U.S. patent application number 10/893888 was filed with the patent office on 2005-03-10 for oxidoreductase molecules.
This patent application is currently assigned to INCYTE CORPORATION. Invention is credited to Au-Young, Janice, Azimzai, Yalda, Bandman, Olga, Baughn, Mariah R., Corley, Neil C., Gorgone, Gina A., Guegler, Karl J., Hillman, Jennifer L., Lal, Preeti, Lu, Dyung Aina M., Tang, Y. Tom, Yang, Junming, Yue, Henry.
Application Number | 20050053987 10/893888 |
Document ID | / |
Family ID | 33160123 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050053987 |
Kind Code |
A1 |
Lal, Preeti ; et
al. |
March 10, 2005 |
Oxidoreductase molecules
Abstract
The invention provides human oxidoreductase molecules (OXRE) and
polynucleotides which identify and encode OXRE. The invention also
provides expression vectors, host cells, antibodies, agonists, and
antagonists. The invention also provides methods for diagnosing,
treating, or preventing disorders associated with expression of
OXRE.
Inventors: |
Lal, Preeti; (Santa Clara,
CA) ; Guegler, Karl J.; (Mento Park, CA) ;
Gorgone, Gina A.; (Boulder Creek, CA) ; Corley, Neil
C.; (Castro Valley, CA) ; Baughn, Mariah R.;
(San Leandro, CA) ; Tang, Y. Tom; (San Jose,
CA) ; Hillman, Jennifer L.; (Mountain View, CA)
; Bandman, Olga; (Mountian View, CA) ; Azimzai,
Yalda; (Castro Valley, CA) ; Au-Young, Janice;
(Brisbane, CA) ; Yue, Henry; (Sunnyvale, CA)
; Lu, Dyung Aina M.; (San Jose, CA) ; Yang,
Junming; (San Jose, CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
INCYTE CORPORATION
|
Family ID: |
33160123 |
Appl. No.: |
10/893888 |
Filed: |
July 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10893888 |
Jul 20, 2004 |
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09806536 |
Mar 28, 2001 |
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6808895 |
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09806536 |
Mar 28, 2001 |
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PCT/US99/23434 |
Oct 6, 1999 |
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60123911 |
Mar 10, 1999 |
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60155202 |
Dec 2, 1998 |
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60172227 |
Oct 6, 1998 |
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Current U.S.
Class: |
435/6.14 ;
435/189; 435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
C12N 9/0004 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/189; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/04; C12N 009/02 |
Claims
What is claimed is:
1. A substantially purified polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6. SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, and fragments thereof:
2. A substantially purified variant having at least 90% amino acid
sequence identity to the amino acid sequence of claim 1.
3. An isolated and purified polynucleotide encoding the polypeptide
of claim 1.
4. An isolated and purified polynucleotide variant having at least
70% polynucleotide sequence identity to the polynucleotide of claim
3.
5. An isolated and purified polynucleotide which hybridizes under
stringent conditions to the polynucleotide of claim 3.
6. An isolated and purified polynucleotide having a sequence which
is complementary to the polynucleotide of claim 3.
7. A method for detecting a polynucleotide, the method comprising
the steps of: (a) hybridizing the polynucleotide of claim 6 to at
least one nucleic acid in a sample, thereby forming a hybridization
complex; and (b) detecting the hybridization complex, wherein the
presence of the hybridization complex correlates with the presence
of the polynucleotide in the sample.
8. The method of claim 7 further comprising amplifying the
polynucleotide prior to hybridization.
9. An isolated and purified polynucleotide comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:16-30 and fragments thereof.
10. An isolated and purified polynucleotide variant having at least
70% polynucleotide sequence identity to the polynucleotide of claim
9.
11. An isolated and purified polynucleotide having a sequence which
is complementary to the polynucleotide of claim 9.
12. An expression vector comprising at least a fragment of the
polynucleotide of claim 3.
13. A host cell comprising the expression vector of claim 12.
14. A method for producing a polypeptide, the method comprising the
steps of: a) culturing the host cell of claim 13 under conditions
suitable for the expression of the polypeptide; and b) recovering
the polypeptide from the host cell culture.
15. A pharmaceutical composition comprising the polypeptide of
claim 1 in conjunction with a suitable pharmaceutical carrier.
16. A purified antibody which specifically binds to the polypeptide
of claim 1.
17. A purified agonist of the polypeptide of claim 1.
18. A purified antagonist of the polypeptide of claim 1.
19. A method for treating or preventing a disorder associated with
decreased expression or activity of OXRE, the method comprising
administering to a subject in need of such treatment an effective
amount of the pharmaceutical composition of claim 15.
20. A method for treating or preventing a disorder associated with
increased expression or activity of OXRE, the method comprising
administering to a subject in need of such treatment an effective
amount of the antagonist of claim 18.
Description
TECHNICAL FIELD
[0001] This invention relates to nucleic acid and amino acid
sequences of oxidoreductase molecules and to the use of these
sequences in the diagnosis, treatment, and prevention of cell
proliferative disorders including cancer, endocrine, metabolic,
reproductive, neurological, autoimmune/inflammatory, and viral
disorders.
BACKGROUND OF THE INVENTION
[0002] Many pathways of biogenesis and biodegradation require
oxidoreductase (dehydrogenase or reductase) activity, coupled to
the reduction or oxidation of a donor or acceptor cofactor.
Potential cofactors include cytochromes, oxygen, disulfide,
iron-sulfur proteins, flavin adenine dinucleotide (FAD), and the
nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E. A.
and Leech, A. R. (1983) Biochemistry for the Medical Sciences, John
Wiley and Sons, Chichester, U. K. pp. 779-793).
[0003] Reductase activity catalyzes the transfer of electrons
between substrate(s) and cofactor(s) with concurrent oxidation of
the cofactor. The reverse dehydrogenase reaction catalyzes the
reduction of a cofactor and consequent oxidation of the substrate.
Oxidoreductase enzymes are a broad superfamily of proteins that
catalyze numerous reactions in all cells of organisms ranging from
bacteria to plants to humans. These reactions include metabolism of
sugar, certain detoxification reactions in the liver, and the
synthesis or degradation of fatty acids, amino acids,
glucocorticoids, estrogens, androgens, and prostaglandins.
Different family members are named according to the direction in
which their reactions are typically catalyzed; thus they may be
referred to as oxidoreductases, oxidases, reductases, or
dehydrogenases. In addition, family members often have distinct
cellular localizations, including the cytosol, the plasma membrane,
mitochondrial inner or outer membrane, and peroxisomes.
[0004] Tetrahydrofolate is a derivatized glutamate molecule that
acts as a carrier, providing activated one-carbon units to a wide
variety of biosynthetic reactions, including synthesis of purines,
pyrimidines, and the amino acid methionine. Tetrahydrofolate is
generated by the activity of a holoenzyme complex called
tetrahydrofolate synthase, which includes three enzyme activities:
tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase,
and tetrahydrofolate synthetase. Thus, tetrahydrofolate
dehydrogenase plays an important role in generating building blocks
for nucleic and amino acids, crucial to proliferating cells.
[0005] Intracellular redox status plays a critical role in the
assembly of proteins. A major rate limiting step in protein folding
is the thiol:disulfide exchange necessary for correct protein
assembly. Although incubation of reduced, unfolded proteins in
buffers containing defined ratios of oxidized and reduced thiols
can lead to folding into native conformation, the rate of folding
is slow, and the attainment of the native conformation decreases
proportionately with protein size and the number of cysteine
residues. Certain cellular compartments such as the endoplasmic
reticulum of eukaryotes and the periplasmic space of prokaryotes
are maintained in a more oxidized state than the surrounding
cytosol. Correct disulfide formation can occur in these
compartments, but it occurs at a rate that is insufficient for
normal cell processes and inadequate for synthesizing secreted
proteins.
[0006] Protein disulfide isomerases (PDIs), thioredoxins, and
glutaredoxins are able to catalyze the formation of disulfide bonds
and regulate the redox environment in cells to enable the necessary
thiol:disulfide exchanges. Each of these classes of molecules has a
somewhat different function, but all belong to a group of
disulfide-containing redox proteins that contain a conserved
active-site sequence and are ubiquitously distributed in eukaryotes
and prokaryotes. PDIs are found in the endoplasmic reticulum of
eukaryotes and in the periplasmic space of prokaryotes. PDIs
function by exchanging their own disulfide for thiols in a folding
peptide chain. In contrast, reduced thioredoxins and glutaredoxins
are generally found in the cytoplasm and function by directly
reducing disulfides in the substrate proteins. Thioredoxin (Trx), a
heat-stable, redox-active protein, contains an active site cysteine
disulfide/dithiol. Oxidized thioredoxin, Trx-S, can be reduced to
the dithiol form by NADPH and a specific flavoprotein enzyme,
thioredoxin reductase. Reduced thioredoxin, Trx-(SH), participates
in a number of redox reactions mostly linked to reduction of
protein disulfides. Trx and thioredoxin reductase (TR), together
with NADPH, form a redox complex in which TR catalyzes the electron
transport from NADPH to Trx. The reduced thioredoxin then functions
as an electron donor in a wide variety of different metabolic
processes.
[0007] Disulfide-containing redox proteins not only facilitate
disulfide formation, but also regulate and participate in a wide
variety of physiological processes. The thioredoxin system serves,
for example, as a hydrogen donor for ribonucleotide reductase and
controls the activity of enzymes by redox reactions. Mammalian
thioredoxin (MT) acts as a hydrogen donor for ribonucleotide
reductase and methionine sulfoxide reductase, facilitates refolding
of disulfide-containing proteins, and activates the glucocorticoid
and interleukin-2 receptors. MT also modulates the DNA binding
activity of some transcription factors either directly (TFIIIC,
BZLF1, and NF-kB) or indirectly (AP-1) through the nuclear factor
Ref-1. The importance of the redox regulation of transcription
factors is exemplified by the v-fos oncogene where a point mutation
of the thioredoxin-modulated cysteine residue results in
constitutive activation of the AP-1 complex. Thioredoxin is
secreted by cells using a leaderless pathway and stimulates the
proliferation of lymphoid cells, fibroblasts, and a variety of
human solid tumor cell lines. Furthermore, thioredoxin is an
essential component of early pregnancy factor, inhibits human
immunodeficiency virus expression in macrophages, reduces
H.sub.2O.sub.2, scavenges free radicals, and protects cells against
oxidative stress (Abate, C. et al., (1990) Science 249: 1157-1161;
Rosen, A. et al. (1995) Int. Immunol. 7: 625-633; Tagaya, Y. et al
(1989) EMBO J. 8: 757-764; Newman, G. W. (1994) J. Expt. Med. 180:
359-363; and Makino, Y. (1996) J. Clin. Invest. 98: 2469-2477).
[0008] Short-chain alcohol dehydrogenases (SCADs) are a family of
dehydrogenases that share only 15% to 30% sequence identity, with
similarity predominantly in the coenzyme binding domain and the
substrate binding domain. In addition to the well-known role in
detoxification of ethanol, SCADs are also involved in synthesis and
degradation of fatty acids, steroids, and some prostaglandins, and
are therefore implicated in a variety of disorders such as lipid
storage disease, myopathy, SCAD deficiency, and certain genetic
disorders. For example, retinol dehydrogenase is a SCAD-family
member (Simon, A. et al. (1995) J. Biol. Chem. 270:1107-1112) that
converts retinol to retinal, the precursor of retinoic acid.
Retinoic acid, a regulator of differentiation and apoptosis, has
been shown to down-regulate genes involved in cell proliferation
and inflammation (Chai, X. et al. (1995) J. Biol. Chem.
270:3900-3904). In addition, retinol dehydrogenase has been linked
to hereditary eye diseases such as autosomal recessive
childhood-onset severe retinal dystrophy (Simon, A. et al. (1996)
Genomics 36:424-430).
[0009] Propagation of nerve impulses, modulation of cell
proliferation and differentiation, induction of the immune
response, and tissue homeostasis involve neurotransmitter
metabolism (Weiss, B. (1991) Neurotoxicology 12:379-386; Collins,
S. M. et al. (1992) Ann. N.Y. Acad. Sci. 664:415-424; Brown, J. K.
and Imam, H. (1991) J. Inherit. Metab. Dis. 14:436-458). Many
pathways of neurotransmitter metabolism require oxidoreductase
activity, coupled to reduction or oxidation of a cofactor, such as
NAD.sup.+/NADH (Newsholme, E. A. and Leech, A. R. (1983)
Biochemistry for the Medical Sciences, John Wiley and Sons,
Chichester, U. K. pp. 779-793). Degradation of catecholamines
(epinephrine or norepincphrine) requires alcohol dehydrogenase (in
the brain) or aldehyde dehydrogenase (in peripheral tissue).
NAD.sup.+-dependent aldehyde dehydrogenase oxidizes
5-hydroxyindole-3-acetate (the product of 5-hydroxytryptamine
(serotonin) metabolism) in the brain, blood platelets, liver and
pulmonary endothelium (Newsholme, E. A. and Leech, A. R. (supra) p.
786). Other neurotransmitter degradation pathways that utilize
NAD.sup.+/NADH-dependent oxidoreductase activity include those of
L-DOPA (precursor of dopamine, a neuronal excitatory compound),
glycine (an inhibitory neurotransmitter in the brain and spinal
cord), histamine (liberated from mast cells during the inflammatory
response), and taurine (an inhibitory neurotransmitter of the brain
stem, spinal cord and retina) (Newsholme, E. A. and Leech, A. R.
supra, pp. 790, 792). Epigenetic or genetic defects in
neurotransmitter metabolic pathways can result in a spectrum of
disease states in different tissues including Parkinson's disease
and inherited myoclonus (McCance, K. L. and Huether, S. E. (1994)
Pathophysiology, Mosby-Year Book, Inc., St. Louis, Mo. pp. 402-404;
Gundlach, A. L. (1990) FASEB J. 4:2761-2766).
[0010] 3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty
acid metabolism. It catalyzes the reduction of 3-hydroxyacyl-CoA to
3-oxoacyl-CoA, with concomitant oxidation of NAD to NADH, in the
mitochondria and peroxisomes of eukaryotic cells. In peroxisomes,
3HACD and enoyl-CoA hydratase form an enzyme complex called
bifunctional enzyme, defects in which are associated with
peroxisomal bifunctional enzyme deficiency. This interruption in
fatty acid metabolism produces accumulation of very-long chain
fatty acids, disrupting development of the brain, bone, and adrenal
glands. Infants born with this deficiency typically die within 6
months (Watkins, P. et al. (1989) J. Clin. Invest. 83:771-777;
Online Mendelian Inheritance in Man (OMIM), #261515). The
neurodegeneration that is characteristic of Alzheimer's disease
involves development of extracellular plaques in certain brain
regions. A major protein component of these plaques is the peptide
amyloid-.beta. (A.beta.), which is one of several cleavage products
of amyloid precursor protein (APP). 3HACD has been shown to bind
the A.beta. peptide, and is overexpressed in neurons affected in
Alzheimer's disease. In addition, an antibody against 3HACD can
block the toxic effects of A.beta. in a cell culture model of
Alzheimer's disease (Yan, S. et al. (1997) Nature 389:689-695;
OMIM, #602057).
[0011] 17.beta.-hydroxysteroid dehydrogenase (17.beta.HSD6) plays
an important role in the regulation of the male reproductive
hormone, dihydrotestosterone (DHTT). 17.beta.HSD6 acts to reduce
levels of DHTT by oxidizing a precursor of DHTT, 3.alpha.-diol, to
androsterone which is readily glucuronidated and removed from
tissues. 17.beta.HSD6 is active with both androgen and estrogen
substrates when expressed in embryonic kidney 293 cells. At least
five other isozymes of 17.beta.HSD have been identified that
catalyze oxidation and/or reduction reactions in various tissues
with preferences for different steroid substrates (Biswas, M. G.
and Russell, D. W. (1997) J. Biol. Chem. 272:15959-15966). For
example, 17.beta.HSD1 preferentially reduces estradiol and is
abundant in the ovary and placenta. 17.beta.HSD2 catalyzes
oxidation of androgens and is present in the endometrium and
placenta. 17.beta.HSD3 is exclusively a reductive enzyme in the
testis (Geissler, W. M. et al. (1994) Nature Genet. 7:34-39). An
excess of androgens such as DHTT can contribute to certain disease
states such as benign prostatic byperplasia and prostate
cancer.
[0012] Steroids, such as estrogen, testosterone, corticosterone,
and others, are generated from a common precursor, cholesterol, and
are interconverted into one another. A wide variety of enzymes act
upon cholesterol, including a number of dehydrogenases. One such
dehydrogenase is 3-oxo-5-.alpha.-steroid dehydrogenase (OASD), a
microsomal membrane protein highly expressed in prostate and other
androgen-responsive tissues. OASD catalyzes the conversion of
testosterone into dihydrotestosterone, which is the most potent
androgen. Dihydrotestosterone is essential for the formation of the
male phenotype during embryogenesis, as well as for proper
androgen-mediated growth of tissues such as the prostate and male
genitalia. A defect in OASD that prevents the conversion of
testosterone into dihydrotestosterone leads to a rare form of male
pseudohermaphroditis, characterized by defective formation of the
external genitalia (Andersson, S., et al. (1991) Nature
354:159-161; Labrie, F., et al. (1992) Endocrinology 131:1571-1573;
OMIM #264600). Thus, OASD plays a central role in sexual
differentiation and androgen physiology.
[0013] The discovery of new oxidoreductase molecules 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 cell proliferative disorders including
cancer, endocrine, metabolic, reproductive, neurological,
autoimmune/inflammatory, and viral disorders.
SUMMARY OF THE INVENTION
[0014] The invention features substantially purified polypeptides,
oxidoreductase molecules, referred to collectively as "OXRE' and
individually as "OXRE-1," "OXRE-2," "OXRE-3," "OXRE-4," "OXRE-5,"
"OXRE-6," "OXRE-7," "OXRE-8," "OXRE-9," "OXRE-10," "OXRE-11,"
"OXRE-12," "OXRE-13," "OXRE-14," and "OXRE-15." In one aspect, the
invention provides a substantially purified polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-15 and fragments thereof.
[0015] The invention further provides a substantially purified
variant having at least 90% amino acid identity to at least one of
the amino acid sequences selected from the group consisting of SEQ
ID NO:1-15 and fragments thereof. The invention also provides an
isolated and purified polynucleotide encoding the polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-15 and fragments thereof. The invention
also includes an isolated and purified polynucleotide variant
having at least 70% polynucleotide sequence identity to the
polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-15 and
fragments thereof.
[0016] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-15
and fragments thereof. The invention also provides an isolated and
purified polynucleotide having a sequence which is complementary to
the polynucleotide encoding the polypeptide comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:1-15
and fragments thereof.
[0017] The invention also provides a method for detecting a
polynucleotide in a sample containing nucleic acids, the method
comprising the steps of (a) hybridizing the complement of the
polynucleotide sequence to at least one of the polynucleotides of
the sample, thereby forming a hybridization complex; and (b)
detecting the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of a
polynucleotide in the sample. In one aspect, the method further
comprises amplifying the polynucleotide prior to hybridization.
[0018] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:16-30 and fragments thereof The
invention further provides an isolated and purified polynucleotide
variant having at least 70% polynucleotide sequence identity to the
polynucleotide sequence selected from the group consisting of SEQ
ID NO:16-30 and fragments thereof. The invention also provides an
isolated and purified polynucleotide having a sequence which is
complementary to the polynucleotide comprising a polynucleotide
sequence selected from the group consisting of SEQ ID NO:16-30 and
fragments thereof.
[0019] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-15 and fragments thereof. In
another aspect, the expression vector is contained within a host
cell.
[0020] The invention also provides a method for producing a
polypeptide, the method comprising the steps of: (a) culturing the
host cell containing an expression vector containing at least a
fragment of a polynucleotide under conditions suitable for the
expression of the polypeptide; and (b) recovering the polypeptide
from the host cell culture.
[0021] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO:1-15
and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
[0022] The invention further includes a purified antibody which
binds to a polypeptide selected from the group consisting of SEQ ID
NO:1-15 and fragments thereof. The invention also provides a
purified agonist and a purified antagonist to the polypeptide.
[0023] The invention also provides a method for treating or
preventing a disorder associated with decreased expression or
activity of OXRE, the method comprising administering to a subject
in need of such treatment an effective amount of a pharmaceutical
composition comprising a substantially purified polypeptide having
the amino acid sequence selected from the group consisting of SEQ
ID NO:1-15 and fragments thereof in conjunction with a suitable
pharmaceutical carrier.
[0024] The invention also provides a method for treating or
preventing a disorder associated with increased expression or
activity of OXRE, the method comprising administering to a subject
in need of such treatment an effective amount of an antagonist of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-15 and fragments thereof.
BRIEF DESCRIPTION OF THE TABLES
[0025] Table 1 shows polypeptide and nucleotide sequence
identification numbers (SEQ ID NOs), clone identification numbers
(clone IDs), cDNA libraries, and cDNA fragments used to assemble
full-length sequences encoding OXRE.
[0026] Table 2 shows features of each polypeptide sequence,
including potential motifs, homologous sequences, and methods and
algorithms used for identification of OXRE.
[0027] Table 3 shows the tissue-specific expression patterns of
each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or conditions associated with these tissues; a
useful fragment of each nucleic acid; and the vector into which
each cDNA was cloned.
[0028] Table 4 describes the tissues used to construct the cDNA
libraries from which cDNA clones encoding OXRE were isolated.
[0029] Table 5 shows the tools, programs, and algorithms used to
analyze OXRE, along with applicable descriptions, references, and
threshold parameters.
DESCRIPTION OF THE INVENTION
[0030] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular machines, materials and methods
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
[0031] 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.
[0032] 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.
[0033] Definitions
[0034] "OXRE" refers to the amino acid sequences of substantially
purified OXRE obtained from any species, particularly a mammalian
species, including bovine, ovine, porcine, murine, equine, and
preferably the human species, from any source, whether natural,
synthetic, semi-synthetic, or recombinant.
[0035] The term "agonist" refers to a molecule which, when bound to
OXRE, increases or prolongs the duration of the effect of OXRE.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules which bind to and modulate the effect of OXRE.
[0036] An "allelic variant" is an alternative form of the gene
encoding OXRE. 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. Any given natural or recombinant gene may have none,
one, or many allelic forms. 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.
[0037] "Altered" nucleic acid sequences encoding OXRE include those
sequences with deletions, insertions, or substitutions of different
nucleotides, resulting in a polynucleotide the same as OXRE or a
polypeptide with at least one functional characteristic of OXRE.
Included within this definition are polymorphisms which may or may
not be readily detectable using a particular oligonucleotide probe
of the polynucleotide encoding OXRE, and improper or unexpected
hybridization to allelic variants, with a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
OXRE. 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 OXRE. 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 OXRE is retained. For example, negatively charged amino acids
may include aspartic acid and glutamic acid, positively charged
amino acids may include lysine and arginine, and amino acids with
uncharged polar head groups having similar hydrophilicity values
may include leucine, isoleucine, and valine; glycine and alanine;
asparagine and glutamine; serine and threonine; and phenylalanine
and tyrosine.
[0038] 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. In this context, "fragments," "immunogenic fragments,"
or "antigenic fragments" refer to fragments of OXRE which are
preferably at least 5 to about 15 amino acids in length, most
preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of OXRE. Where "amino
acid sequence" is recited to refer to an amino acid 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.
[0039] "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.
[0040] The term "antagonist" refers to a molecule which, when bound
to OXRE, decreases the amount or the duration of the effect of the
biological or immunological activity of OXRE. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of OXRE.
[0041] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fab, F(ab').sub.2, and Fv fragments,
which are capable of binding the epitopic determinant. Antibodies
that bind OXRE polypeptides can be prepared using intact
polypeptides or using fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can
be derived from the translation of RNA, or synthesized chemically,
and can be conjugated to a carrier protein if desired. Commonly
used carriers that are chemically coupled to peptides include
bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0042] The term "antigenic determinant" refers to that fragment 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 (given regions or three-dimensional structures on he
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.
[0043] The term "antisense" refers to any composition containing a
nucleic acid sequence which is complementary to the "sense" strand
of a specific nucleic acid sequence. Antisense molecules may be
produced by any method including synthesis or transcription. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0044] The term "biologically active" refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" refers to
the capability of the natural, recombinant, or synthetic OXRE, or
of any oligopeptide thereof, to induce a specific immune response
in appropriate animals or cells and to bind with specific
antibodies.
[0045] The terms "complementary" and "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence "5' A-G-T 3'" bonds to the complementary sequence "3'
T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial," such that only some of the nucleic acids bind, or
it may be "complete," such that total complementarity exists
between the single stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of the hybridization between
the nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands, and in the design and use of peptide nucleic acid
(PNA) molecules.
[0046] 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 OXRE or fragments of OXRE may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g.,
sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.).
[0047] "Consensus sequence" refers to a nucleic acid sequence which
has been resequenced to resolve uncalled bases, extended using the
XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3'
direction, and resequenced, or which has been assembled from the
overlapping sequences of more than one Incyte Clone using a
computer program for fragment assembly, such as the GELVIEW
fragment assembly system (GCG, Madison Wis.). Some sequences have
been both extended and assembled to produce the consensus
sequence.
[0048] The term "correlates with expression of a polynucleotide"
indicates that the detection of the presence of nucleic acids, the
same or related to a nucleic acid sequence encoding OXRE, by
northern analysis is indicative of the presence of nucleic acids
encoding OXRE in a sample, and thereby correlates with expression
of the transcript from the polynucleotide encoding OXRE.
[0049] 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.
[0050] The term "derivative" refers to the chemical modification of
a polypeptide sequence, or a polynucleotide sequence. Chemical
modifications of a polynucleotide sequence can include, for
example, replacement of hydrogen by an alkyl, acyl, 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.
[0051] The term "similarity" refers to a degree of complementarity.
There may be partial similarity or complete similarity. The word
"identity" may substitute for the word "similarity." A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to as "substantially similar." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or northern blot, solution hybridization, and the like)
under conditions of reduced stringency. A substantially similar
sequence or hybridization probe will compete for and inhibit the
binding of a completely similar (identical) sequence to the target
sequence under conditions of reduced stringency. This is not to say
that conditions of reduced stringency are such that non-specific
binding is permitted, as reduced stringency conditions require that
the binding of two sequences to one another be a specific (i.e., a
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
similarity or identity). In the absence of non-specific binding,
the substantially similar sequence or probe will not hybridize to
the second non-complementary target sequence.
[0052] The phrases "percent identity" and "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MEGALIGN program
(DNASTAR, Madison Wis.) which creates alignments between two or
more sequences according to methods selected by the user, e.g., the
clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988)
Gene 73:237-244.) Parameters for each method may be the default
parameters provided by MEGALIGN or may be specified by the user.
The clustal algorithm groups sequences into clusters by examining
the distances between all pairs. The clusters are aligned pairwise
and then in groups. The percentage similarity between two amino
acid sequences, e.g., sequence A and sequence B, is calculated by
dividing the length of sequence A, minus the number of gap residues
in sequence A, minus the number of gap residues in sequence B, into
the sum of the residue matches between sequence A and sequence B,
times one hundred. Gaps of low or of no similarity between the two
amino acid sequences are not included in determining percentage
similarity. Percent identity between nucleic acid sequences can
also be counted or calculated by other methods known in the art,
e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods
Enzymol. 183:626-645.) Identity between sequences can also be
determined by other methods known in the art, e.g., by varying
hybridization conditions.
[0053] "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
stable mitotic chromosome segregation and maintenance.
[0054] The term "humanized antibody" refers to antibody molecules
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.
[0055] "Hybridization" refers to any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing.
[0056] 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., C.sub.0t or R.sub.0t 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).
[0057] 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, to the
sequence found in the naturally occurring molecule.
[0058] "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.
[0059] The term "microarray" refers to an arrangement of distinct
polynucleotides on a substrate.
[0060] The terms "element" and "array element" in a microarray
context, refer to hybridizable polynucleotides arranged on the
surface of a substrate.
[0061] The term "modulate" refers to a change in the activity of
OXRE. For example, modulation may cause an increase or a decrease
in protein activity, binding characteristics, or any other
biological, functional, or immunological properties of OXRE.
[0062] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, 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. In this context, "fragments" refers
to those nucleic acid sequences which comprise a region of unique
polynucleotide sequence that specifically identifies SEQ ID
NO:16-30, for example, as distinct from any other sequence in the
same genome. For example, a fragment of SEQ ID NO:16-30 is useful
in hybridization and amplification technologies and in analogous
methods that distinguish SEQ ID NO:16-30 from related
polynucleotide sequences. A fragment of SEQ ID NO:16-30 is at least
about 15-20 nucleotides in length. The precise length of the
fragment of SEQ ID NO:16-30 and the region of SEQ ID NO:16-30 to
which the fragment corresponds are routinely determinable by one of
ordinary skill in the art based on the intended purpose for the
fragment. In some cases, a fragment, when translated, would produce
polypeptides retaining some functional characteristic, e.g.,
antigenicity, or structural domain characteristic, e.g.,
ATP-binding site, of the full-length polypeptide.
[0063] The terms "operably associated" and "operably linked" refer
to functionally related nucleic acid sequences. A promoter is
operably associated or operably linked with a coding sequence if
the promoter controls the translation of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in the same reading frame, certain genetic
elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0064] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to 60 nucleotides, preferably about
15 to 30 nucleotides, and most preferably about 20 to 25
nucleotides, which can be used in PCR amplification or in a
hybridization assay or microarray. "Oligonucleotide" is
substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the
art.
[0065] "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.
[0066] The term "sample" is used in its broadest sense. A sample
suspected of containing nucleic acids encoding OXRE, or fragments
thereof, or OXRE itself, 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.
[0067] The terms "specific binding" and "specifically binding"
refer to that interaction between a protein or peptide and an
agonist, an antibody, or an antagonist. 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 containing 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.
[0068] The term "stringent conditions" refers to conditions which
permit hybridization between polynucleotides and the claimed
polynucleotides. Stringent conditions can be defined by salt
concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0069] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90%
free from other components with which they are naturally
associated.
[0070] A "substitution" refers to the replacement of one or more
amino acids or nucleotides by different amino acids or nucleotides,
respectively.
[0071] "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.
[0072] "Transformation" describes a process by which exogenous DNA
enters and changes 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, 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.
[0073] A "variant" of OXRE polypeptides refers to an amino acid
sequence that is altered by one or more amino acid residues. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example.
LASERGENE software (DNASTAR).
[0074] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to OXRE. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will 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 base. The presence of SNPs
may be indicative of, for example, a certain population, a disease
state, or a propensity for a disease state.
THE INVENTION
[0075] The invention is based on the discovery of new human
oxidoreductase molecules (OXRE), the polynucleotides encoding OXRE,
and the use of these compositions for the diagnosis, treatment, or
prevention of cell proliferative disorders including cancer,
endocrine, metabolic, reproductive, neurological,
autoimmune/inflammatory, and viral disorders.
[0076] Table 1 lists the Incyte clones used to assemble full length
nucleotide sequences encoding OXRE. Columns 1 and 2 show the
sequence identification numbers (SEQ ID NOs) of the polypeptide and
nucleotide sequences, respectively. Column 3 shows the clone IDs of
the Incyte clones in which nucleic acids encoding each OXRE were
identified, and column 4 shows the cDNA libraries from which these
clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA libraries. Clones for which cDNA libraries are
not indicated were derived from pooled cDNA libraries. The clones
in column 5 were used to assemble the consensus nucleotide sequence
of each OXRE and are useful as fragments in hybridization
technologies.
[0077] The columns of Table 2 show various properties of each of
the polypeptides of the invention: column 1 references the SEQ ID
NO; column 2 shows the number of amino acid residues in each
polypeptide; column 3 shows potential phosphorylation sites; column
4 shows potential glycosylation sites; column 5 shows the amino
acid residues comprising signature sequences and motifs; column 6
shows the identity of each polypeptide, and column 7 shows
analytical methods used to identify each polypeptide through
sequence homology and protein motifs.
[0078] The columns of Table 3 show the tissue-specificity and
diseases, disorders, or conditions associated with nucleotide
sequences encoding OXRE. The first column of Table 3 lists the
nucleotide SEQ ID NOs. Column 2 lists a fragment of each nucleotide
that is useful as described below. Column 3 lists tissue categories
which express OXRE as a fraction of total tissues expressing OXRE.
Column 4 lists diseases, disorders, or conditions associated with
those tissues expressing OXRE as a fraction of total tissues
expressing OXRE. Column S lists the vectors used to subclone each
cDNA library.
[0079] The columns of Table 4 show descriptions of the tissues used
to construct the cDNA libraries from which cDNA clones encoding
OXRE were isolated. Column 1 references the nucleotide SEQ ID NOs,
column 2 shows the cDNA libraries from which these clones were
isolated, and column 3 shows the tissue origins and other
descriptive information relevant to the cDNA libraries in column
2.
[0080] Fragments of the nucleotide sequences encoding OXRE, listed
in Table 3, Column 2, are useful, for example, in hybridization or
amplification technologies to identify SEQ ID NO:16-30 and to
distinguish between SEQ ID NO:16-30 and related polynucleotide
sequences. The polypeptides encoded by these fragments are useful,
for example, as immunogenic peptides.
[0081] The invention also encompasses OXRE variants. A preferred
OXRE variant is one which has at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the OXRE amino acid sequence, and which
contains at least one functional or structural characteristic of
OXRE.
[0082] The invention also encompasses polynucleotides which encode
OXRE. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising a sequence selected from the
group consisting of SEQ ID NO:16-30, which encodes OXRE.
[0083] The invention also encompasses a variant of a polynucleotide
sequence encoding OXRE. In particular, such a variant
polynucleotide sequence will have at least about 70%, more
preferably at least about 85%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding OXRE. 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: I &30 which has at
least about 70%, more preferably at least about 85%. and most
preferably at least about 95% polynucleotide sequence identity to a
nucleic acid sequence selected from the group consisting of SEQ ID
NO:16-30. Any one of the polynucleotide variants described above
can encode an amino acid sequence which contains at least one
functional or structural characteristic of OXRE.
[0084] 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 OXRE, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
sequence that could be made by selecting combinations based on
possible codon choices. These combinations are made in accordance
with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally occurring OXRE, and all such
variations are to be considered as being specifically
disclosed.
[0085] Although nucleotide sequences which encode OXRE and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring OXRE under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding OXRE 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 OXRE 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.
[0086] The invention also encompasses production of DNA sequences
which encode OXRE and OXRE 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 OXRE or any fragment thereof.
[0087] 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:16-30 and fragments thereof under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.) For example, stringent salt concentration will
ordinarily be less than about 750 mM NaCl and 75 mM trisodium
citrate, preferably less than about 500 mM NaCl and 50 mM trisodium
citrate, and most preferably less than about 250 mM NaCl and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic solvent, e.g., formamide, while high
stringency hybridization can be obtained in the presence of at
least about 35% formamide. and most preferably at least about 50%
formamide. Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0088] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 42.degree.
C., and most preferably of at least about 68.degree. C. In a
preferred embodiment, wash steps will occur at 25.degree. C. in 30
mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred
embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred
embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on
these conditions will be readily apparent to those skilled in the
art.
[0089] 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 1, SEQUENASE (US Biochemical, Cleveland Ohio), Taq
polymerase (Perkin-Elmer), thermostable T7 polymerase (Amersham
Pharmacia Biotech, Piscataway N.J.), 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 Hamilton MICROLAB 2200 (Hamilton. Reno Nev.), Peltier thermal
cycler 200 (PTC200; MJ Research, Watertown Mass.) and the ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using
either ABI 373 or 377 DNA sequencing systems (Perkin-Elmer), the
MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale
Calif.), 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 Biology, John Wiley & Sons, New York N.Y., unit 7.7;
Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley
VCH, New York N.Y., pp. 856-853.)
[0090] The nucleic acid sequences encoding OXRE 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. 19:3055-306).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries (Clontech, Palo Alto Calif.) 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
Mont.) 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.degree.
C. to 72.degree. C.
[0091] 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.
[0092] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g., GENOTYPER
and SEQUENCE NAVIGATOR, Perkin-Elmer), 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.
[0093] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode OXRE may be cloned in
recombinant DNA molecules that direct expression of OXRE, 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
OXRE.
[0094] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter OXRE-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.
[0095] In another embodiment, sequences encoding OXRE may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 7:215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 7:225-232.) Alternatively, OXRE itself or a
fragment thereof may be synthesized using chemical methods. For
example, peptide synthesis can be performed using various
solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995)
Science 269:202-204.) Automated synthesis may be achieved using the
ABI 43 IA peptide synthesizer (Perkin-Elmer). Additionally, the
amino acid sequence of OXRE, 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.
[0096] 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. 182:392421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See. e.g., Creighton, T. (1984) Proteins,
Structures and Molecular Pronerties, WH Freeman, New York N.Y.)
[0097] In order to express a biologically active OXRE, the
nucleotide sequences encoding OXRE 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 OXRE. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding OXRE. Such
signals include the ATG initiation codon and adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding OXRE 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.)
[0098] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding OXRE 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
N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current
Protocols in Molecular Biology, John Wiley & Sons, New York
N.Y., ch. 9, 13, and 16.)
[0099] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding OXRE. 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. The invention is not
limited by the host cell employed.
[0100] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding OXRE. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding OXRE can be achieved using a multifunctional E. coli
vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or pSPORT1
plasmid (Life Technologies). Ligation of sequences encoding OXRE
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 OXRE are needed, e.g. for the production of
antibodies, vectors which direct high level expression of OXRE may
be used. For example, vectors containing the strong, inducible T5
or T7 bacteriophage promoter may be used.
[0101] Yeast expression systems may be used for production of OXRE.
A number of vectors containing constitutive or inducible promoters,
such as alpha factor, alcohol oxidase, and PGH promoters, may be
used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors direct either the secretion or intracellular
retention of expressed proteins and enable integration of foreign
sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra; Grant et al. (1987) Methods Enzymol.
153:516-54; and Scorer, C. A. et al. (1994) Bio/Technology
12:181-184.)
[0102] Plant systems may also be used for expression of OXRE.
Transcription of sequences encoding OXRE may be driven viral
promoters, e.g., the 35S and 19S promoters of CaMV used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as
the small subunit of RUBISCO or heat shock promoters may be used.
(See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie,
R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991)
Results Probl. Cell Differ. 17:85-105.) These constructs can be
introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York
N.Y., pp. 191-196.)
[0103] 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 OXRE 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 OXRE in host cells. (See,
e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
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.
[0104] 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.)
[0105] For long term production of recombinant proteins in
mammalian systems, stable expression of OXRE in cell lines is
preferred. For example, sequences encoding OXRE 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.
[0106] 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.sup.- or apr.sup.-
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 or pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
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. 85:8047-8051.) Visible markers, e.g., anthocyanins,
green fluorescent proteins (GFP; Clontech), .beta. glucuronidase
and its substrate .beta.-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.)
[0107] 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 OXRE is inserted within a marker gene
sequence, transformed cells containing sequences encoding OXRE can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding OXRE 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.
[0108] In general, host cells that contain the nucleic acid
sequence encoding OXRE and that express OXRE 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.
[0109] Immunological methods for detecting and measuring the
expression of OXRE 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
OXRE 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 Minn., Sect. IV; Coligan, J. E. et al. (1997)
Current Protocols in Immunology, Greene Pub. Associates and
Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0110] 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 OXRE include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding OXRE, 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 Wis.), 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.
[0111] Host cells transformed with nucleotide sequences encoding
OXRE 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 OXRE may be designed to
contain signal sequences which direct secretion of OXRE through a
prokaryotic or eukaryotic cell membrane.
[0112] 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" 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.
[0113] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding OXRE 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 OXRE protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of OXRE 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-myc, and hemagglutinin
(HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the OXRE encoding sequence and the heterologous protein
sequence, so that OXRE may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra,
ch 10). A variety of commercially available kits may also be used
to facilitate expression and purification of fusion proteins.
[0114] In a further embodiment of the invention, synthesis of
radiolabeled OXRE may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract systems (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, preferably .sup.3"S-methionine.
[0115] Fragments of OXRE may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI 431
A peptide synthesizer (Perkin-Elmer). Various fragments of OXRE may
be synthesized separately and then combined to produce the full
length molecule.
[0116] Therapeutics
[0117] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of OXRE and
oxidoreductase molecules. In addition, the expression of OXRE is
closely associated with cell proliferation, cancer, inflammation
and immune response, nervous system tissues, and reproductive
tissues. Of particular note is the exclusive expression of SEQ ID
NO:30 in proliferating brain tissue. Therefore, OXRE appears to
play a role in cell proliferative disorders including cancer,
endocrine, metabolic, reproductive, neurological,
autoimmune/inflammatory, and viral disorders. In the treatment of
cell proliferative disorders including cancer, endocrine,
metabolic, reproductive, neurological, autoimmune/inflammatory, and
viral disorders associated with increased OXRE expression or
activity, it is desirable to decrease the expression or activity of
OXRE. In the treatment of the above conditions associated with
decreased OXRE expression or activity, it is desirable to increase
the expression or activity of OXRE.
[0118] Therefore, in one embodiment, OXRE 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 OXRE. Examples of such disorders include, but are not limited
to, a cell proliferative disorder, such as such as actinic
keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease, myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis,
primary thrombocythemia; a cancer, such as 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; an endocrine disorder
such as disorders of the hypothalamus and pituitary resulting from
lesions such as primary brain tumors, adenomas, infarction
associated with pregnancy, hypophysectomy, aneurysms, vascular
malformations, thrombosis, infections, immunological disorders, and
complications due to head trauma; disorders associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes
insipidus, Kallman's disease, Hand-Schuller-Christian disease,
Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and
dwarfism: disorders associated with hyperpituitarism including
acromegaly, giantism, and syndrome of inappropriate antidiuretic
hormone (ADH) secretion (SIADH) often caused by benign adenoma;
disorders associated with hypothyroidism including goiter,
myxedema, acute thyroiditis associated with bacterial infection,
subacute thyroiditis associated with viral infection, autoimmune
thyroiditis (Hashimoto's disease), and cretinism; disorders
associated with hyperthyroidism including thyrotoxicosis and its
various forms, Grave's disease, pretibial myxedema, toxic
multinodular goiter, thyroid carcinoma, and Plummer's disease;
disorders associated with hyperparathyroidism including Conn
disease (chronic hypercalemia); pancreatic disorders such as Type I
or Type II diabetes mellitus and associated complications;
disorders associated with the adrenals such as hyperplasia,
carcinoma, or adenoma of the adrenal cortex, hypertension
associated with alkalosis, amyloidosis, hypokalemia, Cushing's
disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome,
pheochromocytoma tumors, and Addison's disease; disorders
associated with gonadal steroid hormones such as: in women,
abnormal prolactin production, infertility, endometriosis,
perturbations of the menstrual cycle, polycystic ovarian disease,
hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea,
galactorrhea, hermaphroditism, hirsutism and virilization, breast
cancer, and, in post-menopausal women, osteoporosis; and, in men,
Leydig cell deficiency, male climacteric phase, and germinal cell
aplasia, hypergonadal disorders associated with Leydig cell tumors,
androgen resistance associated with absence of androgen receptors,
syndrome of 5 a-reductase, and gynecomastia; a metabolic disorder,
such as Addison's disease, cystic fibrosis, diabetes, fatty
hepatocirrhosis, galactosemia, goiter, hyperadrenalism,
hypoadrenalism, hyperparathyroidism, hypoparathyroidism,
hypercholesterolemia, hyperthyroidism, hypothyroidism
hyperlipidemia, hyperlipemia, lipid myopathies, obesity,
lipodystrophies, and phenylketonuria, congenital adrenal
hyperplasia, pseudovitamin D-deficiency rickets, cerebrotendinous
xanthomatosis, and coumarin resistance; a reproductive disorder
such as disorders of prolactin production, infertility, including
tubal disease, ovulatory defects, and endometriosis, disruptions of
the estrous cycle, disruptions of the menstrual cycle, polycystic
ovary syndrome, ovarian hyperstimulation syndrome, endometrial and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic
pregnancies, and teratogenesis; cancer of the breast, fibrocystic
breast disease, and galactorrhea; disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of the male breast, and gynecomastia;
a neurological disorder such as epilepsy, ischemic cerebrovascular
disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and
other extrapyramidal disorders, amyotrophic lateral sclerosis and
other motor neuron disorders, progressive neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases, bacterial and viral meningitis, brain
abscess, subdural empyema, epidural abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system disease; prion diseases including kuru,
Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal familial insomnia, nutritional and metabolic
diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other
developmental disorders of the central nervous system, cerebral
palsy, neuroskeletal disorders, autonomic nervous system disorders,
cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis and polymyositis, inherited, metabolic,
endocrine, and toxic myopathies; myasthenia gravis, periodic
paralysis; mental disorders including mood, anxiety, and
schizophrenic disorders; seasonal affective disorder (SAD);
akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia,
and Tourette's disorder; 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: and a viral disorder, such as viral infections, e.g.,
those caused by adenoviruses (acute respiratory disease,
pneumonia), arenaviruses (lymphocytic choriomeningitis),
bunyaviruses (Hantavirus), coronaviruses (pneumonia, chronic
bronchitis), hepadnaviruses (hepatitis), herpesviruses (herpes
simplex virus, varicella-zoster virus, Epstein-Barr virus,
cytomegalovirus), flaviviruses (yellow fever), orthomyxoviruses
(influenza), papillomaviruses (cancer), paramyxoviruses (measles,
mumps), picomoviruses (rhinovirus, poliovirus, coxsackie-virus),
polyomaviruses (BK virus, JC virus), poxviruses (smallpox),
reovirus (Colorado tick fever), retroviruses (human
immunodeficiency virus, human T lymphotropic virus), rhabdoviruses
(rabies), rotaviruses (gastroenteritis), and togaviruses
(encephalitis, rubella).
[0119] In another embodiment, a vector capable of expressing OXRE
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 OXRE including, but not limited to, those
described above.
[0120] In a further embodiment, a pharmaceutical composition
comprising a substantially purified OXRE 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 OXRE including, but not limited to, those provided
above.
[0121] In still another embodiment, an agonist which modulates the
activity of OXRE may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of OXRE including, but not limited to, those listed above.
[0122] In a further embodiment, an antagonist of OXRE may be
administered to a subject to treat or prevent a disorder associated
with increased expression or activity of OXRE. Examples of such
disorders include, but are not limited to, those described above.
In one aspect, an antibody which specifically binds OXRE may be
used directly as an antagonist or indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or
tissue which express OXRE.
[0123] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding OXRE may be administered
to a subject to treat or prevent a disorder associated with
increased expression or activity of OXRE including, but not limited
to, those described above.
[0124] 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.
[0125] An antagonist of OXRE may be produced using methods which
are generally known in the art. In particular, purified OXRE may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind OXRE. Antibodies
to OXRE 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 especially preferred for therapeutic use.
[0126] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with OXRE or with any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0127] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to OXRE have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, 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 and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of OXRE amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0128] Monoclonal antibodies to OXRE 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. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0129] 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.
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
OXRE-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. 88:10134-10137.)
[0130] 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. 86: 3833-3837; Winter, G. et al.
(1991) Nature 349:293-299.)
[0131] Antibody fragments which contain specific binding sites for
OXRE may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (See. e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0132] 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 OXRE and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering OXRE epitopes
is preferred, but a competitive binding assay may also be employed
(Pound, supra).
[0133] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for OXRE. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
OXRE-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple OXRE epitopes,
represents the average affinity, or avidity, of the antibodies for
OXRE. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular OXRE epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
L/mole are preferred for use in immunoassays in which the
OXRE-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 L/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of OXRE, preferably in active form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, DC; Liddell, J. E. and Cryer, A. (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York N.Y.).
[0134] 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 preferred for use in procedures requiring precipitation of
OXRE-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0135] In another embodiment of the invention, the polynucleotides
encoding OXRE, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding OXRE may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding OXRE. Thus, complementary molecules or
fragments may be used to modulate OXRE activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding OXRE.
[0136] 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. Methods which are well known to
those skilled in the art can be used to construct vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding OXRE. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
[0137] Genes encoding OXRE can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding OXRE. Such constructs
may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the absence of integration into the
DNA, such vectors may continue to transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may
last for a month or more with a non-replicating vector, and may
last even longer if appropriate replication elements are part of
the vector system.
[0138] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding OXRE. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. 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
Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0139] 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 OXRE.
[0140] 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.
[0141] 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 OXRE. 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.
[0142] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0143] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
[0144] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0145] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of OXRE, antibodies to OXRE, and mimetics,
agonists, antagonists, or inhibitors of OXRE. The compositions may
be administered alone or in combination with at least one other
agent, such as a stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs, or hormones.
[0146] The pharmaceutical 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, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0147] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing, Easton
Pa.).
[0148] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0149] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0150] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0151] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0152] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0153] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0154] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0155] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acids. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0156] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of OXRE, such
labeling would include amount, frequency, and method of
administration.
[0157] Pharmaceutical 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.
[0158] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, 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.
[0159] A therapeutically effective dose refers to that amount of
active ingredient, for example OXRE or fragments thereof,
antibodies of OXRE, and agonists, antagonists or inhibitors of
OXRE, 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 ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (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
LD.sub.5/ED.sub.50 ratio. Pharmaceutical 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 ED30 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.
[0160] 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 pharmaceutical 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.
[0161] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, 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.
[0162] Diagnostics
[0163] In another embodiment, antibodies which specifically bind
OXRE may be used for the diagnosis of disorders characterized by
expression of OXRE, or in assays to monitor patients being treated
with OXRE or agonists, antagonists, or inhibitors of OXRE.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for OXRE include methods which utilize the antibody and a label to
detect OXRE in human body fluids or in extracts of cells or
tissues. The antibodies may be used with or without modification,
and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A wide variety of reporter molecules, several of
which are described above, are known in the art and may be
used.
[0164] A variety of protocols for measuring OXRE, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of OXRE expression. Normal or
standard values for OXRE expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to OXRE under conditions suitable
for complex formation. The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of OXRE 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.
[0165] In another embodiment of the invention, the polynucleotides
encoding OXRE 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 quantitate gene
expression in biopsied tissues in which expression of OXRE may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of OXRE, and to
monitor regulation of OXRE levels during therapeutic
intervention.
[0166] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding OXRE or closely related molecules may be used
to identify nucleic acid sequences which encode OXRE. 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 (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding OXRE, allelic variants, or related
sequences.
[0167] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the OXRE 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:16-30 or from genomic
sequences including promoters, enhancers, and introns of the OXRE
gene.
[0168] Means for producing specific hybridization probes for DNAs
encoding OXRE include the cloning of polynucleotide sequences
encoding OXRE or OXRE 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 .sup.32O or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0169] Polynucleotide sequences encoding OXRE may be used for the
diagnosis of disorders associated with expression of OXRE. Examples
of such disorders include, but are not limited to, a cell
proliferative disorder, such as such as actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease, myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia; a cancer, such as 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; an endocrine disorder
such as disorders of the hypothalamus and pituitary resulting from
lesions such as primary brain tumors, adenomas, infarction
associated with pregnancy, hypophysectomy, aneurysms, vascular
malformations, thrombosis, infections, immunological disorders, and
complications due to head trauma; disorders associated with
hypopituitarism including hypogonadism, Sheehan syndrome, diabetes
insipidus, Kallman's disease, Hand-Schuller-Christian disease,
Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and
dwarfism; disorders associated with hyperpituitarism including
acromegaly, giantism, and syndrome of inappropriate antidiuretic
hormone (ADH) secretion (SIADH) often caused by benign adenoma;
disorders associated with hypothyroidism including goiter,
myxedema, acute thyroiditis associated with bacterial infection,
subacute thyroiditis associated with viral infection, autoimmune
thyroiditis (Hashimoto's disease), and cretinism; disorders
associated with hyperthyroidism including thyrotoxicosis and its
various forms, Grave's disease, pretibial myxedema, toxic
multinodular goiter, thyroid carcinoma, and Plummer's disease;
disorders associated with hyperparathyroidism including Conn
disease (chronic hypercalemia); pancreatic disorders such as Type I
or Type II diabetes mellitus and associated complications;
disorders associated with the adrenals such as hyperplasia,
carcinoma, or adenoma of the adrenal cortex, hypertension
associated with alkalosis, amyloidosis. hypokalemia, Cushing's
disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome,
pheochromocytoma tumors, and Addison's disease, disorders
associated with gonadal steroid hormones such as: in women,
abnormal prolactin production, infertiliry, 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 .alpha.-reductase, and gynecomastia; a metabolic
disorder, such as Addison's disease, cystic fibrosis, diabetes,
fatty hepatocirrhosis, galactosemia, goiter, hyperadrenalism,
hypoadrenalism, hyperparathyroidism, hypoparathyroidism,
hypercholesterolemia, hyperthyroidism, hypothyroidism
hyperlipidemia, hyperlipemia, lipid myopathies, obesity,
lipodystrophies, and phenylketonuria, congenital adrenal
hyperplasia, pseudovitamin D-deficiency rickets, cerebrotendinous
xanthomatosis, and coumarin resistance; a reproductive disorder
such as disorders of prolactin production, infertility, including
tubal disease, ovulatory defects, and endometriosis, disruptions of
the estrous cycle, disruptions of the menstrual cycle, polycystic
ovary syndrome, ovarian hyperstimulation syndrome, endometrial and
ovarian tumors, uterine fibroids, autoimmune disorders, ectopic
pregnancies, and teratogenesis; cancer of the breast, fibrocystic
breast disease, and galactorrhea; disruptions of spermatogenesis,
abnormal sperm physiology, cancer of the testis, cancer of the
prostate, benign prostatic hyperplasia, prostatitis, Peyronie's
disease, impotence, carcinoma of the male breast, and gynecomastia;
a neurological disorder such as epilepsy, ischemic cerebrovascular
disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and
other extrapyramidal disorders, amyotrophic lateral sclerosis and
other motor neuron disorders, progressive neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases, bacterial and viral meningitis, brain
abscess, subdural empycma, epidural abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system disease; prion diseases including kuru,
Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal familial insomnia, nutritional and metabolic
diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis,
enceplialotrigeminal syndrome, mental retardation and other
developmental disorders of the central nervous system, cerebral
palsy, neuroskeletal disorders, autonomic nervous system disorders,
cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis and polymyositis; inherited, metabolic,
endocrine, and toxic myopathies; myasthenia gravis, periodic
paralysis; mental disorders including mood, anxiety, and
schizophrenic disorders; seasonal affective disorder (SAD);
akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia,
and Tourette's disorder, 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; and a viral disorder, such as viral infections, e.g.,
those caused by adenoviruses (acute respiratory disease,
pneumonia), arenaviruses (lymphocytic choriomeningitis),
bunyaviruses (Hantavirus), coronaviruses (pneumonia, chronic
bronchitis), hepadnaviruses (hepatitis), herpesviruses (herpes
simplex virus, varicella-zoster virus, Epstein-Barr virus,
cytomegalovirus), flaviviruses (yellow fever), orthomyxoviruses
(influenza), papillomaviruses (cancer), paramyxoviruses (measles,
mumps), picornoviruses (rhinovirus, poliovirus, coxsackie-virus),
polyomaviruses (BK virus, JC virus), poxviruses (smallpox),
reovirus (Colorado tick fever), retroviruses (human
immunodeficiency virus, human T lymphotropic virus), rhabdoviruses
(rabies), rotaviruses (gastroenteritis), and togaviruses
(encephalitis, rubella). The polynucleotide sequences encoding OXRE
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 OXRE expression.
Such qualitative or quantitative methods are well known in the
art.
[0170] In a particular aspect, the nucleotide sequences encoding
OXRE may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding OXRE 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
quantitated 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 OXRE 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.
[0171] In order to provide a basis for the diagnosis of a disorder
associated with expression of OXRE, 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 OXRE, 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.
[0172] 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.
[0173] 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.
[0174] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding OXRE 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 OXRE, or a fragment of a
polynucleotide complementary to the polynucleotide encoding OXRE,
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 quantitation of
closely related DNA or RNA sequences.
[0175] Methods which may also be used to quantify the expression of
OXRE 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 an ELISA format
where the oligomer of interest is presented in various dilutions
and a spectrophotometric or colorimetric response gives rapid
quantitation.
[0176] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and 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, and to develop and monitor the activities of
therapeutic agents.
[0177] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweileret al. (1995) PCT application
WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0178] In another embodiment of the invention, nucleic acid
sequences encoding OXRE may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet.
15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B.
J. (1991) Trends Genet. 7:149-154.)
[0179] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp.
965-968.) Examples of genetic map data can be found in various
scientific journals or at the Online Mendelian Inheritance in Man
(OMIM) site. Correlation between the location of the gene encoding
OXRE on a physical chromosomal map and a specific disorder, or a
predisposition to a specific disorder, may help define the region
of DNA associated with that disorder. The nucleotide sequences of
the invention may be used to detect differences in gene sequences
among normal, carrier, and affected individuals.
[0180] 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 number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., ataxia-telangiectasia to 11q22-23. any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. (See, e.g., Gatti, R.
A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of
the subject invention may also be used to detect differences in the
chromosomal location due to translocation, inversion, etc., among
normal, carrier, or affected individuals.
[0181] In another embodiment of the invention, OXRE, 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 OXRE and the agent being tested may be
measured.
[0182] 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 WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate. The test
compounds are reacted with OXRE, or fragments thereof, and washed.
Bound OXRE is then detected by methods well known in the art.
Purified OXRE 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.
[0183] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding OXRE specifically compete with a test compound for binding
OXRE. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
OXRE.
[0184] In additional embodiments, the nucleotide sequences which
encode OXRE may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0185] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever.
[0186] The disclosures of all patents, applications, and
publications mentioned above and below, in particular U.S. Ser. No.
[Attorney Docket No. PF-0610 P. filed Oct. 6, 1998], U.S. Ser. No.
[Attorney Docket No. PF-0616 P, filed Dec. 2, 1998], and U.S. Ser.
No. 60/123,911, are hereby expressly incorporated by reference.
EXAMPLES
[0187] I. Construction of cDNA Libraries
[0188] RNA was purchased from Clontech or isolated from tissues
described in Table 4. Some tissues were homogenized and lysed in
guanidinium isothiocyanate, while others were homogenized and lysed
in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a monophasic solution of phenol and guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCl
cushions or extracted with chloroform. RNA was precipitated from
the lysates with either isopropanol or sodium acetate and ethanol,
or by other routine methods.
[0189] 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 Calif.), 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 Tex.).
[0190] 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 SEPMACRYL S1000, 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), or pINCY (Incyte
Pharmaceuticals, Palo Alto Calif.). Recombinant plasmids were
transformed into competent E. coli cells including XL1-Blue,
XL1-BlueMRF, or SOLR from Stratagene or DH5.alpha., DH10B, or
ElectroMAX DH10B from Life Technologies.
[0191] II. Isolation of cDNA Clones
[0192] Plasmids 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.degree. C.
[0193] 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
Oreg.) and a Fluoroskan II fluorescence scanner (Labsystems Oy,
Helsinki, Finland).
[0194] III. Sequencing and Analysis
[0195] cDNA sequencing reactions were processed using standard
methods or high-throughput instrumentation such as the ABI CATALYST
800 (Perkin-Elmer) 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 (Perkin-Elmer). 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 (Perkin-Elmer) in
conjunction with standard ABI protocols and base calling software;
or other sequence analysis systems known in the art. Reading frames
within the cDNA sequences were identified using standard methods
(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA
sequences were selected for extension using the techniques
disclosed in Example V.
[0196] The polynucleotide sequences derived from cDNA sequencing
were assembled and analyzed using a combination of software
programs which utilize algorithms well known to those skilled in
the art. Table 5 summarizes the tools, programs, and algorithms
used and provides applicable descriptions, references, and
threshold parameters. The first column of Table 5 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, the greater the homology between two sequences).
Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software Engineering, South San Francisco Calif.) and LASERGENE
software (DNASTAR). Polynucleotide and polypeptide sequence
alignments were generated using the 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.
[0197] The polynucleotide sequences were validated by removing
vector, linker, and polyA sequences and by masking ambiguous bases,
using algorithms and programs based on BLAST, dynamic programing,
and dinucleotide nearest neighbor analysis. The sequences were then
queried against a selection of public databases such as the GenBank
primate, rodent, mammalian, vertebrate, and eukaryote databases,
and BLOCKS to acquire annotation using programs based on BLAST,
FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and 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
amino acid sequences, and these full length sequences were
subsequently analyzed by querying against databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS,
Prosite, 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, e.g.,
Eddy, S. R. (1996) Curr. Opin. Str. Biol. 6:361-365.)
[0198] The programs described above for the assembly and analysis
of full length polynucleotide and amino acid sequences were also
used to identify polynucleotide sequence fragments from SEQ ID
NO:16-30. Fragments from about 20 to about 4000 nucleotides which
are useful in hybridization and amplification technologies were
described in The Invention section above.
[0199] IV. Northern Analysis
[0200] 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.)
[0201] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ (Incyte Pharmaceuticals). 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:
% sequence identity.times.% maximum BLAST score/100
[0202] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0203] The results of northern analyses are reported as a
percentage distribution of libraries in which the transcript
encoding OXRE occurred. Analysis involved the categorization of
cDNA libraries by organ/tissue and disease. The organ/tissue
categories included cardiovascular, dermatologic, developmental,
endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal,
nervous, reproductive, and urologic. The disease/condition
categories included cancer, inflammation/trauma, cell
proliferation, neurological, and pooled. For each category, the
number of libraries expressing the sequence of interest was counted
and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or
condition-specific expression are reported in Table 3.
[0204] V. Extension of OXRE Encoding Polynucleotides
[0205] The full length nucleic acid sequences of SEQ ID NO:16-30
were 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, 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.degree. C. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0206] 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.
[0207] 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 Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4, and
.beta.-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech), LONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair PCI A and PCI B: Step 1: 94.degree. C., 3 min; Step 2:
94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times,
Step 6: 68.degree. C., 5 min; Step 7: storage at 4.degree. C. In
the alternative, the parameters for primer pair T7 and SK+ were as
follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15
sec; Step 3: 57.degree. C., 1 min. Step 4: 68.degree. C., 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C.,
5 min; Step 7: storage at 4.degree. C.
[0208] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1.times. TE
and 0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Coming Costar, Acton Mass.), 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 quantity the concentration of DNA. A 5 .mu.l to
10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a 1% agarose mini-gel to determine which
reactions were successful in extending the sequence.
[0209] The extended nucleotides were desalted and concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison Wis.), and
sonicated or sheared prior to religation into pUC 18 vector
(Amersham Pharmacia Biotech). For shotgun sequencing, the digested
nucleotides were separated on low concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar
ACE (Promega). Extended clones were religated using T4 ligase (New
England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction site overhangs, and transfected into competent
E. coli cells. Transformed cells were selected on
antibiotic-containing media, individual colonies were picked and
cultured overnight at 37.degree. C. in 384-well plates in
LB/2.times. carb liquid media.
[0210] The cells were lysed, and DNA was amplified by PCR using Taq
DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C. 2 min; Step 5: steps 2, 3, and 4 repeated 29
times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
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% dimethysulphoxide (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 (Perkin-Elmer).
[0211] In like manner, the nucleotide sequences of SEQ ID NO:16-30
are used to obtain 5' regulatory sequences using the procedure
above, oligonucleotides designed for such extension, and an
appropriate genomic library.
[0212] VI. Labeling and Use of Individual Hybridization Probes
[0213] Hybridization probes derived from SEQ ID NO:16-30 are
employed to screen cDNAs, genomic DNAs, or mRNAs. Although the
labeling of oligonucleotides, consisting of about 20 base pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham
Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN,
Boston Mass.). The labeled oligonucleotides are substantially
purified using a SEPHADEX G-25 superfine size exclusion dextran
bead column (Amersham Pharmacia Biotech). An aliquot containing
10.sup.7 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, Xbal, or Pvu II (DuPont NEN).
[0214] The DNA from each digest is fractionated on a 0.7% agarose
gel and transferred to nylon membranes (Nytran Plus, Schleicher
& Schuell, Durham N.H.). Hybridization is carried out for 16
hours at 40.degree. C. To remove nonspecific signals, blots are
sequentially washed at room temperature under increasingly
stringent conditions up to 0.1.times. saline sodium citrate and
0.5% sodium dodecyl sulfate. Hybridization patterns are visualized
using autoradiography and compared.
[0215] VII. Microarrays
[0216] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array 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 by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0217] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE software (DNASTAR).
Full-length cDNAs, ESTs, or fragments thereof corresponding to one
of the nucleotide sequences of the present invention, or selected
at random from a cDNA library relevant to the present invention,
are arranged on an appropriate substrate, e.g., a glass slide. The
cDNA is fixed to the slide using, e.g., UV cross-linking followed
by thermal and chemical treatments and subsequent drying. (See,
e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et
al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared
and used for hybridization to the elements on the substrate. The
substrate is analyzed by procedures described above.
[0218] VIII. Complementary Polynucleotides
[0219] Sequences complementary to the OXRE-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring OXRE. 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 OXRE. 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 OXRE-encoding transcript.
[0220] IX. Expression of OXRE
[0221] Expression and purification of OXRE is achieved using
bacterial or virus-based expression systems. For expression of OXRE
in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21 (DE3).
Antibiotic resistant bacteria express OXRE upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of OXRE
in eukaryotic cells is achieved by infecting insect or mammalian
cell lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding OXRE by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera 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.)
[0222] In most expression systems, OXRE is synthesized as a fusion
protein with, e.g., glutathione S-transferase (GST) or a peptide
epitope tag, such as FLAG or 6-His, permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from
crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma
japonicum, enables the purification of fusion proteins on
immobilized glutathione under conditions that maintain protein
activity and antigenicity (Amersham Pharmacia Biotech). Following
purification, the GST moiety can be proteolytically cleaved from
OXRE 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 OXRE obtained by these methods can
be used directly in the following activity assay.
[0223] X. Demonstration of OXRE Activity
[0224] For purposes of example, an assay demonstrating the activity
of a short-chain alcohol dehydrogenase is described. Essentially
the same method is used for other types of oxidoreductases, with
suitable substitution of the substrate and electron acceptor. OXRE
activity is demonstrated by the oxidation of NADPH to NADP in the
presence of substrate (Kunau and Dommes (1978) Eur. J. Biochem.
91:533-544). Substrates include, but are not limited to,
all-trans-retinaldehyde and cis-4-dienoyl-CoA. OXRE is preincubated
for 10 minutes at 37.degree. C. in 60 .mu.M potassium phosphate (pH
7.4), 125 nM NADPH, and 0.2 .mu.M CoA (coenzyme A). The reaction is
initiated by addition of the appropriate substrate (12.5 to 150
.mu.M final concentration). The change in absorbance of the
reaction at 340 nm, due to the oxidation of NADPH to NADP, is
measured using a spectrophotometer at 23.degree. C. Units of OXRE
activity are expressed as .mu.moles of NADP formed per minute. A
reaction lacking OXRE is used as a negative control.
[0225] Alternatively, OXRE activity is assayed by measuring the
reduction of insulin. Aliquots of OXRE are preincubated at
37.degree. C. for 20 min with 2 .mu.l of 50 mM Hepes, pH 7.6, 100
pg/ml bovine serum albumin, and 2 mM DTT in a total volume of 70
.mu.l. Then, 40 .mu.l of a reaction mixture composed of 200 .mu.l
of Hepes (1 M), pH 7.6, 40 .mu.l of EDTA (0.2 M), 40 .mu.l of NADPH
(40 mg/ml), and 500 .mu.l of insulin (10 mg/ml) is added. The
reaction is initiated with the addition of 10 .mu.l of thioredoxin
reductase from calf thymus (3.0 A412 unit), and incubation is
continued for 20 min at 37.degree. C. The reaction rate is followed
by monitoring the oxidation of NADPH at 412 nM. The oxidation of
NADPH is proportional to the amount of insulin reduction.
[0226] XI. Functional Assays
[0227] OXRE function is assessed by expressing the sequences
encoding OXRE 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 Calif.), both of
which contain the cytomegalovirus promoter. 5-10 .mu.g of
recombinant vector are transiently transfected into a human cell
line, preferably of endothelial or hematopoietic origin, using
either liposome formulations or electroporation. 1-2 .mu.g of an
additional plasmid containing sequences encoding a marker protein
are co-transfected. Expression of a marker protein provides a means
to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA expression from the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular
properties. FCM detects and quantifies the uptake of fluorescent
molecules that diagnose events preceding or coincident with cell
death. These events include changes in nuclear DNA content as
measured by staining of DNA with propidium iodide; changes in cell
size and granularity as measured by forward light scatter and 90
degree side light scatter; down-regulation of DNA synthesis as
measured by decrease in bromodeoxyuridine uptake; alterations in
expression of cell surface and intracellular proteins as measured
by reactivity with specific antibodies; and alterations in plasma
membrane composition as measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface.
Methods in flow cytometry are discussed in Ormerod, M. G. (1994)
Flow Cytometry, Oxford, New York N.Y.
[0228] The influence of OXRE on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding OXRE 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 N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding OXRE and other genes of interest can be
analyzed by northern analysis or microarray techniques.
[0229] XII. Production of OXRE Specific Antibodies
[0230] OXRE substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488-495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0231] Alternatively, the OXRE 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 15 residues in length are synthesized using an ABI
431 A peptide synthesizer (Perkin-Elmer) 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-KLH complex in complete Freund's
adjuvant. Resulting antisera are tested for antipeptide activity
by, for example, binding the peptide to plastic, blocking with 1%
BSA, reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
[0232] XIII. Purification of Naturally Occurring OXRE Using
Specific Antibodies
[0233] Naturally occurring or recombinant OXRE is substantially
purified by immunoaffinity chromatography using antibodies specific
for OXRE. An immunoaffinity column is constructed by covalently
coupling anti-OXRE 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.
[0234] Media containing OXRE are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of OXRE (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/OXRE 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 OXRE is collected.
[0235] XIV. Identification of Molecules Which Interact with
OXRE
[0236] OXRE, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
OXRE, washed, and any wells with labeled OXRE complex are assayed.
Data obtained using different concentrations of OXRE are used to
calculate values for the number, affinity, and association of OXRE
with the candidate molecules.
[0237] 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 specific preferred 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.
1TABLE 1 Protein Nucleotide SEQ ID NO: SEQ ID NO: Clone ID Library
Fragments 1 16 000746 U937NOT01 000746H, 000746X777, and 002067R1
(U937NOT01), 1304321T1 (PLACNOT02), 967947R1 (BRSTNOT05),
SAPA01383F1, SXAA00973D1 2 17 2472577 THP1NOT03 755151R1
(BRAITUT02), 2472577H1 (THP1NOT03), 2476593F6 (SMCANOT01) 3 18
2160405 ENDCNOT02 1579324T6 (DUODNOT01), 1929782F6 (COLNTUT03),
1984568R6 (LUNGAST01), 1984568T6 (LUNGAST01), 2160405H1
(ENDCNOT02), 3088049F6 (HEAONOT03), SBMA01335F1 4 19 2591695
LUNGNOT22 1293773H1 (PGANNOT03), 1981831H1 (LUNGTUT03), 2591695H1
(LUNGNOT22), 2599633T6 (UTRSNOT10), 3079232H1 (BRAIUNT01),
3388467H1 (LUNGTUT17), 3728027F6 (SMCCNON03) 5 20 474100 MMLR1DT01
474100H1 (MMLR1DT01), 831142H1 and 1543031T1 (PROSTUT04), 1579546F6
(DUODNOT01), 1833969T6 (BRAINON01), 2360323H1 (LUNGFET05),
2571957H1 (HIPOAZT01), 3584211F6 (293TF4T01), SAIA02772F1 6 21
1304767 PLACNOT02 737411X28R1 (TONSNOT01), 1304767H1 (PLACNOT02),
1429123T1 (SINTBST01), 1458173R1 (COLNFET02), 1594353T6 (BRAINOT14)
7 22 1465978 PANCTUT02 872915R1 (LUNGAST01), 1465978F6 and
1465978H1 (PANCTUT02), 1865790F6 (PROSNOT19), 2062093R6 and
2132634T6 (OVARNOT03), 2232155F6 (PROSNOT16), 2521685H1
(BRAITUT21), 3037185H1 (SMCCNOT02) 8 23 1635966 UTRSNOT06 108553F1
(AMLBNOT01), 816384R1 (OVARTUT01), 1635966H1 (UTRSNOT06), 3076973H1
(BONEUNT01), SBBA03935F1, SBBA00704F1, SBBA05518F1 9 24 1638410
UTRSNOT06 986854R1 (LVENNOT03), 1297217T1 (BRSTNOT07), 1442549F1
(THYRNOT03), 1542919R1 (PROSTUT04), 1621602F6 (BRAITUT13),
1638410H1 (UTRSNOT06), 1867420F6 (SKINBIT01), 3721081H1
(PENCNOT10), 3770890H1 (BRSTNOT25) 10 25 1743409 HIPONON01 190557F1
and 190557R1 (SYNORAB01), 1743409H1 and 1743409R6 (HIPONON01),
3725583H1 (BRSTNOT23) 11 26 1803830 SINTNOT13 1611528T6
(COLNTUT06), 1803830H1, 1803830T6, 1803830X12F1 and 1803830X15F1
(SINTNOT13) 12 27 1867333 SKINBIT01 068790F1 and 068790R1
(HUVESTB01), 1209587R1 (BRSTNOT02), 1498674F6 (SINTBST01),
1519693F6 and 1520517F1 (BLADTUT04), 1867333H1 (SKINBIT01),
1958915H1 (CONNNOT01), 2537783H1 (BONRTUT01), 2638196F6
(BONTNOT01), 3039056H1 (BRSTNOT16), 4051183H1 (SINTNOT18),
4228470H1 (BRAMDIT01) 13 28 2906094 THYMNOT05 1442945F6
(THYRNOT03), 1711329X17C1 and 1711654X21C1 (PROSNOT16), 2309777H1
(NGANNOT01), 2906094H1 (THYMNOT05) 14 29 3294314 TLYJINT01
1528021F1 (UCMCL5T01), 2207228T6 (SINTFET03), 3294314H1
(TLYJINT01), 3333445F6 (BRAIFET01) 15 30 4940951 BRAIFEN03
4940951F6, 4940951H1, and 4940951T6 (BRAIFEN03)
[0238]
2TABLE 2 Potential Potential Protein Amino Acid Phosphorylation
Glycosylation Analytical SEQ ID NO: Residues Sites Sites Signature
Sequences Identification Methods 1 280 S68 T119 S128 Thioredoxin
domain: Thioredoxin Motifs S247 T257 S92 R28-E131 PFAM S209 S221
T257 Thioredoxin family PROFILESCAN T277 active site: P36-V78 2 166
S26 S56 S136 S150 Q2-C162 C1-tetrahydrofolate Motifs T158
dehydrogenase BLAST (GI901850) BLOCKS PRINTS PFAM 3 319 S61 S67
S111 T167 V9-D283 Lambda crystallin Motifs S216 S218 S250
(GI164905) BLAST PFAM BLOCKS 4 318 S140 T191 Y149
3-Oxo-5-alpha-steroid Motifs dehydrogenase BLAST (GI401056) 5 330
T158 S247 T253 N204 Transmembrane dTDP-6-deoxy-L- BLAST, HMM, S294
S203 S239 region: F117-F136 mannose dehydrogenase MOTIFS, GI
1314582 PRINTS 6 266 T60 T201 T235 N134 Short-chain Glucose
dehydrogenase BLAST, T257 T72 dehydrogenase: GI 216268 PFAM, HMM,
K10-P186 MOTIFS, G212-V241 BLOCKS, PRINTS 7 302 T79 S131 S135
short-chain Retinal-short-chain BLAST, T222 T72 T106 dehydrogenase:
dehydrogenase PFAM, HMM, T154 T190 T297 E39-T216 GI 3450828 MOTIFS,
BLOCKS, PRINTS 8 300 S33 S286 T52 S95 Short-chain Retinal
short-chain BLAST, PFAM T198 T267 S103 dehydrogenase: dehydrogenase
HMM, T151 S187 E37-G224 GI 3450830 MOTIFS, Signal Peptide: BLOCKS,
M1-S18 PRINTS 9 613 T87 S116 S118 N186 N350 Pyridine nucleotide-
Putative ferredoxin BLAST, S213 S268 T512 N468 disulphide reductase
MOCF PFAM, HMM, S519 S530 S532 oxidoreductase: GI 3411185 MOTIFS,
T534 S27 T87 S149 F134-Q523 BLOCKS, T190 T229 T263 PRINTS S376 S461
Y95 10 325 T150 T154 S268 T6 N243 Short-chain Similar to the BLAST,
T106 S146 Y167 dehydrogenase: insect-type alcohol PFAM, HMM,
A53-N243, S194-R222 dehydrogenase MOTIFS, Transmembrane GI 1125838
BLOCKS, region: F18-L37 PRINTS 11 421 T119 T126 S169
Gamma-Butyrobetaine BLAST, S228 T406 T108 hydroxylase MOTIFS T202
T413 Y247 GI 3746805 12 610 S484 T46 S151 N271 Pyridine nucleotide-
Pyridine nucleotide- HMM, PFAM, S241 T397 T414 disulphide
disulphide MOTIFS S444 T449 T466 oxidoreductase: oxidoreductase
S492 T564 S335 V70-E341 T380 T488 S551 Signal Peptide: M1-C18 13
415 S33 S101 S165 N186 Acyl CoA Acyl CoA reductase BLAST, T318 S175
dehydrogenase: (fadE9) PFAM, HMM, L41-R410 GI 2911026 MOTIFS,
BLOCKS 14 274 S61 S108 T109 Delta 1-pyrroline-5- Pyrroline-5-
BLAST, S259 S7 T44 T216 carboxylate carboxylate reductase PFAM,
HMM, T265 reductase: R9-A256 GI 189498 MOTIFS, BLOCKS 15 283 S83
S28 S90 S129 N114 Aldehyde ferredoxin Aldehyde ferrodoxin HMM,
BLOCKS S137 S252 T46 oxidoreductase: oxidoreductase S239 S244
P102-N114 Transmembrane region: F260-I279
[0239]
3TABLE 3 Nucleotide Useful Tissue Expression Disease or Condition
SEQ ID NO: Fragment (Fraction of Total) (Fraction of Total) Vector
16 112-147 Cell proliferative (0.68) pBLUESCRIPT
Inflammation/immune (0.26) 17 189-218 Cardiovascular (0.500) Cancer
(0.625) Hematopoietic/Immune (0.125) Inflammation (0.250)
Musculoskeletal (0.125) 18 597-626 Reproductive (0.225) Cancer
(0.620) Gastrointestinal (0.197) Inflammation (0.254)
Hematopoietic/Immune (0.127) 19 57-86 Gastrointestinal (0.217)
Cancer (0.652) Cardiovascular (0.174) Inflammation (0.217)
Reproductive (0.130) 20 1028-1072 Nervous (0.202) Cancer (0.395)
PSPORT1 Reproductive (0.177) Inflammation (0.290) Gastrointestinal
(0.129) Cell proliferative (0.161) 21 651-695 Nervous (0.286)
Cancer (0.393) pINCY Hematopoietic/Immune (0.143) Cell
proliferative (0.214) Reproductive (0.143) Inflammation (0.214) 22
619-663 Reproductive (0.296) Cancer (0.526) pINCY Gastrointestinal
(0.185) Cell proliferative (0.185) Nervous (0.141) Inflammation
(0.185) 23 1034-1078 Reproductive (0.211) Cancer (0.469) pINCY
Gastrointestinal (0.203) Inflammation (0.289) Hematopoietic/Immune
(0.148) Cell proliferative (0.180) 24 768-812 Reproductive (0.306)
Cancer (0.500) pINCY Cardiovascular (0.139) Inflammation (0.250)
Nervous (0.139) Cell proliferative (0.139) 25 999-1043 Reproductive
(0.365) Cancer (0.500) PSPORT1 Cardiovascular (0.115) Inflammation
(0.231) Gastrointestinal (0.096) Cell proliferative (0.173) 26
642-686 Reproductive (0.500) Cancer (0.542) pINCY Gastrointestinal
(0.125) Cell proliferative (0.167) Developmental (0.083)
Inflammation (0.167) 27 56-100 Reproductive (0.301) Cancer (0.540)
pINCY Gastrointestinal (0.221) Inflammation (0.248) Nervous (0.106)
Cell proliferative (0.097) 28 1258-1302 Reproductive (0.333) Cancer
(0.583) pINCY Nervous (0.194) Cell proliferative (0.194)
Hematopoietic/Immune (0.139) Inflammation (0.194) 29 57-101
Developmental (0.214) Cancer (0.500) pINCY Nervous (0.214) Cell
proliferative (0.429) Hematopoietic/Immune (0.143) Inflammation
(0.143) 30 35-79 Nervous (1.000) Cell proliferative (1.000)
pINCY
[0240]
4TABLE 4 Polynucleotide SEQ ID NO: Library Library Comment 16
U937NOT01 The U-937 cDNA library, U937NOT01, was constructed at
Stratagene using RNA isolated from the U937 monocyte-like cell
line. This cell line (ATCC CRL1593) was established by C. Sundstrom
and K. Nilsson in 1974 from cells obtained from the pleural
effusion of a 37-year-old Caucasian male with diffuse histiocytic
lymphoma. 17 THP1NOT03 Library was constructed using RNA isolated
from untreated THP-1 cells. THP-1 (ATCC TIB 202) is a human
promonocyte cell line derived from the peripheral blood of a
1-year-old Caucasian male with acute monocytic leukemia. 18
ENDCNOT02 Library was constructed using RNA isolated from dermal
microvascular endothelial cells removed from a 30-year-old
Caucasian female. 19 LUNGNOT22 Library was constructed using RNA
isolated from lung tissue removed from a 58-year-old Caucasian
female. The tissue sample used to construct this library was found
to have tumor contaminant upon microscopic examination. Pathology
for the associated tumor tissue indicated a caseating granuloma.
Family history included congestive heart failure, breast cancer,
secondary bone cancer, acute myocardial infarction and
atherosclerotic coronary artery disease. 20 MMLR1DT01 Library was
constructed using RNA isolated from adherent mononuclear cells,
which came from a pool of male and female donors. The cells were
cultured for 24 hours following Ficoll Hypaque centrifugation. 21
PLACNOT02 Library was constructed using RNA isolated from the
placental tissue of a Hispanic female fetus, who was prematurely
delivered at 21 weeks' gestation. 22 PANCTUT02 Library was
constructed using RNA isolated from pancreatic tumor tissue removed
from a 45-year-old Caucasian female during radical
pancreaticoduodenectomy. Pathology indicated a grade 4 anaplastic
carcinoma. Family history included benign hypertension,
hyperlipidemia and atherosclerotic coronary artery disease. 23
UTRSNOT06 Library was constructed using RNA isolated from
myometrial tissue removed from a 50-year-old Caucasian female
during a vaginal hysterectomy. Pathology indicated residual
atypical complex endometrial hyperplasia. Pathology for the
associated tissue removed during dilation and curettage indicated
fragments of atypical complex hyperplasia and a single microscopic
focus suspicious for grade 1 adenocarcinoma. Patient history
included benign breast neoplasm, hypothyroid disease, polypectomy,
and arthralgia. Family history included cerebrovascular disease,
atherosclerotic coronary artery disease, hyperlipidemia, and
chronic hepatitis. 24 UTRSNOT06 Library was constructed using RNA
isolated from myometrial tissue removed from a 50-year-old
Caucasian female during a vaginal hysterectomy. Pathology indicated
residual atypical complex endometrial hyperplasia. Pathology for
the associated tissue removed during dilation and curettage
indicated fragments of atypical complex hyperplasia and a single
microscopic focus suspicious for grade 1 adenocarcinoma. Patient
history included benign breast neoplasm, hypothyroid disease,
polypectomy, and arthralgia. Family history included
cerebrovascular disease, atherosclerotic coronary artery disease,
hyperlipidemia, and chronic hepatitis. 25 HIPONON01 This normalized
hippocampus library was constructed from 1.13 million independent
clones from a hippocampus library. RNA was isolated from the
hippocampus tissue of a 72-year-old Caucasian female who died from
an intracranial bleed. Patient history included nose cancer,
hypertension, and arthritis. The normalization and hybridization
conditions were adapted from Soares et al. (PNAS (1994) 91: 9928).
26 SINTNOT13 Library was constructed using RNA isolated from ileum
tissue obtained from a 25-year-old Asian female during a partial
colectomy and temporary ileostomy. Pathology indicated moderately
active chronic ulcerative colitis, involving colonic mucosa from
the distal margin to the ascending colon. Family history included
hyperlipidemia, depressive disorder, malignant cervical neoplasm,
viral hepatitis A, and depressive disorder. 27 SKINBIT01 Library
was constructed using RNA isolated from diseased skin tissue of the
left lower leg. Patient history included erythema nodosum of the
left lower leg. 28 THYMNOT05 Library was constructed using RNA
isolated from thymus tissue removed from a 3-year-old Hispanic male
during thymectomy and closure of a patent ductus arteriosus. The
patient presented with severe pulmonary stenosis and cyanosis.
Patient history included cardiac catheterization, echocardiogram,
and corrective cardiac surgeries. 29 TLYJINT01 Library was
constructed using RNA isolated from a Jurkat cell line derived from
the T cells of a male. The cells were treated for 18 hours with 50
ng/ml phorbol ester and 1 .mu.M calcium ionophore. Patient history
included acute T-cell leukemia. 30 BRAIFEN03 This normalized fetal
brain tissue library was constructed from 3.26 million independent
clones from a fetal brain library. Starting RNA was made from brain
tissue removed from a Caucasian male fetus with a hypoplastic left
heart stillborn after 23 weeks' gestation. The library was
normalized in two rounds (with 48 hour reannealing hybridizations)
using conditions adapted from Soares et al. (PNAS (1994) 91: 9228)
and Bonaldo et al. (Genome Research 6 (1996): 791).
[0241]
5TABLE 5 Program Description Reference Parameter Threshold ABI A
program that removes vector sequences and masks Perkin-Elmer
Applied Biosystems, FACTURA ambiguous bases in nucleic acid
sequences. Foster City, CA. ABI/ A Fast Data Finder useful in
Perkin-Elmer Applied Biosystems, Mismatch <50% PARACEL comparing
and annotating amino Foster City, CA; FDF acid or nucleic acid
sequences. Paracel Inc., Pasadena, CA. ABI A program that assembles
nucleic acid sequences. Perkin-Elmer Applied Biosystems,
AutoAssembler Foster City, CA. BLAST A Basic Local Alignment Search
Tool useful in Altschul, S.F. et al. (1990) ESTs: Probability
sequence similarity search for amino acid and nucleic J. Mol. Biol.
215: 403-410; value = 1.0E-8 acid sequences. BLAST includes five
functions: Altschul, S.F. et al. (1997) or less; blastp, blastn,
blastx, tblastn, and tblastx. Nucleic Acids Res. 25: 3389-3402.
Full Length sequences: Probability value = 1.0E-10 or less FASTA A
Pearson and Lipman algorithm that searches for Pearson, W. R. and
ESTs: fasta E similarity between a query sequence and a group of D.
J. Lipman (1988) Proc. Natl. value = 1.06E-6; sequences of the same
type. FASTA comprises as Acad Sci. USA 85: 2444-2448; Assembled
ESTs: fasta least five functions: fasta, tfasta, fastx, tfastx, and
Pearson, W. R. (1990) Methods Enzymol. 183: 63-98; Identity = 95%
or ssearch. and Smith, T. F. and M. S. Waterman (1981) greater and
Adv. Appl. Math. 2: 482-489. Match length = 200 bases or greater;
fastx E value = 1.0E-8 or less; Full Length sequences: fastx score
= 100 or greater BLIMPS A BLocks IMProved Searcher that matches a
Henikoff, S. and J. G. Henikoff, Nucleic Score = 1000 or sequence
against those in BLOCKS and PRINTS Acids Res., 19: 6565-72, 1991.
J. G. Henikoff greater; Ratio of databases to search for gene
families, and S. Henikoff (1996) Methods Score/Strength = 0.75
sequence homology, and structural Enzymol. 266: 88-105; and
Attwood, T. K. et or larger; and fingerprint regions. al. (1997) J.
Chem. Inf. Comput. Sci. 37: 417-424. Probability value = 1.0E-3 or
less PFAM A Hidden Markov Models-based application useful for
Krogh, A. et al. (1994) J. Mol. Biol. Score = 10-50 bits, protein
family search. 235: 1501-1531; Sonnhammer, E. L. L. et al.
depending on (1988) Nucleic Acids Res. 26: 320-322. individual
protein families ProfileScan An algorithm that searches for
structural and Gribskov, M. et al. (1988) CABIOS 4: 61-66; Score =
4.0 or greater sequence motifs in protein sequences that match
Gribskov, M. et al. (1989) Methods sequence patterns defined in
Prosite. Enzymol. 183: 146-159; Bairoch, A. et al. (1997) Nucleic
Acids Res. 25: 217-221. Phred A base-calling algorithm that
examines automated Ewing, B. et al. (1998) Genome Res. 8: 175-185;
sequencer traces with high sensitivity and probability. Ewing, B.
and P. Green (1998) Genome Res. 8: 186-194. Phrap A Phils Revised
Assembly Program including Smith, T. F. and M. S. Waterman (1981)
Adv. Score = 120 or greater; SWAT and CrossMatch, programs based on
efficient Appl. Math. 2: 482-489; Smith, T. F. and Match length =
implementation of the Smith-Waterman algorithm, M. S. Waterman
(1981) J. Mol. Biol. 147: 195-197; 56 or greater useful in
searching sequence homology and and Green, P., University of
assembling DNA sequences. Washington, Seattle, WA. Consed A
graphical tool for viewing and editing Phrap Gordon, D. et al.
(1998) Genome Res. 8: 195-202. assemblies. SPScan A weight matrix
analysis program that scans protein Nielson, H. et al. (1997)
Protein Engineering Score = 5 or greater sequences for the presence
of secretory signal 10: 1-6; Claverie, J. M. and S. Audic (1997)
peptides. CABIOS 12: 431-439. Motifs A program that searches amino
acid sequences for Bairoch et al. supra; Wisconsin patterns that
matched those defined in Prosite. Package Program Manual, version
9, page M51-59, Genetics Computer Group, Madison, WI.
[0242]
Sequence CWU 1
1
30 1 280 PRT Homo sapiens misc_feature Incyte ID No. 000746CD1 1
Met Ala Pro Ser Gly Ser Leu Ala Val Pro Leu Ala Val Leu Val 1 5 10
15 Leu Leu Leu Trp Gly Ala Pro Trp Thr His Gly Arg Arg Ser Asn 20
25 30 Val Arg Val Ile Thr Asp Glu Asn Trp Arg Glu Leu Leu Glu Gly
35 40 45 Asp Trp Met Ile Glu Phe Tyr Ala Pro Trp Cys Pro Ala Cys
Gln 50 55 60 Asn Leu Gln Pro Glu Trp Glu Ser Phe Ala Glu Trp Gly
Glu Asp 65 70 75 Leu Glu Val Asn Ile Ala Lys Val Asp Val Thr Glu
Gln Pro Gly 80 85 90 Leu Ser Gly Arg Phe Ile Ile Thr Ala Leu Pro
Thr Ile Tyr His 95 100 105 Cys Lys Asp Gly Glu Phe Arg Arg Tyr Gln
Gly Pro Arg Thr Lys 110 115 120 Lys Asp Phe Ile Asn Phe Ile Ser Asp
Lys Glu Trp Lys Ser Ile 125 130 135 Glu Pro Val Ser Ser Trp Phe Gly
Pro Gly Ser Val Leu Met Ser 140 145 150 Ser Met Ser Ala Leu Phe Gln
Leu Ser Met Trp Ile Arg Thr Cys 155 160 165 His Asn Tyr Phe Ile Glu
Asp Leu Gly Leu Pro Val Trp Gly Ser 170 175 180 Tyr Thr Val Phe Ala
Leu Ala Thr Leu Phe Ser Gly Leu Leu Leu 185 190 195 Gly Leu Cys Met
Ile Phe Val Ala Asp Cys Leu Cys Pro Ser Lys 200 205 210 Arg Arg Arg
Pro Gln Pro Tyr Pro Tyr Pro Ser Lys Lys Leu Leu 215 220 225 Ser Glu
Ser Ala Gln Pro Leu Lys Lys Val Glu Glu Glu Gln Glu 230 235 240 Ala
Asp Glu Glu Asp Val Ser Glu Glu Glu Ala Glu Ser Lys Glu 245 250 255
Gly Thr Asn Lys Asp Phe Pro Gln Asn Ala Ile Arg Gln Arg Ser 260 265
270 Leu Gly Pro Ser Leu Ala Thr Asp Lys Ser 275 280 2 166 PRT Homo
sapiens misc_feature Incyte ID No. 2472577CD1 2 Met Gln Glu Ile Asn
Gln Asn Leu Ala Glu Glu Ala Gly Leu Asn 1 5 10 15 Ile Thr His Ile
Cys Leu Pro Pro Asp Ser Ser Glu Ala Glu Ile 20 25 30 Ile Asp Glu
Ile Leu Lys Ile Asn Glu Asp Thr Arg Val His Gly 35 40 45 Leu Ala
Leu Gln Ile Ser Glu Asn Leu Phe Ser Asn Lys Val Leu 50 55 60 Asn
Ala Leu Lys Pro Glu Lys Asp Val Asp Gly Val Thr Asp Ile 65 70 75
Asn Leu Gly Lys Leu Val Arg Gly Asp Ala His Glu Cys Phe Val 80 85
90 Ser Pro Val Ala Lys Ala Val Ile Glu Leu Leu Glu Lys Ser Val 95
100 105 Gly Val Asn Leu Asp Gly Lys Lys Ile Leu Val Val Gly Ala His
110 115 120 Gly Ser Leu Glu Ala Ala Leu Gln Cys Leu Phe Gln Arg Lys
Gly 125 130 135 Ser Met Thr Met Ser Ile Gln Trp Lys Thr Arg Gln Leu
Gln Ser 140 145 150 Lys Thr Glu Ser Arg Ser Val Thr Arg Leu Glu Cys
Arg Arg Val 155 160 165 Ile 3 319 PRT Homo sapiens misc_feature
Incyte ID No. 2160405CD1 3 Met Ala Ser Ser Ala Ala Gly Cys Val Val
Ile Val Gly Ser Gly 1 5 10 15 Val Ile Gly Arg Ser Trp Ala Met Leu
Phe Ala Ser Gly Gly Phe 20 25 30 Gln Val Lys Leu Tyr Asp Ile Glu
Gln Gln Gln Ile Arg Asn Ala 35 40 45 Leu Glu Asn Ile Arg Lys Glu
Met Lys Leu Leu Glu Gln Ala Gly 50 55 60 Ser Leu Lys Gly Ser Leu
Ser Val Glu Glu Gln Leu Ser Leu Ile 65 70 75 Ser Gly Cys Pro Asn
Ile Gln Glu Ala Val Glu Gly Ala Met His 80 85 90 Ile Gln Glu Cys
Val Pro Glu Asp Leu Glu Leu Lys Lys Lys Ile 95 100 105 Phe Ala Gln
Leu Asp Ser Ile Ile Asp Asp Arg Val Ile Leu Ser 110 115 120 Ser Ser
Thr Ser Cys Leu Met Pro Ser Lys Leu Phe Ala Gly Leu 125 130 135 Val
His Val Lys Gln Cys Ile Val Ala His Pro Val Asn Pro Pro 140 145 150
Tyr Tyr Ile Pro Leu Val Glu Leu Val Pro His Pro Glu Thr Ala 155 160
165 Pro Thr Thr Val Asp Arg Thr His Ala Leu Met Lys Lys Ile Gly 170
175 180 Gln Cys Pro Met Arg Val Gln Lys Glu Val Ala Gly Phe Val Leu
185 190 195 Asn Arg Leu Gln Tyr Ala Ile Ile Ser Glu Ala Trp Arg Leu
Val 200 205 210 Glu Glu Gly Ile Val Ser Pro Ser Asp Leu Asp Leu Val
Met Ser 215 220 225 Glu Gly Leu Gly Met Arg Tyr Ala Phe Ile Gly Pro
Leu Glu Thr 230 235 240 Met His Leu Asn Ala Glu Gly Met Leu Ser Tyr
Cys Asp Arg Tyr 245 250 255 Ser Glu Gly Ile Lys His Val Leu Gln Thr
Phe Gly Pro Ile Pro 260 265 270 Glu Phe Ser Arg Ala Thr Ala Glu Lys
Val Asn Gln Asp Met Cys 275 280 285 Met Lys Val Pro Asp Asp Pro Glu
His Leu Ala Ala Arg Arg Gln 290 295 300 Trp Arg Asp Glu Cys Leu Met
Arg Leu Ala Lys Leu Lys Ser Gln 305 310 315 Val Gln Pro Gln 4 318
PRT Homo sapiens misc_feature Incyte ID No. 2591695CD1 4 Met Ala
Pro Trp Ala Glu Ala Glu His Ser Ala Leu Asn Pro Leu 1 5 10 15 Arg
Ala Val Trp Leu Thr Leu Thr Ala Ala Phe Leu Leu Thr Leu 20 25 30
Leu Leu Gln Leu Leu Pro Pro Gly Leu Leu Pro Gly Cys Ala Ile 35 40
45 Phe Gln Asp Leu Ile Arg Tyr Gly Lys Thr Lys Cys Gly Glu Pro 50
55 60 Ser Arg Pro Ala Ala Cys Arg Ala Phe Asp Val Pro Lys Arg Tyr
65 70 75 Phe Ser His Phe Tyr Ile Ile Ser Val Leu Trp Asn Gly Phe
Leu 80 85 90 Leu Trp Cys Leu Thr Gln Ser Leu Phe Leu Gly Ala Pro
Phe Pro 95 100 105 Ser Trp Leu His Gly Leu Leu Arg Ile Leu Gly Ala
Ala Gln Phe 110 115 120 Gln Gly Gly Glu Leu Ala Leu Ser Ala Phe Leu
Val Leu Val Phe 125 130 135 Leu Trp Leu His Ser Leu Arg Arg Leu Phe
Glu Cys Leu Tyr Val 140 145 150 Ser Val Phe Ser Asn Val Met Ile His
Val Val Gln Tyr Cys Phe 155 160 165 Gly Leu Val Tyr Tyr Val Leu Val
Gly Leu Thr Val Leu Ser Gln 170 175 180 Val Pro Met Asp Gly Arg Asn
Ala Tyr Ile Thr Gly Lys Asn Leu 185 190 195 Leu Met Gln Ala Arg Trp
Phe His Ile Leu Gly Met Met Met Phe 200 205 210 Ile Trp Ser Ser Ala
His Gln Tyr Lys Cys His Val Ile Leu Gly 215 220 225 Asn Leu Arg Lys
Asn Lys Ala Gly Val Val Ile His Cys Asn His 230 235 240 Arg Ile Pro
Phe Gly Asp Trp Phe Glu Tyr Val Ser Ser Pro Asn 245 250 255 Tyr Leu
Ala Glu Leu Met Ile Tyr Val Ser Met Ala Val Thr Phe 260 265 270 Gly
Phe His Asn Leu Thr Trp Trp Leu Val Val Thr Asn Val Phe 275 280 285
Phe Asn Gln Ala Leu Ser Ala Phe Leu Ser His Gln Phe Tyr Lys 290 295
300 Ser Lys Phe Val Ser Tyr Pro Lys His Arg Lys Ala Phe Leu Pro 305
310 315 Phe Leu Phe 5 330 PRT Homo sapiens misc_feature Incyte ID
No. 474100CD1 5 Met Pro Glu Met Pro Glu Asp Met Glu Gln Glu Glu Val
Asn Ile 1 5 10 15 Pro Asn Arg Arg Val Leu Val Thr Gly Ala Thr Gly
Leu Leu Gly 20 25 30 Arg Ala Val His Lys Glu Phe Gln Gln Asn Asn
Trp His Ala Val 35 40 45 Gly Cys Gly Phe Arg Arg Ala Arg Pro Lys
Phe Glu Gln Val Asn 50 55 60 Leu Leu Asp Ser Asn Ala Val His His
Ile Ile His Asp Phe Gln 65 70 75 Pro His Val Ile Val His Cys Ala
Ala Glu Arg Arg Pro Asp Val 80 85 90 Val Glu Asn Gln Pro Asp Ala
Ala Ser Gln Leu Asn Val Asp Ala 95 100 105 Ser Gly Asn Leu Ala Lys
Glu Ala Asp Phe Phe Phe Phe Phe Val 110 115 120 Ala Ala Val Gly Ala
Phe Leu Ile Tyr Ile Ser Ser Asp Tyr Val 125 130 135 Phe Asp Gly Thr
Asn Pro Pro Tyr Arg Glu Glu Asp Ile Pro Ala 140 145 150 Pro Leu Asn
Leu Tyr Gly Lys Thr Lys Leu Asp Gly Glu Lys Ala 155 160 165 Val Leu
Glu Asn Asn Leu Gly Ala Ala Val Leu Arg Ile Pro Ile 170 175 180 Leu
Tyr Gly Glu Val Glu Lys Leu Glu Glu Ser Ala Val Thr Val 185 190 195
Met Phe Asp Lys Val Arg Phe Ser Asn Lys Ser Ala Asn Met Asp 200 205
210 His Trp Gln Gln Arg Phe Pro Thr His Val Lys Asp Val Ala Thr 215
220 225 Val Cys Arg Gln Leu Ala Glu Lys Arg Met Leu Asp Pro Ser Ile
230 235 240 Lys Gly Thr Phe His Trp Ser Gly Asn Glu Gln Met Thr Lys
Tyr 245 250 255 Glu Met Ala Cys Ala Ile Ala Asp Ala Phe Asn Leu Pro
Ser Ser 260 265 270 His Leu Arg Pro Ile Thr Asp Ser Pro Val Leu Gly
Ala Gln Arg 275 280 285 Pro Arg Asn Ala Gln Leu Asp Cys Ser Lys Leu
Glu Thr Leu Gly 290 295 300 Ile Gly Gln Arg Thr Pro Phe Arg Ile Gly
Ile Lys Glu Ser Leu 305 310 315 Trp Pro Phe Leu Ile Asp Lys Arg Trp
Arg Gln Thr Val Phe His 320 325 330 6 266 PRT Homo sapiens
misc_feature Incyte ID No. 1304767CD1 6 Met Ala Thr Gly Thr Arg Tyr
Ala Gly Lys Val Val Val Val Thr 1 5 10 15 Gly Ile Gly Ala Gly Ile
Val Arg Ala Phe Val Asp Ser Gly Ala 20 25 30 Arg Val Val Ile Cys
Asp Lys Asp Glu Ser Gly Gly Arg Ala Leu 35 40 45 Glu Gln Glu Leu
Pro Gly Ala Val Phe Ile Leu Cys Asp Val Thr 50 55 60 Gln Glu Asp
Asp Val Lys Thr Leu Val Ser Glu Thr Ile Arg Arg 65 70 75 Phe Gly
Arg Leu Asp Cys Val Val Asn Asn Ala Gly His His Pro 80 85 90 Pro
Pro Gln Arg Pro Glu Glu Thr Ser Ala Gln Gly Phe Arg Gln 95 100 105
Leu Leu Glu Leu Asn Leu Leu Gly Thr Tyr Thr Leu Thr Lys Leu 110 115
120 Ala Leu Pro Tyr Leu Arg Lys Ser Gln Gly Asn Val Ile Asn Ile 125
130 135 Ser Ser Leu Val Gly Ala Ile Gly Gln Ala Gln Ala Val Pro Tyr
140 145 150 Val Ala Thr Lys Gly Ala Val Thr Ala Met Thr Lys Ala Leu
Ala 155 160 165 Leu Asp Glu Ser Pro Tyr Gly Val Arg Val Asn Cys Ile
Ser Pro 170 175 180 Gly Asn Ile Trp Thr Pro Leu Trp Glu Glu Leu Ala
Ala Leu Met 185 190 195 Pro Asp Pro Arg Ala Thr Ile Arg Glu Gly Met
Leu Ala Gln Pro 200 205 210 Leu Gly Arg Met Gly Gln Pro Ala Glu Val
Gly Ala Ala Ala Val 215 220 225 Phe Leu Ala Ser Glu Ala Asn Phe Cys
Thr Gly Ile Glu Leu Leu 230 235 240 Val Thr Gly Gly Ala Glu Leu Gly
Tyr Gly Cys Lys Ala Ser Arg 245 250 255 Ser Thr Pro Val Asp Ala Pro
Asp Ile Pro Ser 260 265 7 302 PRT Homo sapiens misc_feature Incyte
ID No. 1465978CD1 7 Met Val Trp Lys Arg Leu Gly Ala Leu Val Met Phe
Pro Leu Gln 1 5 10 15 Met Ile Tyr Leu Val Val Lys Ala Ala Val Gly
Leu Val Leu Pro 20 25 30 Ala Lys Leu Arg Asp Leu Ser Arg Glu Asn
Val Leu Ile Thr Gly 35 40 45 Gly Gly Arg Gly Ile Gly Arg Gln Leu
Ala Arg Glu Phe Ala Glu 50 55 60 Arg Gly Ala Arg Lys Ile Val Leu
Trp Gly Arg Thr Glu Lys Cys 65 70 75 Leu Lys Glu Thr Thr Glu Glu
Ile Arg Gln Met Gly Thr Glu Cys 80 85 90 His Tyr Phe Ile Cys Asp
Val Gly Asn Arg Glu Glu Val Tyr Gln 95 100 105 Thr Ala Lys Ala Val
Arg Glu Lys Val Gly Asp Ile Thr Ile Leu 110 115 120 Val Asn Asn Ala
Ala Val Val His Gly Lys Ser Leu Met Asp Ser 125 130 135 Asp Asp Asp
Ala Leu Leu Lys Ser Gln His Ile Asn Thr Leu Gly 140 145 150 Gln Phe
Trp Thr Thr Lys Ala Phe Leu Pro Arg Met Leu Glu Leu 155 160 165 Gln
Asn Gly His Ile Val Cys Leu Asn Ser Val Leu Ala Leu Ser 170 175 180
Ala Ile Pro Gly Ala Ile Asp Tyr Cys Thr Ser Lys Ala Ser Ala 185 190
195 Phe Ala Phe Met Glu Ser Leu Thr Leu Gly Leu Leu Asp Cys Pro 200
205 210 Gly Val Ser Ala Thr Thr Val Leu Pro Phe His Thr Ser Thr Glu
215 220 225 Met Phe Gln Gly Met Arg Val Arg Phe Pro Asn Leu Phe Pro
Pro 230 235 240 Leu Lys Pro Glu Thr Val Ala Arg Arg Thr Val Glu Ala
Val Gln 245 250 255 Leu Asn Gln Ala Leu Leu Leu Leu Pro Trp Thr Met
His Ala Leu 260 265 270 Val Ile Leu Lys Ser Ile Leu Pro Gln Ala Ala
Leu Glu Glu Ile 275 280 285 His Lys Phe Ser Gly Thr Tyr Thr Cys Met
Asn Thr Phe Lys Gly 290 295 300 Arg Thr 8 300 PRT Homo sapiens
misc_feature Incyte ID No. 1635966CD1 8 Met Lys Phe Leu Leu Asp Ile
Leu Leu Leu Leu Pro Leu Leu Ile 1 5 10 15 Val Cys Ser Leu Glu Ser
Phe Val Lys Leu Phe Ile Pro Lys Arg 20 25 30 Arg Lys Ser Val Thr
Gly Glu Ile Val Leu Ile Thr Gly Ala Gly 35 40 45 His Gly Ile Val
Arg Leu Thr Ala Tyr Glu Phe Ala Lys Leu Lys 50 55 60 Ser Lys Leu
Val Leu Trp Asp Ile Asn Lys His Gly Leu Glu Glu 65 70 75 Thr Ala
Ala Lys Cys Lys Gly Leu Gly Ala Lys Val His Thr Phe 80 85 90 Val
Val Asp Cys Ser Asn Arg Glu Asp Ile Tyr Ser Ser Ala Lys 95 100 105
Lys Val Lys Ala Glu Ile Gly Asp Val Ser Ile Leu Val Asn Asn 110 115
120 Ala Gly Val Val Tyr Thr Ser Asp Leu Phe Ala Thr Gln Asp Pro 125
130 135 Gln Ile Glu Lys Thr Phe Glu Val Asn Val Leu Ala His Phe Trp
140 145 150 Thr Thr Lys Ala Phe Leu Pro Ala Met Thr Lys Asn Asn His
Gly 155 160 165 His Ile Val Thr Val Ala Ser Ala Ala Gly His Val Ser
Val Pro 170 175 180 Phe Leu Leu Ala Tyr Cys Ser Ser Lys Phe Ala Ala
Val Gly Phe 185 190 195 His Lys Thr Leu Thr Asp Glu Leu Ala Ala Leu
Gln Ile Thr Gly 200 205 210 Val Lys Thr Thr Cys Leu Cys Pro Asn Phe
Val Asn Thr Gly Phe 215 220 225 Ile Lys Asn Pro Ser Thr Ser Leu Gly
Pro Thr Leu Glu Pro Glu 230 235 240 Glu Val Val Asn Arg Leu Met His
Gly Ile Leu Thr Glu Gln Lys 245 250 255 Met Ile Phe Ile Pro Ser Ser
Ile Ala Phe Leu Thr
Thr Leu Glu 260 265 270 Arg Ile Leu Pro Glu Arg Phe Leu Ala Val Leu
Lys Arg Lys Ile 275 280 285 Ser Val Lys Phe Asp Ala Val Ile Gly Tyr
Lys Met Lys Ala Gln 290 295 300 9 613 PRT Homo sapiens misc_feature
Incyte ID No. 1638410CD1 9 Met Phe Arg Cys Gly Gly Leu Ala Ala Gly
Ala Leu Lys Gln Lys 1 5 10 15 Leu Val Pro Leu Val Arg Thr Val Cys
Val Arg Ser Pro Arg Gln 20 25 30 Arg Asn Arg Leu Pro Gly Asn Leu
Phe Gln Arg Trp His Val Pro 35 40 45 Leu Glu Leu Gln Met Thr Arg
Gln Met Ala Ser Ser Gly Ala Ser 50 55 60 Gly Gly Lys Ile Asp Asn
Ser Val Leu Val Leu Ile Val Gly Leu 65 70 75 Ser Thr Val Gly Ala
Gly Ala Tyr Ala Tyr Lys Thr Met Lys Glu 80 85 90 Asp Glu Lys Arg
Tyr Asn Glu Arg Ile Ser Gly Leu Gly Leu Thr 95 100 105 Pro Glu Gln
Lys Gln Lys Lys Ala Ala Leu Ser Ala Ser Glu Gly 110 115 120 Glu Glu
Val Pro Gln Asp Lys Ala Pro Ser His Val Pro Phe Leu 125 130 135 Leu
Ile Gly Gly Gly Thr Ala Ala Phe Ala Ala Ala Arg Ser Ile 140 145 150
Arg Ala Arg Asp Pro Gly Ala Arg Val Leu Ile Val Ser Glu Asp 155 160
165 Pro Glu Leu Pro Tyr Met Arg Pro Pro Leu Ser Lys Glu Leu Trp 170
175 180 Phe Ser Asp Asp Pro Asn Val Thr Lys Thr Leu Arg Phe Lys Gln
185 190 195 Trp Asn Gly Lys Glu Arg Ser Ile Tyr Phe Gln Pro Pro Ser
Phe 200 205 210 Tyr Val Ser Ala Gln Asp Leu Pro His Ile Glu Asn Gly
Gly Val 215 220 225 Ala Val Leu Thr Gly Lys Lys Val Val Gln Leu Asp
Val Arg Asp 230 235 240 Asn Met Val Lys Leu Asn Asp Gly Ser Gln Ile
Thr Tyr Glu Lys 245 250 255 Cys Leu Ile Ala Thr Gly Gly Thr Pro Arg
Ser Leu Ser Ala Ile 260 265 270 Asp Arg Ala Gly Ala Glu Val Lys Ser
Arg Thr Thr Leu Phe Arg 275 280 285 Lys Ile Gly Asp Phe Arg Ser Leu
Glu Lys Ile Ser Arg Glu Val 290 295 300 Lys Ser Ile Thr Ile Ile Gly
Gly Gly Phe Leu Gly Ser Glu Leu 305 310 315 Ala Cys Ala Leu Gly Arg
Lys Ala Arg Ala Leu Gly Thr Glu Val 320 325 330 Ile Gln Leu Phe Pro
Glu Lys Gly Asn Met Gly Lys Ile Leu Pro 335 340 345 Glu Tyr Leu Ser
Asn Trp Thr Met Glu Lys Val Arg Arg Glu Gly 350 355 360 Val Lys Val
Met Pro Asn Ala Ile Val Gln Ser Val Gly Val Ser 365 370 375 Ser Gly
Lys Leu Leu Ile Lys Leu Lys Asp Gly Arg Lys Val Glu 380 385 390 Thr
Asp His Ile Val Ala Ala Val Gly Leu Glu Pro Asn Val Glu 395 400 405
Leu Ala Lys Thr Gly Gly Leu Glu Ile Asp Ser Asp Phe Gly Gly 410 415
420 Phe Arg Val Asn Ala Glu Leu Gln Ala Arg Ser Asn Ile Trp Val 425
430 435 Ala Gly Asp Ala Ala Cys Phe Tyr Asp Ile Lys Leu Gly Arg Arg
440 445 450 Arg Val Glu His His Asp His Ala Val Val Ser Gly Arg Leu
Ala 455 460 465 Gly Glu Asn Met Thr Gly Ala Ala Lys Pro Tyr Trp His
Gln Ser 470 475 480 Met Phe Trp Ser Asp Leu Gly Pro Asp Val Gly Tyr
Glu Ala Ile 485 490 495 Gly Leu Val Asp Ser Ser Leu Pro Thr Val Gly
Val Phe Ala Lys 500 505 510 Ala Thr Ala Gln Asp Asn Pro Lys Ser Ala
Thr Glu Gln Ser Gly 515 520 525 Thr Gly Ile Arg Ser Glu Ser Glu Thr
Glu Ser Glu Ala Ser Glu 530 535 540 Ile Thr Ile Pro Pro Ser Thr Pro
Ala Val Pro Gln Ala Pro Val 545 550 555 Gln Gly Glu Asp Tyr Gly Lys
Gly Val Ile Phe Tyr Leu Arg Asp 560 565 570 Lys Val Val Val Gly Ile
Val Leu Trp Asn Ile Phe Asn Arg Met 575 580 585 Pro Ile Ala Arg Lys
Ile Ile Lys Asp Gly Glu Gln His Glu Asp 590 595 600 Leu Asn Glu Val
Ala Lys Leu Phe Asn Ile His Glu Asp 605 610 10 325 PRT Homo sapiens
misc_feature Incyte ID No. 1743409CD1 10 Met Val Ser Pro Ala Thr
Arg Lys Ser Leu Pro Lys Val Lys Ala 1 5 10 15 Met Asp Phe Ile Thr
Ser Thr Ala Ile Leu Pro Leu Leu Phe Gly 20 25 30 Cys Leu Gly Val
Phe Gly Leu Phe Arg Leu Leu Gln Trp Val Arg 35 40 45 Gly Lys Ala
Tyr Leu Arg Asn Ala Val Val Val Ile Thr Gly Ala 50 55 60 Thr Ser
Gly Leu Gly Lys Glu Cys Ala Lys Val Phe Tyr Ala Ala 65 70 75 Gly
Ala Lys Leu Val Leu Cys Gly Arg Asn Gly Gly Ala Leu Glu 80 85 90
Glu Leu Ile Arg Glu Leu Thr Ala Ser His Ala Thr Lys Val Gln 95 100
105 Thr His Lys Pro Tyr Leu Val Thr Phe Asp Leu Thr Asp Ser Gly 110
115 120 Ala Ile Val Ala Ala Ala Ala Glu Ile Leu Gln Cys Phe Gly Tyr
125 130 135 Val Asp Ile Leu Val Asn Asn Ala Gly Ile Ser Tyr Arg Gly
Thr 140 145 150 Ile Met Asp Thr Thr Val Asp Val Asp Lys Arg Val Met
Glu Thr 155 160 165 Asn Tyr Phe Gly Pro Val Ala Leu Thr Lys Ala Leu
Leu Pro Ser 170 175 180 Met Ile Lys Arg Arg Gln Gly His Ile Val Ala
Ile Ser Ser Ile 185 190 195 Gln Gly Lys Met Ser Ile Pro Phe Arg Ser
Ala Tyr Ala Ala Ser 200 205 210 Lys His Ala Thr Gln Ala Phe Phe Asp
Cys Leu Arg Ala Glu Met 215 220 225 Glu Gln Tyr Glu Ile Glu Val Thr
Val Ile Ser Pro Gly Tyr Ile 230 235 240 His Thr Asn Leu Ser Val Asn
Ala Ile Thr Ala Asp Gly Ser Arg 245 250 255 Tyr Gly Val Met Asp Thr
Thr Thr Ala Gln Gly Arg Ser Pro Val 260 265 270 Glu Val Ala Gln Asp
Val Leu Ala Ala Val Gly Lys Lys Lys Lys 275 280 285 Asp Val Ile Leu
Ala Asp Leu Leu Pro Ser Leu Ala Val Tyr Leu 290 295 300 Arg Thr Leu
Ala Pro Gly Leu Phe Phe Ser Leu Met Ala Ser Arg 305 310 315 Ala Arg
Lys Glu Arg Lys Ser Lys Asn Ser 320 325 11 421 PRT Homo sapiens
misc_feature Incyte ID No. 1803830CD1 11 Met Trp Tyr His Arg Leu
Ser His Leu His Ser Arg Leu Gln Asp 1 5 10 15 Leu Leu Lys Gly Gly
Val Ile Tyr Pro Ala Leu Pro Gln Pro Asn 20 25 30 Phe Lys Ser Leu
Leu Pro Leu Ala Val His Trp His His Thr Ala 35 40 45 Ser Lys Ser
Leu Thr Cys Ala Trp Gln Gln His Glu Asp His Phe 50 55 60 Glu Leu
Lys Tyr Ala Asn Thr Val Met Arg Leu Asp Tyr Val Trp 65 70 75 Leu
Arg Asp His Cys Arg Ser Ala Ser Cys Tyr Asn Ser Lys Thr 80 85 90
His Gln Arg Ser Trp Asp Thr Ala Ser Val Asp Leu Cys Ile Lys 95 100
105 Pro Lys Thr Ile Arg Leu Asp Glu Thr Thr Leu Phe Phe Thr Trp 110
115 120 Pro Asp Gly His Val Thr Lys Tyr Asp Leu Asn Trp Leu Val Lys
125 130 135 Asn Ser Tyr Glu Gly Gln Lys Gln Lys Val Ile Gln Pro Arg
Ile 140 145 150 Leu Trp Asn Ala Glu Ile Tyr Gln Gln Ala Gln Val Pro
Ser Val 155 160 165 Asp Cys Gln Ser Phe Leu Glu Thr Asn Glu Gly Leu
Lys Lys Phe 170 175 180 Leu Gln Asn Phe Leu Leu Tyr Gly Ile Ala Phe
Val Glu Asn Val 185 190 195 Pro Pro Thr Gln Glu His Thr Glu Lys Leu
Ala Glu Arg Ile Ser 200 205 210 Leu Ile Arg Glu Thr Ile Tyr Gly Arg
Met Trp Tyr Phe Thr Ser 215 220 225 Asp Phe Ser Arg Gly Asp Thr Ala
Tyr Thr Lys Leu Ala Leu Asp 230 235 240 Arg His Thr Asp Thr Thr Tyr
Phe Gln Glu Pro Cys Gly Ile Gln 245 250 255 Val Phe His Cys Leu Lys
His Glu Gly Thr Gly Gly Arg Thr Leu 260 265 270 Leu Val Asp Gly Phe
Tyr Ala Ala Glu Gln Val Leu Gln Lys Ala 275 280 285 Pro Glu Glu Phe
Glu Leu Leu Ser Lys Val Pro Leu Lys His Glu 290 295 300 Tyr Ile Glu
Asp Val Gly Glu Cys His Asn His Met Ile Gly Ile 305 310 315 Gly Pro
Val Leu Asn Ile Tyr Pro Trp Asn Lys Glu Leu Tyr Leu 320 325 330 Ile
Arg Tyr Asn Asn Tyr Asp Arg Ala Val Ile Asn Thr Val Pro 335 340 345
Tyr Asp Val Val His Arg Trp Tyr Thr Ala His Arg Thr Leu Thr 350 355
360 Ile Glu Leu Arg Arg Pro Glu Asn Glu Phe Trp Val Lys Leu Lys 365
370 375 Pro Gly Arg Val Leu Phe Ile Asp Asn Trp Arg Val Leu His Gly
380 385 390 Arg Glu Cys Phe Thr Gly Tyr Arg Gln Leu Cys Gly Cys Tyr
Leu 395 400 405 Thr Arg Asp Asp Val Leu Asn Thr Ala Arg Leu Leu Gly
Leu Gln 410 415 420 Ala 12 610 PRT Homo sapiens misc_feature Incyte
ID No. 1867333CD1 12 Met Trp Leu Pro Leu Val Leu Leu Leu Ala Val
Leu Leu Leu Ala 1 5 10 15 Val Leu Cys Lys Val Tyr Leu Gly Leu Phe
Ser Gly Ser Ser Pro 20 25 30 Asn Pro Phe Ser Glu Asp Val Lys Arg
Pro Pro Ala Pro Leu Val 35 40 45 Thr Asp Lys Glu Ala Arg Lys Lys
Val Leu Lys Gln Ala Phe Ser 50 55 60 Ala Asn Gln Val Pro Glu Lys
Leu Asp Val Val Val Ile Gly Ser 65 70 75 Gly Phe Gly Gly Leu Ala
Ala Ala Ala Ile Leu Ala Lys Ala Gly 80 85 90 Lys Arg Val Leu Val
Leu Glu Gln His Thr Lys Ala Gly Gly Cys 95 100 105 Cys His Thr Phe
Gly Lys Asn Gly Leu Glu Phe Asp Thr Gly Ile 110 115 120 His Tyr Ile
Gly Arg Met Glu Glu Gly Ser Ile Gly Arg Phe Ile 125 130 135 Leu Asp
Gln Ile Thr Glu Gly Gln Leu Asp Trp Ala Pro Leu Ser 140 145 150 Ser
Pro Phe Asp Ile Met Val Leu Glu Gly Pro Asn Gly Arg Lys 155 160 165
Glu Tyr Pro Met Tyr Ser Gly Glu Lys Ala Tyr Ile Gln Gly Leu 170 175
180 Lys Glu Lys Phe Pro Gln Glu Glu Ala Ile Ile Asp Lys Tyr Ile 185
190 195 Lys Leu Val Lys Val Val Ser Ser Gly Ala Pro His Ala Ile Leu
200 205 210 Leu Lys Phe Leu Pro Leu Pro Val Val Gln Leu Leu Asp Arg
Cys 215 220 225 Gly Leu Leu Thr Arg Phe Ser Pro Phe Leu Gln Ala Ser
Thr Gln 230 235 240 Ser Leu Ala Glu Val Leu Gln Gln Leu Gly Ala Ser
Ser Glu Leu 245 250 255 Gln Ala Val Leu Ser Tyr Ile Phe Pro Thr Tyr
Gly Val Thr Pro 260 265 270 Asn His Ser Ala Phe Ser Met His Ala Leu
Leu Val Asn His Tyr 275 280 285 Met Lys Gly Gly Phe Tyr Pro Arg Gly
Gly Ser Ser Glu Ile Ala 290 295 300 Phe His Thr Ile Pro Val Ile Gln
Arg Ala Gly Gly Ala Val Leu 305 310 315 Thr Lys Ala Thr Val Gln Ser
Val Leu Leu Asp Ser Ala Gly Lys 320 325 330 Ala Cys Gly Val Ser Val
Lys Lys Gly His Glu Leu Val Asn Ile 335 340 345 Tyr Cys Pro Ile Val
Val Ser Asn Ala Gly Leu Phe Asn Thr Tyr 350 355 360 Glu His Leu Leu
Pro Gly Asn Ala Arg Cys Leu Pro Gly Val Lys 365 370 375 Gln Gln Leu
Gly Thr Val Arg Pro Gly Leu Gly Met Thr Ser Val 380 385 390 Phe Ile
Cys Leu Arg Gly Thr Lys Glu Asp Leu His Leu Pro Ser 395 400 405 Thr
Asn Tyr Tyr Val Tyr Tyr Asp Thr Asp Met Asp Gln Ala Met 410 415 420
Glu Arg Tyr Val Ser Met Pro Arg Glu Glu Ala Ala Glu His Ile 425 430
435 Pro Leu Leu Phe Phe Ala Phe Pro Ser Ala Lys Asp Pro Thr Trp 440
445 450 Glu Asp Arg Phe Pro Gly Arg Ser Thr Met Ile Met Leu Ile Pro
455 460 465 Thr Ala Tyr Glu Trp Phe Glu Glu Trp Gln Ala Glu Leu Lys
Gly 470 475 480 Lys Arg Gly Ser Asp Tyr Glu Thr Phe Lys Asn Ser Phe
Val Glu 485 490 495 Ala Ser Met Ser Val Val Leu Lys Leu Phe Pro Gln
Leu Glu Gly 500 505 510 Lys Val Glu Ser Val Thr Ala Gly Ser Pro Leu
Thr Asn Gln Phe 515 520 525 Tyr Leu Ala Ala Pro Arg Gly Ala Cys Tyr
Gly Ala Asp His Asp 530 535 540 Leu Gly Arg Leu His Pro Cys Val Met
Ala Ser Leu Arg Ala Gln 545 550 555 Ser Pro Ile Pro Asn Leu Tyr Leu
Thr Gly Gln Asp Ile Phe Thr 560 565 570 Cys Gly Leu Val Gly Ala Leu
Gln Gly Ala Leu Leu Cys Ser Ser 575 580 585 Ala Ile Leu Lys Arg Asn
Leu Tyr Ser Asp Leu Lys Asn Leu Asp 590 595 600 Ser Arg Ile Arg Ala
Gln Lys Lys Lys Asn 605 610 13 415 PRT Homo sapiens misc_feature
Incyte ID No. 2906094CD1 13 Met Leu Trp Ser Gly Cys Arg Arg Phe Gly
Ala Arg Leu Gly Cys 1 5 10 15 Leu Pro Gly Gly Leu Arg Val Leu Val
Gln Thr Gly His Arg Ser 20 25 30 Leu Thr Ser Cys Ile Asp Pro Ser
Met Gly Leu Asn Glu Glu Gln 35 40 45 Lys Glu Phe Gln Lys Val Ala
Phe Asp Phe Ala Ala Arg Glu Met 50 55 60 Ala Pro Asn Met Ala Glu
Trp Asp Gln Lys Glu Leu Phe Pro Val 65 70 75 Asp Val Met Arg Lys
Ala Ala Gln Leu Gly Phe Gly Gly Val Tyr 80 85 90 Ile Gln Thr Asp
Val Gly Gly Ser Gly Leu Ser Arg Leu Asp Thr 95 100 105 Ser Val Ile
Phe Glu Ala Leu Ala Thr Gly Cys Thr Ser Thr Thr 110 115 120 Ala Tyr
Ile Ser Ile His Asn Met Cys Ala Trp Met Ile Asp Ser 125 130 135 Phe
Gly Asn Glu Glu Gln Arg His Lys Phe Cys Pro Pro Leu Cys 140 145 150
Thr Met Glu Lys Phe Ala Ser Tyr Cys Leu Thr Glu Pro Gly Ser 155 160
165 Gly Ser Asp Ala Ala Ser Leu Leu Thr Ser Ala Lys Lys Gln Gly 170
175 180 Asp His Tyr Ile Leu Asn Gly Ser Lys Ala Phe Ile Ser Gly Ala
185 190 195 Gly Glu Ser Asp Ile Tyr Val Val Met Cys Arg Thr Gly Gly
Pro 200 205 210 Gly Pro Lys Gly Ile Ser Cys Ile Val Val Glu Lys Gly
Thr Pro 215 220 225 Gly Leu Ser Phe Gly Lys Lys Glu Lys Lys Val Gly
Trp Asn Ser 230 235 240 Gln Pro Thr Arg Ala Val Ile Phe Glu Asp Cys
Ala Val Pro Val 245 250
255 Ala Asn Arg Ile Gly Ser Glu Gly Gln Gly Phe Leu Ile Ala Val 260
265 270 Arg Gly Leu Asn Gly Gly Arg Ile Asn Ile Ala Ser Cys Ser Leu
275 280 285 Gly Ala Ala His Ala Ser Val Ile Leu Thr Arg Asp His Leu
Asn 290 295 300 Val Arg Lys Gln Phe Gly Glu Pro Leu Ala Ser Asn Gln
Tyr Leu 305 310 315 Gln Phe Thr Leu Ala Asp Met Ala Thr Arg Leu Val
Ala Ala Arg 320 325 330 Leu Met Val Arg Asn Ala Ala Val Ala Leu Gln
Glu Glu Arg Lys 335 340 345 Asp Ala Val Ala Leu Cys Ser Met Ala Lys
Leu Phe Ala Thr Asp 350 355 360 Glu Cys Phe Ala Ile Cys Asn Gln Ala
Leu Gln Met His Gly Gly 365 370 375 Tyr Gly Tyr Leu Lys Asp Tyr Ala
Val Gln Gln Tyr Val Arg Asp 380 385 390 Ser Arg Val His Gln Ile Leu
Glu Gly Ser Asn Glu Val Met Arg 395 400 405 Ile Leu Ile Ser Arg Ser
Leu Leu Gln Glu 410 415 14 274 PRT Homo sapiens misc_feature Incyte
ID No. 3294314CD1 14 Met Ala Ala Ala Glu Pro Ser Pro Arg Arg Val
Gly Phe Val Gly 1 5 10 15 Ala Gly Arg Met Ala Gly Ala Ile Ala Gln
Gly Leu Ile Arg Ala 20 25 30 Gly Lys Val Glu Ala Gln His Ile Leu
Ala Ser Ala Pro Thr Asp 35 40 45 Arg Asn Leu Cys His Phe Gln Ala
Leu Gly Cys Arg Thr Thr His 50 55 60 Ser Asn Gln Glu Val Leu Gln
Ser Cys Leu Leu Val Ile Phe Ala 65 70 75 Thr Lys Pro His Val Leu
Pro Ala Val Leu Ala Glu Val Ala Pro 80 85 90 Val Val Thr Thr Glu
His Ile Leu Val Ser Val Ala Ala Gly Val 95 100 105 Ser Leu Ser Thr
Leu Glu Glu Leu Leu Pro Pro Asn Thr Arg Val 110 115 120 Leu Arg Val
Leu Pro Asn Leu Pro Cys Val Val Gln Glu Gly Ala 125 130 135 Ile Val
Met Ala Arg Gly Arg His Val Gly Ser Ser Glu Thr Lys 140 145 150 Leu
Leu Gln His Leu Leu Glu Ala Cys Gly Arg Cys Glu Glu Val 155 160 165
Pro Glu Ala Tyr Val Asp Ile His Thr Gly Leu Ser Gly Ser Gly 170 175
180 Val Ala Phe Val Cys Ala Phe Ser Glu Ala Leu Ala Glu Gly Ala 185
190 195 Val Lys Met Gly Met Pro Ser Ser Leu Ala His Arg Ile Ala Ala
200 205 210 Gln Thr Leu Leu Gly Thr Ala Lys Met Leu Leu His Glu Gly
Gln 215 220 225 His Pro Ala Gln Leu Arg Ser Asp Val Cys Thr Pro Gly
Gly Thr 230 235 240 Thr Ile Tyr Gly Leu His Ala Leu Glu Gln Gly Gly
Leu Arg Ala 245 250 255 Ala Thr Met Ser Ala Val Glu Ala Ala Thr Cys
Arg Ala Lys Glu 260 265 270 Leu Ser Arg Lys 15 283 PRT Homo sapiens
misc_feature Incyte ID No. 4940951CD1 15 Met Ala Ala Pro Phe Phe
Ser Thr Pro Phe Gln Pro Tyr Val Tyr 1 5 10 15 Gln Ser Gln Gln Gly
Ser Val Thr Ala Phe Gln Ile Ser Gly Gly 20 25 30 Asp Val Gln Val
Leu Gln Val Met Leu Lys Ser Gln Glu Lys Leu 35 40 45 Thr Ala Lys
Pro Gly Ala Met Cys Tyr Met Ser Gly Asn Met Gln 50 55 60 Met Asp
Asn Asn Tyr Leu Pro Glu Asn Asp Gly Gly Val Trp Gln 65 70 75 Trp
Ile Phe Gly Lys Arg Val Ser Ser Thr Ile Phe Phe Asn Ser 80 85 90
Gly Ser Asp Asp Gly Tyr Val Gly Ile Ala Ala Pro Phe Pro Gly 95 100
105 Arg Ile Leu Pro Val Asp Leu Thr Asn Phe Ser Gly Glu Leu Leu 110
115 120 Cys Gln Ala Asp Ala Phe Leu Cys Ser Val Asn Asp Val Ser Val
125 130 135 Ser Ser Thr Val Glu Pro Arg Pro Arg Asn Ile Glu Ile Gly
Ala 140 145 150 Glu Met Ile Leu Lys Gln Lys Leu Arg Gly Gln Gly Met
Ala Phe 155 160 165 Leu Val Gly Gly Gly Ser Val Met Gln Lys Ile Leu
Ala Pro Arg 170 175 180 Glu Val Ile Thr Val Asp Ala Ala Cys Ile Val
Ala Met Ser Ala 185 190 195 Thr Ile Asn Phe Gln Leu Lys Ser Pro Asn
Gln Leu Arg Arg Ala 200 205 210 Val Phe Gly Gly Asp Asn Gln Leu Thr
Ala Ser Leu Thr Gly Pro 215 220 225 Gly Val Val Phe Ile Gln Ser Leu
Pro Phe His Arg Leu Ser Gln 230 235 240 Arg Ile Ala Ser Ser Arg Ser
Val Ala Gly Pro Ser Leu Arg Asp 245 250 255 Asn Pro Lys Phe Phe Ile
Gln Ile Val Met Phe Phe Phe Leu Ala 260 265 270 Tyr Val Met Ile Val
Ser Ser Ile Ile Leu Thr Asp Val 275 280 16 2471 DNA Homo sapiens
misc_feature Incyte ID No. 000746CB1 16 gcgctcctcg cagcaccgta
gtgcgcttgc gctgagcagc ccgcgagggc ggaagtggga 60 gctgcgaccg
cgctccctgt gaggtgggca agcggcgaaa tggcgccctc cgggagtctt 120
gcagttcccc tggcagtcct ggtgctgttg ctttggggtg ctccctggac gcacgggcgg
180 cggagcaacg ttcgcgtcat cacggacgag aactggagag aactgctgga
aggagactgg 240 atgatagaat tttatgcccc gtggtgccct gcttgtcaaa
atcttcaacc ggaatgggaa 300 agttttgctg aatggggaga agatcttgag
gttaatattg cgaaagtaga tgtcacagag 360 cagccaggac tgagtggacg
gtttatcata actgctcttc ctactattta tcattgtaaa 420 gatggtgaat
ttaggcgcta tcagggtcca aggactaaga aggacttcat aaactttata 480
agtgataaag agtggaagag tattgagccc gtttcatcat ggtttggtcc aggttctgtt
540 ctgatgagta gtatgtcagc actctttcag ctatctatgt ggatcaggac
gtgccataac 600 tactttattg aagaccttgg attgccagtg tggggatcat
atactgtttt tgctttagca 660 actctgtttt ccggactgtt attaggactc
tgtatgatat ttgtggcaga ttgcctttgt 720 ccttcaaaaa ggcgcagacc
acagccatac ccataccctt caaaaaaatt attatcagaa 780 tctgcacaac
ctttgaaaaa agtggaggag gaacaagagg cggatgaaga agatgtttca 840
gaagaagaag ctgaaagtaa agaaggaaca aacaaagact ttccacagaa tgccataaga
900 caacgctctc tgggtccatc attggccaca gataaatcct agttaaattt
tatagttatc 960 ttaatattat gattttgata aaaacagaag attgatcatt
ttgtttggtt tgaagtgaac 1020 tgtgactttt ttgaatattg cagggttcag
tctagattgt cattaaattg aagagtctac 1080 attcagaaca taaaagcact
aggtatacaa gtttgaaata tgatttaagc acagtatgat 1140 ggtttaaata
gttctctaat ttttgaaaaa tcgtgccaag caataagatt tatgtatatt 1200
tgtttaataa taacctattt caagtctgag ttttgaaaat ttacatttcc caagtattgc
1260 attattgagg tatttaagaa gattatttta gagaaaaata tttctcattt
gatataattt 1320 ttctctgttt cactgtgtga aaaaaagaag atatttccca
taaatgggaa gtttgcccat 1380 tgtctcaaga aatgtgtatt tcagtgacaa
tttcgtggtc tttttagagg tatattccaa 1440 aatttccttg tatttttagg
ttatgcaact aataaaaact accttacatt aattaattac 1500 agttttctac
acatggtaat acaggatatg ctactgattt aggaagtttt taagttcatg 1560
gtattctctt gattccaaca aagtttgatt ttctcttgta tttttcttac ttactatggg
1620 ttacattttt tatttttcaa attggatgat aatttcttgg aaacattttt
tatgttttag 1680 taaacagtat ttttttgttg tttcaaactg aagtttactg
agagatccat caaattgaac 1740 aatctgttgt aatttaaaat tttggccact
tttttcagat tttacatcat tcttgctgaa 1800 cttcaacttg aaattgtttt
ttttttcttt ttggatgtga aggtgaacat tcctgatttt 1860 tgtctgatgt
gaaaaagcct tggtatttta cattttgaaa attcaaagaa gcttaatata 1920
aaagtttgca ttctactcag gaaaaagcat cttcttgtat atgtcttaaa tgtatttttg
1980 tcctcatata cagaaagttc ttaattgatt ttacagtctg taatgcttga
tgttttaaaa 2040 taataacatt tttatatttt ttaaaagaca aacttcatat
tatcctgtgt tctttcctga 2100 ctggtaatat tgtgtgggat ttcacaggta
aaagtcagta ggatggaaca ttttagtgta 2160 tttttactcc ttaaagagct
agaatacata gttttcacct taaaagaagg gggaaaatca 2220 taaatacaat
gaatcaactg accattacgt agtagacaat ttctgtaatg tccccttctt 2280
tctaggctct gttgctgtgt gaatccatta gatttacagt atcgtaatat acaagttttc
2340 tttaaagccc tctcctttag aatttaaaat attgtaccat tgaagagttt
ggatgtgtaa 2400 cttgtgatgc cttagaaaaa tatcctaagc acaaaataaa
cctttctaac cacttcatta 2460 aaaaaaaaaa a 2471 17 822 DNA Homo
sapiens misc_feature Incyte ID No. 2472577CB1 17 cggacccagc
gccgcccgcg cgaactccct ggtgttgtgc gcccttcccc gcgcgcggcc 60
cctcctgctg ggctggggtc tgtggctgac gtccgctttt cccggaacgg caaagagaag
120 tcattcagaa ttcaaaagaa gttctaagtt tattgcaaga aaaaaaccct
gccttcaagc 180 cggttcttgc aattatccag gcaggtgacg acaacttgat
gcaggaaatc aaccagaatt 240 tggctgagga ggctggtctg aacatcactc
acatttgcct ccctccagat agcagtgaag 300 ccgagattat agatgaaatc
ttaaagatca atgaagatac cagagtacat ggccttgccc 360 ttcagatctc
tgagaacttg tttagcaaca aagtcctcaa tgccttgaaa ccagaaaaag 420
atgtggatgg agtaacagac ataaacctgg ggaagctggt gcgaggggat gcccatgaat
480 gttttgtttc acctgttgcc aaagctgtaa ttgaacttct tgaaaaatca
gtaggtgtca 540 acctagatgg aaagaagatt ttggtagtgg gggcccatgg
gtctttggaa gctgctctac 600 aatgcctgtt ccagagaaaa gggtccatga
caatgagcat ccagtggaaa acacgccagc 660 ttcaaagcaa gacggagtct
cgttctgtca ccaggctgga gtgcaggcgc gtgatctagg 720 ctcactgcaa
gctctgcctc ccaggttgaa gtgattctcc tgtgaaaggg aattattttt 780
gatgagtcat taaagtatat ccattcccag aaaaaaaaaa aa 822 18 2026 DNA Homo
sapiens misc_feature Incyte ID No. 2160405CB1 18 aggaagccgg
ccacacccct tttggactcg catggggcac tgtccaatat tctttcatca 60
gggcgtgggg tctgtccaat gttcgtaggg gccgtctccc ggtgggggac cagttaaacc
120 agcgggattt agtatggcgg attcacagga tgagccacga tgcattgctt
cacatggacc 180 aagccagcaa acaacttgga aagccatgag acaagaagtg
gaactgctta agatcactcg 240 atcatcaatg atggaatcta actgagcaaa
aatcttcttc ttcagttcta gatcttctgg 300 aacacattcc tgaatgtgca
tggcgccctc tactgcttct tggatattgg gacaaccact 360 gatgagtgac
agctgctctt ccacactcag ggagcctttc agagaacctg cctgctccag 420
caacttcatc tcctttctga tgttttccag ggcgttcctt atctgctgtt gctcaatgtc
480 atagagtttc acctggaagc ctccactggc aaacagcgtg gcccgagttc
gcccaatgac 540 tccactgcca acgatcacca cgcagccgcc cgcggcgccg
cgtccccggc ccaaccatgg 600 cgtcctccgc ggccggctgc gtggtgatcg
ttggcagtgg agtcattggg cgaagctggg 660 ccatgctgtt tgccagtgga
ggcttccagg tgaaactcta tgacattgag caacagcaga 720 taaggaacgc
cctggaaaac atcagaaagg agatgaagtt gctggagcag gcaggttctc 780
tgaaaggctc cctgagtgtg gaagagcagc tgtcactcat cagtggttgt cccaatatcc
840 aagaagcagt agagggcgcc atgcacattc aggaatgtgt tccagaagat
ctagaactga 900 agaagaagat ttttgctcag ttagattcca tcattgatga
tcgagtgatc ttaagcagtt 960 ccacttcttg tctcatgcct tccaagttgt
ttgctggctt ggtccatgtg aagcaatgca 1020 tcgtggctca tcctgtgaat
ccgccatact acatcccgct ggttgagctg gtcccccacc 1080 cggagacggc
ccctacgaca gtggacagaa cccacgccct gatgaagaag attggacagt 1140
gccccatgcg agtccagaag gaggtggccg gcttcgttct gaaccgcctg caatatgcaa
1200 tcatcagcga ggcctggcgg ctagtggagg aaggaatcgt gtctcctagt
gacctggacc 1260 ttgtcatgtc agaagggttg ggcatgcggt atgcattcat
tggacccctg gaaaccatgc 1320 atctcaatgc agaaggtatg ttaagctact
gcgacagata cagcgaaggc ataaaacatg 1380 tcctacagac ttttggaccc
attccagagt tttccagggc cactgctgag aaggttaacc 1440 aggacatgtg
catgaaggtc cctgatgacc cggagcactt agctgccagg aggcagtgga 1500
gggacgagtg cctcatgaga ctcgccaagt tgaagagtca agtgcagccc cagtgaattt
1560 cttgtaatgc agcttccact cctctcattg gaggccctat ttgggaacac
tgcaagccct 1620 taatcagccc tctgtgacat aggtagcagc ccacggagat
cctaagctgg ctgtcttgtg 1680 tgcagcctga gtggggtggt gcaggccggt
agtctgcccg tcactttgga tcatagccct 1740 gggcctggcg gcacagcagc
acttgcgttc tcggggctgt cgatttcctg ccacctgggc 1800 agataacctg
gagattttca ccttttcttt tcagcttgat tgcatttgac tatattttac 1860
agccagtgat tgtagtttca tgttaatatg tggcaaaata tttttgtaat tattttctaa
1920 tccctttctg agtactctgg ggccctgcat ttatgaggca cctaccttca
ttttgctaac 1980 gcttattctg aataaaagtt tttgattcct taaaaaaaaa aaaaaa
2026 19 1243 DNA Homo sapiens misc_feature Incyte ID No. 2591695CB1
19 tccgcgggct agcgggcggt gggggcgcca cagcgcggaa ggcgggcacg
cgggccatgg 60 ctccctgggc ggaggccgag cactcggcgc tgaacccgct
gcgcgcggtg tggctcacgc 120 tgaccgccgc cttcctgctg accctactgc
tgcagctcct gccgcccggc ctgctcccgg 180 gctgcgcgat cttccaggac
ctgatccgct atgggaaaac caagtgtggg gagccgtcgc 240 gccccgccgc
ctgccgagcc tttgatgtcc ccaagagata tttttcccac ttttatatca 300
tctcagtgct gtggaatggc ttcctgcttt ggtgccttac tcaatctctg ttcctgggag
360 caccttttcc aagctggctt catggtttgc tcagaattct cggggcggca
cagttccagg 420 gaggggagct ggcactgtct gcattcttag tgctagtatt
tctgtggctg cacagcttac 480 gaagactctt cgagtgcctc tacgtcagtg
tcttctccaa tgtcatgatt cacgtcgtgc 540 agtactgttt tggacttgtc
tattatgtcc ttgttggcct aactgtgctg agccaagtgc 600 caatggatgg
caggaatgcc tacataacag ggaaaaatct attgatgcaa gcacggtggt 660
tccatattct tgggatgatg atgttcatct ggtcatctgc ccatcagtat aagtgccatg
720 ttattctcgg caatctcagg aaaaataaag caggagtggt cattcactgt
aaccacagga 780 tcccatttgg agactggttt gaatatgttt cttcccctaa
ctacttagca gagctgatga 840 tctacgtttc catggccgtc acctttgggt
tccacaactt aacttggtgg ctagtggtga 900 caaatgtctt ctttaatcag
gccctgtctg cctttctcag ccaccaattc tacaaaagca 960 aatttgtctc
ttacccgaag cataggaaag ctttcctacc atttttgttt taagttaacc 1020
tcagtcatga agaatgcaaa ccaggtgatg gtttcaatgc ctaaggacag tgaagtctgg
1080 agcccaaagt acagtttcag caaagctgtt tgaaactctc cattccattt
ctatacccca 1140 caagttttca ctgaatgagc atggcagtgc cactcaagaa
aatgaatctc caaagtatct 1200 tcaaagaata aatactaatg gcagatctgc
gaaaaaaaaa aaa 1243 20 1921 DNA Homo sapiens misc_feature Incyte ID
No. 474100CB1 20 gtgaagctgc tcctcacgtt ttggcgtgcc tgcgctctct
gcaggcagaa gcgaacaaag 60 acccagcaag agaaggcaga ggctaagacc
catcccgtat ctgctctcct gaaataattc 120 tggagtcatg cctgaaatgc
cagaggacat ggagcaggag gaagttaaca tccctaatag 180 gagggttctg
gttactggtg ccactgggct tcttggcaga gctgtacaca aagaatttca 240
gcagaataat tggcatgcag ttggctgtgg tttcagaaga gcaagaccaa aatttgaaca
300 ggttaatctg ttggattcta atgcagttca tcacatcatt catgattttc
agccccatgt 360 tatagtacat tgtgcagcag agagaagacc agatgttgta
gaaaatcagc cagatgctgc 420 ctctcaactt aatgtggatg cttctgggaa
tttagcaaag gaagcagatt tttttttttt 480 ttttgtagct gctgttggag
catttctcat ctacattagc tcagattatg tatttgatgg 540 aacaaatcca
ccttacagag aggaagacat accagctccc ctaaatttgt atggcaaaac 600
aaaattagat ggagaaaagg ctgtcctgga gaacaatcta ggagctgctg ttttgaggat
660 tcctattctg tatggggaag ttgaaaagct cgaagaaagt gctgtgactg
ttatgtttga 720 taaagtgcgg ttcagcaaca agtcagcaaa catggatcac
tggcagcaga ggttccccac 780 acatgtcaaa gatgtggcca ctgtgtgccg
gcagctagca gagaagagaa tgctggatcc 840 atcaattaag ggaacctttc
actggtctgg caatgaacag atgactaagt atgaaatggc 900 atgtgcaatt
gcagatgcct tcaacctccc cagcagtcac ttaagaccta ttactgacag 960
ccctgtccta ggagcacaac gtccgagaaa tgctcagctt gactgctcca aattggagac
1020 cttgggcatt ggccaacgaa caccatttcg aattggaatc aaagaatcac
tttggccttt 1080 cctcattgac aagagatgga gacaaacggt ctttcattag
tttatttgtg ttgggttctt 1140 tttttttttt aaatgaaaag tatagtatgt
ggcacttttt aaagaacaaa ggaaatagtt 1200 ttgtatgagt actttaattg
tgactcttag gatctttcag gtaaatgatg ctcttgcact 1260 agtgaaattg
tctaaagaaa ctaaagggca gtcatgccct gtttgcagta atttttcttt 1320
ttatcatttt gtttgtcctg gctaaacttg gagtttgagt atagtaaatt atgatcctta
1380 aatatttgag agtcaggatg aagcagatct gctgtagact tttcagatga
aattgttcat 1440 tctcgtaacc tccatatttt caggattttt gaagctgttg
accttttcat gttgattatt 1500 ttaaattgtg tgaaatagta taaaaatcat
tggtgttcat tatttgcttt gcctgagctc 1560 agatcaaaat gtttgaagaa
aggaacttta tttttgcaag ttacgtacag tttttatgct 1620 tgagatattt
caacatgtta tgtatattgg aacttctaca gcttgatgcc tcctgctttt 1680
atagcagttt atggggagca cttgaaagag cgtgtgtaca tgtatttttt ttctaggcaa
1740 acattgaatg caaacgtgta tttttttaat ataaatatat aactgtcctt
ttcatcccat 1800 gttgccgcta agtgatattt catatgtgtg gttatactca
taataatggg ccttgtaagt 1860 cttttcacca ttcatgaata ataataaata
tgtactgctg gcatgtaaaa aaaaaaaaaa 1920 a 1921 21 1261 DNA Homo
sapiens misc_feature Incyte ID No. 1304767CB1 21 ggggcctgga
ctcccgagtc tgagggagga gggactgagg cttctggatt cctgggtctg 60
tgggaggagg gactggggct cctggattcc tgggtctgtg ggaagagagg agtgtctgag
120 ggaggagggg ctggggacaa acttccaggt ctctagcctt cctggcaacg
ccccccgagg 180 ccggacttcc aggatccagc ctctattgag gatttgatgc
gacggcctca cggggctttg 240 gaggtgaaag aggcccagag tagagagaga
gagagaccga cgtacacggg atggctacgg 300 gaacgcgcta tgccgggaag
gtggtggtcg tgaccggcat cggagctggg atcgtgcgcg 360 ccttcgtgga
cagcggggcc cgagtggtta tctgcgacaa ggatgagtct gggggccggg 420
ccctggagca ggagctccct ggagctgtct ttatcctctg tgatgtgact caggaagatg
480 atgtgaagac cctggtttct gagaccatcc gccgatttgg ccgcctggat
tgtgttgtca 540 acaacgctgg ccaccaccca cccccacaga ggcctgagga
gacctctgcc cagggattcc 600 gccagctgct ggagctgaac ctactgggga
cgtacacctt gaccaagctc gccctcccct 660 acctgcggaa gagtcaaggg
aatgtcatca acatctccag cctggtgggg gcaatcggcc 720 aggcccaggc
agttccctat gtggccacca agggggcagt aacagccatg accaaagctt 780
tggccctgga tgaaagtcca tatggtgtcc gagtcaactg tatctcccca ggaaacatct
840 ggaccccgct gtgggaggag ctggcagcct taatgccaga ccctagggcc
acaatccgag 900 agggcatgct ggcccagcca ctgggccgca tgggccagcc
cgctgaggtc ggggctgcgg 960 cagtgttcct ggcctccgaa gccaacttct
gcacgggcat tgaactgctc gtgacggggg 1020 gtgcagagct ggggtacggg
tgcaaggcca gtcggagcac ccccgtggac gcccccgata 1080 tcccttcctg
atttctctca tttctacttg gggccccctt cctaggactc tcccacccca 1140
aactccaacc tgtatcagat gcagccccca agcccttaga ctctaagccc agttagcaag
1200 gtgccgggtc accctgcagg ttcccataaa aacgatttgc agccagaaaa
aaaaaaaaaa 1260
a 1261 22 1801 DNA Homo sapiens misc_feature Incyte ID No.
1465978CB1 22 cagctttttt tttttttttt tttttttttt taatcttgca
ctttgaaacc gcgggaccga 60 ggcagggtgc gcgcgtgtgg ttggtgcctt
tttttttttt tcttcccctc cctaaactcc 120 tctgtcagtc tgtaaacatt
acctgagaat tccccagccg aaacggctgc tggggcaaga 180 aacttcttgt
tagaactttc cacctccggc ttccccctcc acctctttta ccgtcccaac 240
cttaggagac gctttttctc ccccagagga gaatttatct tttttttttt tttttttttc
300 tttttctcac ccggtgcttt gcatttggga agaggtgatt tcaagagtgg
ccaggtggga 360 cgcctctctc ctccttattc ggtttactat ttattgttcg
gggtgttttt taattcctgt 420 attgctcggc ccggggagtt tcgccccctg
cccggctccg cggcgcggag gatggtgtgg 480 aaacggctgg gcgcgctggt
gatgttccct ctacagatga tctatctggt ggtgaaagca 540 gccgtcggac
tggtgctgcc cgccaagctg cgggacctgt cgcgggagaa cgtcctcatc 600
accggcggcg ggagaggcat cgggcgtcag ctcgcccgcg agttcgcgga gcgcggcgcc
660 agaaagattg ttctctgggg ccggactgag aaatgcctga aggagacgac
ggaggagatc 720 cggcagatgg gcactgagtg ccattacttc atctgtgatg
tgggcaaccg ggaggaggtg 780 taccagacgg ccaaggccgt ccgggagaag
gtgggtgaca tcaccatcct ggtgaacaat 840 gccgccgtgg tccatgggaa
gagcctaatg gacagtgatg atgatgccct cctcaagtcc 900 caacacatca
acaccctggg ccagttctgg accaccaagg ccttcctgcc acgtatgctg 960
gagctgcaga atggccacat cgtgtgcctc aactccgtgc tggcactgtc tgccatcccc
1020 ggtgccatcg actactgcac atccaaagcg tcagccttcg ccttcatgga
gagcctgacc 1080 ctggggctgc tggactgtcc gggagtcagc gccaccacag
tgctgccctt ccacaccagc 1140 accgagatgt tccagggcat gagagtcagg
tttcccaacc tctttccccc actgaagccg 1200 gagacggtgg cccggaggac
agtggaagct gtgcagctca accaggccct cctcctcctc 1260 ccatggacaa
tgcatgccct cgttatcttg aaaagcatac ttccacaggc tgcactcgag 1320
gagatccaca aattctcagg aacctacacc tgcatgaaca ctttcaaagg gcggacatag
1380 agacaggatg aagacatgct tgaggagcca cggagtttgg gggccacagc
acctgggcac 1440 acacccgagc acctgtccat tggcatgctt ctgctgggtg
agcaggacag ctcctgtccc 1500 cagcgaagaa tccggctgcc cctgggccag
tcccaggacc tttgcacagg actgatgggt 1560 gtaactgacc cccacaggga
ggcaggaaaa cagccagaag ccaccttgac acttttgaac 1620 atttccagtt
ctgtagagtt tattgtcaat tgcttctcaa gtctaaccag cctcagcagt 1680
gtgcatagac catttccagg agggtctgtc cccagatgct ctgcctcccg ttccaaaacc
1740 cactcatcct cagcttgcac aaactggttg aacggcagga atgaaaaata
aagacgagat 1800 a 1801 23 1711 DNA Homo sapiens misc_feature Incyte
ID No. 1635966CB1 23 tttgatacgg gagttcctcc ttgctctcgc ccctactctt
tctggtgtta gatcgagcta 60 ccctctaaaa gcagtttaga gtggtaaaaa
aaaaaaaaaa acacaccaaa cgctcgcagc 120 cacaaaaggg atgaaatttc
ttctggacat cctcctgctt ctcccgttac tgatcgtctg 180 ctccctagag
tccttcgtga agctttttat tcctaagagg agaaaatcag tcaccggcga 240
aatcgtgctg attacaggag ctgggcatgg aattgtgaga ctgactgcct atgaatttgc
300 taaacttaaa agcaagctgg ttctctggga tataaataag catggactgg
aggaaacagc 360 tgccaaatgc aagggactgg gtgccaaggt tcataccttt
gtggtagact gcagcaaccg 420 agaagatatt tacagctctg caaagaaggt
gaaggcagaa attggagatg ttagtatttt 480 agtaaataat gctggtgtag
tctatacatc agatttgttt gctacacaag atcctcagat 540 tgaaaagact
tttgaagtta atgtacttgc acatttctgg actacaaagg catttcttcc 600
tgcaatgacg aagaataacc atggccatat tgtcactgtg gcttcggcag ctggacatgt
660 ctcggtcccc ttcttactgg cttactgttc aagcaagttt gctgctgttg
gatttcataa 720 aactttgaca gatgaactgg ctgccttaca aataactgga
gtcaaaacaa catgtctgtg 780 tcctaatttc gtaaacactg gcttcatcaa
aaatccaagt acaagtttgg gacccactct 840 ggaacctgag gaagtggtaa
acaggctgat gcatgggatt ctgactgagc agaagatgat 900 ttttattcca
tcttctatag cttttttaac aacattggaa aggatccttc ctgagcgttt 960
cctggcagtt ttaaaacgaa aaatcagtgt taagtttgat gcagttattg gatataaaat
1020 gaaagcgcaa taagcaccta gttttctgaa aactgattta ccaggtttag
gttgatgtca 1080 tctaatagtg ccagaatttt aatgtttgaa cttctgtttt
ttctaattat ccccatttct 1140 tcaatatcat ttttgaggct ttggcagtct
tcatttacta ccacttgttc tttagccaaa 1200 agctgattac atatgatata
aacagagaaa tacctttaga ggtgacttta aggaaaatga 1260 agaaaaagaa
ccaaaatgac tttattaaaa taatttccaa gattatttgt ggctcacctg 1320
aaggctttgc aaaatttgta ccataaccgt ttatttaaca tatattttta tttttgattg
1380 cacttaaatt ttgtataatt tgtgtttctt tttctgttct acataaaatc
agaaacttca 1440 agctctctaa ataaaatgaa ggactatatc tagtggtatt
tcacaatgaa tatcatgaac 1500 tctcaatggg taggtttcat cctacccatt
gccactctgt ttcctgagag atacctcaca 1560 ttccaatgcc aaacatttct
gcacagggaa gctagaggtg gatacacgtg ttgcaagtat 1620 aaaagcatca
ctgggattta aggagaattg agagaatgta cccacaaatg gcagcaataa 1680
taaatggatc acacttaaaa aaaaaaaaaa a 1711 24 2222 DNA Homo sapiens
misc_feature Incyte ID No. 1638410CB1 24 cttccccggc tctagcaggc
cggcttctct gtccaatgcc cacccggagc tgggaggagg 60 agtctgcgta
atgtgcgtgt gaagagactg ggggagctgg ccggggctca cggtgtttga 120
cccgtcggtc gtgcgtgaga ggaaagggaa ggaggaggtc ccgaatagcg gtcgccgaaa
180 tgttccggtg tggaggcctg gcggcgggtg ctttgaagca gaagctggtg
cccttggtgc 240 ggaccgtgtg cgtccgaagc ccgaggcaga ggaaccggct
cccaggcaac ttgttccagc 300 gatggcatgt tcctctagaa ctccagatga
caagacaaat ggctagctct ggtgcatcag 360 ggggcaaaat cgataattct
gtgttagtcc ttattgtggg cttatcaaca gtaggagctg 420 gtgcctatgc
ctacaagact atgaaagagg acgaaaaaag atacaatgaa agaatttcag 480
ggttagggct gacaccagaa cagaaacaga aaaaggccgc gttatctgct tcagaaggag
540 aggaagttcc tcaagacaag gcgccaagtc atgttccttt cctgctaatt
ggtggaggca 600 cagctgcttt tgctgcagcc agatccatcc gggctcggga
tcctggggcc agggtactga 660 ttgtatctga agatcctgag ctgccgtaca
tgcgacctcc tctttcaaaa gaactgtggt 720 tttcagatga cccaaatgtc
acaaagacac tgcgattcaa acagtggaat ggaaaagaga 780 gaagcatata
tttccagcca ccttctttct atgtctctgc tcaggacctg cctcatattg 840
agaatggtgg tgtggctgtc ctcactggga agaaggtagt acagctggat gtgagagaca
900 acatggtgaa acttaatgat ggctctcaaa taacctatga aaagtgcttg
attgcaacag 960 gaggtactcc aagaagtctg tctgccattg atagggctgg
agcagaggtg aagagtagaa 1020 caacgctttt cagaaagatt ggagacttta
gaagcttgga gaagatttca cgggaagtca 1080 aatcaattac gattatcggt
gggggcttcc ttggtagcga actggcctgt gctcttggca 1140 gaaaggctcg
agccttgggc acagaagtga ttcaactctt ccccgagaaa ggaaatatgg 1200
gaaagatcct ccccgaatac ctcagcaact ggaccatgga aaaagtcaga cgagaggggg
1260 ttaaggtgat gcccaatgct attgtgcaat ccgttggagt cagcagtggc
aagttactta 1320 tcaagctgaa agacggcagg aaggtagaaa ctgaccacat
agtggcagct gtgggcctgg 1380 agcccaatgt tgagttggcc aagactggtg
gcctggaaat agactcagat tttggtggct 1440 tccgggtaaa tgcagagcta
caagcacgct ctaacatctg ggtggcagga gatgctgcat 1500 gcttctacga
tataaagttg ggaaggaggc gggtagagca ccatgatcac gctgttgtga 1560
gtggaagatt ggctggagaa aatatgactg gagctgctaa gccgtactgg catcagtcaa
1620 tgttctggag tgatttgggc cccgatgttg gctatgaagc tattggtctt
gtggacagta 1680 gtttgcccac agttggtgtt tttgcaaaag caactgcaca
agacaacccc aaatctgcca 1740 cagagcagtc aggaactggt atccgatcag
agagtgagac agagtccgag gcctcagaaa 1800 ttactattcc tcccagcacc
ccggcagttc cacaggctcc cgtccagggg gaggactacg 1860 gcaaaggtgt
catcttctac ctcagggaca aagtggtcgt ggggattgtg ctatggaaca 1920
tctttaaccg aatgccaata gcaaggaaga tcattaagga cggtgagcag catgaagatc
1980 tcaatgaagt agccaaacta ttcaacattc atgaagactg aagccccaca
gtggaattgg 2040 caaacccact gcagcccctg agaggaggtc gaatgggtaa
aggagcattt ttttattcag 2100 cagactttct ctgtgtatga gtgtgaatga
tcaagtcctt tgtgaatatt ttcaactatg 2160 taggtaaatt cttaatgttc
acatagtgaa ataaattctg attcttctaa attaaaaaaa 2220 aa 2222 25 1278
DNA Homo sapiens misc_feature Incyte ID No. 1743409CB1 25
gaggatgaag ttgattgact atggtctctc cggctaccag gaagagtctg ccgaaggtga
60 aggccatgga cttcatcacc tccacagcca tcctgcccct gctgttcggc
tgcctgggcg 120 tcttcggcct cttccggctg ctgcagtggg tgcgcgggaa
ggcctacctg cggaatgctg 180 tggtggtgat cacaggcgcc acctcagggc
tgggcaaaga atgtgcaaaa gtcttctatg 240 ctgcgggtgc taaactggtg
ctctgtggcc ggaatggtgg ggccctagaa gagctcatca 300 gagaactcac
cgcttctcat gccaccaagg tgcagacaca caagccttac ttggtgacct 360
tcgacctcac agactctggg gccatagttg cagcagcagc tgagatcctg cagtgctttg
420 gctatgtcga catacttgtc aacaatgctg ggatcagcta ccgtggtacc
atcatggaca 480 ccacagtgga tgtggacaag agggtcatgg agacaaacta
ctttggccca gttgctctaa 540 cgaaagcact cctgccctcc atgatcaaga
ggaggcaagg ccacattgtc gccatcagca 600 gcatccaggg caagatgagc
attccttttc gatcagcata tgcagcctcc aagcacgcaa 660 cccaggcttt
ctttgactgt ctgcgtgccg agatggaaca gtatgaaatt gaggtgaccg 720
tcatcagccc cggctacatc cacaccaacc tctctgtaaa tgccatcacc gcggatggat
780 ctaggtatgg agttatggac accaccacag cccagggccg aagccctgtg
gaggtggccc 840 aggatgttct tgctgctgtg gggaagaaga agaaagatgt
gatcctggct gacttactgc 900 cttccttggc tgtttatctt cgaactctgg
ctcctgggct cttcttcagc ctcatggcct 960 ccagggccag aaaagagcgg
aaatccaaga actcctagta ctctgaccag ccagggccag 1020 ggcagagaag
cagcactctt aggcttgctt actctacaag ggacagttgc atttgttgag 1080
actttaatgg agatttgtct cacaagtggg aaagactgaa gaaacacatc tcgtgcagat
1140 ctgctggcag aggacaatca aaaacgacaa caagcttctt cccagggtga
ggggaaacac 1200 ttaaggaata aatatggagc tggggtttaa cactaaaaac
tagaaataaa catctcaaac 1260 agtaaaaaaa aaaaaaaa 1278 26 1614 DNA
Homo sapiens misc_feature Incyte ID No. 1803830CB1 26 gttttggggg
tgaaaaggca aaaggcgggt gaaaggctgc ctcccgagac tctccttgct 60
tggaattctg cccactctgc ggagttagca gtcacgacct ccagcacagg atgtggtacc
120 acagattgtc ccacctacac agcaggcttc aggacttgct gaagggagga
gtcatatatc 180 cggcccttcc acagcccaac ttcaaaagct tacttccttt
agctgtccat tggcaccata 240 cagcctccaa gtctctgact tgtgcttggc
agcaacatga agatcatttt gagctgaaat 300 atgctaatac cgtgatgcgc
cttgattacg tctggcttcg agaccactgc cgctcagcat 360 cgtgctacaa
ctctaagact caccagcgca gctgggatac tgccagtgtg gatttatgta 420
tcaagccaaa gaccattcgt ctggatgaga ccacactctt tttcacttgg ccagatggtc
480 atgtgactaa atatgatttg aattggctgg tgaaaaacag ctatgaaggg
cagaaacaaa 540 aagtcatcca gcctagaata ctatggaatg ctgaaatcta
ccagcaagcc caagttccat 600 cggtagattg ccagagcttc ttagaaacca
acgagggact gaagaagttt ctgcaaaact 660 ttctgctcta tggaattgca
ttcgtagaaa atgtccctcc cactcaagag cacacagaga 720 agttggcaga
aaggatcagc ttaatcagag aaaccattta tgggaggatg tggtatttca 780
cttcagactt ctccagaggt gacactgcgt acaccaagct agctctggat cggcacactg
840 acactaccta ttttcaagag ccctgtggca ttcaagtgtt tcattgtctt
aaacatgaag 900 gaactggtgg caggacactg ctagtagatg gattctatgc
agcagaacag gtacttcaaa 960 aggcacctga ggaatttgaa ctcctcagta
aagtgccatt gaagcatgaa tatattgaag 1020 atgttggaga atgtcacaac
cacatgattg ggattgggcc agtcttaaat atctacccat 1080 ggaataaaga
gctgtatttg atcaggtaca acaactatga ccgggctgtc atcaataccg 1140
ttccttatga tgtcgtccat cgctggtata cagcacaccg gactctaacg atagagttga
1200 ggagacctga gaatgagttt tgggtcaaac taaagcctgg cagggtccta
tttatagaca 1260 actggcgtgt cctacatggc agggaatgct tcactggcta
ccgccaactg tgtggctgct 1320 atttaacaag agatgatgta ttaaacactg
ctcgcctctt ggggcttcag gcttaaaatt 1380 gacagcatct ggattatgaa
tacacctggc accctggcta ccagaatttc atatgggcag 1440 aataatattg
tgtcaaactc tacttcagat tgtctcctta tcccatccca caaaacagaa 1500
tctgtccgtt tctctagtaa gggagacttg ttggagaggc gggactctga gttatctaat
1560 gtcagacatc tagtggggca gctctcttcc tcgtgttata acatgcatac ccgt
1614 27 3136 DNA Homo sapiens misc_feature Incyte ID No. 1867333CB1
27 cgacgccggc gtgatgtggc ttccgctggt gctgctcctg gctgtgctgc
tgctggccgt 60 cctctgcaaa gtttacttgg gactattctc tggcagctcc
ccgaatcctt tctccgaaga 120 tgtcaaacgg cccccagcgc ccctggtaac
tgacaaggag gccaggaaga aggttctcaa 180 acaagctttt tcagccaacc
aagtgccgga gaagctggat gtggtggtaa ttggcagtgg 240 ctttgggggc
ctggctgcag ctgcaattct agctaaagct ggcaagcgag tcctggtgct 300
ggaacaacat accaaggcag ggggctgctg tcataccttt ggaaagaatg gccttgaatt
360 tgacacagga atccattaca ttgggcgtat ggaagagggc agcattggcc
gttttatctt 420 ggaccagatc actgaagggc agctggactg ggctcccctg
tcctctcctt ttgacatcat 480 ggtactggaa gggcccaatg gccgaaagga
gtaccccatg tacagtggag agaaagccta 540 cattcagggc ctcaaggaga
agtttccaca ggaggaagct atcattgaca agtatataaa 600 gctggttaag
gtggtatcca gtggagcccc tcatgccatc ctgttgaaat tcctcccatt 660
gcccgtggtt cagctcctcg acaggtgtgg gctgctgact cgtttctctc cattccttca
720 agcatccacc cagagcctgg ctgaggtcct gcagcagctg ggggcctcct
ctgagctcca 780 ggcagtactc agctacatct tccccactta cggtgtcacc
cccaaccaca gtgccttttc 840 catgcacgcc ctgctggtca accactacat
gaaaggaggc ttttatcccc gagggggttc 900 cagtgaaatt gccttccaca
ccatccctgt gattcagcgg gctgggggcg ctgtcctcac 960 aaaggccact
gtgcagagtg tgttgctgga ctcagctggg aaagcctgtg gtgtcagtgt 1020
gaagaagggg catgagctgg tgaacatcta ttgccccatc gtggtctcca acgcaggact
1080 gttcaacacc tatgaacacc tactgccggg gaacgcccgc tgcctgccag
gtgtgaagca 1140 gcaactgggg acggtgcggc ccggcttagg catgacctct
gttttcatct gcctgcgagg 1200 caccaaggaa gacctgcatc tgccgtccac
caactactat gtttactatg acacggacat 1260 ggaccaggcg atggagcgct
acgtctccat gcccagggaa gaggctgcgg aacacatccc 1320 tcttctcttc
ttcgctttcc catcagccaa agatccgacc tgggaggacc gattcccagg 1380
ccggtccacc atgatcatgc tcatacccac tgcctacgag tggtttgagg agtggcaggc
1440 ggagctgaag ggaaagcggg gcagtgacta tgagaccttc aaaaactcct
ttgtggaagc 1500 ctctatgtca gtggtcctga aactgttccc acagctggag
gggaaggtgg agagtgtgac 1560 tgcaggatcc ccactcacca accagttcta
tctggctgct ccccgaggtg cctgctacgg 1620 ggctgaccat gacctgggcc
gcctgcaccc ttgtgtgatg gcctccttga gggcccagag 1680 ccccatcccc
aacctctatc tgacaggcca ggatatcttc acctgtggac tggtcggggc 1740
cctgcaaggt gccctgctgt gcagcagcgc catcctgaag cggaacttgt actcagacct
1800 taagaatctt gattctagga tccgggcaca gaagaaaaag aattagttcc
atcagggagg 1860 agtcagagga atttgcccaa tggctggggc atctcccttg
acttacccat aatgtctttc 1920 tgcattagtt ccttgcacgt ataaagcact
ctaatttggt tctgatgcct gaagagaggc 1980 ctagtttaaa tcacaattcc
gaatctgggg caatggaatc actgcttcca gctggggcag 2040 gtgagatctt
tacgcctttt ataacatgcc atccctacta ataggatatt gacttggata 2100
gcttgatgtc tcatgacgag cggcgctctg catccctcac ccatgcctcc taactcagtg
2160 atcaaagcga atattccatc tgtggataga acccctggca gtgttgtcag
ctcaacctgg 2220 tgggttcagt tctgtcctga ggcttctgct ctcattcatt
tagtgctacg ctgcacagtt 2280 ctacactgtc aagggaaaag ggagactaat
gaggcttaac tcaaaacctg ggcatggttt 2340 tggttgccat tccataggtt
tggagagctc tagatctctt ttgtgctggg ttcagtggct 2400 cttcagggga
caggaaatgc ctgtgtctgg ccagtgtggt tctggagctt tggggtaaca 2460
gcaggatcca tcagttagta gggtgcatgt cagatgatca tatccaattc atatggaagt
2520 cccgggtctg tcttccttat catcggggtg gcagctggtt ctcaatgtgc
cagcagggac 2580 tcagtacctg agcctcaatc aagccttatc caccaaatac
acagggaagg gtgatgcagg 2640 gaagggtgac atcaggagtc agggcatgga
ctggtaagat gaatactttg ctgggctgaa 2700 gcaggctgca gggcattcca
gccaagggca cagcagggga cagtgcaggg aggtgtgggg 2760 taagggaggg
aagtcacatc agaaaaggga aagccacgga atgtgtgtga agcccagaaa 2820
tggcatttgc agttaattag cacatgtgag ggttagacag gtaggtgaat gcaagctcaa
2880 ggtttggaaa aatgactttt cagttatgtc tttggtatca gacatacgaa
aggtctcttt 2940 gtagttcgtg ttaatgtaac attaataaat ttattgattc
cattgcttta acatttgaaa 3000 tttattttgg ttttttgttc aagaaaacaa
aactattatt gtgatggcat ttgcagaagc 3060 tcagtaaaac actatatact
gaataacacc aaaataagct ttaaaaaaat aaaattaagt 3120 aattataaaa aaaaaa
3136 28 1594 DNA Homo sapiens unsure 1530 a or g or c or t,
unknown, or other 28 cccacgcgtc cgcggacgcg tgggggcgtt cagactctta
gctgaacgcg gactgcggcg 60 gctatgctgt ggagcggctg ccggcgtttc
ggggcgcgcc tcggctgcct gcccggcggt 120 ctccgggtcc tcgtccagac
cggccaccgg agcttgacct cctgcatcga cccttccatg 180 ggacttaatg
aagagcagaa agaatttcaa aaagtggcct ttgactttgc tgcccgagag 240
atggctccaa atatggcaga gtgggaccag aaggagctgt tcccagtgga tgtgatgcgg
300 aaggcagccc agctaggctt cggaggggtc tacatacaaa cagatgtggg
cgggtctggg 360 ctgtcacgtc ttgatacctc tgtcattttt gaagccttgg
ctacaggctg caccagcacc 420 acagcctata taagcatcca caacatgtgt
gcctggatga ttgatagctt cggaaatgag 480 gaacagaggc acaaattttg
cccaccgctc tgtaccatgg agaagtttgc ttcctactgc 540 ctcactgaac
caggaagtgg gagtgatgct gcctctcttc tgacctccgc taagaaacag 600
ggagatcatt acatcctcaa tggctccaag gccttcatca gtggtgctgg tgagtcagac
660 atctatgtgg tcatgtgccg aacaggagga ccaggcccca agggcatctc
atgcatagtt 720 gttgagaagg ggacccctgg cctcagcttt ggcaagaagg
agaaaaaggt ggggtggaac 780 tcccagccaa cacgagctgt gatcttcgaa
gactgtgctg tccctgtggc caacagaatt 840 gggagcgagg ggcagggctt
cctcattgcc gtgagaggac tgaacggagg gaggatcaat 900 attgcttcct
gctccctggg ggctgcccac gcctctgtca tcctcacccg agaccacctc 960
aatgtccgga agcagtttgg agagcctctg gccagtaacc agtacttgca attcacactg
1020 gctgatatgg caacaaggct ggtggccgcg cggctgatgg tccgcaatgc
agcagtggct 1080 ctgcaggagg agaggaagga tgcagtggcc ttgtgctcca
tggccaagct ctttgctaca 1140 gatgaatgct ttgccatctg caaccaggcc
ttgcagatgc acgggggcta cggctacctg 1200 aaggattacg ctgttcagca
gtacgtgcgg gactccaggg tccaccagat tctagaaggt 1260 agcaatgaag
tgatgaggat actgatctct agaagcctgc ttcaggagta gaacccacac 1320
ttgttctggc ctggtgttca gtgcgactgc agtcagtgtt gagtggtgcc atgtgggccg
1380 ctctattcca aaggaatcat ggattagacc caaaggctga gctcctctag
ggcaggacct 1440 gcaccctgtg tgttggcacc agcatcgggt cttggactgg
gggcagatcc ccagtggaac 1500 cggaagagct ggactgatga gaaacatcan
ggaagacaca tactaccctt ggttttccta 1560 atgcccagaa gggtgaccag
tgaaagattc accc 1594 29 1338 DNA Homo sapiens misc_feature Incyte
ID No. 3294314CB1 29 ggtgagcgca gtctgtccga ggcaacaaga tggcagctgc
ggagccgtct ccgcggcgcg 60 tgggcttcgt gggcgcgggc cgcatggcgg
gggccatcgc gcagggcctc atcagagcag 120 gaaaagtgga agctcagcac
atactggcca gtgcaccaac agacaggaac ctatgtcact 180 ttcaagctct
gggttgccgg accacgcact ccaaccagga ggtgctgcag agctgcctgc 240
tcgtcatctt tgccaccaag cctcatgtgc tgccagctgt cctggcagag gtggctcctg
300 tggtcaccac tgaacacatc ttggtgtccg tggctgctgg ggtgtctctg
agcaccctgg 360 aggagctgct gcccccaaac acacgggtgc tgcgggtctt
gcccaacctg ccctgtgtgg 420 tccaggaagg ggccatagtg atggcgcggg
gccgccacgt ggggagcagc gagaccaagc 480 tcctgcagca tctgctggag
gcctgtgggc ggtgtgagga ggtgcctgaa gcctacgtcg 540 acatccacac
tggcctcagt ggcagtggcg tggccttcgt gtgtgcattc tccgaggccc 600
tggctgaagg agccgtcaag atgggcatgc ccagcagcct ggcccaccgc atcgctgccc
660 agaccctgct ggggacggcc aagatgctgc tgcacgaggg ccaacaccca
gcccagctgc 720 gctcagacgt gtgcaccccg ggtggcacca ccatctatgg
actccacgcc ctggagcagg 780 gcgggctgcg agcagccacc atgagcgccg
tggaggctgc cacctgccgg gccaaggagc 840 tcagcagaaa gtaggctggg
ctctggccat cctttcctgc ctctgtgccc ctgcctctcc 900
ctgtgtccct tcccctgagg actgcggctc cctccctcct gcatgagggt ctcctactgc
960 tccttctccc cttgcacagg gaaatgcagg gggcaggact tgggaggttc
cagcaggcgg 1020 gggagccccg accagtgggg acactcctcc ctccccagtg
agcagaaggc accgtggtgg 1080 tggctctgcc ccttgctgca gtgagcccac
cttgctgcaa cattggttct gaggggccca 1140 agagatggcg tcttggtcat
ttgcccgcat ggttgggcag ttggttgagg ccatgaacag 1200 aacttacggt
aacaggcacg gctggcccaa tgcctggtct ggagctggag cttgcctttg 1260
gctttccaag tgggctcgtg cagctacagc caggccggct gcctcatctc agctctaggg
1320 ggcacgagca tatggggt 1338 30 1091 DNA Homo sapiens misc_feature
Incyte ID No. 4940951CB1 30 ccagaaggtc accgccatgg ccgcgccctt
cttttccact cctttccagc cctacgtcta 60 ccagagccag caaggatctg
tgacggcgtt tcagatatcc ggtggagatg tgcaggtcct 120 gcaggtgatg
ctgaagtctc aggagaagct gactgcaaaa ccaggtgcaa tgtgctacat 180
gtctgggaat atgcagatgg acaacaatta cttgcctgaa aatgatggag gcgtgtggca
240 gtggattttt gggaaacgtg taagcagcac cattttcttt aattctggat
ctgatgatgg 300 atatgtcggg attgctgcac catttcctgg gaggatactg
ccggtagatc taacaaactt 360 tagtggagaa cttctttgcc aggcagatgc
ttttctatgt tcggtcaatg atgtctccgt 420 ctctagtaca gttgagccaa
ggccacggaa tattgagatt ggtgcagaga tgatccttaa 480 acaaaaactt
aggggccagg ggatggcttt tcttgttggt ggtggatcag tcatgcagaa 540
aatccttgct cctagagagg tgataactgt tgatgctgct tgtattgtgg ctatgtcggc
600 caccattaac ttccagttga agagccctaa ccagcttaga agagcagttt
ttgggggtga 660 taaccagcta acagcatctc tcacgggacc aggtgttgtt
ttcattcaaa gtctgccatt 720 ccatcgactc tcacagagaa tcgccagcag
tagatcagtg gcaggcccaa gcttgaggga 780 caacccaaag ttcttcatcc
agattgtcat gttcttcttc ctggcctatg ttatgattgt 840 atcatccata
attctgacag atgtttaagc gattcagtga gcttttggtg tattcctaga 900
caagttatcg aagagttaaa gctacctccc caatgttaat gtagatgtaa gagaacgaat
960 ttcacaagct gttgttagaa accttagcag aaggttcttc attttttttt
ctctaacagt 1020 ttggaggggg cggggcgggt ttcttggtta gcgtgtaaag
aggaagacag caaaatcaag 1080 ttgcacagcg a 1091
* * * * *