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G-PROTEIN COUPLED RECEPTORS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of G-protein
coupled receptors
and to the use of these sequences in the diagnosis, treatment, and prevention
of cell proliferative,
neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and
metabolic disorders, and
viral infections, and in the assessment of the effects of exogenous compounds
on the expression of
nucleic acid and amino acid sequences of G-protein coupled receptors. The
present invention further
relates to the use of specific G-protein coupled receptors to identify
molecules that are involved in
l0 modulating taste or olfactory sensation.
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. (1991) Eur.
J. Biochem. 196:1-10;
Coughlin, S.R. (1994) Curr. Opin. Cell Biol. 6:191-197). The amino-terminus of
a GPCR is
extracellular, is of variable length, and is often glycosylated. The carboxy-
terminus is cytoplasmic and
generally phosphorylated. Extracellular loops alternate with intracellular
loops and link the
transmembrane domains. Cysteine disulfide bridges linking the second and third
extracellular loops
may interact with agonists and antagonists. The most conserved domains of
GPCRs are the
transmembrane domains and the first two cytoplasmic loops. The transmembrane
domains account, in
part, for structural and functional features of the receptor. In most cases,
the bundle of a helices
forms a ligand-binding pocket. The extracellular N-terminal segment, or one or
more of the three
extracellular loops, may also participate in ligand binding. Ligand binding
activates the receptor by
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inducing a conformational change in intracellular portions of the receptor. In
turn, the large, third
intracellular loop of the activated receptor interacts with a heterotrimeric
guanine nucleotide binding
(G) protein complex which mediates further intracellular signaling activities,
including the activation of
second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol
triphosphate, and the
interaction of the activated GPCR with ion channel proteins. (See, e.g.,
Watson, S. and S. Arkinstall
(1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego 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
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,
thyrotropin-releasing hormone (TRH), 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 splice variants appear to be functionally distinct, based upon observed
differences in distribution,
signaling, coupling, regulation, and ligand binding profiles (Kilpatrick, G.J.
et al. (1999) Trends
Pharmacol. Sci. 20:294-301).
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
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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,
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
l0 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) Cell Biochem. Biophys. 30:213-242).
The largest subfamily of GPCRs, the olfactory receptors, are also members of
the rhodopsin-
like GPCR family. These receptors function by transducing odorant signals.
Numerous distinct
olfactory receptors are required to distinguish different odors. Each
olfactory sensory neuron
expresses only one type of olfactory receptor, and distinct spatial zones of
neurons expressing distinct
receptors are found in nasal passages. For example, the 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 (Raming, K. et al. ( 1998) Receptors
Channels 6:141-151 ).
However, the expression of olfactory-like receptors is not confined to
olfactory tissues. For example,
three rat genes encoding olfactory-like receptors having typical GPCR
characteristics showed
expression patterns not only in taste and olfactory tissue, but also in male
reproductive tissue (Thomas,
M.B. et al. (1996) Gene 178:1-5).
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
secredn, a peptide
hormone that stimulates the secretion of enzymes and ions in the pancreas and
small intestine
(Watson, supra, pp. 278-283). Secretin receptors are about 450 amino acids in
length and are found in
the plasma membrane of gastrointestinal cells. Binding of secretin to its
receptor stimulates the
production of cAMP.
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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
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~ans and Caenorhabditis brig~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: luteinizing hormone (precocious puberty);
vasopressin VZ (X-linked
nephrogenic diabetes); glucagon (diabetes and hypertension); calcium
(hyperparathyroidism,
hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); (33-
adrenoceptor
(obesity, non-insulin-dependent diabetes mellitus); growth hormone releasing
hormone (dwarfism); and
adrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al. (1998) Br.
J. Pharmocol. 125:1387-
1392; Stadel, J.M. et al. (1997) Trends Pharmacol. Sci. 18:430-437). GPCRs are
also involved in
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depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal
failure, and several
cardiovascular disorders (Horn, F. and G. Vriend (1998) J. Mot. 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, su ra;
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
l0 cardiovascular disorders, and anxiety; muscarinic agonists are used in the
treatment of glaucoma and
tachycardia; serotonin SHT1D 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.
2o As with the vasopressin V2 receptor mutants, these MC4R mutants are
defective in trafficking to the
plasma membrane (Ho, G. and R.G. MacKenzie (1999) J. Biol. Chem. 274:35816-
35822), and thus
might be treated with a similar strategy. The type 1 receptor for parathyroid
hormone (PTH) is a
GPCR that mediates the PTH-dependent regulation of calcium homeostasis in the
bloodstream. Study
of PTH/receptor interactions may enable the development of novel PTH receptor
ligands for the
treatment of osteoporosis (Mannstadt, M. et al. (1999) Am. J. Physiol.
277:F665-F675).
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,
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results in resistance to AIDS, suggesting that CCRS antagonists could be
useful in preventing the
development of AIDS.
The involvement of some GPCRs in taste and olfactory sensation has been
reported.
Complete or partial sequences of numerous human and other eukaryotic sensory
receptors are
currently known. (See, e.g., Pilpel, Y. and D. Lancet (1999) Protein Sci.
8:969-977; Mombaerts, P.
(1999) Annu. Rev. Neurosci. 22:487-509. See also, e.g., patents EP 867508A2;
US 5,874,243; WO
92/17585; WO 95/18140; WO 97/17444; and WO 99/67282.) It has been reported
that the human
genome contains approximately one thousand genes that encode a diverse
repertoire of olfactory
receptors (Rouquier, S. et al. (1998) Nat. Genet. 18:243-250; Trask, B.J. et
al. (1998) Hum. Mol.
Genet.7:2007-2020).
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
collectively as "GCREC" and individually as "GCREC-1," "GCREC-2," "GCREC-3,"
"GCREC-4,"
"GCREC-5," "GCREC-6," "GCREC-7," "GCREC-8," "GCREC-9," "GCREC-10," and "GCREC-
11." 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:1-11, 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-
11, c) a biologically
active fragment of a polypeptide having an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-11, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-11. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-11.
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:1-11, b) a polypeptide comprising a naturally occurnng
amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
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11, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-11, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-11. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
ID NO:1-11. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:12-22.
The invention additionally provides G-protein coupled receptors that are
involved in olfactory
and/or taste sensation. The invention further provides polynucleotide
sequences that encode said G-
protein coupled receptors.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
l0 sequence operably linked to a polynucleotide encoding a polypeptide
selected from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID N0:1-11, 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 N0:1-11, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-11, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ~>D NO:1-I 1. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-11, 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-11, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO: l -11, and d) an immunogenic fragment of a
polypeptide having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-11, 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:1-11, c) a biologically active fragment of a
polypeptide having an
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amino acid sequence selected from the group consisting of SEQ ID NO:1-11, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-11.
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
D7 N0:12-22, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ )D N0:12-22,
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
to 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
ID N0:12-22, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:12-22,
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
2o said target polynucleotide, under conditions whereby a hybridization
complex is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the probe
comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample, said
target polynucleotide having a sequence of a polynucleotide selected from the
group consisting of a) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:12-22, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:12-22, 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.
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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:1-1 l, 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 >D NO:1-1 l, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-11, and d) an
immunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:l-11,
and a pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11. 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 ID NO:1-11, 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-11, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-11, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-11. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional GCREC, comprising
administering to a patient in need of such treatment the composition.
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:1-1 l, 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:1-11, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:l-11,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ 1T7 NO:l-11. 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
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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:1-11, b) a
polypeptide comprising a
naturally occurnng amino acid sequence at least 90% identical to an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-11, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-11, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-11. 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:1-11, 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:1-11, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-11, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-11. The method comprises a) combining the
polypeptide with at least one
test compound under conditions permissive for the activity of the polypeptide,
b) assessing the activity
of the polypeptide in the presence of the test compound, and c) comparing the
activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence of
the test compound, wherein a change in the activity of the polypeptide in the
presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
The invention further provides methods of using G-protein coupled receptors of
the invention
involved in olfactory and/or taste sensation, biologically active fragments
thereof (including those
having receptor activity), and amino acid sequences having at least 90%
sequence identity therewith,
to identify compounds that agonize or antagonize the foregoing receptor
polypepddes. These
compounds are useful for modulating, blocking and/or mimicking specific tastes
and/or odors.
The present invention also relates to the use of olfactory and/or taste
receptors of the
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invention, biologically active fragments thereof (including those having
receptor activity), and
polypeptides having at least 90% sequence identity therewith, in combination
with one or more other
olfactory and/or taste receptor polypeptides, to identify a compound or
plurality of compounds that
modulate, mimic, and/or block a specific olfactory and/or taste sensation.
The invention also relates to cells that express an olfactory or taste
receptor polypeptide of the
invention, a biologically active fragment thereof (including those having
receptor activity), or a
polypeptide having at least 90% sequence identity therewith, and the use of
such cells in cell-based
screens to identify molecules that modulate, mimic, and/or block specific
olfactory or taste sensations.
Still further, the invention relates to a cell that co-expresses at least one
olfactory or taste G-
l0 protein coupled receptor polypeptide of the invention, and a G-protein, and
optionally one or more other
olfactory and/or taste G-protein coupled receptor polypeptides, and the use of
such a cell in screens to
identify molecules that modulate, mimic, and/or block specific olfactory
and/or taste sensations.
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 N0:12-22,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
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 N0:12-22, 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 N0:12-22,
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 polynucleodde sequence selected from the group consisting of SEQ ID N0:12-
22, 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 N0:12-22,
iii) a polynucleotide
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complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability scores for the
matches between each
polypeptide and its homolog(s) are 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,"
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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
to cell lines, protocols, reagents and vectors which are reported in the
publications and which might be
used in connection with the invention. Nothing herein is to be construed as an
admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"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
2o 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.
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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 sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of 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')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind GCREC polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLI-n. 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
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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 "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have modified sugar groups (e.g., the
2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NH2), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer'' refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels
in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci.
USA 96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurnng enzymes, which
normally act on
substrates containing right-handed nucleotides.
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); oligonucleoddes 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 chenucal synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurnng
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
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translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic 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
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Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu 11e, Val
Lys Arg, Gln, Glu
to Met Leu, lle
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, aryl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"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 shuttling" 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.
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A "fragment" is a unique portion of GCREC or the polynucleotide encoding GCREC
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example,. a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:12-22 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:12-22, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID NO:l 2-22 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
ID N0:12-22 from related polynucleotide sequences. The precise length of a
fragment of SEQ ID
N0:12-22 and the region of SEQ ID N0:12-22 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-11 is encoded by a fragment of SEQ ID N0:12-22. A
fragment of SEQ ID NO:l -11 comprises a region of unique amino acid sequence
that specifically
identifies SEQ ID NO:1-11. For example, a fragment of SEQ ID NO:1-11 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-11.
The precise length of a fragment of SEQ ID NO:1-11 and the region of SEQ ID
NO:1-11 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
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standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
l0 follows: Ktuple=2, gap penalty=S, 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 (NCBn 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:/lwww.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: . I
Penalty for mismatch: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off: SO
Expect: 10
Word Size: 11
Filter: on
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Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypepdde sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 3
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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
l0 DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 pg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (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;
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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 ~g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of 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 microarray.
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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
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, racemizadon, 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 oligonucleoddes, which may be annealed to
a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 1 S 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
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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 Cloning: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
also be obtained from their respective sources and modified to meet the user's
specific needs.) The
PrimeGen program (available to the public from the UK Human Genome Mapping
Project Resource
Centre, Cambridge UK) designs primers based on multiple sequence alignments,
thereby allowing
selection of primers that hybridize to either the most conserved or least
conserved regions of aligned
nucleic acid sequences. Hence, this program is useful for identification of
both unique and conserved
oligonucleotides and polynucleotide fragments. The oligonucleotides and
polynucleotide fragments
identified by any of the above selection methods are useful in hybridization
technologies, for example,
as PCR or sequencing primers, microarray elements, or specific probes to
identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods of
oligonucleotide selection are
not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
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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
l0 expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminesccnt, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of
the nitrogenous base thymine are replaced with uracil, and the sugar backbone
is composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing 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.
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The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"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" or "expression profile" refers to the collective pattern
of gene expression
by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
cells" includes stably transformed cells in which the inserted DNA is capable
of replication either as
an autonomously replicating plasmid or as part of the host chromosome, as well
as transiently
transformed cells which express the inserted DNA or RNA for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to animals
and plants, in which one or 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.
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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
polypepdde 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 polynucleodde sequence of a
particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91 %, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human 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
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single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleodde sequence identification number (Polynucleotide
SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide 1D) 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 >D NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
l0 shows the GenBank identification number (GenBank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide
and its homolog(s).
Column S shows the annotation of the GenBank homolog(s) along with relevant
citations where
applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypepddes of the invention.
Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis sofiware package (Genetics Computer
Group, Madison W>].
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are G-protein coupled
receptors. For example, SEQ
ID NO:1 is 32% identical to rat seven transmembrane receptor (GenBank ID
g5525078) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 1.7e-92, which indicates the probability of obtaining the
observed polypeptide
sequence alignment by chance. SEQ ID NO:1 also contains a seven transmembrane
receptor
(Secretin family) domain and a latrophilin/CL-1-like GPS domain (an unusual
family of ubiquitous G-
3o protein-linked receptors) 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 PROFILESCAN analyses provide further corroborative
evidence that SEQ
ID NO:1 is a seven-transmembrane G-protein coupled receptor. In an alternative
example, SEQ ID
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N0:2 is 48% identical to a mouse odorant receptor (GenBank ID 81419016) as
determined by
BLAST, with a probability score of S.Se-74. (See Table 2.) SEQ ID N0:2 also
contains a rhodopsin
family 7-transmembrane receptor domain as determined by searching for
statistically significant
matches in the HMM-based PFAM database. (See Table 3.) Data from BLIMPS,
MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:2
is a G-protein
coupled receptor. In an alternative example, SEQ ID N0:4 is 55% identical to a
human olfactory
receptor protein (GenBank ID 82370145) as determined by BLAST, with a
probability score of 5.9e-
86. (See Table 2.) SEQ ID N0:4 also contains a 7-transmembrane receptor domain
as determined
by searching for statistically significant matches in the HMM-based PFAM
database. (See Table 3.)
Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative evidence
that SEQ ID N0:4 is a G-protein coupled receptor. In an alternative example,
SEQ ID N0:8 is 77%
identical to Mus musculus odorant receptor S25 (GenBank ID 84680264) as
determined by BLAST,
with a probability score of 8.6e-126. (See Table 2.) SEQ ID N0:8 also contains
a 7-transmembrane
receptor (rhodopsin family) domain as determined by searching for
statistically significant matches in
IS the HMM-based PFAM database. (See Table 3.) Data from BLIMPS, MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:8
is a G-protein
coupled receptor. SEQ ID N0:3, SEQ ID NO:S-7, and SEQ ID N0:9-11 were analyzed
and
annotated in a similar manner. The algorithms and parameters for the analysis
of SEQ ID NO:1-11
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. Column 1 lists the polynucleotide
sequence
identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte
polynucleotide
consensus sequence number (Incyte ID) for each polynucleotide of the
invention, and the length of
each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start
(5') and stop (3')
positions of the cDNA and/or genomic sequences used to assemble the full
length polynucleotide
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ ID
N0:12-22 or that
distinguish between SEQ ID N0:12-22 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
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addition, the polynucleotide fragments described in column 2 may identify
sequences derived from the
ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 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 polynucleotide
fragments described in
column 2 may refer to assemblages of both eDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
FL XXXXXX NI NZ_YYYYY N3 N4 represents a "stitched" sequence in which XXXXXX
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 Nl,z,3...> if
present, represent specific exons
that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleodde fragments in column 2 may refer to assemblages of exons brought
together by an
"exon-stretching" algorithm. For example, a polynucleodde sequence identified
as
FLXXXXXX gAAA~AA_gBBBBB_1 N is a "stretched" sequence, with XXXXXX 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 referring to specific exons (See Example V). In instances where a RefSeq
sequence was used
as a protein homolog for the "exon-stretching" algorithm, .a Ret~Seq identif
er (denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
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,
ENST ~ for example,
GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GB1 Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
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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
Table 4 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
l0 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%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the GCREC amino acid sequence, and which contains at
least one functional or
structural characteristic of GCREC.
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 N0:12-22, which encodes GCREC. The
polynucleotide
sequences of SEQ ID N0:12-22, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
2o 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 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 N0:12-
22 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 N0:12-22. 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.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant of a
polynucleotide sequence encoding GCREC. A splice variant may have portions
which have significant
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sequence identity to the polynucleotide sequence encoding GCREC, but will
generally have a greater
or lesser number of polynucleotides due to additions or deletions of blocks of
sequence arising from
alternate splicing of exons during mRNA processing. A splice variant may have
less than about 70%,
or alternatively less than about 60%, or alternatively less than about 50%
polynucleotide sequence
identity to the polynucleotide sequence encoding GCREC over its entire length;
however, portions of
the splice variant will have at least about 70%, or alternatively at least
about 85%, or alternatively at
least about 95%, or alternatively 100% polynucleotide sequence identity to
portions of the
polynucleotide sequence encoding GCREC. Any one of the splice variants
described above can
encode an amino acid sequence which contains at least one functional or
structural characteristic of
to 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 codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring GCREC, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode GCREC and its variants are
generally capable
of hybridizing to the nucleotide sequence of the naturally occurnng GCREC
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences encoding
GCREC or its derivatives possessing a substantially different codon usage,
e.g., inclusion of non-
naturally occurring codons. Codons may be selected to increase the rate at
which expression of the
peptide occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with
which particular codons are utilized by the host. Other reasons for
substantially altering the nucleotide
sequence encoding GCREC and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater half-life,
than transcripts produced from the naturally occurring sequence.
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.
32
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:12-22 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol. 152:507-
511.) Hybridization conditions, including annealing and wash conditions, are
described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA 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 earned 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 algorithms 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 Biology and Biotechnology, 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. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR ampfilication 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
legations 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).
33
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 primer analysis software
(National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in length,
to have a GC content of about 50% or more, and to anneal to the template at
temperatures of about
68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
l0 sequences containing the S' 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
shufi-7ing 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 glycosyladon patterns, change eodon preference, produce splice
variants, and so forth.
34
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of GCREC, such as its biological or enzymatic
activity or its ability to bind to
other molecules or compounds. DNA shuffling is a process by which a library of
gene variants is
produced using PCR-mediated recombination of gene fragments. The library is
then subjected to
selection or screening procedures that identify those gene variants with the
desired properties. These
preferred variants may then be pooled and further subjected to recursive
rounds of DNA shuffling and
l0 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. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
GCREC itself or a fragment thereof may be synthesized using chemical methods.
For example,
peptide synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York 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 occurnng polypepdde.
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, su ra, 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
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding GCREC. Such elements may vary in their
strength and specificity.
Specific initiation signals may also be used to achieve more efficient
translation of sequences encoding
GCREC. Such signals include the ATG initiation codon and adjacent sequences,
e.g. the Kozak
sequence. In cases where sequences encoding GCREC and its initiation codon and
upstream
regulatory sequences are inserted into the appropriate expression vector, no
additional transcriptional
or translational control signals may be needed. However, in cases where only
coding sequence, or a
fragment thereof, is inserted, exogenous translational control signals
including an in-frame ATG
initiation codon should be provided by the vector. Exogenous translational
elements and initiation
codons may be of various origins, both natural and synthetic. The efficiency
of expression may be
enhanced by the inclusion of enhancers appropriate for the particular host
cell system used. (See,
e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding GCREC and appropriate transcriptional
and translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~v, 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 vims, 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 MeGraw 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
Harnngton, 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
36
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di Nicola,
M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-
242.) The
invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding 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
l0 plasmid (Life Technologies). Ligation of sequences encoding GCREC into the
vector's multiple
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of GCREC are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of GCREC may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
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 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311 ). Alternatively, plant promoters such as the small subunit of RUB
ISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y
(1992) MeGraw Hill,
New York NY, pp. 191-196.)
37
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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
l0 DNA than can be contained in and expressed from a plasmid. HACs of about 6
kb to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-
355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
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, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or
herbicide resistance can be used as the basis for selection. For example, dhfr
confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hi~~D, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
38
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
(GFP; Clontech),13 glucuronidase and its substrate f3-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A, (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding 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
l0 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
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 immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on GCREC is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunoloey, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) lmmunochemical
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 polymerise
39
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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 post-
translational activities
(e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type
Culture
Collection (ATCC, Manassas VA) and may be chosen to ensure the correct
modification and
processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding GCREC may be ligated to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric GCREC protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of GCREC
activity. Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available affinity
matrices. Such moieties include, but are not limited to, glutathione S-
transferase (GST), maltose
binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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, su
ra, eh. 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
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
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
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express GCREC,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing GCREC or cell membrane fractions which contain GCREC
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either GCREC or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example, the
assay may comprise the steps of combining at least one test compound with
GCREC, either in solution
or affixed to a solid support, and detecting the binding of GCREC to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test 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
41
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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 No. 5,175,383 and U.S. Patent No.
5,767,337.) For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,
M.R. (1989)_ Science
244:1288-1292). The vector integrates into the corresponding region of the
host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from
the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and
the resulting chimeric progeny are genotyped and bred to produce heterozygous
or homozygous
strains. Transgenic animals thus generated may be tested with potential
therapeutic or toxic agents.
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 sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the blastulae
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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, examples of
tissues expressing
GCREC can be found in Table 6. Therefore, GCREC appears to play a role in cell
proliferative,
neurological, cardiovascular, gastrointestinal, autoimmune/inflammatory, and
metabolic disorders, and
l0 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
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract,
heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; a neurological disorder such as
epilepsy, ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease,
Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, rednitis
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
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disorders, autonomic nervous system disorders, cranial nerve disorders, spinal
cord diseases, muscular
dystrophy and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis
and polymyositis, inherited, metabolic, endocrine, and toxic myopathies,
myasthenia gravis, periodic
paralysis, mental disorders including mood, anxiety, and schizophrenic
disorders, seasonal affective
disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias,
paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive
supranuclear palsy,
corticobasal degeneration, and familial frontotemporal dementia; a
cardiovascular disorder such as
arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's
disease, aneurysms, arterial
dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular
tumors, complications of
thrombolysis, balloon angioplasty, vascular replacement, and coronary artery
bypass graft surgery,
congestive heart failure, ischemic heart disease, angina pectoris, myocardial
infarction, hypertensive
heart disease, degenerative valvular heart disease, calcific aortic valve
stenosis, congenitally bicuspid
aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic
fever and rheumatic heart
disease, infective endocarditis, nonbacterial thrombotic endocarditis,
endocarditis of systemic lupus
erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,
pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation; a gastrointestinal
disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal
stricture, esophageal
carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis, gastroparesis,
antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis,
intestinal obstruction, infections of
the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis,
cholestasis, panereatitis, 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, irntable bowel
syndrome, short
bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired
immunodeliciency
syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal
syndrome, hepatic
steatosis, hemochromatosis, Wilson's disease, alpha,-andtrypsin deficiency,
Reye's syndrome, primary
sclerosing cholangitis, liver infarction, portal vein obstruction and
thrombosis, centrilobular necrosis,
peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease,
preeclampsia, eclampsia, acute fatty
liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors
including nodular
hyperplasias, adenomas, and carcinomas; an autoimmune/inflammatory disorder
such as acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia,
autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
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(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
tongavirus.
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 tiirther embodiment, a composition comprising a substantially purified
GCREC in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or prevent
a disorder associated with decreased expression or activity of GCREC
including, but not limited to,
those provided above.
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,
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.
CA 02430993 2003-06-06
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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
art. In particular, purified GCREC may be used to produce antibodies or to
screen libraries of
l0 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 polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans, BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum 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.)
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CA 02430993 2003-06-06
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In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
GCREC-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton,
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
l0 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')2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
GCREC and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering GCREC epitopes is generally used, but a competitive
binding assay may also be
employed (Pound, s-u~ra).
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
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GCREC epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka
ranging from about 109 to 10'2 L/mole 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' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of GCREC, preferably in
active form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington 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
l0 the quality and suitability of such preparations for certain downstream
applications. For example, a
polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml, preferably 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of GCREC-antibody
complexes. Procedures for evaluating antibody specificity, titer, and avidity,
and guidelines for
antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and
Coligan et al. supra.)
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 oligonucleoddes) 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.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Andsense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado> R.J. et
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al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25( 14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding GCREC may be
used for
somatic or germfine gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeticiency (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
1o Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-
703), thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA
93:11395-11399), hepatitis
B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and
Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Tnrpanosoma 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) 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; Ivies, 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
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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 al. (1995) Science 268:1766-1769; Rossi,
F.M.V. and H.M. Blau
(1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, 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 Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E, et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to 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
ternunal 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
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et
al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5>910>434 to Rigg ("Method
for obtaining
retrovirus packaging cell lines producing high transducing e~ciency 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
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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
l0 described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, 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 No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
this patent are the construction and use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-
532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
51
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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
l0 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 lyric 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 sufticiently 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,
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
identified by
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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, constitudvely 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 associated with
decreased GCREC expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding GCREC may be therapeutically useful.
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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-occurnng 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 polynucleodde
can be carried out, for example, using a Schizosaccharomvces 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 liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechno1.15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
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An additional embodiment of the invention relates to the administration of a
composition which
generally comprises an active ingredient formulated with a pharmaceutically
acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and
proteins. Various
formulations are commonly known and are thoroughly discussed in the latest
edition of Remin~ton'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, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising 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 adnunistration. Such information can then be used to determine useful
doses and routes for
administration in humans.
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
l0 preferably within a range of circulating concentrations that includes the
EDSO with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and response
to therapy. Long-acting compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 ~cg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind GCREC may be used
for the
diagnosis of disorders characterized by expression of GCREC, or in assays to
monitor patients being
treated with GCREC or agonists, antagonists, or inhibitors of GCREC.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for GCREC include methods which utilize the antibody and a
label to detect
GCREC in human body fluids or in extracts of cells or tissues. The antibodies
may be used with or
without modification, and may be labeled by covalent or non-covalent
attachment of a reporter
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molecule. A wide variety of reporter molecules, several of which are described
above, are known in
the art and may be used.
A variety of protocols for measuring GCREC, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of GCREC expression.
Normal or standard values for GCREC expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibodies to
GCREC under conditions suitable for complex formation. The amount of standard
complex formation
may be quantitated by various methods, such as photometric means. Quantities
of GCREC expressed
in subject, control, and disease samples from biopsied tissues are compared
with the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
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
N0:12-22 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
polymerises and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
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such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding GCREC may be used for the diagnosis of
disorders
associated with expression of GCREC. Examples of such disorders include, but
are not limited to, a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
a neurological disorder such
as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,
Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders,
amyotrophic lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial
and viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases including
kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,
fatal familial
insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal
syndrome, mental retardation
and other developmental disorders of the central nervous system, cerebral
palsy, neuroskeletal
disorders, autonomic nervous system disorders, cranial nerve disorders, spinal
cord diseases, muscular
dystrophy and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis
and polymyositis, inherited, metabolic, endocrine, and toxic myopathies,
myasthenia gravis, periodic
paralysis, mental disorders including mood, anxiety, and schizophrenic
disorders, seasonal affective
disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias,
paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive
supranuclear palsy,
corticobasal degeneration, and familial frontotemporal dementia; a
cardiovascular disorder such as
arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's
disease, aneurysms, arterial
dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular
tumors, complications of
thrombolysis, balloon angioplasty, vascular replacement, and coronary artery
bypass graft surgery,
congestive heart failure, ischemic heart disease, angina pectoris, myocardial
infarction, hypertensive
heart disease, degenerative valvular heart disease, calcific aortic valve
stenosis, congenitally bicuspid
aortic valve, mural annular calcification, mural valve prolapse, rheumatic
fever and rheumatic heart
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disease, infective endocarditis, nonbacterial thrombotic endocardids,
endocarditis of systemic lupus
erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,
pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation; a gastrointestinal
disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal
stricture, esophageal
carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis, gastroparesis,
antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis,
intestinal obstruction, infections of
the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis,
cholestasis, pancreatitis, pancreatic
carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis,
passive congestion of the liver,
hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease,
l0 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, alphas-antitrypsin deficiency,
Reye's syndrome, primary
sclerosing cholangitis, liver infarction, portal vein obstruction and
thrombosis, centrilobular necrosis,
peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease,
preeclampsia, eclampsia, acute fatty
liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors
including nodular
hyperplasias, adenomas, and carcinomas; an autoimmune/inflammatory disorder
such as acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia,
autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
(APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome,
multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and helminthic
infections, and trauma; a metabolic disorder such as diabetes, obesity, and
osteoporosis; and an
infection by a viral agent classified as adenovirus, arenavirus, bunyavirus,
calicivirus, coronavirus,
filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus,
papovavirus,
paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovinzs, and
tongavirus. The
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polynucleotide sequences encoding GCREC may be used in Southern or northern
analysis, dot blot, or
other membrane-based technologies; in PCR technologies; in dipstick, pin, and
multiformat ELISA-like
assays; and in microarrays utilizing fluids or tissues from patients to detect
altered GCREC expression.
Such qualitative or quantitative methods are well known in the art.
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
l0 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 tluids 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 may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
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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
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
l0 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. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by conning 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.
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In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, 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 No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell fines, 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
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molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the present
invention, so that transcript levels corresponding to the polynucleotides of
the present invention may be
quantified. The transcript levels in the treated biological sample are
compared with levels in an
untreated biological sample. Differences in the transcript levels between the
two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance 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
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are visualized in the gel as discrete and uniquely positioned spots, typically
by staining the gel with an
agent such as Coomassie Blue or silver or fluorescent stains. The optical
density of each protein spot
is generally proportional to the level of the protein in the sample. The
optical densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass
spectrometry. The identity of the protein in a spot may be determined by
comparing its partial
sequence, preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the
l0 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 (Lucking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed by
a variety of methods known in the art, for example, by reacting the proteins
in the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difticult, 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
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present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared
with the amount in an untreated biological sample. A difference in the amount
of protein between the
two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding 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 mufti-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial Pl
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
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
CA 02430993 2003-06-06
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region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 11 q22-23, any
sequences mapping to that area may represent associated or regulatory genes
for further investigation.
l0 (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide
sequence of the instant
invention may also be used to detect differences in the chromosomal location
due to translocation,
inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, GCREC, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between GCREC and the agent being tested may be measured.
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.
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Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No. 60/254,323, U.S. Ser. No. 60/255,564, U.S. Ser. No.
60/257,716, and U.S. Ser.
No. 60/262,848, are expressly incorporated by reference herein.
EXAMPLES
l0 I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). 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
15 isothiocyanate. The resulting lysates were centrifuged over CsCI cushions
or extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was
2o 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
25 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
30 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.,
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PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte
Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XLl-Blue, XLl-BIueMRF, or
SOLR from
Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation such
as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200
thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
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1997, su ra, 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 eDNA 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; PROTEOME databases with sequences from Homo
saniens, Rattus norvegicus, Mus musculus, Caenorhabditis ele~ans,
Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); and hidden
Markov model (HMM)-based protein family databases such as PFAM. (HMM is a
probabilistic
approach which analyzes consensus primary structures of gene families. See,
for example, Eddy, S.R.
(1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed using
programs based on
BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to
produce
full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank
ESTs, stitched
sequences, stretched sequences, or Genscan-predicted coding sequences (see
Examples IV and V)
were used to extend Incyte cDNA assemblages to full length. Assembly was
performed using
programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened
for open
reading frames using programs based on GeneMark, BLAST, and FASTA. The full
length
polynucleotide sequences were translated to derive the corresponding full
length polypeptide
sequences. Alternatively, a polypeptide of the invention may begin at any of
the methionine residues
of the full length translated polypepdde. Full length polypeptide sequences
were subsequently
analyzed by querying against databases such as the GenBank protein databases
(genpept), SwissProt,
the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden
Markov
model (HMM)-based protein family databases such as PFAM. Full length
polynucleotide sequences
are also analysed 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
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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 1D
N0:12-22. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies are described in Table 4, column 2.
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
foam 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
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
lncyte 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
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"Stitched" Seguences
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
l0 be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for homologous
genomic sequences from the public human genome databases. Partial DNA
sequences were
therefore "stretched" or extended by the addition of homologous genomic
sequences. The resultant
stretched sequences were examined to determine whether it contained a complete
gene.
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V1. Chromosomal Mapping of GCREC Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:12-22 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:12-22 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
l0 of all sequences of that cluster, including its particular SEQ )D 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.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
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BLAST Score x Percent Identity
x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
5 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
l0 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
2o 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/condidon categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding GCREC. cDNA sequences and cDNA
library/dssue
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
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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.
l0 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)2S04,
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 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~1 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 ~cl 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 relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
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(Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to
fill-in restriction
site overhangs, and transfected into competent E. coli cells. Transformed
cells were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37 °C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step l: 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
l0 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
i5 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 N0:12-22 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
20 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 ~Ci of
~,~ 32P1 adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
25 SEPHADEX G-25 superfine size exclusion dextrin 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
30 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 microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink-jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
A typical array may
to be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; 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 nucroarray. 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
2o 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 polynucleodde 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/~1 oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~1 RNase inhibitor, 500 ~M dATP, 500 pM dGTP, 500 ~M
dTTP, 40 pM
dCTP, 40 pM 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
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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
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and resuspended
in 14 pt 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 pg.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 pt of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 nl of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 pt of sample mixture consisting of 0.2 pg
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 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
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larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
p1 of SX SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (IX 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 crosstalk
(due to overlapping emission
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spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the 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 TS 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 eDNA 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
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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 iaponicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from GCREC at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, su ra,
l0 ch. 10 and 16). Purified GCREC obtained by these methods can be used
directly in the assays shown
in Examples XVI, XVII, and XVIII, where applicable.
XIII. Functional Assays
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. S-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a.
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
CA 02430993 2003-06-06
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The influence of GCREC on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding GCREC and either CD64
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions
of human immunoglobulin G (IgG). Transfected cells are efficiently separated
from nontransfected
cells using magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake
Success NY). mRNA can be purified from the cells using methods well known by
those of skill in the
art. Expression of mRNA encoding GCREC and other genes of interest can be
analyzed by northern
analysis or microarray techniques.
XIV. Production of GCREC Specific Antibodies
l0 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 known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an AB1431 A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccininude 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 immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of GCREC (e.g.,
high ionic strength
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WO 02/46230 PCT/USO1/46659
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
Molecules which interact with GCREC may include agonists and antagonists, as
well as
molecules involved in signal transduction, such as G proteins. GCREC, or a
fragment thereof, is
labeled with'ZSI Bolton-Hunter reagent. (See, e.g., Bolton A.E. and W.M.
Hunter (1973) Biochem. J.
133:529-539.) A fragment of GCREC includes, for example, a fragment comprising
one or more of
the three extracellular loops, the extracellular N-terminal region, or the
third intracellular loop.
l0 Candidate molecules previously arrayed in the wells of a mufti-well plate
are incubated with the
labeled GCREC, washed, and any wells with labeled GCREC complex are assayed.
Data obtained
using different concentrations of GCREC are used to calculate values for the
number, affinity, and
association of GCREC with the candidate ligand molecules.
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.
2o Patent No. 6,057,101).
Potential GCREC agonists or antagonists may be tested for activation or
inhibition of GCREC
receptor activity using the assays described in sections XVII and XVIII.
Candidate molecules may be
selected from known GPCR agonists or antagonists, peptide libraries, or
combinatorial chemical
libraries.
Methods for detecting interactions of GCREC with intracellular signal
transduction molecules
such as G proteins are based on the premise that internal segments or
cytoplasmic domains from an
orphan G protein-coupled seven transmembrane receptor may be exchanged with
the analogous
domains of a known G protein-coupled seven transmembrane receptor and used to
identify the G-
proteins and downstream signaling pathways activated by the orphan receptor
domains (Kobilka, B.K.
et al. (1988) Science 240:1310-1316). In an analogous fashion, domains of the
orphan receptor may
be cloned as a portion of a fusion protein and used in binding assays to
demonstrate interactions with
specific G proteins. Studies have shown that the third intracellular loop of G
protein-coupled seven
transmembrane receptors is important for G protein interaction and signal
transduction (Conklin, B.R.
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WO 02/46230 PCT/USO1/46659
et al. (1993) Cel173:631-641). For example, the DNA fragment corresponding to
the third intracellular
loop of GCREC may be amplified by the polymerase chain reaction (PCR) and
subcloned into a fusion
vector such as pGEX (Pharmacia Biotech). The construct is transformed into an
appropriate bacterial
host, induced, and the liision protein is purified from the cell lysate by
glutathione-Sepharose 4B
(Pharmacia Biotech) affinity chromatography.
For in vitro binding assays, cell extracts containing G proteins are prepared
by extraction with
50 mM Tris, pH 7.8, 1 mM EGTA, 5 mM MgCl2, 20 mM CHAPS, 20% glycerol, 10 pg of
both
aprotinin and leupeptin, and 20 p1 of 50 mM phenylmethylsulfonyl fluoride. The
lysate is incubated on
ice for 45 min with constant sdrnng, centrifuged at 23,000 g for 15 min at
4°C, and the supernatant is
collected. 750 pg of cell extract is incubated with glutathione S-transferase
(GST) fusion protein
beads for 2 h at 4°C. The GST beads are washed five times with
phosphate-buffered saline. Bound
G protein subunits are detected by [32P]ADP-ribosylation with pertussis or
cholera toxins. The
reactions are terminated by the addition of SDS sample buffer (4.6% (w/v) SDS,
10% (v/v)
(3-mercaptoethanol, 20% (w/v) glycerol, 95.2 mM Tris-HCI, pH 6.8, 0.01 % (w/v)
bromphenol blue).
The [32P]ADP-labeled proteins are separated on 10% SDS-PAGE gels, and
autoradiographed. The
separated proteins in these gels are transferred to nitrocellulose paper,
blocked with Motto (5% nonfat
dried milk, 50 mM Tris-HCl (pH 8.0), 2 mM CaCl2, 80 mM NaCI, 0.02% NaN3, and
0.2% Nonidet
P-40) for 1 hour at room temperature, followed by incubation for 1.5 hours
with Ga subtype selective
antibodies (1:500; Calbiochem-Novabiochem). After three washes, blots are
incubated with
horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin
(1:2000, Cappel,
Westchester PA) and visualized by the chemiluminescence-based ECL method
(Amersham: Corp.).
XVII. Demonstration of GCREC Activity
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
83
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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.)
i0 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
GCREC is transfected into a mammalian cell line (e.g., Chinese hamster ovary
(CHO) or human
embryonic kidney (HEK-293) cell lines) using methods well-known in the art.
Transfected cells are
grown in 12-well trays in culture medium for 48 hours, then the culture medium
is discarded, and the
attached cells are gently washed with PBS. The cells are then incubated in
culture medium with or
without ligand for 30 minutes, then the medium is removed and cells lysed by
treatment with 1 M
perchloric acid. The cAMP levels in the lysate are measured by
radioimmunoassay using methods
well-known in the art. Changes in the levels of CAMP in the lysate from cells
exposed to ligand
compared to those without ligand are proportional to the amount of GCREC
present in the transfected
cells.
To measure changes in inositol phosphate levels, the cells are grown in 24-
well plates
containing 1x105 cells/well and incubated with inositol-free media and
[3H]myoinositol, 2 ~Ci/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 AG1-X8 (Bio-Rad) anion
exchange resin, and the
total labeled inositol phosphates counted by liquid scintillation. Changes in
the levels of labeled inositol
phosphate from cells exposed to ligand compared to those without ligand are
proportional to the
amount of GCREC present in the transfected cells.
3o XV1II. 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
84
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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 mull-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 Ga,sn6 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+ mobilization. Alternatively, GCREC may be
expressed in
engineered yeast systems which lack endogenous GPCRs, thus providing the
advantage of a null
background for GCREC activation screening. These yeast systems substitute a
human GPCR and Ga
protein for the corresponding components of the endogenous yeast pheromone
receptor pathway.
Downstream signaling pathways are also modified so that the normal yeast
response to the signal is
converted to positive growth on selective media or to reporter gene expression
(Broach, J.R. and J.
Thorner (1996) Nature 384 (supp.):14-16). The receptors are screened against
putative ligands
including known GPCR ligands and other naturally occurring bioactive
molecules. Biological extracts
from tissues, biological fluids and cell supernatants are also screened.
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 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
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104
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
<110> INCYTE GENOMICS, INC.
KALLICK, Deborah A.
BAUGHN, Mariah R.
LU, Dyung Aina M.
YUE, Henry
GRAUL, Richard C.
LU, Yan
DING, Li
TRIBOULEY, Catherine M.
TANG, Y. Tom
GANDHI, Ameena R.
THORNTON, Michael
<120> G-PROTEIN COUPLED RECEPTORS
<130> PI-0315 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/254,323; 60/255,564; 60/251,716; 60/262,848
<151> 2000-12-08; 2000-12-13; 2000-12-21; 2001-01-19
<160> 22
<170> PERL Program
<210> 1
<211> 1018
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7924827CD1
<400> 1
Met Val Cys Ser Ala Ala Pro Leu Leu Leu Leu Ala Thr Thr Leu
1 5 10 15
Pro Leu Leu Gly Ser Pro Val Ala Gln Ala Ser Gln Pro Leu Trp
20 25 30
Pro Met Ala Lys Gly Gln Thr Met Trp Ala Gln Thr Ser Thr Leu
35 40 45
Thr Leu Thr Glu Glu Glu Leu Gly Gln Ser Gln Ala.Gly Gly Glu
50 55 60
Ser Gly Ser Gly Gln Leu Leu Asp Gln Glu Asn Gly Ala Gly Glu
65 70 75
Ser Ala Leu Val Ser Val Tyr Val His Leu Asp Phe Pro Asp Lys
80 85 90
Thr Trp Pro Pro Glu Leu Ser Arg Thr Leu Thr Leu Pro Ala Ala
95 100 105
Ser Ala Ser Ser Ser Pro Arg Pro Leu Leu Thr Gly Leu Arg Leu
110 115 120
Thr Thr Glu Cys Asn Val Asn His Lys Gly Asn Phe Tyr Cys Ala
125 130 135
Cys Leu Ser Gly Tyr Gln Trp Asn Thr Ser Ile Cys Leu His Tyr
140 145 150
1/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
Pro Pro Cys Gln Ser Leu His Asn His Gln Pro Cys Gly Cys Leu
155 160 165
Val Phe Ser His Pro Glu Pro Gly Tyr Cys Gln Leu Leu Pro Pro
170 175 180
Val Pro Gly Ile Leu Asn Leu Asn Ser Gln Leu Gln Met Pro Gly
185 190 195
Asp Thr Leu Ser Leu Thr Leu His Leu Ser Gln Glu Ala Thr Asn
200 205 210
Leu Ser Trp Phe Leu Arg His Pro Gly Ser Pro Ser Pro Ile Leu
215 220 225
Leu Gln Pro Gly Thr Gln Val Ser Val Thr Ser Ser His Gly Gln
230 235 240
Ala Ala Leu Ser Val Ser Asn Met Ser His His Trp Ala Gly Glu
245 250 255
Tyr Met Ser Cys Phe Glu Ala Gln Gly Phe Lys Trp Asn Leu Tyr
260 265 270
Glu Val Val Arg Val Pro Leu Lys Ala Thr Asp Val Ala Arg Leu
275 280 285
Pro Tyr Gln Leu Ser Ile Ser Cys Ala Thr Ser Pro Gly Phe Gln
290 295 300
Leu Ser Cys Cys Ile Pro Ser Thr Asn Leu Ala Tyr Thr Ala Ala
305 310 315
Trp Ser Pro Gly Glu Gly Ser Lys Ala Ser Ser Phe Asn Glu Ser
320 325 330
Gly Ser Gln Cys Phe Val Leu Ala Val Gln Arg Cys Pro Met Ala
335 340 345
Asp Thr Thr Tyr Ala Cys Asp Leu Gln Ser Leu Gly Leu Ala Pro
350 355 360
Leu Arg Val Pro Ile Ser Ile Thr Ile Ile Gln Asp Gly Asp Ile
365 370 375
Thr Cys Pro Glu Asp Ala Ser Val Leu Thr Trp Asn Val Thr Lys
380 385 390
Ala Gly His Val Ala Gln Ala Pro Cys Pro Glu Ser Lys Arg Gly
395 400 405
Ile Val Arg Arg Leu Cys Gly Ala Asp Gly Val Trp Gly Pro Val
410 415 420
His Ser Ser Cys Thr Asp Ala Arg Leu Leu Ala Leu Phe Thr Arg
425 430 435
Thr Lys Leu Leu Gln Ala Gly Gln Gly Ser Pro Ala Glu Glu Val
440 445 450
Pro Gln Ile Leu Ala Gln Leu Pro Gly Gln Ala Ala Glu Ala Ser
455 460 465
Ser Pro Ser Asp Leu Leu Thr Leu Leu Ser Thr Met Lys Tyr Val
470 475 480
Ala Lys Val Val Ala Glu Ala Arg Ile Gln Leu Asp Arg Arg Ala
485 490 495
Leu Lys Asn Leu Leu Ile Ala Thr Asp Lys Val Leu Asp Met Asp
500 505 510
Thr Arg Ser Leu Trp Thr Leu Ala Gln Ala Arg Lys Pro Trp Ala
515 520 525
Gly Ser Thr Leu Leu Leu Ala Val Glu Thr Leu Ala Cys Ser Leu
530 535 540
Cys Pro Gln Asp His Pro Phe Ala Phe Ser Leu Pro Asn Val Leu
545 550 555
Leu Gln Ser Gln Leu Phe Gly Pro Thr Phe Pro Ala Asp Tyr Ser
560 565 570
2/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
Ile Ser Phe Pro Thr Arg Pro Pro Leu Gln Ala Gln Ile Pro Arg
575 580 585
His Ser Leu Ala Pro Leu Val Arg Asn Gly Thr Glu Ile Ser Ile
590 595 600
Thr Ser Leu Val Leu Arg Lys Leu Asp His Leu Leu Pro Ser Asn
605 610 615
Tyr Gly Gln Gly Leu Gly Asp Ser Leu Tyr Ala Thr Pro Gly Leu
620 625 630
Val Leu Val Ile Ser Ile Met Ala Gly Asp Arg Ala Phe Ser Gln
635 640 645
Gly Glu Val Ile Met Asp Phe Gly Asn Thr Asp Gly Ser Pro His
650 655 660
Cys Val Phe Trp Asp His Ser Leu Phe Gln Gly Arg Gly Gly Trp
665 670 675
Ser Lys Glu Gly Cys Gln Ala Gln Val Ala Ser Ala Ser Pro Thr
680 685 690
Ala Gln Cys Leu Cys Gln His Leu Thr Ala Phe Ser Val Leu Met
695 700 705
Ser Pro His Thr Val Pro Glu Glu Pro Ala Leu Ala Leu Leu Thr
710 715 720
Gln Val Gly Leu Gly Ala Ser Ile Leu Ala Leu Leu Val Cys Leu
725 730 735
Gly Val Tyr Trp Leu Val Trp Arg Val Val Val Arg Asn Lys Ile
740 745 750
Ser Tyr Phe Arg His Ala Ala Leu Leu Asn Met Val Phe Cys Leu
755 760 765
Leu Ala Ala Asp Thr Cys Phe Leu Gly Ala Pro Phe Leu Ser Pro
770 775 780
Gly Pro Arg Ser Pro Leu Cys Leu Ala Ala Ala Phe Leu Cys His
785 790 795
Phe Leu Tyr Leu Ala Thr Phe Phe Trp Met Leu Ala Gln Ala Leu
800 805 810
Val Leu Ala His Gln Leu Leu Phe Val Phe His Gln Leu Ala Lys
815 820 825
His Arg Val Leu Pro Leu Met Val Leu Leu Gly Tyr Leu Cys Pro
830 835 840
Leu Gly Leu Ala Gly Val Thr Leu Gly Leu Tyr Leu Pro Gln Gly
845 850 855
Gln Tyr Leu Arg Glu Gly Glu Cys Trp Leu Asp Gly Lys Gly Gly
860 865 870
Ala Leu Tyr Thr Phe Val Gly Pro Val Leu Ala Ile Ile Gly Val
875 880 885
Asn Gly Leu Val Leu Ala Met Ala Met Leu Lys Leu Leu Arg Pro
890 895 900
Ser Leu Ser Glu Gly Pro Pro Ala Glu Lys Arg Gln Ala Leu Leu
905 910 915
Gly Val Ile Lys Ala Leu Leu Ile Leu Thr Pro Ile Phe Gly Leu
920 925 930
Thr Trp Gly Leu Gly Leu Ala Thr Leu Leu Glu Glu Val Ser Thr
935 940 945
Val Pro His Tyr Ile Phe Thr Ile Leu Asn Thr Leu Gln Gly Val
950 955 960
Phe Ile Leu Leu Phe Gly Cys Leu Met Asp Arg Lys Ile Gln Glu
965 970 975
Ala Leu Arg Lys Arg Phe Cys Arg Ala Gln Ala Pro Ser Ser Thr
980 985 990
3/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
Ile Ser Leu Ala Thr Asn Glu Gly Cys Ile Leu Glu His Ser Lys
995 1000 1005
Gly Gly Ser Asp Thr Ala Arg Lys Thr Asp Ala Ser Glu
1010 1015
<210> 2
<211> 309
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7485408CD1
<400> 2
Met Glu Gly Ile Asn Lys Thr Ala Lys Met Gln Phe Phe Phe Arg
1 5 10 15
Pro Phe Ser Pro Asp Pro Glu Val Gln Met Leu Ile Phe Val Val
20 25 30
Phe Leu Met Met Tyr Leu Thr Ser Leu Gly Gly Asn Ala Thr Ile
35 40 45
Ala Val Ile Val Gln Ile Asn His Ser Leu His Thr Pro Met Tyr
50 55 60
Phe Phe Leu Ala Asn Leu Ala Val Leu Glu Ile Phe Tyr Thr Ser
65 70 75
Ser Ile Thr Pro Leu Ala Leu Ala Asn Leu Leu Ser Met Gly Lys
80 85 90
Thr Pro Val Ser Ile Thr Gly Cys Gly Thr Gln Met Phe Phe Phe
95 100 105
Val Phe Leu Gly Gly Ala Asp Cys Val Leu Leu Val Val Met Ala
110 115 120
Tyr Asp Arg Phe Ile Ala Ile Cys His Pro Leu Arg Tyr Arg Leu
125 130 135
Ile Met Ser Trp Ser Leu Cys Val Glu Leu Leu Val Gly Ser Leu
140 145 150
Val Leu Gly Phe Leu Leu Ser Leu Pro Leu Thr Ile Leu Ile Phe
155 160 165
His Leu Pro Phe Cys His Asn Asp Glu Ile Tyr His Phe Tyr Cys
170 175 180
Asp Met Pro Ala Val Met Arg Leu Ala Cys Ala Asp Thr Arg Val
185 190 195
His Lys Thr Ala Leu Tyr Ile Ile Ser Phe Ile Val Leu Ser Ile
200 205 210
Pro Leu Ser Leu Ile Ser Ile Ser Tyr Val Phe Ile Val Val Ala
215 220 225
Ile Leu Arg Ile Arg Ser Ala Glu Gly Arg Gln Gln Ala Tyr Ser
230 235 240
Thr Cys Ser Ser His Ile Leu Val Val Leu Leu Gln Tyr Gly Cys
245 250 255
Thr Ser Phe Ile Tyr Leu Ser Pro Ser Ser Ser Tyr Ser Pro Glu
260 265 270
Met Gly Arg Val Val Ser Val Ala Tyr Thr Phe Ile Thr Pro Ile
275 280 285
Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Glu Leu Lys Asp
290 295 300
Ala Leu Arg Lys Ala Leu Arg Lys Phe
4/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
305
<210> 3
<211> 319
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7485461CD1
<400> 3
Met Arg Ile Gln Ala Leu Gly Lys Tyr Ala His Ser Lys Trp Glu
1 5 10 15
Lys Leu Ala Lys Thr Met Glu Leu Gln Ala Pro Tyr Val Pro Glu
20 25 30
Leu Gln Val Ala Val Phe Thr Phe Leu Phe Leu Ala Tyr Leu Leu
35 40 45
Ser Ile Leu Gly Asn Leu Thr Ile Leu Ile Leu Thr Leu Leu Asp
50 55 60
Ser His Leu Gln Thr Pro Met Tyr Phe Phe Leu Arg Asn Phe Ser
65 70 75
Phe Leu Glu Ile Ser Phe Thr Asn Ile Phe Ile Pro Arg Val Leu
80 85 90
Ile Ser Ile Thr Thr Gly Asn Lys Ser Ile Ser Phe Ala Gly Cys
95 100 105
Phe Thr Gln Tyr Phe Phe Ala Met Phe Leu Gly Ala Thr Glu Phe
110 115 120
Tyr Leu Leu Ala Ala Met Ser Tyr Asp Arg Tyr Val Ala Ile Cys
125 130 135
Lys Pro Leu His Tyr Thr Thr Ile Met Ser Ser Arg Ile Cys Ile
140 145 150
Gln Leu Ile Phe Cys Ser Trp Leu Gly Gly Leu Met Ala Ile Ile
155 160 165
Pro Thr Ile Thr Leu Met Ser Gln Gln Asp Phe Cys Ala Ser Asn
170 175 180
Arg Leu Asn His Tyr Phe Cys Asp Tyr Glu Pro Leu Leu Glu Leu
185 190 195
Ser Cys Ser Asp Thr Ser Leu Ile Glu Lys Val Val Phe Leu Val
200 205 210
Ala Ser Val Thr Leu Val Val Thr Leu Val Leu Val Ile Leu Ser
215 220 225
Tyr Ala Phe Ile Ile Lys Thr Ile Leu Lys Leu Pro Ser Ala Gln
230 235 240
Gln Arg Thr Lys Ala Phe Ser Thr Cys Ser Ser His Met Ile Val
245 250 255
Ile Ser Leu Ser Tyr Gly Ser Cys Met Phe Met Tyr Ile Asn Pro
260 265 270
Ser Ala Lys Glu Gly Asp Thr Phe Asn Lys Gly Val Ala Leu Leu
275 280 285
Ile Thr Ser Val Ala Pro Leu Leu Asn Pro Phe Ile Tyr Thr Leu
290 295 300
Arg Asn Gln Gln Val Lys Gln Pro Phe Lys Asp Met Val Lys Lys
305 310 315
Leu Leu Asn Leu
5/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
<210> 4
<211> 309
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3794336CD1
<400> 4
Met Glu Arg Ile Asn His Thr Ser Ser Val Ser Glu Phe Ile Leu
1 5 10 15
Leu Gly Leu Ser Ser Arg Pro Glu Asp Gln Lys Thr Leu Phe Val
20 25 30
Leu Phe Leu Ile Val Tyr Leu Val Thr Ile Thr Gly Asn Leu Leu
35 40 45
Ile Ile Leu Ala Ile Arg Phe Asn Pro His Leu Gln Thr Pro Met
50 55 60
Tyr Phe Phe Leu Ser Phe Leu Ser Leu Thr Asp Ile Cys Phe Thr
65 70 75
Thr Ser Val Val Pro Lys Met Leu Met Asn Phe Leu Ser Glu Lys
80 85 90
Lys Thr Ile Ser Tyr Ala Gly Cys Leu Thr Gln Met Tyr Phe Leu
95 100 105
Tyr Ala Leu Gly Asn Ser Asp Ser Cys Leu Leu Ala Val Met Ala
110 115 120
Phe Asp Arg Tyr Val Ala Val Cys Asp Pro Phe His Tyr Val Thr
125 130 135
Thr Met Ser His His His Cys Val Leu Leu Val Ala Phe Ser Cys
140 145 150
Ser Phe Pro His Leu His Ser Leu Leu His Thr Leu Leu Leu Asn
155 160 165
Arg Leu Thr Phe Cys Asp Ser Asn Val Ile His His Phe Leu Cys
170 175 180
Asp Leu Ser Pro Val Leu Lys Leu Ser Cys Ser Ser Ile Phe Val
185 190 195
Asn Glu Ile Val Gln Met Thr Glu Ala Pro Ile Val Leu Val Thr
200 205 210
Arg Phe Leu Cys Ile Ala Phe Ser Tyr Ile Arg Ile Leu Thr Thr
215 220 225
Val Leu Lys Ile Pro Ser Thr Ser Gly Lys Arg Lys Ala Phe Ser
230 235 240
Thr Cys Gly Phe Tyr Leu Thr Val Val Thr Leu Phe Tyr Gly Ser
245 250 255
Ile Phe Cys Val Tyr Leu Gln Pro Pro Ser Thr Tyr Ala Val Lys
260 265 270
Asp His Val Ala Thr Ile Val Tyr Thr Val Leu Ser Ser Met Leu
275 280 285
Asn Pro Phe Ile Tyr Sew Leu Arg Asn Lys Asp Leu Lys Gln Gly
290 295 300
Leu Arg Lys Leu Met Ser Lys Arg Ser
305
<210> 5
<211> 314
<212> PRT
6/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 70829011CD1
<400> 5
Met Arg Gly Phe Asn Lys Thr Thr Val Val Thr Gln Phe Ile Leu
1 5 10 15
Val Gly Phe Ser Ser Leu Gly Glu Leu Gln Leu Leu Leu Phe Val
20 25 30
Ile Phe Leu Leu Leu Tyr Leu Thr Ile Leu Val Ala Asn Val Thr
35 40 45
Ile Met Ala Val Ile Arg Phe Ser Trp Thr Leu His Thr Pro Met
50 55 60
Tyr Gly Phe Leu Phe Ile Leu Ser Phe Ser Glu Ser Cys Tyr Thr
65 70 75
Phe Val Ile Ile Pro Gln Leu Leu Val His Leu Leu Ser Asp Thr
80 85 90
Lys Thr Ile Ser Phe Met Ala Cys Ala Thr Gln Leu Phe Phe Phe
95 ' 100 105
Leu Gly Phe Ala Cys Thr Asn Cys Leu Leu Ile Ala Val Met Gly
110 115 120
Tyr Asp Arg Tyr Val Ala Ile Cys His Pro Leu Arg Tyr Thr Leu
125 130 135
Ile Ile Asn Lys Arg Leu Gly Leu Glu Leu Ile Ser Leu Ser Gly
140 145 150
Ala Thr Gly Phe Phe Ile Ala Leu Val Ala Thr Asn Leu Ile Cys
155 160 165
Asp Met Arg Phe Cys Gly Pro Asn Arg Val Asn His Tyr Phe Cys
170 175 180
Asp Met Ala Pro Val Ile Lys Leu Ala Cys Thr Asp Thr His Val
185 190 195
Lys Glu Leu Ala Leu Phe Ser Leu Ser Ile Leu Val Ile Met Val
200 205 210
Pro Phe Leu Leu Ile Leu Ile Ser Tyr Gly Phe Ile Val Asn Thr
215 220 225
Ile Leu Lys Ile Pro Ser Ala Glu Gly Lys Lys Ala Phe Val Thr
230 235 240
Cys Ala Ser His Leu Thr Val Val Phe Val His Tyr Gly Cys Ala
245 250 255
Ser Ile Ile Tyr Leu Arg Pro Lys Ser Lys Ser Ala Ser Asp Lys
260 265 270
Asp Gln Leu Val Ala Val Thr Tyr Thr Val Val Thr Pro Leu Leu
275 280 285
Asn Pro Leu Val Tyr Ser Leu Arg Asn Lys Glu Val Lys Thr Ala
290 295 300
Leu Lys Arg Val Leu Gly Met Pro Val Ala Thr Lys Met Ser
305 310
<210> 6
<211> 317
<212> PRT
<213> Homo Sapiens
<220>
7/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
<221> misc_feature
<223> Incyte ID No: 7485466CD1
<400> 6
Met Glu Gly Lys Asn Gln Thr Ala Pro Ser Glu Phe Ile Ile Leu
1 5 10 15
Gly Phe Asp His Leu Asn Glu Leu Gln Tyr Leu Leu Phe Thr Ile
20 25 30
Phe Phe Leu Thr Tyr Ile Cys Thr Leu Gly Gly Asn Val Phe Ile
35 40 45
Ile Val Val Thr Ile Ala Asp Ser His Leu His Thr Pro Met Tyr
50 55 60
Tyr Phe Leu Gly Asn Leu Ala Leu Ile Asp Ile Cys Tyr Thr Thr
65 70 75
Thr Asn Val Pro Gln Met Met Val His Leu Leu Ser Glu Lys Lys
80 85 90
Il.e Ile Ser Tyr Gly Gly Cys Val Thr Gln Leu Phe Ala Phe Ile
95 100 105
Phe Phe Val Gly Ser Glu Cys Leu Leu Leu Ala Ala Met Ala Tyr
110 115 120
Asp Arg Tyr Ile Ala Ile Cys Lys Pro Leu Arg Tyr Ser Phe Ile
125 130 135
Met Asn Lys Ala Leu Cys Ser Trp Leu Ala Ala Ser Cys Trp Thr
140 145 150
Cys Gly Phe Leu Asn Ser Val Leu His Thr Val Leu Thr Phe His
155 160 165
Leu Pro Phe Cys Gly Asn Asn Gln Ile Asn Tyr Phe Phe Cys Asp
170 175 180
Ile Pro Pro Leu Leu Ile Leu Ser Cys Gly Asp Thr Ser Leu Asn
185 190 195
Glu Leu Ala Leu Leu Ser Ile Gly Ile Leu Ile Ser Trp Thr Pro
200 205 210
Phe Leu Cys Ile Ile Leu Ser Tyr Leu Tyr Ile Ile Ser Thr Ile
215 220 225
Leu Arg Ile Arg Ser Ser Glu Gly Arg His Lys Ala Phe Ser Thr
230 235 240
Cys Ala Ser His Leu Leu Ile Val Ile Leu Tyr Tyr Gly Ser Ala
245 250 255
Ile Phe Thr Tyr Val Arg Pro Ile Ser Ser Tyr Ser Leu Glu Lys
260 265 270
Asp Arg Leu Ile Ser Val Leu Tyr Ser Val Phe Thr Pro Met Leu
275 280 285
Asn Pro Val Ile Tyr Thr Leu Arg Asn Lys Asp Ile Lys Glu Ala
290 295 300
Val Lys Ala Ile Gly Arg Lys Trp Gln Pro Pro Val Phe Ser Ser
305 310 315
Asp Ile
<210> 7
<211> 312
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
8/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
<223> Incyte ID No: 7485914CD1
<400> 7
Met Ala Leu Gly Asn His Ser Thr Ile Thr Glu Phe Leu Leu Leu
1 5 10 15
Gly Leu Ser Ala Asp Pro Asn Ile Arg Ala Leu Leu Phe Val Leu
20. 25 30
Phe Leu Gly Ile Tyr Leu Leu Thr Ile Met Glu Asn Leu Met Leu
35 40 45
Leu Leu Val Ile Arg Ala Asp Ser Cys Leu His Lys Pro Met Tyr
50 55 60
Phe Phe Leu Ser His Leu Ser Phe Val Asp Leu Cys Phe Ser Ser
65 70 75
Val Ile Val Pro Lys Met Leu Glu Asn Leu Leu Ser Gln Arg Lys
80 85 90
Thr Ile Ser Val Glu Gly Cys Leu Ala Gln Val Phe Phe Val Phe
95 100 105
Val Thr Ala Gly Thr Glu Ala Cys Leu Leu Ser Gly Met Ala Tyr
110 115 120
Asp Arg His Ala Ala Ile Arg Arg Pro Leu Leu Tyr Gly Gln Ile
125 130 135
Met Gly Lys Gln Leu Tyr Met His Leu Val Trp Gly Ser Trp Gly
140 145 150
Leu Gly Phe Leu Asp Ala Leu Ile Asn Val Leu Leu Ala Val Asn
155 160 165
Met Val Phe Cys Glu Ala Lys Ile Ile His His Tyr Ser Tyr Glu
170 175 180
Met Pro Ser Leu Leu Pro Leu Ser Cys Ser Asp Ile Ser Arg Ser
185 190 195
Leu Ile Val Leu Leu Cys Ser Thr Leu Leu His Gly Leu Gly Asn
200 205 210
Phe Leu Leu Val Phe Leu Ser Tyr Thr Arg Ile Ile Ser Thr Ile
215 220 225
Leu Ser Ile Ser Ser Thr Ser Gly Arg Ser Lys Ala Phe Ser Thr
230 235 240
Cys Ser Ala His Leu Thr Ala Val Thr Leu Tyr Tyr Gly Ser Gly
245 250 255
Leu Leu Arg His Leu Met Pro Asn Ser Gly Ser Pro Ile Glu Leu
260 265 270
Ile Phe Ser Val Gln Tyr Thr Val Val Thr Pro Met Leu Asn Ser
275 280 285
Leu Ile Tyr Ser Leu Lys Asn Lys Glu Val Lys Val Ala Leu Lys
290 295 300
Arg Thr Leu Glu Lys Tyr Leu Gln Tyr Thr Arg Arg
305 310
<210> 8
<211> 311
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7475184CD1
<400> 8
9/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
Met Gly Thr Gly Asn Asp Ser Thr Val Val Glu Phe Thr Leu Leu
1 5 10 15
Gly Leu Ser Glu Asp Thr Thr Val Cys Ala Ile Leu Phe Leu Val
20 25 30
Phe Leu Gly Ile Tyr Val Val Thr Leu Met Gly Asn Ile Ser Ile
35 40 45
Ile Val Leu Ile Arg Arg Ser His His Leu His Thr Pro Met Tyr
50 55 60
Ile Phe Leu Cys His Leu Ala Phe Val Asp Ile Gly Tyr Ser Ser
65 70 75
Ser Val Thr Pro Val Met Leu Met Ser Phe Leu Arg Lys Glu Thr
80 85 90
Ser Leu Pro Val Ala Gly Cys Val Ala Gln Leu Cys Ser Val Val
95 100 105
Thr Phe Gly Thr Ala Glu Cys Phe Leu Leu Ala Ala Met Ala Tyr
110 115 120
Asp Arg Tyr Val Ala Ile Cys Ser Pro Leu Leu Tyr Ser Thr Cys
125 130 135
Met Ser Pro Gly Val Cys Ile Ile Leu Val Gly Met Ser Tyr Leu
140 145 150
Gly Gly Cys Val Asn Ala Trp Thr Phe Ile Gly Cys Leu Leu Arg
155 160 165
Leu Ser Phe Cys Gly Pro Asn Lys Val Asn His Phe Phe Cys Asp
170 175 180
Tyr Ser Pro Leu Leu Lys Leu Ala Cys Ser His Asp Phe Thr Phe
185 190 195
Glu Ile Ile Pro Ala Ile Ser Ser Gly Ser Ile Ile Val Ala Thr
200 205 210
Val Cys Val Ile Ala Ile Ser Tyr Ile Tyr Ile Leu Ile Thr Ile
215 220 225
Leu Lys Met His Ser Thr Lys Gly Arg His Lys Ala Phe Ser Thr
230 235 240
Cys Thr Ser His Leu Thr Ala Val Thr Leu Phe Tyr Gly Thr Ile
245 250 255
Thr Phe Ile Tyr Val Met Pro Lys Ser Ser Tyr Ser Thr Asp Gln
260 265 270
Asn Lys Val Val Ser Val Phe Tyr Thr Val Val Ile Pro Met Leu
275 280 285
Asn Pro Leu Ile Tyr Ser Leu Arg~Asn Lys Glu Ile Lys Gly Ala
290 ~ 295 300
Leu Lys Arg Glu Leu Arg Ile Lys Ile Phe Ser
305 310
<210> 9
<211> 316
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7478355CD1
<400> 9
Met Glu Ala Ala Asn Glu Ser Ser Glu Gly Ile Ser Phe Val Leu
1 5 10 15
Leu Gly Leu Thr Thr Ser Pro Gly Gln Gln Arg Pro Leu Phe Val
10/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
20 25 30
Leu Phe Leu Leu Leu Tyr Val Ala Ser Leu Leu Gly Asn Gly Leu
35 40 45
Ile Val Ala Ala Ile Gln Ala Ser Pro Ala Leu His Ala Pro Met
50 55 60
Tyr Phe Leu Leu Ala His Leu Ser Phe Ala Asp Leu Cys Phe Ala
65 70 75
Ser Val Thr Val Pro Lys Met Leu Ala Asn Leu Leu Ala His Asp
80 85 90
His Ser Ile Ser Leu Ala Gly Cys Leu Thr Gln Met Tyr Phe Phe
95 100 105
Phe Ala Leu Gly Val Thr Asp Ser Cys Leu Leu Ala Ala Met Ala
110 115 120
Tyr Asp Cys Tyr Val Ala Ile Arg His Pro Leu Pro Tyr Ala Thr
125 130 135
Arg Met Ser Arg Ala Met Cys Ala Ala Leu Val Gly Met Ala Trp
140 145 150
Leu Val Ser His Val His Ser Leu Leu Tyr Ile Leu Leu Met Ala
155 160 165
Arg Leu Ser Phe Cys Ala Ser His Gln Val Pro His Phe Phe Cys
170 175 180
Asp His Gln Pro Leu Leu Arg Leu Ser Cys Ser Asp Thr His His
185 190 195
Ile Gln Leu Leu Ile Phe Thr Glu Gly Ala Ala Val Val Val Thr
200 205 210
Pro Phe Leu Leu Ile Leu Ala Ser Tyr Gly Ala Ile Ala Ala Ala
215 220 225
Val Leu Gln Leu Pro Ser Ala Ser Gly Arg Leu Arg Ala Val Ser
230 235 240
Thr Cys Gly Ser His Leu Ala Val Val Ser Leu Phe Tyr Gly Thr
245 250 255
Val Ile Ala Val Tyr Phe Gln Ala Thr Ser Arg Arg Glu Ala Glu
260 265 270
Trp Gly Arg Val Ala Thr Val Met Tyr Thr Val Val Thr Pro Met
275 280 285
Leu Asn Pro Ile Ile Tyr Ser Leu Trp Asn Arg Asp Val Gln Gly
290 295 300
Ala Leu Arg Ala Leu Leu Ile Gly Arg Arg Ile Ser Ala Ser Asp
305 310 315
Ser
<210> 10
<211> 316
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7485473CD1
<400>
Met Ala Glu Asn Thr Arg Val Glu Phe Ile Leu
Pro Phe Thr Thr
1 5 10 15
Gly Val Ser Cys Glu Leu Gln Pro Leu Phe Leu
Ser Pro Ile Val
20 25 30
11/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
Phe Leu Val Leu Tyr Gly Leu Thr Met Ala Gly Asn Leu Gly Ile
35 40 45
Ile Thr Leu Thr Ser Val Asp Ser Arg,Leu Gln Thr Pro Met Tyr
50 55 60
Phe Phe Leu Gln His Leu Ala Leu Ile Asn Leu Gly Asn Ser Thr
65 70 75
Val Ile Ala Pro Lys Met Leu Ile Asn Phe Leu Val Lys Lys Lys
80 85 90
Thr Thr Ser Phe Tyr Glu Cys Ala Thr Gln Leu Gly Gly Phe Leu
95 100 105
Phe Phe Ile Val Ser Glu Val Ile Met Leu Ala Leu Met Ala Tyr
110 115 120
Asp Arg Tyr Val Ala Ile Cys Asn Pro Leu Leu Tyr Met Val Val
125 130 135
Val Ser Arg Arg Leu Cys Leu Leu Leu Val Ser Leu Thr Tyr Leu
140 145 150
Tyr Gly Phe Ser Thr Ala Ile Val Val Ser Ser Tyr Val Phe Ser
155 160 165
Val Ser Tyr Cys Ser Ser Asn Ile Ile Asn His Phe Tyr Cys Asp
170 175 180
Asn Val Pro Leu Leu Ala Leu Ser Cys Ser Asp Thr Tyr Leu Pro
185 190 195
Glu Thr Val Val Phe Ile Ser Ala Ala Thr Asn Val Val Gly Ser
200 205 210
Leu Ile Ile Val Leu Val Ser Tyr Phe Asn Ile Val Leu Ser Ile
215 220 225
Leu Lys Ile Cys Ser Ser Glu Gly Arg Lys Lys Ala Phe Ser Thr
230 235 240
Cys Ala Ser His Met Met Ala Val Thr Ile Phe Tyr Gly Thr Leu
245 250 255
Leu Phe Met Tyr Val Gln Pro Arg Ser Asn His Ser Leu Asp Thr
260 265 270
Asp Asp Lys Met Ala Ser Val Phe Tyr Thr Leu Val Ile Pro Met
275 280 285
Leu Asn Pro Leu Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Thr
290 295 300
Ala Leu Gln Arg Phe Met Thr Asn Leu Cys Tyr Ser Phe Lys Thr
305 310 315
Met
<210> 11
<211> 314
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7679085CD1
<400> 11
Met Asn Asn Ser Asp Thr Arg Ile Ala Gly Cys Phe Leu Thr Gly
1 5 10 15
Ile Pro Gly Leu Glu Gln Leu His Ile Trp Leu Ser Ile Pro Phe
20 25 30
Cys Ile Met Tyr Ile Thr Ala Leu Glu Gly Asn Gly Ile Leu Ile
12/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
35 40 45
Cys Val Ile Leu Ser Gln Ala Ile Leu His Glu Pro Met Tyr Ile
50 55 60
Phe Leu Ser Met Leu Ala Ser Ala Asp Val Leu Leu Ser Thr Thr
65 70 75
Thr Met Pro Lys Ala Leu Ala Asn Leu Trp Leu Gly Tyr Ser Leu
80 85 90
Ile Ser Phe Asp Gly Cys Leu Thr Gln Met Phe Phe Ile His Phe
95 100 105
Leu Phe Ile His Ser Ala Val Leu Leu Ala Met Ala Phe Asp Arg
110 115 120
Tyr Val Ala Ile Cys Ser Pro Leu Arg Tyr Val Thr Ile Leu Thr
125 130 135
Ser Lys Val Ile Gly Lys Ile Val Thr Ala Ala Leu Ser His Ser
140 145 150
Phe Ile Ile Met Phe Pro Ser Ile Phe Leu Leu Glu His Leu His
155 160 165
Tyr Cys Gln Ile Asn Ile Ile Ala His Thr Phe Cys Glu His Met
170 175 180
Gly Ile Ala His Leu Ser Cys Ser Asp Ile Ser Ile Asn Val Trp
185 190 195
Tyr Gly Leu Ala Ala Ala Leu Leu Ser Thr Gly Leu Asp Ile Met
200 205 210
Leu Ile Thr Val Ser Tyr Ile His Ile Leu Gln Ala Val Phe Arg
215 220 225
Leu Leu Ser Gln Asp Ala Arg Ser Lys Ala Leu Ser Thr Cys Gly
230 235 240
Ser His Ile Cys Val Ile Leu Leu Phe Tyr Val Pro Ala Leu Phe
245 250 255
Ser Val Phe Ala Tyr Arg Phe Gly Gly Arg Ser Ile Pro Cys Tyr
260 265 270
Val His Ile Leu Leu Ala Ser Leu Tyr Val Val Ile Pro Pro Met
275 280 285
Leu Asn Pro Val Ile Tyr Gly Val Arg Thr Lys Pro Ile Leu Glu
290 295 300
Gly Ala Lys Gln Met Phe Ser Asn Leu Ala Lys Gly Ser Lys
305 310
<210> 12
<211> 3486
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7924827CB1
<400> 12
atggtctgtt cggctgcccc actgctgctc ctggccacaa ctcttcccct gctggggtca 60
ccagttgccc aagcatccca acctctttgg ccgatggcca agggccagac aatgtgggcc 120
cagacctcca ccctcaccct gacagaggag gagttgggac agagtcaggc tggaggggaa 180
tctggatctg ggcagctcct ggaccaagag aatggagcag gggaatcagc gctggtctcc 240
gtctatgtac atctggactt tccagataag acctggcccc ctgaactctc caggacactg 300
actctccctg ctgcctcagc ttcctcttcc ccaaggcctc ttctcactgg cctcagactc 360
acaacagagt gtaatgtcaa ccacaagggg aatttctatt gtgcttgcct ctctggctac 420
cagtggaaca ccagcatctg cctccattac cctccttgtc aaagcctcca caaccaccag 480
13/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
ccttgtggct gccttgtctt cagccatccc gaacccgggt actgccagtt gctgccacct 540
gtccccggga tcctcaacct gaactcccag ctgcagatgc ctggtgacac gctgagcctg 600
actctccatc tgagccagga ggccaccaac ctgagctggt tcctgaggca cccagggagc 660
cccagtccca tcctcctgca gccagggaca caggtgtctg tgacttccag ccacggccag 720
gctgccctca gcgtctccaa catgtcccat cactgggcag gtgagtacat gagctgcttc 780
gaggcccagg gcttcaagtg gaacctgtat gaggtggtga gggtgccctt gaaggcgaca 840
gatgtggctc gacttccata ccagctgtcc atctcctgtg ccacctcccc tggcttccag 900
ctgagctgct gcatccccag cacaaacctg gcctacaccg cggcctggag ccctggagag 960
ggcagcaaag cttcctcctt caacgagtca ggctctcagt gctttgtgct ggctgttcag 1020
cgctgcccga tggctgacac cacgtacgct tgtgacctgc agagcctggg cctggctcca 1080
ctcagggtcc ccatctccat caccatcatc caggatggag acatcacctg ccctgaggac 1140
gcctcggtgc tcacctggaa tgtcaccaag gctggccacg tggcacaggc cccatgtcct 1200
gagagcaaga ggggcatagt gaggaggctc tgtggggctg acggagtctg ggggccggtc 1260
cacagcagct gcacagatgc gaggctcctg gccttgttca ctagaaccaa gctgctgcag 1320
gcaggccagg gcagtcctgc tgaggaggtg ccacagatcc tggcacagct gccagggcag 1380
gcggcagagg caagttcacc ctccgactta ctgaccctgc tgagcaccat gaaatacgtg 1440
gccaaggtgg tggcagaggc cagaatacag cttgaccgca gagccctgaa gaatctcctg 1500
attgccacag acaaggtcct agatatggac accaggtctc tgtggaccct ggcccaagcc 1560
cggaagccct gggcaggctc gactctcctg ctggctgtgg agaccctggc atgcagcctg 1620
tgcccacagg accacccctt cgccttcagc ttacccaatg tgctgctgca gagccagctg 1680
tttggaccca cgtttcctgc tgactacagc atctccttcc ctactcggcc cccactgcag 1740
gctcagattc ccaggcactc actggcccca ttggtccgta atggaactga aataagtatt 1800
actagcctgg tgctgcgaaa actggaccac cttctgccct caaactatgg acaagggctg 1860
ggggattccc tctatgccac tcctggcctg gtccttgtca tttccatcat ggcaggtgac 1920
cgggccttca gccagggaga ggtcatcatg gactttggga acacagatgg ttcccctcac 1980
tgtgtcttct gggatcacag tctcttccag ggcagggggg gttggtccaa agaagggtgc 2040
caggcacagg tggccagtgc cagccccact gctcagtgcc tctgccagca cctcactgcc 2100
ttctccgtcc tcatgtcccc acacactgtt ccggaagaac ccgctctggc gctgctgact 2160
caagtgggct tgggagcttc catactggcg ctgcttgtgt gcctgggtgt gtactggctg 2220
gtgtggagag tcgtggtgcg gaacaagatc tcctatttcc gccacgccgc cctgctcaac 2280
atggtgttct gcttgctggc cgcagacact tgcttcctgg gcgccccatt cctctctcca 2340
gggccccgaa gcccgctctg ccttgctgcc gccttcctct gtcatttcct ctacctggcc 2400
acctttttct ggatgctggc gcaggccctg gtgttggccc accagctgct ctttgtcttt 2460
caccagctgg caaagcaccg agttctcccc ctcatggtgc tcctgggcta cctgtgccca 2520
ctggggttgg caggtgtcac cctggggctc tacctacctc aagggcaata cctgagggag 2580
ggggaatgct ggttggatgg gaagggaggg gcgttataca ccttcgtggg gccagtgctg 2640
gccatcatag gcgtgaatgg gctggtacta gccatggcca tgctgaagtt gctgagacct 2700
tcgctgtcag agggaccccc agcagagaag cgccaagctc tgctgggggt gatcaaagcc 2760
ctgctcattc ttacacccat ctttggcctc acctgggggc tgggcctggc cactctgtta 2820
gaggaagtct ccacggtccc tcattacatc ttcaccattc tcaacaccct ccagggcgtc 2880
ttcatcctat tgtttggttg cctcatggac aggaagatac aagaagcttt gcgcaaacgc 2940
ttctgccgcg cccaagcccc cagctccacc atctccctgg ccacaaatga aggctgcatc 3000
ttggaacaca gcaaaggagg aagcgacact gccaggaaga cagatgcttc agagtgaacc 3060
acacacggac ccatgttcct gcaagggagt tgaggctgtg tgcttgaacc caccagatga 3120
gccctggccc aatgctctga actcttcccg cctcccggag ctcagccctt gagaaaggca 3180
ggcttatatt tcccttagtg acactcattt atcttacagc tcaccccttc tcatttctaa 3240
agtatccagc aagaatagca ggaaaaatta gctaaaggca cctaatgaat aagcctgcct 3300
ttgctccaga aataatcgac agatatcaaa gtgcggaata attacaagta aactttctca 3360
accagttttt aactacaaca atacatgttg tgaatgaata tatttgataa aaatggtttt 3420
aattgaccta ttcagcgatt tctgattatt tctttttcaa tagttatgaa gaaaggatga 3480
cttact 3486
<210> 13
<211> 1010
<212> DNA
<213> Homo Sapiens
14/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
<220>
<221> misc_feature
<223> Incyte ID No: 7485408CB1
<400> 13
gaagttggga aagcatcata tgaaaccaga gatgccttga ggcccatgtc atctttcctc 60
ttaggtccaa tactctactc atggaaggaa taaataaaac tgcaaagatg cagtttttct 120
ttcgtccatt ctcacctgac cctgaggtcc agatgctgat ttttgtggtc ttcctgatga 180
tgtatctgac cagcctcggt ggaaatgcta caattgcagt cattgttcag atcaatcatt 240
ccctccacac accgatgtac tttttcctgg ctaatctggc agttctagaa atcttctata 300
catcttccat caccccattg gccttggcaa acctcctttc aatgggcaaa actcctgttt 360
ccatcacggg atgtggcacc cagatgtttt tctttgtctt cttgggtggg gctgattgtg 420
tcctgctggt agtcatggcc tacgaccggt ttatagcgat ctgtcaccct ctgcgataca 480
ggctcatcat gagctggtcc ttgtgtgtgg agctgctggt aggctccttg gtgctggggt 540
tcctgttgtc actgccactc accattttaa tcttccatct cccattctgc cacaatgatg 600
agatctacca cttctactgt gacatgcctg cagtcatgcg cctggcttgt gcagacacac 660
gcgttcacaa gactgctctg tatatcatca gcttcatcgt ccttagcatc cccctctcat 720
tgatctccat ctcctatgtc ttcatcgtgg tagccatttt acggatccgg tcagcagaag 780
ggcgccagca agcctactct acctgctctt ctcacatctt agtggtcctc ctgcagtatg 840
gctgcaccag ctttatatac ttgtccccca gttccagcta ctctcctgag atgggccggg 900
tggtatctgt ggcctacaca tttatcactc ccattttaaa ccccttgatc tatagtttga 960
ggaacaagga actgaaagat gccctaagga aagcattgag aaaattctag 1010
<210> 14
<211> 960
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7485461CB1
<400> 14
atgaggatac aggcattggg gaaatatgct cattccaaat gggaaaaatt ggccaaaaca 60
atggagctac aggccccata tgtccctgaa ctccaggtgg cagttttcac ctttcttttc 120
cttgcgtatt tactcagcat ccttggaaat ctgactatcc tcatcctcac cttgctggac 180
tcccaccttc agactcccat gtatttcttt ctccggaact tctccttctt ggaaatttcc 240
ttcacaaaca tcttcattcc aagggtcctg attagcatca caacagggaa caagagtatc 300
agctttgctg gctgcttcac tcagtatttc tttgccatgt tccttggggc tacagagttt 360
taccttctgg ctgccatgtc ctatgaccgc tatgtggcca tctgcaaacc tctgcattac 420
accaccatca tgagcagcag aatctgcatc cagctgattt tctgctcttg gctgggtggg 480
ctaatggcta ttataccaac aatcaccctg atgagtcagc aggacttttg tgcatccaac 540
agactgaatc attacttctg tgactatgag cctcttctgg aactctcatg ttcagacaca 600
agcctcatag aga~ggttgt ctttcttgtg gcatctgtga ccctggtggt cactctggtg 660
ctagtgattc tctcctatgc attcattatc aagactattc tgaagctccc ctctgcccaa 720
caaaggacaa aagccttttc cacatgttct tcccacatga ttgtcatctc cctctcttac 780
ggaagctgca tgtttatgta cattaatccc tctgcaaaag aaggggatac attcaacaag 840
ggagtagctc tactcattac ttcagttgct cctttgttga acccctttat ttacacccta 900
aggaaccaac aggtaaaaca acccttcaag gatatggtca aaaagcttct gaatctttaa 960
<210> 15
<211> 1801
<212> DNA
<213> Homo Sapiens
<220>
15/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
<221> misc_feature
<223> Incyte ID No: 3794336CB1
<400> 15
caaaggaaaa acgctggata cagagatttc aatataatgc ccttatggag cccatagatt 60
tgggaaggga ggaccccatc aaggaccatg aggtgaacaa gccatcccac aattagcaca 120
gtagctaatt gtgaccaaac agaagaaaca aagacttgct aggttctcat gattggtgtt 180
atgaaatcag agcatgttaa atgacaaaac ttcttgttta taactgagcc caagtcaatg 240
gaaagaatca accacaccag cagtgtctcc gagtttatcc tcctgggact ctcctcccgg 300
cctgaggacc aaaagacact ctttgttctc ttcctcatcg tgtacctggt caccataaca 360
gggaacctgc tcatcatcct ggccattcgc ttcaaccccc atcttcagac ccctatgtat 420
ttcttcttga gttttctgtc tctcactgat atttgcttta caacaagcgt tgtccccaag 480
atgctgatga acttcctgtc agaaaagaag accatctcct atgctgggtg tctgacacag 540
atgtattttc tctatgcctt gggcaacagt gacagctgcc ttctggcagt catggccttt 600
gaccgctatg tggccgtctg tgaccctttc cactatgtca ccaccatgag ccaccaccac 660
tgtgtcctgc tggtggcctt ctcctgctca tttcctcacc tccactcact cctgcacaca 720
cttctgctga atcgtctcac cttctgtgac tccaatgtta tccaccactt tctctgtgac 780
ctcagccctg tgctgaaatt gtcctgctct tccatatttg tcaatgaaat tgtgcagatg 840
acagaagcac ctattgtttt ggtgactcgt tttctctgca ttgctttctc ttatatacga 900
atcctcacta cagttctcaa gattccctct acttctggga aacgcaaagc cttctccacc 960
tgtggttttt acctcaccgt ggtgacgctc ttttatggaa gcatcttctg tgtctattta 1020
cagcccccat ccacctacgc tgtcaaggac cacgtggcaa caattgttta cacagttttg 1080
tcatccatgc tcaatccttt tatctacagc ctgagaaaca aagacctgaa acagggcctg 1140
aggaagctta tgagcaagag atcctaggaa gcaccctctt gaaaaactcg taagtggaat 1200
ctgctcaact tggacgtgtt ttctactggt ttctggtgaa cagtcaaagc tgttggaagc 1260
tagcacttct gacccatgtg agacaaggct attgtgggca cttacatcca ttgatgatga 1320
cccaacaatt cggcctgtat ctcttaaatc acaatcgttt cctgtctgtg tctcctcttt 1380
cttggaaaga tttatttttt ccactttctc attttccaaa aactgcttta atctaatcct 1440
ttccccatga atatttccta aacaaatttc tctcctttta ttaaggcaga tcctccaaaa 1500
ttcttcacat ttcaatatat tgctgaaaaa tgtgtaattt gtagccattg aatgtttttg 1560
caaaaaaatt gaaaagagaa agaatgaagg aagaggagga tatatatttt agctaatttt 1620
ctcttcttga gaatttttat aattttttat ttttctcctt ctaaaaatgt tttattgctt 1680
aaatcttaag cttttacttt tttatctttc tatccttcct ttattatact gctgtagttt 1740
tatttacttt taatttcctc ttatatttta tcatacaatt taaaaatgct aatggtcaga 1800
a 1801
<210> 16
<211> 1205
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 70829011CB1
<400> 16
ttacaaagat agttatcatt ttgtctcttt caaacacatt cacagaaaga agttcttcag 60
atgcgaggtt tcaacaaaac cactgtggtt acacagttca tcctggtggg tttctccagc 120
ctgggggagc tccagctgct gctttttgtc atctttcttc tcctatactt gacaatcctg 180
gtggccaatg tgaccatcat ggccgttatt cgcttcagct ggactctcca cactcccatg 240
tatggctttc tattcatcct ttcattttct gagtcctgct acacttttgt catcatccct 300
cagctgctgg tccacctgct ctcagacacc aagaccatct ccttcatggc ctgtgccacc 360
cagctgttct ttttccttgg ctttgcttgc accaactgcc tcctcattgc tgtgatggga 420
tatgatcgct atgtagcaat ttgtcaccct ctgaggtaca cactcatcat aaacaaaagg 480
ctggggttgg agttgatttc tctctcagga gccacaggtt tctttattgc tttggtggcc 540
accaacctca tttgtgacat gcgtttttgt ggccccaaca gggttaacca ctatttctgt 600
16/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
gacatggcac ctgttatcaa gttagcctgc actgacaccc atgtgaaaga gctggcttta 660
tttagcctca gcatcctggt aattatggtg ccttttctgt taattctcat atcctatggc 720
ttcatagtta acaccatcct gaagatcccc tcagctgagg gcaagaaggc ctttgtcacc 780
tgtgcctcac atctcactgt ggtctttgtc cactatggct gtgcctctat catctatctg 840
cggcccaagt ccaagtctgc ctcagacaag gatcagttgg tggcagtgac ctacacagtg 900
gttactccct tacttaatcc tcttgtctac agtctgagga acaaagaggt aaaaactgca 960
ttgaaaagag ttcttggaat gcctgtggca accaagatga gctaacaaaa aataataata 1020
aaattaacta ggatagtcac agaagaaatc aaaggcataa aattttctga cctttaatgc 1080
atgtctcaga cagtgtttcc aaggattaag actactcttg cctttttatt ttctcctatt 1140
ccaaaaagaa aaaaaatgca agtcaatcta cactctatat tgtccgatgt ctagttaaaa 1200
aaaaa 1205
<210> 17
<211> 1050
<212> DNA
<213> Homo Sapiens
<220>
<221> mist feature
<223> Incyte ID No: 7485466CB1
<400> 17
gtactagcac ttgctttgtt gttttgcaga ctattatcaa tgcacctgtt tgtcaaattc 60
acaaaggcaa accacctgac atcatggaag gaaagaatca aacagctcca tctgaattca 120
tcatcttggg gttcgaccac ctgaatgaat tgcagtattt actcttcacc atcttctttc 180
tgacctacat atgcacttta ggaggcaatg tttttatcat tgtggtgacc atagctgatt 240
cccacctaca cacacccatg tattatttcc taggaaatct tgcccttatt gacatctgct 300
acactactac taatgtcccc cagatgatgg tgcatcttct gtcagagaag aaaatcattt 360
cctatggagg ctgtgtgacc cagctctttg cattcatttt ctttgttggc tcagagtgtc 420
tcctcctggc agcaatggca tatgatcgat atattgctat ctgtaagccg ttaaggtact 480
catttattat gaacaaggcc ctgtgcagct ggttagcagc ctcatgctgg acatgtgggt 540
ttctcaactc agtgttgcac accgttctga ccttccacct gcccttctgt ggtaacaatc 600
agatcaatta tttcttctgt gacatacctc ccttgctcat cttgtcttgt ggtgatactt 660
ccctcaatga actggctttg ctgtccattg ggatcctcat aagctggact cctttcctgt 720
gcatcatcct ttcctacctt tacatcatct ccaccatcct gaggatccgt tcctctgagg 780
ggaggcacaa agccttttcc acctgtgcct cccacctgct cattgttatt ctctattatg 840
gcagtgctat cttcacgtat gtgaggccca tctcatctta ctcgctagag aaagatagat 900
tgatctcagt gctgtatagt gttttcacac ccatgctgaa tcctgtaatt tatacgctaa 960
ggaataagga catcaaagag gctgtgaagg ccatagggag aaagtggcag ccaccagttt 1020
tctcttctga tatataacct ctcttatgtg 1050
<210> 18
<211> 939
<212> DNA
<213> Homo Sapiens
<220>
<221> mist feature
<223> Incyte ID No: 7485914CB1
<400> 18
atggccttgg ggaatcacag caccatcacc gagttcctcc tccttgggct gtctgccgac 60
cccaacatcc gggctctgct ctttgtgctg ttcctgggga tttacctcct gaccataatg 12'0
gaaaacctga tgctgctgct cgtgatcagg gctgattctt gtctccataa gcccatgtat 180
ttcttcctga gtcacctctc ttttgttgat ctctgcttct cttcagtcat tgtgcccaag 240
atgctggaga acctcctgtc acagaggaaa accatttcag tagagggctg cctggctcag 300
17/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
gtcttctttg tgtttgtcac tgcagggact gaagcctgcc ttctctcagg gatggcctat 360
gaccgccatg ctgccatccg ccgcccacta ctttatggac agatcatggg taaacagctg 420
tatatgcacc ttgtgtgggg ctcatgggga ctgggctttc tggacgcact catcaatgtc 480
ctcctagctg taaacatggt cttttgtgaa gccaaaatca ttcaccacta cagctatgag 540
atgccatccc tcctccctct gtcctgctct gatatctcca gaagcctcat cgttttgctc 600
tgctccactc tcctacatgg gctgggaaac ttccttttgg tcttcttatc ctacacccgt 660
ataatctcta ccatcctaag catcagctct acctcgggca gaagcaaggc cttctccacc 720
tgctctgccc acctcactgc agtgacactt tactatggct caggtttgct ccgccatctc 780
atgccaaact caggttcccc catagagttg atcttctctg tgcagtatac tgtagtcact 840
cccatgctga attccctcat ctatagcctg aaaaataagg aagtgaaggt agctctgaaa 900
agaactttgg aaaaatattt gcaatatacc agacgttga 939
<210> 19
<211> 939
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7475184CB1
<400> 19
tagatgggga ctggaaatga ctccactgtg gtagagttta ctcttttggg attatccgag 60
gatactacag tttgtgctat tttatttctt gtgtttctag gaatttatgt tgtcacctta 120
atgggtaata tcagcataat tgtattgatc agaagaagtc atcatcttca tacacccatg 180
tacattttcc tctgccattt ggcctttgta gacattgggt actcctcatc agtcacacct 240
gtcatgctca tgagcttcct aaggaaagaa acctctctcc ctgttgctgg ttgtgtggcc 300
cagctctgtt ctgtagtgac gtttggtacg gccgagtgct tcctgctggc tgccatggcc 360
tatgatcgct atgtggccat ctgctcaccc ctgctctact ctacctgcat gtcccctgga 420
gtctgcatca tcttagtggg catgtcctac ctgggtggat gtgtgaatgc ttggacattc 480
attggctgct tattaagact gtccttctgt gggccaaata aagtcaatca ctttttctgt 540
gactattcac cacttttgaa gcttgcttgt tcccatgatt ttacttttga aataattcca 600
gctatctctt ctggatctat cattgtggcc actgtgtgtg tcatagccat atcctacatc 660
tatatcctca tcaccatcct gaagatgcac tccaccaagg gccgccacaa ggccttctcc 720
acctgcacct cccacctcac tgcagtcact ctgttctatg ggaccattac cttcatttat 780
gtgatgccca agtccagcta ctcaactgac cagaacaagg tggtgtctgt gttctacacc 840
gtggtgattc ccatgttgaa ccccctgatc tacagcctca ggaacaagga gattaagggg 900
gctctgaaga gagagcttag aataaaaata ttttcttga 939
<210> 20
<211> 951
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7478355CB1
<400> 20
atggaggctg ccaatgagtc ttcagaggga atctcattcg ttttattggg actgacaaca 60
agtcctggac agcagcggcc tctctttgtg ctgttcttgc tcttgtatgt ggccagcctc 120
ctgggtaatg gactcattgt ggctgccatc caggccagtc cagcccttca tgcacccatg 180
tacttcctgc tggcccacct gtcctttgct gacctctgtt tcgcctccgt cactgtgccc 240
aagatgttgg ccaacttgtt ggcccatgac cactccatct cgctggctgg ctgcctgacc 300
caaatgtact tcttctttgc cctgggggta actgatagct gtcttctggc ggccatggcc 360
tatgactgct acgtggccat ccggcacccc ctcccctatg ccacgaggat gtcccgggcc 420
18/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
atgtgcgcag ccctggtggg aatggcatgg ctggtgtccc acgtccactc cctcctgtat 480
atcctgctca tggctcgctt gtccttctgt gcttcccacc aagtgcccca cttcttctgt 540
gaccaccagc ctctattaag gctctcgtgc tctgacaccc accacatcca gctgctcatc 600
ttcaccgagg gcgccgcagt ggtggtcact cccttcctgc tcatcctcgc ctcctatggg 660
gccatcgcag ctgccgtgct ccagctgccc tcagcctctg ggaggctccg ggctgtgtcc 720
acctgtggct cccacctggc tgtggtgagc ctcttctatg ggacagtcat tgcagtctac 780
ttccaggcca catcccgacg cgaggcagag tggggccgtg tggccactgt catgtacact 840
gtagtcaccc ccatgctgaa ccccatcatc tacagcctct ggaatcgcga tgtacagggg 900
gcactccgag cccttctcat tgggcgaagg atctcagcta gtgactcctg a 951
<210> 21
<211> 971
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7485473CB1
<400> 21
atgaatttcc aaactctgac atggctcctg aaaatttcac cagagtcact gagtttattc 60
ttacaggtgt ctctagctgt ccagagctcc agattcccct cttcctggtc tttctggtgc 120
tctatgggct gaccatggca gggaacctgg gcatcatcac cctcaccagt gttgactctc 180
gacttcaaac ccccatgtac tttttcctgc aacatctggc tctcattaat cttggtaact 240
ctactgtcat tgcccctaaa atgctgatta actttttagt aaagaagaaa actacctcat 300
tctatgaatg tgccacccaa ctgggagggt tcttgttctt tattgtatcg gaggtaatca 360
tgctggcttt gatggcctat gaccgctatg tggctatttg taaccctctg ctgtacatgg 420
tggtggtgtc tcggcggctc tgcctcctgc tggtctccct cacatacctc tatggctttt 480
ctacagctat tgtggtttca tcttatgtat tctctgtgtc ttattgctct tctaatataa 540
tcaatcattt ttactgtgat aatgttcctc tgttagcatt atcttgctct gatacttact 600
taccagaaac agttgtcttt atatctgcag caacaaatgt ggttggttcc ttgattatag 660
ttctagtatc ttatttcaat attgttttgt ctattttaaa aatatgttca tcagaaggaa 720
ggaaaaaagc cttttctacc tgtgcttcac atatgatggc agtcacaatt ttttatggga 780
cattgctatt catgtatgtg cagccccgaa gtaaccattc actggatact gatgataaga 840
tggcttctgt gttttacacg ttggtaattc ctatgctgaa tcccttgatc tacagcctga 900
ggaataagga tgtgaagact gctctacaga gattcatgac aaatctgtgc tattccttta 960
aaacaatgta a 971
<210> 22
<211> 1092
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7679085CB1
<400> 22
gtcatcgggc ctgtaagtat atgcaggcct tgtgcatatg gacactctac taatgtattc 60
aaaaatctga agctcttttt tccctatggc acaggtgagg gcgctgcata aaatcatggc 120
cctttttctg ctaacagcat aggtgctatg aacaactctg acactcgcat agcaggctgc 180
ttcctcactg gcatccctgg gctggagcaa:ctacatatct ggctgtccat ccccttctgc 240
atcatgtaca tcactgccct ggaaggcaat ggcatcctaa tttgtgtcat cctctcccag 300
gcaatcctgc atgagcccat gtacatattc ttatctatgc tggccagtgc tgatgtcttg 360
ctctctacca ccaccatgcc taaggccctg gccaatttgt ggctaggtta tagcctcatt 420
tcctttgatg gctgcctcac tcagatgttc ttcattcact tcctcttcat tcactctgct 480
19/20
CA 02430993 2003-06-06
WO 02/46230 PCT/USO1/46659
gtcctgctgg ccatggcctt tgaccgctat gtggccatct gctcccccct gcgatatgtc 540
acaatcctca caagcaaggt cattgggaag atcgtcactg ccgccctgag ccacagcttc 600
atcattatgt ttccatccat ctttctcctt gagcacctgc actattgcca gatcaatatc 660
attgcacaca cattttgtga gcacatgggc attgcccatc tgtcctgttc tgatatctcc 720
atcaatgtct ggtatgggtt ggcagctgct cttctctcca caggcctaga catcatgctt 780
attactgttt cctacatcca catcctccaa gcagtcttcc gcctcctttc tcaagatgcc 840
cgctccaagg ccctgagtac ctgtggatcc catatctgtg tcatcctact cttctatgtc 900
cctgcccttt tttctgtctt tgcctacagg tttggtggga gaagcatccc atgctatgtc 960
catattctcc tggccagcct ctacgttgtc attcctccta tgctcaatcc cgttatttat 1020
ggagtgagga ctaagccaat actggaaggg gctaagcaga tgttttcaaa tcttgccaaa 1080
ggatctaaat as
1092
20/20
