Note: Descriptions are shown in the official language in which they were submitted.
WO 00/52151 PCT/US00/05621
HUMAN SECRETORY PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
secretory proteins
and to the use of these sequences in the diagnosis, treatment, and prevention
of cancer, inflammation,
and gastrointestinal, cardiovascular, and neurological disorders.
BACKGROUND OF THE INVENTION
Protein transport and secretion are essential for cellular function. Protein
transport is
mediated by a signal peptide located at the amino terminus of the protein to
be transported or
secreted. The signal peptide is comprised of about ten to twenty hydrophobic
amino acids which
target the nascent protein from the ribosome to a particular membrane bound
compartment such as the
endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed
through the secretory
pathway or remain in any of the secretory organelles such as the ER, Golgi
apparatus, or lysosomes.
Proteins that transit through the secretory pathway are either secreted into
the extracellular space or
retained in the plasma membrane. Secreted proteins are often synthesized as
inactive precursors that
are activated by post-translational processing events during transit through
the secretory pathway.
Such events include glycosylation, proteolysis, and removal of the signal
peptide by a signal
peptidase. Other events that may occur during protein transport include
chaperone-dependent
unfolding and folding of the nascent protein and interaction of the protein
with a receptor or pore
complex. Examples of secreted proteins with amino terminal signal peptides are
discussed below and
include receptors, extracellular matrix molecules, cytokines, hormones, growth
and differentiation
factors, neuropeptides, vasomediators, ion channels, transporters/pumps, and
proteases. (Reviewed in
Alberts, B. et al. (1994) Molecular Biology of The Cell, Garland Publishing,
New York, NY, pp. 557-
560, 582-592.)
G-protein coupled receptors (GPCRs) comprise a superfamily of integral
membrane proteins
which transduce extracellular signals. Not all GPCRs contain N-terminal signal
peptides. GPCRs
include receptors for biogenic amines such as dopamine, epinephrine,
histamine, glutamate
(metabotropic-type), acetylcholine (muscarinic-type), and serotonin; for lipid
mediators of
inflammation such as prostaglandins, platelet activating factor, and
leukotrienes; for peptide
hormones such as calcitonin, CSa anaphylatoxin, follicle stimulating hormone,
gonadotropin releasing
hormone, neurokinin, oxytocin, and thrombin; and for sensory signal mediators
such as retinal
photopigments and olfactory stimulatory molecules. The structure of these
highly conserved
receptors consists of seven hydrophobic transmembrane regions, cysteine
disulfide bridges between
the second and third extracellular loops, an extracellular N-terminus, and a
cytoplasmic C-terminus.
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The N-terminus interacts with ligands, the disulfide bridges interact with
agonists and antagonists,
and the large third intracellular loop interacts with G proteins to activate
second messengers such as
cyclic AMP, phospholipase C, inositol triphosphate, or ion channels. (Reviewed
in Watson, S. and
Arkinstall, S. (1994) The G-protein Linked Receptor Facts Book, Academic
Press, San Diego, CA,
pp. 2-6; and Bolander, F.F. (1994) Molecular Endocrinolo~y, Academic Press,
San Diego, CA, pp.
162-176.)
Other types of receptors include cell surface antigens identified on
leukocytic cells of the
immune system. These antigens have been identified using systematic,
monoclonal antibody (mAb)-
based "shot gun" techniques. These techniques have resulted in the production
of hundreds of mAbs
directed against unknown cell surface leukocytic antigens. These antigens have
been grouped into
"clusters of differentiation" based on common immunocytochemical localization
patterns in various
differentiated and undifferentiated leukocytic cell types. Antigens in a given
cluster are presumed to
identify a single cell surface protein and are assigned a "cluster of
differentiation" or "CD"
designation. Some of the genes encoding proteins identified by CD antigens
have been cloned and
verified by standard molecular biology techniques. CD antigens have been
characterized as both
transmembrane proteins and cell surface proteins anchored to the plasma
membrane via covalent
attachment to fatty acid-containing glycolipids such as
glycosylphosphatidylinositol (GPI).
(Reviewed in Barclay, A. N. et al. (1995) The Leucocvte Antigen Facts Book,
Academic Press, San
Diego, CA, pp. 17-20.)
Matrix proteins (MPs) are transmembrane and extracellular proteins which
function in
formation, growth, remodeling, and maintenance of tissues and as important
mediators and regulators
of the inflammatory response. The expression and balance of MPs may be
perturbed by biochemical
changes that result from congenital, epigenetic, or infectious diseases. In
addition, MPs affect
leukocyte migration, proliferation, differentiation, and activation in the
immune response. MPs are
frequently characterized by the presence of one or more domains which may
include collagen-like
domains, EGF-like domains, immunoglobulin-like domains, and fibronectin-like
domains. In
addition, MPs may be heavily glycosylated and may contain an Arginine-Glycine-
Aspartate (RGD)
tripeptide motif which may play a role in adhesive interactions. MPs include
extracellular proteins
such as fibronectin, collagen, galectin, vitronectin and its proteolytic
derivative somatomedin B; and
cell adhesion receptors such as cell adhesion molecules (CAMs), cadherins, and
integrins. (Reviewed
in Ayad, S. et al. (1994) The Extracellular Matrix Facts Book, Academic Press,
San Diego, CA, pp. 2-
16; Ruoslahti, E. (1997) Kidney Int. 51:1413-1417; Sjaastad, M.D. and Nelson,
W.J. (1997)
BioEssays 19:47-55.)
Cytokines are secreted by hematopoietic cells in response to injury or
infection. Interleukins,
neurotrophins, growth factors, interferons, and chemokines all define cytokine
families that work in
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conjunction with cellular receptors to regulate cell proliferation and
differentiation. In addition,
cytokines effect activities such as leukocyte migration and function,
hematopoietic cell proliferation,
temperature regulation, acute response to infection, tissue remodeling, and
apoptosis.
Chemokines, in particular, are small chemoattractant cytokines involved in
inflammation,
leukocyte proliferation and migration, angiogenesis and angiostasis,
regulation of hematopoiesis, HIV
infectivity, and stimulation of cytokine secretion. Chemokines generally
contain 70-100 amino acids
and are subdivided into four subfamilies based on the presence of conserved
cysteine-based motifs.
(Canard, R. and Gearing, A. (1994) The Cytokine Facts Book, Academic Press,
New York, NY, pp.
181-190, 210-213, 223-227.)
Growth and differentiation factors are secreted proteins which function in
intercellular
communication. Some factors require oligomerization or association with MPs
for activity. Complex
interactions among these factors and their receptors trigger intracellular
signal transduction pathways
that stimulate or inhibit cell division, cell differentiation, cell signaling,
and cell motility. Most
growth and differentiation factors act on cells in their local environment
(paracrine signaling). There
are three broad classes of growth and differentiation factors. The first class
includes the large
polypeptide growth factors such as epidermal growth factor, fibroblast growth
factor, transforming
growth factor, insulin-like growth factor, and platelet-derived growth factor.
The second class
includes the hematopoietic growth factors such as the colony stimulating
factors (CSFs).
Hematopoietic growth factors stimulate the proliferation and differentiation
of blood cells such as B-
lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils,
neutrophils,
macrophages, and their stem cell precursors. The third class includes small
peptide factors such as
bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin II,
vasoactive intestinal peptide,
and bradykinin which function as hormones to regulate cellular functions other
than proliferation.
Growth and differentiation factors play critical roles in neoplastic
transformation of cells in
vitro and in tumor progression in vivo. Inappropriate expression of growth
factors by tumor cells may
contribute to vascularization and metastasis of tumors. During hematopoiesis,
growth factor
misregulation can result in anemias, leukemias, and lymphomas. Certain growth
factors such as
interferon are cytotoxic to tumor cells both in vivo and in vitro. Moreover,
some growth factors and
growth factor receptors are related both structurally and functionally to
oncoproteins. In addition,
growth factors affect transcriptional regulation of both proto-oncogenes and
oncosuppressor genes.
(Reviewed in Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Ann
Arbor, MI, pp. I-9.)
Proteolytic enzymes or proteases either activate or deactivate proteins by
hydrolyzing peptide
bonds. Proteases are found in the cytosol, in membrane-bound compartments, and
in the extracellular
space. The major families are the zinc, serine, cysteine, thiol, and carboxyl
proteases.
Ion channels, ion pumps, and transport proteins mediate the transport of
molecules across
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cellular membranes. Transport can occur by a passive, concentration-dependent
mechanism or can be
linked to an energy source such as ATP hydrolysis. Symporters and antiporters
transport ions and
small molecules such as amino acids, glucose, and drugs. Symporters transport
molecules and ions
unidirectionally, and antiporters transport molecules and ions
bidirectionally. Transporter
superfamilies include facilitative transporters and active ATP-binding
cassette transporters which are
involved in multiple-drug resistance and the targeting of antigenic peptides
to MHC Class I
molecules. These transporters bind to a specific ion or other molecule and
undergo a conformational
change in order to transfer the ion or molecule across the membrane. (Reviewed
in Alberts, B. et al.
(1994) Molecular Biology of The Cell, Garland Publishing, New York, NY, pp.
523-546.)
Ion channels are formed by transmembrane proteins which create a lined
passageway across
the membrane through which water and ions, such as Na+, K+, Ca2+, and Cl-,
enter and exit the cell.
For example, chloride channels are involved in the regulation of the membrane
electric potential as
well as absorption and secretion of ions across the membrane. Chloride
channels also regulate the
internal pH of membrane-bound organelles.
Ion pumps are ATPases which actively maintain membrane gradients. Ion pumps
are
classified as P, V, or F according to their structure and function. All have
one or more binding sites
for ATP in their cytosolic domains. The P-class ion pumps include Caz+ ATPase
and Na+/K+ ATPase
and function in transporting H+, Na+, K+, and Ca2+ ions. P-class pumps consist
of two a and two (3
transmembrane subunits. The V- and F-class ion pumps have similar structures
but transport only H+.
F class H+ pumps mediate transport across the membranes of mitochondria and
chloroplasts, while V-
class H+ pumps regulate acidity inside lysosomes, endosomes, and plant
vacuoles.
A family of structurally related intrinsic membrane proteins known as
facilitative glucose
transporters catalyze the movement of glucose and other selected sugars across
the plasma membrane.
The proteins in this family contain a highly conserved, large transmembrane
domain comprised of 12
a-helices, and several weakly conserved, cytoplasmic and exoplasmic domains.
(Pessin, J. E., and
Bell, G.I. (1992) Annu. Rev. Physiol. 54:911-930.)
Amino acid transport is mediated by Na+ dependent amino acid transporters.
These
transporters are involved in gastrointestinal and renal uptake of dietary and
cellular amino acids and
in neuronal reuptake of neurotransmitters. Transport of cationic amino acids
is mediated by the
system y+ family and the cationic amino acid transporter (CAT) family. Members
of the CAT family
share a high degree of sequence homology, and each contains 12-14 putative
transmembrane
domains. (Ito, K. and Groudine, M. (1997) J. Biol. Chem. 272:26780-26786.)
Hormones are secreted molecules that travel through the circulation and bind
to specific
receptors on the surface of, or within, target cells. Although they have
diverse biochemical
compositions and mechanisms of action, hormones can be grouped into two
categories. One category
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includes small lipophilic hormones that diffuse through the plasma membrane of
target cells, bind to
cytosolic or nuclear receptors, and form a complex that alters gene
expression. Examples of these
molecules include retinoic acid, thyroxine, and the cholesterol-derived
steroid hormones such as
progesterone, estrogen, testosterone, cortisol, and aldosterone. The second
category includes
hydrophilic hormones that function by binding to cell surface receptors that
transduce signals across
the plasma membrane. Examples of such hormones include amino acid derivatives
such as
catecholamines and peptide hormones such as glucagon, insulin, gastrin,
secretin, cholecystokinin,
adrenocorticotropic hormone, follicle stimulating hormone, luteinizing
hormone, thyroid stimulating
hormone, and vasopressin. (See, for example, Lodish et al. (1995) Molecular
Cell Biolo~y, Scientific
American Books Inc., New York, NY, pp. 856-864.)
Neuropeptides and vasomediators (NP/VM) comprise a large family of endogenous
signaling
molecules. Included in this family are neuropeptides and neuropeptide hormones
such as bombesin,
neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin,
somatostatin,
tachykinins, urotensin II and related peptides involved in smooth muscle
stimulation, vasopressin,
vasoactive intestinal peptide, and circulatory system-borne signaling
molecules such as angiotensin,
complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon,
cholecystokinin and
gastrin. NP/VMs can transduce signals directly, modulate the activity or
release of other
neurotransmitters and hormones, and act as catalytic enzymes in cascades. The
effects of NP/VMs
range from extremely brief to long-lasting. (Reviewed in Martin, C. R. et al.
(1985) Endocrine
Physiology, Oxford University Press, New York, NY, pp. 57-62.)
The discovery of new human secretory proteins 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 cancer, inflammation, and gastrointestinal,
cardiovascular, and
neurological disorders.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human secretory proteins,
referred to
collectively as "HSECP" and individually as "HSECP-1," "HSECP-2," "HSECP-3,"
"HSECP-4,"
"HSECP-5," "HSECP-6," "HSECP-7," "HSECP-8," "HSECP-9," "HSECP-10," "HSECP-11,"
"HSECP-12," "HSECP-13," "HSECP-14," "HSECP-15," "HSECP-16," "HSECP-17," "HSECP-
18,"
"HSECP-19," "HSECP-20," "HSECP-21" and "HSECP-22." In one aspect, the
invention provides an
isolated polypeptide comprising a) an amino acid sequence selected from the
group consisting of SEQ
ID NO: l-22, b) a naturally occurring amino acid sequence having at least
90°Io sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
)D NO:1-22, or d) an
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immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-22. In one alternative, the invention provides an isolated polypeptide
comprising the amino
acid sequence of SEQ ID NO:1-22.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
22, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-22, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-22, or d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-22. In one
alternative, the polynucleotide is selected from the group consisting of SEQ
ID N0:23-44.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-22, b) a naturally
occurring amino acid
sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-22, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-22, or d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-22. 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 comprising a)
an amino
acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-22, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-22, or d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-22. 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 comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:l-22, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-22, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-22.
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The invention further provides an isolated polynucleotide comprising a) a
polynucleotide
sequence selected from the group consisting of SEQ ID N0:23-44, b) a naturally
occurring
polynucleotide sequence having at least 70% sequence identity to a
polynucleotide sequence selected
from the group consisting of SEQ ID N0:23-44, c) a polynucleotide sequence
complementary to a),
or d) a polynucleotide sequence complementary to b). In one alternative, the
polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a) a
polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44,
b) a naturally
occurring polynucleotide sequence having at least 70% sequence identity to a
polynucleotide
sequence selected from the group consisting of SEQ ID N0:23-44, c) a
polynucleotide sequence
complementary to a), or d) a polynucleotide sequence complementary to b). The
method comprises a)
hybridizing the sample with a probe comprising at least 16 contiguous
nucleotides comprising a
sequence complementary to said target polynucleotide in the sample, and which
probe specifically
hybridizes to said target polynucleotide, under conditions whereby a
hybridization complex is formed
between said probe and said target polynucleotide, 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 30 contiguous nucleotides. In another alternative, the
probe comprises at least 60
contiguous nucleotides.
The invention further provides a pharmaceutical composition comprising an
effective amount
of a polypeptide comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-22, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an
amino acid sequence selected from the group consisting of SEQ ID NO:1-22, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-22, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-22, and a pharmaceutically acceptable excipient. The invention
additionally provides a method
of treating a disease or condition associated with decreased expression of
functional HSECP,
comprising administering to a patient in need of such treatment the
pharmaceutical composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising a) an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-22, b) a naturally occurring amino acid sequence having at least
90% sequence identity
to an amino acid sequence selected from the group consisting of SEQ ID NO:1-
22, c) a biologically
active fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-22, or
d) an immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ
ID NO:1-22. The method comprises a) exposing a sample comprising the
polypeptide to a
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compound, and b) detecting agonist activity in the sample. In one alternative,
the invention provides
a pharmaceutical 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 HSECP,
comprising administering to a patient in need of such treatment the
pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising a) an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-22, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-22,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-22, or d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-22. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a pharmaceutical 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
HSECP, comprising administering to a patient in need of such treatment the
pharmaceutical
composition.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:23-44, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding HSECP.
Table 2 shows features of each polypeptide sequence, including predicted
signal peptides and
other motifs, and methods, algorithms, and searchable databases used for
analysis of HSECP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific
expression patterns of each nucleic acid sequence as determined by northern
analysis; diseases,
disorders, or conditions associated with these tissues; and the vector into
which each cDNA was
cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
WO 00/52151 PCT/CTS00/05621
encoding HSECP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze HSECP, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"HSECP" refers to the amino acid sequences of substantially purified HSECP
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
HSECP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of HSECP either by
directly interacting with
HSECP or by acting on components of the biological pathway in which HSECP
participates.
An "allelic variant" is an alternative form of the gene encoding HSECP.
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
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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 HSECP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as HSECP or a
polypeptide with at least one functional characteristic of HSECP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding HSECP, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
HSECP. 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 HSECP. 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 HSECP 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 an amino acid
sequence of a naturally
occurnng 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 HSECP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of HSECP either by
directly interacting with HSECP or by acting on components of the biological
pathway in which
HSECP participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
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Antibodies that bind HSECP polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
strand of a specific nucleic acid sequence. Antisense compositions may include
DNA; RNA; peptide
nucleic acid (PNA); oligonucleotides having modified backbone linkages such as
phosphorothioates,
methylphosphonates, or benzylphosphonates; oligonucleotides having modified
sugar groups such as
2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having
modified bases such
as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense
molecules may be
produced by any method including chemical synthesis or transcription. Once
introduced into a cell,
the complementary antisense molecule base-pairs with a naturally occurring
nucleic acid sequence
produced by the cell to form duplexes which block either transcription or
translation. The
designation "negative" or "minus" can refer to the antisense strand, and the
designation "positive" or
"plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" refers to the
capability of the natural, recombinant, or synthetic HSECP, or of any
oligopeptide thereof, to induce
a specific immune response in appropriate animals or cells and to bind with
specific antibodies.
The terms "complementary" and "complementarity" refer to the natural binding
of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." Complementarity between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarity exists between the single stranded molecules. The degree
of complementarity
between nucleic acid strands has significant effects on the efficiency and
strength of the hybridization
between the nucleic acid strands. This is of particular importance in
amplification reactions, which
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depend upon binding between nucleic acid strands, and in the design and use of
peptide nucleic acid
(PNA) molecules.
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 HSECP or fragments
of HSECP 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., DenNardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk
CT) in the 5' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
one or more Incyte Clones and, in some cases, one or more public domain ESTs,
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI). Some sequences have been both extended and assembled to produce the
consensus sequence.
"Conservative amino acid substitutions" are those substitutions that, when
made, least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
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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 the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen by an alkyl, acyl, 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 "fragment" is a unique portion of HSECP or the polynucleotide encoding HSECP
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, 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 N0:23-44 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:23-44, for example, as distinct from any
other sequence in the
same genome. A fragment of SEQ ID N0:23-44 is useful, for example, in
hybridization and
amplification technologies and in analogous methods that distinguish SEQ ID
N0:23-44 from related
polynucleotide sequences. The precise length of a fragment of SEQ ID N0:23-44
and the region of
SEQ ID N0:23-44 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:1-22 is encoded by a fragment of SEQ ID N0:23-44. A
fragment
of SEQ ID NO: l-22 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-22. For example, a fragment of SEQ ID NO:1-22 is useful as an
immunogenic peptide
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WO 00/52151 PCT/US00/05621
for the development of antibodies that specifically recognize SEQ ID NO:1-22.
The precise length of
a fragment of SEQ ID NO:1-22 and the region of SEQ ID NO:1-22 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
The term "similarity" refers to a degree of complementarity. There may be
partial similarity
or complete similarity. The word "identity" may substitute for the word
"similarity." A partially
complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a
target nucleic acid is referred to as "substantially similar." The inhibition
of hybridization of the
completely complementary sequence to the target sequence may be examined using
a hybridization
assay (Southern or northern blot, solution hybridization, and the like) under
conditions of reduced
stringency. A substantially similar sequence or hybridization probe will
compete for and inhibit the
binding of a completely similar (identical) sequence to the target sequence
under conditions of
reduced stringency. This is not to say that conditions of reduced stringency
are such that non-specific
binding is permitted, as reduced stringency conditions require that the
binding of two sequences to
one another be a specific (i.e., a selective) interaction. The absence of non-
specific binding may be
tested by the use of a second target sequence which lacks even a partial
degree of complementarity
(e.g., less than about 30°Io similarity or identity). In the absence of
non-specific binding, the
substantially similar sequence or probe will not hybridize to the second non-
complementary target
sequence.
The phrases "percent identity" and "% identity," as applied to polynucleotide
sequences,
refer to the percentage of residue matches between at least two polynucleotide
sequences aligned
using a standardized algorithm. Such an algorithm may insert, in a
standardized and reproducible
way, gaps in the sequences being compared in order to optimize alignment
between two sequences,
and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
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Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø9 (May-07-1999) 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' S0
Expect: 10
Word Size: 11
Filter: on
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 polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the
hydrophobicity and acidity at the
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
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 2Ø9
(May-07-1999) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: I penalties
Gap x drop-off:' SO
Expect: l0
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"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 identity.
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
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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 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. Generally, such wash temperatures
are selected to be about
5°C to 20°C lower than the thermal melting point (Tm) 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 et al., 1989,
Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1 % SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, denatured salmon sperm DNA at about 100-200 pg/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
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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 HSECP
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 HSECP 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 distinct polynucleotides on
a substrate.
The terms "element" and "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HSECP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of HSECP.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer
to a nucleotide,
oligonucleotide, polynucleotide, or any fragment thereof. These phrases also
refer to DNA or RNA
of genomic or synthetic origin which may be single-stranded or double-stranded
and may represent
the sense or the antisense strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like
material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with the 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. Generally, 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.
"Probe" refers to nucleic acid sequences encoding HSECP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
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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 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2"d
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel et al.,1987, Current Protocols in
Molecular Bioloey,
Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis 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/MTT 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
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identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
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 nucleic
acids encoding HSECP, or fragments thereof, or HSECP itself, may comprise a
bodily fluid; an
extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic DNA,
RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print;
etc.
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
containing the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
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 acids or
nucleotides by
different amino acids or nucleotides, respectively.
WO 00/52151 PCT/US00/05621
"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.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occur under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign
nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is
selected based on the type of host cell being transformed and may include, but
is not limited to, viral
infection, electroporation, heat shock, lipofectiori, 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, and 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),
sue.
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
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
21
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WO 00/52151 PCT/US00/05621
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
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 95%, or at least
98% 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 human secretory proteins
(HSECP),
the polynucleotides encoding HSECP, and the use of these compositions for the
diagnosis, treatment,
or prevention of cancer, inflammation, and gastrointestinal, cardiovascular,
and neurological
disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
HSECP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs)
of the
polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
IDs of the Incyte
clones in which nucleic acids encoding each HSECP were identified, and column
4 shows the cDNA
libraries from which these clones were isolated. Column 5 shows Incyte clones
and their
corresponding cDNA libraries. Clones for which cDNA libraries are not
indicated were derived from
pooled cDNA libraries. The Incyte clones in column 5 were used to assemble the
consensus
nucleotide sequence of each HSECP and are useful as fragments in hybridization
technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; and
column 6 shows analytical methods and in some cases, searchable databases to
which the analytical
methods were applied. The methods of column 6 were used to characterize each
polypeptide through
sequence homology and protein motifs. In column 5, the first line of each cell
lists the amino acid
residues comprising predicted signal peptide sequences located at the amino
terminus of each
22
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WO 00/52151 PCT/US00/05621
HSECP. Additional identifying motifs or signatures, such as a somatomedin B
signature in SEQ ID
N0:16 and seven putative transmembrane domains in SEQ ID N0:18, are also
listed in column 5.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding HSECP. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of
column 1. These
fragments are useful, for example, in hybridization or amplification
technologies to identify SEQ ID
N0:23-44 and to distinguish between SEQ ID N0:23-44 and related polynucleotide
sequences. The
polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides. Column
3 lists tissue categories which express HSECP as a fraction of total tissues
expressing HSECP.
Column 4 lists diseases, disorders, or conditions associated with those
tissues expressing HSECP as a
fraction of total tissues expressing HSECP. Column 5 lists the vectors used to
subclone each cDNA
library. In particular, three out of four cDNA libraries which express SEQ ID
N0:23 are derived
from cartilage and synovia associated with joint inflammation, and four out of
five cDNA libraries
which express SEQ ID N0:29 are derived from intestinal tissue. Furthermore,
about half of the
cDNA libraries expressing SEQ ID N0:34 are associated with inflammation or the
hematopoietic/immune system. Likewise, about half of the cDNA libraries
expressing SEQ ID
N0:35 are associated with inflammation or the hematopoietic/immune system, and
in particular, with
inflammation of the joints. In addition, 82% of the cDNA libraries expressing
SEQ ID N0:37 are
derived from tissues of the nervous system. Finally, expression of SEQ ID
N0:39 is detected solely
in a subtracted prostate tumor cDNA library, and expression of SEQ ID N0:43 is
detected only in two
cDNA libraries derived from heart tissue.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding HSECP were isolated. Column 1 references the
nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
The invention also encompasses HSECP variants. A preferred HSECP 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 HSECP amino acid sequence, and which contains at
least one functional or
structural characteristic of HSECP.
The invention also encompasses polynucleotides which encode HSECP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:23-44, which encodes HSECP. The
polynucleotide
sequences of SEQ ID N0:23-44, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
23
WO 00/52151 PCT/US00/05621
The invention also encompasses a variant of a polynucleotide sequence encoding
HSECP. 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 HSECP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:23-44 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 N0:23-44. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of HSECP.
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 HSECP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring HSECP, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode HSECP and its variants are
generally capable
of hybridizing to the nucleotide sequence of the naturally occurring HSECP
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences encoding
HSECP 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 rtilized by the host. Other reasons for
substantially altering the
nucleotide sequence encoding HSECP 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 HSECP
and
HSECP 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 HSECP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:23-44 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
24
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WO 00/52151 PCT/US00/05621
G33S.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
(Perkin-
Elmer), 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
(Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377
DNA sequencing
system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics,
Sunnyvale CA), or other systems known in the art. The resu-lting 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 Biolo~y and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding HSECP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto 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
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
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
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the
entire process
from loading of samples to computer analysis and electronic data display may
be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode HSECP may be cloned in recombinant DNA molecules that direct
expression of
HSECP, 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 HSECP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter HSECP-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of HSECP, 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
26
WO 00/52151 PCT/US00/05621
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding HSECP 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, HSECP itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. ( 1995) Science 269:202-204.) Automated synthesis may be
achieved using the
ABI 431A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of HSECP, 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.
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, T. (1984) Proteins, Structures and
Molecular ProRerties, WH
Freeman, New York NY.)
In order to express a biologically active HSECP, the nucleotide sequences
encoding HSECP
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding HSECP. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding HSECP. Such signals include the ATG initiation codon and
adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding HSECP 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
27
CA 02363684 2001-08-14
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WO 00/52151 PCT/US00/05621
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 HSECP 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~y, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding HSECP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding HSECP. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding HSECP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORTI
plasmid (Life Technologies). Ligation of sequences encoding HSECP 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 HSECP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of HSECP may be used.
For example, vectors
containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HSECP. 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
28
WO 00/52151 PCT/US00/05621
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 HSECP. Transcription of
sequences
encoding HSECP may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technolo~y
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding HSECP
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 HSECP in host cells. (See, e.g., Logan, J. and
T. Shenk ( 1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of HSECP in cell lines is preferred. For example, sequences encoding HSECP 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
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CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apY cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. ( 1981 )
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),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 HSECP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding HSECP can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding HSECP under the
control of a
single promoter. Expression of the marker gene in response to induction or
selection usually
indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding HSECP
and that express
HSECP 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 HSECP
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 HSECP 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 Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
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 HSECP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding HSECP, 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
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 HSECP 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 HSECP may be designed to contain
signal sequences which
direct secretion of HSECP 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 HSECP 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 HSECP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of HSECP
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),
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maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-rrryc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the HSECP encoding sequence and the
heterologous protein
sequence, so that HSECP may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled I-ISECP
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.
Fragments of HSECP may be produced not only by recombinant means, but also by
direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, su ra
pp. SS-60.) Protein
synthesis may be performed by manual techniques or by automation. Automated
synthesis may be
achieved, for example, using the ABI 431A peptide synthesizer (Perkin-Elmer).
Various fragments of
HSECP may be synthesized separately and then combined to produce the full
length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of HSECP and human secretory proteins. In addition, the
expression of HSECP is
closely associated with cancer, inflammation, and gastrointestinal,
cardiovascular, and neurological
disorders. Therefore, HSECP appears to play a role in cancer, inflammation,
and gastrointestinal,
cardiovascular, and neurological disorders. In the treatment of disorders
associated with increased
HSECP expression or activity, it is desirable to decrease the expression or
activity of HSECP. In the
treatment of disorders associated with decreased HSECP expression or activity,
it is desirable to
increase the expression or activity of HSECP.
Therefore, in one embodiment, HSECP 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 HSECP. Examples of such disorders include, but are not limited to,
a cancer such as
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
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WO 00/52151 PCT/US00/05621
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus ; an
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
gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal
spasm, esophageal
stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric
carcinoma, anorexia, nausea,
emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis, ulcerative
colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic
obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea,
constipation, gastrointestinal
hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice,
hepatic
encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis,
Wilson's disease, alpha,-
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; a
cardiovascular disorder, and in particular, a disorder of the heart such as
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, 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
33
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WO 00/52151 PCT/US00/05621
disease, congenital heart disease, and complications of cardiac
transplantation; and a neurological
disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease
and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron disorders,
progressive neural
muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis
and other demyelinating
diseases, bacterial and viral meningitis, brain abscess, subdural empyema,
epidural abscess,
suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system
disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis,
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, and Tourette's disorder.
In another embodiment, a vector capable of expressing HSECP 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 HSECP including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
HSECP 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
HSECP including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of HSECP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HSECP including, but not limited to, those listed above.
In a further embodiment, an antagonist of HSECP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of HSECP.
Examples of such
disorders include, but are not limited to, those cancer, inflammation, and
gastrointestinal,
cardiovascular, and neurological disorders described above. In one aspect, an
antibody which
specifically binds HSECP 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 HSECP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
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WO 00/52151 PCT/iJS00/05621
encoding HSECP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of HSECP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
An antagonist of HSECP may be produced using methods which are generally known
in the
art. In particular, purified HSECP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind HSECP.
Antibodies to HSECP 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 HSECP 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
HSECP 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 and contain the
entire amino acid sequence of a small, naturally occurring molecule. Short
stretches of HSECP
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 HSECP 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
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
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
HSECP-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. ( 1991 ) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for HSECP may also be
generated.
For example, such fragments include, but are not limited to, F(ab')z fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
HSECP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering HSECP epitopes is generally used, but a
competitive binding assay
may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for HSECP.
Affinity is expressed as an
association constant, K~, which is defined as the molar concentration of HSECP-
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 HSECP epitopes, represents the average affinity, or
avidity, of the antibodies
for HSECP. The Ka determined for a preparation of monoclonal antibodies, which
are monospecific
for a particular HSECP epitope, represents a true measure of affinity. High-
affinity antibody
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CA 02363684 2001-08-14
WO 00/52151 PCT/I1S00/05621
preparations with Ka ranging from about 109 to 10'Z L/mole are preferred for
use in immunoassays in
which the HSECP-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 HSECP,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies,
Volume I: A Practical
Annroach, IRL Press, Washington, DC; Liddell, J.E. and Cryer, A. (1991) A
Practical Guide to
Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of HSECP-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 HSECP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding HSECP may be used in situations in
which it would be
desirable to block the transcription of the mRNA. In particular, cells may be
transformed with
sequences complementary to polynucleotides encoding HSECP. Thus, complementary
molecules or
fragments may be used to modulate HSECP activity, or to achieve regulation of
gene function. Such
technology is now well known in the art, and sense or antisense
oligonucleotides or larger fragments
can be designed from various locations along the coding or control regions of
sequences encoding
HSECP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses, or
from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the targeted
organ, tissue, or cell population. Methods which are well known.to those
skilled in the art can be
used to construct vectors to express nucleic acid sequences complementary to
the polynucleotides
encoding HSECP. (See, e.g., Sambrook, supra; Ausubel, 1995, su ra.)
Genes encoding HSECP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding HSECP. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in
the absence of integration into the DNA, such vectors may continue to
transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient expression may last
for a month or more
with a non-replicating vector, and may last even longer if appropriate
replication elements are part of
the vector system.
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As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or
regulatory regions of the gene encoding HSECP. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, may be employed.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix
pairing is useful because it causes inhibition of the ability of the double
helix to open sufficiently for
the binding of polymerases, transcription factors, or regulatory molecules.
Recent therapeutic
advances using triplex DNA have been described in the literature. (See, e.g.,
Gee, J.E. et al. ( 1994) in
Huber, B.E. and B.I. Carr, Molecular and Immunolo~ic Approaches, Futura
Publishing, Mt. Kisco
NY, pp. 163-177.) A complementary sequence or antisense molecule may also be
designed to block
translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze. the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding HSECP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
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 HSECP. Such DNA sequences may be incorporated into a wide
variety of
vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into
cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
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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.
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.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
or sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of HSECP,
antibodies to HSECP, and mimetics, agonists, antagonists, or inhibitors of
HSECP. The compositions
may be administered alone or in combination with at least one other agent,
such as a stabilizing
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier including,
but not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered
to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing
of the active compounds into preparations which can be used pharmaceutically.
Further details on
techniques for formulation and administration may be found in the latest
edition of Remin tg On'S
Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral administration.
Such carriers enable the pharmaceutical compositions to be formulated as
tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
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compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose, mannitol,
and sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums,
including arabic and
tragacanth; and proteins, such as gelatin and collagen. If desired,
disintegrating or solubilizing agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and
alginic acid or a salt thereof,
such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules,
the active compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks'solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil,
or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or
liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the suspension may
also contain suitable
stabilizers or agents to increase the solubility of the compounds and allow
for the preparation of
highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a manner
that is known in the art, e.g., by means of conventional mixing, dissolving,
granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
WO 00/52151 PC'd'/US00/05621
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and succinic
acids. Salts tend to be more soluble in aqueous or other protonic solvents
than are the corresponding
free base forms. In other cases, the preparation may be a lyophilized powder
which may contain any
or all of the following: 1 mM to 50 mM histidine, 0.1 % to 2% sucrose, and 2%
to 7% mannitol, at a
pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labeled for treatment of an indicated condition. For
administration of HSECP, such
labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, or pigs.
An animal model may also be used to determine the appropriate concentration
range and route of
administration. Such information can then be used to determine useful doses
and routes for
administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
HSECP or fragments thereof, antibodies of HSECP, and agonists, antagonists or
inhibitors of HSECP,
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.
Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the 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 pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half life and clearance rate of
the particular
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WO 00/52151 PCT/US00/05621
formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 fig, 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 HSECP may be used
for the
diagnosis of disorders characterized by expression of HSECP, or in assays to
monitor patients being
treated with HSECP or agonists, antagonists, or inhibitors of HSECP.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for HSECP include methods which utilize the antibody and a
label to detect
HSECP in human body fluids or in extracts of cells or tissues. The antibodies
may be used with or
IS without modification, and may be labeled by covalent or non-covalent
attachment of a reporter
molecule. A wide variety of reporter molecules, several of which are described
above, are known in
the art and may be used.
A variety of protocols for measuring HSECP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
HSECP expression.
Normal or standard values for HSECP expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibody to
HSECP under conditions suitable for complex formation. The amount of standard
complex formation
may be quantitated by various methods, such as photometric means. Quantities
of HSECP 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 HSECP 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 HSECP
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of HSECP, and to monitor regulation of HSECP levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HSECP or closely related
molecules may be used
to identify nucleic acid sequences which encode HSECP. 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
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WO 00/52151 PCT/US00/05621
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding HSECP, 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 HSECP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:23-44 or from
genomic sequences including promoters, enhancers, and introns of the HSECP
gene.
Means for producing specific hybridization probes for DNAs encoding HSECP
include the
cloning of polynucleotide sequences encoding HSECP or HSECP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding HSECP may be used for the diagnosis of
disorders
associated with expression of HSECP. Examples of such disorders include, but
are not limited to, a
cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and
uterus; an 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
gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal
spasm, esophageal
stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric
carcinoma, anorexia, nausea,
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emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis, ulcerative
colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic
obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea,
constipation, gastrointestinal
hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice,
hepatic
encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis,
Wilson's disease, alpha,-
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; a
cardiovascular disorder, and in particular, a disorder of the heart such as
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 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; and 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
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neuralgia, and Tourette's disorder. The polynucleotide sequences encoding
HSECP 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 HSECP expression. Such qualitative or
quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding HSECP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HSECP may be labeled by standard methods and added to a
fluid or tissue
sample from a patient under conditions suitable for the formation of
hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding HSECP 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
HSECP, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding HSECP, 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 progression of the cancer.
WO 00/52151 PCT/LTS00/05621
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
HSECP 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 HSECP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
HSECP, 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.
Methods which may also be used to quantify the expression of HSECP include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer of interest is
presented in various dilutions and a spectrophotometric or colorimetric
response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray
can be used to monitor the expression level of large numbers of genes
simultaneously and to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, and to develop and
monitor the activities of therapeutic agents.
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.)
In another embodiment of the invention, nucleic acid sequences encoding HSECP
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a specific region
of a chromosome, or
to artificial chromosome constructions, e.g., human artificial chromosomes
(HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price,
C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-
154.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (
1995) in Meyers, supra,
pp. 965-968.) Examples of genetic map data can be found in various scientific
journals or at the
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Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation
between the
location of the gene encoding HSECP on a physical chromosomal map and a
specific disorder, or a
predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the number or arm of a particular human
chromosome is not
known. New sequences can be assigned to chromosomal arms by physical mapping.
This provides
valuable information to investigators searching for disease genes using
positional cloning or other
gene discovery techniques. Once the disease or syndrome has been crudely
localized by genetic
linkage to a particular genomic region, e.g., ataxia-telangiectasia to l 1q22-
23, any sequences mapping
to that area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti,
R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the subject
invention may also
be used to detect differences in the chromosomal location due to
translocation, inversion, etc., among
normal, carrier, or affected individuals.
In another embodiment of the invention, HSECP, 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 HSECP 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 HSECP,
or fragments thereof,
and washed. Bound HSECP is then detected by methods well known in the art.
Purified HSECP 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 HSECP specifically compete with a test compound
for binding HSECP.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with HSECP.
In additional embodiments, the nucleotide sequences which encode HSECP may be
used in
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any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/123,117, are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over 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 isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
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1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Pharmaceuticals, Palo Alto
CA). Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XLI-
BIueMRF, or
SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system
(Stratagene) or by cell lysis. Plasmids were purified using at least one of
the following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
QIAWELL 8
Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid
purification kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or
without lyophilization, at 4°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
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Robbins Scientific)
or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing
reactions were
prepared using reagents provided by Amersham Pharmacia Biotech or supplied in
ABI sequencing
kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction
kit (Perkin-Elmer).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example V.
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The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
colmnn presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:23-44. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Northern Analysis
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
CA 02363684 2001-08-14
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WO 00/52151 PCT/US00/05621
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
sera, ch. 7; Ausubel,
1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte
Pharmaceuticals). This
analysis is much faster than multiple membrane-based hybridizations. In
addition, the sensitivity of
the computer search can be modified to determine whether any particular match
is categorized as
exact or similar. The basis of the search is the product score, which is
defined as:
sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a 1 % to 2% error, and, with a product score of 70, the match will be
exact. Similar molecules
are usually identified by selecting those which show product scores between 15
and 40, although
lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding HSECP occurred. Analysis involved the categorization
of cDNA libraries by
organltissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Extension of HSECP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:23-44 were produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
51
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
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+, (NH~,)~S04,
and (3-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (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
I: 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 ~tl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 pl 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 ~1 to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, individual colonies were picked and cultured
overnight at 37 °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 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, I min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, S min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
52
WO 00/52151 PCT/US00/05621
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:23-44 are used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension, and an
appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:23-44 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although. the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~cCi of
~y 32PJ adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An
array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, UV, chemical, or mechanical bonding procedures. A typical array may
be produced by hand
or using available methods and machines and contain any appropriate number of
elements. After
hybridization, nonhybridized probes are removed and a scanner used to
determine the levels and
patterns of fluorescence. The degree of complementarity and the relative
abundance of each probe
which hybridizes to an element on the microarray may be assessed through
analysis of the scanned
images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise
the elements of the microarray. Fragments suitable for hybridization can be
selected using software
well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs,
ESTs, or
53
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
fragments thereof corresponding to one of the nucleotide sequences of the
present invention, or
selected at random from a cDNA library relevant to the present invention, are
arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide
using, e.g., UV cross-linking
followed by thermal and chemical treatments and subsequent drying. (See, e.g.,
Schena, M. et al.
(1995) Science 270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645.)
Fluorescent probes
are prepared and used for hybridization to the elements on the substrate. The
substrate is analyzed by
procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the HSECP-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring HSECP.
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 HSECP. 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
HSECP-encoding
transcript.
IX. Expression of HSECP
Expression and purification of HSECP is achieved using bacterial or virus-
based expression
systems. For expression of HSECP 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 HSECP upon induction with isopropyl beta-
D-
thiogalactopyranoside (1PTG). Expression of HSECP in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Auto. r~aphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding HSECP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
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WO 00/52151 PCT/US00/05621
In most expression systems, HSECP is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
HSECP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10 and 16). Purified HSECP obtained by these methods can be used
directly in the
following activity assay.
X. Demonstration of HSECP Activity
An assay for HSECP activity measures the expression of HSECP on the cell
surface. cDNA
encoding HSECP is subcloned into an appropriate mammalian expression vector
suitable for high
levels of cDNA expression. The resulting construct is transfected into a
nonhuman cell line such as
NIH3T3. Cell surface proteins are labeled with biotin using methods known in
the art.
Immunoprecipitations are performed using HSECP-specific antibodies, and
immunoprecipitated
samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio
of labeled
immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount
of HSECP
expressed on the cell surface.
Alternatively, an assay for HSECP activity measures the amount of HSECP in
secretory,
membrane-bound organelles. Transfected cells as described above are harvested
and lysed. The
lysate is fractionated using methods known to those of skill in the art, for
example, sucrose gradient
ultracentrifugation. Such methods allow the isolation of subcellular
components such as the Golgi
apparatus, ER, small membrane-bound vesicles, and other secretory organelles.
Immunoprecipitations from fractionated and total cell lysates are performed
using HSECP-specific
antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and
immunoblotting
techniques. The concentration of HSECP in secretory organelles relative to
HSECP in total cell
lysate is proportional to the amount of HSECP in transit through the secretory
pathway.
XI. Functional Assays
HSECP function is assessed by expressing the sequences encoding HSECP 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 plasmid (Life Technologies)
and pCR3.1
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10
~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. I-2 ~cg 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 C tometry, Oxford,
New York NY.
The influence of HSECP on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding HSECP 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 HSECP and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XII. Production of HSECP Specific Antibodies
HSECP 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 HSECP 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 IS residues in length are synthesized using
an ABI 431A
56
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
s HSECP activity by, for example, binding the peptide or HSECP to a substrate,
blocking with 1 %
BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated
goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring HSECP Using Specific Antibodies
Naturally occurring or recombinant HSECP is substantially purified by
immunoaffinity
chromatography using antibodies specific for HSECP. An immunoaffinity column
is constructed by
covalently coupling anti-HSECP 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 HSECP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of HSECP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/HSECP 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 HSECP is collected.
XIV. Identification of Molecules Which Interact with HSECP
HSECP, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter
reagent. (See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-
539.) Candidate
molecules previously arrayed in the wells of a mufti-well plate are incubated
with the labeled HSECP,
washed, and any wells with labeled HSECP complex are assayed. Data obtained
using different
concentrations of HSECP are used to calculate values for the number, affinity,
and association of
HSECP with the candidate molecules.
Alternatively, molecules interacting with HSECP 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).
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.
57
CA 02363684 2001-08-14
CA 02363684 2001-08-14
WO 00/52151 PCT/LJS00/05621
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p ~ ~" ~ -d ~ O ~ ~ _'~ ~ 17 O .O ~ ~ ,~ ~ C
G, ~ O (.~ ~ ~ V ~._.>'r V ~ O~ -._.>'~ V U ~C .,'~.., d pp w: OU
.a c C. o a~ -
a, ~ w - o. ~ '~ ~~ ~ a. '~ a. a~ c~ W -o ~ '° '
a d ~ d ~ d dw ~.~ dw ~w d ~ ~.~ a~w
n
w
d
a
O
rd d Q ~ ~ w w
w Q ~ a ~ w ~ x
73
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
U ~
4.
V ~
_~
OU c~
G O ~ y O wNr
7
y
~ t. V1 N
y
N
_
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w O ~ N
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~ W ~ o w ~
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74
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
TANG, Y. Tom
LAL, Preeti
BAUGHN, Mariah R.
YUE, Henry
AU-YOUNG, Janice
LU, Dyung Aina M.
AZIMZAI, Yalda
<120> HUMAN SECRETORY PROTEINS
<130> PF-0675 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/123,117
<151> 1999-03-05
<160> 44
<170> PERL Program
<210> 1
<211> 182
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 078811CD1
<400> 1
Met Arg Ser Thr Ile Leu Leu Phe Cys Leu Leu Gly Ser Thr Arg
1 5 10 15
Ser Leu Pro Val Phe Pro Ser Leu Ser Leu Ile Pro Leu Thr Gln
20 25 30
Met Leu Thr Leu Gly Pro Asp Leu His Leu Leu Asn Pro Ala Ala
35 40 45
Gly Met Thr Pro Gly Thr Gln Thr His Pro Leu Thr Leu Gly Gly
50 55 60
Leu Asn Val Gln Gln Gln Leu His Pro His Val Leu Pro Ile Phe
65 70 75
Val Thr Gln Leu Gly Ala Pro Gly His Tyr Pro Lys Leu Arg Gly
80 85 90
Ile Ala Thr Asn Leu His Glu Pro His His Pro Phe Leu Val Pro
95 100 105
Arg Glu Ala Ser Leu Pro Thr Ser Gln Ala Gly Ala Asn Pro Asp
110 115 120
Val Gln Asp Gly Ser Leu Pro Ala Gly Gly Ala Gly Val Asn Pro
125 130 135
Ala Thr Gln Gly Thr Pro Ala Gly Arg Leu Pro Thr Pro Ser Gly
140 145 150
Thr Asp Asp Asp Phe Ala Val Thr Thr Pro Ala Gly Ile Gln Arg
155 160 165
Ser Thr His Ala Ile Glu Glu Ala Thr Thr Glu Ser Ala Asn Gly
170 175 180
Ile Gln
<210> 2
<211> 125
1/28
CA 02363684 2001-08-14
WO 00/52151 PCT/CTS00/05621
<212> PRT
<213> Homo sapiens
<220> -
<221> misc_feature
<223> Incyte ID No: 371156CD1
<400> 2
Met Val Cys Glu Asp Ala Pro Ser Phe Gln Met Ala Trp Glu Ser
1 5 10 15
Gln Met Ala Trp Glu Arg Gly Pro Ala Leu Leu Cys Cys Val Leu
20 25 30
Ser Ala Ser Gln Leu Ser Ser Gln Asp Gln Asp Pro Leu Gly His
35 40 45
Ile Lys Ser Leu Leu Tyr Pro Phe Gly Phe Pro Val Glu Leu Pro
50 55 60
Arg Pro Gly Pro Thr Gly Ala Tyr Lys Lys Val Lys Asn Gln Asn
65 70 75
Gln Thr Thr Ser Ser Glu Leu Leu Arg Lys Gln Thr Ser His Phe
80 85 90
Asn Gln Arg Gly His Arg Ala Arg Ser Lys Leu Leu Ala Ser Arg
95 100 105
Gln Ile Pro Asp Arg Thr Phe Lys Cys Gly Lys Trp Leu Pro Gln
110 115 120
Val Pro Ser Pro Val
125
<210> 3
<211> 320
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 584050CD1
<400> 3
Met Ala Gly Leu Ala Ala Arg Leu Val Leu Leu Ala Gly Ala Ala
1 5 10 15
Ala Leu Ala Ser Gly Ser Gln Gly Asp Arg Glu Pro Val Tyr Arg
20 25 30
Asp Cys Val Leu Gln Cys Glu Glu Gln Asn Cys Ser Gly Gly Ala
35 40 45
Leu Asn His Phe Arg Ser Arg Gln Pro Ile Tyr Met Ser Leu Ala
50 55 60
Gly Trp Thr Cys Arg Asp Asp Cys Lys Tyr Glu Cys Met Trp Val
65 70 75
Thr Val Gly Leu Tyr Leu Gln Glu Gly His Lys Val Pro Gln Phe
80 85 90
His Gly Lys Trp Pro Phe Ser Arg Phe Leu Phe Phe Gln Glu Pro
95 100 105
Ala Ser Ala Val Ala Ser Phe Leu Asn Gly Leu Ala Ser Leu Val
110 115 120
Met Leu Cys Arg Tyr Arg Thr Phe Val Pro Ala Ser Ser Pro Met
125 130 135
Tyr His Thr Cys Val Ala Phe Ala Trp Val Ser Leu Asn Ala Trp
140 145 150
Phe Trp Ser Thr Val Phe His Thr Arg Asp Thr Asp Leu Thr Glu
155 160 165
Lys Met Asp Tyr Phe Cys Ala Ser Thr Val Ile Leu His Ser Ile
170 175 180
Tyr Leu Cys Cys Val Arg Thr Val Gly Leu Gln His Pro Ala Val
185 190 195
Val Ser Ala Phe Arg Ala Leu Leu Leu Leu Met Leu Thr Val His
2/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
200 205 210
Val Ser Tyr Leu Ser Leu Ile Arg Phe Asp Tyr Gly Tyr Asn Leu
215 220 225
Val Ala Asn Val Ala Ile Gly Leu Val Asn Val Val Trp Trp Leu
230 235 240
Ala Trp Cys Leu Trp Asn Gln Arg Arg Leu Pro His Val Arg Lys
245 250 255
Cys Val Val Val Val Leu Leu Leu Gln Gly Leu Ser Leu Leu Glu
260 265 270
Leu Leu Asp Phe Pro Pro Leu Phe Trp Val Leu Asp Ala His Ala
275 280 285
Ile Trp His Ile Ser Thr Ile Pro Val His Val Leu Phe Phe Ser
290 295 300
Phe Leu Glu Asp Asp Ser Leu Tyr Leu Leu Lys Glu Ser Glu Asp
305 310 315
Lys Phe Lys Leu Asp
320
<210> 4
<211> 234
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 863808CD1
<400> 4
Met Gly Pro Gly Gly Arg Val Ala Arg Leu Leu Ala Pro Leu Met
1 5 10 15
Trp Arg Arg Ala Val Ser Ser Val Ala Gly Ser Ala Val Gly Ala
20 25 30
Glu Pro Gly Leu Arg Leu Leu Ala Val Gln Arg Leu Pro Val Gly
35 40 45
Ala Ala Phe Cys Arg Ala Cys Gln Thr Pro Asn Phe Val Arg Gly
50 55 60
Leu His Ser Glu Pro Gly Leu Glu Glu Arg Ala Glu Gly Thr Val
65 70 75
Asn Glu Gly Arg Pro Glu Ser Asp Ala Ala Asp His Thr Gly Pro
80 85 90
Lys Phe Asp Ile Asp Met Met Val Ser Leu Leu Arg Gln Glu Asn
95 100 105
Ala Arg Asp Ile Cys Val Ile Gln Val Pro Pro Glu Met Arg Tyr
110 115 120
Thr Asp Tyr Phe Val Ile Val Ser Gly Thr Ser Thr Arg His Leu
125 130 135
His Ala Met Ala Phe Tyr Val Val Lys Met Tyr Lys His Leu Lys
140 145 150
Cys Lys Arg Asp Pro His Val Lys Ile Glu Gly Lys Asp Thr Asp
155 160 165
Asp Trp Leu Cys Val Asp Phe Gly Ser Met Val Ile His Leu Met
170 175 180
Leu Pro Glu Thr Arg Glu Ile Tyr Glu Leu Glu Lys Leu Trp Thr
185 190 195
Leu Arg Ser Tyr Asp Asp Gln Leu Ala Gln Ile Ala Pro Glu Thr
200 205 210
Val Pro Glu Asp Phe Ile Leu Gly Ile Glu Asp Asp Thr Ser Ser
215 220 225
Val Thr Pro Val Glu Leu Lys Cys Glu
230
3/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<210> 5
<211> 278
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 978433CD1
<400> 5
Met Gln Pro Ala Ala Ala Ser Glu Arg Gly Gly Ala Asp Ala Asp
1 5 10 15
His Val Pro Leu Leu Gly Leu Leu Arg Leu Gln Leu Arg Ala Ala
20 25 30
Arg Gln Pro Gly Ala Met Arg Pro Gln Gly Pro Ala Ala Ser Pro
35 40 45
Gln Arg Leu Arg Gly Leu Leu Leu Leu Leu Leu Leu Gln Leu Pro
50 55 60
Ala Pro Ser Ser Ala Ser Glu Ile Pro Lys Gly Lys Gln Lys Ala
65 70 75
Gln Leu Arg Gln Arg Glu Val Val Asp Leu Tyr Asn Gly Met Cys
80 85 90
Leu Gln Gly Pro Ala Gly Val Pro Gly Arg Asp Gly Ser Pro Gly
95 100 105
Ala Asn Gly Ile Pro Gly Thr Pro Gly Ile Pro Gly Arg Asp Gly
110 115 120
Phe Lys Gly Glu Lys Gly Glu Cys Leu Arg Glu Ser Phe Glu Glu
125 130 135
Ser Trp Thr Pro Asn Tyr Lys Gln Cys Ser Trp Ser Ser Leu Asn
140 145 150
Tyr Gly Ile Asp Leu Gly Lys Ile Ala Glu Cys Thr Phe Thr Lys
155 160 165
Met Arg Ser Asn Ser Ala Leu Arg Val Leu Phe Ser Gly Ser Leu
170 175 180
Arg Leu Lys Cys Arg Asn Ala Cys Cys Gln Arg Trp Tyr Phe Thr
185 190 195
Phe Asn Gly Ala Glu Cys Ser Gly Pro Leu Pro Ile Glu Ala Ile
200 205 210
Ile Tyr Leu Asp Gln Gly Ser Pro Glu Met Asn Ser Thr Ile Asn
215 220 225
Ile His Arg Thr Ser Ser Val Glu Gly Leu Cys Glu Gly Ile Gly
230 235 240
Ala Gly Leu Val Asp Val Ala Ile Trp Val Gly Thr Cys Ser Asp
245 250 255
Tyr Pro Lys Gly Asp Ala Ser Thr Gly Trp Asn Ser Val Ser Arg
260 265 270
Ile Ile Ile Glu Glu Leu Pro Lys
275
<210> 6
<211> 136
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1655369CD1
<400> 6
Met Pro Pro Gly Gly Leu Gly Ala Cys Ala Val Thr Pro Ala Pro
1 5 10 15
Gly Glu Glu Arg Thr Gln Pro Gly Glu Leu Gly Gln Gly Leu His
20 25 30
Met Ala Gln Gly Gln Gln Met Leu Ala Gly Gln Leu Leu Pro Met
4/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
35 40 45
Leu Thr Leu Leu Pro Pro Ser Phe Pro Leu Pro His Pro Thr Leu
50 55 60
Gly Pro Arg Arg His Ala Ser Leu Thr Gln Leu Gly Pro Ala Phe
65 70 75
Trp Met Ala Trp Gly Arg Pro Trp Ala His Leu Gly Pro Gly Gln
80 85 90
Pro Leu Gly Gln Leu Trp Lys Ser Ser Val Glu Glu His Leu Leu
95 100 105
Ala Ala Trp Leu Gln Pro Leu Ala Leu Leu Glu Trp Ser Leu Gly
110 115 120
Ala Ser Ala Leu Ser Ala Leu Gly Thr Ser His Pro Leu Gly Leu
125 130 135
Gln
<210> 7
<211> 109
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1703244CD1
<400> 7
Met Leu Met Tyr Met Phe Tyr Val Leu Pro Phe Cys Gly Leu Ala
1 5 10 15
Ala Tyr Ala Leu Thr Phe Pro Gly Cys Ser Trp Leu Pro Asp Trp
20 25 30
Ala Leu Val Phe Ala Gly Gly Ile Gly Gln Ala Gln Phe Ser His
35 40 45
Met Gly Ala Ser Met His Leu Arg Thr Pro Phe Thr Tyr Arg Val
50 55 60
Pro Glu Asp Thr Trp Gly Cys Phe Phe Val Cys Asn Leu Leu Tyr
65 70 75
Ala Leu Gly Pro His Leu Leu Ala Tyr Arg Cys Leu Gln Trp Pro
80 85 90
Ala Phe Phe His Gln Pro Pro Pro Ser Asp Pro Leu Ala Leu His
95 100 105
Lys Lys Gln His
<210> 8
<211> 262
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1730819CD1
<400> 8
Met Ala Ala Ala Ser Ala Gly Ala Thr Arg Leu Leu Leu Leu Leu
1 5 10 15
Leu Met Ala Val Ala Ala Pro Ser Arg Ala Arg Gly Ser Gly Cys
20 25 30
Arg Ala Gly Thr Gly Ala Arg Gly Ala Gly Ala Glu Gly Arg Glu
35 40 45
Gly Glu Ala Cys Gly Thr Val Gly Leu Leu Leu Glu His Ser Phe
50 55 60
Glu Ile Asp Asp Ser Ala Asn Phe Arg Lys Arg Gly Ser Leu Leu
65 70 75
5/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
Trp Asn Gln Gln Asp Gly Thr Leu Ser Leu Ser Gln Arg Gln Leu
80 85 90
Ser Glu Glu Glu Arg Gly Arg Leu Arg Asp Val Ala Ala Leu Asn
95 100 105
Gly Leu Tyr Arg Val Arg Ile Pro Arg Arg Pro Gly Ala Leu Asp
110 115 120
Gly Leu Glu Ala Gly Gly Tyr Val Ser Ser Phe Val Pro Ala Cys
125 130 135
Ser Leu Val Glu Ser His Leu Ser Asp Gln Leu Thr Leu His Val
140 145 150
Asp Val Ala Gly Asn Val Val Gly Val Ser Val Val Thr His Pro
155 160 165
Gly Gly Cys Arg Gly His Glu Val Glu Asp Val Asp Leu Glu Leu
170 175 180
Phe Asn Thr Ser Val Gln Leu Gln Pro Pro Thr Thr Ala Pro Gly
185 190 195
Pro Glu Thr Ala Ala Phe Ile Glu Arg Leu Glu Met Glu Gln Ala
200 205 210
Gln Lys Ala Lys Asn Pro Gln Glu Gln Lys Ser Phe Phe Ala Lys
215 220 225
Tyr Trp Met Tyr Ile Ile Pro Val Val Leu Phe Leu Met Met Ser
230 235 240
Gly Ala Pro Asp Thr Gly Gly Gln Gly Gly Gly Gly Gly Cys Gly
245 250 255
Gly Gly Gly Gly Ser Gly Arg
260
<210> 9
<211> 384
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1757161CD1
<400> 9
Met Ala Glu Gln Thr Tyr Ser Trp Ala Tyr Ser Leu Val Asp Ser
1 5 10 15
Ser Gln Val Ser Thr Phe Leu Ile Ser Ile Leu Leu Ile Val Tyr
20 25 30
Gly Ser Phe Arg Ser Leu Asn Met Asp Phe Glu Asn Gln Asp Lys
35 40 45
Glu Lys Asp Ser Asn Ser Ser Ser Gly Ser Phe Asn Gly Asn Ser
50 55 60
Thr Asn Asn Ser Ile Gln Thr Ile Asp Ser Thr Gln Ala Leu Phe
65 70 75
Leu Pro Ile Gly Ala Ser Val Ser Leu Leu Val Met Phe Phe Phe
80 85 90
Phe Asp Ser Val Gln Val Val Phe Thr Ile Cys Thr Ala Val Leu
95 100 105
Ala Thr Ile Ala Phe Ala Phe Leu Leu Leu Pro Met Cys Gln Tyr
110 115 120
Leu Thr Arg Pro Cys Ser Pro Gln Asn Lys Ile Ser Phe Gly Cys
125 130 135
Cys Gly Arg Phe Thr Ala Ala Glu Leu Leu Ser Phe Ser Leu Ser
140 145 150
Val Met Leu Val Leu Ile Trp Val Leu Thr Gly His Trp Leu Leu
155 160 165
Met Asp Ala Leu Ala Met Gly Leu Cys Val Ala Met Ile Ala Phe
170 175 180
Val Arg Leu Pro Ser Leu Lys Val Ser Cys Leu Leu Leu Ser Gly
185 190 195
Leu Leu Ile Tyr Asp Val Phe Trp Val Phe Phe Ser Ala Tyr Ile
6/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
200 205 210
Phe Asn Ser Asn Val Met Val Lys Val Ala Thr Gln Pro Ala Asp
215 220 225
Asn Pro Leu Asp Val Leu Ser Arg Lys Leu His Leu Gly Pro Asn
230 235 240
Val Gly Arg Asp Val Pro Arg Leu Ser Leu Pro Gly Lys Leu Val
245 250 255
Phe Pro Ser Ser Thr Gly Ser His Phe Ser Met Leu Gly Ile Gly
260 265 " 270
Asp Ile Val Met Pro Gly Leu Leu Leu Cys Phe Val Leu Arg Tyr
275 280 285
Asp Asn Tyr Lys Lys Gln Ala Ser Gly Asp Ser Cys Gly Ala Pro
290 295 300
Gly Pro Ala Asn Ile Ser Gly Arg Met Gln Lys Val Ser Tyr Phe
305 310 315
His Cys Thr Leu Ile Gly Tyr Phe Val Gly Leu Leu Thr Ala Thr
320 325 330
Val Ala Ser Arg Ile His Arg Ala Ala Gln Pro Ala Leu Leu Tyr
335 340 345
Leu Val Pro Phe Thr Leu Leu Pro Leu Leu Thr Met Ala Tyr Leu
350 355 360
Lys Gly Asp Leu Arg Arg Met Trp Ser Glu Pro Phe His Ser Lys
365 370 375
Ser Ser Ser Ser Arg Phe Leu Glu Val
380
<210> 10
<211> 244
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1976095CD1
<400> 10
Met Asp Ile Leu Val Pro Leu Leu Gln Leu Leu Val Leu Leu Leu
1 5 10 15
Thr Leu Pro Leu His Leu Met Ala Leu Leu Gly Cys Trp Gln Pro
20 25 30
Leu Cys Lys Ser Tyr Phe Pro Tyr Leu Met Ala Val Leu Thr Pro
35 40 45
Lys Ser Asn Arg Lys Met Glu Ser Lys Lys Arg Glu Leu Phe Ser
50 55 60
Gln Ile Lys Gly Leu Thr Gly Ala Ser Gly Lys Val Ala Leu Leu
65 70 75
Glu Leu Gly Cys Gly Thr Gly Ala Asn Phe Gln Phe Tyr Pro Pro
80 85 90
Gly Cys Arg Val Thr Cys Leu Asp Pro Asn Pro His Phe Glu Lys
95 100 105
Phe Leu Thr Lys Ser Met Ala Glu Asn Arg His Leu Gln Tyr Glu
110 115 120
Arg Phe Val Val Ala Pro Gly Glu Asp Met Arg Gln Leu Ala Asp
125 130 135
Gly Ser Met Asp Val Val Val Cys Thr Leu Val Leu Cys Ser Val
140 145 150
Gln Ser Pro Arg Lys Val Leu Gln Glu Val Arg Arg Val Leu Arg
155 160 165
Pro Gly Gly Val Leu Phe Phe Trp Glu His Val Ala Glu Pro Tyr
170 175 180
Gly Ser Trp Ala Phe Met Trp Gln Gln Val Phe Glu Pro Thr Trp
185 190 195
Lys His Ile Gly Asp Gly Cys Cys Leu Thr Arg Glu Thr Trp Lys
200 205 210
7/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
Asp Leu Glu Asn Ala Gln Phe Ser Glu Ile Gln Met Glu Arg Gln
215 220 225
Pro Pro Pro Leu Lys Trp Leu Pro Val Gly Pro His Ile Met Gly
230 235 240
Lys Ala Val Lys
<210> 11
<211> 326
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2169991CD1
<400> 11
Met Arg Thr Glu Ala Gln Val Pro Ala Leu Gln Pro Pro Glu Pro
1 5 10 15
Gly Leu Glu Gly Ala Met Gly His Arg Thr Leu Val Leu Pro Trp
20 25 30
Val Leu Leu Thr Leu Cys Val Thr Ala Gly Thr Pro Glu Val Trp
35 40 45
Val Gln Val Arg Met Glu Ala Thr Glu Leu Ser Ser Phe Thr Ile
50 55 60
Arg Cys Gly Phe Leu Gly Ser Gly Ser Ile Ser Leu Val Thr Val
65 70 75
Ser Trp Gly Gly Pro Asn Gly Ala Gly Gly Thr Thr Leu Ala Val
80 85 90
Leu His Pro Glu Arg Gly Ile Arg Gln Trp Ala Pro Ala Arg Gln
95 100 105
Ala Arg Trp Glu Thr Gln Ser Ser Ile Ser Leu Ile Leu Glu Gly
110 115 120
Ser Gly Ala Ser Ser Pro Cys Ala Asn Thr Thr Phe Cys Cys Lys
125 130 135
Phe Ala Ser Phe Pro Glu Gly Ser Trp Glu Ala Cys Gly Ser Leu
140 145 150
Pro Pro Ser Ser Asp Pro Gly Leu Ser Ala Pro Pro Thr Pro Ala
155 160 165
Pro Ile Leu Arg Ala Asp Leu Ala Gly Ile Leu Gly Val Ser Gly
170 175 180
Val Leu Leu Phe Gly Cys Val Tyr Leu Leu His Leu Leu Arg Arg
185 190 195
His Lys His Arg Pro Ala Pro Arg Leu Gln Pro Ser Arg Thr Ser
200 205 210
Pro Gln Ala Pro Arg Ala Arg Ala Trp Ala Pro Ser Gln Ala Ser
215 220 225
Gln Ala Ala Leu His Val Pro Tyr Ala Thr Ile Asn Thr Ser Cys
230 235 240
Arg Pro Ala Thr Leu Asp Thr Ala His Pro His Gly Gly Pro Ser
245 250 255
Trp Trp Ala Ser Leu Pro Thr His Ala Ala His Arg Pro Gln Gly
260 265 270
Pro Ala Ala Trp Ala Ser Thr Pro Ile Pro Ala Arg Gly Ser Phe
275 280 285
Val Ser Val Glu Asn Gly Leu Tyr Ala Gln Ala Gly Glu Arg Pro
290 295 300
Pro His Thr Gly Pro Gly Leu Thr Leu Phe Pro Asp Pro Arg Gly
305 310 315
Pro Arg Ala Met Glu Gly Pro Leu Gly Val Arg
320 325
8/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<210>
12
<211>
105
<212>
PRT
<213> Sapiens
Homo
<220>
<221> feature
misc_
<223> No:2616827CD1
Incyte
ID
<400>
12
Met Asn Gly ValSerMet LeuArg IleLeu PheLeuLeu Asp
Leu
1 5 10 15
Val Gly Ala GlnValLeu AlaThr GlyLys ThrProGly Ala
Gly
20 25 30
Glu Ile Phe LysTyrAla LeuIle GlyThr AlaValGly Val
Asp
35 40 45
Ala Ile Ala GlyPheLeu AlaLeu LysIle CysMetIle Arg
Ser
50 55 60
Arg His Phe AspAspAsp SerSer AspLeu LysSerThr Pro
Leu
65 70 75
Gly Gly Ser AspThrIle ProLeu LysLys ArgAlaPro Arg
Leu
80 85 90
Arg Asn Asn PheSerLys ArgAsp AlaGln ValIleGlu Leu
His
95 100 105
<210> 13
<211> 626
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2991370CD1
<400> 13
Met Ala Pro Ser Ala Asp Pro Gly Met Ser Arg Met Leu Pro Phe
1 5 10 15
Leu Leu Leu Leu Trp Phe Leu Pro Ile Thr Glu Gly Ser Gln Arg
20 25 30
Ala Glu Pro Met Phe Thr Ala Val Thr Asn Ser Val Leu Pro Pro
35 40 45
Asp Tyr Asp Ser Asn Pro Thr Gln Leu Asn Tyr Gly Val Ala Val
50 55 60
Thr Asp Val Asp His Asp Gly Asp Phe Glu Ile Val Val Ala Gly
65 70 75
Tyr Asn Gly Pro Asn Leu Val Leu Lys Tyr Asp Arg Ala Gln Lys
80 85 90
Arg Leu Val Asn Ile Ala Val Asp Glu Arg Ser Ser Pro Tyr Tyr
95 100 105
Ala Leu Arg Asp Arg Gln Gly Asn Ala Ile Gly Val Thr Ala Cys
110 115 120
Asp Ile Asp Gly Asp Gly Arg Glu Glu Ile Tyr Phe Leu Asn Thr
125 130 135
Asn Asn Ala Phe Ser Gly Val Ala Thr Tyr Thr Asp Lys Leu Phe
140 145 150
Lys Phe Arg Asn Asn Arg Trp Glu Asp Ile Leu Ser Asp Glu Val
155 160 165
Asn Val Ala Arg Gly Val Ala Ser Leu Phe Ala Gly Arg Ser Val
170 175 180
Ala Cys Val Asp Arg Lys Gly Ser Gly Arg Tyr Ser Ile Tyr Ile
185 190 195
Ala Asn Tyr Ala Tyr Gly Asn Val Gly Pro Asp Ala Leu Ile Glu
200 205 210
Met Asp Pro Glu Ala Ser Asp Leu Ser Arg Gly Ile Leu Ala Leu
9/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
215 220 225
Arg Asp Val Ala Ala Glu Ala Gly Val Ser Lys Tyr Thr Gly Gly
230 235 240
Arg Gly Val Ser Val Gly Pro Ile Leu Ser Ser Ser Ala Ser Asp
245 250 255
Ile Phe Cys Asp Asn Glu Asn Gly Pro Asn Phe Leu Phe His Asn
260 265 270
Arg Gly Asp Gly Thr Phe Val Asp Ala Ala Ala Ser Ala Gly Val
275 280 285
Asp Asp Pro His Gln His Gly Arg Gly Val Ala Leu Ala Asp Phe
290 295 300
Asn Arg Asp Gly Lys Val Asp Ile Val Tyr Gly Asn Trp Asn Gly
305 310 315
Pro His Arg Leu Tyr Leu Gln Met Ser Thr His Gly Lys Val Arg
320 325 330
Phe Arg Asp Ile Ala Ser Pro Lys Phe Ser Met Pro Ser Pro Val
335 340 345
Arg Thr Val Ile Thr Ala Asp Phe Asp Asn Asp Gln Glu Leu Glu
350 355 360
Ile Phe Phe Asn Asn Ile Ala Tyr Arg Ser Ser Ser Ala Asn Arg
365 370 375
Leu Phe Arg Val Ile Arg Arg Glu His Gly Asp Pro Leu Ile Glu
380 385 390
Glu Leu Asn Pro Gly Asp Ala Leu Glu Pro Glu Gly Arg Gly Thr
395 400 405
Gly Gly Val Val Thr Asp Phe Asp Gly Asp Gly Met Leu Asp Leu
410 415 420
Ile Leu Ser His Gly Glu Ser Met Ala Gln Pro Leu Ser Val Phe
425 430 435
Arg Gly Asn Gln Gly Phe Asn Asn Asn Trp Leu Arg Val Val Pro
440 445 450
Arg Thr Arg Phe Gly Ala Phe Ala Arg Gly Ala Lys Val Val Leu
455 460 465
Tyr Thr Lys Lys Ser Gly Ala His Leu Arg Ile Ile Asp Gly Gly
470 475 480
Ser Gly Tyr Leu Cys Glu Met Glu Pro Val Ala His Phe Gly Leu
485 490 495
Gly Lys Asp Glu Ala Ser Ser Val Glu Val Thr Trp Pro Asp Gly
500 505 510
Lys Met Val Ser Arg Asn Val Ala Ser Gly Glu Met Asn Ser Val
515 520 525
Leu Glu Ile Leu Tyr Pro Arg Asp Glu Asp Thr Leu Gln Asp Pro
530 535 540
Ala Pro Leu Glu Cys Gly Gln Gly Phe Ser Gln Gln Glu Asn Gly
545 550 555
His Cys Met Asp Thr Asn Glu Cys Ile Gln Phe Pro Phe Val Cys
560 565 570
Pro Arg Asp Lys Pro Val Cys Val Asn Thr Tyr Gly Ser Tyr Arg
575 580 585
Cys Arg Thr Asn Lys Lys Cys Ser Arg Gly Tyr Glu Pro Asn Glu
590 595 600
Asp Gly Thr Ala Cys Val Gly Trp Trp Ser Pro Val Leu Lys Ile
605 610 615
Val Thr Pro Gln Val Gly Lys Ser Leu Gly Pro
620 625
<210> 14
<211> 296
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3031062CD1
10/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<400> 14
Met Glu Trp Trp Ala Ser Ser Pro Leu Arg Leu Trp Leu Leu Leu
1 5 10 15
Phe Leu Leu Pro Ser Ala Gln Gly Arg Gln Lys Glu Ser Gly Ser
20 25 30
Lys Trp Lys Val Phe Ile Asp Gln Ile Asn Arg Ser Leu Glu Asn
35 40 45
Tyr Glu Pro Cys Ser Ser Gln Asn Cys Ser Cys Tyr His Gly Val
50 55 60
Ile Glu Glu Asp Leu Thr Pro Phe Arg Gly Gly Ile Ser Arg Lys
65 70 75
Met Met Ala Glu Val Val Arg Arg Lys Leu Gly Thr His Tyr Gln
80 85 90
Ile Thr Lys Asn Arg Leu Tyr Arg Glu Asn Asp Cys Met Phe Pro
95 100 105
Ser Arg Cys Ser Gly Val Glu His Phe Ile Leu Glu Val Ile Gly
110 115 120
Arg Leu Pro Asp Met Glu Met Val Ile Asn Val Arg Asp Tyr Pro
125 130 135
Gln Val Pro Lys Trp Met Glu Pro Ala Ile Pro Val Phe Ser Phe
140 145 150
Ser Lys Thr Ser Glu Tyr His Asp Ile Met Tyr Pro Ala Trp Thr
155 160 165
Phe Trp Glu Gly Gly Pro Ala Val Trp Pro Ile Tyr Pro Thr Gly
170 175 180
Leu Gly Arg Trp Asp Leu Phe Arg Glu Asp Leu Val Arg Ser Ala
185 190 195
Ala Gln Trp Pro Trp Lys Lys Lys Asn Ser Thr Ala Tyr Phe Arg
200 205 210
Gly Ser Arg Thr Ser Pro Glu Arg Asp Pro Leu Ile Leu Leu Ser
215 220 225
Arg Lys Asn Pro Lys Leu Val Asp Ala Glu Tyr Thr Lys Asn Gln
230 235 240
Ala Trp Lys Ser Met Lys Asp Thr Leu Gly Lys Pro Ala Ala Lys
245 250 255
Asp Val His Leu Val Asp His Cys Lys Tyr Lys Tyr Leu Phe Asn
260 265 270
Phe Arg Gly Val Leu Gln Val Ser Gly Leu Asn Thr Ser Ser Cys
275 280 285
Val Ala Ile Ile Leu Met Arg Lys Arg Thr Tyr
290 295
<210> 15
<211> 249
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3101617CD1
<400> 15
Met Asp Gly Lys Lys Cys Ser Val Trp Met Phe Leu Pro Leu Val
1 5 10 15
Phe Thr Leu Phe Thr Ser Ala Gly Leu Trp Ile Val Tyr Phe Ile
20 25 30
Ala Val Glu Asp Asp Lys Ile Leu Pro Leu Asn Ser Ala Glu Arg
35 40 45
Lys Pro Gly Val Lys His Ala Pro Tyr Ile Ser Ile Ala Gly Asp
50 55 60
Asp Pro Pro Ala Ser Cys Val Phe Ser Gln Val Met Asn Met Ala
65 70 75
Ala Phe Leu Ala Leu Val Val Ala Val Leu Arg Phe Ile Gln Leu
80 85 90
11/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
Lys Pro Lys Val Leu Asn Pro Trp Leu Asn Ile Ser Gly Leu Val
95 100 105
Ala Leu Cys Leu Ala Ser Phe Gly Met Thr Leu Leu Gly Asn Phe
110 115 120
Gln Leu Thr Asn Asp Glu Glu Ile His Asn Val Gly Thr Ser Leu
125 130 135
Thr Phe Gly Phe Gly Thr Leu Thr Cys Trp Ile Gln Ala Ala Leu
140 145 150
Thr Leu Lys Val Asn Ile Lys Asn Glu Gly Arg Arg Val Gly Ile
155 160 165
Pro Arg Val Ile Leu Ser Ala Ser Ile Thr Leu Cys Val Val Leu
170 175 180
Tyr Phe Ile Leu Met Ala Gln Ser Ile His Met Tyr Ala Ala Arg
185 190 195
Val Gln Trp Gly Leu Val Met Cys Phe Leu Ser Tyr Phe Gly Thr
200 205 210
Phe Ala Val Glu Phe Arg His Tyr Arg Tyr Glu Ile Val Cys Ser
215 220 225
Glu Tyr Gln Glu Asn Phe Leu Ser Phe Ser Glu Ser Leu Ser Glu
230 235 240
Ala Ser Glu Tyr Gln Thr Asp Gln Val
245
<210> 16
<211> 124
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3216178CD1
<400> 16
Met Gly Gly Tyr Leu Lys Thr Arg Pro Trp Thr Leu Gln His Phe
1 5 10 15
Tyr Leu Cys Leu Met Pro Ala Ala Thr Trp Leu Val Leu Leu Leu
20 25 30
Leu Leu Trp Leu Ser Leu Gly Val Lys Thr Gly Ser Cys Ser Gln
35 40 45
Pro Gln Asn Leu Cys Cys Leu Gly Thr Asp His His Cys Lys Arg
50 55 60
Gly Ser Cys Tyr Cys Asp Glu Phe Cys His Val Ala Pro Asp Cys
65 70 75
His Pro Asp His Ser Val Leu Cys Asn Pro Ala Ser Gln Met Thr
80 85 90
Lys Met Val Leu Gln Met Val Leu Arg Met Glu Asn Pro Pro Ser
95 100 105
Pro Ala Arg Ser His Leu Asp Trp Met Gln Ser Met Val Ser Ser
110 115 120
Leu Gln Val Leu
<210> 17
<211> 101
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3406803CD1
<400> 17
12/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
Met Leu Pro Val Gly Ala Gln Pro Arg Ser Pro Pro Trp Val Leu
1 5 10 15
Ala Arg Leu Leu His Pro Arg Gly Pro Ala Ala Thr Ser Leu Val
20 25 30
Pro Phe Leu Pro Trp Gly Ser Leu Glu Ser His Thr Pro Cys Pro
35 40 45
Tyr Arg Ala Cys Ser Pro Gly Trp Glu Leu Thr Leu Ser Thr Phe
50 55 60
Pro Glu Arg Glu Thr Leu Ser Gly Gly Glu Val Arg Lys Arg Gly
65 70 75
Ala Gly Ser Met Val Gly Gly Gly Glu Ser Thr Met Thr Arg Ala
80 85 90
Leu Cys Val Arg Leu Leu Thr Lys Leu Arg Val
95 100
<210> 18
<211> 540
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3468066CD1
<400> 18
Met Ala Thr Ser Gly Ala Ala Ser Ala Glu Leu Val Ile Gly Trp
1 5 10 15
Cys Ile Phe Gly Leu Leu Leu Leu Ala Ile Leu Ala Phe Cys Trp
20 25 30
Ile Tyr Val Arg Lys Tyr Gln Ser Arg Arg Glu Ser Glu Val Val
35 40 45
Ser Thr Ile Thr Ala Ile Phe Ser Leu Ala Ile Ala Leu Ile Thr
50 55 60
Ser Ala Leu Leu Pro Val Asp Ile Phe Leu Val Ser Tyr Met Lys
65 70 75
Asn Gln Asn Gly Thr Phe Lys Asp Trp Ala Asn Ala Asn Val Ser
80 85 90
Arg Gln Ile Glu Asp Thr Val Leu Tyr Gly Tyr Tyr Thr Leu Tyr
95 100 105
Ser Val Ile Leu Phe Cys Val Phe Phe Trp Ile Pro Phe Val Tyr
110 115 120
Phe Tyr Tyr Glu Glu Lys Asp Asp Asp Asp Thr Ser Lys Cys Thr
125 130 135
Gln Ile Lys Thr Ala Leu Lys Tyr Thr Leu Gly Phe Val Val Ile
140 145 150
Cys Ala Leu Leu Leu Leu Val Gly Ala Phe Val Pro Leu Asn Val
155 160 165
Pro Asn Asn Lys Asn Ser Thr Glu Trp Glu Lys Val Lys Ser Leu
170 175 180
Phe Glu Glu Leu Gly Ser Ser His Gly Leu Ala Ala Leu Ser Phe
185 190 195
Ser Ile Ser Ser Leu Thr Leu Ile Gly Met Leu Ala Ala Ile Thr
200 205 210
Tyr Thr Ala Tyr Gly Met Ser Ala Leu Pro Leu Asn Leu Ile Lys
215 220 225
Gly Thr Arg Ser Ala Ala Tyr Glu Arg Leu Glu Asn Thr Glu Asp
230 235 240
Ile Glu Glu Val Glu Gln His Ile Gln Thr Ile Lys Ser Lys Ser
245 250 255
Lys Asp Gly Arg Pro Leu Pro Ala Arg Asp Lys Arg Ala Leu Lys
260 265 270
Gln Phe Glu Glu Arg Leu Arg Thr Leu Lys Lys Arg Glu Arg His
275 280 285
Leu Glu Phe Ile Glu Asn Ser Trp Trp Thr Lys Phe Cys Gly Ala
13/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
290 295 300
Leu Arg Pro Leu Lys Ile Val Trp Gly Ile Phe Phe Ile Leu Val
305 310 315
Ala Leu Leu Phe Val Ile Ser Leu Phe Leu Ser Asn Leu Asp Lys
320 325 330
Ala Leu His Ser Ala Gly Ile Asp Ser Gly Phe Ile Ile Phe Gly
335 340 345
Ala Asn Leu Ser Asn Pro Leu Asn Met Leu Leu Pro Leu Leu Gln
350 355 360
Thr Val Phe Pro Leu Asp Tyr Ile Leu Ile Thr Ile Ile Ile Met
365 370 375
Tyr Phe Ile Phe Thr Ser Met Ala Gly Ile Arg Asn Ile Gly Ile
380 385 390
Trp Phe Phe Trp Ile Arg Leu Tyr Lys Ile Arg Arg Gly Arg Thr
395 400 405
Arg Pro Gln Ala Leu Leu Phe Leu Cys Met Ile Leu Leu Leu Ile
410 415 420
Val Leu His Thr Ser Tyr Met Ile Tyr Ser Leu Ala Pro Gln Tyr
425 430 435
Val Met Tyr Gly Ser Gln Asn Tyr Leu Ile Glu Thr Asn Ile Thr
440 445 450
Ser Asp Asn His Lys Gly Asn Ser Thr Leu Ser Val Pro Lys Arg
455 460 465
Cys Asp Ala Glu Ala Pro Glu Asp Gln Cys Thr Val Thr Arg Thr
470 475 480
Tyr Leu Phe Leu His Lys Phe Trp Phe Phe Ser Ala Ala Tyr Tyr
485 490 495
Phe Gly Asn Trp Ala Phe Leu Gly Val Phe Leu Ile Gly Leu Ile
500 505 510
Val Ser Cys Cys Lys Gly Lys Lys Ser Val Ile Glu Gly Val Asp
515 520 525
Glu Asp Ser Asp Ile Ser Asp Asp Glu Pro Ser Val Tyr Ser Ala
530 535 540
<210> 19
<211> 108
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3592862CD1
<400> 19
Met Thr Pro Ser Arg Leu Pro Trp Leu Leu Ser Trp Val Ser Ala
1 5 10 15
Thr Ala Trp Arg Ala Ala Arg Ser Pro Leu Leu Cys His Ser Leu
20 25 30
Arg Lys Thr Ser Ser Ser Gln Gly Gly Lys Ser Glu Leu Val Lys
35 40 45
Gln Ser Leu Lys Lys Pro Lys Leu Pro Glu Gly Arg Phe Asp Ala
50 55 60
Pro Glu Asp Ser His Leu Glu Lys Glu Pro Leu Glu Lys Phe Pro
65 70 75
Asp Asp Val Asn Pro Val Thr Lys Glu Lys Gly Gly Pro Arg Gly
80 85 90
Pro Glu Pro Thr Arg Tyr Gly Asp Trp Glu Arg Lys Gly Arg Cys
95 100 105
Ile Asp Phe
<210> 20
14/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<211> 114
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3669422CD1
<400> 20
Met Ser Ser Ser Ser Ser Arg Cys Leu Ser Pro Ser Pro Gly Met
1 5 10 15
Ser Leu Trp Ser Cys Leu Leu Phe Leu Cys Thr Pro Ser Pro Thr
20 25 30
Thr Thr Ser Pro Ser Pro Asp Pro Ser Gln Val Ser Thr Leu Pro
35 40 45
Thr Pro Ser Pro Gln Arg Glu Gly Leu Lys Gln Gly Gln Trp Arg
50 55 60
Lys Thr Gly Pro Ser Ser Thr His Pro His Thr Pro Ser Ser Arg
65 70 75
Pro Pro Ser Pro Ser Ser Leu Pro Leu Thr Trp Lys Leu Leu Gln
80 85 90
Pro Ile Pro Ser His Ser Leu Pro His Pro Pro Lys Ile His Thr
95 100 105
Gly Pro Ser Leu Ala Glu Cys Gly His
110
<210> 21
<211> 114
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3688740CD1
<400> 21
Met Arg Gly Glu His Asn Ser Thr Ser Tyr Asp Ser Ala Val Ile
1 5 10 15
Tyr Arg Gly Phe Trp Ala Val Leu Met Leu Leu Gly Val Val Ala
20 25 30
Val Va1 Ile Ala Ser Phe Leu Ile Ile Cys Ala Ala Pro Phe Ala
35 40 45
Ser His Phe Leu Tyr Lys Ala Gly Gly Gly Ser Tyr Ile Ala Ala
50 55 60
Asp Gly Ile Ser Ser Leu Cys Tyr Ser Ser Leu Ser Lys Ser Leu
65 70 75
Leu Ser Gln Pro Leu Arg Glu Thr Ser Ser Ala Ile Asn Asp Ile
80 85 90
Ser Leu Leu Gln Ala Leu Met Pro Leu Leu Gly Trp Thr Ser His
95 100 105
Trp Thr Cys Ile Thr Val Gly Leu Tyr
110
<210> 22
<211> 287
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3742589CD1
15/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<400> 22
Met Glu Leu Glu Arg Ile Val Ser Ala Ala Leu Leu Ala Phe Val
1 5 10 15
Gln Thr His Leu Pro Glu Ala Asp Leu Ser Gly Leu Asp Glu Val
20 25 30
Ile Phe Ser Tyr Val Leu Gly Val Leu Glu Asp Leu Gly Pro Ser
35 40 45
Gly Pro Ser Glu Glu Asn Phe Asp Met Glu Ala Phe Thr Glu Met
50 55 60
Met Glu Ala Tyr Val Pro Gly Phe Ala His Ile Pro Arg Gly Thr
65 70 75
Ile Gly Asp Met Met Gln Lys Leu Ser Gly Gln Leu Ser Asp Ala
80 85 90
Arg Asn Lys Glu Asn Leu Gln Pro Gln Ser Ser Gly Val Gln Gly
95 100 105
Gln Val Pro Ile Ser Pro Glu Pro Leu Gln Arg Pro Glu Met Leu
110 115 120
Lys Glu Glu Thr Arg Ser Ser Ala Ala Ala Ala Ala Asp Thr Gln
125 130 135
Asp Glu Ala Thr Gly Ala Glu Glu Glu Leu Leu Pro Gly Val Asp
140 145 150
Val Leu Leu Glu Val Phe Pro Thr Cys Ser Val Glu Gln Ala Gln
155 160 165
Trp Val Leu Ala Lys Ala Arg Gly Asp Leu Glu Glu Ala Val Gln
170 175 180
Met Leu Val Glu Gly Lys Glu Glu Gly Pro Ala Ala Trp Glu Gly
185 190 195
Pro Asn Gln Asp Leu Pro Arg Arg Leu Arg Gly Pro Gln Lys Asp
200 205 210
Glu Leu Lys Ser Phe Ile Leu Gln Lys Tyr Met Met Val Asp Ser
215 220 225
Ala Glu Asp Gln Lys Ile His Arg Pro Met Ala Pro Lys Glu Ala
230 235 240
Pro Lys Lys Leu Ile Arg Tyr Ile Asp Asn Gln Val Val Ser Thr
245 250 255
Lys Gly Glu Arg Phe Lys Asp Val Arg Asn Pro Glu Ala Glu Glu
260 265 270
Met Lys Ala Thr Tyr Ile Asn Leu Lys Pro Ala Arg Lys Tyr Arg
275 280 285
Phe His
<210> 23
<211> 854
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 078811CB1
<400> 23
attttgcctc gtggacccaa aggtagcaat ttgaaacatg aggagtacga ttctactgtt 60
ttgtcttcta ggatcaactc ggtcattacc agtctttcct tctttaagtc tgataccatt 120
aacacagatg ctcacactgg ggccagatct gcatctgtta aatcctgctg caggaatgac 180
acctggtacc cagacccacc cattgaccct gggagggttg aatgtacaac agcaactgca 240
cccacatgtg ttaccaattt ttgtcacaca acttggagcc ccagggcact atcctaagct 300
cagaggaatt gccacaaatc ttcacgagcc tcatcatcca ttccttgttc cccgggaggc 360
atccttgccc accagtcagg caggggctaa tccagatgtc caggatggaa gccttccagc 420
aggaggagca ggtgtaaatc ctgccaccca gggaacccca gcaggccgcc tcccaactcc 480
cagtggcaca gatgacgact ttgcagtgac cacccctgca ggcatccaaa ggagcacaca 540
tgccatcgag gaagccacca cagaatcagc aaatggaatt cagtaagctg tttcaaattt 600
tttcaactaa gctgcctcga atttggtgat acatgtgaat ctttatcatt gattatatta 660
tggaatagat tgagacacat tggatagtct tagaagaaat taattcttaa tttacctgaa 720
aatattcttg aaatttcaga aaatatgttc tatgtagaga atcccaactt ttaaaaacaa 780
16/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
taattcaatg gataaatctg tctttgaaat ataacattat gctgcctgga tgatatgcat 840
attaaaacga atta 854
<210> 24
<211> 1804
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 371156CB1
<400> 24
gtgataggca gctttccttc ttttcaacag tgatacctac gaaaatcaaa ataaatgcaa 60
gctgaggttt tgtgctcact gaaagggctg tcaaccccag aaggccgaca caaaaaaaat 120
ggtatgtgaa gatgcaccgt cttttcaaat ggcctgggag agtcaaatgg cctgggagag 180
ggggcctgcc cttctctgct gtgtcctttc ggcttcccag ttgagctccc aagaccagga 240
cccactgggg catataaaat ctctgctgta tcctttcggc ttcccagttg agctcccaag 300
accaggaccc actggggcat ataaaaaagt caaaaatcaa aatcaaacaa caagttctga 360
gttacttagg aaacagactt cgcatttcaa tcagagaggc cacagagcaa ggtctaaact 420
tctggcttct agacaaattc ctgatagaac atttaaatgt gggaagtggc ttccccaggt 480
cccatcccct gtttagggat agagttgata tcatttttat aggtgccatg tatgcctctg 540
cctgaatttt tttaattgac ttttgagctt ttgagattgc acgagggaga acaaggcctt 600
tgctgttgtg gataggaaag acttaaccta aaattaaacc agcaagaaag cattagtaaa 660
aatctaacaa tatgaagggc tcttatgagt catttttttc aaaagatgaa aactccagaa 720
acgcacagga acgaaatacc tcccagaaac atgaagcaat catcgaagac tcactggtaa 780
tatttttaaa aagtatacag atcaaagcaa aaagaagcca tgtgtaacaa agagaaatgt 840
gcaaatattt tttaaggcag tattaagtgc aagaggagta acatgaaata aacattcttt 900
cacatggcta ctgggaatat aaatttcgct ccagaaaggc cgtagcagtt tgacgatagg 960
tggcaaaacc ttaagattgt gtactggggc ccagaatttt tatttctagg aatgtatcct 1020
gaggaaatta tccgagatcc ccacaaactg caatgtttag gaattgtcct tatagcattg 1080
catacacaag aaaaacagag aaaagcctga tccctgtcag tggaaaaggg gttcaatgaa 1140
ttacggtgtg tctgcatgag gcttttatga cattaaaaat tgttgaacaa cggccaggca 1200
cagtggctca tgcctgtaat cctaacactt tgggaggcca aggtgggaag attgcctgag 1260
ctcaggagtt tgagaccagc ctgggcaaca cggtgaaacc ccgtctctac taaaatacaa 1320
aaaattagcc gggcgtcgca gcatgcgcct gtagtcccag ctgctcagga ggctgaggca 1380
ggagaattga ttgaacccgg gaggcagagg ttgcactgag ctgagattaa gccaccgcac 1440
tccagcctgg gcgacagagc aagattccgt tcccaagaaa aaaaaattgt tcaacaataa 1500
gggcaaaggg agagaatcat aacatctgat taaacagaaa aagcaagatt tttaaaacta 1560
actatataag gatggtccca gctgtgtcaa aaggaagctt gtttgtaata cgtgtgcata 1620
aaaattaaat agaggtgaac acaattattt taaggcagtt aaattatctc tgtattgtga 1680
actaagactt tctagaattt tacttattca ttctgtactt aaattttttc taatgaacac 1740
atatactttt gtaatcagaa aatattaaat gcatgtattt ttcaaaatca aaaaaaaaaa 1800
aaaa 1804
<210> 25
<211> 2663
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 584050CB1
<400> 25
ggagaaagga tggccggcct ggcggcgcgg ttggtcctgc tagctggggc agcggcgctg 60
gcgagcggct cccagggcga ccgtgagccg gtgtaccgcg actgcgtact gcagtgcgaa 120
gagcagaact gctctggggg cgctctgaat cacttccgct cccgccagcc aatctacatg 180
agtctagcag gctggacctg tcgggacgac tgtaagtatg agtgtatgtg ggtcaccgtt 240
gggctctacc tccaggaagg tcacaaagtg cctcagttcc atggcaagtg gcccttctcc 300
cggttcctgt tctttcaaga gccggcatcg gccgtggcct cgtttctcaa tggcctggcc 360
agcctggtga tgctctgccg ctaccgcacc ttcgtgccag cctcctcccc catgtaccac 420
acctgtgtgg ccttcgcctg ggtgtccctc aatgcatggt tctggtccac agtcttccac 480
accagggaca ctgacctcac agagaaaatg gactacttct gtgcctccac tgtcatccta 540
cactcaatct acctgtgctg cgtcaggacc gtggggctgc agcacccagc tgtggtcagt 600
17/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
gccttccggg ctctcctgct gctcatgctg accgtgcacg tctcctacct gagcctcatc 660
cgcttcgact atggctacaa cctggtggcc aacgtggcta ttggcctggt caacgtggtg 720
tggtggctgg cctggtgcct gtggaaccag cggcggctgc ctcacgtgcg caagtgcgtg 780
gtggtggtct tgctgctgca ggggctgtcc ctgctcgagc tgcttgactt cccaccgctc 840
ttctgggtcc tggatgccca tgccatctgg cacatcagca ccatccctgt ccacgtcctc 900
tttttcagct ttctggaaga tgacagcctg tacctgctga aggaatcaga ggacaagttc 960
aagctggact gaagaccttg gagcgagtct gccccagtgg ggatcctgcc cccgccctgc 1020
tggcctccct tctcccctca acccttgaga tgattttctc ttttcaactt cttgaacttg 1080
gacatgaagg atgtgggccc agaatcatgt ggccagccca ccccctgttg gccctcacca 1140
gccttggagt ctgttctagg gaaggcctcc cagcatctgg gactcgagag tgggcagccc 1200
ctctacctcc tggagctgaa ctggggtgga actgagtgtg ttcttagctc taccgggagg 1260
acagctgcct gtttcctccc caccagcctc ctccccacat ccccagctgc ctggctgggt 1320
cctgaagccc tctgtctacc tgggagacca gggaccacag gccttaggga tacagggggt 1380
ccccttctgt taccaccccc caccctcctc caggacacca ctaggtggtg ctggatgctt 1440
gttctttggc cagccaaggt tcacggcgat tctccccatg ggatcttgag ggaccaagct 1500
gctgggattg ggaaggagtt tcaccctgac cgttgcccta gccaggttcc caggaggcct 1560
caccatactc cctttcaggg ccagggctcc agcaagccca gggcaaggat cctgtgctgc 1620
tgtctggttg agagcctgcc accgtgtgtc gggagtgtgg gccaggctga gtgcataggt 1680
gacagggccg tgagcatggg cctgggtgtg tgtgagctca ggcctaggtg cgcagtgtgg 1740
agacgggtgt tgtcggggaa gaggtgtggc ttcaaagtgt gtgtgtgcag ggggtgggtg 1800
tgttagcgtg ggttagggga acgtgtgtgc gcgtgctggt gggcatgtga gatgagtgac 1860
tgccggtgaa tgtgtccaca gttgagaggt tggagcagga tgagggaatc ctgtcaccat 1920
caataatcac ttgtggagcg ccagctctgc ccaagacgcc acctgggcgg acagccagga 1980
gctctccatg gccaggctgc ctgtgtgcat gttccctgtc tggtgcccct ttgcccgcct 2040
cctgcaaacc tcacagggtc cccacacaac agtgccctcc agaagcagcc cctcggaggc 2100
agaggaagga aaatggggat ggctggggct ctctccatcc tccttttctc cttgccttcg 2160
catggctggc cttcccctcc aaaacctcca ttcccctgct gccagcccct ttgccatagc 2220
ctgattttgg ggaggaggaa ggggcgattt gagggagaag gggagaaagc ttatggctgg 2280
gtctggtttc ttcccttccc agagggtctt actgttccag ggtggcccca gggcaggcag 2340
gggccacact atgcctgcgc cctggtaaag gtgacccctg ccatttacca gcagccctgg 2400
catgttcctg ccccacagga atagaatgga gggagctcca gaaactttcc atcccaaagg 2460
cagtctccgt ggttgaagca gactggattt ttgctctgcc cctgacccct tgtccctctt 2520
tgagggaggg gagctatgct aggactccaa cctcagggac tcgggtggcc tgcgctagct 2580
tcttttgata ctgaaaactt ttaaggtggg agggtggcaa gggatgtgct taataaatca 2640
attccaagcc tcaaaaaaaa aaa 2663
<210> 26
<211> 769
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 863808CB1
<400> 26
gcgacgccga cgcaaggctg ctgctatggg gccgggcggc cgtgtggcgc ggctgctcgc 60
cccactaatg tggcgcaggg cggtttcctc ggtggcgggg tccgcggttg gagccgagcc 120
cgggcttcgg ctgctggccg tgcagcggct tcccgtagga gcagcgttct gccgggcttg 180
ccagacccca aactttgtcc gcggcctgca cagcgagcct gggctggagg agcgggcgga 240
ggggacggtc aacgagggac gcccagaatc ggacgcggca gatcatactg gtcccaagtt 300
tgacatcgat atgatggttt cacttctgag gcaagaaaat gcaagagaca tttgtgtgat 360
ccaggttcct ccagaaatga gatatacaga ttactttgtg attgttagtg gaacttctac 420
ccgacactta catgccatgg ccttctacgt tgtgaaaatg tacaaacacc tgaaatgtaa 480
acgtgaccct catgttaaga tagaagggaa ggacactgat gactggctgt gcgtggattt 540
tggcagcatg gtgattcatt tgatgcttcc agaaaccaga gaaatctatg aattagagaa 600
attatggacc ctacgttctt atgatgacca gttagctcag atagcacctg agacagtacc 660
tgaagacttc attcttggaa tagaagatga tacttcatct gtgactccag tggagttaaa 720
atgtgaataa aatattttat gcactgcgtt agtcaaaaaa aaaaaaaaa 769
<210> 27
<211> 1257
<212> DNA
<213> Homo Sapiens
18/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<220>
<221> misc_feature
<223> Incyte ID No: 978433CB1
<400> 27
gaggcgcgcg ggtgaaaggc gcattgatgc agcctgcggc ggcctcggag cgcggcggag 60
cagacgctga ccacgttcct ctcctcggtc tcctccgcct ccagctccgc gctgcccggc 120
agccgggagc catgcgaccc cagggccccg ccgcctcccc gcagcggctc cgcggcctcc 180
tgctgctcct gctgctgcag ctgcccgcgc cgtcgagcgc ctctgagatc cccaagggga 240
agcaaaaggc gcagctccgg cagagggagg tggtggacct gtataatgga atgtgcttac 300
aagggccagc aggagtgcct ggtcgagacg ggagccctgg ggccaatggc attccgggta 360
cacctgggat cccaggtcgg gatggattca aaggagaaaa gggggaatgt ctgagggaaa 420
gctttgagga gtcctggaca cccaactaca agcagtgttc atggagttca ttgaattatg 480
gcatagatct tgggaaaatt gcggagtgta catttacaaa gatgcgttca aatagtgctc 540
taagagtttt gttcagtggc tcacttcggc taaaatgcag aaatgcatgc tgtcagcgtt 600
ggtatttcac attcaatgga gctgaatgtt caggacctct tcccattgaa gctataattt 660
atttggacca aggaagccct gaaatgaatt caacaattaa tattcatcgc acttcttctg 720
tggaaggact ttgtgaagga attggtgctg gattagtgga tgttgctatc tgggttggca 780
cttgttcaga ttacccaaaa ggagatgctt ctactggatg gaattcagtt tctcgcatca 840
ttattgaaga actaccaaaa taaatgcttt aattttcatt tgctacctct ttttttatta 900
tgccttggaa tggttcactt aaatgacatt ttaaataagt ttatgtatac atctgaatga 960
aaagcaaagc taaatatgtt tacagaccaa agtgtgattt cacactgttt ttaaatctag 1020
cattattcat tttgcttcaa tcaaaagtgg tttcaatatt ttttttagtt ggttagaata 1080
ctttcttcat agtcacattc tctcaaccta taatttggaa tattgttgtg gtcttttgtt 1140
ttttctctta gtatagcatt tttaaaaaaa tataaaagct accaatcttt gtacaatttg 1200
taaatgttaa gaattttttt tatatctgtt aaataaaaat tatttccaac aacctta 1257
<210> 28
<211> 2560
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1655369CB1
<400> 28
ttccagtgaa gagcaagtgc tgcccgaccc aggaccctgt gccaggctag cagccctcca 60
gctccctcca gagaggaaac ctctgtctgg ctgagggtgg gactagctgg gatgtctcac 120
tccagttgct caggttcacc caggaagctc ctccgtggag tggccagcct gattctagcc 180
ctgtcctctc tggcagcaca tgccacacct gcctgggcct tctgctccct gatgcttgat 240
gagcccctgc ctcctcaatg tttctcaaag acagaccccc ctgaggccag cttgaatgtg 300
aagactgctg aagtcagctg gcttcacttg agctgcagaa aaggtggctg ggatggccca 360
ggtgcaccca gaggccccag ccctttggct gcctttgggt tgtgacttgg gttgtctctg 420
aggccctgcc agagctgggc ctgcgggtgg tgggcggtcc gacctcgggc agtcagtgct 480
ccgcagcctc agcactgcat cccagaccca gtgtcctcag agggaagagc cagcctccct 540
gcctcatgga accaggagtc ccaaaaagtc aggagcctgg aggctctgaa aggagcaggg 600
attccatagt gcgtgaagct gaaataggcg ccctcctggg gagcccccag caaaactgtt 660
tttcataccc actcccagaa ctgccccgct ccagctccag cgccagcgcc agctggttgc 720
caggcgtcat tggagaggcc tggctgcccc aggggcagca gggagtggtg gacctgtatg 780
ggctggcagg aggccattgg ccatgctgac aagtgtcacc tgccttccta gcctggagcc 840
acccctcagg tggcctgctt gcacctccta tccggaggta gcctgcccca cctgtaggca 900
gagggggctc ttgcttgagg cctgcacagg aagcaagtat agccccggtg ccccagagtg 960
ggttccactt agccctggcg agatggcctg tcctgagatc tctgctccca gaccccacca 1020
tctggggagc acagtcctta ggctgcctgg tccaggaagg gggtgcggct ctgtcaggaa 1080
acctggactc tcaaggccca ccagcctctc cgtgagtgtt agaaatcaca gatacagtat 1140
atacttaatt acactaaatt attgctggga ttccttataa gcactaatta tacctgatta 1200
taggttaaaa tatttatttt gtcaaaatat tttcttggga atgtgtttaa ccctttctgc 1260
gttcattgtt gctgagatgt gaaaactaac cattccctcc tgcctacctt tttggccact 1320
gggcggcaga gaatggcgct atgtgcagtt gggcccctgg caccatgggc ctttggcctg 1380
cctgctgcag agtagccctg cctgggcagt ctccaggcac tgagcaggcc atctgtggcc 1440
aggctgagag aatgactggc tcgcttacca gcgtgcatgg gacaaggagc tttggagcct 1500
caaggggttg ttgctggcct gggctagagg gaaaggtgac catccgtctg tcctcctgtc 1560
tttctattag cgcctccatg tgagtgatgg tgccttggtt cactagcctt cccccaccac 1620
cccaccatgc cacctggtgg tcttggggcc tgtgctgtca ctccagcccc tggggaggag 1680
aggacccagc ccggagagtt ggggcaaggg ctccacatgg cccaagggca acagatgctc 1740
19/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
gcagggcagc tgctgccgat gctcacgctc ctgcccccct ccttcccgct gccacacccc 1800
accctgggcc cccgcagaca cgcatctcta actcagttgg gcccagcctt ctggatggct 1860
tggggtaggc catgggccca cctggggcca ggccagcccc tggggcagct ctggaagagc 1920
agtgtggagg agcacttgct tgcagcctgg cttcagcctc tggcactgct ggagtggtcc 1980
ctgggagctt ctgcactgtc ggctttgggg acgtctcacc cacttgggtt acagtaggcc 2040
ttccccaccc agagagaagt gtttccaccc cagagacatt gtctgtcagc ccctgaagtg 2100
ctcgcctccc ccagtgcccg tcaccagccc ttcctatctg tggggtccaa gtcaggcttc 2160
ccctgcggcc accagccata gggagcagcc atcagccccc gagtcagaac tgcttctgtc 2220
tgtccatacc tccaggctct cccggagagg gggacggata tttatttcct aaagtttgca 2280
cttaattgtg aggattctca ggattgttgg gggctactga aaagaggaat gtgttgaatg 2340
tcgcgtttgc tgtccactcg tcctagaagt ttagtgtttt tgtcactgtc atgtgtttct 2400
gtgggcagag ctggttctgg agggtgggtc agtgcacccg aggctcagag catccatcca 2460
ccccactggc cctccttcca gataccctct ctctaattgg gttcttgcat gtaaaatact 2520
ccacaataaa taaataattg aacaaattaa aaaaaaaaaa 2560
<210> 29
<211> 614
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1703244CB1
<400> 29
gtgcaagagg aacaaagaaa gggactcctg cagcgtccgg ctgacctggc ccttgtcata 60
tatctcatcc ttgctggctt cttcactctg ttccggggcc tggtggtgct tgattgcccc 120
acagatgcct gctttgtcta tatctaccag tatgagccat acctgcggga ccctgtggcc 180
taccctaagg tgcagatgct gatgtacatg ttttatgtcc tgcctttctg cggcctggct 240
gcctatgctc tcaccttccc tggttgctcc tggcttccag actgggcctt ggtgtttgct 300
ggaggcatcg gccaggcaca gttctcgcac atgggggctt ccatgcacct gcgcacaccc 360
ttcacctacc gtgtgcctga ggacacctgg ggctgcttct tcgtgtgcaa tctgctgtat 420
gcgctgggcc cccacctgct ggcctaccgt tgccttcagt ggcccgcatt cttccaccag 480
ccaccaccct ccgaccccct agccctccac aagaagcagc attgagagag ctgtggactc 540
aggacccagg actctgttta cgtgcccagt cagccctacc tggggaagcg ggggttgggt 600
gttttagaga cagg 614
<210> 30
<211> 1936
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1730819CB1
<400> 30
gactacgggc tcacagccgt cccttcgctg gtgggaagaa gccgagatgg cggcagccag 60
cgctggggca acccggctgc tcctgctctt gctgatggcg gtagcagcgc ccagtcgagc 120
ccggggcagc ggctgccggg ccgggactgg tgcgcgaggg gctggggcgg aaggtcgaga 180
gggcgaggcc tgtggcacgg tggggctgct gctggagcac tcatttgaga tcgatgacag 240
tgccaacttc cggaagcggg gctcactgct ctggaaccag caggatggta ccttgtccct 300
gtcacagcgg cagctcagcg aggaggagcg gggccgactc cgggatgtgg cagccctgaa 360
tggcctgtac cgggtccgga tcccaaggcg acccggggcc ctggatggcc tggaagctgg 420
tggctatgtc tcctcctttg tccctgcgtg ctccctggtg gagtcgcacc tgtcggacca 480
gctgaccctg cacgtggatg tggccggcaa cgtggtgggc gtgtcggtgg tgacgcaccc 540
cgggggctgc cggggccatg aggtggagga cgtggacctg gagctgttca acacctcggt 600
gcagctgcag ccgcccacca cagccccagg ccctgagacg gcggccttca ttgagcgcct 660
ggagatggaa caggcccaga aggccaagaa cccccaggag cagaagtcct tcttcgccaa 720
atactggatg tacatcattc ccgtcgtcct gttcctcatg atgtcaggag cgccagacac 780
cgggggccag ggtgggggtg ggggttgtgg tggtggtggg ggtagtggcc ggtgagggcc 840
caggctggtc agcgtcccgt cttgcacacc caggggcctc cctttctgct ggagtcccct 900
gtgtcctcag ccatcccaag aagggtttgc tggtccctcc tttccccccg tcccacgagg 960
ccacctgggc cagccccttg tcctctgcct tctgctggca gaggagcagc tggactgggg 1020
cctttggcac agcagccggt gtctcctgcg cccgcctccc ccatggcccc atgcagcccc 1080
20/28
CA 02363684 2001-08-14
cctgctgcag agtagccctg cctgggcagt ctccaggcac tgagcaggcc
WO 00/52151 PCT/US00/05621
aggggcttcc cccctgccca tggagtagag cccgagatcc tggccactat gccagttctg 1140
acctcgcatc cccctacccc gagcccatgc agtctgggaa catgccgcct tctctccagc 1200
ctctgtgcct ttgttccagg tggtctcacc ctcctgtccc tggctgggct aggtggtcct 1260
gtccaggctc ctgcagcgcc cccctcactt tgacactgga ctaggatgca gcctcccttc 1320
tgtgtcccct tgagggtacc ctgggtcccc tcatcagggg cagaggcatg aaagagtcgg 1380
ggctggatgg ccgggggctt ctgggcccga cgcctagtgc agcccctggg gtcgtggttt 1440
gacatttgtc tgcctggtgc aaacaaggaa tccttgcctt taaggtgaca ggccctccac 1500
aggcttccag acttgaagga aaaggtttaa gaaagaaaac aaaaccaaca gttagtggag 1560
tcaaagccca gacactgtaa atagaacccc ctccaccacc ccccgccgcc cagcatccta 1620
cctggactgc ggtgctacga gggcctgcgg gcctttgctg tgtgccaccc tccctgtaag 1680
tctatttaaa aacatcgacg atacattgaa atgtgtgaac gttttgaaaa gctacagctt 1740
ccagcagcca aaagcaactg ttgttttggc aagacggtcc tgatgtacaa gcttgattga 1800
aattcactgc tcacttgata cgttattcag aaacccaagg aatggctgtc cccatcctca 1860
tgtggctgtg tggagctcag ctgtgttgtg tggcagttta ttaaactgtc ccccagatcg 1920
acacgcaaaa aaaaaa 1936
<210> 31
<211> 1958
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1757161CB1
<400> 31
gccgcgcctt cagctacggc ccgagcgagc ccgccgccgc cgggcccggc cacagcctgc 60
agcggagccc acgagaggca gcgccatggc ggagcagacc tactcgtggg cctattccct 120
ggtggattcc agtcaagtgt ctacatttct gatttccatt cttcttatag tctatggtag 180
tttcaggtcc cttaatatgg actttgaaaa tcaagataag gagaaagaca gtaatagttc 240
ttctgggtct ttcaatggca acagcaccaa taatagcatc caaacaattg actctaccca 300
ggctctgttc cttccaattg gagcatctgt ctctctttta gtaatgttct tcttctttga 360
ctcagttcaa gtagttttta caatatgtac agcagttctt gcaacgatag cttttgcttt 420
tcttctcctc ccgatgtgcc agtatttaac aagaccctgc tcacctcaga acaagatttc 480
ctttggttgc tgtggacgtt tcactgctgc tgagttgctg tcattctctc tgtctgtcat 540
gctcgtcctc atctgggttc tcactggcca ttggcttctc atggatgcac tggccatggg 600
cctctgtgtc gccatgatcg cctttgtccg cctgccgagc ctcaaggtct cctgcctgct 660
tctctcaggg cttctcatct atgatgtctt ttgggtattt ttctcagcct acatcttcaa 720
tagcaacgtc atggtgaagg tggccactca gccggctgac aatccccttg acgttctatc 780
ccggaagctc cacctggggC ccaatgttgg gcgtgatgtt cctcgcctgt ctctgcctgg 840
aaaactggtc ttcccaagct ccactggcag ccacttctcc atgttgggca tcggagacat 900
cgttatgcct ggtctcctac tatgctttgt ccttcgctat gacaactaca aaaagcaagc 960
cagtggggac tcctgtgggg cccctggacc tgccaacatc tccgggcgca tgcagaaggt 1020
ctcctacttt cactgcaccc tcatcggata ctttgtaggc ctgctcactg ctactgtggc 1080
gtctcgcatt caccgggccg cccagcccgc ccttctctat ttggtgccat ttactttatt 1140
gccactcctc acgatggcct atttaaaggg cgacctccgg cggatgtggt ctgagccttt 1200
ccactccaag tccagcagct cccgattcct ggaagtatga tggatcacgt ggaaagtgac 1260
cagatggccg tcatagtcct tttctctcaa ctcatggttt gtttcctctt agagctggcc 1320
tggtactcag aaatgtacct gtgtttaagg aactgccgtg tgactggatt tggcatttaa 1380
agggagctcg tttgcaggag agaggtgctg gagccctgtt tggttccttc tcttcctgcg 1440
gatgtagagg tggggcccct tccaagaggg acaggcctct ccccagcgcg ccttcctccc 1500
acgtttttat ggatctgcac cagactgtta ccttctgggg gagatggaga tttgactgtt 1560
taaaaactga aaacagcgag gagtctttct agaacttttg aacactaaaa ggatgaaaaa 1620
aattagcaaa ccgaagtttc ttcaatgacc cctcgagaac tttgggacca gtttcctatg 1680
ggggactcag tttcagagaa ctgagacaga agctcttctg tcgttatatt cttctttcct 1740
ttttttggat ttattaaata ttttctgtgg tgtgaagtga cttattaaat ccacagacat 1800
tgagtgactt cttacaacat ccacataaga atttgttgta atgagttcat gtccacccag 1860
atgttgtgtt ggcagtgaac aagggcacgg tttttataca tacgtacata tatatatata 1920
aacacacaca tagatatata tgaataaaca aaaatgat 1958
<210> 32
<211> 1424
<212> DNA
<213> Homo Sapiens
21/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<220>
<221> misc_feature
<223> Incyte ID No: 1976095CB1
<400> 32
gtcaagtagg agacaaggag caaagtccta tcacagcggg aggggacgcc agcgcctgca 60
gaggctgagc agggaaaaag ccagtgcccc agcggaagca cagctcagag ctggtctgcc 120
atggacatcc tggtcccact cctgcagctg ctggtgctgc ttcttaccct gcccctgcac 180
ctcatggctc tgctgggctg ctggcagccc ctgtgcaaaa gctacttccc ctacctgatg 240
gccgtgctga ctcccaagag caaccgcaag atggagagca agaaacggga gctcttcagc 300
cagataaagg ggcttacagg agcctccggg aaagtggccc tactggagct gggctgcgga 360
accggagcca actttcagtt ctacccaccg ggctgcaggg tcacctgcct agacccaaat 420
ccccactttg agaagttcct gacaaagagc atggctgaga acaggcacct ccaatatgag 480
cggtttgtgg tggctcctgg agaggacatg agacagctgg ctgatggctc catggatgtg 540
gtggtctgca ctctggtgct gtgctctgtg cagagcccaa ggaaggtcct gcaggaggtc 600
cggagagtac tgagaccggg aggtgtgctc tttttctggg agcatgtggc agaaccatat 660
ggaagctggg ccttcatgtg gcagcaagtt ttcgagccca cctggaaaca cattggggat 720
ggctgctgcc tcaccagaga gacctggaag gatcttgaga acgcccagtt ctccgaaatc 780
caaatggaac gacagccccc tcccttgaag tggctacctg ttgggcccca catcatggga 840
aaggctgtca aataatcttt cccaagctcc aaggcactca tttgctcctt ccccagcctc 900
caattagaac aagccaccca ccagcctatc tatcttccac tgagagggac ctagcagaat 960
gagagaagac attcatgtac cacctcctag tccctctctc cccaacctct gccagggcaa 1020
tctctaactt caatcccgcc ttcgacagtg aaaaagctct acttctacgc tgacccaggg 1080
aggaaacact aggaccctgt tgtatcctca actgcaagtt tctggactag tctcccaacg 1140
tttgcctccc aatgttgtcc ctttccttcg ttcccatggt aaagctcctc tcgctttcct 1200
cctgaggcta cacccatgcg tctctaggaa ctggtcacaa aagtcatggt gcctgcatcc 1260
ctgccaagcc cccctgaccc tctctcccca ctaccacctt cttcctgagc tgggggcacc 1320
agggagaatc agagatgctg gggatgccag agcaagactc aaagaggcag aggttttgtt 1380
ctcaaatatt ttttaataaa tagacgaaac cacgaaaaaa aaaa 1424
<210> 33
<211> 2238
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2169991CB1
<400> 33
cctgtctgtg cacacctcac ggcaagggcc agcctgtttc ctcccggtca cctccaaatc 60
ttgctgcttt taattcaact cagaggtatg cacttgaggt aggagggcag gggaagtggg 120
gatggcagga catggatggc ccttgaggca ttggctctgg gtgtcatggg ctgtgagagt 180
caagaagggc agtggcctgc ctgacttggg ttcgaaaggg tcactctggc cactgcggtg 240
agaactgaaa ctagccaggc caattctcca ttgttcctgc tcttccaggt aggagaatat 300
tagattaggt ctggagtcag aaggtagcag gggctggggg ttgcaggggg atgttgagaa 360
gaagtggtcc tttggtcagg gtgggaagcc aacaggattt cctggtgcat tggaggtgaa 420
agggaagagg cgctggtggt tgtgctttgg gcctgagcag ccagaagccg ttgccatcac 480
ctatgacaag ggagacagcg cctggggcag agcccagtat ggggtgcatt cagggtagat 540
caggaggtat gagtgggcag tgggcgagcc aggctggggg ttgtggggga ggcctgggtt 600
gcaggtttaa gcgtgggacc cacgtcagat tggtggtggg tcatgcatgc tggggtggct 660
ctcaggtcct ccctgctggc ttcccactcc caggggcttt ctcctcccag attccttagc 720
tgggtgaggg gcaggacaga gccctttcct agggaagccc ggcaccccct gctgtccagg 780
gaaggggagt ccttctagcc cctgacagct tctctgcccc tcccctggcc tccccaggcc 840
tagccagggt tgagttctca cccacctgtg ccgccctgcc ttgttacctg gaagcacagc 900
cttggggact gagcaggccc tcactgtcac tttaagaagg gaatcagcca ctttgtgctc 960
accacctctg gggaaggtgt gagaggagag aaggaagtgg ctgtttggct gctgacaaca 1020
tgaagacttc ctgcgatgag aacagaggca caggtgccgg ccctgcagcc cccagaacct 1080
ggactggagg gggccatggg gcaccggacc ctggtcctgc cctgggtgct gctgaccttg 1140
tgtgtcactg cggggacccc ggaggtgtgg gttcaagttc ggatggaggc caccgagctc 1200
tcgtccttca ccatccgttg tgggttcctg gggtctggct ccatctccct ggtgactgtg 1260
agctgggggg gccccaacgg tgctgggggg accacgctgg ctgtgttgca cccagaacgt 1320
ggcatccggc aatgggcccc tgctcgccag gcccgctggg aaacccagag cagcatctct 1380
ctcatcctgg aaggctctgg ggccagcagc ccctgcgcca acaccacctt ctgctgcaag 1440
tttgcgtcct tccctgaggg ctcctgggag gcctgtggga gcctcccgcc cagctcagac 1500
ccagggctct ctgccccgcc gactcctgcc cccattctgc gggcagacct ggccgggatc 1560
22/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
ttgggggtct caggagtcct cctctttggc tgtgtctacc tccttcatct gctgcgccga 1620
cataagcacc gccctgcccc taggctccag ccgtcccgca ccagccccca ggcaccgaga 1680
gcacgagcat gggcaccaag ccaggcctcc caggctgctc ttcacgtccc ttatgccact 1740
atcaacacca gctgccgccc agctactttg gacacagctc acccccatgg ggggccgtcc 1800
tggtgggcgt cactccccac ccacgctgca caccggcccc agggccctgc cgcctgggcc 1860
tccacaccca tccctgcacg tggcagcttt gtctctgttg agaatggact ctacgctcag 1920
gcaggggaga ggcctcctca cactggtccc ggcctcactc ttttccctga ccctcggggg 1980
cccagggcca tggaaggacc cttaggagtt cgatgagaga gaccatgagg ccactgggct 2040
ttccccctcc caggcctcct gggtgtcacc cccttacttt aattcttggg cctccaataa 2100
gtgtcccata ggtgtctggc caggcccacc tgctgcggat gtggtctctg tgtgtgcgtg 2160
tgtgggcaca ggtgtgagtg tgtgagtgac agttacccca tttcagtcat ttcctgctgc 2220
aactaagtca gcaacgcc 2238
<210> 34
<211> 536
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2616827CB1
<400> 34
gcatgaactt gggggtcagc atgctgagga tcctcttcct cctggatgta ggaggagctc 60
aagtgctggc aacaggcaag acccctgggg ctgaaattga tttcaagtac gccctcatcg 120
ggactgctgt gggtgtcgcc atatctgctg gcttcctggc cctgaagatc tgcatgatca 180
ggaggcactt atttgacgac gactcttccg acctgaaaag cacgcctggg ggcctcagtg 240
acaccatccc gctaaagaag agagccccaa ggcgaaacca caatttctcc aaaagagatg 300
cacaggtgat tgagctgtag gtgagcagtg acgtgaagag gggttctagc cccgtggaaa 360
acagcccatg gttaacatct caggatgttc tgcattcaaa cacccaaggc tggtaatgaa 420
ctttcacatg gactgaatat tggaggcaaa taatagaagg aatagaatat acagtgcctc 480
tgtcctgaag gaaaatatca tgcctcttct ggaagaaacg gactgcacag aggaag 536
<210> 35
<211> 2177
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2991370CB1
<400> 35
cgggaggctc gaggccagcc cgggaccggg gctgggagca agcaggcggc ggcgccggcg 60
gcagaggcgg cagcgagcgc ccgcttccca cgcccctagg cggcggggcc gagagcggga 120
ggatggctcc gagcgctgac cccggcatgt ccaggatgtt accgttcctg ctgctgctct 180
ggtttctgcc catcactgag gggtcccagc gggctgaacc catgttcact gcagtcacca 240
actcagttct gcctcctgac tatgacagta atcccaccca gctcaactat ggtgtggcag 300
ttactgatgt ggaccatgat ggggactttg agatcgtcgt ggcggggtac aatggaccca 360
acctggttct gaagtatgac cgggcccaga agcggctggt gaacatcgcg gtcgatgagc 420
gcagctcacc ctactacgcg ctgcgggacc ggcaggggaa cgccattggg gtcacagcct 480
gcgacatcga cggggacggc cgggaggaga tctacttcct caacaccaat aatgccttct 540
cgggggtggc cacgtacacc gacaagttgt tcaagttccg caataaccgg tgggaagaca 600
tcctgagcga tgaggtcaac gtggcccgtg gtgtggccag cctctttgcc ggacgctctg 660
tggcctgtgt ggacagaaag ggctctggac gctactctat ctacattgcc aattacgcct 720
acggtaatgt gggccctgat gccctcattg aaatggaccc tgaggccagt gacctctccc 780
ggggcattct ggcgctcaga gatgtggctg ctgaggctgg ggtcagcaaa tatacagggg 840
gccgaggcgt cagcgtgggc cccatcctca gcagcagtgc ctcggatatc ttctgcgaca 900
atgagaatgg gcctaacttc cttttccaca accggggcga tggcaccttt gtggacgctg 960
cggccagtgc tggtgtggac gacccccacc agcatgggcg aggtgtcgcc ctggctgact 1020
tcaaccgtga tggcaaagtg gacatcgtct atggcaactg gaatggcccc caccgcctct 1080
atctgcaaat gagcacccat gggaaggtcc gcttccggga catcgcctca cccaagttct 1140
ccatgccctc ccctgtccgc acggtcatca ccgccgactt tgacaatgac caggagctgg 1200
agatcttctt caacaacatt gcctaccgca gctcctcagc caaccgcctc ttccgcgtca 1260
tccgtagaga gcacggagac cccctcatcg aggagctcaa tcccggcgac gccttggagc 1320
23/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
ctgagggccg gggcacaggg ggtgtggtga ccgacttcga cggagacggg atgctggacc 1380
tcatcttgtc ccatggagag tccatggctc agccgctgtc cgtcttccgg ggcaatcagg 1440
gcttcaacaa caactggctg cgagtggtgc cacgcacccg gtttggggcc tttgccaggg 1500
gagctaaggt cgtgctctac accaagaaga gtggggccca cctgaggatc atcgacgggg 1560
gctcaggcta cctgtgtgag atggagcccg tggcacactt tggcctgggg aaggatgaag 1620
ccagcagtgt ggaggtgacg tggccagatg gcaagatggt gagccggaac gtggccagcg 1680
gggagatgaa ctcagtgctg gagatcctct acccccggga tgaggacaca cttcaggacc 1740
cagccccact ggagtgtggc caaggattct cccagcagga aaatggccat tgcatggaca 1800
ccaatgaatg catccagttc ccattcgtgt gccctcgaga caagcccgta tgtgtcaaca 1860
cctatggaag ctacaggtgc cggaccaaca agaagtgcag tcggggctac gagcccaacg 1920
aggatggcac agcctgcgtg ggctggtgga gccctgtgtt gaagatagtg acaccacaag 1980
ttgggaagag ccttggtccc tgaatcactg aatcactgcc ttgaatcacc gcctggaata 2040
cctgttgatc aggaacactt acctggaact tcactgagca ggatacaaac ttctattgta 2100
ttaagctatt aatacattaa gatttggggg tgctacctta cataataaat tcccatttcc 2160
tcttgaaaaa aaaaaaa 2177
<210> 36
<211> 2043
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3031062CB1
<400> 36
cgccacgacg cagcggggaa tctgcagtag gtctgccggc gatggagtgg tgggctagct 60
cgccgcttcg gctctggctg ctgttgttcc tcctgccctc agcgcagggc cgccagaagg 120
agtcaggttc aaaatggaaa gtatttattg accaaattaa caggtctttg gagaattacg 180
aaccatgttc aagtcaaaac tgcagctgct accatggtgt catagaagag gatctaactc 240
ctttccgagg aggcatctcc aggaagatga tggcagaggt agtcagacgg aagctaggga 300
cccactatca gatcactaag aacagactgt accgggaaaa tgactgcatg ttcccctcaa 360
ggtgtagtgg tgttgagcac tttattttgg aagtgatcgg gcgtctccct gacatggaga 420
tggtgatcaa tgtacgagat tatcctcagg ttcctaaatg gatggagcct gccatcccag 480
tcttctcctt cagtaagaca tcagagtacc atgatatcat gtatcctgct tggacatttt 540
gggaaggggg acctgctgtt tggccaattt atcctacagg tcttggacgg tgggacctct 600
tcagagaaga tctggtaagg tcagcagcac agtggccatg gaaaaagaaa aactctacag 660
catatttccg aggatcaagg acaagtccag aacgagatcc tctcattctt ctgtctcgga 720
aaaacccaaa acttgttgat gcagaataca ccaaaaacca ggcctggaaa tctatgaaag 780
ataccttagg aaagccagct gctaaggatg tccatcttgt ggatcactgc aaatacaagt 840
atctgtttaa ttttcgaggc gtactgcaag tttccggttt aaacacctct tcctgtgtgg 900
ccattattct aatgagaaag agaacatact agtatggaaa tttcttaagg gcaggaagtc 960
atgtatttac agatgtttat tgagtacctg ttatatatca ggactaagct gctgggatgt 1020
actagaaaat acgatatttg tcactctgct tccatggaaa ttttagacta gcataatacc 1080
tggaacatag tagatgaaaa aaaatacatg tggttttagt ggttgaaatt taagcatttg 1140
caagttatta tacttatgac tcacaaattt acctttcacc gaattaagcc aaaaagactt 1200
tatccagaaa atagttgagt agagatgaat acataggatc ctgcatgatt taactttctc 1260
tttgcatttg ggataataca ctcaacacat ttctctacca catttaaaat ttttatttat 1320
atcctgcctt cttcccaaaa ggatttaaag cagcttacaa aaatgtatag~cagggcggga 1380
taaaataaag aaacagacta tacaacatgc atactgtgaa gtcctcttca cttccaggta 1440
ccatcatatg gaacccttat cctcccacca caaaatgtat aattctgctt atatctgtac 1500
ctgtgatctc tcttctttca aaaacgtaga cctttcttct tatgaaaaaa acaatccccc 1560
cacatatatt ctggatggca tcctccttca ctttgtctgg gactttggct ctgcccactg 1620
tcctcatcat ccttgtatcc aggaccacag cctttgtcat gcagacatat tcagcttcct 1680
cccatctccc caacctccag caaaagaaaa cccttcattt tcacattccc cttcagccga 1740
gattgaagat gaagatgagg atgagttcaa ggatgaagac caggatgagg acaaggatga 1800
ggatggagtc tagagcctcc cagagcctgg agaggaggcc tcggtcagcc actccgtgga 1860
cgtgggccac ggtgacccac catgaagtcc ccactagcca ctcgattccc tgctctgtca 1920
gagttgctgc acatcacacc agcccctgcc aagagcagga gtcaccacag gctgaatgcc 1980
cacgaggagc tctgctgaga ctctcaaggg agccagtgaa agaaatagaa ataaagcctg 2040
tgc 2043
<210> 37
<211> 1743
<212> DNA
24/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3101617CB1
<400> 37
cagcaggtca cagcccctcg aggcgacagc ggccccgccg caccagagca gtggtacagg 60
catggatggg aagaaatgca gcgtatggat gttcctacct cttgtattta ctttgtttac 120
ttcagctgga ttgtggatag tatacttcat agctgtggaa gatgacaaaa ttttaccatt 180
aaattcagct gaaaggaaac ctggtgtgaa gcatgcacca tatataagca ttgcaggtga 240
tgatcctcct gcaagctgtg tgtttagtca agttatgaac atggcagcct tcctagccct 300
tgtggtagct gttctgcgct tcatacaact gaaaccgaag gttttaaacc cgtggctgaa 360
tattagtgga ttggtggctc tgtgtctggc ttccttcgga atgaccttac ttggtaattt 420
tcagctcaca aatgatgaag aaatccataa cgtcggaact tccttgacct ttggatttgg 480
cacattgacc tgctggatcc aggctgcgct gacactcaag gtcaacatca agaatgaagg 540
acggagagtt ggaattccac gggttattct gtcggcatct atcactctct gtgtggtcct 600
ctacttcatc ctcatggccc aaagcatcca catgtatgca gccagggtcc agtggggcct 660
ggtcatgtgc ttcctgtctt attttggcac ctttgccgtg gagttccggc attaccgcta 720
tgagattgtt tgctctgagt accaggagaa tttcctaagc ttctcagaaa gcctgtcaga 780
agcttctgaa tatcagactg accaggtgta aaccatcagt ttttccttgc tggtgaggtg 840
ggtgtgacag tgggggaggg gccagtagga cacactcaca ggacttgaca tagaacctca 900
tttcacacac acacacacac acacattcat ggccacattt gccaaatgag cttttcaggg 960
cgagttattt ctttaatgaa aaagcacaag cccttatgtg tcgaaataca cgctgttaca 1020
ctgaaaatat atgcacgaca gagcaagaag cttgtgcatg atcacttctt atccgtcccc 1080
ttcccagcac tccctcctct tcccattctc tccacatgtc tcaagcaccc taccgagtag 1140
ggcaggccaa atgttccttg ggagtaatgc caactcccga cgttgccttc aggtccaaag 1200
ggcttggaac cagctcgtga ggaagttctg aatctggcac taatattctt gagtggataa 1260
tagtgtatca tagaatagga cggaaattgt attgagatgt gaccctgtgt cgcctgtgga 1320
aaggcatagt gagaagaact ttcccacgaa agcccccttc atcgttgttc agtggtcggc 1380
tgtgtggatc ccaggagaga catatgccac agactgtgag agcaaagccc gccgctgtga 1440
tctggacttg atgcactgtg actgagaatg atttccaaat gtgaatatgt gtagggacgt 1500
ggtctatcag gcctggaaca agatgggggc agtgaaggta tggtttagtg tttgctttca 1560
tagtatgcca tgtacaatgt tttatatttc atagtttctt ttaagtaact accatgagtc 1620
tctctaagcc tcatggacaa agatgtagac caaatgcaag agctgagctt gctttgggtt 1680
caaccatgat caaagaaaaa ctgaggtcac ctgcaggctt acgtgggaag ctaagacaat 1740
atc 1743
<210> 38
<211> 1306
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3216178CB1
<400> 38
ctgcaaagtt cctgtgagcg ctgtcatttt gtcactctgg tttttcagat tcttcccctg 60
gaggctggag tttccaggat gtcaaaatta cctctgcttg ggtgagctat ttcaagcagc 120
tgggatacct gtgtcactcc tgctgtctgc cagtgactgc ccaggtgtct gctggttcct 180
ccccaggagt agggaggaac caggtgggct ggctgggatg ggtggatatt taaagaccag 240
gccttggacg ctgcagcact tctatctctg cttgatgcct gctgccacgt ggctggtcct 300
cctcctcctg ctgtggctga gccttggggt gaagacaggc agctgctccc aaccccagaa 360
cctttgctgt cttgggacgg atcaccactg caagagggga agttgctact gtgatgaatt 420
ctgccatgtg gcaccagact gccacccaga ccacagtgtc ctctgcaacc ctgcttctca 480
gatgaccaag atggtgctgc agatggtgct gaggatggag aacccaccaa gccccgctag 540
gagccaccta gactggatgc agagcatggt gagctccctg caggttctct gagaaggggt 600
ggatggcagc ctgctccttg cctttgtgcc ctccaggccc caaagtcagg gaaccaaaag 660
aagaaagggg ccgtagctag ggcagagctc cactgcaatg attgttttag gggtaggagc 720
caggattgcc gtctgtggac actgaaattt gaatctcata tacttttgtg acaaaacatt 780
cttcctcttt tgttcttctc ctaccatcta aaaatgtaga aaacattctt agcctatgag 840
ttgcacaaaa acaggcagtg gccagatttg gcccatagac catagtttgc tgacttctgc 900
cctaaatcat cctccatttc tttccttctg tgtccttgtt actgacaaag ccactttccc 960
taaaatgggg tctttccctg tttggtgcca tgaagccaat atgcaaaacc gaaagtgagc 1020
ctcaagcagt gcaggcttta tttggtggcc atggaattga gaagtgagag cttggctcac 1080
25/28
CA 02363684 2001-08-14
WO 00/52151 PCT/LTS00/05621
aaatcaactt ttctgctcgt gagacccagg aagtcacaga tacagggcat ctttagtgaa 1140
ggggctgagc attaaaagca aggggaggag tggccaggtg caatggctca ctcccataaa 1200
cccagaactt tgggaggcca aaatgagagg attgctgaga ccaggagttc gagaccatcc 1260
tggtcaacat agtgatacac ccccatctct acaaaaataa aaatga 1306
<210> 39
<211> 851
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3406803CB1
<400> 39
gggctggcca cactgcaggg gctgcaggaa gcaaaggatg aaactgatct tttcactaac 60
cagcataggg cacaggccaa agaaaacttt ggttactctc ttgtgagcca gttgaagtta 120
gacttgttct ctttggaata ggtcttcctc tgcagaaata aaaacacttg tctgaaagag 180
gctgtaacta tctgtgggct gttgggcaag gacacttcag atactggctt tgagctcact 240
gggtcctggc ccctcatggc tctgtgatca tctctgctcc acattgcagg ccatgctccc 300
tgttggtgct cagcccagga gccctccttg ggtcctggcc agactcctcc accctcgtgg 360
gcctgccgca accagtctag tgccttttct cccatggggg tccctggaat ctcacacacc 420
ctgcccttac agagcctgca gtccagggtg ggagttaacc ctttccactt tcccagagag 480
ggagaccttg agtggagggg aggtcaggaa acgcggggct ggcagcatgg tgggaggagg 540
tgaaagtacg atgaccagag ccttgtgtgt gcggctgttg acaaagctga gggtatgatg 600
ggtgctgtgt gcagggcgtg cctgagaaga ccactcctga tggagtgctg ggggaagttc 660
aaggcttgga ggccaatgag ggggctgatg gcacccatcc agtggagagg ctgtatttgg 720
aggccctcct gagtgggagc aaggctatgt ggatgatgag atgaggtaag tgagatggaa 780
gtggctgtac gaacagccat tgaggctgag gcaggaggat tgcctaagtc catagcccag 840
851
gtgtcgagac t
<210> 40
<211> 2204
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3468066CB1
<400> 40
gagcagtcct tgctggtccc gcccccgctc ggctcgccgc caggggacgc tagtgggtcc 60
agtctccttg gcaaccactt gctcctcccc ctccgcccct ttaaccttta gggtgcgcgg 120
gtgcagtata tctcgcgctc tctccccttt cccccccccc ttttcccacc ccgggcgctc 180
aggttggtct ggaccggaag cgaagatggc gacttctggc gcggcctcgg cggagctggt 240
gatcggctgg tgcatattcg gcctcttact actggctatt ttggcattct gctggatata 300
tgttcgtaaa taccaaagtc ggcgggaaag tgaagttgtc tccaccataa cagcaatttt 360
ttctctagca attgcactta tcacatcagc acttctacca gtggatatat ttttggtttc 420
ttacatgaaa aatcaaaatg gtacatttaa ggactgggct aatgctaatg tcagcagaca 480
gattgaggac actgtattat acggttacta tactttatat tctgttatat tgttctgtgt 540
gttcttctgg atcccttttg tctacttcta ttatgaagaa aaggatgatg atgatactag 600
taaatgtact caaattaaaa cggcactcaa gtatactttg ggatttgttg tgatttgtgc 660
actgcttctt ttagttggtg cctttgttcc attgaatgtt cccaataaca aaaattctac 720
agagtgggaa aaagtgaagt ccctatttga agaacttgga agtagtcatg gtttagctgc 780
attgtcattt tctatcagtt ctctgacctt gattggaatg ttggcagcta taacttacac 840
agcctatggc atgtctgcgt tacctttaaa tctgataaaa ggcactagaa gcgctgctta 900
tgaacgtttg gaaaacactg aagacattga agaagtagaa caacacattc aaacgattaa 960
atcaaaaagc aaagatggtc gacctttgcc agcaagggat aaacgcgcct taaaacaatt 1020
tgaagaaagg ttacgaacac ttaagaagag agagaggcat ttagaattca ttgaaaacag 1080
ctggtggaca aaattttgtg gcgctctgcg tcccctgaag atcgtctggg gaatattttt 1140
catcttagtt gcattgctgt ttgtaatttc tctcttcttg tcaaatttag ataaagctct 1200
tcattcagct ggaatagatt ctggtttcat aatttttgga gctaacctga gtaatccact 1260
gaatatgctt ttgcctttac tacaaacagt tttccctctt gattatattc ttataacaat 1320
tattattatg tactttattt ttacttcaat ggcaggaatt cgaaatattg gcatatggtt 1380
cttttggatt agattatata aaatcagaag aggtagaacc aggccccaag cactcctttt 1440
26/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
tctctgcatg atacttctgc ttattgtcct tcacactagc tacatgattt atagtcttgc 1500
tccccaatat gttatgtatg gaagccaaaa ttacttaata gagactaata taacttctga 1560
taatcataaa ggcaattcaa ccctttctgt gccaaagaga tgtgatgcag aagctcctga 1620
agatcagtgt actgttaccc ggacatacct attccttcac aagttctggt tcttcagtgc 1680
tgcttactat tttggtaact gggcctttct tggggtattt ttgattggat taattgtatc 1740
ctgttgtaaa gggaagaaat cggttattga aggagtagat gaagattcag acataagtga 1800
tgatgagccc tctgtctatt ctgcttgaca gccttctgtc ttaaaggttt tataatgctg 1860
actgaatatc tgttatgcat ttttaaagta ttaaactaac attaggattt gctaactagc 1920
tttcatcaaa aatgggagca tggctataag acaactatat tttattatat gttttctgaa 1980
gtaacattgt atcatagatt aacattttaa attaccataa tcatgctatg taaatataag 2040
actactggct ttgtgaggga atgtttgtgc aaaatttttt cctctaatgt ataatagtgt 2100
taaattgatt aaaaatcttc cagaattaat attccctttt gtcacttttt gaaaacataa 2160
taaatcatct gtatctgtgc cttaggttct ccaaaaaaaa aaaa 2204
<210> 41
<211> 570
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3592862CB1
<400> 41
gcgcggaggc tcggggagtc ggcgccatga ccccatcgag gcttccctgg ttgcttagct 60
gggtctcggc cacggcgtgg agagcggcaa gatcacccct tctgtgtcat tctctgagga 120
aaacaagttc ttctcaagga ggaaagtctg aacttgtcaa acagtccctt aagaagccga 180
agttaccaga aggtcgtttt gatgcaccag aggattccca tttagagaaa gaaccactgg 240
aaaaatttcc agatgatgtt aatccagtga ccaaagaaaa aggtggaccc aggggcccag 300
aacctacccg atatggagat tgggaacgaa aaggacgctg tattgatttt taagtcgcat 360
attctttaac ttcaatattg ttttctgaat atgtacatct gaattaactt atttctgatt 420
attttctttc tttatatcct ttatgtcgtg tagtttgtgt aatgtgttta aatatatata 480
tatatatata tatatatata tatatatatg ggggcttagg aagaaaatat gctgctgtaa 540
attaggaaag ggagaccagc ctgaccaata 570
<210> 42
<211> 802
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3669422CB1
<400> 42
cagggtcaag gtgaagctgg tggtgtctcg aggcgggtga gtgtcatggg ggagcctggg 60
tgggggtcac actggctctc tctagtccca tgtcgtcgtc ctcttcacga tgcctctccc 120
cttccccagg gatgtctctg tggagctgcc ttttgttctt atgcacccca agccccacga 180
ccacatcccc ctccccagac cccagtcagg tgagcacact acccacccca agccctcaga 240
gggagggcct gaagcagggc cagtggagga aaactggccc ttccagcacc cacccccaca 300
ccccctcttc ccgtcccccc agcccctctt cccttcccct cacctggaag cttcttcaac 360
caatcccttc acactctctc ccccatcccc ccaagataca cactggaccc tctcttgctg 420
aatgtgggca ttaatttttt gactgcagct ctgcttctcc agccccgccg tgggtggcaa 480
gctgtgttca tacctaaatt ttctggaagg ggacagtgaa aagaggagtg acaggaggga 540
aagggggaga caaaactcct actctcaacc tcacaccaac acctcccatt atcactctct 600
ctgcccccat tccttcaaga ggagaccctt tggggacaag gccgtttctt tgtgaggaat 660
aaaaaggtta gaagggcccc cctctctgaa ggcccccact ccctgggatg ctacaatcca 720
atgatggaag atggcattag ctacaccacc ctgcgctttc ccgagatgaa cataccacga 780
actggagatg cagagtcctc ag 802
<210> 43
<211> 693
<212> DNA
27/28
CA 02363684 2001-08-14
WO 00/52151 PCT/US00/05621
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3688740CB1
<400> 43
gttggtttaa tgggattgtg gaagagaatg actccaatat ttggaagttc tggtacacca 60
atcagccacc gtccaagaac tgcacacatg cttacctgtc tccgtacccc ttcatgagag 120
gcgagcacaa ctcgacctcc tatgactctg cagttattta ccgtggtttc tgggcagtcc 180
tgatgctcct gggggtagtt gctgtagtca tcgcaagctt tttgatcatc tgtgcagccc 240
ccttcgccag ccattttctc tacaaagctg ggggaggctc atatattgct gcagatggaa 300
tttcttctct ctgttactca agcctctcaa agtccttatt gtcccagcct ctgcgtgaaa 360
cgtcttcagc catcaatgac atctcactcc ttcaagccct tatgccactg ctgggatgga 420
ccagtcactg gacctgcatt acagtgggct tatattgacc tttacctcta cacttgtata 480
taactcgttt tcccatttag ttgcaagaca cttggaagca cagaccaagg cttacatttg 540
tgttttaatg tttttcttgt aaatgcttta tgcctaaatg tttctgtact actcttcttt 600
ccaaatcctt attgtttaaa agtttctctc ctactatacc atgccttata aatattgatt 660
gaatgaatgg atgaaatgca tacctgctta tag 693
<210> 44
<211> 1212
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3742589CB1
<400> 44
ccctcgaggc aacttgccct tctcaaacat ggccgccacg gcgcctctgg aagggaaccg 60
ctctgggccc cgcctttgat ctcgttggtg gggctggggg atgagagctg caccgcgcgg 120
gacaagtcgc cggcggcgcc cgacggagca gaacagagag catggagctg gagaggatcg 180
tcagtgcagc cctccttgcc tttgtccaga cacacctccc ggaggccgac ctcagtggct 240
tggatgaggt catcttctcc tatgtgcttg gggtcctgga ggacctgggc ccctcgggcc 300
catcagagga gaacttcgat atggaggctt tcactgagat gatggaggcc tatgtgcctg 360
gcttcgccca catccccagg ggcacaatag gggacatgat gcagaagctc tcagggcagc 420
tgagcgatgc caggaacaaa gagaacctgc aaccgcagag ctctggtgtc caaggtcagg 480
tgcccatctc cccagagccc ctgcagcggc ccgaaatgct caaagaagag actaggtctt 540
cggctgctgc tgctgcagac acccaagatg aggcaactgg cgctgaggag gagcttctgc 600
caggggtgga tgtactcctg gaggtgttcc ctacctgttc ggtggagcag gcccagtggg 660
tgctggccaa agctcggggg gacttggaag aagctgtgca gatgctggta gagggaaagg 720
aagaggggcc tgcagcctgg gagggcccca accaggacct gcccagacgc ctcagaggcc 780
cccaaaagga tgagctgaag tccttcatcc tgcagaagta catgatggtg gatagcgcag 840
aggatcagaa gattcaccgg cccatggctc ccaaggaggc ccccaagaag ctgatccgat 900
acatcgacaa ccaggtagtg agcaccaaag gggagcgatt caaagatgtg cggaaccctg 960
aggccgagga gatgaaggcc acatacatca acctcaagcc agccagaaag taccgcttcc 1020
attgaggcac tcgccggact ctgcccgagc cttctaggct cagatcccag agggatgcag 1080
gagccctata cccctacaca ggggccccct aactcctgtc ccccttctct actcctttgc 1140
tccatagtgt taacctactc tcggagctgc ctccatgggc acagtaaagg tggcccaagg 1200
aaaaaaaaaa as ' 1212
28/28
CA 02363684 2001-08-14
