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G-PROTEIN COUPLED RECEPTORS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of G-pxotein
coupled
receptors and to the use of these sequences in the diagnosis, treatment, and
prevention of cell
proliferative, neurological, cardiovascular, gastrointestinal,
autoimmunelinflammatory, 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.
l0 BACKGROUND OF THE INVENTION
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.
GPCRs are integral membrane proteins characterized by the presence of seven
hydrophobic
transmembrane domains which together form a bundle of antiparallel alpha (a)
helices. GPCRs range
in size from under 400 to over 1000 amino acids (Strosberg, A.D. (I99I) 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 transmembxane
domains account,
in part, for structural and functional features of the receptor. In most
cases, the bundle of a helices
forms a ligand-binding pocket. The extracellular N-terminal segment, or one or
more of the three
extracellular loops, may also participate in ligand binding. Ligand binding
activates the receptor by
inducing a conformational change in intracellular portions of the receptor. In
turn, the large, third
intracellular Ioop 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
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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 CA, pp. 2-
6; Bolander, F.F. (1994) Molecular Endocrinolo~y, Academic Press, San Diego
CA, pp. 162-176;
Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6:180-190.)
GPCRs include receptors for sensory signal mediators (e.g., light and
olfactory stimulatory
molecules); adenosine, y-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
IO mediators of inflammation (e.g., prostaglandins and prostanoids, platelet
activating factor, and
leukotrienes); and peptide hormones (e.g., bombesin, bradykinin, calcitonin,
CSa anaphylatoxin,
endothelin, follicle-stimulating hormone (FSH), gonadotropic-releasing hormone
(GnRH),
neurokinin, and thyrotropin-releasing hormone (TRIT), and oxytocin). GPCRs
which act as receptors
for stimuli that have yet to be identified are known as orphan receptors.
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
(I~ilpatrick, G.J. et al.
(1999) Trends Pharmacol. Sci. 20:294-301).
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 CA,
pp. 7-9, 19-22,
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32-35, 130-131, 214-216, 221-222; Habert-Ortoli, E. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:9780-9783.)
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 (Chum J. et al.
(1999) CeII Biochem. Biophys. 30:213-242).
The largest subfamily of GPCRs, the olfactory receptors, axe 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
expxesses only one type of olfactory receptor, and distinct spatial zones of
neurons expressing distinct
receptors are found in nasal passages. For example, the RAlc 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 (Raining, 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).
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, su ra, 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.
Examples of secretin-like GPCRs implicated in inflammation and the immune
response
include the EGF module-containing, mucin-like hormone receptor (Emrl) 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).
The third GPCR subfamily is the metabotropic glutamate receptor family.
Glutamate is the
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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 Ca2+-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 GABAB receptors, and the taste receptors.
Other subfamilies of GPCRs include two groups of chemoreceptor genes found in
the
nematodes Caenorhabditis ele_gans and Caenorhabditis bri~~sae, which are
distantly related to the
mammalian olfactory receptor genes. The yeast pheromone receptors STE2 and
STE3, involved in
the response to mating factors on the cell membrane, have their own seven-
transmembrane signature,
as do the CAMP receptors from the slime mold Dictyostelium discoideum, which
are thought to
regulate the aggregation of individual cells and control the expression of
numerous developmentally-
regulated genes.
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: lutenizing hormone (precocious puberty);
vasopressin VZ (X-linked
nephrogenic diabetes); glucagon (diabetes and hypertension); calcium
(hyperparathyroidism,
hypocalcuria, hypercalceniia); parathyroid hormone (short limbed dwarfism);
(33 adrenoceptor
(obesity, non-insulin-dependent diabetes mellitus); growth hormone releasing
hormone (dwa~sm);
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).
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, s-unra;
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
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tachycardia; serotonin 5HT1D antagonists are used against migraine; and
histamine Hl antagonists
are used against allergic and anaphylactic reactions, hay fever, itching, and
motion sickness (Horn,
supra).
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
aI. (I999) Am. J.
Physiol. 277:F665-F675).
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 CCRS, which
acts as a coreceptor for
infection of T-cells by HIV-1, results in resistance to AIDS, suggesting that
CCRS antagonists could
be useful in preventing the development of AmS.
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
The invention features purified polypeptides, G-protein coupled receptors,
referred to
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collectively as "GCREC" and individually as "GCREC-l," "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," and "GCREC-19." In one aspect, the invention provides an isolated
polypeptide selected from
the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally
occurring annino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-19, c) a biologically active fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-19, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ m N0:1-19.
In one alternative, the invention provides an isolated polypeptide comprising
the amino acid sequence
of SEQ )D NO:1-19.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-19, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ll~ NO:1-19, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ m NO:1-19, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ m NO:1-19.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ ID NO:1-19. Tn another alternative, the polynucleotide is selected from
the group consisting of
SEQ m NO:20-38.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ )D NO:1-19, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO: l-19, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m NO:1-19, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ m NO:1-19. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ m N0:1-19, b) a polypeptide comprising a naturally occurring amino acid
sequence at least
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90% identical to an amino acid sequence selected from the group consisting of
SEQ m N0:1-19, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ m N0:1-19, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-19. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ m N0:1-19, b) a polypeptide
comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
19, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ll~ NO:1-19.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ 1D N0:20-38, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ m
N0:20-38, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ m N0:20-38, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ m N0:20-38, c) a polynucleotide complementary to the polynucleotide of a),
d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
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nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ll7 N0:20-38, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ m
N0:20-38, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-19, b) a
polypeptide comprising a
naturally occurring, amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-19, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
19, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-19, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ m NO:1-
19. 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.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m N0:1-19, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-19, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:l-
19, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO: l-19. 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 GCR.EC,
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comprising administering to a patient in need of such treatment the
composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-19, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-19,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ 117 NO:1-19. 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.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-19, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ m NO:1-19, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
19, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-19. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-19, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-19, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-19, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ m NO:1-19. 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
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polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence
of the test compound, wherein a change in the activity of the polypeptide in
the presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:20-38,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, and b)
detecting altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:20-38, ii) a
polynucleotide comprising a naturally occurnng polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:20-38,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide
in the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
NO:20-38, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ >D N0:20-38,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group consisting
of i)-v) above; c) quantifying the amount of hybridization complex; and d)
comparing the amount of
hybridization complex in the treated biological sample with the amount of
hybridization complex in
an untreated biological sample, wherein a difference in the amount of
hybridization complex in the
treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
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Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly 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.
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DEFINITIONS
"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.
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.
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.
"Altered" nucleic acid sequences encoding GCREC include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as GCREC or
a polypeptide with at least one functional characteristic of GCREC. Included
within this definition
are polymorphisms which may or may not be readily detectable using a
particular oligonucleotide
probe of the polynucleotide encoding GCREC, and improper or unexpected
hybridization to allelic
variants, with a locus other than the normal chromosomal locus for the
polynucleotide sequence
encoding GCREC. The encoded protein may also be "altered," and may contain
deletions, insertions,
or substitutions of amino acid residues which produce a silent change and
result in a functionally
equivalent GCREC. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
residues, as long as the biological or immunological activity of GCREC is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
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sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" xelates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of 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.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')z, 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.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
3S designation "positive" or "plus" can refer to the sense strand of a
reference DNA molecule.
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The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "irnmunologically
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.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding 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., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL,-PCR
kit (Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
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His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
retains at least one biological or innmunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
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
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fragment used as a probe, primer, antigen, therapeutic molecule, or for other
purposes, may be at least
5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500
contiguous nucleotides or amino
acid residues in length. Fragments may be preferentially selected from certain
regions of a molecule.
For example, a polypeptide fragment may comprise a certain length of
contiguous amino acids
selected from the first 250 or 500 amino acids (or first 25% or 50%) of a
polypeptide as shown in a
certain defined sequence. Clearly these lengths are exemplary, and any length
that is supported by
the specification, including the Sequence Listing, tables, and figures, may be
encompassed by the
present embodiments.
A fragment of SEQ >D N0:20-38 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ m N0:20-38, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ m N0:20-38 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ m N0:20-38 from related polynucleotide sequences. The precise length of a
fragment of SEQ
m N0:20-38 and the region of SEQ m N0:20-38 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ m NO:1-19 is encoded by a fragment of SEQ ID N0:20-38. A
fragment
of SEQ m N0:1-19 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID N0:1-19. For example, a fragment of SEQ ID NO:1-19 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ m NO:1-19.
The precise length of
a fragment of SEQ m N0:1-19 and the region of SEQ m NO:1-19 to which the
fragment '
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
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molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorF/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Opera Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' SO
Expect: l0
Word Size: 11
Filter: on
Percent identity ma.y be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
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similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and-hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: I~tuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 pezzalties
Gap x drop-off 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in, size and which contain all of the
elements required for
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chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 ~,g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T,~) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
p,g/ml. Organic
solvent, such as formamide at a concentration of about 35-50% vlv, 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.
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The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amnno acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of 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.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microanray.
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.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
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amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an GCREC may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of GCREC.
"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).
Probes and primers as used in the present invention typically compxise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the,
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloni~yA Laboratory Manual,
2°a ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
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Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge LTI~) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(LTTRs). Regulatory elements interact with host or viral proteins which
control transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
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other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing 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.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60%a free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
vixal infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed cells" includes stably transformed cells in which the inserted
DNA is capable of
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replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or 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.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
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the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human 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.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project 117). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide III) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ m NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID)
as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1
and 2 show the polypeptide sequence identification number (SEQ ILK NO:) and
the corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ~) for each polypeptide
of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI]. Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some cases,
searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of each polypeptide of the
invention, and
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these properties establish that the claimed polypeptides are G-protein coupled
receptors. For
example, SEQ ID NO:1 is 40% identical to Meleagris gallo~avo G protein-coupled
P2Y nucleotide
receptor (GenBank ID g2707256) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 4.0e-62, which
indicates the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ m NO: l
also contains a
rhodopsin family 7 transmembrane receptor domain as determined by searching
for statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMPS and BLAST analyses provide
further .
corroborative evidence that SEQ ll~ N0:1 is G-protein coupled receptor. SEQ ID
N0:2 was analyzed
and annotated in a similar manner. These analyses indicate that SEQ ID N0:2 is
a pheromone
receptor (Dulac, C. and R. Axel (1995) Cell 83:195-206).
As a further example, SEQ ID N0:6 is 29% identical to human C-C chemokine
receptor type
1 (GenBank ID g179985) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 1.6e-15, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID N0:6 also contains a
7 transmembrane
receptor (rhodopsin family) domain as determined by searching for
statistically significant matches in
the hidden Markov model (HMM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLIMPS and PROF1LESCAN analyses provide further
corroborative evidence
that SEQ ID NO:6 is a chemokine receptor.
As a further example, SEQ ID N0:9 is 95% identical to rat calcium-independent
alpha-
latrotoxin receptor (GenBank ID g3882981) as determined by the Basic Local
Alignment Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates
the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ )D NO:9
also contains a 7-
transmembrane receptor (secretin family) domain and a latrophilin/CL-1-like
GPS domain, as
2S determined by searching for statistically significant matches in the hidden
Markov model (HMM)-
based PFAM database of conserved protein family domains. (See Table 3.) Data
from BLllVIPS,
MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that
SEQ ID N0:9 is
a latrophilin-related G-protein coupled receptor.
As a further example, SEQ ID N0:12 is 84% identical to Mus musculus G-protein
coupled
receptor GPR73 (GenBank ID g7248884) as determined by the Basic Local
Alignment Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 6.7e-166, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:12 also contains a
7 transmembrane receptor (rhodopsin family) domain as determined by searching
for statistically
significant matches in the hidden Markov model (I~VIM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLIMfS analysis reveals the presence
of a rhodopsin-like
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GPCR supexfamily signature (See Table 3). Additional data from MOTIFS and
PROFILESCAN
analyses provide further corroborative evidence that SEQ m N0:12 is a G-
protein coupled receptor.
As a further example, SEQ ID N0:15 is 80% identical to rat serotonin receptor
(GenBank ID
g310075) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 2.5e-152, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:15 also contains a
rhodopsin family receptor
domain as determined by searching for statistically significant matches in the
hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
BLI1VIPS, analyses provide further corroborative evidence that SEQ ID N0:15 is
a G-protein coupled
receptor.
As a further example, SEQ DJ N0:16 is 71% identical to mouse olfactory
receptor E3
(GenBank ID g3983382) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 1.9e-88, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ m N0:16 also contains a
rhodopsin
family 7-transmembrane receptor domain as determined by searching for
statistically significant
matches in the hidden Markov model (HMM)-based PFAM database of conserved
protein family
domains. (See Table 3.) Data from BLIMPS, MOT1FS, and PROFILESCAN analyses
provide
further corroborative evidence that SEQ ID N0:16 is an olfactory G-protein
coupled receptor.
As a further example, SEQ ID N0:17 is 83% identical to mouse olfactory G-
protein coupled
receptor G3 (GenBank ID g3983398) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is S.Oe-99, which
indicates the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ m N0:17
also contains a
rhodopsin family 7-transmembrane receptor domain as determined by searching
for statistically
significant matches in the hidden Markov model (HMM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from BLM'S, MOT1FS, and PROF1LESCAN
analyses provide
further corroborative evidence that SEQ ID NO:17 is an olfactory G-protein
coupled receptor. SEQ
113 N0:2-5, SEQ ID N0:7-8, SEQ ID NO: IO-1 I, SEQ ID NO: I3-14, and SEQ TD
NO:I8-19 were
analyzed and annotated in a similar manner. The algorithms and parameters for
the analysis of SEQ
ID NO: I-I9 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments
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of the polynucleotide sequences which are useful, for example, in
hybridization or amplification
technologies that identify SEQ ID N0:20-38 or that distinguish between SEQ ID
N0:20-38 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
7075196H1 is the
identification number of an Incyte cDNA sequence, and BRAUTDR04 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., 71906055V1). Alternatively, the identification
numbers in column 5
may refer to GenBank cDNAs or ESTs (e.g., g900324) which contributed to the
assembly of the full
length polynucleotide sequences. In addition, the identification numbers in
column 5 may identify
sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, ITK)
database (i.e., those
sequences including the designation "ENST"). Alternatively, the identification
numbers in column 5
may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.
e., those sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the identification
numbers in column 5
may refer to assemblages of both cDNA and Genscan-predicted exons brought
together by an "exon
stitching" algorithm. For example, FL IiXXXI~X_NI 1Vz YYYYY_N3 1Vø represents
a "stitched"
sequence in which ~XXXX is the identification number of the cluster of
sequences to which the
algorithm was applied, and YYYYY is the number of the prediction generated by
the algorithm, and
2S N1,2,3...~ if present, represent specific exons that may have been manually
edited during analysis (See
Example V). Alternatively, the identification numbers in column 5 may refer to
assemblages of
exons brought together by an "exon-stretching" algorithm. For example,
FLXXX_gAAAAA_gBBBBB_1 IV is the identification number of a "stretched"
sequence, with
XI~XXXX being the Incyte project identification number, gAAAAA being the
GenBank identification
number of the human genomic sequence to which the "exon-stretching" algorithm
was applied,
gBBBBB being the GenBank identification number or NCBI RefSeq identification
number of the
nearest GenBank protein homolog, and N refernng to specific exons (See Example
V). In instances
where a RefSeq sequence was used as a protein homolog for the "exon-
stretching" algorithm, a
RefSeq identifier (denoted by "NM," "NP," or "NT") may be used in place of the
GenBank identifier
(i.e., gBBBBB).
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Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant
Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses GCREC variants. A preferred GCREC variant is
one which
has at least about 80°Io, or alternatively at least about 90%, or even
at least about 95°Io amino acid
sequence identity to the GCREC amino acid sequence, and which contains at
least one functional or
structural characteristic of GCREC.
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 N0:20-38, which encodes GCREC. The
polynucleotide
sequences of SEQ m N0:20-38, as presented in the Sequence Listing, embrace the
equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
GCREC. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
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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
N0:20-38 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:20-38. 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.
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 occurnng
gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible colon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring GCREC, and all such variations
are to be considered
as being specifically disclosed.
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 colon usage,
e.g., inclusion of non-
naturally occurring colons. Colons rnay be selected to increase the rate at
which expression of the
peptide occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with
which particular colons are utilized by the host. Other reasons for
substantially altering the
nucleotide sequence encoding 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.
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.
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 >l7
N0:20-38 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
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"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
I~lenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithW s which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biolo$y and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding 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. (I988) 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
PROMOTERFIIVDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids.the need
to screen libraries
and is useful in finding intronlexon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 primer analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
31
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode 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.
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.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of 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
32
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In. another embodiment, sequences encoding 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. Sex. 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 Pro
erties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of 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.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active 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-
33
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
frame ATG initiation codon should be provided by the vector. Exogenous
translational elements and
initiation codons may be of various origins, both natural and synthetic. The
efficiency of expression
may be enhanced by the inclusion of enhancers appropriate for the particular
host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding 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 aI. (1989)
Molecular Cloning; A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding 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
Technolo~y (1992)
McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA
81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.)
Expression vectors derived
from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from
various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted organ,
tissue, or cell population.
(See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M.
et al. (1993) Proc.
Natl. Acad. Sci. USA 90(13):6340-6344; Butler, R.M. et al. (1985) Nature
317(6040):813-815;
McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and
N. Somia (1997)
Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding 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 CA)
or PSPORTl
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
34
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
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.
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.)
Plant systems may also be used for expression of GCREC. Transcription of
sequences
encoding GCREC may be driven by viral promoters, e.g., the 355 and 195
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 Technoloay
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding GCREC
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses GCREC in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
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.)
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
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.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, T. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dlafr 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
fluorescentproteins
(GFP; Clontech),13 glucuronidase and its substrate I3-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding 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.
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
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CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
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 irrununosorbent 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 MN,
Sect. IV; Coligan, J.E. et al. ( 1997) Current Protocols in Innnunolo~y,
Greene Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) hnmunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding 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 WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding 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.
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
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CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
post-translational activities (e.g., CHO, HeLa, MDCK, HEI~293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding GCR.EC 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.
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
ystems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
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.
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 Immunolo~y 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
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CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
compound can be rationally designed using known techniques. In one embodiment,
screening fox
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.
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
Iabel. 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 compounds) may be free in
solution or affixed to a
solid support.
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.
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. Patent Number 5,175,383 and U.S.
Patent Number
5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line,
are derived from the
early mouse embryo and grown in culture. The ES cells are transformed with a
vector containing the
gene of interest disrupted by a marker gene, e.g., the neomycin
phosphotransferase gene (neo;
Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region
of the host genome by homologous recombination. Alternatively, homologous
recombination takes
place using the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-
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CA 02417195 2003-O1-24
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specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, I~.U.
et al. (1997) Nucleic
Acids Res. 25:4323-4330). Transformed ES cells are identified and
microinjected into mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding 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).
Polynucleotides encoding GCREC can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding GCREC is injected into animal ES cells,
and the injected
15, 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
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 brain tissue, fetal brain tissue, colon polyps,
diseased colon tissue, colon
tumor tissue, diseased gallbladder tissue, heart tissue, diseased breast
tissue, interleukin-5 stimulated
eosinophils, tumor tissue, and reproductive tissues. 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.
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
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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, colon,
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,
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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 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
autoimmunelinflammatory 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.
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.
In a further embodiment, a composition comprising a substantially purified
GCREC in
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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.
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.
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,
IO 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.
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.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of GCREC may be produced using methods which are generally known
in the
are. 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.
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
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polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium~arvum are especially
preferable.
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.
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.)
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., Mornson,
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 laiown 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.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for GCREC may also be
generated.
For example, such fragments include, but are not limited to, F(ab')Z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
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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).
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, Ka, 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 Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple GCREC epitopes, represents the average affinity, or
avidity, of the antibodies
for GCREC. The Ka 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 Ka ranging from about 109 to 1012 Llmole are preferred for
use in immunoassays in
which the GCREC-antibody complex must withstand rigorous manipulations. Low-
affinity antibody
preparations with Ka ranging from about 106 to 10' Llmole 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 T: A Practical
Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A
Practical Guide to
Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibodylml,
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, su~a, and Coligan et al. supra.)
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 NJ.)
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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. (19.98) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Phaxmacol. 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 Morns, M.C. et al. (1997)
Nucleic Acids Res.
25 ( 14):2730-2736.)
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) Cel175:207-216; Crystal, R.G. et al.
(1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falcipaxum 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.
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)
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ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson
(1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of GCREC include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
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 (3-actin
genes), (ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et aI. (I995) Science 268:1766-1769;
Rossi, F.M.V. and
H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-4.56), 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.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitxogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to 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. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
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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.5. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding 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. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding 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.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.5. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
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herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding 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.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo~ic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
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engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding GCREC.
Specific ribozyme cleavage sites within any potential RNA target are initially
identif ed by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding 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.
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.
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
CA 02417195 2003-O1-24
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associated with decreased GCREC expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding GCREC may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding 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
polynueleotide can be carried out, for example, using a Schizosaccharom, ces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposorne injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
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such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and pxoteins.
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.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar xegion of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
. J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the
advantage of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising 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).
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
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routes for administration in humans.
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 EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the
dose lethal to 50°7o of the population) statistics. The dose ratio of
toxic to therapeutic effects is the
therapeutic index, which can be expressed as the LDSOlEDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about O. l ,ug to 100,000 ,ug, 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 arid generally available to
practitioners in the art.
Those skilled in the axt will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind 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
ma.y 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
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the art and may be used.
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, far 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.
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.
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.
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
N0:20-38 or from
genomic sequences including promoters, enhancers, and introns of the GCREC
gene.
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 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding GCREC may be used for the diagnosis of
disorders
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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, colon, 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
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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 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
autoimmunelinflammatory 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
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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.
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.
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.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type rnay
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
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
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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.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding GCREC may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding
GCREC are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
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. hnmunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
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progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, 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.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression~is
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not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oclnews/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel 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
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polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for 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-l I l; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788).
Detection may be performed
by a variety of methods known in the art, for example, by reacting the
proteins in the sample with a
thiol- or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
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USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding 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 (PACs), bacterial artificial
chromosomes
(BACs), bacterial Pl constructions, or single chromosome cDNA libraries. (See,
e.g., Harnngton, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of
the invention may be
used to develop genetic linkage maps, for example, which correlate the
inheritance of a disease state
with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acad. Sci. USA 83:7353-
7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of 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.
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 mannmalian 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
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the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, 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 GC1ZEC and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with 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.
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.
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.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The following preferred
specific embodiments are, therefore, to be construed as merely illustrative,
and not limitative of the
remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/221,478, U.S. Ser. No. 60/223,268, U.S. Ser. No.
60/231,121, U.S. Ser.
No. 60/232,691, U.S. Ser. No. 60/235,146, U.S. Ser. No. 60/227,054, and U.S.
Ser. No. 60/232,243,
are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LlFESEQ GOLD
database
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(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some eases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries Were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DHlOB from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Prornega); 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 QIA.GEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
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Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ.Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
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.
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
I~VIMER. The
Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
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GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention
may begin at any of the methionine residues of the full length translated
polypeptide. Full length
polypeptide sequences were subsequently analyzed by querying against databases
such as the
GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and hidden Markov model (HMM)-based protein family databases such as PFAM.
Full length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide
and polypeptide sequence alignments are generated using default parameters
specified by the
CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment
program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, ,where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
ID N0:20-38. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative 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.
Potential G-protein
coupled receptors were also identified by homology to Incyte cDNA sequences
that had been
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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. Full length
polynucleotide sequences were obtained by assembling Genscan-predicted coding
sequences with
Incyte cDNA sequences and/or public cDNA sequences using the assembly process
described in
Example III. Alternatively, full length polynucleotide sequences were derived
entirely from edited or
unedited Genscan predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example 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" Seguences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
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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 1V. 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
The sequences which were used to assemble SEQ m N0:20-38 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 1D N0:20-38 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 LD NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
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Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or L1FESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
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
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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
CA).
VIII. Extension of GCREC Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~.l
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~,l of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
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agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) 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°C in
384-well plates in LBl2x carb liquid media.
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°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:20-38 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 ,uCi of
[y-3zP] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
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compared.
X. Microarrays
The linkage or synthesis of array elements upon a microaxray 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, LTV, chemical, or
mechanical bonding.
procedures. A typical array may be produced using available methods and
machines well lalown to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)~ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,l oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/~.1 RNase inhibitor, 500 ~,M dATP, 500 ~.M
dGTP, 500 ~,M dTTP, 40
~,M dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)''- RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)~ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85°C to the stop the reaction and degrade
the RNA. Samples are purified
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using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mglml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 p,1 5X SSC/0.2% SDS.
Microarra,~paration
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
~.g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~,l of the array
element DNA, at an average
concentration of 100 ng/~,1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINI~ER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 ,u1 of sample mixture consisting of 0.2 ~,g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cmz coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 ,u1 of 5X SSC in a corner of the chamber. The chamber
containing the arrays is
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incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45° C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45° C in
a second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:.100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstallc
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
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spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the 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
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 Auto~raphica 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 fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, 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 is onp icum, enables the purification of
fusion proteins on
CA 02417195 2003-O1-24
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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 F-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,
su~a, ch. 10 and 16). Purified GCREC obtained by these methods can be used
directly in the assays
shown in Examples XVI,,XVII, and XVITI, where applicable.
XIII. Functional Ass.-,~s
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 CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug 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 ,ug 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 NY.
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
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Success NY). mRNA can be purified from the cells using methods well known by
those of skill in
the art. Expression of mlZNA encoding GCREC and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XIV. Production of GCREC Specific Antibodies
S 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.
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 lrnown to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the
oligopeptide-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
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.
Media containing GCREC are passed over the immunoaffmity 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
GCREC, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled GCREC, washed,
77
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
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 molecules.
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
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. Demonstration of GCREC Activity
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.
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
[3H]thymidine, a radioactive DNA
precursor molecule. Varying amounts of GCREC ligand are then added to the
cultured cells.
Incorporation of [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 [3H]thymidine into acid-precipitable DNA
(McKay, I. and I.
Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University
Press, New York NY, p.
73.)
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 full length
78
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
GCREC is txansfected 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.
To measure changes in inositol phosphate levels, the cells are grown in 24-
well plates
containing 1x105 cellslwell and incubated with inositol-free media and
[3H]myoinositol, 2 ~uCi/well,
for 48 hr. The culture medium is removed, and the cells washed with buffer
containing 10 mM LiCI
followed by addition of ligand. The reaction is stopped by addition of
perchloric acid. Inositol
phosphates are extracted and separated on Dowex AGl-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
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 Ca2+. 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 Ca2+ 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 Gaisne 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 Ca2+
79
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
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 3~4
(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.
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.
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
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CA 02417195 2003-O1-24
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<110> INCYTE GENOMICS, INC.
THORNTON, Michael
PATTERSON, Chandra
LAL, Preeti
BURFORD, Neil
YUE, Henry
GANDHI, Ameena R.
ELLIOTT, Vicki S.
RAMKUMAR, Jayalaxini
BAUGHN, Mariah R.
KALLICK, Deborah A.
WALIA, Narinder K.
HAFALIA,April J.A.
YAO, Monic,~ue G.
LU, Yan
TRIBOULEY, Catherine M.
POLICKY, Jennifer L.
KEARNEY, Liam
GRAUL, Richard
WARREN, Bridget
LEE, Ernestine A.
DING, Li
<120> G-PROTEIN COUPLED RECEPTORS
<130> PI-0176 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/221,478; 60/223,268; 60/227,054; 60/231,121; 60/232,243;
60/232,691; 60/235,146
151> 2000-07-27; 2000-08-03; 2000-08-21; 2000-09-08; 2000-09-23;
2000-09-15; 2000-09-22
<160> 38
<170> PERL Program
<210> 1
<211> 339
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474806CD1
<400> 1
Met Leu Ser Ile Leu Leu Pro Ser Arg Gly Ser Arg Ser Gly Ser
1 5 10 15
Arg Arg Gly Ala Leu Leu Leu Glu Gly A1a Ser Arg Asp Met Glu
20 25 30
Lys Va1 Asp Met Asn Thr Ser Gln Glu Gln Gly Leu Cys Gln Phe
35 40 45
Ser Glu Lys Tyr Lys Gln Val Tyr Leu Ser Leu Ala Tyr Ser Ile
50 55 60
Ile Phe Ile Leu Gly Leu Pro Leu Asn Gly Thr Val Leu Trp His
65 70 75
Ser Trp Gly Gln Thr Lys Arg Trp Ser Cys Ala Thr Thr Tyr Leu
80 85 90
Val Asn Leu Met Val Ala Asp Leu Leu Tyr Va1 Leu Leu Pro Phe
95 100 105
Leu Ile Ile Thr Tyr Ser Leu Asp Asp Arg Trp Pro Phe Gly Glu
1/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
110 115 120
Leu Leu Cys Lys Leu Val His Phe Leu Phe Tyr Ile Asn Leu Tyr
125 130 135
Gly Ser Ile Leu Leu Leu Thr Cys Ile Ser Val His Gln Phe Leu
140 145 150
Gly Val Trp His Pro Leu Cys Ser Leu Pro Tyr Arg Thr Arg Arg
155 160 165
His Ala Trp Leu Gly Thr Ser Thr Thr Trp Ala Leu Va1 Val Leu
170 175 180
Gln Leu Leu Pro Thr Leu A1a Phe Ser His Thr Asp Tyr Ile Asn
185 190 195
Gly Gln Met Ile Trp Tyr Asp Met Thr Ser Gln Glu Asn Phe Asp
200 205 210
Arg Leu Phe Ala Tyr Gly Ile Val Leu Thr Leu Ser Gly Phe Leu
215 220 225
Ser Pro Ser Leu Val Ile Leu Val Cys Tyr Ser Leu Met Val Arg
230 235 240
Ser Leu Ile Lys Pro Glu G1u Asn Leu Met Arg Thr Gly Asn Thr
245 250 255
Ala Arg Ala Arg Ser Ile Arg Thr Ile Leu Leu Val Cys Gly Leu
260 265 270
Phe Thr Leu Cys Phe Val Pro Phe His Ile Thr Arg Ser Phe Tyr
275 280 285
Leu Thr Ile Cys Phe Leu Leu Ser Gln Asp Cys Gln Leu Leu Met
290 295 300
Ala Pro Ser Val Ala Tyr Lys Ile Trp Arg Pro Leu Val Ser Val
305 310 315
Ser Ser Cys Leu Asn Pro Val Leu Tyr Phe Leu Ser Arg Gly Ala
320 325 330
Lys Ile Glu Ser Gly Ser Ser Arg Asn
335
<210> 2
<211> 335
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474840CD1
<400> 2
Met Thr Pro G1y Gly Arg Ala Cys Ser Glu Met Arg Ser Cys His
1 5 10 15
Cys Ala Pro Ala Trp Ala Thr Glu Arg Asp Ser Val Ser Lys Lys
20 25 30
Lys Lys Asn Lys Lys Lys Asn Leu Phe Ser Gln Ala Thr Ile Gly
35 40 45
Leu Leu Ala Asn Thr Phe Phe Leu Phe Phe Asn Ile Phe Ile Phe
50 55 60
Leu Gln Asp Gln Lys Ser Lys Pro His Asp Leu Ile Ser Cys Asn
65 70 75
Ser Ala Phe Ile His Val Val Met Phe Leu Thr Val Val Asp Ala
80 85 90
Trp Pro Pro Asp Met Pro Glu Ser Leu His Leu Gly Asn Glu Phe
95 100 105
Lys Phe Lys Ser Leu Ser Tyr Ile Asn Arg Val Arg Met Gly Leu
110 115 120
Cys Ile Cys Asn Ile Cys Leu Leu Ser Ile His Gln Ala Asn Thr
125 130 135
I1e Ser Pro Asn Asn Phe Cys Leu Ala Arg Leu Lys Gln Lys Phe
140 145 150
Thr Asn Asn Ile Ile Met Ser Ser Phe Phe Ser Phe Phe Phe Trp
2/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
155 160 165
Ser Ile Asn Leu Ser Phe Ser Tyr Asn Ile Va1 Phe Phe Thr Val
170 175 180
Ala Ser Ser Asn Val Thr Gln Asn Ser Leu Pro Lys Gly Ser Asn
185 190 195
Thr Val His Phe Leu Pro Met Lys Ser Phe Met Arg Lys Val Phe
200 205 210
Phe Thr Leu Thr Leu Ser Arg Asp Val Phe Ile Ile Gly Ile Thr
215 220 225
Leu His Ser Ile Ala His Met Val Ile Leu Val Ser Arg His Glu
230 235 240
Thr Gln Ser Gln His Leu His Ser Ile Ser Ile Ser Pro Gln Ala
245 250 255
Phe Pro Glu Lys Arg Ala Ala Gln Thr Ile Pro Leu Leu Val Ser
260 265 270
Tyr Cys Leu Val Met Cys Trp Val Asp Leu Ile Ile Ser Ser Ser
275 280 285
Ser Thr Leu Leu Trp Thr Cys Asn Pro Val Phe Leu Ser Met Gln
290 295 300
Asn Leu Val Gly Asp Val Tyr Ala Thr Val Val Leu Leu Glu Gln
305 310 315
Ile Ser Ser Asp Lys Asn Ile Val Asp Ile Leu Gln Asn Met Gln
320 325 330
Ser Ala Ile Lys Leu
335
<210> 3
<211> 428
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475092CD1
<400> 3
Met Gln Arg Lys G1u Lys Ala Lys Cys Pro Gln Glu A1a Pro Ala
1 5 10 15
Gly Arg Glu Pro Ser Thr Pro Gly Gly Gly Ser Gly G1y Gly Gly
20 25 30
Ala Val Ala Ala A1a Ser Gly Ala Ala Val Pro Gly Ser Val Gln
35 40 45
Leu Ala Leu Ser Val Leu His Ala Leu Leu Tyr Ala Ala Leu Phe
50 55 60
Ala Phe Ala Tyr Leu Gln Leu Trp Arg Leu Leu Leu Tyr Arg Glu
65 70 75
Arg Arg Leu Ser Tyr G1n Ser Leu Cys Leu Phe Leu Cys Leu Leu
80 85 90
Trp Ala Ala Leu Arg Thr Thr Leu Phe Ser Ala Ala Phe Ser Leu
95 100 105
Ser Gly Ser Leu Pro Leu Leu Arg Pro Pro Ala His Leu His Phe
110 115 120
Phe Pro His Trp Leu Leu Tyr Cys Phe Pro Ser Cys Leu Gln Phe
125 130 135
Ser Thr Leu Cys Leu Leu Asn Leu Tyr Leu Ala Glu Val Ile Cys
140 145 150
Lys Val Arg Cys Ala Thr Glu Leu Asp Arg His Lys Ile Leu Leu
155 160 165
His Leu Gly Phe Ile Met Ala Ser Leu Leu Phe Leu Val Val Asn
170 175 180
Leu Thr Cys A1a Met Leu Val His Gly Asp Val Pro Glu Asn Gln
185 190 195
Leu Lys Trp Thr Val Phe Val Arg Ala Leu Zle Asn Asp Ser Leu
3/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
200 205 210
Phe Ile Leu Cys Ala Ile Ser Leu Val Cys Tyr Ile Cys Lys Ile
215 .220 225
Thr Lys Met Ser Ser Ala Asn Val Tyr Leu Glu Ser Lys Gly Met
230 235 240
Ser Leu Cys Gln Thr Val Val Val Gly Ser Val Val Ile Leu Leu
245 250 255
Tyr Ser Ser Arg Ala Cys Tyr Asn Leu Val Val Val Thr Ile Ser
260 265 270
Gln Asp Thr Leu Glu Ser Pro Phe Asn Tyr G1y Trp Asp Asn Leu
275 280 285
Ser Asp Lys Ala His Val Glu Asp Ile Ser Gly Glu Glu Tyr Ile
290 295 300
Val Phe Gly Met Val Leu Phe Leu Trp Glu His Val Pro Ala Trp
305 310 315
Ser Val Val Leu Phe Phe Arg Ala Gln Arg Leu Asn Gln Asn Leu
320 325 330
Ala Pro Ala Gly Met I1e Asn Ser His Ser Tyr Ser Ser Arg Ala
335 340 345
Tyr Phe Phe Asp Asn Pro Arg Arg Tyr Asp Ser Asp Asp Asp Leu
350 355 360
Pro Arg Leu Gly Ser Ser Arg Glu Gly Ser Leu Pro Asn Ser G1n
365 370 375
Ser Leu Gly Trp Tyr Gly Thr Met Thr Gly Cys G1y Ser Ser Ser
380 385 390
Tyr Thr Val Thr Pro His Leu Asn Gly Pro Met Thr Asp Thr Ala
395 400 405
Pro Leu Leu Phe Thr Cys Ser Asn Leu Asp Leu Asn Asn His His
410 415 420
Ser Leu Tyr Val Thr Pro Gln Asn
425
<210> 4
<211> 330
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7341260CD1
<400> 4
Met Thr Pro Asn Ser Thr G1y Glu Val Pro Ser Pro Ile Pro Lys
1 5 10 15
Gly Ala Leu Gly Leu Ser Leu Ala Leu Ala Ser Leu Ile Ile Thr
20 25 30
Ala Asn Leu Leu Leu Ala Leu Gly Ile Ala Trp Asp Arg Arg Leu
35 40 45
Arg Ser Pro Pro Ala Gly Cys Phe Phe Leu Ser Leu Leu Leu Ala
50 55 60
Gly Leu Leu Thr Gly Leu Ala Leu Pro Thr Leu Pro Gly Leu Trp
65 70 75
Asn Gln Ser Arg Arg Gly Tyr Trp Ser Cys Leu Leu Val Tyr Leu
80 85 90
Ala Pro Asn Phe Ser Phe Leu Ser Leu Leu Ala Asn Leu Leu Leu
95 100 105
Va1 His Gly Glu Arg Tyr Met Ala Val Leu Arg Pro Leu Gln Pro
110 115 120
Pro Gly Ser Ile Arg Leu Ala Leu Leu Leu Thr Trp Ala Gly Pro
125 130 135
Leu Leu Phe A1a Ser Leu Pro Ala Leu Gly Trp Asn His Trp Thr
140 145 150
Pro Gly Ala Asn Cys Ser Ser Gln Ala Ile Phe Pro Ala Pro Tyr
4/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
155 160 165
Leu Tyr Leu Glu Val Tyr Gly Leu Leu Leu Pro Ala Val Gly Ala
170 175 180
Ala Ala Phe Leu Ser Val Arg Val Leu Ala Thr Ala His Arg Gln
185 190 195
Leu Gln Asp Ile Cys Arg Leu Glu Arg Ala Val Cys Arg Asp Glu
200 205 210
Pro Ser Ala Leu Ala Arg Ala Leu Thr Trp Arg Gln Ala Arg Ala
215 220 225
Gln Ala Gly Ala Met Leu Leu Phe Gly Leu Cys Trp Gly Pro Tyr
230 235 240
Val Ala Thr Leu Leu Leu Ser Val Leu Ala Tyr Glu Gln Arg Pro
245 250 255
Pro Leu Gly Pro Gly Thr Leu Leu Ser Leu Leu Ser Leu Gly Ser
260 265 270
Ala Ser Ala Ala Ala Val Pro Va1 Ala Met Gly Leu Gly Asp Gln
275 280 285
Arg Tyr Thr Ala Pro Trp Arg Ala Ala Ala Gln Arg Cys Leu Gln
290 295 300
Gly Leu Trp Gly Arg Ala Ser Arg Asp Ser Pro Gly Pro Ser Ile
305 310 315
Ala Tyr His Pro Ser Ser Gln Ser Ser Val Asp Leu Asp Leu Asn
320 325 330
<210> 5
<211> 676
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473911CD1
<400> 5
Met Asn Lys Asn Asn Lys Pro Ser Ser Phe Ile A1a Ile Arg Asn
1 5 10 15
Ala Ala Phe Ser Glu Val Gly Ile Gly Ile Ser A1a Asn Ala Met
20 25 30
Leu Leu Leu Phe His Ile Leu Thr Cys Leu Leu Lys His Arg Thr
35 40 45
Lys Pro Ala Asp Leu Ile Val Cys His Val Ala Leu Ile His Ile
50 55 60
Ile Leu Leu Leu Pro Thr Glu Phe Ile Ala Thr Asp Ile Phe Gly
65 70 75
Ser Gln Asp Ser Glu Asp Asp Ile Lys His Lys Ser Val Ile Tyr
80 85 90
Arg Arg Asn Arg Gln Ser Gln His Phe His Ser Thr Asn Leu Ser
95 100 105
Pro Lys Ala Pro Pro Glu Lys Met Ala Thr Gln Thr Ile Leu Leu
110 115 120
Leu Val Ser Cys Phe Val Ile Val Tyr Val Leu Asp Cys Val Val
125 130 135
Ala Ser Cys Ser Gly Leu Val Trp Asn Ser Asp Pro Val Arg His
140 145 150
Arg Val Gln Met Leu Val Asp Asn Gly Tyr Ala Thr Ile Ser Pro
155 160 165
Ser Val Leu Pro Arg Leu Thr Ala Pro Asn Glu Trp Arg Ala Ser
170 175 180
Val Tyr Leu Asn Asp Ser Leu Asn Lys Cys Ser Asn Gly Arg Leu
185 190 195
Leu Cys Val Asp Arg Gly Leu Asp Glu Gly Pro Arg Ser Val Pro
200 205 210
5/37
CA 02417195 2003-O1-24
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Lys Cys Ser Glu Ser Glu Thr Asp Glu Asp Tyr Ile Va1 Leu Arg
215 220 225
Ala Pro Leu Arg Glu Asp Glu Pro Lys Asp Gly Gly Ser Val Gly
230 235 240
Asn Ala Ala Leu Val Ser Pro Glu Ala Ser Ala Glu Glu Glu Glu
245 250 255
Glu Arg Glu Glu Gly Gly Glu A1a Cys Gly Leu Glu Arg Thr Gly
260 265 270
Ala Gly G1y Glu Gln Val Asp Leu Gly Glu Leu Pro Asp His Glu
275 280 285
Glu Lys Ser Asn Gln Lys Val Ala Ala Ala Thr Leu Glu Asp Arg
290 295 300
Thr Gln Asp Glu Pro Ala Glu Glu Ser Cys Gln Ile Val Leu Phe
305 310 315
Gln Asn Asn Cys Met Asp Asn Phe Val Thr Ser Leu Thr Gly Ser
320 ~ 325 330
Pro Tyr Glu Phe Phe Pro Thr Lys Ser Thr Ser Phe Cys Arg Glu
335 340 345
Ser Cys Ser Pro Phe Ser Glu Ser Val Lys Ser Leu Glu Ser Glu
350 355 360
Gln Ala Pro Lys Leu Gly Leu Cys Ala Glu Glu Asp Pro Val Val
365 370 375
Gly Ala Leu Cys Gly Gln His Gly Pro Leu Gln Asp Gly Val Ala
380 385 390
Glu Gly Pro Thr Ala Pro Asp Val Val Val Leu Pro Lys Glu Glu
395 400 405
Glu Lys Glu Glu Val Ile Val Asp Asp Met Leu Ala Asn Pro Tyr
410 415 420
Val Met Gly Asp Glu Gly Glu Glu Glu Glu Glu Glu Phe Va1 Asp
425 430 435
Asp Thr Leu Ala Asn Pro Tyr Val Met Gly Val Gly Leu Pro Gly
440 445 450
Arg Gly Gly Glu Glu Glu Glu Glu Glu Glu Va1 Val Asp Asp Thr
455 460 465
Leu Ala Ser Leu Tyr Lys Met Gly Glu Glu His Arg His Lys Gly
470 475 480
Leu Ala Pro Leu Trp Glu Gly Gly Gln Lys Pro Ser Gln Lys Leu
485 490 495
Pro Pro Lys Lys Pro Asp Leu Arg Gln Val Pro Gln Pro Leu Ala
500 505 510
Ser Glu Val Pro Gln Arg Arg Gln Glu Arg Ala Val Val Thr Glu
515 520 525
Gly Arg Pro Leu Glu Ala Ser Arg Ala Leu Pro Ala Lys Pro Arg
530 535 540
Ala Phe Thr Leu Tyr Pro Arg Ser Phe Ser Val Glu Gly Gln Glu
545 550 555
I1e Pro Val Ser Ile Ser Val Tyr Trp Glu Pro Glu Gly Ser Gly
560 565 570
Leu Asp Asp His Arg Ile Lys Arg Lys Glu Glu His Leu Ser Val
575 580 585
Val Ser Gly Ser Phe Ser Gln Arg Asn His Leu Pro Ser Ser Gly
590 595 600
Thr Ser Thr Pro Ser Ser Met Val Asp Ile Pro Pro Pro Phe Asp
605 610 615
Leu Ala Cys Ile Thr Lys Lys Pro Ile Thr Lys Ser Ser Pro Ser
620 625 630
Leu Leu Ile Asp Ser Asp Ser Pro Asp Lys Tyr Lys Lys Lys Lys
635 640 645
Ser Ser Phe Lys Arg Phe Leu Ala Leu Met Phe Asn Lys Met Glu
650 655 ~ 660
Arg Pro Gly Thr Met Ala His Ala Cys His Pro Ser Thr Leu Gly
665 670 675
Ser
6/37
CA 02417195 2003-O1-24
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<210> 6
<211> 372
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474767CD1
<400> 6
Met Glu His Thr His Ala His Leu Ala Ala Asn Ser Ser Leu Ser
1 5 10 15
Trp Trp Ser Pro Gly Ser Ala Cys Gly Leu Gly Phe Val Pro Val
20 25 30
Val Tyr Tyr Ser Leu Leu Leu Cys Leu Gly Leu Pro Ala Asn Ile
35 40 45
Leu Thr Val Ile Ile Leu Ser Gln Leu Val Ala Arg Arg Gln Lys
50 55 60
Ser Ser Tyr Asn Tyr Leu Leu Ala Leu Ala Ala Ala Asp Ile Leu
65 70 75
Val Leu Phe Phe Ile Val Phe Va1 Asp Phe Leu Leu Glu Asp Phe
80 85 90
Ile Leu Asn Met Gln Met Pro Gln Val Pro Asp Lys Ile Ile Glu
95 100 105
Val Leu Glu Phe Ser Ser I1e His Thr Ser Ile Trp Ile Thr Val
110 115 120
Pro Leu Thr Ile Asp Arg Tyr Ile Ala Val Cys His Pro Leu Lys
125 130 135
Tyr His Thr Val Ser Tyr Pro Ala Arg Thr Arg Lys Val Ile Val
140 145 150
Ser Val Tyr Ile Thr Cys Phe Leu Thr Ser Ile Pro Tyr Tyr Trp
155 160 165
Trp Pro Asn Ile Trp Thr Glu Asp Tyr Ile Ser Thr Ser Val His
170 175 180
His Val Leu Ile Trp Ile His Cys Phe Thr Val Tyr Leu Val Pro
185 190 195
Cys Ser Ile Phe Phe Ile Leu Asn Ser Ile Ile Val Tyr Lys Leu
200 205 210
Arg Arg Lys Ser Asn Phe Arg Leu Arg Gly Tyr Ser Thr Gly Lys
215 220 225
Thr Thr Ala Ile Leu Phe Thr Ile Thr Ser Ile Phe Ala Thr Leu
230 235 240
Trp Ala Pro Arg Ile Ile Met Ile Leu Tyr His Leu Tyr Gly Ala
245 250 255
Pro Ile Gln Asn Arg Trp Leu Val His Ile Met Ser Asp Ile Ala
260 265 270
Asn Met Leu Ala Leu Leu Asn Thr Ala Ile Asn Phe Phe Leu Tyr
275 280 285
Cys Phe Ile Ser Lys Arg Phe Arg Thr Met Ala Ala Ala Thr Leu
290 295 300
Lys Ala Phe Phe Lys Cys Gln Lys Gln Pro Val Gln Phe Tyr Thr
305 310 315
Asn His Asn Phe Ser Ile Thr Ser Ser Pro Trp Ile Ser Pro Ala
320 325 330
Asn Ser His Cys Ile Lys Met Leu Val Tyr Gln Tyr Asp Lys Asn
335 340 345
Gly Lys Pro I1e Lys Ser Arg Asn Asp Ser Lys Ser Ser Tyr Gln
350 355 360
Phe Glu Asp Ala Ile Gly Ala Cys Val Ile Ile Leu
365 370
7/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
<210> 7
<211> 271
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475815CD1
<400> 7
Met Asn Lys Asn Asn Lys Pro Ser Ser Phe Ile Ala Ile Arg Asn
1 5 10 15
Ala Ala Phe Ser Glu Val G1y Ile Gly Ile Ser Ala Asn A1a Met
20 25 30
Leu Leu Leu Phe His Ile Leu Thr Cys Leu Leu Lys His Arg Thr
35 40 45
Lys Pro Ala Asp Leu Ile Val Cys His Val Ala Leu Ile His Ile
50 55 60
Ile Leu Leu Leu Pro Thr Glu Phe Ile Ala Thr Asp Ile Phe Gly
65 70 75
Ser Gln Asp Ser Glu Asp Asp Tle Lys His Lys Ser Val Ile Tyr
80 85 90
Arg Tyr Arg Leu Met Arg Gly Leu Ser Ile Ser Thr Thr Cys Leu
95 100 105
Leu Ser Ile Leu Pro Ala Ile Thr Cys Ser Pro Arg Ser Ser Cys
110 115 120
Leu Ala Val Phe Lys Asp Ser His Ile Thr Asn His Val Ala Phe
125 130 135
Ser Ser Val Phe His Ile Ser Ile Ser Asp Ser Phe Leu Val Ser
140 145 150
Thr Leu Pro Ile Lys Asn Leu Ala Ser Asn Ser Leu Thr Phe Val
155 160 165
Thr Gln Ser Cys Ser Ala Gly Tle Gly Ser Arg Pro Pro Ser Ser
170 175 180
Gly Tyr Met Val Ile Leu Leu Ser Arg Arg Asn Arg Gln Ser Gln
185 190 195
His Phe His Ser Thr Asn Leu Ser Pro Lys Ala Pro Pro Glu Lys
200 205 210
Met Ala Thr Gln Thr Ile Leu Leu Leu Val Ser Cys Phe Val Ile
215 220 225
Val Tyr Val Leu Asp Cys Va1 Val Ala Ser Cys Ser Gly Leu Val
230 235 240
Trp Asn Ser Asp Pro Va1 Arg His Arg Val Gln Met Leu Val Asp
245 250 255
Asn Gly Tyr Ala Thr Ile Ser Pro Ser Val Leu Va1 Ser Thr Glu
260 265 270
Lys
<210> 8
<211> 611
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 60263275CD1
<400> 8
Met Gln Gly Pro Leu Leu Leu Pro Gly Leu Cys Phe Leu Leu Ser
1 5 10 15
Leu Phe Gly Ala Val Thr Gln Lys Thr Lys Asn Ile Asn Glu Cys
20 25 30
8/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
Thr Pro Pro Tyr Ser Val Tyr Cys Gly Phe Asn Ala Val Cys Tyr
35 40 45
Asn Val Glu Gly Ser Phe Tyr Cys Gln Cys Val Pro Gly Tyr Arg
50 55 60
Leu His Ser Gly Asn Glu Gln Phe Ser Asn Ser Asn Glu Asn Thr
65 70 75
Cys Gln Asp Thr Thr Ser Ser Lys Thr Thr Gln Gly Arg Lys Glu
80 85 90
Leu Gln Lys Ile Val Asp Lys Phe Glu Ser Leu Leu Thr Asn Gln
95 100 105
Thr Leu Trp Arg Thr Glu Gly Arg Gln Glu Ile Ser Ser Thr Ala
1l0 115 120
Thr Thr Ile Leu Arg Asp Val Glu Ser Lys Val Leu Glu Thr Ala
125 130 l35
Leu Lys Asp Pro Glu Gln Lys Val Leu Lys Ile Gln Asn Asp Ser
140 145 150
Val Ala Ile Glu Thr Gln Ala Ile Thr Asp Asn Cys Ser Glu Glu
155 160 165
Arg Lys Thr Phe Asn Leu Asn Val Gln Met Asn Ser Met Asp Ile
170 175 180
Arg Cys Ser Asp Ile Ile Gln Gly Asp Thr Gln Gly Pro Ser Ala
185 190 195
Ile Ala Phe Ile Ser Tyr Ser Ser Leu Gly Asn Ile Ile Asn Ala
200 205 210
Thr Phe Phe Glu Glu Met Asp Lys Lys Asp Gln Val Tyr Leu Asn
215 220 225
Ser Gln Val Val Ser Ala Ala Ile G1y Pro Lys Arg Asn Val Ser
230 235 240
Leu Ser Lys Ser Val Thr Leu Thr Phe Gln His Val Lys Met Thr
245 250 255
Pro Ser Thr Lys Lys Val Phe Cys Val Tyr Trp Lys Ser Thr Gly
260 265 270
Gln Gly Ser Gln Trp Ser Arg Asp Gly Cys Phe Leu Ile His Val
275 280 285
Asn Lys Ser His Thr Met Cys Asn Cys Ser His Leu Ser Ser Phe
290 295 300
Ala Val Leu Met Ala Leu Thr Ser Gln Glu Glu Asp Pro Val Leu
305 310 315
Thr Val Ile Thr Tyr Val Gly Leu Ser Val Ser Leu Leu Cys Leu
320 325 330
Leu Leu Ala Ala Leu Thr Phe Leu Leu Cys Lys Ala Ile Gln Asn
335 340 345
Thr Ser Thr Ser Leu His Leu Gln Leu Ser Leu Cys Leu Phe Leu
350 355 360
Ala His Leu Leu Phe Leu Val Gly Ile Asp Arg Thr G1u Pro Lys
365 370 375
Val Leu Cys Ser Ile Ile Ala Gly Ala Leu His Tyr Leu Tyr Leu
380 385 390
Ala A1a Phe Thr Trp Met Leu Leu Glu Gly Val His Leu Phe Leu
395 400 405
Thr Ala Arg Asn Leu Thr Va1 Val Asn Tyr Ser Ser Ile Asn Arg
410 415 420
Leu Met Lys Trp Ile Met Phe Pro Val Gly Tyr Gly Val Pro Ala
425 430 435
Val Thr Val Ala I1e Ser Ala Ala Ser Trp Pro His Leu Tyr Gly
440 445 450
Thr A1a Asp Arg Cys Trp Leu His Leu Asp Gln Gly Phe Met Trp
455 460 465
Ser Phe Leu Gly Pro Val Cys Ala Ile Phe Ser Ala Asn Leu Val
470 475 480
Leu Phe Ile Leu Val Phe Trp Ile Leu Lys Arg Lys Leu Ser Ser
485 490 495
Leu Asn Ser G1u Val Ser Thr Ile Gln Asn Thr Arg Met Leu A1a
9/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
500 505 510
Phe Lys Ala Thr Ala Gln Leu Phe Ile Leu Gly Cys Thr Trp Cys
515 520 525
Leu Gly Leu Leu Gln Val Gly Pro Ala A1a G1n Val Met Ala Tyr
530 535 540
Leu Phe Thr Ile Ile Asn Ser Leu Gln Gly Phe Phe Ile Phe Leu
545 550 555
Val Tyr Cys Leu Leu Ser Gln Gln Val Gln Lys Gln Tyr Gln Lys
560 565 570
Trp Phe Arg Glu Ile Val Lys Ser Lys Ser Glu Ser Glu Thr Tyr
575 580 585
Thr Leu Ser Ser Lys Met Gly Pro Asp Ser Lys Pro Ser Glu G1y
590 595 600
Asp Val Phe Pro Gly Gln Val Lys Arg Lys Tyr
605 610
<210> 9
<211> 1469
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 60203310CD1
<400> 9
Met Trp Pro Ser Gln Leu Leu Ile Phe Met Met Leu Leu Ala Pro
1 5 10 15
Ile Ile His Ala Phe Ser Arg Ala Pro I1e Pro Met Ala Val Val
20 25 30
Arg Arg Glu Leu Ser Cys Glu Ser Tyr Pro Ile G1u Leu Arg Cys
35 40 45
Pro Gly Thr Asp Val Tle Met Ile Glu Ser Ala Asn Tyr Gly Arg
50 55 60
Thr Asp Asp Lys Ile Cys Asp Ser Asp Pro Ala Gln Met Glu Asn
65 70 75
Ile Arg Cys Tyr Leu Pro Asp Ala Tyr Lys Ile Met Ser Gln Arg
80 85 90
Cys Asn Asn Arg Thr Gln Cys Ala Val Val Ala Gly Pro Asp Val
95 100 105
Phe Pro Asp Pro Cys Pro G1y Thr Tyr Lys Tyr Leu Glu Val Gln
110 115 120
Tyr G1u Cys Val Pro Tyr Lys Val Glu Gln Lys Val Phe Leu Cys
125 130 135
Pro G1y Leu Leu Lys Gly Val Tyr Gln Ser Glu His Leu Phe Glu
140 145 150
Ser Asp His Gln Ser Gly Ala Trp Cys Lys Asp Pro Leu Gln Ala
155 160 165
Ser Asp Lys Ile Tyr Tyr Met Pro Trp Thr Pro Tyr Arg Thr Asp
170 175 180
Thr Leu Thr Glu Tyr Ser Ser Lys Asp Asp Phe Ile Ala Gly Arg
185 190 195
Pro Thr Thr Thr Tyr Lys Leu Pro His Arg Val Asp Gly Thr Gly
200 205 210
Phe Val Val Tyr Asp Gly Ala Leu Phe Phe Asn Lys Glu Arg Thr
215 220 225
Arg Asn Ile Val Lys Phe Asp Leu Arg Thr Arg Ile Lys Ser Gly
230 235 240
Glu Ala Ile Ile Ala Asn Ala Asn Tyr His Asp Thr Ser Pro Tyr
245 250 255
Arg Trp G1y G1y Lys Ser Asp Ile Asp Leu Ala Va1 Asp Glu Asn
260 265 270
Gly Leu Trp Val Ile Tyr Ala Thr Glu Gln Asn Asn Gly Lys Ile
10137
Thr A1a Asp Arg Cys Trp Leu His Leu Asp Gln Gly Phe
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
275 280 285
Val Ile Ser G1n Leu Asn Pro Tyr Thr Leu Arg Ile Glu Gly Thr
290 295 300
Trp Asp Thr Ala Tyr Asp Lys Arg Ser Ala Ser Asn Ala Phe Met
305 310 315
Ile Cys Gly Ile Leu Tyr Val Val Lys Ser Val Tyr Glu Asp Asp
320 325 330
Asp Asn Glu Ala Thr Gly Asn Lys Ile Asp Tyr Ile Tyr Asn Thr
335 340 345
Asp Gln Ser Lys Asp Ser Leu Val Asp Val Pro Phe Pro Asn Ser
350 355 360
Tyr Gln Tyr Ile Ala Ala Val Asp Tyr Asn Pro Arg Asp Asn Leu
365 370 375
Leu Tyr Val Trp Asn Asn Tyr His Va1 Val Lys Tyr Ser Leu Asp
380 385 390
Phe Gly Pro Leu Asp Ser Arg Ser Gly Gln A1a His His Gly Gln
395 400 405
Val Ser Tyr Ile Ser Pro Pro Ile $is Leu Asp Ser Glu Leu Glu
410 415 420
Arg Pro Ser Val Lys Asp Ile Ser Thr Thr Gly Pro Leu Gly Met
425 430 435
Gly Ser Thr Thr Thr Ser Thr Thr Leu Arg Thr Thr Thr Leu Ser
440 445 450
Pro Gly Arg Ser Thr Thr Pro Ser Val Ser Gly Arg Arg Asn Arg
455 460 465
Ser Thr Ser Thr Pro Ser Pro Ala Va1 Glu Val Leu Asp Asp Met
470 475 480
Thr Thr His Leu Pro Ser Ala Ser Ser Gln Ile Pro Ala Leu Glu
485 490 495
Glu Ser Cys Glu Ala Val Glu Ala Arg Glu I1e Met Trp Phe Lys
500 505 510
Thr Arg Gln Gly Gln Ile Ala Lys Gln Pro Cys Pro Ala Gly Thr
515 520 525
I1e Gly Val Ser Thr Tyr Leu Cys Leu Ala Pro Asp Gly Ile Trp
530 535 540
Asp Pro Gln Gly Pro Asp Leu Ser Asn Cys Ser Ser Pro Trp Val
545 550 555
Asn His Ile Thr Gln Lys Leu Lys Ser Gly Glu Thr Ala Ala Asn
560 565 570
Ile Ala Arg Glu Leu Ala Glu Gln Thr Arg Asn His Leu Asn Ala
575 . 580 585
Gly Asp Ile Thr Tyr Ser Val Arg Ala Met Asp Gln Leu Val G1y
590 595 600
Leu Leu Asp Val Gln Leu Arg Asn Leu Thr Pro Gly Gly Lys Asp
605 610 615
Ser Ala Ala Arg Ser Leu Asn Lys Leu Gln Lys Arg Glu Arg Ser
620 625 630
Cys Arg Ala Tyr Val Gln Ala Met Val Glu Thr Val Asn Asn Leu
635 640 645
Leu Gln Pro Gln Ala Leu Asn Ala Trp Arg Asp Leu Thr Thr Ser
650 655 660
Asp Gln Leu Arg Ala A1a Thr Met Leu Leu Ha.s Thr Val Glu Glu
665 670 675
Ser A1a Phe Val Leu Ala Asp Asn Leu Leu Lys Thr Asp Ile Val
680 685 690
Arg Glu Asn Thr Asp Asn Ile Lys Leu Glu Va1 Ala Arg Leu Ser
695 700 705
Thr Glu Gly Asn Leu Glu Asp Leu Lys Phe Pro Glu Asn Met Gly
710 715 720
His G1y Ser Thr Ile Gln Leu Ser Ala Asn Thr Leu Lys Gln Asn
725 730 735
Gly Arg Asn Gly Glu Ile Arg Val Ala Phe Val Leu Tyr Asn Asn
740 745 750
11/37
CA 02417195 2003-O1-24
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Leu Gly Pro Tyr Leu Ser Thr Glu Asn Ala Ser Met Lys Leu Gly
755 760 765
Thr Glu Ala Leu Ser Thr Asn His Ser Val Ile Val Asn Ser Pro
770 775 780
Val Ile Thr Ala Ala Ile Asn Lys Glu Phe Ser Asn Lys Val Tyr
785 790 795
Leu Ala Asp Pro Val Val Phe Thr Val Lys His Ile Lys Gln Ser
800 805 810
Glu Glu Asn Phe Asn Pro Asn Cys Ser Phe Trp Ser Tyr Ser Lys
815 820 825
Arg Thr Met Thr G1y Tyr Trp Ser Thr Gln Gly Cys Arg Leu Leu
830 835 840
Thr Thr Asn Lys Thr His Thr Thr Cys Ser Cys Asn His Leu Thr
845 850 855
Asn Phe Ala Val Leu Met Ala His Val Glu Val Lys His Ser Asp
860 865 870
Ala Val His Asp Leu Leu Leu Asp Val Ile Thr Trp Val Gly Ile
875 880 885
Leu Leu Ser Leu Val Cys Leu Leu I1e Cys Ile Phe Thr Phe Cys
890 895 900
Phe Phe Arg Gly Leu Gln Ser Asp Arg Asn Thr Ile His Lys Asn
905 910 915
Leu Cys Ile Ser Leu Phe Val Ala Glu Leu Leu Phe Leu Ile Gly
920 925 930
Ile Asn Arg Thr Asp Gln Pro Ile Ala Cys Ala Val Phe Ala Ala
935 940 945
Leu Leu His Phe Phe Phe Leu Ala Ala Phe Thr Trp Met Phe Leu
950 955 960
Glu Gly Val Gln Leu Tyr Ile Met Leu Val Glu Val Phe Glu Ser
965 970 975
Glu His Ser Arg Arg Lys Tyr Phe Tyr Leu Val Gly Tyr Gly Met
980 985 990
Pro Ala Leu Ile Val Ala Val Ser Ala Ala Val Asp Tyr Arg Ser
995 1000 1005
Tyr Gly Thr Asp Lys Val Cys Trp Leu Arg Leu Asp Thr Tyr Phe
1010 1015 1020
Ile Trp Ser Phe Ile Gly Pro Ala Thr Leu Ile Ile Met Leu Asn
1025 1030 1035
Val Ile Phe Leu Gly Ile Ala Leu Tyr Lys Met Val His His Thr
1040 1045 1050
A1a Ile Leu Lys Pro Glu Ser Gly Cys Leu Asp Asn Ile Asn Tyr
1055 1060 1065
Glu Asp Asn Arg Pro Phe Ile Lys Ser Trp Val Ile Gly Ala Ile
1070 1075 1080
Ala Leu Leu Cys Leu Leu Gly Leu Thr Trp Ala Phe Gly Leu Met
1085 1090 1095
Tyr Ile Asn Glu Ser Thr Val Ile Met Ala Tyr Leu Phe Thr Ile
1100 1105 1110
Phe Asn Ser Leu Gln Gly Met Phe Ile Phe Ile Phe His Cys Val
1115 1120 1125
Leu Gln Lys Lys Val Arg Lys Glu Tyr Gly Lys Cys Leu Arg Thr
1130 1135 1140
His Cys Cys Ser Gly Lys Ser Thr Glu Ser Ser Ile Gly Ser Gly
1145 1150 1155
Lys Thr Ser G1y Ser Arg Thr Pro Gly Arg Tyr Ser Thr Gly Ser
1160 1165 1170
G1n Ser Arg Ile Arg Arg Met Trp Asn Asp Thr Val Arg Lys Gln
1175 1180 1185
Ser Glu Ser Ser Phe Ile Thr Gly Asp Ile Asn Ser Ser Ala Ser
1190 1195 1200
Leu Asn Arg Glu Gly Leu Leu Asn Asn Ala Arg Asp Thr Ser Val
1205 1210 1215
Met Asp Thr Leu Pro Leu Asn Gly Asn His Gly Asn Ser Tyr Ser
12/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
1220 1225 1230
Ile Ala Ser Gly Glu Tyr Leu Ser Asn Cys Val Gln Ile Ile Asp
1235 1240 1245
Arg Gly Tyr Asn His Asn Glu Thr Ala Leu Glu Lys Lys Ile Leu
1250 1255 1260
Lys Glu Leu Thr Ser Asn Tyr Ile Pro Ser Tyr Leu Asn Asn His
1265 1270 .1275
Glu Arg Ser Ser Glu Gln Asn Arg Asn Leu Met Asn Lys Leu Val
1280 1285 1290
Asn Asn Leu Gly Ser Gly Arg Glu Asp Asp Ala Ile Va1 Leu Asp
1295 1300 1305
Asp Ala Thr Ser Phe Asn His Glu Glu Ser Leu Gly Leu Glu Leu
1310 1315 1320
Ile His Glu Glu Ser Asp Ala Pro Leu Leu Pro Pro Arg Val Tyr
1325 1330 1335
Ser Thr Glu Asn His Gln Pro His His Tyr Thr Arg Arg Arg Ile
1340 1345 1350
Pro Gln Asp His Ser Glu Ser Phe Phi Pro Leu Leu Thr Asn Glu
1355 1360 1365
His Thr Glu Asp Leu Gln Ser Pro His Arg Asp Ser Leu Tyr Thr
1370 1375 1380
Ser Met Pro Thr Leu Ala Gly Val Ala Ala Thr Glu Ser Val Thr
1385 1390 1395
Thr Ser Thr Gln Thr Glu Pro Pro Pro Ala Lys Cys G1y Asp Ala
1400 1405 1410
Glu Asp Val Tyr Tyr Lys Ser Met Pro Asn Leu Gly Ser Arg Asn
1415 1420 1425
His Val His Gln Leu His Thr Tyr Tyr Gln Leu Gly Arg Gly Ser
1430 1435 1440
Ser Asp Gly Phe Ile Val Pro Pro Asn Lys Asp Gly Thr Pro Pro
1445 1450 1455
Glu Gly Ser Ser Lys Gly Pro Ala His Leu Val Thr Ser Leu
1460 1465
<210> 10
<211> 469
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No~: 7477349CD1
<400> 10
Met Asp Pro Ser Val Val Ser Asn Glu Tyr Tyr Asp Val Ala His
1 5 10 15
Gly Ala Lys Asp Pro Val Val Pro Thr Ser Leu Gln Asp Ile Thr
20 25 30
Ala Val Leu Gly Thr Glu Ala Tyr Thr Glu Glu Asp Lys Ser Met
35 40 45
Val Ser His Ala G1n Lys Ser Gln His Ser Cys Leu Ser His Ser
50 55 60
Arg Trp Leu Arg Ser Pro Gln Va1 Thr Gly Gly Ser Trp Asp Leu
65 70 75
Arg Ile Arg Pro Ser Lys Asp Ser Ser Ser Phe Arg Gln Ala Gln
80 85 90
Cys Leu Arg Lys Asp Pro Gly Ala Asn Asn His Leu Glu Ser Gln
95 100 105
Gly Val Arg Gly Thr Ala Gly Asp Ala Asp Arg Glu Leu Arg Gly
110 115 120
Pro Ser Glu Lys Ala Thr Ala Gly Gln Pro Arg Val Thr Leu Leu
125 130 135
Pro Thr Pro Asn Val Ser Gly Leu Ser Gln Glu Phe Glu Ser His
13137
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
140 145 150
Trp Pro Glu Ile Ala Glu Arg Ser Pro Cys Val A1a Gly Val I1e
155 160 165
Pro Val Ile Tyr Tyr Ser Val Leu Leu Gly Leu Gly Leu Pro Val
170 175 180
Ser Leu Leu Thr Ala Val Ala Leu A1a Arg Leu Ala Thr Arg Thr
185 190 195
Arg Arg Pro Ser Tyr Tyr Tyr Leu Leu Ala Leu Thr Ala Ser Asp
200 205 210
Ile Ile Ile Gln Val Val Ile Val Phe Ala Gly Phe Leu Leu G1n
215 220 225
Gly Ala Val Leu Ala Arg Gln Val Pro Gln Ala Val Val Arg Thr
230 235 240
Ala Asn Ile Leu Glu Phe Ala Ala Asn His Ala Ser Va1 Trp Ile
245 250 255
Ala Ile Leu Leu Thr Val Asp Arg Tyr Thr Ala Leu Cys His Pro
260 265 270
Leu His His Arg Ala Ala Ser Ser Pro Gly Arg Thr Arg Arg Ala
275 280 285
Ile Ala Ala Va1 Leu Ser A1a Ala Leu Leu Thr Gly Ile Pro Phe
290 295 300
Tyr Trp Trp Leu Asp Met Trp Arg Asp Thr Asp Ser Pro Arg Thr
305 310 315
Leu Asp Glu Va1 Leu Lys Trp Ala His Cys Leu Thr Val Tyr Phe
320 325 330
Ile Pro Cys Gly Val Phe Leu Val Thr Asn Ser Ala Ile Ile His
335 340 345
Arg Leu Arg Arg Arg Gly Arg Ser Gly Leu Gln Pro Arg Val Gly
350 355 360
Lys Ser Thr Ala Ile Leu Leu Gly Ile Thr Thr Leu Phe Thr Leu
365 370 375
Leu Trp A1a Pro Arg Val Phe Val Met Leu Tyr His Met Tyr Val
380 385 390
Ala Pro Val His Arg Asp Trp Arg Val His Leu Ala Leu Asp Val
395 400 405
Ala Asn Met Val Ala Met Leu His Thr Ala Ala Asn Phe Gly Leu
410 415 420
Tyr Cys Phe Val Ser Lys Thr Phe Arg Ala Thr Val Arg Gln Val
425 430 435
Ile His Asp Ala Tyr Leu Pro Cys Thr Leu Ala Ser Gln Pro Glu
440 445 450
Gly Met Ala Ala Lys Pro Val Met Glu Pro Pro Gly Leu Pro Thr
455 460 465
Gly Ala Glu Val
<210> 11
<211> 335
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55002225CD1
<400> 11
Met Asn Pro Phe His Ala Ser Cys Trp Asn Thr Ser Ala Glu Leu
1 5 10 15
Leu Asn Lys Ser Trp Asn Lys Glu Phe Ala Tyr Gln Thr A1a Ser
20 25 30
Val Val Asp Thr Val Ile Leu Pro Ser Met Ile Gly Ile Ile Cys
35 40 45
Ser Thr Gly Leu Val Gly Asn Ile Leu Ile Val Phe Thr Ile I1e
14/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
50 55 60
Arg Ser Arg Lys Lys Thr Val Pro Asp Ile Tyr Ile Cys Asn Leu
65 70 75
Ala Val Ala Asp Leu Val His Ile Val Gly Met Pro Phe Leu Ile
80 85 90
His Gln Trp Ala Arg Gly Gly Glu Trp Val Phe Gly Gly Pro Leu
95 100 105
Cys Thr Ile Ile Thr Ser Leu Asp Thr Cys Asn Gln Phe Ala Cys
110 115 120
Ser Ala Ile Met Thr Val Met Ser Val Asp Arg Tyr Phe Ala Leu
125 130 135
Val Gln Pro Phe Arg Leu Thr Arg Trp Arg Thr Arg Tyr Lys Thr
140 145 150
Ile Arg Ile Asn Leu Gly Leu Trp Ala Ala Ser Phe Ile Leu Ala
155 160 165
Leu Pro Val Trp Val Tyr Ser Lys Val Ile Lys Phe Lys Asp Gly
170 175 180
Val Glu Ser Cys Ala Phe Asp Leu Thr Ser Pro Asp Asp Val Leu
185 190 195
Trp Tyr Thr Leu Tyr Leu Thr Ile Thr Thr Phe Phe Phe Pro Leu
200 205 210
Pro Leu Ile Leu Val Cys Tyr Ile Leu Ile Leu Cys Tyr Thr Trp
215 220 225
Glu Met Tyr Gln Gln Asn Lys Asp Ala Arg Cys Cys Asn Pro Ser
230 235 240
Val Pro Lys Gln Arg Val Met Lys Leu Thr Lys Met Val Leu Val
245 250 255
Leu Val Va1 Val Phe Ile Leu Ser Ala Ala Pro Tyr His Val Ile
260 265 270
Gln Leu Val Asn Leu Gln Met Glu Gln Pro Thr Leu Ala Phe Tyr
275 280 285
Val Gly Tyr Tyr Leu Ser Ile Cys Leu Ser Tyr Ala Ser Ser Ser
290 295 300
Ile Asn Pro Phe Leu Tyr Ile Leu Leu Ser Gly Thr Pro G1n I1e
305 3l0 315
Gln Arg Arg Ala Thr Glu Lys Glu Ile Asn Asn Met Gly Asn Thr
320 325 330
Leu Lys Ser His Phe
335
<210> 12
<211> 630
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475686CD1
<400> 12
Met Arg Leu Gly Pro Va1 Pro Ala Arg Ala Arg Ala Leu Leu Ser
1 5 10 15
Trp Val Arg Gly Leu Glu Ser Arg Gly Gly Glu Trp Thr Lys Cys
20 25 30
Ile Val Gln Leu Gly His Leu Leu Ala Thr Gln His Pro Ala Ala
35 40 45
Pro Thr Cys Gly Val Val Ser Ser Ala Leu Val Met His Ser Thr
50 55 60
Asp Val Cys Leu Ala Pro Thr Met His Gln Ala Leu Asp Trp Ala
65 70 75
Ala Gly Ile Trp Phe Thr Gly Arg Leu Gly Leu Arg Glu His Lys
80 85 90
Ser Leu Ala Gln Gly Asp Ser Val Cys Pro Cys Glu Ser Glu Leu
15/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
95 100 105
Gly Asp Phe Gln Val Tyr Gly Leu Val Ser Thr Glu G1y Val Va1
110 115 120
Ser Cys Phe Gly Glu Lys Thr Pro Gln His Pro Gly Pro Pro Ala
125 130 135
Ser Leu Ser Leu Ala Asn Arg Cys His Asn Val Val Thr Ala Va1
140 145 150
Gly Ala Trp Pro Ala His Gly Ser I1e Leu Gly Asn Val Pro Glu
155 160 165
Ala Pro Val Gly Ala Asp Val Leu Gly Ala Gly Gly Cys Asp Trp
170 175 180
Ala Asp Lys Glu Ala Leu Ala Pro Gly Gln Arg Ala Lys Val His
185 190 195
Ile Leu Leu Glu Ser Ser Gly Gln Ser Asp Pro Ser Tyr Ala Va1
200 205 210
Leu Pro Asp Ser Trp Ala Ala Thr G1u G1y Phe Pro Thr Tyr Arg
215 220 225
Ser Gln Val Ser Ser Pro Arg Ile Pro Gly Ser Ser Ile Trp Leu
230 235 240
Gly Ser Gly Ser Gly Trp Pro Ile Leu Gly Glu Leu Arg Glu Cys
245 250 255
Asp Gln Met Phe Ser Cys Met Leu Pro Thr Gly Cys Ala Ser Phe
260 265 270
Gln Asp Pro Gly Arg Tyr Gly Asp Tyr Asp Leu Pro Met Asp Glu
275 280 285
Asp Glu Asp Met Thr Lys Thr Arg Thr Phe Phe Ala Ala Lys Ile
290 295 300
Val Ile Gly Ile Ala Leu Ala Gly Ile Met Leu Val Cys Gly Ile
305 310 315
Gly Asn Phe Val Phe Ile Ala Ala Leu Thr Arg Tyr Lys Lys Leu
320 325 330
Arg Asn Leu Thr Asn Leu Leu Ile Ala Asn Leu Ala Ile Ser Asp
335 340 345
Phe Leu Val Ala Ile Ile Cys Cys Pro Phe Glu Met Asp Tyr Tyr
350 355 360
Val Val Arg Gln Leu Ser Trp Glu His Gly His Val Leu Cys Ala
365 370 375
Ser Val Asn Tyr Leu Arg Thr Val Ser Leu Tyr Val Ser Thr Asn
380 385 390
Ala Leu Leu Ala Ile Ala I1e Asp Arg Tyr Leu Ala Ile Val His
395 400 405
Pro Leu Lys Pro Arg Met Asn Tyr Gln Thr Ala Ser Phe Leu Ile
410 415 420
Ala Leu Val Trp Met Val Ser Ile Leu Ile A1a Ile Pro Ser Ala
425 430 435
Tyr Phe Ala Thr Glu Thr Va1 Leu Phe Ile Val Lys Ser Gln Glu
440 445 450
Lys I1e Phe Cys Gly Gln I1e Trp Pro Val Asp Gln G1n Leu Tyr
455 460 465
Tyr Lys Ser Tyr Phe Leu Phe Ile Phe Gly Val Glu Phe Val Gly
470 475 480
Pro Val Val Thr Met Thr Leu Cys Tyr Ala Arg Ile Ser Arg Glu
485 490 495
Leu Trp Phe Lys Ala Val Pro Gly Phe Gln Thr Glu Gln I1e Arg
500 505 510
Lys Arg Leu Arg Cys Arg Arg Lys Thr Val Leu Val Leu Met Cys
515 520 525
Ile Leu Thr Ala Tyr Val Leu Cys Trp Ala Pro Phe Tyr Gly Phe
530 535 540
Thr Ile Val Arg Asp Phe Phe Pro Thr Val Phe Val Lys Glu Lys
545 550 555
His Tyr Leu Thr Ala Phe Tyr Val Val Glu Cys I1e Ala Met Ser
560 565 570
16/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
Asn Ser Met Ile Asn Thr Val Cys Phe Val Thr Val Lys Asn Asn
575 580 585
Thr Met Lys Tyr Phe Lys Lys Met Met Leu Leu His Trp Arg Pro
590 595 600
Ser Gln Arg Gly Ser Lys Ser Ser Ala Asp Leu Asp Leu Arg Thr
605 610 615
Asn Gly Val Pro Thr Thr Glu Glu Val Asp Cys Ile Arg Leu Lys
620 625 630
<210> 13
<211> 695
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7482007CD1
<400> 13
Met Lys Met Lys Ser Gln A1a Thr Met Ile Cys Cys Leu Val Phe
1 5 10 15
Phe Leu Ser Thr Glu Cys Ser His Tyr Arg Ser Lys Ile His Leu
20 25 30
Lys Ala Gly Asp Lys Leu G1n Ser Pro Glu Gly Lys Pro Lys Thr
35 40 45
Gly Arg Ile Gln Glu Lys Cys Glu Gly Pro Cys Ile Ser Ser Ser
50 55 60
Asn Cys Ser Gln Pro Cys Ala Lys Asp Phe His Gly Glu Ile Gly
65 70 75
Phe Thr Cys Asn .Gln Lys Lys Trp Gln Lys Ser Ala Glu Thr Cys
80 85 90
Thr Ser Leu Ser Val Glu Lys Leu Phe Lys Asp Ser Thr Gly Ala
95 100 105
Ser Arg Leu Ser Val Ala A1a Pro Ser Ile Pro Leu His Ile Leu
110 115 120
Asp Phe Arg Ala Pro Glu Thr Ile G1u Ser Val A1a Gln Gly Ile
125 130 135
Arg Lys Asn Cys Pro Phe Asp Tyr Ala Cys Ile Thr Asp Met Val
140 145 150
Lys Ser Ser Glu Thr Thr Ser Gly Asn Ile Ala Phe Ile Val Glu
155 160 165
Leu Leu Lys Asn Ile Ser Thr Asp Leu Ser Asp Asn Va1 Thr Arg
170 175 180
Glu Lys Met Lys Ser Tyr Ser Glu Val Ala Asn His Ile Leu Asp
185 190 195
Thr Ala Ala Ile Ser Asn Trp Ala Phe Ile Pro Asn Lys Asn Ala
200 205 210
Ser Ser Asp Leu Leu Gln Ser Val Asn Leu Phe Ala Arg Gln Leu
215 220 225
His Ile His Asn Asn Ser G1u Asn Ile Val Asn Glu Leu Phe Ile
230 235 240
Gln Thr Lys G1y Phe His I1e Asn His Asn Thr Ser Glu Lys Ser
245 250 255
Leu Asn Phe Ser Met Ser Met Asn Asn Thr Thr Glu Asp Ile Leu
260 265 270
Gly Met Val Gln Ile Pro Arg Gln Glu Leu Arg Lys Leu Trp Pro
275 280 285
Asn Ala Ser Gln Ala Ile Ser Ile Ala Phe Pro Thr Leu Gly Ala
290 295 300
Ile Leu Arg Glu Ala His Leu Gln Asn Val Ser Leu Pro Arg Gln
305 310 315
Val Asn Gly Leu Val Leu Ser Val Val Leu Pro Glu Arg Leu Gln
17/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
320 325 330
Glu Ile Ile Leu Thr Phe Glu Lys Ile Asn Lys Thr Arg Asn Ala
335 340 345
Arg Ala Gln Cys Val Gly Trp His Ser Lys Lys Arg Arg Trp Asp
350 355 360
Glu Lys Ala Cys Gln Met Met Leu Asp Ile Arg Asn Glu Val Lys
365 370 375
Cys Arg Cys Asn Tyr Thr Ser Val Val Met Ser Phe Ser Ile Leu
380 385 390
Met Ser Ser Lys Ser Met Thr Asp Lys Val Leu Asp Tyr Ile Thr
395 400 405
Cys Ile Gly Leu Ser Val Ser Ile Leu Ser Leu Val Leu Cys Leu
410 415 420
Ile Ile Glu Ala Thr Val Trp Ser Arg Val Val Val Thr G1u Ile
425 430 435
Ser Tyr Met Arg His Val Cys Ile Val Asn Ile Ala Val Ser Leu
440 445 450
Leu Thr Ala Asn Val Trp Phe Ile Ile Gly Ser His Phe Asn Ile
455 460 465
Lys Ala Gln Asp Tyr Asn Met Cys Val Ala Val Thr Phe Phe Ser
470 475 480
His Phe Phe Tyr Leu Ser Leu Phe Phe Trp Ile Leu Phe Lys Ala
485 490 495
Leu Leu Ile I1e Tyr Gly Ile Leu Val Ile Phe Arg Arg Met Met
500 505 510
Lys Ser Arg Met Met Val Ile Gly Phe Ala Ile Gly Tyr Gly Cys
515 520 525
Pro Leu Ile Ile Ala Val Thr Thr Val Ala Ile Thr Gly Pro Val
530 535 540
Lys Gly Tyr Met Arg Pro Glu Ala Cys Trp Leu Asn Trp Asp Asn
545 550 555
Thr Lys Ala Leu Leu A1a Phe Ala Ile Pro Ala Phe Val Ile Val
560 565 570
Ala Val Asn Leu Ile Val Val Leu Val Val Ala Val Asn Thr Gln
575 580 585
Arg Pro Ser I1e Gly Ser Ser Lys Ser Gln Asp Val Val Ile Ile
590 595 600
Met Arg Ile Ser Lys Asn Val Ala Ile Leu Thr Pro Leu Leu Gly
605 610 615
Leu Thr Trp G1y Phe Gly Ile Ala Thr Leu Ile Glu Gly Thr Ser
620 625 630
Leu Thr Phe His Ile Ile Phe Ala Leu Leu Asn Ala Phe Gln Gly
635 640 645
Phe Phe Ile Leu Leu Phe Gly Thr Ile Met Asp His Lys Ile Arg
650 655 660
Asp Ala Leu Arg Met Arg Met Ser Ser Leu Lys Gly Lys Ser Arg
665 670 675
Ala Ala Glu Asn Ala Ser Leu Gly Pro Thr Asn Gly Ser Lys Leu
680 685 690
Met Asn Arg Gln Gly
695
<210> 14
<211> 633
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6769042CD1
<400> 14
Met Tyr Phe Thr Ala Ala Ile Gly Lys His Ala Leu Leu Ser Ser
18/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
1 5 10 15
Thr Leu Pro Ser Leu Phe Met Thr Ser Thr Ala Ser Pro Val Met
20 25 30
Pro Thr Asp Ala Tyr His Pro Ile Ile Thr Asn Leu Thr Glu Glu
35 40 45
Arg Lys Thr Phe Gln Ser Pro Gly Val Ile Leu Ser Tyr Leu Gln
50 55 60
Asn Val Ser Leu Ser Leu Pro Ser Lys Ser Leu Ser Glu Gln Thr
65 70 75
Ala Leu Asn Leu Thr Lys Thr Phe Leu Lys Ala Val Gly Glu Ile
80 85 90
Leu Leu Leu Pro Gly Trp Ile Ala Leu Ser Glu Asp Ser Ala Val
95 100 105
Val Leu Ser Leu Ile Asp Thr Ile Asp Thr Val Met Gly His Val
110 115 120
Ser Ser Asn Leu His Gly Ser Thr Pro Gln Val Thr Val G1u Gly
125 130 135
Ser Ser Ala Met A1a Glu Phe Ser Val Ala Lys Ile Leu Pro Lys
140 145 150
Thr Val Asn Ser Ser His Tyr Arg Phe Pro Ala His Gly Gln Ser
155 160 165
Phe Ile Gln Ile Pro His Glu Ala Phe His Arg His Ala Trp Ser
170 175 7.80
Thr Val Val Gly Leu Leu Tyr His Ser Met His Tyr Tyr Leu Asn
185 190 195
Asn Ile Trp Pro Ala His Thr Lys Ile Ala Glu Ala Met His His
200 205 210
Gln Asp Cys Leu Leu Phe Ala Thr Ser His Leu Ile Ser Leu Glu
215 220 225
Val Ser Pro Pro Pro Thr Leu Ser Gln Asn Leu Ser Gly Ser Pro
230 235 240
Leu Ile Thr Val His Leu Lys His Arg Leu Thr Arg Lys Gln His
245 250 255
Ser Glu Ala Thr Asn Ser Ser Asn Arg Val Phe Val Tyr Cys Ala
260 265 270
Phe Leu Asp Phe Ser Ser Gly Glu Gly Val Trp Ser Asn His Gly
275 280 285
Cys Ala Leu Thr Arg Gly Asn Leu Thr Tyr Ser Val Cys Arg Cys
290 295 300
Thr His Leu Thr Asn Phe Ala Ile Leu Met Gln Val Val Pro Leu
305 310 315
Glu Leu Ala Arg G1y His G1n Val Ala Leu Ser Ser Ile Ser Tyr
320 325 330
Val Gly Cys Ser Leu Ser Val Leu Cys Leu Val Ala Thr Leu Val
335 340 345
Thr Phe Ala Val Leu Ser Ser Val Ser Thr Ile Arg Asn Gln Arg
350 355 360
Tyr His Ile His Ala Asn Leu Ser Phe Ala Val Leu Val Ala Gln
365 370 375
Va1 Leu Leu Leu Ile Ser Phe Arg Leu Glu Pro Gly Thr Thr Pro
380 385 390
Cys Gln Val Met Ala Val Leu Leu His Tyr Phe Phe Leu Ser Ala
395 400 405
Phe Ala Trp Met Leu Val Glu Gly Leu His Leu Tyr Ser Met Val
410 415 420
Ile Lys Val Phe Gly Ser Glu Asp Ser Lys His Arg Tyr Tyr Tyr
425 430 435
Gly Met Gly Trp Gly Phe Pro Leu Leu Ile Cys Ile Ile Ser Leu
440 445 450
Ser Phe Ala Met Asp Ser Tyr Gly Thr Ser Asn Asn Cys Trp Leu
455 460 465
Ser Leu Ala Ser Gly Ala Ile Trp Ala Phe Val Ala Pro Ala Leu
470 475 480
19/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
Phe Val Ile Val Val Asn Ile Gly Ile Leu Ile Ala Val Thr Arg
485 490 495
Val Ile Ser Gln Ile Ser A1a Asp Asn Tyr Lys Ile His G1y Asp
500 505 510
Pro Ser Ala Phe Lys Leu Thr Ala Lys Ala Val Ala Val Leu Leu
515 520 525
Pro Ile Leu Gly Thr Ser Trp Val Phe Gly Val Leu Ala Val Asn
530 535 540
Gly Cys Ala Val Val Phe Gln Tyr Met Phe Ala Thr Leu Asn Ser
545 550 555
Leu Gln Gly Leu Phe Ile Phe Leu Phe His Cys Leu Leu Asn Ser
560 565 570
Glu Val Arg Ala Ala Phe Lys His Lys Ile Lys Val Trp Ser Leu
575 580 585
Thr Ser Ser Ser Ala Arg Thr Ser Asn Ala Lys Pro Phe His Ser
590 595 600
Asp Leu Met Asn Gly Thr Arg Pro Gly Met Ala Ser Thr Lys Leu
605 610 615
Ser Pro Trp Asp Lys Ser Ser His Ser Ala His Arg Val Asp Leu
620 625 630
Ser Ala Val
<210> 15
<211> 370
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7476053CD1
<400> 15
Met Glu Ala Ala Ser Leu Ser Val Ala Thr Ala Gly Val Ala Leu
1 5 l0 15
Ala Leu Gly Pro Glu Thr Ser Ser Gly Thr Pro Ser Pro Arg Gly
20 25 30
Ile Leu Gly Ser Thr Pro Ser Gly Ala Val Leu Pro Gly Arg Gly
35 40 45
Pro Pro Phe Ser Val Phe Thr Val Leu Val Val Thr Leu Leu Val
50 55 60
Leu Leu Ile Ala Ala Thr Phe Leu Trp Asn Leu Leu Val Pro Val
65 70 75
Thr Ile Pro Arg Val Arg Ala Phe His Arg Val Pro His Asn Leu
80 85 90
Val Ala Ser Thr Ala Val Ser Asp Glu Leu Val Ala Ala Leu Ala
95 100 105
Met Pro Pro Ser Leu Ala Ser G1u Leu Ser Thr Gly Arg Arg Arg
110 115 120
Leu Leu Gly Arg Ser Leu Cys His Val Trp Ile Ser Phe Asp Ala
125 130 135
Leu Cys Cys Pro Ala Gly Leu Gly Asn Val Ala Ala Ile Ala Leu
140 145 150
Gly Arg Asp Gly A1a Ile Thr Arg His Leu Gln His Thr Leu Arg
155 160 165
Thr Arg Ser Arg Ala Ser Leu Leu Met Ile Ala Leu Ala Arg Val
170 175 180
Pro Ser Ala Leu Ile Ala Leu Ala Pro Leu Leu Phe Gly Arg Gly
185 190 195
Glu Va1 Cys Asp Ala Arg Leu Gln Arg Cys Gln Val Ser Arg Glu
200 205 210
Pro Ser Tyr Ala Ala Phe Ser Thr Arg Gly Ala Phe His Leu Pro
215 220 225
20!37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
Leu Gly Val Val Pro Phe Val Tyr Arg Lys Ile Tyr Glu Ala Ala
230 235 240
Lys Phe Arg Phe Gly Arg Arg Arg Arg Ala Val Leu Pro Leu Pro
245 250 255
Ala Thr Met G1n Val Lys Glu Ala Pro Asp Glu Ala Glu Val Val
260 265 270
Phe Thr Ala His Cys Lys Ala Thr Val Ser Phe Gln Val Ser G1y
275 280 285
Asp Ser Trp Arg Glu Gln Lys Glu Arg Arg Ala Ala Met Met Val
290 295 300
Gly Ile Leu Ile Gly Val Phe Val Leu Cys Trp Ile Pro Phe Phe
305 310 315
Leu Thr Glu Leu Ile Ser Pro Leu Cys Ala Cys Ser Leu Pro Pro
320 325 330
Ile Trp Lys Ser Ile Phe Leu Trp Leu Gly Tyr Ser Asn Ser Phe
335 340 345
Phe Asn Pro Leu Ile Tyr Thr Ala Phe Asn Lys Asn Tyr Asn Asn
350 355 360
Ala Phe Lys Ser Leu Phe Thr Lys Gln Arg
365 370
<210> 16
<211> 324
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480410CD1
<400> 26
Met Gly Met Glu Gly Leu Leu Gln Asn Ser Thr Asn Phe Val Leu
1 5 10 15
Thr Gly Leu Ile Thr His Pro Ala Phe Pro Gly Leu Leu Phe Ala
20 25 30
Ile Val Phe Ser Ile Phe Val Val Ala Ile Thr Ala Asn Leu Val
35 40 45
Met Ile Leu Leu Ile His Met Asp Ser Arg Leu His Thr Pro Met
50 55 60
Tyr Phe Leu Leu Ser Gln Leu Ser Ile Met Asp Thr Ile Tyr Ile
65 70 75
Cys Ile Thr Val Pro Lys Met Leu Gln Asp Leu Leu Ser Lys Asp
80 85 90
Lys Thr Ile Ser Phe Leu Gly Cys A1a Va1 Gln Ile Phe Leu Tyr
95 100 105
Leu Thr Leu Tle Gly Gly Glu Phe Phe Leu Leu G1y Leu Met Ala
110 115 120
Tyr Asp Arg Tyr Val Ala Val Cys Asn Pro Leu Arg Tyr Pro Leu
125 130 135
Leu Met Asn Arg Arg Val Cys Leu Phe Met Val Val Gly Ser Trp
140 145 150
Val Gly Gly Ser Leu Asp Gly Phe Met Leu Thr Pro Val Thr Met
155 160 165
Ser Phe Pro Phe Cys Arg Ser Arg Glu Ile Asn His Phe Phe Cys
170 175 180
Glu Ile Pro Ala Val Leu Lys Leu Ser Cys Thr Asp Thr Ser Leu
185 190 195
Tyr Glu Thr Leu Met Tyr Ala Cys Cys Val Leu Met Leu Leu Ile
200 205 210
Pro Leu Ser Val Ile Ser Val Ser Tyr Thr His Ile Leu Leu Thr
215 220 225
Val His Arg Met Asn Ser Ala Glu Gly Arg Arg Lys Ala Phe Ala
230 235 240
21/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
Thr Cys Ser Ser His Ile Met Val Val Ser Val Phe Tyr Gly Ala
245 250 255
Ala Phe Tyr Thr Asn Val Leu Pro His Ser Tyr His Thr Pro Glu
260 265 270
Lys Asp Lys Val Val Ser Ala Phe Tyr Thr Ile Leu Thr Pro Met
275 280 285
Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val Ala Ala
290 295 300
Ala Leu Arg Lys Val Leu Gly Arg Cys Gly Ser Ser G1n Ser Ile
305 310 315
Arg Val Ala Thr Val Ile Arg Lys Gly
320
<210> 17
<211> 315
<212> PRT
<213> Homo Sapiens
<220>
<22l> misc_feature
<223> Incyte ID No: 55036418CD1
<400> 17
Met Glu Thr Trp Val Asn Gln Ser Tyr Thr Asp Gly Phe Phe Leu
1 5 10 15
Leu Gly I1e Phe Ser His Ser Thr Ala Asp Leu Val Leu Phe Ser
20 25 30
Val Va1 Met Ala Val Phe Thr Val Ala Leu Cys Gly Asn Val Leu
35 40 45
Leu Ile Phe Leu Ile Tyr Met Asp Pro His Leu His Thr Pro Met
50 55 60
Tyr Phe Phe Leu Ser Gln Leu Ser Leu Met Asp Leu Met Leu Val
65 70 75
Cys Thr Asn Val Pro Lys Met Ala Ala Asn Phe Leu Ser Gly Arg
80 85 90
Lys Ser Ile Ser Phe Val Gly Cys Gly Ile Gln Ile Gly Leu Phe
95 100 105
Val Cys Leu Val Gly Ser Glu G1y Leu Leu Leu Gly Leu Met Ala
110 115 120
Tyr Asp Arg Tyr Val Ala Ile Ser His Pro Leu His Tyr Pro Ile
125 130 135
Leu Met Asn Gln Arg Val Cys Leu Gln Ile Thr Gly Ser Ser Trp
140 l45 150
Ala Phe Gly Ile Ile Asp Gly Leu Ile Gln Met Val Val Val Met
155 160 165
Asn Phe Pro Tyr Cys Gly Leu Arg Lys Val Asn His Phe Phe Cys
170 175 180
Glu Met Leu Ser Leu Leu Lys Leu Ala Cys Val Asp Thr Ser Leu
185 190 195
Phe Glu Lys Val Ile Phe Ala Cys Cys Val Phe Met Leu Leu Phe
200 205 210
Pro Phe Ser Ile Ile Val Ala Ser Tyr A1a His Ile Leu Gly Thr
215 220 225
Val Leu Gln Met His Ser Ala Gln Ala Trp Lys Lys Ala Leu Ala
230 235 240
Thr Cys Ser Ser His Leu Thr Ala Val Thr Leu Phe Tyr Gly Ala
245 250 255
Ala Met Phe I1e Tyr Leu Arg Pro Arg His Tyr Arg Ala Pro Ser
260 265 270
His Asp Lys Val Ala Ser Ile Phe Tyr Thr Val Leu Thr Pro Met
275 280 285
Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Arg Glu Val Met Gly
290 295 300
22137
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
Ala Leu Arg Lys Gly Leu Asp Arg Cys Arg Ile Gly Ser Gln His
305 310 315
<210> 18
<211> 324
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7481701CD1
<400> 18
Met Glu Ser Pro Asn Gln Thr Thr Ile Gln Glu Phe Ile Phe Ser
1 5 10 15
Ala Phe Pro Tyr Ser Trp Val Lys Ser Val Val Cys Phe Val Pro
20 25 30
Leu Leu Phe Ile Tyr Ala Phe Ile Val Val Gly Asn Leu Val Ile
35 40 45
Ile Thr Val Val Gln Leu Asn Thr His Leu His Thr Pro Met Tyr
50 55 60
Thr Phe Ile Ser Ala Leu Ser Phe Leu Glu Ile Trp Tyr Thr Thr
65 70 75
Ala Thr Ile Pro Lys Met Leu Ser Ser Leu Leu Ser Glu Arg Ser
80 85 90
I1e Ser Phe Asn Gly Cys Leu Leu Gln Met Tyr Phe Phe His Ser
95 100 105
Thr G1y Ile Cys Glu Val Cys Leu Leu Thr Val Met Ala Phe Asp
110 115 120
His Tyr Leu Ala Ile Cys Ser Pro Leu His Tyr Pro Ser Ile Met
125 130 135
Thr Pro Lys Leu Cys Thr Gln Leu Thr Leu Ser Cys Cys Val Cys
140 145 150
Gly Phe Ile Thr Pro Val Pro Glu Ile Ala Trp Ile Ser Thr Leu
155 160 165
Pro Phe Cys Gly Ser Asn His Leu Glu His Ile Phe Cys Asp Phe
170 175 280
Leu Pro Val Leu Arg Leu Ala Cys Thr Asp Thr Arg Ala Ile Val
185 190 195
Met Ile Gln Val Val Asp Val Ile His Ala Val Glu Ile Ile Thr
200 205 210
Ala Val Met Leu Ile Phe Met Ser Tyr Asp Gly Ile Val Ala Val
215 220 225
Ile Leu Arg Ile His Ser Ala Gly G1y Arg Arg Thr Ala Phe Ser
230 235 240
Thr Cys Val Ser His Phe Ile Val Phe Ser Leu Phe Phe Gly Ser
245 250 255
Val Thr Leu Met Tyr Leu Arg Phe Ser Ala Thr Tyr Ser Leu Phe
260 265 270
Trp Asp Ile Ala Ile Ala Leu Ala Phe Ala Val Leu Ser Pro Phe
275 280 285
Phe Asn Pro Ile Ile Tyr Ser Leu Arg Asn Lys Glu Ile Lys Glu
290 295 300
Ala Ile Lys Lys His Ile Gly Gln Ala Lys Ile Phe Phe Ser Val
305 310 315
Arg Pro Gly Thr Ser Ser Lys Ile Phe
320
<210> 19
<211> 312
<212> PRT
<213> Homo Sapiens
23/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
<220>
<221> misc_feature
<223> Incyte ID No: 7481774CD1
<400> 19
Met Glu Pro Trp Gln His Pro Thr His Phe Ile Leu Leu Gly Phe
1 5 10 15
Ser Asp Arg Pro His Leu Glu Arg IIe Leu Phe Val Val Ile Leu
20 25 30
Ile Ala Tyr Leu Leu Thr Leu Val Gly Asn Thr Thr Ile Ile Leu
35 40 45
Val Ser Arg Leu Asp Pro His Leu His Thr Pro Met Tyr Phe Phe
50 55 60
Leu Ala His Leu Ser Phe Leu Asp Leu Ser Phe Thr Thr Ser Ser
65 70 75
Ile Pro Gln Leu Leu Tyr Asn Leu Asn Gly Cys Asp Lys Thr Ile
80 85 90
Ser Tyr Met Gly Cys Ala I1e Gln Leu Phe Leu Phe Leu Gly Leu
95 100 105
Gly Gly Val Glu Cys Leu Leu Leu Ala Val Met Ala Tyr Asp Arg
110 115 120
Cys Val Ala Ile Cys Lys Pro Leu His Tyr Met Val Ile Met Asn
125 130 135
Pro Arg Leu Cys Arg Gly Leu Va1 Ser Val Thr Trp Gly Cys Gly
140 145 150
Val Ala Asn Ser Leu Ala Met Ser Pro Val Thr Leu Arg Leu Pro
155 160 165
Arg Cys Gly His His Glu Val Asp His Phe Leu Cys Glu Met Pro
170 175 180
Ala Leu Ile Arg Met Ala Cys Ile Ser Thr Val Ala Ile Asp Gly
185 190 195
Thr Val Phe Val Leu Ala Val Gly Val Val Leu Ser Pro Leu Val
200 205 210
Phe Ile Leu Leu Ser Tyr Ser Tyr Ile Val Arg Ala Val Leu Gln
215 220 225
Ile Arg Ser Ala Ser Gly Arg Gln Lys A1a Phe Gly Thr Cys Gly
230 235 240
Ser His Leu Thr Va1 Val Ser Leu Phe Tyr Gly Asn Ile Ile Tyr
245 250 255
Met Tyr Met Gln Pro Gly Ala Ser Ser Ser Gln Asp Gln Gly Lys
260 265 270
Phe Leu Thr Leu Phe Tyr Asn Ile Val Thr Pro Leu Leu Asn Pro
275 280 285
Leu Ile Tyr Thr Leu Arg Asn Arg Glu Val Lys Gly Ala Leu Gly
290 295 300
Arg Leu Leu Leu Gly Lys Arg Glu Leu Gly Lys Glu
305 310
<210> 20
<211> 1076
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474806CB1
<400> 20
caaagttgaa tgccgggttg gggcagaggc tgatgccgtg ctgaggtcat gatgttatgc 60
tgtccatttt gcttccttcc aggggaagca gaagcgggag ccgtcgtgga gctctgctcc 120
tggagggagc ctcccgggac atggagaagg tggacatgaa tacatcacag gaacaaggtc 180
tctgccagtt ctcagagaag tacaagcaag tctacctctc cctggcctac agtatcatct 240
ttatcctagg gctgccacta aatggcactg tcttgtggca ctcctggggc caaaccaagc 300
24/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
gctggagctg tgccaccacc tatctggtga acctgatggt ggccgacctg ctttatgtgc 360
tattgccctt cctcatcatc acctactcac tagatgacag gtggcccttc ggggagctgc 420
tctgcaagct ggtgcacttc ctgttctata tcaaccttta cggcagcatc ctgctgctga 480
cctgcatctc tgtgcaccag ttcctaggtg tgtggcaccc actgtgttcg ctgccctacc 540
ggacccgcag gcatgcctgg ctgggcacca gcaccacctg ggccctggtg gtcctccagc 600
tgctgcccac actggccttc tcccacacgg actacatcaa tggccagatg atctggtatg 660
acatgaccag ccaagagaat tttgatcggc tttttgccta cggcatagtt ctgacattgt 720
ctggctttct ttccccctcc ttggtcattt tggtgtgcta ttcactgatg gtcaggagcc 780
tgatcaagcc agaggagaac ctcatgagga caggcaacac agcccgagcc aggtccatcc 840
ggaccatcct actggtgtgt ggcctcttca ccctctgttt tgtgcccttc catatcactc 300
gCtCCttCta CCtCaCCatC tgCtttCtgC tttCtCagga ctgccagctc ttgatggcac 960
ccagtgtggc ctacaagata tggaggcctc tggtgagtgt gagcagctgc ctcaacccag 1020
tcctgtactt tctttcaagg ggggcaaaaa tagagtcagg ctcctccaga aactga 1076
<210> 21
<211> 1102
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474840CB1
<400> 21
ggaggctgag gcaggagaat ggcatgaccc caggaggcag agcttgcagt gagatgagat 60
catgccactg tgctccagcc tgggcaacag agcgagactc tgtctcaaaa aaaaaaaaaa 120
acaaaaaaaa aaaccttttt tcccaagcta ccattggact tttagccaac acctttttcc 180
ttttcttcaa catcttcata ttccttcagg atcagaaatc gaagccccat gacctcatca 240
gctgtaattc ggccttcatt catgtagtga tgttcctcac tgtggtggat gcttggcctc 300
cagatatgcc tgaatcactg cacttaggga atgagttcaa atttaagtcc ttgtcctaca 360
taaacagagt gaggatgggc ctatgtatct gtaacatctg tctcctgagt atacaccagg 420
ccaacaccat cagccccaac aacttctgtt tggcaaggct taaacagaaa ttcacaaata 480
acattatcat gtcatctttt ttttcttttt ttttttggtc catcaatttg tctttcagtt 540
ataacatagt attctttact gtggcttctt ctaatgtgac ccagaacagt ctacctaagg 600
gcagcaatac tgttcacttt ctccccatga agtccttcat gagaaaagta ttttttactc 660
tgacattatc cagggatgtc ttcattatag gaattacact gcattcaatt gcacacatgg 720
tgatccttgt gtccaggcat gagacgcaat ctcagcacct tcacagcatc agcatctctc 780
cacaagcctt cccagagaaa agggctgctc agaccatccc gctgttagtg agctactgtc 840
tggtcatgtg ctgggtggac ctcatcatct catcttcttc aaccctgctg tggacgtgta 900
acccagtctt cctgagtatg cagaaccttg tgggcgatgt ctatgccact gttgttctac 960
tggaacaaat cagctctgat aaaaatatag ttgacattct ccaaaatatg caaagtgcta 1020
taaagcttta acaagttggc gatggaaaac atttctaaaa aatagtcttc tcctatagtt 1080
caattgttca agtagccctg ga 1102
<210> 22
<211> 2529
<212> DNA '
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475092CB1
<400> 22
ggctccggtg tttcccgccg ttcatgcagc gaaaagagaa agcaaaatgc cctcaggagg 60
ctccagccgg ccgcgagccc tccacgcccg gcgggggcag cggaggcgga ggcgccgtcg 120
ctgcagcctc aggcgccgcg gtgccgggct ccgtgcagtt ggcgctgagc gtcctgcacg 180
ccctgctcta cgccgcgctg ttcgcctttg cctacctgca gctgtggcgg ctgctcctgt 240
accgcgagcg gcggctgagt taccagagcc tctgcctctt cctctgtctc ctgtgggcag 300
cgctcaggac caccctcttc tccgccgcct tctcgctcag cggctccctg cccttgctcc 360
ggccgcccgc tcacctgcac ttcttccccc actggctgct ctactgcttc ccctcctgtc 420
tccagttctc cacgctctgt ctcctcaacc tctacctggc ggaggttata tgtaaagtca 480
gatgtgccac tgaacttgac agacacaaaa ttctactgca tttgggcttt ataatggcaa 540
25/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
gcctgctctt tttagtggtg aacttgactt gcgcaatgct agttcatgga gatgtcccag 600
aaaatcagtt gaagtggact gtgtttgttc gagcattaat taatgatagc ctgtttattc 660
tttgtgccat ctctttagtg tgttacatat gcaaaattac aaaaatgtca tcagctaatg 720
tctacctcga atcaaagggt atgtctctgt gccagactgt cgtcgtgggc tctgtagtca 780
ttcttctgta ctcttccaga gcttgttata atttggtggt ggtcaccata tctcaggata 840
cattagaaag tccatttaat tatggctggg ataatctttc agataaggct catgtagaag 900
acataagtgg agaagagtat atagtatttg gaatggtcct ctttctgtgg gaacatgtgc 960
cagcatggtc ggtggtactg tttttccggg cacagagatt aaaccagaat ttggcacctg 1020
ctggcatgat aaatagtcac agttatagtt ccagagctta ctttttcgac aatccaagac 1080
gatatgatag tgatgatgac ctgccaagac tgggaagttc aagagaagga agtttaccaa 1140
attcgcaaag tttgggctgg tatggcacca tgactgggtg tggcagcagc agttacacag 1200
tcactcccca cctgaatgga cctatgacag atactgctcc tttgctcttt acttgtagta 1260
atttagattt gaacaatcat catagcttat atgtgacacc acaaaactga cagcatcacc 1320
aagtcatgat tcttgagttg tttttcataa atgtgtatat tcaatgtgtt taaattccat 1380
ctacataaac attccattat ctgttgcaac tgaaaacaaa atctggaagt gtggctgtgt 1440
ttggtaaata acacagctat tatttttgac ctcttcatag taaaatgaag taaaatggaa 1500
agtttggagt aggagaaaag agagattaga tcttaaggca cttgatggcc tccaaaaatc 1560
ctgactttgg aacatcaaat gcatatgtgc acttttatct ttgttctgag tcactgcagt 1620
ccccaaagtc atatgccaat gttcacactg aaatactgta ttgtacacca aactggaagg 1680
caattttcct atgaaaatca aagccggtat attcattggt atgctctata cagatatctt 1740
aataaaaatt ttatagtgtg aacagtgcac agagttaagg cataaaaatg tatcattctt 1800
tataaaaatc tactgaaaat gtgtaatcat tgaagacagt tcttttaagc atgattttaa 2860
aatagcaact gaaattcaat cattttaaac aaatgatggt agtaatccat tagttatggc 1920
cagcagtgtt ctttggagag ccacaataat ttcaagagga aaatatacca gtgaaaattg 1980
tgtggctatt ttgagtagaa ttggtcagtt gattattttg tgtaattgag atatatgtag 2040
tagtttaagc atgattcttg aagaaagcaa tagtgacttt tgcataggga gattttggta 2100
gaaacttctt gggactaaac aagtttagag atgcatttaa gaattattca caaaatgtgt 2160
aattctaaat taaaacataa atatattttc aaaagcattt gatttctctg aagcatgata 2220
tagctggtct tacctagtga atcaggattg tcctcaggta aatgaaatca tgatacatta 2280
ttgcagtgaa ctcaagtgca atactttgta agacatataa ttcctatgat tttcacattt 2340
ttatatctta tatatgggaa aagccaaatt aaattgaatt cagattaatt ccagcattag 2400
actaaatgag caaacttaag taaatgtaca aactaggtaa gtataaaacc acaggttaac 2460
aatattggag tacttttaga attacattaa aactgtctta aatgtcctat cccaaatcta 2520
aaaaaaaaa 2529
<210> 23
<211> 1847
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7341260CB1
<400> 23
gggggaggca ggcctcccag ttgctgcagt ttggaatatg tcaggtccca ccctcccaga 60
ggcggggcca gggctgagtc ctgccagcct catttctcta tccctctgag aacccagacg 120
ggcagagcct gggtaggaga gcctggcccc gctgtcccca ctgggtggag acaccatgca 180
cttggtccac ttgtgctctt cagccaggac accagacatg gtccaaaccg ctgcagggct 240
ggctgcagca actccctgac actcaggaag gcccaggctg ggcaggcaat acctgctccc 300
aacagccatg catgccggct gccgctccag gactcccctg tccccaggac caagatgacg 360
cccaacagca ctggcgaggt gcccagcccc attcccaagg gggctttggg gctctccctg 420
gccctggcaa gcctcatcat caccgcgaac ctgctcctag ccctgggcat cgcctgggac 480
cgccgcctgc gcagcccacc tgctggctgc ttcttcctga gcctactgct ggctgggctg 540
ctcacgggtc tggcattgcc cacattgcca gggctgtgga accagagtcg ccggggttac 600
tggtcctgcc tCCtCgtCta CttggCtCCC aaCttCtCCt tCCtCtCCCt gCttgCCaaC 660
ctcttgctgg tgcacgggga gcgctacatg gcagtcctga ggccactcca gccccctggg 720
agcattcggc tggccctgct cctcacctgg gctggtcccc tgCtCtttgC CagtCtgCCC 780
gctctggggt ggaaccactg gacccctggt gccaactgca gctcccaggc tatcttccca 840
gccccctacc tgtacctcga agtctatggg ctcctgctgc ccgccgtggg tgctgctgcc 900
ttcctctctg tccgcgtgct ggccactgcc caccgccagc tgcaggacat ctgccggctg 960
gagcgggcag tgtgccgcga tgagccctcc gccctggccc gggcccttac ctggaggcag 1020
gcaagggcac aggctggagc catgctgctc ttcgggctgt gctgggggcc ctacgtggcc 1080
26/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
acactgctcc tctcagtcct ggcctatgag cagcgcccgc cactggggcc tgggacactg 1140
ttgtccctcc tctccctagg aagtgccagt gcagcggcag tgcccgtagc catggggctg 1200
ggcgatcagc gctacacagc cccctggagg gcagccgccc aaaggtgcct gcaggggctg 1260
tggggaagag cctcccggga cagtcccggc cccagcattg cctaccaccc aagcagccaa 1320
agcagtgtcg acctggactt gaactaaagg aagggcctct gctgactcct accagagcat 1380
ccgtccagct cagccatcca gcctgtctct actgggcccc acttctctgg atcagagacc 1440
ctgcctctgt ttgaccccgc actgactgaa taaagctcct ctggccgtta aaaaaaaaaa 1500
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaac aaacaaacag 1560
aaaaaaaaaa aaaagaacac acaaagaaca cagaacaaac caagcagcac accacacaca 1620
aaaacaatgc acacacagaa caagacacaa tcagagacag agagcacaca gcacggaccc 1680
cagccacgcc cccagcactg accaccacga cccgacacag aaacgaacac tgaagactca 1740
acgcacaaaa cgacaaccag accacaagcc aaccgcctca cgccccagca acgaacacac 1800
atacaaaacc aaaccgagac aacccacata cagccaaaca aaccaca 1847
<210> 24
<211> 2031
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473911CB1
<400> 24
atgaataaaa acaacaaacc ttccagtttc atagccataa gaaatgctgc tttctctgaa 60
gtcggcattg ggatctctgc caatgccatg ctccttctct tccacatcct cacgtgcctt 120
ctcaagcaca ggaccaagcc cgctgacctg atcgtttgtc atgtggctct aatccatatc 180
atattgctgc tacccacaga gttcatagct acagatattt ttgggtctca ggattcagag 240
gatgacatca aacataagtc agttatctac aggcgtaaca ggcagtccca gcattttcac 300
agcaccaacc tttctccaaa agcaccccca gaaaaaatgg ccacgcagac cattcttctg 360
ctcgtgagtt gctttgtgat tgtgtatgtt ttggactgtg ttgtcgcctc ctgctcagga 420
ctggtgtgga acagtgatcc agtccgtcat cgagtccaga tgctggtgga caatggctat 480
gccaccatca gtccttcagt gctacccagg ctgactgccc caaacgagtg gagagccagt 540
gtgtacctga atgacagctt gaacaaatgc agcaacggac ggctgctctg tgtagacagg 600
gggcttgatg aggggccccg gtccgtccca aagtgctctg agtcagagac cgacgaggat 660
tacatcgtcc tcagggctcc gctgagggag gacgaaccca aggacggggg cagtgtgggg 720
aatgcagccc tggtgtctcc cgaggcctct gcagaagagg aagaggagcg tgaggaggga 780
ggcgaggcat gtggcctgga gaggacagga gctggtgggg agcaggttga ccttggtgaa 840
ctacctgacc atgaggagaa aagcaaccag aaagtggcag ctgccaccct ggaggaccgc 900
acacaggatg agcctgctga ggagagctgc cagatcgtcc ttttccagaa caactgcatg 960
gacaactttg tgacttccct cacaggaagc ccctacgagt tcttcccaac caagagcacc 1020
tctttttgca gggagagctg ttctcctttt tctgagtcag tgaaaagctt agaatcagag 1080
caggcaccaa agttggggct gtgtgcggag gaggaccccg tggttggggc tttgtgtggc 1140
cagcatggac ccttgcaaga tggagtggcg gagggtccca cagcccctga tgtggtggtc 1200
ctgccgaagg aggaggagaa ggaggaggtc attgtggatg acatgctggc caacccctat 1260
gtgatgggag atgaggggga ggaggaggag gaggagttcg tggatgacac actggccaac 1320
ccctatgtga tgggagtggg cctgccagga agaggagggg aggaggagga ggaggaggag 1380
gtcgtggatg acacgctggc cagcctctat aagatgggag aagaacatcg acacaagggc 1440
ctggccccac tctgggaagg tggccagaaa ccgtcccaga aactgccccc aaagaaacca 1500
gatctgaggc aggttcctca gcccctggca tcggaggtgc cgcagaggag gcaggaaaga 1560
gctgttgtca ctgaagggag gcccctggaa gccagcaggg ccttgccagc aaagcccagg 1620
gccttcactt tataccctcg gtcgttctcc gtggaaggcc aagagattcc tgtttccatc 1680
tctgtgtact gggagccaga agggtcgggg ttagatgacc acaggataaa gaggaaagag 1740
gaacatctct ctgtcgtgtc tgggagtttc tcccagagaa accaccttcc atccagcggc 1800
acctccacgc cttcttccat ggtcgacatc ccacctcctt tcgacctggc ctgcatcacc 1860
aagaagccca tcacaaagag ctctccctct ctcctgatcg acagcgactc cccggacaag 1920
tacaagaaga agaagtcatc ctttaagcgg ttcctggcgc tgatgtttaa caagatggag 1980
aggccaggca cgatggctca tgcctgtcat cccagcactt tgggaagctg a 2031
<210> 25
<211> 1130
<212> DNA
<213> Homo Sapiens
27/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
<220>
<221> misc_feature
<223> Incyte ID No: 7474767CB1
<400> 25
ggggcgcgct catggagcac acgcacgccc acctcgcagc caacagctcg ctgtcttggt 60
ggtcccccgg ctcggcctgc ggcttgggtt tcgtgcccgt ggtctactac agcctcttgc 120
tgtgcctcgg tttaccagca aatatcttga cagtgatcat cctctcccag ctggtggcaa 180
gaagacagaa gtcctcctac aactatctct tggcactcgc tgctgccgac atcttggtcc 240
tctttttcat agtgtttgtg gacttcctgt tggaagattt catcttgaac atgcagatgc 300
ctcaggtccc cgacaagatc atagaagtgc tggaattctc atccatccac acctccatat 360
ggattactgt accgttaacc attgacaggt atatcgctgt ctgccacccg ctcaagtacc 420
acacggtctc atacccagcc cgcacccgga aagtcattgt aagtgtttac atcacctgct 480
tcctgaccag catcccctat tactggtggc ccaacatctg gactgaagac tacatcagca 540
cctctgtgca tcacgtcctc atctggatcc actgcttcac cgtctacctg gtgccctgct 600
ccatcttctt catcttgaac tcaatcattg tgtacaagct caggaggaag agcaattttc 660
gtctccgtgg ctactccacg gggaagacca ccgccatctt gttcaccatt acctccatct 720
ttgccacact ttgggccccc cgcatcatca tgattcttta ccacctctat ggggcgccca 780
tccagaaccg ctggctggta cacatcatgt ccgacattgc caacatgcta gcccttctga 840
acacagccat caacttcttc ctctactgct tcatcagcaa gcggttccgc accatggcag 900
ccgccacgct caaggctttc ttcaagtgcc agaagcaacc tgtacagttc tacaccaatc 960
ataacttttc cataacaagt agcccctgga tctcgccggc aaactcacac tgcatcaaga 1020
tgctggtgta ccagtatgac aaaaatggaa aacctataaa aagtcgtaat gacagcaaaa 1080
gctcctacca gtttgaagat gccattggag cttgtgtcat catcctgtga 1130
<210> 26
<211> 1202
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475815CB1
<400> 26
caggttctgc aatacaattg gaaacaactc attgctcctg cctatgaaga agtagaacta 60
tggtaaaaat aagaaaatgc cttcccaata atagggagtt gtactatgga agtaatatgg 120
aggcccagca atgggaatca tagtccagcc atgatggcat agtcatggga gaagagagag 180
ggtggggatg gcttccagga gggtgtgaag agggggtcca gtactgaagg gggaaaagat 240
gcatcagaat taattaatgt attttgatga tggcaatagt gttggttgag attggtgaag 300
gtagtaatat ttgtgatatt tttgttgctt ttctccctag acattaacta tgtgcttatt 360
ttccccataa gatgaataaa aacaacaaac cttccagttt catagccata agaaatgctg 420
ctttctctga agtcggcatt gggatctctg ccaatgccat gctccttctc ttccacatcc 480
tcacgtgcct tctcaagcac aggaccaagc ccgctgacct gatcgtttgt catgtggctc 540
taatccatat catattgctg ctacccacag agttcatagc tacagatatt tttgggtctc 600
aggattcaga ggatgacatc aaacataagt cagttatcta caggtacagg ttgatgagag 660
gCCtCtCCat ttCCaCCa.CC tgCCtgCtga gtatcctccc ggccatcacc tgcagcccca 720
gaagctcctg tttggcagtg ttcaaagatt ctcacatcac caaccacgtt gctttctctt 780
ccgtcttcca catatccatt agtgacagct tcttagtctc cactcttccc atcaaaaatc 840
tggcctcaaa tagccttaca tttgtcactc aatcctgctc tgctgggatc ggctcacggc 900
ccccctccag tggatacatg gtgattctct tgtccaggcg taacaggcag tcccagcatt 960
ttcacagcac caacctttct ccaaaagcac ccccagaaaa aatggccacg cagaccattc 1020
ttctgctcgt gagttgcttt gtgattgtgt atgttttgga ctgtgttgtc gcctcctgct 1080
caggactggt gtggaacagt gatccagtcc gtcatcgagt ccagatgctg gtggacaatg 1140
gctatgccac catcagtcct tcagtgctag tcagtactga aaaatgaatg atcaaagtct 1200
ga 1202
<210> 27
<211> 2079
<212> DNA
<213> Homo Sapiens
<220>
28/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
<221> misc_feature
<223> Incyte ID No: 60263275CB1
<400> 27
tacggcacag tagagagctt ccagggctgg ctggcgtggg atacccgtac cacagaaatg 60
cagggaccat tgcttcttcc aggcctctgc tttctgctga gcctctttgg agctgtgact 120
cagaaaacca aaaacattaa tgaatgtaca ccaccctata gtgtatattg tggatttaac 180
gctgtgtgtt acaatgtcga aggaagtttc tactgtcaat gtgtcccagg atatagactg 240
cattctggga atgaacaatt cagtaattcc aatgagaaca cctgtcagga caccacctcc 300
tcaaagacaa cccagggcag gaaagagctg caaaagattg tggacaaatt tgagtcactt 360
ctcaccaatc agactttatg gagaacagaa gggagacaag aaatctcatc cacagctacc 420
actattctcc gggatgtgga atcgaaagtt ctagaaactg ccttgaaaga tccagaacaa 480
aaagtcctga aaatccaaaa cgatagtgta gctattgaaa ctcaagcgat tacagacaat 540
tgctctgaag aaagaaagac attcaacttg aacgtccaaa tgaactcaat ggacatccgt 600
tgcagtgaca tcatccaggg agacacacaa ggtcccagtg ccattgcctt tatctcatat 660
tcttctcttg gaaacatcat aaatgcaact ttttttgaag agatggataa gaaagatcaa 720
gtgtatctga actctcaggt tgtgagtgct gctattggac ccaaaaggaa cgtgtctctc 780
tccaagtctg tgacgctgac tttccagcac gtgaagatga cccccagtac caaaaaggtc 840
ttctgtgtct actggaagag cacagggcag ggcagccagt ggtccaggga tggctgcttc 900
ctgatacacg tgaacaagag tcacaccatg tgtaattgca gtcacctgtc cagcttcgct 960
gtcctgatgg ccctgaccag ccaggaggag gatcccgtgc tgactgtcat cacctacgtg 1020
gggctgagcg tctctctgct gtgcctcctc ctggcggccc tcacttttct cctgtgtaaa 1080
gccatccaga acaccagcac ctcactgcat ctgcagctct cgctctgcct cttcctggcc 1140
cacctcctct tcctcgtggg gattgatcga actgaaccca aggtgctgtg ctccatcatc 1200
gccggtgctt tgcactatct ctacctggcc gccttcacct ggatgctgct ggagggtgtg 1260
cacctcttcc tcactgcacg gaacctgaca gtggtcaact actcaagcat caatagactc 1320
atgaagtgga tcatgttccc agtcggctat ggcgttcccg ctgtgactgt ggccatttct 1380
gcagcctcct ggcctcacct ttatggaact gctgatcgat gctggctcca cctggaccag 1440
ggattcatgt ggagtttcct tggcccagtc tgtgccattt tctctgcgaa tttagtattg 1500
tttatcttgg tcttttggat tttgaaaaga aaactttcct ccctcaatag tgaagtgtca 1560
accatccaga acacaaggat gctggctttc aaagcaacag ctcagctctt catcctgggc 1620
tgcacatggt gtctgggctt gctacaggtg ggtccagctg cccaggtcat ggcctacctc 1680
ttcaccatca tcaacagcct ccaaggcttc ttcatcttct tggtctactg cctcctcagc 1740
cagcaggtcc agaaacaata tcaaaagtgg tttagagaga tcgtaaaatc aaaatctgag 1800
tctgagacat acacactttc cagcaagatg ggtcctgact caaaacccag tgagggggat 1860
gtttttccag gacaagtgaa gagaaaatat taaaactaga atattcaact ccatatggaa 1920
aatcatatcc atggatctct ttggcattat gaagaatgaa gctaaggaaa agggaattca 1980
ttaaacatat catccttgga gaggaagtaa tcaaccttta cttcccaaac tgtttgttct 2040
ccacaatagg tctcaacaaa tgtgtggtaa attgcatta 2079
<210> 28
<211> 5324
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 60203310CB1
<400> 28
ggcggcagca gcagcagcaa gaggaggaga agcagcccca gctatgactg ccgcatgtta 60
atagctgccg ctgggtccct cggctgctgc tggagacaga gcctgactcc gaagttgtgc 120
aactgtggac tgggagagac atttgaaccc tcttttcttt tcgctcccct tttgccccct 180
tggggtgtgt gaagcgagga acgtaaagga aggcgaacat ttggctctct ttttccttcc 240
cctttctccg tggctgtgta gcggaagaaa gggaagagag actttttgtt gttgtttcct 300
tgactggggt ctccaccctc ctgctgcttt ctctgcgctt cgattctcgt tatttgccgc 360
gtgtJggttgg gggtgtctgc acaggggccg gccggtcttt tgccccgggc tcaatggctg 420
gattgtggaa actgcacccg ccttcaggtt gttgagcaac tgatgggacg atctcaggga 480
ccggcgttta cgaaaggttt cagatttggg atattggtgt ttctgttttg gagaaattat 540
tctttttctt tttaatttga agaaaaatca tcagtcttgg aatacagaag agaaactaga 600
aatatacgta ttttgtttca catttgaaca gtcattcttg aggaatactc catacctgag 660
tagacagcca tgtggccatc gcagctacta attttcatga tgctcttagc tccaataatt 720
catgetttca gccgtgcccc aattccaatg gctgtggtcc gcagagagct atcctgtgag 780
29/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
agctatccta tagagcttcg ctgtccagga acagacgtca tcatgataga aagtgccaac 840
tatggcagga ctgatgacaa aatttgtgac tctgaccctg ctcagatgga gaatatccga 900
tgttatctgc cagatgccta taagattatg tctcaaagat gcaataacag aacccagtgt 960
gcagtggtgg caggtcctga tgtttttcca gacccgtgtc caggaaccta taaatacctt 1020
gaagtgcagt atgaatgtgt cccttacaaa gtggaacaaa aagtttttct ttgtcctgga 1080
ctactaaaag gagtatacca gagtgaacat ttgtttgagt ccgaccacca atctggggcg 1140
tggtgcaaag accctctgca ggcatctgac aagatttatt atatgccctg gactccctac 1200
agaactgata ccctgactga gtattcatcc aaggatgact tcattgctgg aagaccaact 1260
acaacctaca agctccctca tagggtggat ggcacaggat ttgtagtgta tgatggagct 1320
ttgttcttca acaaagagcg caccaggaac atagtaaagt ttgatttgcg gactaggata 1380
aagagtggag aggctatcat agcaaatgcc aattaccatg atacctcccc ttaccgatgg 1440
ggaggcaaat ctgacataga cctggcagta gatgagaatg ggctatgggt aatctatgca 150'0
acagaacaaa acaatggtaa aattgtcatt agtcaattga acccttacac cctacggatc 1560
gaaggaacat gggatactgc atatgataaa aggtcagctt ccaatgcctt tatgatttgt 1620
ggaattctgt atgtggtcaa atctgtatat gaggatgatg acaatgaggc tactggaaat 1680
aagattgact acatttacaa cactgaccaa agcaaggata gtttggtgga tgtacccttt 1740
cctaattcat accagtacat tgcagctgtg gattacaacc ccagggacaa cctactttat 1800
gtatggaata actatcacgt cgtgaaatat tctttggatt ttggacctct ggatagtaga 1860
tcagggcagg cacatcatgg acaagtttca tacatttctc cgccaattca ccttgactct 1920
gagctagaaa gaccctctgt taaagatatc tctaccacag gacctcttgg catgggaagc 1980
actaccacca gtaccaccct tcggaccaca actttgagcc caggaaggag taccaccccg 2040
tcagtgtcag gaagaagaaa ccggagtact agtaccccat ctccagctgt cgaggtactt 2100
gatgacatga ccacacacct tccatcagca tcgtcccaaa tcccagctct cgaagagagc 2160
tgtgaggctg tggaagcccg agaaatcatg tggtttaaga ctcgtcaagg acagatagca 2220
aagcagccat gccctgcagg aactataggt gtatcaactt atctatgcct tgctcctgat 2280
ggaatttggg atccccaagg tccagatctc agcaactgtt cttctccttg ggtcaatcat 2340
ataacacaga agttgaaatc tggtgaaaca gctgccaaca ttgctagaga gctggctgaa 2400
cagacaagaa atcacttgaa tgctggggac atcacctact ctgtccgggc catggaccag 2460
ctggtaggcc tcctagatgt acagcttcgg aacttgaccc caggtggaaa agatagtgct 2520
gcccggagtt tgaacaagct tcagaaaaga gagcgctctt gcagagccta tgtccaggca 2580.
atggtcgaga cagttaacaa cctccttcag ccacaagctt tgaatgcatg gagagacctg 2640
actacgagtg atcagctgcg tgcggccacc atgttgcttc atactgtgga ggaaagtgct 2700
tttgtgctgg ctgataacct tttgaagact gacattgtca gggagaacac agacaatatt 2760
aaattggaag ttgcaagact gagcacagaa ggaaacttag aagacctaaa atttccagaa 2820
aacatgggcc atggaagcac tatccagctg tctgcaaata ccttaaagca aaatggccga 2880
aatggagaga tcagagtggc ctttgtcctg tataacaact tgggtcctta tttatccacg 2940
gagaatgcca gtatgaagtt gggaacggaa gctttgtcca caaatcattc tgttattgtc 3000
aattcccctg ttattacggc agcaataaac aaagagttca gtaacaaggt ttatttggct 3060
gatcctgtgg tatttactgt taaacatatc aagcagtcag aggaaaattt caaccctaac 3120
tgttcatttt ggagctactc caagcgtaca atgacaggtt attggtcaac acaaggctgt 3180
cggctcctga caacaaataa gacacatact acatgctctt gtaaccacct aacaaatttt 3240
gcagtactga tggcacatgt ggaagttaag cacagtgatg cggtccatga cctccttctg 3300
gatgtgatca cgtgggttgg aattttgctg tcccttgttt gtctcctgat ttgcatcttc 3360
acattttgct ttttccgcgg gctccagagt gaccgtaaca ccatccacaa gaacctctgc 3420
atcagtctct ttgtagcaga gctgctcttc ctgattggga tcaaccgaac tgaccaaccg 3480
attgcctgtg ctgttttcgc tgccctgtta catttcttct tcttggctgc cttcacctgg 3540
atgttccttg agggggtgca gctttatatc atgctggtgg aggtttttga gagtgaacat 3600
tcacgtagga aatactttta tctggtcggc tatgggatgc ctgcactcat tgtggctgtg 3660
tcagctgcag tagactacag gagttatgga acagataaag tatgttggct ccgacttgac 3720
acctacttca tttggagttt tataggacca gcaactttga taattatgct taatgtaatc 3780
ttccttggga ttgctttata taaaatggtt catcatactg ctatactgaa acctgaatca 3840
ggctgtcttg ataacatcaa ctatgaggat aacagaccct tcatcaagtc atgggttata 3900
ggtgcaatag ctcttctctg cctattagga ttgacctggg cctttggact catgtatatt 3960
aatgaaagca cagtcatcat ggcctatctc ttcaccattt tcaattctct acagggaatg 4020
tttatattta ttttccattg tgtcctacag aagaaggtac gaaaagagta tgggaaatgc 4080
ctgcgaacac attgctgtag tggcaaaagt acagagagtt ccattggttc agggaaaaca 4140
tctggttctc gaactcctgg acgctactcc acaggctcac agagccgaat ccgtagaatg 4200
tggaatgaca cggttcgaaa gcagtcagag tcttccttta ttactggaga cataaacagt 4260
tcagcgtcac tcaacagaga ggggcttctg aacaatgcca gggatacaag tgtcatggat 4320
actctaccac tgaatggtaa ccatggcaat agttacagca ttgccagcgg cgaatacctg 4380
agcaactgtg tgcaaatcat agaccgtggc tataaccata acgagaccgc cctagagaaa 4440
aagattctga aggaactcac ttccaactat atcccttctt acctgaacaa ccatgagcgc 4500
tccagtgaac agaacaggaa tctgatgaac aagctggtga ataaccttgg cagtggaagg 4560
30/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
gaagatgatg ccattgtcct ggatgatgcc acctcgttta accacgagga gagtttgggc 4620
ctggaactca ttcatgagga atctgatgct cctttgctgc ccccaagagt atactccacc 4680
gagaaccacc agccacacca ttataccaga aggcggatcc cccaagacca cagtgagagc,4740
tttttccctt tgctaaccaa cgagcacaca gaagatctcc agtcacccca tagagactct 4800
ctctatacca gcatgccgac actggctggt gtggccgcca cagagagtgt taccaccagc 4860
acccagaccg aacccccacc ggccaaatgt ggtgatgccg aagatgttta ctacaaaagc 4920
atgccaaacc taggctccag aaaccacgtc catcagctgc atacttacta ccagctaggt 4980
cgcggcagca gtgatggatt tatagttcct ccaaacaaag atgggacccc tcccgaggga 5040
agttcaaaag gaccggctca tttggtcact agtctataga agatgacaca gaaattggaa 5100
ccaacaaaac tgctaacacc ttgttgactg ttctgagttg atataagcag tggtaataat 5160
gtgtgtactc ctaaatcttt atgctgtcct ctaaagacaa acacaaactc tcagactttt 5220
ttttttcaac tgggatttaa ggtcagccca ggggagaaag ataactgcta aaattcccct 5280
gtaccccatc ctttcttgtc ctttccccct tcagatggag actt 5324
<210> 29
<211> 1962
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477349CB1
<400> 29
atggatccca gcgttgttag caatgagtat tatgatgttg cccatggagc aaaagatcca 60
gtggtcccca cttccctgca ggacatcact gctgtcctgg gtacagaagc atatactgag 120
gaagacaaat caatggtgtc ccatgcacag aaaagccagc attcttgtct cagccattcc 180
aggtggctga ggtctccaca ggtcacaggg ggaagctggg acctccgaat aaggccatcc 240
aaggactcca gCagtttccg~ccaggctcag tgtctgcgta aggatcctgg ggcaaacaac 300
cacttggaga gccaaggggt gagaggtaca gctggcgatg ctgacaggga gctgcgggga 360
ccctcagaaa aagccacagc tggccagcca cgagtgaccc tgctgcccac gcccaacgtc 420
agcgggctga gccaggagtt tgaaagccac tggccagaga tcgcagagag gtccccgtgt 480
gtggctggcg tcatccctgt catctactac agtgtcctgc tgggcttggg gctgcctgtc 540
agcctcctga ccgcagtggc cctggcgcgc cttgccacca ggaccaggag gccctcctac 600
tactaccttc tggcgctcac agcctcggat atcatcatcc aggtggtcat cgtgttcgcg 660
ggcttcctcc tgcagggagc agtgctggcc cgccaggtgc cccaggctgt ggtgcgcacg 720
gccaacatcc tggagtttgc tgccaaccac gcctcagtct ggatcgccat cctgctcacg 78O
gttgaccgct acactgccct gtgccacccc ctgcaccatc gggccgcctc gtccccaggc 840
cggacccgcc gggccattgc tgctgtcctg agtgctgccc tgttgaccgg catccccttc 900
tactggtggc tggacatgtg gagagacacc gactcaccca gaacactgga cgaggtcctc 960
aagtgggctc actgtctcac tgtctatttc atcccttgtg gcgtgttcct ggtcaccaac 1020
tcggccatca tccaccggct acggaggagg ggccggagtg ggctgcagcc ccgggtgggc 1080
aagagcacag ccatcctcct gggcatcacc acactgttca ccctcctgtg ggcgccccgg 1140
gtcttcgtca tgctctacca catgtacgtg gcccctgtcc accgggactg gagggtccac 1200
ctggccttgg atgtggccaa catggtggcc atgctccaca cggcagccaa cttcggcctc 1260
tactgctttg tcagcaagac tttccgggcc actgtccgac aggtcatcca cgatgcctac 1320
ctgccctgca ctttggcatc acagccagag ggcatggcgg cgaagcctgt gatggagcct 1380
ccgggactcc ccacaggggc agaagtgtag aggagggggc ccagctaggg agctcagggt 1440
ggctcatggc cacatgtact ggggcctttg aggttgtacc caaaacacgt ttatcaacag 1500
cttgctttcc ttgggtgggg gtggaggctc ctcctttggg tgtggctccc aggtagagag 1560
gaggacaact tagccagctc ttatgtttgc ttcaccagca atccctattt cctgggaaga 1620
tgaaagggca ctgccaggca caggctaata gcatcagtgc tgtgggcatt cctttgcggg 180
gggcattttg cctggctcat cgtgaatgcc agattaatgt tggttgaatg gatagaaaaa 1740
cggcctctca ttttcgtaac tgaggcagga gaatcgcttg aacccaggag acggaggttg 1800
cagcgagctg agatcgcgcc atagaaacac catggaactc caacctgggc aacaagagtg 1860
aaacttcgac tcaaaaaaaa aagagaaaaa acacattagg taacagtttc tttttagcat 1920
ttgtgtaacc tttaataaaa taaagtgata atcaaaaaaa as 1962
<210> 30
<211> 1558
<212> DNA
<213> Homo sapiens
31/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
<220>
<221> misc_feature
<223> Incyte ID No: 55002225CB1
<400> 30
attttattcg cgaaggcacc ccacgctcct agaaaagagc acgacgcacc cgatgctcgg 60
attggatgaa gtggcaaagc tttaatccct ggaaagtcca cgaacaatga atccatttca 120
tgcatcttgt tggaacacct ctgccgaact tttaaacaaa tcctggaata aagagtttgc 180
ttatcaaact gccagtgtgg tagatacagt catcctccct tccatgattg ggattatctg 240
ttcaacaggg ctggttggca acatcctcat tgtattcact ataataagat ccaggaaaaa 300
aacagtccct gacatctata tctgcaacct ggctgtggct gatttggtcc acatagttgg 360
aatgcctttt cttattcacc aatgggcccg agggggagag tgggtgtttg gggggcctct 420
ctgcaccatc atcacatccc tggatacttg taaccaattt gcctgtagtg ccatcatgac 480
tgtaatgagt gtggacaggt actttgccct cgtccaacca tttcgactga cacgttggag 540
aacaaggtac aagaccatcc ggatcaattt gggcctttgg gcagcttcct ttatcctggc 600
attgcctgtc tgggtctact cgaaggtcat caaatttaaa gacggtgttg agagttgtgc 660
ttttgatttg acatcccctg acgatgtact ctggtataca ctttatttga cgataacaac 720
tttttttttc cctctaccct tgattttggt gtgctatatt ttaattttat gctatacttg 780
ggagatgtat caacagaata aggatgccag atgctgcaat cccagtgtac caaaacagag 840
agtgatgaag ttgacaaaga tggtgctggt gctggtggta gtctttatcc tgagtgctgc 900
cccttatcat gtgatacaac tggtgaactt acagatggaa cagcccacac tggccttcta 960
tgtgggttat tacctctcca tctgtctcag ctatgccagc agcagcatta acccttttct 1020
ctacatcctg ctgagtggaa cgcctcaaat ccaaagaaga gcgactgaga aggaaatcaa 1080
caatatggga aacactctga aatcacactt ttaggaaagt acatggatca ccatgagtct 1140
agacatgatt gtctatctta ctggtattat tagaaagggc aggtgtaccg atatgtttat 1200
gcccattctt cttgtgtact tgtgactctt agcagcatgg aagagaagtg taaccatgca 1260
aatacaatga gcttaatatg ctaactttag caagatgtaa aatgttgatc tatattgtgg 1320
gtagggaatg ggatagtctg agatacccag gcttcatgat ggtgtatatt atttcagcat 1380
attataaact agtcactaat gaaaatggcc atccatgacc attgactcaa aactcaccaa 1440
ggaacctgac cttgccctcc acactgcggc ctcactgtaa cagtttcctc aaggttccta 1500
ggagggtatc accttagagt gaagtctaaa atttggctat tttttatcta ttaaaaat 1558
<210> 31
<211> 2304
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475686CB1
<400> 31
atgcggctgg gacctgtccc agcccgggcg cgcgccctct tgtcttgggt tagggggctg 60
gaaagccgag gaggggagtg gaccaaatgc attgttcagc tgggtcatct ccttgctacc 120
cagcatcccg cggcgcccac atgtggagtc gtttccagcg ccctggtcat gcactcaaca 180
gatgtctgtc tagcccccac tatgcaccag gcactggact gggcagcagg aatttggttt 240
acaggaagat taggactcag agagcataaa tcactggccc agggtgactc agtctgtcca 300
tgtgaaagtg aacttggtga tttccaagtc tatggcttgg tcagtacaga aggagtggtg 360
tcctgctttg gagagaagac cccgcagcat cctggccctc ctgcttcatt gtccctggcc 420
aacaggtgcc acaacgttgt gacagctgta ggagcctggc cagctcatgg gagcatcctt 480
ggaaatgttc cagaagcccc tgtgggagct gatgtgttgg gggctggagg atgtgactgg 540
gcagacaaag aggccctggc ccctgggcaa agggcaaagg tgcacattct tcttgagagt 600
tctggacagt ctgatccatc ctatgctgtc cttcctgaca gctgggcagc cacggagggt 660
ttcccaactt acagatctca ggtctcctct ccccgcatcc cgggtagttc catctggtta 720
ggcagtgggt ctggttggcc tatacttggg gaactcaggg aatgtgacca gatgttctcc 780
tgcatgttgc ccactggttg tgcctccttc caggatccag gacgttatgg tgattatgac 840
ctccctatgg atgaggatga ggacatgacc aagacccgga ccttcttcgc agccaagatc 900
gtcattggca ttgcactggc aggcatcatg ctggtctgcg gcatcggtaa ctttgtcttt 960
atcgctgccc tcacccgcta taagaagttg cgcaacctca ccaatctgct cattgccaac 1020
ctggccatct ccgacttcct ggtggccatc atctgctgcc ccttcgagat ggactactac 1080
gtggtacggc agctctcctg ggagcatggc cacgtgctct gtgcctccgt caactacctg 1140
cgcaccgtct ccctctacgt ctccaccaat gccttgctgg ccattgccat tgacaggtat 1200
ctcgccatcg ttcacccctt gaaaccacgg atgaattatc aaacggcctc cttcctgatc 1260
32/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
gccttggtct ggatggtgtc cattctcatt gccatcccat cggcttactt tgcaacagaa 1320
acggtcctct ttattgtcaa gagccaggag aagatcttct gtggccagat ctggcctgtg 1380
gatcagcagc tctactacaa gtcctacttc ctcttcatct ttggtgtcga gttcgtgggc 1440
cctgtggtca ccatgaccct gtgctatgcc aggatctccc gggagctctg gttcaaggca 1500
gtccctgggt tccagacgga gcagattcgc aagcggctgc gctgccgcag gaagacggtc 1560
ctggtgctca tgtgcattct cacggcctat gtgctgtgct gggcaccctt,ctacggtttc 1620
accatcgttc gtgacttctt ccccactgtg ttcgtgaagg aaaagcacta cctcactgcc 1680
ttctacgtgg tcgagtgcat cgccatgagc aacagcatga tcaacaccgt gtgcttcgtg 1740
acggtcaaga acaacaccat gaagtacttc aagaagatga tgctgctgca ctggcgtccc 1800
tcccagcggg ggagcaagtc cagtgctgac cttgacctca gaaccaacgg ggtgcccacc 1860
acagaagagg tggactgtat caggctgaag tgacccactg gtgtcacaca attgaaaacc 1920
ccagtccagt actcagagca tcacccacca tcaaccaagt tcataggctg catgggaaat 1980
gacatctgtg ttcatgcctc ccccgtgccc tcaagaagcc gaatgctgca aagtcgtaac 2040
atacaatgag actagacatg aaccaaatca gctgacattt actgatatcc gctcgacacc 2100
tactgtgtcc acaatcccaa caaggagatt agacacaagg agcagcaact gacatggact 2160
gaacatgtac tgtgtgcaag ccaaaccaat gagattaaca gggacagcag gagctgaatt 2220
atcttactat gtatcaaacc tgttgttcac aaattaaact acagtccaac ttgggtcaca 2280
tcgttttatt tcccattcat tttt 2304
<210> 32
<211> 2322
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature '
<223> Incyte ID No: 7482007CB1
<400> 32
cccatttcaa aaatggagaa gacagatcac tgccactgac caggaccgtg ggaggtgcca 60
cgtgatggtg aggcatcatg ctagggagct gagctctgac cttcctgctg ggtgattctc 120
cacctctggg ctgctagatc tacttcctgg atgccgtgaa gatcctcatg tatgaaaatg 180
aagtcccagg caaccatgat ttgctgctta gtgttctttc tgtccacaga atgttcccac 240
tatagatcca agattcacct aaaagctgga gataaacttc aaagccctga agggaaaccc 300
aagactggaa ggatccaaga gaaatgcgaa ggaccttgta tttcttcttc caactgcagc 360
cagccctgtg ctaaggactt tcatggagaa ataggattta catgtaatca aaaaaagtgg 420
caaaaatcag ctgaaacatg tacaagcctt tctgtggaaa aactctttaa ggactcaact 480
ggtgcatctc gcctttctgt agcagcacca tctatacctc tgcatattct agactttcga 540
gctccagaga ccattgagag tgtagctcaa ggaatccgta agaactgccc ctttgattat 600
gcctgcatca ctgacatggt gaaatcatca gaaacaacat ctggaaatat tgcatttata 660
gtggagttat taaaaaatat ttctacagac ttgtctgata atgttactcg agagaaaatg 720
aagagctata gtgaagtggc Caaccacatc ctcgacacag cagccatttc aaactgggct 780
ttcattccca acaaaaatgc cagctcggat ttgttgcagt cagtgaattt gtttgccaga 840
caactccaca tccacaataa ttctgagaac attgtgaatg aactcttcat tcagacaaaa 900
gggtttcaca tcaaccataa tacctcagag aaaagcctca atttctccat gagcatgaac 960
aataccacag aagatatctt aggaatggta cagattccca ggcaagagct aaggaagctg 1020
tggccaaatg catcccaagc cattagcata gctttcccaa ccttgggggc tatcctgaga 1080
gaagcccact tgcaaaatgt gagtcttccc agacaggtaa atggtctggt gctatcagtg 1140
gttttaccag aaaggttgca agaaatcata ctcaccttcg aaaagatcaa taaaacccgc 1200
aatgccagag cccagtgtgt tggctggcac tccaagaaaa ggagatggga tgagaaagcg 1260
tgccaaatga tgttggatat caggaacgaa gtgaaatgcc gctgtaacta caccagtgtg 1320
gtgatgtctt tttccattct catgtcctcc aaatcgatga ccgacaaagt tctggactac 1380
atcacctgca ttgggctcag cgtctcaatc ctaagcttgg ttctttgcct gatcattgaa 1440
gccacagtgt ggtcccgggt ggttgtgacg gagatatcat acatgcgtca cgtgtgcatc 1500
gtgaatatag cagtgtccct tctgactgcc aatgtgtggt ttatcatagg ctctcacttt 1560
aacattaagg cccaggacta caacatgtgt gttgcagtga catttttcag ccactttttc 1620
tacctctctc tgtttttctg gattctcttc aaagcattgc tcatcattta tggaatattg 1680
gtcattttcc gtaggatgat gaagtcccga atgatggtca ttggctttgc cattggctat 1740
gggtgcccat tgatcattgc tgtcactaca gttgctatca cagggccagt gaaaggctac 1800
atgagacctg aggcctgttg gcttaactgg gacaatacca aagccctttt agcatttgcc 1860
atcccggcgt tcgtcattgt ggctgtaaat ctgattgtgg ttttggttgt tgctgtcaac 1920
actcagaggc cctctattgg cagttccaag tctcaggatg tggtcataat tatgaggatc 1980
agcaaaaatg ttgccatcct cactccactg ctgggactga cctggggttt tggaatagcc 2040
33/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
actctcatag aaggcacttc cttgacgttc catataattt ttgccttgct caatgctttc 2100
cagggttttt tcatcctgct gtttggaacc attatggatc acaagataag agatgctttg 2160
aggatgagga tgtcttcact gaaggggaaa tcgagggcag ctgagaatgc atcactaggc 2220
ccaaccaatg gatctaaatt aatgaatcgt caaggatgaa atgctgcccc atttctcatg 2280
gatgtcctga gaccaagagg ggagatccag gagaaagagg cc 2322
<210> 33
<211> 2366
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6769042CB1
<400> 33
atttaggtga cactatagaa gagcccagtg tgctggaaag gagatcgcca tgtacttcac 60
tgctgccatt ggaaagcatg ctttattgtc ttcaacgctg ccaagcctct tcatgacatc 120
cacagcaagc cccgtgatgc ccacagatgc ctaccatccc atcataacca acctgacaga 180
agagagaaaa accttccaaa gtcccggagt gatactgagt tacctccaaa atgtatccct 240
cagcttaccc agtaagtccc tctcggagca gacagccttg aatctcacca agaccttctt 300
aaaagccgtg ggagagatcc ttctactgcc tggttggatt gctctgtcag aggacagcgc 360
cgtggtactg agtctcatcg acactattga caccgtcatg iggccatgtat cctccaacct 420
gcacggcagc acgccccagg tcaccgtgga gggctcctct gccatggcag agttttccgt 480
ggccaaaatc ctgcccaaga ccgtgaattc ctcccattac cgcttcccgg cccacgggca 540
gagcttcatc cagatccccc acgaggcctt ccacaggcac gcctggagca ccgtcgtggg 600
tctgctgtac cacagcatgc actactacct gaacaacatc tggcccgccc acaccaagat 660
cgcggaggcc atgcatcacc aggactgcct gctgttcgcc accagccacc tgatttccct 720
ggaggtgtcc ccaccaccca ccctgtctca gaacctgtcg ggctctccac tcattacggt 780
ccacctcaag cacagattga cacgtaagca gcacagtgag gccaccaaca gcagcaaccg 840
agtcttcgtg tactgcgcct tcctggactt cagctccgga gaaggggtct ggtcgaacca 900
cggctgtgcg ctcacgagag gaaacctcac ctactccgtc tgccgctgca ctcacctcac 960
caactttgcc atcctcatgc aggtggtccc gctggagctt gcacgcggac accaggtggc 1020
gctgtcgtct atcagctatg tgggctgctc cctctccgtg ctctgcctgg tggccacgct 1080
ggtcaccttc gccgtgctgt cctccgtgag caccatccgg aaccagcgct accacatcca 1140
cgccaacctg tccttcgccg tgctggtggc ccaggtcctg ctgctcatta gtttccgcct 1200
cgagccaggc acgaccccct gccaagtgat ggccgtgctc ctacactact tcttcctgag 1260
tgccttcgca tggatgctgg tggaggggct gcacctctac agcatggtga tcaaggtctt 1320
tgggtcggag gacagcaagc accgttacta ctatgggatg ggatggggtt ttcctcttct 1380
gatctgcatc atttcactgt catttgccat ggacagttac ggaacaagca acaattgctg 1440
gctgtcgttg gcgagtggcg ccatctgggc ctttgtagcc cctgccctgt ttgtcatcgt 1500
ggtcaacatt ggcatcctca tcgctgtgac cagagtcatc tcacagatca gcgccgacaa 1560
ctacaagatc catggagacc ccagtgcctt caagttgacg gccaaggcag tggccgtgct 1620
gctgcccatc ctgggtacct cgtgggtctt tggcgtgctt gctgtcaacg gttgtgctgt 1680
ggttttccag tacatgtttg ccacgctcaa ctccctgcag ggactgttca tattcctctt 1740
tcattgtctc ctgaattcag aggtgagagc cgccttcaag cacaaaatca aggtctggtc 1800
gctcacgagc agctccgccc gcacctccaa cgcgaagccc ttccactcgg acctcatgaa 1860
tgggacccgg ccaggcatgg cctccaccaa gctcagccct tgggacaaga gcagccactc 1920
tgcccaccgc gtcgacctgt cagccgtgtg agccgggagg ctgccaacca ggccaggctg 1980
cgctcagaac acaccccccc aaacagaatg aaatgcccca cctttgccca tggaccctct 2040
ccttgctgct gtctggacat gggtgttgtg gccccgagac agctgtcctc ccctgtgact 2100
ctggctgtcg gagcacactg CtCagCCCag cagcctgatg cccaggccag cgtgggccct 2160
cctgccttgc atccacccgt gggctgagtg acttcctcgg gggattccca ggacacagtg 2220
gcctgacttg tgatggtgcc cttgagcctc ccttcatcac tcagcatcag accagcgagg 2280
cagggcatcg gggccggtcc cgcagcccgg agggatgtca gctctgtgct ggggggttgg 2340
ggcccgccCC aagtgtcagg ccccgc 2366
<210> 34
<211> 1458
<212> DNA
<213> Homo Sapiens
<220>
34!37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
<221> misc_feature
<223> Incyte ID No: 7476053CB1
<400> 34
atggaggccg ctagcctttc agtggccacc gccggcgttg cccttgccct gggacccgag 60
accagcagcg ggaccccaag cccgagaggg atactcggtt cgaccccgag cggcgccgtc 120
ctgccgggcc gagggccgcc cttctctgtc ttcacggtcc tggtggtgac gctgctagtg 180
ctgctgatcg ctgccacttt cctgtggaac ctgctggttc cggtcaccat cccgcgggtc 240
CgtgCCttCC aCCgCgtgCC gcataacttg gtggcctcga cggccgtctc ggacgaacta 300
gtggcagcgc tggcgatgcc accgagcctg gcgagtgagc tgtcgaccgg gcgacgtcgg 360
ctgctgggcc ggagcctgtg ccacgtgtgg atctccttcg acgccctgtg CtgCCCCgCC 420
ggcctcggga acgtggcggc catcgccctg ggccgcgacg gggccatcac acggcacctg 480
cagcacacgc tgcgcacccg cagccgcgcc tcgttgctca tgatcgcgct cgcccgggtg 540
ccgtcggcgc tcatcgccct cgcgccgctg ctctttggcc ggggcgaggt gtgcgacgct 600
CggCtCCagC gctgccaggt gagccgggaa ccctcctatg CCgCCttCtC CdCCCgCggC 660
gccttccacc tgccgcttgg cgtggtgccg tttgtctacc ggaagatcta cgaggcggcc 720
aagtttcgtt tcggccgccg ccggagagct gtgctgccgt tgccggccac catgcaggtg 780
aaggaagcac ctgatgaggc tgaagtggtg ttcacggcac attgcaaagc aacggtgtcc 840
ttccaggtga gcggggactc ctggcgggag cagaaggaga ggcgagcagc catgatggtg 900
ggaattctga ttggcgtgtt tgtgctgtgc tggatcccct tcttcctgac ggaactcatc 960
agcccactct gtgcctgcag cctgcccccc atctggaaaa gcatatttct gtggcttggc 1020
tactccaatt ctttcttcaa ccccctgatt tacacagctt ttaacaagaa ctacaacaat 1080
gccttcaaga gcctctttac taagcagaga tgaacacagg ggttagagag acatgggtag 1140
attttaagga ggaaggaact tggacttttt cgtcagtgat ctgagattct tccctccaca 1200
gctgagtgct aatgctgtat tgagagttat accattgggc ctggactgta gaagcagcag 1260
agccaaggtt ctcaagaaag acagcaaagg tctggcagat gttgtaacta tgccttcttc 1320
ccatgtgcat ggcagacatt gccaattggt catggcttgg ctccccactg agcaggaact 1380
tggtctcaga atcctttcca ggacagcacc ctaggcagct actgttgatt atttaaaatt 1440
gatgcaagac ttgaaaaa 1458
<210> 35
<211> 975
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte zD No: 7480410CB1
<400> 35
atgggcatgg agggtcttct ccagaactcc actaacttcg tcctcacagg cctcatcacc 60
CatCCtgCCt tCCCCgggCt tCtCtttgCa atagtCttCt ccatctttgt ggtggctata 120
acagccaact tggtcatgat tctgctcatc Cacatggact cccgcctcca cactcccatg 180
tacttcttgc tcagccagct ctccatcatg gataccatct acatctgtat cactgtcccc 240
aagatgctcc aggacctcct gtccaaggac aagaccattt ccttcctggg ctgtgcagtt 300
cagatcttcc tctacctgac cctgattgga ggggaattct tcctgctggg tctcatggcc 360
tatgaccgct atgtggctgt gtgcaaccct ctacggtacc ctctcctcat gaaccgcagg 420
gtttgcttat tcatggtggt cggctcctgg gttggtggtt ccttggatgg gttcatgctg 480
actcctgtca ctatgagttt ccccttctgt agatcccgag agatcaatca ctttttctgt 540
gagatcccag ccgtgctgaa gttgtcttgc acagacacgt cactctatga gaccctgatg 600
tatgcctgct gcgtgctgat gctgcttatc cctctatctg tcatctctgt ctcctacacg 660
cacatcctcc tgactgtcca caggatgaac tctgctgagg gccggcgcaa agcctttgct 720
acgtgttcct cccacattat ggtggtgagc gttttctacg gggcagcctt ctacaccaac 780
gtgctgcccc actcctacca cactccagag aaagataaag tggtgtctgc cttctacacc 840
atcctcaccc ccatgctcaa cccactcatc tacagcttga ggaataaaga tgtggctgca 900
gctctgagga aagtactagg gagatgtggt tcctcccaga gcatcagggt ggcgactgtg 960
atcaggaagg gctag 975
<210> 36
<211> 948
<212> DNA
<213> Homo Sapiens
35/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
<220>
<221> misc_feature
<223> Incyte ID No: 55036418CB1
<400> 36
atggagacgt gggtgaacca gtcctacaca gatggcttct tcctcttagg catcttctcc 60
cacagtactg ctgaccttgt cctcttctcc gtggttatgg cggtcttcac agtggccctc 120
tgtgggaatg tcctcctcat cttcctcatc tacatggacc ctcaccttca cacccccatg 180
tacttcttcc tcagccagct ctccctcatg gacctcatgt tggtctgtac caatgtgcca 240
aagatggcag ccaacttcct gtctggcagg aagtccatct cctttgtggg ctgtggcata 300
caaattggcc tctttgtctg tcttgtggga tctgaggggc tcttgctggg actcatggct 360
tatgaccgct atgtggccat tagccaccca cttcactatc ccatcctcat gaatcagagg 420
gtctgtctcc agattactgg gagctcctgg gcctttggga taatcgatgg cttgatccag 480
atggtggtag taatgaattt cccctactgt ggcttgagga aggtgaacca tttcttctgt 540
gagatgctat ccttgttgaa gctggcctgt gtagacacat ccctgtttga gaaggtgata 600
tttgcttgct gtgtcttcat gcttctcttc ccattctcca tcatcgtggc ctcctatgct 660
cacattctag ggactgtgct gcaaatgcac tctgctcagg cctggaaaaa ggccctggcc 720
acctgctcct cccacctgac agctgtcacc ctcttctatg gggcagccat gttcatctac 780
ctgaggccta ggcactaccg ggcccccagc catgacaagg tggcctctat cttctacacg 840
gtccttactc ccatgctcaa ccccctcatt tacagcttga ggaacaggga ggtgatgggg 900
gcactgagga aggggctgga ccgctgcagg atcggcagcc agcactga 948
<210> 37
<211> 1086
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature .
<223> Incyte ID No: 7481701CB1
<400> 37
ggctctattc agacgctggc ttcttgtaag tgtattcctt tatccaatag tagatgcctc 60
ctagaaggct tgagtgcact ggaaattgaa actctcactt tcaacttgga gatggagagc 120
cccaatcaaa ccaccattca ggagtttatc ttctccgctt tcccttattc ctgggttaag 180
tctgttgtct gctttgttcc actgctcttc atctatgctt tcattgttgt tggaaacctg 240
gtcatcatca cagtggtcca gttgaatact cacctccaca ctcccatgta tacttttatc 300
agtgctcttt cttttctgga gatttggtat accacagcca caatcccaaa gatgctgtct 360
agcctgctta gtgagaggag catttccttc aatggttgtc tcctgcagat gtatttcttc 420
cattccaccg gcatctgtga ggtgtgtctc ttgacagtta tggcctttga ccactacctg 480
gccatatgca gccctcttca ttatccctct atcatgaccc ccaagctatg tacccaactg 540
actttaagtt gctgtgtttg tggctttatc acacccgttc ctgagattgc ctggatctct 600
acactgccat tttgtggttc gaatcacctt gaacatatct tctgtgactt cctcccagtg 660
ctgcgtctgg cctgcacaga cacacgagcc atcgtcatga ttcaggtagt ggatgtcatt 720
catgcagtgg agattattac agctgtgatg Ctcatcttca tgtcctacga tggtattgtg 780
gctgtaattc tacgtattca ttcagctgga ggccgccgca cagcattttc cacgtgtgtc 840
tctcacttca ttgtcttttc gctcttcttt ggcagtgtga ctctcatgta cctacgcttc 900
tctgccacct actctttgtt ctgggatata gccattgctc tggcctttgc agttttgtct 960
cccttcttca accccattat ctatagcctg aggaataaag aaataaaaga agctataaaa 1020
aagcacatag gtcaagctaa gatatttttt tccgtaagac cagggacctc aagtaagata 1080
ttttag 1086
<210> 38
<211> 1529
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7481774CB1
<400> 38
aagggagacc acagtgagag ggagccctga gcagaagtaa ggctgtcaca aggctggaag 60
36/37
CA 02417195 2003-O1-24
WO 02/10387 PCT/USO1/23433
cagagaacat ccccatggaa ctgaagacag catgctgcat ccctgggagg agggagctct 120
taaggaagtt ccaaggattg atatttctgt tcagctgcag tagagatgga tggaaccatg 180
gcagcaccca acccatttca tcctactggg attctctgac cgaccccatc tggagaggat 240
cctctttgtg gtcatcctga tcgcgtacct cctgaccctc gtaggcaaca ccaccatcat 300
cctggtgtcc cggctggacc cccacctcca cacccccatg tacttcttcc tcgcccacct 360
ttccttcctg gacctcagtt tcaccaccag CtCCatCCCC CagCtgCtCt acaaccttaa 420
tggatgtgac aagaccatca gctacatggg ctgtgccatc cagctcttcc tgttcctggg 480
tctgggtggt gtggagtgcc tgcttctggc tgtcatggcc tatgaccggt gtgtggctat 540
ctgcaagccc ctgcactaca tggtgatcat gaaccccagg ctctgccggg gcttggtgtc 600
agtgacctgg ggctgtgggg tggccaactc cttggccatg tctcctgtga ccctgcgctt 660
accccgctgt gggcaccacg aggtggacca cttcctgtgt gagatgcccg ccctgatccg 720
gatggcctgc atcagcactg tggccatcga cggcaccgtc tttgtcctgg cggtgggtgt 780
tgtgctgtcc cccttggtgt ttatcctgct ctcttacagc tacattgtga gggctgtgtt 840
acaaattcgg tcagcatcag gaaggcagaa ggccttcggc acctgcggct cccatctcac 900
tgtggtctcc cttttctatg gaaacatcat ctacatgtac atgcagccag gagccagttc 960
ttcccaggac cagggcaagt tcctcacgct cttctacaac attgtcaccc ccctcctcaa 1020
tcctctcatc tacaccctca gaaacagaga ggtgaagggg gcactgggaa ggttgcttct 1080
ggggaagaga gagctaggaa aggagtaaag gcatctccac ctgacttcac ctccatccag 1140
ggccactggc agcatctgga acggctgaat tccagctgat attagcccac gactcccaac 1200
ttgccttttt ctggactttt gtgaggctgt ttcagttctg acattatgtg tttttgttgt 1260
tgctcttaaa attgagacgg ggtctcactc tgtcacctag ggtggagtgc agtggtgcca 2320
ccatagctcc ttcgactatt gggcttaagc gatcctcccc cacctcagcc ttccaagtaa 1380
ctgggactac aggtgtgcat cactggcagt gggaattgtg gcttttctgt cttctatgga 1440
gacggggtct tgctgtgttg accaggctgg tcccaaactc ctggcctcat gtgatcctcc 1500
tgccatggcc tcctaaagtt ctgggatta
1529
37/37
