U.S. patent application number 12/078954 was filed with the patent office on 2009-01-15 for g-protein coupled receptors.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Janice Au-Young, Mariah R. Baughn, Neil Burford, Dyung Aina M. Lu, Roopa Reddy, Junming Yang.
Application Number | 20090018068 12/078954 |
Document ID | / |
Family ID | 27496978 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090018068 |
Kind Code |
A1 |
Burford; Neil ; et
al. |
January 15, 2009 |
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.; (Los Angeles, 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 LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Incyte Genomics, Inc.
|
Family ID: |
27496978 |
Appl. No.: |
12/078954 |
Filed: |
April 8, 2008 |
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Application
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11429430 |
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7396663 |
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12078954 |
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10149826 |
Jun 10, 2002 |
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PCT/US00/33382 |
Dec 7, 2000 |
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11429430 |
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60172852 |
Dec 10, 1999 |
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60176148 |
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Current U.S.
Class: |
514/1.1 ;
435/243; 435/325; 435/69.1; 436/501; 436/86; 530/350;
530/387.9 |
Current CPC
Class: |
A61P 1/06 20180101; A61P
25/02 20180101; A61P 1/16 20180101; A61P 33/14 20180101; A61P 41/00
20180101; A61P 5/38 20180101; A61P 1/00 20180101; A61P 31/04
20180101; A61P 7/06 20180101; A61P 9/10 20180101; A61P 11/06
20180101; A61P 1/14 20180101; A61P 29/00 20180101; A61P 7/10
20180101; A61P 1/18 20180101; A61P 13/10 20180101; A61P 13/12
20180101; A61P 9/00 20180101; A61P 5/18 20180101; A61P 19/02
20180101; A61P 33/02 20180101; A61P 19/00 20180101; A61P 15/00
20180101; A61P 21/00 20180101; A61P 25/00 20180101; A61P 43/00
20180101; A61P 3/00 20180101; A61P 17/06 20180101; A61P 25/14
20180101; A61P 1/02 20180101; A01K 2217/05 20130101; A61P 25/22
20180101; A61P 1/10 20180101; A61P 37/00 20180101; A61P 15/08
20180101; A61P 27/02 20180101; A61P 9/14 20180101; A61P 25/16
20180101; A61P 31/12 20180101; A61P 25/18 20180101; A61P 17/00
20180101; A61P 35/02 20180101; A61P 1/04 20180101; A61P 3/04
20180101; A61P 3/10 20180101; A61P 19/10 20180101; A61P 33/00
20180101; A61P 13/08 20180101; A61P 7/00 20180101; A61P 17/16
20180101; A61P 25/20 20180101; A61P 1/08 20180101; C07K 14/705
20130101; A61P 13/02 20180101; A61P 25/28 20180101; A61P 25/04
20180101; A61P 11/00 20180101; A61K 38/00 20130101; A61P 35/00
20180101; A61P 31/22 20180101; A61P 9/04 20180101; A61P 25/08
20180101; A61P 9/12 20180101; A61P 31/16 20180101; C07K 14/78
20130101; A61P 21/04 20180101; A61P 19/04 20180101; A61P 31/18
20180101; C07K 14/47 20130101; A61P 39/00 20180101 |
Class at
Publication: |
514/12 ; 530/350;
435/243; 435/325; 435/69.1; 530/387.9; 436/86; 436/501 |
International
Class: |
C07K 14/00 20060101
C07K014/00; C12N 1/00 20060101 C12N001/00; C12N 5/10 20060101
C12N005/10; C12P 21/02 20060101 C12P021/02; C07K 16/28 20060101
C07K016/28; A61K 38/16 20060101 A61K038/16; G01N 33/53 20060101
G01N033/53; G01N 33/68 20060101 G01N033/68 |
Claims
1.-28. (canceled)
29. An isolated polypeptide comprising an amino acid sequence
having at least about 95% sequence identity to the amino acid
sequence of SEQ ID NO: 24.
30. The isolated polypeptide of claim 29, wherein the polypeptide
has leukotriene receptor activity.
31. An isolated cell expressing the polypeptide of claim 29.
32. A method for producing the polypeptide of claim 29, the method
comprising: (a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide encoding the polypeptide of claim
29, and (b) recovering the polypeptide so expressed.
33. A composition comprising the polypeptide of claim 29 and a
pharmaceutically acceptable excipient.
34. A method of screening a compound for effectiveness as an
agonist of the polypeptide of claim 29, the method comprising: (a)
exposing a sample comprising the polypeptide of claim 29 to the
compound, and (b) detecting agonist activity in the sample.
35. A method of screening a compound for effectiveness as an
antagonist for the polypeptide of claim 29, the method comprising:
(a) exposing a sample comprising the polypeptide of claim 29 to the
compound, and (b) detecting antagonist activity in the sample.
36. A method of screening for a compound that specifically binds to
the polypeptide of claim 29, the method comprising: (a) combining
the polypeptide of claim 29 with at least one test compound under
suitable conditions, and (b) detecting binding of the polypeptide
of claim 29 to the test compound, thereby identifying a compound
that specifically binds to the polypeptide of claim 29.
37. An antibody that specifically binds to a polypeptide comprising
an amino acid sequence of SEQ ID NO: 24.
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 (.alpha.) 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
.alpha. 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 Dictvostelium 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, hypercalcemia); parathyrold hormone (short limbed
dwarfism); .beta..sub.3-adrenoceptor (obesity,
non-insulin-dependent diabetes mellitus); 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.
DEFINITIONS
[0052] "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.
[0053] 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.
[0054] 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.
[0055] "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, hydrophilcity, 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 glutamic acid, and positively charged
amino acids may include lysine and arginine. Amino acids with
uncharged polar side chains having similar hydrophilicity values
may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar
hydrophilicity values may include: leucine, isoleucine, and valine;
glycine and alanine; and phenylalanine and tyrosine.
[0056] 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.
[0057] "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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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 basepairs 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.
[0062] 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.
[0063] "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'.
[0064] 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.).
[0065] "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.
[0066] "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.
TABLE-US-00001 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
[0067] 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.
[0068] 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.
[0069] 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 irrumJllological function of the polypeptide from
which it was derived.
[0070] 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.
[0071] 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.
[0072] A fragment of SEQ ID NO:40-78 comprises a region of unique
polynucleotide sequence that specitically 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.
[0073] 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:I-39 is useful as an
immunogenic peptide.
[0074] 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.
[0075] 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.
[0076] Alternatively, a suite of commonly used and freely available
sequence comparison algorithms is provided by the National Center
for Biotechnology Information (NCBI) Basic Local Alignment Search
Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.
215:403-410), which is available from several sources, including
the NCBI, Bethesda, Md., and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite
includes various sequence analysis programs including "blastn,"
that is used to align a known polynucleotide sequence with other
polynucleotide sequences from a variety of databases. Also
available is a tool called "BLAST 2 Sequences" that is used for
direct pairwise comparison of two nucleotide sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm nih.gov/gorf/b12.html. The "BLAST 2 Sequences"
tool can be used for both blastn and blastp (discussed below).
BLAST programs are commonly used with gap and other parameters set
to default settings. For example, to compare two nucleotide
sequences, one may use blastn with the "BLAST 2 Sequences" tool
Version 2.0.12 (Apr. 21, 2000) set at default parameters. Such
default parameters may be, for example:
[0077] Matrix: BLOSUM62
[0078] Reward for match: 1
[0079] Penalty for mismatch: -2
[0080] Open Gap: 5 and Extension Gap: 2 penalties
[0081] Gap x drop-off: 50
[0082] Expect: 10
[0083] Word Size: 11
[0084] Filter: on
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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:
[0090] Matrix: BLOSUM62
[0091] Open Gap: 11 and Extension Gap: 1 penalties
[0092] Gap x drop-off: 50
[0093] Expect: 10
[0094] Word Size: 3
[0095] Filter: on
[0096] 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.
[0097] "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.
[0098] 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.
[0099] "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.
[0100] 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.
[0101] 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.
[0102] 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).
[0103] 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.
[0104] "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.
[0105] 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.
[0106] The term "microarray" refers to an arrangement of a
plurality of polynucleotides, polypeptides, or other chemical
compounds on a substrate.
[0107] The terms "element" and "array element" refer to a
polynucleotide, polypeptide, or other chemical compound having a
unique and defined position on a microarray.
[0108] 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.
[0109] 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.
[0110] "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.
[0111] "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.
[0112] "Post-translational modification" of an GCREC may involve
lipidation, glycosylation, phosphorylation, acetylation,
racemization, proteoytic 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.
[0113] "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).
[0114] 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.
[0115] 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.).
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] "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.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] A "substitution" refers to the replacement of one or more
amino acid residues or nucleotides by different amino acid residues
or nucleotides, respectively.
[0126] "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.
[0127] 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.
[0128] "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.
[0129] 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.
[0130] 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.
[0131] 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.
The Invention
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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 thymine are replaced with
uracil, and the sugar backbone is composed of ribose instead of
deoxyribose.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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."
[0146] Methods for DNA sequencing are well known in the art and may
be used to practice any of the embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA
polymerase I, SEQUENASE (US Biochemical, Cleveland 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.)
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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, WH 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.
[0154] 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.)
[0155] 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.)
[0156] 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.)
[0157] 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.
[0158] 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.
[0159] 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.)
[0160] 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.)
[0161] 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
nonessential 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.
[0162] 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.)
[0163] 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.
[0164] 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 .beta.-glucuronide, or luciferase and its
substrate luciferin may be used. These markers can be used not only
to identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system. (See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol.
55:121-131.)
[0165] 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.
[0166] 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.
[0167] 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.)
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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/J6 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.
[0178] 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).
[0179] Polynucleotides encoding GCREC can also be used to create
"knockin" humanized animals (pigs) or transgeric 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).
Therapeutics
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.)
[0192] 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.)
[0193] 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.)
[0194] 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').sub.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.)
[0195] 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).
[0196] 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 affinty, 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.).
[0197] 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.)
[0198] 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.)
[0199] 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.)
[0200] 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 (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E.
et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as
Candida albicans and Paracoccidioides brasiliensis; and protozoan
parasites such as Plasmodium falciparum and Trypanosoma cruzi). In
the case where a genetic deficiency in 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.
[0201] 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 transposons (Morgan, R. A. and W.
F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997)
Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin.
Biotechnol. 9:445-450).
[0202] 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.
[0203] 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.
[0204] 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).
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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).
[0215] 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. et al. (1997) Nat. Biotechnol. 15:462-466.)
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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).
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
Diagnostics
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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 (is SNP), 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.).
[0239] 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.
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] 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.
[0246] 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.
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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.
[0251] 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. A. 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.
[0252] 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.)
[0253] 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.
[0254] 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.
[0255] 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.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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
[0261] I. Construction of cDNA Libraries
[0262] 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.
[0263] 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.).
[0264] 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.
II. Isolation of cDNA Clones
[0265] 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.
[0266] 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).
III. Sequencing and Analysis
[0267] 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.
[0268] 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, Phrap, 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 LASER GENE 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.
[0269] 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).
[0270] 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.
IV. Identification and Editing of Coding Sequences from Genomic
DNA
[0271] 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.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
[0272] 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.
"Stretched" Sequences
[0273] Partial DNA sequences were extended to full length with an
algorithm based on BLAST analysis. First, partial cDNAs assembled
as described in Example III were queried against public databases
such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases using the BLAST program. The nearest GenBank
protein homolog was then compared by BLAST analysis to either
Incyte cDNA sequences or GenScan exon predicted sequences described
in Example IV. A chimeric protein was generated by using the
resultant high-scoring segment pairs (HSPs) to map the translated
sequences onto the GenBank protein homolog. Insertions or deletions
may occur in the chimeric protein with respect to the original
GenBank protein homolog. The GenBank protein homolog, the chimeric
protein, or both were used as probes to search for homologous
genomic sequences from the public human genome databases. Partial
DNA sequences were therefore "stretched" or extended by the
addition of homologous genomic sequences. The resultant stretched
sequences were examined to determine whether it contained a
complete gene.
VI. Chromosomal Mapping of GCREC Encoding Polynucleotides
[0274] 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 Genethon were
used to determine if any of the clustered sequences had been
previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment of all sequences of that cluster,
including its particular SEQ ID NO:, to that map location.
[0275] Map locations are represented by ranges, or intervals, or
human chromosomes. The map position of an interval, in
centiMorgans, is measured relative to the terminus of the
chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement
based on recombination frequencies between chromosomal markers. On
average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances are based on genetic markers
mapped by Genethon which provide boundaries for radiation hybrid
markers whose sequences were included in each of the clusters.
Human genome maps and other resources available to the public, such
as the NCBI "GeneMap'99" World Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to
determine if previously identified disease genes map within or in
proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
[0276] 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.)
[0277] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in cDNA databases such as
GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster
than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined
as:
BLAST Score .times. Percent Identity 5 .times. minimum { length (
Seq . 1 ) , length ( Seq . 2 ) } ##EQU00001##
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.
[0278] 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.).
VIII. Extension of GCREC Encoding Polynucleotides
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] 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.
[0284] 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).
[0285] In like manner, full length polynucleotide sequences are
verified using the above procedure or are used to obtain 5'
regulatory sequences using the above procedure along with
oligonucleotides designed for such extension, and an appropriate
genomic library.
IX. Labeling and Use of Individual Hybridization Probes
[0286] 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 kinase (DuPont NEN,
Boston Mass.). The labeled oligonucleotides are substantially
purified using a SEPHADEX G-25 superfine size exclusion dextran
bead column (Amersham Pharmacia Biotech). An aliquot containing
10.sup.7 counts per minute of the labeled probe is used in a
typical membrane-based hybridization analysis of human genomic DNA
digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
[0287] 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.
X. Microarrays
[0288] 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.)
[0289] 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.
Tissue or Cell Sample Preparation
[0290] Total RNA is isolated from tissue samples using the
guanidinium thiocyanate method and poly(A).sup.+ RNA is purified
using the oligo-(d) cellulose method. Each poly(A).sup.+ RNA sample
is reverse transcribed using MMLV reverse-transcriptase, 0.05
pg/.mu.l oligo-(dT) primer (21mer), 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.
Microarray Preparation
[0291] 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).
[0292] 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.
[0293] 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.
[0294] 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.
Hybridization
[0295] 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.
Detection
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] A grid is superimposed over the fluorescence signal image
such that the signal from each spot is centered in each element of
the grid. The fluorescence signal within each element is then
integrated to obtain a numerical value corresponding to the average
intensity of the signal. The software used for signal analysis is
the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides
[0301] 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.
XII. Expression of GCREC
[0302] 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.)
[0303] 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.
XIII. Functional Assays
[0304] 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.
[0305] 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.
XIV. Production of GCREC Specific Antibodies
[0306] 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.
[0307] 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.)
[0308] 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.
XV. Purification of Naturally Occurring GCREC Using Specific
Antibodies
[0309] 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.
[0310] 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.
XVI. Identification of Molecules which Interact with GCREC
[0311] 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.
[0312] 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).
[0313] 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.
[0314] 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 pGEX (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) .mu.-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 G.alpha. 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.).
XVII. Demonstration of GCREC Activity
[0315] 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.
[0316] 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.)
[0317] 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.
[0318] 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.
XVIII. Identification of GCREC Ligands
[0319] 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.
[0320] 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.
TABLE-US-00002 TABLE 1 Poly- Incyte Incyte Polypeptide Incyte
nucleotide Polynucleotide Project ID SEQ ID NO: Polypeptide 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
TABLE-US-00003 TABLE 2 Incyte Polypeptide Polypeptide GenBank
Probability GenBank SEQ ID NO: ID ID NO: Score 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. 1995 Dec 28; 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. 1997 Aug 18; 237(2): 283-287.) 22 2200534CD1 g5051404
4.6e-131 573K1.15 (mm17M1-6) 7-transmembrane olfactory
receptor-like 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]
TABLE-US-00004 TABLE 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): HMMER-PFAM
G42-Y291 BLIMPS- GPCR signature: BLOCKS K91-P130, I208-Y219,
Y283-K299, BLIMPS- Y103-S151 PRINTS Olfactory receptor signature:
ProfileScan 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): HMMER-PFAM T21 S211 S226 L39-Y297 BLIMPS- T307 S332 S367
Rhodopsin-like GPCR superfamily: BLOCKS L24-L48, V57-R78,
F101-I123, ProfileScan V137-R158, V192-F215, T232-V256, BLIMPS-
L279-R305 PRINTS Transmembrane domains: V275-L295 HMMER 4
3291235CD1 609 S228 S229 S396 7 transmembrane receptor (rhodopsin
MOTIFS S456 S324 S328 family): HMMER-PFAM S364 S417 S466 E80-E154
BLIMPS- T506 S568 S590 GPCR signatures: BLOCKS S153 S268 T392
F76-P115, F395-A405,, A442-E458, BLIMPS- S462 S482 S560 E509-P526
PRINTS Y348 Transmembrane domain: V174-L199 HMMER 5 7472001CD1 313
S68 T194 T200 N5 N85 7 transmembrane receptor (rhodopsin MOTIFS
S267 T309 T138 family): HMMER-PFAM T164 T290 S306 G41-I259 BLIMPS-
GPCR signature: BLOCKS K91-P130, T281-K297, Y103-A147 BLIMPS-
Olfactory receptor signature: PRINTS M60-R81, F178-D192, F239-V254,
ProfileScan 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): HMMER-PFAM G59-Y323 BLIMPS- GPCR signature: BLOCKS
W108-P147, Y213-Y224, A256-F282, BLIMPS- N315-R331, N119-I166
PRINTS Neuropeptide Y receptor signature: ProfileScan R69-I81,
L321-F334 Transmembrane domain: A42-Y65 HMMER 7 7472004CD1 369 S228
T94 T218 N12 7 transmembrane receptor (rhodopsin MOTIFS S339 T350
family): HMMER-PFAM G48-Y321 BLIMPS- Rhodopsin-like GPCR signature:
BLOCKS T33-Y57, I66-F87, F111-I133, BLIMPS- R144-V165, V193-L216,
A262-V286, PRINTS 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):
HMMER-PFAM M1-Y171 BLIMPS- Opsins retinal binding site: BLOCKS
Y142-N194 BLIMPS- Olfactory receptor signature: PRINTS F60-D74,
F121-V136, A155-L166, ProfileScan T172-T186 9 7483029CP1 173 T16
S34 T60 N32 N167 7 transmembrane receptor (rhodopsin MOTIFS
family): HMMER-PFAM G8-C146 BLIMPS- Rhodopsin-like GPCR signature:
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): HMMER-PFAM P1-C192 BLIMPS- Olfactory
receptor signature: BLOCKS M2-K23, F120-D134, F181-G196 BLIMPS-
Rhodopsin-like GPCR signature: PRINTS 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): HMMER-PFAM T69 S128 T151 G32-I193 BLIMPS- S282 GPCR
signature: BLOCKS N81-P120, I273-K289, S93-L142 ProfileScan
Olfactory receptor signature: BLIMPS- V50-K71, Y168-S182,
F229-G244, PRINTS S265-L276, S282-T296 Transmembrane domains: HMMER
F19-L39, I188-I207 12 7473909CP1 110 S70 S36 T66 S94 GPCR
signature: MOTIFS I85-K101 BLIMPS- Olfactory receptor signature:
BLOCKS F41-G56, A77-L88, S94-Y108 BLIMPS- PRINTS 13 7475252CP1 178
S66 S151 S136 N4 N64 7 transmembrane receptor (rhodopsin MOTIFS
family): HMMER-PFAM G40-L153 BLIMPS- Rhodopsin-like GPCR signature:
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): HMMER-PFAM M1-Y96 BLIMPS- GPCR signature: BLOCKS V13-Y24,
Q41-Q67 BLIMPS- Olfactory receptor signature: 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): HMMER-PFAM R22-V128 BLIMPS- GPCR
signature: BLOCKS G71-P110 BLIMPS- PRINTS Transmembrane domain:
M82-A100 HMMER 17 7482993CP1 213 S85 S205 S159 N83 7 transmembrane
receptor (rhodopsin MOTIFS T183 family): HMMER-PFAM S2-Y182 BLIMPS-
GPCR signature: BLOCKS R127-R153, S2-A38 ProfileScan Olfactory
receptor signature: BLIMPS- F69-N83, F130-G145, V166-L177, PRINTS
T183-G197 Transmembrane domains: HMMER P102-I120, F130-V152 18
2829053CD1 180 S30 S41 S109 Beta-1 adrenergic receptor MOTIFS S125
S140 S35 signature: BLIMPS- S36 S149 I148-S166 PRINTS Signal
peptide: M1-S67 SPScan 19 3068234CD1 353 T146 T217 T233 N15 N139
N172 7 transmembrane receptor (rhodopsin MOTIFS S321 S17 T21 N349
family): HMMER-PFAM S294 S329 T141 S47-Y293 BLIMPS- S229 T303 Y14
Rhodopsin-like GPCR superfamily BLOCKS signature: BLIMPS- I32-I56,
F65-L86, L109-I131, PRINTS 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): HMMER-PFAM
G57-Y321 BLIMPS- Rhodopsin-like GPCR superfamily BLOCKS signature:
BLIMPS- V42-A66, T74-V95, M118-I140, PRINTS 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): HMMER-PFAM
R8-C251 BLIMPS- GPCR signature: BLOCKS R57-P96 BLIMPS- Olfactory
receptor signature: 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- DM00013(A570690)15-304:
F16-L305 PRINTS DM00013(P47881)20-309: P20-L305 BLIMPS- PD149621:
T246-L305 BLOCKS PD000921: C168-L245 HMMER-PFAM PD002495: N4-L47
MOTIFS 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- S424 S451 T459 PD000009:
L68-F169 BLOCKS S192 BL00237: W97-P136, G201-H212, BLIMPS-
A230-A256, N287-R303 PRINTS GPCR profile: ProfileScan F109-V155
MOTIFS 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- DM00013(P31391)41-326: L29-L304 BLOCKS
DM00013|P35414)22-324: W16-F290 BLIMPS- BL00237: W87-P126,
F190-Y201, PRINTS R217-V243, S280-L296 ProfileScan GPCR profile:
MOTIFS 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- signatures: BLOCKS
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-
M59-K80, F177-D191, F238-S253, PRINTS I274-L285, S291-I305
Melanocortin receptor family: BLIMPS- A5-L63 PRINTS Rhodopsin-like
GPCR superfamily: BLIMPS- L26-I50, M59-K80, F104-I126, PRINTS
F153-V174, A199-L222, A237-R261, K272-K298 G-protein coupled
receptor: BLAST-DOMO DM00013|P23270|18-311: L23-H306 G-protein
coupled receptor BLAST-DOMO DM00013|P23267|20-309: L27-I305
G-protein coupled receptor BLAST-DOMO DM00013|P23275|17-306:
L23-I305 G-protein coupled receptor BLAST-DOMO
DM00013|P30953|18-306: L19-H306 Olfactory receptor PD000921: BLAST-
F168-L246 PRODOM Olfactory receptor PD149621: BLAST- V247-R307
PRODOM 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: S217 T222 T337 G70-Y319
T361 G-protein coupled receptor BLIMPS- signatures: BLOCKS
K119-P158, L236-S247, K264-Q290, T311-H327 Olfactory receptor
signatures: BLIMPS- M88-Q109, V206-D220, F267-G282, PRINTS
L303-L314, T320-K334 Melanocortin receptor family: BLIMPS- V73-L84,
K80-L92 PRINTS Rhodopsin-like GPCR superfamily: BLIMPS- L55-L79,
M88-Q109, F133-V155, PRINTS M228-A251, A266-Q290, K301-H327
Olfactory receptor PD000921: BLAST- L195-L275 PRODOM Olfactory
receptor PD149621: BLAST- V276-K331 PRODOM G-protein coupled
receptor: BLAST-DOMO DM00013|P30955|18-305: L61-L326 G-protein
coupled receptor: BLAST-DOMO DM00013|P23269|15-304: L61-L326
G-protein coupled receptor BLAST-DOMO DM00013|A57069|15-304:
D59-L326 G-protein coupled receptor BLAST-DOMO
DM00013|P23275|17-306: 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-
signatures: BLOCKS 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- M60-Q81, F182-D196, V243-G258, PRINTS A279-A290, S296-L310
Melanocortin receptor family: BLIMPS- W52-L64 PRINTS Rhodopsin-like
GPCR superfamily: BLIMPS- L27-S51, M60-Q81, F105-I127, PRINTS
R141-G162, I204-G227, A242-Q266, M277-K303 GPR3 orphan receptor
signature: BLIMPS- V161-N178 PRINTS G-protein coupled receptor:
BLAST-DOMO DM00013|P23274|18-306: L27-L310 G-protein coupled
receptor: BLAST-DOMO DM00013|P23272|18-306: Y25-L310 G-protein
coupled receptor: BLAST-DOMO DM00013|P30953|18-306: L27-L310
G-protein coupled receptor: BLAST-DOMO DM00013|P30955|18-305:
L27-L310 Olfactory receptor PD000921: BLAST- G174-L250 PRODOM
Olfactory receptor PD149621: BLAST- T251-L310 PRODOM 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-
signatures: BLOCKS 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- S6-L30, S40-L61, V85-I107,
PRINTS V121-G142, V166-L189, A223-V247, E261-R287 G-protein coupled
receptor: BLAST-DOMO DM00013|P41596|137-461: G8-D220 G-protein
coupled receptor: BLAST-DOMO DM00013|P47800|29-338: G8-Y281
G-protein coupled receptor: BLAST-DOMO DM00013|P31388|20-336:
G8-P218 G-protein coupled receptor: BLAST-DOMO
DM00013|JN0591|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- signatures: BLOCKS K90-P129,
F206-Y217, L234-R260, T280-K296 G-protein coupled receptor
ProfileScan signature: Y102-A146 Olfactory receptor signatures:
BLIMPS- M59-K80, F177-D191, F237-G252, PRINTS I272-L283, S289-F303
Rhodopsin-like GPCR superfamily: BLIMPS- F26-C50, M59-K80,
M104-I126 PRINTS S140-L161, M198-F219, A270-K296 G-protein coupled
receptor: BLAST-DOMO DM00013|P23266|17-306: Q20-L302 G-protein
coupled receptor: BLAST-DOMO DM00013|P23274|18-306: E22-L299
G-protein coupled receptor: BLAST-DOMO DM00013|P23269|15-304:
Q21-L299 G-protein coupled receptor: BLAST-DOMO
DM00013|P30955|18-305: E22-L302 Olfactory receptor PD149621: BLAST-
T245-S309 PRODOM Olfactory receptor PD000921: BLAST- L166-L244
PRODOM 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- signatures: BLOCKS
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-
L164-G179, I200-L211, T217-N231 PRINTS Rhodopsin-like GPCR
superfamily: BLIMPS- S38-V60, L125-A148, G163-Q187, PRINTS
K198-K224 Olfactory receptor PD149621: BLAST- V173-T236 PRODOM
Olfactory receptor PD000921: BLAST- C103-I172 PRODOM G-protein
coupled receptor: BLAST-DOMO DM00013|P23269|15-304: L15-L227
G-protein coupled receptor: BLAST-DOMO DM00013|P30953|18-306:
L15-L227 G-protein coupled receptor: BLAST-DOMO
DM00013|A57069|15-304: L15-R228 G-protein coupled receptor:
BLAST-DOMO DM00013|P23275|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- signatures: BLOCKS T72-P111,
F181-S192, R234-T260, K286-R302 Rhodopsin-like GPCR superfamily:
BLIMPS- L7-A31, S41-F62, D86-V108, PRINTS Y122-G143, T173-H196,
A239-A263, G276-R302 P2Y4 purinoceptor signatures: BLIMPS- Y32-L48,
P111-L126 PRINTS G-protein coupled receptor: BLAST-DOMO
DM00013|JN0591|20-336: P3-L305 G-protein coupled receptor:
BLAST-DOMO DM00013|P53452|17-344: L7-F268 G-protein coupled
receptor: BLAST-DOMO DM00013|P50406|20-335: G4-L305 G-protein
coupled receptor: BLAST-DOMO DM00013|P31388|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- signatures: BLOCKS P90-P129, L206-Y217,
L234-K260, T281-K297 G-protein coupled receptor ProfileScan
signature: S102-T147 Olfactory receptor signatures: BLIMPS-
I59-Q80, F176-D190, F237-G252, PRINTS I273-L284, S290-M304
Melanocortin receptor family: BLIMPS- F51-L63 PRINTS Rhodopsin-like
GPCR superfamily: BLIMPS- T26-H50, I59-Q80, S104-I126, PRINTS
V140-L161, I198-A221, K271-K297 G-protein coupled receptor:
BLAST-DOMO DM00013|P23275|17-306: I17-M304 G-protein coupled
receptor: BLAST-DOMO DM00013|P23269|15-304: F27-M304 G-protein
coupled receptor: BLAST-DOMO DM00013|P23266|17-306: I17-M304
G-protein coupled receptor: BLAST-DOMO DM00013|S29707|18-306:
P21-I300 Olfactory receptor PD000921: BLAST-
L165-L244 PRODOM Olfactory receptor PD149621: BLAST- T245-M304
PRODOM 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- signatures: BLOCKS 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- M79-Q100,
F197-Y211, F258-S273, PRINTS F297-L305, S311-L325 Melanocortin
receptor family: BLIMPS- V71-L83 PRINTS Vasopressin receptor
signature: BLIMPS- L75-L86 PRINTS Olfactory receptor PD000921:
BLAST- I186-L265 PRODOM Olfactory receptor PD149621: BLAST-
V267-E328 PRODOM G-protein coupled receptor: BLAST-DOMO
DM00013|P23269|15-304: E40-L325 G-protein coupled receptor:
BLAST-DOMO DM00013|P23275|17-306: S38-L325 G-protein coupled
receptor: BLAST-DOMO DM00013|P23273|18-306: I45-L325 G-protein
coupled receptor: BLAST-DOMO DM00013|P23266|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- signatures: BLOCKS 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- M59-K80, F177-D191,
F237-G252, PRINTS G273-L284, T290-L304 Rhodopsin-like GPCR
superfamily: BLIMPS- F26-C50, Y104-I126, V140-A161, PRINTS
T198-L221, K271-A297 Melanocortin receptor family: BLIMPS- I51-L63,
I126-N137 PRINTS Vasopressin receptor signature: BLIMPS- L55-L66
PRINTS G-protein coupled receptor: BLAST-DOMO
DM00013|P23275|17-306: L25-L304 G-protein coupled receptor:
BLAST-DOMO DM00013|A57069|15-304: L27-L304 G-protein coupled
receptor: BLAST-DOMO DM00013|P23270|18-311: L25-L304 G-protein
coupled receptor: BLAST-DOMO DM00013|P23266|17-306: L27-L304
Olfactory receptor PD149621: BLAST- T245-T310 PRODOM Olfactory
receptor PD000921: BLAST- F168-L244 PRODOM 35 7472010CD1 299 T68
S126 S280 N55 Transmembrane domain: HMMER T293 S10 S57 L186-I205
T156 7 transmembrane receptor (rhodopsin HMMER-PFAM family)
signatures: G31-Y279 G-protein coupled receptor BLIMPS- signatures:
BLOCKS 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-
M49-K70, Y166-S180, F227-G242, PRINTS A263-L274, S280-L294
Melanocortin receptor family: BLIMPS- I41-L53 PRINTS Rhodopsin-like
GPCR superfamily: BLIMPS- Q16-G40, M49-K70, F93-I115, PRINTS
T181-V204, A226-T250, R261-K287 G-protein coupled receptor:
BLAST-DOMO DM00013|S29709|11-299: G23-L294 G-protein coupled
receptor: BLAST-DOMO DM00013|S51356|18-307: I24-K292 G-protein
coupled receptor: BLAST-DOMO DM00013|P23274|18-306: I24-L294
G-protein coupled receptor: BLAST-DOMO DM00013|P30955|18-305:
I24-L294 Olfactory receptor PD149621: BLAST- V237-R296 PRODOM
Olfactory receptor PD000921: BLAST- L155-I235 PRODOM 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-
signatures: BLOCKS K90-P129, R234-R260, T279-Q295 G-protein coupled
receptor ProfileScan signature: F102-T148 Olfactory receptor
signatures: BLIMPS- M59-K80, F177-D191, A237-V252, PRINTS
V271-L282, T288-G302 Melanocortin receptor family: BLIMPS- S51-L63
PRINTS Rhodopsin-like GPCR superfamily: BLIMPS- F26-T50, M59-K80,
F104-I126, PRINTS L140-A161, K199-L222, A236-R260, K269-Q295
Olfactory receptor PD000921: BLAST- L166-I245 PRODOM Olfactory
receptor PD149621: BLAST- V246-R303 PRODOM G-protein coupled
receptor: BLAST-DOMO DM00013|S29710|15-301: L17-L301 G-protein
coupled receptor: BLAST-DOMO DM00013|P23275|17-306: L17-L301
G-protein coupled receptor: BLAST-DOMO DM00013|P23266|17-306:
L17-L301 G-protein coupled receptor: BLAST-DOMO
DM00013|P47881|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- signatures:
BLOCKS K90-P129, T282-K298 G-protein coupled receptor ProfileScan
signature: Y102-M147 G-protein coupled receptor MOTIFS signature:
M110-A125 Olfactory receptor signatures: BLIMPS- M59-K80,
F177-S191, F238-G253, PRINTS I274-L285, S291-M305 Rhodopsin-like
GPCR superfamily: BLIMPS- P26-L50, M59-K80, F104-I126, PRINTS
I199-I222, T237-R261, R272-K298 G-protein coupled receptor:
BLAST-DOMO DM00013|P23266|17-306: I17-K303 G-protein coupled
receptor: BLAST-DOMO DM00013|P23274|18-306: E22-K303 G-protein
coupled receptor: BLAST-DOMO DM00013|S29707|18-306: P21-G299
G-protein coupled receptor: BLAST-DOMO DM00013|P30955|18-305:
P21-K303 Olfactory receptor PD149621: BLAST- T246-T310 PRODOM
Olfactory receptor PD000921: BLAST- L166-L245 PRODOM 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- signatures: BLOCKS 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- M59-K80, F177-S191,
F238-G253, PRINTS A273-L284, S290-M304 Melanocortin receptor
family: BLIMPS- S51-L63 PRINTS Rhodopsin-like GPCR superfamily:
BLIMPS- P26-R50, M59-K80, F104-I126, PRINTS V199-L222, Q271-K297
Olfactory receptor PD000921: BLAST- L166-L245 PRODOM Olfactory
receptor PD149621: BLAST- T246-R306 PRODOM G-protein coupled
receptor: BLAST-DOMO DM00013|P23266|17-306: L17-M304 G-protein
coupled receptor: BLAST-DOMO DM00013|P23274|18-306: E22-M304
G-protein coupled receptor: BLAST-DOMO DM00013|P30955|18-305:
D23-M304 G-protein coupled receptor: BLAST-DOMO
DM00013|P30953|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- signatures: BLOCKS
R116-P155, G233-Y244, S261-T287, T308-Q324 G-protein coupled
receptor ProfileScan signature: F129-V173 Olfactory receptor
signatures: BLIMPS- M85-K106, F203-D217, F264-G279, PRINTS
A300-L311, S317-R331 GPR orphan receptor signature: BLIMPS-
S317-W328 PRINTS Cannabinoid receptor signatures: BLIMPS- M60-L73,
Y316-A326 PRINTS G-protein coupled receptor: BLAST-DOMO
DM00013|P23265|17-306: E45-L327 G-protein coupled receptor:
BLAST-DOMO DM00013|P23268|18-307: S44-L330 G-protein coupled
receptor: BLAST-DOMO DM00013|S29707|18-306: P47-L327 G-protein
coupled receptor: BLAST-DOMO DM00013|P30953|18-306: P47-L330
Olfactory receptor PD000921: BLAST- N197-L271 PRODOM Olfactory
receptor PD149621: BLAST- V273-R333 PRODOM
TABLE-US-00005 TABLE 4 Incyte Polynucleotide Polynucleotide
Sequence Selected Sequence 5' 3' SEQ ID NO: ID 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.v113.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
TABLE-US-00006 TABLE 5 Polynucleotide Incyte Representative SEQ ID
NO: Project ID Library 61 2200534CB1 BRAVTXT04 62 3275821CB1
PROSBPT06 63 3744167CB1 LUNGNOT27
TABLE-US-00007 TABLE 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.
TABLE-US-00008 TABLE 7 Parameter Program Description Reference
Threshold ABI A program that removes vector sequences and masks
Applied Biosystems, Foster City, CA. FACTURA ambiguous bases in
nucleic acid sequences. ABI/ A Fast Data Finder useful in comparing
and annotating Applied Biosystems, Foster City, CA; Mismatch
PARACEL amino acid or nucleic acid sequences. Paracel Inc.,
Pasadena, CA. <50% FDF ABI A program that assembles nucleic acid
sequences. Applied Biosystems, Foster City, CA. Auto- Assembler
BLAST A Basic Local Alignment Search Tool useful in sequence
Altschul, S. F. et al. (1990) J. Mol. Biol. ESTs: similarity search
for amino acid and nucleic acid 215: 403-410; Altschul, S. F. et
al. (1997) Probability sequences. BLAST includes five functions:
blastp, blastn, Nucleic Acids Res. 25: 3389-3402. value = 1.0E-8
blastx, tblastn, and tblastx. or less Full Length sequences:
Probability value = 1.0E-10 or less FASTA A Pearson and Lipman
algorithm that searches for Pearson, W. R. and D. J. Lipman (1988)
Proc. ESTs: fasta E similarity between a query sequence and a group
of Natl. Acad Sci. USA 85: 2444-2448; Pearson, W. R. value =
sequences of the same type. FASTA comprises as least (1990) Methods
Enzymol. 183: 63-98; 1.06E-6 five functions: fasta, tfasta, fastx,
tfastx, and ssearch. and Smith, T. F. and M. S. Waterman (1981)
Assembled Adv. Appl. Math. 2: 482-489. ESTs: fasta Identity = 95%
or greater and Match length = 200 bases or greater; fastx E value =
1.0E-8 or less Full Length sequences: fastx score = 100 or greater
BLIMPS A BLocks IMProved Searcher that matches a sequence Henikoff,
S. and J. G. Henikoff (1991) Nucleic Probability against those in
BLOCKS, PRINTS, DOMO, PRODOM, Acids Res. 19: 6565-6572; Henikoff,
J. G. and value = 1.0E-3 and PFAM databases to search for gene
families, sequence S. Henikoff (1996) Methods Enzymol. or less
homology, and structural fingerprint regions. 266: 88-105; and
Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37:
417-424. HMMER An algorithm for searching a query sequence against
Krogh, A. et al. (1994) J. Mol. Biol., PFAM hits: hidden Markov
model (HMM)-based databases of protein 235: 1501-1531; Sonnhammer,
E. L. L. et al. Probability family consensus sequences, such as
PFAM. (1988) Nucleic Acids Res. 26: 320-322; value = 1.0E-3 Durbin,
R. et al. (1998) Our World View, in a or less Signal Nutshell,
Cambridge Univ. Press, pp. 1-350. peptide hits: Score = 0 or
greater ProfileScan An algorithm that searches for structural and
sequence Gribskov, M. et al. (1988) CABIOS 4: 61-66; Normalized
motifs in protein sequences that match sequence patterns Gribskov,
M. et al. (1989) Methods Enzymol. quality score .gtoreq. defined in
Prosite. 183: 146-159; Bairoch, A. et al. (1997) Nucleic
GCG-specified Acids Res. 25: 217-221. "HIGH" value for that
particular Prosite motif. Generally, score = 1.4-2.1. Phred A
base-calling algorithm that examines automated Ewing, B. et al.
(1998) Genome Res. sequencer traces with high sensitivity and
probability. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res.
8: 186-194. Phrap A Phils Revised Assembly Program including SWAT
and Smith, T. F. and M. S. Waterman (1981) Adv. Appl. Score = 120
or CrossMatch, programs based on efficient implementation Math. 2:
482-489; Smith, T. F. and M. S. Waterman greater; Match of the
Smith-Waterman algorithm, useful in searching (1981) J. Mol. Biol.
147: 195-197; and Green, P., length = 56 or sequence homology and
assembling DNA sequences. University of Washington, Seattle, WA.
greater Consed A graphical tool for viewing and editing Phrap
assemblies. Gordon, D. et al. (1998) Genome Res. 8: 195-202. SPScan
A weight matrix analysis program that scans protein Nielson, H. et
al. (1997) Protein Engineering 10: 1-6; Score = 3.5 or sequences
for the presence of secretory signal peptides. Claverie, J. M. and
S. Audic (1997) CABIOS greater 12: 431-439. TMAP A program that
uses weight matrices to delineate Persson, B. and P. Argos (1994)
J. Mol. Biol. transmembrane segments on protein sequences and 237:
182-192; Persson, B. and P. Argos (1996) determine orientation.
Protein Sci. 5: 363-371. TMHMMER A program that uses a hidden
Markov model (HMM) to Sonnhammer, E. L. et al. (1998) Proc. Sixth
Intl. delineate transmembrane segments on protein sequences Conf.
on Intelligent Systems for Mol. Biol., and 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 sequences for patterns Bairoch, A. et al. (1997) Nucleic
Acids Res. 25: 217-221; that matched those defined in Prosite.
Wisconsin Package Program Manual, version 9, page M51-59, Genetics
Computer Group, Madison, WI.
Sequence CWU 1
1
781311PRTHomo sapiensmisc_featureIncyte ID No 104941CD1 1Met Glu
Ile Lys Asn Tyr Ser Ser Ser Thr Ser Gly Phe Ile Leu 1 5 10 15Leu
Gly Leu Ser Ser Asn Pro Gln Leu Gln Lys Pro Leu Phe Ala 20 25 30Ile
Phe Leu Ile Met Tyr Leu Leu Ala Ala Val Gly Asn Val Leu 35 40 45Ile
Ile Pro Ala Ile Tyr Ser Asp Pro Arg Leu His Thr Pro Met 50 55 60Tyr
Phe Phe Leu Ser Asn Leu Ser Phe Met Asp Ile Cys Phe Thr 65 70 75Thr
Val Ile Val Pro Lys Met Leu Val Asn Phe Leu Ser Glu Thr 80 85 90Lys
Val Ile Ser Tyr Val Gly Cys Leu Ala Gln Met Tyr Phe Phe 95 100
105Met Ala Phe Gly Asn Thr Asp Ser Tyr Leu Leu Ala Ser Met Ala 110
115 120Ile Asp Arg Leu Val Ala Ile Cys Asn Pro Leu His Tyr Asp Val
125 130 135Val Met Lys Pro Arg His Cys Leu Leu Met Leu Leu Gly Ser
Cys 140 145 150Ser Ile Ser His Leu His Ser Leu Phe Arg Val Leu Leu
Met Ser 155 160 165Arg Leu Ser Phe Cys Ala Ser His Ile Ile Lys His
Phe Phe Cys 170 175 180Asp Thr Gln Pro Val Leu Lys Leu Ser Cys Ser
Asp Thr Ser Ser 185 190 195Ser Gln Met Val Val Met Thr Glu Thr Leu
Ala Val Ile Val Thr 200 205 210Pro Phe Leu Cys Ile Ile Phe Ser Tyr
Leu Arg Ile Met Val Thr 215 220 225Val Leu Arg Ile Pro Ser Ala Ala
Gly Lys Trp Lys Ala Phe Ser 230 235 240Thr Cys Gly Ser His Leu Thr
Ala Val Ala Leu Phe Tyr Gly Ser 245 250 255Ile Ile Tyr Val Tyr Phe
Arg Pro Leu Ser Met Tyr Ser Val Val 260 265 270Arg Asp Arg Val Ala
Thr Val Met Tyr Thr Val Val Thr Pro Met 275 280 285Leu Asn Pro Phe
Ile Tyr Ser Leu Arg Asn Lys Asp Met Lys Arg 290 295 300Gly Leu Lys
Lys Leu Gln Asp Arg Ile Tyr Arg 305 3102891PRTHomo
sapiensmisc_featureIncyte ID No 1499408CD1 2Met Asp Gln Pro Glu Ala
Pro Cys Ser Ser Thr Gly Pro Arg Leu 1 5 10 15Ala Val Ala Arg Glu
Leu Leu Leu Ala Ala Leu Glu Glu Leu Ser 20 25 30Gln Glu Gln Leu Lys
Arg Phe Arg His Lys Leu Arg Asp Val Gly 35 40 45Pro Asp Gly Arg Ser
Ile Pro Trp Gly Arg Leu Glu Arg Ala Asp 50 55 60Ala Val Asp Leu Ala
Glu Gln Leu Ala Gln Phe Tyr Gly Pro Glu 65 70 75Pro Ala Leu Glu Val
Ala Arg Lys Thr Leu Lys Arg Ala Asp Ala 80 85 90Arg Asp Val Ala Ala
Gln Leu Gln Glu Arg Arg Leu Gln Arg Leu 95 100 105Gly Leu Gly Ser
Gly Thr Leu Leu Ser Val Ser Glu Tyr Lys Lys 110 115 120Lys Tyr Arg
Glu His Val Leu Gln Leu His Ala Arg Val Lys Glu 125 130 135Arg Asn
Ala Arg Ser Val Lys Ile Thr Lys Arg Phe Thr Lys Leu 140 145 150Leu
Ile Ala Pro Glu Ser Ala Ala Pro Glu Glu Ala Leu Gly Pro 155 160
165Ala Glu Glu Pro Glu Pro Gly Arg Ala Arg Arg Ser Asp Thr His 170
175 180Thr Phe Asn Arg Leu Phe Arg Arg Asp Glu Glu Gly Arg Arg Pro
185 190 195Leu Thr Val Val Leu Gln Gly Pro Ala Gly Ile Gly Lys Thr
Met 200 205 210Ala Ala Lys Lys Ile Leu Tyr Asp Trp Ala Ala Gly Lys
Leu Tyr 215 220 225Gln Gly Gln Val Asp Phe Ala Phe Phe Met Pro Cys
Gly Glu Leu 230 235 240Leu Glu Arg Pro Gly Thr Arg Ser Leu Ala Asp
Leu Ile Leu Asp 245 250 255Gln Cys Pro Asp Arg Gly Ala Pro Val Pro
Gln Met Leu Ala Gln 260 265 270Pro Gln Arg Leu Leu Phe Ile Leu Asp
Gly Ala Asp Glu Leu Pro 275 280 285Ala Leu Gly Gly Pro Glu Ala Ala
Pro Cys Thr Asp Pro Phe Glu 290 295 300Ala Ala Ser Gly Ala Arg Val
Leu Gly Gly Leu Leu Ser Lys Ala 305 310 315Leu Leu Pro Thr Ala Leu
Leu Leu Val Thr Thr Arg Ala Ala Ala 320 325 330Pro Gly Arg Leu Gln
Gly Arg Leu Cys Ser Pro Gln Cys Ala Glu 335 340 345Val Arg Gly Phe
Ser Asp Lys Asp Lys Lys Lys Tyr Phe Tyr Lys 350 355 360Phe Phe Arg
Asp Glu Arg Arg Ala Glu Arg Ala Tyr Arg Phe Val 365 370 375Lys Glu
Asn Glu Thr Leu Phe Ala Leu Cys Phe Val Pro Phe Val 380 385 390Cys
Trp Ile Val Cys Thr Val Leu Arg Gln Gln Leu Glu Leu Gly 395 400
405Arg Asp Leu Ser Arg Thr Ser Lys Thr Thr Thr Ser Val Tyr Leu 410
415 420Leu Phe Ile Thr Ser Val Leu Ser Ser Ala Pro Val Ala Asp Gly
425 430 435Pro Arg Leu Gln Gly Asp Leu Arg Asn Leu Cys Arg Leu Ala
Arg 440 445 450Glu Gly Val Leu Gly Arg Arg Ala Gln Phe Ala Glu Lys
Glu Leu 455 460 465Glu Gln Leu Glu Leu Arg Gly Ser Lys Val Gln Thr
Leu Phe Leu 470 475 480Ser Lys Lys Glu Leu Pro Gly Val Leu Glu Thr
Glu Val Thr Tyr 485 490 495Gln Phe Ile Asp Gln Ser Phe Gln Glu Phe
Leu Ala Ala Leu Ser 500 505 510Tyr Leu Leu Glu Asp Gly Gly Val Pro
Arg Thr Ala Ala Gly Gly 515 520 525Val Gly Thr Leu Leu Arg Gly Asp
Ala Gln Pro His Ser His Leu 530 535 540Val Leu Thr Thr Arg Phe Leu
Phe Gly Leu Leu Ser Ala Glu Arg 545 550 555Met Arg Asp Ile Glu Arg
His Phe Gly Cys Met Val Ser Glu Arg 560 565 570Val Lys Gln Glu Ala
Leu Arg Trp Val Gln Gly Gln Gly Gln Gly 575 580 585Cys Pro Gly Val
Ala Pro Glu Val Thr Glu Gly Ala Lys Gly Leu 590 595 600Glu Asp Thr
Glu Glu Pro Glu Glu Glu Glu Glu Gly Glu Glu Pro 605 610 615Asn Tyr
Pro Leu Glu Leu Leu Tyr Cys Leu Tyr Glu Thr Gln Glu 620 625 630Asp
Ala Phe Val Arg Gln Ala Leu Cys Arg Phe Pro Glu Leu Ala 635 640
645Leu Gln Arg Val Arg Phe Cys Arg Met Asp Val Ala Val Leu Ser 650
655 660Tyr Cys Val Arg Cys Cys Pro Ala Gly Gln Ala Leu Arg Leu Ile
665 670 675Ser Cys Arg Leu Val Ala Ala Gln Glu Lys Lys Lys Lys Ser
Leu 680 685 690Gly Lys Arg Leu Gln Ala Ser Leu Gly Gly Gly Ser Ser
Gln Gly 695 700 705Thr Thr Lys Gln Leu Pro Ala Ser Leu Leu His Pro
Leu Phe Gln 710 715 720Ala Met Thr Asp Pro Leu Cys His Leu Ser Ser
Leu Thr Leu Ser 725 730 735His Cys Lys Leu Pro Asp Ala Val Cys Arg
Asp Leu Ser Glu Ala 740 745 750Leu Arg Ala Ala Pro Ala Leu Thr Glu
Leu Gly Leu Leu His Asn 755 760 765Arg Leu Ser Glu Ala Gly Leu Arg
Met Leu Ser Glu Gly Leu Ala 770 775 780Trp Pro Gln Cys Arg Val Gln
Thr Val Arg Val Gln Leu Pro Asp 785 790 795Pro Gln Arg Gly Leu Gln
Tyr Leu Val Gly Met Leu Arg Gln Ser 800 805 810Pro Ala Leu Thr Thr
Leu Asp Leu Ser Gly Cys Gln Leu Pro Ala 815 820 825Pro Met Val Thr
Tyr Leu Cys Ala Val Leu Gln His Gln Gly Cys 830 835 840Gly Leu Gln
Thr Leu Ser Leu Ala Ser Val Glu Leu Ser Glu Gln 845 850 855Ser Leu
Gln Glu Leu Gln Ala Val Lys Arg Ala Lys Pro Asp Leu 860 865 870Val
Ile Thr His Pro Ala Leu Asp Gly His Pro Gln Pro Pro Lys 875 880
885Glu Leu Ile Ser Thr Phe 8903422PRTHomo sapiensmisc_featureIncyte
ID No 3168839CD1 3Met Leu Ala Asn Ser Ser Ser Thr Asn Ser Ser Val
Leu Pro Cys 1 5 10 15Pro Asp Tyr Arg Pro Thr His Arg Leu His Leu
Val Val Tyr Ser 20 25 30Leu Val Leu Ala Ala Gly Leu Pro Leu Asn Ala
Leu Ala Leu Trp 35 40 45Val Phe Leu Arg Ala Leu Arg Val His Ser Val
Val Ser Val Tyr 50 55 60Met Cys Asn Leu Ala Ala Ser Asp Leu Leu Phe
Thr Leu Ser Leu 65 70 75Pro Val Arg Leu Ser Tyr Tyr Ala Leu His His
Trp Pro Phe Pro 80 85 90Asp Leu Leu Cys Gln Thr Thr Gly Ala Ile Phe
Gln Met Asn Met 95 100 105Tyr Gly Ser Cys Ile Phe Leu Met Leu Ile
Asn Val Asp Arg Tyr 110 115 120Ala Ala Ile Val His Pro Leu Arg Leu
Arg His Leu Arg Arg Pro 125 130 135Arg Val Ala Arg Leu Leu Cys Leu
Gly Val Trp Ala Leu Ile Leu 140 145 150Val Phe Ala Val Pro Ala Ala
Arg Val His Arg Pro Ser Arg Cys 155 160 165Arg Tyr Arg Asp Leu Glu
Val Arg Leu Cys Phe Glu Ser Phe Ser 170 175 180Asp Glu Leu Trp Lys
Gly Arg Leu Leu Pro Leu Val Leu Leu Ala 185 190 195Glu Ala Leu Gly
Phe Leu Leu Pro Leu Ala Ala Val Val Tyr Ser 200 205 210Ser Gly Arg
Val Phe Trp Thr Leu Ala Arg Pro Asp Ala Thr Gln 215 220 225Ser Gln
Arg Arg Arg Lys Thr Val Arg Leu Leu Leu Ala Asn Leu 230 235 240Val
Ile Phe Leu Leu Cys Phe Val Pro Tyr Asn Ser Thr Leu Ala 245 250
255Val Tyr Gly Leu Leu Arg Ser Lys Leu Val Ala Ala Ser Val Pro 260
265 270Ala Arg Asp Arg Val Arg Gly Val Leu Met Val Met Val Leu Leu
275 280 285Ala Gly Ala Asn Cys Val Leu Asp Pro Leu Val Tyr Tyr Phe
Ser 290 295 300Ala Glu Gly Phe Arg Asn Thr Leu Arg Gly Leu Gly Thr
Pro His 305 310 315Arg Ala Arg Thr Ser Ala Thr Asn Gly Thr Arg Ala
Ala Leu Ala 320 325 330Gln Ser Glu Arg Ser Ala Val Thr Thr Asp Ala
Thr Arg Pro Asp 335 340 345Ala Ala Met Ser Pro Gly Phe Arg Pro Leu
Asn Thr His Ala Ile 350 355 360Ala Leu Ser Val Pro Asp Ser Gln Arg
Leu Ser Phe Trp Glu Ala 365 370 375Tyr Arg Val Tyr Thr Gln Glu Gly
Gly Leu Gly Thr Trp Thr Phe 380 385 390Gly Trp Gln Phe Gln Leu Ser
Asn Ala Glu Glu Tyr Lys Val Trp 395 400 405Lys Pro Gly Pro Arg Glu
Gly Ser Ala Ala Gly Asn Gly Phe Phe 410 415 420Lys Leu4609PRTHomo
sapiensmisc_featureIncyte ID No 3291235CD1 4Met Ser Asp Glu Arg Arg
Leu Pro Gly Ser Ala Val Gly Trp Leu 1 5 10 15Val Cys Gly Gly Leu
Ser Leu Leu Ala Asn Ala Trp Gly Ile Leu 20 25 30Ser Val Gly Ala Lys
Gln Lys Lys Trp Lys Pro Leu Glu Phe Leu 35 40 45Leu Cys Thr Leu Ala
Ala Thr His Met Leu Asn Val Ala Val Pro 50 55 60Ile Ala Thr Tyr Ser
Val Val Gln Leu Arg Arg Gln Arg Pro Asp 65 70 75Phe Glu Trp Asn Glu
Gly Leu Cys Lys Val Phe Val Ser Thr Phe 80 85 90Tyr Thr Leu Thr Leu
Ala Thr Cys Phe Ser Val Thr Ser Leu Ser 95 100 105Tyr His Arg Met
Trp Met Val Cys Trp Pro Val Asn Tyr Arg Leu 110 115 120Ser Asn Ala
Lys Lys Gln Ala Val His Thr Val Met Gly Ile Trp 125 130 135Met Val
Ser Phe Ile Leu Ser Ala Leu Pro Ala Val Gly Trp His 140 145 150Asp
Thr Ser Glu Arg Phe Tyr Thr His Gly Cys Arg Phe Ile Val 155 160
165Ala Glu Ile Gly Leu Gly Phe Gly Val Cys Phe Leu Leu Leu Val 170
175 180Gly Gly Ser Val Ala Met Gly Val Ile Cys Thr Ala Ile Ala Leu
185 190 195Phe Gln Thr Leu Ala Val Gln Val Gly Arg Gln Ala Asp His
Arg 200 205 210Ala Phe Thr Val Pro Thr Ile Val Val Glu Asp Ala Gln
Gly Lys 215 220 225Arg Arg Ser Ser Ile Asp Gly Ser Glu Pro Ala Lys
Thr Ser Leu 230 235 240Gln Thr Thr Gly Leu Val Thr Thr Ile Val Phe
Ile Tyr Asp Cys 245 250 255Leu Met Gly Phe Pro Val Leu Val Val Ser
Phe Ser Ser Leu Arg 260 265 270Ala Asp Ala Ser Ala Pro Trp Met Ala
Leu Cys Val Leu Trp Cys 275 280 285Ser Val Ala Gln Ala Leu Leu Leu
Pro Val Phe Leu Trp Ala Cys 290 295 300Asp Arg Tyr Arg Ala Asp Leu
Lys Ala Val Arg Glu Lys Cys Met 305 310 315Ala Leu Met Ala Asn Asp
Glu Glu Ser Asp Asp Glu Thr Ser Leu 320 325 330Glu Gly Gly Ile Ser
Pro Asp Leu Val Leu Glu Arg Ser Leu Asp 335 340 345Tyr Gly Tyr Gly
Gly Asp Phe Val Ala Leu Asp Arg Met Ala Lys 350 355 360Tyr Glu Ile
Ser Ala Leu Glu Gly Gly Leu Pro Gln Leu Tyr Pro 365 370 375Leu Arg
Pro Leu Gln Glu Asp Lys Met Gln Tyr Leu Gln Val Pro 380 385 390Pro
Thr Arg Arg Phe Ser His Asp Asp Ala Asp Val Trp Ala Ala 395 400
405Val Pro Leu Pro Ala Phe Leu Pro Arg Trp Gly Ser Gly Lys Asp 410
415 420Leu Ser Ala Leu Ala His Leu Val Leu Pro Ala Gly Pro Glu Arg
425 430 435Pro Arg Ala Ser Leu Leu Ala Phe Ala Glu Asp Ala Pro Leu
Ser 440 445 450Arg Ala Arg Arg Arg Ser Ala Glu Ser Leu Leu Ser Leu
Arg Pro 455 460 465Ser Ala Val Asp Ser Gly Pro Arg Gly Ala Arg Asp
Ser Pro Pro 470 475 480Gly Ser Pro Arg Arg Arg Pro Gly Pro Gly Pro
Arg Ser Ala Ser 485 490 495Ala Ser Leu Leu Pro Asp Ala Phe Ala Leu
Thr Ala Phe Glu Cys 500 505 510Glu Pro Gln Ala Leu Arg Arg Pro Pro
Gly Pro Phe Pro Ala Ala 515 520 525Pro Ala Ala Pro Asp Gly Ala Asp
Pro Gly Glu Ala Pro Thr Pro 530 535 540Pro Ser Ser Ala Gln Arg Ser
Pro Gly Pro Arg Pro Ser Ala His 545 550 555Ser His Ala Gly Ser Leu
Arg Pro Gly Leu Ser Ala Ser Trp Gly 560 565 570Glu Pro Gly Gly Leu
Arg Ala Ala Gly Gly Gly Gly Ser Thr Ser 575 580 585Ser Phe Leu Ser
Ser Pro Ser Glu Ser Ser Gly Tyr Ala Thr Leu 590 595 600His Ser Asp
Ser Leu Gly Ser Ala Ser 6055313PRTHomo sapiensmisc_featureIncyte ID
No 7472001CD1 5Met Glu Arg Ile Asn Ser Thr Leu Leu Thr Ala Phe Ile
Leu Thr 1 5 10 15Gly Ile Pro Tyr Pro Leu Arg Leu Arg Thr Leu Phe
Phe Val Phe 20 25 30Phe Phe Leu Ile Tyr Ile Leu Thr Gln Leu Gly Asn
Leu Leu Ile 35 40 45Leu Ile Thr Val Trp Ala Asp Pro Arg Leu His Ala
Arg Pro Met
50 55 60Tyr Ile Phe Leu Gly Val Leu Ser Val Ile Asp Met Ser Ile Ser
65 70 75Ser Ile Ile Val Pro Arg Leu Met Met Asn Phe Thr Leu Gly Val
80 85 90Lys Pro Ile Pro Phe Gly Gly Cys Val Ala Gln Leu Tyr Phe Tyr
95 100 105His Phe Leu Gly Ser Thr Gln Cys Phe Leu Tyr Thr Leu Met
Ala 110 115 120Tyr Asp Arg Tyr Leu Ala Ile Cys Gln Pro Leu Arg Tyr
Pro Val 125 130 135Leu Met Thr Ala Lys Leu Ser Ala Leu Leu Val Ala
Gly Ala Trp 140 145 150Met Ala Gly Ser Ile His Gly Ala Leu Gln Ala
Ile Leu Thr Phe 155 160 165Arg Leu Pro Tyr Cys Gly Pro Asn Gln Val
Asp Tyr Phe Phe Cys 170 175 180Asp Ile Pro Ala Val Leu Arg Leu Ala
Cys Ala Asp Thr Thr Val 185 190 195Asn Glu Leu Val Thr Phe Val Asp
Ile Gly Val Val Val Ala Ser 200 205 210Cys Phe Ser Leu Ile Leu Leu
Ser Tyr Ile Gln Ile Ile Gln Ala 215 220 225Ile Leu Arg Ile His Thr
Ala Asp Gly Arg Arg Arg Ala Phe Ser 230 235 240Thr Cys Gly Ala His
Val Thr Val Val Thr Val Tyr Tyr Val Pro 245 250 255Cys Ala Phe Ile
Tyr Leu Arg Pro Glu Thr Asn Ser Pro Leu Asp 260 265 270Gly Ala Ala
Ala Leu Val Pro Thr Ala Ile Thr Pro Phe Leu Asn 275 280 285Pro Leu
Ile Tyr Thr Leu Arg Asn Gln Glu Val Lys Leu Ala Leu 290 295 300Lys
Arg Met Leu Arg Ser Pro Arg Thr Pro Ser Glu Val 305 3106398PRTHomo
sapiensmisc_featureIncyte ID No 7472003CD1 6Met His Thr Val Ala Thr
Ser Gly Pro Asn Ala Ser Trp Gly Ala 1 5 10 15Pro Ala Asn Ala Ser
Gly Cys Pro Gly Cys Gly Ala Asn Ala Ser 20 25 30Asp Gly Pro Val Pro
Ser Pro Arg Ala Val Asp Ala Trp Leu Val 35 40 45Pro Leu Phe Phe Ala
Ala Leu Met Leu Leu Gly Leu Val Gly Asn 50 55 60Ser Leu Val Ile Tyr
Val Ile Cys Arg His Lys Pro Met Arg Thr 65 70 75Val Thr Asn Phe Tyr
Ile Ala Asn Leu Ala Ala Thr Asp Val Thr 80 85 90Phe Leu Leu Cys Cys
Val Pro Phe Thr Ala Leu Leu Tyr Pro Leu 95 100 105Pro Gly Trp Val
Leu Gly Asp Phe Met Cys Lys Phe Val Asn Tyr 110 115 120Ile Gln Gln
Val Ser Val Gln Ala Thr Cys Ala Thr Leu Thr Ala 125 130 135Met Ser
Val Asp Arg Trp Tyr Val Thr Val Phe Pro Leu Arg Ala 140 145 150Leu
His Arg Arg Thr Pro Arg Leu Ala Leu Ala Val Ser Leu Ser 155 160
165Ile Trp Thr Gly Ser Ala Ala Val Ser Ala Pro Val Leu Ala Leu 170
175 180His Arg Leu Ser Pro Gly Pro Arg Ala Tyr Cys Ser Glu Ala Phe
185 190 195Pro Ser Arg Ala Leu Glu Arg Ala Phe Ala Leu Tyr Asn Leu
Leu 200 205 210Ala Leu Tyr Leu Leu Pro Leu Leu Ala Thr Cys Ala Cys
Tyr Ala 215 220 225Ala Met Leu Arg His Leu Gly Arg Val Ala Val Arg
Pro Ala Pro 230 235 240Ala Asp Ser Ala Leu Gln Gly Gln Val Leu Ala
Glu Arg Ala Gly 245 250 255Ala Val Arg Ala Lys Val Ser Arg Leu Val
Ala Ala Val Val Leu 260 265 270Leu Phe Ala Ala Cys Trp Gly Pro Ile
Gln Leu Phe Leu Val Leu 275 280 285Gln Ala Leu Gly Pro Ala Gly Ser
Trp His Pro Arg Ser Tyr Ala 290 295 300Ala Tyr Ala Leu Lys Thr Trp
Ala His Cys Met Ser Tyr Ser Asn 305 310 315Ser Ala Leu Asn Pro Leu
Leu Tyr Ala Phe Leu Gly Ser His Phe 320 325 330Arg Gln Ala Phe Arg
Arg Val Cys Pro Cys Ala Pro Arg Arg Pro 335 340 345Arg Arg Pro Arg
Arg Pro Gly Pro Ser Asp Pro Ala Ala Pro His 350 355 360Ala Glu Leu
Leu Arg Leu Gly Ser His Pro Ala Pro Ala Arg Ala 365 370 375Gln Lys
Pro Gly Ser Ser Gly Leu Ala Ala Arg Gly Leu Cys Val 380 385 390Leu
Gly Glu Asp Asn Ala Pro Leu 3957369PRTHomo
sapiensmisc_featureIncyte ID No 7472004CD1 7Met Ala Pro Thr Gly Leu
Ser Ser Leu Thr Val Asn Ser Thr Ala 1 5 10 15Val Pro Thr Thr Pro
Ala Ala Phe Lys Ser Leu Asn Leu Pro Leu 20 25 30Gln Ile Thr Leu Ser
Ala Ile Met Ile Phe Ile Leu Phe Val Ser 35 40 45Phe Leu Gly Asn Leu
Val Val Cys Leu Met Val Tyr Gln Lys Ala 50 55 60Ala Met Arg Ser Ala
Ile Asn Ile Leu Leu Ala Ser Leu Ala Phe 65 70 75Ala Asp Met Leu Leu
Ala Val Leu Asn Met Pro Phe Ala Leu Val 80 85 90Thr Ile Leu Thr Thr
Arg Trp Ile Phe Gly Lys Phe Phe Cys Arg 95 100 105Val Ser Ala Met
Phe Phe Trp Leu Phe Val Ile Glu Gly Val Ala 110 115 120Ile Leu Leu
Ile Ile Ser Ile Asp Arg Phe Leu Ile Ile Val Gln 125 130 135Arg Gln
Asp Lys Leu Asn Pro Tyr Arg Ala Lys Val Leu Ile Ala 140 145 150Val
Ser Trp Ala Thr Ser Phe Cys Val Ala Phe Pro Leu Ala Val 155 160
165Gly Asn Pro Asp Leu Gln Ile Pro Ser Arg Ala Pro Gln Cys Val 170
175 180Phe Gly Tyr Thr Thr Asn Pro Gly Tyr Gln Ala Tyr Val Ile Leu
185 190 195Ile Ser Leu Ile Ser Phe Phe Ile Pro Phe Leu Val Ile Leu
Tyr 200 205 210Ser Phe Met Gly Ile Leu Asn Thr Leu Arg His Asn Ala
Leu Arg 215 220 225Ile His Ser Tyr Pro Glu Gly Ile Cys Leu Ser Gln
Ala Ser Lys 230 235 240Leu Gly Leu Met Ser Leu Gln Arg Pro Phe Gln
Met Ser Ile Asp 245 250 255Met Gly Phe Lys Thr Arg Ala Phe Thr Thr
Ile Leu Ile Leu Phe 260 265 270Ala Val Phe Ile Val Cys Trp Ala Pro
Phe Thr Thr Tyr Ser Leu 275 280 285Val Ala Thr Phe Ser Lys His Phe
Tyr Tyr Gln His Asn Phe Phe 290 295 300Glu Ile Ser Thr Trp Leu Leu
Trp Leu Cys Tyr Leu Lys Ser Ala 305 310 315Leu Asn Pro Leu Ile Tyr
Tyr Trp Arg Ile Lys Lys Phe His Asp 320 325 330Ala Cys Leu Asp Met
Met Pro Lys Ser Phe Lys Phe Leu Pro Gln 335 340 345Leu Pro Gly His
Thr Lys Arg Arg Ile Arg Pro Ser Ala Val Tyr 350 355 360Val Cys Gly
Glu His Arg Thr Val Val 3658194PRTHomo sapiensmisc_featureIncyte ID
No 7475687CP1 8Met Ala Tyr Asp Arg Tyr Leu Ala Ile Cys Gln Pro Leu
Arg Tyr 1 5 10 15Pro Val Leu Met Asn Gly Arg Leu Cys Thr Val Leu
Val Ala Gly 20 25 30Ala Trp Val Ala Gly Ser Met His Gly Ser Ile Gln
Ala Thr Leu 35 40 45Thr Phe Arg Leu Pro Tyr Cys Gly Pro Asn Gln Val
Asp Tyr Phe 50 55 60Ile Cys Asp Ile Pro Ala Val Leu Arg Leu Ala Cys
Ala Asp Thr 65 70 75Thr Val Asn Glu Leu Val Thr Phe Val Asp Ile Gly
Val Val Ala 80 85 90Ala Ser Cys Phe Met Leu Ile Leu Leu Ser Tyr Ala
Asn Ile Val 95 100 105Asn Ala Ile Leu Lys Ile Arg Thr Thr Asp Gly
Arg Arg Arg Ala 110 115 120Phe Ser Thr Cys Gly Ser His Leu Ile Val
Val Thr Val Tyr Tyr 125 130 135Val Pro Cys Ile Phe Ile Tyr Leu Arg
Ala Gly Ser Lys Gly Pro 140 145 150Leu Asp Gly Ala Ala Ala Val Phe
Tyr Thr Val Val Thr Pro Leu 155 160 165Leu Asn Pro Leu Ile Tyr Thr
Leu Arg Asn Gln Glu Val Lys Ser 170 175 180Ala Leu Lys Arg Ile Thr
Ala Gly Gln Ala Asp Val Asn Asn 185 1909173PRTHomo
sapiensmisc_featureIncyte ID No 7483029CP1 9Met Tyr Leu Val Thr Val
Leu Gly Asn Leu Leu Ile Ile Leu Ala 1 5 10 15Thr Ile Ser Asp Ser
His Leu His Thr Pro Met Tyr Phe Phe Leu 20 25 30Ser Asn Leu Ser Phe
Ala Asp Ile Cys Phe Val Ser Thr Thr Val 35 40 45Pro Lys Met Leu Val
Asn Ile Gln Thr Gln Ser Arg Val Ile Thr 50 55 60Tyr Ala Asp Cys Ile
Thr Gln Met Cys Phe Phe Ile Leu Phe Val 65 70 75Val Leu Asp Ser Leu
Leu Leu Thr Val Met Ala Tyr Asp Arg Phe 80 85 90Val Ala Ile Cys His
Pro Leu His Tyr Thr Val Ile Met Asn Ser 95 100 105Trp Leu Cys Gly
Leu Leu Val Leu Val Ser Trp Ile Val Ser Ile 110 115 120Leu Tyr Ser
Leu Leu Gln Ser Ile Met Ala Leu Gln Leu Ser Phe 125 130 135Cys Thr
Glu Leu Lys Ile Pro His Phe Phe Cys Glu Leu Asn Gln 140 145 150Val
Ile His Leu Ala Cys Ser Asp Thr Phe Ile Asn Asp Met Met 155 160
165Met Asn Phe Thr Ser Val Leu Leu 17010220PRTHomo
sapiensmisc_featureIncyte ID No 7477933CP1 10Pro Met Tyr Phe Phe
Leu Ser Asn Leu Cys Trp Ala Asp Ile Gly 1 5 10 15Leu Thr Ser Ala
Thr Val Pro Lys Val Ile Leu Asp Met Gln Ser 20 25 30His Ser Arg Val
Ile Ser His Val Gly Cys Leu Thr Gln Met Ser 35 40 45Phe Leu Val Leu
Phe Ala Cys Ile Glu Gly Met Leu Leu Thr Val 50 55 60Met Ala Tyr Gly
Cys Phe Val Ala Ile Cys Arg Pro Leu His Tyr 65 70 75Pro Val Ile Val
Asn Pro His Leu Cys Val Phe Phe Val Leu Val 80 85 90Ser Phe Phe Leu
Asn Leu Leu Asp Ser Gln Leu His Ser Trp Ile 95 100 105Val Leu Gln
Phe Thr Ile Ile Lys Asn Val Glu Ile Ser Asn Phe 110 115 120Phe Cys
Asp Pro Ser Gln Leu Leu Asn Leu Ala Cys Ser Asp Ser 125 130 135Val
Ile Asn Ser Ile Phe Ile Tyr Phe Asp Ser Thr Met Phe Gly 140 145
150Phe Leu Pro Ile Ser Gly Ile Leu Leu Ser Tyr Tyr Lys Ile Val 155
160 165Pro Ser Ile Leu Arg Met Ser Ser Ser Asp Gly Lys Tyr Lys Ala
170 175 180Phe Ser Thr Tyr Gly Ser His Leu Gly Val Val Cys Trp Phe
Tyr 185 190 195Gly Thr Val Ile Gly Met Tyr Leu Ala Ser Ala Val Ser
Pro Pro 200 205 210Pro Arg Asn Gly Val Val Ala Ser Val Met 215
22011302PRTHomo sapiensmisc_featureIncyte ID No 7475164CP1 11Ala
Glu Phe Ile Leu Ala Gly Leu Thr Gln Arg Pro Glu Leu Gln 1 5 10
15Leu Pro Leu Phe Leu Leu Phe Leu Gly Ile Tyr Val Val Thr Val 20 25
30Val Gly Asn Leu Gly Met Ile Phe Leu Ile Ala Leu Ser Ser Gln 35 40
45Leu Tyr Pro Pro Val Tyr Tyr Phe Leu Ser His Leu Ser Phe Ile 50 55
60Asp Leu Cys Tyr Ser Ser Val Ile Thr Pro Lys Met Leu Val Asn 65 70
75Phe Val Pro Glu Glu Asn Ile Ile Ser Phe Leu Glu Cys Ile Thr 80 85
90Gln Leu Tyr Phe Phe Leu Ile Phe Val Ile Ala Glu Gly Tyr Leu 95
100 105Leu Thr Ala Met Glu Tyr Asp Arg Tyr Val Ala Ile Cys Arg Pro
110 115 120Leu Leu Tyr Asn Ile Val Met Ser His Arg Val Cys Ser Ile
Met 125 130 135Met Ala Val Val Tyr Ser Leu Gly Phe Leu Trp Ala Thr
Val His 140 145 150Thr Thr Arg Met Ser Val Leu Ser Phe Cys Arg Ser
His Thr Val 155 160 165Ser His Tyr Phe Cys Asp Ile Leu Pro Leu Leu
Thr Leu Ser Cys 170 175 180Ser Ser Thr His Ile Asn Glu Ile Leu Leu
Phe Ile Ile Gly Gly 185 190 195Val Asn Thr Leu Ala Thr Thr Leu Ala
Val Leu Ile Ser Tyr Ala 200 205 210Phe Ile Phe Ser Ser Ile Leu Gly
Ile His Ser Thr Glu Gly Gln 215 220 225Ser Lys Ala Phe Gly Thr Cys
Ser Ser His Leu Leu Ala Val Gly 230 235 240Ile Phe Phe Gly Ser Ile
Thr Phe Met Tyr Phe Lys Pro Pro Ser 245 250 255Ser Thr Thr Met Glu
Lys Glu Lys Val Ser Ser Val Phe Tyr Ile 260 265 270Thr Ile Ile Pro
Met Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn 275 280 285Lys Asp Val
Lys Asn Ala Leu Lys Lys Met Thr Arg Gly Arg Gln 290 295 300Ser
Ser12110PRTHomo sapiensmisc_featureIncyte ID No 7473909CP1 12Gly
Pro Arg Thr Ala Ser Gly Cys Val Ile Met Ile Cys Phe Ala 1 5 10
15Leu Thr Val Leu Ser Tyr Ile Arg Ile Leu Ala Thr Val Val Gln 20 25
30Ile Arg Ser Ala Ala Ser Arg Arg Lys Ala Phe Ser Thr Cys Ser 35 40
45Ser His Leu Gly Met Val Leu Leu Phe Tyr Gly Thr Gly Ser Ser 50 55
60Thr Tyr Met Arg Pro Thr Thr Arg Tyr Ser Pro Leu Glu Gly Arg 65 70
75Leu Ala Ala Val Phe Tyr Ser Ile Leu Ile Pro Thr Leu Asn Pro 80 85
90Leu Ile Tyr Ser Leu Arg Asn Gln Asp Met Lys Arg Ala Leu Trp 95
100 105Lys Leu Tyr Leu Gln 11013178PRTHomo
sapiensmisc_featureIncyte ID No 7475252CP1 13Glu Pro Glu Asn Leu
Thr Gly Val Leu Glu Phe Leu Leu Leu Gly 1 5 10 15Leu Pro Asp Asp
Pro Glu Leu Gln Pro Val Leu Phe Gly Leu Phe 20 25 30Leu Ser Met Tyr
Leu Val Met Val Leu Gly Asn Leu Leu Ile Ile 35 40 45Leu Ala Val Ser
Ser Asp Ser His Leu His Ser Pro Met Tyr Phe 50 55 60Phe Leu Ser Asn
Leu Ser Leu Ala Asp Ile Gly Phe Ala Ser Thr 65 70 75Thr Val Pro Lys
Met Ile Val Asp Ile Gln Ala His Ser Arg Leu 80 85 90Ile Ser Tyr Val
Gly Cys Leu Thr Gln Met Ser Phe Leu Ile Phe 95 100 105Phe Ala Cys
Met Glu Ser Leu Leu Leu Ile Val Met Ala Tyr Asp 110 115 120Arg Phe
Val Ala Ile Cys His Pro Leu His Tyr Gln Val Ile Met 125 130 135Ser
Pro Arg Leu Cys Gly Phe Leu Val Leu Val Ser Phe Phe Leu 140 145
150Ser Leu Leu Asp Ser Gln Leu His Asn Leu Ile Val Leu Gln Leu 155
160 165Thr Cys Phe Asn Asp Val Glu Ile Ser Asn Phe Phe Leu 170
1751492PRTHomo sapiensmisc_featureIncyte ID No 7927572CP1 14Leu Leu
Asp Ala Gln Leu Tyr Asn Leu Ile Ala Leu Gln Met Thr 1 5 10 15Cys
Phe Lys Asp Val Glu Ile Pro Asn Phe Phe Cys Asp Pro Ser 20 25 30Gln
Leu Pro His Leu Ala Cys Cys Asp Thr Phe Asn Asn Asn Ile 35 40 45Ile
Leu Tyr Phe Pro Asp Ala Ile Phe Gly Phe Leu Pro Ile Ser 50 55 60Gly
Thr Leu Phe Ser Tyr Asp Lys Ile Val Ser
Ser Ile Leu Arg 65 70 75Val Ser Ser Ser Gly Gly Lys Tyr Lys Ala Phe
Ser Thr Tyr Gly 80 85 90Ser His1597PRTHomo
sapiensmisc_featureIncyte ID No 7481257CP1 15Met Glu Val Thr Thr
Phe Ala Met Cys Leu Ile Ile Val Leu Val 1 5 10 15Pro Leu Leu Leu
Ile Leu Val Ser Tyr Gly Phe Ile Ala Val Ala 20 25 30Val Leu Lys Ile
Lys Ser Ala Ala Gly Arg Gln Lys Ala Phe Gly 35 40 45Thr Cys Ser Ser
His Leu Val Val Val Ser Ile Phe Cys Gly Thr 50 55 60Val Thr Tyr Met
Tyr Ile Gln Pro Gly Asn Ser Pro Asn Gln Asn 65 70 75Glu Gly Lys Leu
Leu Ser Ile Phe Tyr Ser Ile Val Thr Pro Ser 80 85 90Leu Asn Pro Leu
Ile Tyr Thr 9516133PRTHomo sapiensmisc_featureIncyte ID No
7485790CP1 16Asp Pro Glu Leu Gln Pro Ile Leu Ala Gly Leu Ser Leu
Ser Met 1 5 10 15Tyr Leu Val Thr Val Leu Arg Asn Leu Leu Ile Ser
Leu Ala Val 20 25 30Ser Ser Asp Ser His Leu His Thr Pro Met Cys Phe
Phe Leu Ser 35 40 45Asn Leu Cys Trp Ala Asp Ile Gly Phe Thr Ser Ala
Thr Val Pro 50 55 60Lys Met Ile Val Asp Met Arg Ser His Ser Gly Val
Ile Ser Tyr 65 70 75Ala Asp Cys Leu Thr Arg Met Ser Phe Leu Val Leu
Phe Ala Cys 80 85 90Val Glu Asp Met Leu Leu Thr Val Met Ala Tyr Asp
Cys Phe Val 95 100 105Ala Ile Cys Arg Pro Leu His Tyr Pro Val Ile
Val Asn Pro His 110 115 120Leu Cys Val Phe Leu Val Ser Val Ser Phe
Ser Leu Ala 125 13017213PRTHomo sapiensmisc_featureIncyte ID No
7482993CP1 17Gly Ser Glu Cys Leu Leu Leu Ala Ala Met Ala Tyr Asp
Arg Tyr 1 5 10 15Ile Ala Ile Cys Asn Pro Leu Arg Tyr Ser Val Ile
Leu Ser Lys 20 25 30Val Leu Cys Asn Gln Leu Ala Ala Ser Cys Trp Ala
Ala Gly Phe 35 40 45Leu Asn Ser Val Val His Thr Val Leu Thr Phe Cys
Leu Pro Phe 50 55 60Cys Gly Asn Asn Gln Ile Asn Tyr Phe Phe Cys Asp
Ile Pro Pro 65 70 75Leu Leu Ile Leu Ser Cys Gly Asn Thr Ser Val Asn
Glu Leu Ala 80 85 90Leu Leu Ser Thr Gly Val Phe Ile Gly Trp Thr Pro
Phe Leu Cys 95 100 105Ile Val Leu Ser Tyr Ile Cys Ile Ile Ser Thr
Ile Leu Arg Ile 110 115 120Gln Ser Ser Glu Gly Arg Arg Lys Ala Phe
Ser Thr Cys Ala Ser 125 130 135His Leu Ala Ile Val Phe Leu Phe Tyr
Gly Ser Ala Ile Phe Thr 140 145 150Tyr Val Arg Pro Ile Ser Thr Tyr
Ser Leu Lys Lys Asp Arg Leu 155 160 165Val Ser Val Leu Tyr Ser Val
Val Thr Pro Met Leu Asn Pro Ile 170 175 180Ile Tyr Thr Leu Arg Asn
Lys Asp Ile Lys Glu Ala Val Lys Thr 185 190 195Ile Gly Ser Lys Trp
Gln Pro Pro Ile Ser Ser Leu Asp Ser Lys 200 205 210Leu Thr
Tyr18180PRTHomo sapiensmisc_featureIncyte ID No 2829053CD1 18Met
Ser Glu Ala Ala Thr Arg Trp Ser Cys Gln Gly Ser Cys Gln 1 5 10
15Lys Thr Cys Phe Ser Arg Val Arg Pro Trp Arg Arg Arg Cys Ser 20 25
30Cys Gly Asp Ser Ser Ser Arg Arg Arg Arg Ser Cys Cys Thr Gly 35 40
45Ser Leu Gly Pro Met Pro Arg Leu Pro Ser Leu Trp Pro Leu Ser 50 55
60Leu Pro Leu Arg Ser Leu Ser Ser Pro His Arg Val Gln Gly Leu 65 70
75Gly Pro Pro Arg Arg Leu Lys Ser Gln Leu Leu Pro Arg Phe Phe 80 85
90Trp Arg Arg Gln Gln Glu Pro Leu Ser Ser Phe Pro Gly Arg Asn 95
100 105Glu Gly Gly Ser Glu Met Glu Ile Leu Gly Val Cys Pro Val Ser
110 115 120Pro Gly Ala Leu Ser Tyr Met Glu Ser Pro Thr Gly Phe Trp
Arg 125 130 135Pro Arg Glu Ala Ser Ser Leu Glu Leu Ala Lys Gly Ile
Ser Lys 140 145 150Arg Arg His Phe Leu Pro Ala Pro Ala Leu Cys Pro
Asn Pro Arg 155 160 165Ser Ser Glu Ala Phe Pro Gly Ala Val Cys Val
Thr Leu Ala Ile 170 175 18019353PRTHomo sapiensmisc_featureIncyte
ID No 3068234CD1 19Met Asn Glu Cys His Tyr Asp Lys His Met Asp Phe
Phe Tyr Asn 1 5 10 15Arg Ser Asn Thr Asp Thr Val Asp Asp Trp Thr
Gly Thr Lys Leu 20 25 30Val Ile Val Leu Cys Val Gly Thr Phe Phe Cys
Leu Phe Ile Phe 35 40 45Phe Ser Asn Ser Leu Val Ile Ala Ala Val Ile
Lys Asn Arg Lys 50 55 60Phe His Phe Pro Phe Tyr Tyr Leu Leu Ala Asn
Leu Ala Ala Ala 65 70 75Asp Phe Phe Ala Gly Ile Ala Tyr Val Phe Leu
Met Phe Asn Thr 80 85 90Gly Pro Val Ser Lys Thr Leu Thr Val Asn Arg
Trp Phe Leu Arg 95 100 105Gln Gly Leu Leu Asp Ser Ser Leu Thr Ala
Ser Leu Thr Asn Leu 110 115 120Leu Val Ile Ala Val Glu Arg His Met
Ser Ile Met Arg Met Arg 125 130 135Val His Ser Asn Leu Thr Lys Lys
Arg Val Thr Leu Leu Ile Leu 140 145 150Leu Val Trp Ala Ile Ala Ile
Phe Met Gly Ala Val Pro Thr Leu 155 160 165Gly Trp Asn Cys Leu Cys
Asn Ile Ser Ala Cys Ser Ser Leu Ala 170 175 180Pro Ile Tyr Ser Arg
Ser Tyr Leu Val Phe Trp Thr Val Ser Asn 185 190 195Leu Met Ala Phe
Leu Ile Met Val Val Val Tyr Leu Arg Ile Tyr 200 205 210Val Tyr Val
Lys Arg Lys Thr Asn Val Leu Ser Pro His Thr Ser 215 220 225Gly Ser
Ile Ser Arg Arg Arg Thr Pro Met Lys Leu Met Lys Thr 230 235 240Val
Met Thr Val Leu Gly Ala Phe Val Val Cys Trp Thr Pro Gly 245 250
255Leu Val Val Leu Leu Leu Asp Gly Leu Asn Cys Arg Gln Cys Gly 260
265 270Val Gln His Val Lys Arg Trp Phe Leu Leu Leu Ala Leu Leu Asn
275 280 285Ser Val Val Asn Pro Ile Ile Tyr Ser Tyr Lys Asp Glu Asp
Met 290 295 300Tyr Gly Thr Met Lys Lys Met Ile Cys Cys Phe Ser Gln
Glu Asn 305 310 315Pro Glu Arg Arg Pro Ser Arg Ile Pro Ser Thr Val
Leu Ser Arg 320 325 330Ser Asp Thr Gly Ser Gln Tyr Ile Glu Asp Ser
Ile Ser Gln Gly 335 340 345Ala Val Cys Asn Lys Ser Thr Ser
35020361PRTHomo sapiensmisc_featureIncyte ID No 5029478CD1 20Met
Ser Pro Glu Cys Ala Arg Ala Ala Gly Asp Ala Pro Leu Arg 1 5 10
15Ser Leu Glu Gln Ala Asn Arg Thr Arg Phe Pro Phe Phe Ser Asp 20 25
30Val Lys Gly Asp His Arg Leu Val Leu Ala Ala Val Glu Thr Thr 35 40
45Val Leu Val Leu Ile Phe Ala Val Ser Leu Leu Gly Asn Val Cys 50 55
60Ala Leu Val Leu Val Ala Arg Arg Arg Arg Arg Gly Ala Thr Ala 65 70
75Cys Leu Val Leu Asn Leu Phe Cys Ala Asp Leu Leu Phe Ile Ser 80 85
90Ala Ile Pro Leu Val Leu Ala Val Arg Trp Thr Glu Ala Trp Leu 95
100 105Leu Gly Pro Val Ala Cys His Leu Leu Phe Tyr Val Met Thr Leu
110 115 120Ser Gly Ser Val Thr Ile Leu Thr Leu Ala Ala Val Ser Leu
Glu 125 130 135Arg Met Val Cys Ile Val His Leu Gln Arg Gly Val Arg
Gly Pro 140 145 150Gly Arg Arg Ala Arg Ala Val Leu Leu Ala Leu Ile
Trp Gly Tyr 155 160 165Ser Ala Val Ala Ala Leu Pro Leu Cys Val Phe
Phe Arg Val Val 170 175 180Pro Gln Arg Leu Pro Gly Ala Asp Gln Glu
Ile Ser Ile Cys Thr 185 190 195Leu Ile Trp Pro Thr Ile Pro Gly Glu
Ile Ser Trp Asp Val Ser 200 205 210Phe Val Thr Leu Asn Phe Leu Val
Pro Gly Leu Val Ile Val Ile 215 220 225Ser Tyr Ser Lys Ile Leu Gln
Ile Thr Lys Ala Ser Arg Lys Arg 230 235 240Leu Thr Val Ser Leu Ala
Tyr Ser Glu Ser His Gln Ile Arg Val 245 250 255Ser Gln Gln Asp Phe
Arg Leu Phe Arg Thr Leu Phe Leu Leu Met 260 265 270Val Ser Phe Phe
Ile Met Trp Ser Pro Ile Ile Ile Thr Ile Leu 275 280 285Leu Ile Leu
Ile Gln Asn Phe Lys Gln Asp Leu Val Ile Trp Pro 290 295 300Ser Leu
Phe Phe Trp Val Val Ala Phe Thr Phe Ala Asn Ser Ala 305 310 315Leu
Asn Pro Ile Leu Tyr Asn Met Thr Leu Cys Arg Asn Glu Trp 320 325
330Lys Lys Ile Phe Cys Cys Phe Trp Phe Pro Glu Lys Gly Ala Ile 335
340 345Leu Thr Asp Thr Ser Val Lys Arg Asn Asp Leu Ser Ile Ile Ser
350 355 360Gly21251PRTHomo sapiensmisc_featureIncyte ID No
5102576CD1 21Met Tyr Leu Val Thr Val Leu Arg Asn Leu Phe Ser Ile
Leu Ala 1 5 10 15Val Ser Ser Asp Cys Pro Leu His Thr Pro Met Tyr
Phe Phe Leu 20 25 30Ser Asn Leu Cys Trp Pro Asp Ile Gly Phe Thr Ser
Ala Met Val 35 40 45Pro Lys Met Ile Val Asp Thr Gln Ser His Ser Arg
Val Ile Ser 50 55 60His Ala Gly Cys Leu Thr Gln Met Ser Phe Leu Leu
Leu Val Ala 65 70 75Cys Ile Glu Gly Met Leu Leu Thr Val Met Ala Tyr
Asp Cys Phe 80 85 90Val Ala Ile Cys Arg Pro Leu His Tyr Pro Val Ile
Val Asn Pro 95 100 105His Leu Cys Val Phe Phe Val Leu Val Ser Phe
Phe Leu Ser Leu 110 115 120Leu Asp Ser Gln Leu His Ser Trp Ile Val
Leu Gln Leu Thr Ile 125 130 135Ile Lys Asn Val Glu Ile Ser Asn Leu
Val Cys Asp Pro Ser Gln 140 145 150Leu Leu Asn Leu Ala Cys Ser Asp
Ser Val Ile Asn Asn Ile Phe 155 160 165Ile Tyr Phe Asp Ser Thr Met
Phe Gly Phe Leu Pro Ile Ser Gly 170 175 180Ile Phe Leu Ser Tyr Tyr
Lys Ile Val Pro Ser Ile Leu Arg Ile 185 190 195Ser Ser Ser Asp Gly
Lys Tyr Lys Ala Phe Ser Thr Cys Gly Cys 200 205 210His Leu Ala Val
Val Cys Trp Phe Tyr Gly Thr Gly Ile Gly Met 215 220 225Tyr Leu Thr
Ser Ala Val Ser Pro Pro Pro Arg Asn Gly Val Val 230 235 240Ala Ser
Val Met Tyr Ala Val Val Thr Pro Cys 245 25022315PRTHomo
sapiensmisc_featureIncyte ID No 2200534CD1 22Met Lys Ala Asn Tyr
Ser Ala Glu Glu Arg Phe Leu Leu Leu Gly 1 5 10 15Phe Ser Asp Trp
Pro Ser Leu Gln Pro Val Leu Phe Ala Leu Val 20 25 30Leu Leu Cys Tyr
Leu Leu Thr Leu Thr Gly Asn Ser Ala Leu Val 35 40 45Leu Leu Ala Val
Arg Asp Pro Arg Leu His Thr Pro Met Tyr Tyr 50 55 60Phe Leu Cys His
Leu Ala Leu Val Asp Ala Gly Phe Thr Thr Ser 65 70 75Val Val Pro Pro
Leu Leu Ala Asn Leu Arg Gly Pro Ala Leu Trp 80 85 90Leu Pro Arg Ser
His Cys Thr Ala Gln Leu Cys Ala Ser Leu Ala 95 100 105Leu Gly Ser
Ala Glu Cys Val Leu Leu Ala Val Met Ala Leu Asp 110 115 120Arg Ala
Ala Ala Val Cys Arg Pro Leu Arg Tyr Ala Gly Leu Val 125 130 135Ser
Pro Arg Leu Cys Arg Thr Leu Ala Ser Ala Ser Trp Leu Ser 140 145
150Gly Leu Thr Asn Ser Val Ala Gln Thr Ala Leu Leu Ala Glu Arg 155
160 165Pro Leu Cys Ala Pro Arg Leu Leu Asp His Phe Ile Cys Glu Leu
170 175 180Pro Ala Leu Leu Lys Leu Ala Cys Gly Gly Asp Gly Asp Thr
Thr 185 190 195Glu Asn Gln Met Phe Ala Ala Arg Val Val Ile Leu Leu
Leu Pro 200 205 210Phe Ala Val Ile Leu Ala Ser Tyr Gly Ala Val Ala
Arg Ala Val 215 220 225Cys Cys Met Arg Phe Ser Gly Gly Arg Arg Arg
Ala Val Gly Thr 230 235 240Cys Gly Ser His Leu Thr Ala Val Cys Leu
Phe Tyr Gly Ser Ala 245 250 255Ile Tyr Thr Tyr Leu Gln Pro Ala Gln
Arg Tyr Asn Gln Ala Arg 260 265 270Gly Lys Phe Val Ser Leu Phe Tyr
Thr Val Val Thr Pro Ala Leu 275 280 285Asn Pro Leu Ile Tyr Thr Leu
Arg Asn Lys Lys Val Lys Gly Ala 290 295 300Ala Arg Arg Leu Leu Arg
Ser Leu Gly Arg Gly Gln Ala Gly Gln 305 310 31523470PRTHomo
sapiensmisc_featureIncyte ID No 3275821CD1 23Met Asp Thr Thr Met
Glu Ala Asp Leu Gly Ala Thr Gly His Arg 1 5 10 15Pro Arg Thr Glu
Leu Asp Asp Glu Asp Ser Tyr Pro Gln Gly Gly 20 25 30Trp Asp Thr Val
Phe Leu Val Ala Leu Leu Leu Leu Gly Leu Pro 35 40 45Ala Asn Gly Leu
Met Ala Trp Leu Ala Gly Ser Gln Ala Arg His 50 55 60Gly Ala Gly Thr
Arg Leu Ala Leu Leu Leu Leu Ser Leu Ala Leu 65 70 75Ser Asp Phe Leu
Phe Leu Ala Ala Ala Ala Phe Gln Ile Leu Glu 80 85 90Ile Arg His Gly
Gly His Trp Pro Leu Gly Thr Ala Ala Cys Arg 95 100 105Phe Tyr Tyr
Phe Leu Trp Gly Val Ser Tyr Ser Ser Gly Leu Phe 110 115 120Leu Leu
Ala Ala Leu Ser Leu Asp Arg Cys Leu Leu Ala Leu Cys 125 130 135Pro
His Trp Tyr Pro Gly His Arg Pro Val Arg Leu Pro Leu Trp 140 145
150Val Cys Ala Gly Val Trp Val Leu Ala Thr Leu Phe Ser Val Pro 155
160 165Trp Leu Val Phe Pro Glu Ala Ala Val Trp Trp Tyr Asp Leu Val
170 175 180Ile Cys Leu Asp Phe Trp Asp Ser Glu Glu Leu Ser Leu Arg
Met 185 190 195Leu Glu Val Leu Gly Gly Phe Leu Pro Phe Leu Leu Leu
Leu Val 200 205 210Cys His Val Leu Thr Gln Ala Thr Ala Cys Arg Thr
Cys His Arg 215 220 225Gln Gln Gln Pro Ala Ala Cys Arg Gly Phe Ala
Arg Val Ala Arg 230 235 240Thr Ile Leu Ser Ala Tyr Val Val Leu Arg
Leu Pro Tyr Gln Leu 245 250 255Ala Gln Leu Leu Tyr Leu Ala Phe Leu
Trp Asp Val Tyr Ser Gly 260 265 270Tyr Leu Leu Trp Glu Ala Leu Val
Tyr Ser Asp Tyr Leu Ile Leu 275 280 285Leu Asn Ser Cys Leu Ser Pro
Phe Leu Cys Leu Met Ala Ser Ala 290 295 300Asp Leu Arg Thr Leu Leu
Arg Ser Val Leu Ser Ser Phe Ala Ala 305 310 315Ala Leu Cys Glu Glu
Arg Pro Gly Ser Phe Thr Pro Thr Glu Pro 320 325 330Gln Thr Gln Leu
Asp Ser
Glu Gly Pro Thr Leu Pro Glu Pro Met 335 340 345Ala Glu Ala Gln Ser
Gln Met Asp Pro Val Ala Gln Pro Gln Val 350 355 360Asn Pro Thr Leu
Gln Pro Arg Ser Asp Pro Thr Ala Gln Pro Gln 365 370 375Leu Asn Pro
Thr Ala Gln Pro Gln Ser Asp Pro Thr Ala Gln Pro 380 385 390Gln Leu
Asn Leu Met Ala Gln Pro Gln Ser Asp Ser Val Ala Gln 395 400 405Pro
Gln Ala Asp Thr Asn Val Gln Thr Pro Ala Pro Ala Ala Ser 410 415
420Ser Val Pro Ser Pro Cys Asp Glu Ala Ser Pro Thr Pro Ser Ser 425
430 435His Pro Thr Pro Gly Ala Leu Glu Asp Pro Ala Thr Pro Pro Ala
440 445 450Ser Glu Gly Glu Ser Pro Ser Ser Thr Pro Pro Glu Ala Ala
Pro 455 460 465Gly Ala Gly Pro Thr 47024358PRTHomo
sapiensmisc_featureIncyte ID No 3744167CD1 24Met Ser Val Cys Tyr
Arg Pro Pro Gly Asn Glu Thr Leu Leu Ser 1 5 10 15Trp Lys Thr Ser
Arg Ala Thr Gly Thr Ala Phe Leu Leu Leu Ala 20 25 30Ala Leu Leu Gly
Leu Pro Gly Asn Gly Phe Val Val Trp Ser Leu 35 40 45Ala Gly Trp Arg
Pro Ala Arg Gly Arg Pro Leu Ala Ala Thr Leu 50 55 60Val Leu His Leu
Ala Leu Ala Asp Gly Ala Val Leu Leu Leu Thr 65 70 75Pro Leu Phe Val
Ala Phe Leu Thr Arg Gln Ala Trp Pro Leu Gly 80 85 90Gln Ala Gly Cys
Lys Ala Val Tyr Tyr Val Cys Ala Leu Ser Met 95 100 105Tyr Ala Ser
Val Leu Leu Thr Gly Leu Leu Ser Leu Gln Arg Cys 110 115 120Leu Ala
Val Thr Arg Pro Phe Leu Ala Pro Arg Leu Arg Ser Pro 125 130 135Ala
Leu Ala Arg Arg Leu Leu Leu Ala Val Trp Leu Ala Ala Leu 140 145
150Leu Leu Ala Val Pro Ala Ala Val Tyr Arg His Leu Trp Arg Asp 155
160 165Arg Val Cys Gln Leu Cys His Pro Ser Pro Val His Ala Ala Ala
170 175 180His Leu Ser Leu Glu Thr Leu Thr Ala Phe Val Leu Pro Phe
Gly 185 190 195Leu Met Leu Gly Cys Tyr Ser Val Thr Leu Ala Arg Leu
Arg Gly 200 205 210Ala Arg Trp Gly Ser Gly Arg His Gly Ala Arg Val
Gly Arg Leu 215 220 225Val Ser Ala Ile Val Leu Ala Phe Gly Leu Leu
Trp Ala Pro Tyr 230 235 240His Ala Val Asn Leu Leu Gln Ala Val Ala
Ala Leu Ala Pro Pro 245 250 255Glu Gly Ala Leu Ala Lys Leu Gly Gly
Ala Gly Gln Ala Ala Arg 260 265 270Ala Gly Thr Thr Ala Leu Ala Phe
Phe Ser Ser Ser Val Asn Pro 275 280 285Val Leu Tyr Val Phe Thr Ala
Gly Asp Leu Leu Pro Arg Ala Gly 290 295 300Pro Arg Phe Leu Thr Arg
Leu Phe Glu Gly Ser Gly Glu Ala Arg 305 310 315Gly Gly Gly Arg Ser
Arg Glu Gly Thr Met Glu Leu Arg Thr Thr 320 325 330Pro Gln Leu Lys
Val Val Gly Gln Gly Arg Gly Asn Gly Asp Pro 335 340 345Gly Gly Gly
Met Glu Lys Asp Gly Pro Glu Trp Asp Leu 350 35525314PRTHomo
sapiensmisc_featureIncyte ID No 7472007CD1 25Met Trp Glu Asn Trp
Thr Ile Val Ser Glu Phe Val Leu Val Ser 1 5 10 15Phe Ser Ala Leu
Ser Thr Glu Leu Gln Ala Leu Leu Phe Leu Leu 20 25 30Phe Leu Thr Ile
Tyr Leu Val Thr Leu Met Gly Asn Val Leu Ile 35 40 45Ile Leu Val Thr
Ile Ala Asp Ser Ala Leu Gln Ser Pro Met Tyr 50 55 60Phe Phe Leu Arg
Asn Leu Ser Phe Leu Glu Ile Gly Phe Asn Leu 65 70 75Val Ile Val Pro
Lys Met Leu Gly Thr Leu Ile Ile Gln Asp Thr 80 85 90Thr Ile Ser Phe
Leu Gly Cys Ala Thr Gln Met Tyr Phe Phe Phe 95 100 105Phe Phe Gly
Ala Ala Glu Cys Cys Leu Leu Ala Thr Met Ala Tyr 110 115 120Asp Arg
Tyr Val Ala Ile Cys Asp Pro Leu His Tyr Pro Val Ile 125 130 135Met
Gly His Ile Ser Cys Ala Gln Leu Ala Ala Ala Ser Trp Phe 140 145
150Ser Gly Phe Ser Val Ala Thr Val Gln Thr Thr Trp Ile Phe Ser 155
160 165Phe Pro Phe Cys Gly Pro Asn Arg Val Asn His Phe Phe Cys Asp
170 175 180Ser Pro Pro Val Ile Ala Leu Val Cys Ala Asp Thr Ser Val
Phe 185 190 195Glu Leu Glu Ala Leu Thr Ala Thr Val Pro Phe Ile Leu
Phe Pro 200 205 210Phe Leu Leu Ile Leu Gly Ser Tyr Val Arg Ile Leu
Ser Thr Ile 215 220 225Phe Arg Met Pro Ser Ala Glu Gly Lys His Gln
Ala Phe Ser Thr 230 235 240Cys Ser Ala His Leu Leu Val Val Ser Leu
Phe Tyr Ser Thr Ala 245 250 255Ile Leu Thr Tyr Phe Arg Pro Gln Ser
Ser Ala Ser Ser Glu Ser 260 265 270Lys Lys Leu Leu Ser Leu Ser Ser
Thr Val Val Thr Pro Met Leu 275 280 285Asn Pro Ile Ile Tyr Ser Ser
Arg Asn Lys Glu Val Lys Ala Ala 290 295 300Leu Lys Arg Leu Ile His
Arg Thr Leu Gly Ser Gln Lys Leu 305 31026365PRTHomo
sapiensmisc_featureIncyte ID No 7472008CD1 26Met Glu Gly Ser Val
Glu Ala Thr Pro Glu Ile Pro Ala Gln Met 1 5 10 15Lys Cys His Pro
Ser Arg Pro Ser Thr Leu Asn Gln Leu Ser Phe 20 25 30Tyr Gly Ala Val
Ser Ser Leu Gly Arg Met His Gly Leu Glu Thr 35 40 45Lys Ser Ser Ala
Glu Ile Arg Ala Gly Leu Lys Arg Cys Asp Thr 50 55 60Leu Val Leu Glu
Ala Ser Thr Leu Glu Gly Asn Met Val Ile Val 65 70 75Leu Val Ser Leu
Lys Asp Pro Lys Leu His Ile Pro Met Tyr Phe 80 85 90Phe Leu Ser Asn
Leu Ser Leu Val Asp Leu Cys Leu Thr Ser Ser 95 100 105Cys Val Pro
Gln Met Leu Ile Asn Phe Trp Gly Pro Glu Lys Thr 110 115 120Ile Ser
Tyr Ile Gly Cys Ala Ile Gln Leu Tyr Val Phe Leu Trp 125 130 135Leu
Gly Ala Thr Glu Tyr Val Leu Leu Val Val Met Ala Val Asp 140 145
150Cys Tyr Val Ala Val Cys His Pro Leu Gln Asn Thr Met Ile Met 155
160 165His Pro Lys Leu Cys Leu Gln Leu Ala Ile Leu Ala Trp Gly Thr
170 175 180Gly Leu Ala Gln Ser Leu Ile Gln Ser Pro Ala Thr Leu Arg
Leu 185 190 195Pro Phe Cys Ser Gln Arg Met Val Asp Asp Val Val Cys
Glu Val 200 205 210Pro Ala Leu Ile Gln Leu Ser Ser Thr Asp Thr Thr
Tyr Ser Glu 215 220 225Ile Gln Met Ser Ile Ala Ser Val Val Leu Leu
Val Met Pro Leu 230 235 240Ile Ile Ile Leu Ser Ser Ser Gly Ala Ile
Ala Lys Ala Val Leu 245 250 255Arg Ile Lys Ser Thr Ala Gly Gln Lys
Lys Ala Phe Gly Thr Cys 260 265 270Ile Ser His Leu Leu Val Val Ser
Leu Phe Tyr Gly Thr Val Thr 275 280 285Gly Val Tyr Leu Gln Pro Lys
Asn His Tyr Pro His Glu Trp Gly 290 295 300Lys Phe Leu Thr Leu Phe
Tyr Thr Val Val Thr Pro Thr Leu Asn 305 310 315Pro Leu Ile Tyr Thr
Leu Arg Asn Lys Glu Leu His Pro Trp Leu 320 325 330Lys Glu Ala Lys
Val Gln Thr Ala Ser Glu Ser Ala Ser Pro Lys 335 340 345His Trp Gln
Leu Pro His Gly Val Gly Pro Val Gly Val Gln Lys 350 355 360Thr Arg
Thr Glu Leu 36527317PRTHomo sapiensmisc_featureIncyte ID No
7472013CD1 27Met Ser Phe Ala Pro Asn Ala Ser His Ser Pro Val Phe
Leu Leu 1 5 10 15Leu Gly Phe Ser Arg Ala Asn Ile Ser Tyr Thr Leu
Leu Phe Phe 20 25 30Leu Phe Leu Ala Ile Tyr Leu Thr Thr Ile Leu Gly
Asn Val Thr 35 40 45Leu Val Leu Leu Ile Ser Trp Asp Ser Arg Leu His
Ser Pro Met 50 55 60Tyr Tyr Leu Leu Arg Gly Leu Ser Val Ile Asp Met
Gly Leu Ser 65 70 75Thr Val Thr Leu Pro Gln Leu Leu Ala His Leu Val
Ser His Tyr 80 85 90Pro Thr Ile Pro Ala Ala Arg Cys Leu Ala Gln Phe
Phe Phe Phe 95 100 105Tyr Ala Phe Gly Val Thr Asp Thr Leu Val Ile
Ala Val Met Ala 110 115 120Leu Asp Arg Tyr Val Ala Ile Cys Asp Pro
Leu His Tyr Ala Leu 125 130 135Val Met Asn His Gln Arg Cys Ala Cys
Leu Leu Ala Leu Ser Trp 140 145 150Val Val Ser Ile Leu His Thr Met
Leu Arg Val Gly Leu Val Leu 155 160 165Pro Leu Cys Trp Thr Gly Asp
Ala Gly Gly Asn Val Asn Leu Pro 170 175 180His Phe Phe Cys Asp His
Arg Pro Leu Leu Arg Ala Ser Cys Ser 185 190 195Asp Ile His Ser Asn
Glu Leu Ala Ile Phe Phe Glu Gly Gly Phe 200 205 210Leu Met Leu Gly
Pro Cys Ala Leu Ile Val Leu Ser Tyr Val Arg 215 220 225Ile Gly Ala
Ala Ile Leu Arg Leu Pro Ser Ala Ala Gly Arg Arg 230 235 240Arg Ala
Val Ser Thr Cys Gly Ser His Leu Thr Met Val Gly Phe 245 250 255Leu
Tyr Gly Thr Ile Ile Cys Val Tyr Phe Gln Pro Pro Phe Gln 260 265
270Asn Ser Gln Tyr Gln Asp Met Val Ala Ser Val Met Tyr Thr Ala 275
280 285Ile Thr Pro Leu Ala Asn Pro Phe Val Tyr Ser Leu His Asn Lys
290 295 300Asp Val Lys Gly Ala Leu Cys Arg Leu Leu Glu Trp Val Lys
Val 305 310 315Asp Pro28335PRTHomo sapiensmisc_featureIncyte ID No
7472015CD1 28Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu Ala Val
Leu Ala 1 5 10 15Ser Leu Ile Ile Ala Thr Asn Thr Leu Val Ala Val
Ala Val Leu 20 25 30Leu Leu Ile His Lys Asn Asp Gly Val Ser Leu Cys
Phe Thr Leu 35 40 45Asn Leu Ala Val Ala Asp Thr Leu Ile Gly Val Ala
Ile Ser Gly 50 55 60Leu Leu Thr Asp Gln Leu Ser Ser Pro Ser Arg Pro
Thr Gln Lys 65 70 75Thr Leu Cys Ser Leu Arg Met Ala Phe Val Thr Ser
Ser Ala Ala 80 85 90Ala Ser Val Leu Thr Val Met Leu Ile Thr Phe Asp
Arg Tyr Leu 95 100 105Ala Ile Lys Gln Pro Phe Arg Tyr Leu Lys Ile
Met Ser Gly Phe 110 115 120Val Ala Gly Ala Cys Ile Ala Gly Leu Trp
Leu Val Ser Tyr Leu 125 130 135Ile Gly Phe Leu Pro Leu Gly Ile Pro
Met Phe Gln Gln Thr Ala 140 145 150Tyr Lys Gly Gln Cys Ser Phe Phe
Ala Val Phe His Pro His Phe 155 160 165Val Leu Thr Leu Ser Cys Val
Gly Phe Phe Pro Ala Met Leu Leu 170 175 180Phe Val Phe Phe Tyr Cys
Asp Met Leu Lys Ile Ala Ser Met His 185 190 195Ser Gln Gln Ile Arg
Lys Met Glu His Ala Gly Ala Met Ala Gly 200 205 210Gly Tyr Arg Ser
Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu Arg 215 220 225Thr Val Ser
Val Leu Ile Gly Ser Phe Ala Leu Ser Trp Thr Pro 230 235 240Phe Leu
Ile Thr Gly Ile Val Gln Val Ala Cys Gln Glu Cys His 245 250 255Leu
Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly 260 265
270Asn Ser Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Lys Glu 275
280 285Val Arg Leu Gln Leu Tyr His Met Ala Leu Gly Val Lys Lys Val
290 295 300Leu Thr Ser Phe Leu Leu Phe Leu Ser Ala Arg Asn Cys Gly
Pro 305 310 315Glu Arg Pro Arg Glu Ser Ser Cys His Ile Val Thr Ile
Ser Ser 320 325 330Ser Glu Phe Asp Gly 33529309PRTHomo
sapiensmisc_featureIncyte ID No 7472016CD1 29Met Arg Glu Asn Asn
Gln Ser Ser Thr Leu Glu Phe Ile Leu Leu 1 5 10 15Gly Val Thr Gly
Gln Gln Glu Gln Glu Asp Phe Phe Tyr Ile Leu 20 25 30Phe Leu Phe Ile
Tyr Pro Ile Thr Leu Ile Gly Asn Leu Leu Ile 35 40 45Val Leu Ala Ile
Cys Ser Asp Val Arg Leu His Asn Pro Met Tyr 50 55 60Phe Leu Leu Ala
Asn Leu Ser Leu Val Asp Ile Phe Phe Ser Ser 65 70 75Val Thr Ile Pro
Lys Met Leu Ala Asn His Leu Leu Gly Ser Lys 80 85 90Ser Ile Ser Phe
Gly Gly Cys Leu Thr Gln Met Tyr Phe Met Ile 95 100 105Ala Leu Gly
Asn Thr Asp Ser Tyr Ile Leu Ala Ala Met Ala Tyr 110 115 120Asp Arg
Ala Val Ala Ile Ser His Pro Leu His Tyr Thr Thr Ile 125 130 135Met
Ser Pro Arg Ser Cys Ile Trp Leu Ile Ala Gly Ser Trp Val 140 145
150Ile Gly Asn Ala Asn Ala Leu Pro His Thr Leu Leu Thr Ala Ser 155
160 165Leu Ser Phe Cys Gly Asn Gln Glu Val Ala Asn Phe Tyr Cys Asp
170 175 180Ile Thr Pro Leu Leu Lys Leu Ser Cys Ser Asp Ile His Phe
His 185 190 195Val Lys Met Met Tyr Leu Gly Val Gly Ile Phe Ser Val
Pro Leu 200 205 210Leu Cys Ile Ile Val Ser Tyr Ile Arg Val Phe Ser
Thr Val Phe 215 220 225Gln Val Pro Ser Thr Lys Gly Val Leu Lys Ala
Phe Ser Thr Cys 230 235 240Gly Ser His Leu Thr Val Val Ser Leu Tyr
Tyr Gly Thr Val Met 245 250 255Gly Thr Tyr Phe Arg Pro Leu Thr Asn
Tyr Ser Leu Lys Asp Ala 260 265 270Val Ile Thr Val Met Tyr Thr Ala
Val Thr Pro Met Leu Asn Pro 275 280 285Phe Ile Tyr Ser Leu Arg Asn
Arg Asp Met Lys Ala Ala Leu Arg 290 295 300Lys Leu Phe Asn Lys Arg
Ile Ser Ser 30530236PRTHomo sapiensmisc_featureIncyte ID No
7472017CD1 30Met Gly Met Thr Asn Ser Ser Val Lys Gly Asp Phe Ile
Leu Leu 1 5 10 15Leu Trp Asn Leu Lys Gly Pro Asp Lys Thr Ile Thr
Phe Leu Gly 20 25 30Cys Val Ile Gln Leu Tyr Ile Ser Leu Ala Leu Gly
Ser Thr Glu 35 40 45Cys Val Leu Leu Ala Val Met Ala Phe Asp Arg Tyr
Ala Ala Val 50 55 60Cys Lys Pro Leu His Tyr Thr Ala Val Met Asn Pro
Gln Leu Cys 65 70 75Gln Ala Leu Ala Gly Val Ala Trp Leu Ser Gly Val
Gly Asn Thr 80 85 90Leu Ile Gln Gly Thr Val Thr Leu Trp Leu Pro Arg
Cys Gly His 95 100 105Arg Leu Leu Gln His Phe Phe Leu Ala Cys Val
Asp Ile His Asp 110 115 120Asn Glu Val Gln Leu Phe Val Ala Ser Leu
Val Leu Leu Leu Leu 125 130 135Pro Leu Val Leu Ile Leu Leu Ser Tyr
Gly His Ile
Ala Lys Val 140 145 150Val Ile Arg Ile Lys Ser Val Gln Ala Trp Cys
Lys Gly Leu Gly 155 160 165Thr Cys Gly Ser His Leu Ile Val Val Ser
Leu Phe Cys Gly Thr 170 175 180Ile Thr Ala Val Tyr Ile Gln Ser Asn
Ser Ser Tyr Ala His Ala 185 190 195His Gly Lys Phe Ile Ser Leu Phe
Tyr Thr Val Val Thr Pro Thr 200 205 210Leu Asn Pro Leu Ile Tyr Thr
Leu Arg Asn Asn Asp Val Lys Gly 215 220 225Ala Leu Arg Leu Phe Asn
Arg Asp Leu Gly Thr 230 23531363PRTHomo sapiensmisc_featureIncyte
ID No 7472018CD1 31Met Gly Pro Gly Glu Ala Leu Leu Ala Gly Leu Leu
Val Met Val 1 5 10 15Leu Ala Val Ala Leu Leu Ser Asn Ala Leu Val
Leu Leu Cys Cys 20 25 30Ala Tyr Ser Ala Glu Leu Arg Thr Arg Ala Ser
Gly Val Leu Leu 35 40 45Val Asn Leu Ser Leu Gly His Leu Leu Leu Ala
Ala Leu Asp Met 50 55 60Pro Phe Thr Leu Leu Gly Val Met Arg Gly Arg
Thr Pro Ser Ala 65 70 75Pro Gly Ala Cys Gln Val Ile Gly Phe Leu Asp
Thr Phe Leu Ala 80 85 90Ser Asn Ala Ala Leu Ser Val Ala Ala Leu Ser
Ala Asp Gln Trp 95 100 105Leu Ala Val Gly Phe Pro Leu Arg Tyr Ala
Gly Arg Leu Arg Pro 110 115 120Arg Tyr Ala Gly Leu Leu Leu Gly Cys
Ala Trp Gly Gln Ser Leu 125 130 135Ala Phe Ser Gly Ala Ala Leu Gly
Cys Ser Trp Leu Gly Tyr Ser 140 145 150Ser Ala Phe Ala Ser Cys Ser
Leu Arg Leu Pro Pro Glu Pro Glu 155 160 165Arg Pro Arg Phe Ala Ala
Phe Thr Ala Thr Leu His Ala Val Gly 170 175 180Phe Val Leu Pro Leu
Ala Val Leu Cys Leu Thr Ser Leu Gln Val 185 190 195His Arg Val Ala
Arg Arg His Cys Gln Arg Met Asp Thr Val Thr 200 205 210Met Lys Ala
Leu Ala Leu Leu Ala Asp Leu His Pro Ser Val Arg 215 220 225Gln Arg
Cys Leu Ile Gln Gln Lys Arg Arg Arg His Arg Ala Thr 230 235 240Arg
Lys Ile Gly Ile Ala Ile Ala Thr Phe Leu Ile Cys Phe Ala 245 250
255Pro Tyr Val Met Thr Arg Leu Ala Glu Leu Val Pro Phe Val Thr 260
265 270Val Asn Ala Gln Trp Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser
275 280 285Lys Ala Val Ala Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg
Pro 290 295 300Phe Arg Gln Val Leu Ala Gly Met Val His Arg Leu Leu
Lys Arg 305 310 315Thr Pro Arg Pro Ala Ser Thr His Asp Ser Ser Leu
Asp Val Ala 320 325 330Gly Met Val His Gln Leu Leu Lys Arg Thr Pro
Arg Pro Ala Ser 335 340 345Thr His Asn Gly Ser Val Asp Thr Glu Asn
Asp Ser Cys Leu Gln 350 355 360Gln Thr His32308PRTHomo
sapiensmisc_featureIncyte ID No 7472019CD1 32Met Ala Met Asp Asn
Val Thr Ala Val Phe Gln Phe Leu Leu Ile 1 5 10 15Gly Ile Ser Asn
Tyr Pro Gln Trp Arg Asp Thr Phe Phe Thr Leu 20 25 30Val Leu Ile Ile
Tyr Leu Ser Thr Leu Leu Gly Asn Gly Phe Met 35 40 45Ile Phe Leu Ile
His Phe Asp Pro Asn Leu His Thr Pro Ile Tyr 50 55 60Phe Phe Leu Ser
Asn Leu Ser Phe Leu Asp Leu Cys Tyr Gly Thr 65 70 75Ala Ser Met Pro
Gln Ala Leu Val His Cys Phe Ser Thr His Pro 80 85 90Tyr Leu Ser Tyr
Pro Arg Cys Leu Ala Gln Thr Ser Val Ser Leu 95 100 105Ala Leu Ala
Thr Ala Glu Cys Leu Leu Leu Ala Ala Met Ala Tyr 110 115 120Asp Arg
Val Val Ala Ile Ser Asn Pro Leu Arg Tyr Ser Val Val 125 130 135Met
Asn Gly Pro Val Cys Val Cys Leu Val Ala Thr Ser Trp Gly 140 145
150Thr Ser Leu Val Leu Thr Ala Met Leu Ile Leu Ser Leu Arg Leu 155
160 165His Phe Cys Gly Ala Asn Val Ile Asn His Phe Ala Cys Glu Ile
170 175 180Leu Ser Leu Ile Lys Leu Thr Cys Ser Asp Thr Ser Leu Asn
Glu 185 190 195Phe Met Ile Leu Ile Thr Ser Ile Phe Thr Leu Leu Leu
Pro Phe 200 205 210Gly Phe Val Leu Leu Ser Tyr Ile Arg Ile Ala Met
Ala Ile Ile 215 220 225Arg Ile Arg Ser Leu Gln Gly Arg Leu Lys Ala
Phe Thr Thr Cys 230 235 240Gly Ser His Leu Thr Val Val Thr Ile Phe
Tyr Gly Ser Ala Ile 245 250 255Ser Met Tyr Met Lys Thr Gln Ser Lys
Ser Tyr Pro Asp Gln Asp 260 265 270Lys Phe Ile Ser Val Phe Tyr Gly
Ala Leu Thr Pro Met Leu Asn 275 280 285Pro Leu Ile Tyr Ser Leu Arg
Lys Lys Asp Val Lys Arg Ala Ile 290 295 300Arg Lys Val Met Leu Lys
Arg Thr 30533343PRTHomo sapiensmisc_featureIncyte ID No 7472021CD1
33Met His Phe Leu Pro Thr Val Phe Gly Phe Leu Asn Arg Val Thr 1 5
10 15Leu Gly Ile Phe Arg Glu Thr Met Val Asn Leu Thr Ser Met Ser 20
25 30Gly Phe Leu Leu Met Gly Phe Ser Asp Glu Arg Lys Leu Gln Ile 35
40 45Leu His Ala Leu Val Phe Leu Val Thr Tyr Leu Leu Ala Leu Thr 50
55 60Gly Asn Leu Leu Ile Ile Thr Ile Ile Thr Val Asp Arg Arg Leu 65
70 75His Ser Pro Met Tyr Tyr Phe Leu Lys His Leu Ser Leu Leu Asp 80
85 90Leu Cys Phe Ile Ser Val Thr Val Pro Gln Ser Ile Ala Asn Ser 95
100 105Leu Met Gly Asn Gly Tyr Ile Ser Leu Val Gln Cys Ile Leu Gln
110 115 120Val Phe Phe Phe Ile Ala Leu Ala Ser Ser Glu Val Ala Ile
Leu 125 130 135Thr Val Met Ser Tyr Asp Arg Tyr Ala Ala Ile Cys Gln
Pro Leu 140 145 150His Tyr Glu Thr Ile Met Asp Pro Arg Ala Cys Arg
His Ala Val 155 160 165Ile Ala Val Trp Ile Ala Gly Gly Leu Ser Gly
Leu Met His Ala 170 175 180Ala Ile Asn Phe Ser Ile Pro Leu Cys Gly
Lys Arg Val Ile His 185 190 195Gln Phe Phe Cys Asp Val Pro Gln Met
Leu Lys Leu Ala Cys Ser 200 205 210Tyr Glu Phe Ile Asn Glu Ile Ala
Leu Ala Ala Phe Thr Thr Ser 215 220 225Ala Ala Phe Ile Cys Leu Ile
Ser Ile Val Leu Ser Tyr Ile Arg 230 235 240Ile Phe Ser Thr Val Leu
Arg Ile Pro Ser Ala Glu Gly Arg Thr 245 250 255Lys Val Phe Ser Thr
Cys Leu Pro His Leu Phe Val Ala Thr Phe 260 265 270Phe Leu Ser Ala
Ala Gly Phe Glu Phe Leu Arg Leu Pro Ser Asp 275 280 285Ser Ser Ser
Thr Val Asp Leu Val Phe Ser Val Phe Tyr Thr Val 290 295 300Ile Pro
Pro Thr Leu Asn Pro Val Ile Tyr Ser Leu Arg Asn Asp 305 310 315Ser
Met Lys Ala Ala Leu Arg Lys Met Leu Ser Lys Glu Glu Leu 320 325
330Pro Gln Arg Lys Met Cys Leu Lys Ala Met Phe Lys Leu 335
34034323PRTHomo sapiensmisc_featureIncyte ID No 7472009CD1 34Met
Trp Gln Lys Asn Gln Thr Ser Leu Ala Asp Phe Ile Leu Glu 1 5 10
15Gly Leu Phe Asp Asp Ser Leu Thr His Leu Phe Leu Phe Ser Leu 20 25
30Thr Met Val Val Phe Leu Ile Ala Val Ser Gly Asn Thr Leu Thr 35 40
45Ile Leu Leu Ile Cys Ile Asp Pro Gln Leu His Thr Pro Met Tyr 50 55
60Phe Leu Leu Ser Gln Leu Ser Leu Met Asp Leu Met His Val Ser 65 70
75Thr Thr Ile Leu Lys Met Ala Thr Asn Tyr Leu Ser Gly Lys Lys 80 85
90Ser Ile Ser Phe Val Gly Cys Ala Thr Gln His Phe Leu Tyr Leu 95
100 105Cys Leu Gly Gly Ala Glu Cys Phe Leu Leu Ala Val Met Ser Tyr
110 115 120Asp Arg Tyr Val Ala Ile Cys His Pro Leu Arg Tyr Ala Val
Leu 125 130 135Met Asn Lys Lys Val Gly Leu Met Met Ala Val Met Ser
Trp Leu 140 145 150Gly Ala Ser Val Asn Ser Leu Ile His Met Ala Ile
Leu Met His 155 160 165Phe Pro Phe Cys Gly Pro Arg Lys Val Tyr His
Phe Tyr Cys Glu 170 175 180Phe Pro Ala Val Val Lys Leu Val Cys Gly
Asp Ile Thr Val Tyr 185 190 195Glu Thr Thr Val Tyr Ile Ser Ser Ile
Leu Leu Leu Leu Pro Ile 200 205 210Phe Leu Ile Ser Thr Ser Tyr Val
Phe Ile Leu Gln Ser Val Ile 215 220 225Gln Met Arg Ser Ser Gly Ser
Lys Arg Asn Ala Phe Ala Thr Cys 230 235 240Gly Ser His Leu Thr Val
Val Ser Leu Trp Phe Gly Ala Cys Ile 245 250 255Phe Ser Tyr Met Arg
Pro Arg Ser Gln Cys Thr Leu Leu Gln Asn 260 265 270Lys Val Gly Ser
Val Phe Tyr Ser Ile Ile Thr Pro Thr Leu Asn 275 280 285Ser Leu Ile
Tyr Thr Leu Arg Asn Lys Asp Val Ala Lys Ala Leu 290 295 300Arg Arg
Val Leu Arg Arg Asp Val Ile Thr Gln Cys Ile Gln Arg 305 310 315Leu
Gln Leu Trp Leu Pro Arg Val 32035299PRTHomo
sapiensmisc_featureIncyte ID No 7472010CD1 35Met Glu Leu Glu Gly
Asp Phe Leu Gly Ser Val Gly Glu Leu Gly 1 5 10 15Gln Val Ile Gln
Thr Cys Ser Gly Ile Tyr Val Phe Thr Val Val 20 25 30Gly Asn Leu Gly
Leu Ile Thr Leu Ile Gly Ile Asn Pro Ser Leu 35 40 45His Thr Pro Met
Tyr Phe Phe Leu Phe Asn Leu Ser Phe Ile Asp 50 55 60Leu Cys Tyr Ser
Cys Val Phe Thr Pro Lys Met Leu Asn Asp Phe 65 70 75Val Ser Glu Ser
Ile Ile Ser Tyr Val Gly Cys Met Thr Gln Leu 80 85 90Phe Phe Phe Cys
Phe Phe Val Asn Ser Glu Cys Tyr Val Leu Val 95 100 105Ser Met Ala
Tyr Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Leu 110 115 120Tyr Met
Val Thr Met Ser Pro Arg Val Cys Phe Leu Leu Met Phe 125 130 135Gly
Ser Tyr Val Val Gly Phe Ala Gly Ala Met Ala His Thr Gly 140 145
150Ser Met Leu Arg Leu Thr Phe Cys Asp Ser Asn Val Ile Asp His 155
160 165Tyr Leu Cys Asp Val Leu Pro Leu Leu Gln Leu Ser Cys Thr Ser
170 175 180Thr His Val Ser Glu Leu Val Phe Phe Ile Val Val Gly Val
Ile 185 190 195Thr Met Leu Ser Ser Ile Ser Ile Val Ile Ser Tyr Ala
Leu Ile 200 205 210Leu Ser Asn Ile Leu Cys Ile Pro Ser Ala Glu Gly
Arg Ser Lys 215 220 225Ala Phe Ser Thr Trp Gly Ser His Ile Ile Ala
Val Ala Leu Phe 230 235 240Phe Gly Ser Gly Thr Phe Thr Tyr Leu Thr
Thr Ser Phe Pro Gly 245 250 255Ser Met Asn His Gly Arg Phe Ala Ser
Val Phe Tyr Thr Asn Val 260 265 270Val Pro Met Leu Asn Pro Ser Ile
Tyr Ser Leu Arg Asn Lys Asp 275 280 285Asp Lys Leu Ala Leu Gly Lys
Thr Leu Lys Arg Val Leu Phe 290 29536307PRTHomo
sapiensmisc_featureIncyte ID No 7472011CD1 36Met Glu Thr Gly Asn
Leu Thr Trp Val Ser Asp Phe Val Phe Leu 1 5 10 15Gly Leu Ser Gln
Thr Arg Glu Leu Gln Arg Phe Leu Phe Leu Met 20 25 30Phe Leu Phe Val
Tyr Ile Thr Thr Val Met Gly Asn Ile Leu Ile 35 40 45Ile Ile Thr Val
Thr Ser Asp Ser Gln Leu His Thr Pro Met Tyr 50 55 60Phe Leu Leu Arg
Asn Leu Ala Val Leu Asp Leu Cys Phe Ser Ser 65 70 75Val Thr Ala Pro
Lys Met Leu Val Asp Leu Leu Ser Glu Lys Lys 80 85 90Thr Ile Ser Tyr
Gln Gly Cys Met Gly Gln Ile Phe Phe Phe His 95 100 105Phe Leu Gly
Gly Ala Met Val Phe Phe Leu Ser Val Met Ala Phe 110 115 120Asp Arg
Leu Ile Ala Ile Ser Arg Pro Leu Arg Tyr Val Thr Val 125 130 135Met
Asn Thr Gln Leu Trp Val Gly Leu Val Val Ala Thr Trp Val 140 145
150Gly Gly Phe Val His Ser Ile Val Gln Leu Ala Leu Met Leu Pro 155
160 165Leu Pro Phe Cys Gly Pro Asn Ile Leu Asp Asn Phe Tyr Cys Asp
170 175 180Val Pro Gln Val Leu Arg Leu Ala Cys Thr Asp Thr Ser Leu
Leu 185 190 195Glu Phe Leu Lys Ile Ser Asn Ser Gly Leu Leu Asp Val
Val Trp 200 205 210Phe Phe Leu Leu Leu Met Ser Tyr Leu Phe Ile Leu
Val Met Leu 215 220 225Arg Ser His Pro Gly Glu Ala Arg Arg Lys Ala
Ala Ser Thr Cys 230 235 240Thr Thr His Ile Ile Val Val Ser Met Ile
Phe Val Pro Ser Ile 245 250 255Tyr Leu Tyr Ala Arg Pro Phe Thr Pro
Phe Pro Met Asp Lys Leu 260 265 270Val Ser Ile Gly His Thr Val Met
Thr Pro Met Leu Asn Pro Met 275 280 285Ile Tyr Thr Leu Arg Asn Gln
Asp Met Gln Ala Ala Val Arg Arg 290 295 300Leu Gly Arg His Arg Leu
Val 30537314PRTHomo sapiensmisc_featureIncyte ID No 7472012CD1
37Met Asp Asn Ser Asn Trp Thr Ser Val Ser His Phe Val Leu Leu 1 5
10 15Gly Ile Ser Thr His Pro Glu Glu Gln Ile Pro Leu Phe Leu Val 20
25 30Phe Ser Leu Met Tyr Ala Ile Asn Ile Ser Gly Asn Leu Ala Ile 35
40 45Ile Thr Leu Ile Leu Ser Ala Pro Arg Leu His Ile Pro Met Tyr 50
55 60Ile Phe Leu Ser Asn Leu Ala Leu Thr Asp Ile Cys Phe Thr Ser 65
70 75Thr Thr Val Pro Lys Met Leu Gln Ile Ile Phe Ser Pro Thr Lys 80
85 90Val Ile Ser Tyr Thr Gly Cys Leu Ala Gln Thr Tyr Phe Phe Ile 95
100 105Cys Phe Ala Val Met Glu Asn Phe Ile Leu Ala Val Met Ala Tyr
110 115 120Asp Arg Tyr Ile Ala Ile Cys His Pro Phe His Tyr Thr Met
Ile 125 130 135Leu Thr Arg Met Leu Cys Val Lys Met Val Val Met Cys
His Ala 140 145 150Leu Ser His Leu His Ala Met Leu His Thr Phe Leu
Ile Gly Gln 155 160 165Leu Ile Phe Cys Ala Asp Asn Arg Ile Pro His
Phe Phe Cys Asp 170 175 180Leu Tyr Ala Leu Met Lys Ile Ser Cys Thr
Ser Thr Tyr Leu Asn 185 190 195Thr Leu Met Ile His Thr Glu Gly Ala
Val Val Ile Ser Gly Ala 200 205 210Leu Ala Phe Ile Thr Ala Ser Tyr
Ala Cys Ile Ile Leu Val Val 215 220 225Leu Arg Ile Pro Ser Ala Lys
Gly Arg Trp Lys Thr Phe Ser Thr 230 235 240Cys Gly Ser His Leu
Thr
Val Val Ala Ile Phe Tyr Gly Thr Leu 245 250 255Ser Trp Val Tyr Phe
Arg Pro Leu Ser Ser Tyr Ser Val Thr Lys 260 265 270Gly Arg Ile Ile
Thr Val Val Tyr Thr Val Val Thr Pro Met Leu 275 280 285Asn Pro Phe
Ile Tyr Ser Leu Arg Asn Gly Asp Val Lys Gly Gly 290 295 300Phe Met
Lys Trp Met Ser Arg Met Gln Thr Phe Phe Phe Arg 305 31038310PRTHomo
sapiensmisc_featureIncyte ID No 7472014CD1 38Met Gly Arg Asn Asn
Leu Thr Arg Pro Ser Glu Phe Ile Leu Leu 1 5 10 15Gly Leu Ser Ser
Arg Pro Glu Asp Gln Lys Pro Leu Phe Ala Val 20 25 30Phe Leu Pro Ile
Tyr Leu Ile Thr Val Ile Gly Asn Leu Leu Ile 35 40 45Ile Leu Ala Ile
Arg Ser Asp Thr Arg Leu Gln Thr Pro Met Tyr 50 55 60Phe Phe Leu Ser
Ile Leu Ser Phe Val Asp Ile Cys Tyr Val Thr 65 70 75Val Ile Ile Pro
Lys Met Leu Val Asn Phe Leu Ser Glu Thr Lys 80 85 90Thr Ile Ser Tyr
Gly Glu Cys Leu Thr Gln Met Tyr Phe Phe Leu 95 100 105Ala Phe Gly
Asn Thr Asp Ser Tyr Leu Leu Ala Ala Met Ala Ile 110 115 120Asp Arg
Tyr Val Ala Ile Cys Asn Pro Phe His Tyr Ile Thr Ile 125 130 135Met
Ser His Arg Cys Cys Val Leu Leu Leu Val Leu Ser Phe Cys 140 145
150Ile Pro His Phe His Ser Leu Leu His Ile Leu Leu Thr Asn Gln 155
160 165Leu Ile Phe Cys Ala Ser Asn Val Ile His His Phe Phe Cys Asp
170 175 180Asp Gln Pro Val Leu Lys Leu Ser Cys Ser Ser His Phe Val
Lys 185 190 195Glu Ile Thr Val Met Thr Glu Gly Leu Ala Val Ile Met
Thr Pro 200 205 210Phe Ser Cys Ile Ile Ile Ser Tyr Leu Arg Ile Leu
Ile Thr Val 215 220 225Leu Lys Ile Pro Ser Ala Ala Gly Lys Arg Lys
Ala Phe Ser Thr 230 235 240Cys Gly Ser His Leu Thr Val Val Thr Leu
Phe Tyr Gly Ser Ile 245 250 255Ser Tyr Val Tyr Phe Gln Pro Leu Ser
Asn Tyr Thr Val Lys Asp 260 265 270Gln Ile Ala Thr Ile Ile Tyr Thr
Val Leu Thr Pro Met Leu Asn 275 280 285Pro Phe Ile Tyr Ser Leu Arg
Asn Lys Asp Met Lys Gln Gly Leu 290 295 300Ala Lys Leu Met His Arg
Met Lys Cys Gln 305 31039359PRTHomo sapiensmisc_featureIncyte ID No
7472020CD1 39Met Phe Lys Ala Ile Leu Gly His Val Trp Pro Lys Asp
His Gly 1 5 10 15Leu Asp Lys Leu Val Val Arg Cys Pro Arg His Thr
Glu Pro Trp 20 25 30Asn Leu Thr Gly Ile Ser Glu Phe Leu Leu Leu Gly
Leu Ser Glu 35 40 45Asp Pro Glu Leu Gln Pro Val Leu Pro Gly Leu Ser
Leu Ser Met 50 55 60Tyr Leu Val Thr Val Leu Arg Asn Leu Leu Ile Ile
Leu Ala Val 65 70 75Ser Ser Asp Ser His Leu His Thr Pro Met Cys Phe
Phe Leu Ser 80 85 90Asn Leu Cys Trp Ala Asp Ile Gly Phe Thr Ser Ala
Met Val Pro 95 100 105Lys Met Ile Val Asp Met Gln Ser His Ser Arg
Val Ile Ser Tyr 110 115 120Ala Gly Cys Leu Thr Gln Met Ser Phe Phe
Val Leu Phe Ala Cys 125 130 135Ile Glu Asp Met Leu Leu Thr Val Met
Ala Tyr Asp Arg Phe Val 140 145 150Ala Ile Cys His Pro Leu His Tyr
Pro Val Ile Met Asn Pro His 155 160 165Leu Gly Val Phe Leu Val Leu
Val Ser Phe Phe Leu Ser Leu Leu 170 175 180Asp Ser Gln Leu His Ser
Trp Ile Val Leu Gln Phe Thr Phe Phe 185 190 195Lys Asn Val Glu Ile
Ser Asn Phe Val Cys Asp Pro Ser Gln Leu 200 205 210Leu Asn Leu Ala
Cys Ser Asp Ser Val Ile Asn Ser Ile Phe Ile 215 220 225Tyr Leu Asp
Ser Ile Met Phe Gly Phe Leu Pro Ile Ser Gly Ile 230 235 240Leu Leu
Ser Tyr Ala Asn Asn Val Pro Ser Ile Leu Arg Ile Ser 245 250 255Ser
Ser Asp Arg Lys Ser Lys Ala Phe Ser Thr Cys Gly Ser His 260 265
270Leu Ala Val Val Cys Leu Phe Tyr Gly Thr Gly Ile Gly Val Tyr 275
280 285Leu Thr Ser Ala Val Ser Pro Pro Pro Arg Asn Gly Val Val Ala
290 295 300Ser Val Met Tyr Ala Val Val Thr Pro Met Leu Asn Pro Phe
Ile 305 310 315Tyr Ser Leu Arg Asn Arg Asp Ile Gln Ser Ala Leu Trp
Arg Leu 320 325 330Arg Ser Arg Thr Val Glu Ser His Asp Leu Leu Ser
Gln Asp Leu 335 340 345Leu His Pro Phe Ser Cys Val Gly Glu Lys Gly
Gln Pro His 350 35540936DNAHomo sapiensmisc_featureIncyte ID No
104941CB1 40atggagataa agaactacag cagcagcacc tcaggcttca tcctcctggg
cctctcttcc 60aaccctcagc tgcagaaacc tctctttgcc atcttcctca tcatgtacct
gctcgctgcg 120gtggggaatg tgctcatcat cccggccatc tactctgacc
ccaggctcca cacccctatg 180tacttttttc tcagcaactt gtctttcatg
gatatctgct tcacaacagt catagtgcct 240aagatgctgg tgaattttct
atcagagaca aaggttatct cctatgtggg ctgcctggcc 300cagatgtact
tctttatggc atttgggaac actgacagct acctgctggc ctctatggcc
360atcgaccggc tggtggccat ctgcaacccc ttacactatg atgtggttat
gaaaccacgg 420cattgcctgc tcatgctatt gggttcttgc agcatctccc
acctacattc cctgttccgc 480gtgctactta tgtctcgctt gtctttctgt
gcctctcaca tcattaagca ctttttctgt 540gacacccagc ctgtgctaaa
gctctcctgc tctgacacat cctccagcca gatggtggtg 600atgactgaga
ccttagctgt cattgtgacc cccttcctgt gtatcatctt ctcctacctg
660cgaatcatgg tcactgtgct cagaatcccc tctgcagccg ggaagtggaa
ggccttctct 720acctgtggct cccacctcac tgcagtagcc cttttctatg
ggagtattat ttatgtctat 780tttaggcccc tgtccatgta ctcagtggtt
agggaccggg tagccacagt tatgtacaca 840gtagtgacac ccatgctgaa
ccctttcatc tacagcctga ggaacaaaga tatgaagagg 900ggtttgaaga
aattacagga cagaatttac cggtaa 936413365DNAHomo
sapiensmisc_featureIncyte ID No 1499408CB1 41atggaccagc cagaggcccc
ctgctccagc acggggccgc gcctcgcggt ggcccgcgag 60ctgctcctgg ctgcgctgga
ggaactgagc caagagcagc tgaagcgctt ccgccacaag 120ctgcgcgacg
tgggcccgga cggacgcagc atcccgtggg ggcggctgga gcgcgcggac
180gccgtggacc tcgcggagca gctggcccag ttctacggcc cggagcctgc
cctggaggtg 240gcccgcaaga ccctcaagag ggcggacgcg cgcgacgtgg
cggcgcagct ccaggagcgg 300cggctgcagc ggctcgggct cggctccggg
acgctgctct ccgtgtccga gtacaagaag 360aagtaccggg agcacgtgct
gcagctgcac gctcgggtga aggagaggaa cgcccgctcc 420gtgaagatca
ccaagcgctt caccaagctg ctcatcgcgc ccgagagcgc cgccccggag
480gaggcgctgg ggcccgcgga agagcctgag ccggggcgcg cgcggcgctc
ggacacgcac 540actttcaacc gcctcttccg ccgcgacgag gagggccggc
ggccgctgac cgtggtgctg 600cagggcccgg cgggcatcgg caagaccatg
gcggccaaaa agatcctgta cgactgggcg 660gcgggcaagc tgtaccaggg
ccaggtggac ttcgccttct tcatgccctg cggcgagctg 720ctggagaggc
cgggcacgcg cagcctggct gacctgatcc tggaccagtg ccccgaccgc
780ggcgcgccgg tgccgcagat gctggcccag ccgcagcggc tgctcttcat
cctggacggc 840gcggacgagc tgccggcgct ggggggcccc gaggccgcgc
cctgcacaga ccccttcgag 900gcggcgagcg gcgcgcgggt gctaggcggg
ctgctgagca aggcgctgct gcccacggcc 960ctcctgctgg tgaccacgcg
cgccgccgcc cccgggaggc tgcagggccg cctgtgttcc 1020ccgcagtgcg
ccgaggtgcg cggcttctcc gacaaggaca agaagaagta tttctacaag
1080ttcttccggg atgagaggag ggccgagcgc gcctaccgct tcgtgaagga
gaacgagacg 1140ctgttcgcgc tgtgcttcgt gcccttcgtg tgctggatcg
tgtgcaccgt gctgcgccag 1200cagctggagc tcggtcggga cctgtcgcgc
acgtccaaga ccaccacgtc agtgtacctg 1260cttttcatca ccagcgttct
gagctcggct ccggtagccg acgggccccg gttgcagggc 1320gacctgcgca
atctgtgccg cctggcccgc gagggcgtcc tcggacgcag ggcgcagttt
1380gccgagaagg aactggagca actggagctt cgtggctcca aagtgcagac
gctgtttctc 1440agcaaaaagg agctgccggg cgtgctggag acagaggtca
cctaccagtt catcgaccag 1500agcttccagg agttcctcgc ggcactgtcc
tacctgctgg aggacggcgg ggtgcccagg 1560accgcggctg gcggcgttgg
gacactcctg cgtggggacg cccagccgca cagccacttg 1620gtgctcacca
cgcgcttcct cttcggactg ctgagcgcgg agcggatgcg cgacatcgag
1680cgccacttcg gctgcatggt ttcagagcgt gtgaagcagg aggccctgcg
gtgggtgcag 1740ggacagggac agggctgccc cggagtggca ccagaggtga
ccgagggggc caaagggctc 1800gaggacaccg aagagccaga ggaggaggag
gagggagagg agcccaacta cccactggag 1860ttgctgtact gcctgtacga
gacgcaggag gacgcgtttg tgcgccaagc cctgtgccgg 1920ttcccggagc
tggcgctgca gcgagtgcgc ttctgccgca tggacgtggc tgttctgagc
1980tactgcgtga ggtgctgccc tgctggacag gcactgcggc tgatcagctg
cagattggtt 2040gctgcgcagg agaagaagaa gaagagcctg gggaagcggc
tccaggccag cctgggtggc 2100ggcagttctc aaggcaccac aaaacaactg
ccagcctccc ttcttcatcc actctttcag 2160gcaatgactg acccactgtg
ccatctgagc agcctcacgc tgtcccactg caaactccct 2220gacgcggtct
gccgagacct ttctgaggcc ctgagggcag cccccgcact gacggagctg
2280ggcctcctcc acaacaggct cagtgaggcg ggactgcgta tgctgagtga
gggcctagcc 2340tggccgcagt gcagggtgca gacggtcagg gtacagctgc
ctgaccccca gcgagggctc 2400cagtacctgg tgggtatgct tcggcagagc
cccgccctga ccaccctgga tctcagcggc 2460tgccaactgc ccgcccccat
ggtgacctac ctgtgtgcag tcctgcagca ccagggatgc 2520ggcctgcaga
ccctcagtct ggcctctgtg gagctgagcg agcagtcact acaggagctt
2580caggctgtga agagagcaaa gccggatctg gtcatcacac acccagcgct
ggacggccac 2640ccacaacctc ccaaggaact catctcgacc ttctgaggct
ctggtggcca gagcagggtg 2700gaagacccta gtcaaagtcc ctgtggagag
aacggcccat tccaagggca ggaggatatt 2760gctctcggcc tttgggaaac
ttttgagccg agaggccgca gacaggcatg tgggaggccc 2820agacacggca
ccctgccccg tccaggacag gcccaggacc tgcccctctc tccacacctg
2880gggtacccct tctcccccag ccccaccact actccaccca ccttcctctc
ctgagaccct 2940ccagccattc cccttgaaaa caccccccga ccccaagcca
caataatgac agcgagagct 3000ccaattaact aagcacctac ctgggggcag
aataaccctt cactgcctga tccccatctg 3060cagtgtggcc caacagcccc
cagaactatg cccacataga ctggaggtag gcagttcacc 3120gtccctccct
gttaggaatg agaccatccc tgaggctatg gcccaggccc acaggcgtcc
3180agtgtctgag atctttggga agggagacta gggcaggtgg agacagcgca
gaacccccgt 3240gctgggtggg aagcatgacc acatggtggg tgagcagccc
ccatgcactg acggtaaatt 3300cccctgtgga ctcatttctg ttggtttcta
ttacacctgg ccaggcgtgg tacaatacag 3360gtcga 3365421325DNAHomo
sapiensmisc_featureIncyte ID No 3168839CB1 42catggcatcc ccagcctagc
tcccaatccc actttggcac gatgttagcc aacagctcct 60caaccaacag ttctgttctc
ccgtgtcctg actaccgacc tacccaccgc ctgcacttgg 120tggtctacag
cttggtgctg gctgccgggc tccccctcaa cgcgctagcc ctctgggtct
180tcctgcgcgc gctgcgcgtg cactcggtgg tgagcgtgta catgtgtaac
ctggcggcca 240gcgacctgct cttcaccctc tcgctgcccg ttcgtctctc
ctactacgca ctgcaccact 300ggcccttccc cgacctcctg tgccagacga
cgggcgccat cttccagatg aacatgtacg 360gcagctgcat cttcctgatg
ctcatcaacg tggaccgcta cgccgccatc gtgcacccgc 420tgcgactgcg
ccacctgcgg cggccccgcg tggcgcggct gctctgcctg ggcgtgtggg
480cgctcatcct ggtgtttgcc gtgcccgccg cccgcgtgca caggccctcg
cgttgccgct 540accgggacct cgaggtgcgc ctatgcttcg agagcttcag
cgacgagctg tggaaaggca 600ggctgctgcc cctcgtgctg ctggccgagg
cgctgggctt cctgctgccc ctggcggcgg 660tggtctactc gtcgggccga
gtcttctgga cgctggcgcg ccccgacgcc acgcagagcc 720agcggcggcg
gaagaccgtg cgcctcctgc tggctaacct cgtcatcttc ctgctgtgct
780tcgtgcccta caacagcacg ctggcggtct acgggctgct gcggagcaag
ctggtggcgg 840ccagcgtgcc tgcccgcgat cgcgtgcgcg gggtgctgat
ggtgatggtg ctgctggccg 900gcgccaactg cgtgctggac ccgctggtgt
actactttag cgccgagggc ttccgcaaca 960ccctgcgcgg cctgggcact
ccgcaccggg ccaggacctc ggccaccaac gggacgcggg 1020cggcgctcgc
gcaatccgaa aggtccgccg tcaccaccga cgccaccagg ccggatgccg
1080ccatgtcccc aggattccgc cctctgaaca cacatgccat tgcgctgtcc
gtgcccgact 1140cccaacgcct ctcgttctgg gaggcttaca gggtgtacac
acaagaaggt gggctgggca 1200cttggacctt tgggtggcaa ttccagctta
gcaacgcaga agagtacaaa gtgtggaagc 1260cagggcccag ggaaggcagt
gctgctggaa atggcttctt taaactgtga gcacgcagag 1320caccc
1325432124DNAHomo sapiensmisc_featureIncyte ID No 3291235CB1
43gtcttacctc ttaatagtat tagaatggca attaaatctc gacacgtttt aattaaattt
60caacatgagt tttgcagggg acattcgaag tgtagcgctc gccatctccc atcccaagta
120cctaagggct acaacgctgt cgccagagag gaagtcactg agtgcccact
gccacccccc 180cacatatgct cgtggtcccc agcatccttg agccaccagg
agtgagggct gctgctccct 240gagacctggc tccaaggagg atgccacagc
cgcctgccag ctccggtctg caccatgagt 300gatgagcggc ggctgcctgg
cagtgcagtg ggctggctgg tatgtggggg cctctccctg 360ctggccaatg
cctggggcat cctcagcgtt ggcgccaagc agaagaagtg gaagcccttg
420gagttcctgc tgtgtacgct cgcggccacc cacatgctaa atgtggccgt
gcccatcgcc 480acctactccg tggtgcagct gcggcggcag cgccccgact
tcgagtggaa tgagggtctc 540tgcaaggtct tcgtgtccac cttctacacc
ctcaccctgg ccacctgttt ctctgtcacc 600tccctctcct accaccgcat
gtggatggtc tgctggcctg tcaactaccg gctgagcaat 660gccaagaagc
aggcggtgca cacagtcatg ggtatctgga tggtgtcctt catcctgtcg
720gccctgcctg ccgttggctg gcacgacacc agcgagcgct tctacaccca
tggctgccgc 780ttcatcgtgg ctgagatcgg cctgggcttt ggcgtctgct
tcctgctgct ggtgggcggc 840agcgtggcca tgggcgtgat ctgcacagcc
atcgccctct tccagacgct ggccgtgcag 900gtggggcgcc aggccgacca
ccgcgccttc accgtgccca ccatcgtggt ggaggacgcg 960cagggcaagc
ggcgctcctc catcgatggc tcggagcccg ccaaaacctc tctgcagacc
1020acgggcctcg tgaccaccat agtcttcatc tacgactgcc tcatgggctt
ccctgtgctg 1080gtggtgagct tcagcagcct gcgggccgac gcctcagcgc
cctggatggc actctgcgtg 1140ctgtggtgct ccgtggccca ggccctgctg
ctgcctgtgt tcctctgggc ctgcgaccgc 1200taccgggctg acctcaaagc
tgtccgggag aagtgcatgg ccctcatggc caacgacgag 1260gagtcagacg
atgagaccag cctggaaggt ggcatctccc cggacctggt gttggagcgc
1320tccctggact atggctatgg aggtgatttt gtggccctag ataggatggc
caagtatgag 1380atctccgccc tggagggggg cctgccccag ctctacccac
tgcggccctt gcaggaggac 1440aagatgcaat acctgcaggt cccgcccacg
cggcgcttct cccacgacga tgcggacgtg 1500tgggccgccg tcccgctgcc
cgccttcctg ccgcgctggg gctccggcaa ggacctgtcc 1560gccctggcgc
acctggtgct gcctgccggg cccgagcggc cccgcgccag cctcctggcc
1620ttcgcggagg acgcaccact gtcccgcgcg cgccgccgct cggccgagag
cctgctgtcg 1680ctgcggccct cggccgtgga tagcggcccg cggggagccc
gcgactcgcc ccccggcagc 1740ccgcgccgcc gccccgggcc cggcccccgc
tccgcctcgg cctcgctgct gcccgacgcc 1800ttcgccctga ccgccttcga
gtgcgagcca caggccctgc gccgcccgcc cgggcccttc 1860cccgctgcgc
ccgccgcccc cgacggcgca gatcccggag aggccccgac gcccccaagc
1920agcgcccagc ggagcccagg gccacgcccc tctgcgcact cgcacgccgg
ctctctgcgc 1980cccggcctga gcgcgtcgtg gggcgagccc ggggggctgc
gcgcggcggg cggcggcggc 2040agcaccagca gcttcctgag ttccccctcc
gagtcctcgg gctacgccac gctgcactcg 2100gactcgctgg gctccgcgtc ctag
212444942DNAHomo sapiensmisc_featureIncyte ID No 7472001CB1
44atggaaagaa tcaacagcac actgttgact gcgtttatcc tgacaggaat tccgtatcca
60ctcaggctaa ggacactctt ttttgtgttc ttttttctaa tctacatcct gactcagctg
120ggaaacctgc ttattttaat cactgtctgg gcagacccaa ggctccatgc
ccgccccatg 180tacatctttc ttggtgttct ctcagtcatt gatatgagca
tctcctccat cattgtccct 240cgcctcatga tgaacttcac tttaggtgtc
aaacccatcc catttggtgg ctgtgttgct 300caactctatt tctatcactt
cctgggcagc acccagtgct tcctctacac cctaatggcc 360tatgacaggt
acctggcaat atgtcagccc ctgcgctacc ctgtgctcat gactgctaag
420ctgagcgcct tgcttgtggc tggagcctgg atggcaggat ccatccatgg
ggctctccag 480gccatcctaa ccttccgcct gccctactgt gggcccaatc
aggtggatta cttcttctgt 540gacatccctg cagtgttgag actggcctgt
gctgacacaa cagtcaacga gctggtgacg 600tttgtagaca ttggggtggt
ggttgccagt tgcttctccc tgatcctcct ctcctacata 660cagatcattc
aggccatcct gagaatccac acagctgatg ggcggcgccg ggctttttca
720acttgtggag cccatgtaac cgtggtcacc gtgtactatg tgccctgtgc
cttcatctac 780ctgaggcctg aaaccaacag ccccctggat ggggcagctg
ccctagtccc cacggccatc 840actcctttcc tcaaccccct tatctacact
ctgcggaacc aagaggtgaa gctggccctg 900aaaagaatgc tcagaagccc
aagaactccg agtgaggttt ga 942451197DNAHomo sapiensmisc_featureIncyte
ID No 7472003CB1 45atgcacaccg tggctacgtc cggacccaac gcgtcctggg
gggcaccggc caacgcctcc 60ggctgcccgg gctgtggcgc caacgcctcg gacggcccag
tcccttcgcc gcgggccgtg 120gacgcctggc tcgtgccgct cttcttcgcg
gcgctgatgc tgctgggcct ggtggggaac 180tcgctggtca tctacgtcat
ctgccgccac aagccgatgc ggaccgtgac caacttctac 240atcgccaacc
tggcggccac ggacgtgacc ttcctcctgt gctgcgtccc cttcacggcc
300ctgctgtacc cgctgcccgg ctgggtgctg ggcgacttca tgtgcaagtt
cgtcaactac 360atccagcagg tctcggtgca ggccacgtgt gccactctga
ccgccatgag tgtggaccgc 420tggtacgtga cggtgttccc gttgcgcgcc
ctgcaccgcc gcacgccccg cctggcgctg 480gctgtcagcc tcagcatctg
gacaggctct gcggcggtgt ctgcgccggt gctcgccctg 540caccgcctgt
cacccgggcc gcgcgcctac tgcagtgagg ccttccccag ccgcgccctg
600gagcgcgcct tcgcactgta caacctgctg gcgctgtacc tgctgccgct
gctcgccacc 660tgcgcctgct atgcggccat gctgcgccac ctgggccggg
tcgccgtgcg ccccgcgccc 720gccgatagcg ccctgcaggg gcaggtgctg
gcagagcgcg caggcgccgt gcgggccaag 780gtctcgcggc tggtggcggc
cgtggtcctg ctcttcgccg cctgctgggg ccccatccag 840ctgttcctgg
tgctgcaggc gctgggcccc gcgggctcct ggcacccacg cagctacgcc
900gcctacgcgc ttaagacctg ggctcactgc atgtcctaca gcaactccgc
gctgaacccg 960ctgctctacg ccttcctggg
ctcgcacttc cgacaggcct tccgccgcgt ctgcccctgc 1020gcgccgcgcc
gcccccgccg cccccgccgg cccggaccct cggaccccgc agccccacac
1080gcggagctgc tccgcctggg gtcccacccg gcccccgcca gggcgcagaa
gccagggagc 1140agtgggctgg ccgcgcgcgg gctgtgcgtc ctgggggagg
acaacgcccc tctctga 1197461110DNAHomo sapiensmisc_featureIncyte ID
No 7472004CB1 46atggctccca ctggtttgag ttccttgacc gtgaatagta
cagctgtgcc cacaacacca 60gcagcattta agagcctaaa cttgcctctt cagatcaccc
tttctgctat aatgatattc 120attctgtttg tgtcttttct tgggaacttg
gttgtttgcc tcatggttta ccaaaaagct 180gccatgaggt ctgcaattaa
catcctcctt gccagcctag cttttgcaga catgttgctt 240gcagtgctga
acatgccctt tgccctggta actattctta ctacccgatg gatttttggg
300aaattcttct gtagggtatc tgctatgttt ttctggttat ttgtgataga
aggagtagcc 360atcctgctca tcattagcat agataggttc cttattatag
tccagaggca ggataagcta 420aacccatata gagctaaggt tctgattgca
gtttcttggg caacttcctt ttgtgtagct 480tttcctttag ccgtaggaaa
ccccgacctg cagatacctt cccgagctcc ccagtgtgtg 540tttgggtaca
caaccaatcc aggctaccag gcttatgtga ttttgatttc tctcatttct
600ttcttcatac ccttcctggt aatactgtac tcatttatgg gcatactcaa
cacccttcgg 660cacaatgcct tgaggatcca tagctaccct gaaggtatat
gcctcagcca ggccagcaaa 720ctgggtctca tgagtctgca gagacctttc
cagatgagca ttgacatggg ctttaaaaca 780cgtgccttca ccactatttt
gattctcttt gctgtcttca ttgtctgctg ggccccattc 840accacttaca
gccttgtggc aacattcagt aagcactttt actatcagca caactttttt
900gagattagca cctggctact gtggctctgc tacctcaagt ctgcattgaa
tccgctgatc 960tactactgga ggattaagaa attccatgat gcttgcctgg
acatgatgcc taagtccttc 1020aagtttttgc cgcagctccc tggtcacaca
aagcgacgga tacgtcctag tgctgtctat 1080gtgtgtgggg aacatcggac
ggtggtgtga 111047582DNAHomo sapiensmisc_featureIncyte ID No
7475687CT1 47atggcctatg acaggtacct ggcaatatgt cagcccctgc gctacccagt
gctcatgaat 60gggaggttat gcacagtcct tgtggctgga gcttgggtcg ccggctccat
gcatgggtct 120atccaggcca ccctgacctt ccgcctgccc tactgtgggc
ccaatcaggt agattacttt 180atctgtgaca tccccgcagt attgagactg
gcctgtgctg acacaactgt caatgagctt 240gtgacctttg tggacatcgg
ggtagtggcc gccagttgct tcatgttaat tctgctctcg 300tatgccaaca
tagtaaatgc catcctgaag atacgcacca ctgatgggag gcgccgggcc
360ttctccacct gtggctccca cctaatcgtg gtcacagtct actatgtccc
ctgtattttc 420atctacctta gggctggctc caaaggcccc ctggatgggg
cagcggctgt gttttacact 480gttgtcactc cattactgaa ccccctcatc
tatacactga ggaaccagga agtgaagtct 540gccctgaaga ggataacagc
aggccaggcg gatgtaaata ac 58248519DNAHomo sapiensmisc_featureIncyte
ID No 7483029CT1 48atgtacctgg tcaccgtgct cgggaacctg ctcatcatcc
tggccacaat ctcagactcc 60cacctccaca cccccatgta cttcttcctc tccaacctgt
cctttgcaga catctgtttt 120gtgtctacca ctgtcccaaa gatgctggtg
aacatccaga cacagagcag agtcatcacc 180tatgcagact gcatcaccca
gatgtgcttt tttatactct ttgtagtgtt ggacagctta 240ctcctgactg
tgatggccta tgaccggttt gtggccatct gtcaccccct gcactacaca
300gtcattatga actcctggct ctgtggactg ctggttctgg tgtcctggat
cgtgagcatc 360ctatattctc tgttacaaag cataatggca ttgcagctgt
ccttctgtac agaattgaaa 420atccctcatt ttttctgtga acttaatcag
gtcatccacc ttgcctgttc cgacactttt 480attaatgaca tgatgatgaa
ttttacaagt gtgctgctg 51949663DNAHomo sapiensmisc_featureIncyte ID
No 7477933CT1 49cccatgtact tcttcctctc caacctgtgc tgggctgaca
tcggtctcac ctcggccacg 60gttcccaagg tgattctgga tatgcagtcg catagcagag
tcatctctca tgtgggctgc 120ctgacacaga tgtctttctt ggtccttttt
gcatgtatag aaggcatgct cctgactgtg 180atggcctatg gctgctttgt
agccatctgt cgccctctgc actacccagt catagtgaat 240cctcacctct
gtgtcttctt cgttttggtg tcctttttcc ttaacctgtt ggattcccag
300ctgcacagtt ggattgtgtt acaattcacc atcatcaaga atgtggaaat
ctctaatttt 360ttctgtgacc cctctcagct tctcaacctt gcctgttctg
acagcgtcat caatagcata 420ttcatatatt tcgatagtac tatgtttggt
tttcttccca tttcagggat ccttttgtct 480tactataaaa ttgtcccctc
cattctaagg atgtcatcgt cagatgggaa gtataaagcc 540ttctccacct
atggctctca cctaggagtt gtttgctggt tttatggaac agtcattggc
600atgtacctgg cttcagccgt gtcaccaccc cccaggaatg gtgtggtggc
atcagtgatg 660tag 66350911DNAHomo sapiensmisc_featureIncyte ID No
7475164CT1 50gggctgagtt tatcctggca ggcttgacac aacgcccaga acttcaactg
ccactcttcc 60tcctgttcct tggaatatat gtggtcacag tggtggggaa cctgggcatg
atcttcttaa 120ttgctctcag ttctcaactt taccctccag tgtattattt
tctcagtcat ttgtctttca 180ttgatctctg ctactcctct gtcattaccc
ctaagatgct ggtgaacttt gttccagagg 240agaacattat ctcctttctg
gaatgcatta ctcaacttta tttcttcctt atttttgtaa 300ttgcagaagg
ctaccttctg acagccatgg aatatgaccg ttatgttgct atctgtcgcc
360cactgcttta caatattgtc atgtcccaca gggtctgttc cataatgatg
gctgtggtat 420actcactggg ttttctgtgg gccacagtcc atactacccg
catgtcagtg ttgtcattct 480gtaggtctca tacggtcagt cattattttt
gtgatattct ccccttattg actctgtctt 540gctccagcac ccacatcaat
gagattctgc tgttcattat tggaggagtt aataccttag 600caactacact
ggcggtcctt atctcttatg ctttcatttt ctctagtatc cttggtattc
660attccactga ggggcaatcc aaagcctttg gcacttgtag ctcccatctc
ttggctgtgg 720gcatcttttt tgggtctata acattcatgt atttcaagcc
cccttccagc actactatgg 780aaaaagagaa ggtgtcttct gtgttctaca
tcacaataat ccccatgctg aatcctctaa 840tctatagcct gaggaacaag
gatgtgaaaa atgcactgaa gaagatgact aggggaaggc 900agtcatcctg a
91151332DNAHomo sapiensmisc_featureIncyte ID No 7473909CT1
51caggccccag gacagccagt ggctgtgtca tcatgatctg ctttgccctc actgtcctct
60cttacatccg catcttggcc acagtggttc agatccgttc agcagccagc cgccggaagg
120ccttctccac ctgttcttcc cacctgggca tggtgctcct gttctatggc
accggcagct 180ccacctacat gcgacccacc acccgctact ccccgctgga
agggcgcttg gctgctgtct 240tctactccat cctcataccc accctgaatc
cgctcatcta cagcctgagg aaccaggaca 300tgaagagagc cctgtggaag
ctctatctcc ag 33252538DNAHomo sapiensmisc_featureIncyte ID No
7475252CT1 52agagccagag aatctcacag gtgtcttaga attcctgctc ctgggactcc
cagatgatcc 60agaactgcag cccgtcctct ttgggctgtt cctgtccatg tacctggtca
tggtgctggg 120gaacctgctc atcattctgg ccgtcagctc tgactcccat
ctccacagcc ccatgtactt 180cttcctctcc aacctgtcct tggctgacat
cggttttgcc tctactactg tccccaagat 240gattgtggac atccaggctc
atagtagact catctcttac gtgggctgcc tgactcagat 300gtcttttttg
atctttttcg catgtatgga aagtctgctc ctgattgtga tggcctatga
360ccggttcgtg gccatctgtc accccctgca ctaccaagtc atcatgagcc
cacgactctg 420tggcttctta gttttggtgt ctttttttct tagccttttg
gactctcagc tgcacaattt 480gattgtgtta caacttacct gcttcaacga
tgtggaaatc tctaattttt ttctgtga 53853279DNAHomo
sapiensmisc_featureIncyte ID No 7927572CT1 53tcttttagat gcccagctgt
acaatttgat tgccttacaa atgacctgct tcaaggatgt 60ggaaattcct aatttcttct
gtgacccttc tcaactcccc catcttgcat gttgtgacac 120cttcaacaat
aacataatcc tgtatttccc tgatgccata tttggttttc ttcccatctc
180ggggacactt ttctcttacg ataaaattgt ttcctccatt ctgagggttt
catcatcagg 240tgggaagtat aaagccttct ccacctatgg gtctcacct
27954291DNAHomo sapiensmisc_featureIncyte ID No 7481257CT1
54atggaggtaa ccacatttgc catgtgcctg attatagttc ttgttcctct tcttcttatt
60cttgtgtcat atggtttcat tgctgtggct gtactcaaga tcaagtctgc agcaggaaga
120caaaaagcat ttgggacctg ttcctcccat ctcgttgtgg tatccatctt
ctgtgggaca 180gttacataca tgtatataca gccaggaaac agtccaaatc
agaatgaggg caaacttctc 240agtatatttt actccattgt tactcccagc
ttgaacccat taatttatac g 29155402DNAHomo sapiensmisc_featureIncyte
ID No 7485790CT1 55aggatccaga actgcagccc atcctcgctg ggctgtccct
gtccatgtat ctggtcacgg 60tgctgaggaa cctcctcatc agcctggctg tcagctctga
ctcccacctc cacaccccaa 120tgtgcttctt cctctccaac ctgtgctggg
ctgacatcgg tttcacctcg gccacggttc 180ccaagatgat tgtggacatg
cggtcgcata gcggagtcat ctcttatgcg gactgcctga 240cacggatgtc
tttcttggtc ctttttgcat gtgtagaaga catgctcctg actgtgatgg
300cctatgactg ttttgtagcc atctgtcgcc ctctgcacta cccagtcatc
gtgaatcctc 360acctctgtgt cttcttagtt tcggtgtcct tttccttagc ct
40256639DNAHomo sapiensmisc_featureIncyte ID No 7482993CT1
56ggatcagagt gtctcctact ggcagcaatg gcatatgatc gttacattgc aatctgcaat
60cctttaaggt attcagttat tctgagcaag gttctatgca atcaattagc agcctcatgc
120tgggctgctg gtttccttaa ctcagtggtg catacagtgt tgacattctg
cctgcccttc 180tgtggcaaca atcagattaa ttacttcttc tgtgacatcc
cccctttgct gatcttgtct 240tgtggaaaca cttctgtcaa tgagttggca
ctgctatcca ctggggtctt cattggttgg 300actcctttcc tttgtatcgt
actttcctac atttgcataa tctccaccat cttgaggatc 360cagtcctcag
agggaagacg aaaagccttt tctacatgtg cctcccacct ggccattgtc
420tttctctttt atggcagcgc catctttaca tatgtacggc ccatctcaac
ttactcatta 480aagaaagata ggttggtttc agtgttgtac agtgttgtta
cccccatgct aaaccctata 540atttacacat tgaggaataa ggacatcaaa
gaagctgtca aaactatagg gagcaagtgg 600cagccaccaa tttcctcttt
ggatagtaaa ctcacttat 639571370DNAHomo sapiensmisc_featureIncyte ID
No 2829053CB1 57cggcaccaga aggatataac cagaattctc cagcaacatg
aggaggaaaa gaagaaatgg 60gcacaacagg tggagaagga aagggagcta gagctcgaga
cagactggat gagcagcaaa 120gggtcctgga aggaaagaat gaagaggccc
tgcaagtcct ccgggcctca tatgaacagg 180agaaagaagc gcttacccac
tctttccggg aggccagttc tacccagcag gagaccatag 240acagactgac
ctcacagctg gaggctttcc aggccaaaat gaagagggtg gaggagtcca
300ttctgagccg aaactataag aaacatatcc aggattatgg gagccccagc
cagttctggg 360agcaggagct ggagagctta cactttgtca tcgagatgaa
gaatgagcgt attcatgagc 420tggacaagcg gctgatcctc atggaaacag
tgaaagagaa aaatctgata ttggaggaaa 480aaattacgac cctgcaacag
gaaaatgagg acctccatgt ccgaagccgc aaccaggtgg 540tcctgtcaag
gcagctgtca gaagacctgc ttctcacgcg tgaggccctg gagaaggagg
600tgcagctgcg gcgacagctc cagcaggaga aggaggagct gttgtaccgg
gtccttgggg 660ccaatgcctc gcctgccttc cctctggccc ctgtcactcc
cactgaggtc tctttcctcg 720ccacataggg tgcagggcct gggcccacca
cgacgcctga agtcacagct ccttccaagg 780tttttctgga gaagacagca
ggagcctctc agttcttttc caggaaggaa cgagggtggg 840agcgagatgg
agatcctggg tgtgtgccca gtgagccctg gggccttgag ttacatggaa
900tcacccacag ggttttggag gccccgagaa gcgtcttccc ttgagttggc
caagggaata 960agcaagagga gacatttcct ccctgcccca gcactctgtc
ccaatccgag aagttccgag 1020gctttcccag gggcagtctg tgtcacgctg
gccatttgac ataaaggaga cagcccctgg 1080tcccagcttg tcagctctgc
tgccgacttg ctgacttatc aacttcctct aggtgtttcc 1140actccaccct
ggcctgctca gagcctcagt ttacccctgc attaaaatgg tggggggact
1200ggtcaaagga ctcttatgcc actgcagtgg cccattctag gattgtctga
aggccagagt 1260aggggttggg gggagtgtgg acaaaccccg caaatcagag
tggggaaggt gagtggtaga 1320gagggggtct ctgaaggccc ttggggctga
cagggccagg cagcctcccc 1370581567DNAHomo sapiensmisc_featureIncyte
ID No 3068234CB1 58gagggactgc gttctaatac ggagctccga ggatgttcac
ttcttctcca caatgaatga 60gtgtcactat gacaagcaca tggacttttt ttataatagg
agcaacactg atactgtcga 120tgactggaca ggaacaaagc ttgtgattgt
tttgtgtgtt gggacgtttt tctgcctgtt 180tatttttttt tctaattctc
tggtcatcgc ggcagtgatc aaaaacagaa aatttcattt 240ccccttctac
tacctgttgg ctaatttagc tgctgccgat ttcttcgctg gaattgccta
300tgtattcctg atgtttaaca caggcccagt ttcaaaaact ttgactgtca
accgctggtt 360tctccgtcag gggcttctgg acagtagctt gactgcttcc
ctcaccaact tgctggttat 420cgccgtggag aggcacatgt caatcatgag
gatgcgggtc catagcaacc tgaccaaaaa 480gagggtgaca ctgctcattt
tgcttgtctg ggccatcgcc atttttatgg gggcggtccc 540cacactgggc
tggaattgcc tctgcaacat ctctgcctgc tcttccctgg cccccattta
600cagcaggagt taccttgttt tctggacagt gtccaacctc atggccttcc
tcatcatggt 660tgtggtgtac ctgcggatct acgtgtacgt caagaggaaa
accaacgtct tgtctccgca 720tacaagtggg tccatcagcc gccggaggac
acccatgaag ctaatgaaga cggtgatgac 780tgtcttaggg gcgtttgtgg
tatgctggac cccgggcctg gtggttctgc tcctcgacgg 840cctgaactgc
aggcagtgtg gcgtgcagca tgtgaaaagg tggttcctgc tgctggcgct
900gctcaactcc gtcgtgaacc ccatcatcta ctcctacaag gacgaggaca
tgtatggcac 960catgaagaag atgatctgct gcttctctca ggagaaccca
gagaggcgtc cctctcgcat 1020cccctccaca gtcctcagca ggagtgacac
aggcagccag tacatagagg atagtattag 1080ccaaggtgca gtctgcaata
aaagtacttc ctaaactctg gatgcctctc ggcccaccca 1140ggcctcctct
gggaaaagag ctgttaagaa tgattacctg tctctaacaa agcccatgta
1200cagtgttatt tgaggtctcc attaatcact gctagatttc tttaaaaaat
tttttttcat 1260agtttaaaag catgggcagt aaagagagga cctgctgcat
ttagagaaag cacagaaacg 1320ggagaggttc ggcgggtccc tgcttgtcct
atgaactgct cagagctcct gtcagtccag 1380ctgggccttc tgggttctgg
caccatttcg tagccattct ctttgtattt taaaaggacg 1440ttatgaaagg
gcttagacca aaataaatca taatgttact tgagccacct tatatagctg
1500cttggagagt ctatgtagtt ctttctgcat gcattaaaaa tgtttagaaa
tgcttcaaaa 1560aaaaaaa 1567591321DNAHomo sapiensmisc_featureIncyte
ID No 5029478CB1 59cagaccgctg cgggccgcag gcgccgggaa tgtcccctga
atgcgcgcgg gcagcgggcg 60acgcgccctt gcgcagcctg gagcaagcca accgcacccg
ctttcccttc ttctccgacg 120tcaagggcga ccaccggctg gtgctggccg
cggtggagac aaccgtgctg gtgctcatct 180ttgcagtgtc gctgctgggc
aacgtgtgcg ccctggtgct ggtggcgcgc cgacgacgcc 240gcggcgcgac
tgcctgcctg gtactcaacc tcttctgcgc ggacctgctc ttcatcagcg
300ctatccctct ggtgctggcc gtgcgctgga ctgaggcctg gctgctgggc
cccgttgcct 360gccacctgct cttctacgtg atgaccctga gcggcagcgt
caccatcctc acgctggccg 420cggtcagcct ggagcgcatg gtgtgcatcg
tgcacctgca gcgcggcgtg cggggtcctg 480ggcggcgggc gcgggcagtg
ctgctggcgc tcatctgggg ctattcggcg gtcgccgctc 540tgcctctctg
cgtcttcttc cgagtcgtcc cgcaacggct ccccggcgcc gaccaggaaa
600tttcgatttg cacactgatt tggcccacca ttcctggaga gatctcgtgg
gatgtctctt 660ttgttacttt gaacttcttg gtgccaggac tggtcattgt
gatcagttac tccaaaattt 720tacagatcac aaaggcatca aggaagaggc
tcacggtaag cctggcctac tcggagagcc 780accagatccg cgtgtcccag
caggacttcc ggctcttccg caccctcttc ctcctcatgg 840tctccttctt
catcatgtgg agccccatca tcatcaccat cctcctcatc ctgatccaga
900acttcaagca agacctggtc atctggccgt ccctcttctt ctgggtggtg
gccttcacat 960ttgctaattc agccctaaac cccatcctct acaacatgac
actgtgcagg aatgagtgga 1020agaaaatttt ttgctgcttc tggttcccag
aaaagggagc cattttaaca gacacatctg 1080tcaaaagaaa tgacttgtcg
attatttctg gctaattttt ctttatagca gagtttctca 1140cacctggcga
gctgtggcat gcttttaaac agagttcatt tccagtaccc tccatcagtg
1200gcaccctgct ttaagaaaat gaacttatgc aaatagacat ccacagcgtc
ggtaaattaa 1260ggggtgatca ccaagtttca taatattttc cctttataaa
aggatttgtt ggccaggtgc 1320a 1321601110DNAHomo
sapiensmisc_featureIncyte ID No 5102576CB1 60atgttccttg tgagttgctc
gcatccagca ttaagggctg gtttttatct tttatttttc 60caatcctctt tccttctcaa
ggtgtccaag acacacggag ccacggaatc tcacaggtgt 120ctgagaattc
ctcctcctgg gactctcaga ggatccagaa ctgcagccgg ccctcgcttt
180gctgtccctg tccctgtcca tgtatctggt cacggtgctg aggaacctgt
tcagcatcct 240ggctgtcagc tctgactgcc ccctccacac ccccatgtac
ttcttcctct ccaacctgtg 300ctggcctgac atcggtttca cctcggccat
ggttcccaag atgattgtgg acacgcagtc 360gcatagcaga gtcatctctc
atgcgggctg cctgacacag atgtctttcc tgctccttgt 420tgcatgtata
gaaggcatgc tcctgactgt gatggcctat gactgctttg tagccatctg
480tcgccctctg cactacccag tcatcgtgaa tcctcacctc tgtgtctttt
tcgttttggt 540gtcctttttc cttagcctgt tggattccca gctgcacagt
tggattgtgt tacaattaac 600catcatcaag aatgtggaaa tctctaattt
ggtctgtgac ccctctcaac ttctcaatct 660tgcctgttct gacagcgtca
tcaataacat attcatatat ttcgatagta ctatgtttgg 720ttttcttccc
atttcaggga tctttttgtc ttactataaa attgtcccct ccattctaag
780gatttcatcg tcagatggga agtataaagc cttctccacc tgtggctgtc
atctagcagt 840tgtttgctgg ttttatggaa caggcattgg catgtacctg
acttcagctg tgtcaccacc 900ccccaggaat ggtgtggtgg catcagtgat
gtacgctgtg gtcaccccat gctgaacctt 960ttcatctgca gcctgagaaa
cagggacata caaagtgccc tgcggaggct gggcagcaga 1020gcattcgaat
ctcatgatct gttccatcct ttttcttgtg tgggtgagaa agggcaatca
1080cattaaatct ctttatctgc aaaaaaaaaa 1110611095DNAHomo
sapiensmisc_featureIncyte ID No 2200534CB1 61atgaaggcca actacagcgc
agaggagcgc tttctcctgc tgggtttctc cgactggcct 60tccctgcagc cggtcctctt
cgcccttgtc ctcctgtgct acctcctgac cttgacgggc 120aactcggcgc
tggtgctgct ggcggtgcgc gacccgcgcc tgcacacgcc catgtactac
180ttcctctgcc acctggcctt ggtagacgcg ggcttcacta ctagcgtggt
gccgccgctg 240ctggccaacc tgcgcggacc agcgctctgg ctgccgcgca
gccactgcac ggcccagctg 300tgcgcatcgc tggctctggg ttcggccgaa
tgcgtcctcc tggcggtgat ggctctggac 360cgcgcggccg cagtgtgccg
cccgctgcgc tatgcggggc tcgtctcccc gcgcctatgt 420cgcacgctgg
ccagcgcctc ctggctaagc ggcctcacca actcggttgc gcaaaccgcg
480ctcctggctg agcggccgct gtgcgcgccc cgcctgctgg accacttcat
ctgtgagctg 540ccggcgttgc tcaagctggc ctgcggaggc gacggagaca
ctaccgagaa ccagatgttc 600gccgcccgcg tggtcatcct gctgctgccg
tttgccgtca tcctggcctc ctacggtgcc 660gtggcccgag ctgtctgttg
catgcggttc agcggaggcc ggaggagggc ggtgggcacg 720tgtgggtccc
acctgacagc cgtctgcctg ttctacggct cggccatcta cacctacctg
780cagcccgcgc agcgctacaa ccaggcacgg ggcaagttcg tatcgctctt
ctacaccgtg 840gtcacacctg ctctcaaccc gctcatctac accctcagga
ataagaaagt gaagggggca 900gcgaggaggc tgctgcggag tctggggaga
ggccaggctg ggcagtgagt agttggggag 960gggagaaagt attaagccag
aacccaagga tggaaatacc ccttagtgag tcagtttaga 1020cttcaggctg
ttcatttttg tatgataatc tgcaagattt gtcctaagga gtccaatggg
1080ggatatgttt tcctc 1095621665DNAHomo sapiensmisc_featureIncyte ID
No 3275821CB1 62ttcctacctt cactgattct ctgaaccttc ctgtcctcgc
ctgtaaagta gattgtatga 60ggactccatg aggtcatcca cttcaagtcc ttggcatagg
ataattactc aaaaggtgat 120gacaatggcg cagggaggga tggtgacttg
cctggagatg cacagcaccg tctctcccat 180actcggtcat tcacaccatc
attgattcac caggcaccca ctccgtgtcc agcaggactc 240tggggacccc
aaatggacac taccatggaa gctgacctgg gtgccactgg ccacaggccc
300cgcacagagc ttgatgatga ggactcctac ccccaaggtg gctgggacac
ggtcttcctg 360gtggccctgc tgctccttgg gctgccagcc aatgggttga
tggcgtggct ggccggctcc 420caggcccggc atggagctgg cacgcgtctg
gcgctgctcc tgctcagcct ggccctctct 480gacttcttgt tcctggcagc
agcggccttc cagatcctag agatccggca tgggggacac 540tggccgctgg
ggacagctgc ctgccgcttc tactacttcc tatggggcgt gtcctactcc
600tccggcctct tcctgctggc
cgccctcagc ctcgaccgct gcctgctggc gctgtgccca 660cactggtacc
ctgggcaccg cccagtccgc ctgcccctct gggtctgcgc cggtgtctgg
720gtgctggcca cactcttcag cgtgccctgg ctggtcttcc ccgaggctgc
cgtctggtgg 780tacgacctgg tcatctgcct ggacttctgg gacagcgagg
agctgtcgct gaggatgctg 840gaggtcctgg ggggcttcct gcctttcctc
ctgctgctcg tctgccacgt gctcacccag 900gccacagcct gtcgcacctg
ccaccgccaa cagcagcccg cagcctgccg gggcttcgcc 960cgtgtggcca
ggaccattct gtcagcctat gtggtcctga ggctgcccta ccagctggcc
1020cagctgctct acctggcctt cctgtgggac gtctactctg gctacctgct
ctgggaggcc 1080ctggtctact ccgactacct gatcctactc aacagctgcc
tcagcccctt cctctgcctc 1140atggccagtg ccgacctccg gaccctgctg
cgctccgtgc tctcgtcctt cgcggcagct 1200ctctgcgagg agcggccggg
cagcttcacg cccactgagc cacagaccca gctagattct 1260gagggtccaa
ctctgccaga gccgatggca gaggcccagt cacagatgga tcctgtggcc
1320cagcctcagg tgaaccccac actccagcca cgatcggatc ccacagctca
gccacagctg 1380aaccctacgg cccagccaca gtcggatccc acagcccagc
cacagctgaa cctcatggcc 1440cagccacagt cagattctgt ggcccagcca
caggcagaca ctaacgtcca gacccctgca 1500cctgctgcca gttctgtgcc
cagtccctgt gatgaagctt ccccaacccc atcctcgcat 1560cctaccccag
gggcccttga ggacccagcc acacctcctg cctctgaagg agaaagcccc
1620agcagcaccc cgccagaggc ggccccgggc gcaggcccca cgtga
1665631609DNAHomo sapiensmisc_featureIncyte ID No 3744167CB1
63tctccttttg ccgattagtg gacgtgacag agatgtgaat ggggcaggga tgtcctttga
60tggcatcaag actttagctt ctggtgcgct gtgtcccagc tctgatttca gttgcagccg
120tgatggacag ttgcatggaa gctgagactc tcactgacag tgaaaccctc
aaatgaacac 180aatccctgct ttcctgccaa ggatccttgt agggtccccc
agcttcccca ctttttttct 240gtgtcctgta ggcccagaag gatgtcggtc
tgctaccgtc ccccagggaa cgagacactg 300ctgagctgga agacttcgcg
ggccacaggc acagccttcc tgctgctggc ggcgctgctg 360gggctgcctg
gcaacggctt cgtggtgtgg agcttggcgg gctggcggcc tgcacggggg
420cgaccgctgg cggccacgct tgtgctgcac ctggcgctgg ccgacggcgc
ggtgctgctg 480ctcacgccgc tctttgtggc cttcctgacc cggcaggcct
ggccgctggg ccaggcgggc 540tgcaaggcgg tgtactacgt gtgcgcgctc
agcatgtacg ccagcgtgct gctcaccggc 600ctgctcagcc tgcagcgctg
cctcgcagtc acccgcccct tcctggcgcc tcggctgcgc 660agcccggccc
tggcccgccg cctgctgctg gcggtctggc tggccgccct gttgctcgcc
720gtcccggccg ccgtctaccg ccacctgtgg agggaccgcg tatgccagct
gtgccacccg 780tcgccggtcc acgccgccgc ccacctgagc ctggagactc
tgaccgcttt cgtgcttcct 840ttcgggctga tgctcggctg ctacagcgtg
acgctggcac ggctgcgggg cgcccgctgg 900ggctccgggc ggcacggggc
gcgggtgggc cggctggtga gcgccatcgt gcttgccttc 960ggcttgctct
gggcccccta ccacgcagtc aaccttctgc aggcggtcgc agcgctggct
1020ccaccggaag gggccttggc gaagctgggc ggagccggcc aggcggcgcg
agcgggaact 1080acggccttgg ccttcttcag ttctagcgtc aacccggtgc
tctacgtctt caccgctgga 1140gatctgctgc cccgggcagg tccccgtttc
ctcacgcggc tcttcgaagg ctctggggag 1200gcccgagggg gcggccgctc
tagggaaggg accatggagc tccgaactac ccctcagctg 1260aaagtggtgg
ggcagggccg cggcaatgga gacccggggg gtgggatgga gaaggacggt
1320ccggaatggg acctttgaca gcagacccta caacctgctg cccttccctg
tccctttcca 1380ccccccaccc accctccaga ggtcagtgtt ctgggacatt
tggggaccct tctttgacta 1440gagtttggat ctggctgggt aggattacta
tacacttggg gcaggcccag gctcctccaa 1500actgagggat tatgagggtg
gtgatggtcc ctgttaagga ctattgtgtg cttgcaagtt 1560ggcatgtacc
catgtgccag cattgcttac ttgttgccaa tagctgtta 160964945DNAHomo
sapiensmisc_featureIncyte ID No 7472007CB1 64atgtgggaaa actggacaat
tgtcagtgaa tttgttctcg tgagcttctc agccctgtcc 60actgagcttc aggctctact
gtttctcctt ttcttgacca tttacttggt tactttaatg 120ggcaatgtcc
tcatcatcct ggtcactata gctgactctg cactacaaag tcctatgtac
180ttcttcctca gaaacttgtc cttcctggag ataggtttca acttggtcat
tgtgcccaag 240atgctgggga ccctgatcat tcaagacaca accatctcct
tccttggatg tgccactcag 300atgtatttct tcttcttttt tggggctgct
gagtgctgcc tcctggccac catggcatat 360gaccgctacg tggccatctg
tgaccccttg cactacccag tcatcatggg ccacatatcc 420tgtgcccagc
tggcagctgc ctcttggttc tcagggtttt cagtggccac tgtgcaaacc
480acatggattt tcagtttccc tttttgtggc cccaacaggg tgaaccactt
cttctgtgac 540agccctcctg ttattgcact ggtctgtgct gacacctctg
tgtttgaact ggaggctctg 600acagccactg tcccattcat tctctttcct
ttcttgctga tcctgggatc ctatgtccgc 660atcctctcca ctatcttcag
gatgccgtca gctgagggga aacatcaggc attctccacc 720tgttccgccc
acctcttggt tgtctctctc ttctatagca ctgccatcct cacgtatttc
780cgaccccaat ccagtgcctc ttctgagagc aagaagctgc tgtcactctc
ttccacagtg 840gtgactccca tgttgaaccc catcatctac agctcaagga
ataaagaagt gaaggctgca 900ctgaagcggc ttatccacag gaccctgggc
tctcagaaac tatga 945651098DNAHomo sapiensmisc_featureIncyte ID No
7472008CB1 65atggagggat ctgttgaagc tacacctgaa attccagctc agatgaaatg
tcatccttca 60agacccagta ctttaaatca attatctttc tatggtgctg tgtcctcact
aggaagaatg 120catggtttag aaaccaaaag ctctgctgaa attagagctg
ggctgaagag atgtgataca 180ctggtactag aggcatctac tttagaagga
aatatggtca tagttcttgt gtccttgaag 240gatccaaaac tccacatccc
tatgtatttc tttctttcca acctttcctt ggtagacctc 300tgtttgacca
gcagctgtgt tccacagatg ttgattaact tctggggccc agaaaagacc
360atcagctaca ttggctgtgc cattcaactc tatgtttttt tgtggcttgg
ggccacggaa 420tatgtccttc ttgttgtcat ggctgtggat tgttatgtag
cagtgtgtca tccactgcaa 480aataccatga tcatgcaccc aaaactttgt
ctgcagctgg ctatcttggc atgggggact 540ggcttggccc agtctctgat
ccagtcccct gccaccctcc ggttaccctt ctgctcccag 600cggatggtgg
atgatgttgt ttgtgaagtc ccagctctga ttcagctctc cagtactgat
660actacctaca gtgaaattca gatgtctatc gccagtgttg tcctcctggt
gatgcccttg 720atcattatcc tttcctcttc tggtgctatt gctaaggctg
tgctgagaat taagtcaact 780gcaggacaga agaaagcatt tggcacctgc
atctctcacc ttcttgtggt ttctctcttt 840tatggcactg tcacaggtgt
ctaccttcaa ccaaaaaatc actatcctca tgaatggggc 900aaatttctca
ctcttttcta cactgtagta accccaactc ttaatcccct catctacact
960ctaaggaaca aggagctcca tccttggcta aaagaggcca aggtacagac
cgcttcagag 1020agtgcaagcc ccaagcattg gcagcttcca catggtgttg
gtcctgtggg tgtgcagaag 1080acaagaactg agctttga 109866954DNAHomo
sapiensmisc_featureIncyte ID No 7472013CB1 66atgagctttg cccctaatgc
ttcacactct ccggtttttt tgctccttgg gttctcgaga 60gctaacatct cctacactct
cctcttcttc ctgttcctgg ctatttacct gaccaccata 120ctggggaatg
tgacactggt gctgctcatc tcctgggact ccagactgca ctcacccatg
180tattatctgc ttcgtggcct ctctgtgata gacatggggc tatccacagt
tacactgccc 240cagttgctgg cccatttggt ctctcattac ccaaccattc
ctgctgcccg ctgcttggct 300cagttctttt tcttctatgc atttggggtt
acagatacac ttgtcattgc tgtcatggct 360ctggatcgct atgtggccat
ctgtgacccc ctgcactatg ctttggtaat gaatcaccaa 420cggtgtgcct
gcttactagc cttgagctgg gtggtgtcca tactgcacac catgttgcgt
480gtgggactcg tcctgcctct ttgctggact ggggatgctg ggggcaacgt
taaccttcct 540cacttctttt gtgaccaccg gccacttctg cgagcctctt
gttctgacat acattctaat 600gagctggcca tattctttga gggtggcttc
cttatgctgg gcccctgtgc cctcattgta 660ctctcttatg tccgaattgg
ggccgctatt ctacgtttgc cttcagctgc tggtcgccgc 720cgagcagtct
ccacctgtgg atcccacctc accatggttg gtttcctcta cggcaccatc
780atttgtgtct acttccagcc tcccttccag aactctcagt atcaggacat
ggtggcttca 840gtaatgtata ctgccattac acctttggcc aacccatttg
tgtatagcct ccacaataag 900gatgtcaagg gtgcactctg caggctgctt
gaatgggtga aggtagaccc ctga 954671008DNAHomo
sapiensmisc_featureIncyte ID No 7472015CB1 67atggaatcat ctttctcatt
tggagtgatc cttgctgtcc tggcctccct catcattgct 60actaacacac tagtggctgt
ggctgtgctg ctgttgatcc acaagaatga tggtgtcagt 120ctctgcttca
ccttgaatct ggctgtggct gacaccttga ttggtgtggc catctctggc
180ctactcacag accagctctc cagcccttct cggcccacac agaagaccct
gtgcagcctg 240cggatggcat ttgtcacttc ctccgcagct gcctctgtcc
tcacggtcat gctgatcacc 300tttgacaggt accttgccat caagcagccc
ttccgctact tgaagatcat gagtgggttc 360gtggccgggg cctgcattgc
cgggctgtgg ttagtgtctt acctcattgg cttcctccca 420ctcggaatcc
ccatgttcca gcagactgcc tacaaagggc agtgcagctt ctttgctgta
480tttcaccctc acttcgtgct gaccctctcc tgcgttggct tcttcccagc
catgctcctc 540tttgtcttct tctactgcga catgctcaag attgcctcca
tgcacagcca gcagattcga 600aagatggaac atgcaggagc catggctgga
ggttatcgat ccccacggac tcccagcgac 660ttcaaagctc tccgtactgt
gtctgttctc attgggagct ttgctctatc ctggaccccc 720ttccttatca
ctggcattgt gcaggtggcc tgccaggagt gtcacctcta cctagtgctg
780gaacggtacc tgtggctgct cggcgtgggc aactccctgc tcaacccact
catctatgcc 840tattggcaga aggaggtgcg actgcagctc taccacatgg
ccctaggagt gaagaaggtg 900ctcacctcat tcctcctctt tctctcggcc
aggaattgtg gcccagagag gcccagggaa 960agttcctgtc acatcgtcac
tatctccagc tcagagtttg atggctaa 100868930DNAHomo
sapiensmisc_featureIncyte ID No 7472016CB1 68atgagggaaa ataaccagtc
ctctacactg gaattcatcc tcctgggagt tactggtcag 60caggaacagg aagatttctt
ctacatcctc ttcctgttca tttaccccat cacattgatt 120ggaaacctgc
tcattgtcct agccatttgc tctgatgttc gccttcacaa ccccatgtat
180tttctccttg ccaacctctc cttggttgac atcttcttct catcggtaac
catccctaag 240atgctggcca accatctctt gggcagcaaa tccatctctt
ttgggggatg cctaacgcag 300atgtatttca tgatagcctt gggtaacaca
gacagctata ttttggctgc aatggcatat 360gatcgagctg tggccatcag
ccacccactt cactacacaa caattatgag tccacggtct 420tgtatctggc
ttattgctgg gtcttgggtg attggaaatg ccaatgccct cccccacact
480ctgctcacag ctagtctgtc cttctgtggc aaccaggaag tggccaactt
ctactgtgac 540attaccccct tgctgaagtt atcctgttct gacatccact
ttcatgtgaa gatgatgtac 600ctaggggttg gcattttctc tgtgccatta
ctatgcatca ttgtctccta tattcgagtc 660ttctccacag tcttccaggt
tccttccacc aagggcgtgc tcaaggcctt ctccacctgt 720ggttcccacc
tcacggttgt ctctttgtat tatggtacag tcatgggcac gtatttccgc
780cctttgacca attatagcct aaaagacgca gtgatcactg taatgtacac
ggcagtgacc 840ccaatgttaa atcctttcat ctacagtctg agaaatcggg
acatgaaggc tgccctgcgg 900aaactcttca acaagagaat ctcctcgtaa
93069711DNAHomo sapiensmisc_featureIncyte ID No 7472017CB1
69atgggcatga ccaacagcag tgtcaaggga gacttcatcc tgctgctgtg gaacctaaaa
60ggacctgaca aaacaatcac attcctgggt tgtgtcatcc agctctacat ctccctggca
120ttgggctcca ctgagtgtgt cctcctggct gtaatggctt ttgatcgcta
tgctgcagtt 180tgcaaacctc tccactatac cgccgtaatg aaccctcagc
tgtgccaggc tctggcaggg 240gttgcgtggc tgagtggagt gggaaacact
cttatccagg gcactgtcac cctctggctt 300cctcgctgtg gacaccgatt
gctccaacat ttcttccttg catgtgtgga catccatgat 360aatgaggttc
agctctttgt tgcttcactg gtcttgctcc tcttgccctt agtgctaata
420ctgctgtcct atggacatat agccaaggtg gtcataagga tcaagtcagt
ccaggcctgg 480tgcaaaggcc tggggacatg tggatcccat ttgatagtag
tgtccctctt ctgtgggacc 540atcacagctg tctacatcca gtccaacagt
tcttatgccc atgctcatgg gaagttcatc 600tccctcttct atacagttgt
gaccccgacc ctcaatcctc tcatctacac actgaggaat 660aatgacgtga
aaggagcact gcgattattt aacagagact taggcacata a 711701092DNAHomo
sapiensmisc_featureIncyte ID No 7472018CB1 70atgggccccg gcgaggcgct
gctggcgggt ctcctggtga tggtactggc cgtggcgctg 60ctatccaacg cactggtgct
gctttgttgc gcctacagcg ctgagctccg cactcgagcc 120tcaggcgtcc
tcctggtgaa tctgtctctg ggccacctgc tgctggcggc gctggacatg
180cccttcacgc tgctcggtgt gatgcgcggg cggacaccgt cggcgcccgg
cgcatgccaa 240gtcattggct tcctggacac cttcctggcg tccaacgcgg
cgctgagcgt ggcggcgctg 300agcgcagacc agtggctggc agtgggcttc
ccactgcgct acgccggacg cctgcgaccg 360cgctatgccg gcctgctgct
gggctgtgcc tggggacagt cgctggcctt ctcaggcgct 420gcacttggct
gctcgtggct tggctacagc agcgccttcg cgtcctgttc gctgcgcctg
480ccgcccgagc ctgagcgtcc gcgcttcgca gccttcaccg ccacgctcca
tgccgtgggc 540ttcgtgctgc cgctggcggt gctctgcctc acctcgctcc
aggtgcaccg ggtggcacgc 600agacactgcc agcgcatgga caccgtcacc
atgaaggcgc tcgcgctgct cgccgacctg 660caccccagtg tgcggcagcg
ctgcctcatc cagcagaagc ggcgccgcca ccgcgccacc 720aggaagattg
gcattgctat tgcgaccttc ctcatctgct ttgccccgta tgtcatgacc
780aggctggcgg agctcgtgcc cttcgtcacc gtgaacgccc agtggggcat
cctcagcaag 840tgcctgacct acagcaaggc ggtggccgac ccgttcacgt
actctctgct ccgccggccg 900ttccgccaag tcctggccgg catggtgcac
cggctgctga agagaacccc gcgcccagca 960tccacccatg acagctctct
ggatgtggcc ggcatggtgc accagctgct gaagagaacc 1020ccgcgcccag
cgtccaccca caacggctct gtggacacag agaatgattc ctgcctgcag
1080cagacacact ga 109271927DNAHomo sapiensmisc_featureIncyte ID No
7472019CB1 71atggccatgg acaatgtcac agcagtgttt cagtttctcc ttattggcat
ttctaactat 60cctcaatgga gagacacgtt tttcacatta gtgctgataa tttacctcag
cacattgttg 120gggaatggat ttatgatctt tcttattcac tttgacccca
acctccacac tccaatctac 180ttcttcctta gtaacctgtc tttcttagac
ctttgttatg gaacagcttc catgccccag 240gctttggtgc attgtttctc
tacccatccc tacctctctt atccccgatg tttggctcaa 300acgagtgtct
ccttggcttt ggccacagca gagtgcctcc tactggctgc catggcctat
360gaccgtgtgg ttgctatcag caatcccctg cgttattcag tggttatgaa
tggcccagta 420tgtgtctgct tggttgctac ctcatggggg acatcacttg
tgctcactgc catgctcatc 480ctatccctga ggcttcactt ctgtggggct
aatgtcatca accattttgc ctgtgagatt 540ctctccctca ttaagctgac
ctgttctgat accagcctca atgaatttat gatcctcatc 600accagtatct
tcaccctgct gctaccattt gggtttgttc tcctctccta catacgaatt
660gctatggcta tcataaggat tcgctcactc cagggcaggc tcaaggcctt
taccacatgt 720ggctctcacc tgaccgtggt gacaatcttc tatgggtcag
ccatctccat gtatatgaaa 780actcagtcca agtcctaccc tgaccaggac
aagtttatct cagtgtttta tggagctttg 840acacccatgt tgaaccccct
gatatatagc ctgagaaaaa aagatgttaa acgggcaata 900aggaaagtta
tgttgaaaag gacatga 927721032DNAHomo sapiensmisc_featureIncyte ID No
7472021CB1 72atgcattttc ttcctactgt ctttggcttc ctaaacagag tcacacttgg
tatcttcaga 60gagactatgg tcaatttgac ttcaatgagt ggattccttc ttatggggtt
ttctgatgag 120cgtaagcttc agattttaca tgcattggta tttctggtga
catacctgct ggccttgaca 180ggcaacctcc tcattatcac catcattacc
gtggaccgtc gtctccattc ccccatgtat 240tactttttaa agcacctctc
tcttctggac ctctgcttca tctctgtcac agtcccccag 300tccattgcaa
attcacttat gggcaacggt tacatttctc ttgttcagtg cattcttcag
360gttttcttct tcatagctct ggcctcatca gaagtggcca ttctcacagt
gatgtcttat 420gacaggtacg cagcaatctg tcaaccactt cattatgaga
ctattatgga tccccgtgcc 480tgtaggcatg cagtgatagc tgtgtggatt
gctgggggcc tctctgggct catgcatgct 540gccattaact tctccatacc
tctctgtggg aagagagtca ttcaccaatt cttctgtgat 600gttcctcaga
tgctgaaact agcctgttct tatgaattca ttaatgagat tgcactggct
660gcattcacaa cgtctgcagc atttatctgt ttgatctcca ttgtgctctc
ctacattcgc 720atcttctcta cagtgctgag aatcccatca gctgagggcc
ggaccaaggt cttctccacc 780tgcctaccac acctatttgt agccaccttc
tttctttcag ctgcaggctt tgagtttctc 840agactgcctt ctgattcctc
atcgactgtg gaccttgtat tctccgtatt ctatactgtg 900atacctccaa
cactcaatcc agtcatttat agcttacgga atgattccat gaaggcagca
960ctgaggaaga tgctgtcaaa ggaagagctt cctcagagaa aaatgtgctt
aaaagccatg 1020tttaaactct ga 103273972DNAHomo
sapiensmisc_featureIncyte ID No 7472009CB1 73atgtggcaga agaatcagac
ctctctggca gacttcatcc ttgaggggct cttcgatgac 60tcccttaccc accttttcct
tttctccttg accatggtgg tcttccttat tgcggtgagt 120ggcaacaccc
tcaccattct cctcatctgc attgatcccc agcttcatac accaatgtat
180ttcctgctca gccagctctc cctcatggat ctgatgcatg tctccacaac
catcctgaag 240atggctacca actacctatc tggcaagaaa tctatctcct
ttgtgggctg tgcaacccag 300cacttcctct atttgtgtct aggtggtgct
gaatgttttc tcttagctgt catgtcctat 360gaccgctatg ttgccatctg
tcatccactg cgctatgctg tgctcatgaa caagaaggtg 420ggactgatga
tggctgtcat gtcatggttg ggggcatccg tgaactccct aattcacatg
480gcgatcttga tgcacttccc tttctgtggg cctcggaaag tctaccactt
ctactgtgag 540ttcccagctg ttgtgaagtt ggtatgtggc gacatcactg
tgtatgagac cacagtgtac 600atcagcagca ttctcctcct cctccccatc
ttcctgattt ctacatccta tgtcttcatc 660cttcaaagtg tcattcagat
gcgctcatct gggagcaaga gaaatgcctt tgccacttgt 720ggctcccacc
tcacggtggt ttctctttgg tttggtgcct gcatcttctc ctacatgaga
780cccaggtccc agtgcactct attgcagaac aaagttggtt ctgtgttcta
cagcatcatt 840acgcccacat tgaattctct gatttatact ctccggaata
aagatgtagc taaggctctg 900agaagagtgc tgaggagaga tgttatcacc
cagtgcattc aacgactgca attgtggttg 960ccccgagtgt ag 97274900DNAHomo
sapiensmisc_featureIncyte ID No 7472010CB1 74atggaactgg aaggagactt
ccttggtagt gtgggagaat tgggccaagt gatccagacc 60tgttctggga tctatgtgtt
cactgtggtg ggcaacttgg gcttgatcac cttaattggg 120ataaatccta
gccttcacac ccccatgtac tttttcctct tcaacttgtc ctttatagat
180ctctgttatt cctgtgtgtt tacccccaaa atgctgaatg actttgtttc
agaaagtatc 240atctcttatg tgggatgtat gactcagcta tttttcttct
gtttctttgt caattctgag 300tgctatgtgt tggtatcaat ggcctatgat
cgctatgtgg ccatctgcaa ccccctgctc 360tacatggtca ccatgtcccc
aagggtctgc tttctgctga tgtttggttc ctatgtggta 420gggtttgctg
gggccatggc ccacactgga agcatgctgc gactgacctt ctgtgattcc
480aacgtcattg accattatct gtgtgacgtt ctccccctct tgcagctctc
ctgcaccagc 540acccatgtca gtgagctggt atttttcatt gttgttggag
taatcaccat gctatccagc 600ataagcatcg tcatctctta cgctttgata
ctctccaaca tcctctgtat tccttctgca 660gagggcagat ccaaagcctt
tagcacatgg ggctcccaca taattgctgt tgctctgttt 720tttgggtcag
ggacattcac ctacttaaca acatcttttc ctggctctat gaaccatggc
780agatttgcct cagtctttta caccaatgtg gttcccatgc ttaacccttc
gatctacagt 840ttgaggaata aggatgataa acttgccctg ggcaaaaccc
tgaagagagt gctcttctaa 90075924DNAHomo sapiensmisc_featureIncyte ID
No 7472011CB1 75atggaaacag ggaacctcac gtgggtatca gactttgtct
tcctggggct ctcgcagact 60cgggagctcc agcgtttcct gtttctaatg ttcctgtttg
tctacatcac cactgttatg 120ggaaacatcc ttatcatcat cacagtgacc
tctgattccc agctccacac acccatgtac 180tttctgctcc gaaacctggc
tgtcctagac ctctgtttct cttcagtcac tgctcccaaa 240atgctagtgg
acctcctctc tgagaagaaa accatctctt accagggctg catgggtcag
300atcttcttct tccacttttt gggaggtgcc atggtcttct tcctctcagt
gatggccttt 360gaccgcctca ttgccatctc ccggcccctc cgctatgtca
ccgtcatgaa cactcagctc 420tgggtggggc tggtggtagc cacctgggtg
ggaggctttg tccactctat tgtccagctg 480gctctgatgc tcccactgcc
cttctgtggc cccaacattt tggataactt ctactgtgat 540gttccccaag
tactgagact tgcctgcact gacacctcac tgctggagtt cctcaagatc
600tccaacagtg ggctgctgga tgtcgtctgg ttcttcctcc tcctgatgtc
ctacttattc 660atcctggtga tgctgaggtc acatccaggg gaggcaagaa
ggaaggcagc ttccacctgc 720accacccaca tcatcgtggt
ttccatgatc ttcgttccaa gcatttacct ctatgcccgg 780cccttcactc
cattccctat ggacaagctt gtgtccatcg gccacacagt catgaccccc
840atgctcaacc ccatgatcta taccctgagg aaccaggaca tgcaggcagc
agtgagaaga 900ttagggagac accggctggt ttga 92476945DNAHomo
sapiensmisc_featureIncyte ID No 7472012CB1 76atggacaaca gcaactggac
cagtgtgtcc cattttgttc tcttgggcat ttccacccac 60ccagaagagc aaatcccact
cttccttgtt ttctcactca tgtacgcaat caatatttct 120ggcaacttgg
ccatcatcac actgattctc tctgctccac gcctccacat ccccatgtac
180atcttcctca gtaacttggc cttgacagac atctgcttca cctccaccac
ggtccccaag 240atgctgcaga ttattttctc ccctacaaag gtaatttcct
acacaggctg tttagcccaa 300acttatttct tcatttgctt cgccgtcatg
gaaaacttca tcctggctgt gatggcctat 360gacaggtaca ttgccatctg
ccaccctttc cactacacta tgatcctgac tagaatgctg 420tgtgtgaaga
tggtggtcat gtgccatgct ctctcccacc ttcatgccat gctgcatacc
480tttctcatag gccaactaat cttctgtgca gataacagaa tcccccactt
cttctgtgac 540ctctacgctc tgatgaagat ctcctgcacc agcacctacc
tcaacaccct tatgattcac 600acagaaggtg ctgttgtaat cagtggagct
ctggccttca ttactgcctc ctatgcctgc 660atcatcctgg tggtcctccg
gatcccctca gccaagggca ggtggaaaac cttttctacc 720tgcggctccc
acctcactgt ggtggccata ttctatggca ccctcagttg ggtctacttc
780cggccccttt ccagctattc agtgaccaag ggtcgcatta taacagtcgt
gtacacagtg 840gtgactccca tgctgaaccc cttcatctac agcctgagga
atggggatgt caagggaggc 900ttcatgaaat ggatgagcag aatgcagact
tttttcttta gataa 94577933DNAHomo sapiensmisc_featureIncyte ID No
7472014CB1 77atgggaagaa ataacctaac aagaccctct gaattcatcc tccttggact
ctcctctcga 60cctgaggatc agaagccgct ctttgctgtg ttcctcccca tctaccttat
cacagtgata 120ggaaacctgc ttatcatcct ggccatccgc tcagacactc
gtctccagac gcccatgtac 180ttctttctaa gcatcctgtc ttttgttgac
atttgctatg tgacagtcat tatccctaag 240atgctggtga acttcttatc
agagacaaag accatctctt acggtgagtg tctgacccag 300atgtactttt
tcttagcctt tggaaacaca gacagttacc tgctagcagc catggccatt
360gaccgctatg tggccatatg taatcccttc cactacatca ccattatgag
tcacagatgc 420tgtgtcctgc ttctggttct ctccttctgc attccacatt
ttcactccct cctgcacatt 480cttctgacta atcagctcat cttctgtgcc
tccaatgtca tccatcactt tttctgcgat 540gatcaaccag tgctaaaatt
gtcctgttcc tcccattttg tcaaagaaat cacagtaatg 600acagaaggct
tggctgtcat aatgaccccg ttttcatgca tcatcatctc ttatttaaga
660atcctcatca ctgttctgaa gattccttca gctgctggaa agcgtaaagc
attttctacc 720tgtggctctc atctcacagt ggtgaccctg ttttatggaa
gcattagcta tgtctatttt 780cagcccctgt ccaactatac tgtcaaggat
caaatagcaa caattatcta caccgtactg 840actcctatgc taaatccatt
tatctatagt ctgaggaaca aagacatgaa gcagggtttg 900gcaaagttga
tgcacaggat gaaatgtcag taa 933781080DNAHomo
sapiensmisc_featureIncyte ID No 7472020CB1 78atgttcaaag ccatccttgg
ccatgtgtgg cccaaagacc atgggttgga caagcttgtt 60gtaaggtgtc caagacacac
agagccatgg aatctcacag gtatctcaga attcctcctc 120ctgggactct
cagaggatcc agaactgcag cccgtcctcc ctgggctgtc cctgtccatg
180tacctggtca cggtgctgag gaacctgctc atcatcctgg ctgtcagctc
tgactcccac 240ctccacaccc ccatgtgctt cttcctctcc aacctgtgct
gggctgacat cggtttcacc 300tcggccatgg ttcccaagat gattgtggac
atgcagtcgc atagcagagt catctcttat 360gcgggctgcc tgacacagat
gtctttcttt gtcctttttg catgtataga agacatgctc 420ctgacagtga
tggcctatga ccgatttgtg gccatctgtc accccctgca ctacccagtc
480atcatgaatc ctcaccttgg tgtcttctta gttttggtgt cctttttcct
cagcctgttg 540gattcccagc tgcacagttg gattgtgtta caattcacct
tcttcaagaa tgtggaaatc 600tccaattttg tctgtgaccc atctcaactt
ctcaaccttg cctgttctga cagtgtcatc 660aatagcatat tcatatattt
agatagtatt atgtttggtt ttcttcccat ttcagggatc 720cttttgtctt
acgctaacaa tgtcccctcc attctaagaa tttcatcatc agataggaag
780tctaaagcct tctccacctg tggctctcac ctggcagttg tttgcttatt
ttatggaaca 840ggcattggcg tgtacctgac ttcagctgtg tcaccacccc
ccaggaatgg tgtggtggca 900tcagtgatgt acgctgtggt cacccccatg
ctgaaccctt tcatctacag cctgagaaat 960agggacattc aaagtgccct
gtggaggctg cgcagcagaa cagtcgaatc tcatgatctg 1020ttatctcaag
atctgctcca tcctttttct tgtgtgggtg agaaaggtca accacattaa 1080
* * * * *
References