U.S. patent application number 10/149826 was filed with the patent office on 2004-11-11 for g-protein coupled receptors.
Invention is credited to Au-Young, Janice, Baughn, Mariah R., Burford, Neil, Lu, Dyung Aina M., Reddy, Roopa, Yang, Junming.
Application Number | 20040224314 10/149826 |
Document ID | / |
Family ID | 27496978 |
Filed Date | 2004-11-11 |
United States Patent
Application |
20040224314 |
Kind Code |
A1 |
Burford, Neil ; et
al. |
November 11, 2004 |
G-protein coupled receptors
Abstract
The invention provides human G-protein coupled receptors (GCREC)
and polynucleotides which identify and encode GCREC. 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
aberrant expression of GCREC.
Inventors: |
Burford, Neil; (Durham,
CT) ; Baughn, Mariah R.; (San Leandro, CA) ;
Au-Young, Janice; (Brisbane, CA) ; Yang, Junming;
(San Jose, CA) ; Lu, Dyung Aina M.; (San Jose,
CA) ; Reddy, Roopa; (Sunnyvale, CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Family ID: |
27496978 |
Appl. No.: |
10/149826 |
Filed: |
June 10, 2002 |
PCT Filed: |
December 7, 2000 |
PCT NO: |
PCT/US00/33382 |
Related U.S. Patent Documents
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60172852 |
Dec 10, 1999 |
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60171732 |
Dec 22, 1999 |
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60176148 |
Jan 14, 2000 |
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60177331 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 13/08 20180101; A61P 37/00 20180101; A61P 5/18 20180101; A61P
17/16 20180101; A61P 21/04 20180101; A61P 25/20 20180101; A61P 1/14
20180101; A61P 7/10 20180101; A61P 19/02 20180101; A61P 25/04
20180101; A61P 27/02 20180101; A61P 19/04 20180101; A61P 3/00
20180101; A61P 9/10 20180101; A61P 17/06 20180101; A61P 25/28
20180101; A61P 29/00 20180101; A61P 31/16 20180101; A61P 33/14
20180101; A61P 19/00 20180101; C07K 14/47 20130101; C07K 14/78
20130101; A61P 15/08 20180101; A61P 13/10 20180101; A61P 25/00
20180101; A61P 1/00 20180101; A61P 3/10 20180101; A61P 15/00
20180101; A61P 19/10 20180101; A61P 33/00 20180101; A61P 41/00
20180101; C07K 14/705 20130101; A61P 25/02 20180101; A61P 21/00
20180101; A61P 1/06 20180101; A61P 35/00 20180101; A61P 17/00
20180101; A61P 1/16 20180101; A61P 25/22 20180101; A61P 1/08
20180101; A61P 5/38 20180101; A61P 31/22 20180101; A61P 31/12
20180101; A61P 25/08 20180101; A61P 35/02 20180101; A01K 2217/05
20130101; A61P 3/04 20180101; A61P 7/00 20180101; A61P 11/06
20180101; A61P 1/18 20180101; A61P 13/02 20180101; A61P 1/02
20180101; A61P 1/10 20180101; A61P 9/04 20180101; A61P 39/00
20180101; A61P 13/12 20180101; A61P 43/00 20180101; A61P 25/16
20180101; A61P 25/18 20180101; A61P 31/04 20180101; A61P 33/02
20180101; A61P 1/04 20180101; A61P 7/06 20180101; A61P 9/12
20180101; A61P 9/00 20180101; A61P 9/14 20180101; A61P 11/00
20180101; A61P 25/14 20180101; A61P 31/18 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/705; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: a) an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39, b) a
naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-39, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39.
2. An isolated polypeptide of claim 1 selected from the group
consisting of SEQ ID NO:1-39.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 selected from the group
consisting of SEQ ID NO:40-78.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. An isolated antibody which specifically binds to a polypeptide
of claim 1.
11. An isolated polynucleotide comprising a polynucleotide sequence
selected from the group consisting of: a) a polynucleotide sequence
selected from the group consisting of SEQ ID NO:40-78, b) a
naturally occurring polynucleotide sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the
group consisting of SEQ ID NO:40-78, c) a polynucleotide sequence
complementary to a), d) a polynucleotide sequence complementary to
b), and e) an RNA equivalent of a)-d).
12. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 11.
13. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
14. A method of claim 13, wherein the probe comprises at least 60
contiguous nucleotides.
15. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 11, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
16. A composition comprising an effective amount of a polypeptide
of claim 1 and a pharmaceutically acceptable excipient.
17. A composition of claim 16, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-39.
18. A method for treating a disease or condition associated with
decreased expression of functional GCREC, comprising administering
to a patient in need of such treatment the composition of claim
16.
19. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
20. A composition comprising an agonist compound identified by a
method of claim 19 and a pharmaceutically acceptable excipient.
21. A method for treating a disease or condition associated with
decreased expression of functional GCREC, comprising administering
to a patient in need of such treatment a composition of claim
20.
22. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
23. A composition comprising an antagonist compound identified by a
method of claim 22 and a pharmaceutically acceptable excipient.
24. A method for treating a disease or condition associated with
overexpression of functional GCREC, comprising administering to a
patient in need of such treatment a composition of claim 23.
25. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, said method comprising the steps of: a)
combining the polypeptide of claim 1 with at least one test
compound under suitable conditions, and b) detecting binding of the
polypeptide of claim 1 to the test compound, thereby identifying a
compound that specifically binds to the polypeptide of claim 1.
26. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
27. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
28. A method for assessing toxicity of a test compound, said method
comprising: a) treating a biological sample containing nucleic
acids with the test compound; b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 11 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 11 or fragment thereof; c)
quantifying the amount of hybridization complex; and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
Description
TECHNICAL FIELD
[0001] This invention relates to nucleic acid and amino acid
sequences of G-protein coupled receptors and to the use of these
sequences in the diagnosis, treatment, and prevention of cell
proliferative, neurological, cardiovascular, gastrointestinal,
autoimmune/inflammatory, and metabolic disorders, and viral
infections, and in the assessment of the effects of exogenous
compounds on the expression of nucleic acid and amino acid
sequences of G-protein coupled receptors.
BACKGROUND OF THE INVENTION
[0002] Signal transduction is the general process by which cells
respond to extracellular signals. Signal transduction across the
plasma membrane begins with the binding of a signal molecule, e.g.,
a hormone, neurotransmitter, or growth factor, to a cell membrane
receptor. The receptor, thus activated, triggers an intracellular
biochemical cascade that ends with the activation of an
intracellular target molecule, such as a transcription factor. This
process of signal transduction regulates all types of cell
functions including cell proliferation, differentiation, and gene
transcription. The G-protein coupled receptors (GPCRs), encoded by
one of the largest families of genes yet identified, play a central
role in the transduction of extracellular signals across the plasma
membrane. GPCRs have a proven history of being successful
therapeutic targets.
[0003] GPCRs are integral membrane proteins characterized by the
presence of seven hydrophobic transmembrane domains which together
form a bundle of antiparallel alpha (a) helices. GPCRs range in
size from under 400 to over 1000 amino acids (Strosberg, A. D.
(1991) Eur. J. Biochem. 196:1-10; Coughlin, S. R. (1994) Curr.
Opin. Cell Biol. 6:191-197). The amino-terminus of a GPCR is
extracellular, is of variable length, and is often glycosylated.
The carboxy-terminus is cytoplasmic and generally phosphorylated
Extracellular loops alternate with intracellular loops and link the
transmembrane domains. Cysteine disulfide bridges linking the
second and third extracellular loops may interact with agonists and
antagonists. The most conserved domains of GPCRs are the
transmembrane domains and the first two cytoplasmic loops. The
transmembrane domains account, in part, for structural and
functional features of the receptor. In most cases, the bundle of a
helices forms a ligand-binding pocket. The extracellular N-terminal
segment, or one or more of the three extracellular loops, may also
participate in ligand binding. Ligand binding activates the
receptor by inducing a conformational change in intracellular
portions of the receptor. In turn, the large, third intracellular
loop of the activated receptor interacts with a heterotrimeric
guanine nucleotide binding (G) protein complex which mediates
further intracellular signaling activities, including the
activation of second messengers such as cyclic AMP (cAMP),
phospholipase C, and inositol triphosphate, and the interaction of
the activated GPCR with ion channel proteins. (See, e.g., Watson,
S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts
Book, Academic Press, San Diego Calif., pp. 2-6; Bolander, F. F.
(1994) Molecular Endocrinology, Academic Press, San Diego Calif.,
pp. 162-176; Baldwin, J. M. (1994) Curr. Opin. Cell Biol.
6:180-190.)
[0004] GPCRs include receptors for sensory signal mediators (e.g.,
light and olfactory stimulatory molecules); adenosine,
.gamma.-aminobutyric acid (GABA), hepatocyte growth factor,
melanocortins, neuropeptide Y, opioid peptides, opsins,
somatostatin, tachykinins, vasoactive intestinal polypeptide
family, and vasopressin; biogenic amines (e.g., dopamine,
epinephrine and norepinephrine, histamine, glutamate (metabotropic
effect), acetylcholine (muscarinic effect), and serotonin);
chemokines; lipid mediators of inflammation (e.g., prostaglandins
and prostanoids, platelet activating factor, and leukotrienes); and
peptide hormones (e.g., bombesin, bradykinin, calcitonin, C5a
anaphylatoxin, endothelin, follicle-stimulating hormone (FSH),
gonadotropic-releasing hormone (GnRH), neurokinin, and
thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs which act
as receptors for stimuli that have yet to be identified are known
as orphan receptors.
[0005] The diversity of the GPCR family is further increased by
alternative splicing. Many GPCR genes contain introns, and there
are currently over 30 such receptors for which splice variants have
been identified. The largest number of variations are at the
protein C-terminus. N-terminal and cytoplasmic loop variants are
also frequent, while variants in the extracellular loops or
transmembrane domains are less common. Some receptors have more
than one site at which variance can occur. The splicing variants
appear to be functionally distinct, based upon observed differences
in distribution, signaling, coupling, regulation, and ligand
binding profiles (Kilpatrick, G. J. et al. (1999) Trends Pharmacol.
Sci. 20:294-301).
[0006] GPCRs can be divided into three major subfamilies: the
rhodopsin-like, secretin-like, and metabotropic glutamate receptor
subfamilies. Members of these GPCR subfamilies share similar
functions and the characteristic seven transmembrane structure, but
have divergent amino acid sequences. The largest family consists of
the rhodopsin-like GPCRs, which transmit diverse extracellular
signals including hormones, neurotransmitters, and light. Rhodopsin
is a photosensitive GPCR found in animal retinas. In vertebrates,
rhodopsin molecules are embedded in membranous stacks found in
photoreceptor (rod) cells. Each rhodopsin molecule responds to a
photon of light by triggering a decrease in cGMP levels which leads
to the closure of plasma membrane sodium channels. In this manner,
a visual signal is converted to a neural impulse. Other
rhodopsin-like GPCRs are directly involved in responding to
neurotransmitters. These GPCRs include the receptors for adrenaline
(adrenergic receptors), acetylcholine (muscarinic receptors),
adenosine, galanin, and glutamate (N-methyl-D-aspartate/NMDA
receptors). (Reviewed in Watson, S. and S. Arkinstall (1994) The
G-Protein Linked Receptor Facts Book, Academic Press, San Diego
Calif., pp. 7-9, 19-22, 32-35, 130-131, 214-216, 221-222;
Habert-Ortoli, E. et al. (1994) Proc. Natl. Acad. Sci. USA
91:9780-9783.)
[0007] The galanin receptors mediate the activity of the
neuroendocrine peptide galanin, which inhibits secretion of
insulin, acetylcholine, serotonin and noradrenaline, and stimulates
prolactin and growth hormone release. Galanin receptors are
involved in feeding disorders, pain, depression, and Alzheimer's
disease (Kask, K. et al. (1997) Life Sci. 60:1523-1533). Other
nervous system rhodopsin-like GPCRs include a growing family of
receptors for lysophosphatidic acid and other lysophospholipids,
which appear to have roles in development and neuropathology (Chun,
J. et al. (1999) Cell Biochem. Biophys. 30:213-242).
[0008] The largest subfamily of GPCRs, the olfactory receptors, are
also members of the rhodopsin-like GPCR family. These receptors
function by transducing odorant signals. Numerous distinct
olfactory receptors are required to distinguish different odors.
Each olfactory sensory neuron expresses only one type of olfactory
receptor, and distinct spatial zones of neurons expressing distinct
receptors are found in nasal passages. For example, the RA1c
receptor which was isolated from a rat brain library, has been
shown to be limited in expression to very distinct regions of the
brain and a defined zone of the olfactory epithelium (Raming, K. et
al. (1998) Receptors Channels 6:141-151). However, the expression
of olfactory-like receptors is not confined to olfactory tissues.
For example, three rat genes encoding olfactory-like receptors
having typical GPCR characteristics showed expression patterns not
only in taste and olfactory tissue, but also in male reproductive
tissue (Thomas, M. B. et al. (1996) Gene 178:1-5).
[0009] Members of the secretin-like GPCR subfamily have as their
ligands peptide hormones such as secretin, calcitonin, glucagon,
growth hormone-releasing hormone, parathyroid hormone, and
vasoactive intestinal peptide. For example, the secretin receptor
responds to secretin, a peptide hormone that stimulates the
secretion of enzymes and ions in the pancreas and small intestine
(Watson, supra, pp. 278-283). Secretin receptors are about 450
amino acids in length and are found in the plasma membrane of
gastrointestinal cells. Binding of secretin to its receptor
stimulates the production of cAMP.
[0010] Examples of secretin-like GPCRs implicated in inflammation
and the immune response include the EGF module-containing,
mucin-like hormone receptor (Emr1) and CD97 receptor proteins.
These GPCRs are members of the recently characterized EGF-TM7
receptors subfamily. These seven transmembrane hormone receptors
exist as heterodimers in vivo and contain between three and seven
potential calcium-binding EGF-like motifs. CD97 is predominantly
expressed in leukocytes and is markedly upregulated on activated B
and T cells (McKnight, A. J. and S. Gordon (1998) J. Leukoc. Biol.
63:271-280).
[0011] The third GPCR subfamily is the metabotropic glutamate
receptor family. Glutamate is the major excitatory neurotransmitter
in the central nervous system. The metabotropic glutamate receptors
modulate the activity of intracellular effectors, and are involved
in long-term potentiation (Watson, supra, p.130). The
Ca.sup.2+-sensing receptor, which senses changes in the
extracellular concentration of calcium ions, has a large
extracellular domain including clusters of acidic amino acids which
may be involved in calcium binding. The metabotropic glutamate
receptor family also includes pheromone receptors, the GABA.sub.B
receptors, and the taste receptors.
[0012] Other subfamilies of GPCRs include two groups of
chemoreceptor genes found in the nematodes Caenorhabditis elegans
and Caenorhabditis briggsae, which are distantly related to the
mammalian olfactory receptor genes. The yeast pheromone receptors
STE2 and STE3, involved in the response to mating factors on the
cell membrane, have their own seven-transmembrane signature, as do
the cAMP receptors from the slime mold Dictyostelium discoideum,
which are thought to regulate the aggregation of individual cells
and control the expression of numerous developmentally-regulated
genes.
[0013] GPCR mutations, which may cause loss of function or
constitutive activation, have been associated with numerous human
diseases (Coughlin, supra). For instance, retinitis pigmentosa may
arise from mutations in the rhodopsin gene. Furthermore, somatic
activating mutations in the thyrotropin receptor have been reported
to cause hyperfunctioning thyroid adenomas, suggesting that certain
GPCRs susceptible to constitutive activation may behave as
protooncogenes (Parma, J. et al. (1993) Nature 365:649-651). GPCR
receptors for the following ligands also contain mutations
associated with human disease: luteinizing hormone (precocious
puberty); vasopressin V.sub.2 (X-linked nephrogenic diabetes);
glucagon (diabetes and hypertension); calcium (hyperparathyroidism,
hypocalcuria, hypercalcemina); parathyroid hormone (short limbed
dwarfism); .beta..sub.3-adrenoceptor (obesity,
non-insulin-dependent diabetes melitus); growth hormone releasing
hormone (dwarfism); and adrenocorticotropin (glucocorticoid
deficiency) (Wilson, S. et al. (1998) Br. J. Pharmocol.
125:1387-1392; Stadel, J. M. et al. (1997) Trends Pharmacol. Sci.
18:430-437). GPCRs are also involved in depression, schizophrenia,
sleeplessness, hypertension, anxiety, stress, renal failure, and
several cardiovascular disorders (Horn, F. and G. Vriend (1998) J.
Mol. Med. 76:464-468).
[0014] In addition, within the past 20 years several hundred new
drugs have been recognized that are directed towards activating or
inhibiting GPCRs. The therapeutic targets of these drugs span a
wide range of diseases and disorders, including cardiovascular,
gastrointestinal, and central nervous system disorders as well as
cancer, osteoporosis and endometriosis (Wilson, supra; Stadel,
supra). For example, the dopamine agonist L-dopa is used to treat
Parkinson's disease, while a dopamine antagonist is used to treat
schizophrenia and the early stages of Huntington's disease.
Agonists and antagonists of adrenoceptors have been used for the
treatment of asthma, high blood pressure, other cardiovascular
disorders, and anxiety; muscarinic agonists are used in the
treatment of glaucoma and tachycardia; serotonin 5HT1D antagonists
are used against migraine; and histamine H1 antagonists are used
against allergic and anaphylactic reactions, hay fever, itching,
and motion sickness (Horn, supra).
[0015] Recent research suggests potential future therapeutic uses
for GPCRs in the treatment of metabolic disorders including
diabetes, obesity, and osteoporosis. For example, mutant V2
vasopressin receptors causing nephrogenic diabetes could be
functionally rescued in vitro by co-expression of a C-terminal V2
receptor peptide spanning the region containing the mutations. This
result suggests a possible novel strategy for disease treatment
(Schoneberg, T. et al. (1996) EMBO J. 15:1283-1291). Mutations in
melanocortin-4 receptor (MC4R) are implicated in human weight
regulation and obesity. As with the vasopressin V2 receptor
mutants, these MC4R mutants are defective in trafficking to the
plasma membrane (Ho, G. and R. G. MacKenzie (1999) J. Biol. Chem.
274:35816-35822), and thus might be treated with a similar
strategy. The type 1 receptor for parathyroid hormone (PTH) is a
GPCR that mediates the PTH-dependent regulation of calcium
homeostasis in the bloodstream Study of PTH/receptor interactions
may enable the development of novel PTH receptor ligands for the
treatment of osteoporosis (Mannstadt, M. et al. (1999) Am. J.
Physiol. 277:F665-F675).
[0016] The chemokine receptor group of GPCRs have potential
therapeutic utility in inflammation and infectious disease. (For
review, see Locati, M. and P. M. Murphy (1999) Annu. Rev. Med.
50:425-440.) Chemokines are small polypeptides that act as
intracellular signals in the regulation of leukocyte trafficking,
hematopoiesis, and angiogenesis. Targeted disruption of various
chemokine receptors in mice indicates that these receptors play
roles in pathologic inflammation and in autoimmune disorders such
as multiple sclerosis. Chemokine receptors are also exploited by
infectious agents, including herpesviruses and the human
immunodeficiency virus (HIV-1) to facilitate infection. A truncated
version of chemokine receptor CCR5, which acts as a coreceptor for
infection of T-cells by HIV-1, results in resistance to AIDS,
suggesting that CCR5 antagonists could be useful in preventing the
development of AIDS.
[0017] The discovery of new G-protein coupled receptors 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, neurological,
cardiovascular, gastrointestinal, autoimmune/inflammatory, and
metabolic disorders, and viral infections, and in the assessment of
the effects of exogenous compounds on the expression of nucleic
acid and amino acid sequences of G-protein coupled receptors.
SUMMARY OF THE INVENTION
[0018] The invention features purified polypeptides, G-protein
coupled receptors, referred to collectively as "GCREC" and
individually as "GCREC-1," "GCREC-2," "GCREC-3," "GCREC-4,"
"GCREC-5," "GCREC-6," "GCREC-7," "GCREC-8," "GCREC-9," "GCREC-10,"
"GCREC-11," "GCREC-12," "GCREC-13," "GCREC-14," "GCREC-15,"
"GCREC-16," "GCREC-17," "GCREC-18," "GCREC-19," "GCREC-20,"
"GCREC-21," "GCREC-22," "GCREC-23," "GCREC-24," "GCREC-25,"
"GCREC-26," "GCREC-27," "GCREC-28," "GCREC-29," "GCREC-30,"
"GCREC-31," "GCREC-32," "GCREC-33," "GCREC-34," "GCREC-35,"
"GCREC-36," "GCREC-37," "GCREC-38," and "GCREC-39." In one aspect,
the invention provides an isolated polypeptide comprising an amino
acid sequence selected from the group consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-39,
b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-39, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39. In one
alternative, the invention provides an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:1-39.
[0019] The invention further provides an isolated polynucleotide
encoding a polypeptide comprising an amino acid sequence selected
from the group consisting of a) an amino acid sequence selected
from the group consisting of SEQ ID NO:1-39, b) a naturally
occurring amino acid sequence having at least 90% sequence identity
to an amino acid sequence selected from the group consisting of SEQ
ID NO: 1-39, c) a biologically active fragment of an amino acid
sequence selected from the group consisting of SEQ ID NO:1-39, and
d) an immunogenic fragment of an amino acid sequence selected from
the group consisting of SEQ ID NO:1-39. In one alternative, the
polynucleotide encodes a polypeptide selected from the group
consisting of SEQ ID NO:1-39. In another alternative, the
polynucleotide is selected from the group consisting of SEQ ID
NO:40-78.
[0020] Additionally, the invention provides a recombinant
polynucleotide comprising a promoter sequence operably linked to a
polynucleotide encoding a polypeptide comprising an amino acid
sequence selected from the group consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-39, b) a
naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-39, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39. In one
alternative, the invention provides a cell transformed with the
recombinant polynucleotide. In another alternative, the invention
provides a transgenic organism comprising the recombinant
polynucleotide.
[0021] The invention also provides a method for producing a
polypeptide comprising an amino acid sequence selected from the
group consisting of a) an amino acid sequence selected from the
group consisting of SEQ ID NO:1-39, b) a naturally occurring amino
acid sequence having at least 90% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NO:1-39,
c) a biologically active fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39, and d) an
immunogenic fragment of an amino acid sequence selected from the
group consisting of SEQ ID NO:1-39. The method comprises a)
culturing a cell under conditions suitable for expression of the
polypeptide, wherein said cell is transformed with a recombinant
polynucleotide comprising a promoter sequence operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the
polypeptide so expressed.
[0022] Additionally, the invention provides an isolated antibody
which specifically binds to a polypeptide comprising an amino acid
sequence selected from the group consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-39, b) a
naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-39, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39.
[0023] The invention further provides an isolated polynucleotide
comprising a polynucleotide sequence selected from the group
consisting of a) a polynucleotide sequence selected from the group
consisting of SEQ ID NO:40-78, b) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:40-78, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence complementary to b), and e) an RNA
equivalent of a)-d). In one alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
[0024] Additionally, the invention provides a method for detecting
a target polynucleotide in a sample, said target polynucleotide
having a sequence of a polynucleotide comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide
sequence selected from the group consisting of SEQ ID NO:40-78, b)
a naturally occurring polynucleotide sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the
group consisting of SEQ ID NO:40-78, c) a polynucleotide sequence
complementary to a), d) a polynucleotide sequence complementary to
b), and e) an RNA equivalent of a)-d). The method comprises a)
hybridizing the sample with a probe comprising at least 20
contiguous nucleotides comprising a sequence complementary to said
target polynucleotide in the sample, and which probe specifically
hybridizes to said target polynucleotide, under conditions whereby
a hybridization complex is formed between said probe and said
target polynucleotide or fragments thereof, and b) detecting the
presence or absence of said hybridization complex, and optionally,
if present, the amount thereof. In one alternative, the probe
comprises at least 60 contiguous nucleotides.
[0025] The invention further provides a method for detecting a
target polynucleotide in a sample, said target polynucleotide
having a sequence of a polynucleotide comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide
sequence selected from the group consisting of SEQ ID NO:40-78, b)
a naturally occurring polynucleotide sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the
group consisting of SEQ ID NO:40-78, c) a polynucleotide sequence
complementary to a), d) a polynucleotide sequence complementary to
b), and e) an RNA equivalent of a)-d). The method comprises a)
amplifying said target polynucleotide or fragment thereof using
polymerase chain reaction amplification, and b) detecting the
presence or absence of said amplified target polynucleotide or
fragment thereof, and, optionally, if present, the amount
thereof.
[0026] The invention further provides a composition comprising an
effective amount of a polypeptide comprising an amino acid sequence
selected from the group consisting of a) an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39, b) a
naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-39, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-39, and a
pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:1-39. The invention additionally
provides a method of treating a disease or condition associated
with decreased expression of functional GCREC, comprising
administering to a patient in need of such treatment the
composition.
[0027] The invention also provides a method for screening a
compound for effectiveness as an agonist of a polypeptide
comprising an amino acid sequence selected from the group
consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, b) a naturally occurring amino acid
sequence having at least 90% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO:1-39, c) a
biologically active fragment of an amino acid sequence selected
from the group consisting of SEQ ID NO:1-39, and d) an immunogenic
fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39. The method comprises a) exposing a
sample comprising the polypeptide to a compound, and b) detecting
agonist activity in the sample. In one alternative, the invention
provides a composition comprising an agonist compound identified by
the method and a pharmaceutically acceptable excipient. In another
alternative, the invention provides a method of treating a disease
or condition associated with decreased expression of functional
GCREC, comprising administering to a patient in need of such
treatment the composition.
[0028] Additionally, the invention provides a method for screening
a compound for effectiveness as an antagonist of a polypeptide
comprising an amino acid sequence selected from the group
consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, b) a naturally occurring amino acid
sequence having at least 90% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO:1-39, c) a
biologically active fragment of an amino acid sequence selected
from the group consisting of SEQ ID NO:1-39, and d) an immunogenic
fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39. The method comprises a) exposing a
sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in the sample. In one alternative, the
invention provides a composition comprising an antagonist compound
identified by the method and a pharmaceutically acceptable
excipient. In another alternative, the invention provides a method
of treating a disease or condition associated with overexpression
of functional GCREC, comprising administering to a patient in need
of such treatment the composition.
[0029] The invention further provides a method of screening for a
compound that specifically binds to a polypeptide comprising an
amino acid sequence selected from the group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-39, b) a naturally occurring amino acid sequence having at
least 90% sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NO:1-39, c) a biologically active
fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, and d) an immunogenic fragment of an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-39. The method comprises a) combining the polypeptide with at
least one test compound under suitable conditions, and b) detecting
binding of the polypeptide to the test compound, thereby
identifying a compound that specifically binds to the
polypeptide.
[0030] The invention further provides a method of screening for a
compound that modulates the activity of a polypeptide comprising an
amino acid sequence selected from the group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-39, b) a naturally occurring amino acid sequence having at
least 90% sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NO:1-39, c) a biologically active
fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-39, and d) an immunogenic fragment of an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-39. The method comprises a) combining the polypeptide with at
least one test compound under conditions permissive for the
activity of the polypeptide, b) assessing the activity of the
polypeptide in the presence of the test compound, and c) comparing
the activity of the polypeptide in the presence of the test
compound with the activity of the polypeptide in the absence of the
test compound, wherein a change in the activity of the polypeptide
in the presence of the test compound is indicative of a compound
that modulates the activity of the polypeptide.
[0031] The invention further provides a method for screening a
compound for effectiveness in altering expression of a target
polynucleotide, wherein said target polynucleotide comprises a
sequence selected from the group consisting of SEQ ID NO:40-78, the
method comprising a) exposing a sample comprising the target
polynucleotide to a compound, and b) detecting altered expression
of the target polynucleotide.
[0032] The invention further provides a method for assessing
toxicity of a test compound, said method comprising a) treating a
biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample
with a probe comprising at least 20 contiguous nucleotides of a
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of i) a polynucleotide sequence selected from
the group consisting of SEQ ID NO:40-78, ii) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:40-78, iii) a polynucleotide sequence complementary to i),
iv) a polynucleotide sequence complementary to ii), and v) an RNA
equivalent of i)-iv). Hybridization occurs under conditions whereby
a specific hybridization complex is formed between said probe and a
target polynucleotide in the biological sample, said target
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of i) a polynucleotide sequence selected from
the group consisting of SEQ ID NO:40-78, ii) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:40-78, iii) a polynucleotide sequence complementary to i),
iv) a polynucleotide sequence complementary to ii), and v) an RNA
equivalent of i)-iv). Alternatively, the target polynucleotide
comprises a fragment of a polynucleotide sequence selected from the
group consisting of i)-v) above; c) quantifying the amount of
hybridization complex; and d) comparing the amount of hybridization
complex in the treated biological sample with the amount of
hybridization complex in an untreated biological sample, wherein a
difference in the amount of hybridization complex in the treated
biological sample is indicative of toxicity of the test
compound.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
[0033] FIG. 1 shows the hydrophobicity plot for GCREC-1 (SEQ ID
NO:1; Incyte ID number 104941CD1). The hydrophobicity plot was
generated using the MacDNASIS Pro software. The positive X axis
reflects amino acid position, and the negative Y axis,
hydrophobicity. The numbers indicate the positions of predicted
transmembrane domains.
[0034] FIG. 2 shows the hydrophobicity plot for GCREC-3 (SEQ ID
NO:3; Incyte ID number 3168839CD1).
[0035] FIG. 3 shows the hydrophobicity plot for GCREC-4 (SEQ ID
NO:4; Incyte ID number 3291235CD1).
[0036] FIG. 4 shows the hydrophobicity plot for GCREC-5 (SEQ ID
NO:5; Incyte ID number 7472001CD1).
[0037] FIG. 5 shows the hydrophobicity plot for GCREC-6 (SEQ ID
NO:6; Incyte ID number 7472003CD1).
[0038] FIG. 6 shows the hydrophobicity plot for GCREC-7 (SEQ ID
NO:7; Incyte ID number 7472004CD1).
[0039] FIG. 7 shows the hydrophobicity plot for GCREC-19 (SEQ ID
NO:19; Incyte ID number 3068234CD1).
[0040] FIG. 8 shows the hydrophobicity plot for GCREC-20 (SEQ ID
NO:20; Incyte ID number 5029478CD1).
[0041] FIG. 9 shows the hydrophobicity plot for GCREC-21 (SEQ ID
NO:21; Incyte ID number 5102576CD1).
[0042] Table 1 summarizes the nomenclature for the polynucleotide
and polypeptide sequences of the present invention.
[0043] Table 2 shows the GenBank identification number and
annotation of the nearest GenBank homolog for each polypeptide of
the invention. The probability score for the match between each
polypeptide and its GenBank homolog is also shown.
[0044] Table 3 shows structural features of each polypeptide
sequence, including predicted motifs and domains, along with the
methods, algorithms, and searchable databases used for analysis of
each polypeptide.
[0045] Table 4 lists the cDNA and genomic DNA fragments which were
used to assemble each polynucleotide sequence, along with selected
fragments of the polynucleotide sequences.
[0046] Table 5 shows the representative cDNA library for each
polynucleotide of the invention.
[0047] Table 6 provides an appendix which describes the tissues and
vectors used for construction of the cDNA libraries shown in Table
5.
[0048] Table 7 shows the tools, programs, and algorithms used to
analyze the polynucleotides and polypeptides of the invention,
along with applicable descriptions, references, and threshold
parameters.
DESCRIPTION OF THE INVENTION
[0049] 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.
[0050] 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.
[0051] 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.
[0052] Definitions
[0053] "GCREC" refers to the amino acid sequences of substantially
purified GCREC obtained from any species, particularly a mammalian
species, including bovine, ovine, porcine, murine, equine, and
human, and from any source, whether natural, synthetic,
semi-synthetic, or recombinant.
[0054] The term "agonist" refers to a molecule which intensifies or
mimics the biological activity of GCREC. Agonists may include
proteins, nucleic acids, carbohydrates, small molecules, or any
other compound or composition which modulates the activity of GCREC
either by directly interacting with GCREC or by acting on
components of the biological pathway in which GCREC
participates.
[0055] An "allelic variant" is an alternative form of the gene
encoding GCREC. Allelic variants may result from at least one
mutation in the nucleic acid sequence and may result in altered
mRNAs or in polypeptides whose structure or function may or may not
be altered. A gene may have none, one, or many allelic variants of
its naturally occurring form. Common mutational changes which give
rise to allelic variants are generally ascribed to natural
deletions, additions, or substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the
others, one or more times in a given sequence.
[0056] "Altered" nucleic acid sequences encoding GCREC include
those sequences with deletions, insertions, or substitutions of
different nucleotides, resulting in a polypeptide the same as GCREC
or a polypeptide with at least one functional characteristic of
GCREC. Included within this definition are polymorphisms which may
or may not be readily detectable using a particular oligonucleotide
probe of the polynucleotide encoding GCREC, and improper or
unexpected hybridization to allelic variants, with a locus other
than the normal chromosomal locus for the polynucleotide sequence
encoding GCREC. 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 GCREC. 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 GCREC is retained. For example, negatively charged amino acids
may include aspartic acid and glutaric acid, and positively charged
amino acids may include lysine and arginine. Amino acids with
uncharged polar side chains having similar hydrophilicity values
may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar
hydrophilicity values may include: leucine, isoleucine, and valine;
glycine and alanine; and phenylalanine and tyrosine.
[0057] The terms "amino acid" and "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a
sequence of a naturally occurring protein molecule, "amino acid
sequence" and like terms are not meant to limit the amino acid
sequence to the complete native amino acid sequence associated with
the recited protein molecule.
[0058] "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.
[0059] The term "antagonist" refers to a molecule which inhibits or
attenuates the biological activity of GCREC. Antagonists may
include proteins such as antibodies, nucleic acids, carbohydrates,
small molecules, or any other compound or composition which
modulates the activity of GCREC either by directly interacting with
GCREC or by acting on components of the biological pathway in which
GCREC participates.
[0060] The term "antibody" refers to intact immunoglobulin
molecules as well as to fragments thereof, such as Fab,
F(ab').sub.2, and Fv fragments, which are capable of binding an
epitopic determinant. Antibodies that bind GCREC 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.
[0061] The term "antigenic determinant" refers to that region of a
molecule (i.e., an epitope) that makes contact with a particular
antibody. When a protein or a fragment of a protein is used to
immunize a host animal, numerous regions of the protein may induce
the production of antibodies which bind specifically to antigenic
determinants (particular regions or three-dimensional structures on
the protein). An antigenic determinant may compete with the intact
antigen (i.e., the immunogen used to elicit the immune response)
for binding to an antibody.
[0062] The term "antisense" refers to any composition capable of
base-pairing with the "sense" (coding) strand of a specific nucleic
acid sequence. Antisense compositions may include DNA; RNA; peptide
nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as phosphorothioates, methylphosphonates, or
benzylphosphonates; oligonucleotides having modified sugar groups
such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having modified bases such as 5-methyl cytosine,
2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules
may be produced by any method including chemical synthesis or
transcription. Once introduced into a cell, the complementary
antisense molecule base-pairs with a naturally occurring nucleic
acid sequence produced by the cell to form duplexes which block
either transcription or translation. The designation "negative" or
"minus" can refer to the antisense strand, and the designation
"positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
[0063] The term "biologically active" refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" or
"immunogenic" refers to the capability of the natural, recombinant,
or synthetic GCREC, or of any oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to
bind with specific antibodies.
[0064] "Complementary" describes the relationship between two
single-stranded nucleic acid sequences that anneal by base-pairing.
For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
[0065] 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 GCREC or fragments of GCREC 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.).
[0066] "Consensus sequence" refers to a nucleic acid sequence which
has been subjected to repeated DNA sequence analysis to resolve
uncalled bases, extended using the XL-PCR kit (Applied Biosystems,
Foster City Calif.) in the 5' and/or the 3' direction, and
resequenced, or which has been assembled from one or more
overlapping cDNA, EST, or genomic DNA fragments using a computer
program for fragment assembly, such as the GELVIEW fragment
assembly system (GCG, Madison Wis.) or Phrap (University of
Washington, Seattle Wash.). Some sequences have been both extended
and assembled to produce the consensus sequence.
[0067] "Conservative amino acid substitutions" are those
substitutions that are predicted to least interfere with the
properties of the original protein, i.e., the structure and
especially the function of the protein is conserved and not
significantly changed by such substitutions. The table below shows
amino acids which may be substituted for an original amino acid in
a protein and which are regarded as conservative amino acid
substitutions.
1 Original Residue Conservative Substitution Ala Gly, Ser Arg His,
Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His
Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu
Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile,
Leu, Thr
[0068] Conservative amino acid substitutions generally maintain (a)
the structure of the polypeptide backbone in the area of the
substitution, for example, as a beta sheet or alpha helical
conformation, (b) the charge or hydrophobicity of the molecule at
the site of the substitution, and/or (c) the bulk of the side
chain.
[0069] 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.
[0070] The term "derivative" refers to a chemically modified
polynucleotide or polypeptide. Chemical modifications of a
polynucleotide can include, for example, replacement of hydrogen by
an alkyl, acyl, hydroxyl, or amino group. A derivative
polynucleotide encodes a polypeptide which retains at least one
biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation,
pegylation, or any similar process that retains at least one
biological or immunological function of the polypeptide from which
it was derived.
[0071] A "detectable label" refers to a reporter molecule or enzyme
that is capable of generating a measurable signal and is covalently
or noncovalently joined to a polynucleotide or polypeptide.
[0072] A "fragment" is a unique portion of GCREC or the
polynucleotide encoding GCREC which is identical in sequence to but
shorter in length than the parent sequence. A fragment may comprise
up to the entire length of the defined sequence, minus one
nucleotide/amino acid residue. For example, a fragment may comprise
from 5 to 1000 contiguous nucleotides or amino acid residues. A
fragment used as a probe, primer, antigen, therapeutic molecule, or
for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40,
50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or
amino acid residues in length. Fragments may be preferentially
selected from certain regions of a molecule. For example, a
polypeptide fragment may comprise a certain length of contiguous
amino acids selected from the first 250 or 500 amino acids (or
first 25% or 50%) of a polypeptide as shown in a certain defined
sequence. Clearly these lengths are exemplary, and any length that
is supported by the specification, including the Sequence Listing,
tables, and figures, may be encompassed by the present
embodiments.
[0073] A fragment of SEQ ID NO:40-78 comprises a region of unique
polynucleotide sequence that specifically identifies SEQ ID
NO:40-78, for example, as distinct from any other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID
NO:40-78 is useful, for example, in hybridization and amplification
technologies and in analogous methods that distinguish SEQ ID
NO:40-78 from related polynucleotide sequences. The precise length
of a fragment of SEQ ID NO:40-78 and the region of SEQ ID NO:40-78
to which the fragment corresponds are routinely determinable by one
of ordinary skill in the art based on the intended purpose for the
fragment.
[0074] A fragment of SEQ ID NO:1-39 is encoded by a fragment of SEQ
ID NO:40-78. A fragment of SEQ ID NO:1-39 comprises a region of
unique amino acid sequence that specifically identifies SEQ ID
NO:1-39. For example, a fragment of SEQ ID NO:1-39 is useful as an
immunogenic peptide
[0075] The terms "percent identity" and "% identity," as applied to
polynucleotide sequences, refer to the percentage of residue
matches between at least two polynucleotide sequences aligned using
a standardized algorithm. Such an algorithm may insert, in a
standardized and reproducible way, gaps in the sequences being
compared in order to optimize alignment between two sequences, and
therefore achieve a more meaningful comparison of the two
sequences.
[0076] Percent identity between polynucleotide sequences may be
determined using the default parameters of the CLUSTAL V algorithm
as incorporated into the MEGALIGN version 3.12e sequence alignment
program. This program is part of the LASERGENE software package, a
suite of molecular biological analysis programs (DNASTAR, Madison
Wis.). CLUSTAL V is described in Higgins, D. G. and P. M. Sharp
(1989) CABIOS 5:151-153 and in Higgins, D. G. et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the
default parameters are set as follows: Ktuple=2, gap penalty=5,
window=4, and "diagonals saved"=4. The "weighted" residue weight
table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent similarity" between aligned
polynucleotide sequences.
[0077] Alternatively, a suite of commonly used and freely available
sequence comparison algorithms is provided by the National Center
for Biotechnology Information (NCBI) Basic Local Alignment Search
Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.
215:403-410), which is available from several sources, including
the NCBI, Bethesda, Md., and on the Internet at
http://www.ncbi.nlm.nihgov/BLAST/. The BLAST software suite
includes various sequence analysis programs including "blastn,"
that is used to align a known polynucleotide sequence with other
polynucleotide sequences from a variety of databases. Also
available is a tool called "BLAST 2 Sequences" that is used for
direct pairwise comparison of two nucleotide sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.h- tml. The "BLAST 2
Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST programs are commonly used with gap and other
parameters set to default settings. For example, to compare two
nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version 2.0.12 (Apr. 21, 2000) set at default
parameters. Such default parameters may be, for example:
[0078] Matrix: BLOSUM62
[0079] Reward for match: 1
[0080] Penalty for mismatch: -2
[0081] Open Gap: 5 and Extension Gap: 2 penalties
[0082] Gap x drop-off: 50
[0083] Expect: 10
[0084] Word Size: 11
[0085] Filter: on
[0086] Percent identity may be measured over the length of an
entire defined sequence, for example, as defined by a particular
SEQ ID number, or may be measured over a shorter length, for
example, over the length of a fragment taken from a larger, defined
sequence, for instance, a fragment of at least 20, at least 30, at
least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such lengths are exemplary only, and it is
understood that any fragment length supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be
used to describe a length over which percentage identity may be
measured.
[0087] Nucleic acid sequences that do not show a high degree of
identity may nevertheless encode similar amino acid sequences due
to the degeneracy of the genetic code. It is understood that
changes in a nucleic acid sequence can be made using this
degeneracy to produce multiple nucleic acid sequences that all
encode substantially the same protein.
[0088] The phrases "percent identity" and "% identity," as applied
to polypeptide sequences, refer to the percentage of residue
matches between at least two polypeptide sequences aligned using a
standardized algorithm. Methods of polypeptide sequence alignment
are well-known. Some alignment methods take into account
conservative amino acid substitutions. Such conservative
substitutions, explained in more detail above, generally preserve
the charge and hydrophobicity at the site of substitution, thus
preserving the structure (and therefore function) of the
polypeptide.
[0089] Percent identity between polypeptide sequences may be
determined using the default parameters of the CLUSTAL V algorithm
as incorporated into the MEGALIGN version 3.12e sequence alignment
program (described and referenced above). For pairwise alignments
of polypeptide sequences using CLUSTAL V, the default parameters
are set as follows: Ktuple=1, gap penalty=3, window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default
residue weight table. As with polynucleotide alignments, the
percent identity is reported by CLUSTAL V as the "percent
similarity" between aligned polypeptide sequence pairs.
[0090] Alternatively the NCBI BLAST software suite may be used. For
example, for a pairwise comparison of two polypeptide sequences,
one may use the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21,
2000) with blastp set at default parameters. Such default
parameters may be, for example:
[0091] Matrix: BLOSUM62
[0092] Open Gap: 11 and Extension Gap: 1 penalties
[0093] Gap x drop-off: 50
[0094] Expect: 10
[0095] Word Size: 3
[0096] Filter: on
[0097] Percent identity may be measured over the length of an
entire defined polypeptide sequence, for example, as defined by a
particular SEQ ID number, or may be measured over a shorter length,
for example, over the length of a fragment taken from a larger,
defined polypeptide sequence, for instance, a fragment of at least
15, at least 20, at least 30, at least 40, at least 50, at least 70
or at least 150 contiguous residues. Such lengths are exemplary
only, and it is understood that any fragment length supported by
the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to describe a length over which percentage
identity may be measured.
[0098] "Human artificial chromosomes" (HACs) are linear
microchromosomes which may contain DNA sequences of about 6 kb to
10 Mb in size and which contain all of the elements required for
chromosome replication, segregation and maintenance.
[0099] The term "humanized antibody" refers to an antibody molecule
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
[0100] "Hybridization" refers to the process by which a
polynucleotide strand anneals with a complementary strand through
base pairing under defined hybridization conditions. Specific
hybridization is an indication that two nucleic acid sequences
share a high degree of complementarity. Specific hybridization
complexes form under permissive annealing conditions and remain
hybridized after the "washing" step(s). The washing step(s) is
particularly important in determining the stringency of the
hybridization process, with more stringent conditions allowing less
non-specific binding, i.e., binding between pairs of nucleic acid
strands that are not perfectly matched. Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by
one of ordinary skill in the art and may be consistent among
hybridization experiments, whereas wash conditions may be varied
among experiments to achieve the desired stringency, and therefore
hybridization specificity. Permissive annealing conditions occur,
for example, at 68.degree. C. in the presence of about 6.times.SSC,
about 1% (w/v) SDS, and about 100 .mu.g/ml sheared, denatured
salmon sperm DNA.
[0101] Generally, stringency of hybridization is expressed, in
part, with reference to the temperature under which the wash step
is carried out. Such wash temperatures are typically selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. An equation for
calculating T.sub.m and conditions for nucleic acid hybridization
are well known and can be found in Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Press, Plainview N.Y.; specifically see volume
2, chapter 9.
[0102] High stringency conditions for hybridization between
polynucleotides of the present invention include wash conditions of
68.degree. C. in the presence of about 0.2.times.SSC and about 0.1%
SDS, for 1 hour. Alternatively, temperatures of about 65.degree.
C., 60.degree. C., 55.degree. C., or 42.degree. C. may be used. SSC
concentration may be varied from about 0.1 to 2.times.SSC, with SDS
being present at about 0.1%. Typically, blocking reagents are used
to block non-specific hybridization. Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at
about 100-200 .mu.g/ml. Organic solvent, such as formamide at a
concentration of about 35-50% v/v, may also be used under
particular circumstances, such as for RNA:DNA hybridizations.
Useful variations on these wash conditions will be readily apparent
to those of ordinary skill in the art. Hybridization, particularly
under high stringency conditions, may be suggestive of evolutionary
similarity between the nucleotides. Such similarity is strongly
indicative of a similar role for the nucleotides and their encoded
polypeptides.
[0103] 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).
[0104] 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.
[0105] "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.
[0106] An "immunogenic fragment" is a polypeptide or oligopeptide
fragment of GCREC which is capable of eliciting an immune response
when introduced into a living organism, for example, a mammal. The
term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of GCREC which is useful in any of the
antibody production methods disclosed herein or known in the
art.
[0107] The term "microarray" refers to an arrangement of a
plurality of polynucleotides, polypeptides, or other chemical
compounds on a substrate.
[0108] The terms "element" and "array element" refer to a
polynucleotide, polypeptide, or other chemical compound having a
unique and defined position on a microarray.
[0109] The term "modulate" refers to a change in the activity of
GCREC. For example, modulation may cause an increase or a decrease
in protein activity, binding characteristics, or any other
biological, functional, or immunological properties of GCREC.
[0110] The phrases "nucleic acid" and "nucleic acid sequence" refer
to a nucleotide, oligonucleotide, polynucleotide, or any fragment
thereof. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material.
[0111] "Operably linked" refers to the situation in which a first
nucleic acid sequence is placed in a functional relationship with a
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Operably linked
DNA sequences may be in close proximity or contiguous and, where
necessary to join two protein coding regions, in the same reading
frame.
[0112] "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.
[0113] "Post-translational modification" of an GCREC may involve
lipidation, glycosylation, phosphorylation, acetylation,
racemization, proteolytic cleavage, and other modifications known
in the art. These processes may occur synthetically or
biochemically. Biochemical modifications will vary by cell type
depending on the enzymatic milieu of GCREC.
[0114] "Probe" refers to nucleic acid sequences encoding GCREC,
their complements, or fragments thereof, which are used to detect
identical, allelic or related nucleic acid sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a
detectable label or reporter molecule. Typical labels include
radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are short nucleic acids, usually DNA
oligonucleotides, which may be annealed to a target polynucleotide
by complementary base-pairing. The primer may then be extended
along the target DNA strand by a DNA polymerase enzyme. Primer
pairs can be used for amplification (and identification) of a
nucleic acid sequence, e.g., by the polymerase chain reaction
(PCR).
[0115] Probes and primers as used in the present invention
typically comprise at least 15 contiguous nucleotides of a known
sequence. In order to enhance specificity, longer probes and
primers may also be employed, such as probes and primers that
comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at
least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may be considerably longer than these
examples, and it is understood that any length supported by the
specification, including the tables, figures, and Sequence Listing,
may be used.
[0116] Methods for preparing and using probes and primers are
described in the references, for example Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Press, Plainview N.Y.; Ausubel, F. M. et al.
(1987) Current Protocols in Molecular Biology, Greene Publ. Assoc.
& Wiley-Intersciences, New York N.Y.; Innis, M. et al. (1990)
PCR Protocols, A Guide to Methods and Applications, Academic Press,
San Diego Calif. PCR primer pairs can be derived from a known
sequence, for example, by using computer programs intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for
Biomedical Research, Cambridge Mass.).
[0117] Oligonucleotides for use as primers are selected using
software known in the art for such purpose. For example, OLIGO 4.06
software is useful for the selection of PCR primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and
larger polynucleotides of up to 5,000 nucleotides from an input
polynucleotide sequence of up to 32 kilobases. Similar primer
selection programs have incorporated additional features for
expanded capabilities. For example, the PrimOU primer selection
program (available to the public from the Genome Center at
University of Texas South West Medical Center, Dallas Tex.) is
capable of choosing specific primers from megabase sequences and is
thus useful for designing primers on a genome-wide scope. The
Primer3 primer selection program (available to the public from the
Whitehead Institute/MIT Center for Genome Research, Cambridge
Mass.) allows the user to input a "mispriming library," in which
sequences to avoid as primer binding sites are user-specified
Primer3 is useful, in particular, for the selection of
oligonucleotides for microarrays. The source code for the latter
two primer selection programs may also be obtained from their
respective sources and modified to meet the user's specific needs.)
The PrimeGen program (available to the public from the UK Human
Genome Mapping Project Resource Centre, Cambridge UK) designs
primers based on multiple sequence alignments, thereby allowing
selection of primers that hybridize to either the most conserved or
least conserved regions of aligned nucleic acid sequences. Hence,
this program is useful for identification of both unique and
conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and polynucleotide fragments identified by any of
the above selection methods are useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray
elements, or specific probes to identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods
of oligonucleotide selection are not limited to those described
above.
[0118] A "recombinant nucleic acid" is a sequence that is not
naturally occurring or has a sequence that is made by an artificial
combination of two or more otherwise separated segments of
sequence. This artificial combination is often accomplished by
chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques such as those described in Sambrook,
supra. The term recombinant includes nucleic acids that have been
altered solely by addition, substitution, or deletion of a portion
of the nucleic acid. Frequently, a recombinant nucleic acid may
include a nucleic acid sequence operably linked to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector
that is used, for example, to transform a cell.
[0119] Alternatively, such recombinant nucleic acids may be part of
a viral vector, e.g., based on a vaccinia virus, that could be use
to vaccinate a mammal wherein the recombinant nucleic acid is
expressed, inducing a protective immunological response in the
mammal.
[0120] A "regulatory element" refers to a nucleic acid sequence
usually derived from untranslated regions of a gene and includes
enhancers, promoters, introns, and 5' and 3' untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins
which control transcription, translation, or RNA stability.
[0121] "Reporter molecules" are chemical or biochemical moieties
used for labeling a nucleic acid, amino acid, or antibody. Reporter
molecules include radionuclides; enzymes; fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors;
inhibitors; magnetic particles; and other moieties known in the
art.
[0122] An "RNA equivalent," in reference to a DNA sequence, is
composed of the same linear sequence of nucleotides as the
reference DNA sequence with the exception that all occurrences of
the nitrogenous base thymine are replaced with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
[0123] The term "sample" is used in its broadest sense. A sample
suspected of containing GCREC, nucleic acids encoding GCREC, or
fragments thereof may comprise a bodily fluid; an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a
cell; genomic DNA, RNA, or cDNA, in solution or bound to a
substrate; a tissue; a tissue print; etc.
[0124] The terms "specific binding" and "specifically binding"
refer to that interaction between a protein or peptide and an
agonist, an antibody, an antagonist, a small molecule, or any
natural or synthetic binding composition. The interaction is
dependent upon the presence of a particular structure of the
protein, e.g., the antigenic determinant or epitope, recognized by
the binding molecule. For example, if an antibody is specific for
epitope "A," the presence of a polypeptide comprising the epitope
A, or the presence of free unlabeled A, in a reaction containing
free labeled A and the antibody will reduce the amount of labeled A
that binds to the antibody.
[0125] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least 60%
free, preferably at least 75% free, and most preferably at least
90% free from other components with which they are naturally
associated.
[0126] A "substitution" refers to the replacement of one or more
amino acid residues or nucleotides by different amino acid residues
or nucleotides, respectively.
[0127] "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.
[0128] A "transcript image" refers to the collective pattern of
gene expression by a particular cell type or tissue under given
conditions at a given time.
[0129] "Transformation" describes a process by which exogenous DNA
is introduced into a recipient cell. Transformation may occur under
natural or artificial conditions according to various methods well
known in the art, and may rely on any known method for the
insertion of foreign nucleic acid sequences into a prokaryotic or
eukaryotic host cell. The method for transformation is selected
based on the type of host cell being transformed and may include,
but is not limited to, bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed cells" includes stably transformed cells in
which the inserted DNA is capable of replication either as an
autonomously replicating plasmid or as part of the host chromosome,
as well as transiently transformed cells which express the inserted
DNA or RNA for limited periods of time.
[0130] A "transgenic organism," as used herein, is any organism,
including but not limited to animals and plants, in which one or
more of the cells of the organism contains heterologous nucleic
acid introduced by way of human intervention, such as by transgenic
techniques well known in the art. The nucleic acid is introduced
into the cell, directly or indirectly by introduction into a
precursor of the cell, by way of deliberate genetic manipulation,
such as by microinjection or by infection with a recombinant virus.
The term genetic manipulation does not include classical
cross-breeding, or in vitro fertilization, but rather is directed
to the introduction of a recombinant DNA molecule. The transgenic
organisms contemplated in accordance with the present invention
include bacteria, cyanobacteria, fungi, plants and animals. The
isolated DNA of the present invention can be introduced into the
host by methods known in the art, for example infection,
transfection, transformation or transconjugation. Techniques for
transferring the DNA of the present invention into such organisms
are widely known and provided in references such as Sambrook et al.
(1989), supra.
[0131] A "variant" of a particular nucleic acid sequence is defined
as a nucleic acid sequence having at least 40% sequence identity to
the particular nucleic acid sequence over a certain length of one
of the nucleic acid sequences using blastn with the "BLAST 2
Sequences" tool Version 2.0.9 (May 7, 1999) set at default
parameters. Such a pair of nucleic acids may show, for example, at
least 50%, at least 60%, at least 70%, at least 80%, at least 85%,
at least 90%, at least 95% or at least 98% or greater sequence
identity over a certain defined length. A variant may be described
as, for example, an "allelic" (as defined above), "splice,"
"species," or "polymorphic" variant. A splice variant may have
significant identity to a reference molecule, but will generally
have a greater or lesser number of polynucleotides due to
alternative splicing of exons during mRNA processing. The
corresponding polypeptide may possess additional functional domains
or lack domains that are present in the reference molecule. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides will generally have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) in which the
polynucleotide sequence varies by one nucleotide base. The presence
of SNPs may be indicative of, for example, a certain population, a
disease state, or a propensity for a disease state.
[0132] A "variant" of a particular polypeptide sequence is defined
as a polypeptide sequence having at least 40% sequence identity to
the particular polypeptide sequence over a certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences"
tool Version 2.0.9 (May 7, 1999) set at default parameters. Such a
pair of polypeptides may show, for example, at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, or at
least 98% or greater sequence identity over a certain defined
length of one of the polypeptides.
[0133] The Invention
[0134] The invention is based on the discovery of new human
G-protein coupled receptors (GCREC), the polynucleotides encoding
GCREC, and the use of these compositions for the diagnosis,
treatment, or prevention of cell proliferative, neurological,
cardiovascular, gastrointestinal, autoimmune/inflammatory, and
metabolic disorders, and viral infections.
[0135] Table 1 summarizes the nomenclature for the polynucleotide
and polypeptide sequences of the invention. Each polynucleotide and
its corresponding polypeptide are correlated to a single Incyte
project identification number (Incyte Project ID). Each polypeptide
sequence is denoted by both a polypeptide sequence identification
number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence
number (Incyte Polypeptide ID) as shown. Each polynucleotide
sequence is denoted by both a polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and an Incyte
polynucleotide consensus sequence number (Incyte Polynucleotide ID)
as shown.
[0136] Table 2 shows sequences with homology to the polypeptides of
the invention as identified by BLAST analysis against the GenBank
protein (genpept) database. Columns 1 and 2 show the polypeptide
sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte polypeptide sequence number (Incyte
Polypeptide ID) for each polypeptide of the invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the
nearest GenBank homolog. Column 4 shows the probability score for
the match between each polypeptide and its GenBank homolog. Column
5 shows the annotation of the GenBank homolog along with relevant
citations where applicable, all of which are expressly incorporated
by reference herein.
[0137] Table 3 shows various structural features of each of the
polypeptides of the invention. Columns 1 and 2 show the polypeptide
sequence identification number (SEQ ID NO:) and the corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention. Column 3 shows the number of amino
acid residues in each polypeptide. Column 4 shows potential
phosphorylation sites, and column 5 shows potential glycosylation
sites, as determined by the MOTIFS program of the GCG sequence
analysis software package (Genetics Computer Group, Madison Wis.).
Column 6 shows amino acid residues comprising signature sequences,
domains, and motifs. Column 7 shows analytical methods for protein
structure/function analysis and in some cases, searchable databases
to which the analytical methods were applied.
[0138] As shown in Table 4, the polynucleotide sequences of the
present invention were assembled using cDNA sequences or coding
(exon) sequences derived from genomic DNA, or any combination of
these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence identification number (Polynucleotide SEQ
ID NO:) and the corresponding Incyte polynucleotide consensus
sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention. Column 3 shows the length of each polynucleotide
sequence in basepairs. Column 4 lists fragments of the
polynucleotide sequences which are useful, for example, in
hybridization or amplification technologies that identify SEQ ID
NO:40-78 or that distinguish between SEQ ID NO:40-78 and related
polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA sequences, coding sequences (exons) predicted
from genomic DNA, and/or sequence assemblages comprised of both
cDNA and genomic DNA. These sequences were used to assemble the
polynucleotide sequences of the invention. Columns 6 and 7 of Table
4 show the nucleotide start (5') and stop (3') positions of the
cDNA and genomic sequences in column 5 relative to their respective
sequences.
[0139] The identification numbers in Column 5 of Table 4 may refer
specifically, for example, to Incyte cDNAs along with their
corresponding cDNA libraries. For example, 927003T6 is the
identification number of an Incyte cDNA sequence, and BRAINOT04 is
the cDNA library from which it is derived. Incyte cDNAs for which
cDNA libraries are not indicated were derived from pooled cDNA
libraries (e.g., 70489898V1). Alternatively, the identification
numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g.,
g835247) which contributed to the assembly of the polynucleotide
sequences. Alternatively, the identification numbers in column 5
may refer to coding regions predicted by Genscan analysis of
genomic DNA. For example, g4190944.v113.gs.sub.--10.edit is the
identification number of a Genscan-predicted coding sequence, with
g4190944 being the GenBank identification number of the sequence to
which Genscan was applied. The Genscan-predicted coding sequences
may have been edited prior to assembly. (See Example IV.)
Alternatively, the identification numbers in column 5 may refer to
assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon stitching" algorithm. (See Example V.)
Alternatively, the identification numbers in column 5 may refer to
assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon-stretching" algorithm. (See Example V.) In
some cases, Incyte cDNA coverage redundant with the sequence
coverage shown in column 5 was obtained to confirm the final
consensus polynucleotide sequence, but the relevant Incyte cDNA
identification numbers are not shown.
[0140] Table 5 shows the representative cDNA libraries for those
polynucleotide sequences which were assembled using Incyte cDNA
sequences. The representative cDNA library is the Incyte cDNA
library which is most frequently represented by the Incyte cDNA
sequences which were used to assemble and confirm the above
polynucleotide sequences. The tissues and vectors which were used
to construct the cDNA libraries shown in Table 5 are described in
Table 6.
[0141] The invention also encompasses GCREC variants. A preferred
GCREC variant is one which has at least about 80%, or alternatively
at least about 90%, or even at least about 95% amino acid sequence
identity to the GCREC amino acid sequence, and which contains at
least one functional or structural characteristic of GCREC.
[0142] The invention also encompasses polynucleotides which encode
GCREC. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising a sequence selected from the
group consisting of SEQ ID NO:40-78, which encodes GCREC. The
polynucleotide sequences of SEQ ID. NO:40-78, as presented in the
Sequence Listing, embrace the equivalent RNA sequences, wherein
occurrences of the nitrogenous base thymidine are replaced with
uracil, and the sugar backbone is composed of ribose instead of
deoxyribose.
[0143] The invention also encompasses a variant of a polynucleotide
sequence encoding GCREC. In particular, such a variant
polynucleotide sequence will have at least about 70%, or
alternatively at least about 85%, or even at least about 95%
polynucleotide sequence identity to the polynucleotide sequence
encoding GCREC. 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:40-78 which has at least
about 70%, or alternatively at least about 85%, or even at least
about 95% polynucleotide sequence identity to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:40-78. Any
one of the polynucleotide variants described above can encode an
amino acid sequence which contains at least one functional or
structural characteristic of GCREC.
[0144] 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 GCREC, 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 GCREC, and all such
variations are to be considered as being specifically
disclosed.
[0145] Although nucleotide sequences which encode GCREC and its
variants are generally capable of hybridizing to the nucleotide
sequence of the naturally occurring GCREC under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding GCREC 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 GCREC 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.
[0146] The invention also encompasses production of DNA sequences
which encode GCREC and GCREC 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 GCREC or any fragment thereof.
[0147] 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:40-78 and fragments thereof under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and
wash conditions, are described in "Definitions."
[0148] 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 (Applied Biosystems), 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 MICROLAB 2200 liquid transfer system (Hamilton, Reno
Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI
CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is
then carried out using either the ABI 373 or 377 DNA sequencing
system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system (Molecular Dynamics, Sunnyvale 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.)
[0149] The nucleic acid sequences encoding GCREC may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based methods known in the art to detect upstream sequences,
such as promoters and regulatory elements. For example, one method
which may be employed, restriction-site PCR, uses universal and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries (Clontech, Palo Alto 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
Minn.) 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.
[0150] 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.
[0151] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g., GENOTYPER
and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process
from loading of samples to computer analysis and electronic data
display may be computer controlled. Capillary electrophoresis is
especially preferable for sequencing small DNA fragments which may
be present in limited amounts in a particular sample.
[0152] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode GCREC may be cloned in
recombinant DNA molecules that direct expression of GCREC, 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
GCREC.
[0153] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter GCREC-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.
[0154] The nucleotides of the present invention may be subjected to
DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc.,
Santa Clara Calif.; described in U.S. Pat. No. 5,837,458; Chang,
C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F. C.
et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al.
(1996) Nat. Biotechnol. 14:315-319) to alter or improve the
biological properties of GCREC, such as its biological or enzymatic
activity or its ability to bind to other molecules or compounds.
DNA shuffling is a process by which a library of gene variants is
produced using PCR-mediated recombination of gene fragments. The
library is then subjected to selection or screening procedures that
identify those gene variants with the desired properties. These
preferred variants may then be pooled and further subjected to
recursive rounds of DNA shuffling and selection/screening. Thus,
genetic diversity is created through "artificial" breeding and
rapid molecular evolution. For example, fragments of a single gene
containing random point mutations may be recombined, screened, and
then reshuffled until the desired properties are optimized.
Alternatively, fragments of a given gene may be recombined with
fragments of homologous genes in the same gene family, either from
the same or different species, thereby maximizing the genetic
diversity of multiple naturally occurring genes in a directed and
controllable manner.
[0155] In another embodiment, sequences encoding GCREC may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucleic
Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic
Acids Symp. Ser. 7:225-232.) Alternatively, GCREC itself or a
fragment thereof may be synthesized using chemical methods. For
example, peptide synthesis can be performed using various
solution-phase or solid-phase techniques. (See, e.g., Creighton, T.
(1984) Proteins, Structures and Molecular Properties, W H Freeman,
New York N.Y., pp.55-60; and Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI
431A peptide synthesizer (Applied Biosystems). Additionally, the
amino acid sequence of GCREC, or any part thereof, may be altered
during direct synthesis and/or combined with sequences from other
proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a sequence of a naturally occurring
polypeptide.
[0156] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g., Chiez, R. M.
and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The
composition of the synthetic peptides may be confirmed by amino
acid analysis or by sequencing. (See, e.g., Creighton, supra, pp.
28-53.)
[0157] In order to express a biologically active GCREC, the
nucleotide sequences encoding GCREC 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 GCREC. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding GCREC.
Such signals include the ATG initiation codon and adjacent
sequences, e.g. the Kozak sequence. In cases where sequences
encoding GCREC 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.)
[0158] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding GCREC 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.)
[0159] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding GCREC. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook,
supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509; Engelhard, E. K et al. (1994) Proc.
Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill,
New York N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J. J. et al.
(1997) Nat. Genet 15:345-355.) Expression vectors derived from
retroviruses, adenoviruses, or herpes or vaccinia viruses, or from
various bacterial plasmids, may be used for delivery of nucleotide
sequences to the targeted organ, tissue, or cell population. (See,
e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356;
Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344;
Buller, R. M. et al. (1985) Nature 317(6040):813-815; McGregor, D.
P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, I. M. and
N. Somia (1997) Nature 389:239-242.) The invention is not limited
by the host cell employed.
[0160] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding GCREC. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding GCREC 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 GCREC
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 GCREC are needed, e.g. for the production of
antibodies, vectors which direct high level expression of GCREC may
be used. For example, vectors containing the strong, inducible SP6
or T7 bacteriophage promoter may be used.
[0161] Yeast expression systems may be used for production of
GCREC. A number of vectors containing constitutive or inducible
promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or
Pichia pastoris. In addition, such vectors direct either the
secretion or intracellular retention of expressed proteins and
enable integration of foreign sequences into the host genome for
stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter, G. A.
et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C. A. et
al. (1994) Bio/Technology 12:181-184.)
[0162] Plant systems may also be used for expression of GCREC.
Transcription of sequences encoding GCREC may be driven by viral
promoters, e.g., the 35S and 19S promoters of CaMV used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as
the small subunit of RUBISCO or heat shock promoters may be used.
(See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie,
R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991)
Results Probl. Cell Differ. 17:85-105.) These constructs can be
introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York
N.Y., pp. 191-196.)
[0163] 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 GCREC 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 GCREC in host cells. (See,
e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0164] 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.)
[0165] For long term production of recombinant proteins in
mammalian systems, stable expression of GCREC in cell lines is
preferred. For example, sequences encoding GCREC 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.
[0166] 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.- and 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 and pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA
77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol.
150:1-14.) Additional selectable genes have been described, e.g.,
trpB and hisD, which alter cellular requirements for metabolites.
(See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.
Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins,
green fluorescent proteins (GFP; Clontech), .beta. glucuronidase
and its substrate 13-glucuronide, or luciferase and its substrate
luciferin may be used. These markers can be used not only to
identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system. (See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol.
55:121-131.)
[0167] 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 GCREC is inserted within a marker gene
sequence, transformed cells containing sequences encoding GCREC can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding GCREC 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.
[0168] In general, host cells that contain the nucleic acid
sequence encoding GCREC and that express GCREC 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.
[0169] Immunological methods for detecting and measuring the
expression of GCREC 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
GCREC 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.)
[0170] 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 GCREC include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding GCREC, 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.
[0171] Host cells transformed with nucleotide sequences encoding
GCREC 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 GCREC may be designed to
contain signal sequences which direct secretion of GCREC through a
prokaryotic or eukaryotic cell membrane.
[0172] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" or "pro" form of the protein may also be used to
specify protein targeting, folding, and/or activity. Different host
cells which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and WI38) are available from the American Type
Culture Collection (ATCC, Manassas Va.) and may be chosen to ensure
the correct modification and processing of the foreign protein.
[0173] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding GCREC 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 GCREC protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of GCREC 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 GCREC encoding sequence and the heterologous protein
sequence, so that GCREC 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.
[0174] In a further embodiment of the invention, synthesis of
radiolabeled GCREC may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract system (Promega). These
systems couple transcription and translation of protein-coding
sequences operably associated with the T7, T3, or SP6 promoters.
Translation takes place in the presence of a radiolabeled amino
acid precursor, for example, .sup.35S-methionine.
[0175] GCREC of the present invention or fragments thereof may be
used to screen for compounds that specifically bind to GCREC. At
least one and up to a plurality of test compounds may be screened
for specific binding to GCREC. Examples of test compounds include
antibodies, oligonucleotides, proteins (e.g., receptors), or small
molecules.
[0176] In one embodiment, the compound thus identified is closely
related to the natural ligand of GCREC, e.g., a ligand or fragment
thereof, a natural substrate, a structural or functional mimetic,
or a natural binding partner. (See, e.g., Coligan, J. E. et al.
(1991) Current Protocols in Immunology 1(2): Chapter 5.) Similarly,
the compound can be closely related to the natural receptor to
which GCREC binds, or to at least a fragment of the receptor, e.g.,
the ligand binding site. In either case, the compound can be
rationally designed using known techniques. In one embodiment,
screening for these compounds involves producing appropriate cells
which express GCREC, either as a secreted protein or on the cell
membrane. Preferred cells include cells from mammals, yeast,
Drosophila, or E. coli. Cells expressing GCREC or cell membrane
fractions which contain GCREC are then contacted with a test
compound and binding, stimulation, or inhibition of activity of
either GCREC or the compound is analyzed.
[0177] An assay may simply test binding of a test compound to the
polypeptide, wherein binding is detected by a fluorophore,
radioisotope, enzyme conjugate, or other detectable label. For
example, the assay may comprise the steps of combining at least one
test compound with GCREC, either in solution or affixed to a solid
support, and detecting the binding of GCREC to the compound.
Alternatively, the assay may detect or measure binding of a test
compound in the presence of a labeled competitor. Additionally, the
assay may be carried out using cell-free preparations, chemical
libraries, or natural product mixtures, and the test compound(s)
may be free in solution or affixed to a solid support.
[0178] GCREC of the present invention or fragments thereof may be
used to screen for compounds that modulate the activity of GCREC.
Such compounds may include agonists, antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under
conditions permissive for GCREC activity, wherein GCREC is combined
with at least one test compound, and the activity of GCREC in the
presence of a test compound is compared with the activity of GCREC
in the absence of the test compound. A change in the activity of
GCREC in the presence of the test compound is indicative of a
compound that modulates the activity of GCREC. Alternatively, a
test compound is combined with an in vitro or cell-free system
comprising GCREC under conditions suitable for GCREC activity, and
the assay is performed. In either of these assays, a test compound
which modulates the activity of GCREC may do so indirectly and need
not come in direct contact with the test compound. At least one and
up to a plurality of test compounds may be screened.
[0179] In another embodiment, polynucleotides encoding GCREC or
their mammalian homologs may be "knocked out" in an animal model
system using homologous recombination in embryonic stem (ES) cells.
Such techniques are well known in the art and are useful for the
generation of animal models of human disease. (See, e.g., U.S. Pat.
No. 5,175,383 and U.S. Pat. No. 5,767,337.) For example, mouse ES
cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and grown in culture. The ES cells are
transformed with a vector containing the gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo;
Capecchi, M. R. (1989) Science 244:1288-1292). The vector
integrates into the corresponding region of the host genome by
homologous recombination. Alternatively, homologous recombination
takes place using the Cre-loxP system to knockout a gene of
interest in a tissue- or developmental stage-specific manner
(Marth, J. D. (1996) Clin. Invest. 97:1999-2002; Wagner, K. U. et
al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells
are identified and microinjected into mouse cell blastocysts such
as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred to pseudopregnant dams, and the resulting
chimeric progeny are genotyped and bred to produce heterozygous or
homozygous strains. Transgenic animals thus generated may be tested
with potential therapeutic or toxic agents.
[0180] Polynucleotides encoding GCREC may also be manipulated in
vitro in ES cells derived from human blastocysts. Human ES cells
have the potential to differentiate into at least eight separate
cell lineages including endoderm, mesoderm, and ectodermal cell
types. These cell lineages differentiate into, for example, neural
cells, hematopoietic lineages, and cardiomyocytes (Thomson, J. A.
et al. (1998) Science 282:1145-1147).
[0181] Polynucleotides encoding GCREC can also be used to create
"knockin" humanized animals (pigs) or transgenic animals (mice or
rats) to model human disease. With knockin technology, a region of
a polynucleotide encoding GCREC is injected into animal ES cells,
and the injected sequence integrates into the animal cell genome.
Transformed cells are injected into blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred
lines are studied and treated with potential pharmaceutical agents
to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress GCREC, e.g., by
secreting GCREC in its milk, may also serve as a convenient source
of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev.
4:55-74).
[0182] Therapeutics
[0183] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of GCREC and G-protein
coupled receptors. In addition, the expression of GCREC is closely
associated with cancerous, neurological, gastrointestinal, and lung
tissue. Therefore, GCREC appears to play a role in cell
proliferative, neurological, cardiovascular, gastrointestinal,
autoimmune/inflammatory, and metabolic disorders, and viral
infections. In the treatment of disorders associated with increased
GCREC expression or activity, it is desirable to decrease the
expression or activity of GCREC. In the treatment of disorders
associated with decreased GCREC expression or activity, it is
desirable to increase the expression or activity of GCREC.
[0184] Therefore, in one embodiment, GCREC 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 GCREC. Examples of such disorders include, but are not limited
to, a cell proliferative disorder such as actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; a 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,
Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial frontotemporal dementia; a
cardiovascular disorder such as arteriovenous fistula,
atherosclerosis, hypertension, vasculitis, Raynaud's disease,
aneurysms, arterial dissections, varicose veins, thrombophlebitis
and phlebothrombosis, vascular tumors, complications of
thrombolysis, balloon angioplasty, vascular replacement, and
coronary artery bypass graft surgery, congestive heart failure,
ischemic heart disease, angina pectoris, myocardial infarction,
hypertensive heart disease, degenerative valvular heart disease,
calcific aortic valve stenosis, congenitally bicuspid aortic valve,
mitral annular calcification, mitral valve prolapse, rheumatic
fever and rheumatic heart disease, infective endocarditis,
nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus, carcinoid heart disease, cardiomyopathy,
myocarditis, pericarditis, neoplastic heart disease, congenital
heart disease, and complications of cardiac transplantation; a
gastrointestinal disorder such as dysphagia, peptic esophagitis,
esophageal spasm, esophageal stricture, esophageal carcinoma,
dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis, gastroparesis, antral or pyloric edema, abdominal
angina, pyrosis, gastroenteritis, intestinal obstruction,
infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma,
biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis,
ulcerative colitis, ulcerative proctitis, Crohn's disease,
Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage,
acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice,
hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis,
hemochromatosis, Wilson's disease, alpha.sub.1-antitrypsin
deficiency, Reye's syndrome, primary sclerosing cholangitis, liver
infarction, portal vein obstruction and thrombosis, centrilobular
necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive
disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of pregnancy, and hepatic tumors including
nodular hyperplasias, adenomas, and carcinomas; an
autoimmune/inflammatory disorder such as acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; a metabolic disorder such as
diabetes, obesity, and osteoporosis; and an infection by a viral
agent classified as adenovirus, arenavirus, bunyavirus,
calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus,
flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus,
picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and
togavirus.
[0185] In another embodiment, a vector capable of expressing GCREC
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 GCREC including, but not limited to,
those described above.
[0186] In a further embodiment, a composition comprising a
substantially purified GCREC 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 GCREC including, but not limited to, those provided above.
[0187] In still another embodiment, an agonist which modulates the
activity of GCREC may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of GCREC including, but not limited to, those listed above.
[0188] In a further embodiment, an antagonist of GCREC may be
administered to a subject to treat or prevent a disorder associated
with increased expression or activity of GCREC. Examples of such
disorders include, but are not limited to, those cell
proliferative, neurological, cardiovascular, gastrointestinal,
autoimmune/inflammatory, and metabolic disorders, and viral
infections, described above. In one aspect, an antibody which
specifically binds GCREC may be used directly as an antagonist or
indirectly as a targeting or delivery mechanism for bringing a
pharmaceutical agent to cells or tissues which express GCREC.
[0189] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding GCREC may be administered
to a subject to treat or prevent a disorder associated with
increased expression or activity of GCREC including, but not
limited to, those described above.
[0190] 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.
[0191] An antagonist of GCREC may be produced using methods which
are generally known in the art. In particular, purified GCREC may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
GCREC. Antibodies to GCREC may also be generated using methods that
are well known in the art Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain
antibodies, Fab fragments, and fragments produced by a Fab
expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are generally preferred for therapeutic
use.
[0192] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with GCREC 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.
[0193] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to GCREC have an amino acid
sequence consisting of at least about 5 amino acids, and generally
will consist of at least about 10 amino acids. It is also
preferable that these oligopeptides, peptides, or fragments are
identical to a portion of the amino acid sequence of the natural
protein. Short stretches of GCREC amino acids may be fused with
those of another protein, such as KLH, and antibodies to the
chimeric molecule may be produced.
[0194] Monoclonal antibodies to GCREC may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. USA 80:2026-2030; and Cole, S. P. et al. (1984) Mol.
Cell Biol. 62:109-120.)
[0195] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. USA
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
GCREC-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton, D. R. (1991) Proc.
Natl. Acad. Sci. USA 88:10134-10137.)
[0196] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et
al. (1991) Nature 349:293-299.)
[0197] Antibody fragments which contain specific binding sites for
GCREC may also be generated. For example, such fragments include,
but are not limited to, F(ab').sub.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.)
[0198] 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 GCREC and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering GCREC
epitopes is generally used, but a competitive binding assay may
also be employed (Pound, supra).
[0199] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for GCREC. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
GCREC-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 GCREC epitopes,
represents the average affinity, or avidity, of the antibodies for
GCREC. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular GCREC 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
GCREC-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 GCREC, preferably in active form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington D.C.; Liddell, J. E. and A. Cryer (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York N.Y.).
[0200] The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml,
is generally employed in procedures requiring precipitation of
GCREC-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.)
[0201] In another embodiment of the invention, the polynucleotides
encoding GCREC, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, modifications of gene
expression can be achieved by designing complementary sequences or
antisense molecules (DNA, RNA, PNA, or modified oligonucleotides)
to the coding or regulatory regions of the gene encoding GCREC.
Such technology is well known in the art, and antisense
oligonucleotides or larger fragments can be designed from various
locations along the coding or control regions of sequences encoding
GCREC. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc., Totawa N.J.)
[0202] In therapeutic use, any gene delivery system suitable for
introduction of the antisense sequences into appropriate target
cells can be used. Antisense sequences can be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence complementary to at least a
portion of the cellular sequence encoding the target protein. (See,
e.g., Slater, J. E. et al. (1998) J. Allergy Cli. Immunol.
102(3):469-475; and Scanlon, K. J. et al. (1995) 9(13):1288-1296.)
Antisense sequences can also be introduced intracellularly through
the use of viral vectors, such as retrovirus and adeno-associated
virus vectors. (See, e.g., Miller, A. D. (1990) Blood 76:271;
Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include
liposome-derived systems, artificial viral envelopes, and other
systems known in the art. (See, e.g., Rossi, J. J. (1995) Br. Med.
Bull. 51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.
87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids
Res. 25(14):2730-2736.)
[0203] In another embodiment of the invention, polynucleotides
encoding GCREC may be used for somatic or germline gene therapy.
Gene therapy may be performed to (i) correct a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1
disease characterized by X-linked inheritance (Cavazzana-Calvo, M.
et al. (2000) Science 288:669-672), severe combined
immunodeficiency syndrome associated with an inherited adenosine
deaminase (ADA) deficiency Blaese, R. M. et al. (1995) Science
270:475-480; Bordignon, C. et al. (1995) Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal,
R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G. et
al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or
Factor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;
Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express
a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated cell proliferation), or (iii) express
a protein which affords protection against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency
virus (H) (Baltimore, D. (1988) Nature 335:395-396; Poeschia, E. et
al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B
or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides brasiliensis; and protozoan parasites such as
Plasmodium falciparum and Trypanosoma cruzi). In the case where a
genetic deficiency in GCREC expression or regulation causes
disease, the expression of GCREC from an appropriate population of
transduced cells may alleviate the clinical manifestations caused
by the genetic deficiency.
[0204] In a further embodiment of the invention, diseases or
disorders caused by deficiencies in GCREC are treated by
constructing mammalian expression vectors encoding GCREC and
introducing these vectors by mechanical means into GCREC-deficient
cells. Mechanical transfer technologies for use with cells in vivo
or ex vitro include (i) direct DNA microinjection into individual
cells, (ii) ballistic gold particle delivery, (iii)
liposome-mediated transfection, (iv) receptor-mediated gene
transfer, and (v) the use of DNA trasposons (Morgan, R. A. and W.
F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997)
Cell 91:501-510; Boulay, J-L. and H. Rcipon (1998) Curr. Opin.
Biotechnol. 9:445-450).
[0205] Expression vectors that may be effective for the expression
of GCREC include, but are not limited to, the PCDNA 3.1, EPITAG,
PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad Calif.),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo
Alto Calif.). GCREC may be expressed using (i) a constitutively
active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma
virus (RSV), SV40 virus, thymidine kinase (TK), or .beta.-actin
genes), (ii) an inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551; Gossen, M. et al.
(1995) Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the
T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter
(available in the plasmids PVGRXR and PIND; Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible promoter (Rossi, F. M. V. and Blau, H. M. supra)), or
(iii) a tissue-specific promoter or the native promoter of the
endogenous gene encoding GCREC from a normal individual.
[0206] Commercially available liposome transformation kits (e.g.,
the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen)
allow one with ordinary skill in the art to deliver polynucleotides
to target cells in culture and require minimal effort to optimize
experimental parameters. In the alternative, transformation is
performed using the calcium phosphate method (Graham, F. L. and A.
J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann,
E. et al. (1982) EMBO J. 1:841-845). The introduction of DNA to
primary cells requires modification of these standardized mammalian
transfection protocols.
[0207] In another embodiment of the invention, diseases or
disorders caused by genetic defects with respect to GCREC
expression are treated by constructing a retrovirus vector
consisting of (i) the polynucleotide encoding GCREC under the
control of an independent promoter or the retrovirus long terminal
repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive element (RRE) along with additional
retrovirus cis-acting RNA sequences and coding sequences required
for efficient vector propagation Retrovirus vectors (e.g., PFB and
PFBNEO) are commercially available (Stratagene) and are based on
published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
U.S.A. 92:6733-6737), incorporated by reference herein. The vector
is propagated in an appropriate vector producing cell line (VPCL)
that expresses an envelope gene with a tropism for receptors on the
target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M. A.
et al. (1987) J. Virol. 61:1639-1646; Adam, M. A. and A. D. Miller
(1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880).
U.S. Pat. No. 5,910,434 to Rigg ("Method for obtaining retrovirus
packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses a method for obtaining
retrovirus packaging cell lines and is hereby incorporated by
reference. Propagation of retrovirus vectors, transduction of a
population of cells (e.g., CD4.sup.+ T-cells), and the return of
transduced cells to a patient are procedures well known to persons
skilled in the art of gene therapy and have been well documented
(Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
(1997) Blood 89:2259-2267; Bonyhadi, M. L. (1997) J. Virol.
71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. U.S.A.
95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
[0208] In the alternative, an adenovirus-based gene therapy
delivery system is used to deliver polynucleotides encoding GCREC
to cells which have one or more genetic abnormalities with respect
to the expression of GCREC. The construction and packaging of
adenovirus-based vectors are well known to those with ordinary
skill in the art. Replication defective adenovirus vectors have
proven to be versatile for importing genes encoding
immunoregulatory proteins into intact islets in the pancreas
(Csete, M. E. et al. (1995) Transplantation 27:263-268).
Potentially useful adenoviral vectors are described in U.S. Pat.
No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also
Antinozzi, P. A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and
Verma, I. M. and N. Somia (1997) Nature 18:389:239-242, both
incorporated by reference herein.
[0209] In another alternative, a herpes-based, gene therapy
delivery system is used to deliver polynucleotides encoding GCREC
to target cells which have one or more genetic abnormalities with
respect to the expression of GCREC. The use of herpes simplex virus
(HSV)-based vectors may be especially valuable for introducing
GCREC to cells of the central nervous system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are
well known to those with ordinary skill in the art. A
replication-competent herpes simplex virus (HSV) type 1-based
vector has been used to deliver a reporter gene to the eyes of
primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The
construction of a HSV-1 virus vector has also been disclosed in
detail in U.S. Pat. No. 5,804,413 to DeLuca ("Herpes simplex virus
strains for gene transfer"), which is hereby incorporated by
reference. U.S. Pat. No. 5,804,413 teaches the use of recombinant
HSV d92 which consists of a genome containing at least one
exogenous gene to be transferred to a cell under the control of the
appropriate promoter for purposes including human gene therapy.
Also taught by this patent are the construction and use of
recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV
vectors, see also Goins, W. F. et al. (1999) J. Virol. 73:519-532
and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby
incorporated by reference. The manipulation of cloned herpesvirus
sequences, the generation of recombinant virus following the
transfection of multiple plasmids containing different segments of
the large herpesvirus genomes, the growth and propagation of
herpesvirus, and the infection of cells with herpesvirus are
techniques well known to those of ordinary skill in the art.
[0210] In another alternative, an alphavirus (positive,
single-stranded RNA virus) vector is used to deliver
polynucleotides encoding GCREC to target cells. The biology of the
prototypic alphavirus, Semliki Forest Virus (SFV), has been studied
extensively and gene transfer vectors have been based on the SFV
genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is
generated that normally encodes the viral capsid proteins. This
subgenomic RNA replicates to higher levels than the full length
genomic RNA, resulting in the overproduction of capsid proteins
relative to the viral proteins with enzymatic activity (e.g.,
protease and polymerase). Similarly, inserting the coding sequence
for GCREC into the alphavirus genome in place of the capsid-coding
region results in the production of a large number of GCREC-coding
RNAs and the synthesis of high levels of GCREC in vector transduced
cells. While alphavirus infection is typically associated with cell
lysis within a few days, the ability to establish a persistent
infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN) indicates that the lytic replication of
alphaviruses can be altered to suit the needs of the gene therapy
application (Dryga, S. A. et al. (1997) Virology 228:74-83). The
wide host range of alphaviruses will allow the introduction of
GCREC into a variety of cell types. The specific transduction of a
subset of cells in a population may require the sorting of cells
prior to transduction. The methods of manipulating infectious cDNA
clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known
to those with ordinary skill in the art.
[0211] Oligonucleotides derived from the transcription initiation
site, e.g., between about positions -10 and +10 from the start
site, may also be employed to inhibit gene expression. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (See, e.g., Gee, J. E. et
al. (1994) in Huber, B. E. and B. I. Carr, Molecular and
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.
[0212] 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 GCREC.
[0213] 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.
[0214] 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 GCREC. 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.
[0215] 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.
[0216] An additional embodiment of the invention encompasses a
method for screening for a compound which is effective in altering
expression of a polynucleotide encoding GCREC. Compounds which may
be effective in altering expression of a specific polynucleotide
may include, but are not limited to, oligonucleotides, antisense
oligonucleotides, triple helix-forming oligonucleotides,
transcription factors and other polypeptide transcriptional
regulators, and non-macromolecular chemical entities which are
capable of interacting with specific polynucleotide sequences.
Effective compounds may alter polynucleotide expression by acting
as either inhibitors or promoters of polynucleotide expression.
Thus, in the treatment of disorders associated with increased GCREC
expression or activity, a compound which specifically inhibits
expression of the polynucleotide encoding GCREC may be
therapeutically useful, and in the treatment of disorders
associated with decreased GCREC expression or activity, a compound
which specifically promotes expression of the polynucleotide
encoding GCREC may be therapeutically useful.
[0217] At least one, and up to a plurality, of test compounds may
be screened for effectiveness in altering expression of a specific
polynucleotide. A test compound may be obtained by any method
commonly known in the art, including chemical modification of a
compound known to be effective in altering polynucleotide
expression; selection from an existing, commercially-available or
proprietary library of naturally-occurring or non-natural chemical
compounds; rational design of a compound based on chemical and/or
structural properties of the target polynucleotide; and selection
from a library of chemical compounds created combinatorially or
randomly. A sample comprising a polynucleotide encoding GCREC is
exposed to at least one test compound thus obtained. The sample may
comprise, for example, an intact or permeabilized cell, or an in
vitro cell-free or reconstituted biochemical system. Alterations in
the expression of a polynucleotide encoding GCREC are assayed by
any method commonly known in the art. Typically, the expression of
a specific nucleotide is detected by hybridization with a probe
having a nucleotide sequence complementary to the sequence of the
polynucleotide encoding GCREC. The amount of hybridization may be
quantified, thus forming the basis for a comparison of the
expression of the polynucleotide both with and without exposure to
one or more test compounds. Detection of a change in the expression
of a polynucleotide exposed to a test compound indicates that the
test compound is effective in altering the expression of the
polynucleotide. A screen for a compound effective in altering
expression of a specific polynucleotide can be carried out, for
example, using a Schizosaccharomyces pombe gene expression system
(Atkins, D. et al. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et
al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as
HeLa cell (Clarke, M. L. et al. (2000) Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention
involves screening a combinatorial library of oligonucleotides
(such as deoxyribonucleotides, ribonucleotides, peptide nucleic
acids, and modified oligonucleotides) for antisense activity
against a specific polynucleotide sequence (Bruice, T. W. et al.
(1997) U.S. Pat. No. 5,686,242; Bruice, T. W. et al. (2000) U.S.
Pat. No. 6,022,691).
[0218] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nat. Biotechnol. 15:462-466.)
[0219] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as humans, dogs, cats, cows, horses, rabbits,
and monkeys.
[0220] An additional embodiment of the invention relates to the
administration of a composition which generally comprises an active
ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins. Various formulations are commonly known and are
thoroughly discussed in the latest edition of Remington's
Pharmaceutical Sciences (Maack Publishing, Easton Pa.). Such
compositions may consist of GCREC, antibodies to GCREC, and
mimetics, agonists, antagonists, or inhibitors of GCREC.
[0221] The compositions utilized in this invention may be
administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0222] Compositions for pulmonary administration may be prepared in
liquid or dry powder form. These compositions are generally
aerosolized immediately prior to inhalation by the patient. In the
case of small molecules (e.g. traditional low molecular weight
organic drugs), aerosol delivery of fast-acting formulations is
well-known in the art. In the case of macromolecules (e.g. larger
peptides and proteins), recent developments in the field of
pulmonary delivery via the alveolar region of the lung have enabled
the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton. J. S. et al., U.S. Pat. No.
5,997,848). Pulmonary delivery has the advantage of administration
without needle injection, and obviates the need for potentially
toxic penetration enhancers.
[0223] 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.
[0224] Specialized forms of compositions may be prepared for direct
intracellular delivery of macromolecules comprising GCREC or
fragments thereof. For example, liposome preparations containing a
cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the macromolecule. Alternatively, GCREC
or a fragment thereof may be joined to a short cationic N-terminal
portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to transduce into the cells of all tissues,
including the brain, in a mouse model system (Schwarze, S. R. et
al. (1999) Science 285:1569-1572).
[0225] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models such as mice, rats, rabbits,
dogs, monkeys, or pigs. An animal model may also be used to
determine the appropriate concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans.
[0226] A therapeutically effective dose refers to that amount of
active ingredient, for example GCREC or fragments thereof,
antibodies of GCREC, and agonists, antagonists or inhibitors of
GCREC, 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.50/ED.sub.50 ratio. Compositions which exhibit large
therapeutic indices are preferred. The data obtained from cell
culture assays and animal studies are used to formulate a range of
dosage for human use. The dosage contained in such compositions is
preferably within a range of circulating concentrations that
includes the ED.sub.50 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.
[0227] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting compositions may be administered every 3 to 4 days,
every week, or biweekly depending on the half-life and clearance
rate of the particular formulation.
[0228] 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.
[0229] Diagnostics
[0230] In another embodiment, antibodies which specifically bind
GCREC may be used for the diagnosis of disorders characterized by
expression of GCREC, or in assays to monitor patients being treated
with GCREC or agonists, antagonists, or inhibitors of GCREC.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for GCREC include methods which utilize the antibody and a label to
detect GCREC 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.
[0231] A variety of protocols for measuring GCREC, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of GCREC expression.
Normal or standard values for GCREC expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, for example, human subjects, with antibodies to GCREC
under conditions suitable for complex formation. The amount of
standard complex formation may be quantitated by various methods,
such as photometric means. Quantities of GCREC 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.
[0232] In another embodiment of the invention, the polynucleotides
encoding GCREC may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantify gene expression
in biopsied tissues in which expression of GCREC may be correlated
with disease. The diagnostic assay may be used to determine
absence, presence, and excess expression of GCREC, and to monitor
regulation of GCREC levels during therapeutic intervention.
[0233] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding GCREC or closely related molecules may be used
to identify nucleic acid sequences which encode GCREC. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification will determine whether the probe
identifies only naturally occurring sequences encoding GCREC,
allelic variants, or related sequences.
[0234] Probes may also be used for the detection of related
sequences, and may have at least 50% sequence identity to any of
the GCREC 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:40-78 or from genomic sequences including
promoters, enhancers, and introns of the GCREC gene.
[0235] Means for producing specific hybridization probes for DNAs
encoding GCREC include the cloning of polynucleotide sequences
encoding GCREC or GCREC 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.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0236] Polynucleotide sequences encoding GCREC may be used for the
diagnosis of disorders associated with expression of GCREC.
Examples of such disorders include, but are not limited to, a cell
proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective
tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; a 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,
Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial frontotemporal dementia; a
cardiovascular disorder such as arteriovenous fistula,
atherosclerosis, hypertension, vasculitis, Raynaud's disease,
aneurysms, arterial dissections, varicose veins, thrombophlebitis
and phlebothrombosis, vascular tumors, complications of
thrombolysis, balloon angioplasty, vascular replacement, and
coronary artery bypass graft surgery, congestive heart failure,
ischemic heart disease, angina pectoris, myocardial infarction,
hypertensive heart disease, degenerative valvular heart disease,
calcific aortic valve stenosis, congenitally bicuspid aortic valve,
mitral annular calcification, mitral valve prolapse, rheumatic
fever and rheumatic heart disease, infective endocarditis,
nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus, carcinoid heart disease, cardiomyopathy,
myocarditis, pericarditis, neoplastic heart disease, congenital
heart disease, and complications of cardiac transplantation; a
gastrointestinal disorder such as dysphagia, peptic esophagitis,
esophageal spasm, esophageal stricture, esophageal carcinoma,
dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis, gastroparesis, antral or pyloric edema, abdominal
angina, pyrosis, gastroenteritis, intestinal obstruction,
infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma,
biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis,
ulcerative colitis, ulcerative proctitis, Crohn's disease,
Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage,
acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice,
hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis,
hemochromatosis, Wilson's disease, alpha.sub.1-antitrypsin
deficiency, Reye's syndrome, primary sclerosing cholangitis, liver
infarction, portal vein obstruction and thrombosis, centrilobular
necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive
disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of pregnancy, and hepatic tumors including
nodular hyperplasias, adenomas, and carcinomas; an
autoimmune/inflammatory disorder such as acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; a metabolic disorder such as
diabetes, obesity, and osteoporosis; and an infection by a viral
agent classified as adenovirus, arenavirus, bunyavirus,
calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus,
flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus,
picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and
togavirus. The polynucleotide sequences encoding GCREC 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 GCREC expression. Such
qualitative or quantitative methods are well known in the art.
[0237] In a particular aspect, the nucleotide sequences encoding
GCREC may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding GCREC may be labeled by standard
methods and added to a fluid or tissue sample from a patient under
conditions suitable for the formation of hybridization complexes.
After a suitable incubation period, the sample is washed and the
signal is quantified and compared with a standard value. If the
amount of signal in the patient sample is significantly altered in
comparison to a control sample then the presence of altered levels
of nucleotide sequences encoding GCREC 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.
[0238] In order to provide a basis for the diagnosis of a disorder
associated with expression of GCREC, 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 GCREC, 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.
[0239] 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.
[0240] 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.
[0241] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding GCREC 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 GCREC, or a fragment of a
polynucleotide complementary to the polynucleotide encoding GCREC,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantification of
closely related DNA or RNA sequences.
[0242] In a particular aspect, oligonucleotide primers derived from
the polynucleotide sequences encoding GCREC may be used to detect
single nucleotide polymorphisms (SNPs). SNPs are substitutions,
insertions and deletions that are a frequent cause of inherited or
acquired genetic disease in humans. Methods of SNP detection
include, but are not limited to, single-stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers derived from the polynucleotide sequences
encoding GCREC are used to amplify DNA using the polymerase chain
reaction (PCR). The DNA may be derived, for example, from diseased
or normal tissue, biopsy samples, bodily fluids, and the like. SNPs
in the DNA cause differences in the secondary and tertiary
structures of PCR products in single-stranded form, and these
differences are detectable using gel electrophoresis in
non-denaturing gels. In fSCCP, the oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in
high-throughput equipment such as DNA sequencing machines.
Additionally, sequence database analysis methods, termed in silico
SNP (isSNP), are capable of identifying polymorphisms by comparing
the sequence of individual overlapping DNA fragments which assemble
into a common consensus sequence. These computer-based methods
filter out sequence variations due to laboratory preparation of DNA
and sequencing errors using statistical models and automated
analyses of DNA sequence chromatograms. In the alternative, SNPs
may be detected and characterized by mass spectrometry using, for
example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego Calif.).
[0243] Methods which may also be used to quantify the expression of
GCREC include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993)
Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in a
high-throughput format where the oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric
or colorimetric response gives rapid quantitation.
[0244] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as elements on a microarray. The microarray can be used
in transcript imaging techniques which monitor the relative
expression levels of large numbers of genes simultaneously as
described below. The microarray may also be used to identify
genetic variants, mutations, and polymorphisms. This information
may be used to determine gene function, to understand the genetic
basis of a disorder, to diagnose a disorder, to monitor
progression/regression of disease as a function of gene expression,
and to develop and monitor the activities of therapeutic agents in
the treatment of disease. In particular, this information may be
used to develop a pharmacogenomic profile of a patient in order to
select the most appropriate and effective treatment regimen for
that patient. For example, therapeutic agents which are highly
effective and display the fewest side effects may be selected for a
patient based on his/her pharmacogenomic profile.
[0245] In another embodiment, GCREC, fragments of GCREC, or
antibodies specific for GCREC may be used as elements on a
microarray. The microarray may be used to monitor or measure
protein-protein interactions, drug-target interactions, and gene
expression profiles, as described above.
[0246] A particular embodiment relates to the use of the
polynucleotides of the present invention to generate a transcript
image of a tissue or cell type. A transcript image represents the
global pattern of gene expression by a particular tissue or cell
type. Global gene expression patterns are analyzed by quantifying
the number of expressed genes and their relative abundance under
given conditions and at a given time. (See Seilhamer et al.,
"Comparative Gene Transcript Analysis," U.S. Pat. No. 5,840,484,
expressly incorporated by reference herein.) Thus a transcript
image may be generated by hybridizing the polynucleotides of the
present invention or their complements to the totality of
transcripts or reverse transcripts of a particular tissue or cell
type. In one embodiment, the hybridization takes place in
high-throughput format, wherein the polynucleotides of the present
invention or their complements comprise a subset of a plurality of
elements on a microarray. The resultant transcript image would
provide a profile of gene activity.
[0247] Transcript images may be generated using transcripts
isolated from tissues, cell lines, biopsies, or other biological
samples. The transcript image may thus reflect gene expression in
vivo, as in the case of a tissue or biopsy sample, or in vitro, as
in the case of a cell line.
[0248] Transcript images which profile the expression of the
polynucleotides of the present invention may also be used in
conjunction with in vitro model systems and preclinical evaluation
of pharmaceuticals, as well as toxicological testing of industrial
and naturally-occurring environmental compounds. All compounds
induce characteristic gene expression patterns, frequently termed
molecular fingerprints or toxicant signatures, which are indicative
of mechanisms of action and toxicity (Nuwaysir, E. F. et al. (1999)
Mol. Carcinog. 24:153-159; Steiner, S. and N. L. Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test compound has a signature similar to that of a
compound with known toxicity, it is likely to share those toxic
properties. These fingerprints or signatures are most useful and
refined when they contain expression information from a large
number of genes and gene families. Ideally, a genome-wide
measurement of expression provides the highest quality signature.
Even genes whose expression is not altered by any tested compounds
are important as well, as the levels of expression of these genes
are used to normalize the rest of the expression data. The
normalization procedure is useful for comparison of expression data
after treatment with different compounds. While the assignment of
gene function to elements of a toxicant signature aids in
interpretation of toxicity mechanisms, knowledge of gene function
is not necessary for the statistical matching of signatures which
leads to prediction of toxicity. (See, for example, Press Release
00-02 from the National Institute of Environmental Health Sciences,
released Feb. 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is
important and desirable in toxicological screening using toxicant
signatures to include all expressed gene sequences.
[0249] In one embodiment, the toxicity of a test compound is
assessed by treating a biological sample containing nucleic acids
with the test compound. Nucleic acids that are expressed in the
treated biological sample are hybridized with one or more probes
specific to the polynucleotides of the present invention, so that
transcript levels corresponding to the polynucleotides of the
present invention may be quantified. The transcript levels in the
treated biological sample are compared with levels in an untreated
biological sample. Differences in the transcript levels between the
two samples are indicative of a toxic response caused by the test
compound in the treated sample.
[0250] Another particular embodiment relates to the use of the
polypeptide sequences of the present invention to analyze the
proteome of a tissue or cell type. The term proteome refers to the
global pattern of protein expression in a particular tissue or cell
type. Each protein component of a proteome can be subjected
individually to further analysis. Proteome expression patterns, or
profiles, are analyzed by quantifying the number of expressed
proteins and their relative abundance under given conditions and at
a given time. A profile of a cell's proteome may thus be generated
by separating and analyzing the polypeptides of a particular tissue
or cell type. In one embodiment, the separation is achieved using
two-dimensional gel electrophoresis, in which proteins from a
sample are separated by isoelectric focusing in the first
dimension, and then according to molecular weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner
and Anderson, supra. The proteins are visualized in the gel as
discrete and uniquely positioned spots, typically by staining the
gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical density of each protein spot is generally
proportional to the level of the protein in the sample. The optical
densities of equivalently positioned protein spots from different
samples, for example, from biological samples either treated or
untreated with a test compound or therapeutic agent, are compared
to identify any changes in protein spot density related to the
treatment. The proteins in the spots are partially sequenced using,
for example, standard methods employing chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein
in a spot may be determined by comparing its partial sequence,
preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the present invention. In some cases,
further sequence data may be obtained for definitive protein
identification.
[0251] A proteomic profile may also be generated using antibodies
specific for GCREC to quantify the levels of GCREC expression. In
one embodiment, the antibodies are used as elements on a
microarray, and protein expression levels are quantified by
exposing the microarray to the sample and detecting the levels of
protein bound to each array element (Lueking, A et al. (1999) Anal.
Biochem. 270:103-111; Mendoze, L. G. et al. (1999) Biotechniques
27:778-788). Detection may be performed by a variety of methods
known in the art, for example, by reacting the proteins in the
sample with a thiol- or amino-reactive fluorescent compound and
detecting the amount of fluorescence bound at each array
element.
[0252] Toxicant signatures at the proteome level are also useful
for toxicological screening, and should be analyzed in parallel
with toxicant signatures at the transcript level. There is a poor
correlation between transcript and protein abundances for some
proteins in some tissues (Anderson, N. L. and J. Seilhamer (1997)
Electrophoresis 18:533-537), so proteome toxicant signatures may be
useful in the analysis of compounds which do not significantly
affect the transcript image, but which alter the proteomic profile.
In addition, the analysis of transcripts in body fluids is
difficult, due to rapid degradation of mRNA, so proteomic profiling
may be more reliable and informative in such cases.
[0253] In another embodiment, the toxicity of a test compound is
assessed by treating a biological sample containing proteins with
the test compound. Proteins that are expressed in the treated
biological sample are separated so that the amount of each protein
can be quantified. The amount of each protein is compared to the
amount of the corresponding protein in an untreated biological
sample. A difference in the amount of protein between the two
samples is indicative of a toxic response to the test compound in
the treated sample. Individual proteins are identified by
sequencing the amino acid residues of the individual proteins and
comparing these partial sequences to the polypeptides of the
present invention.
[0254] In another embodiment, the toxicity of a test compound is
assessed by treating a biological sample containing proteins with
the test compound. Proteins from the biological sample are
incubated with antibodies specific to the polypeptides of the
present invention. The amount of protein recognized by the
antibodies is quantified. The amount of protein in the treated
biological sample is compared with the amount in an untreated
biological sample. A difference in the amount of protein between
the two samples is indicative of a toxic response to the test
compound in the treated sample.
[0255] 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. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT
application WO95/251116; Shalon, D. et al. (1995) PCT application
WO95/35505; Heller, R.& et al. (1997) Proc. Natl. Acad. Sci.
USA 94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No.
5,605,662.) Various types of microarrays are well known and
thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999) Oxford University Press, London, hereby
expressly incorporated by reference.
[0256] In another embodiment of the invention, nucleic acid
sequences encoding GCREC may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some
instances, noncoding sequences may be preferable over coding
sequences. For example, conservation of a coding sequence among
members of a multi-gene family may potentially cause undesired
cross hybridization during chromosomal mapping. The sequences may
be mapped to a particular chromosome, to a specific region of a
chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes (HACs), yeast artificial chromosomes (YACs),
bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g.,
Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C.
M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends
Genet. 7:149-154.) Once mapped, the nucleic acid sequences of the
invention may be used to develop genetic linkage maps, for example,
which correlate the inheritance of a disease state with the
inheritance of a particular chromosome region or restriction
fragment length polymorphism (RFLP). (See, for example, Lander, E.
S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA
83:7353-7357.)
[0257] Fluorescent in situ hybridization (FISH) may be correlated
with other physical and genetic map data. (See, e.g., Heinz-Ulrich,
et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic
map data can be found in various scientific journals or at the
Online Mendelian Inheritance in Man (OMIM) World Wide Web site.
Correlation between the location of the gene encoding GCREC on a
physical map and a specific disorder, or a predisposition to a
specific disorder, may help define the region of DNA associated
with that disorder and thus may further positional cloning
efforts.
[0258] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the exact chromosomal locus is not known. This information
is valuable to investigators searching for disease genes using
positional cloning or other gene discovery techniques. Once the
gene or genes responsible for a disease or syndrome have been
crudely localized by genetic linkage to a particular genomic
region, e.g., ataxia-telangiectasia to 11q22-23, any sequences
mapping to that area may represent associated or regulatory genes
for further investigation. (See, e.g., Gatti, R. A. et al. (1988)
Nature 336:577-580.) The nucleotide sequence of the instant
invention may also be used to detect differences in the chromosomal
location due to translocation, inversion, etc., among normal,
carrier, or affected individuals.
[0259] In another embodiment of the invention, GCREC, 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 GCREC and the agent being tested may be
measured.
[0260] 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 GCREC, or fragments thereof, and washed.
Bound GCREC is then detected by methods well known in the art.
Purified GCREC 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.
[0261] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding GCREC specifically compete with a test compound for binding
GCREC. In this manner, antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with GCREC.
[0262] In additional embodiments, the nucleotide sequences which
encode GCREC 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.
[0263] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever.
[0264] The disclosures of all patents, applications and
publications, mentioned above and below, in particular U.S. Ser.
No. 60/172,852, U.S. Ser. No. 60/171,732, U.S. Ser. No. 60/176,148,
and U.S. Ser. No. 60/177,331, are expressly incorporated by
reference herein.
EXAMPLES
[0265] 1. Construction of cDNA Libraries
[0266] Incyte cDNAs were derived from cDNA libraries described in
the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.) and
shown in Table 4, column 5. The Incyte cDNA shown for SEQ ID NO:40
was derived from a cDNA library constructed from bone marrow
tissue. The Incyte cDNAs shown for SEQ ID NO:41 were derived from
cDNA libraries constructed from small intestine, including tissues
associated with Crohn's disease, from large intestine, and from
brain tissues. The Incyte cDNAs shown for SEQ ID NO:42 were derived
from cDNA libraries constructed from prostate tumor, small
intestine, breast, and epidermal tissues. The Incyte cDNAs shown
for SEQ ID NO:43 were derived cDNA libraries constructed from soft
tissue tumor, fetal rib, and brain tissue associated with
Huntington's disease. The Incyte cDNAs shown for SEQ ID NO:57 were
derived from cDNA libraries constructed from lymphocytes and mast
cells, and from breast, uterine, prostate, adrenal gland, spinal
cord, tibial muscle, lung, esophagus, small intestine, and colon
tissues. The Incyte cDNAs shown for SEQ ID NO:58 were derived from
cDNA libraries constructed from a fallopian tube tumor, uterine
endometrium, and bronchial tissue. The Incyte cDNAs shown for SEQ
ID NO:59 were derived from cDNA libraries constructed from colon
tissues, including cecal tumor tissue, as well as from pancreatic
tumor, pituitary gland, and brain tissues. The Incyte cDNAs shown
for SEQ ID NO:60 were derived from cDNA libraries constructed from
brain, including brain tumor tissue and tissues associated with
Huntington's disease, and from prostate tumor, cervical
adenocarcinoma, breast, small intestine, and bladder tissues. 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.
[0267] 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.).
[0268] In some cases, Stratagene was provided with RNA and
constructed the corresponding cDNA libraries. Otherwise, cDNA was
synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using the recommended procedures or similar methods
known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.)
Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double
stranded cDNA, and the cDNA was digested with the appropriate
restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B,
or SEPHAROSE CL4B column chromatography (Amersham Pharmacia
Biotech) or preparative agarose gel electrophoresis. cDNAs were
ligated into compatible restriction enzyme sites of the polylinker
of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene),
PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen,
Carlsbad Calif.), or pINCY (Incyte Genomics, 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.
[0269] II. Isolation of cDNA Clones
[0270] Plasmids obtained as described in Example I were recovered
from host cells by in vivo excision using the UNIZAP vector system
(Stratagene) or by cell lysis. Plasmids were purified using at
least one of the following: a Magic or WIZARD Minipreps DNA
purification system (Promega); an AGTC Miniprep purification kit
(Edge Biosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following
precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or without lyophilization, at 4.degree.
C.
[0271] 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).
[0272] III. Sequencing and Analysis
[0273] Incyte cDNA recovered in plasmids as described in Example II
were sequenced as follows. Sequencing reactions were processed
using standard methods or high-throughput instrumentation such as
the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the
PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA
microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton)
liquid transfer system. cDNA sequencing reactions were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied
in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and
detection of labeled polynucleotides were carried out using the
MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or 377 sequencing system (Applied Biosystems) in
conjunction with standard ABI protocols and base calling software;
or other sequence analysis systems known in the art. Reading frames
within the cDNA sequences were identified using standard methods
(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA
sequences were selected for extension using the techniques
disclosed in Example VIII.
[0274] The polynucleotide sequences derived from Incyte cDNAs were
validated by removing vector, linker, and poly(A) sequences and by
masking ambiguous bases, using algorithms and programs based on
BLAST, dynamic programming, and dinucleotide nearest neighbor
analysis. The Incyte cDNA sequences or translations thereof were
then queried against a selection of public databases such as the
GenBank primate, rodent, mammalian, vertebrate, and eukaryote
databases, and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov
model (HMM)-based protein family databases such as PFAM. (HMM is a
probabilistic approach which analyzes consensus primary structures
of gene families. See, for example, Eddy, S. R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.) The queries were performed using programs
based on BLAST, FASTA, BLIMPS, and HMMR. The Incyte cDNA sequences
were assembled to produce polynucleotide sequences. Alternatively,
GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or Genscan-predicted coding sequences (see Examples IV
and V) were used to extend Incyte cDNA assemblages to full length.
Assembly was performed using programs based on Phred, Pbrap, and
Consed, and cDNA assemblages were screened for open reading frames
using programs based on GeneMark, BLAST, and FASTA. The
polynucleotide sequences were translated to derive the
corresponding polypeptide sequences which were subsequently
analyzed by querying against databases such as the GenBank protein
databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite, and hidden Markov model (HMM)-based protein family
databases such as PFAM. Full length polynucleotide sequences are
also analyzed using MACDNASIS PRO software (Hitachi Software
Engineering, South San Francisco Calif.) and LASERGENE software
(DNASTAR). Polynucleotide and polypeptide sequence alignments are
generated using default parameters specified by the CLUSTAL
algorithm as incorporated into the MEGALIGN multisequence alignment
program (DNASTAR), which also calculates the percent identity
between aligned sequences.
[0275] Table 7 summarizes the tools, programs, and algorithms used
for the analysis and assembly of Incyte cDNA and assembled
polynucleotide sequences and provides applicable descriptions,
references, and threshold parameters. The first column of Table 7
shows the tools, programs, and algorithms used, the second column
provides brief descriptions thereof, the third column presents
appropriate references, all of which are incorporated by reference
herein in their entirety, and the fourth column presents, where
applicable, the scores, probability values, and other parameters
used to evaluate the strength of a match between two sequences (the
higher the score or the lower the probability value, the greater
the identity between two sequences).
[0276] The programs described above for the assembly and analysis
of polynucleotide and polypeptide sequences were also used to
identify polynucleotide sequence fragments from SEQ ID NO:40-78.
Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and amplification technologies are described in
Table 4, column 4.
[0277] IV. Identification and Editing of Coding Sequences from
Genomic DNA
[0278] Putative G-protein coupled receptors were initially
identified by running the Genscan gene identification program
against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-purpose gene identification program which
analyzes genomic DNA sequences from a variety of organisms (See
Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge,
C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The
program concatenates predicted exons to form an assembled cDNA
sequence extending from a methionine to a stop codon. The output of
Genscan is a FASTA database of polynucleotide and polypeptide
sequences. The maximum range of sequence for Genscan to analyze at
once was set to 30 kb. To determine which of these Genscan
predicted cDNA sequences encode G-protein coupled receptors, the
encoded polypeptides were analyzed by querying against PFAM models
for G-protein coupled receptors (7tm.sub.--1, 7tm.sub.--2,
7tm.sub.--3, and 7tm.sub.--4). Potential G-protein coupled
receptors were also identified by homology to Incyte cDNA sequences
that had been annotated as G-protein coupled receptors. These
selected Genscan-predicted sequences were then compared by BLAST
analysis to the genpept and gbpri public databases. Where
necessary, the Genscan-predicted sequences were then edited by
comparison to the top BLAST hit from genpept to correct errors in
the sequence predicted by Genscan, such as extra or omitted exons.
BLAST analysis was also used to find any Incyte cDNA or public cDNA
coverage of the Genscan-predicted sequences, thus providing
evidence for transcription. When Incyte cDNA coverage was
available, this information was used to correct or confirm the
Genscan predicted sequence. Polynucleotide sequences, including SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:61,
SEQ ID NO:62, and SEQ ID NO:63, were obtained by assembling
Genscan-predicted coding sequences with Incyte cDNA sequences
and/or public cDNA sequences using the assembly process described
in Example III. Alternatively, polynucleotide sequences, including
SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:64, SEQ ID
NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ
ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74,
SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78, are
full length coding regions derived entirely from edited or unedited
Genscan-predicted coding sequences. Alternatively, polynucleotide
sequences, including SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ
ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,
SEQ ID NO:55, and SEQ ID NO:56, are partial genes derived from the
assembly and editing of Genscan-predicted sequences only.
[0279] V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
[0280] Partial cDNA sequences were extended with exons predicted by
the Genscan gene identification program described in Example IV.
Partial cDNAs assembled as described in Example III were mapped to
genomic DNA and parsed into clusters containing related cDNAs and
Genscan exon predictions from one or more genomic sequences. Each
cluster was analyzed using an algorithm based on graph theory and
dynamic programming to integrate cDNA and genomic information,
generating possible splice variants that were subsequently
confirmed, edited, or extended to create a full length sequence.
Sequence intervals in which the entire length of the interval was
present on more than one sequence in the cluster were identified,
and intervals thus identified were considered to be equivalent by
transitivity. For example, if an interval was present on a cDNA and
two genomic sequences, then all three intervals were considered to
be equivalent. This process allows unrelated but consecutive
genomic sequences to be brought together, bridged by cDNA sequence.
Intervals thus identified were then "stitched" together by the
stitching algorithm in the order that they appear along their
parent sequences to generate the longest possible sequence, as well
as sequence variants. Linkages between intervals which proceed
along one type of parent sequence (cDNA to cDNA or genomic sequence
to genomic sequence) were given preference over linkages which
change parent type (cDNA to genomic sequence). The resultant
stitched sequences were translated and compared by BLAST analysis
to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan were corrected by comparison to the top BLAST
hit from genpept Sequences were further extended with additional
cDNA sequences, or by inspection of genomic DNA, when
necessary.
[0281] "Stretched" Sequences
[0282] Partial DNA sequences were extended to full length with an
algorithm based on BLAST analysis. First, partial cDNAs assembled
as described in Example m were queried against public databases
such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases using the BLAST program. The nearest GenBank
protein homolog was then compared by BLAST analysis to either
Incyte cDNA sequences or GenScan exon predicted sequences described
in Example IV. A chimeric protein was generated by using the
resultant high-scoring segment pairs (HSPs) to map the translated
sequences onto the GenBank protein homolog. Insertions or deletions
may occur in the chimeric protein with respect to the original
GenBank protein homolog. The GenBank protein homolog, the chimeric
protein, or both were used as probes to search for homologous
genomic sequences from the public human genome databases. Partial
DNA sequences were therefore "stretched" or extended by the
addition of homologous genomic sequences. The resultant stretched
sequences were examined to determine whether it contained a
complete gene.
[0283] VI. Chromosomal Mapping of GCREC Encoding
Polynucleotides
[0284] The sequences which were used to assemble SEQ ID NO:40-78
were compared with sequences from the Incyte LIFESEQ database and
public domain databases using BLAST and other implementations of
the Smith-Waterman algorithm. Sequences from these databases that
matched SEQ ID NO:40-78 were assembled into clusters of contiguous
and overlapping sequences using assembly algorithms such as Phrap
(Table 7). Radiation hybrid and genetic mapping data available from
public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for Genome Research (WIGR), and Gnthon were
used to determine if any of the clustered sequences had been
previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment of all sequences of that cluster,
including its particular SEQ ID NO:, to that map location.
[0285] Map locations are represented by ranges, or intervals, or
human chromosomes. The map position of an interval, in
centiMorgans, is measured relative to the terminus of the
chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement
based on recombination frequencies between chromosomal markers. On
average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances are based on genetic markers
mapped by Gnthon which provide boundaries for radiation hybrid
markers whose sequences were included in each of the clusters.
Human genome maps and other resources available to the public, such
as the NCBI "GeneMap'99" World Wide Web site
(http://www.ncbi.nlm.ni- h.gov/genemap/), can be employed to
determine if previously identified disease genes map within or in
proximity to the intervals indicated above.
[0286] VII. Analysis of Polynucleotide Expression
[0287] 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.)
[0288] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in cDNA databases such as
GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster
than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
1 BLAST Score .times. Percent Identity 5 .times. minimum { length (
Seq . 1 ) , length ( Seq . 2 ) }
[0289] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. The product score is a normalized value between 0 and 100,
and is calculated as follows: the BLAST score is multiplied by the
percent nucleotide identity and the product is divided by (5 times
the length of the shorter of the two sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches
in a high-scoring segment pair (HSP), and -4 for every mismatch.
Two sequences may share more than one HSP (separated by gaps). If
there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score
represents a balance between fractional overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced
only for 100% identity over the entire length of the shorter of the
two sequences being compared. A product score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88%
identity and 100% overlap at the other. A product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79%
identity and 100% overlap.
[0290] Alternatively, polynucleotide sequences encoding GCREC are
analyzed with respect to the tissue sources from which they were
derived. For example, some full length sequences are assembled, at
least in part, with overlapping Incyte cDNA sequences (see Example
III). Each cDNA sequence is derived from a cDNA library constructed
from a human tissue. Each human tissue is classified into one of
the following organ/tissue categories: cardiovascular system;
connective tissue; digestive system; embryonic structures;
endocrine system; exocrine glands; genitalia, female; genitalia,
male; germ cells; hemic and immune system; liver; musculoskeletal
system; nervous system; pancreas; respiratory system; sense organs;
skin; stomatognathic system; unclassified/mixed; or urinary tract.
The number of libraries in each category is counted and divided by
the total number of libraries across all categories. Similarly,
each human tissue is classified into one of the following
disease/condition categories: cancer, cell line, developmental,
inflammation, neurological, trauma, cardiovascular, pooled, and
other, and the number of libraries in each category is counted and
divided by the total number of libraries across all categories. The
resulting percentages reflect the tissue- and disease-specific
expression of cDNA encoding GCREC. cDNA sequences and cDNA
library/tissue information are found in the LIFESEQ GOLD database
(Incyte Genomics, Palo Alto Calif.).
[0291] VIII. Extension of GCREC Encoding Polynucleotides
[0292] Full length polynucleotide sequences were also produced by
extension of an appropriate fragment of the full length molecule
using oligonucleotide primers designed from this fragment. One
primer was synthesized to initiate 5' extension of the known
fragment, and the other primer was synthesized to initiate 3'
extension of the known fragment. The initial primers were designed
using OLIGO 4.06 software (National Biosciences), or another
appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of about 50% or more, and to anneal to the target
sequence at temperatures of about 68.degree. C. to about 72.degree.
C. Any stretch of nucleotides which would result in hairpin
structures and primer-primer dimerizations was avoided.
[0293] 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.
[0294] 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
2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase
(Stratagene), with the following parameters for primer pair PCI A
and PCI B: Step 1: 94.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.
[0295] 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 (Corning 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 quantify 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 gel to determine which reactions
were successful in extending the sequence.
[0296] 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, and individual colonies were picked
and cultured overnight at 37.degree. C. in 384-well plates in
LB/2.times. carb liquid media.
[0297] 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% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (Applied Biosystems).
[0298] In like manner, full length polynucleotide sequences are
verified using the above procedure or are used to obtain 5'
regulatory sequences using the above procedure along with
oligonucleotides designed for such extension, and an appropriate
genomic library.
[0299] IX. Labeling and Use of Individual Hybridization Probes
[0300] Hybridization probes derived from SEQ ID NO:40-78 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 Winase (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, Xba I, or Pvu II (DuPont NEN).
[0301] 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 conditions of up to,
for example, 0.1.times. saline sodium citrate and 0.5% sodium
dodecyl sulfate. Hybridization patterns are visualized using
autoradiography or an alternative imaging means and compared.
[0302] X. Microarrays
[0303] The linkage or synthesis of array elements upon a microarray
can be achieved utilizing photolithography, piezoelectric printing
(ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate
in each of the aforementioned technologies should be uniform and
solid with a non-porous surface (Schena (1999), supra). Suggested
substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a procedure analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced using available
methods and machines well known to those of ordinary skill in the
art and may contain any appropriate number of elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al.
(1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
[0304] Full length cDNAs, Expressed Sequence Tags (ESTs), or
fragments or oligomers thereof may comprise the elements of the
microarray. Fragments or oligomers suitable for hybridization can
be selected using software well known in the art such as LASERGENE
software (DNASTAR). The array elements are hybridized with
polynucleotides in a biological sample. The polynucleotides in the
biological sample are conjugated to a fluorescent label or other
molecular tag for ease of detection. After hybridization,
nonhybridized nucleotides from the biological sample are removed,
and a fluorescence scanner is used to detect hybridization at each
array element. Alternatively, laser desorbtion and mass
spectrometry may be used for detection of hybridization. The degree
of complementarity and the relative abundance of each
polynucleotide which hybridizes to an element on the microarray may
be assessed. In one embodiment, microarray preparation and usage is
described in detail below.
[0305] Tissue or Cell Sample Preparation
[0306] Total RNA is isolated from tissue samples using the
guanidinium thiocyanate method and poly(A).sup.+ RNA is purified
using the oligo-(dT) cellulose method. Each poly(A).sup.+ RNA
sample is reverse transcribed using MMLV reverse-transcriptase,
0.05 pg/.mu.l oligo-(dT) primer (21 mer), 1.times. first strand
buffer, 0.03 units/.mu.l RNase inhibitor, 500 .mu.M dATP, 500 .mu.M
dGTP, 500 .mu.M dTTP, 40 .mu.M dCTP, 40 .mu.M dCTP-Cy3 (BDS) or
dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription
reaction is performed in a 25 ml volume containing 200 ng
poly(A).sup.+ RNA with GEMBRIGHT kits (Incyte). Specific control
poly(A).sup.+ RNAs are synthesized by in vitro transcription from
non-coding yeast genomic DNA. After incubation at 37.degree. C. for
2 hr, each reaction sample (one with Cy3 and another with Cy5
labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and
incubated for 20 minutes at 85.degree. C. to the stop the reaction
and degrade the RNA. Samples are purified using two successive
CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories,
Inc. (CLONTECH), Palo Alto Calif.) and after combining, both
reaction samples are ethanol precipitated using 1 ml of glycogen (1
mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The
sample is then dried to completion using a SpeedVAC (Savant
Instruments Inc., Holbrook N.Y.) and resuspended in 14 .mu.l
5.times.SSC/0.2% SDS.
[0307] Microarray Preparation
[0308] Sequences of the present invention are used to generate
array elements. Each array element is amplified from bacterial
cells containing vectors with cloned cDNA inserts. PCR
amplification uses primers complementary to the vector sequences
flanking the cDNA insert. Array elements are amplified in thirty
cycles of PCR from an initial quantity of 1-2 ng to a final
quantity greater than 5 .mu.g. Amplified array elements are then
purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
[0309] Purified array elements are immobilized on polymer-coated
glass slides. Glass microscope slides (Corning) are cleaned by
ultrasound in 0.1% SDS and acetone, with extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR Scientific Products Corporation (VWR), West
Chester Pa.), washed extensively in distilled water, and coated
with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a 110.degree. C. oven.
[0310] Array elements are applied to the coated glass substrate
using a procedure described in U.S. Pat. No. 5,807,522,
incorporated herein by reference. 1 .mu.l of the array element DNA,
at an average concentration of 100 ng/.mu.l, is loaded into the
open capillary printing element by a high-speed robotic apparatus.
The apparatus then deposits about 5 nl of array element sample per
slide.
[0311] Microarrays are UV-crosslinked using a STRATALINKER
UV-crosslinker (Stratagene). Microarrays are washed at room
temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays
in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc.,
Bedford Mass.) for 30 minutes at 60.degree. C. followed by washes
in 0.2% SDS and distilled water as before.
[0312] Hybridization
[0313] Hybridization reactions contain 9 .mu.l of sample mixture
consisting of 0.2 .mu.g each of Cy3 and Cy5 labeled cDNA synthesis
products in 5.times.SSC, 0.2% SDS hybridization buffer. The sample
mixture is heated to 65.degree. C. for 5 minutes and is aliquoted
onto the microarray surface and covered with an 1.8 cm.sup.2
coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly larger than a microscope slide. The
chamber is kept at 100% humidity internally by the addition of 140
.mu.l of 5.times.SSC in a corner of the chamber. The chamber
containing the arrays is incubated for about 6.5 hours at
60.degree. C. The arrays are washed for 10 min at 45.degree. C. in
a first wash buffer (1.times.SSC, 0.1% SDS), three times for 10
minutes each at 45.degree. C. in a second wash buffer
(0.1.times.SSC), and dried.
[0314] Detection
[0315] Reporter-labeled hybridization complexes are detected with a
microscope equipped with an Innova 70 mixed gas 10 W laser
(Coherent, Inc., Santa Clara Calif.) capable of generating spectral
lines at 488 nm for excitation of Cy3 and at 632 nm for excitation
of Cy5. The excitation laser light is focused on the array using a
20.times. microscope objective (Nikon, Inc., Melville N.Y.). The
slide containing the array is placed on a computer-controlled X-Y
stage on the microscope and raster-scanned past the objective. The
1.8 cm.times.1.8 cm array used in the present example is scanned
with a resolution of 20 micrometers.
[0316] In two separate scans, a mixed gas multiline laser excites
the two fluorophores sequentially. Emitted light is split, based on
wavelength, into two photomultiplier tube detectors (PMT R1477,
Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the
two fluorophores. Appropriate filters positioned between the array
and the photomultiplier tubes are used to filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650
nm for Cy5. Each array is typically scanned twice, one scan per
fluorophore using the appropriate filters at the laser source,
although the apparatus is capable of recording the spectra from
both fluorophores simultaneously.
[0317] The sensitivity of the scans is typically calibrated using
the signal intensity generated by a cDNA control species added to
the sample mixture at a known concentration. A specific location on
the array contains a complementary DNA sequence, allowing the
intensity of the signal at that location to be correlated with a
weight ratio of hybridizing species of 1:100,000. When two samples
from different sources (e.g., representing test and control cells),
each labeled with a different fluorophore, are hybridized to a
single array for the purpose of identifying genes that are
differentially expressed, the calibration is done by labeling
samples of the calibrating cDNA with the two fluorophores and
adding identical amounts of each to the hybridization mixture.
[0318] The output of the photomultiplier tube is digitized using a
12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog
Devices, Inc., Norwood Mass.) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the
signal intensity is mapped using a linear 20-color transformation
to a pseudocolor scale ranging from blue (low signal) to red (high
signal). The data is also analyzed quantitatively. Where two
different fluorophores are excited and measured simultaneously, the
data are first corrected for optical crosstalk (due to overlapping
emission spectra) between the fluorophores using each fluorophore's
emission spectrum.
[0319] A grid is superimposed over the fluorescence signal image
such that the signal from each spot is centered in each element of
the grid. The fluorescence signal within each element is then
integrated to obtain a numerical value corresponding to the average
intensity of the signal. The software used for signal analysis is
the GEMTOOLS gene expression analysis program (Incyte).
[0320] XI. Complementary Polynucleotides
[0321] Sequences complementary to the GCREC-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring GCREC. 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 GCREC. 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 GCREC-encoding transcript.
[0322] XII. Expression of GCREC
[0323] Expression and purification of GCREC is achieved using
bacterial or virus-based expression systems. For expression of
GCREC 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 GCREC upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of GCREC
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 GCREC 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.)
[0324] In most expression systems, GCREC 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
GCREC 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 GCREC obtained by these methods can
be used directly in the assays shown in Examples XVI, XVII, and
XVIII, where applicable.
[0325] XIII. Functional Assays
[0326] GCREC function is assessed by expressing the sequences
encoding GCREC 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, for example, an endothelial or hematopoietic cell line, 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.
[0327] The influence of GCREC on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding GCREC 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 GCREC and other genes of interest can
be analyzed by northern analysis or microarray techniques.
[0328] XIV. Production of GCREC Specific Antibodies
[0329] GCREC 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.
[0330] Alternatively, the GCREC 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.)
[0331] Typically, oligopeptides of about 15 residues in length are
synthesized using an ABI 431A peptide synthesizer (Applied
Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich,
St. Louis Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the oligopeptide-KLH complex in complete Freund's
adjuvant. Resulting antisera are tested for antipeptide and
anti-GCREC activity by, for example, binding the peptide or GCREC
to a substrate, blocking with 1% BSA, reacting with rabbit
antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
[0332] XV. Purification of Naturally Occurring GCREC Using Specific
Antibodies
[0333] Naturally occurring or recombinant GCREC is substantially
purified by immunoaffinity chromatography using antibodies specific
for GCREC. An immunoaffinity column is constructed by covalently
coupling anti-GCREC 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.
[0334] Media containing GCREC are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of GCREC (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/GCREC 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 GCREC is collected.
[0335] XVI. Identification of Molecules which Interact with
GCREC
[0336] Molecules which interact with GCREC may include agonists and
antagonists, as well as molecules involved in signal transduction,
such as G proteins. GCREC, or a fragment thereof, is labeled with
.sup.125I Bolton-Hunter reagent. (See, e.g., Bolton A. E. and W. M.
Hunter (1973) Biochem J. 133:529-539.) A fragment of GCREC
includes, for example, a fragment comprising one or more of the
three extracellular loops, the extracellular N-terminal region, or
the third intracellular loop. Candidate molecules previously
arrayed in the wells of a multi-well plate are incubated with the
labeled GCREC, washed, and any wells with labeled GCREC complex are
assayed. Data obtained using different concentrations of GCREC are
used to calculate values for the number, affinity, and association
of GCREC with the candidate ligand molecules.
[0337] Alternatively, molecules interacting with GCREC are analyzed
using the yeast two-hybrid system as described in Fields, S. and O.
Song (1989) Nature 340:245-246, or using commercially available
kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech). GCREC may also be used in the PATHCALLING process
(CuraGen Corp., New Haven Conn.) which employs the yeast two-hybrid
system in a high-throughput manner to determine all interactions
between the proteins encoded by two large libraries of genes
(Nandabalan, K et al. (2000) U.S. Pat. No. 6,057,101).
[0338] Potential GCREC agonists or antagonists may be tested for
activation or inhibition of GCREC receptor activity using the
assays described in sections XVII and XVIII. Candidate molecules
may be selected from known GPCR agonists or antagonists, peptide
libraries, or combinatorial chemical libraries.
[0339] Methods for detecting interactions of GCREC with
intracellular signal transduction molecules such as G proteins are
based on the premise that internal segments or cytoplasmic domains
from an orphan G protein-coupled seven transmembrane receptor may
be exchanged with the analogous domains of a known G
protein-coupled seven transmembrane receptor and used to identify
the G-proteins and downstream signaling pathways activated by the
orphan receptor domains (Kobilka, B. K et al. (1988) Science
240:1310-1316). In an analogous fashion, domains of the orphan
receptor may be cloned as a portion of a fusion protein and used in
binding assays to demonstrate interactions with specific G
proteins. Studies have shown that the third intracellular loop of G
protein-coupled seven transmembrane receptors is important for G
protein interaction and signal transduction (Conklin, B. R. et al.
(1993) Cell 73:631-641). For example, the DNA fragment
corresponding to the third intracellular loop of GCREC may be
amplified by the polymerase chain reaction (PCR) and subcloned into
a fusion vector such as nGEX (Pharmacia Biotech). The construct is
transformed into saline. Bound G subunits are detected by
[.sup.32P]ADP-ribosylation with pertussis or cholera toxins. The
reactions are terminated by the addition of SDS sample buffer (4.6%
(w/v) SDS, 10% (v/v) .beta.-mercaptoethanol, 20% (w/v) glycerol,
95.2 mM Tris-HCl, pH 6.8, 0.01% (w/v) bromphenol blue). The
[.sup.32P]ADP-labeled proteins are separated on 10% SDS-PAGE gels,
and autoradiographed. These gels are transferred to nitrocellulose
paper, blocked with blotto (5% nonfat dried milk, 50 mM Tris-HCl
(pH 8.0), 2 mM CaCl.sub.2, 80 mM NaCl, 0.02% NaN.sub.3, and 0.2%
Nonidet P-40) for 1 hour at room temperature, followed by
incubation for 1.5 hours with Gm subtype selective antibodies
(1:500; Calbiochem-Novabiochem). After three washes, blots are
incubated with horseradish peroxidase (HRP)-conjugated goat
anti-rabbit immunoglobulin (1:2000, Cappel, Westchester Pa.) and
visualized by the chemiluminescence-based ECL method (Amersham
Corp.).
[0340] XVII. Demonstration of GCREC Activity
[0341] An assay for GCREC activity measures the expression of GCREC
on the cell surface. cDNA encoding GCREC is transfected into an
appropriate mammalian cell line. Cell surface proteins are labeled
with biotin as described (de la Fuente, M. A. et al. (1997) Blood
90:2398-2405). Immunoprecipitations are performed using
GCREC-specific antibodies, and immunoprecipitated samples are
analyzed using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblotting techniques. The ratio
of labeled immunoprecipitant to unlabeled immunoprecipitant is
proportional to the amount of GCREC expressed on the cell
surface.
[0342] In the alternative, an assay for GCREC activity is based on
a prototypical assay for ligand/receptor-mediated modulation of
cell proliferation. This assay measures the rate of DNA synthesis
in Swiss mouse 3T3 cells. A plasmid containing polynucleotides
encoding GCREC is added to quiescent 3T3 cultured cells using
transfection methods well known in the art. The transiently
transfected cells are then incubated in the presence of
[.sup.3H]thymidine, a radioactive DNA precursor molecule. Varying
amounts of GCREC ligand are then added to the cultured cells.
Incorporation of [.sup.3H]thymidine into acid-precipitable DNA is
measured over an appropriate time interval using a radioisotope
counter, and the amount incorporated is directly proportional to
the amount of newly synthesized DNA. A linear dose-response curve
over at least a hundred-fold GCREC ligand concentration range is
indicative of receptor activity. One unit of activity per
milliliter is defined as the concentration of GCREC producing a 50%
response level, where 100% represents maximal incorporation of
[.sup.3H]thymidine into acid-precipitable DNA (McKay, I. and I.
Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford
University Press, New York N.Y., p. 73.)
[0343] In a further alternative, the assay for GCREC activity is
based upon the ability of GPCR family proteins to modulate G
protein-activated second messenger signal transduction pathways
(e.g., cAMP; Gaudin, P. et al. (1998) J. Biol. Chem.
273:4990-4996). A plasmid encoding GCREC is transfected into a
mammalian cell line (e.g., Chinese hamster ovary (CHO) or human
embryonic kidney (HEK-293) cell lines) using methods well-known in
the art. Transfected cells are grown in 12-well trays in culture
medium for 48 hours, then the culture medium is discarded, and the
attached cells are gently washed with PBS. The cells are then
incubated in culture medium with or without ligand for 30 minutes,
then the medium is removed and cells lysed by treatment with 1 M
perchloric acid. The cAMP levels in the lysate are measured by
radioimmunoassay using methods well-known in the art. Changes in
the levels of cAMP in the lysate from cells exposed to ligand
compared to those without ligand are proportional to the amount of
GCREC present in the transfected cells.
[0344] To measure changes in inositol phosphate levels, the cells
are grown in 24-well plates containing 1.times.10.sup.5 cells/well
and incubated with inositol-free media and [.sup.3H]myoinositol, 2
.mu.Ci/well, for 48 hr. The culture medium is removed, and the
cells washed with buffer containing 10 mM LiCl followed by addition
of ligand. The reaction is stopped by addition of perchloric acid.
Inositol phosphates are extracted and separated on Dowex AG1-X8
(Bio-Rad) anion exchange resin, and the total labeled inositol
phosphates counted by liquid scintillation. Changes in the levels
of labeled inositol phosphate from cells exposed to ligand compared
to those without ligand are proportional to the amount of GCREC
present in the transfected cells.
[0345] XVIII. Identification of GCREC Ligands
[0346] GCREC is expressed in a eukaryotic cell line such as CHO
(Chinese Hamster Ovary) or HEK (Human Embryonic Kidney) 293 which
have a good history of GPCR expression and which contain a wide
range of G-proteins allowing for functional coupling of the
expressed GCREC to downstream effectors. The transformed cells are
assayed for activation of the expressed receptors in the presence
of candidate ligands. Activity is measured by changes in
intracellular second messengers, such as cyclic AMP or Ca.sup.2+.
These may be measured directly using standard methods well known in
the art, or by the use of reporter gene assays in which a
luminescent protein (e.g. firefly luciferase or green fluorescent
protein) is under the transcriptional control of a promoter
responsive to the stimulation of protein kinase C by the activated
receptor (Milligan, G. et al. (1996) Trends Pharmacol. Sci.
17:235-237). Assay technologies are available for both of these
second messenger systems to allow high throughput readout in
multi-well plate format, such as the adenylyl cyclase activation
FlashPlate Assay (NEN Life Sciences Products), or fluorescent
Ca.sup.2+ indicators such as Fluo-4 AM (Molecular Probes) in
combination with the FLIPR fluorimetric plate reading system
(Molecular Devices). In cases where the physiologically relevant
second messenger pathway is not known, GCREC may be coexpressed
with the G-proteins G.sub..alpha.15/16 which have been demonstrated
to couple to a wide range of G-proteins (Offermanns, S. and M. I.
Simon (1995) J. Biol. Chem. 270:15175-15180), in order to funnel
the signal transduction of the GCREC through a pathway involving
phospholipase C and Ca.sup.2+ mobilization. Alternatively, GCREC
may be expressed in engineered yeast systems which lack endogenous
GPCRs, thus providing the advantage of a null background for GCREC
activation screening. These yeast systems substitute a human GPCR
and Ga protein for the corresponding components of the endogenous
yeast pheromone receptor pathway. Downstream signaling pathways are
also modified so that the normal yeast response to the signal is
converted to positive growth on selective media or to reporter gene
expression (Broach, J. R. and J. Thorner (1996) Nature 384
(supp.):14-16). The receptors are screened against putative ligands
including known GPCR ligands and other naturally occurring
bioactive molecules. Biological extracts from tissues, biological
fluids and cell supernatants are also screened.
[0347] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with certain embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in molecular biology or related fields are intended
to be within the scope of the following claims.
2TABLE 1 Incyte Poly- Incyte Poly- Incyte Project peptide
Polypeptide nucleotide Polynucleotide ID SE ID NO: ID SEQ ID NO: ID
104941 1 104941CD1 40 104941CB1 1499408 2 1499408CD1 41 1499408CB1
3168839 3 3168839CD1 42 3168839CB1 3291235 4 3291235CD1 43
3291235CB1 7472001 5 7472001CD1 44 7472001CB1 7472003 6 7472003CD1
45 7472003CB1 7472004 7 7472004CD1 46 7472004CB1 7475687 8
7475687CP1 47 7475687CT1 7483029 9 7483029CP1 48 7483029CT1 7477933
10 7477933CP1 49 7477933CT1 7475164 11 7475164CP1 50 7475164CT1
7473909 12 7473909CP1 51 7473909CT1 7475252 13 7475252CP1 52
7475252CT1 7927572 14 7927572CP1 53 7927572CT1 7481257 15
7481257CP1 54 7481257CT1 7485790 16 7485790CP1 55 7485790CT1
7482993 17 7482993CP1 56 7482993CT1 2829053 18 2829053CD1 57
2829053CB1 3068234 19 3068234CD1 58 3068234CB1 5029478 20
5029478CD1 59 5029478CB1 5102576 21 5102576CD1 60 5102576CB1
2200534 22 2200534CD1 61 2200534CB1 3275821 23 3275821CD1 62
3275821CB1 3744167 24 3744167CD1 63 3744167CB1 7472007 25
7472007CD1 64 7472007CB1 7472008 26 7472008CD1 65 7472008CB1
7472013 27 7472013CD1 66 7472013CB1 7472015 28 7472015CD1 67
7472015CB1 7472016 29 7472016CD1 68 7472016CB1 7472017 30
7472017CD1 69 7472017CB1 7472018 31 7472018CD1 70 7472018CB1
7472019 32 7472019CD1 71 7472019CB1 7472021 33 7472021CD1 72
7472021CB1 7472009 34 7472009CD1 73 7472009CB1 7472010 35
7472010CD1 74 7472010CB1 7472011 36 7472011CD1 75 7472011CB1
7472012 37 7472012CD1 76 7472012CB1 7472014 38 7472014CD1 77
7472014CB1 7472020 39 7472020CD1 78 7472020CB1
[0348]
3TABLE 2 Poly- Incyte peptide Polypeptide GenBank Probability SEQ
ID NO: ID ID NO: Score GenBank Homolog 1 104941CD1 g7211316
6.9E-146 Olfactory receptor [Callithrix jacchus] 2 1499408CD1
g202806 5.20E-162 Vasopressin receptor [Rattus norvegicus] 3
3168839CD1 g3618229 2.2E-44 G protein-linked P2Y4 receptor [Rattus
norvegicus] 4 3291235CD1 g3287369 1.40E-126 A-2 [Mus musculus] 5
7472001CD1 g1256393 2.20E-122 Taste bud receptor protein TB 641
[Rattus norvegicus] 6 7472003CD1 g4378765 1.20E-169 Orphan G
protein-coupled receptor GPR54 [Rattus norvegicus] 7 7472004CD1
g1698952 6.30E-118 High-affinity lysophosphatidic acid receptor
[Xenopus laevis] 8 7475687CP1 g1256393 4.70E-90 Taste bud receptor
protein TB 641 [Rattus norvegicus] 9 7483029CP1 g2447219 2.50E-75
OLF4 [Homo sapiens] 10 7477933CP1 g2792016 4.90E-79 Olfactory
receptor [Homo sapiens] 11 7475164CP1 g517366 3.00E-111 Olfactory
receptor [Rattus norvegicus] 12 7473909CP1 g4680264 1.90E-22
Odorant receptor S25 [Mus musculus] 13 7475252CP1 g2447219 6.50E-61
OLF4 [Homo sapiens] 14 7927572CP1 g8100089 6.7E-54 Putative taste
receptor HTR2 [Homo sapiens] 15 7481257CP1 g4826521 4.00E-29
dJ88J8.1 (novel 7 transmembrane receptor (rhodopsin
family)(olfactory receptor like protein (hs6M1-15)) [Homo sapiens]
16 7485790CP1 g2447219 3.00E-40 OLF4 [Homo sapiens] 17 7482993CP1
g1314665 3.10E-54 CfOLF3 [Canis familiaris] 18 2829053CD1 19
3068234CD1 g5922725 3.1E-190 Lysophosphatidic acid G
protein-coupled receptor [Homo sapiens] 20 5029478CD1 g1049072
3.6E-21 Galanin receptor GALR1 [Rattus norvegicus] (Cloning and
characterization of the rat GALR1 galanin receptor from Rin14B
insulinoma cells. Brain Res. Mol. Brain Res. Dec. 28, 1995; 34(2):
179-189.) 21 5102576CD1 g2792016 2.4E-92 Olfactory receptor [Homo
sapiens] (Molecular cloning and chromosomal mapping of olfactory
receptor genes expressed in the male germ line: evidence for their
wide distribution in the human genome. Biochem. Biophys. Res.
Commun. Aug. 18, 1997; 237(2): 283-287.) 22 2200534CD1 g5051404
4.6e-131 573K1.15 (mm17M1-6) 7-transmembrane olfactory receptor-lik
protein (rhodopsin family) [Mus musculus] 23 3275821CD1 g182742
1.5e-29 Formyl peptide receptor [Homo sapiens] (Murphy, P. M. et
al. (1992) J. Biol. Chem. 267: 7637-7643) 24 3744167CD1 g9186902
1.2E-240 Leukotriene B4 receptor, BLT2 [Mus musculus] 25 7472007CD1
g7638409 1.3E-199 Olfactory receptor P2 [Mus musculus] 26
7472008CD1 g4218182 1.0e-89 dJ271M21.2 (hs6M1-12 (7 transmembrane
receptor (rhodopsin family) (olfactory receptor like) protein))
[Homo sapiens] 27 7472013CD1 g205846 2.5e-70 Olfactory protein
[Rattus norvegicus] 28 7472015CD1 g1204095 2.5e-25 Dopamine
receptor [Fugu rubripes] 29 7472016CD1 g6090796 1.1E-215 Olfactory
receptor [Gorilla gorilla] 30 7472017CD1 g3757727 2.0e-61 dJ80I19.7
(olfactory receptor-like protein (hs6M1-3)) [Homo sapiens] 31
7472018CD1 g6644328 2.3E-112 Orphan G protein-coupled receptor
GPR26 [Rattus norvegicus] 32 7472019CD1 g5869916 2.7e-73 Olfactory
receptor [Mus musculus] 33 7472021CD1 g6090804 2.6E-94 Olfactory
receptor [Gorilla gorilla] 34 7472009CD1 g1016362 1.6e-68 OL1
receptor [Rattus norvegicus] 35 7472010CD1 g2317704 7.3e-80
Olfactory receptor [Rattus norvegicus] 36 7472011CD1 g6178008
4.9E-114 Odorant receptor MOR18 [Mus musculus] 37 7472012CD1
g205816 6.8e-84 Olfactory protein [Rattus norvegicus] 38 7472014CD1
g205816 3.9e-88 Olfactory protein [Rattus norvegicus] 39 7472020CD1
g2792016 1.9e-97 Olfactory receptor [Homo sapiens]
[0349]
4TABLE 3 SEQ Incyte Amino Potential Potential Analytical ID
Polypeptide Acid Phosphorylation Glycosylation Signature Sequences,
Methods and NO: ID Residues Sites Sites Domains and Motifs
Databases 1 104941CD1 311 S68 S189 S292 N5 N66 7 transmembrane
receptor (rhodopsin MOTIFS Y310 family): G42-Y291 HMMER-PFAM GPCR
signature: BLIMPS-BLOCKS K91-P130, I208-Y219, Y283-K299,
BLIMPS-PRINTS Y103-S151 ProfileScan Olfactory receptor signature:
M60-K81, F178-D192, F239-G254, A275-L286, S292-Q306 Transmembrane
domains: HMMER I31-I47, P211-I229 2 1499408CD1 891 T148 S686 S114
N378 ATP/GTP binding site (P-loop): MOTIFS S248 S350 S481 G202-T209
S501 T628 T814 S856 T84 S140 T144 T325 T411 T543 S568 S676 T706
T788 Y372 3 3168839CD1 422 T232 S178 T342 N4 N9 N251 7
transmembrane receptor (rhodopsin MOTIFS S363 S371 S397 N323
family): L39-Y297 HMMER-PFAM T21 S211 S226 Rhodopsin-like GPCR
superfamily: BLIMPS-BLOCKS T307 S332 S367 L24-L48, V57-R78,
F101-I123, ProfileScan V137-R158, V192-F215, T232-V256,
BLIMPS-PRINTS L279-R305 Transmembrane domains: V275-L295 HMMER 4
3291235CD1 609 S228 S229 S396 7 transmembrane receptor (rhodopsin
MOTIFS S456 S324 S328 family): E80-E154 HMMER-PFAM S364 S417 S466
GPCR signatures: BLIMPS-BLOCKS T506 S568 S590 F76-P115, F395-A405,,
A442-E458, BLIMPS-PRINTS S153 S268 T392 E509-P526 S462 S482 S560
Y348 Transmembrane domain: V174-L199 HMMER 5 7472001CD1 313 S68
T194 T200 N5 N85 7 transmembrane receptor (rhodopsin MOTIFS S267
T309 T138 family): G41-I259 HMMER-PFAM T164 T290 S306 GPCR
signature: BLIMPS-BLOCKS K91-P130, T281-K297, Y103-A147
BLIMPS-PRINTS Olfactory receptor signature: ProfileScan M60-R81,
F178-D192, F239-V254, A273-L284, T290-L304 Signal peptide: M1-T38
HMMER SPScan Transmembrane domains: HMMER F30-T48, F63-M83 6
7472003CD1 398 S36 T155 N10 N18 N28 7 transmembrane receptor
(rhodopsin MOTIFS family): G59-Y323 HMMER-PFAM GPCR signature:
BLIMPS-BLOCKS W108-P147, Y213-Y224, A256-F282, BLIMPS-PRINTS
N315-R331, N119-I166 ProfileScan Neuropeptide Y receptor signature:
R69-I81, L321-F334 Transmembrane domain: A42-Y65 HMMER 7 7472004CD1
369 S228 T94 T218 N12 7 transmembrane receptor (rhodopsin MOTIFS
S339 T350 family): G48-Y321 HMMER-PFAM Rhodopsin-like GPCR
signature: BLIMPS-BLOCKS T33-Y57, I66-F87, F111-I133, BLIMPS-PRINTS
R144-V165, V193-L216, A262-V286, S303-H329 Transmembrane domains:
HMMER T33-V51, M109-I125, Y189-M213, M256-V275 8 7475687CP1 194
T186 T76 T82 7 transmembrane receptor (rhodopsin MOTIFS T46 T172
family): M1-Y171 HMMER-PFAM Opsins retinal binding site:
BLIMPS-BLOCKS Y142-N194 BLIMPS-PRINTS Olfactory receptor signature:
ProfileScan F60-D74, F121-V136, A155-L166, T172-T186 9 7483029CP1
173 T16 S34 T60 N32 N167 7 transmembrane receptor (rhodopsin MOTIFS
family): G8-C146 HMMER-PFAM Rhodopsin-like GPCR signature:
BLIMPS-BLOCKS M26-K47, F71-I93, L107-I128 ProfileScan BLIMPS-PRINTS
Signal peptide: M1-L22 SPScan HMMER Transmembrane domains: HMMER
M68-A86, M103-L121 10 7477933CP1 220 S172 7 transmembrane receptor
(rhodopsin MOTIFS family): P1-C192 HMMER-PFAM Olfactory receptor
signature: BLIMPS-BLOCKS M2-K23, F120-D134, F181-G196 BLIMPS-PRINTS
Rhodopsin-like GPCR signature: L47-I69, I142-V165 Transmembrane
domains: HMMER M44-A62, V85-T110 11 7475164CP1 302 T296 S58 S84 7
transmembrane receptor (rhodopsin MOTIFS T107 T257 T9 family):
G32-I193 HMMER-PFAM T69 S128 T151 GPCR signature: BLIMPS-BLOCKS
S282 N81-P120, I273-K289, S93-L142 ProfileScan Olfactory receptor
signature: BLIMPS-PRINTS V50-K71, Y168-S182, F229-G244, S265-L276,
S282-T296 Transmembrane domains: HMMER F19-L39, I188-I207 12
7473909CP1 110 S70 S36 T66 S94 GPCR signature: I85-K101 MOTIFS
Olfactory receptor signature: BLIMPS- F41-G56, A77-L88, S94-Y108
BLOCKS BLIMPS- PRINTS 13 7475252CP1 178 S66 S151 S136 N4 N64 7
transmembrane receptor (rhodopsin MOTIFS family): G40-L153
HMMER-PFAM Rhodopsin-like GPCR signature: BLIMPS-BLOCKS V25-S49,
M58-K79, L103-I125, ProfileScan S102-S151 BLIMPS-PRINTS
Transmembrane domains: HMMER L29-I45, M100-M117 14 7927572CP1 92
Olfactory receptor signature: MOTIFS F25-D39 BLIMPS-PRINTS 15
7481257CP1 97 7 transmembrane receptor (rhodopsin MOTIFS family):
M1-Y96 HMMER-PFAM GPCR signature: V13-Y24, Q41-Q67 BLIMPS-BLOCKS
Olfactory receptor signature: BLIMPS-PRINTS F44-G59, L80-L91 Signal
peptide: M1-G27 SPScan HMMER Transmembrane domain: M8-Y24 HMMER 16
7485790CP1 133 S74 7 transmembrane receptor (rhodopsin MOTIFS
family): R22-V128 HMMER-PFAM GPCR signature: G71-P110 BLIMPS-BLOCKS
BLIMPS-PRINTS Transmembrane domain: M82-A100 HMMER 17 7482993CP1
213 S85 S205 S159 N83 7 transmembrane receptor (rhodopsin MOTIFS
T183 family): S2-Y182 HMMER-PFAM GPCR signature: BLIMPS-BLOCKS
R127-R153, S2-A38 ProfileScan Olfactory receptor signature:
BLIMPS-PRINTS F69-N83, F130-G145, V166-L177, T183-G197
Transmembrane domains: HMMER P102-I120, F130-V152 18 2829053CD1 180
S30 S41 S109 Beta-1 adrenergic receptor MOTIFS S125 S140 S35
signature: I148-S166 BLIMPS-PRINTS S36 S149 Signal peptide: M1-S67
SPScan 19 3068234CD1 353 T146 T217 T233 N15 N139 N172 7
transmembrane receptor (rhodopsin MOTIFS S321 S17 T21 N349 family):
S47-Y293 HMMER-PFAM S294 S329 T141 Rhodopsin-like GPCR superfamily
BLIMPS-BLOCKS S229 T303 Y14 signature: BLIMPS-PRINTS I32-I56,
F65-L86, L109-I131, R144-L165, Y187-Y210, L237-L261, K275-Y301
Transmembrane domains: HMMER V36-I56, T146-G166, Y187-L207,
T240-V258 20 5029478CD1 361 T242 S256 S237 N21 N322 7 transmembrane
receptor (rhodopsin MOTIFS S350 family): G57-Y321 HMMER-PFAM
Rhodopsin-like GPCR superfamily BLIMPS-BLOCKS signature:
BLIMPS-PRINTS V42-A66, T74-V95, M118-I140, A154-V175, D208-L231,
L262-L286, F303-E329 Transmembrane domains: HMMER T45-V65,
V124-Q144, V209-I233, L266-N291 21 5102576CD1 251 S119 S196 7
transmembrane receptor (rhodopsin MOTIFS family): R8-C251
HMMER-PFAM GPCR signature: R57-P96 BLIMPS-BLOCKS Olfactory receptor
signature: BLIMPS-PRINTS M26-K47, L144-D158, F205-G220, A241-C252
Transmembrane domains: HMMER T66-D88, V109-T134 22 2200534CD1 315
S6 S136 T291 N4 N154 G-protein coupled receptor: HMMER
DM00013(P232750)17-306: S17-L305 BLIMPS-PRINTS
DM00013(A570690)15-304: F16-L305 BLIMPS-BLOCKS
DM00013(P47881)20-309: P20-L305 HMMER-PFAM PD149621: T246-L305
PD000921: MOTIFS C168-L245 PD002495: N4-L47 BL00237: L89-P128,
L207-Y218, T282-K298 Olfactory receptor PR00245: M58-P79,
F176-G190, V238-G253, V274-L285, T291-L305 G-protein coupled
receptors BLAST-PRODOM BLAST-DOMO Transmembrane domains: HMMER
V204-M228, G40-Y290 23 3275821CD1 470 T3 T18 T326 N47 G-protein
coupled receptor: HMMER-PFAM T332 T340 S350 DM00013(P21462)20-317:
V34-L306 BLIMPS-BLOCKS S424 S451 T459 PD000009: L68-F169
BLIMPS-PRINTS S192 BL00237: W97-P136, G201-H212, ProfileScan
A230-A256, N287-R303 MOTIFS GPCR profile: F109-V155 Rhodopsin GPCR
family PR00237: W31-G55, L66-Q87, W111-A133, L147-V168, L193-Q216,
F235-L259, L277-R303 G-protein coupled receptors BLAST-PRODOM
BLAST-DOMO Transmembrane domains: HMMER T33-A51, L68-L259 24
3744167CD1 358 T291 S15 T18 N10 N38 N342 G-protein coupled
receptor: HMMER-PFAM S215 DM00013(P46092)27-318: S19-F290
BLIMPS-BLOCKS DM00013(P31391)41-326: L29-L304 BLIMPS-PRINTS
DM00013.vertline.P35414)22-324: W16-F290 ProfileScan BL00237:
W87-P126, F190-Y201, MOTIFS R217-V243, S280-L296 GPCR profile:
Y99-V145 Rhodopsin GPCR family PR00237: T22-A46, A57-F78,
C101-V123, L137-V158, L182-L205, V222-L246, R270-L296 G-protein
coupled receptors BLAST-PRODOM BLAST-DOMO Transmembrane domains:
HMMER A138-Y159, G37-Y288 25 7472007CD1 314 S270 S291 S311 N4 N65
Signal peptide: M1-Q56 SPScan T49 S67 S193 Transmembrane domains:
HMMER L29-V48, L208-M228 7 transmembrane receptor (rhodopsin
HMMER-PFAM family) signature: G41-Y290 G-protein coupled receptor
BLIMPS-BLOCKS signatures: T90-P129, I207-Y218, T282-K298 G-protein
coupled receptor ProfileScan signature: Y102-A147 G-protein coupled
receptor MOTIFS signature: A110-A125 Olfactory receptor signatures:
BLIMPS-PRINTS M59-K80, F177-D191, F238-S253, I274-L285, S291-I305
Melanocortin receptor family: BLIMPS-PRINTS A5-L63 Rhodopsin-like
GPCR superfamily: BLIMPS-PRINTS L26-I50, M59-K80, F104-I126,
F153-V174, A199-L222, A237-R261, K272-K298 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23270.vertline.18-311:
L23-H306 G-protein coupled receptor BLAST-DOMO
DM00013.vertline.P23267.vertline.- 20-309: L27-I305 G-protein
coupled receptor BLAST-DOMO
DM00013.vertline.P23275.vertline.17-306: L23-I305 G-protein coupled
receptor BLAST-DOMO DM00013.vertline.P30953- .vertline.18-306:
L19-H306 Olfactory receptor PD000921: BLAST-PRODOM F168-L246
Olfactory receptor PD149621: BLAST-PRODOM V247-R307 26 7472008CD1
365 S78 T192 S199 N94 Transmembrane domain: I226-L244 HMMER T320
S343 S47 7 transmembrane receptor (rhodopsin HMMER-PFAM S66 S78 S96
family) signature: G70-Y319 S217 T222 T337 G-protein coupled
receptor BLIMPS-BLOCKS T361 signatures: K119-P158, L236-S247,
K264-Q290, T311-H327 Olfactory receptor signatures: BLIMPS-PRINTS
M88-Q109, V206-D220, F267-G282, L303-L314, T320-K334 Melanocortin
receptor family: BLIMPS-PRINTS V73-L84, K80-L92 Rhodopsin-like GPCR
superfamily: BLIMPS-PRINTS L55-L79, M88-Q109, F133-V155, M228-A251,
A266-Q290, K301-H327 Olfactory receptor PD000921: BLAST-PRODOM
L195-L275 Olfactory receptor PD149621: BLAST-PRODOM V276-K331
G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P30955.vertline.18-305: L61-L326 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23269.vertline- .15-304:
L61-L326 G-protein coupled receptor BLAST-DOMO
DM00013.vertline.A57069.vertline.115-304: D59-L326 G-protein
coupled receptor BLAST-DOMO DM00013.vertline.P23275-
.vertline.117-306: T67-L326 27 7472013CD1 317 S68 S193 N6 N22 N43
Signal peptide: M1-G42 SPScan Transmembrane domain: I23-L41 HMMER 7
transmembrane receptor (rhodopsin HMMER-PFAM family) signatures:
G42-L155, A279-Y295 G-protein coupled receptor BLIMPS-BLOCKS
signatures: P91-P130, M212-Y223, T287-K303 G-protein coupled
receptor ProfileScan signature: F103-L148 G-protein coupled
receptor MOTIFS signature: T111-A126 Olfactory receptor signatures:
BLIMPS-PRINTS M60-Q81, F182-D196, V243-G258, A279-A290, S296-L310
Melanocortin receptor family: BLIMPS-PRINTS W52-L64 Rhodopsin-like
GPCR superfamily: BLIMPS-PRINTS L27-S51, M60-Q81, F105-I127,
R141-G162, I204-G227, A242-Q266, M277-K303 GPR3 orphan receptor
signature: BLIMPS-PRINTS V161-N178 G-protein coupled receptor:
BLAST-DOMO DM00013.vertline.P23274.vertline.18-306: L27-L310
G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23272.vertline.18-306: Y25-L310 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P30953.vertline- .18-306:
L27-L310 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P30955.vertline.18-305: L27-L310 Olfactory
receptor PD000921: BLAST-PRODOM G174-L250 Olfactory receptor
PD149621: BLAST-PRODOM T251-L310 28 7472015CD1 335 T73 S79 S214
Signal peptide: M1-A20 HMMER S309 T217 S329 Signal peptide: M1-A20
SPScan S331 Transmembrane domains: HMMER F5-V27, L45-T63, M117-I136
7 transmembrane receptor (rhodopsin HMMER-PFAM family) signatures:
T21-Y279 G-protein coupled receptor BLIMPS-BLOCKS signatures:
R71-P110, F174-Y185, P218-T244, N271-R287 G-protein coupled
receptor ProfileScan signature: F84-L129 G-protein coupled receptor
MOTIFS signature: A91-I106 Rhodopsin-like GPCR superfamily:
BLIMPS-PRINTS S6-L30, S40-L61, V85-I107, V121-G142, V166-L189,
A223-V247, E261-R287 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P41596.vertline.137-461: G8-D220 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P47800.vertline- .29-338:
G8-Y281 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P31388.vertline.20-336: G8-P218 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.JN0591.vertline.20-336:
G8-P218 29 7472016CD1 309 S8 S67 S188 N5 N65 N264 Signal peptide:
M1-L55 SPScan S266 S137 S229 Transmembrane domains: HMMER S266 S289
Y28-A48, M199-I218 7 transmembrane receptor (rhodopsin HMMER-PFAM
family) signatures: G41-Y288 G-protein coupled receptor
BLIMPS-BLOCKS signatures: K90-P129, F206-Y217, L234-R260, T280-K296
G-protein coupled receptor ProfileScan signature: Y102-A146
Olfactory receptor signatures: BLIMPS-PRINTS M59-K80, F177-D191,
F237-G252, I272-L283, S289-F303 Rhodopsin-like GPCR superfamily:
BLIMPS-PRINTS F26-C50, M59-K80, M104-I126 S140-L161, M198-F219,
A270-K296 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23266.vertline.17-306: Q20-L302 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23274.vertline- .18-306:
E22-L299 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23269.vertline.15-304: Q21-L299 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P30955.vertline.18-305:
E22-L302 Olfactory receptor PD149621: BLAST-PRODOM T245-S309
Olfactory receptor PD000921: BLAST-PRODOM L166-L244 30 7472017CD1
236 S7 T217 N5 N189 Signal peptide: M1-G42 SPScan Transmembrane
domains: HMMER C31-M52, V123-L141 7 transmembrane receptor
(rhodopsin HMMER-PFAM family) signatures: F12-Y216 G-protein
coupled receptor BLIMPS-BLOCKS signatures: K24-P63, L133-Y144,
C161-Q187, T208-K224 G-protein coupled receptor ProfileScan
signature: Y36-V81 G-protein coupled receptor MOTIFS signature:
T44-A59 Olfactory receptor signatures: BLIMPS-PRINTS L164-G179,
I200-L211, T217-N231 Rhodopsin-like GPCR superfamily: BLIMPS-PRINTS
S38-V60, L125-A148, G163-Q187, K198-K224 Olfactory
receptor PD149621: BLAST-PRODOM V173-T236 Olfactory receptor
PD000921: BLAST-PRODOM C103-I172 G-protein coupled receptor:
BLAST-DOMO DM00013.vertline.P23269.vertline.15-304: L15-L227
G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P30953.vertline- .18-306: L15-L227 G-protein
coupled receptor: BLAST-DOMO
DM00013.vertline.A57069.vertline.15-304: L15-R228 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23275.vertline.17-306:
M1-L227 31 7472018CD1 363 Y294 S321 S325 N47 N348 N355 Signal
peptide: M1-A24 HMMER T353 S157 T210 Signal peptide: M1-A24 SPScan
S223 T240 T316 7 transmembrane receptor (rhodopsin HMMER-PFAM T340
family) signatures: S22-Y294 G-protein coupled receptor
BLIMPS-BLOCKS signatures: T72-P111, F181-S192, R234-T260, K286-R302
Rhodopsin-like GPCR superfamily: BLIMPS-PRINTS L7-A31, S41-F62,
D86-V108, Y122-G143, T173-H196, A239-A263, G276-R302 P2Y4
purinoceptor signatures: BLIMPS-PRINTS Y32-L48, P111-L126 G-protein
coupled receptor: BLAST-DOMO
DM00013.vertline.JN0591.vertline.20-336: P3-L305 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P53452.vertline- .17-344:
L7-F268 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P50406.vertline.20-335: G4-L305 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P31388.vertline.20-336:
P3-L305 32 7472019CD1 308 S162 S290 S67 N5 N65 Transmembrane
domains: HMMER T187 S192 S265 L30-I49, M197-L215 7 transmembrane
receptor (rhodopsin HMMER-PFAM family) signatures: G41-Y289
G-protein coupled receptor BLIMPS-BLOCKS signatures: P90-P129,
L206-Y217, L234-K260, T281-K297 G-protein coupled receptor
ProfileScan signature: S102-T147 Olfactory receptor signatures:
BLIMPS-PRINTS I59-Q80, F176-D190, F237-G252, I273-L284, S290-M304
Melanocortin receptor family: BLIMPS-PRINTS F51-L63 Rhodopsin-like
GPCR superfamily: BLIMPS-PRINTS T26-H50, I59-Q80, S104-I126,
V140-L161, I198-A221, K271-K297 G-protein coupled receptor:
BLAST-DOMO DM00013.vertline.P23275.vertline.17-306: I17-M304
G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23269.vertline- .15-304: F27-M304 G-protein
coupled receptor: BLAST-DOMO
DM00013.vertline.P23266.vertline.17-306: I17-M304 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.S29707.vertline.18-306:
P21-I300 Olfactory receptor PD000921: BLAST-PRODOM L165-L244
Olfactory receptor PD149621: BLAST-PRODOM T245-M304 33 7472021CD1
343 S87 T154 S288 N25 N183 N314 Transmembrane domains: HMMER S326
S311 S316 Y55-L75, I214-I234 7 transmembrane receptor (rhodopsin
HMMER-PFAM family) signatures: G61-Y310 G-protein coupled receptor
BLIMPS-BLOCKS signatures: S38-L64, G110-P149, P302-K318 G-protein
coupled receptor ProfileScan signature: F122-V168 G-protein coupled
receptor MOTIFS signature: S130-A145 Olfactory receptor signatures:
BLIMPS-PRINTS M79-Q100, F197-Y211, F258-S273, F297-L305, S311-L325
Melanocortin receptor family: BLIMPS-PRINTS V71-L83 Vasopressin
receptor signature: BLIMPS-PRINTS L75-L86 Olfactory receptor
PD000921: BLAST-PRODOM I186-L265 Olfactory receptor PD149621:
BLAST-PRODOM V267-E328 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23269.vertline.15-304: E40-L325 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23275.vertline- .17-306:
S38-L325 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23273.vertline.18-306: I45-L325 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23266.vertline.17-306:
S38-S326 34 7472009CD1 323 S87 S232 T290 N5 Transmembrane domains:
HMMER S8 S67 T193 L30-L47, I201-L221 7 transmembrane receptor
(rhodopsin HMMER-PFAM family) signatures: G41-Y289 G-protein
coupled receptor BLIMPS-BLOCKS signatures: K90-P129, L206-Y217,
R234-R260, T281-A297 G-protein coupled receptor ProfileScan
signature: F102-M147 G-protein coupled receptor MOTIFS signature:
A110-A125 Olfactory receptor signatures: BLIMPS-PRINTS M59-K80,
F177-D191, F237-G252, G273-L284, T290-L304 Rhodopsin-like GPCR
superfamily: BLIMPS-PRINTS F26-C50, Y104-I126, V140-A161,
T198-L221, K271-A297 Melanocortin receptor family: BLIMPS-PRINTS
I51-L63, I126-N137 Vasopressin receptor signature: BLIMPS-PRINTS
L55-L66 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23275.vertline.17-306: L25-L304 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.A57069.vertline.15-304:
L27-L304 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23270.vertline- .18-311: L25-L304 G-protein
coupled receptor: BLAST-DOMO
DM00013.vertline.P23266.vertline.17-306: L27-L304 Olfactory
receptor PD149621: BLAST-PRODOM T245-T310 Olfactory receptor
PD000921: BLAST-PRODOM F168-L244 35 7472010CD1 299 T68 S126 S280
N55 Transmembrane domain: L186-I205 HMMER T293 S10 S57 7
transmembrane receptor (rhodopsin HMMER-PFAM T156 family)
signatures: G31-Y279 G-protein coupled receptor BLIMPS-BLOCKS
signatures: S79-P118, F188-S199, S224-T250, V271-K287 G-protein
coupled receptor ProfileScan signature: F91-F135 G-protein coupled
receptor MOTIFS signature: S99-A114 Olfactory receptor signatures:
BLIMPS-PRINTS M49-K70, Y166-S180, F227-G242, A263-L274, S280-L294
Melanocortin receptor family: BLIMPS-PRINTS I41-L53 Rhodopsin-like
GPCR superfamily: BLIMPS-PRINTS Q16-G40, M49-K70, F93-I115,
T181-V204, A226-T250, R261-K287 G-protein coupled receptor:
BLAST-DOMO DM00013.vertline.S29709.vertline.11-299: G23-L294
G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.S51356.vertline- .18-307: I24-K292 G-protein
coupled receptor: BLAST-DOMO
DM00013.vertline.P23274.vertline.18-306: I24-L294 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P30955.vertline.18-305:
I24-L294 Olfactory receptor PD149621: BLAST-PRODOM V237-R296
Olfactory receptor PD000921: BLAST-PRODOM L155-I235 36 7472011CD1
307 S87 T288 S193 N5 Transmembrane domains: HMMER L23-I43,
M98-M118, G204-H228 7 transmembrane receptor (rhodopsin HMMER-PFAM
family) signatures: G41-Y287 G-protein coupled receptor
BLIMPS-BLOCKS signatures: K90-P129, R234-R260, T279-Q295 G-protein
coupled receptor ProfileScan signature: F102-T148 Olfactory
receptor signatures: BLIMPS-PRINTS M59-K80, F177-D191, A237-V252,
V271-L282, T288-G302 Melanocortin receptor family: BLIMPS-PRINTS
S51-L63 Rhodopsin-like GPCR superfamily: BLIMPS-PRINTS F26-T50,
M59-K80, F104-I126, L140-A161, K199-L222, A236-R260, K269-Q295
Olfactory receptor PD000921: BLAST-PRODOM L166-I245 Olfactory
receptor PD149621: BLAST-PRODOM V246-R303 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.S29710.vertline.15-301:
L17-L301 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23275.vertline.17-306: L17-L301 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23266.vertline- .17-306:
L17-L301 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P47881.vertline.20-309: L23-L301 37 7472012CD1 314
T19 S230 S291 N5 N38 7 transmembrane receptor (rhodopsin HMMER-PFAM
family) signatures: G41-Y290 G-protein coupled receptor
BLIMPS-BLOCKS signatures: K90-P129, T282-K298 G-protein coupled
receptor ProfileScan signature: Y102-M147 G-protein coupled
receptor MOTIFS signature: M110-A125 Olfactory receptor signatures:
BLIMPS-PRINTS M59-K80, F177-S191, F238-G253, I274-L285, S291-M305
Rhodopsin-like GPCR superfamily: BLIMPS-PRINTS P26-L50, M59-K80,
F104-I126, I199-I222, T237-R261, R272-K298 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23266.vertline.17-306:
I17-K303 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23274.vertline.18-306: E22-K303 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.S29707.vertline- .18-306:
P21-G299 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P30955.vertline.18-305: P21-K303 Olfactory
receptor PD149621: BLAST-PRODOM T246-T310 Olfactory receptor
PD000921: BLAST-PRODOM L166-L245 38 7472014CD1 310 S19 S67 S93 N5
N265 Transmembrane domains: HMMER T267 S18 S137 V30-I46, M59-I78
S290 7 transmembrane receptor (rhodopsin HMMER-PFAM family)
signatures: G41-Y289 G-protein coupled receptor BLIMPS-BLOCKS
signatures: K90-P129, I207-Y218, R235-Q261, T281-K297 G-protein
coupled receptor ProfileScan signature: Y102-I151 G-protein coupled
receptor MOTIFS signature: T110-A125 Olfactory receptor signatures:
BLIMPS-PRINTS M59-K80, F177-S191, F238-G253, A273-L284, S290-M304
Melanocortin receptor family: BLIMPS-PRINTS S51-L63 Rhodopsin-like
GPCR super family: BLIMPS-PRINTS P26-R50, M59-K80, F104-I126,
V199-L222, Q271-K297 Olfactory receptor PD000921: BLAST-PRODOM
L166-L245 Olfactory receptor PD149621: BLAST-PRODOM T246-R306
G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23266.vertline- .17-306: L17-M304 G-protein
coupled receptor: BLAST-DOMO
DM00013.vertline.P23274.vertline.18-306: E22-M304 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P30955.vertline.18-305:
D23-M304 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P30953.vertline- .18-306: R20-H305 39 7472020CD1
359 S257 S317 S178 N31 Transmembrane domains: HMMER S255 M127-A145,
V168-T193 7 transmembrane receptor (rhodopsin HMMER-PFAM family)
signatures: R67-Y316 G-protein coupled receptor BLIMPS-BLOCKS
signatures: R116-P155, G233-Y244, S261-T287, T308-Q324 G-protein
coupled receptor ProfileScan signature: F129-V173 Olfactory
receptor signatures: BLIMPS-PRINTS M85-K106, F203-D217, F264-G279,
A300-L311, S317-R331 GPR orphan receptor signature: BLIMPS-PRINTS
S317-W328 Cannabinoid receptor signatures: BLIMPS-PRINTS M60-L73,
Y316-A326 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.P23265.vertline.17-306: E45-L327 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P23268.vertline- .18-307:
S44-L330 G-protein coupled receptor: BLAST-DOMO
DM00013.vertline.S29707.vertline.18-306: P47-L327 G-protein coupled
receptor: BLAST-DOMO DM00013.vertline.P30953.vertline.18-306:
P47-L330 Olfactory receptor PD000921: BLAST-PRODOM N197-L271
Olfactory receptor PD149621: BLAST-PRODOM V273-R333
[0350]
5TABLE 4 Polynucleotide Incyte Sequence Selected Sequence 5' 3' SEQ
ID NO: PolynucleotideID Length Fragments Fragments Position
Position 40 104941CB1 936 g4190944.v113.gs_10.edit 1 936 104941H1
(BMARNOT02) 208 429 41 1499408CB1 3365 g2335202.v113.gs_4.edit 1
2105 1499408H1 (SINTBST01) 1068 1325 927003X11 (BRAINOT04) 1092
1764 1632960F6 (COLNNOT19) 1613 2099 4051362F6 (SINTNOT18) 1994
2618 1426416F6 (SINTBST01) 2223 2686 2925035F6 (SININOT04) 2535
3043 927003T6 (BRAINOT04) 2710 3365 42 3168839CB1 1325 3356166H1
(PROSTUT16) 1 281 g4589937.v113.gs_7.edit 42 1188 3700658H1
(SININOT05) 160 463 3168839H1 (BRSTNOT18) 809 1059 4555080H1
(KERAUNT01) 1084 1325 43 3291235CB1 2124 3291235X308F1 (BONRFET01)
1 413 g5578925.v113.gs_2.edit 295 2124 4720927F6 (BRAIHCT02) 315
810 3291235F6 (BONRFET01) 408 1004 3370971H1 (CONNTUT05) 955 1208
1729983H1 (BRSTTUT08) 1073 1293 44 7472001CB1 942
g2121229.v113.gs_4.2.edit 1 942 45 7472003CB1 1197
g3386590.v113.gs_1.edit 1 1197 46 7472004CB1 1110
g4741473.v113.gs_5.edit 1 1110 47 7475687CT1 582
g2121229.v113.gs4.1.nt.edit 1 582 48 7483029CT1 519
g2447218.v113.gs2.nt.edit 1 519 49 7477933CT1 663
g2673897.v113.gs7.nt.edit 1 663 50 7475164CT1 911
g3738097.v113.gs2.nt.edit 1 911 51 7473909CT1 332
g3962498.v113.gs3.nt.edit 1 332 52 7475252CT1 538
g4092817.v113.gs1.nt.edit 1 538 53 7927572CT1 279
g5102597.v113.gs2.nt.edit 1 279 54 7481257CT1 291
g5262456.v113.gs4.nt.edit 1 291 55 7485790CT1 402
g5306302.v113.gs6.nt.edit 1 402 56 7482993CT1 639
g5708153.v113.gs9.nt.edit 1 639 57 2829053CB1 1370 170756F1
(BMARNOR02) 1 534 2829053F6 (TLYMNOT03) 428 989 6098294H1
(UTRENOT09) 849 1143 5279076H1 (MUSLNOT01) 1071 1308 4588915H1
(MASTTXT01) 1132 1370 58 3068234CB1 1567 70489898V1 1 459
70488597V1 353 883 5837294H1 (FTUBTUT01) 730 983 70490272V1 955
1567 59 5029478CB1 1321 6035153H1 (PITUNOT06) 1 582 6558521H1
(BRAFNON02) 504 1190 5076961F6 (COLCTUT03) 742 1321 60 5102576CB1
1110 5496406H1 (BRABDIR01) 1 250 1720010F6 (BLADNOT06) 151 708
6969401U1 602 710 5102576F6 (PROSTUS20) 673 1110 61 2200534CB1 1095
1037-1095, g2905881.v113.gs_2 1 948 372-491 2200534F6 (SPLNFET02)
534 1095 576308R6 (BRAVTXT04) 490 1051 62 3275821CB1 1665
1431-1665, 3275821F6 (PROSBPT06) 1 548 765-1294, g3779013.v13.gs_9
265 1665 240-597 63 3744167CB1 1609 1184-1238, 2762536H1
(BRSTNOT12) 745 994 249-522 g5578767.v113.gs_4 262 1338 3744167H1
(THYMNOT08) 693 977 g835247 1212 1609 3474586H1 (LUNGNOT27) 1 309
64 7472007CB1 945 g2431610.v113.gs_4.nt 1 945 65 7472008CB1 1098
g3093312.v113.gs_10.nt 1 1098 66 7472013CB1 954
g4190944.v113.gs_3.nt 1 954 67 7472015CB1 1008
g4467309.v113.gs_2.nt 1 1008 68 7472016CB1 930
g4567182.v113.gs_19.nt 1 930 69 7472017CB1 711
g5262456.v113.gs_7.nt 1 711 70 7472018CB1 1092
g5523795.v113.gs_12.nt 1 1092 71 7472019CB1 927
g5566548.v113.gs_7.nt 1 927 72 7472021CB1 1032
g5708153.v113.gs_6.nt 1 1032 73 7472009CB1 972
g3213020.v113.gs_4.nt 1 972 74 7472010CB1 900 g3738097.v113.gs_9.nt
1 900 75 7472011CB1 924 g3924656.v113.gs_5.nt 1 924 76 7472012CB1
945 g4190944.v113.gs_1.nt 1 945 77 7472014CB1 933
g4190944.v113.gs_4.nt 1 933 78 7472020CB1 1080
g5706779.v113.gs_3.nt 1 1080
[0351]
6 TABLE 5 Polynucleotide Incyte Representative SEQ ID NO: Project
ID Library 61 2200534CB1 BRAVTXT04 62 3275821CB1 PROSBPT06 63
3744167CB1 LUNGNOT27
[0352]
7TABLE 6 Library Vector Library Description BRAVTXT04 PSPORT1
Library was constructed using RNA isolated from separate
populations of human astrocytes stimulated for 4 to 6 hours with a
combination of cytokines including IL-1. The RNA was pooled for
polyA RNA isolation and library construction. LUNGNOT27 pINCY
Library was constructed using RNA isolated from lung tissue removed
from a 17-year-old Hispanic female. PROSBPT06 pINCY Library was
constructed using RNA isolated from diseased prostate tissue remove
from a 66-year-old Caucasian male during a radical prostatectomy
and lymph node excision. Pathology indicated adenofibromatous
hyperplasia. Pathology for the associated tumor tissue indicated
grade 2 (of 4) adenocarcinoma, Gleason grade 3 + 3. The patient
presented with elevated prostate specific antigen (PSA),
proteinuria, decreased renal function, and urinary frequency.
Patient history included hemiparesis, depressive disorder, sleep
apnea, psoriasis, mitral valve prolapse, cerebrovascular disease,
benign hypertension, and impotence. Family history included benign
hypertension, cerebrovascular disease, and colon cancer.
[0353]
8TABLE 7 Program Description Reference Parameter Threshold
ABIFACTURA A program that removes Applied Biosystems, vector
sequences and masks Foster City, CA. ambiguous bases in nucleic
acid sequences. ABI/PARACEL A Fast Data Finder useful Applied
Biosystems, Mismatch <50% FDF in comparing and annotating Foster
City, CA; amino acid or nucleic Paracel Inc., acid sequences.
Pasadena, CA. ABI A program that assembles Applied Biosystems,
AutoAssembler nucleic acid sequences. Foster City, CA. BLAST A
Basic Local Alignment Altschul, S. F. et al. ESTs: Probability
value = Search Tool useful in (1990) J. Mol. Biol. 1.0E-8 or less
sequence similarity search 215: 403-410; Altschul, Full Length
sequences: for amino acid and nucleic S. F. et al. (1997)
Probability value = acid sequences. BLAST includes Nucleic Acids
Res. 1.0E-10 or less five functions: blastp, blastn, 25: 3389-3402.
blastx, tblastn, and tblastx. FASTA A Pearson and Lipman algorithm
Pearson, W. R. and D. J. Lipman ESTs: fasta E value = that searches
for similarity (1988) Proc. Natl. Acad Sci. USA 1.06E-6 between a
query sequence and a 85: 2444-2448; Pearson, W. R. Assembled ESTs:
fasta group of sequences of the same (1990) Methods Enzymol.
Identity = 95% or type. FASTA comprises as least 183: 63-98; and
Smith, T. F. greater and five functions: fasta, tfasta, and M. S.
Waterman (1981) Match length = fastx, tfastx, and ssearch. Adv.
Appl. Math. 2: 482-489. 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 Henikoff, S. and J. G.
Henikoff Probability value = matches a sequence against those
(1991) Nucleic Acids Res. 1.0E-3 or less in BLOCKS, PRINTS, DOMO,
PRODOM, 19: 6565-6572; Henikoff, J. G. and PFAM databases to search
for and S. Henikoff (1996) Methods gene families, sequence
homology, Enzymol. 266: 88-105; and and structural fingerprint
regions. Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci.
37: 417-424. HMMER An algorithm for searching a query Krogh, A. et
al. (1994) PFAM hits: Probability sequence against hidden Markov J.
Mol. Biol., 235: 1501-1531; value = 1.0E-3 model (HMM)-based
databases of Sonnhammer, E. L. L. et al. (1988) or less protein
family consensus sequences, Nucleic Acids Res. 26: 320-322; Signal
peptide hits: such as PFAM. Durbin, R. et al. (1998) Our World
Score = 0 or View, in a Nutshell, Cambridge Univ. greater Press,
pp. 1-350. ProfileScan An algorithm that searches for Gribskov, M.
et al. (1988) Normalized quality score .gtoreq. structural and
sequence motifs CABIOS 4: 61-66; Gribskov, GCG-specified "HIGH" in
protein sequences that match M. et al. (1989) Methods value for
that particular sequence patterns defined in Enzymol. 183: 146-159;
Prosite motif. Prosite. Bairoch, A. et al. (1997) Generally, score
= Nucleic Acids Res. 1.4-2.1. 25: 217-221. Phred A base-calling
algorithm that Ewing, B. et al. (1998) Genome examines automated
sequencer Res. 8: 175-185; Ewing, B. traces with high sensitivity
and P. Green (1998) Genome and probability. Res. 8: 186-194. Phrap
A Phils Revised Assembly Program Smith, T. F. and M. S. Waterman
Score = 120 or including SWAT and CrossMatch, (1981) Adv. Appl.
Math. greater; programs based on efficient 2: 482-489; Smith, Match
lengths 56 or implementation of the Smith-Waterman T. F. and M. S.
Waterman (1981) greater algorithm, useful in searching sequence J.
Mol. Biol. 147: 195-197; homology and assembling DNA sequences. and
Green, P., University of Washington, Seattle, WA. Consed A
graphical tool for viewing and Gordon, D. et al. (1998) editing
Phrap assemblies. Genome Res. 8: 195-202. SPScan A weight matrix
analysis program Nielsen, H. et al. (1997) Score = 3.5 or that
scans protein sequences for Protein Engineering 10: 1-6; greater
the presence of secretory Claverie, J. M. and S. Audic signal
peptides. (1997) CABIOS 12: 431-439. TMAP A program that uses
weight matrices Persson, B. and P. Argos (1994) to delineate
transmembrane segments J. Mol. Biol. 237: 182-192; on protein
sequences and Persson, B. and P. Argos (1996) determine
orientation. Protein Sci. 5: 363-371. TMHMMER A program that uses a
hidden Markov Sonnhammer, E. L. et al. (1998) model (HMM) to
delineate transmembrane Proc. Sixth Intl. Conf. on segments on
protein sequences and Intelligent Systems for Mol. Biol., determine
orientation. Glasgow et al., eds., The Am. Assoc. for Artificial
Intelligence Press, Menlo Park, CA, pp. 175-182. Motifs A program
that searches amino acid Bairoch, A. et al. (1997) sequences for
patterns that matched Nucleic Acids Res. 25: 217-221; those defined
in Prosite. Wisconsin Package Program Manual, version 9, page
M51-59, Genetics Computer Group, Madison, WI.
[0354]
Sequence CWU 1
1
78 1 311 PRT Homo sapiens misc_feature Incyte ID No 104941CD1 1 Met
Glu Ile Lys Asn Tyr Ser Ser Ser Thr Ser Gly Phe Ile Leu 1 5 10 15
Leu Gly Leu Ser Ser Asn Pro Gln Leu Gln Lys Pro Leu Phe Ala 20 25
30 Ile Phe Leu Ile Met Tyr Leu Leu Ala Ala Val Gly Asn Val Leu 35
40 45 Ile Ile Pro Ala Ile Tyr Ser Asp Pro Arg Leu His Thr Pro Met
50 55 60 Tyr Phe Phe Leu Ser Asn Leu Ser Phe Met Asp Ile Cys Phe
Thr 65 70 75 Thr Val Ile Val Pro Lys Met Leu Val Asn Phe Leu Ser
Glu Thr 80 85 90 Lys Val Ile Ser Tyr Val Gly Cys Leu Ala Gln Met
Tyr Phe Phe 95 100 105 Met Ala Phe Gly Asn Thr Asp Ser Tyr Leu Leu
Ala Ser Met Ala 110 115 120 Ile Asp Arg Leu Val Ala Ile Cys Asn Pro
Leu His Tyr Asp Val 125 130 135 Val Met Lys Pro Arg His Cys Leu Leu
Met Leu Leu Gly Ser Cys 140 145 150 Ser Ile Ser His Leu His Ser Leu
Phe Arg Val Leu Leu Met Ser 155 160 165 Arg Leu Ser Phe Cys Ala Ser
His Ile Ile Lys His Phe Phe Cys 170 175 180 Asp Thr Gln Pro Val Leu
Lys Leu Ser Cys Ser Asp Thr Ser Ser 185 190 195 Ser Gln Met Val Val
Met Thr Glu Thr Leu Ala Val Ile Val Thr 200 205 210 Pro Phe Leu Cys
Ile Ile Phe Ser Tyr Leu Arg Ile Met Val Thr 215 220 225 Val Leu Arg
Ile Pro Ser Ala Ala Gly Lys Trp Lys Ala Phe Ser 230 235 240 Thr Cys
Gly Ser His Leu Thr Ala Val Ala Leu Phe Tyr Gly Ser 245 250 255 Ile
Ile Tyr Val Tyr Phe Arg Pro Leu Ser Met Tyr Ser Val Val 260 265 270
Arg Asp Arg Val Ala Thr Val Met Tyr Thr Val Val Thr Pro Met 275 280
285 Leu Asn Pro Phe Ile Tyr Ser Leu Arg Asn Lys Asp Met Lys Arg 290
295 300 Gly Leu Lys Lys Leu Gln Asp Arg Ile Tyr Arg 305 310 2 891
PRT Homo sapiens misc_feature Incyte ID No 1499408CD1 2 Met Asp Gln
Pro Glu Ala Pro Cys Ser Ser Thr Gly Pro Arg Leu 1 5 10 15 Ala Val
Ala Arg Glu Leu Leu Leu Ala Ala Leu Glu Glu Leu Ser 20 25 30 Gln
Glu Gln Leu Lys Arg Phe Arg His Lys Leu Arg Asp Val Gly 35 40 45
Pro Asp Gly Arg Ser Ile Pro Trp Gly Arg Leu Glu Arg Ala Asp 50 55
60 Ala Val Asp Leu Ala Glu Gln Leu Ala Gln Phe Tyr Gly Pro Glu 65
70 75 Pro Ala Leu Glu Val Ala Arg Lys Thr Leu Lys Arg Ala Asp Ala
80 85 90 Arg Asp Val Ala Ala Gln Leu Gln Glu Arg Arg Leu Gln Arg
Leu 95 100 105 Gly Leu Gly Ser Gly Thr Leu Leu Ser Val Ser Glu Tyr
Lys Lys 110 115 120 Lys Tyr Arg Glu His Val Leu Gln Leu His Ala Arg
Val Lys Glu 125 130 135 Arg Asn Ala Arg Ser Val Lys Ile Thr Lys Arg
Phe Thr Lys Leu 140 145 150 Leu Ile Ala Pro Glu Ser Ala Ala Pro Glu
Glu Ala Leu Gly Pro 155 160 165 Ala Glu Glu Pro Glu Pro Gly Arg Ala
Arg Arg Ser Asp Thr His 170 175 180 Thr Phe Asn Arg Leu Phe Arg Arg
Asp Glu Glu Gly Arg Arg Pro 185 190 195 Leu Thr Val Val Leu Gln Gly
Pro Ala Gly Ile Gly Lys Thr Met 200 205 210 Ala Ala Lys Lys Ile Leu
Tyr Asp Trp Ala Ala Gly Lys Leu Tyr 215 220 225 Gln Gly Gln Val Asp
Phe Ala Phe Phe Met Pro Cys Gly Glu Leu 230 235 240 Leu Glu Arg Pro
Gly Thr Arg Ser Leu Ala Asp Leu Ile Leu Asp 245 250 255 Gln Cys Pro
Asp Arg Gly Ala Pro Val Pro Gln Met Leu Ala Gln 260 265 270 Pro Gln
Arg Leu Leu Phe Ile Leu Asp Gly Ala Asp Glu Leu Pro 275 280 285 Ala
Leu Gly Gly Pro Glu Ala Ala Pro Cys Thr Asp Pro Phe Glu 290 295 300
Ala Ala Ser Gly Ala Arg Val Leu Gly Gly Leu Leu Ser Lys Ala 305 310
315 Leu Leu Pro Thr Ala Leu Leu Leu Val Thr Thr Arg Ala Ala Ala 320
325 330 Pro Gly Arg Leu Gln Gly Arg Leu Cys Ser Pro Gln Cys Ala Glu
335 340 345 Val Arg Gly Phe Ser Asp Lys Asp Lys Lys Lys Tyr Phe Tyr
Lys 350 355 360 Phe Phe Arg Asp Glu Arg Arg Ala Glu Arg Ala Tyr Arg
Phe Val 365 370 375 Lys Glu Asn Glu Thr Leu Phe Ala Leu Cys Phe Val
Pro Phe Val 380 385 390 Cys Trp Ile Val Cys Thr Val Leu Arg Gln Gln
Leu Glu Leu Gly 395 400 405 Arg Asp Leu Ser Arg Thr Ser Lys Thr Thr
Thr Ser Val Tyr Leu 410 415 420 Leu Phe Ile Thr Ser Val Leu Ser Ser
Ala Pro Val Ala Asp Gly 425 430 435 Pro Arg Leu Gln Gly Asp Leu Arg
Asn Leu Cys Arg Leu Ala Arg 440 445 450 Glu Gly Val Leu Gly Arg Arg
Ala Gln Phe Ala Glu Lys Glu Leu 455 460 465 Glu Gln Leu Glu Leu Arg
Gly Ser Lys Val Gln Thr Leu Phe Leu 470 475 480 Ser Lys Lys Glu Leu
Pro Gly Val Leu Glu Thr Glu Val Thr Tyr 485 490 495 Gln Phe Ile Asp
Gln Ser Phe Gln Glu Phe Leu Ala Ala Leu Ser 500 505 510 Tyr Leu Leu
Glu Asp Gly Gly Val Pro Arg Thr Ala Ala Gly Gly 515 520 525 Val Gly
Thr Leu Leu Arg Gly Asp Ala Gln Pro His Ser His Leu 530 535 540 Val
Leu Thr Thr Arg Phe Leu Phe Gly Leu Leu Ser Ala Glu Arg 545 550 555
Met Arg Asp Ile Glu Arg His Phe Gly Cys Met Val Ser Glu Arg 560 565
570 Val Lys Gln Glu Ala Leu Arg Trp Val Gln Gly Gln Gly Gln Gly 575
580 585 Cys Pro Gly Val Ala Pro Glu Val Thr Glu Gly Ala Lys Gly Leu
590 595 600 Glu Asp Thr Glu Glu Pro Glu Glu Glu Glu Glu Gly Glu Glu
Pro 605 610 615 Asn Tyr Pro Leu Glu Leu Leu Tyr Cys Leu Tyr Glu Thr
Gln Glu 620 625 630 Asp Ala Phe Val Arg Gln Ala Leu Cys Arg Phe Pro
Glu Leu Ala 635 640 645 Leu Gln Arg Val Arg Phe Cys Arg Met Asp Val
Ala Val Leu Ser 650 655 660 Tyr Cys Val Arg Cys Cys Pro Ala Gly Gln
Ala Leu Arg Leu Ile 665 670 675 Ser Cys Arg Leu Val Ala Ala Gln Glu
Lys Lys Lys Lys Ser Leu 680 685 690 Gly Lys Arg Leu Gln Ala Ser Leu
Gly Gly Gly Ser Ser Gln Gly 695 700 705 Thr Thr Lys Gln Leu Pro Ala
Ser Leu Leu His Pro Leu Phe Gln 710 715 720 Ala Met Thr Asp Pro Leu
Cys His Leu Ser Ser Leu Thr Leu Ser 725 730 735 His Cys Lys Leu Pro
Asp Ala Val Cys Arg Asp Leu Ser Glu Ala 740 745 750 Leu Arg Ala Ala
Pro Ala Leu Thr Glu Leu Gly Leu Leu His Asn 755 760 765 Arg Leu Ser
Glu Ala Gly Leu Arg Met Leu Ser Glu Gly Leu Ala 770 775 780 Trp Pro
Gln Cys Arg Val Gln Thr Val Arg Val Gln Leu Pro Asp 785 790 795 Pro
Gln Arg Gly Leu Gln Tyr Leu Val Gly Met Leu Arg Gln Ser 800 805 810
Pro Ala Leu Thr Thr Leu Asp Leu Ser Gly Cys Gln Leu Pro Ala 815 820
825 Pro Met Val Thr Tyr Leu Cys Ala Val Leu Gln His Gln Gly Cys 830
835 840 Gly Leu Gln Thr Leu Ser Leu Ala Ser Val Glu Leu Ser Glu Gln
845 850 855 Ser Leu Gln Glu Leu Gln Ala Val Lys Arg Ala Lys Pro Asp
Leu 860 865 870 Val Ile Thr His Pro Ala Leu Asp Gly His Pro Gln Pro
Pro Lys 875 880 885 Glu Leu Ile Ser Thr Phe 890 3 422 PRT Homo
sapiens misc_feature Incyte ID No 3168839CD1 3 Met Leu Ala Asn Ser
Ser Ser Thr Asn Ser Ser Val Leu Pro Cys 1 5 10 15 Pro Asp Tyr Arg
Pro Thr His Arg Leu His Leu Val Val Tyr Ser 20 25 30 Leu Val Leu
Ala Ala Gly Leu Pro Leu Asn Ala Leu Ala Leu Trp 35 40 45 Val Phe
Leu Arg Ala Leu Arg Val His Ser Val Val Ser Val Tyr 50 55 60 Met
Cys Asn Leu Ala Ala Ser Asp Leu Leu Phe Thr Leu Ser Leu 65 70 75
Pro Val Arg Leu Ser Tyr Tyr Ala Leu His His Trp Pro Phe Pro 80 85
90 Asp Leu Leu Cys Gln Thr Thr Gly Ala Ile Phe Gln Met Asn Met 95
100 105 Tyr Gly Ser Cys Ile Phe Leu Met Leu Ile Asn Val Asp Arg Tyr
110 115 120 Ala Ala Ile Val His Pro Leu Arg Leu Arg His Leu Arg Arg
Pro 125 130 135 Arg Val Ala Arg Leu Leu Cys Leu Gly Val Trp Ala Leu
Ile Leu 140 145 150 Val Phe Ala Val Pro Ala Ala Arg Val His Arg Pro
Ser Arg Cys 155 160 165 Arg Tyr Arg Asp Leu Glu Val Arg Leu Cys Phe
Glu Ser Phe Ser 170 175 180 Asp Glu Leu Trp Lys Gly Arg Leu Leu Pro
Leu Val Leu Leu Ala 185 190 195 Glu Ala Leu Gly Phe Leu Leu Pro Leu
Ala Ala Val Val Tyr Ser 200 205 210 Ser Gly Arg Val Phe Trp Thr Leu
Ala Arg Pro Asp Ala Thr Gln 215 220 225 Ser Gln Arg Arg Arg Lys Thr
Val Arg Leu Leu Leu Ala Asn Leu 230 235 240 Val Ile Phe Leu Leu Cys
Phe Val Pro Tyr Asn Ser Thr Leu Ala 245 250 255 Val Tyr Gly Leu Leu
Arg Ser Lys Leu Val Ala Ala Ser Val Pro 260 265 270 Ala Arg Asp Arg
Val Arg Gly Val Leu Met Val Met Val Leu Leu 275 280 285 Ala Gly Ala
Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe Ser 290 295 300 Ala Glu
Gly Phe Arg Asn Thr Leu Arg Gly Leu Gly Thr Pro His 305 310 315 Arg
Ala Arg Thr Ser Ala Thr Asn Gly Thr Arg Ala Ala Leu Ala 320 325 330
Gln Ser Glu Arg Ser Ala Val Thr Thr Asp Ala Thr Arg Pro Asp 335 340
345 Ala Ala Met Ser Pro Gly Phe Arg Pro Leu Asn Thr His Ala Ile 350
355 360 Ala Leu Ser Val Pro Asp Ser Gln Arg Leu Ser Phe Trp Glu Ala
365 370 375 Tyr Arg Val Tyr Thr Gln Glu Gly Gly Leu Gly Thr Trp Thr
Phe 380 385 390 Gly Trp Gln Phe Gln Leu Ser Asn Ala Glu Glu Tyr Lys
Val Trp 395 400 405 Lys Pro Gly Pro Arg Glu Gly Ser Ala Ala Gly Asn
Gly Phe Phe 410 415 420 Lys Leu 4 609 PRT Homo sapiens misc_feature
Incyte ID No 3291235CD1 4 Met Ser Asp Glu Arg Arg Leu Pro Gly Ser
Ala Val Gly Trp Leu 1 5 10 15 Val Cys Gly Gly Leu Ser Leu Leu Ala
Asn Ala Trp Gly Ile Leu 20 25 30 Ser Val Gly Ala Lys Gln Lys Lys
Trp Lys Pro Leu Glu Phe Leu 35 40 45 Leu Cys Thr Leu Ala Ala Thr
His Met Leu Asn Val Ala Val Pro 50 55 60 Ile Ala Thr Tyr Ser Val
Val Gln Leu Arg Arg Gln Arg Pro Asp 65 70 75 Phe Glu Trp Asn Glu
Gly Leu Cys Lys Val Phe Val Ser Thr Phe 80 85 90 Tyr Thr Leu Thr
Leu Ala Thr Cys Phe Ser Val Thr Ser Leu Ser 95 100 105 Tyr His Arg
Met Trp Met Val Cys Trp Pro Val Asn Tyr Arg Leu 110 115 120 Ser Asn
Ala Lys Lys Gln Ala Val His Thr Val Met Gly Ile Trp 125 130 135 Met
Val Ser Phe Ile Leu Ser Ala Leu Pro Ala Val Gly Trp His 140 145 150
Asp Thr Ser Glu Arg Phe Tyr Thr His Gly Cys Arg Phe Ile Val 155 160
165 Ala Glu Ile Gly Leu Gly Phe Gly Val Cys Phe Leu Leu Leu Val 170
175 180 Gly Gly Ser Val Ala Met Gly Val Ile Cys Thr Ala Ile Ala Leu
185 190 195 Phe Gln Thr Leu Ala Val Gln Val Gly Arg Gln Ala Asp His
Arg 200 205 210 Ala Phe Thr Val Pro Thr Ile Val Val Glu Asp Ala Gln
Gly Lys 215 220 225 Arg Arg Ser Ser Ile Asp Gly Ser Glu Pro Ala Lys
Thr Ser Leu 230 235 240 Gln Thr Thr Gly Leu Val Thr Thr Ile Val Phe
Ile Tyr Asp Cys 245 250 255 Leu Met Gly Phe Pro Val Leu Val Val Ser
Phe Ser Ser Leu Arg 260 265 270 Ala Asp Ala Ser Ala Pro Trp Met Ala
Leu Cys Val Leu Trp Cys 275 280 285 Ser Val Ala Gln Ala Leu Leu Leu
Pro Val Phe Leu Trp Ala Cys 290 295 300 Asp Arg Tyr Arg Ala Asp Leu
Lys Ala Val Arg Glu Lys Cys Met 305 310 315 Ala Leu Met Ala Asn Asp
Glu Glu Ser Asp Asp Glu Thr Ser Leu 320 325 330 Glu Gly Gly Ile Ser
Pro Asp Leu Val Leu Glu Arg Ser Leu Asp 335 340 345 Tyr Gly Tyr Gly
Gly Asp Phe Val Ala Leu Asp Arg Met Ala Lys 350 355 360 Tyr Glu Ile
Ser Ala Leu Glu Gly Gly Leu Pro Gln Leu Tyr Pro 365 370 375 Leu Arg
Pro Leu Gln Glu Asp Lys Met Gln Tyr Leu Gln Val Pro 380 385 390 Pro
Thr Arg Arg Phe Ser His Asp Asp Ala Asp Val Trp Ala Ala 395 400 405
Val Pro Leu Pro Ala Phe Leu Pro Arg Trp Gly Ser Gly Lys Asp 410 415
420 Leu Ser Ala Leu Ala His Leu Val Leu Pro Ala Gly Pro Glu Arg 425
430 435 Pro Arg Ala Ser Leu Leu Ala Phe Ala Glu Asp Ala Pro Leu Ser
440 445 450 Arg Ala Arg Arg Arg Ser Ala Glu Ser Leu Leu Ser Leu Arg
Pro 455 460 465 Ser Ala Val Asp Ser Gly Pro Arg Gly Ala Arg Asp Ser
Pro Pro 470 475 480 Gly Ser Pro Arg Arg Arg Pro Gly Pro Gly Pro Arg
Ser Ala Ser 485 490 495 Ala Ser Leu Leu Pro Asp Ala Phe Ala Leu Thr
Ala Phe Glu Cys 500 505 510 Glu Pro Gln Ala Leu Arg Arg Pro Pro Gly
Pro Phe Pro Ala Ala 515 520 525 Pro Ala Ala Pro Asp Gly Ala Asp Pro
Gly Glu Ala Pro Thr Pro 530 535 540 Pro Ser Ser Ala Gln Arg Ser Pro
Gly Pro Arg Pro Ser Ala His 545 550 555 Ser His Ala Gly Ser Leu Arg
Pro Gly Leu Ser Ala Ser Trp Gly 560 565 570 Glu Pro Gly Gly Leu Arg
Ala Ala Gly Gly Gly Gly Ser Thr Ser 575 580 585 Ser Phe Leu Ser Ser
Pro Ser Glu Ser Ser Gly Tyr Ala Thr Leu 590 595 600 His Ser Asp Ser
Leu Gly Ser Ala Ser 605 5 313 PRT Homo sapiens misc_feature Incyte
ID No 7472001CD1 5 Met Glu Arg Ile Asn Ser Thr Leu Leu Thr Ala Phe
Ile Leu Thr 1 5 10 15 Gly Ile Pro Tyr Pro Leu Arg Leu Arg Thr Leu
Phe Phe Val Phe 20 25 30
Phe Phe Leu Ile Tyr Ile Leu Thr Gln Leu Gly Asn Leu Leu Ile 35 40
45 Leu Ile Thr Val Trp Ala Asp Pro Arg Leu His Ala Arg Pro Met 50
55 60 Tyr Ile Phe Leu Gly Val Leu Ser Val Ile Asp Met Ser Ile Ser
65 70 75 Ser Ile Ile Val Pro Arg Leu Met Met Asn Phe Thr Leu Gly
Val 80 85 90 Lys Pro Ile Pro Phe Gly Gly Cys Val Ala Gln Leu Tyr
Phe Tyr 95 100 105 His Phe Leu Gly Ser Thr Gln Cys Phe Leu Tyr Thr
Leu Met Ala 110 115 120 Tyr Asp Arg Tyr Leu Ala Ile Cys Gln Pro Leu
Arg Tyr Pro Val 125 130 135 Leu Met Thr Ala Lys Leu Ser Ala Leu Leu
Val Ala Gly Ala Trp 140 145 150 Met Ala Gly Ser Ile His Gly Ala Leu
Gln Ala Ile Leu Thr Phe 155 160 165 Arg Leu Pro Tyr Cys Gly Pro Asn
Gln Val Asp Tyr Phe Phe Cys 170 175 180 Asp Ile Pro Ala Val Leu Arg
Leu Ala Cys Ala Asp Thr Thr Val 185 190 195 Asn Glu Leu Val Thr Phe
Val Asp Ile Gly Val Val Val Ala Ser 200 205 210 Cys Phe Ser Leu Ile
Leu Leu Ser Tyr Ile Gln Ile Ile Gln Ala 215 220 225 Ile Leu Arg Ile
His Thr Ala Asp Gly Arg Arg Arg Ala Phe Ser 230 235 240 Thr Cys Gly
Ala His Val Thr Val Val Thr Val Tyr Tyr Val Pro 245 250 255 Cys Ala
Phe Ile Tyr Leu Arg Pro Glu Thr Asn Ser Pro Leu Asp 260 265 270 Gly
Ala Ala Ala Leu Val Pro Thr Ala Ile Thr Pro Phe Leu Asn 275 280 285
Pro Leu Ile Tyr Thr Leu Arg Asn Gln Glu Val Lys Leu Ala Leu 290 295
300 Lys Arg Met Leu Arg Ser Pro Arg Thr Pro Ser Glu Val 305 310 6
398 PRT Homo sapiens misc_feature Incyte ID No 7472003CD1 6 Met His
Thr Val Ala Thr Ser Gly Pro Asn Ala Ser Trp Gly Ala 1 5 10 15 Pro
Ala Asn Ala Ser Gly Cys Pro Gly Cys Gly Ala Asn Ala Ser 20 25 30
Asp Gly Pro Val Pro Ser Pro Arg Ala Val Asp Ala Trp Leu Val 35 40
45 Pro Leu Phe Phe Ala Ala Leu Met Leu Leu Gly Leu Val Gly Asn 50
55 60 Ser Leu Val Ile Tyr Val Ile Cys Arg His Lys Pro Met Arg Thr
65 70 75 Val Thr Asn Phe Tyr Ile Ala Asn Leu Ala Ala Thr Asp Val
Thr 80 85 90 Phe Leu Leu Cys Cys Val Pro Phe Thr Ala Leu Leu Tyr
Pro Leu 95 100 105 Pro Gly Trp Val Leu Gly Asp Phe Met Cys Lys Phe
Val Asn Tyr 110 115 120 Ile Gln Gln Val Ser Val Gln Ala Thr Cys Ala
Thr Leu Thr Ala 125 130 135 Met Ser Val Asp Arg Trp Tyr Val Thr Val
Phe Pro Leu Arg Ala 140 145 150 Leu His Arg Arg Thr Pro Arg Leu Ala
Leu Ala Val Ser Leu Ser 155 160 165 Ile Trp Thr Gly Ser Ala Ala Val
Ser Ala Pro Val Leu Ala Leu 170 175 180 His Arg Leu Ser Pro Gly Pro
Arg Ala Tyr Cys Ser Glu Ala Phe 185 190 195 Pro Ser Arg Ala Leu Glu
Arg Ala Phe Ala Leu Tyr Asn Leu Leu 200 205 210 Ala Leu Tyr Leu Leu
Pro Leu Leu Ala Thr Cys Ala Cys Tyr Ala 215 220 225 Ala Met Leu Arg
His Leu Gly Arg Val Ala Val Arg Pro Ala Pro 230 235 240 Ala Asp Ser
Ala Leu Gln Gly Gln Val Leu Ala Glu Arg Ala Gly 245 250 255 Ala Val
Arg Ala Lys Val Ser Arg Leu Val Ala Ala Val Val Leu 260 265 270 Leu
Phe Ala Ala Cys Trp Gly Pro Ile Gln Leu Phe Leu Val Leu 275 280 285
Gln Ala Leu Gly Pro Ala Gly Ser Trp His Pro Arg Ser Tyr Ala 290 295
300 Ala Tyr Ala Leu Lys Thr Trp Ala His Cys Met Ser Tyr Ser Asn 305
310 315 Ser Ala Leu Asn Pro Leu Leu Tyr Ala Phe Leu Gly Ser His Phe
320 325 330 Arg Gln Ala Phe Arg Arg Val Cys Pro Cys Ala Pro Arg Arg
Pro 335 340 345 Arg Arg Pro Arg Arg Pro Gly Pro Ser Asp Pro Ala Ala
Pro His 350 355 360 Ala Glu Leu Leu Arg Leu Gly Ser His Pro Ala Pro
Ala Arg Ala 365 370 375 Gln Lys Pro Gly Ser Ser Gly Leu Ala Ala Arg
Gly Leu Cys Val 380 385 390 Leu Gly Glu Asp Asn Ala Pro Leu 395 7
369 PRT Homo sapiens misc_feature Incyte ID No 7472004CD1 7 Met Ala
Pro Thr Gly Leu Ser Ser Leu Thr Val Asn Ser Thr Ala 1 5 10 15 Val
Pro Thr Thr Pro Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu 20 25 30
Gln Ile Thr Leu Ser Ala Ile Met Ile Phe Ile Leu Phe Val Ser 35 40
45 Phe Leu Gly Asn Leu Val Val Cys Leu Met Val Tyr Gln Lys Ala 50
55 60 Ala Met Arg Ser Ala Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe
65 70 75 Ala Asp Met Leu Leu Ala Val Leu Asn Met Pro Phe Ala Leu
Val 80 85 90 Thr Ile Leu Thr Thr Arg Trp Ile Phe Gly Lys Phe Phe
Cys Arg 95 100 105 Val Ser Ala Met Phe Phe Trp Leu Phe Val Ile Glu
Gly Val Ala 110 115 120 Ile Leu Leu Ile Ile Ser Ile Asp Arg Phe Leu
Ile Ile Val Gln 125 130 135 Arg Gln Asp Lys Leu Asn Pro Tyr Arg Ala
Lys Val Leu Ile Ala 140 145 150 Val Ser Trp Ala Thr Ser Phe Cys Val
Ala Phe Pro Leu Ala Val 155 160 165 Gly Asn Pro Asp Leu Gln Ile Pro
Ser Arg Ala Pro Gln Cys Val 170 175 180 Phe Gly Tyr Thr Thr Asn Pro
Gly Tyr Gln Ala Tyr Val Ile Leu 185 190 195 Ile Ser Leu Ile Ser Phe
Phe Ile Pro Phe Leu Val Ile Leu Tyr 200 205 210 Ser Phe Met Gly Ile
Leu Asn Thr Leu Arg His Asn Ala Leu Arg 215 220 225 Ile His Ser Tyr
Pro Glu Gly Ile Cys Leu Ser Gln Ala Ser Lys 230 235 240 Leu Gly Leu
Met Ser Leu Gln Arg Pro Phe Gln Met Ser Ile Asp 245 250 255 Met Gly
Phe Lys Thr Arg Ala Phe Thr Thr Ile Leu Ile Leu Phe 260 265 270 Ala
Val Phe Ile Val Cys Trp Ala Pro Phe Thr Thr Tyr Ser Leu 275 280 285
Val Ala Thr Phe Ser Lys His Phe Tyr Tyr Gln His Asn Phe Phe 290 295
300 Glu Ile Ser Thr Trp Leu Leu Trp Leu Cys Tyr Leu Lys Ser Ala 305
310 315 Leu Asn Pro Leu Ile Tyr Tyr Trp Arg Ile Lys Lys Phe His Asp
320 325 330 Ala Cys Leu Asp Met Met Pro Lys Ser Phe Lys Phe Leu Pro
Gln 335 340 345 Leu Pro Gly His Thr Lys Arg Arg Ile Arg Pro Ser Ala
Val Tyr 350 355 360 Val Cys Gly Glu His Arg Thr Val Val 365 8 194
PRT Homo sapiens misc_feature Incyte ID No 7475687CP1 8 Met Ala Tyr
Asp Arg Tyr Leu Ala Ile Cys Gln Pro Leu Arg Tyr 1 5 10 15 Pro Val
Leu Met Asn Gly Arg Leu Cys Thr Val Leu Val Ala Gly 20 25 30 Ala
Trp Val Ala Gly Ser Met His Gly Ser Ile Gln Ala Thr Leu 35 40 45
Thr Phe Arg Leu Pro Tyr Cys Gly Pro Asn Gln Val Asp Tyr Phe 50 55
60 Ile Cys Asp Ile Pro Ala Val Leu Arg Leu Ala Cys Ala Asp Thr 65
70 75 Thr Val Asn Glu Leu Val Thr Phe Val Asp Ile Gly Val Val Ala
80 85 90 Ala Ser Cys Phe Met Leu Ile Leu Leu Ser Tyr Ala Asn Ile
Val 95 100 105 Asn Ala Ile Leu Lys Ile Arg Thr Thr Asp Gly Arg Arg
Arg Ala 110 115 120 Phe Ser Thr Cys Gly Ser His Leu Ile Val Val Thr
Val Tyr Tyr 125 130 135 Val Pro Cys Ile Phe Ile Tyr Leu Arg Ala Gly
Ser Lys Gly Pro 140 145 150 Leu Asp Gly Ala Ala Ala Val Phe Tyr Thr
Val Val Thr Pro Leu 155 160 165 Leu Asn Pro Leu Ile Tyr Thr Leu Arg
Asn Gln Glu Val Lys Ser 170 175 180 Ala Leu Lys Arg Ile Thr Ala Gly
Gln Ala Asp Val Asn Asn 185 190 9 173 PRT Homo sapiens misc_feature
Incyte ID No 7483029CP1 9 Met Tyr Leu Val Thr Val Leu Gly Asn Leu
Leu Ile Ile Leu Ala 1 5 10 15 Thr Ile Ser Asp Ser His Leu His Thr
Pro Met Tyr Phe Phe Leu 20 25 30 Ser Asn Leu Ser Phe Ala Asp Ile
Cys Phe Val Ser Thr Thr Val 35 40 45 Pro Lys Met Leu Val Asn Ile
Gln Thr Gln Ser Arg Val Ile Thr 50 55 60 Tyr Ala Asp Cys Ile Thr
Gln Met Cys Phe Phe Ile Leu Phe Val 65 70 75 Val Leu Asp Ser Leu
Leu Leu Thr Val Met Ala Tyr Asp Arg Phe 80 85 90 Val Ala Ile Cys
His Pro Leu His Tyr Thr Val Ile Met Asn Ser 95 100 105 Trp Leu Cys
Gly Leu Leu Val Leu Val Ser Trp Ile Val Ser Ile 110 115 120 Leu Tyr
Ser Leu Leu Gln Ser Ile Met Ala Leu Gln Leu Ser Phe 125 130 135 Cys
Thr Glu Leu Lys Ile Pro His Phe Phe Cys Glu Leu Asn Gln 140 145 150
Val Ile His Leu Ala Cys Ser Asp Thr Phe Ile Asn Asp Met Met 155 160
165 Met Asn Phe Thr Ser Val Leu Leu 170 10 220 PRT Homo sapiens
misc_feature Incyte ID No 7477933CP1 10 Pro Met Tyr Phe Phe Leu Ser
Asn Leu Cys Trp Ala Asp Ile Gly 1 5 10 15 Leu Thr Ser Ala Thr Val
Pro Lys Val Ile Leu Asp Met Gln Ser 20 25 30 His Ser Arg Val Ile
Ser His Val Gly Cys Leu Thr Gln Met Ser 35 40 45 Phe Leu Val Leu
Phe Ala Cys Ile Glu Gly Met Leu Leu Thr Val 50 55 60 Met Ala Tyr
Gly Cys Phe Val Ala Ile Cys Arg Pro Leu His Tyr 65 70 75 Pro Val
Ile Val Asn Pro His Leu Cys Val Phe Phe Val Leu Val 80 85 90 Ser
Phe Phe Leu Asn Leu Leu Asp Ser Gln Leu His Ser Trp Ile 95 100 105
Val Leu Gln Phe Thr Ile Ile Lys Asn Val Glu Ile Ser Asn Phe 110 115
120 Phe Cys Asp Pro Ser Gln Leu Leu Asn Leu Ala Cys Ser Asp Ser 125
130 135 Val Ile Asn Ser Ile Phe Ile Tyr Phe Asp Ser Thr Met Phe Gly
140 145 150 Phe Leu Pro Ile Ser Gly Ile Leu Leu Ser Tyr Tyr Lys Ile
Val 155 160 165 Pro Ser Ile Leu Arg Met Ser Ser Ser Asp Gly Lys Tyr
Lys Ala 170 175 180 Phe Ser Thr Tyr Gly Ser His Leu Gly Val Val Cys
Trp Phe Tyr 185 190 195 Gly Thr Val Ile Gly Met Tyr Leu Ala Ser Ala
Val Ser Pro Pro 200 205 210 Pro Arg Asn Gly Val Val Ala Ser Val Met
215 220 11 302 PRT Homo sapiens misc_feature Incyte ID No
7475164CP1 11 Ala Glu Phe Ile Leu Ala Gly Leu Thr Gln Arg Pro Glu
Leu Gln 1 5 10 15 Leu Pro Leu Phe Leu Leu Phe Leu Gly Ile Tyr Val
Val Thr Val 20 25 30 Val Gly Asn Leu Gly Met Ile Phe Leu Ile Ala
Leu Ser Ser Gln 35 40 45 Leu Tyr Pro Pro Val Tyr Tyr Phe Leu Ser
His Leu Ser Phe Ile 50 55 60 Asp Leu Cys Tyr Ser Ser Val Ile Thr
Pro Lys Met Leu Val Asn 65 70 75 Phe Val Pro Glu Glu Asn Ile Ile
Ser Phe Leu Glu Cys Ile Thr 80 85 90 Gln Leu Tyr Phe Phe Leu Ile
Phe Val Ile Ala Glu Gly Tyr Leu 95 100 105 Leu Thr Ala Met Glu Tyr
Asp Arg Tyr Val Ala Ile Cys Arg Pro 110 115 120 Leu Leu Tyr Asn Ile
Val Met Ser His Arg Val Cys Ser Ile Met 125 130 135 Met Ala Val Val
Tyr Ser Leu Gly Phe Leu Trp Ala Thr Val His 140 145 150 Thr Thr Arg
Met Ser Val Leu Ser Phe Cys Arg Ser His Thr Val 155 160 165 Ser His
Tyr Phe Cys Asp Ile Leu Pro Leu Leu Thr Leu Ser Cys 170 175 180 Ser
Ser Thr His Ile Asn Glu Ile Leu Leu Phe Ile Ile Gly Gly 185 190 195
Val Asn Thr Leu Ala Thr Thr Leu Ala Val Leu Ile Ser Tyr Ala 200 205
210 Phe Ile Phe Ser Ser Ile Leu Gly Ile His Ser Thr Glu Gly Gln 215
220 225 Ser Lys Ala Phe Gly Thr Cys Ser Ser His Leu Leu Ala Val Gly
230 235 240 Ile Phe Phe Gly Ser Ile Thr Phe Met Tyr Phe Lys Pro Pro
Ser 245 250 255 Ser Thr Thr Met Glu Lys Glu Lys Val Ser Ser Val Phe
Tyr Ile 260 265 270 Thr Ile Ile Pro Met Leu Asn Pro Leu Ile Tyr Ser
Leu Arg Asn 275 280 285 Lys Asp Val Lys Asn Ala Leu Lys Lys Met Thr
Arg Gly Arg Gln 290 295 300 Ser Ser 12 110 PRT Homo sapiens
misc_feature Incyte ID No 7473909CP1 12 Gly Pro Arg Thr Ala Ser Gly
Cys Val Ile Met Ile Cys Phe Ala 1 5 10 15 Leu Thr Val Leu Ser Tyr
Ile Arg Ile Leu Ala Thr Val Val Gln 20 25 30 Ile Arg Ser Ala Ala
Ser Arg Arg Lys Ala Phe Ser Thr Cys Ser 35 40 45 Ser His Leu Gly
Met Val Leu Leu Phe Tyr Gly Thr Gly Ser Ser 50 55 60 Thr Tyr Met
Arg Pro Thr Thr Arg Tyr Ser Pro Leu Glu Gly Arg 65 70 75 Leu Ala
Ala Val Phe Tyr Ser Ile Leu Ile Pro Thr Leu Asn Pro 80 85 90 Leu
Ile Tyr Ser Leu Arg Asn Gln Asp Met Lys Arg Ala Leu Trp 95 100 105
Lys Leu Tyr Leu Gln 110 13 178 PRT Homo sapiens misc_feature Incyte
ID No 7475252CP1 13 Glu Pro Glu Asn Leu Thr Gly Val Leu Glu Phe Leu
Leu Leu Gly 1 5 10 15 Leu Pro Asp Asp Pro Glu Leu Gln Pro Val Leu
Phe Gly Leu Phe 20 25 30 Leu Ser Met Tyr Leu Val Met Val Leu Gly
Asn Leu Leu Ile Ile 35 40 45 Leu Ala Val Ser Ser Asp Ser His Leu
His Ser Pro Met Tyr Phe 50 55 60 Phe Leu Ser Asn Leu Ser Leu Ala
Asp Ile Gly Phe Ala Ser Thr 65 70 75 Thr Val Pro Lys Met Ile Val
Asp Ile Gln Ala His Ser Arg Leu 80 85 90 Ile Ser Tyr Val Gly Cys
Leu Thr Gln Met Ser Phe Leu Ile Phe 95 100 105 Phe Ala Cys Met Glu
Ser Leu Leu Leu Ile Val Met Ala Tyr Asp 110 115 120 Arg Phe Val Ala
Ile Cys His Pro Leu His Tyr Gln Val Ile Met 125 130 135 Ser Pro Arg
Leu Cys Gly Phe Leu Val Leu Val Ser Phe Phe Leu 140 145 150 Ser Leu
Leu Asp Ser Gln Leu His Asn Leu Ile Val Leu Gln Leu 155 160 165 Thr
Cys Phe Asn Asp Val Glu Ile Ser Asn Phe Phe Leu 170 175 14 92 PRT
Homo sapiens misc_feature Incyte ID No 7927572CP1 14 Leu Leu Asp
Ala Gln Leu Tyr Asn Leu Ile Ala Leu Gln Met Thr 1
5 10 15 Cys Phe Lys Asp Val Glu Ile Pro Asn Phe Phe Cys Asp Pro Ser
20 25 30 Gln Leu Pro His Leu Ala Cys Cys Asp Thr Phe Asn Asn Asn
Ile 35 40 45 Ile Leu Tyr Phe Pro Asp Ala Ile Phe Gly Phe Leu Pro
Ile Ser 50 55 60 Gly Thr Leu Phe Ser Tyr Asp Lys Ile Val Ser Ser
Ile Leu Arg 65 70 75 Val Ser Ser Ser Gly Gly Lys Tyr Lys Ala Phe
Ser Thr Tyr Gly 80 85 90 Ser His 15 97 PRT Homo sapiens
misc_feature Incyte ID No 7481257CP1 15 Met Glu Val Thr Thr Phe Ala
Met Cys Leu Ile Ile Val Leu Val 1 5 10 15 Pro Leu Leu Leu Ile Leu
Val Ser Tyr Gly Phe Ile Ala Val Ala 20 25 30 Val Leu Lys Ile Lys
Ser Ala Ala Gly Arg Gln Lys Ala Phe Gly 35 40 45 Thr Cys Ser Ser
His Leu Val Val Val Ser Ile Phe Cys Gly Thr 50 55 60 Val Thr Tyr
Met Tyr Ile Gln Pro Gly Asn Ser Pro Asn Gln Asn 65 70 75 Glu Gly
Lys Leu Leu Ser Ile Phe Tyr Ser Ile Val Thr Pro Ser 80 85 90 Leu
Asn Pro Leu Ile Tyr Thr 95 16 133 PRT Homo sapiens misc_feature
Incyte ID No 7485790CP1 16 Asp Pro Glu Leu Gln Pro Ile Leu Ala Gly
Leu Ser Leu Ser Met 1 5 10 15 Tyr Leu Val Thr Val Leu Arg Asn Leu
Leu Ile Ser Leu Ala Val 20 25 30 Ser Ser Asp Ser His Leu His Thr
Pro Met Cys Phe Phe Leu Ser 35 40 45 Asn Leu Cys Trp Ala Asp Ile
Gly Phe Thr Ser Ala Thr Val Pro 50 55 60 Lys Met Ile Val Asp Met
Arg Ser His Ser Gly Val Ile Ser Tyr 65 70 75 Ala Asp Cys Leu Thr
Arg Met Ser Phe Leu Val Leu Phe Ala Cys 80 85 90 Val Glu Asp Met
Leu Leu Thr Val Met Ala Tyr Asp Cys Phe Val 95 100 105 Ala Ile Cys
Arg Pro Leu His Tyr Pro Val Ile Val Asn Pro His 110 115 120 Leu Cys
Val Phe Leu Val Ser Val Ser Phe Ser Leu Ala 125 130 17 213 PRT Homo
sapiens misc_feature Incyte ID No 7482993CP1 17 Gly Ser Glu Cys Leu
Leu Leu Ala Ala Met Ala Tyr Asp Arg Tyr 1 5 10 15 Ile Ala Ile Cys
Asn Pro Leu Arg Tyr Ser Val Ile Leu Ser Lys 20 25 30 Val Leu Cys
Asn Gln Leu Ala Ala Ser Cys Trp Ala Ala Gly Phe 35 40 45 Leu Asn
Ser Val Val His Thr Val Leu Thr Phe Cys Leu Pro Phe 50 55 60 Cys
Gly Asn Asn Gln Ile Asn Tyr Phe Phe Cys Asp Ile Pro Pro 65 70 75
Leu Leu Ile Leu Ser Cys Gly Asn Thr Ser Val Asn Glu Leu Ala 80 85
90 Leu Leu Ser Thr Gly Val Phe Ile Gly Trp Thr Pro Phe Leu Cys 95
100 105 Ile Val Leu Ser Tyr Ile Cys Ile Ile Ser Thr Ile Leu Arg Ile
110 115 120 Gln Ser Ser Glu Gly Arg Arg Lys Ala Phe Ser Thr Cys Ala
Ser 125 130 135 His Leu Ala Ile Val Phe Leu Phe Tyr Gly Ser Ala Ile
Phe Thr 140 145 150 Tyr Val Arg Pro Ile Ser Thr Tyr Ser Leu Lys Lys
Asp Arg Leu 155 160 165 Val Ser Val Leu Tyr Ser Val Val Thr Pro Met
Leu Asn Pro Ile 170 175 180 Ile Tyr Thr Leu Arg Asn Lys Asp Ile Lys
Glu Ala Val Lys Thr 185 190 195 Ile Gly Ser Lys Trp Gln Pro Pro Ile
Ser Ser Leu Asp Ser Lys 200 205 210 Leu Thr Tyr 18 180 PRT Homo
sapiens misc_feature Incyte ID No 2829053CD1 18 Met Ser Glu Ala Ala
Thr Arg Trp Ser Cys Gln Gly Ser Cys Gln 1 5 10 15 Lys Thr Cys Phe
Ser Arg Val Arg Pro Trp Arg Arg Arg Cys Ser 20 25 30 Cys Gly Asp
Ser Ser Ser Arg Arg Arg Arg Ser Cys Cys Thr Gly 35 40 45 Ser Leu
Gly Pro Met Pro Arg Leu Pro Ser Leu Trp Pro Leu Ser 50 55 60 Leu
Pro Leu Arg Ser Leu Ser Ser Pro His Arg Val Gln Gly Leu 65 70 75
Gly Pro Pro Arg Arg Leu Lys Ser Gln Leu Leu Pro Arg Phe Phe 80 85
90 Trp Arg Arg Gln Gln Glu Pro Leu Ser Ser Phe Pro Gly Arg Asn 95
100 105 Glu Gly Gly Ser Glu Met Glu Ile Leu Gly Val Cys Pro Val Ser
110 115 120 Pro Gly Ala Leu Ser Tyr Met Glu Ser Pro Thr Gly Phe Trp
Arg 125 130 135 Pro Arg Glu Ala Ser Ser Leu Glu Leu Ala Lys Gly Ile
Ser Lys 140 145 150 Arg Arg His Phe Leu Pro Ala Pro Ala Leu Cys Pro
Asn Pro Arg 155 160 165 Ser Ser Glu Ala Phe Pro Gly Ala Val Cys Val
Thr Leu Ala Ile 170 175 180 19 353 PRT Homo sapiens misc_feature
Incyte ID No 3068234CD1 19 Met Asn Glu Cys His Tyr Asp Lys His Met
Asp Phe Phe Tyr Asn 1 5 10 15 Arg Ser Asn Thr Asp Thr Val Asp Asp
Trp Thr Gly Thr Lys Leu 20 25 30 Val Ile Val Leu Cys Val Gly Thr
Phe Phe Cys Leu Phe Ile Phe 35 40 45 Phe Ser Asn Ser Leu Val Ile
Ala Ala Val Ile Lys Asn Arg Lys 50 55 60 Phe His Phe Pro Phe Tyr
Tyr Leu Leu Ala Asn Leu Ala Ala Ala 65 70 75 Asp Phe Phe Ala Gly
Ile Ala Tyr Val Phe Leu Met Phe Asn Thr 80 85 90 Gly Pro Val Ser
Lys Thr Leu Thr Val Asn Arg Trp Phe Leu Arg 95 100 105 Gln Gly Leu
Leu Asp Ser Ser Leu Thr Ala Ser Leu Thr Asn Leu 110 115 120 Leu Val
Ile Ala Val Glu Arg His Met Ser Ile Met Arg Met Arg 125 130 135 Val
His Ser Asn Leu Thr Lys Lys Arg Val Thr Leu Leu Ile Leu 140 145 150
Leu Val Trp Ala Ile Ala Ile Phe Met Gly Ala Val Pro Thr Leu 155 160
165 Gly Trp Asn Cys Leu Cys Asn Ile Ser Ala Cys Ser Ser Leu Ala 170
175 180 Pro Ile Tyr Ser Arg Ser Tyr Leu Val Phe Trp Thr Val Ser Asn
185 190 195 Leu Met Ala Phe Leu Ile Met Val Val Val Tyr Leu Arg Ile
Tyr 200 205 210 Val Tyr Val Lys Arg Lys Thr Asn Val Leu Ser Pro His
Thr Ser 215 220 225 Gly Ser Ile Ser Arg Arg Arg Thr Pro Met Lys Leu
Met Lys Thr 230 235 240 Val Met Thr Val Leu Gly Ala Phe Val Val Cys
Trp Thr Pro Gly 245 250 255 Leu Val Val Leu Leu Leu Asp Gly Leu Asn
Cys Arg Gln Cys Gly 260 265 270 Val Gln His Val Lys Arg Trp Phe Leu
Leu Leu Ala Leu Leu Asn 275 280 285 Ser Val Val Asn Pro Ile Ile Tyr
Ser Tyr Lys Asp Glu Asp Met 290 295 300 Tyr Gly Thr Met Lys Lys Met
Ile Cys Cys Phe Ser Gln Glu Asn 305 310 315 Pro Glu Arg Arg Pro Ser
Arg Ile Pro Ser Thr Val Leu Ser Arg 320 325 330 Ser Asp Thr Gly Ser
Gln Tyr Ile Glu Asp Ser Ile Ser Gln Gly 335 340 345 Ala Val Cys Asn
Lys Ser Thr Ser 350 20 361 PRT Homo sapiens misc_feature Incyte ID
No 5029478CD1 20 Met Ser Pro Glu Cys Ala Arg Ala Ala Gly Asp Ala
Pro Leu Arg 1 5 10 15 Ser Leu Glu Gln Ala Asn Arg Thr Arg Phe Pro
Phe Phe Ser Asp 20 25 30 Val Lys Gly Asp His Arg Leu Val Leu Ala
Ala Val Glu Thr Thr 35 40 45 Val Leu Val Leu Ile Phe Ala Val Ser
Leu Leu Gly Asn Val Cys 50 55 60 Ala Leu Val Leu Val Ala Arg Arg
Arg Arg Arg Gly Ala Thr Ala 65 70 75 Cys Leu Val Leu Asn Leu Phe
Cys Ala Asp Leu Leu Phe Ile Ser 80 85 90 Ala Ile Pro Leu Val Leu
Ala Val Arg Trp Thr Glu Ala Trp Leu 95 100 105 Leu Gly Pro Val Ala
Cys His Leu Leu Phe Tyr Val Met Thr Leu 110 115 120 Ser Gly Ser Val
Thr Ile Leu Thr Leu Ala Ala Val Ser Leu Glu 125 130 135 Arg Met Val
Cys Ile Val His Leu Gln Arg Gly Val Arg Gly Pro 140 145 150 Gly Arg
Arg Ala Arg Ala Val Leu Leu Ala Leu Ile Trp Gly Tyr 155 160 165 Ser
Ala Val Ala Ala Leu Pro Leu Cys Val Phe Phe Arg Val Val 170 175 180
Pro Gln Arg Leu Pro Gly Ala Asp Gln Glu Ile Ser Ile Cys Thr 185 190
195 Leu Ile Trp Pro Thr Ile Pro Gly Glu Ile Ser Trp Asp Val Ser 200
205 210 Phe Val Thr Leu Asn Phe Leu Val Pro Gly Leu Val Ile Val Ile
215 220 225 Ser Tyr Ser Lys Ile Leu Gln Ile Thr Lys Ala Ser Arg Lys
Arg 230 235 240 Leu Thr Val Ser Leu Ala Tyr Ser Glu Ser His Gln Ile
Arg Val 245 250 255 Ser Gln Gln Asp Phe Arg Leu Phe Arg Thr Leu Phe
Leu Leu Met 260 265 270 Val Ser Phe Phe Ile Met Trp Ser Pro Ile Ile
Ile Thr Ile Leu 275 280 285 Leu Ile Leu Ile Gln Asn Phe Lys Gln Asp
Leu Val Ile Trp Pro 290 295 300 Ser Leu Phe Phe Trp Val Val Ala Phe
Thr Phe Ala Asn Ser Ala 305 310 315 Leu Asn Pro Ile Leu Tyr Asn Met
Thr Leu Cys Arg Asn Glu Trp 320 325 330 Lys Lys Ile Phe Cys Cys Phe
Trp Phe Pro Glu Lys Gly Ala Ile 335 340 345 Leu Thr Asp Thr Ser Val
Lys Arg Asn Asp Leu Ser Ile Ile Ser 350 355 360 Gly 21 251 PRT Homo
sapiens misc_feature Incyte ID No 5102576CD1 21 Met Tyr Leu Val Thr
Val Leu Arg Asn Leu Phe Ser Ile Leu Ala 1 5 10 15 Val Ser Ser Asp
Cys Pro Leu His Thr Pro Met Tyr Phe Phe Leu 20 25 30 Ser Asn Leu
Cys Trp Pro Asp Ile Gly Phe Thr Ser Ala Met Val 35 40 45 Pro Lys
Met Ile Val Asp Thr Gln Ser His Ser Arg Val Ile Ser 50 55 60 His
Ala Gly Cys Leu Thr Gln Met Ser Phe Leu Leu Leu Val Ala 65 70 75
Cys Ile Glu Gly Met Leu Leu Thr Val Met Ala Tyr Asp Cys Phe 80 85
90 Val Ala Ile Cys Arg Pro Leu His Tyr Pro Val Ile Val Asn Pro 95
100 105 His Leu Cys Val Phe Phe Val Leu Val Ser Phe Phe Leu Ser Leu
110 115 120 Leu Asp Ser Gln Leu His Ser Trp Ile Val Leu Gln Leu Thr
Ile 125 130 135 Ile Lys Asn Val Glu Ile Ser Asn Leu Val Cys Asp Pro
Ser Gln 140 145 150 Leu Leu Asn Leu Ala Cys Ser Asp Ser Val Ile Asn
Asn Ile Phe 155 160 165 Ile Tyr Phe Asp Ser Thr Met Phe Gly Phe Leu
Pro Ile Ser Gly 170 175 180 Ile Phe Leu Ser Tyr Tyr Lys Ile Val Pro
Ser Ile Leu Arg Ile 185 190 195 Ser Ser Ser Asp Gly Lys Tyr Lys Ala
Phe Ser Thr Cys Gly Cys 200 205 210 His Leu Ala Val Val Cys Trp Phe
Tyr Gly Thr Gly Ile Gly Met 215 220 225 Tyr Leu Thr Ser Ala Val Ser
Pro Pro Pro Arg Asn Gly Val Val 230 235 240 Ala Ser Val Met Tyr Ala
Val Val Thr Pro Cys 245 250 22 315 PRT Homo sapiens misc_feature
Incyte ID No 2200534CD1 22 Met Lys Ala Asn Tyr Ser Ala Glu Glu Arg
Phe Leu Leu Leu Gly 1 5 10 15 Phe Ser Asp Trp Pro Ser Leu Gln Pro
Val Leu Phe Ala Leu Val 20 25 30 Leu Leu Cys Tyr Leu Leu Thr Leu
Thr Gly Asn Ser Ala Leu Val 35 40 45 Leu Leu Ala Val Arg Asp Pro
Arg Leu His Thr Pro Met Tyr Tyr 50 55 60 Phe Leu Cys His Leu Ala
Leu Val Asp Ala Gly Phe Thr Thr Ser 65 70 75 Val Val Pro Pro Leu
Leu Ala Asn Leu Arg Gly Pro Ala Leu Trp 80 85 90 Leu Pro Arg Ser
His Cys Thr Ala Gln Leu Cys Ala Ser Leu Ala 95 100 105 Leu Gly Ser
Ala Glu Cys Val Leu Leu Ala Val Met Ala Leu Asp 110 115 120 Arg Ala
Ala Ala Val Cys Arg Pro Leu Arg Tyr Ala Gly Leu Val 125 130 135 Ser
Pro Arg Leu Cys Arg Thr Leu Ala Ser Ala Ser Trp Leu Ser 140 145 150
Gly Leu Thr Asn Ser Val Ala Gln Thr Ala Leu Leu Ala Glu Arg 155 160
165 Pro Leu Cys Ala Pro Arg Leu Leu Asp His Phe Ile Cys Glu Leu 170
175 180 Pro Ala Leu Leu Lys Leu Ala Cys Gly Gly Asp Gly Asp Thr Thr
185 190 195 Glu Asn Gln Met Phe Ala Ala Arg Val Val Ile Leu Leu Leu
Pro 200 205 210 Phe Ala Val Ile Leu Ala Ser Tyr Gly Ala Val Ala Arg
Ala Val 215 220 225 Cys Cys Met Arg Phe Ser Gly Gly Arg Arg Arg Ala
Val Gly Thr 230 235 240 Cys Gly Ser His Leu Thr Ala Val Cys Leu Phe
Tyr Gly Ser Ala 245 250 255 Ile Tyr Thr Tyr Leu Gln Pro Ala Gln Arg
Tyr Asn Gln Ala Arg 260 265 270 Gly Lys Phe Val Ser Leu Phe Tyr Thr
Val Val Thr Pro Ala Leu 275 280 285 Asn Pro Leu Ile Tyr Thr Leu Arg
Asn Lys Lys Val Lys Gly Ala 290 295 300 Ala Arg Arg Leu Leu Arg Ser
Leu Gly Arg Gly Gln Ala Gly Gln 305 310 315 23 470 PRT Homo sapiens
misc_feature Incyte ID No 3275821CD1 23 Met Asp Thr Thr Met Glu Ala
Asp Leu Gly Ala Thr Gly His Arg 1 5 10 15 Pro Arg Thr Glu Leu Asp
Asp Glu Asp Ser Tyr Pro Gln Gly Gly 20 25 30 Trp Asp Thr Val Phe
Leu Val Ala Leu Leu Leu Leu Gly Leu Pro 35 40 45 Ala Asn Gly Leu
Met Ala Trp Leu Ala Gly Ser Gln Ala Arg His 50 55 60 Gly Ala Gly
Thr Arg Leu Ala Leu Leu Leu Leu Ser Leu Ala Leu 65 70 75 Ser Asp
Phe Leu Phe Leu Ala Ala Ala Ala Phe Gln Ile Leu Glu 80 85 90 Ile
Arg His Gly Gly His Trp Pro Leu Gly Thr Ala Ala Cys Arg 95 100 105
Phe Tyr Tyr Phe Leu Trp Gly Val Ser Tyr Ser Ser Gly Leu Phe 110 115
120 Leu Leu Ala Ala Leu Ser Leu Asp Arg Cys Leu Leu Ala Leu Cys 125
130 135 Pro His Trp Tyr Pro Gly His Arg Pro Val Arg Leu Pro Leu Trp
140 145 150 Val Cys Ala Gly Val Trp Val Leu Ala Thr Leu Phe Ser Val
Pro 155 160 165 Trp Leu Val Phe Pro Glu Ala Ala Val Trp Trp Tyr Asp
Leu Val 170 175 180 Ile Cys Leu Asp Phe Trp Asp Ser Glu Glu Leu Ser
Leu Arg Met 185 190 195 Leu Glu Val Leu Gly Gly Phe Leu Pro Phe Leu
Leu Leu Leu Val 200 205 210 Cys His Val Leu Thr Gln Ala Thr Ala Cys
Arg Thr Cys His Arg 215 220 225 Gln Gln Gln Pro Ala Ala Cys Arg Gly
Phe Ala Arg Val Ala Arg 230 235 240 Thr Ile Leu Ser Ala Tyr Val Val
Leu Arg Leu Pro Tyr Gln Leu
245 250 255 Ala Gln Leu Leu Tyr Leu Ala Phe Leu Trp Asp Val Tyr Ser
Gly 260 265 270 Tyr Leu Leu Trp Glu Ala Leu Val Tyr Ser Asp Tyr Leu
Ile Leu 275 280 285 Leu Asn Ser Cys Leu Ser Pro Phe Leu Cys Leu Met
Ala Ser Ala 290 295 300 Asp Leu Arg Thr Leu Leu Arg Ser Val Leu Ser
Ser Phe Ala Ala 305 310 315 Ala Leu Cys Glu Glu Arg Pro Gly Ser Phe
Thr Pro Thr Glu Pro 320 325 330 Gln Thr Gln Leu Asp Ser Glu Gly Pro
Thr Leu Pro Glu Pro Met 335 340 345 Ala Glu Ala Gln Ser Gln Met Asp
Pro Val Ala Gln Pro Gln Val 350 355 360 Asn Pro Thr Leu Gln Pro Arg
Ser Asp Pro Thr Ala Gln Pro Gln 365 370 375 Leu Asn Pro Thr Ala Gln
Pro Gln Ser Asp Pro Thr Ala Gln Pro 380 385 390 Gln Leu Asn Leu Met
Ala Gln Pro Gln Ser Asp Ser Val Ala Gln 395 400 405 Pro Gln Ala Asp
Thr Asn Val Gln Thr Pro Ala Pro Ala Ala Ser 410 415 420 Ser Val Pro
Ser Pro Cys Asp Glu Ala Ser Pro Thr Pro Ser Ser 425 430 435 His Pro
Thr Pro Gly Ala Leu Glu Asp Pro Ala Thr Pro Pro Ala 440 445 450 Ser
Glu Gly Glu Ser Pro Ser Ser Thr Pro Pro Glu Ala Ala Pro 455 460 465
Gly Ala Gly Pro Thr 470 24 358 PRT Homo sapiens misc_feature Incyte
ID No 3744167CD1 24 Met Ser Val Cys Tyr Arg Pro Pro Gly Asn Glu Thr
Leu Leu Ser 1 5 10 15 Trp Lys Thr Ser Arg Ala Thr Gly Thr Ala Phe
Leu Leu Leu Ala 20 25 30 Ala Leu Leu Gly Leu Pro Gly Asn Gly Phe
Val Val Trp Ser Leu 35 40 45 Ala Gly Trp Arg Pro Ala Arg Gly Arg
Pro Leu Ala Ala Thr Leu 50 55 60 Val Leu His Leu Ala Leu Ala Asp
Gly Ala Val Leu Leu Leu Thr 65 70 75 Pro Leu Phe Val Ala Phe Leu
Thr Arg Gln Ala Trp Pro Leu Gly 80 85 90 Gln Ala Gly Cys Lys Ala
Val Tyr Tyr Val Cys Ala Leu Ser Met 95 100 105 Tyr Ala Ser Val Leu
Leu Thr Gly Leu Leu Ser Leu Gln Arg Cys 110 115 120 Leu Ala Val Thr
Arg Pro Phe Leu Ala Pro Arg Leu Arg Ser Pro 125 130 135 Ala Leu Ala
Arg Arg Leu Leu Leu Ala Val Trp Leu Ala Ala Leu 140 145 150 Leu Leu
Ala Val Pro Ala Ala Val Tyr Arg His Leu Trp Arg Asp 155 160 165 Arg
Val Cys Gln Leu Cys His Pro Ser Pro Val His Ala Ala Ala 170 175 180
His Leu Ser Leu Glu Thr Leu Thr Ala Phe Val Leu Pro Phe Gly 185 190
195 Leu Met Leu Gly Cys Tyr Ser Val Thr Leu Ala Arg Leu Arg Gly 200
205 210 Ala Arg Trp Gly Ser Gly Arg His Gly Ala Arg Val Gly Arg Leu
215 220 225 Val Ser Ala Ile Val Leu Ala Phe Gly Leu Leu Trp Ala Pro
Tyr 230 235 240 His Ala Val Asn Leu Leu Gln Ala Val Ala Ala Leu Ala
Pro Pro 245 250 255 Glu Gly Ala Leu Ala Lys Leu Gly Gly Ala Gly Gln
Ala Ala Arg 260 265 270 Ala Gly Thr Thr Ala Leu Ala Phe Phe Ser Ser
Ser Val Asn Pro 275 280 285 Val Leu Tyr Val Phe Thr Ala Gly Asp Leu
Leu Pro Arg Ala Gly 290 295 300 Pro Arg Phe Leu Thr Arg Leu Phe Glu
Gly Ser Gly Glu Ala Arg 305 310 315 Gly Gly Gly Arg Ser Arg Glu Gly
Thr Met Glu Leu Arg Thr Thr 320 325 330 Pro Gln Leu Lys Val Val Gly
Gln Gly Arg Gly Asn Gly Asp Pro 335 340 345 Gly Gly Gly Met Glu Lys
Asp Gly Pro Glu Trp Asp Leu 350 355 25 314 PRT Homo sapiens
misc_feature Incyte ID No 7472007CD1 25 Met Trp Glu Asn Trp Thr Ile
Val Ser Glu Phe Val Leu Val Ser 1 5 10 15 Phe Ser Ala Leu Ser Thr
Glu Leu Gln Ala Leu Leu Phe Leu Leu 20 25 30 Phe Leu Thr Ile Tyr
Leu Val Thr Leu Met Gly Asn Val Leu Ile 35 40 45 Ile Leu Val Thr
Ile Ala Asp Ser Ala Leu Gln Ser Pro Met Tyr 50 55 60 Phe Phe Leu
Arg Asn Leu Ser Phe Leu Glu Ile Gly Phe Asn Leu 65 70 75 Val Ile
Val Pro Lys Met Leu Gly Thr Leu Ile Ile Gln Asp Thr 80 85 90 Thr
Ile Ser Phe Leu Gly Cys Ala Thr Gln Met Tyr Phe Phe Phe 95 100 105
Phe Phe Gly Ala Ala Glu Cys Cys Leu Leu Ala Thr Met Ala Tyr 110 115
120 Asp Arg Tyr Val Ala Ile Cys Asp Pro Leu His Tyr Pro Val Ile 125
130 135 Met Gly His Ile Ser Cys Ala Gln Leu Ala Ala Ala Ser Trp Phe
140 145 150 Ser Gly Phe Ser Val Ala Thr Val Gln Thr Thr Trp Ile Phe
Ser 155 160 165 Phe Pro Phe Cys Gly Pro Asn Arg Val Asn His Phe Phe
Cys Asp 170 175 180 Ser Pro Pro Val Ile Ala Leu Val Cys Ala Asp Thr
Ser Val Phe 185 190 195 Glu Leu Glu Ala Leu Thr Ala Thr Val Pro Phe
Ile Leu Phe Pro 200 205 210 Phe Leu Leu Ile Leu Gly Ser Tyr Val Arg
Ile Leu Ser Thr Ile 215 220 225 Phe Arg Met Pro Ser Ala Glu Gly Lys
His Gln Ala Phe Ser Thr 230 235 240 Cys Ser Ala His Leu Leu Val Val
Ser Leu Phe Tyr Ser Thr Ala 245 250 255 Ile Leu Thr Tyr Phe Arg Pro
Gln Ser Ser Ala Ser Ser Glu Ser 260 265 270 Lys Lys Leu Leu Ser Leu
Ser Ser Thr Val Val Thr Pro Met Leu 275 280 285 Asn Pro Ile Ile Tyr
Ser Ser Arg Asn Lys Glu Val Lys Ala Ala 290 295 300 Leu Lys Arg Leu
Ile His Arg Thr Leu Gly Ser Gln Lys Leu 305 310 26 365 PRT Homo
sapiens misc_feature Incyte ID No 7472008CD1 26 Met Glu Gly Ser Val
Glu Ala Thr Pro Glu Ile Pro Ala Gln Met 1 5 10 15 Lys Cys His Pro
Ser Arg Pro Ser Thr Leu Asn Gln Leu Ser Phe 20 25 30 Tyr Gly Ala
Val Ser Ser Leu Gly Arg Met His Gly Leu Glu Thr 35 40 45 Lys Ser
Ser Ala Glu Ile Arg Ala Gly Leu Lys Arg Cys Asp Thr 50 55 60 Leu
Val Leu Glu Ala Ser Thr Leu Glu Gly Asn Met Val Ile Val 65 70 75
Leu Val Ser Leu Lys Asp Pro Lys Leu His Ile Pro Met Tyr Phe 80 85
90 Phe Leu Ser Asn Leu Ser Leu Val Asp Leu Cys Leu Thr Ser Ser 95
100 105 Cys Val Pro Gln Met Leu Ile Asn Phe Trp Gly Pro Glu Lys Thr
110 115 120 Ile Ser Tyr Ile Gly Cys Ala Ile Gln Leu Tyr Val Phe Leu
Trp 125 130 135 Leu Gly Ala Thr Glu Tyr Val Leu Leu Val Val Met Ala
Val Asp 140 145 150 Cys Tyr Val Ala Val Cys His Pro Leu Gln Asn Thr
Met Ile Met 155 160 165 His Pro Lys Leu Cys Leu Gln Leu Ala Ile Leu
Ala Trp Gly Thr 170 175 180 Gly Leu Ala Gln Ser Leu Ile Gln Ser Pro
Ala Thr Leu Arg Leu 185 190 195 Pro Phe Cys Ser Gln Arg Met Val Asp
Asp Val Val Cys Glu Val 200 205 210 Pro Ala Leu Ile Gln Leu Ser Ser
Thr Asp Thr Thr Tyr Ser Glu 215 220 225 Ile Gln Met Ser Ile Ala Ser
Val Val Leu Leu Val Met Pro Leu 230 235 240 Ile Ile Ile Leu Ser Ser
Ser Gly Ala Ile Ala Lys Ala Val Leu 245 250 255 Arg Ile Lys Ser Thr
Ala Gly Gln Lys Lys Ala Phe Gly Thr Cys 260 265 270 Ile Ser His Leu
Leu Val Val Ser Leu Phe Tyr Gly Thr Val Thr 275 280 285 Gly Val Tyr
Leu Gln Pro Lys Asn His Tyr Pro His Glu Trp Gly 290 295 300 Lys Phe
Leu Thr Leu Phe Tyr Thr Val Val Thr Pro Thr Leu Asn 305 310 315 Pro
Leu Ile Tyr Thr Leu Arg Asn Lys Glu Leu His Pro Trp Leu 320 325 330
Lys Glu Ala Lys Val Gln Thr Ala Ser Glu Ser Ala Ser Pro Lys 335 340
345 His Trp Gln Leu Pro His Gly Val Gly Pro Val Gly Val Gln Lys 350
355 360 Thr Arg Thr Glu Leu 365 27 317 PRT Homo sapiens
misc_feature Incyte ID No 7472013CD1 27 Met Ser Phe Ala Pro Asn Ala
Ser His Ser Pro Val Phe Leu Leu 1 5 10 15 Leu Gly Phe Ser Arg Ala
Asn Ile Ser Tyr Thr Leu Leu Phe Phe 20 25 30 Leu Phe Leu Ala Ile
Tyr Leu Thr Thr Ile Leu Gly Asn Val Thr 35 40 45 Leu Val Leu Leu
Ile Ser Trp Asp Ser Arg Leu His Ser Pro Met 50 55 60 Tyr Tyr Leu
Leu Arg Gly Leu Ser Val Ile Asp Met Gly Leu Ser 65 70 75 Thr Val
Thr Leu Pro Gln Leu Leu Ala His Leu Val Ser His Tyr 80 85 90 Pro
Thr Ile Pro Ala Ala Arg Cys Leu Ala Gln Phe Phe Phe Phe 95 100 105
Tyr Ala Phe Gly Val Thr Asp Thr Leu Val Ile Ala Val Met Ala 110 115
120 Leu Asp Arg Tyr Val Ala Ile Cys Asp Pro Leu His Tyr Ala Leu 125
130 135 Val Met Asn His Gln Arg Cys Ala Cys Leu Leu Ala Leu Ser Trp
140 145 150 Val Val Ser Ile Leu His Thr Met Leu Arg Val Gly Leu Val
Leu 155 160 165 Pro Leu Cys Trp Thr Gly Asp Ala Gly Gly Asn Val Asn
Leu Pro 170 175 180 His Phe Phe Cys Asp His Arg Pro Leu Leu Arg Ala
Ser Cys Ser 185 190 195 Asp Ile His Ser Asn Glu Leu Ala Ile Phe Phe
Glu Gly Gly Phe 200 205 210 Leu Met Leu Gly Pro Cys Ala Leu Ile Val
Leu Ser Tyr Val Arg 215 220 225 Ile Gly Ala Ala Ile Leu Arg Leu Pro
Ser Ala Ala Gly Arg Arg 230 235 240 Arg Ala Val Ser Thr Cys Gly Ser
His Leu Thr Met Val Gly Phe 245 250 255 Leu Tyr Gly Thr Ile Ile Cys
Val Tyr Phe Gln Pro Pro Phe Gln 260 265 270 Asn Ser Gln Tyr Gln Asp
Met Val Ala Ser Val Met Tyr Thr Ala 275 280 285 Ile Thr Pro Leu Ala
Asn Pro Phe Val Tyr Ser Leu His Asn Lys 290 295 300 Asp Val Lys Gly
Ala Leu Cys Arg Leu Leu Glu Trp Val Lys Val 305 310 315 Asp Pro 28
335 PRT Homo sapiens misc_feature Incyte ID No 7472015CD1 28 Met
Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val Leu Ala 1 5 10 15
Ser Leu Ile Ile Ala Thr Asn Thr Leu Val Ala Val Ala Val Leu 20 25
30 Leu Leu Ile His Lys Asn Asp Gly Val Ser Leu Cys Phe Thr Leu 35
40 45 Asn Leu Ala Val Ala Asp Thr Leu Ile Gly Val Ala Ile Ser Gly
50 55 60 Leu Leu Thr Asp Gln Leu Ser Ser Pro Ser Arg Pro Thr Gln
Lys 65 70 75 Thr Leu Cys Ser Leu Arg Met Ala Phe Val Thr Ser Ser
Ala Ala 80 85 90 Ala Ser Val Leu Thr Val Met Leu Ile Thr Phe Asp
Arg Tyr Leu 95 100 105 Ala Ile Lys Gln Pro Phe Arg Tyr Leu Lys Ile
Met Ser Gly Phe 110 115 120 Val Ala Gly Ala Cys Ile Ala Gly Leu Trp
Leu Val Ser Tyr Leu 125 130 135 Ile Gly Phe Leu Pro Leu Gly Ile Pro
Met Phe Gln Gln Thr Ala 140 145 150 Tyr Lys Gly Gln Cys Ser Phe Phe
Ala Val Phe His Pro His Phe 155 160 165 Val Leu Thr Leu Ser Cys Val
Gly Phe Phe Pro Ala Met Leu Leu 170 175 180 Phe Val Phe Phe Tyr Cys
Asp Met Leu Lys Ile Ala Ser Met His 185 190 195 Ser Gln Gln Ile Arg
Lys Met Glu His Ala Gly Ala Met Ala Gly 200 205 210 Gly Tyr Arg Ser
Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu Arg 215 220 225 Thr Val Ser
Val Leu Ile Gly Ser Phe Ala Leu Ser Trp Thr Pro 230 235 240 Phe Leu
Ile Thr Gly Ile Val Gln Val Ala Cys Gln Glu Cys His 245 250 255 Leu
Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly 260 265 270
Asn Ser Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Lys Glu 275 280
285 Val Arg Leu Gln Leu Tyr His Met Ala Leu Gly Val Lys Lys Val 290
295 300 Leu Thr Ser Phe Leu Leu Phe Leu Ser Ala Arg Asn Cys Gly Pro
305 310 315 Glu Arg Pro Arg Glu Ser Ser Cys His Ile Val Thr Ile Ser
Ser 320 325 330 Ser Glu Phe Asp Gly 335 29 309 PRT Homo sapiens
misc_feature Incyte ID No 7472016CD1 29 Met Arg Glu Asn Asn Gln Ser
Ser Thr Leu Glu Phe Ile Leu Leu 1 5 10 15 Gly Val Thr Gly Gln Gln
Glu Gln Glu Asp Phe Phe Tyr Ile Leu 20 25 30 Phe Leu Phe Ile Tyr
Pro Ile Thr Leu Ile Gly Asn Leu Leu Ile 35 40 45 Val Leu Ala Ile
Cys Ser Asp Val Arg Leu His Asn Pro Met Tyr 50 55 60 Phe Leu Leu
Ala Asn Leu Ser Leu Val Asp Ile Phe Phe Ser Ser 65 70 75 Val Thr
Ile Pro Lys Met Leu Ala Asn His Leu Leu Gly Ser Lys 80 85 90 Ser
Ile Ser Phe Gly Gly Cys Leu Thr Gln Met Tyr Phe Met Ile 95 100 105
Ala Leu Gly Asn Thr Asp Ser Tyr Ile Leu Ala Ala Met Ala Tyr 110 115
120 Asp Arg Ala Val Ala Ile Ser His Pro Leu His Tyr Thr Thr Ile 125
130 135 Met Ser Pro Arg Ser Cys Ile Trp Leu Ile Ala Gly Ser Trp Val
140 145 150 Ile Gly Asn Ala Asn Ala Leu Pro His Thr Leu Leu Thr Ala
Ser 155 160 165 Leu Ser Phe Cys Gly Asn Gln Glu Val Ala Asn Phe Tyr
Cys Asp 170 175 180 Ile Thr Pro Leu Leu Lys Leu Ser Cys Ser Asp Ile
His Phe His 185 190 195 Val Lys Met Met Tyr Leu Gly Val Gly Ile Phe
Ser Val Pro Leu 200 205 210 Leu Cys Ile Ile Val Ser Tyr Ile Arg Val
Phe Ser Thr Val Phe 215 220 225 Gln Val Pro Ser Thr Lys Gly Val Leu
Lys Ala Phe Ser Thr Cys 230 235 240 Gly Ser His Leu Thr Val Val Ser
Leu Tyr Tyr Gly Thr Val Met 245 250 255 Gly Thr Tyr Phe Arg Pro Leu
Thr Asn Tyr Ser Leu Lys Asp Ala 260 265 270 Val Ile Thr Val Met Tyr
Thr Ala Val Thr Pro Met Leu Asn Pro 275 280 285 Phe Ile Tyr Ser Leu
Arg Asn Arg Asp Met Lys Ala Ala Leu Arg 290 295 300 Lys Leu Phe Asn
Lys Arg Ile Ser Ser 305 30 236 PRT Homo sapiens misc_feature Incyte
ID No 7472017CD1 30 Met Gly Met Thr Asn Ser Ser Val Lys Gly Asp Phe
Ile Leu Leu 1 5 10 15 Leu Trp Asn Leu Lys Gly Pro Asp Lys Thr Ile
Thr Phe Leu Gly 20 25
30 Cys Val Ile Gln Leu Tyr Ile Ser Leu Ala Leu Gly Ser Thr Glu 35
40 45 Cys Val Leu Leu Ala Val Met Ala Phe Asp Arg Tyr Ala Ala Val
50 55 60 Cys Lys Pro Leu His Tyr Thr Ala Val Met Asn Pro Gln Leu
Cys 65 70 75 Gln Ala Leu Ala Gly Val Ala Trp Leu Ser Gly Val Gly
Asn Thr 80 85 90 Leu Ile Gln Gly Thr Val Thr Leu Trp Leu Pro Arg
Cys Gly His 95 100 105 Arg Leu Leu Gln His Phe Phe Leu Ala Cys Val
Asp Ile His Asp 110 115 120 Asn Glu Val Gln Leu Phe Val Ala Ser Leu
Val Leu Leu Leu Leu 125 130 135 Pro Leu Val Leu Ile Leu Leu Ser Tyr
Gly His Ile Ala Lys Val 140 145 150 Val Ile Arg Ile Lys Ser Val Gln
Ala Trp Cys Lys Gly Leu Gly 155 160 165 Thr Cys Gly Ser His Leu Ile
Val Val Ser Leu Phe Cys Gly Thr 170 175 180 Ile Thr Ala Val Tyr Ile
Gln Ser Asn Ser Ser Tyr Ala His Ala 185 190 195 His Gly Lys Phe Ile
Ser Leu Phe Tyr Thr Val Val Thr Pro Thr 200 205 210 Leu Asn Pro Leu
Ile Tyr Thr Leu Arg Asn Asn Asp Val Lys Gly 215 220 225 Ala Leu Arg
Leu Phe Asn Arg Asp Leu Gly Thr 230 235 31 363 PRT Homo sapiens
misc_feature Incyte ID No 7472018CD1 31 Met Gly Pro Gly Glu Ala Leu
Leu Ala Gly Leu Leu Val Met Val 1 5 10 15 Leu Ala Val Ala Leu Leu
Ser Asn Ala Leu Val Leu Leu Cys Cys 20 25 30 Ala Tyr Ser Ala Glu
Leu Arg Thr Arg Ala Ser Gly Val Leu Leu 35 40 45 Val Asn Leu Ser
Leu Gly His Leu Leu Leu Ala Ala Leu Asp Met 50 55 60 Pro Phe Thr
Leu Leu Gly Val Met Arg Gly Arg Thr Pro Ser Ala 65 70 75 Pro Gly
Ala Cys Gln Val Ile Gly Phe Leu Asp Thr Phe Leu Ala 80 85 90 Ser
Asn Ala Ala Leu Ser Val Ala Ala Leu Ser Ala Asp Gln Trp 95 100 105
Leu Ala Val Gly Phe Pro Leu Arg Tyr Ala Gly Arg Leu Arg Pro 110 115
120 Arg Tyr Ala Gly Leu Leu Leu Gly Cys Ala Trp Gly Gln Ser Leu 125
130 135 Ala Phe Ser Gly Ala Ala Leu Gly Cys Ser Trp Leu Gly Tyr Ser
140 145 150 Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu Pro Pro Glu Pro
Glu 155 160 165 Arg Pro Arg Phe Ala Ala Phe Thr Ala Thr Leu His Ala
Val Gly 170 175 180 Phe Val Leu Pro Leu Ala Val Leu Cys Leu Thr Ser
Leu Gln Val 185 190 195 His Arg Val Ala Arg Arg His Cys Gln Arg Met
Asp Thr Val Thr 200 205 210 Met Lys Ala Leu Ala Leu Leu Ala Asp Leu
His Pro Ser Val Arg 215 220 225 Gln Arg Cys Leu Ile Gln Gln Lys Arg
Arg Arg His Arg Ala Thr 230 235 240 Arg Lys Ile Gly Ile Ala Ile Ala
Thr Phe Leu Ile Cys Phe Ala 245 250 255 Pro Tyr Val Met Thr Arg Leu
Ala Glu Leu Val Pro Phe Val Thr 260 265 270 Val Asn Ala Gln Trp Gly
Ile Leu Ser Lys Cys Leu Thr Tyr Ser 275 280 285 Lys Ala Val Ala Asp
Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro 290 295 300 Phe Arg Gln Val
Leu Ala Gly Met Val His Arg Leu Leu Lys Arg 305 310 315 Thr Pro Arg
Pro Ala Ser Thr His Asp Ser Ser Leu Asp Val Ala 320 325 330 Gly Met
Val His Gln Leu Leu Lys Arg Thr Pro Arg Pro Ala Ser 335 340 345 Thr
His Asn Gly Ser Val Asp Thr Glu Asn Asp Ser Cys Leu Gln 350 355 360
Gln Thr His 32 308 PRT Homo sapiens misc_feature Incyte ID No
7472019CD1 32 Met Ala Met Asp Asn Val Thr Ala Val Phe Gln Phe Leu
Leu Ile 1 5 10 15 Gly Ile Ser Asn Tyr Pro Gln Trp Arg Asp Thr Phe
Phe Thr Leu 20 25 30 Val Leu Ile Ile Tyr Leu Ser Thr Leu Leu Gly
Asn Gly Phe Met 35 40 45 Ile Phe Leu Ile His Phe Asp Pro Asn Leu
His Thr Pro Ile Tyr 50 55 60 Phe Phe Leu Ser Asn Leu Ser Phe Leu
Asp Leu Cys Tyr Gly Thr 65 70 75 Ala Ser Met Pro Gln Ala Leu Val
His Cys Phe Ser Thr His Pro 80 85 90 Tyr Leu Ser Tyr Pro Arg Cys
Leu Ala Gln Thr Ser Val Ser Leu 95 100 105 Ala Leu Ala Thr Ala Glu
Cys Leu Leu Leu Ala Ala Met Ala Tyr 110 115 120 Asp Arg Val Val Ala
Ile Ser Asn Pro Leu Arg Tyr Ser Val Val 125 130 135 Met Asn Gly Pro
Val Cys Val Cys Leu Val Ala Thr Ser Trp Gly 140 145 150 Thr Ser Leu
Val Leu Thr Ala Met Leu Ile Leu Ser Leu Arg Leu 155 160 165 His Phe
Cys Gly Ala Asn Val Ile Asn His Phe Ala Cys Glu Ile 170 175 180 Leu
Ser Leu Ile Lys Leu Thr Cys Ser Asp Thr Ser Leu Asn Glu 185 190 195
Phe Met Ile Leu Ile Thr Ser Ile Phe Thr Leu Leu Leu Pro Phe 200 205
210 Gly Phe Val Leu Leu Ser Tyr Ile Arg Ile Ala Met Ala Ile Ile 215
220 225 Arg Ile Arg Ser Leu Gln Gly Arg Leu Lys Ala Phe Thr Thr Cys
230 235 240 Gly Ser His Leu Thr Val Val Thr Ile Phe Tyr Gly Ser Ala
Ile 245 250 255 Ser Met Tyr Met Lys Thr Gln Ser Lys Ser Tyr Pro Asp
Gln Asp 260 265 270 Lys Phe Ile Ser Val Phe Tyr Gly Ala Leu Thr Pro
Met Leu Asn 275 280 285 Pro Leu Ile Tyr Ser Leu Arg Lys Lys Asp Val
Lys Arg Ala Ile 290 295 300 Arg Lys Val Met Leu Lys Arg Thr 305 33
343 PRT Homo sapiens misc_feature Incyte ID No 7472021CD1 33 Met
His Phe Leu Pro Thr Val Phe Gly Phe Leu Asn Arg Val Thr 1 5 10 15
Leu Gly Ile Phe Arg Glu Thr Met Val Asn Leu Thr Ser Met Ser 20 25
30 Gly Phe Leu Leu Met Gly Phe Ser Asp Glu Arg Lys Leu Gln Ile 35
40 45 Leu His Ala Leu Val Phe Leu Val Thr Tyr Leu Leu Ala Leu Thr
50 55 60 Gly Asn Leu Leu Ile Ile Thr Ile Ile Thr Val Asp Arg Arg
Leu 65 70 75 His Ser Pro Met Tyr Tyr Phe Leu Lys His Leu Ser Leu
Leu Asp 80 85 90 Leu Cys Phe Ile Ser Val Thr Val Pro Gln Ser Ile
Ala Asn Ser 95 100 105 Leu Met Gly Asn Gly Tyr Ile Ser Leu Val Gln
Cys Ile Leu Gln 110 115 120 Val Phe Phe Phe Ile Ala Leu Ala Ser Ser
Glu Val Ala Ile Leu 125 130 135 Thr Val Met Ser Tyr Asp Arg Tyr Ala
Ala Ile Cys Gln Pro Leu 140 145 150 His Tyr Glu Thr Ile Met Asp Pro
Arg Ala Cys Arg His Ala Val 155 160 165 Ile Ala Val Trp Ile Ala Gly
Gly Leu Ser Gly Leu Met His Ala 170 175 180 Ala Ile Asn Phe Ser Ile
Pro Leu Cys Gly Lys Arg Val Ile His 185 190 195 Gln Phe Phe Cys Asp
Val Pro Gln Met Leu Lys Leu Ala Cys Ser 200 205 210 Tyr Glu Phe Ile
Asn Glu Ile Ala Leu Ala Ala Phe Thr Thr Ser 215 220 225 Ala Ala Phe
Ile Cys Leu Ile Ser Ile Val Leu Ser Tyr Ile Arg 230 235 240 Ile Phe
Ser Thr Val Leu Arg Ile Pro Ser Ala Glu Gly Arg Thr 245 250 255 Lys
Val Phe Ser Thr Cys Leu Pro His Leu Phe Val Ala Thr Phe 260 265 270
Phe Leu Ser Ala Ala Gly Phe Glu Phe Leu Arg Leu Pro Ser Asp 275 280
285 Ser Ser Ser Thr Val Asp Leu Val Phe Ser Val Phe Tyr Thr Val 290
295 300 Ile Pro Pro Thr Leu Asn Pro Val Ile Tyr Ser Leu Arg Asn Asp
305 310 315 Ser Met Lys Ala Ala Leu Arg Lys Met Leu Ser Lys Glu Glu
Leu 320 325 330 Pro Gln Arg Lys Met Cys Leu Lys Ala Met Phe Lys Leu
335 340 34 323 PRT Homo sapiens misc_feature Incyte ID No
7472009CD1 34 Met Trp Gln Lys Asn Gln Thr Ser Leu Ala Asp Phe Ile
Leu Glu 1 5 10 15 Gly Leu Phe Asp Asp Ser Leu Thr His Leu Phe Leu
Phe Ser Leu 20 25 30 Thr Met Val Val Phe Leu Ile Ala Val Ser Gly
Asn Thr Leu Thr 35 40 45 Ile Leu Leu Ile Cys Ile Asp Pro Gln Leu
His Thr Pro Met Tyr 50 55 60 Phe Leu Leu Ser Gln Leu Ser Leu Met
Asp Leu Met His Val Ser 65 70 75 Thr Thr Ile Leu Lys Met Ala Thr
Asn Tyr Leu Ser Gly Lys Lys 80 85 90 Ser Ile Ser Phe Val Gly Cys
Ala Thr Gln His Phe Leu Tyr Leu 95 100 105 Cys Leu Gly Gly Ala Glu
Cys Phe Leu Leu Ala Val Met Ser Tyr 110 115 120 Asp Arg Tyr Val Ala
Ile Cys His Pro Leu Arg Tyr Ala Val Leu 125 130 135 Met Asn Lys Lys
Val Gly Leu Met Met Ala Val Met Ser Trp Leu 140 145 150 Gly Ala Ser
Val Asn Ser Leu Ile His Met Ala Ile Leu Met His 155 160 165 Phe Pro
Phe Cys Gly Pro Arg Lys Val Tyr His Phe Tyr Cys Glu 170 175 180 Phe
Pro Ala Val Val Lys Leu Val Cys Gly Asp Ile Thr Val Tyr 185 190 195
Glu Thr Thr Val Tyr Ile Ser Ser Ile Leu Leu Leu Leu Pro Ile 200 205
210 Phe Leu Ile Ser Thr Ser Tyr Val Phe Ile Leu Gln Ser Val Ile 215
220 225 Gln Met Arg Ser Ser Gly Ser Lys Arg Asn Ala Phe Ala Thr Cys
230 235 240 Gly Ser His Leu Thr Val Val Ser Leu Trp Phe Gly Ala Cys
Ile 245 250 255 Phe Ser Tyr Met Arg Pro Arg Ser Gln Cys Thr Leu Leu
Gln Asn 260 265 270 Lys Val Gly Ser Val Phe Tyr Ser Ile Ile Thr Pro
Thr Leu Asn 275 280 285 Ser Leu Ile Tyr Thr Leu Arg Asn Lys Asp Val
Ala Lys Ala Leu 290 295 300 Arg Arg Val Leu Arg Arg Asp Val Ile Thr
Gln Cys Ile Gln Arg 305 310 315 Leu Gln Leu Trp Leu Pro Arg Val 320
35 299 PRT Homo sapiens misc_feature Incyte ID No 7472010CD1 35 Met
Glu Leu Glu Gly Asp Phe Leu Gly Ser Val Gly Glu Leu Gly 1 5 10 15
Gln Val Ile Gln Thr Cys Ser Gly Ile Tyr Val Phe Thr Val Val 20 25
30 Gly Asn Leu Gly Leu Ile Thr Leu Ile Gly Ile Asn Pro Ser Leu 35
40 45 His Thr Pro Met Tyr Phe Phe Leu Phe Asn Leu Ser Phe Ile Asp
50 55 60 Leu Cys Tyr Ser Cys Val Phe Thr Pro Lys Met Leu Asn Asp
Phe 65 70 75 Val Ser Glu Ser Ile Ile Ser Tyr Val Gly Cys Met Thr
Gln Leu 80 85 90 Phe Phe Phe Cys Phe Phe Val Asn Ser Glu Cys Tyr
Val Leu Val 95 100 105 Ser Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys
Asn Pro Leu Leu 110 115 120 Tyr Met Val Thr Met Ser Pro Arg Val Cys
Phe Leu Leu Met Phe 125 130 135 Gly Ser Tyr Val Val Gly Phe Ala Gly
Ala Met Ala His Thr Gly 140 145 150 Ser Met Leu Arg Leu Thr Phe Cys
Asp Ser Asn Val Ile Asp His 155 160 165 Tyr Leu Cys Asp Val Leu Pro
Leu Leu Gln Leu Ser Cys Thr Ser 170 175 180 Thr His Val Ser Glu Leu
Val Phe Phe Ile Val Val Gly Val Ile 185 190 195 Thr Met Leu Ser Ser
Ile Ser Ile Val Ile Ser Tyr Ala Leu Ile 200 205 210 Leu Ser Asn Ile
Leu Cys Ile Pro Ser Ala Glu Gly Arg Ser Lys 215 220 225 Ala Phe Ser
Thr Trp Gly Ser His Ile Ile Ala Val Ala Leu Phe 230 235 240 Phe Gly
Ser Gly Thr Phe Thr Tyr Leu Thr Thr Ser Phe Pro Gly 245 250 255 Ser
Met Asn His Gly Arg Phe Ala Ser Val Phe Tyr Thr Asn Val 260 265 270
Val Pro Met Leu Asn Pro Ser Ile Tyr Ser Leu Arg Asn Lys Asp 275 280
285 Asp Lys Leu Ala Leu Gly Lys Thr Leu Lys Arg Val Leu Phe 290 295
36 307 PRT Homo sapiens misc_feature Incyte ID No 7472011CD1 36 Met
Glu Thr Gly Asn Leu Thr Trp Val Ser Asp Phe Val Phe Leu 1 5 10 15
Gly Leu Ser Gln Thr Arg Glu Leu Gln Arg Phe Leu Phe Leu Met 20 25
30 Phe Leu Phe Val Tyr Ile Thr Thr Val Met Gly Asn Ile Leu Ile 35
40 45 Ile Ile Thr Val Thr Ser Asp Ser Gln Leu His Thr Pro Met Tyr
50 55 60 Phe Leu Leu Arg Asn Leu Ala Val Leu Asp Leu Cys Phe Ser
Ser 65 70 75 Val Thr Ala Pro Lys Met Leu Val Asp Leu Leu Ser Glu
Lys Lys 80 85 90 Thr Ile Ser Tyr Gln Gly Cys Met Gly Gln Ile Phe
Phe Phe His 95 100 105 Phe Leu Gly Gly Ala Met Val Phe Phe Leu Ser
Val Met Ala Phe 110 115 120 Asp Arg Leu Ile Ala Ile Ser Arg Pro Leu
Arg Tyr Val Thr Val 125 130 135 Met Asn Thr Gln Leu Trp Val Gly Leu
Val Val Ala Thr Trp Val 140 145 150 Gly Gly Phe Val His Ser Ile Val
Gln Leu Ala Leu Met Leu Pro 155 160 165 Leu Pro Phe Cys Gly Pro Asn
Ile Leu Asp Asn Phe Tyr Cys Asp 170 175 180 Val Pro Gln Val Leu Arg
Leu Ala Cys Thr Asp Thr Ser Leu Leu 185 190 195 Glu Phe Leu Lys Ile
Ser Asn Ser Gly Leu Leu Asp Val Val Trp 200 205 210 Phe Phe Leu Leu
Leu Met Ser Tyr Leu Phe Ile Leu Val Met Leu 215 220 225 Arg Ser His
Pro Gly Glu Ala Arg Arg Lys Ala Ala Ser Thr Cys 230 235 240 Thr Thr
His Ile Ile Val Val Ser Met Ile Phe Val Pro Ser Ile 245 250 255 Tyr
Leu Tyr Ala Arg Pro Phe Thr Pro Phe Pro Met Asp Lys Leu 260 265 270
Val Ser Ile Gly His Thr Val Met Thr Pro Met Leu Asn Pro Met 275 280
285 Ile Tyr Thr Leu Arg Asn Gln Asp Met Gln Ala Ala Val Arg Arg 290
295 300 Leu Gly Arg His Arg Leu Val 305 37 314 PRT Homo sapiens
misc_feature Incyte ID No 7472012CD1 37 Met Asp Asn Ser Asn Trp Thr
Ser Val Ser His Phe Val Leu Leu 1 5 10 15 Gly Ile Ser Thr His Pro
Glu Glu Gln Ile Pro Leu Phe Leu Val 20 25 30 Phe Ser Leu Met Tyr
Ala Ile Asn Ile Ser Gly Asn Leu Ala Ile 35 40 45 Ile Thr Leu Ile
Leu Ser Ala Pro Arg Leu His Ile Pro Met Tyr 50 55 60 Ile Phe Leu
Ser Asn Leu Ala Leu Thr Asp Ile Cys Phe Thr Ser 65 70 75 Thr Thr
Val Pro Lys Met Leu Gln Ile Ile Phe Ser Pro Thr Lys 80 85 90 Val
Ile Ser Tyr Thr Gly Cys Leu Ala Gln Thr Tyr Phe Phe Ile 95 100
105 Cys Phe Ala Val Met Glu Asn Phe Ile Leu Ala Val Met Ala Tyr 110
115 120 Asp Arg Tyr Ile Ala Ile Cys His Pro Phe His Tyr Thr Met Ile
125 130 135 Leu Thr Arg Met Leu Cys Val Lys Met Val Val Met Cys His
Ala 140 145 150 Leu Ser His Leu His Ala Met Leu His Thr Phe Leu Ile
Gly Gln 155 160 165 Leu Ile Phe Cys Ala Asp Asn Arg Ile Pro His Phe
Phe Cys Asp 170 175 180 Leu Tyr Ala Leu Met Lys Ile Ser Cys Thr Ser
Thr Tyr Leu Asn 185 190 195 Thr Leu Met Ile His Thr Glu Gly Ala Val
Val Ile Ser Gly Ala 200 205 210 Leu Ala Phe Ile Thr Ala Ser Tyr Ala
Cys Ile Ile Leu Val Val 215 220 225 Leu Arg Ile Pro Ser Ala Lys Gly
Arg Trp Lys Thr Phe Ser Thr 230 235 240 Cys Gly Ser His Leu Thr Val
Val Ala Ile Phe Tyr Gly Thr Leu 245 250 255 Ser Trp Val Tyr Phe Arg
Pro Leu Ser Ser Tyr Ser Val Thr Lys 260 265 270 Gly Arg Ile Ile Thr
Val Val Tyr Thr Val Val Thr Pro Met Leu 275 280 285 Asn Pro Phe Ile
Tyr Ser Leu Arg Asn Gly Asp Val Lys Gly Gly 290 295 300 Phe Met Lys
Trp Met Ser Arg Met Gln Thr Phe Phe Phe Arg 305 310 38 310 PRT Homo
sapiens misc_feature Incyte ID No 7472014CD1 38 Met Gly Arg Asn Asn
Leu Thr Arg Pro Ser Glu Phe Ile Leu Leu 1 5 10 15 Gly Leu Ser Ser
Arg Pro Glu Asp Gln Lys Pro Leu Phe Ala Val 20 25 30 Phe Leu Pro
Ile Tyr Leu Ile Thr Val Ile Gly Asn Leu Leu Ile 35 40 45 Ile Leu
Ala Ile Arg Ser Asp Thr Arg Leu Gln Thr Pro Met Tyr 50 55 60 Phe
Phe Leu Ser Ile Leu Ser Phe Val Asp Ile Cys Tyr Val Thr 65 70 75
Val Ile Ile Pro Lys Met Leu Val Asn Phe Leu Ser Glu Thr Lys 80 85
90 Thr Ile Ser Tyr Gly Glu Cys Leu Thr Gln Met Tyr Phe Phe Leu 95
100 105 Ala Phe Gly Asn Thr Asp Ser Tyr Leu Leu Ala Ala Met Ala Ile
110 115 120 Asp Arg Tyr Val Ala Ile Cys Asn Pro Phe His Tyr Ile Thr
Ile 125 130 135 Met Ser His Arg Cys Cys Val Leu Leu Leu Val Leu Ser
Phe Cys 140 145 150 Ile Pro His Phe His Ser Leu Leu His Ile Leu Leu
Thr Asn Gln 155 160 165 Leu Ile Phe Cys Ala Ser Asn Val Ile His His
Phe Phe Cys Asp 170 175 180 Asp Gln Pro Val Leu Lys Leu Ser Cys Ser
Ser His Phe Val Lys 185 190 195 Glu Ile Thr Val Met Thr Glu Gly Leu
Ala Val Ile Met Thr Pro 200 205 210 Phe Ser Cys Ile Ile Ile Ser Tyr
Leu Arg Ile Leu Ile Thr Val 215 220 225 Leu Lys Ile Pro Ser Ala Ala
Gly Lys Arg Lys Ala Phe Ser Thr 230 235 240 Cys Gly Ser His Leu Thr
Val Val Thr Leu Phe Tyr Gly Ser Ile 245 250 255 Ser Tyr Val Tyr Phe
Gln Pro Leu Ser Asn Tyr Thr Val Lys Asp 260 265 270 Gln Ile Ala Thr
Ile Ile Tyr Thr Val Leu Thr Pro Met Leu Asn 275 280 285 Pro Phe Ile
Tyr Ser Leu Arg Asn Lys Asp Met Lys Gln Gly Leu 290 295 300 Ala Lys
Leu Met His Arg Met Lys Cys Gln 305 310 39 359 PRT Homo sapiens
misc_feature Incyte ID No 7472020CD1 39 Met Phe Lys Ala Ile Leu Gly
His Val Trp Pro Lys Asp His Gly 1 5 10 15 Leu Asp Lys Leu Val Val
Arg Cys Pro Arg His Thr Glu Pro Trp 20 25 30 Asn Leu Thr Gly Ile
Ser Glu Phe Leu Leu Leu Gly Leu Ser Glu 35 40 45 Asp Pro Glu Leu
Gln Pro Val Leu Pro Gly Leu Ser Leu Ser Met 50 55 60 Tyr Leu Val
Thr Val Leu Arg Asn Leu Leu Ile Ile Leu Ala Val 65 70 75 Ser Ser
Asp Ser His Leu His Thr Pro Met Cys Phe Phe Leu Ser 80 85 90 Asn
Leu Cys Trp Ala Asp Ile Gly Phe Thr Ser Ala Met Val Pro 95 100 105
Lys Met Ile Val Asp Met Gln Ser His Ser Arg Val Ile Ser Tyr 110 115
120 Ala Gly Cys Leu Thr Gln Met Ser Phe Phe Val Leu Phe Ala Cys 125
130 135 Ile Glu Asp Met Leu Leu Thr Val Met Ala Tyr Asp Arg Phe Val
140 145 150 Ala Ile Cys His Pro Leu His Tyr Pro Val Ile Met Asn Pro
His 155 160 165 Leu Gly Val Phe Leu Val Leu Val Ser Phe Phe Leu Ser
Leu Leu 170 175 180 Asp Ser Gln Leu His Ser Trp Ile Val Leu Gln Phe
Thr Phe Phe 185 190 195 Lys Asn Val Glu Ile Ser Asn Phe Val Cys Asp
Pro Ser Gln Leu 200 205 210 Leu Asn Leu Ala Cys Ser Asp Ser Val Ile
Asn Ser Ile Phe Ile 215 220 225 Tyr Leu Asp Ser Ile Met Phe Gly Phe
Leu Pro Ile Ser Gly Ile 230 235 240 Leu Leu Ser Tyr Ala Asn Asn Val
Pro Ser Ile Leu Arg Ile Ser 245 250 255 Ser Ser Asp Arg Lys Ser Lys
Ala Phe Ser Thr Cys Gly Ser His 260 265 270 Leu Ala Val Val Cys Leu
Phe Tyr Gly Thr Gly Ile Gly Val Tyr 275 280 285 Leu Thr Ser Ala Val
Ser Pro Pro Pro Arg Asn Gly Val Val Ala 290 295 300 Ser Val Met Tyr
Ala Val Val Thr Pro Met Leu Asn Pro Phe Ile 305 310 315 Tyr Ser Leu
Arg Asn Arg Asp Ile Gln Ser Ala Leu Trp Arg Leu 320 325 330 Arg Ser
Arg Thr Val Glu Ser His Asp Leu Leu Ser Gln Asp Leu 335 340 345 Leu
His Pro Phe Ser Cys Val Gly Glu Lys Gly Gln Pro His 350 355 40 936
DNA Homo sapiens misc_feature Incyte ID No 104941CB1 40 atggagataa
agaactacag cagcagcacc tcaggcttca tcctcctggg cctctcttcc 60
aaccctcagc tgcagaaacc tctctttgcc atcttcctca tcatgtacct gctcgctgcg
120 gtggggaatg tgctcatcat cccggccatc tactctgacc ccaggctcca
cacccctatg 180 tacttttttc tcagcaactt gtctttcatg gatatctgct
tcacaacagt catagtgcct 240 aagatgctgg tgaattttct atcagagaca
aaggttatct cctatgtggg ctgcctggcc 300 cagatgtact tctttatggc
atttgggaac actgacagct acctgctggc ctctatggcc 360 atcgaccggc
tggtggccat ctgcaacccc ttacactatg atgtggttat gaaaccacgg 420
cattgcctgc tcatgctatt gggttcttgc agcatctccc acctacattc cctgttccgc
480 gtgctactta tgtctcgctt gtctttctgt gcctctcaca tcattaagca
ctttttctgt 540 gacacccagc ctgtgctaaa gctctcctgc tctgacacat
cctccagcca gatggtggtg 600 atgactgaga ccttagctgt cattgtgacc
cccttcctgt gtatcatctt ctcctacctg 660 cgaatcatgg tcactgtgct
cagaatcccc tctgcagccg ggaagtggaa ggccttctct 720 acctgtggct
cccacctcac tgcagtagcc cttttctatg ggagtattat ttatgtctat 780
tttaggcccc tgtccatgta ctcagtggtt agggaccggg tagccacagt tatgtacaca
840 gtagtgacac ccatgctgaa ccctttcatc tacagcctga ggaacaaaga
tatgaagagg 900 ggtttgaaga aattacagga cagaatttac cggtaa 936 41 3365
DNA Homo sapiens misc_feature Incyte ID No 1499408CB1 41 atggaccagc
cagaggcccc ctgctccagc acggggccgc gcctcgcggt ggcccgcgag 60
ctgctcctgg ctgcgctgga ggaactgagc caagagcagc tgaagcgctt ccgccacaag
120 ctgcgcgacg tgggcccgga cggacgcagc atcccgtggg ggcggctgga
gcgcgcggac 180 gccgtggacc tcgcggagca gctggcccag ttctacggcc
cggagcctgc cctggaggtg 240 gcccgcaaga ccctcaagag ggcggacgcg
cgcgacgtgg cggcgcagct ccaggagcgg 300 cggctgcagc ggctcgggct
cggctccggg acgctgctct ccgtgtccga gtacaagaag 360 aagtaccggg
agcacgtgct gcagctgcac gctcgggtga aggagaggaa cgcccgctcc 420
gtgaagatca ccaagcgctt caccaagctg ctcatcgcgc ccgagagcgc cgccccggag
480 gaggcgctgg ggcccgcgga agagcctgag ccggggcgcg cgcggcgctc
ggacacgcac 540 actttcaacc gcctcttccg ccgcgacgag gagggccggc
ggccgctgac cgtggtgctg 600 cagggcccgg cgggcatcgg caagaccatg
gcggccaaaa agatcctgta cgactgggcg 660 gcgggcaagc tgtaccaggg
ccaggtggac ttcgccttct tcatgccctg cggcgagctg 720 ctggagaggc
cgggcacgcg cagcctggct gacctgatcc tggaccagtg ccccgaccgc 780
ggcgcgccgg tgccgcagat gctggcccag ccgcagcggc tgctcttcat cctggacggc
840 gcggacgagc tgccggcgct ggggggcccc gaggccgcgc cctgcacaga
ccccttcgag 900 gcggcgagcg gcgcgcgggt gctaggcggg ctgctgagca
aggcgctgct gcccacggcc 960 ctcctgctgg tgaccacgcg cgccgccgcc
cccgggaggc tgcagggccg cctgtgttcc 1020 ccgcagtgcg ccgaggtgcg
cggcttctcc gacaaggaca agaagaagta tttctacaag 1080 ttcttccggg
atgagaggag ggccgagcgc gcctaccgct tcgtgaagga gaacgagacg 1140
ctgttcgcgc tgtgcttcgt gcccttcgtg tgctggatcg tgtgcaccgt gctgcgccag
1200 cagctggagc tcggtcggga cctgtcgcgc acgtccaaga ccaccacgtc
agtgtacctg 1260 cttttcatca ccagcgttct gagctcggct ccggtagccg
acgggccccg gttgcagggc 1320 gacctgcgca atctgtgccg cctggcccgc
gagggcgtcc tcggacgcag ggcgcagttt 1380 gccgagaagg aactggagca
actggagctt cgtggctcca aagtgcagac gctgtttctc 1440 agcaaaaagg
agctgccggg cgtgctggag acagaggtca cctaccagtt catcgaccag 1500
agcttccagg agttcctcgc ggcactgtcc tacctgctgg aggacggcgg ggtgcccagg
1560 accgcggctg gcggcgttgg gacactcctg cgtggggacg cccagccgca
cagccacttg 1620 gtgctcacca cgcgcttcct cttcggactg ctgagcgcgg
agcggatgcg cgacatcgag 1680 cgccacttcg gctgcatggt ttcagagcgt
gtgaagcagg aggccctgcg gtgggtgcag 1740 ggacagggac agggctgccc
cggagtggca ccagaggtga ccgagggggc caaagggctc 1800 gaggacaccg
aagagccaga ggaggaggag gagggagagg agcccaacta cccactggag 1860
ttgctgtact gcctgtacga gacgcaggag gacgcgtttg tgcgccaagc cctgtgccgg
1920 ttcccggagc tggcgctgca gcgagtgcgc ttctgccgca tggacgtggc
tgttctgagc 1980 tactgcgtga ggtgctgccc tgctggacag gcactgcggc
tgatcagctg cagattggtt 2040 gctgcgcagg agaagaagaa gaagagcctg
gggaagcggc tccaggccag cctgggtggc 2100 ggcagttctc aaggcaccac
aaaacaactg ccagcctccc ttcttcatcc actctttcag 2160 gcaatgactg
acccactgtg ccatctgagc agcctcacgc tgtcccactg caaactccct 2220
gacgcggtct gccgagacct ttctgaggcc ctgagggcag cccccgcact gacggagctg
2280 ggcctcctcc acaacaggct cagtgaggcg ggactgcgta tgctgagtga
gggcctagcc 2340 tggccgcagt gcagggtgca gacggtcagg gtacagctgc
ctgaccccca gcgagggctc 2400 cagtacctgg tgggtatgct tcggcagagc
cccgccctga ccaccctgga tctcagcggc 2460 tgccaactgc ccgcccccat
ggtgacctac ctgtgtgcag tcctgcagca ccagggatgc 2520 ggcctgcaga
ccctcagtct ggcctctgtg gagctgagcg agcagtcact acaggagctt 2580
caggctgtga agagagcaaa gccggatctg gtcatcacac acccagcgct ggacggccac
2640 ccacaacctc ccaaggaact catctcgacc ttctgaggct ctggtggcca
gagcagggtg 2700 gaagacccta gtcaaagtcc ctgtggagag aacggcccat
tccaagggca ggaggatatt 2760 gctctcggcc tttgggaaac ttttgagccg
agaggccgca gacaggcatg tgggaggccc 2820 agacacggca ccctgccccg
tccaggacag gcccaggacc tgcccctctc tccacacctg 2880 gggtacccct
tctcccccag ccccaccact actccaccca ccttcctctc ctgagaccct 2940
ccagccattc cccttgaaaa caccccccga ccccaagcca caataatgac agcgagagct
3000 ccaattaact aagcacctac ctgggggcag aataaccctt cactgcctga
tccccatctg 3060 cagtgtggcc caacagcccc cagaactatg cccacataga
ctggaggtag gcagttcacc 3120 gtccctccct gttaggaatg agaccatccc
tgaggctatg gcccaggccc acaggcgtcc 3180 agtgtctgag atctttggga
agggagacta gggcaggtgg agacagcgca gaacccccgt 3240 gctgggtggg
aagcatgacc acatggtggg tgagcagccc ccatgcactg acggtaaatt 3300
cccctgtgga ctcatttctg ttggtttcta ttacacctgg ccaggcgtgg tacaatacag
3360 gtcga 3365 42 1325 DNA Homo sapiens misc_feature Incyte ID No
3168839CB1 42 catggcatcc ccagcctagc tcccaatccc actttggcac
gatgttagcc aacagctcct 60 caaccaacag ttctgttctc ccgtgtcctg
actaccgacc tacccaccgc ctgcacttgg 120 tggtctacag cttggtgctg
gctgccgggc tccccctcaa cgcgctagcc ctctgggtct 180 tcctgcgcgc
gctgcgcgtg cactcggtgg tgagcgtgta catgtgtaac ctggcggcca 240
gcgacctgct cttcaccctc tcgctgcccg ttcgtctctc ctactacgca ctgcaccact
300 ggcccttccc cgacctcctg tgccagacga cgggcgccat cttccagatg
aacatgtacg 360 gcagctgcat cttcctgatg ctcatcaacg tggaccgcta
cgccgccatc gtgcacccgc 420 tgcgactgcg ccacctgcgg cggccccgcg
tggcgcggct gctctgcctg ggcgtgtggg 480 cgctcatcct ggtgtttgcc
gtgcccgccg cccgcgtgca caggccctcg cgttgccgct 540 accgggacct
cgaggtgcgc ctatgcttcg agagcttcag cgacgagctg tggaaaggca 600
ggctgctgcc cctcgtgctg ctggccgagg cgctgggctt cctgctgccc ctggcggcgg
660 tggtctactc gtcgggccga gtcttctgga cgctggcgcg ccccgacgcc
acgcagagcc 720 agcggcggcg gaagaccgtg cgcctcctgc tggctaacct
cgtcatcttc ctgctgtgct 780 tcgtgcccta caacagcacg ctggcggtct
acgggctgct gcggagcaag ctggtggcgg 840 ccagcgtgcc tgcccgcgat
cgcgtgcgcg gggtgctgat ggtgatggtg ctgctggccg 900 gcgccaactg
cgtgctggac ccgctggtgt actactttag cgccgagggc ttccgcaaca 960
ccctgcgcgg cctgggcact ccgcaccggg ccaggacctc ggccaccaac gggacgcggg
1020 cggcgctcgc gcaatccgaa aggtccgccg tcaccaccga cgccaccagg
ccggatgccg 1080 ccatgtcccc aggattccgc cctctgaaca cacatgccat
tgcgctgtcc gtgcccgact 1140 cccaacgcct ctcgttctgg gaggcttaca
gggtgtacac acaagaaggt gggctgggca 1200 cttggacctt tgggtggcaa
ttccagctta gcaacgcaga agagtacaaa gtgtggaagc 1260 cagggcccag
ggaaggcagt gctgctggaa atggcttctt taaactgtga gcacgcagag 1320 caccc
1325 43 2124 DNA Homo sapiens misc_feature Incyte ID No 3291235CB1
43 gtcttacctc ttaatagtat tagaatggca attaaatctc gacacgtttt
aattaaattt 60 caacatgagt tttgcagggg acattcgaag tgtagcgctc
gccatctccc atcccaagta 120 cctaagggct acaacgctgt cgccagagag
gaagtcactg agtgcccact gccacccccc 180 cacatatgct cgtggtcccc
agcatccttg agccaccagg agtgagggct gctgctccct 240 gagacctggc
tccaaggagg atgccacagc cgcctgccag ctccggtctg caccatgagt 300
gatgagcggc ggctgcctgg cagtgcagtg ggctggctgg tatgtggggg cctctccctg
360 ctggccaatg cctggggcat cctcagcgtt ggcgccaagc agaagaagtg
gaagcccttg 420 gagttcctgc tgtgtacgct cgcggccacc cacatgctaa
atgtggccgt gcccatcgcc 480 acctactccg tggtgcagct gcggcggcag
cgccccgact tcgagtggaa tgagggtctc 540 tgcaaggtct tcgtgtccac
cttctacacc ctcaccctgg ccacctgttt ctctgtcacc 600 tccctctcct
accaccgcat gtggatggtc tgctggcctg tcaactaccg gctgagcaat 660
gccaagaagc aggcggtgca cacagtcatg ggtatctgga tggtgtcctt catcctgtcg
720 gccctgcctg ccgttggctg gcacgacacc agcgagcgct tctacaccca
tggctgccgc 780 ttcatcgtgg ctgagatcgg cctgggcttt ggcgtctgct
tcctgctgct ggtgggcggc 840 agcgtggcca tgggcgtgat ctgcacagcc
atcgccctct tccagacgct ggccgtgcag 900 gtggggcgcc aggccgacca
ccgcgccttc accgtgccca ccatcgtggt ggaggacgcg 960 cagggcaagc
ggcgctcctc catcgatggc tcggagcccg ccaaaacctc tctgcagacc 1020
acgggcctcg tgaccaccat agtcttcatc tacgactgcc tcatgggctt ccctgtgctg
1080 gtggtgagct tcagcagcct gcgggccgac gcctcagcgc cctggatggc
actctgcgtg 1140 ctgtggtgct ccgtggccca ggccctgctg ctgcctgtgt
tcctctgggc ctgcgaccgc 1200 taccgggctg acctcaaagc tgtccgggag
aagtgcatgg ccctcatggc caacgacgag 1260 gagtcagacg atgagaccag
cctggaaggt ggcatctccc cggacctggt gttggagcgc 1320 tccctggact
atggctatgg aggtgatttt gtggccctag ataggatggc caagtatgag 1380
atctccgccc tggagggggg cctgccccag ctctacccac tgcggccctt gcaggaggac
1440 aagatgcaat acctgcaggt cccgcccacg cggcgcttct cccacgacga
tgcggacgtg 1500 tgggccgccg tcccgctgcc cgccttcctg ccgcgctggg
gctccggcaa ggacctgtcc 1560 gccctggcgc acctggtgct gcctgccggg
cccgagcggc cccgcgccag cctcctggcc 1620 ttcgcggagg acgcaccact
gtcccgcgcg cgccgccgct cggccgagag cctgctgtcg 1680 ctgcggccct
cggccgtgga tagcggcccg cggggagccc gcgactcgcc ccccggcagc 1740
ccgcgccgcc gccccgggcc cggcccccgc tccgcctcgg cctcgctgct gcccgacgcc
1800 ttcgccctga ccgccttcga gtgcgagcca caggccctgc gccgcccgcc
cgggcccttc 1860 cccgctgcgc ccgccgcccc cgacggcgca gatcccggag
aggccccgac gcccccaagc 1920 agcgcccagc ggagcccagg gccacgcccc
tctgcgcact cgcacgccgg ctctctgcgc 1980 cccggcctga gcgcgtcgtg
gggcgagccc ggggggctgc gcgcggcggg cggcggcggc 2040 agcaccagca
gcttcctgag ttccccctcc gagtcctcgg gctacgccac gctgcactcg 2100
gactcgctgg gctccgcgtc ctag 2124 44 942 DNA Homo sapiens
misc_feature Incyte ID No 7472001CB1 44 atggaaagaa tcaacagcac
actgttgact gcgtttatcc tgacaggaat tccgtatcca 60 ctcaggctaa
ggacactctt ttttgtgttc ttttttctaa tctacatcct gactcagctg 120
ggaaacctgc ttattttaat cactgtctgg gcagacccaa ggctccatgc ccgccccatg
180 tacatctttc ttggtgttct ctcagtcatt gatatgagca tctcctccat
cattgtccct 240 cgcctcatga tgaacttcac tttaggtgtc aaacccatcc
catttggtgg ctgtgttgct 300 caactctatt tctatcactt cctgggcagc
acccagtgct tcctctacac cctaatggcc 360 tatgacaggt acctggcaat
atgtcagccc ctgcgctacc ctgtgctcat gactgctaag 420 ctgagcgcct
tgcttgtggc tggagcctgg atggcaggat ccatccatgg ggctctccag 480
gccatcctaa ccttccgcct gccctactgt gggcccaatc aggtggatta cttcttctgt
540 gacatccctg cagtgttgag actggcctgt gctgacacaa cagtcaacga
gctggtgacg 600 tttgtagaca ttggggtggt ggttgccagt tgcttctccc
tgatcctcct ctcctacata 660 cagatcattc aggccatcct gagaatccac
acagctgatg ggcggcgccg ggctttttca 720 acttgtggag cccatgtaac
cgtggtcacc gtgtactatg tgccctgtgc cttcatctac 780 ctgaggcctg
aaaccaacag ccccctggat ggggcagctg ccctagtccc cacggccatc 840
actcctttcc tcaaccccct tatctacact ctgcggaacc aagaggtgaa gctggccctg
900 aaaagaatgc tcagaagccc aagaactccg agtgaggttt ga 942 45 1197 DNA
Homo sapiens misc_feature Incyte ID No
7472003CB1 45 atgcacaccg tggctacgtc cggacccaac gcgtcctggg
gggcaccggc caacgcctcc 60 ggctgcccgg gctgtggcgc caacgcctcg
gacggcccag tcccttcgcc gcgggccgtg 120 gacgcctggc tcgtgccgct
cttcttcgcg gcgctgatgc tgctgggcct ggtggggaac 180 tcgctggtca
tctacgtcat ctgccgccac aagccgatgc ggaccgtgac caacttctac 240
atcgccaacc tggcggccac ggacgtgacc ttcctcctgt gctgcgtccc cttcacggcc
300 ctgctgtacc cgctgcccgg ctgggtgctg ggcgacttca tgtgcaagtt
cgtcaactac 360 atccagcagg tctcggtgca ggccacgtgt gccactctga
ccgccatgag tgtggaccgc 420 tggtacgtga cggtgttccc gttgcgcgcc
ctgcaccgcc gcacgccccg cctggcgctg 480 gctgtcagcc tcagcatctg
gacaggctct gcggcggtgt ctgcgccggt gctcgccctg 540 caccgcctgt
cacccgggcc gcgcgcctac tgcagtgagg ccttccccag ccgcgccctg 600
gagcgcgcct tcgcactgta caacctgctg gcgctgtacc tgctgccgct gctcgccacc
660 tgcgcctgct atgcggccat gctgcgccac ctgggccggg tcgccgtgcg
ccccgcgccc 720 gccgatagcg ccctgcaggg gcaggtgctg gcagagcgcg
caggcgccgt gcgggccaag 780 gtctcgcggc tggtggcggc cgtggtcctg
ctcttcgccg cctgctgggg ccccatccag 840 ctgttcctgg tgctgcaggc
gctgggcccc gcgggctcct ggcacccacg cagctacgcc 900 gcctacgcgc
ttaagacctg ggctcactgc atgtcctaca gcaactccgc gctgaacccg 960
ctgctctacg ccttcctggg ctcgcacttc cgacaggcct tccgccgcgt ctgcccctgc
1020 gcgccgcgcc gcccccgccg cccccgccgg cccggaccct cggaccccgc
agccccacac 1080 gcggagctgc tccgcctggg gtcccacccg gcccccgcca
gggcgcagaa gccagggagc 1140 agtgggctgg ccgcgcgcgg gctgtgcgtc
ctgggggagg acaacgcccc tctctga 1197 46 1110 DNA Homo sapiens
misc_feature Incyte ID No 7472004CB1 46 atggctccca ctggtttgag
ttccttgacc gtgaatagta cagctgtgcc cacaacacca 60 gcagcattta
agagcctaaa cttgcctctt cagatcaccc tttctgctat aatgatattc 120
attctgtttg tgtcttttct tgggaacttg gttgtttgcc tcatggttta ccaaaaagct
180 gccatgaggt ctgcaattaa catcctcctt gccagcctag cttttgcaga
catgttgctt 240 gcagtgctga acatgccctt tgccctggta actattctta
ctacccgatg gatttttggg 300 aaattcttct gtagggtatc tgctatgttt
ttctggttat ttgtgataga aggagtagcc 360 atcctgctca tcattagcat
agataggttc cttattatag tccagaggca ggataagcta 420 aacccatata
gagctaaggt tctgattgca gtttcttggg caacttcctt ttgtgtagct 480
tttcctttag ccgtaggaaa ccccgacctg cagatacctt cccgagctcc ccagtgtgtg
540 tttgggtaca caaccaatcc aggctaccag gcttatgtga ttttgatttc
tctcatttct 600 ttcttcatac ccttcctggt aatactgtac tcatttatgg
gcatactcaa cacccttcgg 660 cacaatgcct tgaggatcca tagctaccct
gaaggtatat gcctcagcca ggccagcaaa 720 ctgggtctca tgagtctgca
gagacctttc cagatgagca ttgacatggg ctttaaaaca 780 cgtgccttca
ccactatttt gattctcttt gctgtcttca ttgtctgctg ggccccattc 840
accacttaca gccttgtggc aacattcagt aagcactttt actatcagca caactttttt
900 gagattagca cctggctact gtggctctgc tacctcaagt ctgcattgaa
tccgctgatc 960 tactactgga ggattaagaa attccatgat gcttgcctgg
acatgatgcc taagtccttc 1020 aagtttttgc cgcagctccc tggtcacaca
aagcgacgga tacgtcctag tgctgtctat 1080 gtgtgtgggg aacatcggac
ggtggtgtga 1110 47 582 DNA Homo sapiens misc_feature Incyte ID No
7475687CT1 47 atggcctatg acaggtacct ggcaatatgt cagcccctgc
gctacccagt gctcatgaat 60 gggaggttat gcacagtcct tgtggctgga
gcttgggtcg ccggctccat gcatgggtct 120 atccaggcca ccctgacctt
ccgcctgccc tactgtgggc ccaatcaggt agattacttt 180 atctgtgaca
tccccgcagt attgagactg gcctgtgctg acacaactgt caatgagctt 240
gtgacctttg tggacatcgg ggtagtggcc gccagttgct tcatgttaat tctgctctcg
300 tatgccaaca tagtaaatgc catcctgaag atacgcacca ctgatgggag
gcgccgggcc 360 ttctccacct gtggctccca cctaatcgtg gtcacagtct
actatgtccc ctgtattttc 420 atctacctta gggctggctc caaaggcccc
ctggatgggg cagcggctgt gttttacact 480 gttgtcactc cattactgaa
ccccctcatc tatacactga ggaaccagga agtgaagtct 540 gccctgaaga
ggataacagc aggccaggcg gatgtaaata ac 582 48 519 DNA Homo sapiens
misc_feature Incyte ID No 7483029CT1 48 atgtacctgg tcaccgtgct
cgggaacctg ctcatcatcc tggccacaat ctcagactcc 60 cacctccaca
cccccatgta cttcttcctc tccaacctgt cctttgcaga catctgtttt 120
gtgtctacca ctgtcccaaa gatgctggtg aacatccaga cacagagcag agtcatcacc
180 tatgcagact gcatcaccca gatgtgcttt tttatactct ttgtagtgtt
ggacagctta 240 ctcctgactg tgatggccta tgaccggttt gtggccatct
gtcaccccct gcactacaca 300 gtcattatga actcctggct ctgtggactg
ctggttctgg tgtcctggat cgtgagcatc 360 ctatattctc tgttacaaag
cataatggca ttgcagctgt ccttctgtac agaattgaaa 420 atccctcatt
ttttctgtga acttaatcag gtcatccacc ttgcctgttc cgacactttt 480
attaatgaca tgatgatgaa ttttacaagt gtgctgctg 519 49 663 DNA Homo
sapiens misc_feature Incyte ID No 7477933CT1 49 cccatgtact
tcttcctctc caacctgtgc tgggctgaca tcggtctcac ctcggccacg 60
gttcccaagg tgattctgga tatgcagtcg catagcagag tcatctctca tgtgggctgc
120 ctgacacaga tgtctttctt ggtccttttt gcatgtatag aaggcatgct
cctgactgtg 180 atggcctatg gctgctttgt agccatctgt cgccctctgc
actacccagt catagtgaat 240 cctcacctct gtgtcttctt cgttttggtg
tcctttttcc ttaacctgtt ggattcccag 300 ctgcacagtt ggattgtgtt
acaattcacc atcatcaaga atgtggaaat ctctaatttt 360 ttctgtgacc
cctctcagct tctcaacctt gcctgttctg acagcgtcat caatagcata 420
ttcatatatt tcgatagtac tatgtttggt tttcttccca tttcagggat ccttttgtct
480 tactataaaa ttgtcccctc cattctaagg atgtcatcgt cagatgggaa
gtataaagcc 540 ttctccacct atggctctca cctaggagtt gtttgctggt
tttatggaac agtcattggc 600 atgtacctgg cttcagccgt gtcaccaccc
cccaggaatg gtgtggtggc atcagtgatg 660 tag 663 50 911 DNA Homo
sapiens misc_feature Incyte ID No 7475164CT1 50 gggctgagtt
tatcctggca ggcttgacac aacgcccaga acttcaactg ccactcttcc 60
tcctgttcct tggaatatat gtggtcacag tggtggggaa cctgggcatg atcttcttaa
120 ttgctctcag ttctcaactt taccctccag tgtattattt tctcagtcat
ttgtctttca 180 ttgatctctg ctactcctct gtcattaccc ctaagatgct
ggtgaacttt gttccagagg 240 agaacattat ctcctttctg gaatgcatta
ctcaacttta tttcttcctt atttttgtaa 300 ttgcagaagg ctaccttctg
acagccatgg aatatgaccg ttatgttgct atctgtcgcc 360 cactgcttta
caatattgtc atgtcccaca gggtctgttc cataatgatg gctgtggtat 420
actcactggg ttttctgtgg gccacagtcc atactacccg catgtcagtg ttgtcattct
480 gtaggtctca tacggtcagt cattattttt gtgatattct ccccttattg
actctgtctt 540 gctccagcac ccacatcaat gagattctgc tgttcattat
tggaggagtt aataccttag 600 caactacact ggcggtcctt atctcttatg
ctttcatttt ctctagtatc cttggtattc 660 attccactga ggggcaatcc
aaagcctttg gcacttgtag ctcccatctc ttggctgtgg 720 gcatcttttt
tgggtctata acattcatgt atttcaagcc cccttccagc actactatgg 780
aaaaagagaa ggtgtcttct gtgttctaca tcacaataat ccccatgctg aatcctctaa
840 tctatagcct gaggaacaag gatgtgaaaa atgcactgaa gaagatgact
aggggaaggc 900 agtcatcctg a 911 51 332 DNA Homo sapiens
misc_feature Incyte ID No 7473909CT1 51 caggccccag gacagccagt
ggctgtgtca tcatgatctg ctttgccctc actgtcctct 60 cttacatccg
catcttggcc acagtggttc agatccgttc agcagccagc cgccggaagg 120
ccttctccac ctgttcttcc cacctgggca tggtgctcct gttctatggc accggcagct
180 ccacctacat gcgacccacc acccgctact ccccgctgga agggcgcttg
gctgctgtct 240 tctactccat cctcataccc accctgaatc cgctcatcta
cagcctgagg aaccaggaca 300 tgaagagagc cctgtggaag ctctatctcc ag 332
52 538 DNA Homo sapiens misc_feature Incyte ID No 7475252CT1 52
agagccagag aatctcacag gtgtcttaga attcctgctc ctgggactcc cagatgatcc
60 agaactgcag cccgtcctct ttgggctgtt cctgtccatg tacctggtca
tggtgctggg 120 gaacctgctc atcattctgg ccgtcagctc tgactcccat
ctccacagcc ccatgtactt 180 cttcctctcc aacctgtcct tggctgacat
cggttttgcc tctactactg tccccaagat 240 gattgtggac atccaggctc
atagtagact catctcttac gtgggctgcc tgactcagat 300 gtcttttttg
atctttttcg catgtatgga aagtctgctc ctgattgtga tggcctatga 360
ccggttcgtg gccatctgtc accccctgca ctaccaagtc atcatgagcc cacgactctg
420 tggcttctta gttttggtgt ctttttttct tagccttttg gactctcagc
tgcacaattt 480 gattgtgtta caacttacct gcttcaacga tgtggaaatc
tctaattttt ttctgtga 538 53 279 DNA Homo sapiens misc_feature Incyte
ID No 7927572CT1 53 tcttttagat gcccagctgt acaatttgat tgccttacaa
atgacctgct tcaaggatgt 60 ggaaattcct aatttcttct gtgacccttc
tcaactcccc catcttgcat gttgtgacac 120 cttcaacaat aacataatcc
tgtatttccc tgatgccata tttggttttc ttcccatctc 180 ggggacactt
ttctcttacg ataaaattgt ttcctccatt ctgagggttt catcatcagg 240
tgggaagtat aaagccttct ccacctatgg gtctcacct 279 54 291 DNA Homo
sapiens misc_feature Incyte ID No 7481257CT1 54 atggaggtaa
ccacatttgc catgtgcctg attatagttc ttgttcctct tcttcttatt 60
cttgtgtcat atggtttcat tgctgtggct gtactcaaga tcaagtctgc agcaggaaga
120 caaaaagcat ttgggacctg ttcctcccat ctcgttgtgg tatccatctt
ctgtgggaca 180 gttacataca tgtatataca gccaggaaac agtccaaatc
agaatgaggg caaacttctc 240 agtatatttt actccattgt tactcccagc
ttgaacccat taatttatac g 291 55 402 DNA Homo sapiens misc_feature
Incyte ID No 7485790CT1 55 aggatccaga actgcagccc atcctcgctg
ggctgtccct gtccatgtat ctggtcacgg 60 tgctgaggaa cctcctcatc
agcctggctg tcagctctga ctcccacctc cacaccccaa 120 tgtgcttctt
cctctccaac ctgtgctggg ctgacatcgg tttcacctcg gccacggttc 180
ccaagatgat tgtggacatg cggtcgcata gcggagtcat ctcttatgcg gactgcctga
240 cacggatgtc tttcttggtc ctttttgcat gtgtagaaga catgctcctg
actgtgatgg 300 cctatgactg ttttgtagcc atctgtcgcc ctctgcacta
cccagtcatc gtgaatcctc 360 acctctgtgt cttcttagtt tcggtgtcct
tttccttagc ct 402 56 639 DNA Homo sapiens misc_feature Incyte ID No
7482993CT1 56 ggatcagagt gtctcctact ggcagcaatg gcatatgatc
gttacattgc aatctgcaat 60 cctttaaggt attcagttat tctgagcaag
gttctatgca atcaattagc agcctcatgc 120 tgggctgctg gtttccttaa
ctcagtggtg catacagtgt tgacattctg cctgcccttc 180 tgtggcaaca
atcagattaa ttacttcttc tgtgacatcc cccctttgct gatcttgtct 240
tgtggaaaca cttctgtcaa tgagttggca ctgctatcca ctggggtctt cattggttgg
300 actcctttcc tttgtatcgt actttcctac atttgcataa tctccaccat
cttgaggatc 360 cagtcctcag agggaagacg aaaagccttt tctacatgtg
cctcccacct ggccattgtc 420 tttctctttt atggcagcgc catctttaca
tatgtacggc ccatctcaac ttactcatta 480 aagaaagata ggttggtttc
agtgttgtac agtgttgtta cccccatgct aaaccctata 540 atttacacat
tgaggaataa ggacatcaaa gaagctgtca aaactatagg gagcaagtgg 600
cagccaccaa tttcctcttt ggatagtaaa ctcacttat 639 57 1370 DNA Homo
sapiens misc_feature Incyte ID No 2829053CB1 57 cggcaccaga
aggatataac cagaattctc cagcaacatg aggaggaaaa gaagaaatgg 60
gcacaacagg tggagaagga aagggagcta gagctcgaga cagactggat gagcagcaaa
120 gggtcctgga aggaaagaat gaagaggccc tgcaagtcct ccgggcctca
tatgaacagg 180 agaaagaagc gcttacccac tctttccggg aggccagttc
tacccagcag gagaccatag 240 acagactgac ctcacagctg gaggctttcc
aggccaaaat gaagagggtg gaggagtcca 300 ttctgagccg aaactataag
aaacatatcc aggattatgg gagccccagc cagttctggg 360 agcaggagct
ggagagctta cactttgtca tcgagatgaa gaatgagcgt attcatgagc 420
tggacaagcg gctgatcctc atggaaacag tgaaagagaa aaatctgata ttggaggaaa
480 aaattacgac cctgcaacag gaaaatgagg acctccatgt ccgaagccgc
aaccaggtgg 540 tcctgtcaag gcagctgtca gaagacctgc ttctcacgcg
tgaggccctg gagaaggagg 600 tgcagctgcg gcgacagctc cagcaggaga
aggaggagct gttgtaccgg gtccttgggg 660 ccaatgcctc gcctgccttc
cctctggccc ctgtcactcc cactgaggtc tctttcctcg 720 ccacataggg
tgcagggcct gggcccacca cgacgcctga agtcacagct ccttccaagg 780
tttttctgga gaagacagca ggagcctctc agttcttttc caggaaggaa cgagggtggg
840 agcgagatgg agatcctggg tgtgtgccca gtgagccctg gggccttgag
ttacatggaa 900 tcacccacag ggttttggag gccccgagaa gcgtcttccc
ttgagttggc caagggaata 960 agcaagagga gacatttcct ccctgcccca
gcactctgtc ccaatccgag aagttccgag 1020 gctttcccag gggcagtctg
tgtcacgctg gccatttgac ataaaggaga cagcccctgg 1080 tcccagcttg
tcagctctgc tgccgacttg ctgacttatc aacttcctct aggtgtttcc 1140
actccaccct ggcctgctca gagcctcagt ttacccctgc attaaaatgg tggggggact
1200 ggtcaaagga ctcttatgcc actgcagtgg cccattctag gattgtctga
aggccagagt 1260 aggggttggg gggagtgtgg acaaaccccg caaatcagag
tggggaaggt gagtggtaga 1320 gagggggtct ctgaaggccc ttggggctga
cagggccagg cagcctcccc 1370 58 1567 DNA Homo sapiens misc_feature
Incyte ID No 3068234CB1 58 gagggactgc gttctaatac ggagctccga
ggatgttcac ttcttctcca caatgaatga 60 gtgtcactat gacaagcaca
tggacttttt ttataatagg agcaacactg atactgtcga 120 tgactggaca
ggaacaaagc ttgtgattgt tttgtgtgtt gggacgtttt tctgcctgtt 180
tatttttttt tctaattctc tggtcatcgc ggcagtgatc aaaaacagaa aatttcattt
240 ccccttctac tacctgttgg ctaatttagc tgctgccgat ttcttcgctg
gaattgccta 300 tgtattcctg atgtttaaca caggcccagt ttcaaaaact
ttgactgtca accgctggtt 360 tctccgtcag gggcttctgg acagtagctt
gactgcttcc ctcaccaact tgctggttat 420 cgccgtggag aggcacatgt
caatcatgag gatgcgggtc catagcaacc tgaccaaaaa 480 gagggtgaca
ctgctcattt tgcttgtctg ggccatcgcc atttttatgg gggcggtccc 540
cacactgggc tggaattgcc tctgcaacat ctctgcctgc tcttccctgg cccccattta
600 cagcaggagt taccttgttt tctggacagt gtccaacctc atggccttcc
tcatcatggt 660 tgtggtgtac ctgcggatct acgtgtacgt caagaggaaa
accaacgtct tgtctccgca 720 tacaagtggg tccatcagcc gccggaggac
acccatgaag ctaatgaaga cggtgatgac 780 tgtcttaggg gcgtttgtgg
tatgctggac cccgggcctg gtggttctgc tcctcgacgg 840 cctgaactgc
aggcagtgtg gcgtgcagca tgtgaaaagg tggttcctgc tgctggcgct 900
gctcaactcc gtcgtgaacc ccatcatcta ctcctacaag gacgaggaca tgtatggcac
960 catgaagaag atgatctgct gcttctctca ggagaaccca gagaggcgtc
cctctcgcat 1020 cccctccaca gtcctcagca ggagtgacac aggcagccag
tacatagagg atagtattag 1080 ccaaggtgca gtctgcaata aaagtacttc
ctaaactctg gatgcctctc ggcccaccca 1140 ggcctcctct gggaaaagag
ctgttaagaa tgattacctg tctctaacaa agcccatgta 1200 cagtgttatt
tgaggtctcc attaatcact gctagatttc tttaaaaaat tttttttcat 1260
agtttaaaag catgggcagt aaagagagga cctgctgcat ttagagaaag cacagaaacg
1320 ggagaggttc ggcgggtccc tgcttgtcct atgaactgct cagagctcct
gtcagtccag 1380 ctgggccttc tgggttctgg caccatttcg tagccattct
ctttgtattt taaaaggacg 1440 ttatgaaagg gcttagacca aaataaatca
taatgttact tgagccacct tatatagctg 1500 cttggagagt ctatgtagtt
ctttctgcat gcattaaaaa tgtttagaaa tgcttcaaaa 1560 aaaaaaa 1567 59
1321 DNA Homo sapiens misc_feature Incyte ID No 5029478CB1 59
cagaccgctg cgggccgcag gcgccgggaa tgtcccctga atgcgcgcgg gcagcgggcg
60 acgcgccctt gcgcagcctg gagcaagcca accgcacccg ctttcccttc
ttctccgacg 120 tcaagggcga ccaccggctg gtgctggccg cggtggagac
aaccgtgctg gtgctcatct 180 ttgcagtgtc gctgctgggc aacgtgtgcg
ccctggtgct ggtggcgcgc cgacgacgcc 240 gcggcgcgac tgcctgcctg
gtactcaacc tcttctgcgc ggacctgctc ttcatcagcg 300 ctatccctct
ggtgctggcc gtgcgctgga ctgaggcctg gctgctgggc cccgttgcct 360
gccacctgct cttctacgtg atgaccctga gcggcagcgt caccatcctc acgctggccg
420 cggtcagcct ggagcgcatg gtgtgcatcg tgcacctgca gcgcggcgtg
cggggtcctg 480 ggcggcgggc gcgggcagtg ctgctggcgc tcatctgggg
ctattcggcg gtcgccgctc 540 tgcctctctg cgtcttcttc cgagtcgtcc
cgcaacggct ccccggcgcc gaccaggaaa 600 tttcgatttg cacactgatt
tggcccacca ttcctggaga gatctcgtgg gatgtctctt 660 ttgttacttt
gaacttcttg gtgccaggac tggtcattgt gatcagttac tccaaaattt 720
tacagatcac aaaggcatca aggaagaggc tcacggtaag cctggcctac tcggagagcc
780 accagatccg cgtgtcccag caggacttcc ggctcttccg caccctcttc
ctcctcatgg 840 tctccttctt catcatgtgg agccccatca tcatcaccat
cctcctcatc ctgatccaga 900 acttcaagca agacctggtc atctggccgt
ccctcttctt ctgggtggtg gccttcacat 960 ttgctaattc agccctaaac
cccatcctct acaacatgac actgtgcagg aatgagtgga 1020 agaaaatttt
ttgctgcttc tggttcccag aaaagggagc cattttaaca gacacatctg 1080
tcaaaagaaa tgacttgtcg attatttctg gctaattttt ctttatagca gagtttctca
1140 cacctggcga gctgtggcat gcttttaaac agagttcatt tccagtaccc
tccatcagtg 1200 gcaccctgct ttaagaaaat gaacttatgc aaatagacat
ccacagcgtc ggtaaattaa 1260 ggggtgatca ccaagtttca taatattttc
cctttataaa aggatttgtt ggccaggtgc 1320 a 1321 60 1110 DNA Homo
sapiens misc_feature Incyte ID No 5102576CB1 60 atgttccttg
tgagttgctc gcatccagca ttaagggctg gtttttatct tttatttttc 60
caatcctctt tccttctcaa ggtgtccaag acacacggag ccacggaatc tcacaggtgt
120 ctgagaattc ctcctcctgg gactctcaga ggatccagaa ctgcagccgg
ccctcgcttt 180 gctgtccctg tccctgtcca tgtatctggt cacggtgctg
aggaacctgt tcagcatcct 240 ggctgtcagc tctgactgcc ccctccacac
ccccatgtac ttcttcctct ccaacctgtg 300 ctggcctgac atcggtttca
cctcggccat ggttcccaag atgattgtgg acacgcagtc 360 gcatagcaga
gtcatctctc atgcgggctg cctgacacag atgtctttcc tgctccttgt 420
tgcatgtata gaaggcatgc tcctgactgt gatggcctat gactgctttg tagccatctg
480 tcgccctctg cactacccag tcatcgtgaa tcctcacctc tgtgtctttt
tcgttttggt 540 gtcctttttc cttagcctgt tggattccca gctgcacagt
tggattgtgt tacaattaac 600 catcatcaag aatgtggaaa tctctaattt
ggtctgtgac ccctctcaac ttctcaatct 660 tgcctgttct gacagcgtca
tcaataacat attcatatat ttcgatagta ctatgtttgg 720 ttttcttccc
atttcaggga tctttttgtc ttactataaa attgtcccct ccattctaag 780
gatttcatcg tcagatggga agtataaagc cttctccacc tgtggctgtc atctagcagt
840 tgtttgctgg ttttatggaa caggcattgg catgtacctg acttcagctg
tgtcaccacc 900 ccccaggaat ggtgtggtgg catcagtgat gtacgctgtg
gtcaccccat gctgaacctt 960 ttcatctgca gcctgagaaa cagggacata
caaagtgccc tgcggaggct gggcagcaga 1020 gcattcgaat ctcatgatct
gttccatcct ttttcttgtg tgggtgagaa agggcaatca 1080 cattaaatct
ctttatctgc aaaaaaaaaa 1110 61 1095 DNA Homo sapiens misc_feature
Incyte ID No 2200534CB1 61 atgaaggcca actacagcgc agaggagcgc
tttctcctgc tgggtttctc cgactggcct 60 tccctgcagc cggtcctctt
cgcccttgtc ctcctgtgct acctcctgac cttgacgggc 120 aactcggcgc
tggtgctgct ggcggtgcgc gacccgcgcc tgcacacgcc catgtactac 180
ttcctctgcc acctggcctt ggtagacgcg ggcttcacta ctagcgtggt gccgccgctg
240 ctggccaacc tgcgcggacc agcgctctgg ctgccgcgca gccactgcac
ggcccagctg 300 tgcgcatcgc tggctctggg ttcggccgaa tgcgtcctcc
tggcggtgat ggctctggac 360 cgcgcggccg cagtgtgccg cccgctgcgc
tatgcggggc tcgtctcccc gcgcctatgt 420 cgcacgctgg ccagcgcctc
ctggctaagc ggcctcacca actcggttgc gcaaaccgcg 480 ctcctggctg
agcggccgct gtgcgcgccc cgcctgctgg accacttcat ctgtgagctg 540
ccggcgttgc tcaagctggc ctgcggaggc gacggagaca ctaccgagaa ccagatgttc
600 gccgcccgcg tggtcatcct gctgctgccg tttgccgtca tcctggcctc
ctacggtgcc 660 gtggcccgag ctgtctgttg catgcggttc agcggaggcc
ggaggagggc ggtgggcacg 720 tgtgggtccc acctgacagc cgtctgcctg
ttctacggct cggccatcta cacctacctg 780 cagcccgcgc agcgctacaa
ccaggcacgg ggcaagttcg tatcgctctt
ctacaccgtg 840 gtcacacctg ctctcaaccc gctcatctac accctcagga
ataagaaagt gaagggggca 900 gcgaggaggc tgctgcggag tctggggaga
ggccaggctg ggcagtgagt agttggggag 960 gggagaaagt attaagccag
aacccaagga tggaaatacc ccttagtgag tcagtttaga 1020 cttcaggctg
ttcatttttg tatgataatc tgcaagattt gtcctaagga gtccaatggg 1080
ggatatgttt tcctc 1095 62 1665 DNA Homo sapiens misc_feature Incyte
ID No 3275821CB1 62 ttcctacctt cactgattct ctgaaccttc ctgtcctcgc
ctgtaaagta gattgtatga 60 ggactccatg aggtcatcca cttcaagtcc
ttggcatagg ataattactc aaaaggtgat 120 gacaatggcg cagggaggga
tggtgacttg cctggagatg cacagcaccg tctctcccat 180 actcggtcat
tcacaccatc attgattcac caggcaccca ctccgtgtcc agcaggactc 240
tggggacccc aaatggacac taccatggaa gctgacctgg gtgccactgg ccacaggccc
300 cgcacagagc ttgatgatga ggactcctac ccccaaggtg gctgggacac
ggtcttcctg 360 gtggccctgc tgctccttgg gctgccagcc aatgggttga
tggcgtggct ggccggctcc 420 caggcccggc atggagctgg cacgcgtctg
gcgctgctcc tgctcagcct ggccctctct 480 gacttcttgt tcctggcagc
agcggccttc cagatcctag agatccggca tgggggacac 540 tggccgctgg
ggacagctgc ctgccgcttc tactacttcc tatggggcgt gtcctactcc 600
tccggcctct tcctgctggc cgccctcagc ctcgaccgct gcctgctggc gctgtgccca
660 cactggtacc ctgggcaccg cccagtccgc ctgcccctct gggtctgcgc
cggtgtctgg 720 gtgctggcca cactcttcag cgtgccctgg ctggtcttcc
ccgaggctgc cgtctggtgg 780 tacgacctgg tcatctgcct ggacttctgg
gacagcgagg agctgtcgct gaggatgctg 840 gaggtcctgg ggggcttcct
gcctttcctc ctgctgctcg tctgccacgt gctcacccag 900 gccacagcct
gtcgcacctg ccaccgccaa cagcagcccg cagcctgccg gggcttcgcc 960
cgtgtggcca ggaccattct gtcagcctat gtggtcctga ggctgcccta ccagctggcc
1020 cagctgctct acctggcctt cctgtgggac gtctactctg gctacctgct
ctgggaggcc 1080 ctggtctact ccgactacct gatcctactc aacagctgcc
tcagcccctt cctctgcctc 1140 atggccagtg ccgacctccg gaccctgctg
cgctccgtgc tctcgtcctt cgcggcagct 1200 ctctgcgagg agcggccggg
cagcttcacg cccactgagc cacagaccca gctagattct 1260 gagggtccaa
ctctgccaga gccgatggca gaggcccagt cacagatgga tcctgtggcc 1320
cagcctcagg tgaaccccac actccagcca cgatcggatc ccacagctca gccacagctg
1380 aaccctacgg cccagccaca gtcggatccc acagcccagc cacagctgaa
cctcatggcc 1440 cagccacagt cagattctgt ggcccagcca caggcagaca
ctaacgtcca gacccctgca 1500 cctgctgcca gttctgtgcc cagtccctgt
gatgaagctt ccccaacccc atcctcgcat 1560 cctaccccag gggcccttga
ggacccagcc acacctcctg cctctgaagg agaaagcccc 1620 agcagcaccc
cgccagaggc ggccccgggc gcaggcccca cgtga 1665 63 1609 DNA Homo
sapiens misc_feature Incyte ID No 3744167CB1 63 tctccttttg
ccgattagtg gacgtgacag agatgtgaat ggggcaggga tgtcctttga 60
tggcatcaag actttagctt ctggtgcgct gtgtcccagc tctgatttca gttgcagccg
120 tgatggacag ttgcatggaa gctgagactc tcactgacag tgaaaccctc
aaatgaacac 180 aatccctgct ttcctgccaa ggatccttgt agggtccccc
agcttcccca ctttttttct 240 gtgtcctgta ggcccagaag gatgtcggtc
tgctaccgtc ccccagggaa cgagacactg 300 ctgagctgga agacttcgcg
ggccacaggc acagccttcc tgctgctggc ggcgctgctg 360 gggctgcctg
gcaacggctt cgtggtgtgg agcttggcgg gctggcggcc tgcacggggg 420
cgaccgctgg cggccacgct tgtgctgcac ctggcgctgg ccgacggcgc ggtgctgctg
480 ctcacgccgc tctttgtggc cttcctgacc cggcaggcct ggccgctggg
ccaggcgggc 540 tgcaaggcgg tgtactacgt gtgcgcgctc agcatgtacg
ccagcgtgct gctcaccggc 600 ctgctcagcc tgcagcgctg cctcgcagtc
acccgcccct tcctggcgcc tcggctgcgc 660 agcccggccc tggcccgccg
cctgctgctg gcggtctggc tggccgccct gttgctcgcc 720 gtcccggccg
ccgtctaccg ccacctgtgg agggaccgcg tatgccagct gtgccacccg 780
tcgccggtcc acgccgccgc ccacctgagc ctggagactc tgaccgcttt cgtgcttcct
840 ttcgggctga tgctcggctg ctacagcgtg acgctggcac ggctgcgggg
cgcccgctgg 900 ggctccgggc ggcacggggc gcgggtgggc cggctggtga
gcgccatcgt gcttgccttc 960 ggcttgctct gggcccccta ccacgcagtc
aaccttctgc aggcggtcgc agcgctggct 1020 ccaccggaag gggccttggc
gaagctgggc ggagccggcc aggcggcgcg agcgggaact 1080 acggccttgg
ccttcttcag ttctagcgtc aacccggtgc tctacgtctt caccgctgga 1140
gatctgctgc cccgggcagg tccccgtttc ctcacgcggc tcttcgaagg ctctggggag
1200 gcccgagggg gcggccgctc tagggaaggg accatggagc tccgaactac
ccctcagctg 1260 aaagtggtgg ggcagggccg cggcaatgga gacccggggg
gtgggatgga gaaggacggt 1320 ccggaatggg acctttgaca gcagacccta
caacctgctg cccttccctg tccctttcca 1380 ccccccaccc accctccaga
ggtcagtgtt ctgggacatt tggggaccct tctttgacta 1440 gagtttggat
ctggctgggt aggattacta tacacttggg gcaggcccag gctcctccaa 1500
actgagggat tatgagggtg gtgatggtcc ctgttaagga ctattgtgtg cttgcaagtt
1560 ggcatgtacc catgtgccag cattgcttac ttgttgccaa tagctgtta 1609 64
945 DNA Homo sapiens misc_feature Incyte ID No 7472007CB1 64
atgtgggaaa actggacaat tgtcagtgaa tttgttctcg tgagcttctc agccctgtcc
60 actgagcttc aggctctact gtttctcctt ttcttgacca tttacttggt
tactttaatg 120 ggcaatgtcc tcatcatcct ggtcactata gctgactctg
cactacaaag tcctatgtac 180 ttcttcctca gaaacttgtc cttcctggag
ataggtttca acttggtcat tgtgcccaag 240 atgctgggga ccctgatcat
tcaagacaca accatctcct tccttggatg tgccactcag 300 atgtatttct
tcttcttttt tggggctgct gagtgctgcc tcctggccac catggcatat 360
gaccgctacg tggccatctg tgaccccttg cactacccag tcatcatggg ccacatatcc
420 tgtgcccagc tggcagctgc ctcttggttc tcagggtttt cagtggccac
tgtgcaaacc 480 acatggattt tcagtttccc tttttgtggc cccaacaggg
tgaaccactt cttctgtgac 540 agccctcctg ttattgcact ggtctgtgct
gacacctctg tgtttgaact ggaggctctg 600 acagccactg tcccattcat
tctctttcct ttcttgctga tcctgggatc ctatgtccgc 660 atcctctcca
ctatcttcag gatgccgtca gctgagggga aacatcaggc attctccacc 720
tgttccgccc acctcttggt tgtctctctc ttctatagca ctgccatcct cacgtatttc
780 cgaccccaat ccagtgcctc ttctgagagc aagaagctgc tgtcactctc
ttccacagtg 840 gtgactccca tgttgaaccc catcatctac agctcaagga
ataaagaagt gaaggctgca 900 ctgaagcggc ttatccacag gaccctgggc
tctcagaaac tatga 945 65 1098 DNA Homo sapiens misc_feature Incyte
ID No 7472008CB1 65 atggagggat ctgttgaagc tacacctgaa attccagctc
agatgaaatg tcatccttca 60 agacccagta ctttaaatca attatctttc
tatggtgctg tgtcctcact aggaagaatg 120 catggtttag aaaccaaaag
ctctgctgaa attagagctg ggctgaagag atgtgataca 180 ctggtactag
aggcatctac tttagaagga aatatggtca tagttcttgt gtccttgaag 240
gatccaaaac tccacatccc tatgtatttc tttctttcca acctttcctt ggtagacctc
300 tgtttgacca gcagctgtgt tccacagatg ttgattaact tctggggccc
agaaaagacc 360 atcagctaca ttggctgtgc cattcaactc tatgtttttt
tgtggcttgg ggccacggaa 420 tatgtccttc ttgttgtcat ggctgtggat
tgttatgtag cagtgtgtca tccactgcaa 480 aataccatga tcatgcaccc
aaaactttgt ctgcagctgg ctatcttggc atgggggact 540 ggcttggccc
agtctctgat ccagtcccct gccaccctcc ggttaccctt ctgctcccag 600
cggatggtgg atgatgttgt ttgtgaagtc ccagctctga ttcagctctc cagtactgat
660 actacctaca gtgaaattca gatgtctatc gccagtgttg tcctcctggt
gatgcccttg 720 atcattatcc tttcctcttc tggtgctatt gctaaggctg
tgctgagaat taagtcaact 780 gcaggacaga agaaagcatt tggcacctgc
atctctcacc ttcttgtggt ttctctcttt 840 tatggcactg tcacaggtgt
ctaccttcaa ccaaaaaatc actatcctca tgaatggggc 900 aaatttctca
ctcttttcta cactgtagta accccaactc ttaatcccct catctacact 960
ctaaggaaca aggagctcca tccttggcta aaagaggcca aggtacagac cgcttcagag
1020 agtgcaagcc ccaagcattg gcagcttcca catggtgttg gtcctgtggg
tgtgcagaag 1080 acaagaactg agctttga 1098 66 954 DNA Homo sapiens
misc_feature Incyte ID No 7472013CB1 66 atgagctttg cccctaatgc
ttcacactct ccggtttttt tgctccttgg gttctcgaga 60 gctaacatct
cctacactct cctcttcttc ctgttcctgg ctatttacct gaccaccata 120
ctggggaatg tgacactggt gctgctcatc tcctgggact ccagactgca ctcacccatg
180 tattatctgc ttcgtggcct ctctgtgata gacatggggc tatccacagt
tacactgccc 240 cagttgctgg cccatttggt ctctcattac ccaaccattc
ctgctgcccg ctgcttggct 300 cagttctttt tcttctatgc atttggggtt
acagatacac ttgtcattgc tgtcatggct 360 ctggatcgct atgtggccat
ctgtgacccc ctgcactatg ctttggtaat gaatcaccaa 420 cggtgtgcct
gcttactagc cttgagctgg gtggtgtcca tactgcacac catgttgcgt 480
gtgggactcg tcctgcctct ttgctggact ggggatgctg ggggcaacgt taaccttcct
540 cacttctttt gtgaccaccg gccacttctg cgagcctctt gttctgacat
acattctaat 600 gagctggcca tattctttga gggtggcttc cttatgctgg
gcccctgtgc cctcattgta 660 ctctcttatg tccgaattgg ggccgctatt
ctacgtttgc cttcagctgc tggtcgccgc 720 cgagcagtct ccacctgtgg
atcccacctc accatggttg gtttcctcta cggcaccatc 780 atttgtgtct
acttccagcc tcccttccag aactctcagt atcaggacat ggtggcttca 840
gtaatgtata ctgccattac acctttggcc aacccatttg tgtatagcct ccacaataag
900 gatgtcaagg gtgcactctg caggctgctt gaatgggtga aggtagaccc ctga 954
67 1008 DNA Homo sapiens misc_feature Incyte ID No 7472015CB1 67
atggaatcat ctttctcatt tggagtgatc cttgctgtcc tggcctccct catcattgct
60 actaacacac tagtggctgt ggctgtgctg ctgttgatcc acaagaatga
tggtgtcagt 120 ctctgcttca ccttgaatct ggctgtggct gacaccttga
ttggtgtggc catctctggc 180 ctactcacag accagctctc cagcccttct
cggcccacac agaagaccct gtgcagcctg 240 cggatggcat ttgtcacttc
ctccgcagct gcctctgtcc tcacggtcat gctgatcacc 300 tttgacaggt
accttgccat caagcagccc ttccgctact tgaagatcat gagtgggttc 360
gtggccgggg cctgcattgc cgggctgtgg ttagtgtctt acctcattgg cttcctccca
420 ctcggaatcc ccatgttcca gcagactgcc tacaaagggc agtgcagctt
ctttgctgta 480 tttcaccctc acttcgtgct gaccctctcc tgcgttggct
tcttcccagc catgctcctc 540 tttgtcttct tctactgcga catgctcaag
attgcctcca tgcacagcca gcagattcga 600 aagatggaac atgcaggagc
catggctgga ggttatcgat ccccacggac tcccagcgac 660 ttcaaagctc
tccgtactgt gtctgttctc attgggagct ttgctctatc ctggaccccc 720
ttccttatca ctggcattgt gcaggtggcc tgccaggagt gtcacctcta cctagtgctg
780 gaacggtacc tgtggctgct cggcgtgggc aactccctgc tcaacccact
catctatgcc 840 tattggcaga aggaggtgcg actgcagctc taccacatgg
ccctaggagt gaagaaggtg 900 ctcacctcat tcctcctctt tctctcggcc
aggaattgtg gcccagagag gcccagggaa 960 agttcctgtc acatcgtcac
tatctccagc tcagagtttg atggctaa 1008 68 930 DNA Homo sapiens
misc_feature Incyte ID No 7472016CB1 68 atgagggaaa ataaccagtc
ctctacactg gaattcatcc tcctgggagt tactggtcag 60 caggaacagg
aagatttctt ctacatcctc ttcctgttca tttaccccat cacattgatt 120
ggaaacctgc tcattgtcct agccatttgc tctgatgttc gccttcacaa ccccatgtat
180 tttctccttg ccaacctctc cttggttgac atcttcttct catcggtaac
catccctaag 240 atgctggcca accatctctt gggcagcaaa tccatctctt
ttgggggatg cctaacgcag 300 atgtatttca tgatagcctt gggtaacaca
gacagctata ttttggctgc aatggcatat 360 gatcgagctg tggccatcag
ccacccactt cactacacaa caattatgag tccacggtct 420 tgtatctggc
ttattgctgg gtcttgggtg attggaaatg ccaatgccct cccccacact 480
ctgctcacag ctagtctgtc cttctgtggc aaccaggaag tggccaactt ctactgtgac
540 attaccccct tgctgaagtt atcctgttct gacatccact ttcatgtgaa
gatgatgtac 600 ctaggggttg gcattttctc tgtgccatta ctatgcatca
ttgtctccta tattcgagtc 660 ttctccacag tcttccaggt tccttccacc
aagggcgtgc tcaaggcctt ctccacctgt 720 ggttcccacc tcacggttgt
ctctttgtat tatggtacag tcatgggcac gtatttccgc 780 cctttgacca
attatagcct aaaagacgca gtgatcactg taatgtacac ggcagtgacc 840
ccaatgttaa atcctttcat ctacagtctg agaaatcggg acatgaaggc tgccctgcgg
900 aaactcttca acaagagaat ctcctcgtaa 930 69 711 DNA Homo sapiens
misc_feature Incyte ID No 7472017CB1 69 atgggcatga ccaacagcag
tgtcaaggga gacttcatcc tgctgctgtg gaacctaaaa 60 ggacctgaca
aaacaatcac attcctgggt tgtgtcatcc agctctacat ctccctggca 120
ttgggctcca ctgagtgtgt cctcctggct gtaatggctt ttgatcgcta tgctgcagtt
180 tgcaaacctc tccactatac cgccgtaatg aaccctcagc tgtgccaggc
tctggcaggg 240 gttgcgtggc tgagtggagt gggaaacact cttatccagg
gcactgtcac cctctggctt 300 cctcgctgtg gacaccgatt gctccaacat
ttcttccttg catgtgtgga catccatgat 360 aatgaggttc agctctttgt
tgcttcactg gtcttgctcc tcttgccctt agtgctaata 420 ctgctgtcct
atggacatat agccaaggtg gtcataagga tcaagtcagt ccaggcctgg 480
tgcaaaggcc tggggacatg tggatcccat ttgatagtag tgtccctctt ctgtgggacc
540 atcacagctg tctacatcca gtccaacagt tcttatgccc atgctcatgg
gaagttcatc 600 tccctcttct atacagttgt gaccccgacc ctcaatcctc
tcatctacac actgaggaat 660 aatgacgtga aaggagcact gcgattattt
aacagagact taggcacata a 711 70 1092 DNA Homo sapiens misc_feature
Incyte ID No 7472018CB1 70 atgggccccg gcgaggcgct gctggcgggt
ctcctggtga tggtactggc cgtggcgctg 60 ctatccaacg cactggtgct
gctttgttgc gcctacagcg ctgagctccg cactcgagcc 120 tcaggcgtcc
tcctggtgaa tctgtctctg ggccacctgc tgctggcggc gctggacatg 180
cccttcacgc tgctcggtgt gatgcgcggg cggacaccgt cggcgcccgg cgcatgccaa
240 gtcattggct tcctggacac cttcctggcg tccaacgcgg cgctgagcgt
ggcggcgctg 300 agcgcagacc agtggctggc agtgggcttc ccactgcgct
acgccggacg cctgcgaccg 360 cgctatgccg gcctgctgct gggctgtgcc
tggggacagt cgctggcctt ctcaggcgct 420 gcacttggct gctcgtggct
tggctacagc agcgccttcg cgtcctgttc gctgcgcctg 480 ccgcccgagc
ctgagcgtcc gcgcttcgca gccttcaccg ccacgctcca tgccgtgggc 540
ttcgtgctgc cgctggcggt gctctgcctc acctcgctcc aggtgcaccg ggtggcacgc
600 agacactgcc agcgcatgga caccgtcacc atgaaggcgc tcgcgctgct
cgccgacctg 660 caccccagtg tgcggcagcg ctgcctcatc cagcagaagc
ggcgccgcca ccgcgccacc 720 aggaagattg gcattgctat tgcgaccttc
ctcatctgct ttgccccgta tgtcatgacc 780 aggctggcgg agctcgtgcc
cttcgtcacc gtgaacgccc agtggggcat cctcagcaag 840 tgcctgacct
acagcaaggc ggtggccgac ccgttcacgt actctctgct ccgccggccg 900
ttccgccaag tcctggccgg catggtgcac cggctgctga agagaacccc gcgcccagca
960 tccacccatg acagctctct ggatgtggcc ggcatggtgc accagctgct
gaagagaacc 1020 ccgcgcccag cgtccaccca caacggctct gtggacacag
agaatgattc ctgcctgcag 1080 cagacacact ga 1092 71 927 DNA Homo
sapiens misc_feature Incyte ID No 7472019CB1 71 atggccatgg
acaatgtcac agcagtgttt cagtttctcc ttattggcat ttctaactat 60
cctcaatgga gagacacgtt tttcacatta gtgctgataa tttacctcag cacattgttg
120 gggaatggat ttatgatctt tcttattcac tttgacccca acctccacac
tccaatctac 180 ttcttcctta gtaacctgtc tttcttagac ctttgttatg
gaacagcttc catgccccag 240 gctttggtgc attgtttctc tacccatccc
tacctctctt atccccgatg tttggctcaa 300 acgagtgtct ccttggcttt
ggccacagca gagtgcctcc tactggctgc catggcctat 360 gaccgtgtgg
ttgctatcag caatcccctg cgttattcag tggttatgaa tggcccagta 420
tgtgtctgct tggttgctac ctcatggggg acatcacttg tgctcactgc catgctcatc
480 ctatccctga ggcttcactt ctgtggggct aatgtcatca accattttgc
ctgtgagatt 540 ctctccctca ttaagctgac ctgttctgat accagcctca
atgaatttat gatcctcatc 600 accagtatct tcaccctgct gctaccattt
gggtttgttc tcctctccta catacgaatt 660 gctatggcta tcataaggat
tcgctcactc cagggcaggc tcaaggcctt taccacatgt 720 ggctctcacc
tgaccgtggt gacaatcttc tatgggtcag ccatctccat gtatatgaaa 780
actcagtcca agtcctaccc tgaccaggac aagtttatct cagtgtttta tggagctttg
840 acacccatgt tgaaccccct gatatatagc ctgagaaaaa aagatgttaa
acgggcaata 900 aggaaagtta tgttgaaaag gacatga 927 72 1032 DNA Homo
sapiens misc_feature Incyte ID No 7472021CB1 72 atgcattttc
ttcctactgt ctttggcttc ctaaacagag tcacacttgg tatcttcaga 60
gagactatgg tcaatttgac ttcaatgagt ggattccttc ttatggggtt ttctgatgag
120 cgtaagcttc agattttaca tgcattggta tttctggtga catacctgct
ggccttgaca 180 ggcaacctcc tcattatcac catcattacc gtggaccgtc
gtctccattc ccccatgtat 240 tactttttaa agcacctctc tcttctggac
ctctgcttca tctctgtcac agtcccccag 300 tccattgcaa attcacttat
gggcaacggt tacatttctc ttgttcagtg cattcttcag 360 gttttcttct
tcatagctct ggcctcatca gaagtggcca ttctcacagt gatgtcttat 420
gacaggtacg cagcaatctg tcaaccactt cattatgaga ctattatgga tccccgtgcc
480 tgtaggcatg cagtgatagc tgtgtggatt gctgggggcc tctctgggct
catgcatgct 540 gccattaact tctccatacc tctctgtggg aagagagtca
ttcaccaatt cttctgtgat 600 gttcctcaga tgctgaaact agcctgttct
tatgaattca ttaatgagat tgcactggct 660 gcattcacaa cgtctgcagc
atttatctgt ttgatctcca ttgtgctctc ctacattcgc 720 atcttctcta
cagtgctgag aatcccatca gctgagggcc ggaccaaggt cttctccacc 780
tgcctaccac acctatttgt agccaccttc tttctttcag ctgcaggctt tgagtttctc
840 agactgcctt ctgattcctc atcgactgtg gaccttgtat tctccgtatt
ctatactgtg 900 atacctccaa cactcaatcc agtcatttat agcttacgga
atgattccat gaaggcagca 960 ctgaggaaga tgctgtcaaa ggaagagctt
cctcagagaa aaatgtgctt aaaagccatg 1020 tttaaactct ga 1032 73 972 DNA
Homo sapiens misc_feature Incyte ID No 7472009CB1 73 atgtggcaga
agaatcagac ctctctggca gacttcatcc ttgaggggct cttcgatgac 60
tcccttaccc accttttcct tttctccttg accatggtgg tcttccttat tgcggtgagt
120 ggcaacaccc tcaccattct cctcatctgc attgatcccc agcttcatac
accaatgtat 180 ttcctgctca gccagctctc cctcatggat ctgatgcatg
tctccacaac catcctgaag 240 atggctacca actacctatc tggcaagaaa
tctatctcct ttgtgggctg tgcaacccag 300 cacttcctct atttgtgtct
aggtggtgct gaatgttttc tcttagctgt catgtcctat 360 gaccgctatg
ttgccatctg tcatccactg cgctatgctg tgctcatgaa caagaaggtg 420
ggactgatga tggctgtcat gtcatggttg ggggcatccg tgaactccct aattcacatg
480 gcgatcttga tgcacttccc tttctgtggg cctcggaaag tctaccactt
ctactgtgag 540 ttcccagctg ttgtgaagtt ggtatgtggc gacatcactg
tgtatgagac cacagtgtac 600 atcagcagca ttctcctcct cctccccatc
ttcctgattt ctacatccta tgtcttcatc 660 cttcaaagtg tcattcagat
gcgctcatct gggagcaaga gaaatgcctt tgccacttgt 720 ggctcccacc
tcacggtggt ttctctttgg tttggtgcct gcatcttctc ctacatgaga 780
cccaggtccc agtgcactct attgcagaac aaagttggtt ctgtgttcta cagcatcatt
840 acgcccacat tgaattctct gatttatact ctccggaata aagatgtagc
taaggctctg 900 agaagagtgc tgaggagaga tgttatcacc cagtgcattc
aacgactgca attgtggttg 960 ccccgagtgt ag 972 74 900 DNA Homo sapiens
misc_feature Incyte ID No 7472010CB1 74 atggaactgg aaggagactt
ccttggtagt gtgggagaat tgggccaagt gatccagacc 60 tgttctggga
tctatgtgtt cactgtggtg ggcaacttgg gcttgatcac cttaattggg 120
ataaatccta gccttcacac ccccatgtac tttttcctct tcaacttgtc ctttatagat
180 ctctgttatt cctgtgtgtt tacccccaaa atgctgaatg actttgtttc
agaaagtatc 240 atctcttatg tgggatgtat gactcagcta tttttcttct
gtttctttgt caattctgag 300 tgctatgtgt tggtatcaat ggcctatgat
cgctatgtgg ccatctgcaa ccccctgctc 360 tacatggtca ccatgtcccc
aagggtctgc tttctgctga tgtttggttc ctatgtggta 420 gggtttgctg
gggccatggc ccacactgga agcatgctgc gactgacctt ctgtgattcc 480
aacgtcattg accattatct gtgtgacgtt ctccccctct tgcagctctc ctgcaccagc
540 acccatgtca gtgagctggt atttttcatt gttgttggag taatcaccat
gctatccagc 600 ataagcatcg tcatctctta cgctttgata ctctccaaca
tcctctgtat tccttctgca 660 gagggcagat ccaaagcctt tagcacatgg
ggctcccaca taattgctgt
tgctctgttt 720 tttgggtcag ggacattcac ctacttaaca acatcttttc
ctggctctat gaaccatggc 780 agatttgcct cagtctttta caccaatgtg
gttcccatgc ttaacccttc gatctacagt 840 ttgaggaata aggatgataa
acttgccctg ggcaaaaccc tgaagagagt gctcttctaa 900 75 924 DNA Homo
sapiens misc_feature Incyte ID No 7472011CB1 75 atggaaacag
ggaacctcac gtgggtatca gactttgtct tcctggggct ctcgcagact 60
cgggagctcc agcgtttcct gtttctaatg ttcctgtttg tctacatcac cactgttatg
120 ggaaacatcc ttatcatcat cacagtgacc tctgattccc agctccacac
acccatgtac 180 tttctgctcc gaaacctggc tgtcctagac ctctgtttct
cttcagtcac tgctcccaaa 240 atgctagtgg acctcctctc tgagaagaaa
accatctctt accagggctg catgggtcag 300 atcttcttct tccacttttt
gggaggtgcc atggtcttct tcctctcagt gatggccttt 360 gaccgcctca
ttgccatctc ccggcccctc cgctatgtca ccgtcatgaa cactcagctc 420
tgggtggggc tggtggtagc cacctgggtg ggaggctttg tccactctat tgtccagctg
480 gctctgatgc tcccactgcc cttctgtggc cccaacattt tggataactt
ctactgtgat 540 gttccccaag tactgagact tgcctgcact gacacctcac
tgctggagtt cctcaagatc 600 tccaacagtg ggctgctgga tgtcgtctgg
ttcttcctcc tcctgatgtc ctacttattc 660 atcctggtga tgctgaggtc
acatccaggg gaggcaagaa ggaaggcagc ttccacctgc 720 accacccaca
tcatcgtggt ttccatgatc ttcgttccaa gcatttacct ctatgcccgg 780
cccttcactc cattccctat ggacaagctt gtgtccatcg gccacacagt catgaccccc
840 atgctcaacc ccatgatcta taccctgagg aaccaggaca tgcaggcagc
agtgagaaga 900 ttagggagac accggctggt ttga 924 76 945 DNA Homo
sapiens misc_feature Incyte ID No 7472012CB1 76 atggacaaca
gcaactggac cagtgtgtcc cattttgttc tcttgggcat ttccacccac 60
ccagaagagc aaatcccact cttccttgtt ttctcactca tgtacgcaat caatatttct
120 ggcaacttgg ccatcatcac actgattctc tctgctccac gcctccacat
ccccatgtac 180 atcttcctca gtaacttggc cttgacagac atctgcttca
cctccaccac ggtccccaag 240 atgctgcaga ttattttctc ccctacaaag
gtaatttcct acacaggctg tttagcccaa 300 acttatttct tcatttgctt
cgccgtcatg gaaaacttca tcctggctgt gatggcctat 360 gacaggtaca
ttgccatctg ccaccctttc cactacacta tgatcctgac tagaatgctg 420
tgtgtgaaga tggtggtcat gtgccatgct ctctcccacc ttcatgccat gctgcatacc
480 tttctcatag gccaactaat cttctgtgca gataacagaa tcccccactt
cttctgtgac 540 ctctacgctc tgatgaagat ctcctgcacc agcacctacc
tcaacaccct tatgattcac 600 acagaaggtg ctgttgtaat cagtggagct
ctggccttca ttactgcctc ctatgcctgc 660 atcatcctgg tggtcctccg
gatcccctca gccaagggca ggtggaaaac cttttctacc 720 tgcggctccc
acctcactgt ggtggccata ttctatggca ccctcagttg ggtctacttc 780
cggccccttt ccagctattc agtgaccaag ggtcgcatta taacagtcgt gtacacagtg
840 gtgactccca tgctgaaccc cttcatctac agcctgagga atggggatgt
caagggaggc 900 ttcatgaaat ggatgagcag aatgcagact tttttcttta gataa
945 77 933 DNA Homo sapiens misc_feature Incyte ID No 7472014CB1 77
atgggaagaa ataacctaac aagaccctct gaattcatcc tccttggact ctcctctcga
60 cctgaggatc agaagccgct ctttgctgtg ttcctcccca tctaccttat
cacagtgata 120 ggaaacctgc ttatcatcct ggccatccgc tcagacactc
gtctccagac gcccatgtac 180 ttctttctaa gcatcctgtc ttttgttgac
atttgctatg tgacagtcat tatccctaag 240 atgctggtga acttcttatc
agagacaaag accatctctt acggtgagtg tctgacccag 300 atgtactttt
tcttagcctt tggaaacaca gacagttacc tgctagcagc catggccatt 360
gaccgctatg tggccatatg taatcccttc cactacatca ccattatgag tcacagatgc
420 tgtgtcctgc ttctggttct ctccttctgc attccacatt ttcactccct
cctgcacatt 480 cttctgacta atcagctcat cttctgtgcc tccaatgtca
tccatcactt tttctgcgat 540 gatcaaccag tgctaaaatt gtcctgttcc
tcccattttg tcaaagaaat cacagtaatg 600 acagaaggct tggctgtcat
aatgaccccg ttttcatgca tcatcatctc ttatttaaga 660 atcctcatca
ctgttctgaa gattccttca gctgctggaa agcgtaaagc attttctacc 720
tgtggctctc atctcacagt ggtgaccctg ttttatggaa gcattagcta tgtctatttt
780 cagcccctgt ccaactatac tgtcaaggat caaatagcaa caattatcta
caccgtactg 840 actcctatgc taaatccatt tatctatagt ctgaggaaca
aagacatgaa gcagggtttg 900 gcaaagttga tgcacaggat gaaatgtcag taa 933
78 1080 DNA Homo sapiens misc_feature Incyte ID No 7472020CB1 78
atgttcaaag ccatccttgg ccatgtgtgg cccaaagacc atgggttgga caagcttgtt
60 gtaaggtgtc caagacacac agagccatgg aatctcacag gtatctcaga
attcctcctc 120 ctgggactct cagaggatcc agaactgcag cccgtcctcc
ctgggctgtc cctgtccatg 180 tacctggtca cggtgctgag gaacctgctc
atcatcctgg ctgtcagctc tgactcccac 240 ctccacaccc ccatgtgctt
cttcctctcc aacctgtgct gggctgacat cggtttcacc 300 tcggccatgg
ttcccaagat gattgtggac atgcagtcgc atagcagagt catctcttat 360
gcgggctgcc tgacacagat gtctttcttt gtcctttttg catgtataga agacatgctc
420 ctgacagtga tggcctatga ccgatttgtg gccatctgtc accccctgca
ctacccagtc 480 atcatgaatc ctcaccttgg tgtcttctta gttttggtgt
cctttttcct cagcctgttg 540 gattcccagc tgcacagttg gattgtgtta
caattcacct tcttcaagaa tgtggaaatc 600 tccaattttg tctgtgaccc
atctcaactt ctcaaccttg cctgttctga cagtgtcatc 660 aatagcatat
tcatatattt agatagtatt atgtttggtt ttcttcccat ttcagggatc 720
cttttgtctt acgctaacaa tgtcccctcc attctaagaa tttcatcatc agataggaag
780 tctaaagcct tctccacctg tggctctcac ctggcagttg tttgcttatt
ttatggaaca 840 ggcattggcg tgtacctgac ttcagctgtg tcaccacccc
ccaggaatgg tgtggtggca 900 tcagtgatgt acgctgtggt cacccccatg
ctgaaccctt tcatctacag cctgagaaat 960 agggacattc aaagtgccct
gtggaggctg cgcagcagaa cagtcgaatc tcatgatctg 1020 ttatctcaag
atctgctcca tcctttttct tgtgtgggtg agaaaggtca accacattaa 1080
* * * * *
References