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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.
1o BACKGROUND OF THE INVENTION
The metabolism of a drug and its movement through the body (pharmacokinetics)
are
important in determining its effects, toxicity, and interactions with other
drugs. The three processes
governing pharmacokinetics are the absorption of the drug, distribution to
various tissues, and
elimination of drug metabolites. These processes are intimately coupled to
drug metabolism, since a
variety of metabolic modifications alter most of the physicochemical and
pharmacological properties of
drugs, including solubility, binding to receptors, and excretion rates. The
metabolic pathways which
modify drugs also accept a variety of naturally occurring substrates such as
steroids, fatty acids,
prostaglandins, leukotrienes, and vitamins. The enzymes in these pathways are
therefore important
sites of biochemical and pharmacological interaction between natural
compounds, drugs, carcinogens,
mutagens, and xenobiotics.
It has long been appreciated that inherited differences in drug metabolism
lead to drastically
different levels of drug efficacy and toxicity among individuals. For drugs
with narrow therapeutic
indices, or drugs which require bioactivation (such as codeine), these
polymorphisms can be critical.
Moreover, promising new drugs are frequently eliminated in clinical trials
based on toxicities which
may only affect a segment of the patient group. Advances in pharmacogenomics
research, of which
drug metabolizing enzymes constitute an important part, are promising to
expand the tools and
information that can be brought to bear on questions of drug efficacy and
toxicity (See Evans, W. E.
and R. V. Relling (1999) Science 286:487-491).
Drug metabolic reactions are categorized as Phase I, which functionalize the
drug molecule
and prepare it for further metabolism, and Phase II, which are conjugative. In
general, Phase I
reaction products are partially or fully inactive, and Phase II reaction
products are the chief excreted
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).
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Additionally, some nontoxic compounds (e.g. aflatoxin, benzo[a]pyrene) are
metabolized to toxic
intermediates through these pathways. Phase I reactions are usually rate-
limiting in drug metabolism.
Prior exposure to the compound, or other compounds, can induce the expression
of Phase I enzymes
however, and thereby increase substrate flux through the metabolic pathways.
(See Klaassen, C. D.,
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. Katzung (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
l0 contrasted to the immune system, where a large and diverse population of
antibodies are highly
specific for their autigens. The ability of DMEs to metabolize a wide variety
of molecules creates the
potential for drug interactions at the level of metabolism. For example, the
induction of a DME by one
compound may affect the metabolism of another compound by the enzyme.
DMEs have been classified according to the type of reaction they catalyze and
the cofactors
involved. The major classes of Phase I enzymes include, but are not limited
to, cytochrome P450 and
flavin-containing monooxygenase. Other enzyme classes involved in Phase I-type
catalytic cycles and
reactions include, but are not limited to, NADPH cytochrome P450 reductase
(CPR), the microsomal
cytochrome b5/NADH cytochrome b5 reductase system, the ferredoxin/ferredoxin
reductase redox
pair, aldo/keto reductases, and alcohol dehydrogenases. The major classes of
Phase )I enzymes
include, but are not limited to, LJDP glucuronyltransferase, sulfotransferase,
glutathione S-transferase,
N-acyltransferase, and N-acetyl transferase.
Cytochrome P450 and P450 catalytic~cle-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 multi-component
electron transfer chains, called P450-containing monooxygenase systems.
Specific reactions
catalyzed include hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-,
S-, and O-dealkylations,
desulfation, deamination, and reduction of azo, vitro, and N-oxide groups.
These reactions are
involved in steroidogenesis of glucocorticoids, cortisols, estrogens, and
androgens in animals;
insecticide resistance in insects; herbicide resistance and flower coloring in
plants; and environmental
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.
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Cytochromes P450 are abundant in the liver, but also occur in other tissues;
the enzymes are located
in microsomes. (See ExPASY ENZYME EC 1.14.14.1; Prosite PDOC00081 Cytochrome
P450
cysteine heme-iron ligand signature; PRINTS EP450I E-Class P450 Group I
signature; Graham-
Lorence, S. and Peterson, J.A. (1996) FASEB J. 10:206-214.)
Four hundred cytochromes P450 have been identified in diverse organisms
including bacteria,
fungi, plants, and animals (Graham-Lorence, su ra). The B-class is found in
prokaryotes and fungi,
while the E-class is found in bacteria, plants, insects, vertebrates, and
mammals. Five subclasses or
groups are found within the larger family of E-class cytochromes P450 (PRINTS
EP450I E-Class
P450 Group I signature).
l0 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
15 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, sera.)
Cytochrome P450 enzymes are involved in cell proliferation and development.
The enzymes
20 have roles in chemical mutagenesis and carcinogenesis by metabolizing
chemicals to reactive
intermediates that form adducts with DNA (Nebert, D.W. and Gonzalez, F.J.
(1987) Ann. Rev.
Biochem. 56:945-993). These adducts can cause nucleotide changes and DNA
rearrangements that
lead to oncogenesis. Cytochrome P450 expression in liver and other tissues is
induced by xenobiotics
such as polycyclic aromatic hydrocarbons, peroxisomal proliferators,
phenobarbital, and the
25 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 (OM1M)
*601771
Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYP1B 1).
Cytochromes P450 are associated with inflammation and infection. Hepatic
cytochxome P450
30 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-
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candidiasis-ectodermal dystrophy (APECED), a polyglandular autoimmune syndrome
(OMIM
*240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy).
Mutations in cytochromes P450 have been linked to metabolic disorders,
including congenital
adrenal hyperplasia, the most common adrenal disorder of infancy and
childhood; pseudovitamin D-
deficiency rickets; cerebrotendinous xanthomatosis, a lipid storage disease
characterized by
progressive neurologic dysfunction, premature atherosclerosis, and cataracts;
and an inherited
resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J.
et al. (1994) 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 Cournarin
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
cytochrorne 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
(CYP,51) which can be efficiently reduced and supported by the microsomal
cytochrome b5/NADPH
. cytochrorne 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
Da), 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 rr»mal periods of skin exposure to
sunlight (reviewed in
Miller, W.L. and Portale, A.A. (2000) Trends Endocrinol. Metab. 11:315-319).
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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 1a-hydroxylase (la-
hydroxylase). Regulation of
1a,25(OH)2D production is primarily at this final step in the synthetic
pathway. The activity of
la-hydroxylase depends upon several physiological factors including the
circulating level of the
enzyme product (1a,25(OH)ZD) and the levels of parathyroid hormone (PTH),
calcitonin, insulin,
calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal 1a-
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 1 a,25(OH)zD to 24,25-
dihydroxyvitamin D
(24,25(OH)ZD), involving the enzyme 25-hydroxyvitamin D 24-hydroxylase (24-
hydroxylase), also
occurs in the kidney. 24-hydroxylase can also use 25(OH)D as a substrate
(Shinki, T. et al. (1997)
Proc. Natl. Acad. Sci. U.S.A. 94:12920-12925; Miller, W.L. and Portale, A.A.
supra; and references
within).
Vitamin. D 25-hydroxylase, 1a-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,
2o A.A. supra; and references within).
The active form of vitamin D (1a,25(OH)ZD).is involved in calcium and
phosphate
homeostasis and promotes the differentiation of myeloid and skin cells.
Vitamin D deficiency resulting
from deficiencies in the enzymes involved in vitamin D metabolism (e.g., la-
hydroxylase) causes
hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive) rickets, a
disease characterized
by loss of bone density and distinctive clinical features, including bandy or
bow leggedness
accompanied by a waddling gait. Deficiencies in vitamin D 25-hydroxylase cause
cerebrotendinous
xanthomatosis, a lipid-storage disease characterized by the deposition of
cholesterol and cholestanol in
the Achilles' tendons, brain, lungs, and many other tissues. The disease
presents with progressive
neurologic dysfunction, including postpubescent cerebellar ataxia,
atherosclerosis, and cataracts.
Vitamin D 25-hydroxylase deficiency does not result in rickets, suggesting the
existence of alternative
pathways for the synthesis of 25(OI~D (Griffin, J.E. and Zerwekh, J.E. (1983)
J. Clip. Invest.
72:1190-1199; Gamblin, G.T. et al. (1985) J. Clip. Invest. 75:954-960; and
W.L. and Portale, A.A.
supra).
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Ferredoxin and ferredoxin reductase are electron transport accessory proteins
which support
at least one human cytochrome P450 species, cytochrome P450c27 encoded by the
CYP27 gene
(Dilworth, F. J. et al. (1996) Biochem. J. 320:267-71). A Streptomyces griseus
cytochrome P450,
CYP104D1, was heterologously expressed in E. coli and found to be reduced by
the endogenous
ferredoxin and ferredoxin reductase enzymes (Taylor, M. et al. (1999) Biochem.
Biophys. Res.
Commun. 263:838-42), suggesting that many cytochrome P450 species may be
supported by the
ferredoxin/ferredoxin reductase pair. Ferredoxin reductase has also been found
in a model drug
metabolism system to reduce actinomycin D, an antitumor antibiotic, to a
reactive free radical species
(Flitter, W. D. and Mason, R. P. (1988) Arch. Biochem. Biophys. 267:632-9).
Flavin-containing monooxy genase (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 02; there is also a great deal of substrate overlap with
cytochromes P450. The
tissue distribution of PMOs includes liver, kidney, and lung.
. There are five different known isoforms of FMO in mammals (FM01, FM02, FMO3,
FM04,
and FMOS), which are expressed in a tissue-specific manner. The isoforrns
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-
terniinal 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 hydroxylarnines 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
cytochrornes 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 IIz-
antagonist widely used
for the treatment of gastric ulcers. Lever-expressed forms of FMO are not
under the same regulatory
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control as cytochrome P450. In rats, for example, phenobarbital treatment
leads to the induction of
cytochrome P450, but the repression of FMOl.
Endogenous substrates of FMO include cysteamine, which is oxidized to the
disulfide,
cystamine, and 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 (OMINI 602079 Trimethylaminuria).
Lysyl oxidase:
Lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidase
involved in the
formation of connective tissue matrices by crosslinking collagen and elastin.
LO is secreted as 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.
67:996S-1002S and Smith-Mungo. L.I. and Kagan, H.M. (1998) Matrix Biol. 16:387-
398).
Dihydrofolate reductases
Dihydrofolate reductases (DHFR) are ubiquitous enzymes that catalyze the
NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential
step in the de novo
synthesis of glycine and purines as well as the conversion of deoxyuridine
monophosphate (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
7
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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
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 kaown reaction catalyzed by a family member, aldose reductase, is the
reduction of
glucose to sorbitol, which is then further metabolized to fructose by sorbitol
dehydrogenase. Under
normal conditions, the reduction of glucose to sorbitol is a minor pathway. In
hyperglycemic states,
however, the accumulation of sorbitol is implicated in the development of
diabetic complications
(OM1M *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 dehydroaenases
Alcohol dehydrogenases (ADHs) oxidize simple alcohols to the corresponding
aldehydes.
ADH is a cytosolic enzyme, prefers the cofactor NAD+, and also binds zinc ion.
Liver contains the
highest levels of ADH, with lower levels in kidney, lung, and the gastric
mucosa.
Known ADH isoforms are dimeric proteins composed of 40 kDa subunits. There are
five
known gene loci which encode these subunits (a, b, g, p, c), and some of the
loci have characterized
allelic variants (b1, b2, b3, gv gz). 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 1I 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
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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, corticosteroi.d 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
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 (LTGTs)~ 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
2Q such as bilirubin, steroid hormones, and thyroid hormones. Products of
glucuronidation are excreted in
urine if the molecular weight of the substrate is less than. about 250 glmol,
whereas larger
glucuronidated substrates are excreted in bile.
UGTs are located in the microsomes of liver, kidney, intestine, skin, brain,
spleen, and nasal
mucosa, where they are on the same side of the endoplasmic reticulum membrane
as cytochrome
P450 enzymes and flavin-containing monooxygenases, and therefore are ideally
located to access
products of Phase I drug metabolism. UGTs have a C-tern~inal membrane-spanning
domain which
anchors them in the endoplasmic reticulum membrane, and a conserved signature
domain of about 50
amino acid residues in their C terminal section (Prosite PDOC00359 UDP-
glycosyltransferase
signature).
UGTs involved in drug metabolism are encoded by two gene families, UGT1 and
UGT2.
Members of the 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
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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 (OM1M #143500 Hyperbilirubinemia I); Crigler-Najjar
syndrome, characterized by
intense hyperbilirubinemia from birth (OMIM #218800 Crigler-Najjar syndrome);
and a milder form of
hyperbilirubinemia termed Gilbert's disease (OMIM *191740 UGT1).
Sulfotransferase
Sulfate conjugation occurs on many of the same substrates which undergo O-
glucuronidation
to produce a highly water-soluble sulfuric acid ester. Sulfotransferases (ST)
catalyze this reaction by
transferring S03 from the cofactor 3'-phosphoadenosine-5'-phosphosulfate
(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. 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, I~. et al. (1984) J. Biol. Chem. 259:13751-7; OMIM
'217800 Macular
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dystrophy, corneal).
Galactosyltransferases
Galactosyltransferases are a subset of glycosyltransferases that transfer
galactose (Gal) to
the terminal N-acetylglucosamine (GlcNAc) oligosaccharide chains that are part
of glycoproteins or
glycolipids that are free in solution (Kolbinger, F. et al. (1998) J. Biol.
Chem. 273:433-440; Amado, M.
et al. (1999) Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases have
been detected on the
cell surface and as soluble extracellular proteins, in addition to being
present in the Golgi. (31,3-
galactosyltransferases form Type I carbohydrate chains with Gal (~31-3)GlcNAc
linkages. Known
human and mouse (31,3-galactosyltransferases appear to have a short cytosolic
domain, a single
transmembrane domain, and a catalytic domain with eight conserved regions.
(Kolbinger, F. supra and
Rennet, T. et al. (1998) J. Biol. Chem. 273:58-65). In mouse UDP-galactose:(3-
N-acetylglucosamine
(31,3-galactosyltransferase-I region 1 is located at amino acid residues 78-
83, region 2 is located at
amino acid residues 93-102, region 3 is located at amino acid residues 116-
119, region 4 is located at
amino acid residues 147-158, region 5 is located at amino acid residues 172-
183, region 6 is located at
amino acid residues 203-206, region 7 is located at amino acid residues 236-
246, and region 8 is
located at amino acid residues 264-275. A variant of a sequence found within
mouse UDP
galactose:(3-N-acetylglucosamine (31,3-galactosyltransferase-I region 8 is
also found in bacterial
galactosyltransferases, suggesting that this sequence defines a
galactosyltransferase sequence motif
(Rennet, T. su ra). Recent work suggests that brainiac protein is a j31,3-
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)GlcNAc
liukages. As is the case with the (31,3-galactosyltransferase, a soluble form
of the enzyme is formed
by cleavage of the membrane-bound form. Amino acids conserved among j31,4-
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). X31,4-galactosyltransferases have
several specialized
roles in addition to synthesizing carbohydrate chains on glycoproteins or
glycolipids. In mammals a
(31,4-galactosyltransferase, as part of a heterodimer with a-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. (Shur,
ii
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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
S localized mainly in the cytosol, but some level of activity is present in
microsomes as well. The major
isozymes share common structural and catalytic properties; in humans they have
been classified into
four major classes, Alpha, Mu, Pi, and Theta. The two largest classes, Alpha
and Mu, are identified
by their respective protein isoelectric points; pI -- 7.5-9.0 (Alpha), and pI
~ 6.6 (Mu). Each GST
possesses a common binding site for GSH and a variable hydrophobic binding
site. The hydrophobic
binding site in each isozyme is specific for particular electrophilic
substrates. Specific amino acid
residues within GSTs have been identified as important for these binding sites
and for catalytic
activity. Residues Q67, T68, D101, E104, and 8131 are important for the
binding of GSH (Lee, H-C
et al. (1995) J. Biol. Chem. 270: 99-109). Residues R23, 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 effects. Some
forms of rat and human GSTs are reliable preneoplastic markers that aid in the
detection of
carcinogenesis. Expression of human GSTs in bacterial strains, such as
Salmonella typhimurium used
in the well-known Ames test for mutagenicity, has helped to establish the role
of these enzymes in
mutagenesis. Dihalomethanes, which produce liver tumors in mice, are believed
to be activated by
GST. This view is supported by the finding that dihalomethanes are more
mutagenic in bacterial cells
expressing human GST than in untransfected cells (Thier, R. et al. (1993)
Proc. Natl. Acad. Sci. USA
90. 8567-80). The mutagenicity of ethylene dibromide and ethylene dichloride
is increased in bacterial
cells expressing the human Alpha GST, A1-1, while the mutagenicity of
aflatoxin B1 is substantially
reduced by enhancing the expression of GST (Simula, T.P. et al. (1993)
Carcinogenesis 14: 1371-6).
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
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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-elutam, l t~peptidase
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.1'hysiol. 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
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
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genome (or the genome in general). In contrast, histone deacetylation results
in a reduction in
transcription by closing the chromatin structure and limiting access of
transcription factors. To this
end, a common means of stimulating cell transcription is the use of chemical
agents that inhibit the
deacetylation of histories (e.g., sodium butyrate), resulting in a global
(albeit artifactual) increase in
gene expression. The modulation of gene expression by acetylation also results
from the acetylation
of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR,
TFII)E, TFLIF and the high
mobility group proteins (HMG). In the case of p53, acetylation results in
increased DNA binding,
leading to the stimulation of transcription of genes regulated by p53. The
prototypic histone acetylase
(HAT) is GcnS from Saccharomyces cerevisiae. GcnS is a member of a family of
acetylases that
includes Tetrahymena p55, human GcnS, and human p300/CBP. Histone acetylation
is reviewed in
(Cheung, W.L. et al. (2000) Curr. Opin. Cell Biol. 12:326-333 and Berger, S.L
(1999) Curr. Opin. Cell
Biol. 11:336-341). Some acetyltransferase enzymes posses the alpha/beta
hydrolase fold (Center of
Applied Molecular Engineering.lnst. of Chemistry and Biochemistry - University
of Salzburg, '
lzttp://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://stop.mrc-lmb.cam.ac.uk/scop/index.html.).
N-acetyltransferase
Aromatic amines and hydrazine-containing compounds are subject to N-
acetylation by the N
acetyltxansferase enzymes of liver and other tissues. Some xenobiotics can be
O-acetylated to some
extent by the same enzymes. N-acetyltransferases are cytosolic enzymes which
utilize the cofactor
acetyl-coenzyme A (acetyl-CoA). to transfer the acetyl group in a two step
process. In the first step;
the acetyl group is transferred from acetyl-CoA to an active site cysteine
residue; in the second step,
the acetyl group is transferred to the substrate amino group and the enzyme is
regenerated.
In contrast to most other DME classes, there are a limited number of known N-
acetyltransferases. In humans, there are two highly similar enzymes, 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 (pare-aminobenzoic acid, pare-
aminosalicylic acid,
sulfamethoxazole, and sulfanilamide), while NAT2 prefers others (isoniazid,
hydralazine, procainamide,
dapsone, aminoglutethirnide, 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
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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 maybe 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
anninotransferase, branched-chain
15. 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. Iu the absence of AGT,
glyoxylate is oxidized to
oxalate rather than being transaminated to glycine. The result is the
deposition of insoluble calcium
oxalate in the kidneys and urinary tract, ultimately causing renal failure
(Lumb, M.J. et al. ( 1999) J.
Biol. Chem. 274:20587-20596).
Kynurenine aminotransferase catalyzes the irreversible transatnination 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).
Copper-zinc superoxide dismutases
Copper-zinc superoxide dismutases are compact homodimeric metalloenzymes
involved in
n
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 IIz02. 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. sme~rnatis, 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,
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which are essential for cell growth and replication, and topoisomerases, which
break and rejoin nucleic
acid strands during topological rearrangement of DNA. A Tyr-DNA
phosphodiesterase functions in
DNA repair by hydrolyzing dead-end covalent intermediates formed between
topoisomerase I and
DNA (Pouliot, J.J. et al. (1999) Science 286:552-555; Yang, S.-W. (1996) Proc.
Natl. Acad. Sci.
USA 93:11534-11539).
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
l0 concentration in neural tissue. Defective acid sphingomyelinase leads to a
build-up of sphingomyelin
molecules in lysosomes, resulting in Niemann-Pick disease (Schuchmau, 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 bxoad specificity for glycerophosphodiester
substrates (Larson,
T.J. et a1 (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 trausduction.
Due to their roles as
regulators of signal transduction, PDEs have been extensively studied as
chemotherapeutic targets
(Petty, M.J. and G.A. Higgs (1998) Curt. 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,
17
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isozymes, and splice variants is an indication of the variety and complexity
of the regulatory pathways
involving cyclic nucleotides (Houslay, M.D. and G. Milligan (1997) Trends
Biochem. Sci. 22:217-224).
Type 1 PDEs (PDEls) are Ca2+/calmodulin-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 cahnodulin, decreases PDE
activity, and increases
steady state levels of cAMP (Kakkar, supra). PDEls 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 (Petty,
M.J. and G.A. Higgs
(1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE2s are cGMP-stimulated PDEs that have been found in the cerebellum,
neocortex, heart,
kidney, lung, pulmonary artery, and skeletal muscle (Sadhu, K. et al. (1999)
J. Histochem. Cytochem.
47:895-906). PDE2s are thought to mediate the effects of cAMP on catecholamine
secretion,
participate in the regulation of aldosterone (Beavo, supra), anal play a role
in olfactory signal
transduction (Juilfs, D.M. et al. (1997) Proc. Natl. Aced. Sci. USA 94:3388-
3395).
PDE3 s 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 cilostanude, 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 (Bared,
M. et al. (1998) Proc. Natl. Aced. Sci. USA 95:15020-15025). PDE4 inhibitors
have also been
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studied as possible therapeutic agents against acute lung injury, endotoxemia,
rheumatoid arthritis,
multiple sclerosis, and various neurological and gastrointestinal indicatious
(Doherty, A.M. (1999)
C~rr. Opin. Chem. Biol. 3:466-473).
PDES is highly selective for cGMP as a substrate (Turko, LV. et al. (1998)
Biochemistry
37:4200-4205), and has two allosteric cGMP-specific binding sites (McAllister-
Lucas, L.M. et al.
( 1995) J. Biol. Chem. 270:30671-30679). Binding of cGMP to these allosteric
binding sites seems to
be important for phosphorylation of 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 (ferry, 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;
ferry, 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).
PDE8s 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-
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hydrolyzing activity of PDE8s 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
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.
to Biol. Chem. 273:15559-15564; Soderling, S.H. et al. (1998) J. Biol. Chem.
273:15553-15558).
PDElOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDElOs are
expressed
in brain, thyroid, and testis. (Soderling, S.H. et al. (1999) Proc. Natl.
Acad. Sci. USA 96:7071-7076;
Fujishige, K. et al. (1999) J. Biol. Chem. 274:18438-18445; Loughuey, K. et al
(1999) Gene 234:109-
117).
PDEs are composed of a catalykic domain of about 270-300 amino acids, an N-
ternlinal
regulatory domain responsible for binding cofactors, and, iu some cases, a
hydrophilic C-terminal
domain of unknown function (Coati, M. and S.-L.C. Jin (1999) Prog. Nucleic
Acid Res. Mol. Biol.
63:1-38). A conserved, putative zinc-binding motif, HDXXI3XGXXN, has been
identified in the
catalytic domain of all PDEs. N-terminal regulatory domains include non-
catalytic cGMP binding
2o 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(R/K)XnFX3DE (McAllister-Lucas, L.M. et al. (1993) J. Biol.
Chem. 268:22863-
22873). The NKXnD motif has been shown by mutagenesis to be importaut for cGMP
binding
(Turko, LV. et al. (1996) J. Biol. Chem. 271:22240-22244). PDE families
display approximately 30%
amino acid identity within the catalytic domain; however, isozymes within the
same family typically
display about 85-95% identity in this region (e.g. PDE4A vs PDE4B).
Furthermore, within a family
there is extensive similarity (>60%) outside the catalytic domain; while
across families, there is little or
no sequence similarity outside this domain.
Many of the constituent functions of immune and inflammatory responses are
inhibited by
agents that increase intracellular levels of cAMP (Verghese, M.W. et al.
(1995) Mol. Pharmacol.
47:1164-1171). A variety of diseases have been attributed to increased PDE
activity and associated .
with decreased levels of cyclic nucleotides. For example, a form of diabetes
insipidus in mice has
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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 (Petty,
M.J. and G.A. Higgs (1998) C~rr. Opin. Chem. Biol. 2:472-481; Torphy, T.J.
(1998) Am. J. Respir.
Crit. Care Med. 157:351-370). PDE3 inhibitors are being developed as
antithrombotic agents,
antihypertensive agents, and as cardiotonic agents useful in the treatment of
congestive heart failure.
Rolipram, a PDE4 inhibitor, has been used in the treatment of depression, and
other inhibitors of PDE4
are undergoing evaluation as anti-inflammatory agents. Rolipram has also been
shown to inhibit
lipopolysaccharide (LPS) induced TNF-a which has been shown to enhance HIV-1
replication in vitro.
to 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. Theophylline is believed to act on airway smooth muscle
function and in an
anti-inflammatory or i_m_m__unomodulatory 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 HIV-1 replication
(Angel et al., supra).
PDEs have been reported to affect cellular proliferation of a variety of cell
types (Coati et al.
(1995) Endocrine Rev. 16:370-389) and have been implicated in various cancers.
Growth of prostate
carcinoma cell lines DU145 and LNCaP was inhibited by delivery of cAMP
derivatives and PDE
inhibitors (Bang, Y.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334).
These cells also
showed a permanent conversion in phenotype from epithelial to neuronal
morphology. It has also been
suggested that PDE inhibitors have the potential to regulate mesangial cell
proliferation (Matousovic,
K. et al. (1995) J. Clip. Invest. 96:401-410) and lymphocyte proliferation
(Joulain, C. et al. (1995) J.
Lipid Mediat. Cell Signal. 11:63-79). A cancer treatment has been described
that involves intracellular
delivery of PDEs to particular cellular compartments of tumors, resulting in
cell death (Deonarain,
M.P. and A.A. Epenetos (1994) Br. J. Cancer 70:786-794).
Phosphotriesterases
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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, explain 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 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 novo biosynthesis of fatty acids. Chain
termination involves the
hydrolysis of the thioester bond which links the fatty acyl chain to the 4'-
phosphopantetheine prosthetic
group of he 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., su ra). For that reason, Naggert et al. su ra)
proposed that the
physiological substrates for E. coli TEII may be coenzyme A (CoA)-fatty acid
esters instead of ACP-
phosphopanthetheine-fatty acid esters.
Carboxylesterases
Mammalian carboxylesterases constitute a multigene family expressed in a
variety of tissues
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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,
palinitoyl-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 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).
Squalene epoxidase
Squalene epoxidase (squalene monooxygenase, SE) is a microsomal membrane-
bound, FAD-
dependent oxidoreductase that catalyzes the first oxygenation step in the
sterol biosynthetic pathway
of eukaryotic cells. Cholesterol is an essential structural component of
cytoplasmic membranes
acquired via the LDL receptor-mediated pathway or the biosynthetic pathway. In
the latter case, all
27 carbon atoms in the cholesterol molecule are derived from acetyl-CoA
(Stryer, L., su ra). SE
converts squalene to 2,3(S)-oxidosqualene, which is then converted to
lanosterol and then cholesterol.
The steps involved in cholesterol biosynthesis are summarized below (Stryer, L
(1988) Biochemistry.
W.H Freeman and Co., Inc. New York. pp. 554-560 and Sakakibara, J. et al.
(1995) 270:17-20):
acetate (from Acetyl-CoA) -- 3-hydoxy-3-methyl-glutaryl CoA ~ mevalonate ~ 5-
phosphomevalonate
S-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
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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).
Epoxide hydrolases
Epoxide hydrolases catalyze the addition of water to epoxide-containing
compounds, thereby
hydrolyzing epoxides to their corresponding 1,2-diols. They are related to
bacterial haloalkane
dehalogenases and show sequence similarity to other members of the aJ(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
hydrolase proceeds via a covalently bound ester intermediate initiated by the
nucleophilic attack of one
of the Asp residues on the primary carbon atom of the epoxide ring of the
target molecule, leading to a
covalently bound ester intermediate (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).
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Catechol-O-methyltransferase:
Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group
of S-
adenosyl-z-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
S of the 4'-hydroxyl group and the membrane bound isoform of COMT is more
regiospecific than the
soluble forth. Translation of the soluble form of the enzyme results from
utilization of an internal start
codon in a full-length mRNA (1.5 kb) or from the translation of a shorter mRNA
(1.3 kb), transcribed
from an internal promoter. The proposed SN2-like methylation reaction requires
Mg++ and is inhibited
by Ca'~'. The binding of the donor and substrate to COMT occurs sequentially.
AdoMet first binds
COMT in a Mg**-independent manner, followed by the binding of Mg+'' and the
binding of the catechol
substrate.
The amount of COMT in tissues is relatively high compared to the amount of
activity normally
required, thus inhibition is problematic. Nonetheless, inhibitors have been
developed for in vitro use
(e.g., gallates, tropolone, U-0521, and 3 ;4'-dihydroxy-2-methyl-
propiophetropolone) and for clinical use
(e.g., nitrocatechol-based compounds and tolcapone). Administration of these
inhibitors results in the
increased half life of L-dopa and the consequent formation of dopamine.
Inhibition of COMT is also
likely to increase the half life of various other catechol-structure
compounds, including but not limited
to epinephrine/norepinephrine, isoprenaline, rimiterol, dobutamiue,
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).
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
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collectively as "DIvIE" and individually as "DME-1," "DME-2," "DME-3," "DME-
4," "DIvIE-5,"
«DME-6~» «DME-7~» <'DME-8>» «DME-9~» «DME-10,» «DME-11~» «DME-12,» «DME-13~»
<~ME-
14," "DME-15," "DME-16," "DIME-17," "D1VIE-18," and "DME-19." In one aspect,
the invention
provides an isolated polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ >D NO:1-19 , b)
a polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ 1D N0:1-19 , c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-19 ,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ 1D N0:1-19 . In one alternative, the invention
provides an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:1-19 .
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ B7 N0:1-19 , b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID N0:1-19 , c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ m N0:1-19 , and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ~ N0:1-19 .
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of SEQ
)D N0:1-19 . In another alternative, the polynucleotide is selected from the
group consisting of SEQ
ID N0:20-3 8.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ll~ NO:1-19 , b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ )D N0:1-19 , c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-19 , and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ )D NO:1-19 . 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 selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
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of SEQ m N0:1-19 , b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ )D NO:1-19 , c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-19 , and d) an i_m_m__unogenic fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ ID N0:1-19 . 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 selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the soup consisting of SEQ ID NO:1-19 , b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90%o identical to an amino acid
sequence selected from the
group consisting of SEQ 1D N0:1-19 , c) a biologically active fragment of a
polypeptide having an
arntno acid sequence selected from the group consisting of SEQ ID NO:I-I9 ,
and d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:l-19 .
The invention further provides an isolated polynucleotide selected from the
group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ll~ N0:20-38, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ B7 N0:20-38,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). Iu 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 selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
m N0:20-38, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:20-38,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). The method
comprises a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
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WO 02/12467 PCT/USO1/24382
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 selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ~
N0:20-38, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:20-38, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
l0 polynucleotide of b), and e) an RNA equivalent of a)-d). The method
comprises a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-19 , b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical to.an amino acid sequence selected
from the group
consisting of SEQ m NO:1-19 , c) a biologically active fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ m NO:1-19 , and d) an
immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
m N0:1-19 , and a pharmaceutically acceptable excipient. In one embodiment,
the composition
comprises an amino acid sequence selected from the group consisting of SEQ ID
N0:1-19 . The
invention additionally provides a method of treating a disease or condition
associated with decreased
expression of functional DME, comprising administering to a patient in need of
such treatment the
composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID N0:1-19 , b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ n7 N0:1-19 , c) a biologically active
fragment of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-19 , and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID N0:1-19 . The method comprises a) exposing a sample
comprising the
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polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional DME, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ m N0:1-19 , b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ 1D N0:1-19 , c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ll~ NO:1-19 ,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ m NO:l-19 . 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 condition associated with overexpression of functional
DME, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds to.
a polypeptide selected from the group consisting of a), a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ m NO:1-19 , b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-19 , c) a biologically active
fragment of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
19 , and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO:1-19 . The method comprises a) combining the
polypeptide with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ D7 N0:1-19 , b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID N0:1-19 , c) a biologically active
fragment of a polypeptide
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having an amino acid sequence selected from the group consisting of SEQ ll~
NO:1-19 , and d) an
?mmunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m N0:1-19 . The method comprises a) combining the
polypeptide with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ )D N0:20-38,
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 selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
m N0:20-38, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:20-3
8, iii) a polynucleotide
having a sequence complementary to i), iv) a polynucleotide complementary to
the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide selected from the group consisting of i) a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of SEQ m N0:20-
38, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ 1D N0:20-38,
iii) a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
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toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
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.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
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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
S 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 occurring 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,
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 D1V1E. 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
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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 DN1E. 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')Z, 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. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLI~. 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;
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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.
"Complementat~' 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
2D 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., NaCl), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the 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 are
predicted to least
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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, 115
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.
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.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
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absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of DME or the polynucleotide encoding DME
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, 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 m N0:20-38 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ )D N0:20-38, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:20-38 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
ID N0:20-3 8 from related polynucleotide sequences. The precise length of a
fragment of SEQ )D
N0:20-38 and the region of SEQ ll~ N0:20-38 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 N0:1-19 is encoded by a fragment of SEQ ID N0:20-3 8. A
fragment of SEQ ID N0:1-19 comprises a region of unique amino acid sequence
that specifically
identifies SEQ )D N0:1-19 . For example, a fragment of SEQ ll~ N0:1-19 is
useful as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ D7 N0:1-19 .
The precise length of a fragment of SEQ ID N0:1-19 and the region of SEQ ID
N0:1-19 to which
the fragment corresponds are routinely determinable by one of ordinary skill
in the art based on the
intended purpose for the fragment.
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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 "full
length" polynucleotide sequence encodes a "full length" polypeptlde 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
l0 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 WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=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/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlin.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
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Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 arid Extensiorv Gap: 2 penalties
Gap x drop-off. 50
Expect: l0
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
i
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
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2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 asad Extension Gap: 1 penalties
Gap x drop-off. SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEC2 ID number, or may be measuxed
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 lengtbs 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.
'I'he 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 xetains 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
determ;ni"g 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 occui, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 ~Cglml sheared, denatured salmon sperm DNA.
39
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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
S°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory.Manual, 2"d ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 ~,g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for 1ZIVA: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.
"hnmune 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
CA 02417769 2003-O1-29
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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
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or inununological 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
25 functionalrelationship 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 terminahlysine 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-taranslational 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, chenuluminescent 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
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DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specibcity, 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, ftgures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
to example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo , Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
cau 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 specibc 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 fbrary," 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
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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
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
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protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or auy 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 ox 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
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 trausiently
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
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fertilization, but rather is directed to the introduction of a recombinant DNA
molecule. The transgenic
organisms contemplated in accordance with the present invention include
bacteria, cyanobacteria,
fungi, plants and animals. The isolated DNA of the present invention can be
introduced into the host
by methods known in the art, for example infection, transfection,
transformation or transconjugation.
Techniques for transferring the DNA of the present invention into such
organisms are widely known
and provided in references such as Sambrook et al. (1989), su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
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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.
S 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 (Iucyte Project m). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide m) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ B7 NO:) and an
Iucyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
Table 2 shows. sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
1S polypeptide sequence number (Incyte Polypeptide )D) for polypeptides 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 along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
2o Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ 117 NO:) and the
corresponding Incyte
polypeptlde sequence number (Incyte Polypeptide m) 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 MOT1FS
25 program of the GCG sequence analysis software pacT~age (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
30 properties establish that the claimed polypeptides are drug metabolizing
enzymes. For example, SEQ
>D N0:3 is 40% identical to a mouse cytochrome P450 monooxygenase (GenBank ll~
g2653663) as
determined by the Basic Local Alignment Search Tovl (BLAST, see Table 2). The
BLAST
probability score is 5.3e-91, which indicates the probability of obtaining the
observed polypeptide
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sequence alignment by chance. SEQ )D N0:3 also contains cytochrome P450
signature sequences as
determined by searching for statistically significant matches in the hidden
Markov model (HIVIM)-
based PFAM database of conserved protein family domains (see Table 3). Data
from BLIIVVIPS and
PROF1LESCAN analyses provide further corroborative evidence that SEQ ID N0:3
is a member of
the cytochrome P450 family.
In an alternative example, SEQ ID N0:1 is 58% identical to a lysyl oxidase
from the yellow
perch Perca flavescens; GenBank ID 84929199) as determined by BLAST analysis
The BLAST
probability score is 1.9e-248. SEQ ID N0:1 also contains cytochrome P450
signature sequences as
determined by searching for statistically significant matches in the IWM-based
PFAM database of
l0 conserved protein family domains and by BLIMPS analyses.
In an alternative example, SEQ ID N0:2 is 61% identical to human flavin-
containing
monooxygenase 5 (GenBank 1D 8559046) as determined by BLAST analysis, with a
probability score
of 4.5e-181. SEQ )D N0:2 also contains flavin-containing monooxygenase
signature sequences as
determined by searching for statistically significant matches in the I-hVIM-
based PFAM database of
is conserved protein family domains and by BLllVII'S and PROF1LESCAN analyses.
In an alternative example, SEQ ID N0:4 is 39% identical to a Pseudomonas 2,3-
butanediol
dehydrogenase (GenBank )D 8529564) as determined by BLAST analysis, with a
probability score of
2.0e-61. SEQ )D N0:4 also contains dehydrogenase signature sequences as
determined by searching
for statistically significant matches in the HIvIM-based PFAM database of
conserved protein family
2o domains. Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative
evidence that SEQ ID N0:4 is a dehydrogenase.
SEQ ID N0:5 is 54% identical to a Bacillus quinone oxidase (GenBank ID
82633069) as
determined by BLAST analysis, with a probability score of 7.1e-96. Data
obtained by searching the
IEVIM-based PFAM database of conserved protein family domains and by BLllVIPS
analyses provide
25 further corroborative evidence that SEQ D7 N0:5 is a quinone oxidase.
In an alternative example, SEQ ll~ N0:6 is 92% identical to mouse heparan
sulfate 6-
sulfotransferase 2 (GenBank 1D 86683558) as determined by BLAST analysis, with
a probability
score of 2.3e-255.
In an alternative example, SEQ ID N0:7 is 90% identical to a human glutathione
S-
3o transferase subunit (GenBank 1D 8242749) as determined by BLAST analysis,
with a probability
score of 1.3e-101. SEQ ID N0:7 also contains glutathione S-transferase
signature sequences as
determined by searching for statistically significant matches in the I-hVIM-
based PFAM database of
conserved protein family domains and by BLI1VVIPS analyses.
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In an alternative example, SEQ m N0:8 is 40% identical to a human steriod
dehydrogenase
(GenBank B7 85531815) as determined by BLAST analysis, with a probability
score of 1.9e-56. SEQ
ID N0:8 also contains dehydrogenase signature sequences as determined by
searching for statistically
significant matches in the 1=nVIM based PFAM database of conserved protein
family domains.
SEQ B7 N0:9 is 47% identical to a rabbit liver carboxylesterase (GenBank ID
83219695) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 6.3e-72, which indicates the probability of obtaining the
observed polypeptide
sequence alignment by chance. SEQ B~ N0:9 also contains carboxylesterase
domains as determined
by searching for statistically significant matches in the hidden Markov model
(I~VIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from
BLIZVVIPS, MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ 117 N0:9
is a
carboxylesterase.
SEQ m N0:10 is 45% identical to human carboxylesterase (GenBank ID 8180950) as
determined by the Basic Local Alignment Search Tool (BLAST, see Table 2). The
BLAST
probability score is 8.7e-130, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ ll~ N0:10 also contains carboxylesterase
domains as
determined by searching for statistically significant matches in the hidden
Markov model (FEVVIM)-
based PFAM database of conserved protein family domains (see Table 3). Data
from BLnVIPS,
MOTIFS, and PROF1LESCAN analyses provide further corroborative evidence that
SEQ m N0:10
is a carboxylesterase.
In an alternative example, SEQ 117 N0:11 is 89% identical to murine heparan
sulfate 6-
sulfotransferase 2 (GenBank ID 86683558) as determined by the Basic Local
Alignment Search Tool
(BLAST, see Table 2). The BLAST probability score is 1.8e-236, which indicates
the probability of
obtaining the observed polypeptide sequence alignment by chance, and provides
evidence that SEQ ID
N0:11 is a DME, and specifically that SEQ m NO:l 1 is a sulfotransferase.
In an alternative example, SEQ ID N0:12 is 25% identical to a Bacillus
subtilis epoxide
hydrolase (GenBank m 82633182) as determined by the Basic Local Alignment
Search Tool
(BLAST, see Table 2). The BLAST probability score is 1.3e-11, which indicates
the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ m N0:12
also contains
hydxolase domains as determined by searching for statistically significant
matches in the hidden
Markov model (FEVIM) based PFAM database of conserved protein family domains
(see Table 3).
Data from BLllVIPS analyses provide further corroborative evidence that SEQ ID
N0:12 is a
hydrolase.
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In an alternative example, SEQ ID N0:13 is 83 % identical to a rat beta-
alanine-pyruvate
aminotransferase (GenBank ll~ 81944136) as determined by the BLAST analysis
(see Table 2). The
BLAST probability score is 1.1e-234. SEQ m N0:13 also contains
aminotransferase domains as
determined by searching for statistically significant matches in the hidden
Markov model (FIIVIM)-
based PFAM database of conserved protein family domains (see Table 3). Data
from BLIMPS and
PROF1LESCAN analyses provide further corroborative evidence that SEQ ID N0:13
is an
aminotransferase.
In an alternative example, SEQ 117 N0:14 is 50% identical to a guinea pig
hyroxysteroid
sulfotransferase (GenBank 1D 81151081) as determined by the BLAST analysis
(see Table 2). The
BLAST probability score is 5.4e-34, and provides evidence that SEQ ID N0:14 is
a sulfotransferase.
In an alternative example, SEQ ID N0:15 is 52% identical to a guinea pig
copper/zinc
superoxide dismutase (GenBank m 81066120) as determined by the BLAST analysis
(see Table 2).
The BLAST probability score is 2.1e-25. SEQ 1D N0:15 also contains copper/zinc
superoxide
dismutase domains as determined by searching for statistically significant
matches in the hidden
Markov model (fIlVIM)-based PFAM database of conserved protein family domains
(see Table 3).
Data from BLllVIPS analyses provide further corroborative evidence that SEQ ID
NO:15 is a
copper/zinc superoxide dismutase.
SEQ ID NO:16 is 37% identical to human 3'-phosphoadenylylsulfate-
galactosylceramide
3 =sulfotransferase (cerebroside sulfotransferase, GenBank )D 81871141) as
determined by the Basic
Local Alignment Search Tool (BLAST, see Table 2). The BLAST probability score
is 2.8e-60, which
indicates the probability of obtaining the observed polypeptide sequence
alignment by chance.
In an alternative example, SEQ ID N0:17 is 38% identical to a putative C.
eleg_ans
monoamine oxidase (GenBank 117 86782275) as determined by BLAST analysis with
a probability
score of 3.0e-99. SEQ ID N0:17 also contains a monoamine oxidase domain as
determined by
searching for statistically significant matches in the hidden Markov model
(FEVIM)-based PFAM
database of conserved protein family domains (see Table 3). Data from BLIMPS
analysis provide
further corroborative evidence that SEQ ID N0:17 is a monoamine oxidase.
In an alternative example, SEQ ID N0:18 is 36% identical to human catechol-O-
methyltransferase (GenBank m 8179955) as determined by BLAST analysis with a
probability score
of 9.5e-41. SEQ m N0:18 is also 36% identical to murine catechol-0-
methyltransferase (GenBank
ID 83493253) as determined by BLAST analysis with a probability score of 1.3e-
41.
In an alternative example, SEQ ID N0:19 is 44% identical to Fundulus
heteroclitus
cytochrome P450 2N1 (GenBank ID 85852342) as determined by the Basic Local
Alignment Search
49
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Tool (BLAST, see Table 2). The BLAST probability score is 4.6e-99, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:19 also contains
cytochrome P450 domains as determined by searching for statistically
significant matches in the
hidden Markov model (PIIVIM)-based PFAM database of conserved protein family
domaitts (see Table
3). Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative
evidence that SEQ ID N0:19 is a cytochrome P450.
The algorithms and parameters for the analysis of SEQ ID NO:1-19 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 N0:20-38 or that distinguish between SEQ 117
N0:20-38 and
related polynucleotide sequences. Column 5 shows identification numbexs
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/or 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,
7690384J1 is the
identification number of an Incyte cDNA sequence, and PROSTME06 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA fbraries are not indicated
were derived from
pooled cDNA libraries (e.g., 55017748J1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs (e.g., g1203094) which contributed to the
assembly of the full
length polynucleotide sequences. In addition, the identification numbers in
column 5 may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database
(i.e., those
sequences including the designation "ENST"). Alteratively, the identification
numbers in column 5
rnay be derived from the NCBI RefSeq Nucleotide Sequence Records Database
(i.e., those
sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein
Sequence Records
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(i.e., those sequences including the designation "NP"). Alternatively, the
identification numbers in
column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, FL_XXXXXX NI N~ YYYYY N3 NQ
represents a
"stitched" sequence in which XXXXXX is the identification number of the
cluster of sequences to
which the algorithm was applied, and YYYYY is the number of the prediction
generated by the
algorithm, and Nl,z,3..., if present, represent specific exons that may have
been manually edited during
analysis (See Example V). Alternatively, the identification numbers in column
5 may refer to
assemblages of exons brought together by an "exon-stretching" algorithm. For
example,
FIJXXI~XXX_gAAAAA~BBBBB_1 N is the identification number of a "stretched"
sequence, with
l0 XXXXXX being the Incyte project identification number, gAAAAA being the
GenBank identification
number of the human genomic sequence to which the "exon-stretching" algorithm
was applied,
gBBBBB being the GenBank identification number or NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N referring to specific exons (See
Example V). In instances
where'a RefSeq sequence was used as a protein homolog for the "exon-
stretching" algorithm, a
RefSeq identifier (denoted by "NM," "NP," or "NT") may be used in place of the
GenBank identifier
(i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example 1V and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences
using, for example,
ENST GENSCAN (Stanford University, CA, USA)
or FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons
and resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column S was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Iucyte
cDNA identification numbers are not shown.
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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
structuxal 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 N0:20-38, which encodes DME. The
polynucleotide sequences
of SEQ ID N0:20-38, 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 D7 N0:20-
38 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
D7 NO:20-38. 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 occurnng DME under
appropriately selected
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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
N0:20-38 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; I~irnmel, 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
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 Biolo~y and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding DME may be extended utilizing a partial
nucleotide
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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 Appfic. 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 intron/exon junctions. For all PCR based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 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 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-
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
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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
2o produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene .variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular
evolution. For example, fragments of a single gene containing random point
mutations may be
recombined, screened, and then reshuffled until the desired properties are
optimized. Alternatively,
fragments of a given gene may be recombined with fragments of homologous genes
in the same gene
family, either from the same or different species, thereby maximizing the
genetic diversity of multiple
naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding DME may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
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.,
CA 02417769 2003-O1-29
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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 maybe 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
codon should be provided
by the vector. Exogenous translational elements and initiation codons may be
of various origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding DME and appropriate transcriptional and
translational control
elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plai_nview NY, ch. 4, 8, anal 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
56
CA 02417769 2003-O1-29
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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 Technology (1992) McGraw
Hill, New
1o York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
USA 81:3655-3659; and
Harrington, J.J: et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di Nicola,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. Tmmunol. 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 PBLUESCR1PT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding DME into the
vector's multiple. cloning
site disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of DME are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of DME may be used. For
example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of DME. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
57
CA 02417769 2003-O1-29
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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; Brogue, 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 Technoloay
(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
3o 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 trausformed cells may be
propagated using tissue
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culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltrausferase genes, for use in tk' and apt' 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; rceo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltrausferase,
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), !3 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformauts, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may.need to be
confirmed. For example, if
the sequence encoding DME is inserted within a marker gene sequence,
transformed cells containing
sequences encoding DME can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding DME under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding DME and
that express
DME may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
T_m_m__unological methods for detecting and measuring the expression of DME
using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal ant'bodies reactive to two non-interfering epitopes on DME is
preferred, but a competitive
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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 Itnmunolo~y, Greene Pub.
Associates and Wiley-
Interscience, New York NY; and Pound, J.D. (1998) Inltnunochemical 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 polymerise
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 Phaxmacia Biotech, Promega
(Madison WI), and
US Biochemical. Suitable reporter molecules or labels which rnay 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
and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode DME may be designed to contain signal sequences
which direct
secretion of DME through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted 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
modification and
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
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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), calinodulin binding peptide (CBP), 6-
His, FLAG, c-myc, 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,
1o 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
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
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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
1o 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
(Marth, J.D. (1996) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997)
Nucleic Acids Res.
25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell blastocysts such
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as those from the C57BL/6 mouse strain. The blastocysts are surgically
transferred to
pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred
to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding DME may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding DME can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a region
of a polynucleotide encoding DME is injected into animal ES cells, and the
injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred lines are
studied and treated with
potential pharmaceutical agents to obtain information on treatment of a human
disease. Alternatively,
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 a variety of diseased tissues, including that of the brain,
prostate, bone, intestine, and
breast. Therefore, DME appears to play a role in autoimmune/inflammatory, cell
proliferative,
developmental, endocrine, eye, metabolic, and gastrointestinal disorders,
including liver disorders. In
the treatment of disorders associated with increased DME expression or
activity, it is desirable to
decrease the expression or activity of DME. In the treatment of disorders
associated with decreased
DME expression or activity, it is desirable to increase the expression or
activity of DME.
Therefore, in one embodiment, DME or a fragment or derivative thereof may be
administered
to a subject to treat or prevent a disorder associated with decreased
expression or activity of DME.
Examples of such disorders include, but are not limited to, an
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), bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis,
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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, polyrnyositis, 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 helininthic
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 vets, 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
dwarftsm, 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
20, neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spins bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; an
endocrine disorder, such as disorders of the hypothalamus and pituitary
resulting from lesions such as
primary brain tumors, adenomas, infarction associated with pregnancy,
hypophysectomy, aneurysms,
vascular malformations, thrombosis, infections, immunological disorders, and
complications due to head
trauma; disorders associated with hypopituitarism including hypogonadism,
Sheehan syndrome,
diabetes insipidus, Kalltnan's disease, Hand-Schuller-Christian disease,
Letterer-Siwe disease,
sarcoidosis, empty sells syndrome, and dwarfism; disorders associated with
hyperpituitarism including
acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH)
secretion (SIAI7H)
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 (Hashirnoto's disease), and cretinism;
disorders associated with
hyperthyroidism including thyrotoxicosis and its various forms, Grave's
disease, pretibial myxedema,
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toxic multinodular goiter, thyroid carcinoma, and Plummer's disease; disorders
associated with
hyperparathyroidism including Coon disease (chronic hypercalemia); pancreatic
disorders such as
Type I or Type II diabetes mellitus and associated complications; disorders
associated with the
adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex,
hypertension associated
with alkalosis, amyloidosis, hypokalemia, 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;
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'syndrorne (AIDS)
enteropathy, jaundice,
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hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis,
hemochromatosis, Wilson's disease,
alphai 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 cattier 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 I7ME.
Examples of such
disorders include, but are not limited to, those autoimmune/inflammatory, cell
proliferative,
developmental, endocrine, eye, metabolic, and gastrointestinal disorders,
including liver disorders
described above. In one aspect, an antibody which specifically binds DME may
be used directly as an
antagonist or indirectly as a targeting ox 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.
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In particular, purified DME may be used to produce antibodies or to screen
libraries of pharmaceutical
agents to identify those which specifically bind DME. Antibodies to DME may
also be generated
using methods that are well known in the art. Such antibodies may include, but
are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and fragments produced
by a Fab expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are
generally preferred for therapeutic use.
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
ZO 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 Calinette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to DME
have an amino acid sequence consisting of at least about 5 amino acids, and
generally will consist of at
least about 10 amino acids. It is also preferable that these oligopeptides,
peptides, or fragments are
identical to a portion of the amino acid sequence of the natural protein.
Short stretches of DME amino
acids may be fused with those of another protein, such as KL,H, 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.
Im_m__unol. 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.)
Tn af~f~lt1(1h tPrl,ninnPC rlPVPlnnPrl fnr tW? Y~Tnr~l7t~fintv n~ ~~r~lmPrin
anti~r~r~iAC ~~ emn~, ac ly,a
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D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte population
or by screening immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in
the literature. (See, e.g., Orlandi, R. et 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 maybe
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 maybe 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, su ra).
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, K~, 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 Ka ranging
from about 109 to 101a L/mole are preferred for use in immunoassays in which
the DME-antibody
complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ke 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 Auproach, IRh Press, Washington DC;
Liddell, J.E. and A.
Cryer (1991) A Practical Guide to Monoclonal Antl'bodies, John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
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the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
anti'body/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of DME-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, su ra, 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 Therapeutics, 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. Allexgy Clin. Tmmunol. 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 gerrnline gene therapy. Gene therapy may be performed to (i)
correct a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCm)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) 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
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hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciuarum and
Tryhanosoma 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; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998)
Curr. Opin. Biotechnol.
9:445-450).
Expression vectors that may be effective for the expression of DME include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). 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, su ra)), 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
CA 02417769 2003-O1-29
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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.
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 trausducing 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).
Iu 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"),
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hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding DME to target cells which have one or more genetic
abnormalities with
respect to the expression of DME. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing DME to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1 based vector has
l0 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 herpesvirus
genomes, the growth and
propagation of herpesvirus, and the infection of cells with herpesvirus are
techniques well known to
those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding DME to target cells. The biology of the
prototypic alphavirus, Semlik_i
Forest Virus (SFV), has been studied extensively and gene transfer vectors
have been based on the
SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469).
During alphavirus
RNA replication, a subgenomic RNA is generated that normally encodes the viral
capsid proteins.
This subgenomic RNA replicates to higher levels than the full length genomic
RNA, resulting in the
overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g., protease
and polymerase). Similarly, inserting the coding sequence for DME into the
alphavirus genome in
place of the capsid-coding region results in the production of a large number
of DME-coding RNAs
and the synthesis of 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
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hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN)
indicates that the lytic
replication of alphaviruses can be altered to suit the needs of the gene
therapy application (Dryga,
S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses
will allow the
introduction of DME into a variety of cell types. The specific transduction of
a subset of cells in a
population may require the sorting of cells prior to transduction. The methods
of manipulating
infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and
performing alphavirus infections, are well known to those with ordinary skill
in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
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 hnmunolo~~ic Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A
complementary sequence or antisense molecule rnay 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
3o 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
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with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines, cells,
or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified
forms of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding DME. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased DME
. expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding DME may be therapeutically useful, and in the treatment of disorders
associated with
decreased DME expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding DME rnay 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 DIME 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
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by hybridization with a probe having a nucleotide sequence complementary to
the sequence of the
polynucleotide encoding DME. The amount of hybridization may be quantified,
thus forming the basis
for a comparison of the expression of the polynucleotide both with and without
exposure to one or
more test compounds. Detection of a change in the expression of a
polynucleotide exposed to a test
compound indicates that the test compound is effective in altering the
expression of the polynucleotide.
A screen for a compound effective in altering expression of a specific
polynucleotide can be carried
out, for example, using a Schizosaccharomyces pombe gene expression system
(Atkins, D. et al.
(1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res.
28:E15) or a human
cell line such as Heha cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
Commun. 268:8-13).
A particular embodiment of the present invention involves screening a
combinatorial library of
oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide
nucleic acids, and modified
oligonucleotides) for antisense activity against a specific polynucleotide
sequence (Bruice, T.W. et al.
(1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No.
6,022,691).
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
3o including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,.
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
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These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising DME or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, DME or a fragment thereof may be joined to a
short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, monkeys,
or pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example DME
or fragments thereof, antibodies of DME, and agonists, antagonists or
inhibitors of DME, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in SO% of the
population) or LDSo (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDSO ratio. Compositions
which exhibit large
3o 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
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patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiriug 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 ~g to 100,000 ~tg, up to a total
dose of
l0 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,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind DME may be used for
the diagnosis
of disorders characterized by expression of DME, or in assays to monitor
patients being treated with
DME or agonists, antagonists, or inhibitors of DME. Antibodies useful for
diagnostic purposes may be
prepared in the same manner as described above for therapeutics. Diagnostic
assays for DME
include methods which utilize the antibody and a label to detect DME in human
body fluids or in
extracts of cells or tissues. The antibodies may be used with or without
modification, and may be
labeled by covalent or non-covalent attachment of a reporter molecule. A wide
variety of reporter
molecules, several of which are described above, are known in the art and may
be used.
A variety of protocols for measuring DME, including ELISAs, RIAs, and FAGS,
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,
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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 occurring sequences encoding DME, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the DME encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ )D
N0:20-38 or from
genomic sequences including pxomoters, 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 3sS,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding DME may be used for the diagnosis of
disorders
associated with expression of DME. Examples of such disorders include, but are
not limited to, an
autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoirnmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic
lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
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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 heltninthic 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 (Wilrns' tumor, aniridia, genitourinary
abnormalities, and mental
retardation), Smith-Magenis syndronc~,e, 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 (SIADIT) 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 myxederna, toxic multinodular goiter, thyroid
carcinoma, and Plummer's
disease; disorders associated with hyperparathyroidism including Cone disease
(chronic
hypercalemia); pancreatic disorders such as Type I or Type II diabetes
mellitus and associated
complications; disorders associated with the adrenals such as hyperplasia,
carcinoma, or adenoma of
the adrenal cortex, hypertension associated with alkalosis, amyloidosis,
hypokalemia, C~xshing's
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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 virifization, 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 xanthornatosis,
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; and a gastrointestinal
disorder, such as
dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture,
esophageal carcinoma,
dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea,
emesis, gastroparesis, antral or
pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal
obstruction, infections of the
intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma,
biliary tract disease, hepatitis, hyperbilirubinemia, hereditary
hyperbilirubinemia, cirrhosis, passive
congestion of the liver, hepatoma, infectious colitis, ulcerative colitis,
ulcerative proctitis, Crohn's
disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic
obstruction, irntable
bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal
hemorrhage, acquired
immunodeficiency syndrome (AIDS enteropathy, jaundice, hepatic encephalopathy,
hepatorenal
syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, 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
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including nodular hyperplasias, adenomas, and carcinomas. The polynucleotide
sequences encoding
DME may be used in Southern or northern analysis, dot blot, or other membrane-
based technologies;
in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and
in microarrays utilizing
fluids or tissues from patients to detect altered DME expression. Such
qualitative or quantitative
methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding DME may be useful in
assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding DME may be labeled by standard methods and added to a fluid
or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a suitable
incubation period, the sample is washed and the signal is quantified and
compared with a standard
value. If the amount of signal in the patient sample is significantly altered
in comparison to a control
sample then the presence of altered levels of nucleotide sequences encoding
DME in the sample
indicates the presence of the associated disorder. Such assays may also be
used to evaluate the
efficacy of a particular therapeutic treatment regimen in animal studies, in
clinical trials, or to monitor
the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of DME,
a normal or standard profile for expression is established. This may be
accomplished by combining
body fluids or cell extracts taken from normal subjects, either animal or
human, with a sequence, or a
fragment thereof, encoding DME, under conditions suitable for hybridization or
amplification.
~ Standard hybridization may be quantified by comparing the values obtained
from normal subjects with
values from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation from standard values is
used to establish the
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
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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.
to 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
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 MASSARRAY 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. Tmmunol. 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 spectrophotornetric or
colorimetric response gives rapid
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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 descn'bed 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 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 Bell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
rnay 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
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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/newsltoxchip.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 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
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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 (Lueking, A. et al. (1999)
Anal. Biochem 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol- or
amino-reactive fluorescent compound and detecting the amount of fluorescence
bound at each array
element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
3o 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
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residues of the individual proteins and comparing these partial sequences to
the polypeptades 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.,
l0 Brennau, T.M. et al. (1995) U.S. Patent No. 5,474,796; 5chena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalon, D. et
al. (1995) PCT applieation 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 Approach,
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 genexate 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.
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, fox 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, Larder, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
3o Fluorescent in situ hybridization (FISH) may be correlated with other
physical and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, su ra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding DME on a
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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 11q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant
invention may also be used to detect differences in the chromosomal location
due to translocation,
inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, DME, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used 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.,
Geysers, et al. (1984) PCT
application W084103564.) In this method, large numbers, of different small
test compounds are
synthesized on a solid substrate. The test compounds are reacted with DME, or
fragments thereof,
and washed. Bound DME is then detected by methods well known in the art.
Purified DME can also
be coated directly onto plates for use in the aforementioned drug screening
techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and immobilize
it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding DME specifically compete with a test compound
for binding DME. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or 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.
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Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
S The disclosures of all patents, applications and publications, mentioned
above and below,
incuding U.S. Ser. No. 60/223>055, U.S. Ser. No. 60/224,728, U.S. Ser.
No.60/226,440, U.S. Ser.
No.60/228,067, U.S. Ser. No.60/230,063, U.S. Ser. No.60/232,244, and U.S. Ser.
No.60/234,269, are
expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some
tissues were
homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed in
phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCl
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)'+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Arnbion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the
UNIZAP vector system (Stratagene) or SUPERSCRTPT plasmid system (Life
Technologies), using
the recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra,
units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
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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.,
PBLUESCR1PT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA
(Invitrogen), or
pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including ~1-Blue, XL1-BlueMRF, 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 lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Mtniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
~.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at 4
°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
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 1I 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
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
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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
S 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 (I~vIM)-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
HIV1IVIER.
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
Iucyte 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, PRTNTS, DOMO, PRODOM, Prosite, and
hidden
Markov model (HIVIM)-based protein family databases such as PFAM. Full length
polynucleotide
sequences are also analyzed using MACDNASIS PRO software (Hitachi Software
Engineering,
South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and
polypeptide
sequence alignments are generated using default parameters specified by the
CLUSTAL algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
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applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:20-38. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplil'xcation technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genoznic DNA
Putative drug metabolizing enzymes were initially identified by ntnning 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 geuomic DNA
sequences from a
variety of organisms (See Burge, C. and S. Karliu (1997) J. Mol. Biol. 268:78-
94, and Surge, C. and
S. Karlin (1998) Curr. Opin. Struct. Biol. $:346-354). The program
concatenates predicted exons to
form au 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 Iucyte cDNA or public eDNA coverage of the
Genscan-predicted
sequences, thus providing evidence fox 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" Seguences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
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identification program described in Example 1V. Partial cDNAs assembled as
described in Example
Ill 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
2o 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 1V. 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 ll~ N0:20-38 were compared with
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sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:20-3S 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 )D NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances
are based on genetic markers mapped by Genethon which provide boundaries for
radiation hybrid
markers whose sequences were included in each of the clusters. Human genome
maps and other
resources available to the public, such as the NCBI "GeneMap'99" World Wide
Web site ,
(http://www.ncbi.nlm.nih.gov/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,
suura, 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 LIF'ESEQ (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
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length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being 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 1B). Each
cDNA sequence is
derived from a cDNA library constructed from a human tissue. Each human tissue
is classified into
one of the following organ/tissue categories: cardiovascular system;
connective tissue; digestive
system; embryonic structures; endocrine system; exocrine glands; genitalia,
female; genitalia, male;
germ cells; heroic and immune system; liver; musculoskeletal system; nervous
system; pancreas;
respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or urinary tract.
The number of libraries in each category is counted and divided by the total
number of libraries across
all categories. Similarly, each human tissue is classified into one of the
following disease/condition
categories: cancer, cell line, developmental, inflammation, neurological,
trauma, cardiovascular, pooled,
and other, and the number of libraries in each category is counted and divided
by the total number of
libraries across all categories. The resulting percentages reflect the tissue-
and disease-specific
expression of cDNA encoding DME. cDNA sequences and cDNA library/tissue
information are
found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of DME Encoding Polynucleotides
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
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nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72 °C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well 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 rimol of each primer, reaction buffer
containing Mga+, (NH4)zS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68°C, 5 min; Step 7: storage
at 4°C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94 °C,
3 min; Step 2: 94 °C, 15 sec; Step 3: 57 °C, 1 min; Step 4: 68
°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 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 ,u1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in 384-
well plates in LB/2x carb liquid media.
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The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polyrnerase (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°!o dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction 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 ID N0:20-38 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 /,cCi of
[Y szp] 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 are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, Sae, e.g.,
Baldeschweiler, supra.), mechanical
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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; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to au element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 unitslpl RNase inhibitor, 500 p.M dATP, 500 p,M dGTP, 500
~M dTTP, 40 p.M
dCTP, 40 p.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 trauscription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85° C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories,. Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
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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 ~,l SX SSC/0.2% SDS.
Microarra~reparation
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 ~,g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
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 of the array
element DNA, at an average
concentration of 100 ng/pl, 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-speciftc binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ~,1 of sample mixture consisting of 0.2 ~,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
~1 of SX SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer ( 1X SSC, 0.1
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
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Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, lnc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photornultiplier 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
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
spectra) between the fluorophores using each fluorophore's emission specti um.
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
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for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the DME-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring DME. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of DME. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the DME-encoding transcript.
XII. Expression of DME
Expression and purification of DME is achieved using bacterial or virus-based
expression
systems. For expression of DME in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
ttp-lac (tac) hybrid
promoter and the TS 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 Auto~raphica californica nuclear polyhedrosis virus
(AcMNPV), commonly
known as baculovirus. The nonessential polyhedrin gene of baculovirus is
replaced with cDNA
encoding DME by either homologous recombination or 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,
afFnity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma 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
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specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffiuity 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, sera,
ch. 10 and 16). Purified DME obtained by these methods can be used directly in
the assays shown in
Examples XVI, XVII, and XVI)I, 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
1o vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 /.cg of recombinant vector are
trausiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side lift
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow C, ometry, 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). Trausfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
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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 ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
hT.H (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (IvlBS) to
increase irnmunogenicity. (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
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DME, or biologically active fragments thereof, are labeled with 1~I 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. 6,0S7,101).
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, su ra). This assay is a convenient measure, but underestimates the
total hydroxylation, which
also occurs at the 2- and 3- positions. Assays are performed at 37 °C
and contain an aliquot of the
enzyme and a suitable amount of aniline (approximately 2 mM) in reaction
buffer. For this reaction,
the buffer must contain NADPH or an NADPH-generating cofactor system. One
formulation for this.
reactionbuffer includes 85 mM Tris pH 7.4, 15 mM MgC)2, 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.
1x,25-dihydroxyvitamin D 24-hydroxylase activity of DME is determined by
monitoring the
conversion of 3H-labeled 1a,25-dihydroxyvitamin D (1a,25(OH)ZD) to 24,25-
dihydroxyvitamin D
(24,25(OI~ZD) in transgenic rats expressing DME. 1 ~tg 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
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homogenate is then incubated with 0.25 nM 1a,25(OI-~2[1 3H]D, with a specific
activity of
approximately 3.5 GBq/mmol, for 15 min at 37 °C under oxygen with
constant shaking. Total lipids
are extracted as described (Bligh, E.G. and W.J. Dyer (1959) Can. J. Biochem.
Physiol. 37: 911-917)
and the chloroform phase is analyzed by HPLC using a FINEPAK S1L 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)a[1 3H]D) and product
(24,25(0H)2[1 3H]D) are
determined and correlated with the fractions collected. The amount of
24,25(0H)2[1 3H]D produced
in control rats is subtracted from that of transgenic rats expressing DME. The
difference in the
production of 24,25(0H)2[1 3H]D in the transgenic and control animals is
proportional to the amount
of 25-hydrolase activity of DME present in the sample. Confirmation of the
identity of the substrate
and products) is confirmed by means of mass spectroscopy (Miyamoto, Y. et al.
(1997) J. Biol.
Chem. 272:14115-14119).
Flavin-containing monooxygenase activity of DME is measured by chromatographic
analysis
of metabolic products. For example, Ring, B. J. et al. (1999; Drug Metab. Dis.
27:1099-1103)
incubated FMO in 0.1 M sodium phosphate buffer (pH 7.4 or 8.3) and 1 mM NADPH
at 37 °C,
stopped the reaction with an organic solvent, and determined product formation
by HPLC.
. Alternatively, activity is measured by monitoring oxygen uptake using a
Clark-type electrode. For
example, Ziegler, D. M. and Poulsen, L. L. (1978; Methods Enzymol. 52:142-151)
incubated the
enzyme at 37 °C in an NADPH-generating cofactor system (similar to the
orle 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
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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 constructed using known concentrations of aniline, which will
form a chromophore with
similar properties to 2-aminophenol glucuronide.
Sulfotransferase activity of DME is measured using the incorporation of 35S
from [35S]PAPS
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
l0 6.4, 50 ~M phenol, 0.4-4.0 ~.M [35S]PAPS. After sufficient time for 5-20%
of the radiolabel to be
transferred to the substrate, 0.2 mL of 0.1 M barium acetate is added to
precipitate protein and
phosphate buffer. Then 0.2 mL of 0.1 M Ba(OH)Z 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.
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
substarate preferences of the
GST of interest. Assays are performed at ambient temperature aild contain an
aliquot of the enzyme
in a suitable reaction buffer (for-example, 1 mM glutathione, 1 mM
dinitrochlorobenzene, 90 mM
potassium phosphate buffer pH 6.5). Reactions are carried out in an optical
cuvette, and the
absorbance at 340 nm is measured. The rate of increase in absorbance is
proportional to the enzyme
activity in the assay. .
N-acyltransferase activity of DME is measured using radiolabeled amino acid
substrates and
measuring radiolabel incorporation into conjugated products. Enzyme is
incubated in a reaction buffer
containing an unlabeled acyl-CoA compound and radiolabeled amino acid, and the
radiolabeled acyl-
conjugates are separated from the unreacted amino acid by extraction into n-
butanol or other
appropriate organic solvent. For example, Johnson, M. R. et al. (1990; J.
Biol. Chem. 266:10227-
10233) measured bile acid-CoA:amino acid N-acyltransferase activity by
incubating the enzyme with
cholyl-CoA and 3H-glycine or 3H-taurine, separating the tritiated cholate
conjugate by extraction into
n-butanol, and measuring the radioactivity in the extracted product by
scintillation. Alternatively, N-
acyltransferase activity is measured using the spectrophotometric
determination of reduced CoA
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(CoASH) described below.
N-acetyltransferase activity of DME is measured using the transfer of
radiolabel from
[iaC~acetyl-CoA to a substrate molecule (for example, see Deguchi, T. (1975)
J. Neurochem.
24:1083-5). Alternatively, a newer 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.
l0 Protein arginine methyltransferase activity of DME is measured at 37
°C for various periods
of time. S-adenosyl-L-[methyl-3H]methionine ([3H]AdoMet; specific activity =
75 Cilmmol; NEN
Life Science Products) is used as the methyl-donor substrate. Useful methyl-
accepting substrates
include glutathione S-transferase fibrillarin glycine-arginine domain fusion
protein (GST-GAR), '
heterogeneous nuclear nbonucleoprotein (hnRNP), or hypomethylated proteins
present in lysates from
adenosine dialdehyde-treated cells. Methylation reactions are stopped by
adding SDS-PAGE sample
buffer. The products of the reactions are resolved by SDS-PAGE and visualized
by fluorography.
The presence of 3H-labeled methyl-donor substrates is indicative of protein
arginine methyltransferase
activity of DME (Tang, J. et al. (2000) J. Biol. Chem. 275:7723-7730 and Tang,
J. et al. (2000) J. Biol.
Chem.275:19866-19876).
Aldo/keto reductase activity of DME is measured using the decrease in
absorbance at 340 nm
as NADPH is consumed. A standard reaction mixture 1s 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 1s incubated at 30°C and
the reaction is monitored
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
absorbauce 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 / dug of enzyme.
3o DME activity is determined using 4-methylumbelliferyl acetate as a
substrate. The
enzymatic reaction is initiated by adding approximately 10 ~,1 of DME-
containing sample to 1 ml of
reaction buffer (90 mM KHzPO~, 40 mM KCl, pH 7.3) with 0.5 mM 4-
methylumbelliferyl acetate
at 37 °C. The production of 4-methylumbelliferone is monitored with a
spectrophotometer (s3so =
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12.2 mM-1 crri 1) 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 3.3 mM cocaine in reaction buffer
(50 mM NaHZP04,
pH 7.4) with 1 mM benzamidine, 1 mM EDTA, and 1 mM dithiothreitol at 37
°C. The reaction is
incubated for 1 h in a total volume of 0.4 ml then terminated with an equal
volume of 5%
trichloroacetic acid. 0.1 ml of the internal standard 3,4-dimethylbenzoic acid
(10 p.g/ml) is added.
Precipitated protein is separated by centrifugation at 12,000 x g for 10 rpm.
The supernatant is
1o transferred to a clean tube and extracted twice with 0.4 ml of rnethylene
chloride. The two extracts
are combined and dried under a stream of nitrogen. The residue is resuspended
in 14% acetonitrile,
25O mM KHZPO4, pH 4.0, with 8 p,1 of diethylamine per 100 ml and ) and
injected onto a C18
reverse-phase HPLC colunmn for separation. The column eluate was monitored at
235 rm. DME
activity is quantified by comparing peak area ratios of the analyte to the
internal standard. A
standard curve was 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
gara-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 p.g/ml in sodium
acetate, pH 5.0). Carboxyl esterase activity was then monitored and compared
with control
autohydrolysis of the substrate using an spectrophotometer set at 405 nm (Wan,
L. et al. (2000) J.
Biol. Chem 275:10041-10046).
Heparan sulfate 6-sulfotransferase activity of DME is measured in vitro by
incubating a
sample containing DME along with 2.5 ,umol imidazole HCl (pH 6.8), 3.75 ug 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 heal
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 p,mol (as glucuronic acid) of chondroitin sulfate A is added to the
reaction mixture as a
carrier. 35S-labeled polysaccharides are precipitated with 3 volumes of cold
ethanol containing 1.3%
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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 deftned as
the amount required to transfer I pmol of sulfate/min. as determined by the
amount of [35S]PAPS
incorporated into the precipitated polysaccharides (Habuchi, H. et al. (1995)
J. Biol. Chem.
270:4172-4179).
In the alternative, heparan sulfate 6-sulfotrausferase 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 0.05
M Tris-HCl, pH 8.0, cut into 3-5 mm segments and subjected to agitation at 4
°C with 100 ~,1 of 0,05
M Tris-HCl, pH 8.0 containing 0.15 M NaCl for 48 h. The eluted enzyme is
collected by centrifugation
and assayed for the sulfotransferase activity as 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. In one example, the
peptide
(QATEYEYLDYDFLPEC) represents the N-terminal 15 residues of the mature P-
selectin
glycoprotein ligand-1 polypeptide to which is added C-terminal cysteine
residue. 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 ~,mol peptide/ml of
resin. The enzyme assay is
performed by combining 10 ~,1 of peptide-derivitized beads with 2-20 p1 of DME-
containing sample in
40 mM Pipes (pH 6.8), 0.3 M NaCl, 20 mM MnCh, 50 mM NaF, I % Triton X-I00, and
1 mM
5'-AMP in a final volume of 130 p.1. The assay is initiated by. addition of
0.5 ~.Ci of [35S]PAPS (1.7
~.M; 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, contains 50 mM Hepes-NaOH (pH 7.0),
250 mM sucrose,
1 mM dithiothreitol, 14 ~.M[35S]PAPS (15 Ci/mmol), and dopamine (25 ,uM), 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
108
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
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
relates of migration of reaction products (Sakakibara, Y. et al. (1998) J.
Biol. Chem
273:6242-6247).
Squalene epoxidase activity of DME is assayed in a mixture comprising purified
DME (or a
crude mixture comprising DME), 20 mM Tris-HCl (pH 7.5), 0.01 mM FAD, 0.2 unit
of
NADPH-cytochrome C (P-450) reductase, 0.01 mM [14C]squalene (dispersed with
the aid of 20 ~Cl 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
epode hydrolase control sample is incubated in 10 mM Tris-HCl (pH 8.0), 1 mM
ethylenediaminetetraacetate (EDTA), and 5 mM epoxide substrate (e.g., ethylene
oxide, styrene
oxide, propylene oxide, isoprene monoxide, epichlorohydrin, epibromohydrin,
epifluorohydrin, glycidol,
1,2-epoxybutane, 1,2-epoxyhexane, or 1,2-epoxyoctanea). A portion of the
sample is withdrawn from
the reaction mixture at various time points, and added to 1 ml of ice-cold
acetone containing an internal
standard for GC analysis (e.g., 1-nonanol). Protein and salts are removed by
centrifugation (15 min,
4000 x g) and the extract is analyzed by GC using a 0.2 mm x 25-m CP-Wax57-CB
column
(CHROMPACK, Middelburg, The Netherlands) and a flame-ionization detector. The
identification of
GC products is performed using appropriate standards and controls well known
to those skilled in the
art. 1 Unit of DME activity is defined as the amount of enzyme that catalyzes
the production of 1
~mol of diol/min (Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657).
Aminotransferase activity of DME is assayed by incubating samples containing
DME for 1
hour at 37 °C in the presence of 1 mM L-kynurenine and 1 mM 2-
oxoglutarate in a final volume of
200 p1 of 150 mM Tris acetate buffex (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
standards and controls well known to those skilled in the art. In the
alternative,
109
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
L-3-hydroxykynurenine is used as substrate and the production of xanthurenic
acid is determined by
HPLC analysis of the products with IJV detection at 340 nm. The production of
kynurenic acid
xanthurenic acid, respectively, is indicative of aminotransferase acitity
(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, PLP. The reactions are performed at 25
°C in 50 mM
4-methylinorpholine (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
wm due to the conversion of enzyme-bound PLP to 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
puri~ted protein preparations. Samples or lysates are resolved by
electrophoresis on 15%
non-denaturing polyacrylarnide gels. The gels are incubated for 30 min in 2.5
mM vitro blue'
tetrazolium, followed by incubation for 20 min in 30 mM potassium phosphate,
30 mM TEMED, and
30 p,M 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).
Catechol-O-methyltransferase activity of DME is measured in a reaction mixture
consisting
of 50 mM Tris-HCl (pH 7.4), 1.2 mM MgCl2, 200 ~,M SAM (S-adenosyl-z-
methionine) iodide
(containing 0.5 ~Ci of [methyl-[3H]SAM), 1 mM dithiothreitol, and varying
concentrations of
catechol substrate (e.g., z-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
3o 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 activity
110
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
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 (E34o = 11,800 Mwcm'1). The standard assay
mixture contains
100 p,M NADPH, 14 mM 2-mercaptoethanol, MTEN buffer (50 mM 2-
morpholinoethanesulfonic acid,
25 mM tris(hydroxymethyl)aminornethane, 25 mM ethanolamine, and 100 mM NaCl,
pH 7.0), and
DME in a final volume of 2.0 ml. The reaction is started by the addition of 50
p,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).
1o Sulfotransferase activity of DME is measured using the incorporation of 355
from [355]PAPS
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, 0.4-4.0 p,M [355]PAPS. After sufficient time for 5-20%
of the radiolabel to
be transferred to the substrate, 0.2 mL of 0.1 M barium acetate is added to
precipitate protein and
i5 phosphate buffer. Then 0.2 mL of 0.1 M Ba(OH)Z is added, followed by 0.2 mL
ZnS04. The
supernatant is cleared by centrifugation, which removes proteins as well as
unreacted [355]PAPS.
Radioactivity in the supernatant is measured by scintillation. The enzyme
activity is determined from
the number of moles of radioactivity in the reaction product.
XVIII. Identification of DME Inhibitors
20 Compounds to be tested are arrayed in the wells of a multi-well plate in
varying
concentrations along with an appropriate buffer and substrate, as described in
the assays in Example
XVII. DME activity is measured for each well and the ability of each compound
to inhibit DME
activity can be determined, as well as the dose-response profiles. This assay
could also be used to
identify molecules which enhance DME activity.
Various modifications and variations of the described 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 descn'bed 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.
111
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
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CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<110> INCYTE GENOMICS, INC.
BAUGHN, Mariah R.
BRUNS, Christopher M.
0A5, Debopriya
DELEGEANE, Angelo M.
DING, Li
ELLIOT, Vicki S.
GANDHI, Ameena R.
GRIFFIN, Jennifer A.
HAFALIA, April J.A.
KHAN, Farrah A.
LAL, Preeti
LEE, Sally
LU, Dyung Aina M.
LU, Yan
PATTERSON, Chandra
RAMKUMAR, Jayala~an.i
RING, Huijun Z.
SANJANWALA, Madhu S.
TANG, Y. Tom
THANGAVELU, Kavitha
THORNTON, Michael
TRIBOULEY, Catherine M.
WALIA, Narinder K.
WARREN, Bridget A.
YANG, Junming
YAO, Monique G.
YUE, Henry
<120> DRUG METABOLIZING ENZYMES
<130> PI-0185 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/223,055; 60/224,728; 60/226,440; 60/228,067; 60/230,063; 60/232,244;
60/234,269
<151> 2000-08-04; 2000-08-11; 2000-08-18; 2000-08-24; 2000-08-31; 2000-09-13;
2000-09-20
<160> 38
<170> PERL Program
<210> 1
<211> 756
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7248285C01
<400> 1
Met Ala Trp Ser Pro Pro Ala Thr Leu Phe Leu Phe Leu Leu Leu
1 5 10 15
Leu Gly Gln Pro Pro Pro Ser Arg Pro Gln Ser Leu Gly Thr Thr
20 25 30
Lys Leu Arg Leu Val Gly Pro Glu Ser Lys Pro Glu Glu Gly Arg
35 40 45
Leu Glu Val Leu His Gln Gly Gln Trp Gly Thr Val Cys Asp Asp
50 55 60
1/36
CA 02417769 2003-O1-29
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Asn Phe Ala Ile Gln Glu Ala Thr Val Ala Cys Arg Gln Leu Gly
65 70 75
Phe Glu Ala Ala Leu Thr Trp Ala His Ser Ala Lys Tyr Gly Gln
80 85 90
Gly Glu Gly Pro Ile Trp Leu Asp Asn Val Arg Cys Val Gly Thr
95 100 105
Glu Ser Ser Leu Asp Gln Cys Gly Ser Asn Gly Trp Gly Val Ser
110 115 120
Asp Cys Ser His Ser Glu Asp Val Gly Val Ile Cys His Pro Arg
125 130 135
Arg His Arg Gly Tyr Leu Ser Glu Thr Val Ser Asn Ala Leu Gly
140 145 150
Pro Gln Gly Gln Arg Leu Glu Glu Val Arg Leu Lys Pro Ile Leu
155 160 165
Ala Ser Ala Lys Gln His Ser Pro Val Thr Glu Gly Ala Val Glu
170 175 180
Val Lys Tyr Glu Gly His Trp Arg Gln Val Cys Asp G1n Gly Trp
185 190 195
Thr Met Asn Asn Ser Arg Val Val Cys Gly Met Leu Gly Phe Pro
200 205 210
Ser Glu Val Pro Val Asp Ser His Tyr Tyr Arg Lys Val Trp Asp
215 220 225
Leu Lys Met Arg Asg Pro Lys Ser Arg Leu Lys Ser Leu Thr Asn
230 235 240
Lys Asn Ser Phe Trp Ile His Gln Val Thr Cys Leu Gly Thr Glu
245 250 255
Pro His Met Ala Asn Cys Gln Val Gln Val Ala Pro Ala Arg Gly
260 265 270
Lys Leu Arg Pro Ala Cys Pro Gly Gly Met His Ala Va1 Val Ser
275 280 285
Cys Val Ala Gly Pro His Phe Arg Pro Pro Lys Thr Lys Pro Gln
290 295 300
Arg Lys Gly Ser Trp Ala Glu Glu Pro Arg Val Arg Leu Arg Ser
305 310 315
Gly Ala Gln Val Gly Glu Gly Arg Val Glu Val Leu Met Asn Arg
320 325 330
Gln Trp Gly Thr Val Cys Asp His Arg Trp Asn Leu Ile Ser Ala
335 340 345
Ser Val Val Cys Arg Gln Leu Gly Phe Gly Ser Ala Arg Glu Ala
350 355 360
Leu Phe G1y Ala Arg Leu Gly Gln Gly Leu Gly Pro Ile His Leu
365 370 375
Ser Glu Val Arg Cys Arg Gly Tyr Glu Arg Thr Leu Ser Asp Cys
380 385 390
Pro Ala Leu Glu Gly Ser Gln Asn Gly Cys Gln His Glu Asn Asp
395 400 405
Ala Ala Val Arg Cys Asn Val Pro Asn Met Gly Phe G1n Asn Gln
410 415 420
Val Arg Leu Ala Gly Gly Arg Ile Pro Glu Glu Gly Leu Leu Glu
425 430 435
Val Gln Val Glu Va1 Asn Gly Val Pro Arg Trp Gly Ser Val Cys
440 445 450
Ser G1u Asn Trp Gly Leu Thr Glu Ala Met Val Ala Cys Arg Gln
455 460 465
Leu Gly Leu Gly Phe Ala Ile His Ala Tyr Lys Glu Thr Trp Phe
470 475 480
Trp Ser Gly Thr Pro Arg Ala Gln Glu Val Val Met Ser Gly Val
485 490 495
Arg Cys Ser Gly Thr Glu Leu Ala Leu Gln Gln Cys Gln Arg His
500 505 510
Gly Pro Val His Cys Ser His Gly Gly Gly Arg Phe Leu Ala Gly
515 520 525
2136
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Val Ser Cys Met Asp Ser Ala Pro Asp Leu Val Met Asn Ala Gln
530 535 540
Leu Val Gln Glu Thr A1a Tyr Leu Glu Asp Arg Pro Leu Ser Gln
545 550 555
Leu Tyr Cys Ala His G1u Glu Asn Cys Leu Ser Lys Ser Ala Asp
560 565 570
His Met Asp Trp Pro Tyr Gly Tyr Arg Arg Leu Leu Arg Phe Ser
575 580 585
Thr Gln Ile Tyr Asn Leu Gly Arg Thr Asp Phe Arg Pro Lys Thr
590 595 600
Gly Arg Asp Ser Trp Val Trp His Gln Cys His Arg His Tyr His
605 610 615
Ser Ile Glu Val Phe Thr His Tyr Asp Leu Leu Thr Leu Asn G1y
620 625 630
Ser Lys Val Ala Glu Gly His Lys Ala Ser Phe Cys Leu Glu Asp
635 640 645
Thr Asn Cys Pro Thr G1y Leu Gln Arg Arg Tyr Ala Cys Ala Asn
650 655 660
Phe Gly G1u Gln Gly Val Thr Val Gly Cys Trp Asp Thr Tyr Arg
665 670 675
His Asp Ile Asp Cys G1n Trp Val Asp Ile Thr Asp Val Gly Pro
680 685 690
Gly Asn Tyr Ile Phe Gln Val Ile Val Asn Pro His Tyr Glu Val
695 700 705
Ala Glu Ser Asp Phe Ser Asn Asn Met Leu G1n Cys Arg Cys Lys
710 715 720
Tyr Asp Gly His Arg Val Trp Leu His Asn Cys His Thr Gly Asn
725 730 735
Ser Tyr Pro Ala Asn Ala Glu Leu Ser Leu Glu Gln Glu Gln Arg
740 745 750
Leu Arg Asn Asn Leu I1e
755
<210> 2
<211> 544
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472835CD1
<400> 2
Met Ala Lys Lys Ala I1e Ala Val Ile Gly A1a Gly Ile Ser Gly
1 5 10 15
Leu Gly Ala Ile Lys Cys Cys Leu Asp Glu Asp Leu Glu Pro Thr
20 25 30
Cys Phe Glu Arg Asn Asp Asp Ile Gly His Leu Trp Lys Phe Gln
35 40 45
Lys Asn Thr Ser Glu Lys Met Pro Ser Ile Tyr Lys Ser Val Thr
50 55 60
Ile Asn Thr Ser Lys G1u Met Met Cys Phe Ser Asp Phe Pro Val
65 70 75
Pro Asp His Phe Pro Asn Tyr Met His Asn Ser Lys Leu Met Asp
80 85 90
Tyr Phe Gly Met Tyr Ala Thr His Phe Gly Leu Leu Asn Tyr Ile
95 100 105
Arg Phe Lys Thr Glu Val Gln Ser Val Arg Lys His Pro Asp Phe
110 115 120
Ser Ile Asn Gly Gln Trp Asp Val Val Val Glu Thr Glu Glu Lys
125 130 135
Gln Glu Thr Leu Val Phe Asp Gly Val Leu Val Cys Ser Gly His
3/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
140 145 ' 150
His Thr Asp Pro Tyr Leu Pro Leu Gln Ser Phe Pro Gly Met Glu
155 160 165
Lys Phe Glu Gly Cys Tyr Phe His Ser Arg Glu Tyr Lys Ser Pro
170 175 180
Glu Asp Phe Ser Gly Lys Arg Ile Ile Val Ile Gly Ile Gly Asn
185 190 195
Ser Gly Val Asp Ile Ala Val Glu Leu Ser Arg Val Ala Lys Gln
200 205 210
Val Ile Phe Leu Ser Thr Arg Arg Gly Ser Trp Ile Leu His Arg
215 220 225
Val Trp Asp Asn Gly Tyr Pro Met Asp Ser Ser Phe Phe Thr Arg
230 235 240
Phe Asn Ser Phe Leu Gln Lys Ile Leu Thr Thr Pro Gln Ile Asn
245 250 255
Asn Gln Leu Glu Lys Ile Met Asn Ser Arg Phe Asn His Ala His
260 265 270
Cys Gly Leu Gln Pro Gln His Arg Ala Leu Ser Gln His Pro Thr
275 2S0 285
Val Ser Asp Asp Leu Pro Asn His Ile Ile Ser Gly Lys Val Gln
290 295 300
Val Lys Pro Ser Val Lys Glu Phe Thr Glu Thr Asp Ala Ile Phe
305 310 315
Glu Asp Ser Thr Val Glu Glu Asn Ile Asp Val Val Ile Phe Ala
320 325 330
Thr Gly Tyr Ser Phe Ser Phe Ser Phe Leu Asp G1y Leu Ile Lys
335 340 345
Val Thr Asn Asn Glu Val Ser Leu Tyr Lys Leu Met Phe Pro Pro
350 355 360
Asp Leu Glu Lys Pro Thr Leu Ala Val Ile Gly Leu Ile Gln Pro
365 370 375
Leu Gly Ile Ile Leu Pro Ile Ala Glu Leu Gln Ser Arg Trp Ala
380 385 390
Thr Arg Val Phe Lys Gly Leu Ile Lys Leu Pro Ser Ala Glu Asn
395 400 405
Met Met Ala Asp Ile Ala Gln Arg Lys Arg Ala Met Glu Lys Arg
410 415 420
Tyr Val Lys Thr Pro Arg His Thr Ile Gln Val Asp His Ile Glu
425 430 435
Tyr Met Asp Glu Ile Ala Met Pro Ala Gly Val Lys Pro Asn Leu
440 445 450
Leu Phe Leu Phe Leu Ser Asp Pro Lys Leu Ala Met G1u Val Phe
455 460 465
Phe Gly Pro Cys Thr Pro Tyr Gln Tyr His Leu His Gly Pro Glu
470 475 480
Lys Trp Asp Gly Ala Arg Arg Ala Asn Leu Thr Gln Arg Glu Arg
485 490 495
Ile Ile Lys Pro Leu Arg Thr Arg Ile Thr Ser Glu Asp Ser His
500 5o-5 510
Pro Ser Ser Gln Leu Ser Trp Ile Lys Met Ala Pro Val Ser Leu
515 520 525
Ala Phe Leu Ala Ala Gly Leu Ala Tyr Phe Arg Tyr Thr Pro Tyr
530 535 540
Gly Lys Trp Lys
<210> 3
<211> 501
<212> PRT
<213> Homo sapiens
<220>
4/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<221> misc_feature
<223> Incyte ID No: 7476203CD1
<400> 3
Met Leu Ser Leu Leu Ser Gly Leu Ala Leu Leu Ala Ile Ser Phe
1 5 10 15
Leu Leu Leu Lys Leu Gly Thr Phe Cys Trp Asp Arg Ser Cys Leu
20 25 30
Pro Pro Gly Pro Leu Pro Phe Pro Ile Leu Gly Asn Leu Trp Gln
35 40 45
Leu Cys Phe Gln Gln Pro His Leu Ser Leu Lys Asn Phe Gln Lys
50 55 60
Lys Ile Gly Asn Ile Phe Met Asn Leu Gly Ser Ser Val Val Pro
65 70 75
Leu Ala Leu Pro Leu Leu Pro Val Thr Phe His Pro Leu Asn Gln
80 85 90
Gly Val Leu Cys Lys Pro Leu Ile Thr Phe Pro Lys Pro Phe Pro
95 100 105
Thr Arg Asn Pro Gly Ile Ile Cys Ser Ser Gly His Thr Trp Arg
110 115 120
Gln Lys Arg Arg Phe Cys Leu Val Met Ile Arg Gly Leu Gly Leu
125 130 135
Gly Lys Leu Ala Leu Glu Val Gln Leu Gln Lys Glu Ala Ala Glu
140 145 150
Leu Ala Glu Ala Phe Arg Gln Glu Gln Gly Lys Arg Pro Phe Asp
155 160 165
Pro Gln Val Ser Ile Val Arg Ser Thr Val Arg Val Ile Gly Ala
170 175 180
Leu Val Phe Gly His His Phe Leu Leu Glu Asp Pro Ile Phe Gln
185 , 190 195
Glu Leu Thr Gln Ala Ile Asp Phe Gly Leu Ala Phe Val Ser Thr
200 205 210
Val Trp Arg Gln Leu Tyr Asp Val Phe Pro Trp Ala Leu Cys His
215 220 225
Leu Pro Gly Pro His Gln Glu I1e Phe Arg Tyr Gln Glu Val Val
230 235 240
Leu Ser Leu Ile His Gln Glu Ile Thr Arg His Lys Leu Arg Ala
245 250 255
Pro Glu Ala Pro Arg Asp Phe Ile Ser Cys Tyr Leu Ala Gln Ile
260 265 270
Ser Lys Ala Met Asp Asp Pro Val Ser Thr Phe Asn Gln Glu Asn
275 280 285
Leu Val Gln Val Val Ile Asp Leu Phe Leu Gly Gly Thr Asp Thr
290 295 300
Thr Ala Thr Thr Leu Cys Trp Ala Leu Ile His Met Ile Gln His
305 310 315
Gly Ala Val Gln Glu Thr Val Gln Leu Glu Leu Asp Glu Val Leu
320 325 330
Gly Ala Ala Pro Val Val Cys Tyr Glu Asp Arg Lys Arg Leu Pro
335 340 345
Tyr Thr Met Ala Val Leu His Asp Val Gln Arg Leu Ser Ser Val
350 355 360
Met Ala Met Gly Ala Val Arg Gln Cys Val Thr Ser Thr Arg Val
365 370 375
Cys Ser Tyr Pro Val Ser Lys Gly Thr Ile Ile Leu Pro Asn Leu
380 385 390
Ala Ser Val Leu Tyr Asp Pro Glu Cys Trp Glu Thr Pra Arg Gln
395 400 405
Phe Asn Pro Gly His Phe Ser Asp Lys Asp Gly Asn Phe Val Ala
410 415 420
Asn Glu Ala Phe Leu Pro Phe Ser Ala Gly Thr Arg Val Tyr Pro
425 430 435
5/36
CA 02417769 2003-O1-29
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Ala Asp Gln Leu Ala Gln Met Glu Leu Phe Leu Met Phe Ala Thr
440 445 450
Leu Leu Arg Thr Phe Arg Phe Gln Leu Pro Glu Gly Ser Pro Gly
455 460 465
Leu Lys Leu Glu Tyr Ile Phe Gly Gly Thr Trp Gln Pro Gln Pro
470 475 480
Gln Glu Ile Cys Ala Val Pro Arg Leu Ser Ser Pro Ser Pro Gly
485 490 495
Pro Arg Glu Asp Gly Leu
500
<210> 4
<211> 345
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID~ No: 7478583CD1
<400> 4
Met Lys Ala Ala Val Trp Tyr Gly Gln Lys Asp Val Arg Val Glu
1 5 10 15
Glu Arg Glu Pro Lys Glu Leu Gln Asp Asn Glu Val Lys Va1 Lys
20 25 30
Val Ser Trp Ala Gly 21e Cys Gly Thr Asp Leu His Glu Tyr Leu
35 40 45
Glu Gly Pro Ile Phe Ile Ser Thr Glu Lys Pro Asp Pro Phe Leu
50 55 60
Gly Gln Lys Ala Pro Val Thr Leu Gly His Glu Phe Ala Gly Val
65 70 75
Val Glu Glu Thr Gly Ser Gln Val Thr Lys Phe Asn Lys Gly Asp
80 85 90
Arg Val Val Val Asn Pro Thr Val Ser Lys Arg Glu Lys Glu Glu
95 100 105
Asn Ile Asp Leu Tyr Asp Gly Tyr Ser Phe Ile Gly Leu Gly Ser
110 115 120
Asp Gly Gly Phe Ala Glu Phe Thr Asn Ala Pro G1u Glu Asn Val
125 130 135
Tyr Lys Leu Pro Asp Asn Val Ser Asp Lys Glu Gly Ala Leu Val
140 145 150
Glu Pro Thr Ala Val Ala Val Gln Ala Ile Lys Glu Gly Glu Val
155 160 165
Leu Phe Gly Asp Thr Val Ala Ile Phe Gly Ala G1y Pro Ile Gly
170 175 180
Leu Leu Thr Val Val Ala Ala Lys Ala Ala Gly Ala Ser Lys Ile
185 190 195
Phe Val Phe Asp Leu Ser Glu Glu Arg Leu Ser Lys Ala Lys Ala
200 205 210
Leu Gly Ala Thr His A1a Ile Asn Ser Gly Lys Thr Asp Pro Val
215 220 225
Asp Val Ile Asn Glu Tyr Thr Glu Asn Gly Val Asp Val Ser Phe
230 235 240
Glu Val Ala Gly Val Ala Pro Thr Leu Lys Ser Ser Ile Asp Val
245 250 255
Thr Lys Ala Arg Gly Thr Val Val Ile Val Ser Ile Phe Gly His
260 265 270
Pro Ile Glu Trp Asn Pro Met Gln Leu Thr Asn Thr Gly Val Lys
275 280 285
Leu Thr Ser Thr Ile Ala Tyr Thr Pro Thr Thr Phe Gln Gln Thr
290 295 300
Ile Asp Leu Ile Asn Glu Gly Asn Leu Asn Val Lys Asp Val Val
6/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
305 310 315
Thr Asp Glu Ile Glu Leu Glu Asn Ile Val Glu Ser Gly Phe Glu
320 325 330
Gln Leu Val Asn Asp Lys Ser Gln Ala Lys Ile Leu I1e Lys Leu
335 340 345
<210> 5
<211> 361
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478585CD1
<400> 5
Met Ser Ala Gln Phe Glu Asn Val Gln Asn Pro Ser Ile Thr Arg
1 5 10 15
Glu Asp Val Ala Glu Val Leu Val Ser Val Leu Thr Asp Glu Thr
20 25 30
Leu Gln Val Val Leu Ala Lys Arg Pro Gln Ser Ile Pro Gln Asp
35 40 45
Asp Val Phe Arg Phe Glu Thr Ile Glu Thr Arg Glu Pro His Ala
50 55 60
Gly Glu Val Gln Val Glu Ser Ile Tyr Val Ser Val Asp Pro Tyr
65 70 75
Met Arg Gly Arg Met Asn Asp Thr Lys Ser Tyr Val Gln Pro Phe
80 85 90
Gln Val Asn Glu Pro Leu Gln Gly His Ile Val Gly Lys Val Thr
95 100 105
Gln Ser Asn Asp Glu Arg Leu Ser Val Gly Asp Tyr Val Thr Gly
110 115 120
Ile Leu Pro Trp Lys Lys Ile Asn Thr Val Asn Gly Asp Asp Val
125 130 135
Thr Pro Val Pro Ser Lys Asp Val Pro Leu His Leu Tyr Leu Ser
140 145 150
Val Leu Gly Met Pro Gly Met Thr Ala Tyr Thr Gly Leu Leu Gln.
155 160 165
Ile Gly Gln Pro Gln Ser Gly Glu Thr Val Val Val Ser Ala Ala
170 175 180
Ser Gly Ala Val Gly Ser Val Val Gly Gln Ile Ala Lys Ile Lys
185 190 195
Gly Ala Lys Val Val Gly Ile Ala Gly Gly Lys Gln Lys Thr Thr
200 205 210
Tyr Leu Thr Asp Glu Leu Gly Phe Asp Ala Ala Ile Asp Tyr Lys
215 220 225
Gln Asp Asp Phe Ala Gln Gln Leu Glu Ala Ala Val Pro Asp Gly
230 235 240
Ile Asp Val Tyr Phe Glu Asn Val Gly Gly Val Ile Ser Asp Glu
245 250 255
Val Phe Lys His Leu Asn Arg Phe Ala Arg Val Pro Val Cys Gly
260 265 270
Ala Ile Ser Ala Tyr Asn Asn Glu Lys Asp Asp Ile Gly Pro Arg
275 280 285
Ile Gln Gly Thr Leu I1e Lys Asn Gln Ala Leu Met Gln Gly Phe
290 295 300
Val Val Ala Gln Phe Ala Asp His Phe Lys Glu Ala Ser Glu Gln
305 310 315
Leu Ala Gln Trp Val Ser Glu Gly Lys Ile Lys Phe Glu Val Thr
320 325 330
Ile Asp Glu Gly Phe Asp Asn Leu Pro Ser Ala Phe Arg Lys Leu
7/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
335 340 345
Phe Thr Gly Asp Asn Phe Gly Lys Gln Val Val Lys Ile Lys Glu
350 355 360
Glu
<210> 6
<211> 499
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7479904CD1
<400> 6
Met Asp Glu Lys Ser Asn Lys Leu Leu Leu Ala Leu Val Met Leu
1 5 10 15
Phe Leu Phe Ala Val Ile Val Leu Gln Tyr Val Cys Pro Gly Thr
20 25 30
Glu Cys Gln Leu Leu Arg Leu Gln Ala Phe Ser Ser Pro Val Pro
35 40 45
Asp Pro Tyr Arg Ser Glu Asp Glu Ser Ser Ala Arg Phe Val Pro
50 55 60
Arg Tyr Asn Phe Thr Arg Gly Asp Leu Leu Arg Lys Val Asp Phe
65 70 75
Asp Ile Lys Gly Asp Asp Leu Ile Val Phe Leu His Ile Gln Lys
80 85 90
Thr Gly Gly Thr Thr Phe Gly Arg His Leu Val Arg Asn Ile Gln
95 100 105
Leu Glu Gln Pro Cys Glu Cys Arg Val Gly Gln Lys Lys Cys Thr
110 115 120
Cys His Arg Pro Gly Lys Arg GIu Thr Trp Leu Phe Ser Arg Phe
125 130 135
Ser Thr Gly Trp Ser Cys Gly Leu His Ala Asp Trp Thr Glu Leu
140 145 150
Thr Ser Cys Val Pro Ser Val Val Asp Gly Lys Arg Asp Ala Arg
155 160 165
Leu Arg Pro Ser Arg Trp Arg Ile Phe Gln Ile Leu Asp Ala Ala
170 175 180
Ser Lys Asp Lys Arg Gly Ser Pro Asn Thr Asn Ala Gly A1a Asn
185 190 195
Ser Pro Ser Ser Thr Lys Thr Arg Asn Thr Ser Lys Ser G1y Lys
200 205 210
Asn Phe His Tyr Ile Thr Ile Leu Arg Asp Pro Val Ser Arg Tyr
215 220 225
Leu Ser Glu Trp Arg His Val Gln Arg Gly Ala Thr Trp Lys Ala
230 235 240
Ser Leu His Val Cys Asp Gly Arg Pro Pro Thr Ser Glu Glu Leu
245 250 255
Pro Ser Cys Tyr Thr Gly Asp Asp Trp Ser Gly Cys Pro Leu Lys
260 265 270
Glu Phe Met Asp Cys Pro Tyr Asn Leu Ala Asn Asn Arg Gln Val
275 280 285
Arg Met Leu Ser Asp Leu Thr Leu Val Gly Cys Tyr Asn Leu Ser
290 295 300
Val Met Pro Glu Lys Gln Arg Asn Lys Val Leu Leu Glu Ser Ala
305 310 315
Lys Ser Asn Leu Lys His Met Ala Phe Phe Gly Leu Thr Glu Phe
320 325 330
Gln Arg Lys Thr Gln Tyr Leu Phe Glu Lys Thr Phe Asn Met Asn
335 340 345
8/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Phe Ile Ser Pro Phe Thr Gln Tyr Asn Thr Thr Arg Ala Ser Ser
350 355 360
Val Glu Ile Asn Glu Glu Ile Gln Lys Arg Ile Glu Gly Leu Asn
365 370 375
~Phe Leu Asp Met Glu Leu Tyr Ser Tyr Ala Lys Asp Leu Phe Leu
380 385 390
Gln Arg Tyr Gln Phe Met Arg Gln Lys Glu His G1n Glu A1a Arg
395 400 405
Arg Lys Arg Gln Glu Gln Arg Lys Phe Leu Lys Gly Arg Leu Leu
410 415 420
Gln Thr His Phe Gln Ser Gln Gly Gln Gly Gln Ser Gln Asn Pro
425 430 435
Asn Gln Asn Gln Ser Gln Asn Pro Asn Pro Asn Ala Asn Gln Asn
440 445 450
Leu Thr Gln Asn Leu Met Gln Asn Leu Thr Gln Ser Leu Ser Gln
455 46D 465
Lys Glu Asn Arg Glu Ser Pro Lys Gln Asn Ser Gly Lys Glu G1n
470 475 480
Asn Asp Asn Thr Ser Asn Gly Thr Asn Asp Tyr Ile Gly Ser Val
485 490 495
Glu Lys Trp Arg
<210> 7
<211> 222
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480367CD1
<400> 7
Met Ala Glu Lys Pro Lys Leu His Tyr Ser Asn Ala Arg Gly Ser
1 5 10 15
Met Glu Ser Ile Arg Trp Leu Leu Ala Ala Ala Gly Val Glu Leu
20 25 30
Glu Glu Lys Phe Leu Glu Ser Ala Glu Asp Leu Asp Lys Leu Arg
35 40 45
Asn Asp Gly Ser Leu Leu Phe Gln Gln Val Pro Met Val Glu Ile
50 55 60
Asp Gly Met Lys Leu Val Gln Thr Arg Ala Ile Leu Asn Tyr Ile
65 70 75
Ala Ser Lys Tyr Asn Leu Tyr Gly Lys Asp Met Lys Glu Arg Ala
80 85 90
Leu Ile Asp Met Tyr Thr Glu Gly Ile Val Asp Leu Thr Glu Met
95 100 105
Ile Leu Leu Leu Leu Ile Cys G1n Pro Glu Glu Arg Asp Ala Lys
110 115 120
Thr Ala Leu Val Lys Glu Lys Ile Lys Asn Arg Tyr Phe Pro Ala
125 130 135
Phe Glu Lys Val Leu Lys Ser His Arg Gln Asp Tyr Leu Val Gly
140 145 150
Asn Lys Leu Ser Trp Ala Asp Ile His Leu Val Glu Leu Phe Tyr
155 160 165
Tyr Val Glu Glu Leu Asp Ser Ser Leu Ile Ser Ser Phe Pro Leu
170 175 180
Leu Lys Ala Leu Lys Thr Arg Ile Ser Asn Leu Pro Thr Val Lys
185 190 195
Lys Phe Leu Gln Pro Gly Ser Gln Arg Lys Pro Pro Met Asp Glu
200 205 210
Lys Ser Leu Glu Glu Ala Arg Lys Ile Phe Arg Phe
9/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
215 220
<210> 8
<211> 330
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8069390CD1
<400> 8
Met Ala Ala Val Asp Ser Phe Tyr Leu Leu Tyr Arg Glu Ile Ala
1 5 10 15
Arg Ser Cys Asn Cys Tyr Met Glu Ala Leu Ala Leu Val Gly Ala
20 25 30
Trp Tyr Thr Ala Arg Lys Ser Ile Thr Va1 Ile Cys Asp Phe Tyr
35 40 45
Ser Leu Ile Arg Leu His Phe Ile Pro Arg Leu Gly Ser Arg Ala
50 55 60
Asp Leu Ile Lys Gln Tyr Gly Arg Trp Ala Val Val Ser Gly Ala
65 70 75
Thr Asp Gly Ile Gly Lys Ala Tyr Ala Glu Glu Leu Ala Ser Arg
80 85 90
Gly Leu Asn Ile Ile Leu Ile Ser Arg Asn G1u Glu Lys Leu Gln
95 100 105
Val Va1 Ala Lys Asp Ile Ala Asp Thr Tyr Lys Val Glu Thr Asp
110 115 120
Ile Ile Val Ala Asp Phe Ser Ser Gly Arg Glu I1e Tyr Leu Pro
125 130 135
Ile Arg Glu Ala Leu Lys Asp Lys Asp Va1 Gly Ile Leu Va1 Asn
140 145 150
Asn Va1 Gly Val Phe Tyr Pro Tyr Pro Gln Tyr Phe Thr GIn Leu
155 160 165
Ser Glu Asp Lys Leu Trp Asp Ile Ile Asn Val Asn Ile AIa Ala
170 175 180
Ala Ser Leu Met Val His Val Val Leu Pro Gly Met Val Glu Arg
185 190 295
Lys Lys Gly Ala Ile Val Thr Ile Ser Ser Gly Ser Cys Cys Lys
200 205 210
Pro Thr Pro Gln Leu Ala Ala Phe Ser Ala Ser Lys Ala Tyr Leu
215 220 225
Asp His Phe Ser Arg Ala Leu Gln Tyr Glu Tyr Ala Ser Lys Gly
230 235 240
Ile Phe Val Gln Ser Leu Ile Pro Phe Tyr Val A1a Thr Ser Met
245 250 255
Thr Ala Pro Ser Asn Phe Leu His Arg Cys Ser Trp Leu Val Pro
260 265 270
Ser Pra Lys Va1 Tyr AIa His His Ala Va1 Ser Thr Leu Gly Ile
275 280 285
Ser Lys Arg Thr Thr Gly Tyr Trp Ser His Ser Ile Gln Phe Leu
290 295 300
Phe Ala Gln Tyr Met Pro Glu Trp Leu Trp Val Trp Gly Ala Asn
305 310 315
Ile Leu Asn Arg Ser Leu Arg Lys Glu Ala Leu Ser Cys Thr Ala
320 325 330
<210> 9
<211> 303
<212> PRT
<213> Homo Sapiens
10/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<220>
<221> misc_feature
<223> Incyte ID No: 7473869CD1
<400> 9
Met Tyr Val Ser Thr Arg G1u Arg Tyr Lys Trp Leu Arg Phe Ser
1 5 10 15
Glu Asp Cys Leu Tyr Leu Asn Val Tyr Ala Pro Ala Arg Ala Pro
20 25 30
Gly Asp Pro Gln Leu Pro Val Met Val Trp Phe Pro Gly Gly Ala
35 40 45
Phe Ile Val Gly Ala Ala Ser Ser Tyr Glu G1y Ser Asp Leu Ala
50 55 60
Ala Arg Glu Lys Val Val Leu Val Phe Leu Gln His Arg Leu Gly
65 70 75
Ile Phe Gly Phe Leu Ser Thr Asp Asp Ser His Ala Arg Gly Asn
80 85 90
Trp Gly Leu Leu Asp Gln Met Ala Ala Leu Arg Trp Val Gln Glu
95 100 105
Asn Ile Ala Ala Phe Gly Gly Asp Pro Gly Asn Val Thr Leu Phe
110 115 120
Gly Gln Ser Ala Gly Ala Met Ser Ile Ser Gly Leu Met Met Ser
125 130 135
Pro Leu Ala Ser Gly Leu Phe His Arg Ala Ile Ser Gln Ser Gly
140 145 150
Thr Ala Leu Phe Arg Leu Phe Ile Thr Ser Asn Pro Leu Lys Val
155 160 165
Ala Lys Lys Val Ala His Leu Ala G1y Cys Asn His Asn Ser Thr
170 175 180
Gln Ile Leu Val Asn Cys Leu Arg Ala Leu Ser Gly Thr Lys Val
185 190 195
Met Arg Val Ser Asn Lys Met Arg Phe Leu Gln Leu Asn Phe Gln
200 205 210
Arg Asp Pro Glu Glu Ile Ile Trp Ser Met Ser Pro Val Val Asp
215 220' 225
Gly Val Val Ile Pro Asp Asp Pro Leu Val Leu Leu Thr Gln Gly
230 235 240
Lys Val Ser Ser Val Pro Tyr Leu Leu Gly Val Asn Asn Leu Glu
245 250 255
Phe Asn Trp Leu Leu Pro Tyr Ile Met Lys Phe Pro Leu Asn Arg
260 265 270
Gln Ala Met Arg Lys Glu Thr Ile Thr Lys Met Leu Trp Ser Thr
275 280 285
Arg Thr Leu Leu Val Arg Asp Pro Ala Gly Arg Gly Ala Gln Phe
290 295 300
Gly Gln Gly
<210> 10
<211> 584
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478588CD1
<400> 10
Met Pro Ser Thr Val Leu Pro Ser Thr Val Leu Pro Ser Leu Leu
1 5 10 15
Pro Thr Ala Gly Ala Gly Trp Ser Met Arg Trp Ile Leu Cys Trp
20 25 30
11/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Ser Leu Thr Leu Cys Leu Met Ala Gln Thr Ala Leu Gly Ala Leu
35 40 45
His Thr Lys Arg Pro Gln Val Val Thr Lys Tyr Gly Thr Leu Gln
50 55 60
Gly Lys Gln Met His Val Gly Lys Thr Pro Ile Gln Val Phe Leu
65 70 75
Gly Val Pro Phe Ser Arg Pro Pro Leu Gly Ile Leu Arg Phe Ala
80 85 90
Pro Pro Glu Pro Pro Glu Pro Trp Lys Gly Ile Arg Asp Ala Thr
95 100 105
Thr Tyr Pro Pro Gly Cys Leu Gln Glu Ser Trp Gly Gln Leu Ala
110 ' 115 120
Ser Met Tyr Val Ser Thr Arg Glu Arg Tyr Lys Trp Leu Arg Phe
125 130 135
Ser Glu Asp Cys Leu Tyr Leu Asn Val Tyr Ala Pro Ala Arg Ala
140 145 150
Pro Gly Asp Pro Gln Leu Pro Val Met Val Trp Phe Pro Gly Gly
155 160 165
Ala Phe Ile Val Gly Ala Ala Ser Ser Tyr Glu Gly Ser Asp Leu
170 175 180
Ala Ala Arg Glu Lys Val Val Leu Val Phe Leu Gln His Arg Leu
185 190 195
Gly Ile Phe Gly Phe Leu Ser Thr Asp Asp Ser His Ala Arg Gly
200 205 210
Asn Trp Gly Leu Leu Asp Gln Met Ala Ala Leu Arg Trp Val Gln
215 220 225
Glu Asn Ile Ala Ala Phe Gly Gly Asp Pro Gly Asn Val Thr Leu
230 235 240
Phe Gly Gln Ser Ala Gly Ala Met Ser I1e Ser Gly Leu Met Met
245 250 255
Ser Pro Leu Ala Ser Gly Leu Phe His Arg Ala Ile Ser Gln Ser
260 265 270
Gly Thr Ala Leu Phe Arg Leu Phe Ile Thr Ser Asn Pro Leu Lys
275 280 285
Val Ala Lys Lys Val Ala His Leu Ala Gly Cys Asn His Asn Ser
290 295 300
Thr Gln Ile Leu Val Asn Cys Leu Arg Ala Leu Ser Gly Thr Lys
305 310 315
Val Met Arg Val Ser Asn Lys Met Arg Phe Leu Gln Leu Asn Phe
320 325 330
Gln Arg Asp Pro Glu Glu Ile Ile Trp Ser Met Ser Pro Val Val
335 340 345
Asp Gly Val Val Ile Pro Asp Asp Pro Leu Val Leu Leu Thr Gln
350 355 360
Gly Lys Val Ser Ser Val Pro Tyr Leu Leu Gly Val Asn Asn Leu
365 370 375
Glu Phe Asn Trp Leu Leu Pro Tyr Ile Met Lys Phe Pro Leu Asn
380 385 390
Arg Gln Ala Met Arg Lys Glu Thr Ile Thr Lys Met Leu Trp Ser
395 400 405
Thr Arg Thr Leu Leu Asn Ile Thr Lys Glu Gln Val Pro Leu Val
410 415 420
Val Glu Glu Tyr Leu Asp Asn Val Asn Glu His Asp Trp Lys Met
425 430 435
Leu Arg Asn Arg Met Met Asp Ile Val Gln Asp Ala Thr Phe Val
440 445 450
Tyr Ala Thr Leu Gln Thr Ala His Tyr His Arg Asp Ala Gly Leu
455 460 465
Pro Val Tyr Leu Tyr Glu Phe Glu His His Ala Arg Gly Ile Ile
470 475 480
Val Lys Pro Arg Thr Asp Gly Ala Asp His Gly Asp Glu Met Tyr
485 490 495
12/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Phe Leu Phe Gly Gly Pro Phe Ala Thr Gly Leu Ser Met Gly Lys
500 505 510
Glu Lys Ala Leu Ser Leu Gln Met Met Lys Tyr Trp Ala Asn Phe
515 520 525
Ala Arg Thr G1y Asn Pro Asn Asp Gly Asn Leu Pro Cys Trp Pro
530 535 540
Arg Tyr Asn Lys Asp Glu Lys Tyr Leu Gln Leu Asp Phe Thr Thr
545 550 555
Arg Val Gly Met Lys Leu Lys Glu Lys Lys Met Ala Phe Trp Met
560 565 570
Ser Leu 'I'yr Gln Ser Gln Arg Pro Glu Lys Gln Arg Gln Phe
575 580
<210> 11
<211> 508
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55046125CD1
<400> 11
Met His Val Leu Arg Arg Arg Trp Asp Leu Gly Ser Leu Cys Arg
1 5 10 15
Ala Leu Leu Thr Arg Gly Leu Ala Ala Leu Gly His Ser Leu Lys
20 25 30
His Val Leu Gly Ala Ile Phe Ser Lys Ile Phe Gly Pro Met Ala
35 40 45
Ser Val Gly Asn Met Asp Glu Lys Ser Asn Lys Leu Leu Leu Ala
50 55 60
Leu Val Met Leu Phe Leu Phe Ala Val Ile Val Leu Gln Tyr Val
65 70 75
Cys Pro Gly Thr Glu Cys Gln Leu Leu Arg Leu Gln Ala Phe Ser
80 85 90
Ser Pro Val Pro Asp Pro Tyr Arg Ser Glu Asp Glu Ser Ser Ala
95 100. 105
Arg Phe Val Pro Arg Tyr Asn Phe Thr Arg Gly Asp Leu Leu Arg
110 115 120
Lys Val Asp Phe Asp Ile Lys Gly Asp Asp Leu Ile Val Phe Leu
125 130 135
His Ile Gln Lys Thr Gly Gly Thr Thr Phe Gly Arg His Leu Val
140 145 150
Arg Asn Ile Gln Leu Glu Gln Pro Cys Glu Cys Arg Val Gly Gln
155 160 165
Lys Lys Cys Thr Cys His Arg Pro Gly Lys Arg Glu Thr Trp Leu
170 175 180
Phe Ser Arg Phe Ser Thr Gly Trp Ser Cys Gly Leu His Ala Asp
185 190 195
Trp Thr Glu Leu Thr Ser Cys Val Pro Ser Val Val Asp Gly Lys
200 205 210
Arg Asp Ala Arg Leu Arg Pro Ser Arg Asn Phe His Tyr Ile Thr
215 220' 225
Ile Leu Arg Asp Pro Val Ser Arg Tyr Leu Ser Glu Trp Arg His
230 235 240
Val Gln Arg Gly Ala Thr Trp Lys Ala Ser Leu His Val Cys Asp
245 250 255
Gly Arg Pro Pro Thr Ser Glu Glu Leu Pro Ser Cys Tyr Thr Gly
260 265 270
Asp Asp Trp Ser Gly Cys Pro Leu Lys Glu Phe Met Asp Cys Pro
275 280 285
Tyr Asn Leu Ala Asn Asn Arg Gln Val Arg Met Leu Ser Asp Leu
13/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
290 295 300
Thr Leu Val Gly Cys Tyr Asn Leu Ser Val Met Pro Glu Lys Gln
305 310 315
Arg Asn Lys Val Leu Leu Glu Ser Ala Lys Ser Asn Leu Lys His
320 325 330
Met Ala Phe Phe Gly Leu Thr Glu Phe Gln Arg Lys Thr Gln Tyr
335 340 345
Leu Phe Glu Lys Thr Phe Asn Met Asn Phe Ile Ser Pro Phe Thr
350 355 360
Gln Tyr Asn Thr Thr Arg Ala Ser Ser Val Glu Ile Asn Glu Glu
365 370 375
Ile Gln Lys Arg Ile Glu Gly Leu Asn Phe Leu Asp Met Glu Leu
380 385 390
Tyr Ser Tyr Ala Lys Asp Leu Phe Leu Gln Arg Tyr Gln Phe Met
395 400 405
Arg Gln Lys Glu His Gln Glu Ala Arg Arg Lys Arg Gln Glu Gln
410 415 420
Arg Lys Phe Leu Lys Gly Arg Leu Leu Gln Thr His Phe Gln Ser
425 430 435
Gln Gly Gln Gly Gln Ser Gln Asn Pro Asn Gln Asn Gln Ser Gln
440 445 450
Asn Pro Asn Pro Asn Ala Asn Gln Asn Leu Thr Gln Asn Leu Met
455 460 465
Gln Asn Leu Thr Gln Ser Leu Ser Gln Lys Glu Asn Arg Glu Ser
470 475 480
Pro Lys Gln Asn Ser Gly Lys Glu Gln Asn Asp Asn Thr Ser Asn
485 490 495
Gly Thr Asn Asp Tyr Ile Gly Ser Val Glu Lys Trp Arg
500 505
<210> 12
<211> 439
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3538709CD1
<400> 12
Met Leu Thr Gly Val Thr Asp Gly Ile Phe Cys Cys Leu Leu Gly
1 5 10 15
Thr Pro Pro Asn Ala Val Gly Pro Leu Glu Ser Val Glu Ser Ser
20 25 30
Asp Gly Tyr Thr Phe Val Glu Val Lys Pro Gly Arg Val Leu Arg
35 40 45
Val Lys His Ala G1y Pro Ala Pro Ala Ala Ala Pro Pro Pro Pro
50 55 60
Ser Ser Ala Ser Ser Asp Ala Ala Gln Gly Asp Leu Ser Gly Leu
65 70 75
Val Arg Cys Gln Arg Arg Ile Thr Val Tyr Arg Asn Gly Arg Leu
80 85 90
Leu Val Glu Asn Leu Gly Arg Ala Pro Arg Ala Asp Leu Leu His
95 100 105
Gly Gln Asn Gly Ser Gly Glu Pro Pro Ala Ala Leu Glu Val Glu
110 115 120
Leu Ala Asp Pro Ala Gly Ser Asp Gly Arg Leu Ala Pro Gly Ser
125 130 135
Ala Gly Ser Gly Ser Gly Ser Gly Ser G1y Gly Arg Arg Arg Arg
140 145 150
Ala Arg Arg Pro Lys Arg Thr Ile His Ile Asp Cys Glu Lys Arg
155 160 165
14/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Ile Thr Ser Cys Lys Gly Ala Gln Ala Asp Val Val Leu Phe Phe
170 175 180
Ile His Gly Val Gly Gly Ser Leu Ala Ile Trp Lys Glu Gln Leu
185 190 195
Asp Phe Phe Val Arg Leu Gly Tyr Glu Val Val Ala Pro Asp Leu
200 205 210
Ala Gly His Gly Ala Ser Ser Ala Pro Gln Val Ala Ala Ala Tyr
215 220 225
Thr Phe Tyr A1a Leu Ala Glu Asp Met Arg Ala Ile Phe Lys Arg
230 235 240
Tyr Ala Lys Lys Arg Asn Val Leu Ile Gly His Ser Tyr Gly Val
245 250 255
Ser Phe Cys Thr Phe Leu Ala His Glu Tyr Pro Asp Leu Val His
260 265 270
Lys Val Ile Met Ile Asn Gly Gly Gly Pro Thr Ala Leu Glu Pro
275 280 285
Ser Phe Cys Ser Ile Phe Asn Met Pro Thr Cys Val Leu His Cys
290 295 300
Leu Ser Pro Cys Leu Ala Trp Ser Phe Leu Lys Ala Gly Phe Ala
305 310 315
Arg Gln Gly Ala Lys Glu Lys Gln Leu Leu Lys Glu Gly Asn Ala
320 325 330
Phe Asn Val Ser Ser Phe Val Leu Arg Ala Met Met Ser Gly Gln
335 340 345
Tyr Trp Pro Glu Gly Asp Glu Val Tyr His Ala Glu Leu Thr Val
350 355 360
Pro Val Leu Leu Val His Gly Met His Asp Lys Phe Val Pro Val
365 370 375
Glu Glu Asp Gln Arg Met Ala Glu Ile Leu Leu Leu Ala Phe Leu
380 385 390
Lys Leu Ile Asp Glu Gly Ser His Met Val Met Leu Glu Cys Pro
395 400 405
Glu Thr Val Asn Thr Leu Leu His Glu Phe Leu Leu Trp Glu Pro
410 415 420
Glu Pro Ser Pro Lys Ala Leu Pro Glu Pro Leu Pro Ala Pro Pro
425 430 435
Glu Asp Lys Lys
<210> 13
<211> 514
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71563101CD1
<400> 13
Met Thr Leu Ile Trp Arg His Leu Leu Arg Pro Leu Cys Leu Va2
1 5 10 15
Thr Ser Ala Pro Arg Ile Leu Glu Met His Pro Phe Leu Ser Leu
20 25 30
Gly Thr Ser Arg Thr Ser Val Thr Lys Leu Ser Leu His Thr Lys
35 - 40 45
Pro Arg Met Pro Pro Cys Asp Phe Met Pro Glu Arg Tyr Gln Ser
50 55 60
Leu Gly Tyr Asn Arg Val Leu Glu Ile His Lys Glu His Leu Ser
65 70 75
Pro Val Val Thr Ala Tyr Phe Gln Lys Pro Leu Leu Leu His Gln
80 85 90
Gly His Met Glu Trp Leu Phe Asp Ala Glu Gly Asn Arg Tyr Leu
15/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
95 100 105
Asp Phe Phe Ser Gly Ile Val Thr Val Ser Val Gly His Cys His
110 115 120
Pro Lys Val Asn Ala Val Ala Gln Lys Gln Leu G1y Arg Leu Trp
125 130 135
His Thr Ser Thr Val Phe Phe His Pro Pro Met His Glu Tyr Ala
140 145 150
Glu Lys Leu Ala Ala Leu Leu Pro Glu Pro Leu Lys Val Ile Phe
155 160 165
Leu Val Asn Ser Gly Ser Glu Ala Asn Glu Leu Ala Met Leu Met
170 175 180
Ala Arg Ala His Ser Asn Asn Ile Asp I1e Ile Ser Phe Arg Gly
185 190 195
Ala Tyr His Gly Cys Ser Pro Tyr Thr Leu Gly Leu Thr Asn Val
200 205 210
Gly Ile Tyr Lys Met Glu Leu Pro Gly Gly Thr Gly Cys Gln Pro
215 220 225
Thr Met Cys Pro Asp Val Phe Arg Gly Pro Trp Gly Gly Ser His
230 235 240
Cys Arg Asp Ser Pro Val Gln Thr Ile Arg Lys Cys Ser Cys Ala
245 250 255
Pro Asp Cys Cys Gln Ala Lys Asp G1n Tyr Ile Glu Gln Phe Lys
260 265 270
Asp Thr Leu Ser Thr Ser Val Ala Lys Ser Ile Ala Gly Phe Phe
275 280 285
Ala Glu Pro Ile Gln Gly Val Asn G1y Val Val Gln Tyr Pro Lys
290 295 300
Gly Phe Leu Lys Glu Ala Phe Glu Leu Val Arg Ala Arg Gly Gly
305 310 315
Val Cys Ile Ala Asp Glu Val Gln Thr Gly Phe Gly Arg Leu Gly
320 325 330
Ser His Phe Trp Gly Phe Gln Thr His Asp Val Leu Pro Asp Ile
335 340 345
Val Thr Met Ala Lys Gly Ile Gly Asn Gly Phe Pro Met Ala Ala
350 355 360
Val Ile Thr Thr Pro Glu Ile Ala Lys Ser Leu Ala Lys Cys Leu
365 370 375
Gln His Phe Asn Thr Phe Gly Gly Asn Pro Met Ala Cys Ala Ile
380 385 390
Gly Ser Ala Val Leu Glu Val Ile Lys Glu Glu Asn Leu Gln Glu
395 400 405
Asn Ser Gln Glu Val Gly Thr Tyr Met Leu Leu Lys Phe Ala Lys
410 415 420
Leu Arg Asp Glu Phe Glu Ile Val Gly Asp Val Arg Gly Lys Gly
425 430 435
Leu Met Ile Gly Ile Glu Met Val Gln Asp Lys Ile Ser Cys Arg
440 445 450
Pro Leu Pro Arg Glu Glu Val Asn Gln Ile His Glu Asp Cys Lys
455 460 465
His Met Gly Leu Leu Val Gly Arg Gly Ser Ile Phe Ser Gln Thr
470 475 480
Phe Arg Ile Ala Pro Ser Met Cys Ile Thr Lys Pro Glu Val Asp
485 490 495
Phe Ala Val Glu Val Phe Arg Ser Ala Leu Thr Gln His Met Glu
500 505 510
Arg Arg Ala Lys
<210> 14
<211> 226
<212> PRT
<213> Homo Sapiens
16/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<220>
<221> misc_feature
<223> Incyte ID No: 7472027CD1
<400> 14
Met Arg Leu Cys Glu Lys Thr Glu Leu Gln Leu Ile Gly Val Pro
1 5 10 15
Glu Ser Asp Arg Glu Asn Gly Thr Lys Leu Glu Asn Thr Phe Gln
20 25 30
Asp Ile Ile Gln Glu Asn Phe Pro Asn Leu Ala Arg Gln Ala Asn
35 40 45
Ile Gln Ile Gln Met Ala Gly Gly Ser Ile Trp Ile Glu Gly Ile
50 55 60
Pro Phe Pro Ser Asn Asn Phe Thr Asp Leu Arg Arg Leu Gln Asp
65 70 75
Glu Ile Val Leu Arg Asp Glu Asp Val Ile Thr Leu Ser Tyr Pro
80 85 90
Lys Ser Gly Ser Phe Trp Ile Val Glu Ile Ile Ser Leu Ile His
95 100 105
Ser Lys Gly Asp Pro Ser Trp Val Gln Ser Val Val Pro Trp Asp
110 115 120
Arg Ser Pro Trp Ile Glu Val Lys Arg Lys Lys Ala Gly Leu Glu
125 130 135
Ser Gln Lys Gly Pro His Leu Tyr Thr Ser His Leu Pro Ile Gln
140 145 150
Leu Phe Pro Lys Ser Phe Leu Asn Ser Lys Ala Lys Cys Ile Tyr
155 160 165
Pro His Val Leu Met Leu Val Val Leu Ile Leu Gly His Lys Ser
170 175 180
Gln Trp Ser Ile Ala Ile Lys Ile Ser Glu Asn Ala G1u Ala Thr
185 190 195
Ser Lys Leu Gly Asn Gly Gln Arg Leu Glu Glu Phe Gly Gly Leu
200 205 210
Arg Arg Arg Gln Glu Asp Glu Arg Ser Leu Glu Phe Leu Arg Asp
215 220 225
Cys
<210> 15
<211> 121
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 748035BCD1
<400> 15
Met Met Lys Val Met Tyr Met Leu Lys Gly Gln Ser Pro Val Gln
1 5 10 15
Gly Thr Ile His Phe Glu Gln Lys Glu Asn Glu Pro Phe Met Val
20 25 30
Ser Glu Cys Ile Thr Gly Leu Thr Glu Arg Gln His Arg Phe His
35 40 45
Val His Gln Phe G1y Asp Asn Thr P-ro Gly Cys Thr Arg Ala Val
5D 55 60
Pro Tyr Phe Asn Pro Leu Thr Lys Asn His Ser Gly Pro Arg Ile
65 70 75
Lys Arg Gly Arg Leu Glu Thr Trp Val Met Trp Pro Leu Ala Lys
80 85 90
Met Cys Arg His Met Ser Val Glu Asp Ser Leu Val Ser Leu Ser
95 100 105
17/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Gly His Tyr Ser Ile Thr Ala His Thr Met Val Ser Met Thr Thr
110 115 120
Arg
<210> 16
<211> 486
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Tncyte ID No: 1618256CD1
<400> 16
Met Gly Pro Leu Ser Pro Ala Arg Thr Leu Arg Leu Trp Gly Pro
1 5 10 15
Arg Ser Leu Gly Val Ala Leu G1y Val Phe Met Thr Ile Gly Phe
20 25 30
Ala Leu Gln Leu Leu Gly Gly Pro Phe Gln Arg Arg Leu Pro Gly
35 40 45
Leu Gln Leu Arg Gln Pro Ser A1a Pro Ser Leu Arg Pro Ala Leu
50 55 60
Pro Ser Cys Pro Pro Arg Gln Arg Leu Val Phe Leu Lys Thr His
65 70 75
Lys Ser Gly Ser Ser Ser Val Leu Ser Leu Leu His Arg Tyr Gly
80 85 90
Asp Gln His Gly Leu Arg Phe A1a Leu Pro Ala Arg Tyr Gln Phe
95 100 105
Gly Tyr Pro Lys Leu Phe Gln Ala Ser Arg Val Lys Gly Tyr Arg
110 115 120
Pro Gln Gly G1y Gly Thr Gln Leu Pro Phe His Ile Leu Cys His
125 130 135
His Met Arg Phe Asn Leu Lys Glu Val Leu Gln Val Met Pro Ser
140 245 150
Asp Ser Phe Phe Phe Ser Ile Val Arg Asp Pro Ala Ala Leu Ala
155 160 165
Arg Ser Ala Phe Ser Tyr Tyr Lys Ser Thr Ser Ser Ala Phe Arg
170 175 180
Lys Sex- Pro Ser Leu Ala Ala Phe Leu Ala Asn Pro Arg Gly Phe
185 190 195
Tyr Arg Pro Gly Ala Arg Gly Asp His Tyr Ala Arg Asn Leu Leu
200 205 210
Trp Phe Asp Phe Gly Leu Pro Phe Pro Pro Glu Lys Arg Ala Lys
215 220 225
Arg Gly Asn Ile His Pro Pro Arg Asp Pro Asn Pro Pro Gln Leu
230 235 240
Gln Val Leu Pro Ser Gly Ala Gly Pro Arg Ala Gln Thr Leu Asn
245 250 255
Pro Asn Ala Leu Ile His Pro Val Ser Thr Val Thr Asp His Arg
260 265 270
Ser Gln Ile Ser Ser Pro Ala Ser Phe Asp Leu Gly Ser Ser Ser
275 280 285
Phe Ile Gln Trp Gly Leu Ala Trp Leu Asp Ser Val Phe Asp Leu
290 295 300
Val Met Val Ala Glu Tyr Phe Asp Glu Ser Leu Val Leu Leu Ala
305 310 315
Asp Ala Leu Cys Trp Gly Leu Asp Asp Val Val Gly Phe Met His
320 325 330
Asn Ala Gln A1a Gly His Lys Gln Gly Leu Ser Thr Val Ser Asn
335 340 345
Ser Gly Leu Thr AIa Glu Asp Arg Gln Leu Thr Ala Arg Ala Arg
18/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
350 355 360
Ala Trp Asn Asn Leu Asp Trp Ala Leu Tyr Val His Phe Asn Arg
365 370 375
Ser Leu Trp Ala Arg Ile Glu Lys Tyr Gly Gln Gly Arg Leu G1n
380 385 390
Thr Ala Val Ala Glu Leu Arg Ala Arg Arg Glu Ala Leu Ala Lys
395 400 405
His Cys Leu Val Gly Gly Glu Ala Ser Asp Pro Lys Tyr Ile Thr
410 415 420
Asp Arg Arg Phe Arg Pro Phe Gln Phe Gly Ser Ala Lys Val Leu
425 430 435
Gly Tyr Ile Leu Arg Ser Gly Leu Ser Pro Gln Asp Gln Glu Glu
440 445 450
Cys Glu Arg Leu Ala Thr Pro Glu Leu Gln Tyr Lys Asp Lys Leu
455 460 465
Asp Val Lys Gln Phe Pro Pro Thr Val Ser Leu Pro Leu Lys Thr
470 475 480
Ser Arg Pro Leu Ser Pro
485
<214> 17
<211> 649
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3387823CD1
<400> 17
Met Tyr Ile Ser Cys Leu Ser Leu Ser Leu Phe Phe Leu Ser Gly
1 5 10 15
Pro Leu Gln Arg Val Leu Glu Val Ser Asn His Trp Trp Tyr Ser
20 25 30
Met Leu Ile Leu Pro Pro Leu Leu Lys Asp Ser Val Ala Ala Pro
35 40 45
Leu Leu Ser Ala Tyr Tyr Pro Asp Cys Val Gly Met Ser Pro Ser
50 55 60
Cys Thr Ser Thr Asn Arg Ala Ala Ala Thr Gly Asn Ala Ser Pro
65 70 75
G1y Lys Leu Glu His Ser Lys Ala Ala Leu Ser Val His Val Pro
80 85 90
Gly Met Asn Arg Tyr Phe Gln Pro Phe Tyr Gln Pro Asn Glu Cys
95 100 105
Gly Lys Ala Leu Cys Val Arg Pro Asp Val Met Glu Leu Asp Glu
110 115 120
Leu Tyr Glu Phe Pro Glu Tyr Ser Arg Asp Pro Thr Met Tyr Leu
125 130 135
Ala Leu Arg Asn Leu Ile Leu Ala Leu Trp Tyr Thr Asn Cys Lys
140 145 150
Glu Ala Leu Thr Pro Gln Lys Cys Ile Pro His Ile Ile Val Arg
155 160 165
Gly Leu Val Arg Ile Arg Cys Val Gln Glu Val Glu Arg Ile Leu
170 175 180
Tyr Phe Met Thr Arg Lys Gly Leu Ile Asn Thr Gly Val Leu Ser
185 190 195
Val Gly Ala Asp Gln Tyr Leu Leu Pro Lys Asp Tyr His Asn Lys
200 205 210
Ser Val Ile Ile Ile Gly Ala Gly Pro Ala Gly Leu Ala Ala Ala
215 220 225
Arg Gln Leu His Asn Phe Gly Ile Lys Val Thr Val Leu Glu Ala
230 235 240
19/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Lys Asp Arg Ile Gly Gly Arg Val Trp Asp Asp Lys Ser Phe Lys
245 250 255
Gly Val Thr Val Gly Arg Gly Ala Gln Ile Val Asn Gly Cys Ile
260 265 270
Asn Asn Pro Val Ala Leu Met Cys Glu Gln Leu Gly Ile Ser Met
275 280 285
His Lys Phe Gly Glu Arg Cys Asp Leu Ile Gln Glu Gly Gly Arg
290 295 300
Ile Thr Asp Pro Thr Ile Asp Lys Arg Met Asp Phe His Phe Asn
305 310 315
Ala Leu Leu Asp Val Val Ser Glu Trp Arg Lys Asp Lys Thr Gln
320 325 330
Leu Gln Asp Val Pro Leu Gly Glu Lys Ile Glu Glu Ile Tyr Lys
335 340 345
Ala Phe Ile Lys Glu Ser Gly Ile Gln Phe Ser Glu Leu Glu Gly
350 355 360
Gln Val Leu Gln Phe His Leu Ser Asn Leu Glu Tyr Ala Cys Gly
365 370 375
Ser Asn Leu His Gln Val Ser Ala Arg Ser Trp Asp His Asn Glu
380 385 390
Phe Phe Ala Gln Phe Ala Gly Asp His Thr Leu Leu Thr Pro Gly
395 400 405
Tyr Ser Val Ile Ile Glu Lys Leu Ala Glu Gly Leu Asp I1e Gln
410 415 420
Leu Lys Ser Pro Val Gln Cys Ile Asp Tyr Ser Gly Asp Glu Val
425 430 435
Gln Val Thr Thr Thr Asp Gly Thr Gly Tyr Ser Ala Gln Lys Val
440 445 450
Leu Va1 Thr Val Pro Leu Ala Leu Leu Gln Lys Gly Ala Ile Gln
455 460 465
Phe Asn Pro Pro Leu Ser Glu Lys Lys Met Lys Ala Ile Asn Ser
470 475 480
Leu Gly Ala Gly Ile Ile Glu Lys Ile Ala Leu Gln Phe Pro Tyr
485 490 495
Arg Phe Trp Asp Ser Lys Val Gln Gly Ala Asp Phe Phe Gly His
500 505 510
Val Pro Pro Ser Ala Ser Lys Arg Gly Leu Phe Ala Val Phe Tyr
515 520 525
Asp Met Asp Pro Gln Lys Lys His Ser Val Leu Met Ser Val Ile
530 535 540
Ala Gly Glu Ala Val Ala Ser Val Arg Thr Leu Asp Asp Lys Gln
545 550 555
Val Leu Gln Gln Cys Met Ala Thr Leu Arg Glu Leu Phe Lys Glu
560 565 570
Gln Glu Val Pro Asp Pro Thr Lys Tyr Phe Val Thr Arg Trp Ser
575 580 585
Thr Asp Pro Trp Ile Gln Met Ala Tyr Ser Phe Val Lys Thr Gly
590 595 600
Gly Ser Gly Glu Ala Tyr Asp Ile Ile Ala Glu Asp Ile Gln Gly
605 610 615
Thr Val Phe Phe Ala Gly Glu Ala Thr Asn Arg His Phe Pro Gln
620 625 630
Thr Val Thr Gly Ala Tyr Leu Ser Gly Val Arg Glu Ala Ser Lys
635 640 645
Ile Ala Ala Phe
<210> 18
<211> 258
<212> PRT
<213> Homo Sapiens
20/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<220>
<221> misc_feature
<223> Incyte ID No: 55142051CD1
<400> 18
Met Ser Pro Ala Ile Ala Leu Ala Phe Leu Pro Leu Val Val Thr
1 5 10 15
Leu Leu Val Arg Tyr Arg His Tyr Phe Arg Leu Leu Val Arg Thr
20 25 30
Val Leu Leu Arg Ser Leu Arg Asp Cys Leu Ser Gly Leu Arg Ile
35 40 45
Glu Glu Arg Ala Phe Ser Tyr Val Leu Thr His Ala Leu Pro Gly
50 55 60
Asp Pro Gly His I1e Leu Thr Thr Leu Asp His Trp Ser Ser Arg
65 70 75
Cys Glu Tyr Leu Ser His Met Gly Pro Val Lys Gly Gln Ile Leu
80 85 90
Met Arg Leu Val Glu Glu Lys Ala Pro Ala Cys Val Leu Glu Leu
95 100 105
Gly Thr Tyr Cys Gly Tyr Ser Thr Leu Leu Ile Ala Arg Ala Leu
110 115 120
Pro Pro Gly Gly Arg Leu Leu Thr Val Glu Arg Asp Pro Arg Thr
125 130 135
Ala Ala Val Ala Glu Lys Leu Ile Arg Leu Ala Gly Phe Asp Glu
140 145 150
His Met Val Glu Leu Ile Val Gly Ser Ser Glu Asp Val Ile Pro
155 160 165
Cys Leu Arg Thr Gln Tyr Gln Leu Ser Arg Ala Asp Leu Val Leu
170 175 180
Leu Ala His Arg Pro Arg Cys Tyr Leu Arg Asp Leu Gln Leu Leu
185 190 195
Glu Ala His Ala Leu Leu Pro Ala Gly Ala Thr Val Leu Ala Asp
200 205 210
His Val Leu Phe Pro Gly Ala Pro Arg Phe Leu Gln Tyr Ala Lys
215 220 225
Ser Cys Gly Arg Tyr Arg Cys Arg Leu His His Thr Gly Leu Pro
230 235 240
Asp Phe Pro Ala Ile Lys Asp Gly Ile Ala Gln Leu Thr Tyr Ala
245 250 255
Gly Pro Gly
<210> 19
<211> 544
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7395274CD1
<400> 19
Met Ser Ser Pro G1y Pro Ser Gln Pro Pro Ala Glu Asp Pro Pro
1 5 10 15
Trp Pro Ala Arg Leu Leu Arg Ala Pro Leu Gly Leu Leu Arg Leu
20 25 30
Asp Pro Ser Gly Gly Ala Leu Leu Leu Cys Gly Leu Val Ala Leu
35 40 45
Leu Gly Trp Ser Trp Leu Arg Arg Arg Arg Ala Arg Gly Ile Pro
50 55 60
Pro Gly Pro Thr Pro Trp Pro Leu Val Gly Asn Phe Gly His Val
65 70 75
21/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Leu Leu Pro Pro Phe Leu Arg Arg Arg Ser Trp Leu Ser Ser Arg
80 85 90
Thr Arg Ala Ala Gly Ile Asp Pro Ser Val Ile Gly Pro Gln Val
95 100 105
Leu Leu Ala His Leu Ala Arg Val Tyr Gly Ser Ile Phe Ser Phe
110 115 120
Phe Ile Gly His Tyr Leu Val Val Val Leu Ser Asp Phe His Ser
125 130 135
Val Arg Glu Ala Leu Val Gln Gln Ala Glu Val Phe Ser Asp Arg
140 145 150
Pro Arg Val Pro Leu Ile Ser Ile Val Thr Lys Glu Lys Gly Val
155 160 165
Val Phe Ala His Tyr Gly Pro Val Trp Arg Gln Gln Arg Lys Phe
170 175 180
Ser His Ser Thr Leu Arg His Phe Gly Leu Gly Lys Leu Ser Leu
185 190 195
Glu Pro Lys Ile Ile Glu Glu Phe Lys Tyr Val Lys Ala Glu Met
200 205 210
Gln Lys His Gly Glu Asp Pro Phe Cys Pro Phe Ser Ile Ile Ser
215 220 225
Asn Ala Val Ser Asn Ile Ile Cys Ser Leu Cys Phe Gly Gln Arg
230 235 240
Phe Asp Tyr Thr Asn Ser Glu Phe Lys Lys Met Leu Gly Phe Met
245 250 255
Ser Arg Gly Leu Glu Ile Cys Leu Asn Ser Gln Val Leu Leu Val
260 265 270
Asn Ile Cys Pro Trp Leu Tyr Tyr Leu Pro Phe Gly Pro Phe Lys
275 280 285
Glu Leu Arg Gln Ile Glu Lys Asp Ile Thr Ser Phe Leu Lys Lys
290 295 300
Ile Ile Lys Asp His Gln Glu Ser Leu Asp Arg Glu Asn Pro Gln
305 310 315
Asp Phe Ile Asp Met Tyr Leu Leu His Met Glu Glu Glu Arg Lys
320 325 330
Asn Asn Ser Asn Ser Ser Phe Asp Glu Glu Tyr Leu Phe Tyr Ile
335 34D 345
Ile Gly Asp Leu Phe Ile Ala Gly Thr Asp Thr Thr Thr Asn Ser
350 355 360
Leu Leu Trp Cys Leu Leu Tyr Met Ser Leu Asn Pro Asp Val Gln
365 37D 375
Glu Lys Val His Glu Glu Ile Glu Arg Val I1e G1y Ala Asn Arg
380 385 390
Ala Pro Ser Leu Thr Asp Lys Ala G1n Met Pro Tyr Thr Glu Ala
395 400 405
Thr Ile Met Glu Val Gln Arg Leu Thr Va1 Val Val Pro Leu Ala
410 415 420
Ile Pro His Met Thr Ser Glu Asn Thr Val Leu Gln Gly Tyr Thr
425 430 435
Ile Pro Lys Gly Thr Leu Ile Leu Pro Asn Leu Trp Ser Va1 His
440 445 450
Arg Asp Pro Ala I1e Trp Glu Lys Pro G1u Asp Phe Tyr Pro Asn
455 460 465
Arg Phe Leu Asp Asp Gln Gly G1n Leu Ile Lys Lys Glu Thr Phe
470 475 480
Ile Pro Phe Gly Ile Gly Lys Arg Val Cys Met Gly Glu Gln Leu
485 490 495
Ala Lys Met Glu Leu Phe Leu Met Phe Va1 Ser Leu Met Gln Ser
500 505 520
Phe Ala Phe Ala Leu Pro Glu Asp Ser Lys Lys Pro Leu Leu Thr
515 520 525
Gly Arg Phe Gly Leu Thr Leu Ala Pro His Pro Phe Asn Ile Thr
530 535 540
22/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
Ile Ser Arg Arg
<210> 20
<211> 2603
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7248285CB1
<400> 20
atggcgtggt ccccaccagc caccctcttt ctgttcctgc tgctgctagg ccagccccct 60
cccagcaggc cacagtcact gggcaccact aagctccggc tggtgggccc agagagcaag 120
ccagaggagg gccgcctgga ggtgctgcac cagggccagt ggggcaccgt gtgtgatgac 180
aactttgcta tccaggaggc cacagtggct tgccgccagc tgggcttcga agctgccttg 240
acctgggccc acagtgccaa gtacggccaa ggggagggac ccatctggct ggacaatgtg 300
cgctgtgtgg gcacagagag ctccttggac cagtgcgggt ctaatggctg gggagtcagt 36D
gactgcagtc actcagaaga cgtaggggtg atatgccacc cccggcgcca tcgtggctac 420
ctttctgaaa ctgtctccaa tgcccttggg ccccagggcc agcggctgga ggaggtgcgg 480
ctcaagccca tccttgccag tgccaagcag catagcccag tgaccgaggg agccgtggag 540
gtgaagtatg agggccactg gcggcaggtg tgtgaccagg gctggaccat gaacaacagc 600
agggtggtgt gcgggatgct gggcttcccc agcgaggtgc ctgtcgacag ccactactac 660
aggaaagtct gggatctgaa gatgagggac cctaagtcta ggctgaagag cctgacgaat 720
aagaactcct tctggatcca ccaggtcacc tgcctgggga cagagcccca catggccaac 780
tgccaggtgc aggtggctcc agcccggggc aagctgcggc cagcctgccc aggtggcatg 840
catgctgtgg tcagctgtgt ggcagggcct cacttccgcc caccgaagac aaagccacaa 900
cgcaaagggt cctgggcaga ggagccgagg gtgcgcctgc gctccggggc ccaggtgggc 960
gagggccggg tggaagtgct catgaaccgc cagtggggca cggtctgtga ccacaggtgg 1020
aacctcatct ctgccagtgt cgtgtgtcgt cagctgggct ttggctctgc tcgggaggcc 1080
ctctttgggg cccggctggg ccaagggcta gggcccatcc acctgagtga ggtgcgctgc 1140
aggggatatg agcggaccct cagcgactgc cctgccctgg aagggtccca gaatggttgc 120D
caacatgaga atgatgctgc tgtcaggtgc aatgtcccta acatgggctt tcagaatcag 1260
gtgcgcttgg ctggtgggcg tatccctgag gaggggctat tggaggtgca ggtggaggtg 1320
aacggggtcc cacgctgggg gagcgtgtgc agtgaaaact gggggctcac cgaagccatg 1380
gtggcctgcc gacagctcgg cctgggtttt gccatccatg cctacaagga aacctggttc 1440
tggtcgggga cgccaagggc ccaggaggtg gtgatgagtg gggtgcgctg ctcaggcaca 1500
gagctggccc tgcagcagtg ccagaggcac gggccggtgc actgctccca cggtggcggg 1560
cgcttcctgg ctggagtctc ctgcatggac agtgcaccag acctggtgat gaacgcccag 1620
ctagtgcagg agacggccta cttggaggac cgcccgctca gccagctgta ttgtgcccac 1680
gaggagaact gcctctccaa gtctgcggat cacatggact ggccctacgg ataccgccgc 1740
ctattgcgct tctccacaca gatctacaat ctgggccgga ctgactttcg tccaaagact 1800
ggacgcgata gctgggtttg gcaccagtgc cacaggcatt accacagcat tgaggtcttc 1860
acccactacg acctcctcac tctcaatggc tccaaggtgg ctgaggggca caaggccagc 1920
ttctgtctgg aggacacaaa ctgccccaca ggactgcagc ggcgctacgc atgtgccaac 1980
tttggagaac agggagtgac tgtaggctgc tgggacacct accggcatga cattgattgc 2040
cagtgggtgg atatcacaga tgtgggcccc gggaattata tcttccaggt gattgtgaac 2100
ccccactatg aagtggcaga gtcagatttc tccaacaata tgctgcagtg ccgctgcaag 2160
tatgatgggc accgggtctg gctgcacaac tgccacacag ggaattcata cccagccaat 2220
gcagaactct ccctggagca ggaacagegt ctcaggaaca acctcatctg aagctgtcac 2280
tgcacactcc tagctgctgc cgatacacca gatacctcag cttattggag ccatgccctt 2340
cacagagtcc caactcagag gaaaagggcc agtgccaagg ggcaccaaga acctgctcag 2400
gaagcctttt gatggcaaga tcaccaatcc agatggtatt gctccctcag gatggctctg 2460
ggcctgcccc taagggcctg tggcctatgg aatatgtcct ecaggctttg ctcagctgag 2520
ctcctcttct gtaaggaaac ccagtcatcc ctgaatcttg ccacagagat ccgggattca 2580
ggagctctca gtttcttaag gag 2603
<210> 21
<211> 1745
<212> DNA
<213> Homo Sapiens
23136
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<220>
<221> misc_feature
<223> Incyte ID No: 7472835CB1
<400> 21
gtaatttttt ttaaatggga tggatgacag tgacagagct ctaaatttct actaaccagc 60
tgagacacaa tggccaaaaa agcgattgct gtgattggag ctggaattag cggactgggg 120
gccatcaagt gctgcctgga tgaagatctg gagcccacct gctttgaaag aaatgatgat 180
attggacatc tctggaaatt tcaaaaaaat acttcagaga aaatgcctag tatctacaaa 240
tctgtgacca tcaatacttc caaggagatg atgtgcttca gtgacttcec tgtccctgat 300
cattttccca actacatgca caactccaaa ctcatggact acttcgggat gtatgccaca 360
cactttggcc tcctgaatta cattcgtttt aagactgaag tgcaaagtgt gaggaagcac 420
ccagattttt ctatcaatgg acaatgggat gttgttgtgg agactgaaga gaaacaagag 480
actttggtct ttgatggggt cttagtttgc agtggacacc acacagatcc ctacttacca 540
cttcagtcct tcccaggtat ggagaaattt gaaggctgtt atttccatag tcgggaatac 600
aaaagtcceg aggacttttc agggaaaaga atcatagtga tcggcattgg aaattctgga 660
gtggatattg cggtggagct cagtcgtgta gcaaaacagg ttatattcct tagtactaga 720
cgtggatcat ggattttaca ccgtgtttgg gataatgggt atcccatgga tagttcattt 780
ttcactcggt tcaatagttt tctccagaaa atactaacta caccacaaat aaataaccag 840
ctagagaaaa taatgaactc aagatttaat catgcgcact gtggcctgca gcctcagcac 900
agagctttaa gtcagcatcc aactgtcagt gatgacctgc caaatcacat aatttctgga 960
aaagtccaag taaagcccag cgtgaaggag ttcacagaaa cagatgccat ttttgaagac 1020
agcactgtag aggagaatat tgatgttgtc atctttgcta caggatacag tttttctttt 1080
tetttccttg atggtctgat caaggttact aacaatgaag tatetctgta taagcttatg 1140
ttccctcctg acctggagaa gccaaccttg gctgtcatcg gtcttatcca accactgggc 1200
atcatcttac ctattgcaga gctccaatct cgttgggcta cacgagtgtt caaaggtctg 1260
atcaaattac cctcagcgga gaacatgatg gcagatattg cccagaggaa aagggctatg 1320
gaaaaacggt atgtaaagac accccgccac acaatccaag tggatcacat tgagtacatg 1380
gatgagattg ccatgccagc aggggtgaaa cccaacctgc tcttcctct.t tctctcagat 1440
ccaaagctgg ccatggaggt tttctttggc ccctgcaccc cataccagta ccacctccat 1500
gggcccgaga aatgggatgg ggcccggaga gctaacctga cccagagaga gaggatcatc 1560
aageccctga ggactcgcat tactagtgag gacagccacc catectcaca gctctcttgg 1620
ataaagatgg ccccagtgag cctggcattt ctggctgctg gcttggcata ctttcgatat 1680
actccttacg gtaaatggaa ataaatgaaa gaacactgag ggggaaaagc atggaatagt 1740
ttcta 1745
<210> 22
<211> 1587
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476203CB1
<400> 22
atgctctccc tgctcagtgg gctggcttta ctggccatct ecttcctgct cctgaaactt 60
ggcaccttct getgggacag gagctgtett ectcctggec cactcccctt ececatcctt 120
ggaaacctgt ggcagctatg ctttcagcag cctcaccttt cacttaaaaa ctttcagaag 180
aagataggaa atatctttat gaacttggga agcagtgtgg ttccactggc attgccattg 240
ttaccggtga etttccatcc cttgaaccaa ggcgtgttat gtaagccact tattactttc 300
ectaaacect tccetaccag aaatecaggc atcatetgca gcagegggca cacgtggegg 360
caaaagagac gcttctgcct ggtgatgatt cgagggctgg gcctaggcaa gctggcgctg 420
gaggtgcagc tgcagaaaga ggcagcagag ctggcagaag ccttccgcca ggagcagggt 480
aagagaccct tegaccctca ggtatccatt gtcaggtcca cagtcagagt catcggggcc 540
cttgtgtttg gccaccactt cctcttagag gatcccatct tccaggaact gactcaagcc 600
atcgactttg gcctggcatt tgtcagcact gtgtggcgcc agctgtatga cgtgtttccc 660
tgggccctct gccacctccc aggaccccac caggagatat ttaggtacca agaggtcgtg 720
ctgagcttaa tccaccagga gatcaccagg cacaaactca gggcaccgga ggcccccagg 780
gacttcatca gctgctacct ggcccagatc tccaaggcca tggatgaccc tgtctccaca 840
ttcaaccagg agaacctggt ccaggtggtg atcgacctgt ttctgggagg caccgacacc 900
acagccacca ccctgtgctg ggcactcatc cacatgatcc agcacggagc tgtccaggag 960
24/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
acggtgcagc tggagctgga cgaggtgctg ggtgctgccc cggttgtctg ctatgaagac 1020
cgcaagcgac tgccttacac catgctgtcc tccatgacgt gcagcgcctc agcagcgtca 1080
tggccatggg tgccgtgcgc cagtgtgtga cctccacccg tgtgtgcagc tatcccgtga 1140
gcaagggcac catcatctta cccaacctgg cctctgtgct ctatgaccct gagtgctggg 1200
agacccctcg acagttcaac cctggccact tctcggacaa ggatggaaac tttgtggcca 1260
atgaggcctt cctgccattc tctgcaggta ctagggtcta cccagcagac cagctggctc 132D
aaatggagct cttcctgatg tttgccaccc tcctcaggac ctttcggttc caactgccag 138D
aagggagccc ggggctcaag ctggagtaca tctttggcgg cacttggcaa ccccagcccc 144D
aggagatctg cgcagtgccc cgcctgagca gccccagccc tggtcctagg gaggatggcc 1500
tgtagccact gggggtctgg aggcctgtcc cccatgaagt ccttcctcag tctcttttgg 1560
ttcctgcaaa gttagaaaag aggagga 1587
<210> 23
<211> 1038
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478583CB1
<400> 23
atgaaagcag cagtttggta tggtcaaaaa gatgtaagag ttgaagaacg tgaacctaaa 60
gaattacagg acaatgaagt taaggttaaa gtatcttggg etggcatttg tggtacagat 120
ttacatgaat acttagaagg ecctatattt atttcaacag aaaagccaga tccattctta 180
ggtcaaaaag cgccagttac attgggtcat gaatttgcag gtgtagtaga agaaactggt 240
tcccaagtta caaaatttaa taaaggcgat cgagttgtag ttaaccctac agtttcaaaa 300
cgtgaaaaag aagaaaatat tgacctttat gatggttatt catttatagg cttaggttct 360
gatggtggat ttgcagagtt tacaaatgcg ccggaagaaa atgtttataa actaccagat 420
aatgtttctg ataaagaagg tgcgcttgtc gaaccaacag ccgttgcagt tcaagcaatt 480
aaagaaggtg aagttctatt tggtgatact gtagctattt ttggtgcagg accaattgga 540
ttattaacag tcgtagcagc caaagcagct ggtgcaagta aaatatttgt tttcgattta 600
tcagaagaaa gactaagtaa agctaaagca ctaggcgcaa ctcatgctat aaactctggt 660
aagacagatc cagttgatgt tattaatgag tatacagaaa atggtgtaga tgtatctttt 720
gaagtggctg gtgtagcacc aacacttaaa tcttctatag atgttacaaa agcaagaggt 780
acagttgtta tcgtttctat ttttggtcat cctatcgagt ggaatccaat gcaattaact 840
aatacaggag taaaacttac ttetacaatt gcatacacac ctactacatt ccaacaaaca 900
attgacttaa tcaacgaagg taatttaaac gttaaagatg tagttactga tgaaattgag 960
ttagaaaata tcgtagaatc aggatttgaa caacttgtaa atgataaatc tcaagcaaaa 1020
atattaatta aattataa 1038
<210> 24
<211> 1584
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478585CB1
<400> 24
atgaaatcat taatgattgg cgctaatggt ggcgtcggtc aacatctcgt acgtaaacta 60
aatgcacgag atgttgattt tacggccggt gttagaaaag aagaacaagt tgaagcttta 120
aaagcagatg gtatcgatgc aacttacatt gatgttgcaa aacaatctat tgatgaatta 180
atagaattat ttaaaccgta tgaccaaatc cttttttetg tcggttctgg tggaagcaca 240
ggtgacgatc aaacaatcat agtagattta gacggttcag tgaaagcaat taaagcaagt 300
gaacacgtcg gtcgtcaaca ctttgttatg gtatcaacgt acgattcacg tcgagaagct 360
tttgatgcgt caggcgactt gaaaccatac accattgcta aacattatgc ggatgactac 420
ttaagacatg caaatttaaa atataccatc gtacatccag gcgcattaac taacaatcat 480
gaaacgcaac aattcaatat gagtgctcaa tttgaaaatg tacaaaatcc gtctatcacg 540
agagaagatg tagcagaagt gcttgtttct gtgttaactg atgaaacatt acaagttgta 600
cttgcaaaac gaccacaaag tatccctcaa gacgatgtat ttagatttga aacaatagaa 660
25/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
actcgagaac cacatgcagg tgaggttcaa gtagaatcca tttatgtatc tgtagatcct 720
tacatgagag gcagaatgaa tgatacaaaa agttatgttc aacctttcca agtgaatgaa 780
ccattacaag gtcatattgt tggaaaagtc acacaatcga acgatgaacg tctatctgtt 840
ggcgattatg tcacaggcat attaccatgg aaaaagataa atacagtgaa tggagacgat 900
gtgacccctg tgccatcaaa agatgtacca ttacatttat atttgagtgt tttaggcatg 960
ccgggaatga cggcatatac aggattgctt caaattggtc aaccacaatc tggcgagacg 1020
gttgtcgtgt cagctgcatc aggtgcagta ggatctgtcg taggacaaat tgctaagatt 1080
aaaggcgcaa aagttgtcgg tattgctggt ggtaagcaga aaacaacata tttaacagat 1140
gaattaggat ttgatgcggc cattgactat aaacaagatg atttcgcaca gcaactcgaa 1200
gcggctgtac cagatggtat tgatgtgtat tttgaaaatg taggcggcgt aatttctgat 1260
gaagtgttta aacacttaaa tcgatttgca cgcgttccgg tatgtggtgc aatttcagca 1320
tataataatg aaaaagacga tattggacca cgtatccaag gaacgttgat taaaaatcaa 1380
gcattgatgc aaggttttgt agtagcacaa ttcgctgatc attttaaaga agcaagcgaa 1440
caactcgcac aatgggtgtc tgaaggtaaa attaaatttg aagtgacgat agatgaaggt 1500
tttgacaatt taccttctgc attcagaaag ttatttacag gtgataattt cggtaaacaa 1560
gttgtcaaaa tcaaagaaga atag 1584
<210> 25
<211> 15D0
<212> DNA
<213> Homo Sapiens
<220>
<2-21> misc_feature
<223> Incyte ID No: 7479904CB1
<400> 25
atggatgaga aatecaacaa gctgctgcta gctttggtga tgctcttcct atttgccgtg 60
atcgtcctcc aatacgtgtg ccccggcaca gaatgccagc tcctccgcct gcaggcgttc 120
agctccccgg tgccggaccc gtaccgctcg gaggatgaga gctccgccag gttcgtgccc 180
cgctacaatt tcacccgcgg cgacctcctg cgcaaggtag acttcgacat caagggcgat 240
gacctgatcg tgttcctgca catccagaag accgggggca ccactttcgg ccgccacttg 300
gtgcgtaaca tccagctgga gcagccgtgc gagtgccgcg tgggtcagaa gaaatgcact 360
tgccaccggc cgggtaagcg ggaaacctgg ctcttctcca.ggttctccac gggctggagc 420
tgcgggttgc acgccgactg gaccgagctc accagctgtg tgccctccgt ggtggacggc 480
aagcgcgacg ccaggctgag accgtccagg tggaggattt ttcagattct agatgcagca 540
agtaaggata aacggggttc tccaaacact aacgcaggcg ccaactctcc gtcatccaca 600
aagacccgga acacatctaa gagtgggaag aacttccact acatcaccat cctccgagac 660
ccagtgtccc ggtacttgag tgagtggagg catgtccaga gaggggcaac atggaaagca 720
tccctgcatg tctgcgatgg aaggcctcca acctccgaag agctgcccag ctgctacact 780
ggcgatgact ggtctggctg ccccctcaaa gagtttatgg actgtcccta caatctagcc 840
aacaaccgcc aggtgcgcat gctctccgac ctgaccctgg taggctgcta caacctctct 900
gtcatgcctg aaaagcaaag aaacaaggtc cttctggaaa gtgccaagtc aaatctgaag 960
cacatggcgt tcttcggcct cactgagttt cagcggaaga cccaatatct gtttgagaaa 1020
accttcaaca tgaactttat ttcgccattt acccagtata ataccactag ggcctctagt 1080
gtagagatca atgaggaaat tcaaaagcgt attgagggac tgaattttct ggatatggag 1140
ttgtacagct atgccaaaga cctttttttg cagaggtatc agtttatgag gcagaaagag 1200
catcaggagg ccaggcgaaa gcgtcaggaa caacgcaaat ttctgaaggg aaggctcctt 1260
cagacccatt tccagagcca gggtcagggc cagagccaga atccgaatca gaatcagagt 1320
cagaacccaa atccgaatgc caatcagaac ctgactcaga. atctgatgca gaatctgact 1380
cagagtttga gccagaagga gaaccgggaa agcccgaagc agaactcagg caaggagcag 1440
aatgataaca ccagcaatgg caccaacgac tacataggca gtgtagagaa atggcgttaa 1500
<210> 26
<211> 669
<212> DNA
<213> Homo Sapiens
<220>
<221> misc-feature ,
<223> Incyte ID No: 7480367CB1
26/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<400> 26
atggcagaga agcccaagct ccactactcc aatgcacggg gcagtatgga gtccattcgg 60
tggctcctgg ctgcagctgg agtagagttg gaagagaaat ttctagaatc tgcagaagat 120
ttggacaagt taagaaatga tgggagtttg ctgttccagc aagtaccaat ggttgagatt 180
gacgggatga agctggtgca gaccagagcc attcttaact aeattgccag caaatacaac 240
ctttatggga aagacatgaa ggagagagcc ctgattgata tgtacacaga aggtatagta 300
gatttgactg aaatgatcct tcttctgctc atatgtcaac cagaggaaag agatgccaag 360
actgccttgg tcaaagagaa aataaaaaat cgctacttcc ctgcctttga aaaagtatta 420
aagagccaca gacaagacta ccttgttggc aacaagctga gctgggctga cattcacctg 480
gtggaacttt tctactacgt ggaagagctt gactcgagtc ttatctccag cttccctctg 540
ctgaaggccc tgaaaaccag aatcagcaac ctgcccacgg tgaagaagtt tctgcagcct 600
ggcagccaga gaaagcctcc catggatgag aaatctttag aagaagcaag gaagattttc 660
aggttttaa 669
<210> 27
<211> 3551
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 8069390CB1
<400> 27
ggcgcggagc agctccggcg gcgagacggg ggcggcgccg cgcgggtctg gcgggaccgg 60
tttggaagac tttgccggcc tgcagattgg ccttaagaga aggacggagc cacatactgc 120
tgacggccca gaactggcag agagaaggtt gccatggctg ctgttgacag tttctacctc 180
ttgtacaggg aaatcgccag gtcttgcaat tgctatatgg aagctctagc tttggttgga 240
gcctggtata cggccagaaa aagcatcact gtcatctgtg acttttacag cctgatcagg 300
ctgcatttta tcccccgcct ggggagcaga gcagacttga tcaagcagta tggaagatgg 360
gccgttgtca gcggtgcaac agatgggatt ggaaaagcct acgctgaaga gttagcaagc 420
cgaggtctca atataatcct gattagtcgg aacgaggaga agttgcaggt tgttgctaaa 480
gacatagccg acacgtacaa agtggaaact gatattatag ttgcggactt cagcagcggt 540
cgtgagatct accttccaat tcgagaagcc ctgaaggaca aagacgttgg catcttggta 600
aataacgtgg gtgtgtttta tccctacccg cagtatttca ctcagctgtc cgaggacaag 660
ctctgggaca tcataaatgt gaacattgcc gccgctagtt.tgatggtcca tgttgtgtta 720
ccgggaatgg tggagagaaa gaaaggtgcc atcgtcacga tctcttctgg ctcctgctgc 780
aaacccactc ctcagctggc tgcattttct gcttctaagg cttatttaga ccacttcagc 840
agagccttgc aatatgaata tgcctctaaa ggaatctttg tacagagtct aatccctttc 900
tatgtagcca ccagcatgac agcacccagc aactttctgc acaggtgctc gtggttggtg 960
ccttcgccaa aagtctatgc acatcatgct gtttctactc ttgggatttc caaaaggacc 1020
acaggatatt ggtcccattc tattcagttt ctttttgcac agtatatgcc tgaatggctc 1080
tgggtgtggg gagcaaatat tctcaaccgt tcactacgta aggaagcctt atcctgcaca 1140
gcctgagtct ggatggccac ttgagaagtt ttgccaactc ctgggaacct cgatattctg 1200
acatttggaa aaacacattt aatttatctc ctgtgtttca ttgctgatta ttcagcatac 1260
tgttgattcg tcatttgcaa aacacacata ataccgtcag agtgctgtga aaaaccttaa 1320
gggtgtgtgg atggcacagg atcaataatg cctgaggctg attgacgaca tctacatttc 1380
agtgcttttt ccctaagctg tttgaaagtt acgcttttct gttgttctag agecacagca 1440
gtctaatatt gaaatataat atgatttgtc aggtcttata atttcagatg ttgtttttta 1500
agggaaattg accatttcac tagaggagtt gtgctggttt ttaaatgtgc atcaagaaag 1560
actactgaaa agtattattt tgtaactaag attgctggta ctattaggaa aaatctgtgt 1620
gtattgtata gctctagctg tttgactatc tgtaatgaaa atgctgcact tcaactggta 1680
tttcattaga gaaccgtgtg tgtgcgtgtg tgtggtgcct ttgagcaact ttatttatgg 1740
ttaccatatt tttaaaaaga ttttttgtca gggtgactta acatggactc ttatagggta 1800
ttaaaacaat ctagattatt ccttttcatc ctaaataagc ctaccaaatt tcatgctgtt 1860
ggtttgccat gaatgatatt acttcctaca ttatatttgt gttttttcaa atctgctatg 1920
gaatgaactt attcctagat ttggatatgt aagagaaacc tgcagtcatc ttttgattta 1980
taaggcaatt ettgtggata aatagtgatt tctcagcctc tgacccattt tataactgaa 2040
atttagccct ttagagcttg ttatatctgg ttttcctacg tttttctatg taatattatt 2100
ccattccagt agcattattg atagaaatag taagtattta tggaatagta aaatatggac 2160
aaattacgtg tgtgacatat ctgtcaaaat aagttagaag cttattcttg gtttgtgtaa 2220
tgaatttatg tattgtagtg aataccttta ctggtgtgaa gataattatg cacaaaccct 2280
27/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
cacaatacgc gttaacattg aaacctgtga aatgtcctta ggttgggtca tataaagcca 2340
accatttttg aggaccatgt acctagtgct ttgaaaactg taagtcacta tatgaatatg 2400
acaatatgtg cacatttaaa attcagagct cggcattgtg atactgatgc agaagctagt 2460
agattggtta aaagtctgga cttctgtggc atttttttcg tgacgtgata atctatcata 2520
agcagaccta agcacagttt tatgaacaca attttgccca tgacattgcc tacaggattt 2580
ccagatgtga cttgcactca gaagatcagt ggtcaacttc agaagttctt ccacgcttag 2640
atcatgtctt cagaacttag atgtgaaaat ctacacactg ggagatgctg tgagccccaa 2700
ggttttgatg gagtttgctt ggaatcctct tgacttcatg ccacattgac gtgaactttg 2760
atgtataata agcagcagca acttcatgtg aaaatatggt caggtagtta tatgtaaggt 2820
tacgtggtcc agtaatgtct tagattgata aattaggtat ggaatccatc agtgttacgt 2880
gatgagaata ggtgaacaca ccttgtcagt gatgatgtaa acttctctcc ttggcaggac 2940
atgggcaaac atgctgattg gtgcaaatgt ggtgccgagc tgtccatagc tgcagtgaaa 3000
gatgaagagc aagaccttct ctaggttttc tagctttcat taaatgtatt tttttcecca 3060
gagctaattt gaaagttgat tggaccactg tggatggggt ctcattaaga atgtgggaaa 3120
taggggccga gtgcggtggc tcacacctgt aatcccagca gtttggaagg ccagggcagg 3180
tggatcgctt gatcccagga ggtcgagacc agcctgggga acacatcctg tctctacaaa 3240
aaatacaaaa attagccagg cagggtggtg catgcctgta gtcccagcta cttgggaggc 3300
tgaggcagga gaattttttg agcccaggat gcagaggttg aagtgagcca agatcgtgcc 3360
actgcactcc agccttgaga cagagcgaga ccctgtctca aaaaaaaaaa aagaacgtgg 3420
gaaatatgaa cctttgaaag ttaatctgtg aattgaaagt ttaacaataa aagtagttgt 3480
ttgtttcctt tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaatggcgg 3540
tcgcaagctt a 3551
<210> 28
<211> 2178
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Inc~tte ID No: 7473869CB1
<4,00> 28
tcgggtgggg agtagagtag gtaagcgtgt ttctttattg gcggagggta tgtgtaaccg 60
agagggtttc actgccaatt atggaagtag cctctccttg atattgttga ggtgggattg 120
tgcaacaaca ttgtactgtt gtgggttgta atttcgccta gatggcataa gtgagaactg 180
aagacttcag gtaggatcag aggeacgggt cttgttagtt atgctcttgg gatcagtgtc 240
gcctgctgag ctgaaaagga aatggattca atctcttcaa cctttaaggt gatagatagt 300
ttgagcagac tggagaatgg acacactatg aagctgtggc tagaaaggga ctggtcatgt 360
cccatcctct ggccagattg actggggatg tccggacaga tgcctgcatg ggtggtgagg ,420
gccacatctg cacacgagcc agtggctgct tgcagttcac tgctgtgatg ccagagtgtg 480
ttcaaaggtg actctcctgc tcttctggac tcttctctca ggcaagaaag gctgcaggct 540
gcctgctatg tgatgcctga gcacaaagcc aaggaactga actaagtctt tctgttaagt 600
cctgagtttg tcattggcag gtttacttgt ggccagctct ctctgccctt gggggttccg 660
tcttctcact gcggaccctg gattgaaacg atctccccgc ggccgccgcc gctacctggt 720
gcccgcaggt gcctgcagga gtcctggggc cagctggcct cgatgtacgt cagcacgcgg 780
gaacggtaca agtggctgcg cttcagcgag gactgtctgt acctgaacgt gtacgcgccg 840
gcgcgcgcgc ccggggatcc ccagctgcca gtgatggtct ggttcccggg aggcgccttc 900
atcgtgggcg etgcttctte gtacgagggc tctgacttgg cegeccgcga gaaagtggtg 960
ctggtgtttc tgcagcacag gctcggcatc ttcggcttcc tgagcacgga cgacagccac 1020
gcgcgcggga actgggggct gctggaccag atggcggctc tgcgctgggt gcaggagaac 108D
atcgcagcet tcgggggaga cccaggaaat gtgaccctgt tcggccagtc ggcgggggcc 1140
atgagcatct caggactgat gatgtcaccc ctagcctcgg gtctcttcca tcgggccatt 1200
tcccagagtg gcaccgcgtt attcagactt ttcatcacta gtaacccact gaaagtggcc 1260
aagaaggttg cccacctggc tggatgcaac cacaacagca cacagatcct ggtaaactgc 1320
ctgagggcac tatcagggac caaggtgatg cgtgtgtcca acaagatgag attcctccaa 1380
ctgaacttcc agagagaccc ggaagagatt atctggtcca tgagccctgt ggtggatggt 1440
gtggtgatcc cagatgaccc tttggtgctc ctgacccagg ggaaggtttc atctgtgccc 1500
taccttctag gtgtcaacaa cctggaattc aattggctct tgccttatat catgaagttc 1560
ccgctaaacc ggcaggcgat gagaaaggaa accatcacta agatgctetg gagtacccgc 1620
accctgttgg tgagggaccc agctggcagg ggtgctcagt tcggacaggg ttgacccccc 1680
tgtttttttt aacctagtag ctgctctttg caaaggggct cccagccagg gtaaggatct 1740
28/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
ttcttggagg gtctggggtt tgctgtggga tcagatgact gcttacaggt aaggtgctca 1800
gggtcacagg ggcagttatg cagcaaaatc aggggttaca atcagcagag acagaaactt 1860
tcccaggaag ctccctttct ccccctccca ggccaaaaac tectgggggg ctgagcatgg 1920
atccaagtca ctggtgggcc cacctctggc ccagctggca cccaggcctc aggtaagtgt 1980
ggcctcctca ctcagaatat caccaaggag caggtaccac ttgeggtgga ggagtacctg 2040
gacaatgtca atgagcatga ctggaagatg ctacgaaacc gtatgatgga catagttcaa 2100
gatgccactt tcgtgtatgc cacactgcag actgctcact accaccgaga tgccggcctt 2160
cctgtctacc tgtatgaa 2178
<210> 29
<211> 2081
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7478588CB1
<400> 29
gctgtgggct ggtcagaagc tggttacaat tccccccgcc ccagtacttg ctggcaggga 60
ttaagagcag ataaaagtgt gctcacacac tgtagacacg gctaccatgc catccacagt 120
gttgccatcc acagtgttgc catcactcct gcccacagca ggagctggct ggagcatgag 180
gtggattctg tgctggagcc tcaccctctg cctgatggcg cagacggcct tgggtgcctt 240
gcacaccaag aggcctcaag tggtcaccaa atatggaacc ctgcaaggaa aacagatgca 300
tgtggggaag acacecatcc aagtcttttt aggagtcccc ttctccagac ctcctctagg 360
tatcctcagg tttgcacctc cagaaccccc ggagccctgg aaaggaatca gagatgctac 420
cacctacccg cctgggtgcc tgcaggagtc ctggggccag ctggcctcga tgtacgtcag 480
cacgcgggaa cggtacaagt ggctgcgctt cagcgaggac tgtctgtacc tgaacgtgta 540
cgcgccggcg cgcgcgcccg gggatcccca gctgccagtg atggtctggt tcecgggagg 600
cgccttcatc gtgggcgctg cttcttcgta cgagggctct gacttggccg cccgcgagaa 660
agtggtgctg gtgtttctgc agcacaggct cggcatcttc ggcttcctga gcacggacga 720
cagccacgcg cgcgggaact gggggctgct ggaccagatg gcggctctgc gctgggtgca 780
ggagaacatc gcagccttcg ggggagaccc aggaaatgtg accctgttcg gccagtcggc 840
gggggccatg agcatctcag gactgatgat gtcaccccta gcctcgggtc tcttccatcg 900
ggccatttcc cagagtggca ccgcgttatt cagacttttc atcactagta acccactgaa 960
agtggccaag aaggttgccc acctggctgg atgcaaccac aacagcacac agatcctggt 1020
aaactgcctg agggcactat cagggaccaa ggtgatgcgt gtgtccaaca agatgagatt 1080
cctccaactg aacttccaga gagacccgga agagattatc tggtccatga gccctgtggt 1140
ggatggtgtg gtgatcccag atgacccttt ggtgctcctg acccagggga aggtttcatc 1200
tgtgccctac cttctaggtg tcaacaacct ggaattcaat tggctcttgc ettatatcat 1260
gaagttcccg ctaaaccggc aggcgatgag aaaggaaacc atcactaaga tgctctggag 1320
tacccgcacc ctgttgaata tcaccaagga gcaggtacca cttgtggtgg aggagtacct 1380
ggacaatgtc aatgagcatg actggaagat gctacgaaac cgtatgatgg acatagttca 1440
agatgccact ttcgtgtatg ccacactgca gactgctcac taccaccgag atgccggcct 1500
ccctgtctac ctgtatgaat ttgagcacca cgctcgtgga ataatcgtca aaccccgcac 1560
tgatggggca gaccatgggg atgagatgta cttcctcttt gggggcccct tcgccacagg 1620
cctttccatg ggtaaggaga aggcacttag cctccagatg atgaaatact gggccaactt 1680
tgcccgcaca ggaaacccca atgatgggaa tctgccctgc tggccacgct acaacaagga 1740
tgaaaagtac ctgcagctgg attttaccac aagagtgggc atgaagctca aggagaagaa 1800
gatggctttt tggatgagtc tgtaccagtc tcaaagacct gagaagcaga ggcaattcta 1860
agggtggcta tgcaggaagg agccaaagag gggtttgccc ccaccatcca ggccctgggg 1920
agactagcca tggacatacc tggggacaag agttctaccc accccagttt agaactgcag 1980
gagctccctg ctgcctccag gccaaagcta gagcttttgc ctgttgtgtg ggacctgcac 2040
tgccctttcc agcctgacat cccatgatgc ccctctactt c 2081
<210> 30
<211> 2642
<212> DNA
<213> Homo Sapiens
<220>
<221> mist feature
29/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<223> Incyte ID No: 55046125CB1
<400> 30
gagccggccc gggtcggtcg ccgcctcagt tcgcgcgggc cctcctaggg gtgtgtctca 60
cggattccac acccggcegc tcctggacaa gccccgaaag gcgtcttctt ccctggcggg 120
agccgcgtgc gccccgcttt tcgcgctgct gtcccggggc cgccgcaggc ggatgcacgt 180
cctcaggcga cgctgggacc tgggctccct ctgccgggcc ctgctcactc ggggcctggc 240
cgccctgggc cactcgctga agcacgtgct cggtgcgatc ttctecaaga ttttcggccc 30D
catggccagc gtcgggaaca tggatgagaa atccaacaag ctgctgctag ctttggtgat 360
gctcttccta tttgccgtga tcgtcctcca atacgtgtgc cccggcacag aatgccagct 420
cctccgcctg caggcgttca gctccccggt gccggacccg taccgctcgg aggatgagag 480
ctccgccagg ttcgtgcccc gctacaattt cacccgcggc gacctcctgc gcaaggtaga 540
cttcgacatc aagggcgatg acctgatcgt gttcctgcac atccagaaga ccgggggcac 600
cactttcggc cgccacttgg tgcgtaacat ccagctggag cagccgtgcg agtgccgcgt 660
gggtcagaag aaatgcactt gccaccggcc gggtaagcgg gaaacctggc tcttctccag 720
gttctecacg ggctggagct gcgggttgca cgcegactgg accgagctca ccagctgtgt 780
gccctecgtg gtggacggca agcgcgacgc caggctgaga ccgtccagga acttccacta 84D
catcaccatc ctccgagacc cagtgtcccg gtacttgagt gagtggaggc atgtccagag 90D
aggggcaaca tggaaagcat ccctgcatgt ctgcgatgga aggcctccaa cctccgaaga 960
gctgcccagc tgctacactg gcgatgactg gtctggctgc cccctcaaag agtttatgga 1020
ctgtccctac aatctageca acaaccgeca ggtgcgcatg ctctccgacc tgaccctggt 1080
aggctgctac aacctctctg tcatgcctga aaagcaaaga aacaaggtcc ttctggaaag 1140
tgccaagtca aatctgaagc acatggcgtt cttcggcctc actgagtttc agcggaagac 1200
ccaatatctg tttgagaaaa ccttcaacat gaactttatt tcgccattta cccagtataa 1260
taccactagg gcctctagtg tagagatcaa tgaggaaatt caaaagcgta ttgagggact 1320
gaattttctg gatatggagt tgtacagcta tgccaaagac ctttttttgc agaggtatca 1380
gtttatgagg cagaaagagc atcaggaggc caggcgaaag cgtcaggaac aacgcaaatt 1440
tetgaaggga aggctccttc agacccattt ccagagccag ggtcagggcc agagccagaa 15D0
tccgaatcag aatcagagtc agaacccaaa tccgaatgcc aatcagaacc tgactcagaa 1560
tctgatgcag aatctgactc agagtttgag ccagaaggag aaccgggaaa gcccgaagca 1620
gaactcaggc aaggagcaga atgataacac cagcaatggc accaacgact acataggcag 1680
tgtagagaaa tggcgttaaa tggctcaaaa aggcctgtac atacttctcc caaagcgcca 1740
ctgaaaagat ggcatagctt aaaagatgaa agtgtccaaa cacatcctgc ttecttcatt 1800
ggggaagttt taaaaaaaag tttagatgtt gcctttacag ttgcctttca attcagtgtt 1860
atactgtgtg taggtaaaac aaatctcaat atggaattaa attgtctttt tggggttgga 1920
ctaaatatga aatcegaaag ccaaaccaga ctcaccagaa attgctgttt agatatttta 1980
agaagttctt aaattagtta tggagacaaa gtgaaaacat aaaatgtgac catttaactt 2040
atggctaaga aatggacttt aaattattca tgatacactg ttaaaaccca atcttggaat 2100
caaatatttt ttccaggggt gagaataagt ataaacataa agcaactaaa atgaaacata 2160
aaacctttta ttttcttctg attttaacaa ggaatctatt taaatagaat aacaactgat 2220
ggtgaatctt accgagctgt agaaaataaa aaattcctct ccaaacatgg gtagttttat 2280
gtcaaaatat tggcttttca agaacaggac tcatatcttg atatttaaga gatgtttaaa 2340
attttaaact ttttetacct tctactgttt aaaggtttta cacagggtgt atctcacatt 2400
aaacaaaaca cctttttttc aaaatgaaat accaatgtaa agatetaatt tccaggcgct 2460
ttcagggcac tgtaatttca acaatactgg aatcattttg gcgctgcttc tcattcattt 2520
taaggcttct ctgaattgtg ctcattccaa attaacccat gtatagaatc tttcttcatc 2580
atctaaatgg tgtgttgetg aagttattgt ggtatataat cctggattaa agtcaggact 2640
tt 2642
<210> 31
<211> 2080
<212> DNA
<213> Homo Sapiens
<220> -
<221> misc_feature
<223> Incyte ID No: 3538709CB1
<400> 31
tgtttcggcg gccgcgggat gcccctgcgc tgaccgccag gggcaggtgc ccgcccgcgt 60
agacgcaccc ggcctgaccc cgcgccacca tgtaaacagc gccagcaggc ggacgctggc 120
ttctccgcct gggacccctc cgccccgacc cgggccccgc ggccctcgat gaggacacac 180
30/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
catgctgacc ggggtgaccg acggtatctt ctgttgcctg ctgggcacgc cccccaacgc 240
cgtggggcca ctggagagcg tcgagtccag cgatggctac acctttgtag aggtcaagcc 300
cggccgcgtg ctgcgggtga agcatgca.gg acccgcccca gccgctgccc cacetccacc 360
atcatccgca tcctcggatg cagcccaggg ggacctctcc ggcttggtcc gctgtcageg 420
ccggatcacc gtgtaccgca atgggcggtt gctggtggaa aacctgggcc gagcccctcg 480
agccgacctc ctacacgggc agaatggctc tggggagccg ccggccgccc tggaggtgga 540
gctggcagat ecggcgggca gcgatggccg cttggccccc ggcagcgcag gcagcggcag 600
cggcagtggc agtggtgggc ggcggcggcg agccaggcgc cccaagagga ccatccatat 660
tgactgtgag aagcgcatca ctagctgcaa aggcgcccag gccgacgtgg tgctcttttt 720
catccatggt gtcggcggtt ccctggccat ctggaaggag cagctggact tctttgtgcg 780
cctaggctat gaggtggtgg ctcctgacct ggccggccac ggggccagct ctgegcccca 840
ggtggccgca gcctacacct tctatgcgct ggctgaggac atgcgagcaa tcttcaagcg 900
ctatgccaag aagcgaaatg tgctcattgg ccattcctac ggtgtctctt tctgcacatt 960
cctggcacat gagtacccag acctagtgca caaggtgatc atgatcaatg gcgggggccc 1020
tacggcgctg gagcccagct tctgctcaat cttcaacatg cccacctgcg tcctgcactg 1080
cttgtcgccc tgcctggcct ggagcttcct caaggccggc ttcgcccgcc aaggagccaa 1140
ggagaagcag ctgttaaagg agggcaacgc tttcaacgtg tcatccttcg tactccgggc 12D0
catgatgagc ggccagtact ggcccgaggg cgacgaggtc taccacgccg agctcaccgt 1260
gcccgtcctg cttgtccacg gcatgcacga taagtttgtg ccggtggagg aagaccagcg 1320
catggccgag atcctgctcc tggcattcct gaagctcatc gacgagggca gccacatggt 1380
gatgctggaa tgccctgaga cggtcaacac gctgctccac gaattcctgc tctgggagcc 1440
cgagccctcg cccaaggctc taccggagcc actgccggeg cctccagaag acaagaagta 1500
gccgctgggccggeggggca tcgcttggtg agcacagccg cagcaggagg aggcccgagc 1560
ctgcgccagg tctgcagcgc agaccacctg ggcgggccgt tcgctccggt gggcggggcc 1620
aggtcaggga gacgccccca ggctgcctgg gcggggcgtg gcatccgagg gagcccagcg 1680
gacattccgc tctccgcttc cgtcecgcgg ggcccatcgg cgttttgggg ccgcagccgg 1740
gaccctcacg gaagatgacc ttgtacagaa gctctccctc accttccccc caacgccacg 1800
gccaaggcag gccccccacc ccgctgtctt ccgtgtcagc cgtgcttgat cctgggaccc 1860
acgagcccca cagggaccct cgaggcccca tcccgttatc cgagaccctt cctacccccc 1920
attccteggc gctgggagct atttttgccc aagggggggg gatggggggg ctggcgecac 1980
cgaacctgca catetcaact tgtaactcaa taaacagaag tgacaatcgg aaaaaaaaaa 2040
aaaaaaaaaa aaaaaaaaaa aaaaaaaaag aaaaaaaaaa 2080
<210> 32 .
<211> 2219
<212> DNA
<213> Homo saprens
<220>
<221> misc_feature
<223> Incyte ID No: 71563101CB1
<400> 32
ggcctccaat ctgcttccat gggggttggc tttctgagtg ggagaaatga ctctaatctg 60
gagacatttg ctgagaccct tgtgcctggt cacttccgct cccaggatcc ttgagatgca 120
tcctttcctg agcctaggta cttcccggac atcagtaacc aagctcagtc ttcatacaaa 180
gcccagaatg cctccatgtg acttcatgcc tgaaagatac cagtcccttg gctacaaccg 240
tgtcctggaa atccacaagg aacatctttc tcctgtggtg acggcatatt tccagaaacc 300
cctgctgctc caccaggggc acatggagtg gctctttgat gctgaaggaa acagatacct 360
ggatttcttt tccgggattg ttactgtcag tgttggccac tgccacccaa aggtgaatgc 420
agtggcacaa aagcagctcg gccgcctgtg gcatacaagc accgtcttct tccaccctcc 480
aatgcatgaa tatgcagaga agcttgccgc acttcttcct gagcctctta aggtcatttt 540
cttggtgaac agtggctcag aagccaatga gctggccatg ctgatggcca gggcgcactc 600
aaacaacata gacatcattt ctttcagagg agcctaccat ggatgcagtc cttacacact 66D
tggcttgaca aacgtaggga tctacaagat ggaactccct ggtgggacag gttgccaacc 720
aacaatgtgt ccagatgttt ttcgtggccc ttggggagga agccactgtc gagattctcc 780
agtgcaaaca atcaggaagt gcagctgtgc accagactgc tgccaagcta aagatcagta 840
tattgagcaa ttcaaagata cgctgagcac atctgtggcc aagtcaattg ctggattttt 900
cgcagaacct attcaaggtg tgaatggagt tgtccagtac ccaaaggggt ttctaaagga 960
agcctttgag ctggtgcgag caaggggagg cgtgtgcatt gcagatgaag tgcagacagg 1020
atttggaagg ttgggctctc acttctgggg cttccaaacc cacgatgtcc tgcctgacat 1080
tgtcaccatg gctaaaggga ttgggaatgg ctttcccatg gcagcagtca taaccactcc 1140
31/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
agagattgcc aaatctttgg cgaaatgcct gcagcacttc aacacctttg gagggaaccc 1200
catggcctgt gccattggat ctgctgtgct tgaggtgatt aaagaagaaa atctacagga 1260
aaacagtcaa gaagttggga cctacatgtt actaaagttt gctaagctgc gggatgaatt 1320
tgaaattgtt ggagacgtcc gaggcaaagg cctcatgata ggcatagaaa tggtgcagga 1380
taagataagc tgtcggcctc ttccccgtga agaagtaaat cagatecatg aggactgcaa 1440
gcacatggga ctcctcgttg gcagaggcag cattttttct cagacatttc gcattgcgcc 1500
ctcaatgtgc atcactaaac cagaagttga ttttgcagta gaagtatttc gttctgcctt 1560
aacccaacac atggaaagaa gagctaagta acattgtcag aaataaataa aaccacaagt 1620
ctcaagaatt tgccacgtat gttcaagggt gaatttgaag aatttcagaa ccactggtat 1680
ccagagaaag cctgcagctc tccacaggag ctgtaaaagt catggttgac tgcctaccaa 1740
ccatatttgt tagcagagcc cctcttatct tgagaactcc attcttcagg gaaaggatct 1800
ccctagctca gagaataaat cctaattagt ttatgttagg tatggtaatt tgattcccct 1860
ttgcagtgat tggtttatgc atgaatatgt gatgtatttt tgtccagtga atcttgaaga 1920
aaaatctttt ggtggaggtg ccttcaggga aagttttctt caccctcact cttcagttca 1980
agaagagatg tcttcttgtt gegctgagaa caccatatgt tcatgacgag attcctggca 2040
ccatgtcagc cggcttgtag tcatgaggac aacccttttt ggtgaggttg gaagatggat 2100
ggaagccaag tgcttagtga tgtcaaagaa gcaotcactt aagcattcct ggagecaccc 2160
tacctcaggg cctcttgata tttgaggtaa taaaattcat tgttctgtat aaaaaaaaa 2219
<210> 33
<211> 681
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472027CB1
<400> 33
atgagactat gtgaaaagac cgaactacag ttgattggtg tacctgaaag tgacagggag 60
aatggaacca agttggaaaa cacatttcag gatattatcc aggagaactt ccccaaccta 120
gcaagacagg ccaacattca aattcagatg gctggtggat ctatctggat tgaagggatt 180
ccttttccca gcaataattt tacagacctg agacgtttgc aagatgaaat tgtgttgcgg 240
gatgaagatg tcattacact ttcttaccca aagtcaggaa gcttttggat agtggaaatc 300
atcagtctga tccactccaa gggagatcct agttgggtcc aatctgttgt tccctgggat 360
cgttcaccat ggatagaagt taaacgtaag aaagcaggtt tagagagtca gaagggccca 420
cacctctaca cctcccacct tcccattcag ctcttcccca agtcattctt gaattccaag 480
gccaagtgta tttatcctca tgttctcatg cttgtggttc tcatcctagg acataagagc 540
cagtggagta ttgctataaa gatatctgaa aatgcagaag caacttcaaa actgggtaat 600
gggcaaagat tggaagagtt tggagggctc agaagaagac aggaagatga aagaagtttg 660
gaatttctta gagactgtta a 681
<210> 34
<211> 399
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480358CB1
<400> 34
agcccagacc agagtgtggc ccctggagtc ataatgatga aggtcatgta catgttgaag 60
ggccagagcc eggtgcaggg caccatccac tttgagcaga aggaaaatga accatttatg 120
gtgtcagaat gcattacagg attgactgaa cgccagcaca gattccatgt tcatcagttt 180
ggagataata caccaggctg taccagggca gttccttact ttaatccttt aaccaaaaac 240
cacagtgggc caaggatcaa gagaggcagg ttggagacct gggtaatgtg gccgctggca 300
aagatgtgtc gccacatgtc tgttgaagat tctctggtct cactctcagg acactattcc 360
atcactgccc acacaatggt gtccatgaca accagatga 399
<210> 35
<211> 2302
32/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1618256CB1
<400> 35
taactaatcg gaaggcgctg tgaatttcac gtttctcgcg tagctgtaac gcgcggtata 60
gttcttactc aagtcgtgcc ctgccagaaa ggggaagatg cagcgaggga gagacctaga 120
accgcgatca aagctgcagc agctgctggg gctggcggca caaagggagg agggaggagc 180
ctgggcgctg agaaagttct tggggaaagt tgagctgagc caagagtcgg gcggtggctg 240
ggatgggcgg gagggcccgg cccgatttcc cttgctgctc cccttgtggc ctgacgctga 300
cagaggcaaa aatctgctaa ctcagggggc agactcaacc aagactgtga gcaggcctgg 360
ggaatgaccc cccgatctcc aaccagtgcc ttccgcagct gcacggctgt ctccagctgt 420
ctctgcccct cttcctggcc ctggctccat ctctctgtca cctcaccctt ccctgtgcca 480
catgggccct ctetctcctg ccaggacgct gcggctctgg ggacctcgga gcctgggggt 540
ggctctggga gtcttcatga ccattggctt tgcactccag ctcttgggag ggcccttcca 600
gaggaggcta cctgggctac agctccgaca gccctcggcc ccatccctac gaccagccct 660
tccgtcctgc ccaccccggc agcgactggt gttcctgaag acacataaat ccgggagcag 720
ctctgtgctg agcctgcttc accgctatgg ggaccagcac gggctgcgct tcgccctccc 780
tgcccgctac cagtttggct acccaaagct cttccaggcc tctagggtaa aaggctaccg 840
cccacagggt ggaggcaccc agctcccctt ccacatcctc tgtcaccaca tgaggttcaa 900
cctgaaagag gtacttcagg tcatgccttc tgacagcttc tttttttcca ttgtccgaga 960
cccagcggct ctggctcgct ctgccttctc ctactataaa tccacctcat cagccttccg 1020
caagtcacca tctttggctg ccttcctggc caatcctcga ggcttctaca ggcctggggc 1080
ccgtggggac cactacgctc gcaacttact atggtttgac tttggcctgc cctttccccc 1140
agagaagagg gccaagagag ggaatattca tccccccaga gaccccaacc ccccacagct 1200
gcaggtcttg ccttctggtg ctggccctcg agcccaaacc ctcaatccca atgccctcat 1260
ccatcctgtt tccactgtta ctgatcatcg,cagccagata tcaagccctg cctctttcga 1320
tttggggtct tcatccttca tccagtgggg tctggcctgg ctggactctg tctttgacct 1380
ggtcatggtg gctgagtact tcgatgagtc attggttctg ctggcagatg ccctgtgctg 1440
gggtctagat gacgtggtgg gcttcatgca.caatgcccag gctggacata agcagggcct 1500
cagcactgtc agcaacagtg gactgactgc ggaggaccgg cagctgactg cacgggcccg 1560
agcctggaac aacctggact gggctctcta tgtccaettc aaccgcagtc tctgggcacg 1620
gatagagaaa tacggecagg gccggctgca gacagctgtg gccgagctcc gggctcgccg 1680
agaggcccta gcgaaacatt gtctggtagg gggtgaggct tctgacccca aatacatcac 1740
tgatcgecgg ttccgcccct tccagtttgg gtcagctaag gttttgggct atatacttcg 1800
gagtggattg agcccccaag accaagagga atgtgagcgc ctagctaccc ctgagetcca 1860
gtacaaggac aagctggatg tcaagcagtt cccccctacc gtetcactgc ccctcaagac 1920
ttcaaggcca ctctccccat aaacatcaga ctacagattt aggtggaaga gcagccatgt 1980
ttgaagggca catgtgatga gtggggggca gcaagatgcc atttctgcat ctcccagaag 2040
ggatgagtet ttgtecegat gcaagcccec tcttegctgg geteccagca gtgcttecct 2100
cctccaccct ccactcattt tgttctttcc ccccaacttt tttttttttg aaacggagtc 2160
ttgctctgtc ccccaggctg gagtgcagtg gcatgatctc ggctcactgc aacctctgcc 2220
tcccaggttc aagcgattct cctgcctcag cctccagagt agctaggatt acagatacgt 2280
gccaccatac ccggctaatt tt 2302
<210> 36
<211> 3341
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3387823CB1
<400> 36
gaaacgaaga gaaccctetg tccctggcag aatctgcatg tacatttctt gtctgtcctt 60
gtctctcttc ttcctgtctg gcccattgca gagagtattg gaagtttcca accattggtg 120
gtactctatg ctcatcctac ctcctttgct gaaagacagt gtggcagcgc ccctgctgtc 180
tgcctactac cctgactgtg ttggcatgag cccctcctgc accagcacaa accgcgccgc 240
33/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
tgccactggc aatgccagcc ctgggaagct ggagcactcc aaggctgccc tctccgtgca 300
cgttccaggc atgaaccgat acttccagcc tttctaccag cccaatgagt gtggcaaagc 360
cctctgtgtg aggccggatg tgatggaact ggatgagctc tatgagtttc cagagtattc 420
ccgagacccc accatgtacc tggctttgag aaacctcatc ctcgcactgt ggtatactaa 480
ctgcaaagaa gctcttactc ctcagaaatg tattcctcac atcatcgtcc ggggtctcgt 540
gcgtattcga tgcgttcagg aagtggagag aatactgtat tttatgacca gaaaaggtct 600
catcaacact ggagttctca gcgtgggagc cgaccagtat cttctcccta aggactacca 660
caataaatca gtcatcatta tcggggctgg tccagcagga ttagcagctg ctaggcaact 720
gcataacttt ggaattaagg tgactgtcct ggaagccaaa gacagaattg gaggccgagt 780
ctgggatgat aaatctttta aaggcgtcac agtgggaaga ggagctcaga ttgtcaatgg 840
gtgtattaac aacccagtag cattaatgtg tgaacaactt ggcatcagca tgcataaatt 900
tggagaaaga tgtgacttaa ttcaggaagg tggaagaata actgacccca ctattgacaa 960
gcgcatggat tttcatttta atgctctctt ggatgttgtc tctgagtgga gaaaggataa 1020
gactcagctc caagatgtcc ctttaggaga aaagatagaa gaaatctaca aggcatttat 1080
taaggaatct ggtatccaat'tcagtgagct ggagggacag gtgcttcagt tccatctcag 1140
taacctggag tacgcctgtg gcagcaacct tcaccaggta tctgctcgct cgtgggacca 1200
caatgaattc tttgcccagt ttgctggtga ccacactctg ctaactcccg ggtactcggt 1260
gataattgaa aaactggcag aagggcttga cattcaactc aaatctccag tgcagtgtat 1320
tgattattct ggagatgaag tgcaggttac cactacagat ggcacagggt attctgcaca 1380
aaaggtatta gtcactgtac cactggcttt actacagaaa ggtgccattc agtttaatcc 1440
accgttgtca gagaagaaga tgaaggctat caacagctta ggcgcaggca tcattgaaaa 1500
gattgccttg caatttccgt atagattttg ggacagtaaa gtacaagggg ctgacttttt 1560
tggtcacgtt cctcccagtg ccagcaagcg agggcttttt gccgtgttct atgacatgga 1620
tccccagaag aagcacagcg tgctgatgtc tgtgattgcc ggggaggctg tcgcatccgt 1680
gaggaccctg gacgacaaac aggtgctgca gcagtgcatg gccacgctcc gggagctgtt 1740
caaggagcag gaggtcccag atcccacaaa gtattttgtc actcggtgga gcacagaccc 1800
atggatccag atggcataca gttttgtgaa gacaggtgga agtggggagg cctacgatat 1860
cattgctgaa gacattcaag gaaccgtctt tttcgctggt gaggcaacaa acaggcattt 1920
cccacaaact gttacagggg catatttgag tggcgttcga gaagcaagca agattgcagc 1980
attttaagaa ttcggtggac ccagctttct tctgtacccc agatggggaa atttgaatca 2040
catgttaaac ctcagtttta taagaggggg aaaaaaccgt ctctacatag taaaactgaa 2100
atgtttctaa.ggcgatatga taatgcaaac ctatttcatc actctaaaag cactgacctc 2160
aaaaaacctt ataagcactt agatttaatt gcattttcca taggttcaac tactgctgaa 2220
agtctggatt tcagaataaa gcagaatgta agtttcagtt gaggccatgg atttgattgt 2280
tccatggctg gaagttccct ttagatttca cattttatat ggctgatcaa ttttcataca 2340
ttgagaaacc aagtcaatca agcaggaatc atttaaaaac cagataaagc catgtttttc 2400
ttctgtgaca atttatcagt atctttacca atgagcctta atttttatat aggtccaata 2460
ttgagctttt acttaaaatt tagatagaac ttttttttgg atacagcaca aactccagtt 2520
gacagtaaaa tgaagcttct aggtattttg tattgtacat atttcctcct actgggtgtt 2580
caaaagaaat ttaaattcaa gtaccttttg tgataaaatg ttttagattt gtgcacccat 2640
tggcaaaaca ggaaagtttc cagataggta ttgtatcatt gagaatgcag cacagatagt 2700
gtgggcttca cactatagac acagaatata gctttttctt aaagccaaat ttgggtgata 2760
ggacacttta aatatcctta attttggcaa ccactagcaa aaaaaacttg tcagaataat 2820
ttaaccaagc ccctctccac ttcttttatt taaaagcact gattcaattg ctaggaatat 2880
ttttgcagat ttttctttac agtattccat aggcaggtcc actggaaaac tgcagaaaaa 2940
tgtgagctct cctggtaaat agtatacatt ttataagcta tattttaaag gcctaagaac 3000
atggcgagta tttactttta tctttttttt aaaaacactc atgacagaaa acagtctaat 3060
aatatctcat tctaaaataa aacactggtt gcagggtctt caggatgcct attttgccag 3120
aaacttcagt atacaggtta gaaatatgct tttgtttttg aacataatat actggtttgc 3180
tttaaagaag ggactaaata tgactttaaa gagacttcaa atattgagta ttttaaaaat 3240
ttaaaagtag gtcagtttat aacgagtaaa tacetaacac ccaagaatgt gcagtgaacc 3300
tcaggcgggg atccttagtt ctaacggccg ccccgtaggc g 3341
<210> 37
<211> 777
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55142051CB1
34/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
<4D0> 37
atgtcccctg ccattgcatt ggccttcctg ccactggtgg taacattgct ggtgcggtac 60
cggcactact tccgattgct ggtgcgcacg gtcttgctgc gaagcctccg agactgcctg 120
tcagggctgc ggatcgagga gcgggccttc agctacgtgc tcacccatgc cctgcccggt 180
gaccctggtc acatcctcac caccctggac cactggagca gccgctgcga gtacttgagc 240
cacatggggc ctgtcaaagg tcagatcctg atgcggctgg tggaggagaa ggcccctgct 300
tgtgtgctgg aattgggaac ctactgtgga tactctaccc tgcttattgc ccgagccctg 360
ccccctgggg gtcgccttct tactgtggag cgggacccac gcacggcagc agtggctgaa 420
aaactcatcc gcctggccgg ctttgatgag cacatggtgg agctcatcgt gggcagctca 480
gaggacgtga tcccgtgcct acgcacccag tatcagctga gtcgggcaga cctggtgctc 540
ctggcacacc ggccacgatg ttacctgagg gacctgcagc tgctggaggc ccatgcccta 600
ctgccagcag gtgccaccgt gctggctgac catgtgctct tccctggtgc accccgcttc 660
ttgcagtatg ctaagagctg tggccgctac cgctgccgcc tccaccacac tggccttcca 720
gacttccctg ccatcaagga tggaatagct cagctcacct atgctggacc aggctga 777
<210> 38
<211> 3600
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7395274CB1
<400> 38
gagagcagag caggacactg gcgccgcggg tcaggcagct gcgtgcgcgt ctcctccagg 60
cagcaagggg aacccgaggc cgccggcgcc cggaccatgt cgtctccggg gccgtcgcag 120
ccgccggccg aggacccgcc ctggcccgcg cgcctcctgc gtgcgcctct ggggctgctg 180
cggctggacc ecagcggggg cgcgctgctg ctatgcggcc tcgtagcgct gctgggctgg 240.
agctggctgc ggaggcgccg ggcgcggggc atcccgcccg ggcccacgcc ctggcctctg 300
gtgggcaact tcggtcacgt gctgctgcct cccttcctcc ggcggcggag ctggctgagc 360
agcaggacca gggcegcagg gattgatccc tcggtcatag gcccgcaggt gctcctggct 420
cacctagccc gcgtgtacgg cagcatcttc agcttcttta tcggccacta cctggtggtg 480
gtcctcagcg acttccacag cgtgcgcgag gcgctggtgc agcaggccga ggtcttcagc 540
gaccgcccgc gggtgccgct catctecatc gtgaccaagg agaagggggt tgtgtttgca 600
cattatggtc ccgtctggag acaacaaagg aagttctctc attcaactct tcgtcatttt 660
gggttgggaa aacttagctt ggagcccaag attattgagg agttcaaata tgtgaaagca 720
gaaatgcaaa agcacggaga agaccccttc tgccctttct ccatcatcag caatgccgtc 780
tctaacatca tttgctcctt gtgctttggc cagcgctttg attacactaa tagtgagttc 840
aagaaaatgc ttggttttat gtcacgaggc ctagaaatct gtctgaacag tcaagtcctc 900
ctggtcaaca tatgcccttg gctttattac cttccctttg gaccatttaa ggaattaaga 960
caaattgaaa aggatataac cagtttcctt aaaaaaatca tcaaagacca tcaagagtct 1020
ctggatagag agaaccctca ggactteata gacatgtacc ttctccacat ggaagaggag 1080
aggaaaaata atagtaacag cagttttgat gaagagtact tattttatat cattggggat 1140
ctctttattg ctgggactga taccacaact aactctttgc tctggtgcct gctgtatatg 1200
tcgctgaacc ccgatgtaca agaaaaggtt catgaagaaa ttgaaagagt cattggcgcc 1260
aaccgagctc cttccctcac agacaaggcc cagatgccct acacagaagc caccatcatg 1320
gaagtgcaga ggctaactgt ggtggtgccg cttgccattc ctcatatgac ctcagagaac 1380
acagtgctcc aagggtatac cattcctaaa ggcacattga tcttacccaa cctgtggtca 1440
gtacatagag acccagccat ttgggagaaa ccggaggatt tctaccctaa tcgatttctg 1500
gatgaccaag gacaactaat taaaaaagaa acctttattc cttttgggat agggaagcgg 1560
gtgtgtatgg gagaacaact ggcaaagatg gaattattcc taatgtttgt gagcctaatg 1620
cagagtttcg catttgcttt acctgaggat tctaagaagc ccctcctgac tggaagattt 1680
ggtctaactt tagccccaca tccatttaat ataactattt caaggagatg aagagcatct 1740
ccaagaagag atggtaaaaa gatatataaa tacatatcct tctaagcaga ttcttcctac 1800-
tgcaaaggac agtgaatcca gcaactcagt ggatccaagc tgggctcaga ggtcggaagg 1860
agggtagagc acactgggag gtttcatctt ggaggattcc tcagcaggat acttcagcca 1920
ttttagtaat gcaggtctgt gatttggggg atagaaaaca aagtacctat gaaacgggat 1980
atctggattt tacttgcagt ggcttccacc gatgggccaa tcttctcatt tcttagtgcc 2040
tcagacatcc catatgtaaa atgagagtaa taaaacttgg cttctctcta cctctcagca 2100
ctaatgatgg tcaaatgcct tacatctttt ctgatatctc taaaatgctg ttaagttctg 2160
gagaagaact tcaggagaag aagatctatc agctggcttt taaagaccta tgacaacatg 2220
35/36
CA 02417769 2003-O1-29
WO 02/12467 PCT/USO1/24382
aaagtggtgt tcagcctgga atgctttgtc agagatgggt gtggatttag gttatactgg 2280
gggagaactt ttctcagcac agattctatg ccagcttctt tgggcttgtt ctgtcactat 2340
ctttttgttt atgattttag tttttacttt ttgtagatgt gggatgaagt ggactctgtc 2400
gtgtatattg aggaaaaaag aaattataat tttaaaaaat cccttgtagg attattatct 2460
aaatttatat gtctaacttc tactacaact acaggaacag tgagccttgc tacttcttta 2520
gtagcttctt ggcagaattc ctttctactg agttatttgc aaagatgcag ctctaccttt 2580
ttacttaagg cctgaatggt gagcatgggg attttgatac tgggactcat caggaaagga 2640
ttctgctttc aaactatact gaacattcct gtcctagcgt ccctgccacc aggcccaatg 2700
catctgatcc ttgaatatac tctcaaagaa ttcactctct ttttattaag agaactaaat 2760
tgtttctaaa tgtagatggt ccctctggaa aagcagtttt cagcaggggt ggtaacccct 2820
tcagagggag tttggaaatg tgtgggtatg attcttggtt atcataatga tgggggtgct 2880
actggccttc tgctgccatg ggaccaggat gctaaatgtc aaggtagtcc tatacagtga 2940
agaattgtcc tgctcaagat gccaggattt cccccagtga gaacatgctc taaggaatga 3000
ccaccccttt cttttattct cccacagtgc tccatgtaca gaagtaagca tagcagtcat 3060
atgagcaacc acattcctga acetttcctc atgetggctc tacacttaat cctttacttg 3120
tatgtttctg taattcttac ataaattcta ttaagagggt ggcatactgt agtggatgaa 3180
gctgaggctt atagtaggta aggcacaaag ttaaaaagta acatcactgg gtttcaaacc 3240
tactggtctc tgtgactaaa gaacactttc agaaccactt cttgattctg ccaccacttg 3300
atcccataac aggetacccc ttggcctcat gctggagttg tgtgtgtctg tcttcatccc 3360
aggctgagct ccttgaggtg aggatgttgt gctgtttgcc tcectcacag tgccttggtc 3420
ttagtggatg cccagttgtc ttgtgaatga cttttaagaa gtgtacttaa gagaaaaatc 3480
ctaccttatt tgaataatta caagtcatgt ttttgttgct taaaggtgat aaatcagtgt 3540
atattatttg ttaatgtcca ttaaagccag tttttaaaaa aaaaaaaaaa aaaaaaaaaa 3600
36/36
