Note: Descriptions are shown in the official language in which they were submitted.
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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
autoimmunelinflanmlatory, cell proliferative, developmental, endocrine, eye,
metabolic, and
gastrointestinal disorders, including liver disorders, and in the assessment
of the effects of exogenous
compounds on the expression of nucleic acid and amino acid sequences of drug
metabolizing enzymes.
BACKGROUND OF THE INVENTION
The metabolism of a drug and its movement through the body (pharmacokinetics)
are
important in determining its effects, toxicity, and interactions with other
drugs. The three processes
governing pharmacokinetics are the absorption of the drug, distribution to
various tissues, and
elimination of drug metabolites. These processes are intimately coupled to
drug metabolism, since a
variety of metabolic modifications alter most of the physicochemical and
pharmacological properties
of drugs, including solubility, binding to receptors, and excretion rates. The
metabolic pathways
which modify drugs also accept a variety of naturally occurring substrates
such as steroids, fatty
acids, prostaglandins, leukotrienes, and vitamins. The enzymes in these
pathways are therefore
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 bioactivadon (such as codeine), these
polymotphisms can be critical.
Moreover, promising new drugs are frequently eliminated in clinical trials
based on toxicities which
may only affect a segment of the patient group. Advances in pharmacogenomics
research, of which
drug metabolizing enzymes constitute an important part, are promising to
expand the tools and
information that can be brought to bear on questions of drug efficacy and
toxicity (See Evans, W. E.
and R. V. Relling (1999) Science 286:487-491).
Drug metabolic reactions are categorized as Phase I, which functionalize the
drug molecule
and prepare it for further metabolism, and Phase II, which are conjugative. In
general, Phase I
reaction products are partially or fully inactive, and Phase II reaction
products are the chief excreted
species. However, Phase I reaction products are sometimes more active than the
original
administered drugs; this metabolic activation principle is exploited by pro-
drugs (e.g. L-dopa).
Additionally, some nontoxic compounds (e.g. atlatoxin, benzo[a]pyrene) are
metabolized to toxic
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intermediates through these pathways. Phase I reactions are usually rate-
limiting in drug metabolism.
Prior exposure to the compound, or other compounds, can induce the expression
of Phase I enzymes
however, and thereby increase substrate flux through the metabolic pathways.
(See Klaassen, C. D.,
Amdur, M. O. and J. Doull (1996) Casarett and Doull's Toxicolo~,,y: 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 Drub
Metabolism, Blackie Academic and Professional, London.)
Drug metabolizing enzymes (DMEs) have broad substrate specificities. This can
be
contrasted to the immune system, where a large and diverse population of
antibodies are highly
specific for their antigens. The ability of DMEs to metabolize a wide variety
of molecules creates the
potential for drug interactions at the level of metabolism. For example, the
induction of a DME by
one compound may affect the metabolism of another compound by the enzyme.
DMEs have been classified according to the type of reaction they catalyze and
the cofactors
involved. The major classes of Phase I enzymes include, but are not limited
to, cytochrome P450 and
llavin-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 reduclase system, the
ferredoxin/ferredoxin
reductase redox pair, aldo/keto reductases, and alcohol dehydrogenases. The
major classes of Phase II
enzymes include, but are not lin>;ted to, UDP glucuronyltransferase,
sulfotransferase, glutathione S-
transferase, N-acyltransferase, and N-acetyl transferase.
Cytochrome P450 and P450 catalytic cycle-associated enzymes
Members of the cytochrome P450 superfamily of enzymes catalyze the oxidative
metabolism
of a variety of substrates, including natural compounds such as steroids,
fatty acids, prostaglandins,
leukotrienes, and vitamins, as well as drugs, carcinogens, mutagens, and
xenobiotics. Cytochromes
P450, also known as P450 heme-thiolate proteins, usually act as terminal
oxidases in
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, nitro, and N-
oxide groups. These
reactions are involved in steroidogenesis of glucocorticoids, cortisols,
estrogens, and androgens in
animals; insecticide resistance in insects; herbicide resistance and flower
coloring in plants; and
environmental bioremediation by microorganisms. Cytochrome P450 actions on
drugs, carcinogens,
mutagens, and xenobiotics can result in detoxification or in conversion of the
substance to a more
toxic product. Cytochromes P450 are abundant in the liver, but also occur in
other tissues; the
enzymes are located in microsomes. (See ExPASY ENZYME EC 1.14.14.1; Prosite
PDOC00081
Cytochrome P450 cysteine heme-iron ligand signature; PRINTS EP450I E-Class
P450 Group I
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signature; Graham-Lorence, S. and Peterson, J.A. (1996) FASEB J. 10:206-214.)
Four hundred cytochromes P450 have been identified in diverse organisms
including bacteria,
fungi, plants, and animals (Graham-Lorence, supra). The B-class is found in
prokaryotes and fungi,
while the E-class is found in bacteria, plants, insects, vertebrates, and
mammals. Five subclasses or
groups are found within the larger family of E-class cytochromes P450 (PRINTS
EP450I E-Class
P450 Group I signature).
All cytochromes P450 use a heme cofactor and share structural attributes. Most
cytochromes
P450 are 400 to 530 amino acids in length. The secondary structure of the
enzyme is about 70%
alpha-helical and about 22% beta-sheet. The region around the heme-binding
site in the C-terminal
part of the protein is conserved among cytochromes P450. A ten amino acid
signature sequence in
this heme-iron ligand region has been identified which includes a conserved
cysleine 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, s-upra; Graham-Lorence, supra.)
Cytochrome P450 enzymes are involved in cell proliferation and development.
The enzymes
have roles in chemical mutagenesis and carcinogenesis by metabolizing
chemicals to reactive
intermediates that form adducts with DNA (Nebert, D.W. and Gonzalez, F.J.
(1987) Ann. Rev.
Biochem. 56:945-993). These adducts can cause nucleotide changes and DNA
rearrangements that
lead to oncogenesis. Cytochrome P450 expression in liver and other tissues is
induced by xenobiotics
such as polycyclic aromatic hydrocarbons, peroxisomal proliferators,
phenobarbital, and the
glucocorticoid dexamethasone (Dogra, S.C. et al. (1998) 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 (OMIM)
*601771
Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYP1B1).
Cytochromes P450 are associated with inflammation and infection. Hepatic
cytochrome
P450 activities are profoundly affected by various infections and inflammatory
stimuli, some of
which are suppressed and some induced (Morgan, E.T. (1997) Drug Metab. Rev.
29:1129-1188).
Effects observed in viva can be mimicked by proinflammatory cytokines and
interferons.
Autoantibodies to two e;ytochrome P450 proteins were found in patients with
autoinmmne
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), a polyglandular
autoimmune
syndrome (OMIM *240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal
dystrophy).
Mutations in cytochromes P450 have been linked to metabolic disorders,
including congenital
adrenal hyperplasia, the most common adrenal disorder of infancy and
childhood; pseudovitamin D-
deficiency rickets; cerebrotendinous xanthomatosis, a lipid storage disease
characterized by
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progressive neurologic dysfunction, premature atherosclerosis, and cataracts;
and an inherited
resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J.
et al. (1994) Harrison's
Principles of Internal Medicine, McGraw-Hill, Inc. New York, NY, pp. 1968-
1970; Takeyama, K. et
al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998) N. Engl. J. Med.
338:653-661; OMIM
*213700 Cerebrotendinous xanthomatosis; and OMIM #122700 Coumarin resistance).
Extremely
high levels of expression of the cytochrome P450 protein aromatase were found
in a librolamellar
hepatocellular carcinoma from a boy with severe gynecomastia (feminization)
(Agarwal, V.R. (1998)
J. Clin. Endocrinol. Metab. 83:1797-1800).
The cytochrome P450 catalytic cycle is completed through reduction of
cytochrome P450 by
NADPH cytochrome P450 reductase (CPR). Another microsomal electron transport
system
consisting of cytochrome b5 and NADPH cytochrome b5 reductase has been widely
viewed as a
minor contributor of electrons to the cytochrome P450 catalytic cycle.
However, a recent report by
Lamb, D. C. et al. (1999; FEBS Lett. 462:283-8) identifies a Candida albicans
cytochrome P450
(CYP51) which can be efficiently reduced and supported by the microsomal
cytochrome b5/NADPH
cytochrome b5 reductase system. Therefore, there are likely many cylochromes
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 asscx;iated with
vitamin D synthesis
and catabolism. Vitamin D exists as two biologically equivalent prohormones,
ergocalciferol (vitamin
Dz), produced in plant tissues, and cholecalciferol (vitamin D3), produced in
animal tissues. The latter
form, cholecalciferol, is formed upon the exposure of 7-dehydrocholesterol to
near ultraviolet light (i.e.,
290-310 nm), normally resulting from even minimal periods of skin exposure to
sunlight (reviewed in
Miller, W.L. and Portale, A.A. (2000) Trends in Endocrinology and Metabolism
11:315-319).
Both prohormone forms are further metabolized in the liver to 25-
hydroxyvitamin D
(25(OH)D) by the enzyme 25-hydroxylase. 25(OH)D is the most abundant precursor
form of vitamin
D which must be further metabolized in the kidney to the active form, 1 a,25-
dihydroxyvitamin D
(1a,25(OH)ZD), by the enzyme 25-hydroxyvitamin D la-hydroxylase (la-
hydroxylase). Regulation of
1a,25(OH)ZD production is primarily at this final step in the synthetic
pathway. The activity of
1 a-hydroxylase depends upon several physiological factors including the
circulating level of the enzyme
4
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product (1 a,25(OH)ZD) and the levels of parathyroid hormone (PTH),
calcitonin, insulin, calcium,
phosphorus, growth hormone, and prolactin. Furthermore, extrarenal la-
hydroxylase activity has been
reported, suggesting that tissue-specific, local regulation of 1a,25(OH)2D
production may also be
biologically important. The catalysis of 1 a,25(OH)2D 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, 1 a-hydroxylase, and 24-hydroxylase are all NADPH-
dependent,
type I (mitochondrial) cytochrome P450 enzymes that show a high degree of
homology with other
members of the family. Vitamin D 25-hydroxylase also shows a broad substrate
specificity and may
also perform 26-hydroxylation of bile acid intermediates and 25, 26, and 27-
hydroxylation of
cholesterol (Dilworth, F.J. et al. (1995) J. Biol. Chem. 270:16766-16774;
Miller, W.L. and Portale,
A. A. supra; and references within).
The active form of vitamin D (1 a,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., 1 a-
hydroxylase) causes
hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive) rickets, a
disease characterized
by loss of bone density and distinctive clinical features, including bandy or
bow leggedness
accompanied by a waddling gait. Deficiencies in vitamin D 25-hydroxylase cause
cerebrotendinous
xanthomatosis, a lipid-storage disease characterized by the deposition of
cholesterol and cholestanol in
the Achilles' tendons, brain, lungs, and many other tissues. The disease
presents with progressive
neurologic dysfunction, including postpubescent cerebellar ataxia,
atherosclerosis, and cataracts.
Vitamin D 25-hydroxylase deficiency does not result in rickets, suggesting the
existence of alternative
pathways for the synthesis of 25(OH)D (Griffin, J.E. and Zerwekh, J.E. (1983)
J. Clin. Invest.
72:1190-1199; Gamblin, G.T. et al. (1985) J. Clin. Invest. 75:954-960; and
W.L. and Portale, A.A.
supra).
Ferredoxin and ferredoxin reductase are electron transport accessory proteins
which support at
least one human cytochrome P450 species, cytochrome P450c27 encoded by the
CYP27 gene
(Dilworth, F. J. et al. (1996) Biochem. J. 320:267-71). A Streptomyces ~riseus
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 tree radical species
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(Flitter, W. D. and Mason, R. P. (1988) Arch. Biochem. Biophys. 267:632-9).
Flavin-containing monoox,~~enase (FMO)
Flavin-containing monooxygenases oxidize the nucleophilic nitrogen, sulfur,
and phosphorus
heteroatom of an exceptional range of substrates. Like cytochromes P450, FMOs
are microsomal and
use NADPH and O2; there is also a great deal of substrate overlap with
cytochromes P450. The tissue
distribution of FMOs includes liver, kidney, and lung.
There are Live different known isoforms of FMO in mammals (FMOl, FM02, FM03,
FM04, and FMOS), which are expressed in a tissue-specific manner. The isoforms
differ in their
substrate specificities and other properties such as inhibition by various
compounds and
stereospecificity of reaction. FMOs have a 13 amino acid signature sequence,
the components of
which span the N-ternlinal two-thirds of the sequences and include the FAD
binding region and the
FATGY motif which has been found in many N-hydroxylating enzymes (Stehr, M. et
al. (1998)
Trends Biochem. Sci. 23:56-57; PRINTS FMOXYGENASE Flavin-containing
monooxygenase
signature).
Specific reactions include oxidation of nucleophilic tertiary amines to N-
oxides, secondary
amines to hydroxylamines and nitrones, primary amines to hydroxylamines and
oximes, and sulfur-
containing compounds and phosphines to S- and P-oxides. Hydrazines, iodides,
selenides, and boron-
containing compounds are also substrates. Although FMOs appear similar to
cytochromes P450 in
their chemistry, they can generally be distinguished from cytochromes P450 in
vitro based on, for
example, the higher heat lability of FMOs and the nonionic detergent
sensitivity of cytochromes
P450; however, use of these properties in identification is complicated by
further variation among
FMO isoforms with respect to thermal stability and detergent sensitivity.
FMOs play important roles in the metabolism of several drugs and xenobiotics.
FMO
(FM03 in liver) is predominantly responsible for metabolizing (S)-nicotine to
(S)-nicotine N-1'-
oxide, which is excreted in urine. FMO is also involved in S-oxygenation of
cimetidine, an HZ
antagonist widely used for the treatment of gastric ulcers. Liver-expressed
forms of FMO are not
under the same regulatory control as cytochrome P450. In rats, for example,
phenobarbital treatment
leads to the induction of cytochrome P450, but the repression of FM01.
Endogenous substrates of FMO include cysteamine, which is oxidized to the
disulfide,
cystamine, and trimethylamine (TMA), which is metabolized to trimethylan>ine N-
oxide. TMA
smells like rotting fish, and mutations in the FM03 isoform lead to large
amounts of the malodorous
free amine being excreted in sweat, urine, and breath. These symptoms have led
to the designation
fish-odor syndrome (OMIM 602079 Trimethylaminuria).
Lysyl oxidase:
Lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidase
involved in the
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formation of connective tissue matrices by crosslinking collagen and elastin.
LO is secreted as a N-
glycosylated precuror protein of approximately 50 kDa Levels and cleaved to
the mature form of the
enzyme by a metalloprotease, although the precursor form is also active. The
copper atom in LO is
involved in the transport of electron to and from oxygen to facilitate the
oxidative deanunation 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, cadnuum, 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
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
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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 known reaction catalyzed by a family member, aldose reductase, is the
reduction of
glucose to sorbitol, which is then further metabolized to Iructose by sorbitol
dehydrogenase. Under
normal conditions, the reduction of glucose to sorbitol is a minor pathway. In
hyperglycemic states,
however, the accumulation of sorbitol is implicated in the development of
diabetic complications
(OMIM *103880 Aldo-keto reductase family 1, member Bl). 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 dehydro e~ nases
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, g,, g2). The subunits can form homodimers and
heterodimers; the subunit
composition determines the specific properCies 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 1II (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 carbons and longer) and aromatic alcohols, and
are not inhibited by
pyrazole.
The short-chain alcohol dehydrogenases include a number of related enzymes
with a variety
of substrate specificities. Included in this group are the mammalian enzymes D-
beta-hydroxybutyrate
dehydrogenase, (R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin
dehydrogenase,
NADPH-dependent carbonyl reductase, corticosteroid 11-beta-dehydrogenase, and
estradiol 17-beta-
dehydrogenase, as well as the bacterial enzymes acetoacetyl-CoA reductase,
glucose 1-
dehydrogenase, 3-beta-hydroxysteroid dehydrogenase, 20-beta-hydroxysteroid
dehydrogenase, ribitol
dehydrogenase, 3-oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate
dehydrogenase, sorbitol-6-
phosphate 2-dehydrogenase, 7-alpha-hydroxysteroid dehydrogenase, cis-1,2-
dihydroxy-3,4-
cyclohexadiene-1-carboxylate dehydrogenase, cis-toluene dihydrodiol
dehydrogenase, cis-benzene
glycol dehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase, N-
acylmannosamine 1-
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dehydrogenase, and 2-deoxy-D-gluconate 3-dehydrogenase (lCrozowski, 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 (UGTs) catalyze the transfer
of a
glucuronic acid group from the cofactor uridine diphosphate-glucuronic acid
(UDP-glucuronic acid)
to a substrate. The transfer is generally to a nucleophilic heteroatom (O, N,
or S). Substrates include
xenobiotics which have been functionalized by Phase I reactions, as well as
endogenous compounds
such as bilirubin, steroid hormones, and thyroid hormones. Products of
glucuronidation are excreted
in urine if the molecular weight of the substrate is less than about 250
g/mol, whereas larger
glucuronidated substrates are excreted in bile.
UGTs are located in the microsomes of liver, kidney, intestine, skin, brain,
spleen, and nasal
mucosa, where they are on the same side of the endoplasmic reticulum membrane
as cytochrome
P450 enzymes and flavin-containing monooxygenases, and therefore are ideally
located to access
products of Phase I drug metabolism. UGTs have a C-terminal membrane-spanning
domain which
anchors them in the endoplasmic reticulum membrane, and a conserved signature
domain of about 50
amino acid residues in their C terminal section (Prosite PDOC00359 UDP-
glycosyltransferase
signature).
UGTs involved in drug metabolism are encoded by two gene families, UGT1 and
UGT2.
Members of the UGT1 family result from alternative splicing of a single gene
locus, which has a
variable substrate binding domain and constant region involved in cofactor
binding and membrane
insertion. Members of the UGT2 family are encoded by separate gene loci, and
are divided into two
families, UGT2A and UGT2B. The 2A subfamily is expressed in olfactory
epithelium, and the 2B
subfamily is expressed in liver microsomes. Mutations in UGT genes are
associated with
hyperbilirubinemia (OMIM #143500 Hyperbilirubinemia I); Crigler-Najjar
syndrome, characterized
by intense hyperbilirubinemia from birth (OMIM #218800 Crigler-Najjar
syndrome); and a milder
form of hyperbilirubinemia termed Gilbert's disease (OMIM *191740 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,
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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 monoanune substrates such as dopamine,
epinephrine, and levadopa.
Other cloned STs include an estrogen sulfotransferase and an N-
acetylglucosamine-6-O-
sulfotransferase. This last enzyme is illustrative of the other major role of
STs in cellular
biochemistry, the modification of carbohydrate structures that may be
important in cellular
differentiation and maturation of proteoglycans. Indeed, an inherited defect
in a sulfotransferase has
been implicated in macular corneal dystrophy, a disorder characterized by a
failure to synthesize
mature keratan sulfate proteoglycans (Nakazawa, K. et al. (1984) J. Biol.
Chem. 259:13751-7; OMIM
*217800 Macular dystrophy, corneal).
Galactosyltransferases
Galactosyltransferases are a subset of glycosyltransferases that transfer
galactose (Gal) to the
terminal N-acetylglucosamine (GlcNAc) oligosaccharide chains that are part of
glycoproteins or
glycolipids that are tiee 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 X31,3-galactosyltransferases appear to have a short cytosolic
domain, a single
transmembrane domain, and a catalytic domain with eight conserved regions.
(Kolbinger, F. su ra
and Hennet, T. et al. (1998) J. Biol. Chem. 273:58-65). 1n mouse UDP-
galactose:~i-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
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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. supra). Recent work suggests that brainiac protein
is a (31,3-
galactosyltransferase. (Yuan, Y. et al. (1997) Cell 88:9-11; and Rennet, T.
s_upra).
UDP-Gal:GlcNAc-1,4-galactosyltransferase (-1,4-Gall) (Sato, T. et al., (1997)
EMBO J.
16:1850-1857) catalyzes the formation of Type II carbohydrate chains with Gal
(/31-4)GlcNAc
linkages. As is the case with the (31,3-galactosyltransferase, a soluble form
of the enzyme is formed
by cleavage of the membrane-bound form. Amino acids conserved among (31,4-
galactosyltransferases include two cysteines linked through a disulfide-bonded
and a putative UDP-
galactose-binding site in the catalytic domain (Yadav, S. and Brew, K. (1990)
J. Biol. Chem.
265:14163-14169; Yadav, S.P. and Brew, K. (1991) J. Biol. Chem. 266:698-703;
and Shaper, N.L. et
al. (1997) J. Biol. Chem. 272:31389-31399). (31,4-galactosyltransferases have
several specialized
roles in addition to synthesizing carbohydrate chains on glycoproteins or
glycolipids. In mammals a
(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 X31,4-
galactosyltransferases also
function in cell adhesion, cell/basal lamina interaction, and normal and
metastatic cell migration.
(Shur, B. (1993) Curr. Opin. Cell Biol. 5:854-863; and Shaper, J. (1995) Adv.
Exp. Med. Biol.
376:95-104).
Glutathione S-transferase
The basic reaction catalyzed by glutathione S-transferases (GST) is the
conjugation of an
electrophile with reduced glutathione (GSH). GSTs are homodimeric or
heterodimeric proteins
localized mainly in the cytosol, but some level of activity is present in
microsomes as well. The
major isozymes share common structural and catalytic properties; in humans
they have been classified
into four major classes, Alpha, Mu, Pi, and Theta. The two largest classes,
Alpha and Mu, are
identified by their respective protein isoelectric points; pI ~ 7.5-9.0
(Alpha), and pI -- 6.6 (Mu). Each
GST possesses a common binding site for GSH and a variable hydrophobic binding
site. The
hydrophobic binding site in each isozyme is specific for particular
electrophilic substrates. Specific
amino acid residues within GSTs have been identified as important for these
binding sites and for
catalytic activity. Residues Q67, T68, D101, E104, and 8131 are important for
the binding of GSH
(Lee, H-C et al. (1995) J. Biol. Chem. 270: 99-109). Residues R13, R20, and
R69 are important for
the catalytic activity of GST (Stenberg G et al. (1991) Biochem. J. 274: 549-
55).
In most cases, GSTs perform the beneficial function of deactivation and
detoxification of
potentially mutagenic and carcinoge~uc chemicals. However, in some cases their
action is detrimental
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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 t'phimurium
used in the well-known Ames test for mutagenicity, has helped to establish the
role of these enzymes
in mutagenesis. Dihalomethanes, which produce liver tumors in mice, are
believed to be activated by
GST. This view is supported by the finding that dihalomethanes are more
mutagenic in bacterial cells
expressing human GST than in untransfected cells (Thier, R. et al. (1993)
Proc. Natl. Acad. Sci. USA
90: 8567-80). The mutagenicity of ethylene dibromide and ethylene dichloride
is increased in
bacterial cells expressing the human Alpha GST, A1-1, while the mutagenicity
of allatoxin 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
mutagencsis and
carcinogenesis.
GST has been implicated in the acquired resistance of many cancers to drug
treatment, the
phenomenon known as multi-drug resistance (MDR). MDR occurs when a cancer
patient is treated
with a cytotoxic drug such as cyclophosphamide and subsequently becomes
resistant to this drug and
to a variety of other cytotoxic agents as well. Increased GST levels are
associated with some of these
drug resistant cancers, and it is believed that this increase occurs in
response to the drug agent which
is then deactivated by the GST catalyzed GSH conjugation reaction. The
increased GST levels then
protect the cancer cells from other cytotoxic agents which bind to GST.
Increased levels of Al-1 in
tumors has been linked to drug resistance induced by cyclophosphamide
treatment (Dirven H.A. et al.
(1994) Cancer Res. 54: 6215-20). Thus control of GST activity in cancerous
tissues may be useful in
treating MDR in cancer patients.
Gamma-~lutamyl transpeptidase
Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes that
initiate extracellular
glutathione (GSH) breakdown by cleaving gamma-glutamyl amide bonds. The
breakdown of GSH
provides cells with a regional cysteine pool for biosynthetic pathways. Gamma-
glutamyl
transpeptidases also contribute to cellular antioxidant defenses and
expression is induced by oxidative
steress. The cell surface-localized glycoproteins.are expressed at high levels
in cancer cells. Studies
have suggested that the high level of gamma-glutamyl transpeptidases activity
present on the surface of
cancer cells could be exploited to activate precursor drugs, resulting in high
local concentrations of anti-
cancer therapeutic agents (Hanigan, M.H. (1998) Chem. Biol. Interact. 111-
112:333-42; Taniguchi, N.
and Ikeda, Y. (1998) Adv. Enzymol. Relat. Areas Mol. Biol. 72:239-78; Chikhi,
N. et al. (1999) Comp.
Biochem. Physiol. B. Biochem. Mol. Biol. 122:367-80).
Acyltransferase
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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 aryl-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 alter partial hepatectomy (Furutani, M. et
al. (1996) Hepatology
24:1441-5).
Acetvltransferases
Acetyltransferases have been extensively studied for their role in histone
acetylation. Histone
acetylation results in the relaxing of the chromatin structure in eukaryotic
cells, allowing transcription
factors to gain access to promoter elements of the DNA templates in the
affected region of the genome
(or the genome in general). In contrast, histone deacetylation results in a
reduction in transcription by
closing the chromatin structure and limiting access of transcription factors.
To this end, a common
means of stimulating cell transcription is the use of chemical agents that
inhibit the deacetylation of
histones (e.g., sodium butyrate), resulting in a global (albeit ardfactual)
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, TFIIE, TFI1F and the
high mobility group
proteins (HMG). In the case of p53, acetylation results in increased DNA
binding, leading to the
stimulation of transcription of genes regulated by p53. The prototypic histone
acetylase (HAT) is GcnS
from Saccharomyces cerevisiae. GcnS is a member of a family of acetylases that
includes Tetrahymena
p55, human GcnS, and human p300/CBP. Histone acetylation is reviewed in
(Cheung, W.L. et al.
(2000) Current Opinion in Cell Biology 12:326-333 and Bergen S.L (1999)
Current Opinion in Cell
Biology 11:336-341). Some acetyltransferase enzymes posses the alpha/beta
hydrolase fold (Center of
Applied Molecular Engineering Inst. of Chenustry and Biochemistry - University
of Salzburg,
http://predict.sanger.ac.uk/irbm-course97/Docs/ms/) common to several other
major classes of
enzymes, including but not limited to, acetylcholinesterases and
carboxylesterases (Structural
Classification of Proteins, http://scop.mrc-lmb.cam.ac.uk/scop/index.htn~l).
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N-acetyltransferase
Aromatic amines and hydrazine-containing compounds are subjeca to N-
acetylation by the N-
acetyltransferase enzymes of liver and other tissues. Some xenobiotics can be
O-acetylated to some
extent by the same enzymes. N-acetyltransferases are cytosolic enzymes which
utilize the cofactor
acetyl-coenzyme A (acetyl-CoA) to transfer the acetyl group in a two step
process. In the first step, the
acetyl group is transferred from acetyl-CoA to an active site cysteine
residue; in the second step, the
acetyl group is transferred to the substrate amino group and the enzyme is
regenerated.
In contrast to most other DME classes, there are a limited number of known N-
acetyltransferases. In humans, there are two highly similar enzymes, NAT1 and
NAT2; nuce appear to
have a third form of the enzyme, NAT3. The human forms of N-acetyltransferase
have independent
regulation (NAT1 is widely-expressed, whereas NAT2 is in liver and gut only)
and overlapping
substrate preferences. Both enzymes appear to accept most substrates to some
extent, but NAT1 does
prefer some substrates (para-aminobenzoic acid, para-aminosalicylic acid,
sulfamethoxazole, and
sulfanilanude), while NAT2 prefers others (isoniazid, hydralazine,
procainamide, dapsone,
aminoglutethimide, and sulfamethazine).
Clinical observations of patients taking the antituberculosis drug isoniazid
in the 1950s led to the
description of fast and slow acetylators of the compound. These phenotypes
were shown subsequently
to be due to mutations in the NAT2 gene which affected enzyme activity or
stability. The slow
isoniazid acetylator phenotype is very prevalent in Middle Eastern populations
(approx. 70%), and is
less prevalent in Caucasian (approx. 50%) and Asian (<25%) populations. More
recently, functional
polymorphism in NAT1 has been detected, with approximately 8% of the
population tested showing a
slow acetylator phenotype (Butcher, N. J. et al. (1998) Pharmacogenetics 8:67-
72). Since NAT1 can
activate some known aromatic amine carcinogens, polymorphism in the widely-
expressed NATl
enzyme may be important in determining cancer risk (OMIM *108345 N-
acetyltransferase 1).
Aminotransferases
Aminotransferases comprise a family of pyridoxal 5'-phosphate (PLP) -dependent
enzymes that
catalyze transformations of amino acids. Aspartate aminotransferase (AspAT) is
the most extensively
studied PLP-containing enzyme. It catalyzes the reversible transamination of
dicarboxylic L-amino
acids, aspartate and glutamate, and the corresponding 2-oxo acids, oxalacetate
and 2-oxoglutarate.
Other members of the fanuly included pyruvate aminotransferase, branched-chain
amino acid
aminotransferase, tyrosine aminotransferase, aromatic aminotransferase,
alanine:glyoxylate
anunotransferase (AGT), and kynurenine anunotransferase (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
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liver-specific peroxisomal enzyme, alanine:glyoxylate anunotransferase-1. The
phenotype of the
disorder is a deficiency in glyoxylate metabolism. In the absence of AGT,
glyoxylate is oxidized to
oxalate rather than being transaminated to glycine. The result is the
deposition of insoluble calcium
oxalate in the kidneys and urinary tract, ultimately causing renal failure
(Lumb, M.J. et al. (1999) J.
Biol. Chem. 274:20587-20596).
Kynurenine aminotransferase catalyzes the irreversible transamination of the L-
tryptophan
metabolite L-kynurenine to form kynurenic acid. The enzyme may also catalyzes
the reversible
transanunation reaction between L-2-anunoadipate 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).
C atechol-O-methvltransferase:
Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group
of S-adenosyl-
L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the catechol
substrate (e.g., L-
dopa, dopamine, or DBA). Methylation of the 3'-hydroxyl group is favored over
methylation of the 4'
hydroxyl group and the membrane bound isoform of COMT is more regiospecific
than the soluble
form. Translation of the soluble form of the enzyme results from utilization
of an internal start colon 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 epinephrinelnorepinephrine, isoprenaline, rimiterol, dobutamine,
fenoldopam, apomorphine,
and a-methyldopa. A deficiency in norepinephrine has been linked to clinical
depression, hence the use
of COMT inhibitors could be usefull in the treatment of depression. COMT
inhibitors are generally
well tolerated with minimal side effects and are ultimately metabolized in the
liver with only minor
accumulation of metabolites in the body (Mannisto, P.T. and Kaakkola, S.
(1999) Pharmacological
Reviews 51:593-628).
Copper-zinc superoxide dismutases
CA 02399873 2002-08-09
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Copper-zinc superoxide dismutases are compact homodimeric metalloenzymes
involved in
cellular defenses against oxidative damage. The enzymes contain one atom of
zinc and one atom of
copper per subunit and catalyze the dismutation of superoxide anions into 02
and H202. The rate of
dismutation is diffusion-linuted 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
fieezing tolerance of
transgenic Alfalfa as well as providing resistance to enviromnental toxins
such as the diphenyl ether
herbicide, acifluorfen (McKersie, B.D. et al. (1993) Plant Physiol. 103:1155-
1163). In adddon, 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~matis, 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. sme~matis,
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
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ester bonds in a phosphodiester compound. Phosphodiesterases are therefore
crucial to a variety of
cellular processes. Phosphodiesterases include DNA and RNA endonucleases and
exonucleases, which
are essential for cell growth and replication, and topoisomerases, which break
and rejoin nucleic acid
strands during topological rearrangement of DNA. A Tyr-DNA phosphodiesterase
functions in DNA
repair by hydrolyzing dead-end covalent intermediates formed between
topoisomerase I and DNA
(Pouliot, J.J. et al. (1999) Science 286:552-555; Yang, S.-W. (1996) Proc.
Natl. Acad. Sci. USA
93:11534-11539).
Acid sphingomyelinase is a phosphodiesterase which hydrolyzes the membrane
phospholipid
sphingomyelin to produce ceramide and phosphorylcholine. Phosphorylcholine is
used in the synthesis
of phosphatidylcholine, which is involved in numerous intracellular signaling
pathways, while ceramide
is an essential precursor for the generation of gangliosides, membrane lipids
found in high concentration
in neural tissue. Defective acid sphingomyelinase leads to a build-up of
sphingomyelin molecules in
lysosomes, resulting in Niemann-Pick disease (Schuclnnan, E.H. and S.R.
Miranda (1997) Genet. Tesl.
1:13-19).
Glycerophosphoryl diester phosphodiesterase (also known as
glycerophosphodiester
phosphodiesterase) is a phosphodiesterase which hydrolyzes deacetylated
phospholipid
glycerophosphodiesters to produce sn-glycerol-3-phosphate and an alcohol.
Glycerophosphocholine,
glycerophosphoethanolamine, glycerophosphoglycerol, and glycerophosphoinositol
are examples of
substrates for glycerophosphoryl diester phosphodiesterases. A
glycerophosphoryl diester
phosphodiesterase from E. coli has broad specificity for glycerophosphodiester
substrates (Larson, T.J.
et al. (1983) J. Biol. Chem. 248:5428-5432).
Cyclic nucleotide phosphodieslerases (PDEs) are crucial enzymes in the
regulation of the cyclic
nucleotides cAMP and cGMP. cAMP and cGMP function as intracellular second
messengers to
transduce a variety of extracellular signals including hormones, light, and
neurotransmitters. PDEs
degrade cyclic nucleotides to their corresponding monophosphates, thereby
regulating the intracellular
concentrations of cyclic nucleotides and their effects on signal transduction.
Due to their roles as
regulators of signal transduction, PDEs have been extensively studied as
chemotherapeutic targets
(ferry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481; Torphy,
J.T. (1998) Am. J.
Resp. Crit. Care Med. 157:351-370).
Families of manunalian 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.-
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L.C. Jin (1999) Prog. Nucleic Acid Res. Mot. Biol. 63:1-38). The existence of
multiple PDE fanulies,
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 (PDE 1 s) 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 PDEl isozymes
decreases the affinity of the enzyme for calmodulin, decreases PDE activity,
and increases steady state
levels of cAMP (Kakkar, supra). PDEls may provide useful therapeutic targets
for disorders of the
central nervous system, and the cardiovascular and inunune systems due to the
involvement of PDE I s
in both cyclic nucleotide and calcium signaling (Perry, M.J. and G.A. Higgs
(1998) Curr. Opin. Chem.
Biol. 2:472-481 ).
PDE2s are cGMP-stimulated PDEs that have been found in the cerebellum,
neocortex, heart,
kidney, lung, pulmonary artery, and skeletal muscle (Sadhu, K. et al. (1999)
J. Histochem. Cytochem.
47:895-906). PDE2s are thought to mediate the effects of CAMP on catecholamine
secretion,
participate in the regulation of aldosterone (Beavo, su ra), and play a role
in olfactory signal
transduction (Juilfs, D.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3388-
3395).
PDE3s have high affinity for both cGMP and CAMP, and so these cyclic
nucleotides act as
competitive substrates for PDE3s. PDE3s play roles in stimulating myocardial
contractility, inhibiting
platelet aggregation, relaxing vascular and airway smooth muscle, inhibiting
proliferation of T-
lymphocytes and cultured vascular smooth muscle cells, and regulating
calec:holanune-induced release
of free fatty acids from adipose tissue. The PDE3 fanuly of phosphodiesterases
are sensitive to specific
inhibitors such as cilostamide, enoximone, and lixazinone. Isozymes of PDE3
can be regulated by
cAMP-dependent protein kinase, or by insulin-dependent kinases (Degerman, E.
et al. (1997) J. Biol.
Chem. 272:6823-6826).
PDE4s are specific for cAMP; are localized to airway smooth muscle, the
vascular endothelium,
and all inflammatory cells; and can be activated by cAMP-dependent
phosphorylation. Since elevation
of cAMP levels can lead to suppression of inflammatory cell activation and to
relaxation of bronchial
smooth muscle, PDE4s have been studied extensively as possible targets for
novel anti-inflanunatory
agents, with special emphasis placed on the discovery of asthma treatments.
PDE4 inhibitors are
currently undergoing clinical trials as treatments for asthma, chronic
obstructive pulmonary disease,
and atopic eczema. All four known isozymes of PDE4 are susceptible to the
inhibitor rolipram, a
compound which has been shown to improve behavioral memory in mice (Barad, M.
et al. (1998) Proc.
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Natl. Acad. Sci. USA 95:15020-15025). PDE4 inhibitors have also been studied
as possible
therapeutic agents against acute lung injury, endotoxenua, rheumatoid
arthritis, multiple sclerosis, and
various neurological and gastrointestinal indications (Doherty, A.M. (1999)
Curr. Opin. Chem. Biol.
3:466-473).
PDES is highly selective for cGMP as a substrate (Turko, LV. et al. (1998)
Biochemistry
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 sildenaf7l
(VIAGRA; Pfizer, Inc., New
York NY), a treatment for male erectile dysfunction (Terrett, N. et al. (1996)
Bioorg. Med. Chem. Lett.
6:1819-1824). Inhibitors of PDES are currently being studied as agents for
cardiovascular therapy
(Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE6s, the photoreceptor cyclic nucleotide phosphodiesterases, are crucial
components of the
phototransduction cascade. In association with the G-protein transducin, PDE6s
hydrolyze eGMP to
regulate cGMP-gated canon channels in photoreceptor membranes. In addition to
the cGMP-binding
active site, PDE6s also have two high-affinity cGMP-binding sites which are
thought to play a
regulatory role in PDE6 function (Artemyev, N.O. et al. (1998) Methods 14:93-
104). Defects in
PDE6s have been associated with retinal disease. Retinal degeneration in the
rd mouse (Yan, W. et al.
(1998) Invest. Opthalmol. Vis. Sci. 39:2529-2536), autosomal recessive
retinitis pigmentosa in humans
(Danciger, M. et al. (1995) Genomics 30:1-7), and rod/cone dysplasia 1 in
Irish Setter dogs (Suber,
M.L. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3968-3972) have been
attributed to mutations in the
PDE6B gene.
The PDE7 family of PDEs consists of only one known member having multiple
splice variants
(Bloom, T.J. and J.A. Beavo (1996) Proc. Natl. Acad. Sci. USA 93:14188-14192).
PDE7s are CAMP
specific, but little else is known about their physiological function.
Although mRNAs encoding PDE7s
are found in skeletal muscle, heart, brain, lung, kidney, and pancreas,
expression of PDE7 proteins is
restricted to specific tissue types (Han, P. et al. (1997) J. Biol. Chem.
272:16152-16157; Perry, M.J.
and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481). PDE7s are very
closely related to the
PDE4 family; however, PDE7s are not inhibited by rolipram, a specific
inhibitor of PDE4s (Beavo,
supra).
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 PDEBs is not inhibited by the PDE inhibitors rolipram,
vinpocetine, milrinone,
IBMX (3-isobutyl-1-methylxanthine), or zaprinast, but PDEBs are inhibited by
dipyridamole (Fisher,
D.A. et al. (1998) Biochem. Biophys. Res. Commun. 246:570-577; Hayashi, M. et
al. (1998) Biochem.
Biophys. Res. Commun. 250:751-756; Soderling, S.H. et al. (1998) Proc. Natl.
Acad. Sci. USA
95:8991-8996).
PDE9s are cGMP specific and most closely resemble the PDE8 fanuly of PDEs.
PDE9s are
expressed in kidney, liver, lung, brain, spleen, and small intestine. PDE9s
are not inhibited by sildenalil
(VIAGRA; Pfizer, Inc., New York NY), rolipram, vinpocetine, dipyridamole, or
IBMX (3-isobutyl-1-
methylxanthine), but they are sensitive to the PDES inhibitor zaprinast
(Fisher, D.A. et al. (1998) J.
Biol. Chem. 273:15559-15564; Soderling, S.H. et al. (1998) J. Biol. Chem.
273:15553-15558).
PDElOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDEI Os are
expressed
in brain, thyroid, and testis. (Soderling, S.H. et al. (1999) Proc. Natl.
Acad. Sci. USA 96:7071-7076;
Fujishige, K. et al. (1999) J. Biol. Chem. 274:18438-18445; Loughney, K. et al
(1999) Gene 234:109-
117).
PDEs are composed of a catalytic domain of about 270-300 anuno acids, an N-
terminal
regulatory domain responsible for binding cofactors, and, in some cases, a
hydrophilic C-terminal
domain of unknown function (Conti, M. and S.-L.C. Jin (1999) Prog. Nucleic
Acid Res. Mol. Biol.
63:1-38). A conserved, putative zinc-binding motif, HDXXHXGXXN, has been
identified in the
catalytic domain of all PDEs. N-terminal regulatory domains include non-
catalytic cGMP-binding
domains in PDE2s, PDESs, and PDE6s; calmodulin-binding domains in PDEls; and
domains
containing phosphorylation sites in PDE3s and PDE4s. In PDES, the N-terminal
cGMP-binding
domain spans about 380 amino acid residues and comprises tandem repeats of the
conserved sequence
motif N(R/K)XnFX3DE (McAllister-Lucas, L.M. et al. (1993) J. Biol. Chem.
268:22863-22873). The
NKXnD motif has been shown by mutagenesis to be important for cGMP binding
(Turko, I. V. 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 catalyeic domain; while across
families, there is little or no
sequence similarity outside this domain.
Many of the constituent functions of immune and inflammatory responses are
inhibited by agents
that increase intracellular levels of cAMP (Verghese, M.W. et al. (1995) Mol.
Pharmacol.
47:1164-1171). A variety of diseases have been attributed to increased PDE
activity and associated
with decreased levels of cyclic nucleotides. For example, a form of diabetes
insipidus in mice has been
associated with increased PDE4 activity, an increase in low-K", CAMP PDE
activity has been reported
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in leukocytes of atopic patients, and PDE3 has been associated with cardiac
disease.
Many inhibitors of PDEs have been identified and have undergone clinical
evaluation (Perry,
M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481; Torphy, T.J.
(1998) Am. J. Respir.
Crit. Care Med. 157:351-370). PDE3 inhibitors are being developed as
antithrombotic agents,
antihypertensive agents, and as cardiotonic agents useful in the treatment of
congestive heart failure.
Rolipram, a PDE4 inhibitor, has been used in the treatment of depression, and
other inhibitors of PDE4
are undergoing evaluation as anti-inflammatory agents. Rolipram has also been
shown to inhibit
lipopolysaccharide (LPS) induced TNF-a which has been shown to enhance HIV-'1
replication in vitro.
Therefore, rolipram may inhibit HIV-1 replication (Angel, J.B. et al. (1995)
AIDS 9:1137-1144).
Additionally, rolipram, based on its ability to suppress the production of
cytokines such as TNF-a and b
and interferon g, has been shown to be effective in the treatment of
encephalomyelitis. Rolipram may
also be effective in treating tardive dyskinesia and was effective in treating
multiple sclerosis in an
experimental animal model (Summer, N. et al. (1995) Nat. Med. 1:244-248;
Sasaki, H. et al. (1995)
Eur. J. Pharmacol. 282:71-76).
Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial
asthma and other
respiratory diseases. Theophylline is believed to act on airway smooth muscle
function and in an
anti-inflammatory or immunomodulatory capacity in the treatment of respiratory
diseases (Banner,
K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000). Pentoxifylline is
another nonspecific PDE
inhibitor used in the treatment of intermittent claudication and diabetes-
induced peripheral vascular
disease. Pentoxifylline is also known to block TNF-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 (Conti et al.
(1995) Endocrine Rev. 16:370-389) and have been implicated in various cancers.
Growth of prostate
carcinoma cell lines DU145 and LNCaP was inhibited by delivery of cAMP
derivatives and PDE
inhibitors (Bang, Y.J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334).
These cells also
showed a permanent conversion in phenotype from epithelial to neuronal
morphology. It has also been
suggested that PDE inhibitors have the potential to regulate mesangial cell
proliferation (Matousovic,
K. et al. (1995) J. Clin. Invest. 96:401-410) and lymphocyte proliferation
(Joulain, C. et al. (1995) J.
Lipid Mediat. Cell Signal. 11:63-79). A cancer treatment has been described
that involves intracellular
delivery of PDEs to particular cellular compartments of tumors, resulting in
cell death (Deonarain,
M.P. and A.A. Epenetos (1994) Br. J. Cancer 70:786-794).
Pho~hotriesterases
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
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and insects and abundant in mammals, explaining the reduced tolerance of birds
and insects to
organophosphorus compound (Vilanova, E. and Sogorb, M.A. (1999) Crit. Rev.
Toxicol. 29:21-57).
Phosphotriesterases play a central role in the detoxification of insecticides
by mammals.
Phosphotriesterase activity varies among individuals and is lower in infants
than adults. Knockout mice
are markedly more sensitive to the organophosphate-based toxins diazoxon and
chlorpyrifos oxon
(Furlong, C.E., et al. (2000) Neurotoxicology 21:91-100). PTEs have attracted
interest as enzymes
capable of the detoxification of organophosphate-containing chemical waste and
warfare 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-
ternunadng step in the de novo biosynthesis of fatty acids. Chain tern>ination
involves the hydrolysis of
the thioester bond which links the fatty acyl chain to the 4'-
phosphopantetheine prosthetic group of the
acyl carrier protein (ACP) subunit of the fatty acid synthase (Smith, S.
(1981a) Methods Enzymol.
7'1:181-188; 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
laity 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 funceion as a chain-terminating
enzyme in fatty acid biosynthesis
(Naggers et al., supra). For that reason, Naggert et al. su ra) proposed that
the physiological
substrates for E. coli TEII may be coenzyme A (CoA)-fatty acid esters instead
of ACP-
phosphopanthetheine-fatty acid esters.
Carboxylesterases
Mammalian carboxylesterases constitute a multigene family expressed in a
variety of tissues and
cell types. lsozymes 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,
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thrombin, Factor IX, gliotactin, and plasnunogen. Carboxylesterases catalyze
the hydrolysis of ester-
and amide- groups from molecules and are involved in detoxification of drugs,
environmental toxins,
and carcinogens. Substrates for carboxylesterases include short- and long-
chain acyl-glycerols,
acylcarnitine, carbonates, dipivefrin hydrochloride, cocaine, salicylates,
capsaicin, palmitoyl-coenzyme
A, inudapril, 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-ternunal 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).
Sctualene epoxidase
Squalene epoxidase (squalene monooxygenase, SE) is a microsomal membrane-
bound, FAD-
dependent oxidoreductase that catalyzes the first oxygenation step in the
sterol biosynthetic pathway of
eukaryotic cells. Cholesterol is an essential structural component of
cytoplasmic membranes acquired
via the LDL receptor-mediated pathway or the biosynthetic pathway. In the
latter case, all 27 carbon
atoms in the cholesterol molecule are derived from acetyl-CoA (Stryer, L.,
supra). SE converts
squalene to 2,3(S)-oxidosqualene, which is then converted to lanosterol and
then cholesterol. The steps
involved in cholesterol biosynthesis are summarized below (Stryer, L (1988)
Biochemistry. W.H
Freeman and Co., Inc. New York. pp. 554-560 and Sakakibara, J. et al. (1995)
270:17-20):
acetate (from Acetyl-CoA) ~ 3-hydoxy-3-methyl-glutaryl CoA ~ mevalonate
5-phosphomevalonate ~ 5-pyrophosphomevalonate ~ isopentenyl pyrophosphate ~
dimethylallyl
pyrophosphate ~ geranyl pyrophosphate ~ farnesyl pyrophosphate
squalene ~ squalene epoxide ~ lanosterol ~ cholesterol
While cholesterol is essential for the viability of eukaryotic cells,
inordinately high serum
cholesterol levels results in the formation of atherosclerotic plaques in the
arteries of higher organisms.
This deposition of highly insoluble lipid material onto the walls of essential
blood vessels (e.g.,
coronary arteries) results in decreased blood flow and potential necrosis of
the tissues deprived of
adequate blood flow. HMG-CoA reductase is responsible for the conversion of 3-
hydroxyl-3-methyl-
glutaryl CoA (HMG-CoA) to mevalonate, which represents the first committed
step in cholesterol
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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 cxcurs 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 sinularity to other members of the a/(3
hydrolase fold family of
enzymes (e.g., bromoperoxidase A2 from Streptomvces aureofaciens,
hydroxymuconic senualdehyde
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-epoxycx;tadec-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 prcx;eeds via a covalently bound ester intermediate initiated by the
nucleophilic attack of one
of the Asp residues on the primary carbon atom of the epoxide ring of the
target molecule, leading to a
covalently bound ester intermediate (Michael Arand, M. et al. (1996) J. Biol.
Chem. 271:4223-4229;
Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657; Argiriadi, M.A. et al.
(2000) J. Biol. Chem.
275:15265-15270).
Enzymes involved in tyrosine catal
The degradation of the anuno acid tyrosine to either succinate and pyruvate or
fumarate and
acetoacetate, requires a large number of enzymes and generates a large number
of intermediate
compounds. 1n addition, many xenobiotic compounds may be metabolized using one
or more reactions
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that are part of the tyrosine catabolic pathway. While the pathway has been
studied primarily in
bacteria, tyrosine degradation is known to occur in a variety of organisms and
is likely to involve many
of the same biological reactions.
The enzymes involved in the degradation of tyrosine to succinate and pyruvate
(e.g., in
Arthrobacter species) include 4-hydroxyphenylpyruvate oxidase, 4-
hydroxyphenylacetate
3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxymethyl-2-
hydroxymuconic
semialdehyde dehydrogenase, traps,cis-5-carboxymethyl-2-hydroxymuconate
isomerase,
homoprotocatechuate isomerase/decarboxylase, cis-2-oxohept-3-ene-1,7-dioate
hydratase,
2,4-dihydroxyhept-traps-2-ene-1,7-dioate aldolase, and succinic semialdehyde
dehydrogenase.
The enzymes involved in the degradation of tyrosine to fumarate and
acetoacetate (e.g., in
Pseuclnntonas species) include 4-hydroxyphenylpyruvate dioxygenase,
homogentisate 1,2-dioxygenase,
maleylacetoacetate isomerase, and fumarylacetoacetase. 4-hydroxyphenylacetate
1-hydroxylase may
also be involved if intermediates from the succinate/pyruvate pathway are
accepted.
Additional enzymes associated with tyrosine metabolism in different organisms
include
4-chlorophenylacetate-3,4-dioxygenase, aromatic aminotransferase,
5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase, 2-oxo-kept-3-ene-1,7-
dioate hydratase, and
5-carboxymethyl-2-hydroxymuconate isomerase (Elks, L.B.M. et al. (1999)
Nucleic Acids Res.
27:373-376; Wackett, L.P. and Ellis, L.B.M. (1996) J. Microbial. Meth. 25:91-
93; and Schmidt, M.
(1996) Amer. Sac. Microbial. News 62:102).
In humans, acquired or inherited genetic defects in enzymes of the tyrosine
degradation
pathway may result in hereditary tyrosinemia. One form of this disease,
hereditary tyrosinemia 1
(HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase,
the last enzyme in the
pathway in organisms that metabolize tyrosine to fumarate and acetoacetate.
HT1 is characterized by
progressive liver damage beginning at infancy, and increased risk for liver
cancer (Endo, F. et al.
(1997) J. Biol. Chem. 272:24426-24432).
The discovery of new drug metabolizing enzymes and the polynucleotides
encoding them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis, prevention,
and treatment of autoimmune/inflammatory, cell proliferative, developmental,
endocrine, eye,
metabolic, and gastrointestinal disorders, including liver disorders, and in
the assessment of the effects
of exogenous compounds on the expression of nucleic acid and amino acid
sequences of drug
metabolizing enzymes.
SUMMARY OF THE INVENTION
The invention features puritied polypeptides, drug metabolizing enzymes,
referred to
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collectively as "DME" and individually as ''DME-1,'' ''DME-2,'' "DME-3," "DME-
4," "DME-5,"
"DME-6," "DME-7," "DME-8," "DME-9," "DME-10,'' "DME-11,'' and "DME-12." In one
aspect,
the invention provides an isolated polypeptide comprising an amino acid
sequence selected from the
group consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID NO:1-12,
b) a naturally occurring amino acid sequence having at least 90% sequence
identity to an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12, c) a
biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID NO:1-12,
and d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ 1D
NO:1-12. 1n one alternative, the invention provides an isolated polypeptide
comprising the amino acid
sequence of SEQ ID NO:1-12.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid
sequence having at least
90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
12, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12. In one alternative, the polynucleotide encodes a
polypeptide selected
from the group consisting of SEQ ID NO:1-12. In another alternative, the
polynucleotide is selected
from the group consisting of SEQ ID N0:13-24.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypepiide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ 1D NO:1-12, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-12. In one alternative, the invention provides a
cell transformed with the
recombinant polynucleotide. In another alternative, the invention provides a
transgenic organism
comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-'12, b) a naturally occurring an uno acid sequence
having at least 90%
sequence identity to an amino acid sequence selected lrom the group consisting
of SEQ ID NO:l-12, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
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NO:1-12, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12. 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 polypeplide, and b)
recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an anuno acid sequence selected from the group
consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12, b) a naturally
occurring amino acid
sequence having at least 90% sequence identity to an anuno acid sequence
selected from the group
consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, and d) an imnmnogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected liom the group consisting of a) a polynucleodde sequence
selected from the group
consisting of SEQ ID N0:13-24, b) a naturally occurring polynucleotide
sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID N0:13-
24, c) a polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary to
b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide
comprises at least 60
contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting
of SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:13-24, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and
e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample
with a probe
comprising at least 20 contiguous nucleotides comprising a sequence
complementary to said target
polynucleotide in the sample, and which probe specifically hybridizes to said
target polynucleotide,
under conditions whereby a hybridization complex is formed between said probe
and said target
polynucleotide or fragments thereof, and b) detecaing the presence or absence
of said hybridization
complex, and optionally, if present, the amount thereof. In one alternative,
the probe comprises at least
60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide sequence
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selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting
of SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:13-24, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and
e) an RNA equivalent of a)-d). The method comprises a) amplifying said target
polynucleotide or
fragment thereof using 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 comprising an amino acid sequence selected from the group
consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID N0:1-12, b) a naturally
occurring an>ino acid
sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12, c) a biologically active liagment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12, and a
pharmaceutically acceptable
excipient. In one embodiment, the composition comprises an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-12. The invention additionally provides a
method of treating a
disease or condition asscx:iated with decreased expression of functional DME,
comprising
administering to a patient in need of such treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-12,
b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-12, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:l-l 2, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-12. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
agonist activity in the sample. In one alternative, the invention provides a
composition comprising an
agonist compound identified by the method and a pharmaceutically acceptable
excipient. In another
alternative, the invention provides a method of treating a disease or
condition associated with
decreased expression of functional DME, comprising administering to a patient
in need of such
treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
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of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
12, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
anuno acid sequence
selected from the group consisting of SEQ ID NO:1-12, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an
immunogenic
fragment of an amino acid sequence selected Iiom the group consisting of SEQ
ID NO:1-12. 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 comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selecaed from the group consisting of SEQ ID NO:1-12, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an anuno acid sequence
selected from the group
consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino
acid sequence selected
fiom the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12. The method
comprises a)
combining the polypeptide with at least one test compound under suitable
conditions, and b)
detecting binding of the polypeptide to the test compound, thereby identifying
a compound that
specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected tiom the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-12,
b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-12, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12, and d) an
immunogenic
fragment of an amino acid sequence selecaed from the group consisting of SEQ
ID NO:1-12. 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.
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The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected Iiom the group consisting of SEQ ID N0:13-24, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide. .
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:13-24, ii) a naturally occurring polynucleotide sequence having at least
90% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-
24, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleodde comprising a polynucleotide sequence
selected from the group
consisting of i) a polynucleotide sequence selected from the group consisting
of SEQ ID NO: '13-24,
ii) a naturally occurring polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24,
iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide
comprises a fragment of
a polynucleotide sequence selected from the group consisting of i)-v) above;
c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the
treated biological sample with the amount of hybridization complex in an
untreated biological
sample, wherein a difference in the amount of hybridization complex in the
treated biological sample
is indicative of toxicity of the test compound.
BRIEF DESCRIPT10N 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 armotation 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
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analysis of the polypeptides.
Table 4 lists the cDNA and 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 understcx~d by one of ordinary skill in the art to which this
invention belongs. Although
any machines, materials, and methods similar or equivalent to those described
herein can be used to
practice or test the present invention, the preferred machines, materials and
methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing the
cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the .
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINIT10NS
"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
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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
polypeplides 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. Conunon 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 DME. Deliberate
amino acid substitutions may be made on the basis of similarity in polarity,
charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues,
as long as the biological
or immunological activity of DME is retained. For example, negatively charged
amino acids may
include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side
chains having similar hydrophilicity values may include: leucine, isoleucine,
and valine; glycine and
alanine; and phenylalanine and tyrosine.
The terms ''amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the anuno 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.
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The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of DME. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of DME either by
directly interacting with DME or by acting on components of the biological
pathway in which DME
participates.
The term "antibody" refers to intact inununoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind DME polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the inununizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
inununize 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 antigenc determinant may compete with the intact antigen
(i.e., the immunogen used
to elicit the inunune response) for binding to an antibody.
The term ''antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or ''minus" can refer to the antisense
strand, and the
designation ''positive" or ''plus" can refer to the sense strand of a
reference DNA molecule.
The term ''biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "inmmnologically
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
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antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A ''composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding DME or fragments of
DME may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry nulk, 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 liagment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
assembled to produce the consensus sequence.
"Conservative anuno acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
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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 anuno 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.
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 ID N0:13-24 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:13-24, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:13-24 is
useful, for
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example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:13-24 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:13-24 and the region of SEQ ID N0:13-24 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO: l-12 is encoded by a fragment of SEQ ID N0:13-24. A
fragment of SEQ 1D NO:1-12 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID NO:1-12. For example, a fragment of SEQ ID NO:l-12 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-
12. The precise length of a fragment of SEQ 1D NO:1-12 and the region of SEQ
ID NO:l-12 to
which the fragment corresponds are routinely determinable by one of ordinary
skill in the art based
on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms ''percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison 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 coreunonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Aligmnent
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
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http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
''BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are conumonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: 10
Word Size: Il
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ 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 Iiagment of at least
20, at least 30, at least 40, at least 50, at least 70, at least 100, or at
least 200 contiguous nucleotides.
Such lengths are exemplary only, and it is understood that any fragment length
supported by the
sequences shown herein, in the tables, llgures, 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.
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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 sinularity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(April-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: Il and Extension Gap: 1 penalties
Gap x drop-off 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
a fragment of at least 15, at least 20, at least 30, at least 40, at least 50,
at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is understood
that any Iiagment length
supported by the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the prcxess by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
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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 deternunable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 ~~n~l sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (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 T", 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°~ 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 pg/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
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.
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"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression of
various factors, e.g., cytokines, chemokines, and other signaling molecules,
which may affect cellular
and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of DME
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of DME which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chenucal compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a muque and defined position on a microarray.
The term "modulate" refers to a change in the activity of DME. For example,
modulation may
cause an increase or a decrease in protein activity, binding characteristics,
or any other biological,
functional, or immunological properties of DME.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleolide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
anuno acid residues ending in lysine. The ternunal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an DME may involve lipidation,
glycosylation,
phosphorylation, acetylation, racen lization, proteolytic cleavage, and other
modifications known in the
art. These prcx;esses may occur synthetically or bicxhemically. Biochemical
modifications will vary
by cell type depending on the enzymatic milieu of DME.
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"Probe" refers to nucleic acid sequences encoding DME, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes. "Primers''
are short nucleic acids, usually DNA oligonucleotides, which may be annealed
to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerise enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
BiolcWV, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; lnnis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead InstitutelMIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useiial, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
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also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, nucroarray 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 cxcurring 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,
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nucleic acids encoding DME, or fragments thereof may comprise a bodily fluid;
an extract from a cell,
chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA,
RNA, or cDNA, in
solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding'' refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic deternunant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope ''A," the presence of a
polypeptide comprising the
epitope A, or the presence of free unlabeled A, in a reaction containing free
labeled A and the antibody
will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or anuno 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 senu-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 plasnud or as part of the host chromosome, as well as
transiently
transformed cells which express the inserted DNA or RNA for limited periods of
lime.
A "transgenic organism," as used herein, is any orgatusm, 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
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art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
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 95% or
at least 98% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), ''splice," "species,'' or "polymorphic''
variant. A splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
liom one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
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THE INVENTION
The invention is based on the discovery of new human drug metabolizing enzymes
(DME), the
polynucleotides encoding DME, and the use of these compositions for the
diagnosis, treatment, or
prevention of autoimmunelintlammatory, cell proliferative, developmental,
endocrine, eye, metabolic,
and gastrointestinal disorders, including liver disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identitication number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide 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
polypeptide sequence number (Incyte Polypeptide ID) 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 polypeplide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column 3
shows the number of amino acid residues in each polypeplide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structurelfunction analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are drug metabolizing
enzymes. For example, SEQ
ID N0:9 is 99% identical, from residue M'1 to residue V512, to human
cytoc;hrome P450 retinoid
metabolizing protein P450RAI-2 (GenBank ID 88515441) as determined by the
Basic Local
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is
0, which indicates
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the probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:9
also contains a cytochrome P450 domain as determined by searching for
statistically significant
matches in the hidden Markov model (HMM)-based PFAM database of conserved
protein family
domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses
provide
further corroborative evidence that SEQ ID N0:9 is a cytochrome P450. SEQ 1D
NO:1, SEQ ID
N0:2, SEQ ID N0:3, SEQ 1D N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID
N0:8,
SEQ ID N0:10, SEQ ID NO:11, and SEQ ID N0:12 were analyzed and annotated in a
similar
manner. The algorithms and parameters for the analysis of SEQ ID NO:1-12 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 genonuc
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 Polynucleot:ide 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 N0:13-24 or that distinguish between SEQ ID
N0:13-24 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and genomic sequences in column 5 relative
to their respective full
length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
45600181 is the
identification number of an Incyte cDNA sequence, and KERANOTO1 is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.8., 70683296V1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs (e.8., 83250572) which contributed to the
assembly of the full
length polynucleotide sequences. Alternatively, the identification numbers in
column 5 may refer to
coding regions predicted by Genscan analysis of genomic DNA. For example,
GNN.g5091644.edit is
the identification number of a Genscan-predicted coding sequence, with
85091644 being the GenBank
identification number of the sequence to which Genscan was applied. The
Genscan-predicted coding
sequences may have been edited prior to assembly. (See Example IV.)
Alternatively, the identification
numbers in column 5 may refer to assemblages of both cDNA and Genscan-
predicted exons brought
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together by an "exon stitching" algorithm. For example, FL7256116_00002
represents a "stitched"
sequence in which 7256116 is the identification number of the cluster of
sequences to which the
algorithm was applied, and 00002 is the number of the prediction generated by
the algorithm. (See
Example V.) Alternatively, the identification numbers in column 5 may refer to
assemblages of both
cDNA and Genscan-predicted exons brought together by an "exon-stretching"
algorithm. (See
Example V.) In some cases, Incyte cDNA coverage redundant with the sequence
coverage shown in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide sequences
which were assembled using Incyte cDNA sequences. The representative cDNA
library is the Incyte
cDNA library which is most frequently represented by the Incyte cDNA sequences
which were used to
assemble and confirm the above polynucleodde 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 anuno acid sequence, and which contains at least one
functional or structural
characteristic of DME.
The invention also encompasses polynucleotides which encode DME. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID NO:I 3-24, which encodes DME. The
polynucleotide sequences
of SEQ ID NO:l 3-24, as presented in the Sequence Listing, embrace the
equivalent RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
DME. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding DME. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID N0:13-
24 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
ID N0:13-24. 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
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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 colon choices. These
combinations are made in
accordance with the standard triplet genetic code as applied to the
polynucleodde sequence of naturally
occurring DME, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode DME and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring DME under
appropriately selecaed
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding DME or its
derivatives possessing a substantially different ccxion usage, e.g., inclusion
of non-naturally occurring
ccxlons. Colons 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
colons 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 chenustry. 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
N0:13-24 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of
DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises 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
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(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
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 Iiagments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-'I 19.) 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
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sequencing may employ flowable polymers for elecarophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, 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 Iiagments and
synthetic
oligonucleolides 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 ccxlon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999)
Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.
14:315-319) to alter or
improve the biological properties of DME, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
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
JO
CA 02399873 2002-08-09
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manner.
In another embodiment, sequences encoding DME may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.)
Alternatively, DME itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis may
be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino
acid sequence of DME, or any part thereof, may be altered during direct
synthesis and/or combined
with sequences from other proteins, or any part thereof, to produce a variant
polypeptide or a
polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, su ra, pp. 28-53.)
Tn 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 transcriplional 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
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elements. These methods include in vitro recombinant DNA techniques, synthetic
techniques, and in
vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel,
F.M. et al. (1995)
Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding DME. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987)
EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992)
McGraw Hill,
New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
USA
81:3655-3659; and Harrington, J.J. et al. ( 1997) Nat. Genet. 15:345-355.)
Expression vectors
derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or
from various bacterial
plasmids, may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell
population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-
356; Yu, M. et al.
(1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985)
Nature
317(6040):813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226;
and Verma, LM.
and N. Somia ( 1997) Nature 389:239-242.) The invention is not limited by the
host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding DME. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding DME can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORTl
plasmid (Life Technologies). Ligation of sequences encoding DME into the
vector's multiple cloning
site disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for in
vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of nested
deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem.
264:5503-5509.) When large quantities of DME are needed, e.g. for the
production of 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.
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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~astoris. 1n addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of DME. Transcription of
sequences encoding
DME may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; 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 Technolo~y (1992) McGraw Hill,
New York NY, pp.
191-196.)
1n 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 EI 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.) 1n 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
vecaor. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
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being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thynudine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et al.
(1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfr
confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), Ii glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the 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 prcxedures known to those of skill in
the art. These procedures
include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and
protein bioassay or immunoassay techniques which include membrane, solution,
or chip based
technologies for the detection and/or quantification of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of DME using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RlAs),
and Iluorescence
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activated cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering epitopes on DME is preferred, but a
competitive binding
assay may be employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. IV; Coligan,
J.E. et al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and
Wiley-Interscience,
New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press,
Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding DME
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding DME, or any fragments thereof, may be cloned into a vector
for the production of
an mRNA probe. Such vectors are known in the art, are commercially available,
and may be used to
synthesize RNA probes in vitro by addition of an appropriate RNA polymerase
such as T7, T3, or
SP6 and labeled nucleotides. These procedures may be conducted using a variety
of commercially
available kits, such as those provided by Amersham Pharmacia Biotech, Promega
(Madison WI), and
US Biochemical. Suitable reporter molecules or labels which may be used for
ease of detection include
radionuclides, enzymes, fluorescent, chenuluminescent, 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 linuted 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
CA 02399873 2002-08-09
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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), calmodulin binding peptide (CBP), 6-
His, FLAG, c-rnyc,
and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of
their cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using conunercially 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, eh. 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 fiagment 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. coli. Cells expressing DME or cell membrane fractions which contain DME are
then contacted
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with a test compound and binding, stimulation, or inhibition of activity of
either DME or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
DME, either in
solution or affixed to a solid support, and detecting the binding of DME to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to
a solid support.
DME of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of DME. Such compounds may include agoW sts,
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 wish 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) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997)
Nucleic Acids Res.
25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell blastocysts
such as those from the C57BL/6 mouse strain. The blastocysts are surgically
transferred to
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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 blastoc:ysts. 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) Biotec:hnol. 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 normal tissues such as rib bone, brain, hippocampus,
bronchial, testicular,
breast, lymph node, lung, and ovarian tissues, and diseased tissues such as
brain tumor, ovarian
tumor, lung tumor, breast tumor, asthmatic lung, and diseased breast tissues.
Therefore, DME
appears to play a role in autoimmune/inflammatory, cell proliferative,
developmental, endocrine,
eye, metabolic, and gastrointestinal disorders, including liver disorders. In
the treatment of disorders
associated with increased DME expression or activity, it is desirable to
decrease the expression or
activity of DME. In the treatment of disorders associated with decreased DME
expression or
activity, it is desirable to increase the expression or activity of DME.
Therefore, in one embodiment, DME or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of DME. Examples of such disorders include, but are not limited to,
an
autoimmune/inflammatory disorder, such as acquired immunodeliciency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
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bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis
fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout,
Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma, Sjogren's
syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminlhic infections, and trauma; a cell proliferative disorder, such as
actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed
connective tissue disease
(MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,
psoriasis,
primary thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal
gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal
tract, heart, kidney, liver,
lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands,
skin, spleen, testis,
thymus, thyroid, and uterus; a developmental disorder, such as renal tubular
acidosis, anemia,
Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular
dystrophy,
epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia,
genitourinary.
abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic syndrome,
hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary
neuropathies such as
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure
disorders such as Syndenham's chorea and cerebral palsy, spina bifida,
anencephaly,
craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing
loss; an endocrine
disorder, such as disorders of the hypothalamus and pituitary resulting from
lesions such as primary
brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy,
aneurysms,
vascular malformations, thrombosis, infections, immunological disorders, and
complications due to
head trauma; disorders associated with hypopituitarism including hypogonadism,
Sheehan syndrome,
diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease,
Letterer-Siwe disease,
sarcoidosis, empty sella syndrome, and dwarfism; disorders associated with
hyperpituitarism
including acromegaly, giantism, and syndrome of inappropriate antidiuretic
hormone (ADH)
secretion (SIADH) often caused by benign adenoma; disorders associated with
hypothyroidism
including goiter, myxedema, acute thyroiditis associated with bacterial
infection, subacute thyroiditis
associated with viral infection, autoimmune thyroiditis (Hashimoto's disease),
and cretinism;
disorders associated with hyperthyroidism including thyrotoxicosis and its
various forms, Grave's
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disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
Plummer's disease;
disorders associated with hyperparathyroidism including Conn disease (chronic
hypercalemia);
pancreatic disorders such as Type I or Type II diabetes mellitus and
associated complications;
disorders associated with the adrenals such as hyperplasia, carcinoma, or
adenoma of the adrenal
cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia,
Cushing's disease,
Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors,
and Addison's
disease; disorders associated with gonadal steroid hormones such as: in women,
abnormal prolactin
production, infertility, endometriosis, perturbations of the menstrual cycle,
polycystic ovarian
disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea,
hermaphroditism, hirsutism and virilization, breast cancer, and, in post-
menopausal women,
osteoporosis; and, in men, Leydig cell deficiency, male climacteric phase, and
germinal cell aplasia,
hypergonadal disorders associated with Leydig cell tumors, androgen resistance
associated with
absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia; an
eye disorder, such
as conjunctivitis, keratoconjunctivitis sicca, keratitis, episclerids, iritis,
posterior uveitis, glaucoma,
amaurosis fugax, ischemic optic neuropathy, optic neuritis, Lcber'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,
hyperparalhyroidism, hypoparathyroidism, hypercholesterolemia,
hyperthyroidism, hypoglycemia,
hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies,
lipodystrophies, lysosomal storage
diseases, Menkes syndrome, occipital horn syndrome, mannosidosis,
neuraminidase deficiency,
obesity, pentosuria phenylketonuria, pseudovitamin D-deficiency rickets;
hypocalcemia,
hypophosphatemia, and postpubescent cerebellar ataxia, tyrosinemia, and a
gastrointestinal disorder,
such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture,
esophageal
carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis,
gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, hereditary
hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma,
infectious colitis, ulcerative
colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-
Weiss syndrome, colonic
carcinoma, colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea,
constipation, gastrointestinal hemorrhage, acquired immunodeiiciency syndrome
(AIDS)
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enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic
steatosis,
hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye's
syndrome, primary
sclerosing cholangitis, liver infarction, portal vein obstruction and
thrombosis, centrilobular necrosis,
peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease,
preeclampsia, eclampsia, acute
fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic
tumors including nodular
hyperplasias, adenomas, and carcinomas.
In another embodiment, a vector capable of expressing DME or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of DME including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified DME
in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of DME including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of DME
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of DME including, but not limited to, those listed above.
In a further embodiment, an antagonist of DME may be administered to a subject
to treat or
prevent a disorder associated with increased expression or activity of DME.
Examples of such
disorders include, but are not limited to, those autoimmune/inilammatory, cell
proliferative,
developmental, endocrine, eye, metabolic, and gastrointestinal disorders,
including liver disorders
described above. In one aspect, an antibody which specifically binds DME may
be used directly as an
antagonist or indirectly as a targeting or delivery mechanism for bringing a
pharmaceutical agent to
cells or tissues which express DME.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding DME may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of DME including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be adnunistered 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 inununized by injection with DME or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase irmnunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to DME
have an amino acid sequence consisting of at least about 5 anuno acids, and
generally will consist of at
least about 10 amino acids. It is also preferable that these oligopeptides,
peptides, or fragments are
identical to a portion of the amino acid sequence of the natural protein.
Short stretches of DME amino
acids may be fused with those of another protein, such as KLH, and antibodies
to the chimeric
molecule may be produced.
Monoclonal antibodies to DME may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Prcx;. Natl. Acad. Sci.
USA 80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
DME-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Nail. Acad. Sci. USA 88:10134-10137.)
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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 may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D. et
al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
DME and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering DME epitopes is generally used, but a competitive binding
assay may also be employed
(Pound, 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 liee 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 affuuty, or avidity, of the
antibodies for DME. The l~
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 I~ ranging
from about 10~ to 10'2 L/mole are preferred for use in immunoassays in which
the DME-antibody
complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka ranging
from about 106 to 10' L/mole are preferred for use in immunopurilication and
similar procedures
which ultimately require dissociation of DME, preferably in active form, from
the antibody (Catty, D.
(1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC;
Liddell, J.E. and A.
Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably
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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,
supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding DME, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modiFcations 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. Allergy Cli. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of
viral vectors, such as retrovirus and adeno-associated virus vectors. (See,
e.g., Miller, A.D. (1990)
Blood 76:271; Ausubel, su ra; 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 gern~line gene therapy. Gene therapy may be performed to (f)
correct a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeliciency 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),
thalassanuas,
fanulial hypercholesterolemia, and hemophilia resulting from Factor V11I or
Factor 1X deficiencies
(Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997)
Nature 389:239-242)),
(ii) express a conditionally lethal gene product (e.g., in the case of cancers
which result from
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unregulated cell proliferation), or (iii) express a protein which affords
protection against intracellular
parasites (e.g., against human retroviruses, such as human immunodeficiency
virus (HIV) (Baltimore,
D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad.
Sci. USA. 93:11395-
11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida
albicans and
Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium
falciparum and
Trypanosoma cruzi). In the case where a genetic deficiency in DME expression
or regulation causes
disease, the expression of DME from an appropriate population of transduced
cells may alleviate the
clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in DME
are treated by constructing mammalian expression vectors encoding DME and
introducing these
vectors by mechanical means into DME-deficient cells. Mechanical transfer
technologies for use with
cells in vivo or ex vitro include (i) direca DNA nucroinjection 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;
Ivies, 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
linuted to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-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), conunercially available in the T-
REX plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible promoter
(Rossi, F.M.V. and Blau, H.M. supra)), or (iii) a tissue-specific promoter or
the native promoter of the
endogenous gene encoding DME from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require nunimal 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.
CA 02399873 2002-08-09
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(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
respeca 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. Viral. 62:3802-3806; Dull, T. et al. (1998) J. Viral.
72:8463-8471; Zufferey, R.
et al. (1998) J. Viral. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtainng 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. Viral.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Viral. 71:4707-4716;
Ranga, U. et al.
(1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding DME to cells which have one or more genetic
abnormalities with respect to
the expression of DME. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544 and Verma, LM. and N. Son ua (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
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respect to the expression of DME. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing DME to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent 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,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based on
the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
DME into the alphavirus
genome in place of the capsid-coding region results in the production of a
large number of DME-
coding RNAs and the synthesis of high levels of DME in vector transduced
cells. While alphavirus
infection is typically. associated with cell lysis within a few days, the
ability to establish a persistent
infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis
virus (SIN) indicates that
the lytic replication of alphaviruses can be altered to suit the needs of the
gene therapy application
(Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of
alphaviruses will allow the
introduction of DME into a variety of cell types. The specific transduction of
a subset of cells in a
population may require the sorting of cells prior to transduction. The methods
of manipulating
infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and
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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 polymerises,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding DME.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chenucal synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding DME. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerise 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
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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 sinularly 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 polynucleo
tide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased DME
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding DME may be therapeutically useful, and in the treament of disorders
associated with
decreased DME expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding DME may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective
in altering polynucleotide expression; selection from an existing,
commercially-available or
proprietary library of naturally-occurring or non-natural chemical compounds;
rational design of a
compound based on chemical and/or structural properties of the target
polynucleotide; and selection
from a library of chemical compounds created combinatorially or randomly. A
sample comprising a
polynucleotide encoding DME is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
DME are assayed by
any method commonly known in the art. Typically, the expression of a specific
nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding DME. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
polynucleolide. A screen for a compound effective in altering expression of a
specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
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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 info 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.
B iotechnol. 15 :462-466. )
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin~ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of DME,
antibodies to DME, and mimetics, agonists, antagonists, or inhibitors of DME.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathec;al, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal, enteral,
topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination of
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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
adn unistration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example DME
or fragments thereof, antibodies of DME, and agonists, antagonists or
inhibitors of DME, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDso (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained Iiom cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or biweekly
depending on the half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 ~cg, up to a
total dose of
about 1 gram, depending upon the route of adn>inistration. Guidance as to
particular dosages and
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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. Sinularly, 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 FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
DME expression. Normal or
standard values for DME expression are established by combining body fluids or
cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibodies
to DME under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of DME
expressed in subject,
control, and disease samples from biopsied tissues are compared with the
standard values. Deviation
between standard and subject values establishes the parameters for diagnosing
disease.
In another embodiment of the invention, the polynucleotides encoding DME may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect and
quantify gene expression in biopsied tissues in which expression of DME may be
correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
DME, and to monitor regulation of DME levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding DME or closely related
molecules may be used to
identify nucleic acid sequences which encode DME. The specificity of the
probe, whether it is made
from a highly specific region, e.g., the 5'regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding DME, allelic
variants, or related
sequences.
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Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the DME encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:13-24 or from
genomic sequences including promoters, enhancers, and introns of the DME gene.
Means for producing specific hybridization probes for DNAs encoding DME
include the
cloning of polynucleotide sequences encoding DME or DME derivatives into
vecaors for the production
of mRNA probes. Such vectors are known in the art, are conunercially
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 ~ZP or ASS, 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/inllammatory disorder, such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, alherosclerosis, autoimmune hemolytic anemia,
auloimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblaslosis
fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout,
Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma, Sjogrcn's
syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections, and trauma; a cell proliferative disorder, such as
actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed
connective tissue disease
(MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythenlia
vera, psoriasis,
primary thrombocythemia, and cancers including adcnocarcinoma, 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,
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Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular
dystrophy,
epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia,
genitourinary
abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic syndrome,
hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary
neuropathies such as
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure
disorders such as Syndenham's chorea and cerebral palsy, spina bilida,
anencephaly,
craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing
loss; an endocrine
disorder, such as disorders of the hypothalamus and pituitary resulting from
lesions such as primary
brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy,
aneurysms,
vascular malformations, thrombosis, infections, immunological disorders, and
complications due to
head trauma; disorders associated with hypopituitarism including hypogonadism,
Sheehan syndrome,
diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease,
Letterer-Siwe disease,
sarcoidosis, empty sella syndrome, and dwarfism; disorders associated with
hyperpituitarism
including acromegaly, giantism, and syndrome of inappropriate antidiuretic
hormone (ADH)
secretion (SIADH) often caused by benign adenoma; disorders associated with
hypothyroidism
including goiter, myxedema, acute thyroiditis associated with bacterial
infection, subacute thyroiditis
associated with viral infection, autoimmune thyroiditis (Hashimoto's disease),
and cretinism;
disorders associated with hyperthyroidism including thyrotoxicosis and its
various forms, Grave's
disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
Plummer's disease;
disorders associated with hyperparathyroidism including Conn disease (chronic
hypercalemia);
pancreatic disorders such as Type I or Type II diabetes mellitus and
associated complications;
disorders associated with the adrenals such as hyperplasia, carcinoma, or
adenoma of the adrenal
cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia,
Cushing's disease,
Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors,
and Addison's
disease; disorders associated with gonadal steroid hormones such as: in women,
abnormal prolactin
production, infertility, endometriosis, perturbations of the menstrual cycle,
polycystic ovarian
disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea,
hermaphroditism, hirsutism and virilization, breast cancer, and, in post-
menopausal women,
osteoporosis; and, in men, Leydig cell deficiency, male climacteric phase, and
germinal cell aplasia,
hypergonadal disorders associated with Leydig cell tumors, androgen resistance
associated with
absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia; an
eye disorder, such
as conjunctivitis, keratoconjunctivitis sicca, keratitis, episcleritis,
iritis, posterior uveitis, glaucoma,
amaurosis fugax, ischemic optic neuropathy, optic neuritis, Leber's hereditary
optic neuropathy,
toxic optic neuropathy, vitreous detachment, retinal detachment, cataract,
macular degeneration,
central serous chorioretinopalhy, retintis pigmentosa, melanoma of the
choroid, retrobulbar tumor,
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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;
hypocalcenua,
hypophosphatemia, and postpubescent cerebellar ataxia, tyrosinemia, and a
gastrointestinal disorder,
such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture,
esophageal
carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis,
gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, hereditary
hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma,
infectious colitis, ulcerative
colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-
Weiss syndrome, colonic
carcinoma, colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea,
constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome
(AIDS)
enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic
steatosis,
hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency, Reye's
syndrome, primary
sclerosing cholangitis, liver infarction, portal vein obstruction and
thrombosis, centrilobular necrosis,
peliosis hepatic, hepatic vein thrombosis, veno-occlusive disease,
preeclampsia, eclampsia, acute
fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic
tumors including nodular
hyperplasias, adenomas, and carcinomas. The polynucleotide sequences encoding
DME may be used
in Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR
technologies; in dipstick, pin, and multiformat ELISA-like assays; and in
nucroarrays utilizing tluids
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
CA 02399873 2002-08-09
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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.
1n 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 Irom 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 fiom 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
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding DME
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding DME, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
DME, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding DME may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic disease
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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
polymerise chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the
secondary and tertiary structures of PCR products in single-stranded form, and
these differences are
detectable using gel electrophoresis in non-denaturing gels. In fSCCP,
the~oligonucleotide primers are
lluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silica 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. Inmmnol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Bicxhem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
calorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to deternune 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 pharmacogenonuc 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
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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
polynucleoddes of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share those
toxic properties. These fingerprints or signatures are most useful and refined
when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is not
altered by any tested compounds are important as well, as the levels of
expression of these genes are
used to normalize the rest of the expression data. The normalization procedure
is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
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http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared
with levels in an untreated biological sample. Differences in the transcript
levels between the two
samples are indicative of a toxic response caused by the test compound in the
treated sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating and
analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel elecarophoresis in the second dimension (Steiner and
Anderson, supra). The proteins
are visualized in the gel as discrete and muquely 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 chenucal or enzymatic cleavage followed by
mass specarometry.
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 proteonuc 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.
Bioc;hem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Deter ion may be
performed by a variety of
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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 proteonlic profiling may be more reliable and
informative in such
. cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological sample.
A difference in the amount of protein between the two samples is indicative of
a toxic response to the
test compound in the treated sample. Individual proteins are identified by
sequencing the anuno acid
residues of the individual proteins and comparing these partial sequences to
the polypeptides of the
present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D.
et al. (1995) PCT application W095/35505; Holler, R.A. et al. (1997) Proc.
Natl. Acad. Sci. USA
94:2150-2155; and Holler, 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
generate hybridization probes useful in mapping the naturally occurring
genonuc sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
CA 02399873 2002-08-09
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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 Pl
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, for
example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA
83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding DME on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to l 1q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
(See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant
invention may also be used to detect differences in the chromosomal location
due to translocation,
inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, DME, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be flee 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
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having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) 1n 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.
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 preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the
remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/181,856, U.S. Ser. No. 60/183,684 , U.S. Ser. No.
60/185,141, U.S. Ser.
No. 60/186,818, U.S. Ser. No. 60/188,345 , and U.S. Ser. No. 60/189,997 are
hereby expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothicxyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TR1ZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repealed as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
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using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (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
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
elecarophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasnuds obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasnlid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
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 plasnud 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
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Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the AB1 CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Bobbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such
as the ABI PRISM BIGDYE Ternunator cycle sequencing ready reaction kit
(Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the eDNA sequences were selected for extension
using the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from lncyte cDNAs were validated by
removing vector,
linker, and poly(A) sequences and by masking ambiguous bases, using algorithms
and programs based
on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The
lncyte cDNA
sequences or translations thereof were then queried against a selection of
public databases such as the
GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and
BLOCKS, PRINTS,
DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases
such as
PFAM. (HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-
365.) The queries
were performed using programs ,based on BLAST, FASTA, BLIMPS, and HMMER. The
Incyte
cDNA sequences were assembled to prcxluce full length polynucleotide
sequences. Alternatively,
GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or
Genscan-predicted
coding sequences (see Examples IV and V) were used to extend Incyte cDNA
assemblages to full
length. Assembly was performed using programs based on Phred, Phrap, and
Consed, and cDNA
assemblages were screened for open reading frames using programs based on
GeneMark, BLAST, and
FASTA. The full length polynucleotide sequences were translated to derive the
corresponding full
length polypeptide sequences. Alternatively, a polypeptide of the invention
may begin at any of the
methionine residues of the full length translated polypeptide. Full length
polypeptide sequences were
subsequently analyzed by querying against databases such as the GenBank
protein databases
(genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov
model
(HMM)-based protein family databases such as PFAM. Full length polynucleotide
sequences are also
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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 sununarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ 1D
N0:13-24. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative drug metabolizing enzymes were initially identified by running the
Genscan gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan is
a general-purpose gene identification program which analyzes genomic DNA
sequences from a variety
of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and
Burge, C. and S.
Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted exons to
form an assembled cDNA sequence extending from a methionine to a stop codon.
The output of
Genscan is a FASTA database of polynucleotide and polypeptide sequences. The
maximum range of
sequence for Genscan to analyze at once was set to 30 kb. To determine which
of these Genscan
predicted cDNA sequences encode drug metabolizing enzymes, the encoded
polypeptides were
analyzed by querying against PFAM models for drug metabolizing enzymes.
Potential drug
metabolizing enzymes were also identified by homology to Incyte cDNA sequences
that had been
annotated as drug metabolizing enzymes. These selected Genscan-predicted
sequences were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or
c.>mitted exons. BLAST analysis
was also used to find any lncyte cDNA or public cDNA coverage of the Genscan-
predicted sequences,
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thus providing evidence for transcription. When Incyte cDNA coverage was
available, this
information was used to correct or confirm the Genscan predicted sequence.
Full length polynucleotide
sequences were obtained by assembling Genscan-predicted coding sequences with
lncyte cDNA
sequences and/or public cDNA sequences using the assembly process described in
Example III.
Alternatively, full length polynucleotide sequences were derived entirely from
edited or unedited
Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
I1I 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 genonuc
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 genonuc sequence) were given preference over linkages
which change parent type
(eDNA 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" Seguences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in Example
IV. A chimeric protein was generated by using the resultant high-scoring
segment pairs (HSPs) to
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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 genonlic
sequences from the public human genome databases. Partial DNA sequences were
therefore
"stretched" or extended by the addition of homologous genomic sequences. The
resultant stretched
sequences were examined to determine whether it contained a complete gene.
VI. Chromosomal Mapping of DME Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:13-24 were compared with
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 N0:13-24 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and GenEthon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, or human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, yupra,
eh. 7; Ausubel (1995)
5-upra, eh. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (lncyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
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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
x nunimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of sinularity between two
sequences and the
length of the sequence match. 'The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps).
If there is more than one HSP, then the pair with the highest BLAST score is
used to calculate the
product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap al 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 III). Each
cDNA sequence is
derived from a eDNA library constructed from a human tissue. Each human tissue
is classified into
one of the following organ/tissue categories: cardiovascular system;
connective tissue; digestive
system; embryonic structures; endocrine system; exocrine glands; genitalia,
female; genitalia, male;
germ cells; hemic and inunune 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 diseaseJcondition
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 (lncyte Genomics, Palo Alto CA).
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VIII. Extension of DME Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the Lull length molecule using oligonucleodde primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known liagment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target sequence
at temperatures of about 68°C to about 72°C. Any stretch of
nucleotides which would result in
hairpin structures and primer-primer dimerizations was avoided.
Selecaed 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,
lnc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)2S04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 nun; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
P1COGREEN
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 lluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~.cl to 10 ~cl aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotec;h). 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
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were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotec;h), 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 colonel were picked and cultured overnight at
37 °C in 384-well
plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA pc.>lymerase (Stratagene) with the
following parameters:
Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C,
1 nun; Step 4: 72°C, 2 nun; Step 5: steps 2,
3, and 4 repeated 29 times; Step 6: 72°C, 5 nun; Step 7: storage at
4°C. DNA was quantified by
PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries
were reamplilied using the same conditions as described above. Samples were
diluted with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing primers and
the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE
Ternunator 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 1D N0:13-24 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 prcxedure is used with
larger nucleotide fragments.
Oligonucleotides are designed using state-oI'-the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of [y-
32P~ adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleolide 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 I1,
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
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compared.
X. Microarrays
The linkage or synthesis of array elements upon a nlicroarray can be achieved
utilizing
photolithography, piezoelectric printing (ink-jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively,
a procedure analogous to a dot or slot blot may also be used to arrange and
link elements to the surface
of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
A typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M, et al.
(1995) Science
270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
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
complementarily and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described in
detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/NI RNase inhibitor, 500 pM dATP, 500 NM dGTP,
500 EiNI dTTP, 40
NM dCTP, 40 pM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro
transcription from non-coding yeast genomic DNA. After incubation at
37° C for 2 hr, each reaction
sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of
O.SM sodium
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hydroxide and incubated for 20 minutes at 85° C to the stop the
reaction and degrade the RNA.
Samples are purified using two successive CHROMA SPIN 30 gel filtration spin
columns
(CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) and after combining,
both reaction
samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml
sodium acetate, and 300
. ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC
(Savant Instruments
Inc., Holbrook NY) and resuspended in 14 E~l SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
Iinal quantity greater than 5
pg. 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 ~l of the array
element DNA, at an
average concentration of 100 ng/Nl, is loaded into the open capillary printing
element by a high-
speed robotic apparatus. The apparatus then deposits about 5 n1 of array
element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°C
followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 Nl of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 nunutes 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
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incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45 ° C in a first wash
buffer (1 X SSC, 0.1 % SDS), three times for 10 minutes each at 45 ° C
in a second wash buffer (0.1X
SSC), and dried.
Detection
S Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral
lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light
is focused on the array using a 20X microscope objective (Nikon, Inc.,
Melville NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores.
Appropriate filters positioned between the array and the photomultiplier tubes
are used to filter the
signals. The enussion 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 Iiom
both Iluorophores
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
Iluorophores are excited and
measured simultaneously, the data are Iirst corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emissic.>n
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
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spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the DME-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring DME. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of DME. To
inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5' sequence and
used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is
designed to prevent ribosomal binding to the DME-encoding transcript.
XI1. Expression of DME
Expression and purification of DME is achieved using bacterial or virus-based
expression
systems. For expression of DME in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the 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
(TPTG). Expression of DME in eukaryotic cells is achieved by infecting insect
or mammalian cell
lines with recombinant Autogr~hica 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 infectivily is maintained and the strong
polyhedrin promoter
drives high levels of cDNA transcription. Recombinant baculovirus is used to
infect Spodoptera
fru~iperda (Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter
requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945.)
In most expression systems, DME is synthesized as a fusion protein with, e.g.,
glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma japonicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
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Biotech). Following purification, the GST moiety can be proteolytically
cleaved from DME at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, supra,
eh. 10 and 16). Purified DME obtained by these methods can be used directly in
the assays shown in
Examples XVI, XVII, and XVIII where applicable.
XIII. Functional Assays
DME function is assessed by expressing the sequences encoding DME at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives 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 ~.g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporaeion. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfec;ted cells liom 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;
Clontec;h), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify lransfected cells expressing GFP or CD64-
GFP and to evaluate the
apoptotic state of the cells and other cellular properties. FCM detects and
quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
iluorescein-conjugated Annexin V protein to the cell surface. Methods in Ilow
cytometry are discussed
in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of DME on gene expression can be assessed using highly purified
populations of
cells transfected with sequences encoding DME and either CD64 or CD64-GFP.
CD64 and CD64-
GFP are expressed on the surface of transfected cells and bind to conserved
regions of human
inununoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfec;ted cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success NY).
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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, eh. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 43'I A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KL,H (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-DME activity by, for example, binding the peptide or DME
to a substrate,
blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XV. Purification of Naturally Occurring DME Using Specific Antibodies
Naturally cxcurring 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 thicx:yanate
ion), and DME is collected.
XV1. Identification of Molecules Which Interact with DME
DME, or biologically active Iragments thereof, are labeled wilh'25I Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Bicx;hem. J. 133:529-539.)
Candidate molecules
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previously arrayed in the wells of a multi-well plate are incubated with the
labeled DME, washed, and
any wells with labeled DME complex are assayed. Data obtained using different
concentrations of
DME are used to calculate values for the number, affinity, and association of
DME with the candidate
molecules.
Alternatively, molecules interacting with DME are analyzed using the yeast two-
hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
DME may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
detern line all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6,057,101 ).
XVII. Demonstration of DME Activity
Cytochrome P450 activity of DME is measured using the 4-hydroxylation of
aniline. Aniline
is converted to 4-aminophenol by the enzyme, and has an absorption maximum at
630 nm (Gibson
and Skett, supra). This assay is a convenient measure, but underestimates the
total hydroxylation,
which also occurs at the 2- and 3- positions. Assays are performed at 37
°C and contain an aliquot of
the enzyme and a suitable amount of aniline (approximately 2 mM) in reaction
buffer. For this
reaction, the buffer must contain NADPH or an NADPH-generating cofactor
system. One
formulation for this reaction buffer includes 85 mM Tris pH 7.4, 15 mM MgCl2,
50 mM
nicotinamide, 40 mg trisodium isocitrate, and 2 units isocitrate
dehydrogenase, with 8 mg NADP+
added to a 10 mL reaction buffer stock just prior to assay. Reactions are
carried out in an optical
cuvette, and the absorbance at 630 nm is measured. The rate of increase in
absorbance is proportional
to the enzyme activity in the assay. A standard curve can be constructed using
known concentrations
of 4-aminophenol.
1a,25-dihydroxyvitamin D 24-hydroxylase activity of ABBR is determined by
monitoring the
conversion of 3H-labeled 1a,25-dihydroxyvitamin D (1a,25(OH)2D) to 24,25-
dihydroxyvitanun D
(24,25(OH)ZD) in transgenic rats expressing ABBR. 1 ~g of 1a,25(OH)ZD
dissolved in ethanol (or
ethanol alone as a control) is administered intravenously to approximately 6-
week-old male transgenic
rats expressing ABBR or otherwise identical control rats expressing either a
defective variant of
ABBR or not expressing ABBR. The rats are killed by decapitation after 8 hrs,
and the kidneys are
rapidly removed, rinsed, and homogenized in 9 volumes of ice-cold buffer (15
mM Tris-acetate (pH
7.4), 0.19 M sucrose, 2 mM magnesium acetate, and 5 mM sodium succinate). A
portion (e.g., 3 ml)
of each homogenate is then incubated with 0.25 nM 1a,25(OH)z[1 ~H]D, with a
specific activity of
approximately 3.5 GBq/nunol, for 15 nun at 37 °C under oxygen with
constant shaking. Total lipids
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are extracted as described (Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem.
Physiol. 37: 911-917)
and the chloroform phase is analyzed by HPLC using a FINEPAK SIL column
(JASCO, Tokyo,
Japan) with a n-hexane/chloroform/methanol (10:2.5:1.5) solvent system at a
flow rate of 1 ml/min. In
the alternative, the chloroform phase is analyzed by reverse phase HPLC using
a J SPHERE ODS-AM
column (YMC Co. Ltd., Kyoto, Japan) with an acetonitrile buffer system (40 to
100%, in water, in 30
min) at a flow rate of 1 ml/min. The eluates are collected in fractions of 30
seconds (or less) and the
amount of'H present in each fraction is measured using a scintillation
counter. By comparing the
chromatograms of control samples (i.e., samples comprising 1a,25-
dihydroxyvitamin D or
24,25-dihydroxyvitamin D (24,25(OH)ZD), with the chromatograms of the reaction
products, the
relative mobilities of the substrate (1a,25(OH)2[1-3H]D) and product
(24,25(0H)2[1 3H]D) are
determined and correlated with the fractions collected. The amount of
24,25(OH)2[1-3H]D produced in
control rats is subtracted from that of transgenic rats expressing ABBR. The
difference in the
production of 24,25(OH)2[1-3H]D in the transgenic and control animals is
proportional to the amount
of 25-hydrolase activity of ABBR 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 foniiation by HPLC.
Alternatively, activity is measured by monitoring oxygen uptake using a Clark-
type electrode. For
example, Ziegler, D. M. and Poulsen, L. L. (1978; Methods Enzymol. 52:142-151)
incubated the
enzyme at 37°C in an NADPH-generating cofactor system (similar to the
one described above)
containing the substrate methimazole. The rate of oxygen uptake is
proportional to enzyme activity.
UDP glucuronyltransferase activity of DME is measured using a colorimetric
determination
of free amine groups (Gibson and Skett, supra). An amine-containing substrate,
such as 2-
aminophenol, is incubated at 37 °C with an aliquot of the enzyme in a
reaction buffer containing the
necessary cofactors (40 mM Tris pH 8.0, 7.5 mM MgCl2, 0.025% Triton X-100, 1
mM ascorbic acid,
0.75 mM UDP-glucuronic acid). After sufficient time, the reaction is stopped
by addition of ice-cold
20% trichloroacetic acid in 0.1 M phosphate buffer pH 2.7, incubated on ice,
and centrifuged to
clarify the supernatant. Any unreacted 2-aminophenol is destroyed in this
step. Sufficient freshly-
prepared sodium nitrite is then added; this step allows formation of the
diazonium salt of the
glucuronidated product. Excess nitrite is removed by addition of sufficient
ammonium sulfamate,
and the diazoaum salt is reacted with an aromatic amine (for example, N-
naphthylethylene diamine)
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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-anunophenol glucuronide.
Glutathione S-transferase activity of DME is measured using a model substrate,
such as 2,4-
dinitro-1-chlorobenzene, which reacts with glutathione to form a product, 2,4-
dinitrophenyl-
glutathione, that has an absorbance maximum at 340 nm. It is important to note
that GSTs have
differing substrate specificities, and the model substrate should be selected
based on the substrate
preferences of the GST of interest. Assays are performed at ambient
temperature and contain an
aliquot of the enzyme in a suitable reaction buffer (for example, 1 mM
glutathione, 1 mM
dinitrochlorobenzene, 90 mM potassium phosphate buffer pH 6.5). Reactions are
carried out in an
optical cuvette, and the absorbance at 340 nm is measured. The rate of
increase in absorbance is
proportional to the enzyme activity in the assay.
N-acyltransferase activity of DME is measured using radiolabeled amino acid
substrates and
measuring radiolabel incorporation into conjugated products. Enzyme is
incubated in a reaction
buffer containing an unlabeled acyl-CoA compound and radiolabeled amino acid,
and the
radiolabeled acyl-conjugates are separated from the unreacted amino acid by
extraction into n-
butanol or other appropriate organic solvent. For example, Johnson, M. R. et
al. (1990; J. Biol.
Chem. 266:10227-10233) measured bile acid-CoA:amino acid N-acyltransferase
activity by
incubating the enzyme with cholyl-CoA and 3H-glycine or 3H-taurine, separating
the tritiated cholate
2U conjugate by extraction into n-butanol, and measuring the radioactivity in
the extracted product by
scintillation. Alternatively, N-acyltransferase activity is measured using the
spectrophotometric
determination of reduced CoA (CoASH) described below.
N-acetyltransferase activity of DME is measured using the transfer of
radiolabel from
['4C]acetyl-CoA to a substrate molecule (for example, see Deguchi, T. (1975)
J. Neurochem.
24:1083-5). Alternatively, a spectrophotometric assay based on DTNB (5,5'-
dithio-bis(2-nitrobenzoic
acid; Ellman's reagent) reaction with CoASH may be used. Free thiol-containing
CoASH is formed
during N-acetyltransferase catalyzed transfer of an acetyl group to a
substrate. CoASH is detected
using the absorbance of DTNB conjugate at 412 nm (De Angelis, J. et al. (1997)
J. Biol. Chem.
273:3045-3050). Enzyme activity is proportional to the rate of radioactivity
incorporation into
3U substrate, or the rate of absorbance increase in the spectrophotometric
assay.
Catechol-O-methyltransferase activity of DME is measured in a reaction mixture
consisting of
50 mM Tris-HCl (pH 7.4), 1.2 n~lVl MgCl2, 200 ~M SAM (S-adenosyl-L-methionine)
iodide
(containing 0.5 pCi of methyl-[H3]SAM), 1 mM dithiothreitol, and varying
concentrations of catechol
substrate (e.g., L-dopa, dopamine, or DBA) in a final volume of 1.0 ml. The
reaction is initiated by
the addition of 250-500 pg of purified DME or crude DME-containing sample and
performed at 37 °C
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for 30 min. The reaction is arrested by rapidly cooling on ice and
inunediately extracting with 7 ml of
ice-cold n-heptane. Following centrifugation at 1000 x g for 10 min, 3-ml
aliquots of the organic
extracts are analyzed for radioactivity content by liquid scintillation
counting. The level of catechol-
associated radioactivity in the organic phase is proportional to the catechol-
0-methyltransferase
activity of DME (Zhu, B.T. Liehr, J.G. (1996) 271:1357-1363).
DHFR activity of ABBR is determined spectrophotometrically at 15 °C by
following the
disappearance of NADPH at 340 nm (s3ao = 11,800 M-'~cni'). The standard assay
mixture contains
100 pM NADPH, 14 mM 2-mercaptoethanol, MTEN buffer (50 nllVl 2-
morpholinoethanesulfonic
acid, 25 mM tris(hydroxymethyl)anunomethane, 25 mM ethanolamine, and 100 mM
NaCl, pH 7.0),
and ABBR in a final volume of 2.0 ml. The reaction is started by the addition
of 50 pM dihydrofolate
(as substrate). The oxidation of NADPH to NADP+ corresponds to the reduction
of dihydrofolate in
the reaction and is proportional to the amount of DHFR activity in the sample
(Nakamura, T. and
Iwakura, M. (1999) J. Biol. Chem. 274:19041-19047).
Aldo/keto reductase activity of DME is measured using the decrease in
absorbance at 340
nm as NADPH is consumed. A standard reaction mixture is 135 mM sodium
phosphate buffer (pH
6.2-7.2 depending on enzyme), 0.2 mM NADPH, 0.3 M lithium sulfate, 0.5-2.5 pg
enzyme and an
appropriate level of substrate. The reaction is incubated at 30°C and
the reaction is monitored
continuously with a spectrophotometer. Enzyme activity is calculated as mot
NADPH consumed /
pg of enzyme.
Alcohol dehydrogenase activity of DME is measured using the increase in
absorbance at 340
nm as NAD+ is reduced to NADH. A standard reaction mixture is 50 mM sodium
phosphate, pH
7.5, and 0.25 mM EDTA. The reaction is incubated at 25 °C and monitored
using a
spectrophotometer. Enzyme activity is calculated as mot NADH produced / pg of
enzyme.
Carboxylesterase activity of DME activity is determined using 4-
methylumbelliferyl acetate as
a substrate. The enzymatic reaction is initiated by adding approximately 10 pt
of DME-containing
sample to 1 ml of reaction buffer (90 mM KHZPO4, 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 (s35o = 12.2 mM-' cni') for 1.5 min. Specific activity is
expressed as micromoles of
product formed per minute per milligram of protein and corresponds to the
activity of DME in the
sample (Evgenia, V. et al. (1997) J. Biol. Chem. 272:14769-14775).
In the alternative, the cocaine benzoyl ester hydrolase activity of DME is
measured by
incubating approximately 0.1 nil of enzyme and 3.3 mM cocaine in reaction
buffer (50 n~Ivl NaH2P04,
pH 7.4) with 1 mM benzamidine, 1 nlNl EDTA, and 1 mM dithiothreitol at 37
°C. The reaction is
incubated for 1 h in a total volume of 0.4 ml then ternunated with an equal
volume of 5%
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trichloroacetic acid. 0.1 ml of the internal standard 3,4-dimcthylbenzoic acid
(10 pg/n~l) is added.
Precipitated protein is separated by centrifugation at 12,000 x g for 10 min.
The supernatant is
transferred to a clean tube and extracted twice with 0.4 ml of methylene
chloride. The two extracts are
combined and dried under a stream of nitrogen. The residue is resuspended in
14% acctonitrile, 250
mM KHzP04, pH 4.0, with 8 pt of diethylamine per '100 ml and injected onto a
C18 reverse-phase
HPLC column for separation. The column eluate is monitored at 235 nm. DME
activity is quantified
by comparing peak area ratios of the analyte to the internal standard. A
standard curve is generated
with benzoic acid standards prepared in a trichloroacetic acid-treated protein
matrix (Evgenia, V. et al.
(1997) J. Biol. Chem. 272:14769-14775).
In another alternative, DME carboxyl esterase activity against the water-
soluble substrate
para-nitrophenyl butyric acid is determined by spectrophotomctric 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 taurocholatc. The
assay is intiated by adding
a freshly prepared para-nitrophenyl butyric acid solution (100 p~ml in sodium
acetate, pH 5.0).
Carboxyl esterase activity is then monitored and compared with control
autohydrolysis of the substrate
using a spectrophotometer set at 405 nm (Wan, L. et al. (2000) J. Biol. Chem.
275:10041-10046).
Sulfotransferase activity of DME is measured using the incorporation of 35S
liom [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
6.4, 50 pM phenol, and 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)2 is added, followed by 0.2 mL
ZnS04. The
supernatant is cleared by centrifugation, which removes proteins as well as
unreacted [ASS]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.
Heparin sulfate 6-sulfotransferase activity of DME is measured in vitro by
incubating a
sample containing DME along with 2.5 pmol inudazole HCl (pH 6.8), 3.75 pg of
protamine chloride,
25 nmol (as hexosamine) of completely desulfated and N-resulfated heparin, and
50 pmol (about 5 x
105 cpm) of [35S] adenosine 3'-phosphate 5'-phosphosulfate (PAPS) in a final
reaction volume of 50 pt
at 37 °C for 20 min. The reaction is stopped by immersing the reaction
tubes in a boiling water bath
for 1 min. 0.1 pmol (as glucuronic acid) of chondroitin sulfate A is added to
the reaction mixture as a
carrier. ASS-Labeled polysaccharides are precipitated with 3 volumes of cold
ethanol containing 1.3%
potassium acetate and separated completely from unincorporated [35S]PAPS and
its degradation
products by gel chromatography using desalting columns. One unit of enzyme
activity is defined as
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the amount required to transfer 1 pmol of sulfateJmin., determined by the
amount of [35S]PAPS
incorporated into the precipitated polysaccharides (Habuchi, H.et al. (1995)
J. Biol. Chem.
270:4172-4179).
In the alternative, heparan sulfate 6-sulfotransferase activity of DME is
measured by
extraction and renaturation of enzyme from gels following separation by sodium
dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). Following separation, the gel
is washed with buffer
(0.05 M Tris-HCI, pH 8.0), cut into 3-5 mm segments and subjected to agitation
at 4 °C with 100 p1
of the same buffer containing 0.15 M NaCl for 48 h. The eluted enzyme is
collected by centrifugation
and assayed for the sulfotransferase activity as described above (Habuchi,
H.et al. (1995) J. Biol.
Chem.270:4172-4179).
In another alternative, DME sulfotransferase activity is determined by
measuring the transfer
of [35S]sulfate from [ASS]PAPS to an immobilized peptide that represents the N-
terminal 15 residues of
the mature P-selectin glycoprotein ligand-1 polypeptide to which a C-terminal
cysteine residue is
added. The peptide spans three potential tyrosine sulfation sites. The peptide
is linked via the cysteine
residue to iodoacetamide-activated resin at a density of 1.5-3.0 pmol
peptide/ml of resin. The enzyme
assay is performed by combining 10 p1 of peptide-derivitized beads with 2-20
p1 of DME-containing
sample in 40 mM Pipes (pH 6.8), 0.3 M NaCI, 20 mM MnClz, 50 n 11VI NaF, 1 %
Triton X-100, and 1
mM 5'-AMP in a final volume of 130 ~tl. The assay is initiated by addition of
0.5 pCi of [35S]PAPS
( 1.7 pM; 1 Ci = 37 GBq). After 30 min at 37°C, the reaction beads are
washed with 6 M guanidine at
65°C and the radioactivity incorporated into the beads is determined by
liquid scintillation counting.
Transfer of [ASS]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
nun (Ouyang, Y-B. et al.
(1998) Biochemistry 95:2896-2901).
In another alternative, DME sulfotransferase assays are performed using
[~'S]PAPS as the
sulfate donor in a final volume of 30 p1, containing 50 mM Hepes-NaOH (pH
7.0), 250 mM sucrose,
1 mM dithiothreitol, 14 pM[35S]PAPS (15 Ci/mmol), and dopamine (25 pM), p-
nitrophenol (5 pM),
or other candidate substrates. Assay reactions are started by the addition of
a purified DME enzyme
preparation or a sample containing DME activity, allowed to proceed for 15 min
at 37 °C, and
terminated by heating at 100 °C for 3 min. The precipitates formed are
cleared by centrifugation. The
supernatants are then subjected to the analysis of ASS-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 3'S-
sulfated products and determine
the enzyme specificity of the DME-containing samples based on relative rates
of migration of reaction
products (Sakakibara, Y. et al. (1998) J. Biol. Chem. 273:6242-6247).
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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), O.OI mM FAD, 0.2 unit
of
NADPH-cytochrome C (P-450) reductase, 0.01 mM ['4C]squalene (dispersed with
the aid of 20 N1 of
Tween 80), and 0.2% Triton X-100. 1 nllVl 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 acetatelbenzene (0.5:99.5, v/v). The reaction products are compared
to those from a
reaction mixture without DME. The presence of 2,3(S)-oxidosqualene is
confirmed using appropriate
lipid standards (Sakakibara, J. et al. (1995) 270:17-20).
Epoxide hydrolase activity of DME is determined by following substrate
depletion using gas
chromatographic (GC) analysis of ethereal extracts or by following substrate
depletion and diol
production by GC analysis of reaction mixtures quenched in acetone. A sample
containing DME or an
epoxide hydrolase control sample is incubated in 10 mM Tris-HCl (pH 8.0), 1
n~lVl
ethylenedianunetetraacetate (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-epoxycx;tane). A portion of the
sample is withdrawn from
the reaction mixture at various time points, and added to 1 nil 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
colurrn~
(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
pmol of diol/nun (Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657).
Aminotransferase activity of DME is assayed by incubating samples containing
DME for 1
hour at 37 °C in the presence of 1 mM L-kynurenine and 1 mM 2-
oxoglutarate in a final volume of
200 p1 of 150 mM Tris acetate buffer (pH 8.0) containing 70 p M PLP. The
formation of kynurenic
acid is quantified by HPLC with spectrophotometric detection at 330 nm using
the appropriate
standards and controls well known to those skilled in the art. In the
alternative,
L-3-hydroxykynurenine is used as substrate and the production of xanthurenic
acid is determined by
HPLC analysis of the products with UV detection at 340 run. The production of
kynurenic acid and
xanthurenic acid, respectively, is indicative of aminotransferase activity
(Buchli, R. et al. (1995) J.
Biol. Chem. 270:29330-29335).
In another alternative, aminotransferase activity of DME is measured by
determining the
activity of purified DME or crude samples containing DME toward various amino
and oxo acid
substrates under single turnover conditions by monitoring the changes in the
UV/VIS absorption
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spectrum of the enzyme-bound cofactor, pyridoxal 5'-phosphate (PLP). The
reactions are performed
at 25 °C in 50 mM 4-methylmorpholine (pH 7.5) containing 9 pM purified
DME or DME containing
samples and substrate to be tested (amino and oxo acid substrates). The half-
reaction from amino acid
to oxo acid is followed by measuring the decrease in absorbance at 360 nm and
the increase in
absorbance at 330 nm due to the conversion of enzyme-bound PLP to pyridoxamine
5' phosphate
(PMP). The specificity and relative activity of DME is determined by the
activity of the enzyme
preparation against specific substrates (Vacca, R.A. et al. (1997) J. Biol.
Chem. 272:21932-21937).
Superoxide dismutase activity of DME is assayed from cell pellets, culture
supernatants, or
purified protein preparations. Samples or lysates are resolved by
electrophoresis on 15%
non-denaturing polyacrylamide gels. The gels are incubated for 30 nun in 2.5
mM nitro blue
tetrazolium, followed by incubation for 20 nun in 30 mM potassium phosphate,
30 mM TEMED, and
30 pM riboflavin (pH 7.8). Superoxide dismutase activity is visualized as
white bands against a blue
background, following illumination of the gels on a lightbox. Quantitation of
superoxide dismutase
activity is performed by densitometric scanning of the activity gels using the
appropriate superoxide
dismutase positive and negative controls (e.g., various amounts of
commercially available E. coli
superoxide dismutase (Harth, G. and Horwitz, M.A. (1999) J. Biol. Chem.
274:4281-4292).
XVIII. Identification of DME Inhibitors
Compounds to be tested are arrayed in the wells of a mufti-well place in
varying
concentrations along with an appropriate buffer and substrate, as described in
the assays in Example
XV1I. 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 linuted to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
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CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
BAUGHN, Mariah R.
YAO, Monique G.
BANDMAN, Olga
AZIMZAI, Yalda
LAL, Preeti
GANDHI, Ameena R.
RING, Huijun Z.
SHIH, Leo L.
YANG, Junming ,
POLICKY, Jennifer L.
<120> DRUG METABOLIZING ENZYMES
<130> PI-0033 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/181,856; 10/183,684; 60/185,141; 60/186,818; 60/188,345; 60/189,997
<151> 2000-02-11; 2000-02-17; 2000-02-25; 2000-03-03; 2000-03-09; 2000-03-17
<160> 24
<170> PERL Program
<210> 1
<211> 208
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1642862CD1
<400> 1
Met Trp Phe Leu Leu Tyr Cys Glu Gly Thr Arg Phe Thr Glu Thr
1 5 10 15
Lys His Arg Val Ser Met Glu Val Ala Ala Ala Lys Gly Leu Pro
20 25 30
Val Leu Lys Tyr His Leu Leu Pro Arg Thr Lys Gly Phe Thr Thr
35 40 45
Ala Val Lys Cys Leu Arg Gly Thr Val Ala Ala Val Tyr Asp Val
50 55 60
Thr Leu Asn Phe Arg Gly Asn Lys Asn Pro Ser Leu Leu Gly Ile
65 70 75
Leu Tyr Gly Lys Lys Tyr Glu Ala Asp Met Cys Val Arg Arg Phe
80 85 90
Pro Leu Glu Asp Ile Pro Leu Asp Glu Lys Glu Ala Ala Gln Trp
95 100 105
Leu His Lys Leu Tyr Gln Glu Lys Asp Ala Leu Gln Glu Ile Tyr
110 115 120
Asn Gln Lys Gly Met Phe Pro Gly Glu Gln Phe Lys Pro Ala Arg
125 130 135
Arg Pro Trp Thr Leu Leu Asn Phe Leu Ser Trp Ala Thr Ile Leu
140 145 150
Leu Ser Pro Leu Phe Ser Phe Val Leu Gly Val Phe Ala Ser Gly
155 160 165
Ser Pro Leu Leu Ile Leu Thr Phe Leu Gly Phe Val Gly Ala Ala
170 175 180
Ser Phe Gly Val Arg Arg Leu Ile Gly Val Thr Glu Ile Glu Lys
185 190 195
Gly Ser Ser Tyr Gly Asn Gln Glu Phe Lys Lys Lys Glu
200 205
<210> 2
<211> 294
1/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3861612CD1
<400> 2
Met Leu Val Leu His Asn Ser Gln Lys Leu Gln Ile Leu Tyr Lys
1 5 10 15
Ser Leu Glu Lys Ser Ile Pro Glu Ser Ile Lys Val Tyr Gly Ala
20 25 30
Ile Phe Asn Ile Lys Asp Lys Asn Pro Phe Asn Met Glu Val Leu
35 40 45
Val Asp Ala Trp Pro Asp Tyr Gln Ile Val Ile Thr Arg Pro Gln
50 55 60
Lys Gln Glu Met Lys Asp Asp Gln Asp His Tyr Thr Asn Thr Tyr
65 70 75
His Ile Phe Thr Lys Ala Pro Asp Lys Leu Glu Glu Val Leu Ser
80 85 90
Tyr Ser Asn Val Ile Ser Trp Glu Gln Thr Leu Gln Ile Gln Gly
95 100 105
Cys Gln Glu Gly Leu Asp Glu Ala Ile Arg Lys Val Ala Thr Ser
110 115 120
Lys Ser Val Gln Val Asp Tyr Met Lys Thr Ile Leu Phe Ile Pro
125 130 135
Glu Leu Pro Lys Lys His Lys Thr Ser Ser Asn Asp Lys Met Glu
140 145 150
Leu Phe Glu Val Asp Asp Asp Asn Lys Glu Gly Asn Phe Ser Asn
155 160 165
Met Phe Leu Asp Ala Ser His Ala Gly Leu Val Asn Glu His Trp
170 175 180
Ala Phe Gly Lys Asn Glu Arg Ser Leu Lys Tyr Ile Glu Arg Cys
185 190 195
Leu Gln Asp Phe Leu Gly Phe Gly Val Leu Gly Pro Glu Gly Gln
200 205 210
Leu Val Ser Trp Ile Val Met Glu Gln Ser Cys Glu Leu Arg Met
215 220 225
Gly Tyr Thr Val Pro Lys Tyr Arg His Gln Gly Asn Met Leu Gln
230 235 240
Ile Gly Tyr His Leu Glu Lys Tyr Leu Ser Gln Lys Glu Ile Pro
245 250 255
Phe Tyr Phe His Val Ala Asp Asn Asn Glu Lys Ser Leu Gln Ala
260 265 270
Leu Asn Asn Leu Gly Phe Lys Ile Cys Pro Cys Gly Trp His Gln
275 280 285
Trp Lys Cys Thr Pro Lys Lys Tyr Cys
290
<210> 3
<211> 241
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472055CD1
<400> 3
Met Ala Leu Glu Leu Tyr Met Asp Leu Leu Ser Ala Pro Cys Arg
1 5 10 15
Ala Val Tyr Ile Phe Ser Lys Lys His Asp Ile Gln Phe Asn Phe
20 25 30
Gln Phe Val Asp Leu Leu Lys Gly His His His Ser Lys Glu Tyr
35 40 45
Ile Asp Ile Asn Pro Leu Arg Lys Leu Pro Ser Leu Lys Asp Gly
50 55 60
2/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
Lys Phe Ile Leu Ser Glu Ser Pro Gln Leu Leu Tyr Tyr Leu Cys
65 70 75
Arg Lys Tyr Ser Ala Pro Ser His Trp Cys Pro Pro Asp Pro His
80 85 90
Ala Arg Ala Arg Val Asp Glu Phe Val Ala Trp Gln His Thr Ala
95 100 105
Phe Gln Leu Pro Met Lys Lys Ile Val Trp Leu Lys Leu Leu Ile
110 115 120
Pro Lys Ile Thr Gly Glu Glu Val Ser Ala Glu Lys Met Glu His
125 130 135
Ala Val Glu Glu Val Lys Asn Ser Leu Gln Leu Phe Glu Glu Tyr
140 145 150
Phe Leu Gln Asp Lys Met Phe Ile Thr Gly Asn Gln Ile Ser Leu
155 160 165
Ala Asp Leu Val Ala Val Val Glu Met Met Gln Pro Met Ala Ala
170 175 180
Asn Tyr Asn Val Phe Leu Asn Ser Ser Lys Leu Ala Glu Trp Arg
185 190 195
Met Gln Val Glu Leu Asn Ile Gly Ser Gly Leu Phe Arg Glu Ala
200 205 210
His Asp Arg Leu Met Gln Leu Ala Asp Trp Asp Phe Ser Thr Leu
215 220 225
Asp Ser Met Val Lys Glu Asn Ile Ser Glu Leu Leu Lys Lys Ser
230 235 240
Arg
<210> 4
<211> 640
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1923521CD1
<400> 4
Met Pro Cys Gly Glu Asp Trp Leu Ser His Pro Leu Gly Ile Val
1 5 10 15
Gln Gly Phe Phe Ala Gln Asn Gly Val Asn Pro Asp Trp Glu Lys
20 25 30
Lys Val Ile Glu Tyr Phe Lys Glu Lys Leu Lys Glu Asn Asn Ala
35 40 45
Pro Lys Trp Val Pro Ser Leu Asn Glu Val Pro Leu His Tyr Leu
50 55 60
Lys Pro Asn Ser Phe Val Lys Phe Arg Cys Met Ile Gln Asp Met
65 70 75
Phe Asp Pro Glu Phe Tyr Met Gly Val Tyr Glu Thr Val Asn Gln
80 85 90
Asn Thr Lys Ala His Val Leu His Phe Gly Lys Tyr Arg Asp Val
95 100 105
Ala Glu Cys Gly Pro Gln Gln Glu Leu Asp Leu Asn Ser Pro Arg
110 115 120
Asn Thr Thr Leu Glu Arg Gln Thr Phe Tyr Cys Val Pro Val Pro
125 130 135
Gly Glu Ser Thr Trp Val Lys Glu Ala Tyr Val Asn Ala Asn Gln
140 145 150
Ala Arg Val Ser Pro Ser Thr Ser Tyr Thr Pro Ser Arg His Lys
155 160 165
Arg Ser Tyr Glu Asp Asp Asp Asp Met Asp Leu Gln Pro Asn Lys
170 175 180
Gln Lys Asp Gln His Ala Gly Ala Arg Gln Ala Gly Ser Val Gly
185 190 195
Gly Leu Gln Trp Cys Gly Glu Pro Lys Arg Leu Glu Thr Glu Ala
200 205 210
Ser Thr Gly Gln Gln Leu Asn Ser Leu Asn Leu Ser Ser Pro Phe
215 220 225
3/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
Asp Leu Asn Phe Pro Leu Pro Gly Glu Lys Gly Pro Ala Cys Leu
230 235 240
Val Lys Val Tyr Glu Asp Trp Asp Cys Phe Lys Val Asn Asp Ile
245 250 255
Leu Glu Leu Tyr Gly Ile Leu Ser Val Asp Pro Val Leu Ser Ile
260 265 270
Leu Asn Asn Asp Glu Arg Asp Ala Ser Ala Leu Leu Asp Pro Met
275 280 285
Glu Cys Thr Asp Thr Ala Glu Glu Gln Arg Val His Ser Pro Pro
290 295 300
Ala Ser Leu Val Pro Arg Ile His Val Ile Leu Ala Gln Lys Leu
305 310 315
Gln His Ile Asn Pro Leu Leu Pro Ala Cys Leu Asn Lys Glu Glu
320 325 330
Ser Lys Thr Phe Val Ser Ser Phe Met Ser Glu Leu Ser Pro Val
335 340 345
Arg Ala Glu Leu Leu Gly Phe Leu Thr His Ala Leu Leu Gly Asp
350 355 360
Ser Leu Ala Ala Glu Tyr Leu Ile Leu His Leu Ile Ser Thr Val
365 370 375
Tyr Thr Arg Arg Asp Val Leu Pro Leu Gly Lys Phe Thr Val Asn
380 385 390
Leu Ser Gly Cys Pro Arg Asn Ser Thr Phe Thr Glu His Leu Tyr
395 400 405
Arg Ile Ile Gln His Leu Val Pro Ala Ser Phe Arg Leu Gln Met
410 415 420
Thr Ile Glu Asn Met Asn His Leu Lys Phe Ile Pro His Lys Asp
425 430 435
Tyr Thr Ala Asn Arg Leu Val Ser Gly Leu Leu Gln Leu Pro Ser
440 445 450
Asn Thr Ser Leu Val Ile Asp Glu Thr Leu Leu Glu Gln Gly Gln
455 460 465
Leu Asp Thr Pro Gly Val His Asn Val Thr Ala Leu Ser Asn Leu
470 475 480
Ile Thr Trp Gln Lys Val Asp Tyr Asp Phe Ser Tyr His Gln Met
485 490 495
Glu Phe Pro Cys Asn Ile Asn Val Phe Ile Thr Ser Glu Gly Arg
500 SOS 510
Ser Leu Leu Pro Ala Asp Cys Gln Ile His Leu Gln Pro Gln Leu
515 520 525
Ile Pro Pro Asn Met Glu Glu Tyr Met Asn Ser Leu Leu Ser Ala
530 535 540
Val Leu Pro Ser Val Leu Asn Lys Phe Arg Ile Tyr Leu Thr Leu
545 550 555
Leu Arg Phe Leu Glu Tyr Ser Ile Ser Asp Glu Ile Thr Lys Ala
560 565 570
Val Glu Asp Asp Phe Val Glu Met Arg Lys Asn Asp Pro Gln Ser
575 580 585
Ile Thr Ala Asp Asp Leu His Gln Leu Leu Val Val Ala Arg Cys
590 595 600
Leu Ser Leu Ser Ala Gly Gln Thr Thr Leu Ser Arg Glu Arg Trp
605 610 615
Leu Arg Ala Lys Gln Leu Glu Ser Leu Arg Arg Thr Arg Leu Gln
620 625 630
Gln Gln Lys Cys Val Asn Gly Asn Glu Leu
635 640
<210> 5
<211> 870
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1558210CD1
<400> 5
4/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
Met Gly Pro Pro Ser Leu Val Leu Cys Leu Leu Ser Ala Thr Val
1 5 10 15
Phe Ser Leu Leu Gly Gly Ser Ser Ala Phe Leu Ser His His Arg
20 25 30
Leu Lys Gly Arg Phe Gln Arg Asp Arg Arg Asn Ile Arg Pro Asn
35 40 45
Ile Ile Leu Val Leu Thr Asp Asp Gln Asp Val Glu Leu Gly Ser
50 55 60
Met Gln Val Met Asn Lys Thr Arg Arg Ile Met Glu Gln Gly Gly
65 70 75
Ala His Phe Ile Asn Ala Phe Val Thr Thr Pro Met Cys Cys Pro
80 85 90
Ser Arg Ser Ser Ile Leu Thr Gly Lys Tyr Val His Asn His Asn
95 100 105
Thr Tyr Thr Asn Asn Glu Asn Cys Ser Ser Pro Ser Trp Gln Ala
110 115 120
Gln His Glu Ser Arg Thr Phe Ala Val Tyr Leu Asn Ser Thr Gly
125 130 135
Tyr Arg Thr Ala Phe Phe Gly Lys Tyr Leu Asn Glu Tyr Asn Gly
140 145 150
Ser Tyr Val Pro Pro Gly Trp Lys Glu Trp Val Gly Leu Leu Lys
155 160 165
Asn Ser Arg Phe Tyr Asn Tyr Thr Leu Cys Arg Asn Gly Val Lys
170 175 180
Glu Lys His Gly Ser Asp Tyr Ser Lys Asp Tyr Leu Thr Asp Leu
185 190 195
Ile Thr Asn Asp Ser Val Ser Phe Phe Arg Thr Ser Lys Lys Met
200 205 210
Tyr Pro His Arg Pro Val Leu Met Val Ile Ser His Ala Ala Pro
215 220 225
His Gly Pro Glu Asp Ser Ala Pro Gln Tyr Ser Arg Leu Phe Pro
230 235 240
Asn Ala Ser Gln His Ile Thr Pro Ser Tyr Asn Tyr Ala Pro Asn
245 250 255
Pro Asp Lys His Trp Ile Met Arg Tyr Thr Gly Pro Met Lys Pro
260 265 270
Ile His Met Glu Phe Thr Asn Met Leu Gln Arg Lys Arg Leu Gln
275 280 285
Thr Leu Met Ser Val Asp Asp Ser Met Glu Thr Ile Tyr Asn Met
290 295 300
Leu Val Glu Thr Gly Glu Leu Asp Asn Thr Tyr Ile Val Tyr Thr
305 310 315
Ala Asp His Gly Tyr His Ile Gly Gln Phe Gly Leu Val Lys Gly
320 325 330
Lys Ser Met Pro Tyr Glu Phe Asp Ile Arg Val Pro Phe Tyr Val
335 340 345
Arg Gly Pro Asn Val Glu Ala Gly Cys Leu Asn Pro His Ile Val
350 355 360
Leu Asn Ile Asp Leu Ala Pro Thr Ile Leu Asp Ile Ala Gly Leu
365 370 375
Asp Ile Pro Ala Asp Met Asp Gly Lys Ser Ile Leu Lys Leu Leu
380 385 390
Asp Thr Glu Arg Pro Val Asn Arg Phe His Leu Lys Lys Lys Met
395 400 405
Arg Val Trp Arg Asp Ser Phe Leu Val Glu Arg Gly Lys Leu Leu
410 415 420
His Lys Arg Asp Asn Asp Lys Val Asp Ala Gln Glu Glu Asn Phe
425 430 435
Leu Pro Lys Tyr Gln Arg Val Lys Asp Leu Cys Gln Arg Ala Glu
440 445 450
Tyr Gln Thr Ala Cys Glu Gln Leu Gly Gln Lys Trp Gln Cys Val
455 460 465
Glu Asp Ala Thr Gly Lys Leu Lys Leu His Lys Cys Lys Gly Pro
470 475 480
Met Arg Leu Gly Gly Ser Arg Ala Leu Ser Asn Leu Val Pro Lys
485 490 495
Tyr Tyr Gly Gln Gly Ser Glu Ala Cys Thr Cys Asp Ser Gly Asp
5/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
500 505 510
Tyr Lys Leu Ser Leu Ala Gly Arg Arg Lys Lys Leu Phe Lys Lys
515 520 525
Lys Tyr Lys Ala Ser Tyr Val Arg Ser Arg Ser Ile Arg Ser Val
530 535 540
Ala Ile Glu Val Asp Gly Arg Val Tyr His Val Gly Leu Gly Asp
545 550 555
Ala Ala Gln Pro Arg Asn Leu Thr Lys Arg His Trp Pro Gly Ala
560 565 570
Pro Glu Asp Gln Asp Asp Lys Asp Gly Gly Asp Phe Ser Gly Thr
575 580 585
Gly Gly Leu Pro Asp Tyr Ser Ala Ala Asn Pro Ile Lys Val Thr
590 595 600
His Arg Cys Tyr Ile Leu Glu Asn Asp Thr Val Gln Cys Asp Leu
605 610 615
Asp Leu Tyr Lys Ser Leu Gln Ala Trp Lys Asp His Lys Leu His
620 625 630
Ile Asp His Glu Ile Glu Thr Leu Gln Asn Lys Ile Lys Asn Leu
635 640 645
Arg Glu Val Arg Gly His Leu Lys Lys Lys Arg Pro Glu Glu Cys
650 655 660
Asp Cys His Lys Ile Ser Tyr His Thr Gln His Lys Gly Arg Leu
665 670 675
Lys His Arg Gly Ser Ser Leu His Pro Phe Arg Lys Gly Leu Gln
680 685 690
Glu Lys Asp Lys Val Trp Leu Leu Arg Glu Gln Lys Arg Lys Lys
695 700 705
Lys Leu Arg Lys Leu Leu Lys Arg Leu Gln Asn Asn Asp Thr Cys
710 715 720
Ser Met Pro Gly Leu Thr Cys Phe Thr His Asp Asn Gln His Trp
725 730 735
Gln Thr Ala Pro Phe Trp Thr Leu Gly Pro Phe Cys Ala Cys Thr
740 745 750
Ser Ala Asn Asn Asn Thr Tyr Trp Cys Met Arg Thr Ile Asn Glu
755 760 765
Thr His Asn Phe Leu Phe Cys Glu Phe Ala Thr Gly Phe Leu Glu
770 775 780
Tyr Phe Asp Leu Asn Thr Asp Pro Tyr Gln Leu Met Asn Ala Val
785 790 795
Asn Thr Leu Asp Arg Asp Val Leu Asn Gln Leu His Val Gln Leu
800 805 810
Met Glu Leu Arg Ser Cys Lys Gly Tyr Lys Gln Cys Asn Pro Arg
815 820 825
Thr Arg Asn Met Asp Leu Gly Leu Lys Asp Gly Gly Ser Tyr Glu
830 835 840
Gln Tyr Arg Gln Phe Gln Arg Arg Lys Trp Pro Glu Met Lys Arg
845 850 855
Pro Ser Ser Lys Ser Leu Gly Gln Leu Trp Glu Gly Trp Glu Gly
860 865 870
<210> 6
<211> 488
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5629033CD1
<400> 6
Met Pro Glu Glu Met Asp Lys Pro Leu Ile Ser Leu His Leu Val
1 5 10 15
Asp Ser Asp Ser Ser Leu Ala Lys Val Pro Asp Glu Ala Pro Lys
20 25 30
Val Gly Ile Leu Gly Ser Gly Asp Phe Ala Arg Ser Leu Ala Thr
35 40 45
6/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
Arg Leu Val Gly Ser Gly Phe Lys Val Val Val Gly Ser Arg Asn
50 55 60
Pro Lys Arg Thr Ala Arg Leu Phe Pro Ser Ala Ala Gln Val Thr
65 70 75
Phe Gln Glu Glu Ala Val Ser Ser Pro Glu Val Ile Phe Val Ala
80 85 90
Val Phe Arg Glu His Tyr Ser Ser Leu Cys Ser Leu Ser Asp Gln
95 100 105
Leu Ala Gly Lys Ile Leu Val Asp Val Ser Asn Pro Thr Glu Gln
110 115 120
Glu His Leu Gln His Arg Glu Ser Asn Ala Glu Tyr Leu Ala Ser
125 130 135
Leu Phe Pro Thr Cys Thr Val Val Lys Ala Phe Asn Val Ile Ser
140 145 150
Ala Trp Thr Leu Gln Ala Gly Pro Arg Asp Gly Asn Arg Gln Val
155 160 165
Pro Ile Cys Gly Asp Gln Pro Glu Ala Lys Arg Ala Val Ser Glu
170 175 180
Met Ala Leu Ala Met Gly Phe Met Pro Val Asp Met Gly Ser Leu
185 190 195
Ala Ser Ala Trp Glu Val Glu Ala Met Pro Leu Arg Leu Leu Pro
200 205 210
Ala Trp Lys Val Pro Thr Leu Leu Ala Leu Gly Leu Phe Val Cys
215 220 225
Phe Tyr Ala Tyr Asn Phe Val Arg Asp Val Leu Gln Pro Tyr Val
230 235 240
Gln Glu Ser Gln Asn Lys Phe Phe Lys Leu Pro Val Ser Val Val
245 250 255
Asn Thr Thr Leu Pro Cys Val Ala Tyr Val Leu Leu Ser Leu Val
260 265 270
Tyr Leu Pro Gly Val Leu Ala Ala Ala Leu Gln Leu Arg Arg Gly
275 280 285
Thr Lys Tyr Gln Arg Phe Pro Asp Trp Leu Asp His Trp Leu Gln
290 295 300
His Arg Lys Gln Ile Gly Leu Leu Ser Phe Phe Cys Ala Ala Leu
305 310 315
His Ala Leu Tyr Ser Phe Cys Leu Pro Leu Arg Arg Ala His Arg
320 325 330
Tyr Asp Leu Val Asn Leu Ala Val Lys Gln Val Leu Ala Asn Lys
335 340 345
Ser His Leu Trp Val Glu Glu Glu Val Trp Arg Met Glu Ile Tyr
350 355 360
Leu Ser Leu Gly Val Leu Ala Leu Gly Thr Leu Ser Leu Leu Ala
365 370 375
Val Thr Ser Leu Pro Ser Ile Ala Asn Ser Leu Asn Trp Arg Glu
380 385 390
Phe Ser Phe Val Gln Ser Ser Leu Gly Phe Val Ala Leu Val Leu
395 400 405
Ser Thr Leu His Thr Leu Thr Tyr Gly Trp Thr Arg Ala Phe Glu
410 415 420
Glu Ser Arg Tyr Lys Phe Tyr Leu Pro Pro Thr Phe Thr Leu Thr
425 430 435
Leu Leu Val Pro Cys Val Val Ile Leu Ala Lys Ala Leu Phe Leu
440 445 450
Leu Pro Cys Ile Ser Arg Arg Leu Ala Arg Ile Arg Arg Gly Trp
455 460 465
Glu Arg Glu Ser Thr Ile Lys Phe Thr Leu Pro Thr Asp His Ala
470 475 480
Leu Ala Glu Lys Thr Ser His Val
485
<210> 7
<211> 402
<212> PRT
<213> Homo Sapiens
<220>
7/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
<221> misc_feature
<223> Incyte ID No: 2750679CD1
<400> 7
Met Thr Ala Pro His Leu Cys Ser Cys Leu Pro Ala Ile Leu Arg
1 5 10 15
Pro Leu Ala Met Gly Gly Cys Phe Ser Lys Pro Lys Pro Val Glu
20 25 30
Leu Lys Ile Glu Val Val Leu Pro Glu Lys Glu Arg Gly Lys Glu
35 40 45
Glu Leu Ser Ala Ser Gly Lys Gly Ser Pro Arg Ala Tyr Gln Gly
50 55 60
Asn Gly Thr Ala Arg His Phe His Thr Glu Glu Arg Leu Ser Thr
65 70 75
Pro His Pro Tyr Pro Ser Pro Gln Asp Cys Val Glu Ala Ala Val
80 85 90
Cys His Val Lys Asp Leu Glu Asn Gly Gln Met Arg Glu Val Glu
95 100 105
Leu Gly Trp Gly Lys Val Leu Leu Val Lys Asp Asn Gly Glu Phe
110 115 120
His Ala Leu Gly His Lys Cys Pro His Tyr Gly Ala Pro Leu Val
125 130 135
Lys Gly Val Leu Ser Arg Gly Arg Val Arg Cys Pro Trp His Gly
140 145 150
Ala Cys Phe Asn Ile Ser Thr Gly Asp Leu Glu Asp Phe Pro Gly
155 160 165
Leu Asp Ser Leu His Lys Phe Gln Val Lys Ile Glu Lys Glu Lys
170 175 180
Val Tyr Val Arg Ala Ser Lys Gln Ala Leu Gln Leu Gln Arg Arg
185 190 195
Thr Lys Val Met Ala Lys Cys Ile Ser Pro Ser Ala Gly Tyr Ser
200 205 210
Ser Ser Thr Asn Val Leu Ile Val Gly Ala Gly Ala Ala Gly Leu
215 220 225
Val Cys Ala Glu Thr Leu Arg Gln Glu Gly Phe Ser Asp Arg Ile
230 235 240
Val Leu Cys Thr Leu Asp Arg His Leu Pro Tyr Asp Arg Pro Lys
245 250 255
Leu Ser Lys Ser Leu Asp Thr Gln Pro Glu Gln Leu Ala Leu Arg
260 265 270
Pro Lys Glu Phe Phe Arg Ala Tyr Gly Ile Glu Val Leu Thr Glu
275 280 285
Ala Gln Val Val Thr Val Asp Val Arg Thr Lys Lys Val Val Phe
290 295 300
Lys Asp Gly Phe Lys Leu Glu Tyr Ser Lys Leu Leu Leu Ala Pro
305 310 315
Gly Glu Gln Pro Gln Asp Ser Glu Leu Gln Arg Gln Arg Ser Gly
320 325 330
Glu Arg Val His Tyr Pro Asp Ala Arg Gly Cys Gln Ser Arg Gly
335 340 345
Glu Ala Gly Pro Arg Pro Gln Arg Gly Arg Arg Gly Ser Arg Leu
350 355 360
Pro Gly Asp Gly Gly Gly Arg Leu Pro Asp Gly Glu Gly Pro Leu
365 370 375
Cys Val Cys Gly Gly Ala Gly Gly Asp Ala Leu Gln Glu Val Pro
380 385 390
Gly Gly Ala Arg Gly Ser Cys Pro His Glu Asp Val
395 400
<210> 8
<211> 276
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1570911CD1
8/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
<400> 8
Met Asn Ser Arg Arg Arg Glu Pro Ile Thr Leu Gln Asp Pro Glu
1 5 10 15
Ala Lys Tyr Pro Leu Pro Leu Ile Glu Lys Glu Lys Ile Ser His
20 25 30
Asn Thr Arg Arg Phe Arg Phe Gly Leu Pro Ser Pro Asp His Val
35 40 45
Leu Gly Leu Pro Val Gly Asn Tyr Val Gln Leu Leu Ala Lys Ile
50 55 60
Asp Asn Glu Leu Val Val Arg Ala Tyr Thr Pro Val Ser Ser Asp
65 70 75
Asp Asp Arg Gly Phe Val Asp Leu Ile Ile Lys Ile Tyr Phe Lys
80 85 90
Asn Val His Pro Gln Tyr Pro Glu Gly Gly Lys Met Thr Gln Tyr
95 100 105
Leu Glu Asn Met Lys Ile Gly Glu Thr Ile Phe Phe Arg Gly Pro
110 115 120
Arg Gly Arg Leu Phe Tyr His Gly Pro Gly Asn Leu Gly Ile Arg
125 130 135
Pro Asp Gln Thr Ser Glu Pro Lys Lys Thr Leu Ala Asp His Leu
140 145 150
Gly Met Ile Ala Gly Gly Thr Gly Ile Thr Pro Met Leu Gln Leu
155 160 165
Ile Arg His Ile Thr Lys Asp Pro Ser Asp Arg Thr Arg Met Ser
170 175 180
Leu Ile Phe Ala Asn Gln Thr Glu Glu Asp Ile Leu Val Arg Lys
185 190 195
Glu Leu Glu Glu Ile Ala Arg Thr His Pro Asp Gln Phe Asp Leu
200 205 210
Trp Tyr Thr Leu Asp Arg Pro Pro Ile Gly Trp Lys Tyr Ser Ser
215 220 225
Gly Phe Val Thr Ala Asp Met Ile Lys Glu His Leu Pro Pro Pro
230 235 240
Ala Lys Ser Thr Leu Ile Leu Val Cys Gly Pro Pro Pro Leu Ile
245 250 255
Gln Thr Ala Ala His Pro Asn Leu Glu Lys Leu Gly Tyr Thr Gln
260 265 270
Asp Met Ile Phe Thr Tyr
275
<210> 9
<211> 512
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1959720CD1
<400> 9
Met Leu Phe Glu Gly Leu Asp Leu Val Ser Ala Leu Ala Thr Leu
1 5 10 15
Ala Ala Cys Leu Val Ser Val Thr Leu Leu Leu Ala Val Ser Gln
20 25 30
Gln Leu Trp Gln Leu Arg Trp Ala Ala Thr Arg Asp Lys Ser Cys
35 40 45
Lys Leu Pro Ile Pro Lys Gly Ser Met Gly Phe Pro Leu Ile Gly
50 55 60
Glu Thr Gly His Trp Leu Leu Gln Val Ser Gly Phe Gln Ser Ser
65 70 75
Arg Arg Glu Lys Tyr Gly Asn Val Phe Lys Thr His Leu Leu Gly
80 85 90
Arg Pro Leu Ile Arg Val Thr Gly Ala Glu Asn Val Arg Lys Ile
95 100 105
Leu Met Gly Glu His His Leu Val Ser Thr Glu Trp Pro Arg Ser
110 115 120
Thr Arg Met Leu Leu Gly Pro Asn Thr Val Ser Asn Ser Ile Gly
9/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
125 130 135
Asp Ile His Arg Asn Lys Arg Lys Val Phe Ser Lys Ile Phe Ser
140 145 150
His Glu Ala Leu Glu Ser Tyr Leu Pro Lys Ile Gln Leu Val Ile
155 160 165
Gln Asp Thr Leu Arg Ala Trp Ser Ser His Pro Glu Ala Ile Asn
170 175 180
Val Tyr Gln Glu Ala Gln Lys Leu Thr Phe Arg Met Ala Ile Arg
185 190 195
Val Leu Leu Gly Phe Ser Ile Pro Glu Glu Asp Leu Gly His Leu
200 205 210
Phe Glu Val Tyr Gln Gln Phe Val Asp Asn Val Phe Ser Leu Pro
215 220 225
Val Asp Leu Pro Phe Ser Gly Tyr Arg Arg Gly Ile Gln Ala Arg
230 235 240
Gln Ile Leu Gln Lys Gly Leu Glu Lys Ala Ile Arg Glu Lys Leu
245 250 255
Gln Cys Thr Gln Gly Lys Asp Tyr Leu Asp Val Leu Asp Leu Leu
260 265 270
Ile Glu Ser Ser Lys Glu His Gly Lys Glu Met Thr Met Gln Glu
275 280 285
Leu Lys Asp Gly Thr Leu Glu Leu Ile Phe Ala Ala Tyr Ala Thr
290 295 300
Thr Ala Ser Ala Ser Thr Ser Leu Ile Met Gln Leu Leu Lys His
305 310 315
Pro Thr Val Leu Glu Lys Leu Arg Asp Glu Leu Arg Ala His Gly
320 325 330
Ile Leu His Ser Gly Gly Cys Pro Cys Glu Gly Thr Leu Arg Leu
335 340 345
Asp Thr Leu Ser Gly Leu Arg Tyr Leu Asp Cys Val Ile Lys Glu
350 355 360
Val Met Arg Leu Phe Thr Pro Ile Ser Gly Gly Tyr Arg Thr Val
365 370 375
Leu Gln Thr Phe Glu Leu Asp Gly Phe Gln Ile Pro Lys Gly Trp
380 385 390
Ser Val Met Tyr Ser Ile Arg Asp Thr His Asp Thr Ala Pro Val
395 400 405
Phe Lys Asp Val Asn Val Phe Asp Pro Asp Arg Phe Ser Gln Ala
410 415 420
Arg Ser Glu Asp Lys Asp Gly Arg Phe His Tyr Leu Pro Phe Gly
425 430 435
Gly Gly Val Arg Thr Cys Leu Gly Lys His Leu Ala Lys Leu Phe
440 445 450
Leu Lys Val Leu Ala Val Glu Leu Ala Ser Thr Ser Arg Phe Glu
455 460 465
Leu Ala Thr Arg Thr Phe Pro Arg Ile Thr Leu Val Pro Val Leu
470 475 480
His Pro Val Asp Gly Leu Ser Val Lys Phe Phe Gly Leu Asp Ser
485 490 495
Asn Gln Asn Glu Ile Leu Pro Glu Thr Glu Ala Met Leu Ser Ala
500 505 510
Thr Val
<210> 10
<211> 524
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6825202CD1
<400> 10
Met Pro Gln Leu Ser Leu Ser Trp Leu Gly Leu Gly Pro Val Ala
1 5 10 15
Ala Ser Pro Trp Leu Leu Leu Leu Leu Val Gly Gly Ser Trp Leu
10/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
20 25 30
Leu Ala Arg Val Leu Ala Trp Thr Tyr Thr Phe Tyr Asp Asn Cys
35 40 45
Arg Arg Leu Gln Cys Phe Pro Gln Pro Pro Lys Gln Asn Trp Phe
50 55 60
Trp Gly His Gln Gly Leu Val Thr Pro Thr Glu Glu Gly Met Lys
65 70 75
Thr Leu Thr Gln Leu Val Thr Thr Tyr Pro Gln Gly Phe Lys Leu
80 85 90
Trp Leu Gly Pro Thr Phe Pro Leu Leu Ile Leu Cys His Pro Asp
95 100 105
Ile Ile Arg Pro Ile Thr Ser Ala Ser Ala Ala Val Ala Pro Lys
110 115 120
Asp Met Ile Phe Tyr Gly Phe Leu Lys Pro Trp Leu Gly Asp Gly
125 130 135
Leu Leu Leu Ser Gly Gly Asp Lys Trp Ser Arg His Arg Arg Met
140 145 150
Leu Thr Pro Ala Phe His Phe Asn Ile Leu Lys Pro Tyr Met Lys
155 160 165
Ile Phe Asn Lys Ser Val Asn Ile Met His Asp Lys Trp Gln Arg
170 175 180
Leu Ala Ser Glu Gly Ser Ala Arg Leu Asp Met Phe Glu His Ile
185 190 195
Ser Leu Met Thr Leu Asp Ser Leu Gln Lys Cys Val Phe Ser Phe
200 205 210
Glu Ser Asn Cys Gln Glu Lys Pro Ser Glu Tyr Ile Ala Ala Ile
215 220 225
Leu Glu Leu Ser Ala Phe Val Glu Lys Arg Asn Gln Gln Ile Leu
230 235 240
Leu His Thr Asp Phe Leu Tyr Tyr Leu Thr Pro Asp Gly Gln Arg
245 250 255
Phe Arg Arg Ala Cys His Leu Val His Asp Phe Thr Asp Ala Val
260 265 270
Ile Gln Glu Arg Arg Arg Thr Leu Pro Thr Gln Gly Ile Asp Asp
275 280 285
Phe Leu Lys Asn Lys Ala Lys Ser Lys Thr Leu Asp Phe Ile Asp
290 295 300
Val Leu Leu Leu Ser Lys Asp Glu Asp Gly Lys Glu Leu Ser Asp
305 310 315
Glu Asp Ile Arg Ala Glu Ala Asp Thr Phe Met Phe Glu Gly His
320 325 330
Asp Thr Thr Ala Ser Gly Leu Ser Trp Val Leu Tyr His Leu Ala
335 340 345
Lys His Pro Glu Tyr Gln Glu Gln Cys Arg Gln Glu Val Gln Glu
350 355 360
Leu Leu Lys Asp Arg Glu Pro Ile Glu Ile Glu Trp Asp Asp Leu
365 370 375
Ala Gln Leu Pro Phe Leu Thr Met Cys Ile Lys Glu Ser Leu Arg
380 385 390
Leu His Pro Pro Val Pro Val Ile Ser Arg Cys Cys Thr Gln Asp
395 400 405
Phe Val Leu Pro Asp Gly Arg Val Ile Pro Lys Gly Ile Val Cys
410 415 420
Leu Ile Asn Ile Ile Gly Ile His Tyr Asn Pro Thr Val Trp Pro
425 430 435
Asp Pro Glu Val Tyr Asp Pro Phe Arg Phe Asp Gln Glu Asn Ile
440 445 450
Lys Glu Arg Ser Pro Leu Ala Phe Ile Pro Phe Ser Ala Gly Pro
455 460 465
Arg Asn Cys Ile Gly Gln Ala Phe Ala Met Ala Glu Met Lys Val
470 475 480
Val Leu Ala Leu Thr Leu Leu His Phe Arg Ile Leu Pro Thr His
485 490 495
Thr Glu Pro Arg Arg Lys Pro Glu Leu Ile Leu Arg Ala Glu Gly
500 505 510
Gly Leu Trp Leu Arg Val Glu Pro Leu Gly Ala Asn Ser Gln
515 520
11/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
<210> 11
<211> 369
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7256116CD1
<400> 11
Met Leu Pro Ile Thr Asp Arg Leu Leu His Leu Leu Gly Leu Glu
1 5 10 15
Lys Thr Ala Phe Arg Ile Tyr Ala Val Ser Thr Leu Leu Leu Phe
20 25 30
Leu Leu Phe Phe Leu Phe Arg Leu Leu Leu Arg Phe Leu Arg Leu
35 40 45
Cys Arg Ser Phe Tyr Ile Thr Cys Arg Arg Leu Arg Cys Phe Pro
50 55 60
Gln Pro Pro Arg Arg Asn Trp Leu Leu Gly His Leu Gly Met Tyr
65 70 75
Leu Pro Asn Glu Ala Gly Leu Gln Asp Glu Lys Lys Val Leu Asp
80 85 90
Asn Met His His Val Leu Leu Val Trp Met Gly Pro Val Leu Pro
95 100 105
Leu Leu Val Leu Val His Pro Asp Tyr Ile Lys Pro Leu Leu Gly
110 115 120
Ala Ser Ala Ala Ile Ala Pro Lys Asp Asp Leu Phe Tyr Gly Phe
125 130 135
Leu Lys Pro Trp Leu Gly Asp Gly Leu Leu Leu Ser Lys Gly Asp
140 145 150
Lys Trp Ser Arg His Arg Arg Leu Leu Thr Pro Ala Phe His Phe
155 160 165
Asp Ile Leu Lys Pro Tyr Met Lys Ile Phe Asn Gln Ser Ala Asp
170 175 180
Ile Met His Ala Lys Trp Arg His Leu Ala Glu Gly Ser Ala Val
185 190 195
Ser Leu Asp Met Phe Glu His Ile Ser Leu Met Thr Leu Asp Ser
200 205 210
Leu Gln Lys Cys Val Phe Ser Tyr Asn Ser Asn Cys Gln Glu Lys
215 220 225
Met Ser Asp Tyr Ile Ser Ala Ile Ile Glu Leu Ser Ala Leu Ser
230 235 240
Val Arg Arg Gln Tyr Arg Leu His His Tyr Leu Asp Phe Ile Tyr
245 250 255
Tyr Arg Ser Ala Asp Gly Arg Arg Phe Arg Gln Ala Cys Asp Met
260 265 270
Val His His Phe Thr Thr Glu Val Ile Gln Glu Arg Arg Arg Ala
275 280 285
Leu Arg Gln Gln Gly Ala Glu Ala Trp Leu Lys Ala Lys Gln Gly
290 295 300
Lys Thr Leu Asp Phe Ile Asp Val Leu Leu Leu Ala Arg Asp Glu
305 310 315
Asp Gly Lys Glu Leu Ser Asp Glu Asp Ile Arg Ala Glu Ala Asp
320 325 330
Thr Phe Met Phe Glu Gly His Asp Thr Thr Ile Gln Trp Asp Leu
335 340 345
Leu Gly Cys Cys Ser Ile Trp Gln Ser Ile Arg Asn Thr Arg Arg
350 355 360
Asn Ala Glu Lys Arg Phe Arg Lys Ser
365
<210> 12
<211> 144
<212> PRT
<213> Homo sapiens
<220>
12/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
<221> misc_feature
<223> Incyte ID No: 4210675CD1
<400> 12
Met Tyr Val Glu Gly Leu Lys Asp Leu Ser Asp Met Ile Met Phe
1 5 10 15
Gln Pro Leu Ser Leu Pro Glu Glu Lys Met Asn Leu Ala Tyr Ile
20 25 30
Leu Glu Arg Ala Thr Thr Arg Leu Phe Pro Val Cys Glu Lys Ala
35 40 45
Leu Arg Asp His Arg Gln Asp Phe Leu Val Gly Asn Arg Leu Ser
50 55 60
Trp Ala Asp Thr Gln Gln Pro Glu Val Ile Leu Met Thr Glu Glu
65 70 75
Cys Lys Pro Ser Val Leu Leu Gly Phe Pro Leu Leu Gln Lys Phe
80 85 90
Lys Ala Arg Ile Ile His Ile Pro Thr Ile Asn Lys Cys Leu Gln
95 100 105
Pro Gly Ser Gln Arg Lys Pro Pro Leu Asp Glu Glu Ser Ile Glu
110 115 120
Thr Val Lys Asn Ile Phe Lys Phe Glu His Gly Leu Phe Leu Lys
125 130 135
Asn Met Ile Thr Thr Leu Ala Glu Tyr
140
<210> 13
<211> 3878
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1642862CB1
<400> 13
ctttctcatc atggccttgc ctttgagatg accccacctg cgtccctgca gaaccacttc 60
cgttagctaa gctgcctcag atgaaaccta aactactccc cgatgctggc agaagaattt 120
cattgcagtc aaagcccctg tgtgaggcag cacccccagg ccaccccccg gaagcctggc 180
agcctctgca tccggctcat ccaccttccc tgagggccct cccagccaag cctgagcctc 240
agtttcctca tttctggggc gacccactca ccctcagaag ccgggtcctg cttcacagca 300
gaccccctga gccacaaagc cgtgactcct agagcgacac cacacaggag ctgggtgcag 360
cgggagcctg gccaagcccc tggcctctgt ccgacgctga agtgccaggt gcccctcctt 420
ctcctccctc cagagctcca aggtcctcgc taagaaggag ctgctctacg tgcccctcat 480
cggctggacg tggtactttc tggagattgt gttctgcaag cggaagtggg aggaggaccg 540
ggacaccgtg gtcgaagggc tgaggcgcct gtcggactac cccgagtaca tgtggtttct 600
cctgtactgc gaggggacgc gcttcacgga gaccaagcac cgcgttagca tggaggtggc 660
ggctgctaag gggcttcctg tcctcaagta ccacctgctg ccgcggacca agggcttcac 720
caccgcagtc aagtgcctcc gggggacagt cgcagctgtc tatgatgtaa ccctgaactt 780
cagaggaaac aagaacccgt ccctgctggg gatcctctac,gggaagaagt acgaggcgga 840
catgtgcgtg aggagatttc ctctggaaga catcccgctg gatgaaaagg aagcagctca 900
gtggcttcat aaactgtacc aggagaagga cgcgctccag gagatatata atcagaaggg 960
catgtttcca ggggagcagt ttaagcctgc ccggaggccg tggaccctcc tgaacttcct 1020
gtcctgggcc accattctcc tgtctcccct cttcagtttt gtcttgggcg tctttgccag 1080
cggatcacct ctcctgatcc tgactttctt ggggtttgtg ggagcagctt cctttggagt 1140
tcgcagactg ataggagtaa ctgagataga aaaaggctcc agctacggaa accaagagtt 1200
taagaaaaag gaataattaa tggctgtgac tgaacacacg cggccctgac ggtggtatcc 1260
agttaactca aaaccaacac acagagtgca ggaaaagaca attagaaact atttttctta 1320
ttaactggtg actaatatta acaaaacttg agccaagagt aaagaattca gaaggcctgt 1380
caggtgaagt cttcagcctc ccacagcgca gggtcccagc atctccacgc gcgcccgtgg 1440
gaggtgggtc cggccggaga ggcctcccgc ggacgccgtc tctccagaac tccgcttcca 1500
agagggagcc tttggctgct ttctctcctt aaacttagat caaatttttt ggtttttaat 1560
cagttatctt gggaacttaa cctggcccct cacctcttct gcaccccccg cccccgaaac 1620
tgtctcgtaa tgaatttctg ctgtcctcct gggagtggac ggccgggtcc cgtcccccgg 1680
gagcatcgct cggctcagca ccttggctcc cagtgggggc cccgtggagg gcgcccgtag 1740
tgataagcac accggcacga acgtcaggtc cattcctcga agtcggagcc ctcactctgc 1800
cctgtcctgg ggctggctga gggcgaacgc cccacctcac tttctagagc cctgtctgtc 1860
ctagctccta tctgaccttg tgtgtaaata cgtacatctg tttttaaagt ggatgggccc 1920
13/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
ctgagaactc agtgaaatgc agagttctcc atgcacctaa agctcctttg tcgctctcat 1980
ggctgtcaga tcctggtccc tccacactgg gtgctgggga gggaggaccc tcggggctac 2040
cgcgcgcccc cccatcccac agatcaggag ccaaggaggg agaacagggc agcctgtggg 2100
actctaggat gcttcagaag aagcgacggc accgtcaacc ctctgttttt taaaggtggt 2160
tggagactgt taacactgag ctcattgact tctagagatt ttatttttac tggttgatct 2220
cttggtggtt ttcaacttcc tgctggaaac tagaggtggg gcacccccca ccccccagcc 2280
tcgcactgtg tccttgggga aggcccgccc ccatcctggc cggtgtcact gtggcccggc 2340
cacccctgag cgcccagctc cctacctcct ggacgtctct gagagtccag gcagagcaga 2400
gggcagcgct cggccggtca tgctggctcc cttggccttg cagcgagccc ctggcccacg 2460
ccgagcgagg gatgcttctc cctacagcat gtccactccc ccggcatggc caggtggggc 2520
ccctggggca atggcagtgg tagaacgctc aacttggttg cggtaccatc agcccacctg 2580
catttggctt tcgacttgct tgttctaagt cacagcgccc tcatcttttt agcaaggtaa 2640
aaaaaccaaa atgggtgtta tctctgatat cttgaaacca gcgttctgaa tagaggtagg 2700
ttgagttttc taggggaaaa caaatggaga aaagaggcat gaagaaaagt aaaccgagaa 2760
cataattagg catcgggcct aagtgtcctg gggagattgg aggggacggc agcgttctgc 2820
atgatggagg cgctgccggg ccccgggtct gtgggggccg tgctctcagg gcgtgtgcgg 2880
gacgccacct gtgcacacct gctcagagca cggctcctcg caggggtgaa ggggcagacc 2940
aacgaaacca gatgagacca acgacaccat gcgagacacg cttgcagaca ctgttgtttt 3000
ggaaatgtgc ttccctccat ctgaaatctc atccctccac ccgcccactc gggcagctgt 3060
gctgtgggca gggcatgcgc tcccctggct gagcacccca gagattctcc tgcaccttcc 3120
tcatgccgca cgctgctcat ccgtctccat gtgtgtttag atccatgcca ttcactgact 3180
cactaacacc tgcaaaatct ttaaggaaaa aagctgaagg gtacgaccat gcacatatgt 3240
gacctggaaa atgcaaattt agatctttta tgatttaatt attattgttt cccatagaag 3300
ttccctccct ttgaaattaa tatataatgt ataaattctg cactgagcca tggcggagct 3360
gggcagcccc taggttagag tggagacgga ggcccaggcg caggggtcac acctcatctg 3420
gtttccttcc catctcacag cttagcttgt gcttctcaac accaagtctt taagagcaat 3480
aaaaactaca ccatgaatgt ttgaattttt ttttttgggg ggggggaggg tggattttgc 3540
ttttcatcca gaaggaaaag gggaggagag ctcctttaca ttttttaaat taaattcata 3600
aatcccagaa cagtcttttt tttttccttt tccctttaca ccctatttct gagcttaatc 3660
cagttgatgt tttgtccaat ttcaggctga gtgcccaggc tgaagcaatt ctgtagccca 3720
cagtccgtgc tggccactgt cggggtgagg cactttctag gcctggaatc gttgatgccc 3780
tctgtgccca gtctttgagc caggccgagg acaggaaggg cattgctggc ctgtagcccc 3840
tgttacccac ccagagccag gggccacacg tgaaggct 3878
<210> 14
<211> 1645
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3861612CB1
<400> 14
attgacttaa tattgttcta gaatagcctt tcagctacaa gaggttatat ataaatcaaa 60
agcttcttga gtagaacttc ttagaattgt agaagctgct caatacggaa catattctca 120
gtcctcctct ggtctacaaa gcctgtgatt tcttgtctat ggacagaacg tctggtttaa 180
tctacaggaa cccataactt cctgaagctt tatgcttaac agtgacaacg tgagtcagtt 240
gaattttatt gtgtttcagt ccgtagagta ttagctacag aaacctttcc attgccatac 300
tgagaaactg cagcaggcag tgtgcctaca ggtctacaaa gaaacttcag atcatcttct 360
tgagggaaag aagctgaagt gctacataag atgcttgtgc ttcataactc tcagaagctg 420
cagattctgt ataaatcctt agaaaagagc atccctgaat ccataaaggt atatggcgcc 480
attttcaaca taaaagataa aaaccctttc aacatggagg tgctggtaga tgcctggcca 540
gattaccaga tcgtcattac ccggcctcag aaacaggaga tgaaagatga ccaggatcat 600
tataccaaca cttaccacat cttcaccaaa gctcctgaca aattagagga agtcctgtca 660
tactccaatg taatcagctg ggagcaaact ttgcagatcc aaggttgcca agagggcttg 720
gatgaagcaa taagaaaggt tgcaacttca aaatcagtgc aggtagatta catgaaaacc 780
atcctcttta taccggaatt accaaagaaa cacaagacct caagtaatga caagatggag 840
ttatttgaag tggatgatga taacaaggaa ggaaactttt caaacatgtt cttagatgct 900
tcacatgcag gtcttgtgaa tgaacactgg gcctttggga aaaatgagag gagcttgaaa 960
tatattgaac gctgcctcca ggattttcta ggatttggtg tgctgggtcc agagggccag 1020
cttgtctctt ggattgtgat ggaacagtcc tgtgagttga gaatgggtta tactgtcccc 1080
aaatacagac accaaggcaa catgttgcaa attggttatc atcttgaaaa gtatctttct 1140
cagaaagaaa tcccatttta tttccatgtg gcagataata atgagaaaag cctacaggca 1200
ctgaacaatt tggggtttaa gatttgtcct tgtggctggc atcagtggaa atgcaccccc 1260
aagaaatatt gttgattgat tccactgtcc atttcaaatc tttcttatca gtaaaaaaac 1320
attaattcaa acacaagcat tgtgatctac attagcacaa aatgcaactg attatctagg 1380
14/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
atctgtgtat tacttaagct cacccttaac agttttacct tccttctcct ctgtattctt 1440
acagaaaatt agaagctcaa ttttatggtc tcataatttc ctttatgaca gacatctcag 1500
aattaaaatc acccaaagcc aatcattagt gccaagataa ccctttaacg gcaacacttt 1560
cttaaatgaa gactatttct ttcatgaaaa aattcacttt tatgactttc ttgttaaaat 1620
aaaaagtctg cttttaaaaa aaaaa 1645
<210> 15
<211> 798
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472055CB1
<400> 15
atggccctgg agctctacat ggacctgctg tcagcaccct gccgtgccgt ctacatcttc 60
tcgaagaagc atgacatcca gttcaacttt cagtttgtgg atctgctgaa aggtcaccac 120
cacagcaaag aatacattga catcaacccc ctcaggaagc tgcccagcct caaagatggg 180
aaatttatct taagtgaaag cccccaactc ctttactacc tgtgccgcaa gtacagcgca 240
ccatcgcact ggtgcccgcc agacccgcac gcacgtgccc gtgtggatga gttcgtggct 300
tggcaacaca cggcctttca gctgcccatg aagaagatag tctggctcaa gttgctgatc 360
ccaaagataa caggggagga agtttcagct gagaagatgg agcatgcagt ggaagaggtg 420
aagaacagcc tgcagctctt tgaggagtat tttctgcagg ataagatgtt catcaccggg 480
aaccaaatct cactggctga cctggtggcc gtggtggaga tgatgcagcc catggcagcc 540
aactataatg tcttcctcaa cagctccaag ctagctgagt ggcgtatgca ggtggagctg 600
aatattggct ctggcctctt tagggaggcc catgatcgac taatgcagtt ggccgactgg 660
gacttttcaa cattggattc aatggtcaag gagaatattt ctgagttgct gaagaagagc 720
aggtgaccct aggcgcagcc tgtcccgcag ggcctggctg gcttagcaat ttgagccacc 780
ttccttaaag gaaatgtt 798
<210> 16
<211> 2478
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1923521CB1
<400> 16
ccggtcttcg ccggccccgg cccctggcga gatgccgtgt ggggaggatt ggctcagcca 60
cccgctggga atcgtgcagg gattcttcgc ccaaaatgga gttaatcctg actgggagaa 120
gaaagtaatt gagtatttta aggagaagct gaaggaaaat aatgctccta agtgggtacc 180
atcactgaac gaagttcccc ttcattattt gaaacctaat agttttgtga aatttcgttg 240
catgattcag gatatgtttg accctgagtt ttacatggga gtttatgaaa cggttaacca 300
aaacacaaaa gcacatgttc ttcattttgg aaaatataga gatgtagcag agtgtgggcc 360
tcaacaagaa cttgatttaa actctccacg aaataccact ttggaaagac agactttcta 420
ttgtgttccg gtgcctgggg aatctacgtg ggtaaaagaa gcctatgtta atgcaaacca 480
agctcgagtc agtccctcaa catcctacac tcctagtcgc cacaagagga gttatgaaga 540
tgatgacgat atggacctac agcccaataa gcagaaagac caacatgcag gtgccagaca 600
agcagggagt gttggtggtc ttcaatggtg tggagagcca aaacgtttag aaactgaagc 660
ttctactggg caacagctga actctctgaa cttgtcttct ccttttgatt tgaattttcc 720
attgccagga gagaagggcc ctgcatgcct tgtgaaggtt tatgaagatt gggattgttt 780
caaagtaaat gacattcttg agctatatgg catactgtct gtggatcctg tgctgagtat 840
actgaataat gatgaaaggg atgcctctgc actgctggat ccgatggagt gcacagacac 900
agcagaggag cagagagtac acagtcctcc tgcttcatta gtgccgagaa ttcatgtgat 960
cttagcccag aagttgcaac acatcaaccc attattgcct gcctgcctta acaaagagga 1020
gagcaaaacc tttgtttcaa gtttcatgtc cgaattgtct ccagtcagag cagaacttct 1080
tgggttcctt actcatgccc ttctggggga tagtttggct gctgaatacc ttatattaca 1140
tctcatctcc acagtatata caagaagaga tgtccttcca ctaggaaaat ttacagttaa 1200
cttgagtggt tgcccacgga atagtacctt cacagaacac ttgtatcgaa ttattcaaca 1260
tcttgttcca gcatcttttc gtctgcagat gactatagag aacatgaacc atttgaaatt 1320
cattccccac aaagactaca cagccaatcg cttggtcagt gggctcctcc agctgcccag 1380
caatacttcc cttgtaatcg atgagactct cctggaacag gggcagctgg ataccccagg 1440
tgttcataat gtgacagccc tgagcaacct cataacgtgg cagaaggtgg attatgactt 1500
cagctaccat cagatggaat tcccctgcaa tattaacgtt ttcattactt cggaggggag 1560
15/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
gtcactcctc ccggcagact gccagattca cttacagccc cagctaattc caccaaacat 1620
ggaggagtac atgaacagcc ttctctcagc ggtgctgcct tccgtgctga acaaattccg 1680
catttatcta actcttttga gattcttgga atatagcata tctgatgaaa taaccaaggc 1740
agttgaagat gactttgtgg aaatgcggaa gaacgaccct cagagcatca ctgctgatga 1800
tcttcaccag ctgctcgtgg tggctcggtg tctgtctctc agtgctggtc agacaacgct 1860
gtcaagagaa cgatggctga gagcaaagca gctagagtct ttaagaagaa cgaggcttca 1920
gcagcaaaaa tgtgtgaatg gaaatgaact ttaaagatgt aatacctatg aagagtaatg 1980
ggcaaactgt agccacataa ttgtaaaatt cagatattca tttataccac attgttttat 2040
aggtaatttc tatcacaaac cagtgacatt tcctgaaatc aagcctggta acacctgatg 2100
tttatatgat attcagtaag gacttttacc ttactgattt catggagctt ttgaagtttg 2160
ttttataata attatataaa ttagtaatga tgtaaaaaaa gtatttgata ttaaaagttt 2220
aatattgata atgttgctga ttgtaccatt tccttagctt cagctgagtc ataggccaga 2280
ctgttgaaat gctgaaatga agaaggttgt tgcagtttca aagtcagagg aatcgtgctt 2340
cggatttctt atgttttcta gttctctgtt tttccagttc acagtgggtt ggggtgcatt 2400
cagtagtcca tctttgggga acggaggcgt acttgccatt gattcacatg actacatgaa 2460
attctgtact gtcatttc 2478
<210> 17
<211> 3348
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1558210CB1
<400> 17
cccaaaagaa gcaccagatc agcaaaaaaa gaagatgggc cccccgagcc tcgtgctgtg 60
cttgctgtcc gcaactgtgt tctccctgct gggtggaagc tcggccttcc tgtcgcacca 120
ccgcctgaaa ggcaggtttc agagggaccg caggaacatc cgccccaaca tcatcctggt 180
gctgacggac gaccaggatg tggagctggg ttccatgcag gtgatgaaca agacccggcg 240
cattatggag cagggcgggg cgcacttcat caacgccttc gtgaccacac ccatgtgctg 300
cccctcacgc tcctccatcc tcactggcaa gtacgtccac aaccacaaca cctacaccaa 360
caatgagaac tgctcctcgc cctcctggca ggcacagcac gagagccgca cctttgccgt 420
gtacctcaat agcactggct accggacagc tttcttcggg aagtatctta atgaatacaa 480
cggctcctac gtgccacccg gctggaagga gtgggtcgga ctccttaaaa actcccgctt 540
ttataactac acgctgtgtc ggaacggggt gaaagagaag cacggctccg actactccaa 600
ggattacctc acagacctca tcaccaatga cagcgtgagc ttcttccgca cgtccaagaa 660
gatgtacccg cacaggccag tcctcatggt catcagccat gcagcccccc acggccctga 720
ggattcagcc ccacaatatt cacgcctctt cccaaacgca tctcagcaca tcacgccgag 780
ctacaactac gcgcccaacc cggacaaaca ctggatcatg cgctacacgg ggcccatgaa 840
gcccatccac atggaattca ccaacatgct ccagcggaag cgcttgcaga ccctcatgtc 900
ggtggacgac tccatggaga cgatttacaa catgctggtt gagacgggcg agctggacaa 960
cacgtacatc gtatacaccg ccgaccacgg ttaccacatc ggccagtttg gcctggtgaa 1020
agggaaatcc atgccatatg agtttgacat cagggtcccg ttctacgtga ggggccccaa 1080
cgtggaagcc ggctgtctga atccccacat cgtcctcaac attgacctgg cccccaccat 1140
cctggacatt gcaggcctgg acatacctgc ggatatggac gggaaatcca tcctcaagct 1200
gctggacacg gagcggccgg tgaatcggtt tcacttgaaa aagaagatga gggtctggcg 1260
ggactccttc ttggtggaga gaggcaagct gctacacaag agagacaatg acaaggtgga 1320
cgcccaggag gagaactttc tgcccaagta ccagcgtgtg aaggacctgt gtcagcgtgc 1380
tgagtaccag acggcgtgtg agcagctggg acagaagtgg cagtgtgtgg aggacgccac 1440
ggggaagctg aagCtgCata agtgcaaggg ccccatgcgg ctgggcggca gcagagccct 1500
ctccaacctc gtgcccaagt actacgggca gggcagcgag gcctgcacct gtgacagcgg 1560
ggactacaag ctcagcctgg ccggacgccg gaaaaaactc ttcaagaaga agtacaaggc 1620
cagctatgtc cgcagtcgct ccatccgctc agtggccatc gaggtggacg gcagggtgta 1680
ccacgtaggc ctgggtgatg ccgcccagcc ccgaaacctc accaagcggc actggccagg 1740
ggcccctgag gaccaagatg acaaggatgg tggggacttc agtggcactg gaggccttcc 1800
cgactactca gccgccaacc ccattaaagt gacacatcgg tgctacatcc tagagaacga 1860
cacagtccag tgtgacctgg acctgtacaa gtccctgcag gcctggaaag accacaagct 1920
gcacatcgac cacgagattg aaaccctgca gaacaaaatt aagaacctga gggaagtccg 1980
aggtcacctg aagaaaaagc ggccagaaga atgtgactgt cacaaaatca gctaccacac 2040
ccagcacaaa ggccgcctca agcacagagg ctccagtctg catcctttca ggaagggcct 2100
gcaagagaag gacaaggtgt ggctgttgcg ggagcagaag cgcaagaaga aactccgcaa 2160
gctgctcaag cgcctgcaga acaacgacac gtgcagcatg ccaggcctca cgtgcttcac 2220
ccacgacaac cagcactggc agacggcgcc tttctggaca ctggggcctt tctgtgcctg 2280
caccagcgcc aacaataaca cgtactggtg catgaggacc atcaatgaga ctcacaattt 2340
cctcttctgt gaatttgcaa ctggcttcct agagtacttt gatctcaaca cagaccccta 2400
16/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
ccagctgatg aatgcagtga acacactgga cagggatgtc ctcaaccagc tacacgtaca 2460
gctcatggag ctgaggagct gcaagggtta caagcagtgt aacccccgga ctcgaaacat 2520
ggacctggga cttaaagatg gaggaagcta tgagcaatac aggcagtttc agcgtcgaaa 2580
gtggccagaa atgaagagac cttcttccaa atcactggga caactgtggg aaggctggga 2640
aggttaagaa acaacagagg tggacctcca aaaacataga ggcatcacct gactgcacag 2700
gcaatgaaaa accatgtggg tgatttccag cagacctgtg ctattggcca ggaggcctga 2760
gaaagcaagc acgcactctc agtcaacatg acagattctg gaggataacc agcaggagca 2820
gagataactt caggaagtcc atttttgccc ctgcttttgc tttggattat acctcaccag 2880
ctgcacaaaa tgcatttttt cgtatcaaaa agtcaccact aaccctcccc cagaagctca 2940
caaaggaaaa cggagagagc gagcgagaga gatttccttg gaaatttctc ccaagggcga 3000
aagtcattgg aatttttaaa tcatagggga aaagcagtcc tgttctaaat cctcttattc 3060
ttttggtttg tcacaaagaa ggaactaaga agcaggacag aggcaacgtg gagaggctga 3120
aaacagtgca gagacgtttg acaatgagtc agtagcacaa aagagatgac atttacctag 3180
cactataaac cctggttgcc tctgaagaaa ctgccttcat tgtatatatg tgactattta 3240
catgtaatca acatgggaac ttttagggga acctaataag aaatcccaat tttcaggagt 3300
ggtggtgtca ataaacgctc tgtggccagt gtaaaagaaa aaaaaaaa 3348
<210> 18
<211> 3844
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5629033CB1
<400> 18
gaccttcagc tgccgcggtc gctccgagcg gcgggccgca gagccaccaa aatgccagaa 60
gagatggaca agccactgat cagcctccac ctggtggaca gcgatagtag ccttgccaag 120
gtccccgatg aggcccccaa agtgggcatc ctgggtagcg gggactttgc ccgctccctg 180
gccacacgcc tggtgggctc tggcttcaaa gtggtggtgg ggagccgcaa ccccaaacgc 240
acagccaggc tgtttccctc agcggcccaa gtgactttcc aagaggaggc agtgagctcc 300
ccggaggtca tctttgtggc tgtgttccgg gagcactact cttcactgtg cagtctcagt 360
gaccagctgg cgggcaagat cctggtggat gtgagcaacc ctacagagca agagcacctt 420
cagcatcgtg agtccaatgc tgagtacctg gcctccctct tccccacttg cacagtggtc 480
aaggccttca atgtcatctc tgcctggacc ctgcaggctg gcccaaggga tggtaacagg 540
caggtgccca tctgcggtga ccagccagaa gccaagcgtg ctgtctcgga gatggcgctc 600
gccatgggct tcatgcccgt ggacatggga tccctggcgt cagcctggga ggtggaggcc 660
atgcccctgc gcctcctccc ggcctggaag gtgcccaccc tgctggccct ggggctcttc 720
gtctgcttct atgcctacaa cttcgtccgg gacgttctgc agccctatgt gcaggaaagc 780
cagaacaagt tcttcaagct gcccgtgtcc gtggtcaaca ccacactgcc gtgcgtggcc 840
tacgtgctgc tgtcactcgt gtacttgccc ggcgtgctgg cggctgccct gcagctgcgg 900
cgcggcacca agtaccagcg cttccccgac tggctggacc actggctaca gcaccgcaag 960
cagatcgggc tgctcagctt cttctgcgcc gccctgcacg ccctctacag cttctgcttg 1020
ccgctgcgcc gcgcccaccg ctacgacctg gtcaacctgg cagtcaagca ggtcttggcc 1080
aacaagagcc acctctgggt ggaggaggag gtctggcgga tggagatcta cctctccctg 1140
ggagtgctgg ccctcggcac gttgtccctg ctggccgtga cctcactgcc gtccattgca 1200
aactcgctca actggaggga gttcagcttc gttcagtcct cactgggctt tgtggccctc 1260
gtgctgagca cactgcacac gctcacctac ggctggaccc gcgccttcga ggagagccgc 1320
tacaagttct acctgcctcc caccttcacg ctcacgctgc tggtgccctg cgtcgtcatc 1380
ctggccaaag ccctgtttct cctgccctgc atcagccgca gactcgccag gatccggaga 1440
ggctgggaga gggagagcac catcaagttc acgctgccca cagaccacgc cctggccgag 1500
aagacgagcc acgtatgagg tgcctgccct gggctctgga ccccgggcac acgagggacg 1560
gtgccctgag cccgttaggt tttcttttct tggtggtgca aagtggtata actgtgtgca 1620
aataggaggt ttgaggtcca aattcctggg actcaaatgt atgcagtact attcagaatg 1680
atatacacac atatgtgtat atgtatttac atatattcca catatataac aggatttgca 1740
attatacata gctagctaaa aagttgggtc tctgagattt caacttgtag atttaaaaac 1800
aagtgccgta cgttaagaga agagcagatc atgctattgt gacatttgca gagatataca 1860
cacacttttt gtacagaaga ggcttgtgct gtggtgggtt cgatttatcc ctgcccaccc 1920
catccccaca acttcccttt tgctacttcc ccaaggctct tgcagagcta gggctctgaa 1980
ggggagggaa ggcaacggct ctgcccagag ccatccctgg agcatgtgag cagcggctgg 2040
tctcttccct ccacctgggg cagcagcagg aggcctgggg aggaggaaaa tcaggcagtc 2100
ggcctggagt ctgtgcctgg tcctttgccc ggtggtggga ggatggaggg attgggctga 2160
agctgctcca cctcatcctt gctgagtggg ggagacattt tccctgaaag tcagaagtca 2220
ccatagagcc tgcaaatgga tcctcctgtg agagtgacgt cacctccttt ccagagccat 2280
tagtgagcct ggcttgggaa caagtgtaat ttccttccct cctttaacct ggcgatgagc 2340
gtcctttaaa ccactgtgcc ttctcaccct ttccatcttc agtttgaacg actcccagga 2400
17/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
aggcctagag cagacccttt agaaatcagc ccaaggggga gagcaagaga aaacactcta 2460
gggagtaaag ctccccgggc gtcagagttg agccctgcct gggctgaagg actgtcttca 2520
cgaagtcagt cctgaggaaa aatattgggg actccaaatg tcctctggca gaggacccag 2580
aaaaccacac tggctccaac ttcctcctca tggggcatta cacttcaaaa cagtggggag 2640
caacttttcc accaaagcta caaacctaaa atgctgctgc cccaaagcac aagagggaag 2700
agcaccgccg gggccacagg acgtctgtcc tccagtcaca ggccatcctt gctgctccct 2760
actgactcta gcttacttcc cctgtgaaga aacaggtgtt ctcggctgag cccccaaccc 2820
tctgcagaac caggttgatc tgccacagaa aaagcatctt tgaagacaaa gagggtgagg 2880
tcttcatgag tctcctgggc ccaaagccat cttctgatgg aaggaagaga gtagggccag 2940
tgaaggctgc ccagagagaa tgtcacagat gaggctgccc ctgccccccc tccgccaggg 3000
aggtttcatg agctcatgtc tatgcagcac ataagggttc ttcagtgaaa agcaggagaa 3060
gagcccactg caaggatagc tcattaggca catgaccgat gcagggaagg ccatgccggg 3120
gaagctcttc ctgcaggtat tttccatctg ctgtgccaag gctgagcggc agaaacttgt 3180
ctcataaatt ggcactgatg gagcatcagc tgtggcccac agagagcctt gctgagaagg 3240
gggcaggtaa agcagagatt ttagcattgc cttggcataa caagggccca tcgattccct 3300
actaatgaga ggcagggaga gcatgggcaa tggagaccca ccaatgatcc ccaaccccgg 3360
tgggtactgg ctgcctgccc tgggccaggg aatggctcct tataccaaag atgctggcac 3420
atagcagaac ccagtgcacg tcctcccctt cccacccacc tctggctgaa ggtgctcaag 3480
agggaagcaa ttataaggtg ggtggcagga gggaacaggt gccacctgct ggacaatcac 3540
acgaaaggca ggcgggctgt gtactgggcc ctgactgtgc gtccactgct gtcttcccta 3600
cctcaccagg ctactggcag cagcatcccg agagcacatc atctccacag cctggtaaat 3660
tccatgtgcc tctgggtaca aaagtgcctc aacgacatgc tctggaaatc ccaaatgcca 3720
cagtctgagg ttgatatcta aaatctatgc cttcaaaaga gtctctgttt ttttttttta 3780
acctggtaga cggtataaaa gcagtgcaaa taaacaccta accttctgca aaaaaaaaaa 3840
aaaa 3844
<210> 19
<211> 2278
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2750679CB1
<400> 19
ccaaggcccg gcagcctcag tccactgctg ggcctggaac acggagcagt ggctgccctg 60
cgaggaggtc ctagagcagc tccagcagga tgacagctcc ccatctgtgc tcctgcctgc 120
cggccatcct caggccactc gccatgggcg gctgcttctc caaacccaaa ccagtggagc 180
tcaagatcga ggtggtgctg cctgagaagg agcgaggcaa ggaggagctg tcggccagtg 240
ggaagggcag cccccgggcc taccagggca atggcacggc ccgccacttc cacacggagg 300
agcgcctgtc cacccctcac ccctacccca gccctcagga ttgcgtggag gctgctgtct 360
gccacgtcaa ggacctcgag aatggccaga tgcgggaagt ggagctgggc tgggggaagg 420
tgttgctggt gaaggacaat ggggagttcc acgccctggg ccataagtgt ccgcactacg 480
gcgcacccct ggtgaaaggc gttctgtccc gtggtcgggt gcgctgcccc tggcacggcg 540
cctgcttcaa catcagcact ggggacctgg aggacttccc tggcctggac agtctacaca 600
agttccaggt gaagattgag aaggagaagg tgtacgtccg ggccagcaag caggccctac 660
agctgcagcg aaggaccaag gtgatggcca agtgtatctc tccaagtgct gggtacagca 720
gtagcaccaa tgtgctcatt gtgggtgcag gtgcagctgg cctggtgtgt gcagagacac 780
tgcggcagga gggcttctcc gaccggatcg tcctgtgcac gctagaccgg caccttccct 840
acgaccgtcc caagctcagc aagtccctgg acacacagcc tgagcagctg gccctgaggc 900
ccaaggagtt tttccgagcc tatggcatcg aggtgctcac cgaggctcag gtggtcacag 960
tggacgtgag aactaagaag gtcgtgttca aggatggctt caagctggag tacagcaagc 1020
tgctgctggc accaggggag cagccccaag actctgagct gcaaaggcaa agaagtggag 1080
aacgtgttca ctatccggac gccagaggat gccaatcgcg tggtgaggct ggcccgaggc 1140
cgcaacgtgg tcgtcgtggg agccggcttc ctggggatgg aggtggccgc ttacctgacg 1200
gagaaggccc actctgtgtc tgtggtggag ctggaggaga cgcccttcag gaggttcctg 1260
ggggagcgcg tgggtcgtgc cctcatgaag atgtttgaga acaaccgggt gaagttctac 1320
atgcagacgg aggtgtctga gctgcggggc caggagggaa agctgaagga ggttgtgctg 1380
aagagcagca aggtcgtgcg ggctgacgtc tgcgtggtgg gcattggtgc agtgcccgcc 1440
acaggcttcc tgaggcaaag cggcatcggt ttggattccc gaggcttcat ccctgtcaac 1500
aagatgatgc agaccaatgt cccaggcgtg tttgcagctg gcgatgctgt caccttcccc 1560
cttgcctgga ggaacaaccg caaagtgaac attccacatt ggcagatggc tcatgctcag 1620
gggcgcgtgg cagcccagaa catgttggcg caggaggcgg agatgagcac tgtgccctac 1680
ctctggaccg ccatgtttgg caagagcctg cgctacgcgg gctacggaga aggcttcgac 1740
gacgtcatca tccaggggga tctggaggag ctgaagtttg tggcttttta cactaaaggc 1800
gacgaggtga tcgccgtggc cagcatgaac tacgatccca ttgtgtccaa ggtcgctgag 1860
18/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
gtgctggcct caggccgtgc catccggaag cgggaggtgg agactggcga catgtcctgg 1920
cttacgggga aaggatcctg agctcacatg cagtagactt gggcaggcaa agggggcacc 1980
aagggcacag gccaagcctt gggggcaggt gccaatctcc agtcccagga tcccccaggg 2040
cagaacctga gccctcccag tgcttgcctt cagccacctg gctcccctcc tgggaggcct 2100
ctgctggatc cagaagatgc tcaaccctca aggcctctgc tgccactgac agctggcact 2160
ggaggcagga caagccctgc ctcttctccc tctattggga ctggtcccct gaagaaccct 2220
gcaacatgtt agacattacc gtaaaattaa aacgcacaaa tttgcagaaa aaaaaaaa 2278
<210> 20
<211> 1288
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1570911CB1
<400> 20
tgaatatatt cgcgcgctct ttgcagctgc ctgaattctt ccttccccag catccccctc 60
cgcccggtca cccagacggc cttctccagc cttgccgagc ttaagacccg tccctgctcc 120
tgaccatcac cgtcactggg gtcactgtgc tcgtgttggt cctgaagagc atgaactcca 180
ggaggagaga gccaatcacc ttacaggacc ctgaagccaa gtacccgctg cccttgattg 240
agaaagagaa aatcagccac aacacccgga ggttccgctt tggactgcct tcgccggacc 300
atgtcttagg gcttcctgta ggtaactatg tccagctctt ggcaaaaatc gataatgaat 360
tggtggtcag ggcttacacc cctgtctcca gtgatgatga cagaggcttt gtggacctaa 420
ttataaagat ctacttcaaa aatgtacacc cccaatatcc tgaaggtggg aagatgactc 480
agtatttgga gaacatgaaa atcggggaga ccatcttttt tcgagggcca aggggacgct 540
tgttttacca tgggccaggg aatcttggaa tcagaccaga ccagacgagt gagcctaaaa 600
aaacactggc cgatcacctg ggaatgattg ctgggggcac aggcatcaca cccatgttgc 660
agctcattcg ccacatcacc aaggacccca gtgacaggac caggatgtcc ctcatctttg 720
ccaaccagac agaggaggat atcttggtca gaaaagagct tgaagaaatt gccaggactc 780
acccagacca gttcgacctg tggtacaccc tggacaggcc tcccattggc tggaagtaca 840
gctcaggctt cgttactgcc gacatgatca aggagcacct tcctcctcca gcgaagtcca 900
cgctcatcct ggtgtgtggc ccgccaccac tgatccagac ggcggctcac cctaacctgg 960
agaagctggg ttatacccag gacatgattt tcacctacta acaaacacct ccatgtgc.tc 1020
agcaaatttg catgtccctt ttcatctgtt tcagagtaag ttcaatttca ccacggtaaa 1080
ctgggatgtt ttcaaaagtg ccttgccatg taccttcgcg cacacactgg ttctcctctt 1140
ttgggtgtgg gcctaacaaa aagggctcaa ggggctggag actggctgct ggggcctcct 1200
tgcttggagg ctggcaagag ctccatttca gtatctttct ccgtggtttt gtgaaataaa 1260,.
ctcaagtaca aagcagaaaa aaaaaaaa 1288
<210> 21
<211> 4660
<212> DNA
<213> Homo Sapiens
<220> '
<221> misc_feature
<223> Incyte ID No: 1959720CB1
<400> 21
cgccgctccg gtcccctccc gtcgggccct cccctccccc gccgcggccg gcacagccaa 60
tcccccgagc ggccgccaac atgctctttg agggcttgga tctggtgtcg gcgctggcca 120
ccctcgccgc gtgcctggtg tccgtgacgc tgctgctggc cgtgtcgcag cagctgtggc 180
agctgcgctg ggccgccact cgcgacaaga gctgcaagct gcccatcccc aagggatcca 240
tgggcttccc gctcatcgga gagaccggcc actggctgct gcaggtttct ggcttccagt 300
cgtcgcggag ggagaagtat ggcaacgtgt tcaagacgca tttgttgggg cggccgctga 360
tacgcgtgac cggcgcggag aacgtgcgca agatcctcat gggcgagcac cacctcgtga 420
gcaccgagtg gcctcgcagc acccgcatgt tgctgggccc caacacggtg tccaattcca 480
ttggcgacat ccaccgcaac aagcgcaagg tcttctccaa gatcttcagc cacgaggccc 540
tggagagtta cctgcccaag atccagctgg tgatccagga cacactgcgc gcctggagca 600
gccaccccga ggccatcaac gtgtaccagg aggcgcagaa gctgaccttc cgcatggcca 660
tccgggtgct gctgggcttc agcatccctg aggaggacct tgggcacctc tttgaggtct 720
accagcagtt tgtggacaat gtcttctccc tgcctgtcga cctgcccttc agtggctacc 780
ggcggggcat tcaggctcgg cagatcctgc agaaggggct ggagaaggcc atccgggaga 840
agctgcagtg cacacagggc aaggactact tggacgtcct ggacctcctc attgagagca 900
gcaaggagca cgggaaggag atgaccatgc aggagctgaa ggacgggacc ctggagctga 960
19/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
tctttgcggc ctatgccacc acggcCagcg ccagcacctc actcatcatg cagc'tgctga 1020
agcaccccac tgtgctggag aagctgcggg atgagctgcg ggctcatggc atcctgcaca 1080
gtggcggctg cccctgcgag ggcacactgc gcctggacac gctcagtggg ctgcgctacc 1140
tggactgcgt catcaaggag gtcatgcgcc tgttcacgcc catttccggc ggctaccgca 1200
ctgtgctgca gaccttcgag cttgatggtt tccagatccc caaaggctgg agtgtcatgt 1260
atagcatccg ggacacccat gacacagcgc ccgtgttcaa agacgtgaac gtgttcgacc 1320
ccgatcgctt cagccaggcg cggagcgagg acaaggatgg ccgcttccat tacctcccgt 1380
tcggtggcgg tgtccggacc tgcctgggca agcacctggc caagctgttc ctgaaggtgc 1440
tggcggtgga gctggctagc accagccgct ttgagctggc cacacggacc ttcccccgca 1500
tcaccttggt ccccgtcctg caccccgtgg atggcctcag cgtcaagttc tttggcctgg 1560
actccaacca gaacgagatc ctgccggaga cggaggccat gctgagcgcc acagtctaac 1620
ccaagaccca cccgcctcag cccagcccag gcagcggggt ggtgcttgtg ggaggtagaa 1680
acctgtgtgt gggagggggc cggaacgggg agggcgagtg gcccccatac ttgccctccc 1740
ttgctccccc ttcctggcaa accctaccca aagccagtgg gccccattcc tagggctggg 1800
ctccccttct ggctccagct tccctccagc cactccccat ttaccatcag ctcagcccct 1860
gggaagggcg tggcaggggc tctgcatgcc cgtgacagtg ttaggtgtca gcgcgtgcta 1920
cagtgttttt gtgatgttct gaactgctcc cttccctccg ttcctttcgg acccttttag 1980
ctggggttgg gggacgggaa gagccgtgcc cccttgggcg cactcttcag cgtctcctcc 2040
tcctgcgccc ccactgcgtc tgcccaggaa cagcatcctg ggtagcagaa caggagtcaa 2100
ccttggcggg gcgggggctg cgtccaacct ggagattgcc cttccctatg ccacggttcc 2160
caccctccct caccagtttg gacaatttga aattacctat tgctgctact tgttctgtcc 2220
tctgaccttg gggcaaagga gccccaggcc ctgtctcccc agcatcctcc ctggtggccc 2280
tgggcaggtg cactgacacc cccaccttcc catcccctgc tgaaccaggc cctgttacac 2340
acagccgcct aaggcccgcg gctcatgtgc tgcccgcccc catatttatt cactgataga 2400
gaatcttggg gatgctgggg tctggagtga acatctcctc cccttcatgc cctagcctgt 2460
gttctagctg tcctggcgag acttctgtga gtgaagagga aggggtctct ggtcaaaccc 2520
agcccccagg gcctagggtt gaaagccttc cccggctccg ggcattattt gggtttaatc 2580
tcggagcctc actcctggac tgaagtccgg tgcctctgcc ttatccctgg tggagatgga 2640
atgtggccca ttgcctcctc cctctcctgt caaaaaccct gatcaggtag atttggaggc 2700
ggccacgatt tcctgtttgg cccctgttca ccccagtgca ctggccctga ctccaggcgt 2760
gagtatgggg aaggatacgg gttcttctga cggggagcaa gggcctccgt cttcccttcc 2820
ttaactctcc ccctttgccc tccgccctga aaaaggtgtc cttgaagtcc cttccacctc 2880
tatgccactg tctgcttagc ccagctcagg ggtggggaag aggcgaaagc gtgggggagg 2940
tgagcgcagc ggcagttctg cctcggagct gatttcaggg ccctgtgtgg tttccggaca 3000
gctgcgggaa ggctgccgca gctgaagctg aagaggcggc tacgtgcggt ttgtcagggg 3060
gattgggttg aaaactggcc agtcgggatg actgggtgaa agaggagtag ctcctgccac 3120
tggcgttttg agtgttggca atttgggatg cctcctgggg aaggtttccg ggcgtttggt 3180
gagtctctag atttttcctt gctttctgtg tttattggtt tttgatgttg taaaagcaat 3240.
gaatcccctt tacaagaaaa tcgaaaacac agaagaatga aggacatgcc agtccccgat 3300:
cgctgctgtg agcacctcag tggctccctc agaccagatc ccgtaggcag ccccacagac 3360
cgaccctgac cccactcaca gccaccctga agatagacta taggaacggg cccataccac 3420
acagactgct ctccaatccc tgagtctcag atgtttcatt tatttcctac ttttccacta 3480
ctaaaaaaca gtgtggaata gacattattg gcaaaattgc tcatccctaa tcctgaaaaa 3540
caggccagaa tgggtaaaga cttgtcaaag cttgcaacat agctacatgg tgcacccgga 3600
cctgtacccc ctccccccaa cacaaaacca gtgtctggga ggttcatttt cctttaaact 3660
gatccagctg gccctgaacc aattgttttt gactgagtat ctaggagagc agtaagtgga.3720
acttcagaca agcccactgg gtctggtcca ggtgaggggc agggggcatg gggctgggag 3780
gtctcagggg ccttccctgg gggtggccag cctggtaggg ggcagagaag gaaaagctga 3840
ggggggtccc tgtgagggag gaaagaagga tcatttgccc cgctgggtct caaaggcagt 3900
gagaagagag ctgaagaaag ctctggctgg ctgacaggat ccctgtgttg taattggtcc 3960
ctcctttcag ctctctagtg agatgcccgt gtctgtgcgt gtgcgtgtgt gtttcataca 4020
gctagcatta gatgggtgat gtttcttact tatcatccct aactattgca acttgacctt 4080
aaaaagacaa aaccccacaa aactcttcct gccacgggct tgcagattga agcactttcg 4140
atgttgggcg ctggcgtttg tgttctgggc accaccgtga ccctgcccag atggctataa 4200
tattatttta tacacaaacc ttttttttcc ataaatgtta taattttgtg tctgtcttta 4260
taaactatta taagtactat ttttgttata attcaaaata gatatttagt ataaagtttt 4320
tgctgttaaa tatttgttat ttagtaaact atgaattttg ctctattgta aacatggttc 4380
aaaatattaa tatgttttta tcacagtcgt tttaatattg aaaaagcact tgtgtgtttt 4440
gttttgatat gaaactggta ccgtgtgagt gtttttgctg tcgtggtttt aatctgtata 4500
taatattcca tgttgcatat taaaaacatg aatgttgtgc attttgtgat tttggaaata 4560
ctcaatgtgg ctcttctata ggcttctaga ataaaccgtg gggacccgca aaaaaaaaaa 4620
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac 4660
<210> 22
<211> 1669
<212> DNA
<213> Homo Sapiens
20/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
<220>
<221> misc_feature
<223> Incyte ID No: 6825202CB1
<400> 22
ctagcagagg gggagaggag ggatgccgca gctgagcctg tcctggctgg gcctcgggcc 60
cgtggcagca tccccgtggc tgcttctgct gctggttggg ggctcctggc tcctggcccg 120
cgtcctggcc tggacctaca ccttctatga caactgccgc cgcctccagt gttttcctca 180
acccccgaaa cagaactggt tttggggaca ccagggcctg gtcactccca cggaagaggg 240
catgaagaca ttgacccagc tggtgaccac atatccccag ggctttaagt tgtggctggg 300
tcctaccttc cccctcctca ttttatgcca ccctgacatt atccggccta tcaccagtgc 360
ctcagctgct gtcgcaccca aggatatgat tttctatggc ttcctgaagc cctggctggg 420
ggatgggctc ctgctgagtg gtggtgacaa gtggagccgc caccgtcgga tgttgacgcc 480
tgccttccat ttcaacatct tgaagcctta tatgaagatt ttcaacaaga gtgtgaacat 540
catgcacgac aagtggcagc gcctggcctc agagggcagc gccagactgg acatgtttga 600
aca.catcagc ctcatgacct tggacagtct gcagaaatgt gtcttcagct ttgaaagcaa 660
ttgtcaggag aagcccagtg aatatattgc cgccatcttg gagctcagtg cctttgtaga 720
aaagagaaac cagcagattc tcttgcacac ggacttcctg tattatctca ctcctgatgg 780
gcagcgcttc cgcagggcct gccacctggt gcacgacttc acagatgccg tcatccagga 840
gcggcgccgc accctcccca ctcagggtat tgatgatttc ctcaagaaca aggcaaagtc 900
caagacttta gacttcattg atgtgcttct gctgagcaag gatgaagatg ggaaggaatt 960
gtctgatgag gacataagag cagaagctga caccttcatg tttgagggcc atgacactac 1020
agccagtggt ctctcctggg tcctatacca ccttgcaaag cacccagaat accaggaaca 1080
gtgccggcaa gaagtgcaag agcttctgaa ggaccgtgaa cctatagaga ttgaatggga 1140
cgacctggcc cagctgccct tcctgaccat gtgcattaag gagagcctgc ggttgcatcc 1200
cccagtcccg gtcatctccc gatgttgcac gcaggacttt gtgctcccag acggccgcgt 1260
catccccaaa ggcattgtct gcctcatcaa tattatcggg atccattaca acccaactgt 1320
gtggccagac cctgaggtct acgacccctt ccgtttcgac caagagaaca tcaaggagag 1380
gtcacctctg gcttttattc ccttctcggc agggcccaga aactgcatcg ggcaggcgtt 1440
cgccatggct gagatgaagg tggtcctggc gctcacgctg ctgcacttcc gcatcctgcc 1500
gacccacact gaaccccgca ggaaacccga gctgatattg cgcgcagagg gtggactttg 1560
gctgcgggtg gagcccctgg gtgcgaactc acagtgactg tcctacccac ccacccacct 1620
ctgtagagtc ccagaaacaa aactatgctg acaaaaaata taaaaaaaa 1669
<210> 23
<211> 1882
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7256116CB1
<400> 23
gcgccggtgg atccggatcg agggcaggag gctgagaccc gcgggagctg gccctaaagc 60
aaggacctga gtgcaagtaa tttttttggg aagtaataac agaaaatacc agcaaggaag 120
aagacagtga acccaaaaga attgaaaaca ggatgctgcc catcacagac cgcctgctgc 180
acctcctggg gctggagaag acggcgttcc gcatatacgc ggtgtccacc cttctcctct 240
tcctgctctt cttcctgttc cgcctgctgc tgcggttcct gaggctctgc aggagcttct 300
acatcacctg ccgccggctg cgctgcttcc cccagcctcc ccggcgcaac tggctgctgg 360
gccacctggg catgtacctt ccaaatgagg cgggccttca agatgagaag aaggtactgg 420
acaacatgca ccatgtactc ttggtatgga tgggacctgt cctgccgctg ttggttctgg 480
tgcaccctga ttacatcaaa ccccttttgg gagcctcagc tgccatcgcc cccaaggatg 540
acctcttcta tggcttccta aaaccttggc taggggatgg gctgctgctc agcaaaggtg 600
acaagtggag ccggcaccgt cgcctgctga cacccgcctt ccactttgac atcctgaagc 660
cttacatgaa gatcttcaac cagagcgctg acattatgca tgctaaatgg cggcatctgg 720
cagagggctc agcggtctcc cttgatatgt ttgagcatat cagcctcatg accctggaca 780
gtcttcagaa atgtgtcttc agctacaaca gcaactgcca agagaagatg agtgattata 840
tctccgctat cattgaactg agcgctctgt ctgtccggcg ccagtatcgc ttgcaccact 900
acctcgactt catttactac cgctcggcgg atgggcggag gttccggcag gcctgtgaca 960
tggtgcacca cttcaccact gaagtcatcc aggaacggcg gcgggcactg cgtcagcagg 1020
gggccgaggc ctggcttaag gccaagcagg ggaagacctt ggactttatt gatgtgctgc 1080
tcctggccag ggatgaagat ggaaaggaac tgtcagacga ggatatccga gccgaagcag 1140
acaccttcat gtttgagggt cacgacacaa ccatccagtg ggatcttctt ggatgctgtt 1200
caatttggca aagtatccgg aataccagga gaaatgccga gaagagattc aggaagtcat 1260
gaaaggccgg gagctggagg agctggagtg ggacgatctg actcagctgc cctttacaac 1320
tatgtgcatt aaggagagcc tgcgccagta cccacctgtc aactcttgtc tctcgccaat 1380
21/22
CA 02399873 2002-08-09
WO 01/59127 PCT/USO1/04423
gcacggagga catcaagctc ccagatgggc gcatcatccc caaaggaatc ,atctgc,ttgg 1440
tcagcatcta tggaacccac cacaacccca cagtgtggcc tgactccaag gtgtacaacc 1500
cctaccgctt tgacccggac aacccacagc agcgctctcc actggcctat gtgcccttct 1560
ctgcaggacc caggaattgc atcggacaga gcttcgccat ggccgagttg cgcgtggttg 1620
tggcactaac actgctacgt ttccgcctga gcgtggaccg aacgcgcaag gtgcggcgga 1680
agccggagct catactgcgc acggagaacg ggctctggct caaggtggag ccgctgcctc 1740
cgcgggcctg agcgtgggcg cgcccctgcg gctcccgagg gtccaggccc cgcccccaaa 1800
ggaccaggac tcgccccaaa gatcccgagg gcataggcac ccccctcgaa gttcaggtta 1860
gctcctggat gacaggcacc gc 1882
<210> 24
<211> 880
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4210675CB1
<400> 24
atgtggttct gtctcccagc tagaccctga aacaatggaa aggagaactg cctcaacttc 60
aggtggaacc ctgatgtatg gacaagtgcc catggtcgaa actcatggaa tgaattaggt 120
agaaaccaga gccttcctaa gatacatagc tgcaaaatat gacttgtatg gaaggaacat 180
gaaggaacaa gcctgatgca tcttccctaa tatttcaaag gaacagcatg cctctgaaaa 240
cacttggctt cagttcctgg aacaatgttc catgaaaaca cctgataact aagcaggatt 300
cacatgtatg tagaaggctt gaaggacctg agtgacatga ttatgttcca gccactctct 360
ctgcctgaag agaagatgaa tcttgcatac atccttgaaa gagccactac aagattattc 420
cctgtctgtg agaaggcact gagagaccac agacaagatt ttcttgtggg caatcggctg 480
agctgggctg atacacagca acctgaagtc atcttaatga ctgaagagtg caaacccagt 540
gtcctcttgg gctttcctct gctacagaaa ttcaaggcca gaatcatcca catccccaca 600
attaataaat gtctccaacc tggaagccaa aggaagcctc cactggatga agaatccatt 660
gagactgtga agaatatatt taaatttgaa catggcctgt ttcttaaaaa catgatcact 720
acattagctg agtattaaca aatgaaacaa agtctaagaa acgtagtaaa tatttcacta 780
ttcattgtta tcatacccga ggagaatatc ataaatccac attaatgtaa taaagtaata 840
aggcatttgg tgtgtttttt ttacatgtaa tcgcgtggca 880
22/22
