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
autoimmune/inflammatory, cell proliferative, developmental, endocrine, eye,
metabolic, and
gastrointestinal disorders, including liver disorders, and in the assessment
of the effects of exogenous
compounds on the expression of nucleic acid and amino acid sequences of drug
metabolizing
enzymes.
BACKGROUND OF THE INVENTION
The metabolism of a drug and its movement through the body (pharmacokinetics)
are
important in determining its effects, toxicity, and interactions with other
drugs. The three processes
governing pharmacokinetics are the absorption of the drug, distribution to
various tissues, and
elimination of drug metabolites. These processes are intimately coupled to
drug metabolism, since a
variety of metabolic modifications alter most of the physicochemical and
pharmacological properties
of drugs, including solubility, binding to receptors, and excretion rates. The
metabolic pathways
which modify drugs also accept a variety of naturally occurring substrates
such as steroids, fatty
acids, prostaglandins, leukotrienes, and vitamins. The enzymes in these
pathways are therefore
important sites of biochemical and pharmacological interaction between natural
compounds, drugs,
carcinogens, mutagens, and xenobiotics.
It has long been appreciated that inherited differences in drug metabolism
lead to drastically
different levels of drug efficacy and toxicity among individuals. For drugs
with narrow therapeutic
indices, or drugs which require bioactivation (such as codeine), these
polymorphisms can be critical.
Moreover, promising new drugs are frequently eliminated in clinical trials
based on toxicities which
may only affect a segment of the patient group. Advances in pharmacogenomics
research, of which
drug metabolizing enzymes constitute an important part, are promising to
expand the tools and
information that can be brought to bear on questions of drug efficacy and
toxicity (See Evans, W.E.
and R.V. Relling (1999) Science 286:487-491).
Drug metabolic reactions are categorized as Phase I, which functionalize the
drug molecule
and prepare it for further metabolism, and Phase II, which are conjugative. In
general, Phase I
reaction products are partially or fully inactive, and Phase II reaction
products are the chief excreted
species. However, Phase I reaction products are sometimes more active than the
original
administered drugs; this metabolic activation principle is exploited by pro-
drugs (e.g. L-dopa).
Additionally, some nontoxic compounds (e.g. aflatoxin, benzo[a]pyrene) are
metabolized to toxic
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intermediates through these pathways. Phase I reactions are usually rate-
limiting in drug metabolism.
Prior exposure to the compound, or other compounds, can induce the expression
of Phase I enzymes
however, and thereby increase substrate flux through the metabolic pathways.
(See Klaassen, C.D.,
Amdur, M.O. and J. Doull (1996) Casarett and Doull's Toxicol~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 Drug
Metabolism, Blackie Academic and Professional, London.)
Drug metabolizing enzymes (DMEs) have broad substrate specificities. This can
be
contrasted to the immune system, where a large and diverse population of
antibodies are highly
specific for their antigens. The ability of DMEs to metabolize a wide variety
of molecules creates the
potential for drug interactions at the level of metabolism. For example, the
induction of a DME by
one compound may affect the metabolism of another compound by the enzyme.
DMEs have been classified according to the type of reaction they catalyze and
the cofactors
involved. The major classes of Phase I enzymes include, but are not limited
to, cytochrome P450 and
flavin-containing monooxygenase. Other enzyme classes involved in Phase I-type
catalytic cycles
and reactions include, but are not limited to, NADPH cytochrome P450 reductase
(CPR), the
microsomal cytochrome b5/NADH cytochrome b5 reductase system, the
ferredoxin/ferredoxin
reductase redox pair, aldo/keto reductases, and alcohol dehydrogenases. The
major classes of Phase
II enzymes include, but are not limited to, UDP glucuronyltransferase,
sulfotransferase, glutathione S-
transferase, N-acyltransferase, and N-acetyl transferase.
Cytochrome P450 and P450 catalytic cvcle-associated enz
Members of the cytochrome P450 superfamily of enzymes catalyze the oxidative
metabolism
of a variety of substrates, including natural compounds such as steroids,
fatty acids, prostaglandins,
leukotrienes, and vitamins, as well as drugs, carcinogens, mutagens, and
xenobiotics. Cytochromes
P450, also known as P450 heme-thiolate proteins, usually act as terminal
oxidases in
multi-component electron transfer chains, called P450-containing monooxygenase
systems. Specific
reactions catalyzed include hydroxylation, epoxidation, N-oxidation,
sulfooxidation, N-, S-, and O-
dealkylations, desulfation, deamination, and reduction of azo, 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
cysteine involved in
binding the heme iron in the fifth coordination site. In eukaryotic
cytochromes P450, a
membrane-spanning region is usually found in the first 15-20 amino acids of
the protein, generally
consisting of approximately 15 hydrophobic residues followed by a positively
charged residue. (See
Prosite PDOC00081, supra; Graham-Lorence, supra.)
Cytochrome P450 enzymes are involved in cell proliferation and development.
The enzymes
have roles in chemical mutagenesis and carcinogenesis by metabolizing
chemicals to reactive
intermediates that form adducts with DNA (Nebert, D.W. and Gonzalez, F.J.
(1987) Ann. Rev.
Biochem. 56:945-993). These adducts can cause nucleotide changes and DNA
rearrangements that
lead to oncogenesis. Cytochrome P450 expression in liver and other tissues is
induced by xenobiotics
such as polycyclic aromatic hydrocarbons, peroxisomal proliferators,
phenobarbital, and the
glucocorticoid dexamethasone (Dogra, S.C. et al. (1998) Clin. Exp. Pharmacol.
Physiol. 25:1-9). A
cytochrome P450 protein may participate in eye development as mutations in the
P450 gene CYP1B1
cause primary congenital glaucoma (Online Mendelian Inheritance in Man (OMIM)
*601771
Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYP1B1).
Cytochromes P450 are associated with inflammation and infection. Hepatic
cytochrome
P450 activities are profoundly affected by various infections and inflammatory
stimuli, some of
which are suppressed and some induced (Morgan, E.T. (1997) Drug Metab. Rev.
29:1129-1188).
Effects observed in vivo can be mimicked by proinflammatory cytokines and
interferons.
Autoantibodies to two cytochrome P450 proteins were found in patients with
autoimmune
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), a polyglandular
autoimmune
syndrome (OMIM *240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal
dystrophy).
Mutations in cytochromes P450 have been linked to metabolic disorders,
including congenital
adrenal hyperplasia, the most common adrenal disorder of infancy and
childhood; pseudovitamin D-
deficiency rickets; cerebrotendinous xanthomatosis, a lipid storage disease
characterized by
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progressive neurologic dysfunction, premature atherosclerosis, and cataracts;
and an inherited
resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J.
et al. ( 1994) Harrison's
Principles of Internal Medicine, McGraw-Hill, Inc. New York, NY, pp. 1968-
1970; Takeyama, K. et
al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998) N. Engl. J. Med.
338:653-661; OMIM
*213700 Cerebrotendinous xanthomatosis; and OMIM #122700 Coumarin resistance).
Extremely
high levels of expression of the cytochrome P450 protein aromatase were found
in a fibrolamellar
hepatocellular carcinoma from a boy with severe gynecomastia (feminization)
(Agarwal, V.R. ( 1998)
J. Clip. Endocrinol. Metab. 83:1797-1800).
The cytochrome P450 catalytic cycle is completed through reduction of
cytochrome P450 by
NADPH cytochrome P450 reductase (CPR). Another microsomal electron transport
system
consisting of cytochrome b5 and NADPH cytochrome b5 reductase has been widely
viewed as a
minor contributor of electrons to the cytochrome P450 catalytic cycle.
However, a recent report by
Lamb, D.C. et al. (1999; FEBS Lett. 462:283-288) identifies a Candida albicans
cytochrome P450
(CYP51) which can be efficiently reduced and supported by the microsomal
cytochrome b5/NADPH
cytochrome b5 reductase system. Therefore, there are likely many cytochromes
P450 which are
supported by this alternative electron donor system.
Cytochrome b5 reductase is also responsible for the reduction of oxidized
hemoglobin
(methemoglobin, or ferrihemoglobin, which is unable to carry oxygen) to the
active hemoglobin
(ferrohemoglobin) in red blood cells. Methemoglobinemia results when there is
a high level of
oxidant drugs or an abnormal hemoglobin (hemoglobin M) which is not
efficiently reduced.
Methemoglobinemia can also result from a hereditary deficiency in red cell
cytochrome b5 reductase
(Reviewed in Mansour, A. and Lurie, A.A. (1993) Am. J. Hematol. 42:7-12).
Members of the cytochrome P450 family are also closely associated with vitamin
D synthesis
and catabolism. Vitamin D exists as two biologically equivalent prohormones,
ergocalciferol
(vitamin DZ), produced in plant tissues, and cholecalciferol (vitamin D3),
produced in animal tissues.
The latter form, cholecalciferol, is formed upon the exposure of 7-
dehydrocholesterol to near
ultraviolet light (i.e., 290-310 nm), normally resulting from even minimal
periods of skin exposure to
sunlight (reviewed in Miller, W.L. and Portale, A.A. (2000) Trends Endocrinol.
Metab. 11:315-319).
Both prohormone forms are further metabolized in the liver to 25-
hydroxyvitamin D
(25(OH)D) by the enzyme 25-hydroxylase. 25(OH)D is the most abundant precursor
form of vitamin
D which must be further metabolized in the kidney to the active form, 1a,25-
dihydroxyvitamin D
(1a,25(OH)ZD), by the enzyme 25-hydroxyvitamin D la-hydroxylase (la-
hydroxylase). Regulation
of 1a,25(OH)ZD production is primarily at this final step in the synthetic
pathway. The activity of
la-hydroxylase depends upon several physiological factors including the
circulating level of the
enzyme product (1a,25(OH)ZD) and the levels of parathyroid hormone (PTH),
calcitonin, insulin,
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calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal la-
hydroxylase
activity has been reported, suggesting that tissue-specific, local regulation
of 1a,25(OH)2D
production may also be biologically important. The catalysis of 1a,25(OH)ZD to
24,25-dihydroxyvitamin D (24,25(OH)ZD), involving the enzyme 25-hydroxyvitamin
D
24-hydroxylase (24-hydroxylase), also occurs in the kidney. 24-hydroxylase can
also use 25(OH)D as
a substrate (Shinki, T. et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12920-
12925; Miller, W.L. and
Portale, A.A. supra; and references within).
Vitamin D 25-hydroxylase, 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 (1a,25(OH)ZD) is involved in calcium and
phosphate
homeostasis and promotes the differentiation of myeloid and skin cells.
Vitamin D deficiency
resulting from deficiencies in the enzymes involved in vitamin D metabolism
(e.g., la-hydroxylase)
causes hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive)
rickets, a disease
characterized by loss of bone density and distinctive clinical features,
including bandy or bow
leggedness accompanied by a waddling gait. Deficiencies in vitamin D 25-
hydroxylase cause
cerebrotendinous xanthomatosis, a lipid-storage disease characterized by the
deposition of cholesterol
and cholestanol in the Achilles' tendons, brain, lungs, and many other
tissues. The disease presents
with progressive neurologic dysfunction, including postpubescent cerebellar
ataxia, atherosclerosis,
and cataracts. Vitamin D 25-hydroxylase deficiency does not result in rickets,
suggesting the
existence of alternative pathways for the synthesis of 25(OH)D (Griffin, J.E.
and Zerwekh, J.E.
(1983) J. Clin. Invest. 72:1190-1199; Gamblin, G.T. et al. (1985) J. Clin.
Invest. 75:954-960; and
W.L. and Portale, A.A. supra).
Ferredoxin and ferredoxin reductase are electron transport accessory proteins
which support
at least one human cytochrome P450 species, cytochrome P450c27 encoded by the
CYP27 gene
(Dilworth, F.J. et al. (1996) Biochem. J. 320:267-71). A Streptomyces ng ~seus
cytochrome P450,
CYP104D1, was heterologously expressed in E. coli and found to be reduced by
the endogenous
ferredoxin and ferredoxin reductase enzymes (Taylor, M. et al. (1999) Biochem.
Biophys. Res.
Commun. 263:838-42), suggesting that many cytochrome P450 species may be
supported by the
ferredoxin/ferredoxin reductase pair. Ferredoxin reductase has also been found
in a model drug
metabolism system to reduce actinomycin D, an antitumor antibiotic, to a
reactive free radical species
(Flitter, W.D. and Mason, R.P. (1988) Arch. Biochem. Biophys. 267:632-639).
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Flavin-containing monooxygenase (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 OZ; there is also a great deal of substrate overlap with
cytochromes P450. The tissue
distribution of FMOs includes liver, kidney, and lung.
There are five different known isoforms of FMO in mammals (FMO1, FM02, FM03,
FM04,
and FM05), which are expressed in a tissue-specific manner. The isoforms
differ in their substrate
specificities and other properties such as inhibition by various compounds and
stereospecificity of
reaction. FMOs have a 13 amino acid signature sequence, the components of
which span the N-
terminal two-thirds of the sequences and include the FAD binding region and
the FATGY motif
which has been found in 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 FMO1.
Endogenous substrates of FMO include cysteamine, which is oxidized to the
disulfide,
cystamine, and trimethylamine (TMA), which is metabolized to trimethylamine N-
oxide. TMA
smells like rotting fish, and mutations in the FM03 isoform lead to large
amounts of the malodorous
free amine being excreted in sweat, urine, and breath. These symptoms have led
to the designation
fish-odor syndrome (OMIM 602079 Trimethylaminuria).
Lysyl oxidase:
Lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidase
involved in the
formation of connective.tissue matrices by crosslinking collagen and elastin.
LO is secreted as a N-
glycosylated precuror protein of approximately 50 kDa Levels and cleaved to
the mature form of the
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enzyme by a metalloprotease, although the precursor form is also active. The
copper atom in LO is
involved in the transport of electron to and from oxygen to facilitate the
oxidative deamination of
lysine residues in these extracellular matrix proteins. While the coordination
of copper is essential to
LO activity, insufficient dietary intake of copper does not influence the
expression of the apoenzyme.
However, the absence of the functional LO is linked to the skeletal and
vascular tissue disorders that
are associated with dietary copper deficiency. LO is also inhibited by a
variety of semicarbazides,
hydrazines, and amino nitrites, as well as heparin. Beta-aminopropionitrile is
a commonly used
inhibitor. LO activity is increased in response to ozone, cadmium, and
elevated levels of hormones
released in response to local tissue trauma, such as transforming growth
factor-beta, platelet-derived
growth factor, angiotensin II, and fibroblast growth factor. Abnormalities in
LO activity has been
linked to Menkes syndrome and occipital horn syndrome. Cytosolic forms of the
enzyme hae been
implicated in abnormal cell proliferation (reviewed in Rucker, R.B. et al.
(1998) Am. J. Clin. Nutr.
67:9965-1002S 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 (dLTMP) to
deoxythymidine monophosphate (dTMP). The basic reaction is as follows:
7,8-dihydrofolate + NADPH -~ 5,6,7,8-tetrahydrofolate + NADP+
The enzymes can be inhibited by a number of dihydrofolate analogs, including
trimethroprim and
methotrexate. Since an abundance of TMP is required for DNA synthesis, rapidly
dividing cells
require the activity of DHFR. The replication of DNA viruses (i.e.,
herpesvirus) also requires high
levels of DHFR activity. As a result, drugs that target DHFR have been used
for cancer
chemotherapy and to inhibit DNA virus replication. (For similar reasons,
thymidylate synthetases are
also target enzymes.) Drugs that inhibit DHFR are preferentially cytotoxic for
rapidly dividing cells
(or DNA virus-infected cells) but have no specificity, resulting in the
indiscriminate destruction of
dividing cells. Furthermore, cancer cells may become resistant to drugs such
as methotrexate as a
result of acquired transport defects or the duplication of one or more DHFR
genes (Stryer, L. ( 1988)
Biochemistry. W.H Freeman and Co., Inc. New York. pp. 511-5619).
Aldo/keto reductases
Aldo/keto reductases are monomeric NADPH-dependent oxidoreductases with broad
substrate specificities (Bohren, K.M. et al. (1989) J. Biol. Chem. 264:9547-
9551). These enzymes
catalyze the reduction of carbonyl-containing compounds, including carbonyl-
containing sugars and
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aromatic compounds, to the corresponding alcohols. Therefore, a variety of
carbonyl-containing
drugs and xenobiotics are likely metabolized by enzymes of this class.
One known reaction catalyzed by a family member, aldose reductase, is the
reduction of
glucose to sorbitol, which is then further metabolized to fructose by sorbitol
dehydrogenase. Under
normal conditions, the reduction of glucose to sorbitol is a minor pathway. In
hyperglycemic states,
however, the accumulation of sorbitol is implicated in the development of
diabetic complications
(OMIM * 103880 Aldo-keto reductase family 1, member B1). Members of this
enzyme family are
also highly expressed in some liver cancers (Cao, D. et al. (1998) J. Biol.
Chem. 273:11429-11435).
Alcoholdehydro eg eases
Alcohol dehydrogenases (ADHs) oxidize simple alcohols to the corresponding
aldehydes.
ADH is a cytosolic enzyme, prefers the cofactor NAD+, and also binds zinc ion.
Liver contains the
highest levels of ADH, with lower levels in kidney, lung, and the gastric
mucosa.
Known ADH isoforms are dimeric proteins composed of 40 kDa subunits. There are
five
known gene loci which encode these subunits (a, b, g, p, c), and some of the
loci have characterized
allelic variants (b" b2, b3, g,, g2). The subunits can form homodimers and
heterodimers; the subunit
composition determines the specific properties of the active enzyme. The
holoenzymes have
therefore been categorized as Class I (subunit compositions aa, ab, ag, bg,
gg), Class II (pp), and
Class III (cc). Class I ADH isozymes oxidize ethanol and other small aliphatic
alcohols, and are
inhibited by pyrazole. Class II isozymes prefer longer chain aliphatic and
aromatic alcohols, are
unable to oxidize methanol, and are not inhibited by pyrazole. Class III
isozymes prefer even longer
chain aliphatic alcohols (five carbons and longer) and aromatic alcohols, and
are not inhibited by
pyrazole.
The short-chain alcohol dehydrogenases include a number of related enzymes
with a variety
of substrate specificities. Included in this group are the mammalian enzymes D-
beta-hydroxybutyrate
dehydrogenase, (R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin
dehydrogenase,
NADPH-dependent carbonyl reductase, corticosteroid 11-beta-dehydrogenase, and
estradiol 17-beta-
dehydrogenase, as well as the bacterial enzymes acetoacetyl-CoA reductase,
glucose 1-
dehydrogenase, 3-beta-hydroxysteroid dehydrogenase, 20-beta-hydroxysteroid
dehydrogenase, ribitol
dehydrogenase, 3-oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate
dehydrogenase, sorbitol-6-
phosphate 2-dehydrogenase, 7-alpha-hydroxysteroid dehydrogenase, cis-1,2-
dihydroxy-3,4-
cyclohexadiene-1-carboxylate dehydrogenase, cis-toluene dihydrodiol
dehydrogenase, cis-benzene
glycol dehydrogenase, biphenyl-2,3-dihydro-2,3-diol dehydrogenase, N-
acylmannosamine 1-
dehydrogenase, and 2-deoxy-D-gluconate 3-dehydrogenase (Krozowski, Z. (1994)
J. Steroid
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).
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UDP glucuronyltransferase
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,
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,
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alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester
sulfotransferase, and bile salt
sulfotransferase.
ST enzyme activity varies greatly with sex and age in rats. The combined
effects of
developmental cues and sex-related hormones are thought to lead to these
differences in ST
expression profiles, as well as the profiles of other DMEs such as cytochromes
P450. Notably, the
high expression of STs in cats partially compensates for their low level of
UDP glucuronyltransferase
activity.
Several forms of ST have been purified from human liver cytosol and cloned.
There are two
phenol sulfotransferases with different thermal stabilities and substrate
preferences. The
thermostable enzyme catalyzes the sulfation of phenols such as para-
nitrophenol, minoxidil, and
acetaminophen; the thermolabile enzyme prefers monoamine substrates such as
dopamine,
epinephrine, and levadopa. Other cloned STs include an estrogen
sulfotransferase and an N-
acetylglucosamine-6-O-sulfotransferase. This last enzyme is illustrative of
the other major role of
STs in cellular biochemistry, the modification of carbohydrate structures that
may be important in
cellular differentiation and maturation of proteoglycans. Indeed, an inherited
defect in a
sulfotransferase has been implicated in macular corneal dystrophy, a disorder
characterized by a
failure to synthesize mature keratan sulfate proteoglycans (Nakazawa, K. et
al. (1984) J. Biol. Chem.
259:13751-13757; OMIM *217800 Macular dystrophy, corneal).
Galactosyltransferases
Galactosyltransferases are a subset of glycosyltransferases that transfer
galactose (Gal) to the
terminal N-acetylglucosamine (GIcNAc) oligosaccharide chains that are part of
glycoproteins or
glycolipids that are free in solution (Kolbinger, F. et al. (1998) J. Biol.
Chem. 273:433-440; Amado,
M. et al. (1999) Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases
have been detected on
the cell surface and as soluble extracellular proteins, in addition to being
present in the Golgi. (31,3-
galactosyltransferases form Type I carbohydrate chains with Gal (ail-3)GIcNAc
linkages. Known
human and mouse (31,3-galactosyltransferases appear to have a short cytosolic
domain, a single
transmembrane domain, and a catalytic domain with eight conserved regions.
(Kolbinger, F., supra
and Hennet, T. et al. (1998) J. Biol. Chem. 273:58-65). In mouse UDP-
galactose:(3-N-
acetylglucosamine (31,3-galactosyltransferase-I region 1 is located at amino
acid residues 78-83,
region 2 is located at amino acid residues 93-102, region 3 is located at
amino acid residues 116-119,
region 4 is located at amino acid residues 147-158, region 5 is located at
amino acid residues 172-
183, region 6 is located at amino acid residues 203-206, region 7 is located
at amino acid residues
236-246, and region 8 is located at amino acid residues 264-275. A variant of
a sequence found
within mouse UDP-galactose:(3-N-acetylglucosamine (31,3-galactosyltransferase-
I region 8 is also
found in bacterial galactosyltransferases, suggesting that this sequence
defines a galactosyltransferase
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sequence motif (Hennet, T. supra). Recent work suggests that brainiac protein
is a (31,3-
galactosyltransferase (Yuan, Y. et al. (1997) Cell 88:9-11; and Hennet, T.
supra).
UDP-Gal:GlcNAc-1,4-galactosyltransferase (-1,4-GaIT) (Sato, T. et al., (1997)
EMBO J.
16:1850-1857) catalyzes the formation of Type II carbohydrate chains with Gal
((31-4)GIcNAc
linkages. As is the case with the (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
~i1,4-
galactosyltransferase, as part of a heterodimer with a-lactalbumin, functions
in lactating mammary
gland lactose production. A ~i1,4-galactosyltransferase on the surface of
sperm functions as a
receptor that specifically recognizes the egg. Cell surface (31,4-
galactosyltransferases also function in
cell adhesion, cell/basal lamina interaction, 'and normal and metastatic cell
migration. (Shur, B.
(1993) Curr. Opin. Cell Biol. 5:854-863; and Shaper, J. (1995) Adv. Exp. Med.
Biol. 376:95-104).
Glutathione S-transferase
The basic reaction catalyzed by glutathione S-transferases (GST) is the
conjugation of an
electrophile with reduced glutathione (GSH). GSTs are homodimeric or
heterodimeric proteins
localized mainly in the cytosol, but some level of activity is present in
microsomes as well. The
major isozymes share common structural and catalytic properties; in humans
they have been
classified into four major classes, Alpha, Mu, Pi, and Theta. The two largest
classes, Alpha and Mu,
are identified by their respective protein isoelectric points; pI ~ 7.5-9.0
(Alpha), and pI ~ 6.6 (Mu).
Each GST possesses a common binding site for GSH and a variable hydrophobic
binding site. The
hydrophobic binding site in each isozyme is specific for particular
electrophilic substrates. Specific
amino acid residues within GSTs have been identified as important for these
binding sites and for
catalytic activity. Residues Q67, T68, D101, E104, and 8131 are important for
the binding of GSH
(Lee, H.-C. et al. (1995) J. Biol. Chem. 270:99-109). Residues R13, R20, and
R69 are important for
the catalytic activity of GST (Stenberg, G. et al. (1991) Biochem. J. 274:549-
555).
In most cases, GSTs perform the beneficial function of deactivation and
detoxification of
potentially mutagenic and carcinogenic chemicals. However, in some cases their
action is detrimental
and results in activation of chemicals with consequent mutagenic and
carcinogenic effects. Some
forms of rat and human GSTs are reliable preneoplastic markers that aid in the
detection of
carcinogenesis. Expression of human GSTs in bacterial strains, such as
Salmonella t~phimurium
used in the well-known Ames test for mutagenicity, has helped to establish the
role of these enzymes
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in mutagenesis. Dihalomethanes, which produce liver tumors in mice, are
believed to be activated by
GST. This view is supported by the finding that dihalomethanes are more
mutagenic in bacterial cells
expressing human GST than in untransfected cells (Thier, R. et al. ( 1993)
Proc. Natl. Acad. Sci. USA
90:8567-8580). The mutagenicity of ethylene dibromide and ethylene dichloride
is increased in
bacterial cells expressing the human Alpha GST, A1-1, while the mutagenicity
of aflatoxin B1 is
substantially reduced by enhancing the expression of GST (Simula, T.P. et al.
(1993) Carcinogenesis
14:1371-1376). Thus, control of GST activity may be useful in the control of
mutagenesis and
carcinogenesis.
GST has been implicated in the acquired resistance of many cancers to drug
treatment, the
phenomenon known as multi-drug resistance (MDR). MDR occurs when a cancer
patient is treated
with a cytotoxic drug such as cyclophosphamide and subsequently becomes
resistant to this drug and
to a variety of other cytotoxic agents as well. Increased GST levels are
associated with some of these
drug resistant cancers, and it is believed that this increase occurs in
response to the drug agent which
is then deactivated by the GST catalyzed GSH conjugation reaction. The
increased GST levels then
protect the cancer cells from other cytotoxic agents which bind to GST.
Increased levels of A1-1 in
tumors has been linked to drug resistance induced by cyclophosphamide
treatment (Dirven H.A. et al.
(1994) Cancer Res. 54: 6215-6220). Thus control of GST activity in cancerous
tissues may be useful
in treating MDR in cancer patients.
Gamma-. lug tamvl 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. 122:367-380).
Acyltransferase
N-acyltransferase enzymes catalyze the transfer of an amino acid conjugate to
an activated
carboxylic group. Endogenous compounds and xenobiotics are activated by acyl-
CoA synthetases in
the cytosol, microsomes, and mitochondria. The acyl-CoA intermediates are then
conjugated with an
amino acid (typically glycine, glutamine, or taurine, but also ornithine,
arginine, histidine, serine,
aspartic acid, and several dipeptides) by N-acyltransferases in the cytosol or
mitochondria to form a
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metabolite with an amide bond. This reaction is complementary to O-
glucuronidation, but amino acid
conjugation does not produce the reactive and toxic metabolites which often
result from
glucuronidation.
One well-characterized enzyme of this class is the bile acid-CoA:amino acid N-
acyltransferase (BAT) responsible for generating the bile acid conjugates
which serve as detergents in
the gastrointestinal tract (Falany, C.N. et al. (1994) J. Biol. Chem.
269:19375-19379; Johnson, M.R.
et al. (1991) J. Biol. Chem. 266:10227-10233). BAT is also useful as a
predictive indicator for
prognosis of hepatocellular carcinoma patients after partial hepatectomy
(Furutani, M. et al. (1996)
Hepatology 24:1441-1445).
Acetyltransferases
Acetyltransferases have been extensively studied for their role in histone
acetylation. Histone
acetylation results in the relaxing of the chromatin structure in eukaryotic
cells, allowing transcription
factors to gain access to promoter elements of the DNA templates in the
affected region of the
genome (or the genome in general). In contrast, histone deacetylation results
in a reduction in
transcription by closing the chromatin structure and limiting access of
transcription factors. To this
end, a common means of stimulating cell transcription is the use of chemical
agents that inhibit the
deacetylation of histones (e.g., sodium butyrate), resulting in a global
(albeit artifactual) increase in
gene expression. The modulation of gene expression by acetylation also results
from the acetylation
of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR,
TFIIE, TFIIF and the
high mobility group proteins (HMG). In the case of p53, acetylation results in
increased DNA
binding, leading to the stimulation of transcription of genes regulated by
p53. The prototypic histone
acetylase (HAT) is GcnS from Saccharomyces cerevisiae. GcnS is a member of a
family of acetylases
that includes Tetrahymena p55, human GcnS, and human p300/CBP. Histone
acetylation is reviewed
in (Cheung, W.L. et al. (2000) Curr. Opin. Cell Biol. 12:326-333 and Bergen
S.L (1999) Curr. Opin.
Cell Biol. 11:336-341). Some acetyltransferase enzymes posses the alpha/beta
hydrolase fold (Center
of Applied Molecular Engineering Inst. of Chemistry 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-Imb.cam.ac.uk/scop/index.html).
N-acetyltransferase
Aromatic amines and hydrazine-containing compounds are subject to N-
acetylation by the N-
acetyltransferase enzymes of liver and other tissues. Some xenobiotics can be
O-acetylated to some
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,
13
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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, NATI and
NAT2; mice appear
to have a third form of the enzyme, NAT3. The human forms of N-
acetyltransferase have
independent regulation (NAT1 is widely-expressed, whereas NAT2 is in liver and
gut only) and
overlapping substrate preferences. Both enzymes appear to accept most
substrates to some extent, but
NAT1 does prefer some substrates (para-aminobenzoic acid, para-aminosalicylic
acid,
sulfamethoxazole, and sulfanilamide), while NAT2 prefers others (isoniazid,
hydralazine,
procainamide, dapsone, aminoglutethimide, and sulfamethazine).
Clinical observations of patients taking the antituberculosis drug isoniazid
in the 1950s led to
the description of fast and slow acetylators of the compound. These phenotypes
were shown
subsequently to be due to mutations in the NAT2 gene which affected enzyme
activity or stability.
The slow isoniazid acetylator phenotype is very prevalent in Middle Eastern
populations (approx.
70%), and is less prevalent in Caucasian (approx. 50%) and Asian (<25%)
populations. More
recently, functional polymorphism in NAT1 has been detected, with
approximately 8% of the
population tested showing a slow acetylator phenotype (Butcher, N. J. et al.
(1998) Pharmacogenetics
8:67-72). Since NAT1 can activate some known aromatic amine carcinogens,
polymorphism in the
widely-expressed NAT1 enzyme may be important in determining cancer risk (OMIM
*108345 N-
acetyltransferase 1).
Aminotransferases
Aminotransferases comprise a family of pyridoxal 5 =phosphate (PLP) -dependent
enzymes
that catalyze transformations of amino acids. Aspartate aminotransferase
(AspAT) is the most
extensively studied PLP-containing enzyme. It catalyzes the reversible
transamination of dicarboxylic
L-amino acids, aspartate and glutamate, and the corresponding 2-oxo acids,
oxalacetate and
2-oxoglutarate. Other members of the family included pyruvate
aminotransferase, branched-chain
amino acid aminotransferase, tyrosine aminotransferase, aromatic
aminotransferase,
alanine:glyoxylate aminotransferase (AGT), and kynurenine aminotransferase
(Vacca, R.A. et al.
(1997) J. Biol. Chem. 272:21932-21937).
Primary hyperoxaluria type-1 is an autosomal recessive disorder resulting in a
deficiency in
the liver-specific peroxisomal enzyme, alanine:glyoxylate aminotransferase-1.
The phenotype of the
disorder is a deficiency in glyoxylate metabolism. In the absence of AGT,
glyoxylate is oxidized to
oxalate rather than being transaminated to glycine. The result is the
deposition of insoluble calcium
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
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metabolite L-kynurenine to form kynurenic acid. The enzyme may also catalyzes
the reversible
transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce
2-oxoadipate and
L-glutamate. Kynurenic acid is a putative modulator of glutamatergic
neurotransmission, thus a
deficiency in kynurenine aminotransferase may be associated with pleotrophic
effects (Buchli, R. et
al. (1995) J. Biol. Chem. 270:29330-29335).
Catechol-O-methyltransferase
Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group
of S-
adenosyl-L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the
catechol substrate
(e.g., L-dopa, dopamine, or DBA). Methylation of the 3 =hydroxyl group is
favored over methylation
of the 4'-hydroxyl group and the membrane bound isoform of COMT is more
regiospecific than the
soluble form. Translation of the soluble form of the enzyme results from
utilization of an internal
start codon in a full-length mRNA (1.5 kb) or from the translation of a
shorter mRNA (1.3 kb),
transcribed from an internal promoter. The proposed SN2-like methylation
reaction requires Mg++ and
is inhibited by Ca''-". The binding of the donor and substrate to COMT occurs
sequentially. AdoMet
first binds COMT in a Mg**-independent manner, followed by the binding of Mg**
and the binding of
the catechol substrate.
The amount of COMT in tissues is relatively high compared to the amount of
activity
normally required, thus inhibition is problematic. Nonetheless, inhibitors
have been developed for in
vitro use (e.g., gallates, tropolone, U-0521, and 3',4=dihydroxy-2-methyl-
propiophetropolone) and for
clinical use (e.g., nitrocatechol-based compounds and tolcapone).
Administration of these inhibitors
results in the increased half-life of L-dopa and the consequent formation of
dopamine. Inhibition of
COMT is also likely to increase the half-life of various other catechol-
structure compounds, including
but not limited to epinephrine/norepinephrine, isoprenaline, rimiterol,
dobutamine, fenoldopam,
apomorphine, and a-methyldopa. A deficiency in norepinephrine has been linked
to clinical
depression, hence the use of COMT inhibitors could be usefull in the treatment
of depression.
COMT inhibitors are generally well tolerated with minimal side effects and are
ultimately
metabolized in the liver with only minor accumulation of metabolites in the
body (Mannisto, P.T. and
Kaakkola, S. (1999) Pharmacol. Rev. 51:593-628).
Copper-zinc superoxide dismutases
Copper-zinc superoxide dismutases are compact homodimeric metalloenzymes
involved in
cellular defenses against oxidative damage. The enzymes contain one atom of
zinc and one atom of
copper per subunit and catalyze the dismutation of superoxide anions into OZ
and HZO2. The rate of
dismutation is diffusion-limited and consequently enhanced by the presence of
favorable electrostatic
interactions between the substrate and enzyme active site. Examples of this
class of enzyme have
been identified in the cytoplasm of all the eukaryotic cells as well as in the
periplasm of several
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bacterial species. Copper-zinc superoxide dismutases are robust enzymes that
are highly resistant to
proteolytic digestion and denaturing by urea and SDS. In addition to the
compact structure of the
enzymes, the presence of the metal ions and intrasubunit disulfide bonds is
believed to be responsible
for enzyme stability. The enzymes undergo reversible denaturation at
temperatures as high as 70°C
(Battistoni; A. et al. (1998) J. Biol. Chem. 273:5655-5661).
Overexpression of superoxide dismutase has been implicated in enhancing
freezing tolerance
of transgenic Alfalfa as well as providing resistance to environmental toxins
such as the diphenyl
ether herbicide, acifluorfen (McKersie, B.D. et al. ( 1993) Plant Physiol.
103:1155-1163). In addtion,
yeast cells become more resistant to freeze-thaw damage following exposure to
hydrogen peroxide
which causes the yeast cells to adapt to further peroxide stress by
upregulating expression of
superoxide dismutases. In this study, mutations to yeast superoxide dismutase
genes had a more
detrimental effect on freeze-thaw resistance than mutations which affected the
regulation of
glutathione metabolism, long suspected of being important in determining an
organisms survival
through the process of cryopreservation (Jong-In Park, J.-I. et al. (1998) J.
Biol. Chem.
273:22921-22928).
Expression of superoxide dismutase is also associated with Mycobacterium
tuberculosis, the
organism that causes tuberculosis. Superoxide dismutase is one of the ten
major proteins secreted by
M. tuberculosis and its expression is upregulated approximately 5-fold in
response to oxidative stress.
M. tuberculosis expresses almost two orders of magnitude more superoxide
dismutase than the
nonpathogenic mycobacterium M. smegmatis, and secretes a much higher
proportion of the expressed
enzyme. The result is the secretion of --350-fold more enzyme by M.
tuberculosis than M. smegmaris,
providing substantial resistance to oxidative stress (Harth, G. and Horwitz,
M.A. (1999) J. Biol.
Chem. 274:4281-4292).
The reduced expression of copper-zinc superoxide dismutases, as well as other
enzymes with
anti-oxidant capabilities, has been implicated in the early stages of cancer.
The expression of copper-
zinc superoxide dismutases has been shown to be lower in prostatic
intraepithelial neoplasia and
prostate carcinomas, compared to normal prostate tissue (Bostwick, D.G. (2000)
Cancer 89:123-134).
Phosphodiesterases
Phosphodiesterases make up a class of enzymes which catalyze the hydrolysis of
one of the
two ester bonds in a phosphodiester compound. Phosphodiesterases are therefore
crucial to a variety
of cellular processes. Phosphodiesterases include DNA and RNA endonucleases
and exonucleases,
which are essential for cell growth and replication, and topoisomerases, which
break and rejoin
nucleic acid strands during topological rearrangement of DNA. A Tyr-DNA
phosphodiesterase
functions in DNA repair by hydrolyzing dead-end covalent intermediates formed
between
topoisomerase I and DNA (Pouliot, J.J. et al. (1999) Science 286:552-555;
Yang, S.-W. (1996) Proc.
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Natl. Acad. Sci. USA 93:11534-11539).
Acid sphingomyelinase is a phosphodiesterase which hydrolyzes the membrane
phospholipid
sphingomyelin to produce ceramide and phosphorylcholine. Phosphorylcholine is
used in the
synthesis of phosphatidylcholine, which is involved in numerous intracellular
signaling pathways,
while ceramide is an essential precursor for the generation of gangliosides,
membrane lipids found in
high concentration in neural tissue. Defective acid sphingomyelinase leads to
a build-up of
sphingomyelin molecules in lysosomes, resulting in Niemann-Pick disease
(Schuchman, E.H. and
S.R. Miranda (1997) Genet. Test. 1:13-19).
Glycerophosphoryl diester phosphodiesterase (also known as
glycerophosphodiester
phosphodiesterase) is a phosphodiesterase which hydrolyzes deacetylated
phospholipid
glycerophosphodiesters to produce sn-glycerol-3-phosphate and an alcohol.
Glycerophosphocholine,
glycerophosphoethanolamine, glycerophosphoglycerol, and glycerophosphoinositol
are examples of
substrates for glycerophosphoryl diester phosphodiesterases. A
glycerophosphoryl diester
phosphodiesterase from E. coli has broad specificity for glycerophosphodiester
substrates (Larson,
T.J. et al. (1983) J. Biol: Chem. 248:5428-5432).
Cyclic nucleotide phosphodiesterases (PDEs) are crucial enzymes in the
regulation of the
cyclic nucleotides cAMP and cGMP. cAMP and cGMP function as intracellular
second messengers
to transduce a variety of extracellular signals including hormones, light, and
neurotransmitters. PDEs
degrade cyclic nucleotides to their corresponding monophosphates, thereby
regulating the
intracellular concentrations of cyclic nucleotides and their effects on signal
transduction. Due to their
roles as regulators of signal transduction, PDEs have been extensively studied
as chemotherapeutic
targets (Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481;
Torphy, J.T. (1998)
Am. J. Resp. Crit. Care Med. 157:351-370).
Families of mammalian PDEs have been classified based on their substrate
specificity and
affinity, sensitivity to cofactors, and sensitivity to inhibitory agents
(Beavo, J.A. (1995) Physiol. Rev.
75:725-748; Conti, M. et al. (1995) Endocrine Rev. 16:370-389). Several of
these families contain
distinct genes, many of which are expressed in different tissues as splice
variants. Within PDE
families, there are multiple isozymes and multiple splice variants of these
isozymes (Conti, M. and S.-
L.C. Jin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63:1-38). The existence of
multiple PDE
families, isozymes, and splice variants is an indication of the variety and
complexity of the regulatory
pathways involving cyclic nucleotides (Houslay, M.D. and G. Milligan (1997)
Trends Biochem. Sci.
22:217-224).
Type 1 PDEs (PDEls) are Caz+/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). PDEIs have been found in the lung, heart, and
brain. Some PDE1
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isozymes are regulated in vitro by phosphorylation/dephosphorylation.
Phosphorylation of these
PDE1 isozymes decreases the affinity of the enzyme for calmodulin, decreases
PDE activity, and
increases steady state levels of cAMP (Kakkar, supra). PDEls may provide
useful therapeutic targets
for disorders of the central nervous system, and the cardiovascular and immune
systems due to the
involvement of PDEls in both cyclic nucleotide and calcium signaling (Perry,
M.J. and G.A. Higgs
(1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE2s are cGMP-stimulated PDEs that have been found in the cerebellum,
neocortex, heart,
kidney, lung, pulmonary artery, and skeletal muscle (Sadhu, K. et al. (1999)
J. Histochem. Cytochem.
47:895-906). PDE2s are thought to mediate the effects of cAMP on catecholamine
secretion,
participate in the regulation of aldosterone (Beavo, supra), and play a role
in olfactory signal
transduction (Juilfs, D.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3388-
3395).
PDE3s have high affinity for both cGMP and cAMP, and so these cyclic
nucleotides act as
competitive substrates for PDE3s. PDE3s play roles in stimulating myocardial
contractility,
inhibiting platelet aggregation, relaxing vascular and airway smooth muscle,
inhibiting proliferation
of T-lymphocytes and cultured vascular smooth muscle cells, and regulating
catecholamine-induced.
release of free fatty acids from adipose tissue. The PDE3 family of
phosphodiesterases are sensitive
to specific inhibitors such as cilostamide, enoximone, and lixazinone.
Isozymes of PDE3 can be
regulated by cAMP-dependent protein kinase, or by insulin-dependent kinases
(Degerman, E. et al.
(1997) J. Biol. Chem. 272:6823-6826).
PDE4s are specific for cAMP; are localized to airway smooth muscle, the
vascular
endothelium, and all inflammatory cells; and can be activated by cAMP-
dependent phosphorylation.
Since elevation of cAMP levels can lead to suppression of inflammatory cell
activation and to
relaxation of bronchial smooth muscle, PDE4s have been studied extensively as
possible targets for
novel anti-inflammatory agents, with special emphasis placed on the discovery
of asthma treatments.
PDE4 inhibitors are currently undergoing clinical trials as treatments for
asthma, chronic obstructive
pulmonary disease, and atopic eczema. All four known isozymes of PDE4 are
susceptible to the
inhibitor rolipram, a compound which has been shown to improve behavioral
memory in mice (Barad,
M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:15020-15025). PDE4 inhibitors
have also been
studied as possible therapeutic agents against acute lung injury, endotoxemia,
rheumatoid arthritis,
multiple sclerosis, and various neurological and gastrointestinal indications
(Doherty, A.M. (1999)
Curr. Opin. Chem. Biol. 3:466=473).
PDES is highly selective for cGMP as a substrate (Turko, LV. et al. (1998)
Biochemistry
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
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regulation of catalytic activity. High levels of PDES are found in vascular
smooth muscle, platelets,
lung, and kidney. The inhibitor zaprinast is effective against PDES and PDEls.
Modification of
zaprinast to provide specificity against PDES has resulted in sildenafil
(VIAGRA; Pfizer, Inc., New
York NY), a treatment for male erectile dysfunction (Terrett, N. et al. (1996)
Bioorg. Med. Chem.
Lett. 6:1819-1824). Inhibitors of PDES are currently being studied as agents
for cardiovascular
therapy (Perry, M.J. and G.A. Higgs (1998) Curr. Opin. Chem. Biol. 2:472-481).
PDE6s, the photoreceptor cyclic nucleotide phosphodiesterases, are crucial
components of
the phototransduction cascade. In association with the G-protein transducin,
PDE6s hydrolyze cGMP
to regulate cGMP-gated 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).
PDEBs are cAMP specific, and are closely related to the PDE4 family. PDEBs are
expressed
in thyroid gland, testis, eye, liver, skeletal muscle, heart, kidney, ovary,
and brain. The cAMP-
hydrolyzing activity of PDEBs is not inhibited by the PDE inhibitors rolipram,
vinpocetine, milrinone,
IBMX (3-isobutyl-1-methylxanthine), or zaprinast, but PDEBs are inhibited by
dipyridamole (Fisher,
D.A. et al. (1998) Biochem. Biophys. Res. Commun. 246:570-577; Hayashi, M. et
al. (1998)
Biochem. Biophys. Res. Commun. 250:751-756; Soderling, S.H. et al. (1998)
Proc. Natl. Acad. Sci.
USA 95:8991-8996).
PDE9s are cGMP specific and most closely resemble the PDEB family of PDEs.
PDE9s are
expressed in kidney, liver, lung, brain, spleen, and small intestine. PDE9s
are not inhibited by
sildenafil (VIAGRA; Pfizer, Inc., New York NY), rolipram, vinpocetine,
dipyridamole, or IBMX (3-
isobutyl-1-methylxanthine), but they are sensitive to the PDES inhibitor
zaprinast (Fisher, D.A. et al.
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(1998) J. Biol. Chem. 273:15559-15564; Soderling, S.H. et al. (1998) J. Biol.
Chem. 273:15553-
15558).
PDEIOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP. PDElOs are
expressed
in brain, thyroid, and testis. (Soderling, S.H. et al. (1999) Proc. Natl.
Acad. Sci. USA 96:7071-7076;
Fujishige, K. et al. (1999) J. Biol. Chem. 274:18438-18445; Loughney, K. et al
(1999) Gene 234:109
117).
PDEs are composed of a catalytic domain of about 270-300 amino acids, an N-
terminal
regulatory domain responsible for binding cofactors, and, in some cases, a
hydrophilic C-terminal
domain of unknown function (Conti, M. and S.-L.C. Jin (1999) Prog. Nucleic
Acid Res. Mol. Biol.
63:1-38). A conserved, putative zinc-binding motif, 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, LV. et al.
(1996) J. Biol. Chem. 271:22240-22244). PDE families display approximately 30%
amino acid
identity within the catalytic domain; however, isozymes within the same family
typically display
about 85-95% identity in this region (e.g. PDE4A vs PDE4B). Furthermore,
within a family there is
extensive similarity (>60%) outside the catalytic domain; while across
families, there is little or no
sequence similarity outside this domain.
Many of the constituent functions of immune and inflammatory responses are
inhibited by
agents that increase intracellular levels of cAMP (Verghese, M.W. et al.
(1995) Mol. Pharmacol.
47:1164-1171). A variety of diseases have been attributed to increased PDE
activity and associated
with decreased levels of cyclic nucleotides. For example, a form of diabetes
insipidus in mice has
been associated with increased PDE4 activity, an increase in low-Km cAMP PDE
activity has been
reported in leukocytes of atopic patients, and PDE3 has been associated with
cardiac disease.
Many inhibitors of PDEs have been identified and have undergone clinical
evaluation (Perry,
M.J. and G.A. Higgs (1998) 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
CA 02403644 2002-09-26
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9:1137-1144). Additionally, rolipram, based on its ability to suppress the
production of cytokines
such as TNF-a and b and interferon g, has been shown to be effective in the
treatment of
encephalomyelitis. Rolipram may also be effective in treating tardive
dyskinesia and was effective in
treating multiple sclerosis in an experimental animal model (Sommer, N. et al.
(1995) Nat. Med.
1:244-248; Sasaki, H. et al. ( 1995) Eur. J. Pharmacol. 282:71-76).
Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial
asthma and
other respiratory diseases. Theophylline is believed to act on airway smooth
muscle function and in
an anti-inflammatory or 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).
Phosphotriesterases
Phosphotriesterases (PTE, paraoxonases) are enzymes that hydrolyze toxic
organophosphorus
compounds and have been isolated from a variety of tissues. The enzymes appear
to be lacking in
birds and insects and abundant in mammals, explaining the reduced tolerance of
birds and insects to
organophosphorus compound (Vilanova, E. and Sogorb, M.A. (1999) Crit. Rev.
Toxicol. 29:21-57).
Phosphotriesterases play a central role in the detoxification of insecticides
by mammals.
Phosphotriesterase activity varies among individuals and is lower in infants
than adults. Knockout
mice are markedly more sensitive to the organophosphate-based toxins diazoxon
and chlorpyrifos
oxon (Furlong, C.E., et al. (2000) Neurotoxicology 21:91-100). PTEs have
attracted interest as
enzymes capable of the detoxification of organophosphate-containing chemical
waste and warfare
reagents (e.g., parathion), in addition to pesticides and insecticides. Some
studies have also
implicated phosphotriesterase in atherosclerosis and diseases involving
lipoprotein metabolism.
Thioesterases
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Two soluble thioesterases involved in fatty acid biosynthesis have been
isolated from
mammalian tissues, one which is active only toward long-chain fatty-acyl
thioesters and one which is
active toward thioesters with a wide range of fatty-acyl chain-lengths. These
thioesterases catalyze
the chain-terminating step in the de novo biosynthesis of fatty acids. Chain
termination involves the
hydrolysis of the thioester bond which links the fatty acyl chain to the 4'-
phosphopantetheine
prosthetic group of the acyl Garner protein (ACP) subunit of the fatty acid
synthase (Smith, S. (1981a)
Methods Enzymol. 71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-200).
~E. coli contains two soluble thioesterases, thioesterase I which is active
only toward long-
chain acyl thioesters, and thioesterase II (TEII) which has a broad chain-
length specificity (Naggert, J.,
et al. (1991) J. Biol. Chem. 266:11044-11050). E. coli TEII does not exhibit
sequence similarity with
either of the two types of mammalian thioesterases which function as chain-
terminating enzymes in
de novo fatty acid biosynthesis. Unlike the mammalian thioesterases, E. coli
TEII lacks the
characteristic serine active site gly-X-ser-X-gly sequence motif and is not
inactivated by the serine
modifying agent diisopropyl fluorophosphate. However, modification of
histidine 58 by
iodoacetamide and diethylpyrocarbonate abolished TEII activity. Overexpression
of TEII did not
alter fatty acid content in E. coli, which suggests that it does not function
as a chain-terminating
enzyme in fatty acid biosynthesis (Naggert et al., supra). For that reason,
Naggert et al. (supra)
proposed that the physiological substrates for E. coli TEII may be coenzyme A
(CoA)-fatty acid esters
instead of ACP-phosphopanthetheine-fatty acid esters.
Carboxylesterases
Mammalian carboxylesterases constitute a multigene family expressed in a
variety of tissues
and cell types. Isozymes have significant sequence homology and are classified
primarily on the
basis of amino acid sequence. Acetylcholinesterase, butyrylcholinesterase, and
carboxylesterase are
grouped into the serine super family of esterases (B-esterases). Other
carboxylesterases included
thyroglobulin, thrombin, Factor IX, gliotactin, and plasminogen.
Carboxylesterases catalyze the
hydrolysis of ester- and amide- groups from molecules and are involved in
detoxification of drugs,
environmental toxins, and carcinogens. Substrates for carboxylesterases
include short- and
long-chain acyl-glycerols, acylcarnitine, carbonates, dipivefrin
hydrochloride, cocaine, salicylates,
capsaicin, palmitoyl-coenzyme A, imidapril, haloperidol, pyrrolizidine
alkaloids, steroids,
p-nitrophenyl acetate, malathion, butanilicaine, and isocarboxazide. The
enzymes often demonstrate
low substrate specificity. Carboxylesterases are also important for the
conversion of prodrugs to their
respective free acids, which may be the active form of the drug (e.g.,
lovastatin, used to lower blood
cholesterol) (reviewed in Satoh, T. and Hosokawa, M. ( 1998) Annu. Rev.
Pharmacol.
Toxico1.38:257-288).
Neuroligins are a class of molecules that (i) have N-terminal signal
sequences, (ii) resemble
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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).
Sgualene 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 biosynthesis. HMG-CoA is the target of a number of pharmaceutical
compounds
designed to lower plasma cholesterol levels. However, inhibition of MHG-CoA
also results in the
reduced synthesis of non-sterol intermediates (e.g., mevalonate) required for
other biochemical
pathways. SE catalyzes a rate-limiting reaction that occurs later in the
sterol synthesis pathway and
cholesterol in the only end product of the pathway following the step
catalyzed by SE. As a result,
SE is the ideal target for the design of anti-hyperlipidemic drugs that do not
cause a reduction in other
necessary intermediates (Nakamura, Y. et al. (1996) 271:8053-8056).
Epoxide hydrolases
Epoxide hydrolases catalyze the addition of water to epoxide-containing
compounds, thereby
hydrolyzing epoxides to their corresponding 1,2-diols. They are related to
bacterial haloalkane
dehalogenases and show sequence similarity to other members of the aJ~i
hydrolase fold family of
enzymes (e.g., bromoperoxidase A2 from Streptomyces aureofaciens,
hydroxymuconic semialdehyde
hydrolases from Pseudomonas putida, and haloalkane dehalogenase from
Xanthobacter
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autotro~hicus). Epoxide hydrolases are ubiquitous in nature and have been
found in mammals,
invertebrates, plants, fungi, and bacteria. This family of enzymes is
important for the detoxification
of xenobiotic epoxide compounds which are often highly electrophilic and
destructive when
introduced into an organism. Examples of epoxide hydrolase reactions include
the hydrolysis of
cis-9,10-epoxyoctadec-9(Z)-enoic acid (leukotoxin) to form its corresponding
diol,
threo-9,10-dihydroxyoctadec-12(Z)-enoic acid (leukotoxin diol), and the
hydrolysis of
cis-12,13-epoxyoctadec-9(Z)-enoic acid (isoleukotoxin) to form its
corresponding diol
threo-12,13-dihydroxyoctadec-9(Z)-enoic acid (isoleukotoxin diol). Leukotoxins
alter membrane
permeability and ion transport and cause inflammatory responses. In addition,
epoxide carcinogens
are known to be produced by cytochrome P450 as intermediates in the
detoxification of drugs and
environmental toxins.
The enzymes possess a catalytic triad composed of Asp (the nucleophile), Asp
(the
histidine-supporting acid), and His (the water-activating histidine). The
reaction mechanism of
epoxide hydrolase proceeds via a covalently bound ester intermediate initiated
by the nucleophilic
attack of one of the Asp residues on the primary carbon atom of the epoxide
ring of the target
molecule, leading to a covalently bound ester intermediate (Michael Arand, M.
et al. (1996) J. Biol.
Chem. 271:4223-4229; Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657;
Argiriadi, M.A. et al.
(2000) J. Biol. Chem. 275:15265-15270).
Enzymes involved in tyrosine catalysis
The degradation of the amino acid tyrosine to either succinate and pyruvate or
fumarate and
acetoacetate, requires a large number of enzymes and generates a large number
of intermediate
compounds. In addition, many xenobiotic compounds may be metabolized using one
or more
reactions that are part of the tyrosine catabolic pathway. While the pathway
has been studied
primarily in bacteria, tyrosine degradation is known to occur in a variety of
organisms and is likely to
involve many of the same biological reactions.
The enzymes involved in the degradation of tyrosine to succinate and pyruvate
(e.g., in
Arthrobacter species) include 4-hydroxyphenylpyruvate oxidase, 4-
hydroxyphenylacetate
3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxymethyl-2-
hydroxymuconic
semialdehyde dehydrogenase, traps,cis-5-carboxymethyl-2-hydroxymuconate
isomerase,
homoprotocatechuate isomerase/decarboxylase, cis-2-oxohept-3-ene-1,7-dioate
hydratase,
2,4-dihydroxyhept-traps-2-ene-1,7-dioate aldolase, and succinic semialdehyde
dehydrogenase.
The enzymes involved in the degradation of tyrosine to fumarate and
acetoacetate (e.g., in
Pseudomonas species) include 4-hydroxyphenylpyruvate dioxygenase,
homogentisate
1,2-dioxygenase, maleylacetoacetate isomerase, and fumarylacetoacetase. 4-
hydroxyphenylacetate
1-hydroxylase may also be involved if intermediates from the
succinate/pyruvate pathway are
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accepted.
Additional enzymes associated with tyrosine metabolism in different organisms
include
4-chlorophenylacetate-3,4-dioxygenase, aromatic aminotransferase,
5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase, 2-oxo-kept-3-ene-1,7-
dioate hydratase, and
5-carboxymethyl-2-hydroxymuconate isomerase (Elks, L.B.M. et al. (1999)
Nucleic Acids Res.
27:373-376; Wackett, L.P. and Ellis, L.B.M. (1996) J. Microbiol. Meth. 25:91-
93; and Schmidt, M.
(1996) Amer. Soc. Microbiol. News 62:102).
In humans, acquired or inherited genetic defects in enzymes of the tyrosine
degradation
pathway may result in hereditary tyrosinemia. One form of this disease,
hereditary tyrosinemia 1
(HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase,
the last enzyme in the
pathway in organisms that metabolize tyrosine to fumarate and acetoacetate.
HT1 is characterized
by progressive liver damage beginning at infancy, and increased risk for liver
cancer (Endo, F. et al.
(1997) J. Biol. Chem. 272:24426-24432).
The discovery of new drug metabolizing enzymes and the polynucleotides
encoding them
satisfies a need in the art by providing new compositions which are useful in
the diagnosis,
prevention, and treatment of autoimmune/inflammatory, cell proliferative,
developmental, endocrine,
eye, metabolic, and gastrointestinal disorders, including liver disorders, and
in the assessment of the
effects of exogenous compounds on the expression of nucleic acid and amino
acid sequences of drug
metabolizing enzymes.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, drug metabolizing enzymes,
referred to
collectively as "DME" and individually as "DME-1," "DME-2," "DME-3," "DME-4,"
"DME-5,"
"DME-6," "DME-7," "DME-8," "DME-9," and "DME-10." In one aspect, the invention
provides an
isolated polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: l-10, b) a naturally
occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-10,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10. In one alternative, the invention provides
an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:1-10.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-10, b) a naturally occurring polypeptide
comprising an amino acid
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sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
117 NO:1-10, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-10.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ )D NO:1-10. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ ID NO:11-20.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:I-10, b) a naturally occurring polypeptide comprising an amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-10, c)
a biologically active fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-10. In
one alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative,
the invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-10, b) a naturally occurring polypeptide comprising an amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO:1-10, c)
a biologically active fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-10. The
method comprises
a) culturing a cell under conditions suitable for expression of the
polypeptide, wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-10, b) a naturally
occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-10,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10.
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The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ )D NO:11-20, b) a naturally occurring polynucleotide comprising a
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ m
NO:11-20, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative,
the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ )D NO:11-20, b) a naturally occurring polynucleotide
comprising a polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ ID NO:11-20, c) a polynucleotide complementary to the polynucleotide of
a), d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ID NO:11-20, b) a naturally occurring polynucleotide comprising a
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ l~
NO:11-20, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-10, b) a naturally
occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
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selected from the group consisting of SEQ ID NO:1-10, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:I-10,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition comprises an amino acid sequence selected from the
group consisting
of SEQ ID NO:l-10. The invention additionally provides a method of treating a
disease or condition
associated with decreased expression of functional DME, comprising
administering to a patient in
need of such treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-10,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional DME, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-10, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-10,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10. 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
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to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ )D NO:I-10, b) a naturally
occurring
polypeptide cmoprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ >D NO:1-10, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:l-10,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10. The method comprises a) combining the
polypeptide with at
least one test compound under suitable conditions, and b) detecting binding of
the polypeptide to the
test compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-10, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-10, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:I-10,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-10. The method comprises a) combining the
polypeptide with at
least one test compound under conditions permissive for the activity of the
polypeptide, b) assessing
the activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of
the polypeptide in the presence of the test compound with the activity of the
polypeptide in the
absence of the test compound, wherein a change in the activity of the
polypeptide in the presence of
the test compound is indicative of a compound that modulates the activity of
the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID NO:11-20, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID NO:11-20, ii) a
naturally occurring polynucleotide comprising a polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID NO:11-20,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
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polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide
in the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
NO:11-20, ii) a naturally occurnng polynucleotide comprising a polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID NO:11-20,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group
consisting of i)-v) above; c) quantifying the amount of hybridization complex;
and d) comparing the
amount of hybridization complex in the treated biological sample with the
amount of hybridization
complex in an untreated biological sample, wherein a difference in the amount
of hybridization
complex in the treated biological sample is indicative of toxicity of the test
compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and 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
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understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can
be used to practice or test the present invention, the preferred machines,
materials and methods are
now described. All publications mentioned herein are cited for the purpose of
describing and
disclosing the cell lines, protocols, reagents and vectors which are reported
in the publications and
which might be used in connection with the invention. Nothing herein is to be
construed as an
admission that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
DEFINITIONS
"DME" refers to the amino acid sequences of substantially purified DME
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
DME. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of DME either by directly
interacting with
DME or by acting on components of the biological pathway in which DME
participates.
An "allelic variant" is an alternative form of the gene encoding DME. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more
times in a given sequence.
"Altered" nucleic acid sequences encoding DME include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as DME or a
polypeptide with at least one functional characteristic of DME. Included
within this definition are
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polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding DME, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
DME. The encoded protein may also be "altered," and may contain deletions,
insertions, or
substitutions of amino acid residues which produce a silent change and result
in a functionally
equivalent DME. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of DME is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
, amino acids may include lysine and arginine. Amino acids with uncharged
polar side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or
synthetic molecules. Where "amino acid sequence" is recited to refer to a
sequence of a naturally
occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino
acid sequence to the complete native amino acid sequence associated with the
recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological
activity of DME. Antagonists may include proteins such as antibodies, nucleic
acids, carbohydrates,
small molecules, or any other compound or composition which modulates the
activity of DME either
by directly interacting with DME or by acting on components of the biological
pathway in which
DME participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic
determinant. Antibodies that bind DME polypeptides can be prepared using
intact polypeptides or
using fragments containing small peptides of interest as the immunizing
antigen. The polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
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
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immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or
biochemical functions of a naturally occurring molecule. Likewise,
"immunologically active" or
"immunogenic" refers to the capability of the natural, recombinant, or
synthetic DME, or of any
oligopeptide thereof, to induce a specific immune response in appropriate
animals or cells and to
bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a given amino acid sequence" refer broadly to any composition
containing the given
polynucleotide or amino acid sequence. The composition may comprise a dry
formulation or an
aqueous solution. Compositions comprising polynucleotide sequences encoding
DME or fragments
of DME may be employed as hybridization probes. The probes may be stored in
freeze-dried form
and may be associated with a stabilizing agent such as a carbohydrate. In
hybridizations, the probe
may be deployed in an aqueous solution containing salts (e.g., NaCI),
detergents (e.g., sodium
dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry
milk, salmon sperm
DNA, etc.).
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"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to
repeated DNA sequence analysis to resolve uncalled bases, extended using the
XL-PCR kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
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
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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 NO:11-20 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID NO:11-20, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID NO:11-20 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID NO:11-20 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID NO:11-20 and the region of SEQ ID NO:11-20 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-10 is encoded by a fragment of SEQ ID NO:11-20. A
fragment of SEQ ID NO:1-10 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID NO:1-10. For example, a fragment of SEQ ID NO:1-10 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-
10. The precise length of a fragment of SEQ ID NO:1-10 and the region of SEQ
ID NO:1-10 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.
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"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 commonly used and freely available sequence
comparison
algorithms is provided by the National Center for Biotechnology Information
(NCBI) Basic Local
Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol.
215:403-410), which is
available from several sources, including the NCBI, Bethesda, MD, and on the
Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST
2 Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
heward for match: I
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: l0
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Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
' Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that
changes in a nucleic acid sequence can be made using this degeneracy to
produce multiple nucleic
acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer
to the percentage of residue matches between at least two polypeptide
sequences aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at
the site of substitution, thus preserving the structure (and therefore
function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Cap x drop-off. 50
Expect: 10
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Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ m number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of
complementarity. Specific hybridization complexes form under permissive
annealing conditions and
remain hybridized after the "washing" step(s). The washing steps) is
particularly important in
determining the stringency of the hybridization process, with more stringent
conditions allowing less
non-specific binding, i.e., binding between pairs of nucleic acid strands that
are not perfectly
matched. Permissive conditions for annealing of nucleic acid sequences are
routinely determinable
by one of ordinary skill in the art and may be consistent among hybridization
experiments, whereas
wash conditions may be varied among experiments to achieve the desired
stringency, and therefore
hybridization specificity. Permissive annealing conditions occur, for example,
at 68°C in the
presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 pg/ml sheared,
denatured salmon
sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50°l0 of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
( 1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
38
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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.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of DME
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of DME which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of DME. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
39
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biological, functional, or immunological properties of DME.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide, polynucleotide, or any fragment thereof. These phrases also
refer to DNA or RNA
of genomic or synthetic origin which may be single-stranded or double-stranded
and may represent
the sense or the antisense strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like
material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an DME may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of DME.
"Probe" refers to nucleic acid sequences encoding DME, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 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
CA 02403644 2002-09-26
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example Sambrook, J. et al. (1989) Molecular Cloning: A Laborator~Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in
hybridization technologies, for example, as PCR or sequencing primers,
microarray elements, or
specific probes to identify fully or partially complementary polynucleotides
in a sample of nucleic
acids. Methods of oligonucleotide selection are not limited to those described
above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, s-bra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently,
41
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a recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing DME,
nucleic acids encoding DME, or fragments thereof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
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"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a
recipient cell. Transformation may occur under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign
nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is
selected based on the type of host cell being transformed and may include, but
is not limited to,
bacteriophage or viral infection, electroporation, heat shock, lipofection,
and particle bombardment.
The term "transformed cells" includes stably transformed cells in which the
inserted DNA is capable
of replication either as an autonomously replicating plasmid or as part of the
host chromosome, as
well as transiently transformed cells which express the inserted DNA or RNA
for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or mote of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a
precursor of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by
infection with a recombinant virus. The term genetic manipulation does not
include classical
cross-breeding, or in vitro fertilization, but rather is directed to the
introduction of a recombinant
DNA molecule. The transgenic organisms contemplated in accordance with the
present invention
include bacteria, 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), s_ upra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence
having at least 40% sequence identity to the particular nucleic acid sequence
over a certain length of
one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences"
tool Version 2Ø9
(May-07-1999) set at default parameters. Such a pair of nucleic acids may
show, for example, at
least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least
90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least
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99% or greater sequence identity over a certain defined length. A variant may
be described as, for
example, an "allelic" (as defined above), "splice," "species," or
"polymorphic" variant. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or
lesser number of polynucleotides due to alternative splicing of exons during
mRNA processing. The
corresponding polypeptide may possess additional functional domains or lack
domains that are
present in the reference molecule. Species variants are polynucleotide
sequences that vary from one
species to another. The resulting polypeptides will generally have significant
amino acid identity
relative to each other. A polymorphic variant is a variation in the
polynucleotide sequence of a
particular gene between individuals of a given species. Polymorphic variants
also may encompass
"single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence
varies by one
nucleotide base. The presence of SNPs may be indicative of, for example, a
certain population, a
disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human drug metabolizing enzymes
(DME),
the polynucleotides encoding DME, and the use of these compositions for the
diagnosis, treatment,
or prevention of autoimmune/inflammatory, cell proliferative, developmental,
endocrine, eye,
metabolic, and gastrointestinal disorders, including liver disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to
a single Incyte project identification number (Incyte Project m). Each
polypeptide sequence is
denoted by both a polypeptide sequence identification number (Polypeptide SEQ
ID NO:) and an
Incyte polypeptide sequence number (Incyte Polypeptide 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
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polypeptide sequence identification number (Polypeptide SEQ )D 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 polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
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 ff~) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some
cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and
these properties establish that the claimed polypeptides are drug metabolizing
enzymes. For
example, SEQ ID N0:9 is 32% identical to human putative N-acetyltransferase
Camello 2 (GenBank
ID 86651438) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.)
The BLAST probability score is 6.50E-23, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:9 also contains a signal
peptide signature
sequence as determined by the SPScan program, and an acetyltransferase domain
as determined by
searching for statistically significant matches in the hidden Markov model
(HMM)-based PFAM
database of conserved protein family domains. The probability value of the HMM
comparison to
acetyltransferase domain is 3.2e-12. (See Table 3.) Based on BLAST and HMM
analyses, the
protein of SEQ )D N0:9 is an N-acetyltransferase which N-acetylates aromatic
amines and
hvrlra~ina-rnntainino rnmnnnnrlc inrlnrlina hnt not limitPrl tn nara-
aminnhPn~nir arid nara-
CA 02403644 2002-09-26
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database (See Table 7 for descriptions). All of these signature sequence
identifications are highly
statistically significant (see Table 7 for threshold values); for example, the
probability value of the
HMM comparison to the aldo-keto reductase family signature sequence is 5.6e-
170. Based on
BLAST, HMM, BLIMPS, MOTIFS, and ProfileScan analyses, the protein of SEQ ID
NO:10 is an
aldo/keto reductase which reduces carbonyl-containing sugars and aromatic
compounds, including,
but not limited to, glucose, and carbonyl-containing drug molecules and
xenobiotics. SEQ ID NO:1-
8 were analyzed and annotated in a similar manner. The algorithms and
parameters for the analysis
of SEQ ID NO:1-10 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide )D) 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 NO:I 1-20 or that distinguish between SEQ ID
NO:11-20 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
(S') 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,
3700065F6 is the
identification number of an Incyte cDNA sequence, and SININOTOS is the cDNA
library from
which it is derived. Incyte cDNAs for which cDNA libraries are not indicated
were derived from
pooled cDNA libraries (e.g., 71447910V 1 ). Alternatively, the identification
numbers in column 5
may refer to GenBank cDNAs or ESTs (e.g., g5663459) 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.g7025744_000055 002 is the identification number of a Genscan-predicted
coding sequence,
with g7025744 being the GenBank identification number of the sequence to which
Genscan was
applied. The Genscan-predicted coding sequences may have been edited prior to
assembly. (See
Example IV.) Alternatively, the identification numbers in column 5 may refer
to assemblages of
both cDNA and Genscan-predicted exons brought together by an "exon stitching"
algorithm. For
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CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
example, FL,152824_00001 represents a "stitched" sequence in which 152824 is
the identification
number of the cluster of sequences to which the algorithm was applied, and
00001 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 polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses DME variants. A preferred DME variant is one
which has
at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the DME amino acid sequence, and which contains at least
one functional or
structural characteristic of DME.
The invention also encompasses polynucleotides which encode DME. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID NO:11-20, which encodes DME. The
polynucleotide
sequences of SEQ ID NO:11-20, 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
NO:11-20 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 NO:11-20. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of DME.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding DME, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
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produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring DME, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode DME and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring DME under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding DME or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding DME and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half-life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode DME
and
DME derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding DME or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
NO:11-20 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno
NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800
thermal
cycler (Applied Biosystems). Sequencing is then carried out using either the
ABI 373 or 377 DNA
48
CA 02403644 2002-09-26
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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 BioloQV, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and BiotechnoloQV, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding DME may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments
comprising a known genomic locus and surrounding sequences. (See, e.g.,
Triglia, T. et al. (1988)
Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR
amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome
DNA. (See, e.g.,
Lagerstrom, M. et al. ( 1991 ) PCR Methods Applic. 1:111-119.) In this method,
multiple restriction
enzyme digestions and ligations may be used to insert an engineered double-
stranded sequence into a
region of unknown sequence before performing PCR. Other methods which may be
used to retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 primer analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of
sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
49
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode DME may be cloned in recombinant DNA molecules that direct
expression of DME,
or fragments or functional equivalents thereof, in appropriate host cells. Due
to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express DME.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter DME-encoding sequences for a
variety of purposes
including, but not limited to, modification of the cloning, processing, and/or
expression of the gene
product. DNA shuffling by random fragmentation and PCR reassembly of gene
fragments and
synthetic oligonucleotides may be used to engineer the nucleotide sequences.
For example,
oligonucleotide-mediated site-directed mutagenesis may be used to introduce
mutations that create
new restriction sites, alter glycosylation patterns, change codon preference,
produce splice variants,
and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of DME, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding DME may be synthesized, in whole or
in part,
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
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, supra, pp. 28-53.)
In order to express a biologically active DME, the nucleotide sequences
encoding DME or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding DME. Such elements may vary in their
strength and specificity.
Specific initiation signals may also be used to achieve more efficient
translation of sequences
encoding DME. Such signals include the ATG initiation codon and adjacent
sequences, e.g. the
Kozak sequence. In cases where sequences encoding DME and its initiation codon
and upstream
regulatory sequences are inserted into the appropriate expression vector, no
additional transcriptional
or translational control signals may be needed. However, in cases where only
coding sequence, or a
fragment thereof, is inserted, exogenous translational control signals
including an in-frame ATG
initiation codon should be provided by the vector. Exogenous translational
elements and initiation
codons may be of various origins, both natural and synthetic. The efficiency
of expression may be
enhanced by the inclusion of enhancers appropriate for the particular host
cell system used. (See,
e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct
expression vectors containing sequences encoding DME and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook,
J. et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
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WO 01/79468 PCT/USO1/11869
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 PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding DME into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of DME are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of DME may be used. For
example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of DME. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
52
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
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, suura; 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.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding DME
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses DME in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of DME in cell lines is preferred. For example, sequences encoding DME can be
transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer
resistance to a selective agent, and its presence allows growth and recovery
of cells which
successfully express the introduced sequences. Resistant clones of stably
transformed cells may be
propagated using tissue culture techniques appropriate to the cell type.
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CA 02403644 2002-09-26
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Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), (3 glucuronidase and its substrate f3-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding DME is inserted within a marker gene sequence,
transformed cells containing
sequences encoding DME can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding DME under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding DME and
that express
DME may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution,
or chip based technologies for the detection and/or quantification of nucleic
acid or protein
sequences.
Immunological methods for detecting and measuring the expression of DME using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on DME is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art.
(See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory
Manual, APS Press, St. Paul
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MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in ImmunoloQV,
Greene Pub. Associates
and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding DME
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding DME, or any fragments
thereof, may be cloned
into a vector for the production of an mRNA probe. Such vectors are known in
the art, are
commercially available, and may be used to synthesize RNA probes in vitro by
addition of an
appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures may
be conducted using a variety of commercially available kits, such as those
provided by Amersham
Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter
molecules or
labels which may be used for ease of detection include radionuclides, enzymes,
fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates, cofactors,
inhibitors, magnetic
particles, and the like.
Host cells transformed with nucleotide sequences encoding DME may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the
sequence and/or the vector used. As will be understood by those of skill in
the art, expression
vectors containing polynucleotides which encode DME may be designed to contain
signal sequences
which direct secretion of DME through a prokaryotic or eukaryotic cell
membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
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
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
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-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-niyc, and hemagglutinin (HA)
enable immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the DME encoding sequence and the
heterologous protein
sequence, so that DME may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel ( 1995, s. unra, ch.
10). A variety of commercially available kits may also be used to facilitate
expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled DME may be
achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with
the T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
DME of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to DME. At least one and up to a plurality of test
compounds may be screened
for specific binding to DME. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
DME, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which DME
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express DME, either
as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing DME or cell membrane fractions which contain DME are
then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either DME or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
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CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
DME, either in
solution or affixed to a solid support, and detecting the binding of DME to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to
a solid support.
DME of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of DME. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for DME
activity, wherein DME is combined with at least one test compound, and the
activity of DME in the
presence of a test compound is compared with the activity of DME in the
absence of the test
compound. A change in the activity of DME in the presence of the test compound
is indicative of a
compound that modulates the activity of DME. Alternatively, a test compound is
combined with an
in vitro or cell-free system comprising DME under conditions suitable for DME
activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of DME
may do so indirectly and need not come in direct contact with the test
compound. At least one and
up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding DME or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S.
Patent Number
5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line,
are derived from the
early mouse embryo and grown in culture. The ES cells are transformed with a
vector containing the
gene of interest disrupted by a marker gene, e.g., the neomycin
phosphotransferase gene (neo;
Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region
of the host genome by homologous recombination. Alternatively, homologous
recombination takes
place using the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-
specific manner (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 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
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human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
( 1998) Science 282:1145-1147).
Polynucleotides encoding DME can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding DME is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress DME, e.g., by secreting DME in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of DME and drug metabolizing enzymes. In addition, the
expression of DME is
closely associated with a liver tumor cell line, liver tumor tissue,
pancreatic tissue, pituitary gland
tissue, brain tissue, small intestine tissue, fetal brain tissue, and
allocortex brain tissue. 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 immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
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systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; a cell proliferative disorder, such as actinic keratosis,
arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),
myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma,
and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow,
brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus; a
developmental disorder, such as renal tubular acidosis, anemia, Cushing's
syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation),
Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as
Syndenham's chorea
and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss; an endocrine disorder, such as disorders of the
hypothalamus and
pituitary resulting from lesions such as primary brain tumors, adenomas,
infarction associated with
pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis,
infections,
immunological disorders, and complications due to head trauma; disorders
associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus,
Kallman's disease,
Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty
sella syndrome, and
dwarfism; disorders associated with hyperpituitarism including acromegaly,
giantism, and syndrome
of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by
benign adenoma;
disorders associated with hypothyroidism including goiter, myxedema, acute
thyroiditis associated
with bacterial infection, subacute thyroiditis associated with viral
infection, autoimmune thyroiditis
(Hashimoto's disease), and cretinism; disorders associated with
hyperthyroidism including
thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema,
toxic multinodular goiter,
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
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hormones such as: in women, abnormal prolactin production, infertility,
endometriosis,
perturbations of the menstrual cycle, polycystic ovarian disease,
hyperprolactinemia, isolated
gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism
and virilization,
breast cancer, and, in post-menopausal women, osteoporosis; and, in men,
Leydig cell deficiency,
male climacteric phase, and germinal cell aplasia, hypergonadal disorders
associated with Leydig
cell tumors, androgen resistance associated with absence of androgen
receptors, syndrome of 5 a-
reductase, and gynecomastia; an eye disorder, such as conjunctivitis,
keratoconjunctivitis sicca,
keratitis, episcleritis, iritis, posterior uveitis, glaucoma, amaurosis fugax,
ischemic optic neuropathy,
optic neuritis, Leber's hereditary optic neuropathy, toxic optic neuropathy,
vitreous detachment,
retinal detachment, cataract, macular degeneration, central serous
chorioretinopathy, retinitis
pigmentosa, melanoma of the choroid, retrobulbar tumor, and chiasmal tumor; a
metabolic disorder,
such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal
hyperplasia,
coumarin resistance, cystic fibrosis, diabetes, fatty hepatocirrhosis,
fructose-1,6-diphosphatase
deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases,
hereditary fructose
intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism,
hypoparathyroidism,
hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism,
hyperlipidemia,
hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases,
Menkes syndrome,
occipital horn syndrome, mannosidosis, neuraminidase deficiency, obesity,
pentosuria
phenylketonuria, pseudovitamin D-deficiency rickets; hypocalcemia,
hypophosphatemia, and
postpubescent cerebellar ataxia, tyrosinemia, and a gastrointestinal disorder,
such as dysphagia,
peptic esophagitis, esophageal spasm, esophageal stricture, esophageal
carcinoma, dyspepsia,
indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,
gastroparesis, antral or pyloric
edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction,
infections of the intestinal
tract, peptic ulcer, cholelithiasis, cholecysfitis, cholestasis, pancreatitis,
pancreatic carcinoma, biliary
tract disease, hepatitis, hyperbilirubinemia, hereditary hyperbilirubinemia,
cirrhosis, passive
congestion of the liver, hepatoma, infectious colitis, ulcerative colitis,
ulcerative proctitis, Crohn's
disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic
obstruction,
irritable bowel syndrome, short bowel syndrome, diarrhea, constipation,
gastrointestinal hemorrhage,
acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic
encephalopathy,
hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease,
alpha,-antitrypsin
deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction,
portal vein obstruction
and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein
thrombosis, veno-occlusive
disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic
cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas.
In another embodiment, a vector capable of expressing DME or a fragment or
derivative
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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/inflammatory, cell
proliferative,
developmental, endocrine, eye, metabolic, and gastrointestinal disorders,
including liver disorders,
described above. In one aspect, an antibody which specifically binds DME may
be used directly as
an antagonist or indirectly as a targeting or delivery mechanism for bringing
a pharmaceutical agent
to cells or tissues which express DME.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding DME may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of DME including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of DME may be produced using methods which are generally known
in the
art. In particular, purified DME may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind DME.
Antibodies to DME may also
be generated using methods that are well known in the art. Such antibodies may
include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with DME or with any fragment or
oligopeptide thereof
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which has immunogenic properties. Depending on the host species, various
adjuvants may be used
to increase immunological response. Such adjuvants include, but are not
limited to, Freund's,
mineral gels such as aluminum hydroxide, and surface active substances such as
lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
Among adjuvants
used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium narvum are
especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
DME have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of DME amino acids may be fused with those of another protein, such as KLH,
and antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to DME may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-
hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;
Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl.
Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of
single chain antibodies may be adapted, using methods known in the art, to
produce DME-specific
single chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition,
may be generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See,
e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
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
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easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
DME and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering DME epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for DME. Affinity
is expressed as an
association constant, K~, which is defined as the molar concentration of DME-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The K~ determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
IS affinities for multiple DME epitopes, represents the average affinity, or
avidity, of the antibodies for
DME. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular DME epitope, represents a true measure of affinity. High-affinity
antibody preparations
with K~ ranging from about 109 to 10'2 L/mole are preferred for use in
immunoassays in which the
DME-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ka ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and
similar procedures which ultimately require dissociation of DME, preferably in
active form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991 ) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
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. s_u~ra.)
In another embodiment of the invention, the polynucleotides encoding DME, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
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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 Theraueutics,
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 germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias,
familial hypercholesterolemia, and hemophilia resulting from Factor VIII or
Factor IX deficiencies
(Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997)
Nature 389:239-242)),
(ii) express a conditionally lethal gene product (e.g., in the case of cancers
which result from
unregulated cell proliferation), or (iii) express a protein which affords
protection against intracellular
parasites (e.g., against human retroviruses, such as human immunodeficiency
virus (HIV)
(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc.
Natl. Acad. Sci. USA.
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as
Candida albicans
and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium
falciparum and
Trypanosoma cruzi). In the case where a genetic deficiency in DME expression
or regulation causes
disease, the expression of DME from an appropriate population of transduced
cells may alleviate the
clinical manifestations caused by the genetic deficiency.
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In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
DME are treated by constructing mammalian expression vectors encoding DME and
introducing
these vectors by mechanical means into DME-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson
(1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of DME include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX 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 ~i-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau
(1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible
promoter (Rossi, F.M.V. and Blau, H.M. supra)), or (iii) a tissue-specific
promoter or the native
promoter of the endogenous gene encoding DME from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to DME expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding DME under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
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WO 01/79468 PCT/USO1/11869
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding DME to cells which have one or more genetic
abnormalities with respect
to the expression of DME. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven
to be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the
pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially
useful adenoviral
vectors are described in U.S. Patent Number 5,707,618 to Armentano
("Adenovirus vectors for gene
therapy"), hereby incorporated by reference. For adenoviral vectors, see also
Antinozzi, P.A. et al.
(1999) Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242,
both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding DME to target cells which have one or more genetic
abnormalities with
respect to the expression of DME. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing DME to cells of the central nervous
system, for which HSV has a
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
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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 performing alphavirus infections, are well known to those
with ordinary skill in
the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo i~c Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence oc antisense molecule may also be designed to
block translation of
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mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding DME.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include
techniques for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite
chemical synthesis. Alternatively, RNA molecules may be generated by in vitro
and in vivo
transcription of DNA sequences encoding DME. Such DNA sequences may be
incorporated into a
wide variety of vectors with suitable RNA polymerase promoters such as T7 or
SP6. Alternatively,
these cDNA constructs that synthesize complementary RNA, constitutively or
inducibly, can be
introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as
inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thin-, and
similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as easily
recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding DME. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
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compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased DME
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding DME may be therapeutically useful, and in the treament of disorders
associated with
decreased DME expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding DME may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective
in altering polynucleotide expression; selection from an existing,
commercially-available or
proprietary library of naturally-occurring or non-natural chemical compounds;
rational design of a
compound based on chemical and/or structural properties of the target
polynucleotide; and selection
from a library of chemical compounds created combinatorially or randomly. A
sample comprising a
polynucleotide encoding DME is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
DME are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding DME. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific
polynucleotide can be carried out, for example, using a 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:E 15) or a human cell line such as HeLa cell (Clarke,
M.L. et al. (2000)
Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the
present invention
involves screening a combinatorial library of oligonucleotides (such as
deoxyribonucleotides,
ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for
antisense activity against a
specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No.
5,686,242; Bruice,
T.W. et al. (2000) U.S. Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be
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achieved using methods which are well known in the art. (See, e.g., Goldman,
C.K. et al. (1997)
Nat. Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits,
and monkeys.
An additional embodiment of the invention.relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of DME, antibodies to DME, and mimetics, agonists, antagonists, or
inhibitors of DME.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g.,
Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the
advantage of
administration without needle injection, and obviates the need for potentially
toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising DME or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the 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
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culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example DME
or fragments thereof, antibodies of DME, and agonists, antagonists or
inhibitors of DME, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDso/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no
toxicity. The dosage varies within this range depending upon the dosage form
employed, the
sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of
the active moiety or to maintain the desired effect. Factors which may be
taken into account include
the severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 fig, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
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
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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.
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
NO:11-20 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
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and
may be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
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such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding DME may be used for the diagnosis of
disorders
associated with expression of DME. Examples of such disorders include, but are
not limited to, an
autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; a cell proliferative disorder, such as actinic keratosis,
arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),
myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma,
and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow,
brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus; a
developmental disorder, such as renal tubular acidosis, anemia, Cushing's
syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation),
Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
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,
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Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty
sella syndrome, and
dwarfism; disorders associated with hyperpituitarism including acromegaly,
giantism, and syndrome
of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by
benign adenoma;
disorders associated with hypothyroidism including goiter, myxedema, acute
thyroiditis associated
with bacterial infection, subacute thyroiditis associated with viral
infection, autoimmune thyroiditis
(Hashimoto's disease), and cretinism; disorders associated with
hyperthyroidism including
thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema,
toxic multinodular goiter,
thyroid carcinoma, and Plummer's disease; disorders associated with
hyperparathyroidism including
Conn disease (chronic hypercalemia); pancreatic disorders such as Type I or
Type II diabetes
mellitus and associated complications; disorders associated with the adrenals
such as hyperplasia,
carcinoma, or adenoma of the adrenal cortex, hypertension associated with
alkalosis, amyloidosis,
hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon
syndrome,
pheochromocytoma tumors, and Addison's disease; disorders associated with
gonadal steroid
hormones such as: in women, abnormal prolactin production, infertility,
endometriosis,
perturbations of the menstrual cycle, polycystic ovarian disease,
hyperprolactinemia, isolated
gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism
and virilization,
breast cancer, and, in post-menopausal women, osteoporosis; and, in men,
Leydig cell deficiency,
male climacteric phase, and germinal cell aplasia, hypergonadal disorders
associated with Leydig
cell tumors, androgen resistance associated with absence of androgen
receptors, syndrome of 5 a-
reductase, and gynecomastia; an eye disorder, such as conjunctivitis,
keratoconjunctivitis sicca,
keratitis, episcleritis, iritis, posterior uveitis, glaucoma, amaurosis fugax,
ischemic optic neuropathy,
optic neuritis, Leber's hereditary optic neuropathy, toxic optic neuropathy,
vitreous detachment,
retinal detachment, cataract, macular degeneration, central serous
chorioretinopathy, retinitis
pigmentosa, melanoma of the choroid, retrobulbar tumor, and chiasmal tumor; a
metabolic disorder,
such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal
hyperplasia,
coumarin resistance, cystic fibrosis, diabetes, fatty hepatocirrhosis,
fructose-1,6-diphosphatase
deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases,
hereditary fructose
intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism,
hypoparathyroidism,
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
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edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction,
infections of the intestinal
tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis,
pancreatic carcinoma, biliary
tract disease, hepatitis, hyperbilirubinemia, hereditary hyperbilirubinemia,
cirrhosis, passive
congestion of the liver, hepatoma, infectious colitis, ulcerative colitis,
ulcerative proctitis, Crohn's
disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic
obstruction,
irritable bowel syndrome, short bowel syndrome, diarrhea, constipation,
gastrointestinal hemorrhage,
acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic
encephalopathy,
hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease,
alpha,-antitrypsin
deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction,
portal vein obstruction
and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein
thrombosis, veno-occlusive
disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic
cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas. The
polynucleotide sequences encoding DME may be used in Southern or northern
analysis, dot blot, or
other membrane-based technologies; in PCR technologies; in dipstick, pin, and
multiformat ELISA-
like assays; and in microarrays utilizing fluids or tissues from patients to
detect altered DME
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding DME may be useful in
assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding DME may be labeled by standard methods and added to a fluid
or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding DME in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
DME, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding DME, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
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Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained
from successive assays may be used to show the efficacy of treatment over a
period ranging from
several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding DME
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a
polynucleotide encoding DME, or a fragment of a polynucleotide complementary
to the
polynucleotide encoding DME, and will be employed under optimized conditions
for identification
of a specific gene or condition. Oligomers may also be employed under less
stringent conditions for
detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding DME may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding DME
are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory
preparation of DNA and sequencing errors using statistical models and
automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and
characterized by mass
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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. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide
of interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The
microarray can be used in transcript imaging techniques which monitor the
relative expression levels
of large numbers of genes simultaneously as described below. The microarray
may also be used to
identify genetic variants, mutations, and polymorphisms. This information may
be used to determine
gene function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be
used to develop a pharmacogenomic profile of a patient in order to select the
most appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, DME, fragments of DME, or antibodies specific for DME
may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern
of gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed
by quantifying the number of expressed genes and their relative abundance
under given conditions
and at a given time. (See Seilhamer et al., "Comparative Gene Transcript
Analysis," U.S. Patent
Number 5,840,484, expressly incorporated by reference herein.) Thus a
transcript image may be
generated by hybridizing the polynucleotides of the present invention or their
complements to the
totality of transcripts or reverse transcripts of a particular tissue or cell
type. In one embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
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Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they
contain expression information from a large number of genes and gene families.
Ideally, a genome-
wide measurement of expression provides the highest quality signature. Even
genes whose
expression is not altered by any tested compounds are important as well, as
the levels of expression
of these genes are used to normalize the rest of the expression data. The
normalization procedure is
useful for comparison of expression data after treatment with different
compounds. While the
assignment of gene function to elements of a toxicant signature aids in
interpretation of toxicity
mechanisms, knowledge of gene function is not necessary for the statistical
matching of signatures
which leads to prediction of toxicity. (See, for example, Press Release 00-02
from the National
Institute of Environmental Health Sciences, released February 29, 2000,
available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared
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
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under given conditions and at a given time. A profile of a cell's proteome may
thus be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample
are separated by isoelectric focusing in the first dimension, and then
according to molecular weight
by sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
suera). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for DME to
quantify the
levels of DME expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. ( 1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
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each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount
of protein between the two samples is indicative of a toxic response to the
test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See,
e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl.
Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116;
Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997)
Proc. Natl. Acad.
Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No.
5,605,662.) Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical 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
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harnngton,
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
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genetic map data can be found in various scientific journals or at the Online
Mendelian Inheritance
in Man (OMIM) World Wide Web site. Correlation between the location of the
gene encoding DME
on a physical map and a specific disorder, or a predisposition to a specific
disorder, may help define
the region of DNA associated with that disorder and thus may further
positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information
is valuable to investigators searching for disease genes using positional
cloning or other gene
discovery techniques. Once the gene or genes responsible for a disease or
syndrome have been
crudely localized by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to
l 1q22-23, any sequences mapping to that area may represent associated or
regulatory genes for
further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-
580.) The nucleotide
sequence of the instant invention may also be used to detect differences in
the chromosomal location
due to translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, DME, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between DME and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with DME, or
fragments thereof,
and washed. Bound DME is then detected by methods well known in the art.
Purified DME can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively,.non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing antibodies capable of binding DME specifically compete with a
test compound for
binding DME. In this manner, antibodies can be used to detect the presence of
any peptide which
shares one or more antigenic determinants with DME.
In additional embodiments, the nucleotide sequences which encode DME may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
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properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/197,590, U.S. Ser. No. 601198,403, U.S. Ser. No.
60/200,185, U.S. Ser.
No. 60/202,234, and U.S. Ser. No. 60/203,509, are expressly incorporated by
reference herein.
EXAMPLES
I. ~ Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and~precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles
(QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively,
RNA was isolated directly from tissue lysates using other RNA isolation kits,
e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (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 S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
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(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
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega);
an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and
QIAWELL 8
Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems
or the
R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following
precipitation, plasmids were
resuspended in 0.1 ml of distilled water and stored, with or without
lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such
as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit
(Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
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
iri the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, s-unra, unit 7.7). Some of the cDNA sequences were selected for
extension using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
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programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases,
and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein
family databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus
primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.
6:361-365.) The queries were performed using programs based on BLAST, FASTA,
BLIMPS, and
HMMER. The Incyte cDNA sequences were assembled to produce full length
polynucleotide
sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,
stretched
sequences, or Genscan-predicted coding sequences (see Examples IV and V) were
used to extend
Incyte cDNA assemblages to full length. Assembly was performed using programs
based on Phred,
Phrap, and Consed, and cDNA assemblages were screened for open reading frames
using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences
were
translated to derive the corresponding full length polypeptide sequences.
Alternatively, a
polypeptide of the invention may begin at any of the methionine residues of
the full length translated
polypeptide. Full length polypeptide sequences were subsequently analyzed by
querying against
databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS,
PRINTS, DOMO,
PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases
such as
PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS
PRO software
(Hitachi Software Engineering, South San Francisco CA) and LASERGENE software
(DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default
parameters
specified by the CLUSTAL algorithm as incorporated into the MEGALIGN
multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly
of Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
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
B7 NO:11-20. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
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and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative drug metabolizing enzymes were initially identified by running the
Genscan gene
identification program against public genomic sequence databases (e.g., gbpri
and gbhtg). Genscan
is a general-purpose gene identification program which analyzes genomic DNA
sequences from a
variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-
94, and Burge, C. and
S. Karlin ( 1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to
form an assembled cDNA sequence extending from a methionine to a stop codon..
The output of
Genscan is a FASTA database of polynucleotide and polypeptide sequences. The
maximum range of
sequence for Genscan to analyze at once was set to 30 kb. To determine which
of these Genscan
predicted cDNA sequences encode drug metabolizing enzymes, the encoded
polypeptides were
analyzed by querying against PFAM models for drug metabolizing enzymes.
Potential drug
metabolizing enzymes were also identified by homology to Incyte cDNA sequences
that had been
annotated as drug metabolizing enzymes. These selected Genscan-predicted
sequences were then
compared by BLAST analysis to the genpept and gbpri public databases. Where
necessary, the
Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra or omitted
exons. BLAST
analysis was also used to find any Incyte cDNA or public cDNA coverage of the
Genscan-predicted
sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available,
this information was used to correct or confirm the Genscan predicted
sequence. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences 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 1V. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an
algorithm based on graph theory and dynamic programming to integrate cDNA and
genomic
information, generating possible splice variants that were subsequently
confirmed, edited, or
extended to create a full length sequence. Sequence intervals in which the
entire length of the
interval was present on more than one sequence in the cluster were identified,
and intervals thus
identified were considered to be equivalent by transitivity. For example, if
an interval was present
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on a cDNA and two genomic sequences, then all three intervals were considered
to be equivalent.
This process allows unrelated but consecutive genomic sequences to be brought
together, bridged by
cDNA sequence. Intervals thus identified were then "stitched" together by the
stitching algorithm in
the order that they appear along their parent sequences to generate the
longest possible sequence, as
well as sequence variants. Linkages between intervals which proceed along one
type of parent
sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given
preference over
linkages which change parent type (cDNA to genomic sequence). The resultant
stitched sequences
were translated and compared by BLAST analysis to the genpept and gbpri public
databases.
Incorrect exons predicted by Genscan were corrected by comparison to the top
BLAST hit from
genpept. Sequences were further extended with additional cDNA sequences, or by
inspection of
genomic DNA, when necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of DME Encoding Polynucleotides
The sequences which were used to assemble SEQ ID NO:11-20 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 NO:11-20 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, of human chromosomes.
The map
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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,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or
related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte
Genomics). This
analysis is much faster than multiple membrane-based hybridizations. In
addition, the sensitivity of
the computer search can be modified to determine whether any particular match
is categorized as
exact or similar. The basis of the search is the product score, which is
defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in
a BLAST alignment. For example, a product score of 100 is produced only for
100% identity over
the entire length of the shorter of the two sequences being compared. A
product score of 70 is
produced either by 100% identity and 70% overlap at one end, or by 88%
identity and 100% overlap
at the other. A product score of 50 is produced either by 100% identity and
50% overlap at one end,
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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 cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective
tissue; digestive system; embryonic structures; endocrine system; exocrine
glands; genitalia, female;
genitalia, male; germ cells; heroic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and
divided by the total number of libraries across all categories. The resulting
percentages reflect the
tissue- and disease-specific expression of cDNA encoding DME. cDNA sequences
and cDNA
library/tissue information are found in the LIFESEQ GOLD database (Incyte
Genorriics, Palo Alto
CA).
VIII. Extension of DME Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68°C, 5 min; Step 7: storage
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at 4°C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C,
3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4:
68°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 ~l aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in
384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure
or are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:11-20 are employed to screen
cDNAs,
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genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ,uCi of
[y-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25' superfine size exclusion dextran bead column (Amersham
Pharmacia Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, 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),
su ra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof
may comprise the elements of the microarray. Fragments or oligomers suitable
for hybridization can
be selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in
the biological sample are conjugated to a fluorescent label or other molecular
tag for ease of
detection. After hybridization, nonhybridized nucleotides from the biological
sample are removed,
and a fluorescence scanner is used to detect hybridization at each array
element. Alternatively, laser
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desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/pl RNase inhibitor, 500 E.~M dATP, 500 pM
dGTP, 500 NM dTTP, 40
E.~M dCTP, 40 l.~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
The reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro
transcription from non-coding yeast genomic DNA. After incubation at 37
° C for 2 hr, each reaction
sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of
O.SM sodium
hydroxide and incubated for 20 minutes at 85°C to the stop the reaction
and degrade the RNA.
Samples are purified using two successive CHROMA SPIN 30 gel filtration spin
columns
(CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) and after combining,
both reaction
samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml
sodium acetate, and 300
ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC
(Savant Instruments
Inc., Holbrook NY) and resuspended in 14 p1 SX SSC/0.2% SDS.
Microarrav Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 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
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Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°C
followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65°C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an I .8 cmz coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 p1 of SX SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60°C. The arrays are washed for 10 min
at 45°C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a
second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral
lines 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 emission maxima of the fluorophores used are 565 nm for Cy3 and
650 nm for CyS.
Each array is typically scanned twice, one scan per fluorophore using the
appropriate filters at the
laser source, although the apparatus is capable of recording the spectra from
both fluorophores
simultaneously.
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The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are _
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the DME-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring DME. Although
use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of DME. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a
complementary oligonucleotide is designed to prevent ribosomal binding to the
DME-encoding
transcript.
XII. Expression of DME
Expression and purification of DME is achieved using bacterial or virus-based
expression
systems. For expression of DME in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
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element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express DME upon induction with isopropyl beta-D-
thiogalactopyranoside (IPTG). Expression of DME in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Auto~raphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding DME by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect ~odoptera frugiperda (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 Laponicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
DME at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies
(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,
enables purification on
metal-chelate resins (QIAGEN). Methods for protein expression and purification
are discussed in
Ausubel (1995, supra, ch. 10 and 16). Purified DME obtained by these methods
can be used directly
in the assays shown in Examples XVI, XVII, and 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 electroporation. 1-2 ~cg of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
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Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser
optics-based technique, is used to identify transfected cells expressing GFP
or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the
uptake of fluorescent molecules that diagnose events preceding or coincident
with cell death. These
events include changes in nuclear DNA content as measured by staining of DNA
with propidium
iodide; changes in cell size and granularity as measured by forward light
scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine
uptake; alterations in expression of cell surface and intracellular proteins
as measured by reactivity
with specific antibodies; and alterations in plasma membrane composition as
measured by the
binding of fluorescein-conjugated Annexin V protein to the cell surface.
Methods in flow cytometry
are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of DME on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding DME and either CD64 or CD64-GFP.
CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind to
conserved regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding DME and other genes of interest can be analyzed by
northern
analysis or microarray techniques.
XIV. Production of DME Specific Antibodies
DME substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harnngton, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used
to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the DME amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are
well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (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-KL,H 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°lo BSA, reacting with rabbit antisera, washing, and
reacting with radio-iodinated
CA 02403644 2002-09-26
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goat anti-rabbit IgG.
XV. Purification of Naturally Occurring DME Using Specific Antibodies
Naturally occurring or recombinant DME is substantially purified by
immunoaffinity
chromatography using antibodies specific for DME. An immunoaffinity column is
constructed by
covalently coupling anti-DME antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing DME are passed over the immunoaffinity column, and the column
is
washed under conditions that allow the preferential absorbance of DME (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/DME binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such
as urea or thiocyanate ion), and DME is collected.
XVI. Identification of Molecules Which Interact with DME
DME, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled DME, washed,
and any wells with labeled DME complex are assayed. Data obtained using
different concentrations
of DME are used to calculate values for the number, affinity, and association
of DME with the
candidate molecules.
Alternatively, molecules interacting with DME are analyzed using the yeast two-
hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
DME may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all
interactions between the proteins encoded by two large libraries of genes
(Nandabalan, K. et al.
(2000) U.S. Patent No. 6,057,101).
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 MgClz,
50 mM
nicotinamide, 40 mg trisodium isocitrate, and 2 units isocitrate
dehydrogenase, with 8 mg NADP+
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added to a 10 mL reaction buffer stock just prior to assay. Reactions are
carried out in an optical
cuvette, and the absorbance at 630 nm is measured. The rate of increase in
absorbance is
proportional to the enzyme activity in the assay. A standard curve can be
constructed using known
concentrations of 4-aminophenol.
1a,25-dihydroxyvitamin D 24-hydroxylase activity of DME is determined by
monitoring the
conversion of 3H-labeled 1a,25-dihydroxyvitamin D (1a,25(OH)ZD) to 24,25-
dihydroxyvitamin D
(24,25(OH)zD) in transgenic rats expressing DME. 1 pg of 1a,25(OH)ZD dissolved
in ethanol (or
ethanol alone as a control) is administered intravenously to approximately 6-
week-old male
transgenic rats expressing DME or otherwise identical control rats expressing
either a defective
variant of DME or not expressing DME. The rats are killed by decapitation
after 8 hrs, and the
kidneys are rapidly removed, rinsed, and homogenized in 9 volumes of ice-cold
buffer (15 mM Tris-
acetate (pH 7.4), 0.19 M sucrose, 2 mM magnesium acetate, and 5 mM sodium
succinate). A portion
(e.g., 3 ml) of each homogenate is then incubated with 0.25 nM 1a,25(OH)2[1
3H]D, with a specific
activity of approximately 3.5 GBq/mmol, for 15 min at 37 °C under
oxygen with constant shaking.
Total lipids are extracted as described (Bligh, E.G. and W.J. Dyer (1959) Can.
J. Biochem. Physiol.
37: 911-917) and the chloroform phase is analyzed by HPLC using a FINEPAK SIL
column
(JASCO, Tokyo, Japan) with a n-hexane/chloroform/methanol (10:2.5:1.5) solvent
system at a flow
rate of 1 ml/min. In the alternative, the chloroform phase is analyzed by
reverse phase HPLC using a
J SPHERE ODS-AM column (YMC Co. Ltd., Kyoto, Japan) with an acetonitrile
buffer system (40
to 100%, in water, in 30 min) at a flow rate of 1 ml/min. The eluates are
collected in fractions of 30
seconds (or less) and the amount of 3H present in each fraction is measured
using a scintillation
counter. By comparing the chromatograms of control samples (i.e., samples
comprising
1a,25-dihydroxyvitamin D or 24,25-dihydroxyvitamin D (24,25(OH)zD), with the
chromatograms of
the reaction products, the relative mobilities of.the substrate (1a,25(OH)z[1-
;H]D) and product
(24,25(0H)2[1 3H]D) are determined and correlated with the fractions
collected. The amount of
24,25(0H)2[1 3H]D produced in control rats is subtracted from that of
transgenic rats expressing
DME. The difference in the production of 24,25(0H)2[1 3H]D in the transgenic
and control animals
is proportional to the amount of 25-hydrolase activity of DME present in the
sample. Confirmation
of the identity of the substrate and products) is confirmed by means of mass
spectroscopy
(Miyamoto, Y. et al. (1997) J. Biol. Chem. 272:14115-14119).
Flavin-containing monooxygenase activity of DME is measured by chromatographic
analysis
of metabolic products. For example, Ring, B.J. et al. (1999; Drug Metab. Dis.
27:1099-1103)
incubated FMO in 0.1 M sodium phosphate buffer (pH 7.4 or 8.3) and 1 mM NADPH
at 37°C,
stopped the reaction with an organic solvent, and determined product formation
by HPLC.
Alternatively, activity is measured by monitoring oxygen uptake using a Clark-
type electrode. For
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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, suera). 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 MgCIZ, 0.025% Triton X-100, 1
mM ascorbic acid,
0.75 mM UDP-glucuronic acid). After sufficient time, the reaction is stopped
by addition of ice-cold
20% trichloroacetic acid in 0.1 M phosphate buffer pH 2.7, incubated on ice,
and centrifuged to
clarify the supernatant. Any unreacted 2-aminophenol is destroyed in this
step. Sufficient freshly-
prepared sodium nitrite is then added; this step allows formation of the
diazonium salt of the
glucuronidated product. Excess nitrite is removed by addition of sufficient
ammonium sulfamate,
and the diazonium salt is reacted with an aromatic amine (for example, N-
naphthylethylene diamine)
to produce a colored azo compound which can be assayed spectrophotometrically
(at 540 nm for the
example). A standard curve can be constructed using known concentrations of
aniline, which will
form a chromophore with similar properties to 2-aminophenol glucuronide.
Glutathione S-transferase activity of DME is measured using a model substrate,
such as 2,4-
dinitro-1-chlorobenzene, which reacts with glutathione to form a product, 2,4-
dinitrophenyl-
glutathione, that has an absorbance maximum at 340 nm. It is important to note
that GSTs have
differing substrate specificities, and the model substrate should be selected
based on the substrate
preferences of the GST of interest. Assays are performed at ambient
temperature and contain an
aliquot of the enzyme in a suitable reaction buffer (for example, 1 mM
glutathione, 1 mM
dinitrochlorobenzene, 90 mM potassium phosphate buffer pH 6.5). Reactions are
carried out in an
optical cuvette, and the absorbance at 340 nm is measured. The rate of
increase in absorbance is
proportional to the enzyme activity in the assay.
N-acyltransferase activity of DME is measured using radiolabeled amino acid
substrates and
measuring radiolabel incorporation into conjugated products. Enzyme is
incubated in a reaction
buffer containing an unlabeled acyl-CoA compound and radiolabeled amino acid,
and the
radiolabeled acyl-conjugates are separated from the unreacted amino acid by
extraction into n-
butanol or other appropriate organic solvent. For example, Johnson, M.R. et
al. (1990; J. Biol. Chem.
266:10227-10233) measured bile acid-CoA:amino acid N-acyltransferase activity
by incubating the
enzyme with cholyl-CoA and 3H-glycine or 3H-taurine, separating the tritiated
cholate conjugate by
extraction into n-butanol, and measuring the radioactivity in the extracted
product by scintillation.
Alternatively, N-acyltransferase activity is measured using the
spectrophotometric determination of
reduced CoA (CoASH) described below.
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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
substrate, or the rate of absorbance increase in the spectrophotometric assay.
Protein arginine methyltransferase activity of DME is measured at 37 °C
for various periods
of time. S-adenosyl-L-[methyl 3H]methionine ([3H]AdoMet; specific activity =
75 Ci/mmol; NEN
Life Science Products) is used as the methyl-donor substrate. Useful methyl-
accepting substrates
include glutathione S-transferase fibrillarin glycine-arginine domain fusion
protein (GST-GAR),
heterogeneous nuclear ribonucleoprotein (hnRNP), or hypomethylated proteins
present in lysates
from adenosine dialdehyde-treated cells. Methylation reactions are stopped by
adding SDS-PAGE
sample buffer. The products of the reactions are resolved by SDS-PAGE and
visualized by
fluorography. The presence of 3H-labeled methyl-donor substrates is indicative
of protein arginine
methyltransferase activity of DME (Tang, J. et al. (2000) J. Biol. Chem.
275:7723-7730 and Tang, J.
et al. (2000) J. Biol. Chem.275:19866-19876).
Catechol-O-methyltransferase activity of DME is measured in a reaction mixture
consisting
of 50 mM Tris-HCl (pH 7.4), 1.2 mM MgCIZ, 200 pM 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 for 30 min. The reaction is arrested by rapidly
cooling on ice and immediately
extracting with 7 ml of ice-cold n-heptane. Following centrifugation at 1000 x
g for 10 min, 3-ml
aliquots of the organic extracts are analyzed for radioactivity content by
liquid scintillation counting.
The level of catechol-associated radioactivity in the organic phase is
proportional to the catechol-O-
methyltransferase activity of DME (Zhu, B.T. and J.G. Liehr (1996) 271:1357-
1363).
DHFR activity of DME is determined spectrophotometrically at 15 °C by
following the
disappearance of NADPH at 340 nm (e3ao = 11,800 M-'~crri'). The standard assay
mixture contains
100 pM NADPH, 14 mM 2-mercaptoethanol, MTEN buffer (50 mM 2-
morpholinoethanesulfonic
acid, 25 mM tris(hydroxymethyl)aminomethane, 25 mM ethanolamine, and 100 mM
NaCI, pH 7.0),
and DME in a final volume of 2.0 ml. The reaction is started by the addition
of 50 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
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Iwakura, M. (1999) J. Biol. Chem. 274:19041-19047).
Aldo/keto reductase activity of DME is measured using the decrease in
absorbance at 340
nm as NADPH is consumed. A standard reaction mixture is 135 mM sodium
phosphate buffer (pH
6.2-7.2 depending on enzyme), 0.2 mM NADPH, 0.3 M lithium sulfate, 0.5-2.5 mg
enzyme and an
appropriate level of substrate. The reaction is incubated at 30°C and
the reaction is monitored
continuously with a spectrophotometer. Enzyme activity is calculated as mol
NADPH consumed /
mg of enzyme.
Alcohol dehydrogenase activity of DME is measured using the increase in
absorbance at 340
nm as NAD+ is reduced to NADH. A standard reaction mixture is 50 mM sodium
phosphate, pH 7.5,
and 0.25 mM EDTA. The reaction is incubated at 25°C and monitored using
a spectrophotometer.
Enzyme activity is calculated as mol NADH produced / mg of enzyme.
Carboxylesterase activity of DME activity is determined using 4-
methylumbelliferyl acetate
as a substrate. The enzymatic reaction is initiated by adding approximately 10
p1 of DME-
containing sample to 1 ml of reaction buffer (90 mM KHZP04, 40 mM KCI, pH 7.3)
with 0.5 mM
4-methylumbelliferyl acetate at 37°C. The production of 4-
methylumbelliferone is monitored with a
spectrophotometer (s3so = 12.2 mM-' cm') for 1.5 min. Specific activity is
expressed as micromoles
of product formed per minute per milligram of protein and corresponds to the
activity of DME in the
sample (Evgenia, V. et al. (1997) J. Biol. Chem. 272:14769-14775).
In the alternative, the cocaine benzoyl ester hydrolase activity of DME is
measured by
incubating approximately 0.1 ml of enzyme and 3.3 mM cocaine in reaction
buffer (50 mM
NaH2P04, pH 7.4) with 1 mM benzamidine, 1 mM EDTA, and 1 mM dithiothreitol at
37°C. The
reaction is incubated for 1 h in a total volume of 0.4 ml then terminated with
an equal volume of 5%
trichloroacetic acid. 0.1 ml of the internal standard 3,4-dimethylbenzoic acid
(10 pg/ml) is added.
Precipitated protein is separated by centrifugation at 12,000 x g for 10 min.
The supernatant is
transferred to a clean tube and extracted twice with 0.4 ml of methylene
chloride. The two extracts
are combined and dried under a stream of nitrogen. The residue is resuspended
in 14% acetonitrile,
250 mM KHzP04, pH 4.0, with 8 p1 of diethylamine per 100 ml and injected onto
a C18 reverse-
phase HPLC column for separation. The column eluate is monitored at 235 nm.
DME activity is
quantified by comparing peak area ratios of the analyte to the internal
standard. A standard curve is
generated with benzoic acid standards prepared in a trichloroacetic acid-
treated protein matrix
(Evgenia, V. et al. (1997) J. Biol. Chem. 272:14769-14775).
In another alternative, DME carboxyl esterase activity against the water-
soluble substrate
para-nitrophenyl butyric acid is determined by spectrophotometric methods well
known to those
skilled in the art. In this procedure, the DME-containing samples are diluted
with 0.5 M Tris-HCl
(pH 7.4 or 8.0) or sodium acetate (pH 5.0) in the presence of 6 mM
taurocholate. The assay is
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initiated by adding a freshly prepared para-nitrophenyl butyric acid solution
(100 pg/ml in sodium
acetate, pH 5.0). Carboxyl esterase activity is then monitored and compared
with control
autohydrolysis of the substrate using a spectrophotometer set at 405 nm (Wan,
L. et al. (2000) J.
Biol. Chem. 275:10041-10046).
Sulfotransferase activity of DME is measured using the incorporation of 35S
from [35S]PAPS
into a model substrate such as phenol (Folds, A. and J.L. Meek (1973) Biochim.
Biophys. Acta
327:365-374). An aliquot of enzyme is incubated at 37°C with 1 mL of 10
mM phosphate buffer, pH
6.4, 50 mM phenol, and 0.4-4.0 mM [35S]PAPS. After sufficient time for 5-20%
of the radiolabel to
be transferred to the substrate, 0.2 mL of 0.1 M barium acetate is added to
precipitate protein and
phosphate buffer. Then 0.2 mL of 0.1 M Ba(OH)Z-is added, followed by 0.2 mL
ZnS04. The
supernatant is cleared by centrifugation, which removes proteins as well as
unreacted [35S]PAPS.
Radioactivity in the supernatant is measured by scintillation. The enzyme
activity is determined from
the number of moles of radioactivity in the reaction product.
Heparan sulfate 6-sulfotransferase activity of DME is measured in vitro by
incubating a
sample containing DME along with 2.5 pmol imidazole HCl (pH 6.8), 3.75 ~tg of
protamine chloride,
nmol (as hexosamine) of completely desulfated and N-resulfated heparin, and 50
pmol (about 5 x
105 cpm) of ['SS] adenosine 3'-phosphate 5'-phosphosulfate (PAPS) in a final
reaction volume of 50
p1 at 37°C for 20 min. The reaction is stopped by immersing the
reaction tubes in a boiling water
bath for 1 min. 0.1 pmol (as glucuronic acid) of chondroitin sulfate A is
added to the reaction
20 mixture as a carrier. 35S-Labeled polysaccharides are precipitated with 3
volumes of cold ethanol
containing 1.3% potassium acetate and separated completely from unincorporated
[35S]PAPS and its
degradation products by gel chromatography using desalting columns. One unit
of enzyme activity
is defined as the amount required to transfer 1 pmol of sulfate/min.,
determined by the amount of
[35S]PADS incorporated into the precipitated polysaccharides (Habuchi, H. et
al. (1995) J. Biol.
25 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-S mm segments and subjected to
agitation at 4 °C with
100 ~1 of the same buffer containing 0.15 M NaCI for 48 h. The eluted enzyme
is collected by
centrifugation and assayed for the sulfotransferase activity as described
above (Habuchi, H. et al.
(1995) J. Biol. Chem. 270:4172-4179).
In another alternative, DME sulfotransferase activity is determined by
measuring the transfer
of [35S]sulfate from [35S]PAPS to an immobilized peptide that represents the N-
terminal 15 residues
of the mature P-selectin glycoprotein ligand-1 polypeptide to which a C-
terminal cysteine residue is
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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 MnCl2, 50 mM
NaF, 1 %
Triton X-100, and I mM 5'-AMP in a final volume of 130 p1. 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 [35S]sulfate to the bead-associated
peptide is measured to
determine the DME activity in the sample. One unit of activity is defined as 1
pmol of product
formed per min (Ouyang, Y.-B. et al. (1998) Biochemistry 95:2896-2901).
In another alternative, DME sulfotransferase assays are performed using
[35S]PAPS as the
sulfate donor in a final volume of 30 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 'SS-sulfated product by
either thin-layer
chromatography or a two-dimensional thin layer separation procedure.
Appropriate standards are
run in parallel with the supernatants to allow the identification of the 35S-
sulfated products and
determine the enzyme specificity of the DME-containing samples based on
relative rates of
migration of reaction products (Sakakibara, Y. et al. (1998) J. Biol. Chem.
273:6242-6247).
Squalene epoxidase activity of DME is assayed in a mixture comprising purified
DME (or a
crude mixture comprising DME), 20 mM Tris-HCI (pH 7.5), 0.01 mM FAD, 0.2 unit
of
NADPH-cytochrome C (P-450) reductase, 0.01 mM ['4C]squalene (dispersed with
the aid of 20 p1 of
Tween 80), and 0.2% Triton X-100. 1 mM NADPH is added to initiate the reaction
followed by
incubation at 37 °C for 30 min. The nonsaponifiable lipids are analyzed
by silica gel TLC developed
with ethyl acetate/benzene (0.5:99.5, v/v). The reaction products are compared
to those from a
reaction mixture without DME. The presence of 2,3(S~-oxidosqualene is
confirmed using
appropriate lipid standards (Sakakibara, J. et al. (1995) 270:17-20).
Epoxide hydrolase activity of DME is determined by following substrate
depletion using gas
chromatographic (GC) analysis of ethereal extracts or by following substrate
depletion and diol
production by GC analysis of reaction mixtures quenched in acetone. A sample
containing DME or
an epoxide hydrolase control sample is incubated in 10 mM Tris-HCl (pH 8.0), 1
mM
ethylenediaminetetraacetate (EDTA), and 5 mM epoxide substrate (e.g., ethylene
oxide, styrene
oxide, propylene oxide, isoprene monoxide, epichlorohydrin, epibromohydrin,
epifluorohydrin,
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glycidol, 1,2-epoxybutane, 1,2-epoxyhexane, or 1,2-epoxyoctane). A portion of
the sample is
withdrawn from the reaction mixture at various time points, and added to 1 ml
of ice-cold acetone
containing an internal standard for GC analysis (e.g., 1-nonanol). Protein and
salts are removed by
centrifugation (15 min, 4000 x g) and the extract is analyzed by GC using a
0.2 mm x 25-m
CP-Wax57-CB column (CHROMPACK, Middelburg, The Netherlands) and a flame-
ionization
detector. The identification of GC products is performed using appropriate
standards and controls
well known to those skilled in the art. 1 Unit of DME activity is defined as
the amount of enzyme
that catalyzes the production of 1 pmol of diol/min (Rink, R. et al. (1997) J.
Biol. Chem.
272:14650-14657).
Aminotransferase activity of DME is assayed by incubating samples containing
DME for 1
hour at 37°C in the presence of 1 mM L-kynurenine and 1 mM 2-
oxoglutarate in a final volume of
200 p1 of 150 mM Tris acetate buffer (pH 8.0) containing 70 pM PLP. The
formation of kynurenic
acid is quantified by HPLC with spectrophotometric detection at 330 nm using
the appropriate
standards and controls well known to those skilled in the art. In the
alternative,
L-3-hydroxykynurenine is used as substrate and the production of xanthurenic
acid is determined by
HPLC analysis of the products with UV detection at 340 nm. The production of
kynurenic acid and
xanthurenic acid, respectively, is indicative of aminotransferase activity
(Buchli, R. et al. (1995) J.
Biol. Chem. 270:29330-29335).
In another alternative, aminotransferase activity of DME is measured by
determining the
activity of purified DME or crude samples containing DME toward various amino
and oxo acid
substrates under single turnover conditions by monitoring the changes in the
UV/VIS absorption
spectrum of the enzyme-bound cofactor, pyridoxal 5'-phosphate (PLP). The
reactions are performed
at 25°C in SO 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 min in 2.5
mM nitro blue
tetrazolium, followed by incubation for 20 min in 30 mM potassium phosphate,
30 mM TEMED,
and 30 pM riboflavin (pH 7.8). Superoxide dismutase activity is visualized as
white bands against a
blue background, following illumination of the gels on a lightbox.
Quantitation of superoxide
dismutase activity is performed by densitometric scanning of the activity gels
using the appropriate
103
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
superoxide dismutase positive and negative controls (e.g., various amounts of
commercially
available E. coli superoxide dismutase (Harth, G. and Horwitz, M.A. (1999) J.
Biol. Chem.
274:4281-4292).
XVIII. Identification of DME Inhibitors
Compounds to be tested are arrayed in the wells of a multi-well plate in
varying
concentrations along with an appropriate buffer and substrate, as described in
the assays in Example
XVII. DME activity is measured for each well and the ability of each compound
to inhibit DME
activity can be determined, as well as the dose-response profiles. This assay
could also be used to
identify molecules which enhance DME activity.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
104
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
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CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
1
<110> INCYTE GENOMICS, INC.
POLICKY, Jennifer L.
HAFALIA, April
BURFORD, Neil
RING, Huijun Z.
LAL, Preeti
TRIBOULEY, Catherine M.
YAO, Monique G.
YUE, Henry
TANG, Y. Tom
PATTERSON, Chandra
DAS, Debopriya
SANJANWALA, Madhu S.
GANDHI, Ameena R.
REDDY, Roopa
KHAN, Farrah A.
BAUGHN, Mariah R.
RAMKUMAR, Jaya
GRIFFIN, Jennifer A.
AU-YOUNG, Janice
<120> DRUG METABOLIZING ENZYMES
<130> PI-0070 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/197,590; 60/198,403; 60/200,185; 60/202,234; 60/203,509
<151> 2000-04-13; 2000-04-19; 2000-04-28; 2000-05-05; 2000-05-11
<160> 20
<170> PERL Program
<210> 1
<211> 527
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2434655CD1
<400> 1
Met Arg Ser Asp Lys Ser Ala Leu Val Phe Leu Leu Leu Gln Leu
1 5 10 15
Phe Cys Val Gly Cys Gly Phe Cys Gly Lys Val Leu Val Trp Pro
20 25 30
Cys Asp Met Ser His Trp Leu Asn Val Lys Val Ile Leu Glu Glu
35 40 45
Leu Ile Val Arg Gly His Glu Val Thr Val Leu Thr His Ser Lys
50 55 60
Pro Ser Leu Ile Asp Tyr Arg Lys Pro Ser Ala Leu Lys Phe Glu
65 70 75
Val Val His Met Pro Gln Asp Arg Thr Glu Glu Asn Glu Ile Phe
80 85 90
Val Asp Leu Ala Leu Asn Val Leu Pro,Gly Leu Ser Thr Trp Gln
95 100 105
Ser Val Ile Lys Leu Asn Asp Phe Phe Val Glu Ile Arg Gly Thr
110 115 120
Leu Lys Met Met Cys Glu Ser Phe Ile Tyr Asn Gln Thr Leu Met
125 130 135
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
2
Lys Lys Leu Gln Glu Thr Asn Tyr Asp Val Met Leu Ile Asp Pro
140 145 150
Val Ile Pro Cys Gly Asp Leu Met Ala Glu Leu Leu Ala Val Pro
155 160 165
Phe Val Leu Thr Leu Arg Ile Ser Val Gly Gly Asn Met Glu Arg
170 175 180
Ser Cys Gly Lys Leu Pro Ala Pro Leu Ser Tyr Val Pro Val Pro
185 190 195
Met Thr Gly Leu Thr Asp Arg Met Thr Phe Leu Glu Arg Val Lys
200 205 210
Asn Ser Met Leu Ser Val Leu Phe His Phe Trp Ile Gln Asp Tyr
215 220 225
Asp Tyr His Phe Trp Glu Glu Phe Tyr Ser Lys Ala Leu Gly Arg
230 235 240
Pro Thr Thr Leu Cys Glu Thr Val Gly Lys Ala Glu Ile Trp Leu
245 250 255
Ile Arg Thr Tyr Trp Asp Phe Glu Phe Pro Gln Pro Tyr Gln Pro
260 265 270
Asn Phe Glu Phe Val Gly Gly Leu His Cys Lys Pro Ala Lys Ala
275 280 285
Leu Pro Lys Glu Met Glu Asn Phe Val Gln Ser Ser Gly Glu Asp
290 295 300
Gly Ile Val Val Phe Ser Leu Gly Ser Leu Phe Gln Asn Val Thr
305 310 315
Glu Glu Lys Ala Asn Ile Ile Ala Ser Ala Leu Ala Gln Ile Pro
320 325 330
Gln Lys Val Leu Trp Arg Tyr Lys Gly Lys Lys Pro Ser Thr Leu
335 340 345
Gly Ala Asn Thr Arg Leu Tyr Asp Trp Ile Pro Gln Asn Asp Leu
350 355 360
Leu Gly His Pro Lys Thr Lys Ala Phe Ile Thr His Gly Gly Met
365 370 375
Asn Gly Ile Tyr Glu Ala Ile Tyr His Gly Val Pro Met Val Gly
380 385 390
Val Pro Ile Phe Gly Asp Gln Leu Asp Asn Ile Ala His Met Lys
395 400 405
Ala Lys Gly Ala Ala Val Glu Ile Asn Phe Lys Thr Met Thr Ser
410 415 420
Glu Asp Leu Leu Arg Ala Leu Arg Thr Val Ile Thr Asp Ser Ser
425 430 435
Tyr Lys Glu Asn Ala Met Arg Leu Ser Arg Ile His His Asp Gln
440 445 450
Pro.Val Lys Pro Leu Asp Arg Ala Val Phe Trp Ile Glu Phe Val
455 460 465
Met Arg His Lys Gly Ala Lys His Leu Arg Ser Ala Ala His Asp
470 475 480
Leu Thr Trp Phe Gln His Tyr Ser Ile Asp Val Ile Gly Phe Leu
485 490 495
Leu Thr Cys Val Ala Thr Ala Ile Phe Leu Phe Thr Lys Cys Phe
500 505 510
Leu Phe Ser Cys Gln Lys Phe Asn Lys Thr Arg Lys Ile Glu Lys
515 520 525
Arg Glu
<210> 2
<211> 523
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2516747CD1
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
3
<400> 2
Met Val Gly Gln Arg Val Leu Leu Leu Val Ala Phe Leu Leu Ser
1 5 10 15
Gly Val Leu Leu Ser Glu Ala Ala Lys Ile Leu Thr Ile Ser Thr
20 25 30
Leu Gly Gly Ser His Tyr Leu Leu Leu Asp Arg Val Ser Gln Ile
35 40 45
Leu Gln Glu His Gly His Asn Val Thr Met Leu His Gln Ser Gly
50 55 60
Lys Phe Leu Ile Pro Asp Ile Lys Glu Glu Glu Lys Ser Tyr Gln
65 70 75
Val Ile Arg Trp Phe Ser Pro Glu Asp His Gln Lys Arg Ile Lys
80 85 90
Lys His Phe Asp Ser Tyr Ile Glu Thr Ala Leu Asp Gly Arg Lys
95 100 105
Glu Ser Glu Ala Leu Val Lys Leu Met Glu Ile Phe Gly Thr Gln
110 115 120
Cys Ser Tyr Leu Leu Ser Arg Lys Asp Ile Met Asp Ser Leu Lys
125 130 135
Asn Glu Asn Tyr Asp Leu Val Phe Val Glu Ala Phe Asp Phe Cys
140 145 150
Ser Phe Leu Ile Ala Glu Lys Leu Val Lys Pro Phe Val Ala Ile
155 160 165
Leu Pro Thr Thr Phe Gly Ser Leu Asp Phe Gly Leu Pro Ser Pro
170 175 180
Leu Ser Tyr Val Pro Val Phe Pro Ser Leu Leu Thr Asp His Met
185 190 195
Asp Phe Trp Gly Arg Val Lys Asn Phe Leu Met Phe Phe Ser Phe
200 205 210
Ser Arg Ser Gln Trp Asp Met Gln Ser Thr Phe Asp Asn Thr Ile
215 220 225
Lys Glu His Phe Pro Glu Gly Ser Arg Pro Val Leu Ser His Leu
230 235 240
Leu Leu Lys Ala Glu Leu Trp Phe Val Asn Ser Asp Phe Ala Phe
245 250 255
Asp Phe Ala Arg Pro Leu Leu Pro Asn Thr Val Tyr Ile Gly Gly
260 265 270
Leu Met Glu Lys Pro Ile Lys Pro Val Pro Gln Asp Leu Asp Asn
275 280 285
Phe Ile Ala Asn Phe Gly Asp Ala Gly Phe Val Leu Val Ala Phe
290 295 300
Gly Ser Met Leu Asn Thr His Gln Ser Gln Glu Val Leu Lys Lys
305 310 315
Met His Asn Ala Phe Ala His Leu Pro Gln Gly Val Ile Trp Thr
320 325 330
Cys Gln Ser Ser His Trp Pro Arg Asp Val His Leu Ala Thr Asn
335 340 345
Val Lys Ile Val Asp Trp Leu Pro Arg Ser Asp Leu Leu Ala His
350 355 360
Pro Ser Ile Arg Leu Phe Val Thr His Gly Gly Gln Asn Ser Val
365 370 375
Met Glu Ala Ile Arg His Gly Val Pro Met Val Gly Leu Pro Val
380 385 390
Asn Gly Asp Gln His Gly Asn Met Val Arg Val Val Ala Lys Asn
395 400 405
Tyr Gly Val Ser Ile Arg Leu Asn Gln Val Thr Ala Asp Thr Leu
410 415 420
Thr Leu Thr Met Lys Gln Val Ile Glu Asp Lys Arg Tyr Lys Ser
425 430 435
Ala Val Val Ala Ala Ser Val Ile Leu His Ser Gln Pro Leu Ser
440 445 450
Pro Ala Gln Arg Leu Val Gly Trp Ile Asp His Ile Leu Gln Thr
455 460 465
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
4
Gly Gly Ala Thr His Leu Lys Pro Tyr Ala Phe Gln Gln Pro Trp
470 475 480
His Glu Gln Tyr Leu Ile Asp Val Phe Val Phe Leu Leu Gly Leu
485 490 495
Thr Leu Gly Thr Met Trp Leu Cys Gly Lys Leu Leu Gly Val Val
500' 505 510
Ala Arg Trp Leu Arg Gly Ala Arg Lys Val Lys Lys Thr
515 520
<210> 3
<211> 358
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472775CD1
<400> 3
Met Pro Glu Asp Val Arg Glu Lys Lys Glu Asn Leu Leu Leu Asn
1 5 10 15
Ser Glu Arg Ser Thr Arg Leu Leu Thr Lys Thr Ser His Ser Gln
20 25 30
Gly Gly Asp Gln Ala Leu Ser Lys Ser Thr Gly Ser Pro Thr Glu
35 40 45
Lys Leu Ile Glu Lys Arg Gln Gly Ala Lys Thr Val Phe Asn Lys
50 55 60
Phe Ser Asn Met Asn Trp Pro Val Asp Ile His Pro Leu Asn Lys
65 70 75
Ser Leu Val Lys Asp Asn Lys Trp Lys Lys Thr Glu Glu Thr Gln
80 85 90
Glu Lys Arg Arg Ser Phe Leu Gln Glu Phe Cys Lys Lys Tyr Gly
95 100 105
Gly Val Ser His His Gln Ser His Leu Phe His Thr Val Ser Arg
110 115 120
Ile Tyr Val Glu Asp Lys His Lys Ile Leu Tyr Cys Glu Val Pro
125 130 135
Lys Ala Gly Cys Ser Asn Trp Lys Arg Ile Leu Met Val Leu Asn
140 145 150
Gly Leu Ala Ser Ser Ala Tyr Asn Ile Ser His Asn Ala Val His
155 160 165
Tyr Gly Lys His Leu Lys Lys Leu Asp Ser Phe Asp Leu Lys Gly
170 175 180
Ile Tyr Thr Arg Leu Asn Thr Tyr Thr Lys Ala Val Phe Val Arg
185 190 195
Asp Pro Met Glu Arg Leu Val Ser Ala Phe Arg Asp Lys Phe Glu
200 205 210
His Pro Asn Ser Tyr Tyr His Pro Val Phe Gly Lys Ala Ile Ile
215 220 225
Lys Lys Tyr Arg Pro Asn Ala Cys Glu Glu Ala Leu Ile Asn Gly
230 235 240
Ser Gly Val Lys Phe Lys Glu Phe Ile His Tyr Leu Leu Asp Ser
245 250 255
His Arg Pro Val Gly Met Asp Ile His Trp Glu Lys Val Ser Lys
260 265 270
Leu Cys Tyr Pro Cys Leu Ile Asn Tyr Asp Phe Val Gly Lys Phe
275 280 285
Glu Thr Leu Glu Glu Asp Ala Asn Tyr Phe Leu Gln Met Ile Gly
290 295 300
Ala Pro Lys Glu Leu Lys Phe Pro Asn Phe Lys Asp Arg His Ser
305 310 315
Ser Asp Glu Arg Thr Asn Ala Gln Val Val Arg Gln Tyr Leu Lys
320 325 330
CA 02403644 2002-09-26
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Asp Leu Thr Arg Thr Glu Arg Gln Leu Ile Tyr Asp Phe Tyr Tyr
335 340 345
Leu Asp Tyr Leu Met Phe Asn Tyr Thr Thr Pro Phe Leu
350 355
<210> 4
<211> 396
<212> PRT
<213> Homo Sapiens
<220>
<221> misC_feature
<223> Incyte ID No: 7473323CD1
<400> 4
Met Ala Ile Asp Ala Leu Val Ser Leu Cys Leu Pro Glu Val Ile
1 5 10 15
Arg Ile Lys Phe Asn Ile Arg Pro Arg Gln Pro His His Asp Leu
20 25 30
Pro Pro Gly Gly Ser Gln Asp Gly Asp Leu Lys Glu Pro Thr Glu
35 40 45
Arg Val Thr Arg Asp Leu Ser Ser Gly Ala Pro Arg Gly Arg Asn
50 55 60
Leu Pro Ala Pro Asp Gln Pro Gln Pro Pro Leu Gln Arg Gly Thr
65 70 75
Arg Leu Arg Leu Arg Gln Arg Arg Arg Arg Leu Leu Ile Lys Lys
80 85 90
Met Pro Ala Ala Ala Thr Ile Pro Ala Asn Ser Ser Asp Ala Pro
95 100 105
Phe Ile Arg Pro Gly Pro Gly Thr Leu Asp Gly Arg Trp Val Ser
110 115 120
Leu His Arg Ser Gln Gln Glu Arg Lys Arg Val Met Gln Glu Ala
125 130 135
Cys Ala Lys Tyr Arg Ala Ser Ser Ser Arg Arg Ala Val Thr Pro
140 145 150
Arg His Val Ser Arg Ile Phe Val Glu Asp Arg His Arg Val Leu
155 160 165
Tyr Cys Glu Val Pro Lys Ala Gly Cys Ser Asn Trp Lys Arg Val
170 175 180
Leu Met Val Leu Ala Gly Leu Ala Ser Ser Thr Ala Asp Ile Gln
185 190 195
His Asn Thr Val His Tyr Gly Ser Ala Leu Lys Arg Leu Asp Thr
200 205 210
Phe Asp Arg Gln Gly Ile Leu His Arg Leu Ser Thr Tyr Thr Lys
215 220 225
Met Leu Phe Val Arg Glu Pro Phe Glu Arg Leu Val Ser Ala Phe
230 235 240
Arg Asp Lys Phe Glu His Pro Asn Ser Tyr Tyr His Pro Val Phe
245 250 255
Gly Lys Ala Ile Leu Ala Arg Tyr Arg Ala Asn Ala Ser Arg Glu
260 265 270
Ala Leu Arg Thr Gly Ser Gly Val Arg Phe Pro Glu Phe Val Gln
275 280 285
Tyr Leu Leu Asp Val His Arg Pro Val Gly Met Asp Ile His Trp
290 295 300
Asp His Val Ser Arg Leu Cys Ser Pro Cys Leu Ile Asp Tyr Asp
305 310 315
Phe Val Gly Lys Phe Glu Ser Met Glu Asp Asp Ala Asn Phe Phe
320 325 330
Leu Ser Leu Ile Arg Ala Pro Arg Asn Leu Thr Phe Pro Arg Phe
335 340 345
Lys Asp Arg His Ser Gln Glu Ala Arg Thr Thr Ala Arg Ile Ala
350 355 360
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
6
His Gln Tyr Phe Ala Gln Leu Ser Ala Leu Gln Arg Gln Arg Thr
365 370 375
Tyr Asp Phe Tyr Tyr Met Asp Tyr Leu Met Phe Asn Tyr Ser Lys
380 385 390
Pro Phe Ala Asp Leu Tyr
395
<210> 5
<211> 395
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472777CD1
<400> 5
Met Trp Leu Pro Arg Val Ser Ser Thr Ala Val Thr Ala Leu Leu
1 5 10 15
Leu Ala Gln Thr Phe Leu Leu Leu Phe Leu Val Ser Arg Pro Gly
20 25 30
Pro Ser Ser Pro Ala Gly Gly Glu Ala Arg Val His Val Leu Val
35 40 45
Leu Ser Ser Trp Arg Ser Gly Ser Ser Phe Val Gly Gln Leu Phe
50 55 60
Asn Gln His Pro Asp Val Phe Tyr Leu Met Glu Pro Ala Trp His
65 70 75
Val Trp Thr Thr Leu Ser Gln Gly Ser Ala Ala Thr Leu His Met
80 85 90
Ala Val Arg Asp Leu Val Arg Ser Val Phe Leu Cys Asp Met Asp
95 100 105
Val Phe Asp Ala Tyr Leu Pro Trp Arg Arg Asn Leu Ser~Asp Leu
110 115 120
Phe Gln Trp Ala Val Ser Arg Ala Leu Cys Ser Pro Pro Ala Cys
125 130 135
Ser Ala Phe Pro Arg Gly Ala Ile Ser Ser Glu Ala Val Cys Lys
140 145 150
Pro Leu Cys Ala Arg Gln Ser Phe Thr Leu Ala Arg Glu Ala Cys
155 160 165
Arg Ser Tyr Ser His Val Val Leu Lys Glu Val Arg Phe Phe Asn
170 175 180
Leu Gln Val Leu Tyr Pro Leu Leu Ser Asp Pro Ala Leu Asn Leu
185 190 195
Arg Ile Val His Leu Val Arg Asp Pro Arg Ala Val Leu Arg Ser
200 205 210
Arg Glu Gln Thr Ala Lys Ala Leu Ala Arg Asp Asn Gly Ile Val
215 220 225
Leu Gly Thr Asn Gly Thr Trp Val Glu Ala Asp Pro Gly Leu Arg
230 235 240
Val Val Arg Glu Val Cys Arg Ser His Val Arg Ile Ala Glu Ala
245 250 255
Ala Thr Leu Lys Pro Pro Pro Phe Leu Arg Gly Arg Tyr Arg Leu
260 265 270
Val Arg Phe Glu Asp Leu Ala Arg Glu Pro Leu Ala Glu Ile Arg
275 280 285
Ala Leu Tyr Ala Phe Thr Gly Leu Ser Leu Thr Pro Gln Leu Glu
290 295 300
Ala Trp Ile His Asn Ile Thr His Gly Ser Gly Pro Gly Ala Arg
305 310 315
Arg Glu Ala Phe Lys Thr Ser Ser Arg Asn Ala Leu Asn Val Ser
320 325 330
Gln Ala Trp Arg His Ala Leu Pro Phe Ala Lys Ile Arg Arg Val
335 340 345
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
7
Gln Glu Leu Cys Ala Gly Ala Leu Gln Leu Leu Gly Tyr Arg Pro
350 355 360
Val Tyr Ser Glu Asp Glu Gln Arg Asn Leu Ala Leu Asp Leu Val
365 370 375
Leu Pro Arg Gly Leu Asn Gly Phe Thr Trp Ala Ser Ser Thr Ala
380 385 390
Ser His Pro Arg Asn
395
<210> 6
<211> 504
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1272843CD1
<400> 6
Met Glu Ala Thr Gly Thr Trp Ala Leu Leu Leu Ala Leu Ala Leu
1 5 10 15
Leu Leu Leu Leu Thr Leu Ala Leu Ser Gly Thr Arg Ala Arg Gly
20 25 30
His Leu Pro Pro Gly Pro Thr Pro Leu Pro Leu Leu Gly Asn Leu
35 40 45
Leu Gln Leu Arg Pro Gly Ala Leu Tyr Ser Gly Leu Met Arg Leu
50 55 60
Ser Lys Lys Tyr Gly Pro Val Phe Thr Ile Tyr Leu Gly Pro Trp
65 70 75
Arg Pro Val Val Val Leu Val Gly Gln Glu Ala Val Arg Glu Ala
80 85 90
Leu Gly Gly Gln Ala Glu Glu Phe Ser Gly Arg Gly Thr Val Ala
95 100 105
Met Leu Glu Gly Thr Phe Asp Gly His Gly Val Phe Phe Ser Asn
110 115 120
Gly Glu Arg Trp Arg Gln Leu Arg Lys Phe Thr Met Leu Ala Leu
125 130 135
Arg Asp Leu Gly Met Gly Lys Arg Glu Gly Glu Glu Leu Ile Gln
140 145 150
Ala Glu Ala Arg Cys Leu Val Glu Thr Phe Gln Gly Thr Glu Gly
155 160 165
Arg Pro Phe Asp Pro Ser Leu Leu Leu Ala Gln Ala Thr Ser Asn
170 175 180
Val Val Cys Ser Leu Leu Phe Gly Leu Arg Phe Ser Tyr Glu Asp
185 190 195
Lys Glu Phe Gln Ala Val Val Arg Ala Ala Gly Gly Thr Leu Leu
200 205 210
Gly Val Ser Ser Gln Gly Gly Gln Thr Tyr Glu Met Phe Ser Trp
215 220 225
Phe Leu Arg Pro Leu Pro Gly Pro His Lys Gln Leu Leu His His
230 235 240
Val Ser Thr Leu Ala Ala Phe Thr Val Arg Gln Val Gln Gln His
245 250 255
Gln Gly Asn Leu Asp Ala Ser Gly Pro Ala Arg Asp Leu Val Asp
260 265 270
Ala Phe Leu Leu Lys Met Ala Gln Glu Glu Gln Asn Pro Gly Thr
275 280 285
Glu Phe Thr Asn Lys Asn Met Leu Met Thr Val Ile Tyr Leu Leu
290 295 300
Phe Ala Gly Thr Met Thr Val Ser Thr Thr Val Gly Tyr Thr Leu
305 310 315
Leu Leu Leu Met Lys Tyr Pro His Val Gln Lys Trp Val Arg Glu
320 325 330
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
8
Glu Leu Asn Arg Glu Leu Gly Ala Gly Gln Ala Pro Ser Leu Gly
335 340 345
Asp Arg Thr Arg Leu Pro Tyr Thr Asp Ala Val Leu His Glu Ala
350 355 360
Gln Arg Leu Leu Ala Leu Val Pro Met Gly Ile Pro Arg Thr Leu
365 370 375
Met Arg Thr Thr Arg Phe Arg Gly Tyr Thr Leu Pro Gln Gly Thr
380 385 390
Glu Val Phe Pro Leu Leu Gly Ser Ile Leu His Asp Pro Asn Ile
395 400 405
Phe Lys His Pro Glu Glu Phe Asn Pro Asp Arg Phe Leu Asp Ala
410 415 420
Asp Gly Arg Phe Arg Lys His Glu Ala Phe Leu Pro Phe Ser Leu
425 430 435
Gly Lys Arg Val Cys Leu Gly Glu Gly Leu Ala Lys Ala Glu Leu
440 445 450
Phe Leu Phe Phe Thr Thr Ile Leu Gln Ala Phe Ser Leu Glu Ser
455 460 465
Pro Cys Pro Pro Asp Thr Leu Ser Leu Lys Pro Thr Val Ser Gly
470 475 480
Leu Phe Asn Ile Pro Pro Ala Phe Gln Leu Gln Val Arg Pro Thr
485 490 495
Asp Leu His Ser Thr Thr Gln Thr Arg
500
<210> 7
<211> 229
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472790CD1
<400> 7
Met Asn Ile Arg Asn Ala Gln Pro Asp Asp Leu Met Asn Met Gln
1 5 10 15
His Cys Asn Leu Leu Cys Leu Pro Glu Asn Tyr Gln Met Lys Tyr
20 25 30
Tyr Leu Tyr His Gly Leu Ser Trp Pro Gln Leu Ser Tyr Ile Ala
35 40 45
Glu Asp Glu Asp Gly Lys Ile Val Gly Tyr Val Leu Ala Lys Met
50 55 60
Glu Glu Glu Pro Asp Asp Val Pro His Gly His Ile Thr Ser Leu
65 70 75
Ala Val Lys Arg Ser His Arg Arg Leu Gly Leu Ala Gln Lys Leu
80 85 90
Met Asp Gln Ala Ser Arg Ala Met Ile Glu Asn Phe Asn Ala Lys
95 100 105
Tyr Val Ser Leu His Val Arg Lys Ser Asn Arg Pro Ala Leu His
110 115 120
Leu Tyr Ser Asn Thr Leu Asn Phe Gln Ile Ser Glu Va1 Glu Pro
125 130 135
Lys Tyr Tyr Ala Asp Gly Glu Asp Ala Tyr Ala Met Lys Arg Asp
140 145 150
Leu Ser Gln Met Ala Asp Glu Leu Arg Arg Gln Met Asp Leu Lys
155 160 165
Lys Gly Gly Tyr Val Val Leu Gly Ser Arg Glu Asn Gln Glu Thr
170 175 180
Gln Gly Ser Thr Leu Ser Asp Ser Glu Glu Ala Cys Gln Gln Lys
185 190 195
Asn Pro Ala Thr Glu Glu Ser Gly Ser Asp Ser Lys Glu Pro Lys
200 205 210
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
9
Glu Ser Val Glu Ser Thr Asn Val Gln Asp Ser Ser Glu Ser Ser
215 220 225
Asp Ser Thr Ser
<210> 8
<211> 347
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473944CD1
<400> 8
Met Lys Pro Ala Leu Leu Glu Val Met Arg Met Asn Arg Ile Cys
1 5 10 15
Arg Met Val Leu Ala Thr Cys Leu Gly Ser Phe Ile Leu Val Ile
20 25 30
Phe Tyr Phe Gln Ile Met Arg Arg Asn Pro Phe Gly Val Asp Ile
35 40 45
Cys Cys Arg Lys Gly Ser Arg Ser Pro Leu Gln Glu Leu Tyr Asn
50 55 60
Pro Ile Gln Leu Glu Leu Ser Asn Thr Ala Val Leu His Gln Met
65 70 75
Arg Arg Asp Gln Val Thr Asp Thr Cys Arg Ala Asn Ser Ala Thr
80 85 90
Ser Arg Lys Arg Arg Val Leu Thr Pro Asn Asp Leu Lys His Leu
95 100 105
Val Val Asp Glu Asp His Glu Leu Ile Tyr Cys Tyr Val Pro Lys
110 115 120
Val Ala Cys Thr Asn Trp Lys Arg Leu Met Met Val Leu Thr Gly
125 130 135
Arg Gly Lys Tyr Ser Asp Pro Met Glu Ile Pro Ala Asn Glu Ala
140 145 150
His Val Ser Ala Asn Leu Lys~Thr Leu Asn Gln Tyr Ser Ile Pro
155 160 165
Glu Ile Asn His Arg Leu Lys Ser Tyr Met Lys Phe Leu Phe Val
170 175 180
Arg Glu Pro Phe Glu Arg Leu Val Ser Ala Tyr Arg Asn Lys Phe
185 190 195
Thr Gln Lys Tyr Asn Ile Ser Phe His Lys Arg Tyr Gly Thr Lys
200 205 210
Ile Ile Lys Arg Gln Arg Lys Asn Ala Thr Gln Glu Ala Leu Arg
215 220 225
Lys Gly Asp Asp Val Lys Phe Glu Glu Phe Val Ala Tyr Leu Ile
230 235 240
Asp Pro His Thr Gln Arg Glu Glu Pro Phe Asn Glu His Trp Gln
245 250 255
Thr Val Tyr Ser Leu Cys His Pro Cys His Ile His Tyr Asp Leu
260 265 270
Val Gly Lys Tyr Glu Thr Leu Glu Glu Asp Ser Asn Tyr Val Leu
275 280 285
Gln Leu Ala Gly Val Gly Ser Tyr Leu Lys Phe Pro Thr Tyr Ala
290 295 300
Lys Ser Thr Arg Thr Thr Asp Glu Met Thr Thr Glu Phe Phe Gln
305 310 315
Asn Ile Ser Ser Glu His Gln Thr Gln Leu Tyr Glu Val Tyr Lys
320 325 330
Leu Asp Phe Leu Met Phe Asn Tyr Ser Val Pro Ser Tyr Leu Lys
335 340 345
Leu Glu
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
<210> 9
<211> 218
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2244136CD1
<400> 9
Met Thr Pro Ala Pro Pro Pro Gly Ala Arg Pro Gly Ala Ala Ser
1 5 10 15
Leu Ala Gly Phe Ala Gly Val Ala Ser Leu Gly Pro Gly Asp Pro
25 30
Arg Arg Ala Ala Asp Pro Arg Pro Leu Pro Pro Ala Leu Cys Phe
35 40 45
Ala Val Ser Arg Ser Leu Leu Leu Thr Cys Leu Val Pro Ala Ala
50 55 60
Leu Leu Gly Leu Arg Tyr Tyr Tyr Ser Arg Lys Val Ile Arg Ala
65 70 75
Tyr Leu Glu Cys Ala Leu His Thr Asp Met Ala Asp Ile Glu Gln
80 85 90
Tyr Tyr Met Lys Pro Pro Gly Ser Cys Phe Trp Val Ala Val Leu
95 100 105
Asp Gly Asn Val Val Gly Ile Val Ala Ala Arg Ala His Glu Glu
110 115 120
Asp Asn Thr Val Glu Leu Leu Arg Met Ser Val Asp Ser Arg Phe
125 130 135
Arg Gly Lys Gly Ile Ala Lys Ala Leu Gly Arg Lys Val Leu Glu
140 145 150
Phe Ala Val Val His Asn Tyr Ser Ala Val Val Leu Gly Thr Thr
155 160 165
Ala Val Lys Val Ala Ala His Lys Leu Tyr Glu Ser Leu Gly Phe
170 175 180
Arg His Met Gly Ala Ser Asp His Tyr Val Leu Pro Gly Met Thr
185 190 195
Leu Ser Leu Ala Glu Arg Leu Phe Phe Gln Val Arg Tyr His Arg
200 205 210
Tyr Arg Leu Gln Leu Arg Glu Glu
215
<210> 10
<211> 318
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474327CD1
<400> 10
Met Ala Ser His Ile Val Leu Asn Asn Gly Thr Lys Met Pro Ile
1 5 10 15
Leu Gly Leu Gly Thr Trp Asn Ser Pro Pro Gly Gln Val Thr Glu
20 25 30
Ala Val Lys Val Ala Ile Asn Val Gly Tyr Cys His Ile Asp Cys
35 40 45
Ala His Val Tyr Gln Asn Glu Asn Asp Val Gly Val Ala Ile Arg
50 55 60
Glu Lys Leu Arg Glu Gln Val Val Lys Cys Glu Glu Leu Phe Ile
65 70 75
Thr Ser Lys Leu Trp Cys Ala Tyr His Glu Lys Gly Leu Val Lys
80 85 90
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
11
Gly Ala Cys Gln Lys Met Leu Ile Asp Leu Lys Leu Asp Tyr Leu
95 100 105
Asp Leu Tyr Leu Ile Arg Trp Pro Thr Ser Phe Lys Pro Gly Lys
110 115 120
Glu Phe Phe Pro Leu Asp Glu Pro Gly Asn Gly Asn Val Val Pro
125 130 135
Ser Asn Ser Asn Ile Leu Asp Thr Trp Ala Gly Met Glu Glu Leu
140 145 150
Val Asp Glu Gly Leu Val Lys Ala Ile Gly Ile Ser Asn Phe Asn
155 160 165
His Leu Gln Val Glu Arg Ile Leu Asn Lys Pro Asp Leu Lys Tyr
170 175 180
Lys Pro Val Val Asn Gln Ile Glu Cys His Pro Tyr Leu Thr Gln
185 190 195
Glu Lys Leu Ile Gln Tyr Cys Gln Ser Lys Gly Ile Met Val Thr
200 205 210
Ala Tyr Ser Ser Phe Ser Ser Pro Asp Arg Pro Trp Ala Lys Pro
215 220 225
Glu Asp Pro Ser Leu Leu Glu Asp Pro Arg Ile Lys Ala Ile Thr
230 235 240
Ala Lys His Asn Lys Thr Thr Ala Gln Val Leu Ile Trp Phe Pro
245 250 255
Met Gln Arg Asn Leu Val Val Ile Pro Lys Ser Val Thr Pro Glu
260 265 270
Cys Ile Ala Glu Asn Phe Lys Val Phe Asn Phe Glu Leu Asn Ser
275 280 285
Gln Asp Met Thr Thr Leu Phe Ser Tyr Asn Arg Asn Trp Arg Val
290 295 300
Cys Ala Leu Val Ser Cys Ala Ser His Lys Asp Tyr Pro Phe His
305 310 315
Glu Glu Phe
<210> 11
<211> 1636
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2434655CB1
<400> 11
gatcagtgtg tgagggaact gccatcatga ggtctgacaa gtcagctttg gtatttctgc 60
tcctgcagct cttctgtgtt ggctgtggat tctgtgggaa agtcctggtg tggccctgtg 120
acatgagcca ttggcttaat gtcaaggtca ttctagaaga gctcatagtg agaggccatg 180
aggtaacagt attgactcac tcaaagcctt cgttaattga ctacaggaag ccttctgcat 240
tgaaatttga ggtggtccat atgccacagg acagaacaga agaaaatgaa atatttgttg 300
acctagctct gaatgtcttg ccaggcttat caacctggca atcagttata aaattaaatg 360
atttttttgt tgaaataaga ggaactttaa aaatgatgtg tgagagcttt atctacaatc 420
agacgcttat gaagaagcta caggaaacca actacgatgt aatgcttata gaccctgtga 480
ttccctgtgg agacctgatg gctgagttgc ttgcagtccc ttttgtgctc acacttagaa 540
tttctgtagg aggcaatatg gagcgaagct gtgggaaact tccagctcca ctttcctatg 600
tacctgtgcc tatgacagga ctaacagaca gaatgacctt tctggaaaga gtaaaaaatt 660
caatgctttc agttttgttc cacttctgga ttcaggatta cgactatcat ttttgggaag 720
agttttatag taaggcatta ggaaggccca ctacattatg tgagactgtg ggaaaagctg 780
agatatggct aatacgaaca tattgggatt ttgaatttcc tcaaccatac caacctaact 840
ttgagtttgt tggaggattg cactgtaaac ctgccaaagc tttgcctaag gaaatggaaa 900
attttgtcca gagttcaggg gaagatggta ttgtggtgtt ttctctgggg tcactgtttc 960
aaaatgttac agaagaaaag gctaatatca ttgcttcagc ccttgcccag atcccacaga 1020
aggtgttatg gaggtacaaa ggaaaaaaac catccacatt aggagccaat actcggctgt 1080
atgattggat accccagaat gatcttcttg gtcatcccaa aaccaaagct tttatcactc 1140
atggtggaat gaatgggatc tatgaagcta tttaccatgg ggtccctatg gtgggagttc 1200
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
12
ccatatttgg tgatcagctt gataacatag ctcacatgaa ggccaaagga gcagctgtag 1260
aaataaactt caaaactatg acaagcgaag atttactgag ggctttgaga acagtcatta 1320
ccgattcctc ttataaagag aatgctatga gattatcaag aattcaccat gatcaacctg 1380
taaagcccct agatcgagca gtcttctgga tcgagtttgt catgcgccac aaaggagcca 1440
agcacctgcg atcagctgcc catgacctca cctggttcca gcactactct atagatgtga 1500
ttgggttcct gctgacctgt gtggcaactg ctatattctt gttcacaaaa tgttttttat 1560
tttcctgtca aaaatttaat aaaactagaa agatagaaaa gagggaatag atctttccaa 1620
attcaagaaa gacctg 1636
<210> 12
<211> 2086
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2516747CB1
<400> 12
cctacctctt cctaggccca cagccagtgc ctttggagta ctgaggcgcg cacagagtcc 60
ttagcccggc gcagggcgcg cagcccaggc tgagatccgc tgcttctgtg gaagtgagca 120
tggttgggca gcgggtgctg cttctagtgg ccttccttct ttctggggtc ctgctctcag 180
aggctgccaa aatcctgaca atatctacac tgggtggaag ccattaccta ctgttggacc 240
gggtgtctca gattcttcaa gagcatggtc ataatgtgac tatgcttcat cagagtggaa 300
agtttttgat cccagatatt aaagaggagg aaaaatcata ccaagttatc aggtggtttt 360
cacctgaaga tcatcaaaaa agaattaaga agcattttga tagctacata gaaacagcat 420
tggatggcag aaaagaatct gaagcccttg taaagctaat ggaaatattt gggactcaat 480
gtagttattt gctaagcaga aaggatataa tggattcctt aaagaatgag aactatgatc 540
tggtatttgt tgaagcattt gatttctgtt ctttcctgat tgctgagaag cttgtgaaac 600
catttgtggc cattcttccc accacattcg gctctttgga ttttgggcta ccaagcccct 660
tgtcttatgt tccagtattc ccttccttgc tgactgatca catggacttc tggggccgag 720
tgaagaattt tctgatgttc tttagtttct ccaggagcca atgggacatg cagtctacat 780
ttgacaacac catcaaggag catttcccag aaggctctag gccagttttg tctcatcttc 840
tactgaaagc agagttgtgg tttgttaact ctgattttgc ctttgatttt gcccggcccc 900
tgcttcccaa cactgtttat attggaggct tgatggaaaa acctattaaa ccagtaccac 960
aagacttgga caacttcatt gccaactttg gggatgcagg gtttgtcctt gtggcctttg 1020
gctccatgtt gaacacccat cagtcccagg aagtcctcaa gaagatgcac aatgcctttg 1080
cccacctccc tcaaggagtg atatggacat gtcagagttc tcattggccc agagatgttc 1140
atttggccac aaatgtgaaa attgtggact ggcttcctcg gagtgacctc ctggctcacc 1200
ccagcatccg tctttttgtc actcatggtg ggcagaacag cgtaatggag gccatccgtc 1260
atggtgtgcc catggtggga ttaccagtca atggagacca gcatggaaac atggtccgag 1320
tagtagccaa aaattatggt gtctctatcc ggttgaatca ggtcacagcc gacacactga 1380
cacttacaat gaaacaagtc atagaagaca agaggtacaa gtcggcagtg gtggcagcca 1440
gtgtcatcct gcactctcag cccctgagcc ccgcacagcg gctggtgggc tggatcgacc 1500
acatcctcca gactggggga gcgacgcacc tcaagcccta tgccttccag cagccttggc 1560
atgagcagta cctcattgat gtctttgtgt ttctgctggg gctcactctg ggcactatgt 1620
ggctttgtgg gaagctgctg ggtgtggtgg ccaggtggct gcgtggggcc aggaaggtga 1680
agaagacatg aggctaggtg tagccttggg tgaggggagg gcatccctgg tcctttgaag 1740
gttctcccca ccccagcaca cgccacccct ctgttctctc ttcagctcca cctgccactg 1800
atcctgcaac ttgcttcttt ctattctctg cctctgttta gaaatcttca cacaccactg 1860
aggcttcttg acttgcccct tgtgacttga attcccagct cagatacaaa ttttcacctg 1920
ccagccctgc ctcctccttt ctcccttttc ctagacacag gactctgaca acttcatcct 1980
ccttgtttag atgacttccc agtttccagt ccccatttct ccttctatca cttttcataa 2040
aaaaactcag gaaatatttg acatatcttc catttcaaat tcttcc 2086
<210> 13
<211> 1814
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472775CB1
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
13
<400> 13
cttggacgag ggggagagtg gagaagagaa gagaacaaaa agtaacttca ggatggggac 60
cagtgaagta cttgcggcct gtacccagaa tcaaacccca agtttcacat gcctgaggat 120
gtacgagaaa aaaaggaaaa tcttctactc aattctgaga gatctactag gctcttaaca 180
aagaccagtc attcacaagg aggggatcaa gctttaagta agtccacagg gtcaccaaca 240
gagaagttga ttgaaaaacg tcaaggagct aagactgttt ttaacaagtt cagcaacatg 300
aattggccag tggacattca ccctttaaac aaaagtttag tcaaagataa taaatggaag 360
aaaactgagg agacccaaga gaaacgaagg tctttccttc aggagttttg caagaaatac 420
ggtggggtga gtcatcatca gtcacatctt ttccatacag tatccagaat ctatgtagaa 480
gataaacaca aaatcttata ttgtgaggta cctaaggctg gctgttccaa ttggaaaaga 540
attctgatgg tactaaatgg attggcttcc tctgcataca acatctccca caatgctgtc 600
cactacpgga agcatttgaa gaagctagat agctttgacc taaaagggat atatacccgc 660
ttaaatactt acaccaaagc tgtgtttgtt cgtgatccca tggaaagatt agtatcagcc 720
tttagggaca aatttgaaca ccccaatagt tattaccatc cagtattcgg aaaggcaatt 780
atcaagaaat atcgaccaaa tgcctgtgaa gaagcattaa ttaatggatc tggagtcaag 840
ttcaaagagt ttatccacta cttgctggat tcccaccgtc cagtaggaat ggacattcac 900
tgggaaaagg tcagcaaact ctgctatccg tgtttgatca actatgattt tgtagggaaa 960
tttgagactt tggaagaaga tgccaattac tttttacaga tgatcggtgc tccaaaggag 1020
ctgaaatttc ccaactttaa ggataggcac tcttccgatg aaagaaccaa tgctcaagtc 1080
gtgagacagt atttaaagga tctgactaga actgagagac aattaatcta tgacttttat 1140
tacttggact atttaatgtt taattataca actccatttt tgtagtttgc attcattttc 1200
taaaaccctg tatatactta atgatgataa gttcaaatca gctgtaattt ttctataatt 1260
ctctgtatga cagaaattta accaagtgca gttgtcttga tttaatgtag atttttacca 1320
aatagtatga caccaattgg cacaaagtta taggaaaatc acctacagga gatgtaaaca 1380
acttgagttg ctctaaaatg tttggaaaag agctgctttt gcattatgaa ttatattgtt 1440
gaagcaataa cctagccagc tgttgcatta gctaaagcag cctcttgcaa tggtaggaaa 1500
aaaggatctc aaatagcatg agtgtatgtc tatatcctga aatttattgt ctaaaatgca 1560
tgaatatatt tttagcagtc tgtggcatat taatcaaact gttgaattgt tttcttacac 1620
cctggaaatc tttctatcaa ctataatgat aaatccattt tgaagtgata ttttggactt 1680
aggcatttta ctttagattg gaaggcatta tgtgatttac aatatgagaa tatagcagaa 1740
aaaccagatg aggctgtggc tttttatatt caacagccaa taaaaaatgc acaacatgct 1800
aagatcaaag caaa 1814
<210> 14
<211> 1650
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473323CB1
<400> 14
atggccattg atgcactggt ctctctgtgt cttcctgagg tcatcagaat aaagttcaac 60
atcaggccaa ggcagcccca ccacgacctc ccaccaggcg gctcccagga tggtgacttg 120
aaggaaccca cagagagggt cactcgggac ttatccagtg gggccccgag gggccgcaac 180
ctgccagcgc ctgaccagcc tcaacccccg ctgcagaggg gaacccgtct gcggctccgc 240
cagcgccgtc gccgtctgct catcaagaaa atgccagctg cggcgaccat cccggccaac 300
agctcggacg cgcccttcat ccggccggga cccgggacgc tggatggccg ctgggtcagc 360
ctgcaccgga gccagcagga gcgcaagcgg gtgatgcagg aggcctgcgc caagtaccgg 420
gcgagcagca gccgccgggc cgtcacgccc cgccacgtgt cccgtatctt cgtggaggac 480
cgccaccgcg tgctctactg cgaggtgccc aaggccggct gctccaattg gaagcgggtg 540
ctcatggtgc tggccggcct ggcctcgtcc actgccgaca tccagcacaa caccgtccac 600
tatggcagcg ctctcaagcg cctggacacc ttcgaccgcc agggtatctt gcaccgtctc 660
agcacctaca ccaagatgct ctttgtccgc gagcccttcg agaggctggt gtccgccttc 720
cgcgacaagt ttgagcaccc caacagctac tatcacccgg tcttcggcaa ggccatcctg 780
gcccggtacc gcgccaatgc ctctcgggag gccctgcgga ccggctctgg ggtgcgtttt 840
cccgagttcg tccagtacct gctggacgtg caccggcccg tggggatgga cattcactgg 900
gaccatgtca gccggctctg cagcccctgc ctcatcgact acgatttcgt aggcaagttc 960
gagagcatgg aggacgatgc caacttcttc ctgagcctca tccgcgcgcc gcggaacctg 1020
accttccccc ggttcaagga ccggcactcg caggaggcgc ggaccacagc gaggatcgcc 1080
caccagtact tcgcccaact ctcggccctg caaaggcagc gcacctacga cttctactac 1140
atggattacc tgatgttcaa ctattccaag ccctttgcag atctgtactg aggggcgccg 1200
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
14
cagctggccg gggccgccct gccccggtca ctcacctgtg ctcccgggca tcctcctgtc 1260
cctggctcct catcctggga gcaacagggc tctgaggacg tgaggagcca tcgctgtggg 1320
aggcagcagg ccccgggtgg ggggcagagg cgcccagcct tggatgggga ccccagcccc 1380
tggcctgtac ctgtttcctc attccttggc tgagggagag gctgagaact gggcagacac 1440
ccctggagct cagccgacag ttttgatgag cagggaagtc tgaggcccag aggacggggg 1500
gcccagcggt aagggatgtc ccgcactccc ttagccattg ccttggacca aaccacgtgg 1560
tttgcagctt ttctacgagc caggggggag gttcccttgg attaaggttc caaataaagc 1620
acatggtttc cagaacaaaa aaaaacaaaa 1650
<210> 15
<211> 1647
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472777CB1
<400> 15
atgtggctgc cgcgcgtctc cagcacagca gtgaccgcgc tcctcctggc gcagaccttc 60
ctcctcctct ttctggtttc ccggccaggg ccctcgtccc cagcaggcgg cgaggcgcgc 120
gtgcatgtgc tggtgctgtc ctcgtggcgc tcgggctcgt ccttcgtggg ccaactcttc 180
aaccagcacc ccgacgtctt ctacctaatg gagcccgcgt ggcacgtgtg gaccaccctg 240
tcgcagggca gcgccgcaac gctgcacatg gctgtgcgcg acctggtgcg ctccgtcttc 300
ctgtgcgaca tggacgtgtt tgatgcctat ctgccttggc gccgcaacct gtccgacctc 360
ttccagtggg ccgtgagccg tgcactgtgc tcgccacccg cctgcagtgc ctttccccga 420
ggcgccatca gcagcgaggc cgtgtgcaag ccactgtgcg cgcggcagtc cttcaccctg 480
gcccgggagg cctgccgctc ctacagccac gtggtgctca aggaggtgcg cttcttcaac 540
ctgcaggtgc tctacccgct gctcagcgac cccgcgctca acctacgcat cgtgcacctg 600
gtgcgcgacc cgcgggccgt gctgcgctcc cgggagcaga cagccaaggc tctggcgcgt 660
gacaacggca tcgtgctggg caccaacggc acgtgggtgg aggccgaccc cggcctgcgc 720
gtggtgcgcg aggtgtgccg tagccacgta cgcatcgccg aggccgccac actcaagccg 780
ccaccctttc tgcgcggccg ctaccgcctg gtgcgcttcg aggacctggc gcgggagccg 840
ctggcagaaa tccgtgcgct ctacgccttc actgggctca gtctcacgcc acagctcgag 900
gcctggatcc ataacatcac ccacggatct ggacctggtg cgcgccgcga agccttcaag 960
acttcgtcca ggaatgcgct caacgtctcc caggcctggc gccatgcgct gccctttgcc 1020
aagatccgcc gcgtgcagga actgtgcgct ggtgcgctgc agctgctggg ctaccggcct 1080-
gtgtactctg aggacgagca gcgcaacctc gcccttgatc tggtgctgcc acgaggcctg 1140
aacggcttca cttgggcatc atccaccgcc tcgcaccccc gaaattagtg gaggccacag 1200
ttgtagcagg cgctaggccc gggaggagag tgcatggtgc agagggggct ggggcgcacg 1260
gagaagcagg tccctatatt gaccaaggag tttgtgagaa cctgcgtgct gctcctttgc 1320
ttcggacctc cgcctctgcc cgggagaaag cccaggccag cctgctggac aagcagagac 1380
catgagaagg agagttcagg ggtcccaaac caggccatcc tagaccagcc agctccagct 1440
gatccgcacg cagccacttc ggctaccttc tactggccaa agggagtccc agggctcacc 1500
cagattcaga ggtggggaaa ctgagtccac cacttgagaa gagtagctat aaagacatat 1560
gagcgaggcc agctgagccc agcactgcgg ccaagtcgaa gctttaggag caataaaagt 1620
gcttattgtg tttcagtcaa aaaaaaa 1647
<210> 16
<211> 2620
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1272843CB1
<400> 16
cgcggagacc tgggagagga gaaggagccg acctgccgag atggaggcga ccggcacctg 60
ggcgctgctg ctggcgctgg cgctgctcct gctgctgacg ctggcgctgt ccgggaccag 120
ggcccgaggc cacctgcccc ccgggcccac gccgctacca ctgctgggaa acctcctgca 180
gctacggccc ggggcgctgt attcagggct catgcggctg agtaagaagt acggaccggt 240
gttcaccatc tacctgggac cctggcggcc tgtggtggtc ctggttgggc aggaggctgt 300
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
gcgggaggcc ctgggaggtc aggctgagga gttcagcggc cggggaaccg tagcgatgct 360
ggaagggact tttgatggcc atggggtttt cttctccaac ggggagcggt ggaggcagct 420
gaggaagttt accatgcttg ctctgcggga cctgggcatg gggaagcgag aaggcgagga 480
gctgatccag gcggaggccc ggtgtctggt ggagacattc caggggacag aaggacgccc 540
attcgatccc tccctgctgc tggcccaggc cacctccaac gtagtctgct ccctcctctt 600
tggcctccgc ttctcctatg aggataagga gttccaggcc gtggtccggg cagctggtgg 660
taccctgctg ggagtcagct cccagggggg tcagacctac gagatgttct cctggttcct 720
gcggcccctg ccaggccccc acaagcagct cctccaccac gtcagcacct tggctgcctt 780
cacagtccgg caggtgcagc agcaccaggg gaacctggat gcttcgggcc ccgcacgtga 840
ccttgtcgat gccttcctgc tgaagatggc acaggaggaa caaaacccag gcacagaatt 900
caccaacaag aacatgctga tgacagtcat ttatttgctg tttgctggga cgatgacggt 960
cagcaccacg gtcggctata ccctcctgct cctgatgaaa taccctcatg tccaaaagtg 1020
ggtacgtgag gagctgaatc gggagctggg ggctggccag gcaccaagcc taggggaccg 1080
tacccgcctc ccttacaccg acgcggttct gcatgaggcg cagcggctgc tggcgctggt 1140
gcccatggga ataccccgca ccctcatgcg gaccacccgc ttccgagggt acaccctgcc 1200
ccagggcacg gaggtcttcc ccctccttgg ctccatcctg catgacccca acatcttcaa 1260
gcacccagaa gagttcaacc cagaccgttt cctggatgca gatggacggt tcaggaagca 1320
tgaggcgttc ctgcccttct ccttagggaa gcgtgtctgc cttggagagg gcctggcaaa 1380
agcggagctc ttcctcttct tcaccaccat cctacaagcc ttctccctgg agagcccgtg 1440
cccgccggac accctgagcc tcaagcccac cgtcagtggc cttttcaaca ttcccccagc 1500
cttccagctg caagtccgtc ccactgacct tcactccacc acgcagacca gatgaaggaa 1560
ggcaacttgg aagtggtggg tgcccaggac ggtgcctcca gcctcaacag tgggcatgga 1620
cagggttaat gtctccagag tgtacactgc aggcagccac atttacacgc ctgcagttgt 1680
tttccggagt ctgtcccacg gcccacacgc tcacttgact catgctgcta agatgcacaa 1740
ccgcacaccc atacacaact acaagggcca caaagcaact gctgggttag ctttccacag 1800
acataaatat agtccatctg caatcacaag cacatagcca ggtaacccac caactcccct 1860
ggatctgcag cccacacgtg ggagtctggc tgtcaccttc acaagccaca gaaacggcca 1920
cacatgttca cagctcacac gccctctcca ttcatcgaac ttctcagtgt ccctgtccct 1980
ggtgcctggc acagggaaca gcatgccccc tccggggtca tgccacccag agactgtcgc 2040
tgtctatggc cccaactcat gctccctctc ttggctacac cactctccca gcctgtgacc 2100
accgatgtcc acacaccccc aaccacttgt ccacacagct acccacgtac gacatcgtcc 2160
tggctcccca gagtatcttc ccactgagac acgccgcccc cacagaggca cagtccccag 2220
ccacctctgc aactgcagcc ctcagtcacc cctttttaag caccctgatt ctaccaaatg 2280
caaacacatc tgggtctgcg attatgcaca gagactttgg acatacgagg accctcagac 2340
cggaggaaca cctgcccaac cccaacacgt gcttatgtaa ccacgtggaa agcggcccct 2400
gctgcccctc cacacacaca tacacactca ctgatctaca gcccctgttc ggcgtcagag 2460
tccccactag acccagtgga aggggttaga gaccaagtag gggccagttt ccaattcacc 2520
ctgtcaggga gtgagccgga tctgacgttc cttgtgactt aagggtccgg cttgggaatt 2580
aaagtttgtt tctggccttt agcctaaaaa aaaaaaaaaa 2620
<210> 17
<211> 690
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472790CB1
<400> 17
atgaacatcc gcaacgctca gccagacgac ctgatgaata tgcaacactg caacctcctt 60
tgccttcctg agaactacca gatgaaatac tatttatatc atggcctttc ctggccccag 120
ctttcttaca tcgctgagga tgaggacggg aagattgtgg gctatgttct ggccaaaatg 180
gaggaggaac cagatgatgt cccgcatggc catatcacct cactggccgt gaagcgttca 240
caccggcgcc tcggcctggc ccagaagctg atggaccagg cctccagggc catgatagag 300
aactttaacg ccaaatacgt gtctctgcac gtcaggaaga gtaaccggcc agccttgcac 360
ctttattcta acaccctcaa ctttcagatt agtgaggtgg aacctaaata ctatgcagat 420
ggggaagatg cttatgctat gaagcgggat ctctcgcaga tggcagatga gctgagacga 480
caaatggacc tgaagaaggg cgggtatgtg gtcctgggct ccagggagaa ccaggagacc 540
cagggcagca cactttctga ttctgaagag gcctgtcagc aaaagaaccc ggctaccgaa 600
gaaagtggca gtgacagcaa agaacctaag gagtctgtgg agagcaccaa cgtccaggac 660
agctcagaaa gctcggattc cacctcctag 690
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
16
<210> 18
<211> 1510
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473944CB1
<400> 18
gcggccgcga agcgactccg atcctccctc tgagccttgc tcagctctgc cccgcgcctc 60
ccgggctccg gtccgcgcgg cggggtccct gctcctgcgc cccgggcgcg cttcccggac 120
atcccggtcc ccgcagccag gacaaagcca tgaagccagc gctgctggaa gtgatgagga 180
tgaacagaat ctgccggatg gtgctggcca cttgcttggg atcctttatc ctggtcatct 240
tctatttcca aatcatgcgg aggaatccct ttggtgtgga catctgctgc cggaaggggt 300
cccgaagccc cctgcaggaa ctctacaacc caatccagct ggagctctca aacactgctg 360
tcctgcacca gatgcggcgg gaccaggtga cagacacgtg ccgagccaac agcgccacaa 420
gccgtaagcg gagggtgctg acccccaacg acctgaagca cttggtggtg gatgaggacc 480
acgagctcat ctactgctac gtgcccaagg tggcctgcac caactggaag cggctcatga 540
tggtcctgac cgggcggggg aagtacagcg accccatgga gatcccggcc aacgaggcac 600
acgtctccgc caacctgaag accctgaacc agtacagcat cccagaaatc aaccaccgct 660
tgaaaagcta catgaagttc ctgtttgtcc gggagccctt cgagaggcta gtgtccgcct 720
accgcaacaa gttcacccag aagtacaaca tctccttcca caagcggtac ggcaccaaga 780
tcatcaaacg ccagcggaag aacgccaccc aggaggccct gcgcaaaggg gacgatgtca 840
aattcgagga gtttgtggcc tatctcatcg acccacacac ccagcgggag gagcctttca 900
acgaacactg gcaaaccgtc tactcactct gccatccctg ccacatccac tatgacctcg 960
tgggcaagta cgagacactg gaagaggatt ctaattacgt cctgcagctg gcaggagtgg 1020
gcagctacct gaagttcccc acctatgcaa agtctacgag aactactgat gaaatgacca 1080
cagaattctt ccagaacatc agctcagagc accaaacgca gctgtacgaa gtctacaaac 1140
tcgatttttt aatgttcaat tactcagtgc caagctacct gaaattggaa taaagggggt 1200
ggggagaggg agagaatcat gctttttaat ttaagatttt tatttgtcaa aagaattata 1260,
tggatattgg gttattttgt aaattaatat ttctttgggg atgatgctgc gagcagcata 1320
gtgagaatta tttaaaatcc ttcgtaggga aggacagctg tctttgcagg ggaaatagga 1380
tgggtcgtcc ttgtctgtag aagtgaatac tgcaacactg tctcaaaggt ttcttgtgtt 1440
ctggtgaatt ccatgaattg tgcattccat aaattctaat taatattatt tatagttatt 1500
taaaaaaaaa 1510
<210> 19
<211> 3701
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2244136CB1
<400> 19
gagctcggct cactatgtac gtgcgcagtg tgctggcaag gggagaagta gaaagcaccc 60
tgagatgacc ccggctcctc caccaggagc gcggccgggc gcggcgtccc tagcgggctt 120
cgccggggtg gcgtctctgg ggcctgggga cccccgccgc gccgctgacc cgcgccctct 180
gcccccagcg ctgtgcttcg ccgtgagccg ctcgctgctg ctgacgtgcc tggtgccggc 240
cgcgctgctg ggcctgcgct actactacag ccgcaaggtg atccgcgcct acctggagtg 300
cgcgctgcac acggacatgg cggacatcga gcagtactac atgaagccgc ccggctcctg 360
cttctgggtg gccgtgctgg atggcaacgt ggtgggcatt gtggctgcac gggcccacga 420
ggaggacaac acggtggagc tgctgcggat gtctgtggac tcacgtttcc gaggcaaggg 480
catcgccaag gcgctgggcc ggaaggtgct ggagttcgcc gtggtgcaca actactccgc 540
ggtggtgctg ggcacgacgg ccgtcaaggt ggccgcccac aagctctacg agtcgctggg 600
cttcagacac atgggcgcca gtgaccacta cgtgctgccg ggcatgaccc tctcgctggc 660
tgagcgcctc ttcttccagg tccgctacca ccgctaccgc ctgcagctgc gcgaggagtg 720
accgccgccg ctcgcccgcc cgcccccccg gccgccctgt ccgcctttgc ccgcctgccc 780
gccgcccggc gcggcctgct ttcagacgct cacttggcgt ttgtgttggg tttccccttt 840
tcaacatcct gcggttgtct ggctggttcc ggggggtgcg gggctgttgt tcttcggcga 900
cactttggtg ggggtgggtt gtttgtcgcc atagcccctc gcccttcccc acctgcctgg 960
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
17
gcggcttgcc acctgaagag tggcatcttg gaccaccgcg gctgtccatg acgctgccct 1020
gcccgccgcc actggggaag gcccagcctt gctcaccaag cacagaacct ctgcagcaga 1080
tcccggggcc aggctccggc cccgcctgcg gccccagcgc cactgcctgt ggaggcccga 1140
gctggccacg gcgctgcttt gctccgcgca tgccgagggt gtggcccggc tgagcatgcc 1200
gcatgcacac agccccgccc tgccgccctg cccagactgg acccggagac ccgggctggt 1260
gagcgcccct gtccccagcc cccagctggc tgtgggaggg cctgcccctg cccccacctc 1320
ctggagggcc tggtctgccc cgcgccgccc ggctctgtcc acacctgctt tgctctgacg 1380
ccctccattt ctctggctcc ggcccctccc ctgcctgggc tgtgctgact ggtgtcatca 1440
cccaggtgac tcccatggcg tccgtggcac agccagggtg ggggtccatg ggacccctct 1500
ccccagtgcc cactggatcg tgctggcctc tcccagatgt ccccggggac ctcctgcctc 1560
tggctgacgg tccaccctgt gaatcttatc agcccaggct gctgccaaca gcgcccagcc 1620
cacagcttct cccagcctga aaccaacaca ttttctaata agttatttag acagaatagc 1680
actctgcatg actttaattc ttgggacaaa acggtagttt gtaccctaag acacagtttc 1740
tggcccagtg tgatgggggt gggtggccgg gtgggtggag cgttttgctg ttggaaacct 1800
ggagggaaac cctgattgga tgtcatttcc tgccatggag cacgcctccc agccctggcc 1860
tgcagggtgg gcagggtggg gggcaaggga gtccgcagcc tccgggagga ggggcagggc 1920
gctgccttgg gctgggtggg aagaggggtg gccgcctcgg cttccgctgg ccatgctcct 1980
ggtctctcct tcctgaggtc acaggcaggg gctgccctgg acggggggcg gggggggtgg 2040
cctggaaggg gagacagagg tggagggtgg cacaggctgc acattcagct tagaagtgga 2100
cctggctttg gtggcaggag aagaataaac acttgcccag acccctttgt gtgggggaat 2160
tggggagggg tcgtggcagg cagggtgggc cacggaactg ggtcccaggc atcaaggcca 2220
cgtgcagggc catggaggga tgcttctcac gaggcgcttc agaagcgagc gaagggacag 2280
agaagccctg cgtccaaggg ccttttgtcc tgttagcaat tgaggtgtgc agagcactgt 2340
acagacccca ctcccctgta cattcctccc tggaggtgcc cggtccccgc.ttggggatgg 2400
gagttttgta gactgtacag aaatcggcac cctattttct tgcagctcag attttgttaa 2460
tctggaatat acagacagac gtaaagtgtt ttagcaaaat ggaaacaaac agttgtgcct 2520
ttttcctctt ttggtttggt ttgggtttgg cctggggctg gtcccagttg gtgcggggca 2580
tgctgggggc aggaggggca gggcgggcca ggtggagtca ggtcttgggg gtgtcatgtc 2640
ggggtgctgc cagcgtccct tggtcctgtg cctttggagc ctcgggctcc tggggtgcag 2700
ggtgcttggg ggtggctgtt ggagcccacc gacgccaggg cagggcctgg aggcccaggg 2760
actgccagtg tctccttgat attgatccta gcagatcccc ctcctggggg ttctgagagt 2820:
cctgggcagt gtggccttct ctcattctgg tggcatctgc gcccgtgagt gacctcttcc 2880'
ttggctgcac tgccctgtgg gtggtgagac gcttggcctt ttttgttgct gccaggactt 2940
ggtagagatg gcaggaaggg tgtgcggggt ggttgtgtgc agaaccctgc cgcccctgag 3000
gtgagcagag gcactggtgt gcctgccagg ctggggcgga gctgcccgga acccttgcca 3060
ggccaatgtg tagcttggtg gcgcccttga aggccactgg ggggtaggtg tgtcctcccc 3120.
cggggctgga ggccgggtgc ctggtgggtg ggcctgacct ggcccacctc atccctccag 3180
cctgggatct cactgctctg cacctgtgca ctgacgtgaa ttttatacgt ttgtagaaac 3240
tctgatgtaa cttcttctac ctctgaagcg ccctcctggg ccctgctgtc acgtggctgg 3300'
tgcggcttcc ccgagtgctg gccccctgcc cgctccccat gggcacccac cactcaaact 3360
ctgcgtggtg aggcccgggg aaagccaggc cgggccctag cactgctcag gggcctgggg 3420
ctcctgggat ttctgtgtgt ttggaagcct ctgtttttgg agtggggggc gtggaaggcg 3480
ggaggggctg acagtgcctg gggccagagc tgggcgaagg aggacatcct cactggacga 3540
gcactgcagg cctgacgaga ggctcccggc agctgagggc ttaagcgcct cgtgatgtcc 3600
ccaatggccc aagcccccgt tctgctttgc tggggtgggt ttgcccccgc gattgcactg 3660
tcccctggtg ttcttcgacc agctcctccg gggagggcct c 3701
<210> 20
<211> 960
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474327CB1
<400> 20
gcagccatgg ccagccacat tgtgctcaac aatggcacca agatgcccat cctggggcta 60
ggcacctgga attcccctcc aggccaggta actgaggctg tgaaggtggc cattaatgtt 120
gggtactgcc acatcgactg tgcccacgtg taccagaatg agaatgacgt gggggtggcc 180
attcgggaga agctcaggga gcaggtggtg aagtgtgagg agctcttcat caccagcaag 240
ctgtggtgcg cgtaccatga gaagggcctg gtgaaaggag cctgccagaa gatgctcatt 300
gacctgaagc tggactacct ggacctctac cttattcgct ggccaaccag cttcaagcct 360
CA 02403644 2002-09-26
WO 01/79468 PCT/USO1/11869
18
gggaaggaat ttttcccatt ggatgagcca ggtaatggta atgtggttcc cagtaacagt 420
aacattctgg acacatgggc gggcatggaa gagctggtgg atgaagggct ggtgaaagct 480
attggcatct ccaacttcaa ccatctccag gttgagagga tcttaaacaa acctgactta 540
aagtataagc cggtggttaa tcagattgag tgccacccgt acctcactca ggagaagtta 600
atccagtact gccagtccaa aggcatcatg gtgactgcct acagctcctt cagctccccc 660
gacaggccct gggccaagcc tgaggaccct tccctcctgg aagatcccag gatcaaggcg 720
atcacagcca aacacaataa aactacagcc caggttctga tctggttccc catgcagagg 780
aacttggtgg tgatccccaa gtctgtgaca ccagaatgca ttgctgagaa ctttaaggtc 840
ttcaactttg aactgaacag ccaggatatg accaccttat tcagttacaa cagaaactgg 900
agggtctgtg ccttggtgag ctgtgcctcc cacaaggatt accccttcca tgaagagttt 960
