttggct actgtaaagc atttagagaa agtaacaagg agggagcgca ttcttccacc 720
ttctgggtgc tgctgagtat ctttctggga gcagtggcca tgctgtgcaa agagcaaggg 780
atcactgtgc tgggtttaaa tgcggtattt gacatcttgg tgataggcaa attcaatgtt 840
ctggaaattg tccagaaggt actacataag gacaagtcat tagagaatct cggcatgctc 900
aggaacgggg gcctcctctt cagaatgacc ctgctcacct ctggaggggc tgggatgctc 960
35/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
tacgtgcgct ggaggatcat gggcacgggc ccgccggcct tcaccgaggt ggacaacccg 1020
gcctcctttg ctgacagcat gctggtgagg gccgtaaact acaattacta ctattcattg 1080
aatgcctggc tgctgctgtg tccctggtgg ctgtgttttg attggtcaat gggctgcatc 1140
cccctcatta agtccatcag cgactggagg gtaattgcac ttgcagcact ctggttctgc 1200
ctaattggcc tgatatgcca agccctgtgc tctgaagacg gccacaagag aaggatcctt 1260
actctgggcc tgggatttct cgttatccca tttctccccg cgagtaacct gttcttccga 1320
gtgggcttcg tggtcgcgga gcgtgtcctc tacctcccca gcattgggta ctgtgtgctg 1380
ctgacttttg gattcggagc cctgagcaaa cataccaaga aaaagaaact cattgccgct 1440
gtcgtgctgg gaatcttatt catcaacacg ctgagatgtg tgctgcgcag cggcgagtgg 2500
cggagtgagg aacagctttt cagaagtgct ctgtctgtgt gtcccctcaa tgctaaggtt 1560
cactacaaca ttggcaaaaa cctggctgat aaaggcaacc agacagctgc catcagatac 1620
taccgggaag ctgtaagatt aaatcccaag tatgttcatg ccatgaataa tcttggaaat 1680
atcttaaaag aaaggaatga gctacaggaa gctgaggagc tgctgtcttt ggctgttcaa 1740
atacagccag actttgccgc tgcgtggatg aatctaggca tagtgcagaa tagcctgaaa 1800
cggtttgaag cagcagagca aagttaccgg acagcaatta aacacagaag gaaataccca 1860
gactgttact acaacctcgg gcgtctgtat gcagatctca atcgccacgt ggatgccttg 1920
aatgcgtgga gaaatgccac cgtgctgaaa ccagagcaca gcctggcctg gaacaacatg 1980
attatactcc tcgacaatac aggtaattta gcccaagctg aagcagttgg aagagaggca 2040
ctggaattaa tacctaatga tcactctctc atgttctcgt tggcaaacgt gctggggaaa 2100
tcccagaaat acaaggaatc tgaagcttta ttcctcaagg caattaaagc aaatccaaat 2160
gctgcaagtt accatggtaa tttggctgtg ctttatcatc gttggggaca tctagacttg 2220
gccaagaaac actatgaaat ctccttgcag cttgacccca cggcatcagg aactaaggag 2280
aattacggtc tgctgagaag aaagctagaa ctaatgcaaa agaaagctgt ctgatcctgt 2340
ttccttcatg ttttgagttt gagtgtgtgt gtgcatgagg catatcatta atagtatgtg 2400
gttacattta accatttaaa agtcttagac atgttatttt actgattttt ttctatgaaa 2460
acaaagacat gcaaaaagat tatagcacca gcaatatact cttgaatgcg tgatatgatt 2520
tttcattgaa attgtatttt ttcagacaac tcaaatgtaa ttctaaaatt ccaaaaatgt 2580
cttttttaat taaacagaaa aagagaaaaa attatcttga gcaactttta gtagaattga 2640
gcttacattt gggatctgag ccttgtcgtg tatggactag cactattaaa cttcaattat 2700
gaccaagaaa ggatacactg gcccctacaa tttgtataaa tattgaacat gtctatatat 2760
tagcattttt atttaatgac aaagcaaatt aagttttttt atctcttttt tttaaaacaa 2820
catactgtga actttgtaag gaaatattta tttgtatttt tatgttttga atagggcaaa 2880
taatcgaatg aggaatggaa gttttaacat agtatatcta tatgcttttc cccataggaa 2940
gaaattgact cttgcagttt ttggatgctc tgacttgtgc aatttcaata cacaggagat 3000
tatgtaatgt aatatttttc ataagcggtt actatcaatt gaaagttcaa gccatgcttt 3060
aggcaagagc aggcagcctc acatctttat ttttgttaca tccaaggtga agagggcaac 3120
acatctgtgt aagctgcttt ttagtgtgtt tatctgaagg ccgttttcca ttttgcttaa 3180
tgtaactaca gacattatcc agaaaatgca aaattttcta tcaaatggag ccacattcgg 3240
ggaattcgtg gtatttttaa gaattgagtt gttcctgctg ttttttattt gatccaaaca 3300
atgttttgtt ttgttcttct ctgtatgctg ttgacctaat gatttatgca atctctgtaa 3360
tttcttatgc agtaaaatta ctacacaaac tagcatgaaa atgtcatatt gccttcttaa 3420
tcaattattt tcaagtagtg aactttgtat cctcctttac cttaaaatga aatcaaactg 3480
accaaatcat catttatgtg gcttctgtgt gacttgg 3517
<210> 44
<211> 2339
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_~eature
<223> Incyte ID No: 2925182CB1
<400> 44
ggcagccgcg ggagcacggc gacgccagcg gggtgaaggg aaaaggccga ggcatcagcg 60
tgtgaagacc gcaaagacga tcccgagtac agttgtgaac agcattgctg ctaggctcct 120
cctgcagatc atctgaaatg aacctctctt attgattttt attggcctag agccaggagt 180
actgcattca gttgactttc agggtaaaaa gaaaacagtc ctggttgttg tcatcataaa 240
catatggacc agtgtgatgg tgaaatgaga tgaggctccg caatggaact gtagccactg 300
ctttagcatt tatcacttcc ttccttactt tgtcttggta tactacatgg caaaatggga 360
aagaaaaact gattgcttat caacgagaat tccttgcttt gaaagaacgt cttcgaatag 420
ctgaacacag aatctcacag cgctcttctg aattaaatac gattgtgcaa cagttcaagc 480
gtgtaggagc agaaacaaat ggaagtaagg atgcgttgaa taagttttca gataataccc 540
taaagctgtt aaaggagtta acaagcaaaa aatctcttca agtgccaagt atttattatc 600
36/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
atttgcctca tttattgaaa aatgaaggaa gtcttcaacc tgctgtacag attggcaacg 660
gaagaacagg agtttcaata gtcatgggca ttcccacagt gaagagagaa gttaaatctt 720
acctcataga aactcttcat tcccttattg ataacctgta tcctgaagag aagttggact 780
gtgttatagt agtcttcata ggagagacag atattgatta tgtacatggt gttgtagcca 840
acctggagaa agaattttct aaagaaatca gttctggctt ggtggaagtc atatcacccc 900
ctgaaagcta ttatcctgac ttgacaaacc taaaggagac atttggagac tccaaagaaa 960
gagtaagatg gagaacaaag caaaacctag attactgttt tctaatgatg tatgctcaag 1020
aaaagggcat atattacatt cagcttgaag atgatattat tgtcaaacaa aattatttta 1080
ataccataaa aaattttgca cttcaacttt cttctgagga atggatgatt ctagagtttt 1140
cccagctggg cttcattggt aaaatgtttc aagcgccgga tcttactctg attgtagaat 1200
tcatattcat gttttacaag gagaaaccca ttgattggct cctggaccat attctctggg 1260
tgaaagtctg caaccctgaa aaagatgcaa aacattgtga tagacagaaa gcaaatctgc 1320
gaattcgctt cagaccttcc cttttccaac atgttggtct gcactcatca ctatcaggaa 1380
aaatccaaaa actcacggat aaagattata tgaaaccatt acttcttaaa atccatgtaa 1440
acccacctgc ggaggtatct acttccttga aggtctacca agggcatacg ctggagaaaa 1500
cttacatggg agaggatttc ttctgggcta tcacaccgat agctggagac tacatcttgt 1560
ttaaatttga taaaccagtc aatgtagaaa gttatttgtt ccatagcggc aaccaagaac 1620
atcctggaga tattctgcta aacacaactg tggaagtttt gccttttaag agtgaaggtt 1680
tggaaataag caaagaaacc aaagacaaac gattagaaga tggctatttc agaataggaa 1740
aatttgagaa tggtgttgca gaaggaatgg tggatccaag tctcaatccc atttcagcct 1800
ttcgactttc agttattcag aattctgctg tttgggccat tcttaatgag attcatatta 1860
aaaaagccac caactgatca tctgagaaac caacacattt tttcctgtga atttgttaat 1920
taaagatagt taagcatgta tctttttttt atttctactt gaacactacc tcttgtgaag 1980
tctactgtag ataagacgat tgtcgtttcc acttggaaag tgaatctccc ataataattg 2040
tatttgtttg aaactaagct gtcctcagat tttaacttga ctcaaacatt tttcaattat 2100
gacagcctgt taatatgact tgtactattt tgggtattat actaataaca taagagttgt 2160
acatattgtt acattcttta aatttgagaa aaactaatgt tacatacatt ttatgaaggg 2220
gggtactttt gagattcact tattttacta ttatagaccc tcttttatag attatcaggg 2280
attatatata taaatatata aatatacata aaaatgttat ggattaattt attagaaca 2339
<210> 45
<211> 1955
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3271838CB1
<400> 45
gccccagaag ccccacgacg atggcggcaa tggcggtggc gctgcgggga ttaggagggc 60
gcttccggtg gcggacgcag gccgtggcgg gcggggtgcg gggcgcggcg cggggcgcag 120
cagcaggtca gcgggactat gatctcctgg tggtcggcgg gggatctggt ggcctggctt 180
gtgccaagga ggccgctcag ctgggaagga aggtgtccgt ggtggactac gtggaacctt 240
ctccccaagg cacccggtgg ggccttggcg gcacctgcgt caacgtgggc tgcatcccca 300
agaagctgat gcaccaggcg gcactgctgg gaggcctgat ccaagatgcc cccaactatg 360
gctgggaggt ggcccagccc gtgccgcatg actggaggaa gatggcagaa gctgttcaaa 420
atcacgtgaa atccttgaac tggggccacc gtgtccagct tcaggacaga aaagtcaagt 480
actttaacat caaagccagc tttgttgacg agcacacggt ttgcggcgtt gccaaaggtg 540
ggaaagagat tctgctgtca gccgatcaca tcatcattgc tactggaggg cggccgagat 600
accccacgca catcgaaggt gccttggaat atggaatcac aagtgatgac atcttctggc 660
tgaaggaatc ccctggaaaa acgttggtgg tcggggccag ctatgtggcc ctggagtgtg 720
ctggcttcct caccgggatt gggctggaca ccaccatcat gatgcgcagc atccccctcc 780
gcggcttcga ccagcaaatg tcctccatgg tcatagagca catggcatct catggcaccc 840
ggttcctgag gggctgtgcc ccctcgcggg tcaggaggct ccctgatggc cagctgcagg 9,00
tcacctggga ggaccgcacc accggcaagg aggacacggg cacctttgac accgtcctgt 960
gggccatagg tcgagtccca gacaccagaa gtctgaattt ggagaaggct ggggtagata 1020
ctagccccga cactcagaag atcctggtgg actcccggga agccacctct gtgccccaca 1080
tctacgccat tggtgacgtg gtggaggggc ggcctgagct gacacccaca gcgatcatgg 1140
ccgggaggct cctggtgcag cggctcttcg gcgggtcctc agatctgatg gactacgaca 1200
atgttcccac gaccgtcttc accccgctgg agtatggctg tgtggggctg tccgaggagg 1260
aggcagtggc tcgccacggg caggagcatg ttgaggtcta tcacgcccat tataaaccac 1320
tggagttcac ggtggctgga cgagatgcat cccagtgtta tgtaaagatg gtgtgcctga 1380
gggagccccc acagctggtg ctgggcctgc atttccttgg ccccaacgca ggcgaagtta 1440
37/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
ctcaaggatt tgctctgggg atcaagtgtg gggcttccta tgcgcaggtg atgcggaccg 1500
tgggtatcca tcccacatgc tctgaggagg tagtcaagct gcgcatctcc aagcgctcag 1560
gcctggaccc cacggtgaca ggctgctgag ggtaagcgcc atccctgcag gccagggcac 1620
acggtgcgcc cgccgccagc tcctcggagg ccagacccag gatggctgca ggccaggttt 1680
ggggggcctc aaccctctcc tggagcgcct gtgagatggt cagcgtggag cgcaagtgct 1740
ggacaggtgg cccgtgtgcc ccacagggat ggctcagggg actgtccacc tcacccctgc 1800
acctctcagc ctctgccgcc gggcaccccc ccccaggctc ctggtgccag atgatgacga 1860
cctgggtgga aacctaccct gtgggcaccc atgtccgagc cccctggcat ttctgcaatg 1920
caaataaaga gggtactttt tctgaaaata aaaaa 1955
<210> 46
<211> 2065
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3292871CB1
<400> 46
ctaggcccta cttcgcagtt cttgtgcacg ctatgaaaaa taaaacctgc gtgctcgtct 60
gtgtgagtgt gtttggtggg gagagggggc aggtgactgt accccgggtt ggggtccgcc 120
gcccctccct cgcgggccct ctgcagaagt gcaccctgag agagacccgg gtgtggctcc 180
cgcagggttc tggcttccag tcgtcgcgga gggagaagta tggcaacgtg ttcaagacgc 240
atttgttggg gcggccgctg atacgcgtga ccggcgcgga gaacgtgcgc aagatcctca 300
tgggcgagca ccacctcgtg agcaccgagt ggcctcgcag cacccgcatg ttgctgggcc 360
ccaacacggt gtccaattcc attggcgaca tccaccgcaa caagcgcaag gtcttctcca 420
agatcttcag ccacgaggcc ctggagagtt acctgcccaa gatccagctg gtgatccagg 480
acacactgcg cgcctggagc agccaccccg aggccatcaa cgtgtaccag gaggcgcaga 540
agctgacctt ccgcatggcc atccgggtgc tgctgggctt cagcatccct gaggaggacc 600
ttgggcacct ctttgaggtc taccagcagt ttgtggacaa tgtcttctcc ctgcctgtcg 660
acctgccctt cagtggctac cggcggggca ttcaggctcg gcagatcctg cagaaggggc 720
tggagaaggc catccgggag aagctgcagt gcacacaggg caaggactac ttggacgccc 780
tggacctcct cattgagagc agcaaggagc acgggaagga gatgaccatg caggagctga 840
aggacgggac cctggagctg atctttgcgg cctatgccac cacggccagc gccagcacct 900
cactcatcat gcagctgctg aagcacccca ctgtgctgga gaagctgcgg gatgagctgc 960
gggctcatgg catcctgcac agtggcggct gcccctgcga gggcacactg cgcctggaca 1020
cgctcagtgg gctgcgctac ctggactgcg tcatcaagga ggtcatgcgc ctgttcacgc 1080
ccatttccgg cggctaccgc actgtgctgc agaccttcga gcttgatggt ttccagatcc 1140
ccaaaggctg gagtgtcatg tatagcatcc gggacaccca tgacacagcg cccgtgttca 1200
aagacgtgaa cgtgttcgac cccgatcgct tcagccaggc gcggagcgag gacaaggatg 1260
gccgcttcca ttacctcccg.ttcggtggcg gtgtccggac ctgcctgggc aagcacctgg 1320
ccaagctgtt cctgaaggtg ctggcggtgg agctggctag caccagccgc tttgagctgg 1380
CCaCdCggaC cttcccccgc atCaCCttgg tCCCCgtCCt gCaCCCCgtg gatggcctca 1440
gcgtcaagtt ctttggcctg gactccaacc agaacgagat cctgccggag acggaggcca 1500
tgctgagcgc cacagtctaa cccaagaccc acccgcctca gCCCagCCCa ggcagcgggg 1560
tggtgcttgt gggaggtaga aacctgtgtg tgggaggggg ccggaacggg gagggcgagt 1620
ggcccccata cttgccctcc cttgctcccc cttcctggca aaccctaccc aaagccagtg 1680
ggccccattc ctagggctgg gctccccttc tggctccagc ttccctccag ccactcccca 1740
tttaccatca gctcagcccc tgggaagggc gtggcagggg ctctgcatgc ccgtgacagt 1800
gttaggtgtc agcgcgtgct acagtgtttt tgtgatgttc tgaactgctc ccttccctcc 1860
gttcctttcg gaccctttta gctggggttg ggggacggga agagccgtgc ccccttgggc 1920
gcactcttca gcgtctcctc ctcctgcgcc cccactgcgt ctgcccagga acagcatcct 1980
gggtagcaga acaggagtca accttggcgg ggcgggggct gcgtccaacc tggagattgc 2040
ccttccctat gccacggttc.ccacc 2065
<210> 47
<211> 866
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4109179CB1
38/39
CA 02397340 2002-07-11
WO 01/51638 PCT/USO1/01174
<400> 47
ttcaaaagag gtttctggtc actcctaatc atcgcagcat aactctgctt tttaagctat 60
tgttttctgc atttgtaggg gcacgataca actgcagctg caataaactg gtccttatac 120
ctgttgggtt ctaacccaga agtccagaaa aaagtggatc atgaattgga tgacgtgttt 180
gggaagtctg accgtcccgc tacagtagaa gacctgaaga aacttcggta tctggaatgt 240
gttattaagg agacccttcg cctttttcct tctgttcctt tatttgcccg tagtgttagt 300
gaagattgtg aagtggcagg ttacagagtt ctaaaaggca ctgaagccgt catcattccc 360
tatgcattgc acagagatcc gagatacttc cccaaccccg aggagttcca gcctgagcgg 420
ttcttccccg agaatgcaca agggcgccat ccatatgcct acgtgccctt ctctgctggc 480
cccaggaact gtataggtca aaagtttgct gtgatggaag aaaagaccat tctttcgtgc 540
atcctgaggc acttttggat agaatccaac cagaaaagag aagagcttgg tctagaagga 600
cagttgattc ttcgtccaag taatggcatc tggatcaagt tgaagaggag aaatgcagat 660
gaacgctaac tatattattg ggttgtgcct ttatcatgag aaaggtcttt attttaagag 720
atccttgtca tttacaattt acagatcatg agttcaatat gcttgaatcc cctagaccta 780
atttttcctt gatcccactg atcttgacat caagtctaac aaagaaaaag ttttgagttt 840
tgtattttct tttttctttt ttcttt 866
<210> 48
<211> 1593
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4780365CB1
<400> 48
ttacggcgca gtgtgctgga cagcggtctc ccagggaagg gggtgctgag tggaaggagg 60
tcaatgggaa gccggggtgg ctctcagagt cggcaggagc agtcgggctg atgagctggg 120
aggagcagac cgcctccctc ttctctgagt gggaggaggg ccagatctgg actgggtttg 180
gagatgctca ggtggggctc agagcatcac ctgtggggca gagggaccat cttggcagat 240
gaaggcccgt cgcagggtgt gatgcctgaa ttacaaggcg ggacaggtaa agtggggcag 300
gtgagagaag gagggtgagt gatgtgattt ttctactcct gttttccagg aaaaccaaaa 360
tgccacgcac ttcgacctat gatccttttc ctaataatgc ttgtcttggt cttgtttggt 420
tacggggtcc taagccccag aagtctaatg ccaggaagcc tggaacgggg gttctgcatg 480
gctgttaggg aacctgacca tctgcagcgc gtctcgttgc caaggatggt ctacccccag 540
ccaaaggtgc tgacaccgtg taggaaggat gtcctcgtgg tgaccccttg gctggctccc 600
attgtctggg agggcacatt caacatcgac atcctcaacg agcagttcag gctccagaac 660
accaccattg ggttaactgt gtttgccatc aagaaatacg tggctttcct gaagctgttc 720.
ctggagacgg cggagaagca cttcatggtg ggccaccgtg tccactacta tgtcttcacc 780
gaccagccgg ccgcggtgcc ccgcgtgacg ctggggaccg gtcggcagct gtcagtgctg 840
gaggtgcgcg cctacaagcg ctggcaggac gtgtccatgc gccgcatgga gatgatcagt 900
gacttctgcg agcggcgctt cctcagcgag gtggattacc tggtgtgcgt ggacgtggac 960
atggagttcc gcgaccacgt gggcgtggag atcctgactc cgctgttcgg caccctgcac 1020
cccggcttct acggaagcag ccgggaggcc ttcacctacg agcgccggcc ccagtcccag 1080
gcctacatcc ccaaggacga gggcgatttc tactacctgg gggggttctt cggggggtcg 1140
gtgcaagagg tgcagcggct caccagggcc tgccaccagg ccatgatggt cgaccaggcc 1200
aacggcatcg aggccgtgtg gcacgacgag agccacctga acaagtacct gctgcgccac 1260
aaacccacca aggtgctctc ccccgagtac ttgtgggacc agcagctgct gggctggccc 1320
gccgtcctga ggaagctgag gttcactgcg gtgcccaaga accaccaggc ggtccggaac 1380
ccgtgagcgg ctgccagggg ctctgggagg gctgccggca gCCCCgtCCC CCtCCCgCCC 1440
ttggttttag cagaacgggt aaactctgtt tcctttgtcc gtcctgttgt gagtaactga 1500
agcctaggcc ccgtccccac ctcaaatcac acacaccccc tccccaccac agagacacca 1560
ttacatacac agacacacac agaaagacac aca 1593
39/39
