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SECRETED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of secreted
proteins and to
the use of these sequences in the diagnosis, treatment, and prevention of cell
proliferative,
autoimmune/inflammatory, cardiovascular, neurological, and developmental
disorders, and in the
assessment of the effects of exogenous compounds on the expression of nucleic
acid and amino acid
sequences of secreted proteins.
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. Proteins that are retained in the plasma
membrane contain one or
more transmembrane domains, each comprised of about 20 hydrophobic amino acid
residues.
Secreted proteins are generally 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 proteins
with important roles in
cell-to-cell signaling. Such proteins include transmembrane receptors and cell
surface markers,
extracellular matrix molecules, cytokines, hormones, growth and
differentiation factors, enzymes,
neuropeptides, vasomediators, cell surface markers, and antigen recognition
molecules. (Reviewed in
Alberts, B. et al. (1994) Molecular Biology of The Cell, Garland Publishing,
New York, NY, pp. 557-
560, 582-592.)
Cell surface markers 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
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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 Leucocyte 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
IS 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.)
Mucins are highly glycosylated glycoproteins that are the major structural
component of the
mucus gel. The physiological functions of mucins are cytoprotection,
mechanical protection,
maintenance of viscosity in secretions, and cellular recognition. MUC6 is a
human gastric mucin that
is also found in gall bladder, pancreas, seminal vesicles, and female
reproductive tract (Toribara,
N.W. et al. (1997) J. Biol. Chem. 272:16398-16403). The MUC6 gene has been
mapped to human
chromosome 11 (Toribara, N.W. et al. (1993) J. Biol. Chem. 268:5879-5885).
Hemomucin is a novel
Drosophila surface mucin that may be involved in the induction of
antibacterial effector molecules
(Theopold, U. et al. (1996) J. Biol. Chem. 217:12708-12715).
Tuftelins are one of four different enamel matrix proteins that have been
identified so far.
The other three known enamel matrix proteins are the amelogenins, enamelin and
ameloblastin.
Assembly of the enamel extracellular matrix from these component proteins is
believed to be critical
in producing a matrix competent to undergo mineral replacement. (Paine C.T. et
al. (1998) Connect
Tissue Res.38:257-267). Tuftelin mRNA has been found to be expressed in human
ameloblastoma
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tumor, a non-mineralized odontogenic tumor (Deutsch D. et al. (1998) Connect
Tissue Res.
39:177-184).
Olfactomedin-related proteins are extracellular matrix, secreted glycoproteins
with
conserved C-terminal motifs. They are expressed in a wide variety of tissues
and in broad range of
species, from Caenorlzabditis elegans to Homo Sapiens. Olfactomedin-related
proteins comprise a
gene family with at Least 5 family members in humans. One of the five,
TIGR/myocilin protein, is
expressed in the eye and is associated with the pathogenesis of glaucoma
(Kulkarni, N.H. et al.,
(2000) Genet. Res. 76:41-50). Research by Yokoyama et aI. (1996) found a 135-
amino acid protein,
termed AMY, having 96% sequence identity with rat neuronal olfactomedin-
releated ER localized
protein in a neuroblastoma cell line cDNA library, suggesting an essential
role for AMY in nerve
tissue (Yokoyama, M. et al., (1996) DNA Res. 3:311-320). Neuron-specific
olfactomedin-related
glycoproteins isolated from rat brain cDNA libraries show strong sequence
similarity with
olfactomedin. This similarity is suggestive of a matrix-related function of
these glycoproteins in
neurons and neurosecretory cells (Danielson, P.E. et al., (1994) J. Neurosci.
Res. 38:468-478).
Mac-2 binding protein is a 90-kD serum protein (90K) and another secreted
glycoprotein,
isolated from both the human breast carcinoma cell line SK-BR-3, and human
breast milk. It
specifically binds to a human macrophage-associated lectin, Mac-2.
Structurally, the mature protein
is 567 amino acids in length and is proceeded by an 18-amino acid leader.
There are 16 cysteines and
seven potential N-linked glycosylation sites. The first 106 amino acids
represent a domain very
similar to an ancient protein superfamily defined by a macrophage scavenger
receptor cysteine-rich
domain (Koths,K. et al., (1993) J. Biol. Chem. 268:14245-14249). 90K is
elevated in the serum of
subpopulations of AIDS patients and is expressed at varying levels in primary
tumor samples and
tumor cell lines. Ullrich et al. (1994) have demonstrated that 90K stimulates
host defense systems
and can induce interleukin-2 secretion. This immune stimulation is proposed to
be a result of
oncogenic transformation, viral infection or pathogenic invasion (UIIrich,A.,
et al. (1994) J. Biol.
Chem. 269:18401-18407).
Semaphorins are a large group of axonal guidance molecules consisting of at
least 30
different members and are found in vertebrates, invertebrates, and even
certain viruses. All
semaphorins contain the sema domain which is approximately 500 amino acids in
length. Neuropilin,
a semaphorin receptor has been shown to promote neurite outgrowth in vitro.
The extracellular
region of neuropilins consists of three different domains: CUB, discoidin, and
MAM domains. The
CUB and the MAM motifs of neuropilin have been suggested as having roles in
protein-protein
interactions and are suggested to be involved in the binding of semaphorins
through the sema and the
C-terminal domains (reviewed in Raper, J.A. (2000) Curr. Opin. Neurobiol.
10:88-94). Plexins are
neuronal cell surface molecules that mediate cell adhesion via a homophilic
binding mechanism in the
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presence of calcium ions. Plexins have been shown to be expressed in the
receptors and neurons of
particular sensory systems (Ohta, K. et al. (1995) Cell 14:1189-1199). There
is evidence that
suggests that some plexins function to control motor and CNS axon guidance in
the developing
nervous system. Plexins, which themselves contain complete semaphorin domains,
may be both the
ancestors of classical semaphorins and binding partners for semaphorins
(Winberg, M.L. et al (1998)
Cell 95:903-916).
Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely
related
glycoproteins of molecular weights of 72 KDa, 64KDa, 62KDa, and 54KDa.
Together with the
carcinoembryonic antigen, they comprise a subfamily within the immunoglobulin
superfamily
(Plouzek C.A. and Chou J.Y., Endocrinology 129:950-958) Different
subpopulations of PSG have
been found to be produced by the trophoblasts of the human placenta, and the
amnionic, and
chorionic membranes (Plouzek C.A. et al. (1993) Placenta 14:277-285).
Autocrine motility factor (AMF) is one of the motility cytokines regulating
tumor cell
migration, therefore identification of the signaling pathway coupled with it
has critical importance.
Autocrine motility factor receptor (AMFR) expression has been found to be
associated with tumor
progression in thymoma (Ohta Y. et al. (2000) Int. J. Oncol. 17:259-264). AMFR
is a cell surface
glycoprotein of molecular weight 78KDa.
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
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 (epinephrine, norepinephrine) and histamine, 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.)
Pro-opiomelanocortin (POMC) is the precursor polypeptide of corticotropin
(ACTH) a
hormone synthesized by the anterior pituitary gland, which functions in the
stimulation of the adrenal
cortex. POMC is also the precursor polypeptide of the hormone, beta-lipotropin
(beta-LPH),. Each
hormone includes smaller peptides with distinct biological activities: alpha-
melanotropin (alpha-
MSH) and corticotropin-like intermediate lobe peptide (CLIP) are formed from
ACTH; gamma-
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lipotropin (gamma-LPH) and beta-endorphin are peptide components of beta-LPH,
while beta-MSH
is contained within gamma-LPH. Adrenal insufficiency due to ACTH deficiency,
resulting from a
genetic mutation in exons 2 and 3 of POMC results in an endocrine disorder
characterized by early-
onset obesity, adrenal insufficiency, and red hair pigmentation (Chretien, M.
et al., (1979) Canad. J.
Biochem. 57:1111-1121, Krude, H, et al., (1998) Nature Genet. 19:155-157,
Online Mendelian
Inheritance in Man, OMIM. Johns Hopkins University, Baltimore, MD. OMIM
Number: 176830:
August l, 2000. World Wide Web URL: www.ncbi.nlm.nih.gov/omim/).
Growth and differentiation factors are secreted proteins which function in
intercellular
communication. Some factors require oligomerization or association with
membrane proteins 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. 1-9.)
The Slit protein, first identified in Drosophila, is critical in central
nervous system midline
formation and potentially in nervous tissue histogenesis and axonal
pathfinding. Itoh et al. have
identified mammalian homologues of the slit gene (human Slit-1, Slit-2, Slit-3
and rat Slit-1). The
encoded proteins are putative secreted proteins containing EFG-like motifs and
leucine-rich repeats,
both are conserved protein-protein interaction domains. Slit-l, -2, and -3
mRNAs are expressed in
the brain, spinal cord, and thyroid, respectively (Itoh, A. et al., (1998)
Brain Res. Mol. Brain Res.
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62:175-186). The Slit family of proteins are indicated to be functional
ligands of glypican-1 in
nervous tissue and suggests that their interactions may be critical in certain
stages during central
nervous system histogenesis (Liang, Y. et al., (1999) J. Biol. Chem. 274:17885-
17892).
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
Ph s~gy, Oxford University Press, New York, NY, pp. 57-62.)
NP/VMs are involved in numerous neurological and cardiovascular disorders. For
example,
neuropeptide Y is involved in hypertension, congestive heart failure,
affective disorders, and appetite
regulation. Somatostatin inhibits secretion of growth hormone and prolactin in
the anterior pituitary,
as well as inhibiting secretion in intestine, pancreatic acinar cells, and
pancreatic beta-cells. A
reduction in somatostatin levels has been reported in Alzheimer's disease and
Parkinson's disease.
Vasopressin acts in the kidney to increase water and sodium absorption, and in
higher concentrations
stimulates contraction of vascular smooth muscle, platelet activation, and
glycogen breakdown in the
liver. Vasopressin and its analogues are used clinically to treat diabetes
insipidus. Endothelia and
angiotensin are involved in hypertension, and drugs, such as captopril, which
reduce plasma levels of
angiotensin, are used to reduce blood pressure (Watson, S. and S. Arkinstall
(1994) The G-protein
Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 194; 252; 284;
55; 111).
Neuropeptides have also been shown to have roles in nociception (pain).
Vasoactive
intestinal peptide appears to play an important role in chronic neuropathic
pain. Nociceptin, an
endogenous ligand for for the opioid receptor-like 1 receptor, is thought to
have a predominantly anti-
nociceptive effect, and has been shown to have analgesic properties in
different animal models of
tonic or chronic pain (Dickinson, T. and Fleetwood-Walker, S.M. (1998) Trends
Pharmacol. Sci.
19:346-348).
Other proteins that contain signal peptides include secreted proteins with
enzymatic activity.
Such activity includes, for example, oxidoreductase/dehydrogenase activity,
transferase activity,
hydrolase activity, lyase activity, isomerase activity, or ligase activity.
For example, matrix
metalloproteinases are secreted hydrolytic enzymes that degrade the
extracellular matrix and thus
play an important role in tumor metastasis, tissue morphogenesis, and
arthritis (Reponen, P. et al.
6
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(1995) Dev. Dyn. 202:388-396; Firestein, G.S. (1992) Curr. Opin. Rheumatol.
4:348-354; Ray, J.M.
and Stetler-Stevenson, W.G. (1994) Eur. Respir. J. 7:2062-2072; and Mignatti,
P. and Rifkin, D.B.
(1993) Physiol. Rev. 73:161-195). Additional examples are the acetyl-CoA
synthetases which
activate acetate for use in lipid synthesis or energy generation (Luong, A. et
al. (2000) J. Biol. Chem.
275:26458-26466). The result of acetyl-CoA synthetase activity is the
formation of acetyl-CoA from
acetate and CoA. Acetyl-CoA sythetases share a region of sequence similarity
identified as the AMP-
binding domain signature. Acetyl-CoA synthetase has been shown to be
associated with hypertension
(H. Toh (1991) Protein Seq. Data Anal. 4:111-117 and Iwai, N. et al., (1994)
Hypertension 23:375-
380).
Other proteins that contain signal peptides include enzymes involved in the
glycosylation of
proteins in transit through the secretory pathway. Mucin-type O-linked
glycosylation is a dominant
form of protein glycosylation. Initiation of mucin-type glycosylation occurs
by the addition of the
monosaccharide N-acetylgalactosamine to the hydroxyl group of serine and
threonine amino acids
(GaINAc~l-O-Ser/Thr). GalNAc O-glycosylation is more prominent on high
molecular weight
secretory glycoproteins such as mucins, but is also found on a variety of
glycoproteins (White, T. et.
al., J. Biol. Chem. (1995) 270:24156-24165). Additionally, serine/threonine-
rich tandem repeats are a
characteristic of human mucin core proteins. The tandem repeat region also
contains numerous
antigenic determinants as recognized by the monoclonal antibodies HMFG-1, HMFG-
1, and SM-3.
Glycosylation sites within the tandem repeat region were found to be
differentially glycosylated
depending on the organ from which Mucl was isolated. The finding of variable
glycosylation activity
may be critical to further understanding of the molecular basis of cancer-
associated epitopes which
map to the Mucl tandem repeat (Gendler, S.J. et al. (1990) J. Biol. Chem.
265:15286-15293):
Antigen recognition molecules are key players in the sophisticated and complex
immune
systems which all vertebrates have developed to provide protection from viral,
bacterial, fungal, and
parasitic infections. A key feature of the immune system is its ability to
distinguish foreign
molecules, or antigens, from "self" molecules. This ability is mediated
primarily by secreted and
transmembrane proteins expressed by leukocytes (white blood cells) such as
lymphocytes,
granulocytes, and monocytes. Most of these proteins belong to the
immunoglobulin (Ig) superfamily,
members of which contain one or more repeats of a conserved structural domain.
This Ig domain is
comprised of antiparallel (3 sheets joined by a disulfide bond in an
arrangement called the Ig fold.
Members of the Ig superfamily include T-cell receptors, major
histocompatibility (MHC) proteins,
antibodies, and immune cell-specific surface markers such as the "cluster of
differentiation" or CD
antigens. 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
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"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 Leucocyte Antigen Facts Book,
Academic Press, San
Diego, CA, pp. 17-20.)
MHC proteins are cell surface markers that bind to and present foreign
antigens to T cells.
MHC molecules are classified as either class I or class II. Class I MHC
molecules (MHC I) are
expressed on the surface of almost all cells and are involved in the
presentation of antigen to
cytotoxic T cells. For example, a cell infected with virus will degrade
intracellular viral proteins and
express the protein fragments bound to MHC I molecules on the cell surface.
The MHC I/antigen
complex is recognized by cytotoxic T-cells which destroy the infected cell and
the virus within.
Class II MHC molecules are expressed primarily on specialized antigen-
presenting cells of the
immune system, such as B-cells and macrophages. These cells ingest foreign
proteins from the
extracellular fluid and express MHC II/antigen complex on the cell surface.
This complex activates
helper T-cells, which then secrete cytokines and other factors that stimulate
the immune response.
MHC molecules also play an important role in organ rejection following
transplantation. Rejection
occurs when the recipient's T-cells respond to foreign MHC molecules on the
transplanted organ in
the same way as to self MHC molecules bound to foreign antigen. (Reviewed in
Alberts, B. et al.
(1994) Molecular Biology of the Cell, Garland Publishing, New York, NY, pp.
1229-1246.)
Antibodies, or immunoglobulins, are either expressed on the surface of B-cells
or secreted by
B-cells into the circulation. Antibodies bind and neutralize foreign antigens
in the blood and other
extracellular fluids. The prototypical antibody is a tetramer consisting of
two identical heavy
polypeptide chains (H-chains) and two identical light polypeptide chains (L-
chains) interlinked by
disulfide bonds. This arrangement confers the characteristic Y-shape to
antibody molecules.
Antibodies are classified based on their H-chain composition. The five
antibody classes, IgA, IgD,
IgE, IgG and IgM, are defined by the a, b, e, y, and p H-chain types. There
are two types of L-
chains, tc and ~,, either of which may associate as a pair with any H-chain
pair. IgG, the most
common class of antibody found in the circulation, is tetrameric, while the
other classes of antibodies
are generally variants or multimers of this basic structure.
H-chains and L-chains each contain an N-terminal variable region and a C-
terminal constant
region. The constant region consists of about 110 amino acids in L-chains and
about 330 or 440
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amino acids in H-chains. The amino acid sequence of the constant region is
nearly identical among
H- or L-chains of a particular class. The variable region consists of about
110 amino acids in both H-
and L-chains. However, the amino acid sequence of the variable region differs
among H- or L-chains
of a particular class. Within each H- or L-chain variable region are three
hypervariable regions of
extensive sequence diversity, each consisting of about 5 to 10 amino acids. In
the antibody molecule,
the H- and L-chain hypervariable regions come together to form the antigen
recognition site.
(Reviewed in Alberts, supra, pp. 1206-1213 and 1216-1217.)
Both H-chains and L-chains contain repeated Ig domains. For example, a typical
H-chain
contains four Ig domains, three of which occur within the constant region and
one of which occurs
within the variable region and contributes to the formation of the antigen
recognition site. Likewise,
a typical L-chain contains two Ig domains, one of which occurs within the
constant region and one of
which occurs within the variable region.
The immune system is capable of recognizing and responding to any foreign
molecule that
enters the body. Therefore, the immune system must be armed with a full
repertoire of antibodies
against all potential antigens. Such antibody diversity is generated by
somatic rearrangement of gene
segments encoding variable and constant regions. These gene segments are
joined together by site-
specific recombination which occurs between highly conserved DNA sequences
that flank each gene
segment. Because there are hundreds of different gene segments, millions of
unique genes can be
generated combinatorially. In addition, imprecise joining of these segments
and an unusually high
rate of somatic mutation within these segments further contribute to the
generation of a diverse
antibody population.
A number of isomerases catalyze steps in protein folding, phototransduction,
and various
anabolic and catabolic pathways. One class of isomerases is known as peptidyl-
prolyl cis-tr-ans
isomerases (PPIases). PPIases catalyze the cis to traps isomerization of
certain proline imidic bonds
in proteins. Two families of PPIases are the FK506 binding proteins (FKBPs),
and cyclophilins
(CyPs). FKBPs bind the potent immunosuppressants FK506 and rapamycin, thereby
inhibiting
signaling pathways in T-cells. Specifically, the PPIase activity of FKBPs is
inhibited by binding of
FK506 or rapamycin. There are five members of the FKBP family which are named
according to
their calculated molecular masses (FKBP12, FKBP13, FKBP2S, FKBP52, and
FKBP65), and
localized to different regions of the cell where they associate with different
protein complexes (Coss,
M. et al. (1995) J. Biol. Chem. 270:29336 - 29341; Schreiber, S.L. (1991)
Science 251:283 - 287).
The peptidyl-prolyl isomerase activity of CyP may be part of the signaling
pathway that leads
to T-cell activation. CyP isomerase activity is associated with protein
folding and protein trafficking,
and may also be involved in assemblyldisassembly of protein complexes and
regulation of protein
activity. For example, in Drosophila, the CyP NinaA is required for correct
localization of
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rhodopsins, while a mammalian CyP (Cyp40) is part of the Hsp90/Hsc70 complex
that binds steroid
receptors. The mammalian CypA has been shown to bind the gag protein from
human
immunodeficiency virus 1 (HIV-1), an interaction that can be inhibited by
cyclosporin. Since
cyclosporin has potent anti-HIV-1 activity, CypA may play an essential
function in HIV-1 replication.
Finally, Cyp40 has been shown to bind and inactivate the transcription factor
c-Myb, an effect that is
reversed by cyclosporin. This effect implicates CyPs in the regulation of
transcription,
transformation, and differentiation (Bergsma, D.J. et al (1991) J. Biol. Chem.
266:23204 - 23214;
Hunter, T. (f998) Cell 92: 141-143; and Leverson, J.D. and Ness, S.A. (1998)
Mol. Cell. 1:203-211).
Gamma-carboxyglutamic acid (Gla) proteins rich in proline (PRGPs) are members
of a family
of vitamin K-dependent single-pass integral membrane proteins. These proteins
are characterized by
an extracellular amino terminal domain of approximately 45 amino acids rich in
Gla. The
intracellular carboxyl terminal region contains one or two copies of the
sequence PPXY, a motif
present in a variety of proteins involved in such diverse cellular functions
as signal transduction, cell
cycle progression, and protein turnover (Kulman, J.D. et al., (2001) Proc.
Natl. Acad. Sci. U.S.A.
98:1370-1375). The process of post-translational modification of glutamic
residues to form Gla is
Vitamin K-dependent carboxylation. Proteins which contain Gla include plasma
proteins involved in
blood coagulation. These proteins are prothrombin, proteins C, S, and Z, and
coagulation factors VII,
IX, and X. Osteocalcin (bone-Gla protein, BGP) and matrix Gla-protein (MGP)
also contain Gla
(Friedman, P.A., and C.T. Przysiecki (1987) Int. J. Biochem. 19:1-7; C.
Vermeer (1990) Biochem. J.
266:625-636).
The Drosophila sp. gene crossveinless 2 is characterized as having a putative
signal or
transmembrane sequence, and a partial Von Willebrand Factor D domain similar
to those domains
known to regulate the formation of intramolecular and intermolecular bonds and
five cysteine-rich
domains, known to bind BMP-like (bone morphogenetic proteins) ligands. These
features suggest
that crossveinless 2 may act extracelluarly or in the secretory pathway to
directly potentiate ligand
signaling and hence, involvement in the BMP-like signaling pathway known to
play a role in vein
specification (Conley, C.A. et al., (2000) Development 127:3947-3959). The
dorsal-ventral
patterning in both vertebrate and Drosophila embryos requires a conserved
system of extracellular
proteins to generate a positional informational gradient.
Another protein that contains a signal peptide is encoded by the seizure-
related gene, SEZ-6,
a brain specific cDNA whose expression is increased by the convulsant drug
pentylentetrazole. The
SEZ-6 protein is expressed in the cerebrum and cerebellum. SEZ-6 contains five
short consensus
repeats (SCR, or sushi domains) and two CUB (complement Clr/s-like repeat)
domains in addition to
a signal peptide and a single transmembrane domain (Shimizu-Nishikawa, K. et
al. (1995) Bioehem.
Biophys. Res. Connmun. 216:382-389).
CA 02409778 2002-11-25
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The discovery of new secreted 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 cell proliferative, autoimmunelinflammatory, cardiovascular,
neurological, and
developmental disorders, and in the assessment of the effects of exogenous
compounds on the
expression of nucleic acid and amino acid sequences of secreted proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, secreted proteins, referred to
collectively as
"SECP" and individually as "SECP-1," "SECP-2," "SECP-3," "SECP-4," "SECP-5,"
"SECP-6,"
"SECP-7," "SECP-8," "SECP-9," "SECP-10," "SECP-11," "SECP-12," "SECP-13,"
"SECP-14,"
"SECP-15," "SECP-16," "SECP-17," "SECP-18," "SECP-19," "SECP-20," "SECP-21,"
"SECP-22,"
"SECP-23," "SECP-24," "SECP-25," "SECP-26," "SECP-27," "SECP-28," "SECP-29,"
"SECP-30,"
"SECP-31," "SECP-32," "SECP-33," "SECP-34," "SECP-35," "SECP-36," "SECP-37,"
"SECP-38,"
"SECP-39," "SECP-40," "SECP-41," "SECP-42," "SECP-43," and "SECP-44." In one
aspect, the
invention provides an isolated polypeptide selected from the group consisting
of a) a polypeptide
comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:l-44, b) a '
naturally occurring polypeptide comprising an amino acid sequence at least 90%
identical to an
amino acid sequence selected from the group consisting of SEQ ID NO:1-44, c) a
biologically active
fiagment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO: l-44, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-44. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-44.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-44, b) a naturally occurring polypeptide
comprising an amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID NO: l-44, c) a biologically active fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-44, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-44.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ ID NO:1-44. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ ID N0:45-88.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
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of SEQ m NO:1-44, b) a naturally occurring polypeptide comprising an amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ )D NO:1-44, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ >D NO:1-44, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ >D NO:1-44. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO: I-44, b) a naturally occurring polypeptide comprising an amino
acid sequence at Least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID NO: l-44, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ >D NO:1-44, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:l-44. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ )D NO:1-44, b) a naturally
occurring polypeptide
comprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-44, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ >D NO:1-44, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
)D NO:l-44.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:45-88, b) a naturally occurring polynucleotide comprising a
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ff~
N0:45-88, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
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WO 01/98353 PCT/USO1/19862
consisting of SEQ ID N0:45-88, b) a naturally occurring polynucleotide
comprising a polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:45-88, c) a polynucleotide complementary to the polynucleotide of
a), d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:45-88, b) a naturally occurring polynucleotide comprising a
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:45-88, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ID NO:l-44, b) a naturally
occurring polypeptide
comprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from
the group consisting of SEQ ID NO:l-44, c) a biologically active fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-44, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-44, and a pharmaceutically acceptable excipient. In one embodiment,
the composition
comprises an amino acid sequence selected from the group consisting of SEQ ID
NO:1-44. The
invention additionally provides a method of treating a disease or condition
associated with decreased
expression of functional SECP, comprising administering to a patient in need
of such treatment the
composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
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acid sequence selected from the group consisting of SEQ ID NO:l-44, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-44, c) a biologically active
fragment of a .
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-44,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-44. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional SECP, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID NO:1-44, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-44, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-44,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-44. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
SECP, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-44, b) a naturally
occurring polypeptide
comprising an amino acid sequence at least 90% identical to an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-44, c) a biologically active fragment of a
polypeptide having an
annino acid sequence selected from the group consisting of SEQ ID NO:1-44, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-44. The method comprises a) combining the polypeptide with at least
one test compound
under suitable conditions, and b) detecting binding of the polypeptide to the
test compound, thereby
identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
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activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-44, b) a
naturally occurring
polypeptide comprising an amino acid sequence at least 90% identical to an
amino acid sequence
selected from the group consisting of SEQ ID NO:l-44, c) a biologically active
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-44,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-44. The method comprises a) combining the
polypeptide with at
least one test compound under conditions permissive for the activity of the
polypeptide, b) assessing
the activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of
the polypeptide in the presence of the test compound with the activity of the
polypeptide in the
absence of the test compound, wherein a change in the activity of the
polypeptide in the presence of
the test compound is indicative of a compound that modulates the activity of
the polypeptide.
The invention further provides 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 N0:45-88, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
TD N0:45-88, ii) a
naturally occurring polynucleotide comprising a polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:45-88,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide
in the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:45-88, ii) a naturally occurring polynucleotide comprising a polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:45-88,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group consisting
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
of i)-v) above; c) quantifying the amount of hybridization complex; and d)
comparing the amount of
hybridization complex in the treated biological sample with the amount of
hybridization complex in
an untreated biological sample, wherein a difference in the amount of
hybridization complex in the
treated biological sample is indicative of toxicity of the test compound.
S
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog for polypeptides of the invention. The probability score for the match
between each
polypeptide and its GenBank homolog is also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table S shows the representative cDNA library for polynucleotides of the
invention
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
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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
"SECP" refers to the amino acid sequences of substantially purified SECP
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
SECP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of SECP either by
directly interacting with
SECP or by acting on components of the biological pathway in which SECP
participates.
An "allelic variant" is an alternative form of the gene encoding SECP. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding SECP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as SECP or a
polypeptide with at least one functional characteristic of SECP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding SECP, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
SECP. 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 SECP. 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 SECP is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
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amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of SECP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of SECP either by
directly interacting with SECP or by acting on components of the biological
pathway in which SECP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind SECP 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 (I~LH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
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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" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic SECP, or of
any oligopeptide thereof,
to induce a specific immune response in appropriate animals or cells and to
bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding SECP or fragments of
SECP may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCl), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' andlor the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended and
assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
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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
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change,in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
retains at least one biological or immunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
A "fragment" is a unique portion of SECP or the polynucleotide encoding SECP
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
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to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example,
a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths axe 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 N0:45-88 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:45-88, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:45-88 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:45-88 from related polynueleotide sequences. The precise length of a
fragment of SEQ
ID N0:45-88 and the region of SEQ ID N0:45-88 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:l-44 is encoded by a fragment of SEQ ID N0:45-88. A
fragment
of SEQ ID NO:1-44 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-44. For example, a fragment of SEQ ID NO:l-44 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-44.
The precise length of
a fragment of SEQ ID NO:1-44 and the region of SEQ ID NO:1-44 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 "full length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
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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 Wn. CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS .
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. BioI. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http:l/www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Operz Gap: 5 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
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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 charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 afad Extetasio~z Gap: 1 pe~talties
Gap x drop-off' SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
far 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.
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"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 pglml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (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, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
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
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WO 01/98353 PCT/USO1/19862
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of SECP
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 SECP which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of SECP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of SECP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
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"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an SECP may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of SECP.
"Probe" refers to nucleic acid sequences encoding SECP, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerise enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise 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, J. et al. (1989) Molecular Cloning: A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Bioloav, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
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selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
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amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
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 SECP,
nucleic acids encoding SECP, or fragments thereof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90°7o free
from other components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
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viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed cells" includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91
%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to .
another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
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A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human secreted proteins (SECP),
the
polynucleotides encoding SECP, and the use of these compositions for the
diagnosis, treatment, or
prevention of cell proliferative, autoimmune%inflammatory, cardiovascular,
neurological, and
developmental disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to a
single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is denoted
by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and
an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID)
as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the
invention. Column 3
shows the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog.
Column 4 shows the probability score for the match between each polypeptide
and its GenBank
homolog. Column 5 shows the annotation of the GenBank homolog along with
relevant citations
where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1 and
2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of
the invention. Column
3 shows the number of amino acid residues in each polypeptide. Column 4 shows
potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
motifs. In particular, the locations of signal peptides (as indicated by
"Signal peptide" or
"Signal cleavage") are shown. Column 7 shows analytical methods for protein
structurelfunction
analysis and in some cases, searchable databases to which the analytical
methods were applied.
Together, tables 2 and 3 summarize the properties of each polypeptide of the
invention, and
these properties establish that the claimed polypeptides are secreted
proteins. For example, SEQ ID
NO:l is 51% identical to human UDP-GalNAc:polypeptide N-
acetylgalactosaminyltransferase
(GenBank ID g971461).as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 1.5e-141, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ ID NO:l also contains a
signal peptide
and a transmembrane domain as determined by hidden Markov model (HMM)-based
methods. (See
Table 3.) Likewise, SPScan analysis also indicates the presence of an N-
terminal signal peptide in
SEQ ID NO:1. Taken together, the evidence shows that SEQ ID NO:1 is present in
the secretory
pathway as an N-acetylgalactosaminyl transferase.
For example, SEQ )D N0:2 is 90% identical to mouse seizure-related gene
product 6 type 2
precursor (GenBank ID g1139548) as determined by the Basic Local Alignment
Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates
the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:2
also contains five
sushi domains and two CUB domains as determined by searching for statistically
significant matches
in the hidden Markov model (HMM)-based PFAM database of conserved protein
family domains.
(see Table 3.) In addition, SEQ ID N0:2 contains a signal peptide and a single
transmembrane
domain, as identified by HMMER analysis.
For example, SEQ ID N0:3 is 43% identical to Gallus gallus lysozyme (GenBank
ID
g4467410) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 5.2e-40, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:3 also contains a G-
lysozyme signature
domain as determined by searching for statistically significant matches in the
BLIMPS analysis of the
PRINTS database of conserved protein motifs. (See Table 3.) Data from the
PFAM, PRODOM and
DOMO databases provide further corroborative evidence that SEQ ID N0:3 is a
lysozyme.
For example, SEQ ID N0:17 has a signal peptide, as determined by SPScan and
hidden
Markov model (HMM) based analyses. SEQ ID N0:17 is 86% identical to human
immunoglobulin
lambda light chain (GenBank ID g33702) as determined by the Basic Local
Alignment Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 2.2e-106, which
indicates the probability
of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:17 also contains
an immunoglobulin domain as determined by searching for statistically
significant matches in the
HMM-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
31
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WO 01/98353 PCT/USO1/19862
BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ
ID N0:17 is a secreted immunoglobulin. The available evidence shows that SEQ
ID N0:19 is also a
secreted immunoglobulin.
For example, SEQ ID N0:38 shows 95% identity to human immunoglobulin lambda
light
chain (GenBank ID g33718) as determined by the Basic Local Alignment Search
Tool (BLAST).
(See Table 2.) The BLAST probability score is 5.2e-114, which indicates the
probability of obtaining
the observed polypeptide sequence alignment by chance. SEQ ID N0:38 also
contains an
immunoglobulin domain as determined by searching for statistically significant
matches in the hidden
Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative evidence
that SEQ ID N0:38 is a secreted protein, and more specifically an
immunoglobulin. SEQ ID N0:4-
16, SEQ ID N0:18-37, and SEQ ID N0:39-44 were analyzed and annotated in a
similar manner. The
algorithms and parameter s for the analysis of SEQ ID NO:1-44 are described in
Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments
of the polynucleotide sequences which are useful, for example, in
hybridization or amplification
technologies that identify SEQ ID N0:45-88 or that distinguish between SEQ ID
N0:45-88 and
related polynucleotide sequences. Column 5 shows identification numbers
corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or
sequence assemblages
comprised of both cDNA and genomic DNA. These sequences were used to assemble
the full length
polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the
nucleotide start (5')
and stop (3') positions of the cDNA and/or genomic sequences in column 5
relative to their respective
full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for
example, to
Incyte cDNAs along with their corresponding cDNA libraries. For example,
6735891H1 is the
identification number of an Incyte cDNA sequence, and LIVRTUT13 is the cDNA
library from which
it is derived. Incyte cDNAs for which cDNA libraries are not indicated were
derived from pooled
cDNA libraries (e.g., 71013085V1). Alternatively, the identification numbers
in column 5 may refer
to GenBank cDNAs or ESTs (e.g., g1496797) which contributed to the assembly of
the full length
polynucleotide sequences. Alternatively, the identification numbers in column
5 may refer to coding
regions predicted by Genscan analysis of genomic DNA. The Genscan-predicted
coding sequences
32
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WO 01/98353 PCT/USO1/19862
may have been edited prior to assembly. (See Example IV.) Alternatively, the
identification numbers
in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together
by an "exon stitching" algorithm. (See Example V.) Alternatively, the
identification numbers in
column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon-stretching" algorithm. (See Example V.) In some cases, Incyte cDNA
coverage redundant
with the sequence coverage shown in column 5 was obtained to confirm the final
consensus
polynucleotide sequence, but the relevant Incyte cDNA identification numbers
are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
The invention also encompasses SECP variants. A preferred SECP 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 SECP amino acid sequence, and which contains at least
one functional or
structural characteristic of SECP.
The invention also encompasses polynucleotides which encode SECP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:45-88, which encodes SECP. The
polynucleotide sequences
of SEQ ID N0:45-88, as presented in the Sequence Listing, embrace the
equivalent RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
SECP. 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 SECP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:45-88 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 )D N0:45-88. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of SECP.
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 SECP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally occurnng
gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
33
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
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 SECP, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode SECP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring SECP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding SECP or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding SECP 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 SECP
and
SECP 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 SECP 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
N0:45-88 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
34
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding SECP 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
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
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode SECP may be cloned in recombinant DNA molecules that direct
expression of SECP,
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 SECP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter SECP-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 SECP, such as its biological or enzymatic
activity or its ability to
bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding SECP 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.)
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WO 01/98353 PCT/USO1/19862
Alternatively, SECP itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of SECP, or any part thereof, may be altered during direct
synthesis and/or
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide or
a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active SECP, the nucleotide sequences
encoding SECP 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 SECP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
SECP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding SECP and its initiation codon and upstream regulatory
sequences are inserted
into the appropriate expression vector, no additional transcriptional or
translational control signals
may be needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted,
exogenous translational control signals including an in-frame ATG initiation
codon should be
provided by the vector. Exogenous translational elements and initiation codons
may be of various
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
enhancers appropriate for the particular host cell system used. (See, e.g.,
Scharf, D. et al. (1994)
Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding SECP 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
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CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
encoding SECP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasxnid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Butler, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding SECP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding SECP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRll'T (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding SECP 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 SECP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of SECP may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of SECP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
38
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of SECP. Transcription of
sequences
encoding SECP may be driven by viral promoters, e.g., the 35S and 195
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technolo~y
(1992) 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 SECP
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 SECP 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 SECP in cell lines is preferred. For example, sequences encoding SECP can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apo cells, respectively.
(See, e.g., Wigler, M. et
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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 hasp, 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 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presencelabsence 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 SECP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding SECP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding SECP 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 SECP
and that express
SECP 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 SECP 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 SECP 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
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
CA 02409778 2002-11-25
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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 SECP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding SECP, 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 SECP 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 SECP may be designed to contain signal
sequences which
direct secretion of SECP 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 SECP 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 SECP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of SECP activity.
Heterologous protein and
peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
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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 SECP encoding sequence and the
heterologous protein
sequence, so that SECP 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 SECP 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.
SECP of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to SECP. At least one and up to a plurality of test
compounds may be screened
for specific binding to SECP. Examples of test compounds include antibodies,
oligonucleotides,
proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
SECP, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which SECP
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express SECP,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing SECP or cell membrane fractions which contain SECP
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either SECP or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
SECP, either ~in
solution or affixed to a solid support, and detecting the binding of SECP to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
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libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
SECP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of SECP. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for SECP
activity, wherein SECP is combined with at least one test compound, and the
activity of SECP in the
presence of a test compound is compared with the activity of SECP in the
absence of the test
compound. A change in the activity of SECP in the presence of the test
compound is indicative of a
compound that modulates the activity of SECP. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising SECP under conditions suitable for
SECP activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of SECP
may do so indirectly and need not come in direct contact with the test
compound. At least one and up
to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding SECP or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S.
Patent Number
5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line,
are derived from the
early mouse embryo and grown in culture. The ES cells are transformed with a
vector containing the
gene of interest disrupted by a marker gene, e.g., the neomycin
phosphotransferase gene (neo;
Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region
of the host genome by homologous recombination. Alternatively, homologous
recombination takes
place using the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-
specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U.
et al. (1997) Nucleic
Acids Res. 25:4323-4330). Transformed ES cells are identified and
microinjected into mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding SECP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding SECP can also be used to create "knockin" humanized
animals
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(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding SECP is injected into animal ES cells, and
the injected sequence
integrates into the animal cell genome. Transformed cells are injected into
blastulae, and the
blastulae are implanted as described above. Transgenic progeny or inbred lines
are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress SECP, e.g., by secreting SECP in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of SECP and secreted proteins. In addition, the expression of
SECP is closely
associated with reproductive, endocrine, immune system, gastrointestinal,
fibroblastic, lung, brain
and neurological tissue. Therefore, SECP appears to play a role in cell
proliferative,
autoimmune/inflammatory, cardiovascular, neurological, and developmental
disorders. In the
treatment of disorders associated with increased SECP expression or activity,
it is desirable to
decrease the expression or activity of SECP. In the treatment of disorders
associated with decreased
SECP expression or activity, it is desirable to increase the expression or
activity of SECP.
Therefore, in one embodiment, SECP 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 SECP. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, a cancer 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
autoimmune/inflammatory
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease,
adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
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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
cardiovascular disorder 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
disease, congenital heart disease, complications of cardiac transplantation,
arteriovenous fistula,
atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms,
arterial dissections, varicose
veins, thrombophlebitis and phlebothrombosis, vascular tumors, and
complications of thrombolysis,
balloon angioplasty, vascular replacement, and coronary artery bypass graft
surgery; congenital lung
anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism,
pulmonary
hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis,
obstructive
pulmonary disease, restrictive pulmonary disease, chronic obstructive
pulmonary disease,
emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial
pneumonia, viral and
mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse
interstitial diseases,
pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative
interstitial pneumonitis,
hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-
organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic
pulmonary
hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary
alveolar
proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions,
pneumothorax,
pleural tumors, drug-induced lung disease, radiation-induced lung disease, and
complications of lung
transplantation; 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 arid 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 including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic
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nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
~postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal degeneration,
and familial frontotemporal dementia and siezures; and a developmental
disorder such as renal
tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism,
Duchenne and Becker
muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor,
aniridia,
genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic
syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas,
hereditary neuropathies such
as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure
disorders such as Syndenham's chorea and cerebral palsy, spina bifida,
anencephaly,
craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing
loss.
In another embodiment, a vector capable of expressing SECP 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 SECP including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
SECP 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 SECP including,
but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of SECP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of SECP including, but not limited to, those listed above.
In a further embodiment, an antagonist of SECP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of SECP.
Examples of such
disorders include, but are not limited to, those cell proliferative,
autoimmunelinflammatory,
cardiovascular, neurological, and developmental disorders described above. In
one aspect, an
antibody which specifically binds SECP 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
SECP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding SECP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of SECP including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
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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 SECP may be produced using methods which are generally known
in the art.
In particular, purified SECP may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind SECP.
Antibodies to SECP 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 SECP 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
SECP have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of SECP 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 SECP may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
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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
SECP-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 SECP may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
SECP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering SECP 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 SECP. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of SECP-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ira determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple SECP epitopes, represents the average affinity, or
avidity, of the antibodies for
SECP. The Ira determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular SECP epitope, represents a true measure of affinity. High-affinity
antibody preparations
with Ka ranging from about 10~ to 10'Z L/mole are preferred for use in
immunoassays in which the
SECP-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ira ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of SECP, preferably in active
form, from the
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CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine 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 SECP-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 SECP, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
modifications of gene
expression can be achieved by designing complementary sequences or antisense
molecules (DNA,
RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of
the gene encoding
SECP. Such technology is well known in the art, and antisense oligonucleotides
or larger fragments
can be designed from various locations along the coding or control regions of
sequences encoding
SECP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press
Inc., Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Cli. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morns, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding SECP may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
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cystic fibrosis (Zabner, J. et al. (1993) Cel175:207-216; Crystal, R.G. et al.
(1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in SECP expression or regulation causes
disease, the expression of
SECP from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
SECP are treated by constructing mammalian expression vectors encoding SECP
and introducing
these vectors by mechanical means into SECP-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson
(1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of SECP include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON,
PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). SECP may be expressed
using (i) a
constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous
sarcoma virus (RSV), SV40
virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter
(e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding SECP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
CA 02409778 2002-11-25
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TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to SECP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding SECP under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding SECP to cells which have one or more genetic
abnormalities with respect to
the expression of SECP. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. ( 1999)
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Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding SECP to target cells which have one or more genetic
abnormalities with
respect to the expression of SECP. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing SECP to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding SECP to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
SECP into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
SECP-coding RNAs and the synthesis of high levels of SECP in vector transduced
cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
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allow the introduction of SECP into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt.
Disco 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 endonucleolytie cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding SECP.
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 SECP. 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
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modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding SECP. Compounds
which may be effective in altering expression of a specific polynucleotide may
include, but are not
limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming
oligonucleotides,
transcription factors and other polypeptide transcriptional regulators, and
non-macromolecular
chemical entities which are capable of interacting with specific
polynucleotide sequences. Effective
compounds may alter polynucleotide expression by acting as either inhibitors
or promoters of
polynucleotide expression. Thus, in the treatment of disorders associated with
increased SECP
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding SECP may be therapeutically useful, and in the treatment of disorders
associated with
decreased SECP expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding SECP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical andlor structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding SECP is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
SECP are assayed by
any method commonly known in the art. Typically, the expression of a specific
nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding SECP. The amount of hybridization may be
quantified, thus forming
the basis for a comparison of the expression of the polynucleotide both with
and without exposure to
one or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to
a test compound indicates that the test compound is effective in altering the
expression of the
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WO 01/98353 PCT/USO1/19862
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosaccharomyces pombe gene
expression system
(Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000)
Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000)
Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves
screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechno1.15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of SECP, antibodies to SECP, and mimetics, agonists, antagonists, or
inhibitors of SECP.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
CA 02409778 2002-11-25
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without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising SECP or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, SECP or a fragment thereof may be joined to
a short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
~, For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example SECP
or fragments thereof, antibodies of SECP, and agonists, antagonists or
inhibitors of SECP, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDso ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half-life and clearance rate of the particular
formulation.
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Normal dosage amounts may vary from about 0.1 ,ug to 100,000 ~cg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind SECP may be used for
the
diagnosis of disorders characterized by expression of SECP, or in assays to
monitor patients being
treated with SECP or agonists, antagonists, or inhibitors of SECP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for SECP include methods which utilize the antibody and a label to detect SECP
in human body
fluids or in extracts of cells or tissues. The antibodies may be used with or
without modification, and
may be labeled by covalent or non-covalent attachment of a reporter molecule.
A wide variety of
reporter molecules, several of which are described above, are known in the art
and may be used.
A variety of protocols for measuring SECP, including ELISAs, RIAs, and FRCS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
SECP expression. Normal
or standard values for SECP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibodies to SECP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of SECP
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 SECP 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 SECP
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
SECP, and to monitor regulation of SECP levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding SECP or closely related
molecules may be used to
identify nucleic acid sequences which encode SECP. The specificity of the
probe, whether it is made
from a highly specific region, e.g., the 5'regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding SECP, allelic
variants, or related
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sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the SECP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ m
N0:45-88 or from
genomic sequences including promoters, enhancers, and introns of the SECP
gene.
Means for producing specific hybridization probes for DNAs encoding SECP
include the
cloning of polynucleotide sequences encoding SECP or SECP 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 355,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding SECP may be used for the diagnosis of
disorders
associated with expression of SECP. Examples of such disorders include, but
are not limited to, a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, a cancer 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
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS), Addison's
disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma; a
cardiovascular disorder such as congestive heart failure, ischemic heart
disease, angina pectoris,
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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 disease, congenital heart disease,
complications of cardiac
transplantation, arteriovenous fistula, atherosclerosis, hypertension,
vasculitis, Raynaud's disease,
aneurysms, arterial dissections, varicose veins, thrombophlebitis and
phlebothrombosis, vascular
tumors, and complications of thrombolysis, balloon angioplasty, vascular
replacement, and coronary
artery bypass graft surgery; congenital lung anomalies, atelectasis, pulmonary
congestion and edema,
pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary
hypertension,
vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary
disease, chronic obstructive
pulmonary disease, emphysema, chronic bronchitis, bronchial asthma,
bronchiectasis, bacterial
pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary
tuberculosis, diffuse
interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative
interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia
bronchiolitis
obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,
Goodpasture's
syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in
collagen-vascular
disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and
noninflammatory pleural
effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-
induced lung disease,
and complications of lung transplantation; 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,
eneephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system
including Down
syndrome, 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,
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diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,
postherpetic neuralgia,
Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia and siezures; and a developmental disorder such as
renal tubular acidosis,
anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker
muscular dystrophy,
epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia,
genitourinary abnormalities,
and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,
hereditary
mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies
such as Charcot-Marie-
Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure
disorders such as
Syndenham's chorea and cerebral palsy, spina bifida, anencephaly,
craniorachischisis, congenital
glaucoma, cataract, and sensorineural hearing loss. The polynucleotide
sequences encoding SECP
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 SECP expression. Such
qualitative or quantitative
methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding SECP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding SECP 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 SECP 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 SECP,
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 SECP, 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
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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.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding SECP
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 SECP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
SECP, 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 ox RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding SECP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding
SECP are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of SECP include
radiolabeling or
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biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progressionlregression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, SECP, fragments of SECP, or antibodies specific for
SECP may be
used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
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Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
axe used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
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separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for SECP
to quantify the
levels of SECP expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting
the levels of protein bound to each array element (Lueking, A. et al. (1999)
Anal. Biochem. 270:103-
111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a
variety of methods known in the art, for example, by reacting the proteins in
the sample with a thiol-
or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at each
array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
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the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Ap ru
oach, M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding SECP
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop
genetic linkage maps, for example, which correlate the inheritance of a
disease state with the
inheritance of a particular chromosome region or restriction fragment length
polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding SECP on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
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region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to 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 instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, SECP, 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 SECP 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 W084103564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with SECP, or
fragments thereof,
and washed. Bound SECP is then detected by methods well known in the art.
Purified SECP 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 SECP specifically compete with a test compound
for binding SECP. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with SECP.
In additional embodiments, the nucleotide sequences which encode SECP may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
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embodiments are, therefore, to be construed as merely illustrative, and not
!imitative 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.60/214,601, U.S. Ser. No. 60/212,890, U.S. Ser. No.
60/222,372, U.S. Ser.
No. 60/213,466, U.S. Ser. No. 60/231,435, and U.S. Ser. No. 60/232,889, are
hereby expressly
incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues
were homogenized
and lysed in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a
suitable mixture of denaturants, such as TRIZOL (Life Technologies), a
monophasic solution of
phenol and guanidine isothiocyanate. The resulting lysates were centrifuged
over CsCI cushions or
extracted with chloroform. RNA was precipitated from the lysates with either
isopropanol or sodium
acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some 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, s_ upra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
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including XLl-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or
ElectroMAX
DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, su ra, unit 7.7). Some of the cDNA sequences were selected for extension
using the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
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databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein
family
databases such as PFAM. (HMM is a probabilistic approach which analyzes
consensus primary
structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.)
The queries were performed using programs based on BLAST, FASTA, BLIMPS, and
HMMER. The
Incyte cDNA sequences were assembled to produce full length polynucleotide
sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched
sequences, or
Genscan-predicted coding sequences (see Examples IV and V) were used to extend
Incyte cDNA
assemblages to full length. Assembly was performed using programs based on
Phred, Phrap, and
Consed, and cDNA assemblages were screened for open reading frames using
programs based on
GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were
translated to derive
the corresponding full length polypeptide sequences. Alternatively, a
polypeptide of the invention
may begin at any of the methionine residues of the full length translated
polypeptide. Full length
polypeptide sequences were subsequently analyzed by querying against databases
such as the
GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and hidden Markov model (HMM)-based protein family databases such as PFAM.
Full length
polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide
and polypeptide sequence alignments are generated using default parameters
specified by the
CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment
program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the wore or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
ID N0:45-88. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative secreted proteins were initially identified by running the Genscan
gene identification
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program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-
purpose gene identification program which analyzes genomic DNA sequences from
a variety of
organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and
Burge, C. and S. Karlin
(1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates
predicted exons to form an
assembled cDNA sequence extending from a methionine to a stop codon. The
output of Genscan is a
FASTA database of polynucleotide and polypeptide sequences. The maximum range
of sequence for
Genscan to analyze at once was set to 30 kb. To determine which of these
Genscan predicted cDNA
sequences encode secreted proteins, the encoded polypeptides were analyzed by
querying against
PFAM models for secreted proteins. Potential secreted proteins were also
identified by homology to
Incyte cDNA sequences that had been annotated as secreted proteins. These
selected Genscan-
predicted sequences were then compared by BLAST analysis to the genpept and
gbpri public
databases. Where necessary, the Genscan-predicted sequences were then edited
by comparison to the
top BLAST hit from genpept to correct errors in the sequence predicted by
Genscan, such as extra or
omitted exons. BLAST analysis was also used to find any Incyte cDNA or public
cDNA coverage of
the Genscan-predicted sequences, thus providing evidence for transcription.
When Incyte cDNA
coverage was available, this information was used to correct or confirm the
Genscan predicted
sequence. Full length polynucleotide sequences were obtained by assembling
Genscan-predicted
coding sequences with Incyte cDNA sequences and/or public cDNA sequences using
the assembly
process described in Example III. Alternatively, full length polynucleotide
sequences were derived
entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example IV. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an algorithm
based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
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Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change. parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of SECP Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:45-88 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:45-88 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
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hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:48 was mapped to chromosome 15 within the interval
from 72.3
to 77.4 centiMorgans.
In this manner, SEQ ID N0:54 was mapped to chromosome 20 within the interval
from 6.20
to 9.40 centiMorgans. SEQ ID N0:61 was mapped to chromosome 22 within the
interval from 0.00
to 19.50 centiMorgans.
In this manner, SEQ ID N0:82 was mapped to chromosome 22 within the interval
from 0.0 to
19.5 centiMorgans. SEQ ID N0:85 was mapped to chromosome 12 within the
interval from 84.7 to
92.5 centiMorgans and from 137.5 to 145.7 centiMorgans. More than one map
location is reported
for SEQ ID N0:85, indicating that sequences having different map locations
were assembled into a
single cluster. This situation occurs, for example, when sequences having
strong similarity, but not
complete identity, are assembled into a single cluster.
In this manner, SEQ ID N0:66 was mapped to chromosome 16 within the interval
from
65.60 to 72.60 centiMorgans. In this manner, SEQ ID NO:67 was mapped to
chromosome 11 within
the interval from 59.50 to 65.00 centiMorgans. In this manner, SEQ ID N0:69
was mapped to
chromosome 6 within the interval from 132.70 to 144.40 centiMorgans.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
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calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. Fox example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding SECP are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
IS sequence is derived from a cDNA library constructed from a human tissue.
Each human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding SECP. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
~A).
VIII. Extension of SECP Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72 °C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
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Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p l of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ,u1 to 10 ,u1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
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WO 01/98353 PCT/USO1/19862
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In Iike manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes '
Hybridization probes derived from SEQ ID N0:45-88 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 ~Ci of
~Y 32P7 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.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
CA 02409778 2002-11-25
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Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson ( 1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preuaration
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~1 oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/~.~1 RNase inhibitor, 500 E.~M dATP, 500 ~M
dGTP, 500 EtM dTTP, 40
E.~M dCTP, 40 l.~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
The reverse .
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85°C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 p1 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
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Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 E.~l of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 ~.,~1 of 5X SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60° C: The arrays are washed for 10
min at 45 ° C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a
second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
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CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the SECP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring SECP. 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 SECP. 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 SECP-encoding transcript.
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XII. Expression of SECP
Expression and purification of SECP is achieved using bacterial or virus-based
expression
systems. For expression of SECP in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express SECP upon induction with isopropyl beta-
D-
thiogalactopyranoside (Il'TG). Expression of SECP in eukaryotic cells is
achieved by infecting insect
or mannmalian cell lines with recombinant Auto_g~raphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding SECP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, SECP 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
SECP 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 SECP obtained by these methods can be used
directly in the assays
shown in Examples XVI and XVII, where applicable.
XIII. Functional Assays
SECP function is assessed by expressing the sequences encoding SECP at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
79
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ,ug of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of SECP on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding SECP 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 SECP and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIV. Production of SECP Specific Antibodies
SECP 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 SECP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, s, unra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-SECP activity by, for example, binding the peptide or
SECP to a substrate,
blocking with 1 % BSA, reacting with rabbit antisera; washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XV. Purification of Naturally Occurring SECP Using Specific Antibodies
Naturally occurring or recombinant SECP is substantially purified by
immunoaffinity
chromatography using antibodies specific fox SECP. An immunoaffinity column is
constructed by
covalently coupling anti-SECP 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 SECP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of SECP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/SECP 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 SECP is collected.
XVI. Identification of Molecules Which Interact with SECP
SECP, or biologically active fragments thereof, are labeled with'zsI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled SECP, washed,
and any wells with labeled SECP complex are assayed. Data obtained using
different concentrations
of SECP are used to calculate values for the number, affinity, and association
of SECP with the
candidate molecules.
Alternatively, molecules interacting with SECP 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).
SECP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K,
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. Demonstration of SECP Activity
An assay for the determination of SECP activity consists of an enzyme reaction
mixture
consisting of 25 mM Tris-HCl (pH 7.4), 0.25% Triton X-100, 5 MM MnClz, 5 mM
CDP-choline, 5
81
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
mM 2-mercaptoethanol, 0.05 mM UDP-[14C]GaINAc (4,000 cpm/nmol), 250 pM
peptide, and varying
amounts of SECP in a final volume of 100 ~1. The reaction mixture is incubated
for 10 min. at 37° C
followed by Dowex 1 ion exchange (formic acid form) chromatography. Eluted
peptide-containing
fractions are subjected to scintillation counting. The amount of ['4C]GaINAc
present in the peptide-
s containing fractions is proportional to SECP activity. Confirmation of
substrate and SECP source can
be evaluated by C-18 chromatography (C2C18 3.2 Smart System, Pharmacia Biotech
Inc.) to ensure
peptide stability and that incorporated [14C]GalNAc is associated with the
peptide (SgSrensen,T. et al.
(1995) J. Biol. Chem. 270:24166-24173).
Alternatively, an assay for growth stimulating or inhibiting activity of SECP
measures the
amount of DNA synthesis in Swiss mouse 3T3 cells (McKay, I. and Leigh, L, eds.
(1993) Growth
Factors: A Practical Approach, Oxford University Press, New York, NY). In this
assay, varying
amounts of SECP are added to quiescent 3T3 cultured cells in the presence of
[3H]thymidine, a
radioactive DNA precursor. SECP for this assay can be obtained by recombinant
means or from
biochemical preparations. Incorporation of [3H]thymidine into acid-
precipitable DNA is measured
over an appropriate time interval, and the amount incorporated is directly
proportional to the amount
of newly synthesized DNA. A linear dose-response curve over at least a hundred-
fold SECP
concentration range is indicative of growth modulating activity. One unit of
activity per milliliter is
defined as the concentration of SECP producing a 50% response level, where
100% represents
maximal incorporation of [3H]thymidine into acid-precipitable DNA.
Alternatively, an assay for SECP activity measures the stimulation or
inhibition of
neurotransmission in cultured cells. Cultured CHO fibroblasts are exposed to
SECP. Following
endocytic uptake of SECP, the cells are washed with fresh culture medium, and
a whole cell voltage-
clamped Xenopus myocyte is manipulated into contact with one of the
fibroblasts in SECP-free
medium. Membrane currents are recorded from the myocyte. Increased or
decreased current relative
to control values are indicative of neuromodulatory effects of SECP (Morimoto,
T. et al. (1995)
Neuron 15:689-696).
Alternatively, an assay for SECP activity measures the amount of SECP 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 SECP-specific
antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and
immunoblotting
techniques. The concentration of SECP in secretory organelles relative to SECP
in total cell lysate is
proportional to the amount of SECP in transit through the secretory pathway.
82
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
In another alternative, SECP recognizes and precipitates antigen from serum.
This activity
can be measured by the quantitative precipitin reaction. (Golub, E. S. et al.
(1987) Immunolo~y~A
Synthesis, Sinauer Associates, Sunderland, MA, pages 113-115.) SECP is
isotopically labeled using
methods known in the art. Various serum concentrations are added to constant
amounts of labeled
SECP. SECP-antigen complexes precipitate out of solution and are collected by
centrifugation. The
amount of precipitable SECP-antigen complex is proportional to the amount of
radioisotope detected
in the precipitate. The amount of precipitable SECP-antigen complex is plotted
against the serum
concentration. For various serum concentrations, a characteristic
precipitation curve is obtained, in
which the amount of precipitable SECP-antigen complex initially increases
proportionately with
increasing serum concentration, peaks at the equivalence point, and then
decreases proportionately
with further increases in serum concentration. Thus, the amount of
precipitable SECP-antigen
complex is a measure of SECP activity which is characterized by sensitivity to
both limiting and
excess quantities of antigen.
Alternatively, an assay fox SECP activity measures the expression of SECP on
the cell
surface. cDNA encoding SECP is transfected into a non-leukocytic cell line.
Cell surface proteins
are labeled with biotin (de la Fuente, M.A. et.al. (1997) Blood 90:2398-2405).
Immunoprecipitations
are performed using SECP-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 SECP expressed on the cell
surface.
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.
83
CA 02409778 2002-11-25
WO 01/98353 . PCT/USO1/19862
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CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<110> INCYTE GENOMICS, INC.
HILLMAN, Jennifer L.
TANG, Y. Tom
YUE, Henry
ELLIOTT, Vicki S.
TRIBOULEY, Catherine M.
LEE, Ernestine A.
RAMKUMAR, Jayala~ni
LAL, Preeti
XU, Yuming
WARREN, Bridget A.
HAFALIA, April J. A.
BAUGHN, Mariah R.
AZIMZAI, Yalda
BATRA, Sajeev
BURFORD, Neil
YAO, Monique G.
NGUYEN, Danniel B.
LU, Dyung Aina M.
WALIA, Narinder K.
GANDHI, Ameena R.
AU-YOUNG, Janice
PATTERSON, Chandra
<120> SECRETED PROTEINS
<130> PI-0133 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/212,890; 60/213,466; 60/214,601; 60/222,372; 60/231,435; 60/232,889
<151> 2000-06-20; 2000-06-23; 2000-06-27; 2000-07-31; 2000-09-08; 2000-09-15
<160> 88
<170> PERL Program
<210> 1
<211> 552
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2101688CD1
<400> 1
Met Arg Arg Leu Thr Arg Arg Leu Val Leu Pro Val Phe Gly Val
1 5 10 15
Leu Trp Ile Thr Val Leu Leu Phe Phe Trp Val Thr Lys Arg Lys
20 25 30
Leu Glu Val Pro Thr Gly Pro Glu Val Gln Thr Pro Lys Pro Ser
35 40 45
Asp Ala Asp Trp Asp Asp Leu Trp Asp Gln Phe Asp Glu Arg Arg
50 55 60
Tyr Leu Asn Ala Lys Lys Trp Arg Val Gly Asp Asp Pro Tyr Lys
65 70 75
Leu Tyr Ala Phe Asn Gln Arg Glu Ser Glu Arg Ile Ser Ser Asn
80 85 90
Arg Ala Ile Pro Asp Thr Arg His Leu Arg Cys Thr Leu Leu Val
95 100 105
Tyr Cys Thr Asp Leu Pro Pro Thr Ser Ile Ile Ile Thr Phe His
1/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
110 115 120
Asn Glu Ala Arg Ser Thr Leu Leu Arg Thr Ile Arg Ser Val Leu
125 130 135
Asn Arg Thr Pro Thr His Leu Ile Arg Glu Ile Ile Leu Val Asp
140 145 150
Asp Phe Ser Asn Asp Pro Asp Asp Cys Lys Gln Leu Ile Lys Leu
155 160 165
Pro Lys Val Lys Cys Leu Arg Asn Asn Glu Arg Gln Gly Leu Val
170 275 180
Arg Ser Arg Ile Arg Gly Ala Asp Ile Ala Gln Gly Thr Thr Leu
185 190 195
Thr Phe Leu Asp Ser His Cys Glu Val Asn Arg Asp Trp Leu Gln
200 205 210
Pro Leu Leu His Arg Val Lys Glu Asp Tyr Thr Arg Val Val Cys
215 220 225
Pro Val Ile Asp Ile Ile Asn Leu Asp Thr Phe Thr Tyr Ile Glu
230 235 240
Ser Ala Ser Glu Leu Arg Gly Gly Phe Asp Trp Ser Leu His Phe
245 250 255
Gln Trp Glu Gln Leu Ser Pro Glu Gln Lys Ala Arg Arg Leu Asp
260 265 270
Pro Thr Glu Pro Ile Arg Thr Pro Ile Ile Ala Gly Gly Leu Phe
275 280 285
Val Ile Asp Lys Ala Trp Phe Asp Tyr Leu G1y Lys Tyr Asp Met
290 295 300
Asp Met Asp Ile Trp Gly Gly Glu Asn Phe Glu Ile Ser Phe Arg
305 310 315
Val Trp Met Cys Gly Gly Ser Leu Glu Ile Val Pro Cys Ser Arg
320 325 330
Val Gly His Val Phe Arg Lys Lys His Pro Tyr Val Phe Pro Asp
335 340 345
Gly Asn Ala Asn Thr Tyr Ile Lys Asn Thr Lys Arg Thr Ala Glu
350 355 360
Val Trp Met Asp Glu Tyr Lys Gln Tyr Tyr Tyr Ala Ala Arg Pro
365 370 375
Phe Ala Leu Glu Arg Pro Phe Gly Asn Val Glu Ser Arg Leu Asp
380 385 390
Leu Arg Lys Asn Leu Arg Cys Gln Ser Phe Lys Trp Tyr Leu Glu
395 400 405
Asn Ile Tyr Pro Glu Leu Ser Ile Pro Lys Glu Ser Ser Ile Gln
410 415 420
Lys Gly Asn Ile Arg Gln Arg Gln Lys Cys Leu Glu Ser Gln Arg
425 430 435
Gln Asn Asn Gln Glu Thr Pro Asn Leu Lys Leu Ser Pro Cys Ala
440 445 450
Lys Val Lys Gly Glu Asp Ala Lys Ser Gln Val Trp Ala Phe Thr
455 460 465
Tyr Thr Gln Lys Ile Leu Gln Glu Glu Leu Cys Leu Ser Val Ile
470 475 480
Thr Leu Phe Pro Gly Ala Pro Val Val Leu Val Leu Cys Lys Asn
485 490 495
Gly Asp Asp Arg Gln Gln Trp Thr Lys Thr Gly Ser His Ile Glu
500 505 510
His Ile Ala Ser His I~eu Cys Leu Asp Thr Asp Met Phe Gly Asp
515 520 525
Gly Thr Glu Asn Gly Lys Glu Ile Val Val Asn Pro Cys Glu Ser
530 535 540
Ser Leu Met Ser Gln His Trp Asp Met Val Ser Ser
545 550
<210> 2
<211> 994
<212> PRT
2/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5452330CD1
<400> 2
Met Arg Pro Val Ala Leu Leu Leu Leu Pro Ser Leu Leu Ala Leu
1 5 10 15
Leu Ala His Gly Leu Ser Leu Glu Ala Pro Thr Val Gly Lys Gly
20 25 30
Gln Ala Pro Gly Ile Glu Glu Thr Asp Gly Glu Leu Thr Ala Ala
35 40 45
Pro Thr Pro Glu Gln Pro Glu Arg Gly Val His Phe Val Thr Thr
50 55 60
Ala Pro Thr Leu Lys Leu Leu Asn His His Pro Leu Leu Glu Glu
65 70 75
Phe Leu Gln Glu Gly Leu Glu Lys Gly Asp Glu Glu Leu Arg Pro
80 85 90
Ala Leu Pro Phe Gln Pro Asp Pro Pro Ala Pro Phe Thr Pro Ser
95 100 105
Pro Leu Pro Arg Leu Ala Asn Gln Asp Ser Arg Pro Val Phe Thr
110 115 120
Ser Pro Thr Pro Ala Met Ala Ala Val Pro Thr Gln Pro Gln Ser
125 130 135
Lys Glu Gly Pro Trp Ser Pro Glu Ser Glu Ser Pro Met Leu Arg
140 145 150
Ile Thr Ala Pro Leu Pro Pro Gly Pro Ser Met Ala Val Pro Thr
155 160 165
Leu Gly Pro Gly Glu Ile Ala Ser Thr Thr Pro Pro Ser Arg Ala
170 175 180
Trp Thr Pro Thr Gln Glu Gly Pro Gly Asp Met Gly Arg Pro Trp
185 190 195
Val Ala Glu Val Val Ser Gln Gly Ala Gly Ile Gly Ile Gln Gly
200 205 210
Thr Ile Thr Ser Ser Thr Ala Ser Gly Asp Asp G1u Glu Thr Thr
215 220 225
Thr Thr Thr Thr Ile Ile Thr Thr Thr I1e Thr Thr Val Gln Thr
230 235 240
Pro Gly Pro Cys Ser Trp Asn Phe Ser Gly Pro Glu Gly Ser Leu
245 250 255
Asp Ser Pro Thr Asp Leu Ser Ser Pro Thr Asp Val Gly Leu Asp
260 265 270
Cys Phe Phe Tyr Ile Ser Val Tyr Pro Gly Tyr Gly Val Glu Ile
275 280 285
Lys Val Gln Asn Ile Ser Leu Arg Glu Gly Glu Thr Val Thr Val
290 295 300
Glu Gly Leu Gly Gly Pro Asp Pro Leu Pro Leu Ala Asn Gln Ser
305 310 315
Phe Leu Leu Arg Gly Gln Val Ile Arg Ser Pro Thr His Gln Ala
320 325 330
Ala Leu Arg Phe Gln Ser Leu Pro Pro Pro Ala Gly Pro Gly Thr
335 340 345
Phe His Phe His Tyr Gln Ala Tyr Leu Leu Ser Cys His Phe Pro
350 355 360
Arg Arg Pro Ala Tyr Gly Asp Val Thr Val Thr Ser Leu His Pro
365 370 375
Gly Gly Ser Ala Arg Phe His Cys Ala Thr Gly Tyr Gln Leu Lys
380 385 390
Gly Ala Arg His Leu Thr Cys Leu Asn Ala Thr Gln Pro Phe Trp
395 400 405
Asp Ser Lys Glu Pro Val Cys Ile Ala Ala Cys Gly Gly Val Ile
410 415 420
3/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Arg Asn Ala Thr Thr Gly Arg Ile Val Ser Pro G1y Phe Pro Gly
425 430 435
Asn Tyr Ser Asn Asn Leu Thr Cys His Trp Leu Leu Glu Ala Pro
440 445 450
Glu Gly Gln Arg Leu His Leu His Phe Glu Lys Va1 Ser Leu Ala
455 460 465
Glu Asp Asp Asp Arg Leu Ile Ile Arg Asn Gly Asp Asn Val Glu
470 475 480
Ala Pro Pro Val Tyr Asp Ser Tyr Glu Val Glu Tyr Leu Pro Ile
485 490 495
Glu Gly Leu Leu Ser Ser Gly Lys His Phe Phe Val Glu Leu Ser
500 505 510
Thr Asp Ser Ser Gly Ala Ala Ala Gly Met Ala Leu Arg Tyr Glu
515 520 525
Ala Phe Gln Gln Gly His Cys Tyr Glu Pro Phe Val Lys Tyr Gly
530 535 540
Asn Phe Ser Ser Ser Thr Pro Thr Tyr Pro Val Gly Thr Thr Val
545 550 555
G1u Phe Ser.Cys Asp Pro Gly Tyr Thr Leu Glu Gln Gly Ser Ile
560 565 570
Ile Ile Glu Cys Val Asp Pro His Asp Pro Gln Trp Asn Glu Thr
575 580 585
Glu Pro Ala Cys Arg Ala Val Cys Ser Gly Glu Ile Thr Asp Ser
590 595 600
Ala Gly Val Val Leu Ser Pro Asn Trp Pro Glu Pro Tyr Gly Arg
605 610 625
Gly Gln Asp Cys Ile Trp Gly Val His Val Glu Glu Asp Lys Arg
620 625 630
Ile Met Leu Asp Ile Arg Val Leu Arg Ile Gly Pro Gly Asp Val
635 640 645
Leu Thr Phe Tyr Asp Gly Asp Asp Leu Thr Ala Arg Val Leu Gly
650 655 660
Gln Tyr Ser Gly Pro Arg Ser His Phe Lys Leu Phe Thr Ser Met
665 670 675
Ala Asp Val Thr Ile Gln Phe Gln Ser Asp Pro Gly Thr Ser Val
680 685 690
Leu Gly Tyr Gln Gln Gly Phe Va1 Ile His Phe Phe Glu Val Pro
695 700 705
Arg Asn Asp Thr Cys Pro Glu Leu Pro Glu Ile Pro Asn Gly Trp
710 715 720
Lys Ser Pro Ser Gln Pro Glu Leu Val His Gly Thr Val Val Thr
725 730 735
Tyr Gln Cys Tyr Pro Gly Tyr Gln Val Val Gly Ser Ser Val Leu
740 745 750
Met Cys Gln Trp Asp Leu Thr Trp Ser G1u Asp Leu Pro Ser Cys
755 760 765
Gln Arg Val Thr Ser Cys His Asp Pro Gly Asp Val Glu His Ser
770 775 780
Arg Arg Leu Ile Ser Ser Pro Lys Phe Pro Val Gly Ala Thr Val
785 790 795
Gln Tyr Ile Cys Asp Gln Gly Phe Val Leu Met Gly Ser Ser Ile
800 805 810
Leu Thr Cys His Asp Arg Gln Ala Gly Ser Pro Lys Trp Ser Asp
815 820 825
Arg Ala Pro Lys Cys Leu Leu Glu Gln Leu Lys Pro Cys His Gly
830 835 840
Leu Ser Ala Pro Glu Asn Gly Ala Arg Ser Pro Glu Lys Gln Leu
845 850 855
His Pro Ala Gly Ala Thr Ile His Phe Ser Cys Ala Pro Gly Tyr
860 865 870
Val Leu Lys Gly G1n Ala Ser Ile Lys Cys Val Pro Gly His Pro
875 880 885
Ser His Trp Ser Asp Pro Pro Pro Ile Cys Arg Ala Ala Ser Leu
4/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
890 895 900
Asp Gly Phe Tyr Asn Ser Arg Ser Leu Asp Val Ala Lys Ala Pro
905 910 915
Ala Ala Ser Ser Thr Leu Asp Ala Ala His Ile Ala Ala Ala Ile
920 925 930
Phe Leu Pro Leu Val Ala Met Val Leu Leu Val Gly Gly Val Tyr
935 940 945
Phe Tyr Phe Ser Arg Leu Gln Gly Lys Ser Ser Leu Gln Leu Pro
950 955 960
Arg Pro Arg Pro Arg Pro Tyr Asn Arg Ile Thr Ile Glu Ser A1a
965 970 975
Phe Asp Asn Pro Thr Tyr Glu Thr Gly Ser Leu Ser Phe Ala Gly
980 985 990
Asp Glu Arg Ile
<210> 3
<211> 212
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4362432CD1
<400> 3
Met Leu Ser Ser Val Val Phe Trp Gly Leu Ile A1a Leu Ile Gly
1 5 10 15
Thr Ser Arg Gly Ser Tyr Pro Phe Ser His Ser Met Lys Pro His
20 25 30
Leu His Pro Arg Leu Tyr His Gly Cys Tyr Gly Asp Ile Met Thr
35 40 45
Met Lys Thr Ser Gly A1a Thr Cys Asp Ala Asn Ser Val Met Asn
50 55 60
Cys Gly Ile Arg Gly Ser Glu Met Phe Ala Glu Met Asp Leu Arg
65 70 75
Ala Ile Lys Pro Tyr Gln Thr Leu Ile Lys Glu Val Gly Gln Arg
80 85 90
His Cys Val Asp Pro Ala Val Ile Ala Ala Ile Ile Ser Arg Glu
95 100 105
Ser His Gly Gly Ser Val Leu Gln Asp Gly Trp Asp His Arg Gly
110 115 120
Leu Lys Phe Gly Leu Met Gln Leu Asp Lys Gln Thr Tyr His Pro
125 130 135
Val Gly Ala Trp Asp Ser Lys Glu His Leu Ser Gln Ala Thr Gly
140 145 150
Ile Leu Thr Glu Arg Ile Lys Ala Ile Gln Lys Lys Phe Pro Thr
155 160 165
Trp Ser Val Ala Gln His Leu Lys Gly Gly Leu Ser Ala Phe Lys
170 175 180
Ser Gly Ile G1u Ala Ile Ala Thr Pro Ser Asp Ile Asp Asn Asp
185 190 195
Phe Val Asn Asp Ile Ile Ala Arg Ala Lys Phe Tyr Lys Arg Gln
200 205 210
Ser Phe
<210> 4
<211> 308
<212> PRT
<213> Homo Sapiens
<220>
5/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<221> misc_feature
<223> Incyte ID No: 5308104CD1
<400> 4
Met Asn Gly Leu Ser Leu Ser Glu Leu Cys Cys Leu Phe Cys Cys
1 5 10 15
Pro Pro Cys Pro Gly Arg Ile Ala Ala Lys Leu Ala Phe Leu Pro
20 25 30
Pro Glu Ala Thr Tyr Ser Leu Val Pro Glu Pro Glu Pro Gly Pro
35 40 45
Gly Gly Ala Gly Ala Ala Pro Leu Gly Thr Leu Arg Ala Ser Ser
50 55 60
Gly Ala Pro Gly Arg Trp Lys Leu His Leu Thr Glu Arg Ala Asp
65 70 75
Phe Gln Tyr Ser Gln Arg Glu Leu Asp Thr Ile Glu Val Phe Pro
80 85 90
Thr Lys Ser Ala Arg Gly Asn Arg Val Ser Cys Met Tyr Val Arg
95 100 105
Cys Val Pro Gly Ala Arg Tyr Thr Val Leu Phe Ser His Gly Asn
110 115 120
Ala Val Asp Leu Gly Gln Met Ser Ser Phe Tyr Ile Gly Leu Gly
125 130 135
Ser Arg Leu His Cys Asn Ile Phe Ser Tyr Asp Tyr Ser Gly Tyr
140 145 150
Gly Ala Ser Ser Gly Arg Pro Ser Glu Arg Asn Leu Tyr Ala Asp
155 160 165
Ile Asp Ala Ala Trp Gln Ala Leu Arg Thr Arg Tyr Gly Ile Ser
170 175 180
Pro Asp Ser Ile Ile Leu Tyr Gly Gln Ser Ile Gly Thr Val Pro
185 190 195
Thr Val Asp Leu Ala Ser Arg Tyr Glu Cys Ala Ala Val Ile Leu
200 205 210
His Ser Pro Leu Met Ser Gly Leu Arg Val Ala Phe Pro Asp Thr
215 220 225
Arg Lys Thr Tyr Cys Phe Asp Ala Phe Pro Ser Ile Asp Lys Ile
230 235 240
Ser Lys Val Thr Ser Pro Val Leu Val Ile His Gly Thr Glu Asp
245 250 255
Glu Val Ile Asp Phe Ser His Gly Leu Ala Met Tyr Glu Arg Cys
260 265 270
Pro Arg Ala Val Glu Pro Leu Trp Val Glu Gly Ala Gly His Asn
275 280 285
Asp Ile Glu Leu Tyr Ala Gln Tyr Leu Glu Arg Leu Lys Gln Phe
290 295 300
Ile Ser His Glu Leu Pro Asn Ser
305
<210> 5
<211> 328
<222> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3092736CD1
<400> 5
Met Trp Cys Ser Phe Leu Ala Pro Val Ser Ser Ser Cys Leu Cys
1 5 10 15
Trp Val Trp Ala Cys Trp Gly Glu Gly His Cys Cys Gln Arg Gly
20 25 30
Thr Asp Phe Leu Met Val Leu Pro Lys Val Asn Val Gly Asp Thr
35 40 45
6/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Val Ala Met Leu Pro Lys Ser Arg Arg Ala Leu Thr Ile Gln Glu
50 55 60
Ile Ala Ala Leu Ala Arg Ser Ser Leu His Gly Ile Ser Gln Val
65 70 75
Val Lys Asp His Val Thr Lys Pro Thr Ala Met Ala Gln Gly Arg
80 85 90
Val Ala His Leu Ile Glu Trp Lys Gly Trp Ser Lys Pro Ser Asp
95 100 l05
Ser Pro Ala Ala Leu Glu Ser Ala Phe Ser Ser Tyr Ser Asp Leu
110 115 120
Ser Glu Gly Glu Gln Glu Ala Arg Phe Ala Ala Gly Val Ala Glu
125 130 135
Gln Phe Ala Ile Ala Glu Ala Lys Leu Arg Ala Trp Ser Ser Val
140 145 150
Asp Gly Glu Asp Ser Thr Asp Asp Ser Tyr Asp Glu Asp Phe Ala
155 160 165
Gly Gly Met Asp Thr Asp Met Ala Gly G1n Leu Pro Leu Gly Pro
170 175 180
His Leu Gln Asp Leu Phe Thr Gly His Arg Phe Ser Arg Pro Val
l85 190 195
Arg Gln Gly Ser Val Glu Pro Glu Ser Asp Cys Ser Gln Thr Val
200 205 210
Ser Pro Asp Thr Leu Cys Ser Ser Leu Cys Ser Leu Glu Asp Gly
2l5 220 225
Leu Leu Gly Ser Pro Ala Arg Leu Ala Ser Gln Leu Leu Gly Asp
230 235 240
Glu Leu Leu Leu Ala Lys Leu Pro Pro Ser Arg Glu Ser Ala Phe
245 250 255
Arg Ser Leu Gly Pro Leu Glu Ala Gln Asp Ser Leu Tyr Asn Ser
260 265 270
Pro Leu Thr Glu Ser Cys Leu Ser Pro Ala Glu Glu Glu Pro Ala
275 280 285
Pro Cys Lys Asp Cys Gln Pro Leu Cys Pro Pro Leu Thr Gly Ser
290 295 300
Trp Glu Arg Gln Arg G1n Ala Ser Asp Leu Ala Ser Ser Gly Val
305 310 315
Val Ser Leu Asp Glu Asp Glu Ala Glu Pro Glu Glu Gln
320 325
<210> 6
<211> 69
<212> PRT
<213> Homo Sapiens
<220>
<22l> misC_feature
<223> Incyte ID No: 3580257CD1
<400> 6
Met Ala Met Ala Val Asp Val Ala Ala Ser Ala Asp Gly Val Leu
1 5 10 15
Ala Val Ala Met Glu Ala Thr Asp Met Ala Leu Ala Leu Glu Ala
20 25 30
Thr Asp Met Ala Leu Ala Leu Glu Ala Thr Asp Met Ala Leu Ala
35 40 45
Leu Glu Ala Met Asp Met Ala Ala Ala Ala His Arg Thr Met Glu
50 55 60
Asp Thr Asp Ser Leu Ala Phe Ile Lys
<210> 7
<211> 158
<212> PRT
7/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3634758CD1
<400> 7
Met Ala Asp Glu Ala Leu Phe Leu Leu Leu His Asn Glu Met Val
1 5 10 15
Ser Gly Val Tyr Lys Ser Ala Glu Gln Gly Glu Val Glu Asn Gly
20 25 30
Arg Cys Ile Thr Lys Leu Glu Asn Met Gly Phe Arg Val Gly Gln
35 40 45
Gly Leu Ile Glu Arg Phe Thr Lys Asp Thr Ala Arg Phe Lys Asp
50 55 60
Glu Leu Asp Ile Met Lys Phe Ile Cys Lys Asp Phe Trp Thr Thr
65 70 75
Val Phe Lys Lys Gln Tle Asp Asn Leu Arg Thr Asn His Gln G1y
80 85 90
Ile Tyr Val Leu Gln Asp Asn Lys Phe Arg Leu Leu Thr Gln Met
95 100 105
Ser Ala Gly Lys Gln Tyr Leu Glu His Ala Ser Lys Tyr Leu Ala
110 115 120
Phe Thr Cys Gly Leu Tle Arg Gly Gly Leu Ser Asn Leu Gly Ile
125 130 135
Lys Ser Ile Va1 Thr A1a Glu Val Ser Ser Met Pro Ala Cys Lys
140 145 150
Phe Gln Val Met Ile Gln Lys Leu
155
<210> 8
<211> 463
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4027923CD1
<400> 8
Met Arg Ala Gly Pro Glu Pro Gln Ala Leu Val Gly Gln Lys Arg
1 5 10 15
Gly Ala Leu Arg Leu Leu Val Pro Arg Leu Val Leu Thr Val Ser
20 25 30
Ala Pro Ala Glu Val Arg Arg Arg Val Leu Arg Pro Val Leu Ser
35 40 45
Trp Met Asp Arg Glu Thr Arg Ala Leu Ala Asp Ser His Phe Arg
50 55 60
Gly Leu Gly Val Asp Val Pro Gly Va1 Gly Gln Ala Pro Gly Arg
65 70 75
Val Ala Phe Val Ser Glu Pro Gly Ala Phe Ser Tyr Ala Asp Phe
80 85 90
Val Arg Gly Phe Leu Leu Pro Asn Leu Pro Cys Val Phe Ser Ser
95 100 105
Ala Phe Thr Gln Gly Trp Gly Ser Arg Arg Arg Trp Val Thr Pro
110 115 120
Ala Gly Arg Pro Asp Phe Asp His Leu Leu Arg Thr Tyr Gly Asp
125 130 135
Val Val Val Pro Val Ala Asn Cys Gly Val Gln Glu Tyr Asn Ser
140 145 150
Asn Pro Lys G1u His Met Thr Leu Arg Asp Tyr Ile Thr Tyr Trp
155 160 165
Lys Glu Tyr Ile Gln Ala Gly Tyr Ser Ser Pro Arg Gly Cys Leu
g/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
170 175 180
Tyr Leu Lys Asp Trp His Leu Cys Arg Asp Phe Pro Val Glu Asp
185 190 195
Val Phe Thr Leu Pro Val Tyr Phe Ser Ser Asp Trp Leu Asn Glu
200 205 210
Phe Trp Asp Ala Leu Asp Val Asp Asp Tyr Arg Phe Val Tyr Ala
215 220 225
Gly Pro Ala Gly Ser Trp Ser Pro Phe His Ala Asp Ile Phe Arg
230 235 240
Ser Phe Ser Trp Ser Val Asn Val Cys Gly Arg Lys Lys Trp Leu
245 250 255
Leu Phe Pro Pro Gly Gln Glu Glu Ala Leu Arg Asp Arg His Gly
260 265 270
Asn Leu Pro Tyr Asp Val Thr Ser Pro Ala Leu Cys Asp Thr His
275 280 285
Leu His Pro Arg Asn Gln Leu Ala Gly Pro Pro Leu Glu Ile Thr
290 295 300
Gln Glu Ala Gly Glu Met Val Phe Val Pro Ser Gly Trp His His
305 310 315
Gln Val His Asn Leu Asp Asp Thr Ile Ser Ile Asn His Asn Trp
320 325 330
Val Asn Gly Phe Asn Leu Ala Asn Met Trp Arg Phe Leu Gln Gln
335 340 345
Glu Leu Cys Ala Val Gln Glu Glu Val Ser Glu Trp Arg Asp Ser
350 355 360
Met Pro Asp Trp His His His Cys Gln Val Ile Met Arg Ser Cys
365 370 375
Ser Gly Ile Asn Phe Glu G1u Phe Tyr His Phe Leu Lys Val Ile
380 385 390
Ala Glu Lys Arg Leu Leu Val Leu Arg Glu Ala Ala Ala Glu Asp
395 400 405
Gly Ala Gly Leu Gly Phe Glu Gln Ala Ala Phe Asp Val Gly Arg
410 415 420
Ile Thr Glu Val Leu Ala Ser Leu Val Ala His Pro Asp Phe Gln
425 430 435
Arg Val Asp Thr Ser Ala Phe Ser Pro Gln Pro Lys Glu Leu Leu
440 445 450
Gln G1n Leu Arg Glu Ala Val Asp Ala Ala Ala Ala Pro
455 460
<210> 9
<211> 648
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4348533CD1
<400> 9
Met Glu Lys Ala Arg Arg Gly Gly Asp Gly Val Pro Arg Gly Pro
1 5 10 15
Val Leu His Ile Val Val Val Gly Phe His His Lys Lys Gly Cys
20 25 30
Gln Val Glu Phe Ser Tyr Pro Pro Leu Ile Pro Gly Asp Gly His
35 40 45
Asp Ser His Thr Leu Pro Glu Glu Trp Lys Tyr Leu Pro Phe Leu
50 55 60
Ala Leu Pro Asp Gly Ala His Asn Tyr Gln Glu Asp Thr Val Phe
65 70 75
Phe His Leu Pro Pro Arg Asn Gly Asn Gly Ala Thr Val Phe Gly
80 85 90
Ile Ser Cys Tyr Arg Gln Ile Glu Ala Lys Ala Leu Lys Val Arg
9/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
95 100 105
Gln Ala Asp Ile Thr Arg Glu Thr Val Gln Lys Ser Val Cys Val
110 115 120
Leu Ser Lys Leu Pro Leu Tyr Gly Leu Leu Gln Ala Lys Leu Gln
125 130 135
Leu Ile Thr His Ala Tyr Phe Glu Glu Lys Asp Phe Ser Gln Ile
140 145 150
Ser Ile Leu Lys Glu Leu Tyr Glu His Met Asn Ser Ser Leu Gly
155 160 165
Gly Ala Ser Leu Glu Gly Ser Gln Val Tyr Leu Gly Leu Ser Pro
170 175 180
Arg Asp Leu Val Leu His Phe Arg His Lys Val Leu Ile Leu Phe
185 190 195
Lys Leu Ile Leu Leu Glu Lys Lys Val Leu Phe Tyr Ile Ser Pro
200 205 210
Val Asn Lys Leu Val Gly Ala Leu Met Thr Val Leu Ser Leu Phe
215 220 225
Pro Gly Met Ile Glu His Gly Leu Ser Asp Cys Ser Gln Tyr Arg
230 235 240
Pro Arg Lys Ser Met Ser Glu Asp Gly Gly Leu Gln Glu Ser Asn
245 250 255
Pro Cys Ala Asp Asp Phe Val Ser Ala Ser Thr Ala Asp Val Ser
260 265 270
His Thr Asn Leu Gly Thr Ile Arg Lys Val Met Ala Gly Asn His
275 280 285
Gly Glu Asp Ala Ala Met Lys Thr Glu Glu Pro Leu Phe Gln Val
290 295 300
Glu Asp Ser Ser Lys G1y Gln Glu Pro Asn Asp Thr Asn Gln Tyr
305 310 315
Leu Lys Pro Pro Ser Arg Pro Ser Pro Asp Ser Ser Glu Ser Asp
320 325 330
Trp Glu Thr Leu Asp Pro Ser Val Leu Glu Asp Pro Asn Leu Lys
335 340 345
Glu Arg Glu Gln Leu Gly Ser Asp Gln Thr Asn Leu Phe Pro Lys
350 355 360
Asp Ser Val Pro Ser Glu Ser Leu Pro Ile Thr Val G1n Pro Gln
365 370 375
Ala Asn Thr Gly Gln Val Val Leu Ile Pro Gly Leu Ile Ser Gly
380 385 390
Leu Glu Glu Asp Gln Tyr Gly Met Pro Leu Ala Ile Phe Thr Lys
395 400 405
Gly Tyr Leu Cys Leu Pro Tyr Met Ala Leu G1n Gln His His Leu
410 415 420
Leu Ser Asp Val Thr Val Arg Gly Phe Val Ala Gly Ala Thr Asn
425 430 435
Ile Leu Phe Arg Gln Gln Lys His Leu Ser Asp Ala Ile Val Glu
440 445 450
Val Glu Glu Ala Leu Ile Gln Ile His Asp Pro Glu Leu Arg Lys
455 460 465
Leu Leu Asn Pro Thr Thr Ala Asp Leu Arg Phe Ala Asp Tyr Leu
470 475 480
Val Arg His Val Thr Glu Asn Arg Asp Asp Val Phe Leu Asp Gly
485 490 495
Thr Gly Trp Glu Gly Gly Asp Glu Trp Ile Arg Ala Gln Phe Ala
500 505 510
Val Tyr Ile His Ala Leu Leu Ala Ala Thr Leu Gln Leu Asp Asn
515 520 525
Glu Lys Ile Leu Ser Asp Tyr Gly Thr Thr Phe Val Thr Ala Trp
530 535 540
Lys Asn Thr His Asn Tyr Arg Val Trp Asn Ser Asn Lys His Pro
545 550 555
Ala Leu Ala Glu Ile Asn Pro Asn His Pro Phe Gln Gly Gln Tyr
560 565 570
10171
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Ser Val Ser Asp Met Lys Leu Arg Phe Ser His Ser Val Gln Asn
575 580 585
Ser Glu Arg Gly Lys Lys Ile Gly Asn Val Met Val Thr Thr Ser
590 595 600
Arg Asn Val Val Gln Thr Gly Lys Ala Val Gly Gln Ser Val Gly
605 610 615
Gly Ala Phe Ser Ser Ala Lys Thr Ala Met Ser Ser Trp Leu Ser
620 625 630
Thr Phe Thr Thr Ser Thr Ser Gln Ser Leu Thr Glu Pro Pro Asp
635 640 645
Glu Lys Pro
<210> 10
<211> 130
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4521857CD1
<400> 10
Met Tyr Leu Gln Val Glu Thr Arg Thr Ser Ser Arg Leu His Leu
1 , 5 10 15
Lys Arg Ala Pro Gly Ile Arg Ser Trp Ser Leu Leu Val Gly Ile
20 25 30
Leu Ser Ile Gly Leu Ala Ala Ala Tyr Tyr Ser Gly Asp Ser Leu
35 40 45
Gly Trp Lys Leu Phe Tyr Val Thr Gly Cys Leu Phe Val Ala Val
50 55 60
Gln Asn Leu Glu Asp Trp Glu Glu Ala Ile Phe Asp Lys Ser Thr
65 70 75
Gly Lys Val Val Leu Lys Thr Phe Ser Leu Tyr Lys Lys Leu Leu
80 85 90
Thr Leu Phe Arg Ala Gly His Asp Gln Val Val Val Leu Leu His
95 100 105
Val Val Pro Asp Thr Ala Ser Ser Pro Trp Trp Thr Ser Pro Ala
110 115 120
Val Arg Cys Phe Pro Lys Gly Ser Glu Gly
125 130
<210> 11
<211> 279
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4722253CD1
<400> 11
Met Gly Arg Gly Leu Arg Trp Trp Gly Gly Arg Gly Arg Arg His
1 5 10 15
Gly Gln Ala Pro Glu Trp Gly Pro Leu Val Gly Ala Arg Leu Lys
20 25 30
Gly Val Ala Arg Ala Ala Ser Leu Val Gly Arg Arg Arg Ala Gly
35 40 45
Thr Gly Met Ala Leu Leu Leu Cys Leu Val Cys Leu Thr Ala Ala
50 55 60
Leu Ala His Gly Cys Leu His Cys His Ser Asn Phe Ser Lys Lys
' 65 70 75
Phe Ser Phe Tyr Arg His His Val Asn Phe Lys Ser Trp Trp Val
11!71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
80 85 90
Gly Asp Ile Pro Val Ser Gly Ala Leu Leu Thr Asp Trp Ser Asp
95 100 105
Asp Thr Met Lys Glu Leu His Leu Ala Ile Pro Ala Lys Ile Thr
110 115 120
Arg Glu Lys Leu Asp Gln Val Ala Thr Ala Val Tyr Gln Met Met
125 130 135
Asp Gln Leu Tyr Gln Gly Lys Met Tyr Phe Pro Gly Tyr Phe Pro
140 145 150
Asn Glu Leu Arg Asn Ile Phe Arg Glu Gln Val His Leu Ile Gln
155 160 165
Asn Ala Ile Ile Glu Ser Arg Ile Asp Cys Gln His Arg Cys Gly
170 175 180
Ile Phe Gln Tyr Glu Thr Ile Ser Cys Asn Asn Cys Thr Asp Ser
185 190 195
His Val Ala Cys Phe Gly Tyr Asn Cys Glu Ser Ser Ala Gln Trp
200 205 210
Lys Ser Ala Val Gln Gly Leu Leu Asn Tyr Ile Asn Asn Trp His
215 220 225
Lys Gln Asp Thr Ser Met Arg Pro Arg Ser Ser Ala Phe Ser Trp
230 235 240
Pro Gly Thr His Arg Ala Thr Pro Ala Phe Leu Val Ser Pro Ala
245 250 255
Leu Arg Cys Leu Glu Pro Pro His Leu Ala Asn Leu Thr Leu Glu
260 265 270
Asp Ala Ala Glu Cys Leu Lys Gln His
275
<210> 12
<211> 458
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4878134CD1
<400> 12
Met Pro Thr Ile Leu Trp Leu Met Asp Trp Ser Asp Met Asn Ser
1 5 10 15
Asn Leu Asp Leu Leu Ala Leu Leu Gly Leu Gly Ile Ser Ser Phe
20 25 30
Val Leu Ile Thr Gly Cys Ala Asn Met Leu Leu Met Ala Ala Leu
35 40 45
Trp Gly Leu Tyr Met Ser Leu Val Asn Val Gly His Val Trp Tyr
50 55 60
Ser Phe Gly Trp Glu Ser Gln Leu Leu Glu Thr Gly Phe Leu Gly
65 70 75
Ile Phe Leu Cys Pro Leu Trp Thr Leu Ser Arg Leu Pro Gln His
80 85 90
Thr Pro Thr Ser Arg Ile Val Leu Trp Gly Phe Arg Trp Leu Ile
95 100 105
Phe Arg Ile Met Leu Gly Ala Gly Leu Ile Lys Ile Arg Gly Asp
110 115 120
Arg Cys Trp Arg Asp Leu Thr Cys Met Asp Phe His Tyr Glu Thr
125 130 135
Gln Pro Met Pro Asn Pro Val Ala Tyr Tyr Leu His His Ser Pro
140 145 150
Trp Trp Phe His Arg Phe Glu Thr Leu Ser Asn His Phe Ile Glu
155 160 165
Leu Leu Val Pro Phe Phe Leu Phe Leu Gly Arg Arg Ala Cys Ile
170 175 180
Ile His G1y Val Leu Gln Ile Leu Phe Gln Ala Val Leu Ile Val
12/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
185 190 195
Ser Gly Asn Leu Ser Phe Leu Asn Trp Leu Thr Met Val Pro Ser
200 205 210
Leu Ala Cys Phe Asp Asp Ala Thr Leu Gly Phe Leu Phe Pro Ser
215 220 225
Gly Pro Gly Ser Leu Lys Asp Arg Val Leu Gln Met Gln Arg Asp
230 235 240
Ile Arg Gly Ala Arg Pro Glu Pro Arg Phe Gly Ser Val Val Arg
245 250 255
Arg Ala Ala Asn Val Ser Leu Gly Val Leu Leu Ala Trp Leu Ser
260 265 270
Val Pro Val Val Leu Asn Leu Leu Ser Ser Arg Gln Val Met Asn
275 280 285
Thr His Phe Asn Ser Leu His Ile Val Asn Thr Tyr Gly Ala Phe
290 295 300
Gly Ser Ile Thr Lys Glu Arg Ala Glu Val Ile Leu Gln Gly Thr
305 310 315
Ala Ser Ser Asn Ala Ser Ala Pro Asp Ala Met Trp Glu Asp Tyr
320 325 330
Glu Phe Lys Cys Lys Pro Gly Asp Pro Ser Arg Arg Pro Cys Leu
335 340 345
Ile Ser Pro Tyr His Tyr Arg Leu Asp Trp Leu Met Trp Phe Ala
350 355 360
Ala Phe Gln Thr Tyr Glu His Asn Asp Trp Ile Ile His Leu Ala
365 370 375
Gly Lys Leu Leu Ala Ser Asp Ala Glu Ala Leu Ser Leu Leu Ala
380 385 390
His Asn Pro Phe Ala Gly Arg Pro Pro Pro Arg Trp Val Arg Gly
395 400 405
Glu His Tyr Arg Tyr Lys Phe Ser Arg Pro Gly Gly Arg His Ala
410 415 420
Ala Glu Gly Lys Trp Trp Val Arg Lys Arg Ile Gly Ala Tyr Phe
425 430 435
Pro Pro Leu Ser Leu Glu Glu Leu Arg Pro Tyr Phe Arg Asp Arg
440 445 450
Gly Trp Pro Leu Pro Gly Pro Leu
455
<210> 13
<211> 173
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5050133CD1
<400> 13
Met Leu Leu Val Asp Ala Asp Gln Pro Glu Pro Met Arg Ser Gly
1 5 10 15
Ala Arg Glu Leu Ala Leu Phe Leu Thr Pro Glu Pro Gly Ala Glu
20 25 30
Ala Lys Glu Val Glu Glu Thr Ile Glu Gly Met Leu Leu Arg Leu
35 40 45
Glu Glu Phe Cys Ser Leu Ala Asp Leu Ile Arg Ser Asp Thr Ser
50 55 60
Gln Ile Leu Glu Glu Asn Ile Pro Val Leu Lys Ala Lys Leu Thr
65 70 75
Glu Met Arg Gly Ile Tyr Ala Lys Val Asp Arg Leu Glu Ala Phe
80 85 90
Val Lys Met Val G1y His His Val Ala Phe Leu Glu Ala Asp Val
95 100 105
Leu Gln Ala Glu Arg Asp His Gly Ala Phe Pro Gln Ala Leu Arg
13/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
110 115 120
Arg Trp Leu Gly Ser Ala Gly Leu Pro Ser Phe Arg Asn Lys Ser
125 130 135
Pro Ala Pro Val Pro Val Thr Tyr Glu Leu Pro Thr Leu Tyr Arg
140 145 150
Thr G1u Asp Tyr Phe Pro Val Asp Ala Gly Glu Ala Gln His His
155 160 165
Pro Arg Thr Cys Pro Arg Pro Leu
170
<210> 14
<211> 335
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5630124CD1
<400> 14
Met Gly Ala Ser Ser Ser Ser Ala Leu Ala Arg Leu Gly Leu Pro
1 5 10 15
Ala Arg Pro Trp Pro Arg Trp Leu Gly Val Ala Ala Leu Gly Leu
20 25 30
Ala Ala Val Ala Leu Gly Thr Val Ala Trp Arg Arg Ala Trp Pro
35 40 45
Arg Arg Arg Arg Arg Leu Gln Gln Val Gly Thr Val Ala Lys Leu
50 55 60
Trp Ile Tyr Pro Val Lys Ser Cys Lys Gly Val Pro Val Ser Glu
65 70 75
Ala Glu Cys Thr Ala Met Gly Leu Arg Ser Gly Asn Leu Arg Asp
80 85 90
Arg Phe Trp Leu Val Ile Lys Glu Asp Gly His Met Val Thr Ala
95 100 105
Arg Gln Glu Pro Arg Leu Val Leu Ile Ser Ile Ile Tyr Glu Asn
110 115 120
Asn Cys Leu Ile Phe Arg Ala Pro Asp Met Asp Gln Leu Val Leu
125 130 135
Pro Ser Lys Gln Pro Ser Ser Asn Lys Leu His Asn Cys Arg Ile
140 . 145 150
Phe Gly Leu Asp Ile Lys Gly Arg Asp Cys Gly Asn Glu Ala Ala
155 160 165
Lys Trp Phe Thr Asn Phe Leu Lys Thr Glu Ala Tyr Arg Leu Val
170 175 180
Gln Phe Glu Thr Asn Met Lys Gly Arg Thr Ser Arg Lys Leu Leu
185 190 195
Pro Thr Leu Asp Gln Asn Phe Gln Val Ala Tyr Pro Asp Tyr Cys
200 205 210
Pro Leu Leu Ile Met Thr Asp Ala Ser Leu Val Asp Leu Asn Thr
215 220 225
Arg Met Glu Lys Lys Met Lys Met Glu Asn Phe Arg Pro Asn Ile
230 235 240
Val Val Thr Gly Cys Asp Ala Phe Glu G1u Asp Thr Trp Asp Glu
245 250 255
Leu Leu Ile Gly Ser Val Glu Val Lys Lys Val Met Ala Cys Pro
260 265 270
Arg Cys Ile Leu Thr Thr Val Asp Pro Asp Thr Gly Val Ile Asp
275 280 285
Arg Lys Gln Pro Leu Asp Thr Leu Lys Ser Tyr Arg Leu Cys Asp
290 295 300
Pro Ser Glu Arg Glu Leu Tyr Lys Leu Ser Pro Leu Phe Gly Ile
305 310 315
Tyr Tyr Ser Val Glu Lys Ile Gly Ser Leu Arg Val Gly Asp Pro
14/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
320 325 330
Val Tyr Arg Met Va1
335
<210> 15
<211> 71
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 5677286CD1
<400> 15
Met His Ser Pro Ala Ser G1y Pro Leu Leu Pro Pro Leu Arg Val
1 5 10 15
Pro Trp Leu Pro Pro Val Val Leu Gly Asn Leu Gly Pro Ser Pro
20 25 30
Ala Ser Pro Ala Ser His Ser Ser Ser Leu Val Thr Leu Arg Glu
35 40 45
Leu Arg Ala Arg Leu Val Ala Gly Leu Leu Cys Phe Cys Pro Arg
50 55 60
Leu Leu Trp Ser Leu Ala Gly Asn Ser Met Ile
65 70
<210> 16
<211> 148
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6436791CD1
<400> 16
Met Leu Pro Arg Gly Leu Lys Met Ala Pro Arg Gly Lys Arg Leu
1 5 10 15
Ser Ser Thr Pro Leu Glu Ile Leu Phe Phe Leu Asn Gly Trp Tyr
20 25 30
Asn Ala Thr Tyr Phe Leu Leu Glu Leu Phe Ile Phe Leu Tyr Lys
35 40 45
Gly Val Leu Leu Pro Tyr Pro Thr Ala Asn Leu Val Leu Asp Val
50 55 60
Val Met Leu Leu Leu Tyr Leu Gly Ile Glu Val Ile Arg Leu Phe
65 70 75
Phe Gly Thr Lys Gly Asn Leu Cys Gln Arg Lys Met Pro Leu Ser
80 85 90
Ile Ser Val Ala Leu Thr Phe Pro Ser Ala Met Met Ala Ser Tyr
95 100 105
Tyr Leu Leu Leu Gln Thr Tyr Val Leu Arg Leu Glu Ala Ile Met
110 115 120
Asn Gly Ile Leu Leu Phe Phe Cys Gly Ser Glu Leu Leu Leu Glu
125 130 135
Val Leu Thr Leu Ala Ala Phe Ser Ser Met Asp Thr Ile
140 145
<210> 17
<211> 231
<212> PRT
<213> Homo Sapiens
<220>
<221> misC-feature
15/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<223> Incyte ID No: 1820972CD1
<400> 17
Met Ala Trp Ile Pro Leu Phe Leu Gly Val Leu Ala Tyr Cys Thr
1 5 10 15
Gly Ser Met Asp Ser Phe Glu Leu Thr Gln Ala Pro Ser Thr Ser
20 25 30
Val Ser Pro Gly Gln Thr Ala Thr Ile Ser Cys Ser Gly Glu Lys
35 40 45
Val Gly Ser Lys Phe Phe Ser Trp Tyr Gln Gln Lys Glu Gly Gln
50 55 60
Ser Pro Val Val Ile Ile Tyr Gln Asn Gly Lys Arg Pro Ser Glu
65 70 75
Ile Ala Asp Arg Phe Ser Gly Ser Lys Ser Gly Asp Thr Ala Thr
80 85 90
Leu Thr Ile Ser Arg Ala Gln Ala Gly Asp Glu Ala Asp Tyr Phe
95 100 105
Cys Gln Val Trp Asp Ser Ser Thr Ala Val Phe Gly Gly Gly Thr
110 115 120
Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr
125 130 135
Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr
140 145 150
Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val
155 160 165
Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr
170 175 180
Thr Thr Pro Ser Lys Gln Cys Asn Asn Lys Tyr Ala Ala Ser Ser
185 190 195
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr
200 205 210
Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
215 220 225
Ala Pro Thr Glu Cys Ser
230
<210> 18
<211> 716
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3286805CD1
<400> 18
Met Asn Asn Phe Arg Ala Thr Ile Leu Phe Trp Ala Ala Ala Ala
1 5 10 15
Trp Ala Lys Ser Gly Lys Pro Ser Gly Glu Met Asp Glu Val Gly
20 25 30
Val Gln Lys Cys Lys Asn Ala Leu Lys Leu Pro Val Leu Glu Val
35 40 45
Leu Pro Gly Gly Gly Trp Asp Asn Leu Arg Asn Val Asp Met Gly
50 55 60
Arg Val Met Glu Leu Thr Tyr Ser Asn Cys Arg Thr Thr Glu Asp
65 70 75
Gly Gln Tyr Ile Ile Pro Asp Glu Ile Phe Thr Ile Pro Gln Lys
80 85 90
Gln Ser Asn Leu Glu Met Asn Ser Glu I1e Leu Glu Ser Trp Ala
95 100 105
Asn Tyr Gln Ser Ser Thr Ser Tyr Ser Ile Asn Thr Glu Leu Ser
110 115 120
Leu Phe Ser Lys Val Asn Gly Lys Phe Ser Thr Glu Phe Gln Arg
16/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
125 130 135
Met Lys Thr Leu Gln Val Lys Asp Gln Ala Ile Thr Thr Arg Val
140 145 150
Gln Val Arg Asn Leu Val Tyr Thr Val Lys Ile Asn Pro Thr Leu
155 160 165
Glu Leu Ser Ser Gly Phe Arg Lys Glu Leu Leu Asp Tle Ser Asp
170 175 180
Arg Leu Glu Asn Asn Gln Thr Arg Met Ala Thr Tyr Leu Ala Glu
185 190 195
Leu Leu Val Leu Asn Tyr Gly Thr His Val Thr Thr Ser Val Asp
200 205 210
Ala Gly Ala Ala Leu Ile Gln Glu Asp His Leu Arg Ala Ser Phe
215 220 225
Leu Gln Asp Ser Gln Ser Ser Arg Ser Ala Val Thr Ala Ser Ala
230 235 240
Gly Leu Ala Phe Gln Asn Thr Val Asn Phe Lys Phe Glu Glu Asn
245 250 255
Tyr Thr Ser Gln Asn Val Leu Thr Lys Ser Tyr Leu Ser Asn Arg
260 265 270
Thr Asn Ser Arg Val Gln Ser Ile Gly Gly Val Pro Phe Tyr Pro
275 280 285
Gly Ile Thr Leu Gln Ala Trp Gln Gln Gly Ile Thr Asn His Leu
290 295 300
Val Ala Ile Asp Arg Ser Gly Leu Pro Leu His Phe Phe Ile Asn
305 310 315
Pro Asn Met Leu Pro Asp Leu Pro Gly Pro Leu Val Lys Lys Val
320 325 330
Ser Lys Thr Val Glu Thr Ala Val Lys Arg Tyr Tyr Thr Phe Asn
335 340 345
Thr Tyr Pro Gly Cys Thr Asp Leu Asn Ser Pro Asn Phe Asn Phe
350 355 360
Gln Ala Asn Thr Asp Asp Gly Ser Cys Glu Gly Lys Met Thr Asn
365 370 375
Phe Ser Phe Gly Gly Val Tyr Gln Glu Cys Thr Gln Leu Ser Gly
380 385 390
Asn Arg Asp Val Leu Leu Cys Gln Lys Leu Glu Gln Lys Asn Pro
395 400 405
Leu Thr Gly Asp Phe Ser Cys Pro Ser Gly Tyr Ser Pro Val His
410 415 420
Leu Leu Ser Gln I1e His Glu Glu Gly Tyr Asn His Leu Glu Cys
425 430 435
His Arg Lys Cys Thr Leu Leu Val Phe Cys Lys Thr Val Cys Glu
440 445 450
Asp Val Phe Gln Val Ala Lys Ala Glu Phe Arg Ala Phe Trp Cys
455 460 465
Val Ala Ser Ser Gln Val Pro Glu Asn Ser Gly Leu Leu Phe Gly
470 475 480
Gly Leu Phe Ser Ser Lys Ser Ile Asn Pro Met Thr Asn Ala Gln
485 490 495
Ser Cys Pro Ala Gly Tyr Phe Pro Leu Arg Leu Phe Glu Asn Leu
500 505 510
Lys Val Cys Val Ser Gln Asp Tyr Glu Leu Gly Ser Arg Phe Ala
515 520 525
Val Pro Phe Gly Gly Phe Phe Ser Cys Thr Val Gly Asn Pro Leu
530 535 540
Val Asp Pro Ala Ile Ser Arg Asp Leu Gly Ala Pro Ser Leu Lys
545 550 555
Lys Cys Pro Gly Gly Phe Ser Gln His Pro Ala Leu Ile Ser Asp
560 565 570
Gly Cys Gln Val Ser Tyr Cys Val Lys Ser Gly Leu Phe Thr Gly
575 580 585
Gly Ser Leu Pro Pro Ala Arg Leu Pro Pro Phe Thr Arg Pro Pro
590 595 600
17/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Leu Met Ser Gln Ala Ala Thr Asn Thr Val Ile Val Thr Asn Ser
605 610 615
Glu Asn Ala Arg Ser Trp Ile Lys Asp Ser Gln Thr His Gln Trp
620 625 630
Arg Leu Gly Glu Pro Ile Glu Leu Arg Arg Ala Met Asn Val Ile
635 640 645
His Gly Asp Gly Gly Gly Leu Ser Gly Gly Ala Ala Ala Gly Val
650 655 660
Thr Val Gly Val Thr Thr Ile Leu Ala Val Val Ile Thr Leu Ala
665 670 675
Ile Tyr Gly Thr Arg Lys Phe Lys Lys Lys Ala Tyr Gln Ala Ile
680 685 690
Glu Glu Arg Gln Ser Leu Val Pro Gly Thr Ala Ala Thr Gly Asp
695 700 705
Thr Thr Tyr Gln Glu Gln Gly Gln Ser Pro Ala
710 715
<210> 19
<211> 519
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3506590CD1
<400> 19
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg
1 5 10 15
Gly Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
20 25 30
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
35 40 45
Phe Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro
50 55 60
Gly Lys Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser
65 70 75
Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
80 85 90
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
95 100 105
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Gly Glu
110 115 120
Gly Ser Pro Asp Thr Leu Val Ala Phe Asp Ile Trp Gly Gln Gly
125 130 135
Thr Met Val Thr Va1 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
140 145 150
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Gly Gly Thr Ala
155 160 165
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
170 175 180
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
185 190 195
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
200 205 210
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys
215 220 225
Asn Val Asn His Lys Pro Ser Asn Thr Lys Va1 Asp Lys Arg Val
230 235 240
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg
245 250 255
Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
260 265 270
18/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
275 280 285
Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg
290 295 300
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
305 310 315
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
320 325 330
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
335 340 345
Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
350 355 360
Thr Lys Leu Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val
365 370 375
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
380 385 390
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
395 400 405
Lys Thr Ile Ser Lys A1a Lys Gly Gln Pro Arg Glu Pro Gln Val
410 415 420
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
425 430 435
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
440 445 450
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Asn Thr
455 460 465
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
470 475 480
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe
485 490 495
Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln
500 505 510
Lys Ser Leu Ser Leu Ser Pro Gly Lys
515
<210> 20
<211> 172
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 003600CD1
<400> 20
Met Leu Thr Glu Val Met Glu Val Trp His Gly Leu Val Ile Ala
1 5 10 15
Val Val Ser Leu Phe Leu Gln Ala Cys Phe Leu Thr Ala Ile Asn
20 25 30
Tyr Leu Leu Ser Arg His Met Ala His Lys Ser Glu Gln Ile Leu
35 40 45
Lys Ala Ala Ser Leu Gln Val Pro Arg Pro Ser Pro Gly His His
50 55 60
His Pro Pro Ala Val Lys Glu Met Lys Glu Thr Gln Thr Glu Arg
65 70 75
Asp Ile Pro Met Ser Asp Ser Leu Tyr Arg His Asp Ser Asp Thr
80 85 90
Pro Ser Asp Ser Leu Asp Ser Ser Cys Ser Ser Pro Pro Ala Cys
95 100 105
Gln Ala Thr Glu Asp Val Asp Tyr Thr Gln Val Val Phe Ser Asp
110 115 120
Pro Gly Glu Leu Lys Asn Asp Ser Pro Leu Asp Tyr Glu Asn Ile
125 130 135
19171
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Lys Glu Tle Thr Asp Tyr Val Asn Val Asn Pro Glu Arg His Lys
140 14S 150
Pro Ser Phe Trp Tyr Phe Val Asn Pro Ala Leu Ser Glu Pro Ala
155 160 165
Glu Tyr Asp Gln Val Ala Met
170
<210> 21
<211> 314
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1251534CD1
<400> 21
Met Gly Leu Leu Asp Ser Glu Pro Gly Ser Val Leu Asn Val Val
1 5 10 15
Ser Thr Ala Leu Asn Asp Thr Val Glu Phe Tyr Arg Trp Thr Trp
20 25 30
Ser Ile Ala Asp Lys Arg Val Glu Asn Trp Pro Leu Met Gln Ser
35 40 45
Pro Trp Pro Thr Leu Ser Ile Ser Thr Leu Tyr Leu Leu Phe Val
50 55 60
Trp Leu Gly Pro Lys Trp Met Lys Asp Arg Glu Pro Phe Gln Met
65 70 75
Arg Leu Val Leu Ile Ile Tyr Asn Phe Gly Met Val Leu Leu Asn
80 85 90
Leu Phe Ile Phe Arg Glu Leu Phe Met Gly Ser Tyr Asn Ala Gly
95 100 105
Tyr Ser Tyr Ile Cys Gln Ser Val Asp Tyr Ser Asn Asn Val His
110 115 120
Glu Val Arg Ile Ala Ala Ala Leu Trp Trp Tyr Phe Val Ser Lys
125 130 135
Gly Val Glu Tyr Leu Asp Thr Val Phe Phe Ile Leu Arg Lys Lys
140 145 150
Asn Asn Gln Val Ser Phe Leu His Val Tyr His His Cys Thr Met
155 160 165
Phe Thr Leu Trp Trp Ile Gly Ile Lys Trp Val Ala Gly Gly Gln
170 175 180
Ala Phe Phe Gly Ala Gln Leu Asn Ser Phe Ile His Val Ile Met
185 190 195
Tyr Ser Tyr Tyr Gly Leu Thr Ala Phe Gly Pro Trp Ile Gln Lys
200 205 210
Tyr Leu Trp Trp Lys Arg Tyr Leu Thr Met Leu Gln Leu Ile Gln
215 220 225
Phe His Val Thr Ile Gly His Thr Ala Leu Ser Leu Tyr Thr Asp
230 235 240
Cys Pro Phe Pro Lys Trp Met His Trp Ala Leu Ile Ala Tyr Ala
245 250 255
Ile Ser Phe Ile Phe Leu Phe Leu Asn Phe Tyr Ile Arg Thr Tyr
260 265 270
Lys Glu Pro Lys Lys Pro Lys AIa Gly Lys Thr Ala Met Asn Gly
275 280 285
Ile Ser Ala Asn Gly Val Ser Lys Ser Glu Lys Gln Leu Met Ile
290 295 300
GIu Asn GIy Lys Lys GIn Lys Asn Gly Lys Ala Lys Gly Asp
305 310
<210> 22
<211> 542
<212> PRT
20/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1402211CD1
<400> 22
Met Asn Gly Lys Arg Pro Ala Glu Pro Gly Pro Ala Arg Val Gly
1 5 10 15
Lys Lys Gly Lys Lys Glu Val Met Ala Glu Phe Ser Asp Ala Val
20 25 30
Thr Glu Glu Thr Leu Lys Lys Gln Val Ala Glu Ala Trp Ser Arg
35 40 45
Arg Thr Pro Phe Ser His Glu Val Ile Val Met Asp Met Asp Pro
50 55 60
Phe Leu His Cys Val Ile Pro Asn Phe Ile Gln Ser Gln Asp Phe
65 70 75
Leu Glu Gly Leu Gln Lys Glu Leu Met Asn Leu Asp Phe His Glu
80 85 90
Lys Tyr Asn Asp Leu Tyr Lys Phe Gln Gln Ser Asp Asp Leu Lys
95 100 105
Lys Arg Arg Glu Pro His Ile Ser Thr Leu Arg Lys I1e Leu Phe
110 115 120
Glu Asp Phe Arg Ser Trp Leu Ser Asp Ile Ser Lys Ile Asp Leu
125 130 135
Glu Ser Thr Ile Asp Met Ser Cys Ala Lys Tyr Glu Phe Thr Asp
140 145 150
Ala Leu Leu Cys His Asp Asp Glu Leu Glu Gly Arg Arg Ile Ala
155 160 165
Phe Ile Leu Tyr Leu Val Pro Pro Trp Asp Arg Ser Met Gly Gly
170 175 180
Thr Leu Asp Leu Tyr Ser Ile Asp Glu His Phe Gln Pro Lys Gln
185 190 195
Ile Val Lys Ser Leu Ile Pro Ser Trp Asn Lys Leu Val Phe Phe
200 205 210
Glu Val Ser Pro Val Ser Phe His Gln Val Ser Glu Val Leu Ser
215 220 225
Glu Glu Lys Ser Arg Leu Ser Ile Ser Gly Trp Phe His Gly Pro
230 235 240
Ser Leu Thr Arg Pro Pro Asn Tyr Phe Glu Pro Pro Ile Pro Arg
245 250 255
Ser Pro His Ile Pro Gln Asp His Glu Ile Leu Tyr Asp Trp Ile
260 265 270
Asn Pro Thr Tyr Leu Asp Met Asp Tyr Gln Val Gln Ile Gln Glu
275 280 285
Glu Phe Glu Glu Ser Ser Glu Ile Leu Leu Lys Glu Phe Leu Lys
290 295 300
Pro Glu Lys Phe Thr Lys Val Cys Glu Ala Leu Glu His Gly His
305 310 315
Val Glu Trp Ser Ser Arg Gly Pro Pro Asn Lys Arg Phe Tyr Glu
320 325 330
Lys Ala Glu Glu Ser Lys Leu Pro Glu Ile Leu Lys Glu Cys Met
335 340 345
Lys Leu Phe Arg Ser Glu Ala Leu Phe Leu Leu Leu Ser Asn Phe
350 355 360
Thr Gly Leu Lys Leu His Phe Leu Ala Pro Ser Glu Glu Asp Glu
365 370 375
Met Asn Asp Lys Lys Glu Ala Glu Thr Thr Asp Ile Thr Glu Glu
380 385 390
Gly Thr Ser His Ser Pro Pro Glu Pro Glu Asn Asn Gln Met Ala
395 400 405
Ile Ser Asn Asn Ser Gln Gln Ser Asn Glu Gln Thr Asp Pro Glu
410 415 420
21/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Pro Glu Glu Asn Glu Thr Lys Lys Glu Ser Ser Val Pro Met Cys
425 430 435
Gln Gly Glu Leu Arg His Trp Lys Thr Gly His Tyr Thr Leu Ile
440 445 450
His Asp His Ser Lys Ala Glu Phe Ala Leu Asp Leu Ile Leu Tyr
455 460 465
Cys Gly Cys Glu Gly Trp Glu Pro Glu Tyr Gly Gly Phe Thr Ser
470 475 480
Tyr Ile Ala Lys Gly Glu Asp Glu Glu Leu Leu Thr Val Asn Pro
485 490 495
Glu Ser Asn Ser Leu Ala Leu Val Tyr Arg Asp Arg Glu Thr Leu
500 505 510
Lys Phe Val Lys His Ile Asn His Arg Ser Leu Glu Gln Lys Lys
515 520 525
Thr Phe Pro Asn Arg Thr Gly Phe Trp Asp Phe Ser Phe Ile Tyr
530 535 540
Tyr Glu
<210> 23
<211> 715
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1623474CD1
<400> 23
Met Pro Ala Glu Ser Gly Lys Arg Phe Lys Pro Ser Lys Tyr Val
1 5 10 15
Pro Val Ser Ala Ala Ala Ile Phe Leu Val Gly Ala Thr Thr Leu
20 25 30
Phe Phe Ala Phe Thr Cys Pro Gly Leu Ser Leu Tyr Val Ser Pro
35 40 45
Ala Val Pro Ile Tyr Asn Ala Ile Met Phe Leu Phe Val Leu Ala
50 55 60
Asn Phe Ser Met Ala Thr Phe Met Asp Pro Gly Ile Phe Pro Arg
65 70 75
Ala Glu Glu Asp Glu Asp Lys Glu Asp Asp Phe Arg Ala Pro Leu
80 85 90
Tyr Lys Thr Val Glu Ile Lys Gly Ile Gln Val Arg Met Lys Trp
95 100 105
Cys Ala Thr Cys Arg Phe Tyr Arg Pro Pro Arg Cys Ser His Cys
110 115 120
Ser Val Cys Asp Asn Cys Val Glu Glu Phe Asp His His Cys Pro
125 130 135
Trp Val Asn Asn Cys Ile Gly Arg Arg Asn Tyr Arg Tyr Phe Phe
140 145 150
Leu Phe Leu Leu Ser Leu Thr Ala His Ile Met Gly Val Phe Gly
155 160 165
Phe Gly Leu Leu Tyr Val Leu Tyr His Ile Glu Glu Leu Ser Gly
170 175 180
Val Arg Thr Ala Val Thr Met Ala Val Met Cys Val Ala Gly Leu
185 190 195
Phe Phe Ile Pro Val Ala Gly Leu Thr Gly Phe His Val Val Leu
200 205 210
Val Ala Arg Gly Arg Thr Thr Asn Glu Gln Val Thr Gly Lys Phe
215 220 225
Arg Gly Gly Val Asn Pro Phe Thr Asn Gly Cys Cys Asn Asn Val
230 235 240
Ser Arg Val Leu Cys Ser Ser Pro Ala Pro Arg Tyr Leu Gly Arg
245 250 255
22/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Pro Lys Lys Glu Lys Thr Ile Val Ile Arg Pro Pro Phe Leu Arg
260 265 270
Pro Glu Val Ser Asp Gly Gln Ile Thr Val Lys Ile Met Asp Asn
275 280 285
Gly Ile Gln Gly Glu Leu Arg Arg Thr Lys Ser Lys Gly Ser Leu
290 295 300
G1u Ile Thr Glu Ser G1n Ser Ala Asp Ala Glu Pro Pro Pro Pro
305 310 315
Pro Lys Pro Asp Leu Ser Arg Tyr Thr Gly Leu Arg Thr His Leu
320 325 330
Gly Leu Ala Thr Asn Glu Asp Ser Ser Leu Leu Ala Lys Asp Ser
335 340 345
Pro Pro Thr Pro Thr Met Tyr Lys Tyr Arg Pro Gly Tyr Ser Ser
350 355 360
Ser Ser Thr Ser Ala Ala Met Pro His Ser Ser Ser Ala Lys Leu
365 370 375
Ser Arg Gly Asp Ser Leu Lys Glu Pro Thr Ser Ile Ala Glu Ser
380 385 390
Ser Arg His Pro Ser Tyr Arg Ser Glu Pro Ser Leu Glu Pro Glu
395 400 405
Ser Phe Arg Ser Pro Thr Phe Gly Lys Ser Phe His Phe Asp Pro
410 415 420
Leu Ser Ser Gly Ser Arg Ser Ser Ser Leu Lys Ser Ala Gln Gly
425 430 435
Thr Gly Phe Glu Leu Gly Gln Leu Gln Ser Ile Arg Ser Glu Gly
440 445 450
Thr Thr Ser Thr Ser Tyr Lys Ser Leu Ala Asn Gln Thr Arg Asn
455 460 465
Gly Ser Leu Ser Tyr Asp S.er Leu Leu Thr Pro Ser Asp Ser Pro
470 475 480
Asp Phe Glu Ser Val Gln Ala Gly Pro Glu Pro Asp Pro Pro Leu
485 490 495
Gly Tyr Thr Ser Pro Phe Leu Ser Ala Arg Leu Ala Gln Gln Arg
500 505 510
Glu Ala Glu Arg His Pro Arg Leu Val Pro Thr Gly Pro Thr His
515 520 525
Arg Glu Pro Ser Pro Val Arg Tyr Asp Asn Leu Ser Arg His I1e
530 535 540
Val Ala Ser Leu Gln Glu Arg Glu Lys Leu Leu Arg Gln Ser Pro
545 550 555
Pro Leu Pro Gly Arg Glu Glu Glu Pro Gly Leu Gly Asp Ser Gly
560 565 570
Ile Gln Ser Thr Pro Gly Ser Gly His Ala Pro Arg Thr Ser Ser
575 580 585
Ser Ser Asp Asp Ser Lys Arg Ser Pro Leu Gly Lys Thr Pro Leu
590 595 600
Gly Arg Pro Ala Val Pro,Arg Phe Gly Lys Pro Asp Gly Leu Arg
605 610 615
Gly Arg Gly Val Gly Ser Pro Glu Pro Gly Pro Thr Ala Pro Tyr
620 625 630
Leu Gly Arg Ser Met Ser Tyr Ser Ser Gln Lys Ala Gln Pro Gly
635 640 645
Val Ser GIu Thr GIu Glu Val Ala Leu Gln Pro Leu Leu Thr Pro
650 655 660
Lys Asp Glu Val Gln Leu Lys Thr Thr Tyr Ser Lys Ser Asn Gly
665 670 675
Gln Pro Lys Ser Leu Gly Ser Ala Ser Pro Gly Pro Gly Gln Pro
680 685 690
Pro Leu Ser Ser Pro Thr Arg Gly Gly Val Lys Lys Val Ser Gly
695 700 705
Val Gly Gly Thr Thr Tyr Glu Ile Ser Val
710 715
23/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<210> 24
<211> 469
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1706443CD1
<400> 24
Met Gly Arg Val Arg Arg Ile Tyr Pro Gln Leu Leu Leu Ala Leu
1 5 10 15
Leu Ile Gln Val His Tyr His Ile Gly Leu Asn Leu Pro Gly Cys
20 25 30
Val Ala Pro Pro Lys Asp Thr Lys Lys Gly Ala Gln Pro Ser Pro
35 40 45
Phe Val Pro Val Arg Trp Val Val Lys Val Val Lys Thr Leu Leu
50 55 60
Leu Arg Met Gly Cys Ser Tyr Glu Thr Thr Phe Leu Glu Asp Gln
65 70 75
Gly Gly Trp Glu Leu Met Glu Gln Val Glu Ser His His Arg Gly
80 85 90
Val Ala Leu Leu Ala Arg Ala Met Val Gln Tyr Ser Cys Gln Glu
95 100 105
Leu Cys Arg Ile Leu Tyr Leu Leu Ile Pro Leu Leu Glu Arg Gly
110 115 120
Asp Glu Lys His Arg Ile Thr Ala Thr Ala Phe Phe Val Glu Leu
125 130 135
Leu Gln Met Glu Gln Va1 Arg Arg Ile Pro Glu Glu Tyr Ser Leu
140 145 150
Gly Arg Met Ala Glu Gly Leu Ser His His Asp Pro Ile Met Lys
155 160 165
Val Leu Ser Ile Arg Gly Leu Val Ile Leu Ala Arg Arg Ser Glu
170 175 180
Lys Thr Ala Lys Val Lys Ala Leu Leu Pro Ser Met Val Lys Gly
185 190 195
Leu Lys Asn Met Asp Gly Met Leu Val Val Glu Ala Val His Asn
200 205 210
Leu Lys Ala Val Phe Lys Gly Arg Asp Gln Lys Leu Met Asp Ser
215 220 225
Ala Val Tyr Val Glu Met Leu Gln Ile Leu Leu Pro His Phe Ser
230 235 240
Asp Ala Arg Glu Asp Val Arg Ser Ser Cys Ile Asn Leu Tyr Gly
245 250 255
Lys Val Val Gln Lys Leu Arg Ala Pro Arg Thr Gln Ala Met Glu
260 265 270
Glu Gln Leu Val Ser Thr Leu Val Pro Leu Leu Leu Thr Met Gln
275 280 285
Glu Gly Asn Ser Lys Val Ser Gln Lys Cys Val Lys Thr Leu Leu
290 295 300
Arg Cys Ser Tyr Phe Met Ala Trp Glu Leu Pro Lys Arg Ala Tyr
305 310 315
Ser Arg Lys Pro Trp Asp Asn Gln Gln Gln Thr Val Ala Lys Ile
320 325 330
Cys Lys Cys Leu Val Asn Thr His Arg Asp Ser Ala Phe Ile Phe
335 340 345
Leu Ser Gln Ser Leu Glu Tyr Ala Lys Asn Ser Arg Ala Ser Leu
350 355 360
Arg Lys Cys Ser Val Met Phe Ile Gly Ser Leu Val Pro Cys Met
365 370 375
Glu Ser Ile Met Thr Glu Asp Arg Leu Asn Glu Val Lys Ala Ala
380 385 390
Leu Asp Asn Leu Arg His Asp Pro Glu Ala Ser Val Cys Ile Tyr
24/71
CA 02409778 2002-11-25
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395 400 405
Ala Ala Gln Val Gln Asp His Ile Leu Ala Ser Cys Trp Gln Asn
410 415 420
Ser Trp Leu Pro His Gly Asn Ser Trp Val Cys Tyr Ser Ala Thr
425 430 435
Thr His Arg Trp Ser Pro Ser Cys Glu Asn Leu Pro Thr Ser His
440 445 450
Gln Arg Arg Ser Trp Ile Met Gln Ala Leu Gly Ser Trp Lys Met
455 460 465
Ser Leu Lys Lys
<210> 25
<211> 274
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1748627CD1
<400> 25
Met Pro Arg Ala Glu Pro Arg Ala Thr Leu Gly Glu Gln Glu Lys
1 5 10 15
Ala Gly Leu Pro Leu Gly Ala Trp Arg Leu Tyr Leu Leu Arg His
20 25 30
Phe Arg Lys Gln Thr Glu Leu Arg Arg Ser Gly Ser Arg Asp Val
35 40 45
Thr Gly Ala Leu Leu Val Ala Ala Ala Val Ala Ser Glu Ala Val
50 55 60
Gly Ser Leu Arg Val Ala Glu Gly Gly Pro Asn Thr Leu Leu Leu
65 70 75
Gln Val Leu Arg Ser Trp Pro Trp Cys Asn Lys Glu Leu Lys Thr
80 85 90
Met Glu Glu Arg Lys Val Lys Arg Arg Ser Pro Lys Ser Phe Ser
95 100 105
Ala His Cys Thr Gln Val Val Asn Ala Lys Lys Asn Ala Ile Pro
110 115 120
Val Ser Lys Ser Thr Gly Phe Ser Asn Pro Ala Ser Gln Ser Thr
125 130 135
Ser Gln Arg Pro Lys Leu Lys Arg Val Met Lys Glu Lys Thr Lys
140 145 150
Pro Gln Gly Gly Glu Gly Lys Gly Ala Gln Ser Thr Pro Ile Gln
155 160 165
His Ser Phe Leu Thr Asp Val Ser Asp Val Gln Glu Met Glu Arg
170 175 180
Gly Leu Leu Ser Leu Leu Asn Asp Phe His Ser Gly Lys Leu Gln
185 190 195
Ala Phe Gly Asn Glu Cys Ser Ile Glu Gln Met Glu His Val Arg
200 205 210
Gly Met Gln Glu Lys Leu Ala Arg Leu Asn Leu Glu Leu Tyr Gly
215 220 225
Glu Leu Glu Glu Leu Pro Glu Asp Lys Arg Lys Thr Ala Ser Asp
230 235 240
Ser Asn Leu Asp Arg Leu Leu Ser Asp Leu Glu Glu Leu Asn Ser
245 250 255
Ser Ile Gln Lys Leu His Leu Ala Asp Ala Gln Asp Val Pro Asn
260 265 270
Thr Ser Ala Ser
<210> 26
<211> 154
25/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1818332CD1
<400> 26
Met Ala Gly Pro Val Lys Asp Arg Glu Ala Phe Gln Arg Leu Asn
1 5 10 15
Phe Leu Tyr Gln Ala Ala His Cys Val Leu Ala Gln Asp Pro Glu
20 25 30
Asn Gln Ala Leu Ala Arg Phe Tyr Cys Tyr Thr Glu Arg Thr Ile
35 40 45
Ala Lys Arg Leu Val Leu Arg Arg Asp Pro Ser Val Lys Arg Thr
50 55 60
Leu Cys Arg Gly Cys Ser Ser Leu Leu Val Pro Gly Leu Thr Cys
65 70 75
Thr Gln Arg Gln Arg Arg Cys Arg Gly Gln Arg Trp Thr Val Gln
80 85 90
Thr Cys Leu Thr Cys Gln Arg Ser Gln Arg Phe Leu Asn Asp Pro
95 100 105
Gly His Leu Leu Trp Gly Asp Arg Pro Glu Ala Gln Leu Gly Ser
110 115 120
Gln Ala Asp Ser Lys Pro Leu Gln Pro Leu Pro Asn Thr Ala His
125 130 135
Ser Ile Ser Asp Arg Leu Pro Glu Glu Lys Met Gln Thr Gln Gly
140 145 150
Ser Ser Asn Gln
<210> 27
<211> 102
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1822832CD1
<400> 27
Met Lys Phe Asp Trp Val Met Gly Leu Arg Ser Ile Thr Leu Lys
1 5 10 15
Asn Ser Ser Thr Gly Arg Gly Asp Gly Pro Lys Gln His Leu Gln
20 25 30
Ala Asp Pro Met Leu Ile Ile Arg Ala Arg Thr Leu Ser Leu Ser
35 40 45
Val Ser Leu Ser Val Ser Pro Leu Gly Leu Thr Pro His Trp Thr
50 55 60
Pro Leu His Pro Cys Pro Ser His Asn Thr Ala Ala Val Ser Ser
65 70 75
Ala Cys Leu Trp Glu Ser Pro Leu Phe Ser Ser Val Phe Phe Ser
80 85 90
Ser Cys Pro Ile Thr Pro Cys Thr Ser Pro Phe Pro
95 100
<210> 28
<211> 113
<212> PRT
<213> Homo Sapiens
<220>
<221> mist feature
26/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<223> Incyte ID No: 1832219CD1
<400> 28
Met Ala Gly Pro Ala Ala Ala Phe Arg Arg Leu Gly Ala Leu Ser
1 5 10 15
Gly Ala Ala Ala Leu Gly Phe Ala Ser Tyr Gly Ala His Gly Ala
20 25 30
Gln Phe Pro Asp Ala Tyr Gly Lys Glu Leu Phe Asp Lys Ala Asn
35 40 45
Lys His His Phe Leu His Ser Leu Ala Leu Leu Gly Val Pro His
50 55 60
Cys Arg Lys Pro Leu Trp Ala Gly Leu Leu Leu Ala Ser Gly Thr
65 70 75
Thr Leu Phe Cys Thr Ser Phe Tyr Tyr G1n Ala Leu Ser Gly Asp
80 85 90
Pro Ser Ile Gln Thr Leu Ala Pro Ala Gly Gly Thr Leu Leu Leu
95 100 105
Leu Gly Trp Leu Ala Leu Ala Leu
110
<210> 29
<211> 313
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1899010CD1
<400> 29
Met Ala Leu Leu Val Asp Arg Val Arg Gly His Trp Arg Ile Ala
1 5 10 15
Ala Gly Leu Leu Phe Asn Leu Leu Val Ser Ile Cys Ile Val Phe
20 25 30
Leu Asn Lys Trp Ile Tyr Val Tyr His Gly Phe Pro Asn Met Ser
35 40 45
Leu Thr Leu Val His Phe Val Val Thr Trp Leu Gly Leu Tyr Ile
50 55 60
Cys Gln Lys Leu Asp Ile Phe Ala Pro Lys Ser Leu Pro Pro Ser
65 70 75
Arg Leu Leu Leu Leu Ala Leu Ser Phe Cys Gly Phe Val Val Phe
80 85 90
Thr Asn Leu Ser Leu Gln Asn Asn Thr Ile Gly Thr Tyr Gln Leu
95 100 105
Ala Lys Ala Met Thr Thr Pro Val Ile Ile Ala Ile Gln Thr Phe
110 115 120
Cys Tyr Gln Lys Thr Phe Ser Thr Arg Ile Gln Leu Thr Leu Ile
125 130 135
Pro Ile Thr Leu Gly Val Tle Leu Asn Ser Tyr Tyr Asp Val Lys
140 145 150
Phe Asn Phe Leu Gly Met Val Phe Ala Ala Leu Gly Val Leu Val
155 160 165
Thr Ser Leu Tyr Gln Val Trp Val Gly Ala Lys Gln His Glu Leu
170 175 180
Gln Val Asn Ser Met Gln Leu Leu Tyr Tyr Gln Ala Pro Met Ser
185 190 195
Ser Ala Met Leu Leu Val Ala Val Pro Phe Phe Glu Pro Val Phe
200 205 210
Gly Glu Gly Gly Ile Phe Gly Pro Trp Ser Val Ser Ala Leu Leu
215 220 225
Met Val Leu Leu Ser Gly Val Ile Ala Phe Met Val Asn Leu Ser
230 235 240
Ile Tyr Trp Ile Ile Gly Asn Thr Ser Pro Val Thr Tyr Asn Met
27/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
245 250 255
Phe Gly His Phe Lys Phe Cys Ile Thr Leu Phe Gly Gly Tyr Val
260 265 270
Leu Phe Lys Asp Pro Leu Ser I1e Asn Gln Ala Leu Gly Ile Leu
275 280 285
Cys Thr Leu Phe Gly Ile Leu Ala Tyr Thr His Phe Lys Leu Ser
290 295 300
Glu Gln Glu Gly Ser Arg Ser Lys Leu Ala Gln Arg Pro
305 310
<210> 30
<211> 195
<212> PRT
<213> Homojsapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2008768CD1
<400> 30
Met Ala Pro Lys Ala Ala Lys Gly Ala Lys Pro Glu Pro Ala Pro
1 5 10 15
Ala Pro Pro Pro Pro Gly Ala Lys Pro Glu Glu Asp Lys Lys Asp
20 25 30
Gly Lys Glu Pro Ser Asp Lys Pro Gln Lys Ala Val Gln Asp His
35 40 45
Lys Glu Pro Ser Asp Lys Pro Gln Lys Ala Val Gln Pro Lys His
50 55 60
Glu Val Gly Thr Arg Arg Gly Cys Arg Arg Tyr Arg Trp Glu Leu
65 70 75
Lys Asp Ser Asn Lys Glu Phe Trp Leu Leu Gly His Ala Glu Ile
80 85 90
Lys Ile Arg Ser Leu Asp Leu Phe Asn Asp Leu Ile Ala Cys Ala
95 100 105
Phe Leu Val Gly Ala Val Val Phe Ala Val Arg Ser Arg Arg Ser
110 115 120
Met Asn Leu His Tyr Leu Leu Ala Val Ile Leu Ile Gly Ala Ala
125 130 135
Gly Val Phe Ala Phe Tle Asp Val Cys Leu Gln Arg Asn His Phe
140 145 150
Arg Gly Lys Lys Ala Lys Lys His Met Leu Val Pro Pro Pro Gly
155 160 165
Lys Glu Lys Gly Pro Gln Gln Gly Lys Gly Pro Glu Pro Ala Lys
170 175 180
Pro Pro Glu Pro Gly Lys Pro Pro Gly Pro Ala Lys Gly Lys Lys
185 190 195
<210> 31
<211> 350
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2070984CD1
<400> 31
Met Asn Leu GlyLeu Phe Gln Asp Phe Pro Ser Lys
Arg Asn Phe
1 5 10 15
Leu Ile Tyr CysLeu Leu Leu Phe Ser Leu Leu Ala
Ala Val Leu
20 25 30
Arg Leu Asp IleIle Gln Trp Ser Tyr Ala Val Phe
Gly Trp Ala
28/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
35 40 45
Pro Ile Trp Leu Trp Lys Leu Met Val Ile Val Gly Ala Ser Val
50 55 60
Gly Thr Gly Val Trp Ala Arg Asn Pro Gln Tyr Arg Ala Glu Gly
65 70 75
Glu Thr Cys Val Glu Phe Lys Ala Met Leu Ile Ala Val Gly Ile
80 85 90
His Leu Leu Leu Leu Met Phe Glu Val Leu Val Cys Asp Arg Ile
95 100 105
Glu Arg Gly Ser His Phe Trp Leu Leu Val Phe Met Pro Leu Phe
110 115 120
Phe Val Ser Pro Val Ser Val Ala Ala Cys Val Trp Gly Phe Arg
125 130 135
His Asp Arg Ser Leu Glu Leu Glu Ile Leu Cys Ser Val Asn Ile
140 145 150
Leu Gln Phe Ile Phe Ile Ala Leu Arg Leu Asp Lys Ile Ile His
155 160 165
Trp Pro Trp Leu Val Val Cys Val Pro Leu Trp Ile Leu Met Ser
170 175 180
Phe Leu Cys Leu Val Val Leu Tyr Tyr Ile Val Trp Ser Val Leu
185 190 195
Phe Leu Arg Ser Met Asp Val Ile Ala Glu Gln Arg Arg Thr His
200 205 210
Ile Thr Met Ala Leu Ser Trp Met Thr Ile Val Val Pro Leu Leu
215 220 225
Thr Phe Glu Ile Leu Leu Val His Lys Leu Asp Gly His Asn Ala
230 235 240
Phe Ser Cys Ile Pro Ile Phe Val Pro Leu Trp Leu Ser Leu Ile
245 250 255
Thr Leu Met Ala Thr Thr Phe Gly Gln Lys Gly Gly Asn His Trp
260 265 270
Trp Phe Gly Ile Arg Lys Asp Phe Cys Gln Phe Leu Leu Glu Ile
275 280 285
Phe Pro Phe Leu Arg Glu Tyr Gly Asn Ile Ser Tyr Asp Leu His
290 295 300
His Glu Asp Asn Glu Glu Thr Glu Glu Thr Pro Val Pro Glu Pro
305 310 315
Pro Lys Ile Ala Pro Met Phe Arg Lys Lys Ala Arg Val Val Ile
320 325 330
Thr Gln Ser Pro Gly Lys Tyr Val Leu Pro Pro Pro Lys Leu Asn
335 340 345
Ile Glu Met Pro Asp
350
<210> 32
<211> 360
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2193240CD1
<400> 32
Met Ser Leu Leu Ala Val Ser Arg Arg Ala Gln Lys His Ala Leu
1 5 10 15
Lys Ala Asn Leu Ile Asp Asn Cys Met Glu Gln Met Lys His Ile
20 25 30
Asn Ala Gln Leu Asn Leu Asp Ser Leu Arg Pro Gly Lys Ala Ala
35 40 45
Leu Lys Lys Lys Glu Asp Gly Val Ile Lys Glu Leu Ser Ile Ala
50 55 60
Met Gln Leu Leu Arg Asn Cys Leu Tyr Gln Asn Glu Glu Cys Lys
29/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
65 70 75
Glu Ala Ala Leu Glu Ala His Leu Val Pro Val Leu His Ser Leu
80 85 90
Trp Pro Trp Ile Leu Met Asp Asp Ser Leu Met Gln Ile Ser Leu
95 100 105
Gln Leu Leu Cys Val Tyr Thr Ala Asn Phe Pro Asn Gly Cys Ser
110 115 120
Ser Leu Cys Trp Ser Ser Cys Gly Gln His Pro Val Gln Ala Thr
125 130 135
His Arg Gly Ala Val Ser Asn Ser Leu Met Leu Cys Ile Leu Lys
140 145 150
Leu Ala Ser Gln Met Pro Leu Glu Asn Thr Thr Val Gln Gln Met
155 160 165
Val Phe Met Leu Leu Ser Asn Leu Ala Leu Ser His Asp Cys Lys
170 175 180
Gly Val Ile Gln Lys Ser Asn Phe Leu Gln Asn.Phe Leu Ser Leu
185 190 195
Ala Leu Pro Lys Gly Gly Asn Lys His Leu Ser Asn Leu Thr Ile
200 205 210
Leu Trp Leu Lys Leu Leu Leu Asn Ile Ser Ser Gly Glu Asp Gly
215 220 225
Gln Gln Met Ile Leu Arg Leu Asp Gly Cys Leu Asp Leu Leu Thr
230 235 240
Glu Met Ser Lys Tyr Lys His Lys Ser Ser Pro Leu Leu Pro Leu
245 250 255
Leu Ile Phe His Asn Val Cys Phe Ser Pro Ala Asn Lys Pro Lys
260 265 270
Ile Leu Ala Asn Glu Lys Val Ile Thr Val Leu Ala Ala Cys Leu
275 280 285
Glu Ser Glu Asn Gln Asn Ala Gln Arg Ile Gly Ala Ala Ala Leu
290 295 300
Trp Ala Leu Ile Tyr Asn Tyr Gln Lys Ala Lys Thr Ala Leu Lys
305 310 315
Ser Pro Ser Val Lys Arg Arg Val Asp Glu Ala Tyr Ser Leu Ala
320 325 330
Lys Lys Thr Phe Pro Asn Ser Glu Ala Asn Pro Leu Asn Ala Tyr
335 340 345
Tyr Leu Lys Cys Leu Glu Asn Leu Val Gln Leu Leu Asn Ser Ser
350 355 360
<210> 33
<211> 559
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2235177CD1
<400> 33
Met Gly Ser Arg Ile Lys Gln Asn Pro Glu Thr Thr Phe Glu Val
1 5 10 15
Tyr Val Glu Val Ala Tyr Pro Arg Thr Gly Gly Thr Leu Ser Asp
20 25 30
Pro Glu Val Gln Arg Gln Phe Pro Glu Asp Tyr Ser Asp Gln Glu
35 40 45
Val Leu Gln Thr Leu Thr Lys Phe Cys Phe Pro Phe Tyr Val Asp
50 55 60
Ser Leu Thr Val Ser Gln Val Gly Gln Asn Phe Thr Phe Val Leu
65 70 75
Thr Asp Ile Asp Ser Lys Gln Arg Phe Gly Phe Cys Arg Leu Ser
80 85 90
30/71
CA 02409778 2002-11-25
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Ser Gly Ala Lys Ser Cys Phe Cys Ile Leu Ser Tyr Leu Pro Trp
95 100 105
Phe Glu Val Phe Tyr Lys Leu Leu Asn Ile Leu Ala Asp Tyr Thr
110 115 120
Thr Lys Arg Gln Glu Asn Gln Trp Asn Glu Leu Leu Glu Thr Leu
125 130 135
His Lys Leu Pro Ile Pro Asp Pro Gly Val Ser Val His Leu Ser
140 145 150
Val His Ser Tyr Phe Thr Val Pro Asp Thr Arg Glu Leu Pro Ser
155 160 165
Ile Pro Glu Asn Arg Asn Leu Thr Glu Tyr Phe Val Ala Val Asp
170 175 180
Val Asn Asn Met Leu His Leu Tyr Ala Ser Met Leu Tyr Glu Arg
185 190 195
Arg Ile Leu Ile Ile Cys Ser Lys Leu Ser Thr Leu Thr Ala Cys
200 205 210
Ile His Gly Ser Ala Ala Met Leu Tyr Pro Met Tyr Trp Gln His
215 220 225
Val Tyr Ile Pro Val Leu Pro Pro His Leu Leu Asp Tyr Cys Cys
230 235 240
Ala Pro Met Pro Tyr Leu Ile Gly Ile His Leu Ser Leu Met Glu
245 250 255
Lys Val Arg Asn Met Ala Leu Asp Asp Val Val Ile Leu Asn Val
260 265 270
Asp Thr Asn Thr Leu Glu Thr Pro Phe Asp Asp Leu Gln Ser Leu
275 280 285
Pro Asn Asp Val Ile Ser Ser Leu Lys Asn Arg Leu Lys Lys Val
290 295 300
Ser Thr Thr Thr Gly Asp Gly Val Ala Arg Ala Phe Leu Lys Ala
305 310 315
Gln Ala Ala Phe Phe Gly Ser Tyr Arg Asn Ala Leu Lys Ile Glu
320 325 330
Pro Glu Glu Pro Ile Thr Phe Cys Glu Glu Ala Phe Val Ser His
335 340 345
Tyr Arg Ser Gly Ala Met Arg Gln Phe Leu Gln Asn Ala Thr Gln
350 355 360
Leu Gln Leu Phe Lys Gln Phe Ile Asp Gly Arg Leu Asp Leu Leu
365 370 375
Asn Ser Gly Glu Gly Phe Ser Asp Val Phe Glu Glu Glu Ile Asn
380 385 390
Met Gly Glu Tyr Ala Gly Ser Asp Lys Leu Tyr His Gln Trp Leu
395 400 405
Ser Thr Val Arg Lys Gly Ser Gly Ala Ile Leu Asn Thr Val Lys
410 415 420
Thr Lys Ala Asn Pro Ala Met Lys Thr Val Tyr Lys Phe Ala Lys
425 430 435
Asp His Ala Lys Met Gly Ile Lys Glu Val Lys Asn Arg Leu Lys
440 445 450
Gln Lys Asp Ile Ala G1u Asn Gly Cys Ala Pro Thr Pro Glu Glu
455 460 465
Gln Leu Pro Lys Thr Ala Pro Ser Pro Leu Val Glu Ala Lys Asp
470 475 480
Pro Lys Leu Arg Glu Asp Arg Arg Pro Ile Thr Val His Phe Gly
485 490 495
Gln Val Arg Pro Pro Arg Pro His Val Val Lys Arg Pro Lys Ser
500 505 510
Asn Ile Ala Val Glu Gly Arg Arg Thr Ser Val Pro Ser Pro Glu
515 520 525
Gln Asn Thr Ile Ala Thr Pro Ala Thr Leu His Ile Leu Gln Lys
530 535 540
Ser Ile Thr His Phe Ala Ala Lys Phe Pro Thr Arg Gly Trp Thr
545 550 555
Ser Sex Ser His
31/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<210> 34
<211> 198
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2416227CD1
<400> 34
Met Ala Leu Arg His Leu Ala Leu Leu Ala Gly Leu Leu Val Gly
1 5 10 15
Val Ala Ser Lys Ser Met Glu Asn Thr Ala Gln Leu Pro Glu Cys
20 25 30
Cys Val Asp Val Val Gly Val Asn Ala Ser Cys Pro Gly Ala Ser
35 40 45
Leu Cys Gly Pro Gly Cys Tyr Arg Arg Trp Asn Ala Asp Gly Ser
50 55 60
Ala Ser Cys Val Arg Cys Gly Asn Gly Thr Leu Pro Ala Tyr Asn
65 70 75
Gly Ser Glu Cys Arg Ser Phe Ala Gly Pro Gly Ala Pro Phe Pro
80 85 90
Met Asn Arg Ser Ser Gly Thr Pro Gly Arg Pro His Pro Gly Ala
95 100 105
Pro Arg Val Ala Ala Ser Leu Phe Leu Gly Thr Phe Phe I1e Ser
110 115 120
Ser Gly Leu Ile Leu Ser Val Ala Gly Phe Phe Tyr Leu Lys Arg
125 130 135
Ser Ser Lys Leu Pro Arg Ala Cys Tyr Arg Arg Asn Lys Ala Pro
140 145 150
Ala Leu Gln Pro Gly Glu Ala Ala Ala Met Ile Pro Pro Pro Gln
155 160 165
Ser Ser Val Arg Lys Pro Arg Tyr Val Arg Arg Glu Arg Pro Leu
170 175 180
Asp Arg Ala Thr Asp Pro Ala Ala Phe Pro Gly Glu Ala Arg Ile
185 190 195
Ser Asn Val
<210> 35
<211> 73
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2461076CD1
<400> 35
Met Lys Leu Pro Leu Ser. Leu Leu Phe Leu Arg Thr Leu Gly Phe
1 5 10 15
Tyr Ile Pro Val Lys Gly Asp Leu Ser Ser Gly Cys Glu Asp Lys
20 25 30
Ala Cys Leu Tyr Val Leu Lys Arg Val Thr Thr Asp Lys Val Phe
35 40 45
Phe Asp Pro Phe Lys Ile Tyr Phe Arg Pro Val Ile Pro Gly Leu
50 55 60
Trp Glu Ala Glu Ala Gly Gly Ser Leu Gly Leu Gly Val
65 70
<210> 36
32/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<211> 376
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1957517CD1
<400> 36
Met Asp Gly Glu Glu Gln Gln Pro Pro His Glu Ala Asn Val Glu
1 5 10 15
Pro Val Val Pro Ser Glu Ala Ser Glu Pro Val Pro Arg Val Leu
20 25 30
Ser Gly Asp Pro Gln Asn Leu Ser Asp Val Asp Ala Phe Asn Leu
35 40 45
Leu Leu Glu Met Lys Leu Lys Arg Arg Arg Gln Arg Pro Asn Leu
50 55 60
Pro Arg Thr Val Thr Gln Leu Val Ala Glu Asp Gly Ser Arg Val
65 70 75
Tyr Val Val Gly Thr Ala His Phe Ser Asp Asp Ser Lys Arg Asp
80 85 90
Val Val Lys Thr Ile Arg Glu Val Gln Pro Asp Val Val Val Val
95 100 105
Glu Leu Cys G1n Tyr Arg Val Ser Met Leu Lys Met Asp Glu Ser
110 115 120
Thr Leu Leu Arg Glu Ala Gln Glu Leu Ser Leu Glu Lys Leu Gln
125 130 135
Gln Ala Val Arg Gln Asn Gly Leu Met Ser Gly Leu Met Gln Met
140 145 150
Leu Leu Leu Lys Val Ser Ala His Ile Thr Glu Gln Leu Gly Met
155 160 165
Ala Pro Gly G1y Glu Phe Arg Glu Ala Phe Lys Glu Ala Ser Lys
170 175 180
Val Pro Phe Cys Lys Phe His Leu Gly Asp Arg Pro Ile Pro Val
185 190 195
Thr Phe Lys Arg Ala Ile Ala Ala Leu Ser Phe Trp G1n Lys Val
200 205 210
Arg Leu Ala Trp Gly Leu Cys Phe Leu Ser Asp Pro Ile Ser Lys
215 220 225
Asp Asp Val Glu Arg Cys Lys Gln Lys Asp Leu Leu Glu Gln Met
230 235 240
Met Ala Glu Met Ile Gly Glu Phe Pro Asp Leu His Arg Thr Ile
245 250 255
Val Ser Glu Arg Asp Val Tyr Leu Thr Tyr Met Leu Arg Gln Ala
260 265 270
Ala Arg Arg Leu Glu Leu Pro Arg Ala Ser Asp Ala Glu Pro Arg
275 280 285
Lys Cys Val Pro Ser Val Val Val Gly Val Val Gly Met GIy His
290 295 300
Val Pro Gly Ile Glu Lys Asn Trp Ser Thr Asp Leu Asn Ile Gln
305 310 315
Glu Tle Met Thr Val Pro Pro Pro Ser Val Ser Gly Arg Val Ser
320 325 330
Arg Leu Ala Val Lys Ala Ala Phe Phe Gly Leu Leu Gly Tyr Ser
335 340 345
Leu Tyr Trp Met Gly Arg Arg Thr Ala Ser Leu Val Leu Ser Leu
350 355 360
Pro Ala Ala Gln Tyr Cys Leu Gln Arg Val Thr Glu Ala Arg His
365 370 375
Lys
<210> 37
33/71
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<211> 216
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 866038CD1
<400> 37
Met Met Tyr Trp Ile Val Phe Ala Phe Phe Thr Thr Ala Glu Thr
1 5 10 15
Leu Thr Asp Ile Val Leu Ser Trp Phe Pro Phe Tyr Phe Glu Leu
20 25 30
Lys Ile Ala Phe Val Ile Trp Leu Leu Ser Pro Tyr Thr Lys Gly
35 40 45
Ser Ser Val Leu Tyr Arg Lys Phe Val His Pro Thr Leu Ser Asn
50 55 60
Lys Glu Lys Glu Ile Asp Glu Tyr Ile Thr Gln Ala Arg Asp Lys
65 70 75
Ser Tyr Glu Thr Met Met Arg Val Gly Lys Arg Gly Leu Asn Leu
80 85 90
Ala Ala Asn Ala Ala Val Thr Ala Ala Ala Lys Gly Gln Gly Val
95 100 105
Leu Ser Glu Lys Leu Arg Ser Phe Ser Met Gln Asp Leu Thr Leu
110 115 120
Ile Arg Asp Glu Asp Ala Leu Pro Leu Gln Arg Pro Asp Gly Arg
125 130 135
Leu Arg Pro Ser Pro Gly Ser Leu Leu Asp Thr Ile Glu Asp Leu
140 145 150
Gly Asp Asp Pro Ala Leu Ser Leu Arg Ser Ser Thr Asn Pro Ala
155 160 165
Asp Ser Arg Thr Glu Ala Ser Glu Asp Asp Met Gly Asp Lys Ala
170 175 180
Pro Lys Arg Ala Lys Pro Ile Lys Lys Ala Pro Lys Ala Glu Pro
185 190 195
Leu A1a Ser Lys Thr Leu Lys Thr Arg Pro Lys Lys Lys Thr Ser
200 205 210
Gly Gly Gly Asp Ser Ala
215
<210> 38
<211> 233
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3869704CD1
<400> 38
Met Ala Trp Thr Pro Leu Leu Leu Pro Leu Leu Thr Phe Cys Thr
1 5 10 15
Val Ser Glu Ala 5er Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser
20 25 30
Val Ser Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala
35 40 45
Leu Pro Lys Lys Tyr Ala Tyr Trp Tyr Gln Gln Lys Ser Gly Gln
50 55 60
Ala Pro Val Leu Val Ile Tyr Glu Asp Asn Lys Arg Pro Ser Gly
65 70 75
Ile Pro Glu Arg Phe Phe Gly Ser Ser Ser Gly Thr Met Ala Thr
80 85 90
Leu Thr Ile Ser Gly A1a Gln Val Glu Asp Glu Ala Asp Tyr Tyr
34/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
95 100 105
Cys Tyr Ser Thr Asp Ser Ser Gly Asn Asp Arg Val Phe Gly Gly
110 115 120
Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser
125 130 135
Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys
140 145 150
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val
155 160 165
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val
170 175 180
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
185 190 195
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys
200 205 210
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
215 220 225
Thr Val Ala Pro Thr Glu Cys Ser
230
<210> 39
<211> 163
<212> PRT
<213> Homo Sapiens ,
<220>
<221> misc_feature
<223> Incyte ID No: 1415179CD1
<400> 39
Met Leu Cys Pro Leu Ser His Ala Arg Val Val Arg Gly Ala Gly
1 5 10 15
Ser Glu Gly Gly Arg Ile Leu Leu Ser Leu Cys Phe Ser Phe Cys
20 25 30
Pro Ser Gly Leu Ser Cys Trp Cys Ser Arg His Cys Leu Pro Ala
35 40 45
Leu Ala Pro Arg Cys Ser Pro Gln Pro Tyr Leu Ser Cys Phe Pro
50 55 60
Gly Ala Thr His Pro Cys Pro Thr Pro Ser Ala Cys Ser His Gly
65 70 75
Arg Gly Arg Thr His Ser Leu His Thr His Thr Pro Arg Leu His
80 85 90
Pro Val Ser Ile Tyr Lys His Val Arg Ala Arg Val His Thr Ser
95 100 105
Arg Phe Ser Thr Ala Tyr Gln Ala Leu Leu Leu Pro Cys Leu Ser
110 115 ~ 120
Ala Trp Arg Gly Pro Pro Leu Leu Thr Pro Ser Val Pro Pro Pro
125 130 135
Glu Leu Ile Arg Met Arg Met Val Val Pro Ala Ser Glu Gly Leu
140 145 150
Leu Gly Leu Leu Gly Ala Lys Pro Leu Cys Pro Lys Gln
155 160
<210> 40
<211> 235
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1664792CD1
<400> 40
35/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Met Arg Leu Lys Leu Phe Ser Ile Leu Ser Thr Val Leu Leu Arg
1 5 10 15
Ala Thr Asp Thr Ile Asn Ser Gln Gly Gln Phe Pro Ser Tyr Leu
20 25 30
Glu Thr Val Thr Lys Asp Ile Leu Ala Pro Asn Leu Gln Trp His
35 40 45
Ala Gly Arg Thr Ala Ala Ala Ile Arg Thr Ala Ala Val Ser Cys
50 55 60
Leu Trp Ala Leu Thr Ser Ser Glu Val Leu Ser Ala Glu Gln Ile
65 70 75
Arg Asp Val Gln Glu Thr Leu Met Pro Gln Val Leu Thr Thr Leu
80 85 90
Glu Glu Asp Ser Lys Met Thr Arg Leu Ile Ser Cys Arg Ile Ile
95 100 105
Asn Thr Phe Leu Lys Thr Ser Gly Gly Met Thr Asp Pro Glu Lys
110 115 120
Leu Ile Lys Ile Tyr Pro Glu Leu Leu Lys Arg Leu Asp Asp Val
125 130 135
Ser Asn Asp Val Arg Met Ala Ala Ala Ser Thr Leu Val Thr Trp
140 145 150
Leu Gln Cys Val Lys Gly Ala Asn Ala Lys Ser Tyr Tyr Gln Ser
155 160 165
Ser Val Gln Tyr Leu Tyr Arg Glu Leu Leu Val His Leu Asp Asp
170 175 180
Pro Glu Arg Ala Tle Gln Asp Ala Ile Leu Glu Val Leu Lys G1u
185 190 195
Gly Ser Gly Leu Phe Pro Asp Leu Leu Val Arg Glu Thr Glu Ala
200 205 210
Val Ile His Lys His Arg Ser Ala Thr Tyr Cys Glu Gln Leu Leu
215 220 225
Gln His Val Gln Ala Val Pro Ala Thr Gln
230 235
<210> 41
<211> 94
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2079396CD1
<400> 41
Met Ser Pro Leu Ser Pro Thr Gly Leu Asn Leu Trp Gly Gly Glu
1 5 10 15
Gly Ser Ser Leu His Ser Ala Leu Asp His Gln Gly Arg Gly Ile
20 25 30
Thr Leu Ala Ile Gly Ile Ile Ser Ser Ser Phe Ser Ser Pro Ser
35 40 45
Pro Arg Ile Arg Pro Ser Ser Gln His Cys Val Gly Leu Ile Leu
50 55 60
Arg Ile Leu Tyr His His Pro Gly Leu Gly Gly Cys Arg Ser Trp
65 70 75
Val Leu Leu Leu Arg Asp Arg Val Ser Leu Cys His Pro Gly Trp
80 85 90
Ser Ala Val Ala fs
<210> 42
<211> 85
<212> PRT
<213> Homo sapiens
36/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<220>
<221> misc_feature
<223> Incyte ID No: 5390115CD1
<400> 42
Met Ala Ser Asp Leu Asp Phe Ser Pro Pro Glu Val Pro Glu Pro
1 5 10 15
Thr Phe Leu Glu Asn Leu Leu Arg Tyr Gly Leu Phe Leu Gly Ala
20 25 30
Ile Phe Gln Leu Ile Cys Va1 Leu Ala Ile Ile Val Pro Ile Pro
35 40 45
Lys Ser His Glu Ala Glu Ala Glu Pro Ser Glu Pro Arg Ser Ala
50 55 60
Glu Val Thr Arg Lys Pro Lys Ala Ala Val Pro Ser Val Asn Lys
65 70 75
Arg Pro Lys Lys Glu Thr Lys Lys Lys Arg
80 85
<210> 43
<211> 901
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1403326CD1
<400> 43
Met Glu Ala Asn Gln Cys Pro Leu Val Val Glu Pro Ser Tyr Pro
1 5 10 15
Asp Leu Val Ile Asn Val Gly Glu Val Thr Leu Gly Glu Glu Asn
20 25 30
Arg Lys Lys Leu Gln Lys Ile Gln Arg Asp Gln Glu Lys Glu Arg
35 40 45
Val Met Arg Ala Ala Cys Ala Leu Leu Asn Ser Gly Gly Gly Val
50 55 60
Ile Arg Met Ala Lys Lys Val Glu His Pro Val Glu Met Gly Leu
65 70 75
Asp Leu Glu Gln Ser Leu Arg Glu Leu Ile Gln Ser Ser Asp Leu
80 85 90
Gln Ala Phe Phe Glu Thr Lys Gln Gln Gly Arg Cys Phe Tyr Ile
95 100 105
Phe Val Lys Ser Trp Ser Ser Gly Pro Phe Pro Glu Asp Arg Ser
110 ~ 115 120
Phe Lys Pro Arg Leu Cys Ser Leu Ser Ser Ser Leu Tyr Arg Arg
125 130 135
Ser Glu Thr Ser Va1 Arg Ser Met Asp Ser Arg Glu Ala Phe Cys
140 145 150
Phe Leu Lys Thr Lys Arg Lys Pro Lys Ile Leu Glu Glu Gly Pro
155 160 165
Phe His Lys Ile His Lys Gly Val Tyr Gln Glu Leu Pro Asn Ser
170 175 180
Asp Pro Ala Asp Pro Asn Ser Asp Pro Ala Asp Leu Ile Phe Gln
185 190 195
Lys Asp Tyr Leu Glu Tyr Gly Glu Ile Leu Pro Phe Pro Glu Ser
200 205 210
Gln Leu Va1 Glu Phe Lys Gln Phe Ser Thr Lys His Phe Gln Glu
215 220 225
Tyr Val Lys Arg Thr Ile Pro Glu Tyr Val Pro Ala Phe Ala Asn
230 235 240
Thr Gly Gly Gly Tyr Leu Phe Ile Gly Val Asp Asp Lys Ser Arg
245 250 255
Glu Val Leu Gly Cys Ala Lys Glu Asn Val Asp Pro Asp Ser Leu
37/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
260 265 270
Arg Arg Lys Ile Glu G1n Ala Ile Tyr Lys Leu Pro Cys Val His
275 280 285
Phe Cys Gln Pro Gln Arg Pro Ile Thr Phe Thr Leu Lys Ile Val
290 295 300
Asp Val Leu Lys Arg Gly G1u Leu Tyr Gly Tyr Ala Cys Met Ile
305 310 315
Arg Val Asn Pro Phe Cys Cys Ala Val Phe Ser Glu Ala Pro Asn
320 325 330
Ser Trp Ile Val Glu Asp Lys Tyr Val Cys Ser Leu Thr Thr Glu
335 340 345
Lys Trp Val Gly Met Met Thr Asp Thr Asp Pro Asp Leu Leu Gln
350 355 360
Leu Ser Glu Asp Phe Glu Cys Gln Leu Ser Leu Ser Ser Gly Pro
365 370 375
Pro Leu Ser Arg Pro Val Tyr Ser Lys Lys Gly Leu Glu His Lys
380 385 390
Ala Asp Leu Gln Gln His Leu Phe Pro Val Pro Pro Gly His Leu
395 400 405
Glu Cys Thr Pro Glu Ser Leu Trp Lys Glu Leu Ser Leu Gln His
410 415 420
Glu Gly Leu Lys Glu Leu Ile His Lys Gln Met Arg Pro Phe Ser
425 430 435
Gln Gly Ile Val Ile Leu Ser Arg Ser Trp Ala Val Asp Leu Asn
440 445 450
Leu Gln Glu Lys Pro Gly Va1 Ile Cys Asp Ala Leu Leu Ile Ala
455 ' 460 465
Gln Asn Ser Thr Pro Ile Leu Tyr Thr Ile Leu Arg Glu Gln Asp
470 475 480
Ala Glu Gly Gln Asp Tyr Cys Thr Arg Thr Ala Phe Thr Leu Lys
485 490 495
Gln Lys Leu Val Asn Met Gly Gly Tyr Thr Gly Lys Va1 Cys Val
500 505 510
Arg Ala Lys Val Leu Cys Leu Ser Pro Glu Ser Ser Ala Glu Ala
515 520 525
Leu Glu Ala Ala Val Ser Pro Met Asp Tyr Pro Ala Ser Tyr Ser
530 535 540
Leu Ala Gly Thr Gln His Met Glu Ala Leu Leu Gln Ser Leu Val
545 550 555
Ile Val Leu Leu Gly Phe Arg Ser Leu Leu Ser Asp Gln Leu Gly
560 565 570
Cys Glu Val Leu Asn Leu Leu Thr Ala Gln Gln Tyr Glu Ile Phe
575 580 585
Ser Arg Ser Leu Arg Lys Asn Arg Glu Leu Phe Val His Gly Leu
590 595 600
Pro Gly Ser Gly Lys Thr Ile Met Ala Met Lys Ile Met Glu Lys
605 610 615
Ile Arg Asn Val Phe His Cys Glu Ala His Arg Ile Leu Tyr Val
620 625 630
Cys Glu Asn Gln Pro Leu Arg Asn Phe Ile Ser Asp Arg Asn Ile
635 640 645
Cys Arg Ala Glu Thr Arg Lys Thr Phe Leu Arg Glu Asn Phe Glu
650 655 660
His Ile Gln His Ile Val Ile Asp Glu Ala Gln Asn Phe Arg Thr
665 670 675
Glu Asp Gly Asp Trp Tyr Gly Lys Ala Lys Ser Ile Thr Arg Arg
680 685 690
Ala Lys GIy Gly Pro Gly IIe Leu Trp Ile Phe Leu Asp Tyr Phe
695 700 705
Gln Thr Ser His Leu Asp Cys Ser Gly Leu Pro Pro Leu Ser Asp
710 715 72p
Gln Tyr Pro Arg Glu Glu Leu Thr Arg Ile Val Arg Asn Ala Asp
725 730 735
38/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Pro Ile Ala Lys Tyr Leu Gln Lys Glu Met Gln Val Ile Arg Ser
740 745 750
Asn Pro Ser Phe Asn Ile Pro Thr Gly Cys Leu Glu Val Phe Pro
755 760 765
Glu Ala Glu Trp Ser Gln Gly Val Gln Gly Thr Leu Arg Ile Lys
770 775 780
Lys Tyr Leu Thr Val Glu Gln Ile Met Thr Cys Val Ala Asp Thr
785 ' 790 795
Cys Arg Arg Phe Phe Asp Arg Gly Tyr Ser Pro Lys Asp Val Ala
800 805 810
Val Leu Val Ser Thr Ala Lys Glu Val Glu His Tyr Lys Tyr Glu
815 820 825
Leu Leu Lys Ala Met Arg Lys Lys Arg Val Val Gln Leu Ser Asp
830 835 840
Ala Cys Asp Met Leu Gly Asp His Ile Val Leu Asp Ser Val Arg
845 850 855
Arg Phe Ser Gly Leu Glu Arg Ser Ile Val Phe Gly Ile His Pro
860 865 870
Arg Thr Ala Asp Pro Ala Ile Leu Pro Asn Val Leu Ile Cys Leu
875 880 885
Ala Ser Arg Ala Lys Gln His Leu Tyr Ile Phe Pro Trp Gly Gly
890 895 900
His
<210> 44
<211> 1040
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7690129CD1
<400> 44
Met Ala Ser Thr Gly Gly Thr Lys Val Val Ala Met Gly Val Ala
1 5 10 15
Pro Trp Gly Val Val Arg Asn Arg Asp Thr Leu Ile Asn Pro Lys
20 25 30
Gly Ser Phe Pro Ala Arg Tyr Arg Trp Arg Gly Asp Pro Glu Asp
35 40 45
Gly Val Gln Phe Pro Leu Asp Tyr Asn Tyr Ser Ala Phe Phe Leu
50 55 60
Val Asp Asp Gly Thr His Gly Cys Leu Gly Gly Glu Asn Arg Phe
65 70 75
Arg Leu Arg Leu Glu Ser Tyr Ile Ser Gln Gln Lys Thr Gly Val
80 85 90
Gly Gly Thr Gly Ile Asp Ile Pro Val Leu Leu Leu Leu Ile Asp
95 100 105
Gly Asp Glu Lys Met Leu Thr Arg Ile Glu Asn Ala Thr Gln Ala
110 115 120
Gln Leu Pro Cys Leu Leu Val Ala Gly Ser Gly Gly Ala Ala Asp
125 130 135
Cys Leu Ala Glu Thr Leu Glu Asp Thr Leu Ala Pro Gly Ser Gly
140 145 150
Gly Ala Arg Gln Gly Glu Ala Arg Asp Arg Ile Arg Arg Phe Phe
155 160 165
Pro Lys Gly Asp Leu Glu Val Leu Gln Ala Gln Val Glu Arg Ile
170 175 180
Met Thr Arg Lys Glu Leu Leu Thr Val Tyr Ser Ser Glu Asp Gly
185 190 195
Ser Glu Glu Phe Glu Thr Ile Val Leu Lys Ala Leu Val Lys Ala
200 205 210
39/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
Cys Gly Ser Ser Glu Ala Ser Ala Tyr Leu Asp Glu Leu Arg Leu
215 220 225
Ala Val Ala Trp Asn Arg Val Asp Ile Ala Gln Ser Glu Leu Phe
230 235 240
Arg Gly Asp Ile Gln Trp Arg Ser Phe His Leu Glu Ala Ser Leu
245 250 ~ 255
Met Asp Ala Leu Leu Asn Asp Arg Pro Glu Phe Val Arg Leu Leu
260 265 270
Ile Ser His Gly Leu Ser Leu Gly His Phe Leu Thr Pro Met Arg
275 280 285
Leu Ala Gln Leu Tyr Ser Ala Ala Pro Ser Asn Ser Leu Ile Arg
290 295 300
Asn Leu Leu Asp Gln Ala Ser His Ser Ala Gly Thr Lys Ala Pro
305 310 315
Ala Leu Lys Gly Gly Ala Ala Glu Leu Arg Pro Pro Asp Val Gly
320 325 330
His Val Leu Arg Met Leu Leu Gly Lys Met Cys Ala Pro Arg Tyr
335 340 345
Pro Ser Gly Gly Ala Trp Asp Pro His Pro Gly Gln Gly Phe Gly
350 355 360
Glu Ser Met Tyr Leu Leu Ser Asp Lys Ala Thr Ser Pro Leu Ser
365 370 375
Leu Asp Ala Gly Leu Gly Gln Ala Pro Trp Ser Asp Leu Leu Leu
380 385 390
Trp Ala Leu Leu Leu Asn Arg Ala Gln Met Ala Met Tyr Phe Trp
395 400 405
Glu Met Gly Ser Asn Ala Val Ser Ser Ala Leu Gly Ala Cys Leu
410 415 420
Leu Leu Arg Val Met Ala Arg Leu Glu Pro Asp Ala Glu Glu Ala
425 430 435
Ala Arg Arg Lys Asp Leu Ala Phe Lys Phe Glu Gly Met Gly Val
440 445 450
Asp Leu Phe Gly Glu Cys Tyr Arg Ser Ser Glu Val Arg Ala Ala
455 460 465
Arg Leu Leu Leu Arg Arg Cys Pro Leu Trp Gly Asp Ala Thr Cys
470 475 480
Leu Gln Leu Ala Met Gln Ala Asp Ala Arg Ala Phe Phe Ala Gln
485 490 495
Asp Gly Val Gln Ser Leu Leu Thr G1n Lys Trp Trp Gly Asp Met
500 505 510
Ala Ser Thr Thr Pro Ile Trp Ala Leu Val Leu Ala Phe Phe Cys
515 520 525
Pro Pro Leu Ile Tyr Thr Arg Leu Ile Thr Phe Arg Lys Ser Glu
530 535 540
Glu Glu Pro Thr Arg Glu Glu Leu Glu Phe Asp Met Asp Ser Val
545 550 555
Ile Asn Gly Glu Gly Pro Val Gly Thr Ala Asp Pro Ala Glu Lys
560 565 570
Thr Pro Leu Gly Val Pro Arg Gln Ser Gly Arg Pro Gly Cys Cys
575 580 585
Gly Gly Arg Cys Gly Gly Arg Arg Cys Leu Arg Arg Trp Phe His
590 595 600
Phe Trp Gly Ala Pro Val Thr Ile Phe Met Gly Asn Val Va1 Ser
605 610 615
Tyr Leu Leu Phe Leu Leu Leu Phe Ser Arg Val Leu Leu Val Asp
620 625 630
Phe Gln Pro Ala Pro Pro Gly Ser Leu Glu Leu Leu Leu Tyr Phe
635 640 645
Trp Ala Phe Thr Leu Leu Cys Glu Glu Leu Arg Gln Gly Leu Ser
650 655 660
Gly Gly Gly Gly Ser Leu Ala Ser Gly Gly Pro Gly Pro Gly His
665 670 675
Ala Ser Leu Ser Gln Arg Leu Arg Leu Tyr Leu Ala Asp Ser Trp
40/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
680 685 690
Asn Gln Cys Asp Leu Val Ala Leu Thr Cys Phe Leu Leu Gly Val
695 700 705
Gly Cys Arg Leu Thr Pro Gly Leu Tyr His Leu Gly Arg Thr Val
710 715 720
Leu Cys Ile Asp Phe Met Val Phe Thr Val Arg Leu Leu His Ile
725 730 735
Phe Thr Val Asn Lys Gln Leu Gly Pro Lys Ile Val Ile Val Ser
740 745 750
Lys Met Met Lys Asp Val Phe Phe Phe Leu Phe Phe Leu Gly Val
755 760 765
Trp Leu Val Ala Tyr Gly Val Ala Thr Glu Gly Leu Leu Arg Pro
770 775 780
Arg Asp Ser Asp Phe Pro Ser Ile Leu Arg Arg Val Phe Tyr Arg
785 790 795
Pro Tyr Leu Gln Ile Phe Gly Gln Ile Pro Gln Glu Asp Met Asp
800 805 810
Val Ala Leu Met Glu His Ser Asn Cys Ser Ser Glu Pro Gly Phe
815 820 825
Trp Ala His Pro Pro Gly Ala Gln Ala Gly Thr Cys Val Ser Gln
830 835 840
Tyr Ala Asn Trp Leu Val Val Leu Leu Leu Val Ile Phe Leu Leu
845 850 855
Val Ala Asn Ile Leu Leu Val Asn Leu Leu Ile Ala Met Phe Ser
860 865 870
Tyr Thr Phe Gly Lys Val Gln Gly Asn Ser Asp Leu Tyr Trp Lys
875 880 885
Ala Gln Arg Tyr Arg Leu Ile Arg Glu Phe His Ser Arg Pro Ala
890 895 900
Leu Ala Pro Pro Phe Ile Val Ile Ser His Leu Arg Leu Leu Leu
905 910 915
Arg Gln Leu Cys Arg Arg Pro Arg Ser Pro Gln Pro Ser Ser Pro
920 925 930
Ala Leu Glu His Phe Arg Val Tyr Leu Ser Lys Glu Ala Glu Arg
935 940 945
Lys Leu Leu Thr Trp Glu Ser Val His Lys Glu Asn Phe Leu Leu
950 955 960
Ala Arg Ala Arg Asp Lys Arg Glu Ser Asp Ser Glu Arg Leu Lys
965 970 975
Arg Thr Ser Gln Lys Val Asp Leu Ala Leu Lys Gln Leu Gly His
980 985 990
Ile Arg Glu Tyr Glu Gln Arg Leu Lys Val Leu Glu Arg Glu Val
995 1000 1005
Gln Gln Cys Ser Arg Val Leu Gly Trp Val Ala Glu Ala Leu Ser
1010 1015 1020
Arg Ser Ala Leu Leu Pro Pro Gly Gly Pro Pro Pro Pro Asp Leu
1025 1030 1035
Pro Gly Ser Lys Asp
1040
<210> 45
<211> 2508
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2101688CB1
<400> 45
cgtgctcctc ccggggtgct tggcacagcc tcggattcct ccctctcgct gctcgagtca 60
gtttccctat cggcggcagc gggcaaggcg gcggcggcgg cggcggcagc cgcggtggcg 120
gcgtggggaa catctcggca gccaccgcgc ttctcccgct ggagcgggcg tccagcttgg 180
41/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
ctgccctcgg tccttccctg ccaogtttcg ggtogccctg caccccccac ccaggctcgc 240
ttctcttcga agcgggaagg gcgccttgca ggatcctgcc gcccctccaa ccggatcctg 300
ggtctagagc tccccagagc gaggcgctcg ccaggactcc tgccccgcca accctgaccg 360
ccggggggtg cccccgggac gtagcgccgc ggagaggaag cggcaaaggg gaccatgcgg 420
cgcctgactc gtcggctggt tctgccagtc ttcggggtgc tctggatcac ggtgctgctg 480
ttcttctggg taaccaagag gaagttggag gtgccgacgg gacctgaagt gcagacccct 540
aagccttcgg acgctgactg ggacgacctg tgggaccagt ttgatgagcg gcggtatctg 600
aatgccaaaa agtggcgcgt tggtgacgac ccctataagc tgtatgcttt caaccagcgg 660
gagagtgagc ggatctccag caatcgggcc atcccggaca ctcgccatct gagatgcaca 720
ctgctggtgt attgcacgga ccttccaccc actagcatca tcatcacctt ccacaacgag 780
gcccgctcca cgctgctcag gaccatccgc agtgtattaa accgcacccc tacgcatctg 840
atocgggaaa tcatattagt ggatgacttc agcaatgacc ctgatgactg taaacagctc 900
atcaagttgc ccaaggtgaa atgcttgcgc aataatgaac ggcaaggtct ggtccggtcc 960
cggattcggg gcgctgacat cgcccagggc accactctga ctttcctcga cagccaotgt 1020
gaggtgaaca gggactggct ccagcctctg ttgcacaggg tcaaagagga ctacacgcgg 1080
gtggtgtgcc ctgtgatcga tatcattaac ctggacacct tcacctacat cgagtctgcc 1140
tcggagctca gaggggggtt tgactggagc ctccacttcc agtgggagca gctctcccca 1200
gagcagaagg ctcggcgcct ggacccoacg gagcccatca ggactcctat catagctgga 1260
gggctcttcg tgatcgacaa agcttggttt gattacctgg ggaaatatga tatggacatg 1320
gacatctggg gtggggagaa ctttgaaatc tccttccgag tgtggatgtg cgggggcagc 1380
ctagagatcg tcccctgcag ccgagtgggg cacgtcttcc ggaagaagca cccctacgtt 1440
ttccctgatg gaaatgccaa cacgtatata aagaacacca agcggacagc tgaagtgtgg 1500
atggatgaat acaagcaata ctattacgct gcccggccat tcgccctgga gaggcccttc 1560
gggaatgttg agagcagatt ggacctgagg aagaatctgc gctgccagag cttcaagtgg 1620
tacctggaga atatctaccc tgaactcagc atcccoaagg agtcctccat ccagaagggc 1680
aatatccgac agagacagaa gtgcctggaa tctcaaaggc agaacaacoa agaaacccca 1740
aacctaaagt tgagcccctg tgccaaggtc aaaggcgaag atgcaaagtc ccaggtatgg 1800
gccttcacat acacccagaa gatcctccag gaggagctgt gcctgtcagt catcaccttg 1860
ttccctggog CCCCagtggt tCttgtCCtt tgcaagaatg gagatgaccg acagcaatgg 1920
accaaaactg gttcccacat cgagcacata gcatcccacc tctgcctcga tacagatatg 1980
ttoggtgatg gcaccgagaa cggcaaggaa atcgtcgtca acccatgtga gtcctcactc 2040
atgagccagc actgggacat ggtgagctct tgaggacccc tgccagaagc agcaagggcc 2100
atggggtggt gcttccctgg accagaacag actggaaact gggcagcaag cagcctgcaa 2160
ccacctcaga catcctggac tgggaggtgg aggcagagcc ccccaggaca ggagcaactg 2220
tctcagggag gacagaggaa aacatcacaa gccaatgggg ctcaaagaca aatcccacat 2280
gttctcaagg ccgttaagtt ccagtcctgg ccagtcattc cctgattggt atctggagac 2340
agaaacctaa tgggaagtgt ttattgttcc ttttcctaca aaggaagcag tctctggagg 2400
ccagaaagaa aagccttctt tttcactagg ccaggactac attgagagat gaagaatgga 2460
ggttgtttcc aaaagaaata aagagaaact tagaaaaaaa aaaaaaaa 2508
<210> 46
<211> 4034
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5452330CB1
<400> 46
aaccagcacc atgcgcccgg tagccctgct gctcctgccc tcgctgctgg cgctcctggc 60
tcacggactc tctttagagg ccccaaccgt ggggaaagga caagccccag gcatcgagga 120
gacagatggc gagctgacag cagcccccac acctgagcag ccagaacgag gcgtccactt 180
tgtcacaaca gCCCCCaCCt tgaagCtgCt caaccaccac ccgctgcttg aggaattcct 240
acaagagggg ctggaaaagg gagatgagga gctgaggcca gcactgccct tccagcctga 300
cccacctgca cccttcaccc caagtcccct tccccgcctg gccaaccagg acagccgccc 360
tgtctttacc agccccactc cagccatggc tgcggtaccc actcagcccc agtccaagga 420
gggaccctgg agtccggagt cagagtcccc tatgcttcga atcacagctc ccctacctcc 480
agggcccagc atggcagtgc ccaccctagg cccaggggag atagccagca ctacaccccc 540
cagcagagcc tggacaccaa cccaagaggg tcctggagac atgggaaggc cgtgggttgc 600
agaggttgtg tcccagggcg cagggatcgg gatccagggg accatcacct cctccacagc 660
ttcaggagat gatgaggaga ccaccactac cacoaccatc atcaccacca ccatoaccac 720
agtccagaca ccaggccctt gtagctggaa tttctcaggc ccagagggct ctctggactc 780
42/71
CA 02409778 2002-11-25
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ccctacagac ctcagctccc ccactgatgt tggcctggac tgcttcttct acatctctgt 840
ctaccctggc tatggcgtgg aaatcaaggt ccagaatatc agcctccggg aaggggagac 900
agtgactgtg gaaggcctgg gggggcctga cccactgccc ctggccaacc agtctttcct 960
gctgcggggc caagtcatcc gcagccccac ccaccaagcg gccctgaggt tccagagcct 1020
cccgccaccg gctggccctg gcaccttcca tttccattac caagcctatc tcctgagctg 1080
ccactttccc cgtcgtccag cttatggaga tgtgactgtc accagcctcc acccaggggg 1140
tagtgcccgc ttccattgtg ccactggcta ccagctgaag ggcgccaggc atctcacctg 1200
tctcaatgcc acccagccct tctgggattc aaaggagccc gtctgcatcg ctgcttgcgg 1260
cggagtgatc cgcaatgcca ccaccggccg catcgtctct ccaggcttcc cgggcaacta 1320
cagcaacaac ctcacctgtc actggctgct tgaggctcct gagggccagc ggctacacct 1380
gcactttgag aaggtttccc tggcagagga tgatgacagg ctcatcattc gcaatgggga 1440
caacgtggag gccccaccag tgtatgattc ctatgaggtg gaatacctgc ccattgaggg 1500
cctgctcagc tctggcaaac acttctttgt tgagctcagt actgacagca gcggggcagc 1560
tgcaggcatg gccctgcgct atgaggcctt ccagcagggc cattgctatg agccctttgt 1620
caaatacggt aacttcagca gcagcacacc cacctaccct gtgggtacca ctgtggagtt 1680
cagctgcgac cctggctaca ccctggagca gggctccatc atcatcgagt gtgttgaccc 1740
ccacgacccc cagtggaatg agacagagcc agcctgccga gccgtgtgca gcggggagat 1800
cacagactcg gctggcgtgg tactctctcc caactggcca gagccctacg gtcgtgggca 1860
ggattgtatc tggggtgtgc atgtggaaga ggacaagcgc atcatgctgg acatccgagt 1920
gctgcgcata ggccctggtg atgtgcttac cttctatgat ggggatgacc tgacggcccg 1980
ggttctgggc cagtactcag ggccccgtag ccacttcaag Ctctttacct ccatggctga 2040
tgtcaccatt cagttccagt cggaccccgg gacctcagtg ctgggctacc agcagggctt 2100
cgtcatccac ttctttgagg tgccccgcaa tgacacatgt ccggagctgc ctgagatccc 2160
caatggctgg aagagcccat cgcagcctga gctagtgcac ggcaccgtgg tcacttacca 2220
gtgctaccct ggctaccagg tagtgggatc cagtgtcctc atgtgccagt gggacctaac 2280
ttggagtgag gacctgccct catgccagag ggtgacttcc tgccacgatc ctggagatgt 2340
ggagcacagc cgacgcctca tatccagccc caagtttccc gtgggggcca ccgtgcaata 2400
tatctgtgac cagggttttg tgctgatggg CagCtCCatC CtCaCCtgCC atgatCgCCa 2460
ggctggcagc cccaagtgga gtgaccgggc ccctaaatgt ctcctggaac agctcaagcc 2520
atgccatggt ctcagtgccc ctgagaatgg tgcccgaagt cctgagaagc agctacaccc 2580
agcaggggcc accatccact tctcgtgtgc ccctggctat gtgctgaagg gccaggccag 2640
catcaagtgt gtgcctgggc acccctcgca ttggagtgac cccccaccca tctgtagggc 2700
tgcctctctg gatgggttct acaacagtcg cagcctggat_gttgccaagg cacctgctgc 2760
ctccagcacc ctggatgctg cccacattgc agctgccatc ttcttgccac tggtggcgat 2820
ggtgttgttg gtaggaggtg tatacttcta cttctccagg ctccagggaa aaagctccct 2880
gcagctgccc cgcccccgcc cccgccccta caaccgcatt accatagagt cagcgtttga 2940
caatccaact tacgagactg gatctctttc ctttgcagga gacgagagaa tatgaagtct 3000
ccatctaggt gggggcagtc tagggaagtc aactcagact tgcaccacag tccagcagca 3060
aggctccttg cttcctgctg tccctccacc tcctgtatat accacctagg aggagatgcc 3120
accaagccct caagaagttg tgcccttccc cgcctgcgat gcccaccatg gcctattttc 3180
ttggtgtcat tgcccacttg gggcccttca ttgggcccat gtcagggggc atctacctgt 3240
gggaagaaca tagctggagc acaagcatca acagccagca tcctgagcct cctcatgccc 3300
tggaccagcc tggaacacac tagcagagca ggagtacctt tctccacatg accaccatcc 3360
cgccctggca tggcaacctg cagcaggatt aacttgacca tggtgggaac tgcaccaggg 3420
tactcctcac agcgccatca ccaatggcca aaactcctct caacggtgac ctctgggtag 3480
tcctggcatg ccaacatcag cctcttggga ggtctctagt tctctaaagt tctggacagt 3540
tctgcctcct gccctgtccc agtggaggca gtaattctag gagatcctaa ggggttcagg 3600
gggaccctac ccccacctca ggttgggctt ccctgggcac tcatgctcca caccaaagca 3660
ggacacgcca ttttccactg accaccctat accctgagga aagggagact ttcctccgat 3720
gtttatttag ctgttgcaaa catcttcacc ctaatagtcc ctcctccaat tccagccact 3780
tgtcaggctc tcctcttgac cactgtgtta tgggataagg ggagggggtg ggcatattct 3840
ggagaggagc agaggtccaa ggacccagga atttggcatg gaacaggtgg taggagagcc 3900
ccagggagac gcccaggagc tggctgaaag ccactttgta catgtaatgt attatatggg 3960
gtctgggctc cagccagaga acaatctttt atttctgttg tttccttatt aaaatggtgt 4020
ttttggaaaa aaaa 4034
<210> 47
<211> 845
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
43/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<223> Incyte ID No: 4362432CB1
<400> 47
ggtagaaaag ccagcctatg ttacaggaca aaggccgtcg ctttgtaaaa gcttgaagtg 60
cagtttgctg ctgagtacag aagacctttg caaacagaga ggggagattt tctctgtaag 120
gttgcaaaca agagcaggtc ctggaagata agattccccg ccatgttatc ctccgtggtg 180
ttttggggac taattgccct cattggcact tccaggggct catacccctt cagtcactca 240
atgaagcctc acctacatcc acgcctgtac cacggctgct atggggacat catgaccatg 300
aagacctctg gggccacttg tgatgcaaac agtgtgatga actgcgggat ccgtggttct 360
gaaatgtttg ctgagatgga tttgagggcc ataaaacctt accagactct gatcaaagaa 420
gtcgggcaga gacattgcgt ggaccctgct gtcatcgcag ccatcatctc cagggaaagc 480
catggcggat ctgtcctgca agacggctgg gaccacaggg gacttaaatt tggcttgatg 540
cagcttgata aacaaacgta ccaccctgtc ggtgcctggg atagcaaaga gcacctttca 600
caggctactg ggattctaac agagagaatt aaggcaatcc agaaaaaatt ccccacgtgg 660
agtgttgctc agcacctcaa aggtggtctc tcagctttta agtcaggaat tgaagcgatt 720
gCCaCCCCat cggacataga caatgacttc gtcaatgata tcattgctcg agctaagttc 780
tataaaagac aaagcttcta ggcaaagctc tgtgggtggg ccaggttggc agagtgctca 840
gatgt 845
<210> 48
<211> 2300
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5308104CB1
<400> 48
tttacggcgc agtgtgctgg acaagcggtt caggccgggc ggcgttgttg gcgggggccc 60
cggtggaggc ccggcctggg cggcgcccgc catgaatggg ctgtcgctga gtgagctctg 120
ctgcctcttc tgctgcccgc cctgccccgg ccgcatcgct gccaagctcg ccttcctgcc 180
gccggaggcc acctactccc tggtgcctga gcccgagccg gggcctggtg gggccggggc 240
cgcccccttg gggaccctga gagcctcctc gggcgcaccc gggcgctgga agctgcacct 300
gacggagcgt gccgacttcc agtacagcca gcgcgagctg gacaccatcg aggtcttccc 360
caccaagagc gcccgcggca accgcgtctc ctgcatgtat gttcgctgcg tgcctggtgc 420
caggtacacg gtcctcttct cgcacggcaa tgccgtggac ctgggccaga tgagcagctt 480
ctacattggc ctgggctccc gcctccactg caacatcttc tcctacgact actccggcta 540
cggtgccagc tcgggcaggc cttccgagag gaacctctat gccgacatcg acgccgcctg 600
gcaggccctg cgcaccaggt acggcatcag cccggacagc atcatcctgt acgggcagag 660
catcggcacg gtgcccaccg tggacctggc ctcgcgctac gagtgtgccg cggtaattct 720
ccattcccct ctgatgtctg gtttgcgtgt ggcttttccg gataccagga aaacatactg 780
ctttgatgct ttccccagca ttgacaagat atctaaagtc acctctcctg tgttggtcat 840
tcatggtaca gaggatgagg tcatcgattt ctcccatggc ctagcgatgt acgagcgctg 900
tccccgagcc gtggagcccc tttgggttga aggggctggg cataatgaca tagagcttta 960
tgcacaatac ctagaaagac taaaacagtt catatctcac gaacttccta attcctgaag 1020
acaacaactt gatcttacct catttactgt gaacagaaga gtcctctgtt ttgcacatgc 1080
tttaactggg tagctgtaaa ggcttgataa ccatgaagaa gtgcccaacc tttagggtgt 1140
tctaatcaaa gagctgatga aatctcagtc ttttgtatct agaggtggtt ctgctaattc 1200
acacaacacg ttaaactgaa Cagtcgtgat tcccagcttc attaccttgc aggaatggga 1260
atgagagctg aatgtaggga caattttcta gtgctgtata aagtagcctc gcatctgttt 1320
ctcaacctta tccatcattt ctgacattca tgcaggactt gccctgttgc caccaatgtt 1380
ctcggtattt cacatgcagc tctctttctg ccactggata catgggttca atccatttgt 1440
gaagctgtga tagtgtaact ggaaagctag tgtggtgaaa attcctttat tattttttgt 1500
taacatgctg atctttcccg gacaaatgaa ctgaagggta atttactgga actctcgtgt 1560
acagcttcat caactgtaac catataaata taactggaat attcttaaac aaaaagaaac 1620
taggggtttt tttaagtgta aatttattac tagccaacag agttttacta ttttgattgt 1680
ctggttggtt taacaaagag,cctagctgac tttccttctg taaagtcctc cttgtaggct 1740
tttttaaagt actgtacata tttgcaatca cattgtgcat agattcttaa tggtagatat 1800
gatttctttt gtcaggctac aacaatgaac tgcagattcc ttgtttgtaa tgtaaatgat 1860
tgaatacatt ttgttaatat gtttttattc ctatgttttg ctattaaaaa ttttataaca 1920
tttccaagac aaaaattcca agtttatgct ttgaagaatt tatgtaatta aaatttcact 1980
aaactaatct ttttagttta ggaattattt gggttttgac actggaagtt gcgccaaata 2040
44/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
agcatcagaa ataggagatg cttaacattg ctatactact tgtgttggtt aggggtttgg 2100
atttgggggt tctttggttt taattttttt ttccacattt aaaagcctta aatgtactgt 2160
aagcctcaga tcgttgtaca actggactgc ggttgattgc cagtttgtgt actgttgctt 2220
ggatgcggca cagtggttgg taatggaata aaggatgcat ggatcagaaa aaaaaaaaaa 2280
aaaaaaaaaa aaaaaaaaaa 2300
<210> 49
<211> 1587
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3092736CB1
<400> 49
ggaggacata attcacctgt cctagcttct tatcatctta catttccctg tagccactgg 60
gacatatgtg gtgttccttc ctagctcctg tctcctcctc atgcctttgc tgggtatggg 120
catgttgggg ggaaggtcat tgctgtcaga ggggcactga ctttctaatg gtgttaccca 180
aggtgaatgt tggagacaca gtcgcgatgc tgcccaagtc ccggcgagcc ctaactatcc 240
aggagatcgc tgcgctggcc aggtcctccc tgcatggtat ttcccaggtg gtgaaggacc 300
acgtgaccaa gcctaccgcc atggcccagg gccgagtggc tcacctcatt gagtggaagg 360
gctggagcaa gccgagtgac tcacctgctg ccctggaatc agccttttcc tcctattcag 420
acctcagcga gggcgaacaa gaggctcgct ttgcagcagg agtggctgag cagtttgcca 480
tcgcggaagc caagctccga gcatggtctt cggtggatgg cgaggactcc actgatgact 540
cctatgatga ggactttgct gggggaatgg acacagacat ggctgggcag ctgcccctgg 600
ggccgcacct ccaggacctg ttcaccggcc accggttctc ccggcctgtg cgccagggct 660
ccgtggagcc tgagagcgac tgctcacaga ccgtgtcccc agacaccctg tgctctagtc 720
tgtgcagcct ggaggatggg ttgttgggct ccccggcccg gctggcctcc cagctgctgg 780
gcgatgagct gcttctcgcc aaactgcccc ccagccggga aagtgccttc cgcagcctgg 840
gcccactgga ggcccaggac tcactctaca actcgcccct cacagagtcc tgcctttccc 900
ccgcggagga ggagccagcc ccctgcaagg actgccagcc actctgccca ccactaacgg 960
gcagctggga acggcagcgg caagcctctg acctggcctc ttctggggtg gtgtccttag 1020
atgaggatga ggcagagcca gaggaacagt gacccacatc atgcctggca gtggcatgca 1080
tcccccggct gctgccaggg gcagagcctc tgtgcccaag tgtgggctca aggctcccag 1140
cagagctcca cagcctagag ggctcctggg agcgctcgct tctccgttgt gtgttttgca 1200
tgaaagtgtt tggagaggag gcaggggctg ggctgggggc gcatgtcctg cccccactcc 1260
cggggcttgc cgggggttgc ccggggcctc tggggcatgg ctacagctgt ggcagacagt 1320
gatgttcatg ttcttaaaat gccacacaca catttcctcc tcggataatg tgaaccacta 1380
agggggttgt gactgggctg tgtgatggtg gggtgggagg gggcccagca accccccacc 1440
ctccccatgc ctctctcttc tctgcttttc ttctcacttc cgagtccatg tgcagtgctt 1500
gatagaatca accccacctg gaggggctgg ctcctgccct cccggagcct atgggttgag 1560
ccgtccctca agggccctgc cagctgg 1587
<210> 50
<211> 669
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3580257CB1
<400> 50
cttgcctgaa caacaaacca actcaccact cctgacacca tgagtcacta cggcagctac 60
tacggaggcc tgggctacag ctgtggaggc ttcggtggcc tgggctatgg ctatggctgt 120
ggatgtggca gcttctgcag acggggttct ggctgtggct atggaggcta cggatatggc 180
tctggctttg gaagctacgg atatggctct ggctttggag gctacggata tggctctggc 240
tttggaggct atggatatgg ctgctgccgc ccatcgtaca atggaggata cggattctct 300
ggcttttatt aaatgaattg ctgaaattgg aagcagagga gaaacctcca aatgtgtttg 360
gtcctgtccc gtgctttcat tccaaaaatc cattctattg ccttcagcat caatggagag 420
atatttagct atgttaaatc tttaaaatag atttaagctg cttctgtgaa tatttgttgt 480
ctttttactt tcggaatccg tcacctgaaa tcatattcat gtataactac tctaggttgt 540
45/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tcttctaaat ttgctttatt tttcttacca ccattacctt gatgttatct atcaagatcc 600
tagcaaaaac ttgtattttg cttttatcta ataaatgaag taaagtataa atattttaaa 660
aaaaaaaaa 669
<210> 51
<211> 1463
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3634758CB1
<400> 51
atcgcatctc agctggttgg ctttggttag agctcccgtc agactttcgt tcggccctag 60
gatttggtag ccccgaagtg tgggctctct ccagtaccag actcatttca gtaccagcct 120
ttgggaagtc gtgtgaatac ctcggtctct tagccacagg gatagaatgg cggcctgacg 180
gagccgcggc gccggcgaag tcgctgaggc gcgagctgga acccccagac cagctcaaac 240
gggagccaaa actcgaagct tggaagaatt agcaggaaat ggcggatgag gcgttgtttt 300
tgcttctcca taacgagatg gtgtctggag tgtacaagtc cgcggagcag ggggaggtgg 360
aaaacggacg atgtattact aagctggaaa acatggggtt tcgagtggga caaggattga 420
tagaaaggtt tacaaaagat actgcaaggt tcaaggatga gttagatatc atgaagttca 480
tttgtaaaga tttttggact acggtattca agaaacaaat cgacaatcta aggacaaatc 540
atcagggcat ctatgtactt caggacaaca aatttcgcct gcttactcag atgtctgcag 600
gaaaacagta tttagaacat gcatctaagt atttagcatt tacgtgtggc ttaatcagag 660
gtggcttatc aaacttggga ataaaaagta ttgtaacagc tgaagtgtct tcaatgcctg 720
Cttgcaaatt tcaggtgatg atacagaagc tgtagaacat actgaaatgc aaggcttcaa 780
cagtgtaaag agataaatta ttcatgtaaa agtatttcaa gtagtgatga tttaattaca 840
ttgttcgatg tttgtacagg agtaagcatg tatttttatc aatttaacac agatcaaagg 900
agatgaaggg acattctgcc atgacataca cttaaccaaa actattcaaa atgaaaaccg 960
gatttcaaat aaccagacac caagatgcag ggcccttatt ttaaaccttt ttatttggtt 1020
agagtgatat gtatttagcc atagatggag aaacaaagct cagggtttgt tgaattagca 1080
tgagagaaaa ttatgtacca acagaattat ttgtgagaag aatgaacaaa ttttgataaa 1140
gtatgaattt gttttatttt aaaaagcaaa catactaaat tttttttatt ttattgctta 1200
taatttatta agaatgttta cacctgtata aggatttcat atatacattg tatgtgtgta 1260
tatataaata catatatgac tgcctaaatt gtttataaat ttaatttttc tttaataggt 1320
ttcattcctt cagagctcca ttaatgtaat caaaatgaaa tacagattag tttaaatgtg 1380
aattcagtga ctctagggcc aaagaatatt aggtatgttt ggaaagaatt tttgtattta 1440
ttcctgttac agttttgact ttc 1463
<210> 52
<211> 1686
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4027923CB1
<400> 52
gtaacccatc gccacttggg atccaggtag taggtgcacg gccaggtgtg gatgcagaca 60
tccccgctca tctgttcctg ggtctgtgtg cgccaaggtc caggcctctt ctgcctctga 120
gggacgccag acgagtccca gagccgaccg cggcgcctcg gccgctcggc gcctgcgcag 180
tgaagaccgc ggcgagccgc gcgcatgcgt gcaggcccgg agccccaggc gctggtgggg 240
cagaaacgcg gcgccctgcg tcttctggtt ccgaggctgg tcctcaccgt ttccgctccg 300
gcggaagtga ggaggagggt CCttCgaCCC gtgctgagct ggatggaccg cgagacgcgc 360
gCCCtCgCCg acagccactt ccgaggcctg ggggtcgatg tccccggcgt cggccaggct 420
ccgggccggg tagccttcgt ctcggagccg ggcgccttct cctacgccga ctttgtgcgg 480
ggcttcttgc tgcccaacct gccctgcgtg ttttccagcg ccttcacgca gggctggggc 540
agccggcggc gctgggtgac gcccgcgggg aggcccgact tcgaccacct gctacggacc 600
tacggagacg tggttgtacc agttgcaaac tgtggggtcc aggaatacaa ctcgaacccc 660
aaagagcaca tgactctcag agactacatc acctactgga aagagtacat acaggcgggc 720
tactcctctc ccaggggctg tctctacctc aaagactggc acttgtgcag ggactttccg 780
46/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
gtggaggacg ttttcaccct gcctgtgtac ttctcgtccg actggctgaa tgagttctgg 840
gatgcactgg atgtggatga ctaccgcttt gtctacgcgg ggcctgcggg cagctggtcc 900
ccgttccatg ctgacatctt ccgctccttc agctggtctg tcaatgtctg tgggaggaag 960
aagtggctcc tcttcccccc agggcaggaa gaggccctgc gggaccgcca cggcaacctg 1020
ccctacgacg tgacctcccc agcactctgc gacacacacc tgcacccacg gaaccagctt 1080
gctggcccac ccttggagat cacgcaggaa gcgggcgaga tggtgtttgt gcccagtggc 1140
tggcaccacc aggtgcacaa cctggatgac accatctcca tcaaccacaa ctgggtcaat 1200
ggcttcaacc tggccaacat gtggcgcttc ttgcagcagg agctatgcgc cgtgcaggag 1260
gaggtcagcg agtggaggga ctccatgccc gactggcacc accactgcca ggtcatcatg 1320
aggtcctgct cgggcatcaa ctttgaagag ttttaccact tcctcaaggt catcgctgag 1380
aagaggctcc tggtcctgag ggaggcagcc gctgaggacg gtgctgggtt gggtttcgaa 1440
caggcagcct ttgatgttgg gcgcatcaca gaggtgctgg cctccttggt tgcgcacccc 1500
gacttccaga gagtggacac cagcgcgttc tcaccacagc ccaaagagct gctgcagcag 1560
ctgagagagg ctgttgatgc tgctgcggcc ccatagcacc tgtcgtgagg atagaaggac 1620
gggtggacga gaggcagcct cctgctccgg ggcccttcca gaaataaaga ccgccctccc 1680
tgtgaa 1686
<210> 53
<211> 2497
<212> DNA
<213> Homo Sapiens
<220>
<2'21> misc_feature
<223> Incyte ID No: 4348533CB1
<400> 53
gggcggcggg agctgctttg cctccaccga tctccctgtg cggccctcat gtgctgtgct 60
cgctgacacc cgaagtccgc ggctttccgc acacggtggg gtcgtcagac ccgctgccct 120
tggcggtcga agtcgtcgtg cgggcccgcg gcggccgccc atggagaagg ccaggagagg 180
cggggatggc gtcccccggg ggcccgtact gcacatcgtg gtggtcggat ttcaccacaa 240
gaagggctgc caggttgaat tctcttaccc gcccctgatt ccaggagatg gacatgacag 300
ccacacttta cctgaagaat ggaagtattt gcccttcctt gccttaccag atggcgcaca 360
caactaccag gaagatactg tgttttttca cttgccaccc agaaatggaa atggagccac 420
agtatttggt atctcttgct atcgacaaat tgaagccaag gcactgaaag taaggcaagc 480
agatatcacc agagagactg ttcagaaaag tgtctgtgtt ctaagcaagc tgcctctgta 540
tggtttactt caagcaaaac ttcaactcat tacacatgca tattttgaag agaaggattt 600
ttcccaaatt tctattctaa aggagcttta tgaacatatg aatagttcct tgggaggtgc 660
ttcattagaa ggatcccaag tatatcttgg tctgtcacct cgagatcttg tccttcattt 720
tcgacacaag gtcttaatcc tatttaagct aattcttctt gaaaaaaagg ttctttttta 780
tatttctcca gtgaataaat tggtgggtgc actgatgact gtgttatccc tttttccagg 840
catgattgaa catggtctca gtgactgttc tcagtataga ccccggaaaa gtatgtctga 900
agatggtggg cttcaggaaa gtaacccatg tgcagatgat tttgtttctg catccactgc 960
tgatgtttca cataccaact tgggaactat caggaaagtc atggcaggaa accatggaga 1020
agatgctgcc atgaagactg aggagccttt gttccaagtg gaagacagca gcaaagggca 1080
ggaacccaat gataccaatc aatatttgaa acctccatct cgcccatctc cagattcttc 1140
agaaagtgac tgggaaactt tggatcctag tgtcttagag gaccccaact tgaaagaaag 1200
ggaacagctg ggatcagacc agacaaattt gtttccaaag gactctgtcc cctcagagag 1260
tcttccaatt actgtacaac ctcaagctaa tacgggacag gtagtcctga taccagggct 1320
catttcgggt ttggaagagg atcagtatgg catgcccctg gccatcttca caaagggata 1380
tctgtgtttg ccttacatgg cattgcagca gcatcatctt ctctccgatg tcaccgttcg 1440
ggggtttgtt gctggagcta ctaacatcct ttttcgacaa cagaaacacc tcagtgatgc 1500
cattgtggaa gtagaagaag ctctgatcca gatccatgat ccagaactca ggaagctgct 1560
taacccaacc actgcagacc taaggttcgc agactaccta gtgaggcacg tgactgagaa 1620
tcgggatgac gtcttcctag atggcacggg ctgggaggga ggtgacgaat ggatccgggc 1680
ccagtttgcg gtctacattc atgccctgct ggctgccacg ctgcaattag acaatgaaaa 1740
gatattatcg gactatggga caacttttgt tacagcatgg aagaatactc acaactacag 1800
ggtgtggaac agcaacaagc atccagcact tgcagaaata aatccaaacc atccatttca 1860
aggccaatac tcagtatcag acatgaagtt aaggttctca cattctgttc agaatagtga 1920
acgtggcaaa aaaattggaa acgtcatggt cacaactagc cggaatgttg tacaaacagg 1980
aaaagctgtt ggccagtcag ttggaggagc tttttccagt gcaaagacag ctatgtcttc 2040
atggctttcc actttcacca cttccacctc ccaaagtctc actgagccac cagatgagaa 2100
gccttgagca aggcgtcaga ggctgctatt gctttctgag gtttaagtgt cccctgtctg 2160
47/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tctgctgctc ccaggctgtt actagccaca gatccacagc aggggaccat atgtcgaact 2220
gtttacatgg atgttgctct aagtgaatgt ttcgggatgc cgaaatgatg aaatcacagc 2280
catagcaggg atggctttcc aggttggggt ttcaattgac tacttttatt tcagtctgag 2340
cctgattaaa acatacagtg aaccttctaa tgaattttga gttttggcat ttgaagtttc 2400
gtacattggt ttactttaga agagttttta cttatgtaaa ttttgttctg ttttggtgtt 2460
tgaatatcta gatggtcact gtaatttata cttgcta 2497
<210> 54
<211> 1783
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4521857CB1
<400> 54
ccgctgtgct ctcgggaaag ccctgagaaa ctaccagaac ggggtccctg gggccagtgt 60
tgggagcgta cggtgaagtg gcagggctgc tgcgcccgtg cggaactccg ctgtggaaga 120
ttagcccctt tggtgactcg tcggttctgg ccgctttggg accctctact gtgcagtctg 180
aactttcagc aggtggagga gggcagagag agagctgaga tcgttggtcc atgccagtgc 240
gagagccagt acggccggaa gccatgatgg gactttcagt tcctttcttg gtggaggcca 300
gcacagggga gagggggtgc ccacagatgc tagaagctta aaggctgctg ggggctagcg 360
gcctcaggac ataaggtttc cagacaggac ggggcaggtg gtgatgcagc tgtagctcgt 420
gagtctgagc gggaaacagc ccccgcgcag tcggtgtcgc tgtgaacgga aaagggcctt 480
ccttacacag gaaacggggt ctctggggga tccagtgacg tgcccaacca cagagaccag 540
acccctcctc ctgtagagag tggtgctgcc ctctcgggat gtacctgcag gtggagaccc 600
gcaccagctc ccgcctccat ctgaagaggg ctccaggcat ccggtcctgg tccctgctgg 660
ttggaatctt gtcgattggc ctggctgctg cctactacag cggagatagc ctgggctgga 720
agctcttcta cgtcacaggc tgcctgtttg tggctgtgca gaacttggag gactgggagg 780
aagccatctt cgacaagagc acagggaagg ttgttttgaa gacgttcagc ctctacaaga 840
agctgctgac tcttttcaga gctggccacg accaggtggt ggtcctgctc catgttgtcc 900
ccgacacagc gtcctctccc tggtggacat ccccagcggt caggtgcttc cccaagggca 960
gtgaggggtg aacatccagg gcctacctgg ctgtgcacgc tgcagccaca ctgtggaagc 1020
tgcccctccc cgaggacccg cctcccttgc tgatgccagg atctcggcgc atagaccact 1080
ctgccccagc ggtcgtcaca gaaaggtctc tctgttcctc acactcagct tcagcataag 1140
ctgtgaggcc agaaaaaagg tcagctcttc tagtatcgtg cagtgcttaa aaaccgggag 1200
ctccagccgg gcgcagtggt tcatgccagt aatcccagca ctttcggagg ccgaggtggg 1260
aggattgctt gaggccagga gttcaagacc agcctgggca acacagcaag atcctgtctt 1320
tgtaaaaaaa ctaaccaaac aggaaaaact gggagatttt ctgcagaaat tgagttccag 1380
cctctctcga acctgggaag acctggcagg agggggctgg gctctcggct acagacttct 1440
ccccaccccg taggagctga acgccaacca tcctgacccg ccagtgctct tggtctcctg 1500
agtgtaccca ggtcctccca ggtgcggtgt gcaccgagcg cgcctggcct gatgccctgg 1560
cctgtgagct ggggactcct gggccctgtg agcccctagg cggcaggccc aggaatggct 1620
gggtaggaca gggaacacct ttgccccacg tctggctgtg acctcggtga aagccgacag 1680
gagagagatg ggaccctcct cctcagtagt ggctgccagt ccctcgttgc aggacagggt 1740
catcataacc ataaataacc cttcacgtgt caaaaaaaaa aaa 1783
<210> 55
<211> 1461
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4722253CB1
<400> 55
ccagactcgg tcccgcgggc tttaaggggc ccgggcggcg aaggcgcacg gagccaagtt 60
ccgggcagca gccctgggat gccctgagcc gggccgggcg gaggtctccg ttgcctagca 120
accggggccg cggcctgtgg gcggagcctg caccgtggct gcgacacggg gcggggcctc 180
agcgggagcc ggccgaggag cgggcaccgg ccattggcac aggcacgggc cattggcgca 240
tgcgtagggc gcggccttgg ggcggggccc acccagtggc ggagtcttct gaggggcggg 300
48/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tcgtgggcgg ggcctggagg cggagccggc gccgtcagta gtcggctgag gacggaggcg 360
gggctgggcc tgggatgggg cggggcctgc gctggtgggg cgggcggggg aggagacacg 420
gccaagcgcc cgagtggggg ccgttggttg gtgcgcggct gaagggtgtg gcgcgagcag 480
cgtcgttggt tggccggcgg cgggccggga cgggcatggc cctgctgctg tgcctggtgt 540
gcctgacggc ggcgctggcc cacggctgtc tgcactgcca cagcaacttc tccaagaagt 600
tctccttcta ccgccaccat gtgaacttca agtcctggtg ggtgggcgac atccccgtgt 660
caggggcgct gctcaccgac tggagcgacg acacgatgaa ggagctgcac ctggccatcc 720
ccgccaagat cacccgggag aagctggacc aagtggcgac agcagtgtac cagatgatgg 780
atcagctgta ccaggggaag atgtacttcc ccgggtattt ccccaacgag ctgcgaaaca 840
tcttccggga gcaggtgcac ctcatccaga acgccatcat cgaaagccgc atcgactgtc 900
agcaccgctg tggcatcttc cagtacgaga ccatctcctg caacaactgc acagactcgc 960
acgtcgcctg ctttggctat aactgcgagt cctcggcgca gtggaagtca gctgtccagg 1020
gcctcctgaa ctacataaat aactggcaca aacaggacac gagcatgaga ccacgctcct 1080
ctgccttctc ctggcctggg acacacagag ccaccccggc cttcctggta tcgccagcct 1140
taaggtgtct ggagccccca cacttggcca acctgacctt ggaagatgct gctgagtgtc 1200
tcaagcagca ctgacagcag ctgggcctgc cccagggcaa cgtgggggcg gagactcagc 1260
tggacagccc ctgcctgtca ctctggagct gggctgctgc tgcctcagga ccccctctcc 1320
gaccccggac agagctgagc tggccagggc caggagggcg ggagggaggg aatgggggtg 1380
ggctgtgcgc agcatcagcg cctgggcagg tccgcagagc tgcgggatgt gattaaagtc 1440
cctgatgttt ctcaaaaaaa a 1461
<210> 56
<211> 2116
<212> DNA
<213> fiomo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4878134CB1
<400> 56
cgcccgcgga gtcgctgagg aggcggaaga ctgggtactc ggatccggag cctgagtcgc 60
CgCCCgCgCC ggggcgtggc cccgcaggct CtCCggCCCa tCtCCdCa.Cg ggCaCCttCt 12O
ggctgacccg gatcgtgctc ctgaaggccc tagccttcgt gtacttcgtg gcattcctgg 180
tggctttcca tcagaacaag cagctcatcg gtgacagggg gctgcttccc tgcagagtgt 240
tcctgaagaa cttccagcag tacttccagg acaggacgag ctgggaagtc ttcagctaca 300
tgcccaccat cctctggctg atggactggt cagacatgaa ctccaacctg gacttgctgg 360
ctcttctcgg actgggcatc tcgtctttcg tactgatcac gggctgcgcc aacatgcttc 420
tCatggCtgC CCtgtggggC ctctacatgt ccctggttaa tgtgggccat gtctggtact 480
ctttcggatg ggagtcccag cttctggaga cggggttcct ggggatcttc ctgtgccctc 540
tgtggacgct gtcaaggctg CCCCagCata CCCCCaCatC CCggattgtC CtgtggggCt 600
tccggtggct gatcttcagg atcatgcttg gagcaggcct gatcaagatc cggggggacc 660
ggtgctggcg agacctcacc tgcatggact tccactatga gacccagccg atgcccaatc 720
ctgtggcgta ctacctgcac cactcaccct ggtggttcca tcgcttcgag acgctcagca 780
aCCaCttCat cgagctcctg gtgcccttct tCCtCttCCt CggCCggCgg gcgtgcatca 840
tccacggggt gctgcagatc ctgttccagg ccgtcctcat cgtcagcggg aacctcagct 900
tcctgaactg gctgactatg gtgcccagcc tggcctgctt tgatgacgcc accctgggat 960
tcttgttccc ctctgggcca ggcagcctga aggaccgagt tctgcagatg cagagggaca 1020
tccgaggggc ccggcccgag cccagattcg gctccgtggt gcggcgtgca gccaacgtct 1080
cgctgggcgt cctgctggcc tggctcagcg tgcccgtggt cctcaacttg ctgagctcca 1140
ggcaggtcat gaacacccac ttcaactctc ttcacatcgt caacacttac ggggccttcg 1200
gaagcatcac caaggagcgg gcggaggtga tcctgcaggg cacagccagc tccaacgcca 1260
gCgCCCCCga tgccatgtgg gaggactacg agttcaagtg caagccaggt gaccccagca 1320
gacggccctg CCtCatCtCC CCgtaCCaCt accgcctgga ctggctgatg tggttcgcgg 1380
ccttccagac ctacgagcac aacgactgga tcatccacct ggctggcaag ctcctggcca 1440
gcgacgccga ggccttgtcc ctgctggcac acaacccctt cgcgggcagg cccccgccca 1500
ggtgggtccg aggagagcac tacaggtaca agttcagccg tcctgggggc aggcacgccg 1560
ccgagggcaa gtggtgggtg cggaagagga tcggagccta cttccctccg ctcagcctgg 1620
aggagctgag gccctacttc agggaccgtg ggtggcctct gcccgggccc ctctagacgt 1680
gcaccagaaa taaaggcgaa gacccagccc ctcggcggct cagcaacgtt tgcccttccc 1740
tgcgcccagc ccaagctggg catcgccaag agagacgtgg agaggagagc ggtgggaccc 1800
agcccccagc acgggggtcc agggtggggt ctgttgtcac atactgtggc ggctcccagg 1860
ccctgcccac ctggggcccc acatccaggc caacccttgt gcccaggcgc caggggctct 1920
49/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
gatctcccat ccatcccacc ctcctcccag aggcccagcc tggggctgtg ccgcccacag 1980
gagttgagac aatggccatc ctgacacctt cctccactac agccctgacc atagacccag 2040
ccaggtagct cttggggtct ctagcgtccc agggcctggt ttctgttccc tcttcaatgg 2100
tgtgttccca gccagg 2116
<210> 57
<211> 702
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5050133CB1
<400> 57
ccgaggcccg ggcgcaacca cgggctccca ggcagcctcc gccagccgga ccccgtcgcc 60
ctcctgatgc tgctcgtgga cgctgatcag ccggagccca tgcgcagcgg ggcgcgcgag 120
ctcgcgctct tcctgacccc cgagcctggg gccgaggcga aggaggtgga ggagaccatc 180
gagggcatgc tcctcaggct ggaagagttt tgcagcctgg ctgacctgat caggagtgat 240
acttcacaga tcctggagga aaacatccca gtccttaagg ccaaactgac agaaatgcgt 300
ggcatctatg ccaaagtgga ccggctagag gccttcgtca agatggttgg acaccacgtc 360
gccttcctgg aagcagacgt gcttcaggct gagcgggacc atggggcctt ccctcaggcc 420
ctgcggaggt ggctgggatc cgcagggctc ccctccttca ggaacaagtc acctgcaccg 480
gtgcccgtga cgtacgagct gcccacactg tataggacgg aggactattt tcctgtggac 540
gccggggaag cacagcacca cccccgcacc tgccctcggc ctttgtgagc tttgtggtct 600
tcccatcagg aacgctggaa agtgacattg tgtacacgct gcagcttggg ggttttttct 660
ttgtattgct gtttatttta tattttaaaa atatttaaaa as 702
<210> 58
<211> 2613
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5630124CB1
<400> 58
tctcggaaca cattttactg ggctcgagtt tagccgccac cggagggtgg gggaccttga 60
gtcatgctcc tataaccacc gctagagttc ctcgtctttg agtgcagagg tttagactgt 120
gtctttgtgt gcagaaagtc ctgcagttct cacagcgacc tgccagaaaa agtcgttccc 180
aaatgtttgt aaatcctccg ttgggcaacc cgccttcacg ttctgcggtg atcttgtcga 240
gcgactaagc gtgcagtatt agcagagaag ggggtggcag agtgctggcg ctgaaggtca 300
tgttgcatgg gtaactgtcg tgttgtaggg gcggggaaga gggaggagac actgaccacc 360
ccagaggccg ccccattagc tcgcttgctt tgggcggcgt cgctcccacg gcgcccaggg 420
taCCCCCCJCC gctgtctgcc tgtcttcctc cattaccgcg caggcttggt caccgcatta 480
aggcattccc gctctccgcg gaactgctct gccgtctcgg cggtgaaagt gtgagagggt 540
ccgtagttgg gtcaactttg actcctctcg cctgcccgga tccttaaggg cctcctcgtc 600
ctcccggtct ccggtcgctg ccgggtctgt gcgccggtcc gCgCCCgCCC tcgctctgcc 660
atgggcgctt ccagctcctc cgcgctggcc cgcctcggcc tCCCagCCCg gCCCtggCCC 720
aggtggctcg gggtcgccgc gctaggactg gccgccgtgg ccctggggac tgtcgcctgg 780
cgccgcgcat ggcccaggcg gcgccggcgg ctgcagcagg tgggcaccgt ggcgaagctc 840
tggatctacc cggtgaaatc ctgcaaaggg gtgccggtga gcgaggctga gtgcacggcc 900
atggggctgc gcagcggcaa cctgcgggac aggttttggc tggtgattaa ggaagatgga 960
cacatggtca ctgcccgaca ggagcctcgc ctcgtgctca tctccatcat ttatgagaat 1020
aactgcctga tcttcagggc tccagacatg gaccagctgg ttttgcctag caagcagcct 1080
tcctcaaaca aactccacaa ctgcaggata tttggccttg acattaaagg cagagactgt 1140
ggcaatgagg cagctaagtg gttcaccaac ttcttgaaaa ctgaagcgta tagattggtt 1200
caatttgaga caaacatgaa gggaagaaca tcaagaaaac ttctccccac tcttgatcag 1260
aatttccagg tggcctaccc agactactgc ccgctcctga tcatgacaga tgcctccctg 1320
gtagatttga ataccaggat ggagaagaaa atgaaaatgg agaatttcag gccaaatatt 1380
gtggtgaccg gctgtgatgc ttttgaggag gatacctggg atgaactcct aattggtagt 1440
gtagaagtga aaaaggtaat ggcatgcccc aggtgtattt tgacaacggt ggacccagac 1500
50/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
actggagtca tagacaggaa acagccactg gacaccctga agagctaccg cctgtgtgat 1560
ccttctgaga gggaattgta caagttgtct ccactttttg ggatctatta ttcagtggaa 1620
aaaattggaa gcctgagagt tggtgaccct gtgtatcgga tggtgtagtg atgagtgatg 1680
gatccactag ggtgatatgg taaagggctt cagcaaccag gagggattga ctgagatctt 1740
aacaacagca gcaacgatac atcagcaaat ccttattatc cagccttcaa ctatctttac 1800
cctggaaaac aatctcgatt tttgactttt caaagttgtg tatgctccag gttaatgcaa 1860
ggaaagtatt agagggggga atatgaaagt atatatataa attttaggta ctgaaggctt 1920
taaaaataat taagatcatc aaaaatgcta ttttgaatgt tatcatggct attacacttt 1980
tacttcctga ctttaatatt gatgaataaa gcaagtttaa tgaatcaact aaaaagctgc 2040
aaaaatgttt ttaaaatgtg tgccttttat tacctatcag tctatgtttt gggagaaatg 2100
ggaagcaaca gatcactgtg tcctgatgtg caggacgcat gttaccacac tcacaaatgc 2160
ctaatattgg tctttatgtg gccattgagt cctgttgact ttccactcat gtgcttttta 2220
ctctagcatt atggaatctg ggctgtactt gagtatggaa attctcttat agacttagtt 2280
ttagtactct attacacctt tactaagcca cataaaagta atctgtttgt gtgtaactgc 2340
cagatatacc acctggaatt ccaagtaaga taaggaagag gatgacattt aaaagagaat 2400
ggaattttga gagtaggaat gcaaggaaga cagcatgaac atattttttt cagtgcaaat 2460
aattttttcg taacaaagaa acgaacaact ttggtatgat cttaagcaaa aatactcact 2520
gaaatagtat gtggatgaat tcacctactt acaattttat ggtttctttg taaataataa 2580
atgtgaatct caatcctgaa aaaaaaaaaa aaa 2613
<210> 59
<211> 1778
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5677286CB1
<400> 59
gtctgccctt tgaatggtgt gatctgggct caccgcaacc tCCgCCtCCa gggttcaagc 60
gattctcctg CCtCagCCtC cgagtagctg ggattacagg catgcgccac cacgcccgga 120
taattttgta tttttagtag agacagggtt tctccatgtt ggtcaggctg gtctcaaact 180
gacctcaggt gatccgccca cctcggcctc ccaaagtgct gggattacag gcatgagcca 240
ctgcgcctgg ccggacaata atgttttgag ctgttgctga gtctggcttt tgtgacggga 300
acttttagca ctcaaggagt ggccttgtct ctggcctgtc cccgagtgct catgggcagc 360
caaggcctgg ggactgctca ggcagggtag atgtatttgc caggccagct cccgtccctg 420
ggacctcagg gagtagcttc atccctgaag gctgagtgtc ttctgcctca tgtgggtggc 480
ccctctgcag ggttctcagg catccaggga agtgggtgcc acagcgttgc ctccagcctc 540
actggggctg gctggacttt gctctgacaa cacagcagtg gctgggcccc tgcaactctc 600
cagggctcag tttccctgtt ggccgcacta tggcataggc ccctgtgtgg acaccgatga 660
gctgacccca caaatgccac ccggccgctc ccccaggctt cagtggccta acagctgttg 720
tgtcaggaac cagcttaaag aatctttctt gctttctcaa actctccagg aattcctggt 780
gactggaagg gggagtgact caggccctca acctctagca ggtaagggtg ctacctcctg 840
ggtgggcatg cccagttcct cagtgggccc agcgcggccc agcctccgag gaagctctga 900
gcagctggct tggctccgag gtatttttag CtaCagatCC agCCCCCCtC CatCdCCaCa 960
acagtcgagt ccaaatcaaa cgcgctccag gcaggtgggg ctggggttct gccagcctcc 1020
tggccagcag gggtgggtgg gcagactggg gccagtatca gctgttctgc ctggacccag 1080
gccgggctgg gaaggcacac ttgtgcttat ttcccgcctc cacttctgtg caagcttgtg 1140
ctgtcataag cagagatcac agccccattt cttggatgga gaaagtggac actgaggtct 1200
gaggcttttg aggacagtca ggagCCCtCC tatgggctcc agtgatgcac tcaccagctt 1260
ctggtcctct tcttccacct cttagagtgc cttggctccc tcctgtcgtc ctggggaacc 1320
tcggccccag ccctgcctcc ccagccagtc acagctcctc cctggtcacc ctgagggagc 1380
tcagggcccg gctggtagct gggttgctct gcttctgtcc ccgactcctg tggagcctgg 1440
caggcaactc catgatctga ccccggttac cttgacagcc ctgcctggcc tcccctctca 1500
tggcccagcc accccagaac ctgaagaggt tttctagctg ccatgcattt gccaggctgg 1560
gttacccacc ctactttccc tgcctgccct ccagtgctgc caggcctagt gtgccagcca 1620
gcgctcagcc ttcagtaaag ggttcccctg cttccaacct ccattgcact gcttccccta 1680
agactgtgac ctcctggaag gctggagcac aactgcctct caataaacgt gttgcaaaaa 1740
aggaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1778
<210> 60
<211> 1234
51/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6436791CB1
<400> 60
cctgcagacg acggcgtcgt gggtggtcac cgttatccct taggtctgga gaggggacat 60
ccgagcgagg gccacttgcg gccaggcccg agctcgtcca gctccgggtg accacagagt 120
gccgcgggcg ggcagagggg ccggaaaccc aggccgcttc gtccctgttt ccggcagcgc 180
cgcgctgctc cgggagccgc tgtggcagcg tatgctgcca cggggactga agatggcgcc 240
gcgaggtaaa cggttgtcct ccaccccgct ggaaatcctg ttctttctga acgggtggta 300
taatgctacc tatttcctgc tggaactttt catatttctg tataaaggtg tcctgctacc 360
atatccaaca gctaacctag tactggatgt ggtgatgctc ctcctttatc ttggaattga 420
agtaattcgc ctgttttttg gtacaaaggg aaacctctgc cagcgaaaga tgccactcag 480
tattagcgtg gccttgacct tcccatctgc catgatggcc tcctattacc tgctgctgca 540
gacctacgta ctccgcctgg aagccatcat gaatggcatc ttgctcttct tctgtggctc 600
agagctttta cttgaggtgc tcaccttggc tgctttctcc agtatggaca cgatttgaag 660
tacagaattt cagccagcag cccatcaggc tgacaccaca catattgctt ctggtacttt 720
agccacacca gtgagaattg gtggggcaag ttgtcctgag aaaggctgtg tggcttttct 780
tcagcacaga catttgggca agcaactcag cataaggcca gtgggtacca tcttctaaac 840
caggaccatc agcccaagag actcttctac actccagtat agggaggggc aaggttattc 900
ccatcctgcc ccttctcaga accagtcccc tgctgacctc aagttctcct ccttgatcac 960
cgtggccaga gcatctcgtg tggaccatct aggctccttg ggcttcaagc aggacctgag 1020
ccacatgctc cctgtacgag ctgtgctata cctgtcccac atgagcacgg agagcctcat 1080
gttggtgggt ttccagagtg atgtgaaagc ctctcacccc aatcctcgga gactgagttc 1140
cacaactttt ttagtagctc atagtgttat ttttctactc tcttcatgaa actaacttta 1200
ttttataata aatatatatt ttctgttaaa aaaa 1234
<210> 61
<211> 863
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1820972CB1
<400> 61
ggaggtgcct cagccatggc atggatccct ctcttcctcg gcgtccttgc ttactgcaca 60
ggatccatgg actcctttga attgactcag gcaccgtcaa cgtccgtgtc cccaggacag 120
acagccacca tctcctgctc tggcgagaag gtgggaagta aattcttttc gtggtatcaa 180
cagaaggaag gccagtcccc tgtcgtaatc atctatcaga atgggaagcg gccctcagag 240
attgctgacc gattctctgg ctccaagtct ggggacacgg ccaccctgac catcagcaga 300
gcccaagccg gggatgaggc tgactatttc tgtcaggtgt gggacagcag cactgcggtg 360
ttcggcggag ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 420
ctgttcccgc cctcctctga ggagcttcaa gccaacaagg ccacactggt gtgtctcata 480
agtgacttct acccgggagc cgtgacagtg gcctggaagg cagatagcag ccccgtcaag 540
gcgggagtgg agaccaccac accctccaaa caatgcaaca acaagtacgc ggccagcagc 600
tacctgagcc tgacgcctga gcagtggaag tcccacagaa gctacagctg ccaggtcacg 660
catgaaggga gcaccgtgga gaagacagtg gcccctacag aatgttcata ggttctaaac 720
CCtCaCCCCC CCCdCgggag actagagctg caggatcccc aggggagggg tctctcctcc 780
caccccaagg catcaagccc ttctcccgtg cactcaataa accctcaata aatattctca 840
tttgtcactc aaaaaaaaaa aaa 863
<210> 62
<211> 2521
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
52171
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<223> Incyte ID No: 3286805CB1
<400> 62
gcttgctggc cgctgtgtag ggctggtgag tggctggggc tgtctgagcc atgaacaact 60
tcagggccac catcctcttc tgggcagcgg cagcatgggc taaatcaggc aagccttcgg 120
gagagatgga cgaagttgga gttcaaaaat gcaagaatgc cttgaaacta cctgtcctgg 180
aagtcctacc tggagggggc tgggacaatc tgcggaatgt ggacatggga cgagttatgg 240
aattgactta ctccaactgc aggacaacag aggatggaca gtatatcatc cctgatgaaa 300
tcttcaccat tccccagaaa cagagcaacc tggagatgaa ctcagaaatc ctggaatcct 360
gggcaaatta ccagagtagc acctcctact ccatcaacac agaactctct cttttttcca 420
aagtcaatgg caagttttcc actgagttcc agaggatgaa gaccctccaa gtgaaggacc 480
aagctataac tacccgagtt caggtaagaa acctcgtcta cacagtcaaa atcaacccaa 540
ctttagagct aagctcaggt tttaggaagg aactccttga catctctgac cgtctagaga 600
acaaccagac gaggatggcc acctacctgg cagaactcct ggtgctcaac tatggcaccc 660
acgtcaccac cagtgtcgac gctggggctg ctcttattca ggaggaccac ctcagggcct 720
ccttcctcca agacagccag agcagtcgta gtgccgtgac cgcctctgct ggacttgcct 780
ttcaaaacac cgtgaacttc aaatttgagg aaaactatac ctcgcagaat gtcctcacca 840
agagctacct ctcaaaccga accaactcca gggtgcagag cattggaggg gttccttttt 900
acccaggcat caccctccag gcctggcagc agggtatcac caaccacctg gtggccatcg 960
accgctctgg cctgccgctg catttcttca tcaaccccaa catgctacct gacttgccag 1020
gccccctggt gaagaaggtg tcaaagacag tggaaactgc tgtgaagcgc tattatacat 1080
tcaacaccta ccctggctgc acagatctca attctcccaa cttcaatttt caggccaaca 1140
cggatgatgg ctcctgcgag gggaaaatga ccaacttctc tttcggtggg gtttatcagg 1200
aatgcactca gctctcaggg aatagggatg tcctcctctg ccaaaagttg gagcagaaga 1260
atccactcac tggtgatttc tcctgcccct ctggctactc cccggtgcac~ctgttatccc 1320
agatccacga ggagggttac aaccacctgg agtgtcatcg aaagtgcact ctcctcgtct 1380
tctgcaagac cgtgtgtgaa gatgtgttcc aggtggcaaa agctgaattt agggcttttt 1440
ggtgtgtggc cagcagccaa gtacctgaaa actcaggact gctttttggg ggcctcttca 1500
gcagcaagag cataaacccc atgacaaatg cacagtcatg cccagccggc tactttccac 1560
tgagactctt tgaaaacctc aaggtatgtg tttctcagga ctatgagttg ggaagcaggt 1620
ttgcggtccc ctttggcggg ttctttagct gcacagttgg gaaccccctg gtagatcctg 1680
ctatatccag agatttaggg gcaccgtctc tgaaaaagtg ccccgggggc ttcagccagc 1740
acccagccct catcagcgat ggatgccaag tgtcctattg cgtcaaatcc gggctcttca 1800
caggagggtc cctgccccct gccaggctcc cacctttcac ccggccaccc ctcatgagtc 1860
aggctgccac caatactgtc atagtgacca attctgagaa tgcgagatcc tggattaaag 1920
actcccagac ccaccagtgg aggctgggag aaccgataga gctgcggagg gccatgaatg 1980
tcatccatgg ggatggtggt ggtctgtcag gaggggctgc agctggggtc acagtggggg 2040
tcaccaccat tctggctgtt gttatcacct tggccatcta cggcacccgg aagttcaaga 2100
agaaagcata tcaggcaatt gaggaaaggc agagtttggt tccaggcact gcagcaactg 2160
gagacaccac ttaccaagag caggggcaga gtccagctta aatctctccc cgaaaatggt 2220
ttctctcatc tccagtgtgg tcattgctga ccactctgtt ttcctaagca ttgaaatggc 2280
aagtgcaacc aaaaatcttg ggctgagaag aggcccttca cccaaggagt ttgaagtatc 2340
acagcgtgtg ggaaggtggg aaccaggata cccattcatt tccaaccgag acacagagaa 2400
gtgagtcaca gaatttgagc ccgctctctt gactgcccag ccagagacac tgatttctgt 2460
aacctcttca cttgatcctg cctcttaagc attaaaacat tctcctaaaa aaaaaaaaaa 2520
a 2521
<210> 63
<211> 1765
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3506590CB1
<400> 63
gcactggaag tcgccggtgt ttccattcgg tgatcatcac tgaacacaga ggactcacca 60
tggagtttgg gctgagctgg gttttcctcg ttgctctttt aagaggtgtc cagtgtcagg 120
tgcagctggt ggagtctggg ggaggcgtgg tccagcctgg gaggtccctg agactctcct 180
gtgcagcctc tggattcacc ttcagtagct atgctatgca ctgggtccgc caggctccag 240
gcaaggggct ggagtgggtg gcagttatat catatgatgg aagcaataaa tactacgcag 300
actccgtgaa gggccgattc accatctcca gagacaattc caagaacacg ctgtatctgc 360
53/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
aaatgaacag cctgagagct gaggacacgg ctgtgtatta ctgtgcgaga gcgggagaag 420
ggagccccga tactttggtt gcttttgata tctggggcca agggacaatg gtcaccgtct 480
cttcagcttc caccaagggc ccatcggtct tccccctggc gccctgctcc aggagcacct 540
ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg 600
tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt 660
cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc 720
agacctacac ctgcaacgtg aatcacaagc ccagcaacac caaggtggac aagagagttg 780
agctcaaaac cccacttggt gacacaactc acacatgccc acggtgccca gagcccaaat 840
cttgtgacac acctcccccg tgcccacggt gcccagagcc caaatcttgt gacacacctc 900
ccccatgccc acggtgccca gagcccaaat cttgtgacac acctcccccg tgcccaaggt 960
gcccagcacc tgaactcctg ggaggaccgt cagtcttcct cttcccccca aaacccaagg 1020
atacccttat gatttcccgg acccctgagg tcacgtgcgt ggtggtggac gtgagccacg 1080
aagaccccga ggtccagttc aagtggtacg tggacggcgt ggaggtgcat aatgccaaga 1140
caaagctgcg ggaggagcag tacaacagca cgttccgtgt ggtcagcgtc ctcaccgtcc 1200
tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc 1260
cagcccccat cgagaaaacc atctccaaag ccaaaggaca gccccgagaa ccacaggtgt 1320
acaccctgcc cccatcccgg gaggagatga ccaagaacca ggtcagcctg acctgcctgg 1380
tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg cagccggaga 1440
acaactacaa caccacgcct cccatgctgg actccgacgg ctccttcttc ctctacagca 1500
agctcaccgt ggacaagagc aggtggcagc aggggaacat cttctcatgc tccgtgatgc 1560
atgaggctct gcacaaccgc tacacgcaga agagcctctc cctgtctccg ggtaaatgag 1620
tgccatggcc ggcaagcccc cgctccccgg gctctcgggg tcgcgcgagg atgcttggca 1680
CgtaCCCCgt gtaCataCtt cccaggcacc cagcatggaa ataaagcacc cagcgctgcc 1740
ctgggcccct gcaaaaaaaa aaaaa 1765
<210> 64
<211> 1264
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 003600CB1
<400> 64
ccctgctcca gtcacacccg gaagctgact ggtccacgca cagctgaagc atgaggaaac 60
tcatcgcggg actaattttc cttaaaattt agacttgcac agtaaggact tcaactgacc 120
ttcctcagac tgagaactgt ttccagtata tacatcaagt cactgagatc tccagcaccc 180
tgccggtggc actactgaga gacgaggtgc cagggtggtt cctgaaagtg cctgagcccc 240
aacttatcag caaggagctc atcatgctga cagaagtcat ggaggtctgg catggcttag 300
tgatcgcggt ggtgtccctc ttcctgcagg cctgcttcct caccgccatc aactacctgc 360
tcagcaggca catggcccac aagagtgaac agatactgaa agcggccagt ctccaggttc 420
ccaggcccag ccctggccac catcatccac ctgctgtcaa agagatgaag gagactcaga 480
cagagagaga catcccaatg tctgattccc tttacaggca tgacagcgac acaccctcag 540
atagcttgga tagctcctgc agttcgcctc ctgcctgcca ggccacagag gatgtggatt 600
acacacaagt cgtcttttct gaccctggag aactaaaaaa tgactccccg ctggactatg 660
agaacataaa ggaaatcaca gattatgtca atgtcaatcc agaaagacac aagcccagtt 720
tctggtattt tgtcaaccct gctctgtctg agccagcgga atatgatcaa gtggccatgt 780
gaattccaaa tatttttaat ggggtccagt tctctatgga ttcttacatt taatttgtag 840
ggaaatgcca tttttccccc ttaaacaagg catggggctc acaagtctat ggagacaggc 900
caaaaagaat gtggagaaga aaactgataa atacacagag gtcctcaaga cccatggact 960
cctggtctgt acccaaaaaa gctgttcgtt cctcaaaaac aaaaacaagg cttggctggg 1020
aaaacaggcc aatgccccgg caagaaaggt tgagatcaga tgttaggaag aactttcagg 1080
taaagtatga gaactatgga gtccatcagc agagatagta gtgaagtctc tccccaggga 1140
aaattttaaa aaggttgaat cagctgttgt agagttctat ttggcaatct catggttaaa 1200
tgacttccct ttgagctctt taattattgg caataaacaa cttctttaaa agttttaaat 1260
aaaa 1264
<210> 65
<211> 3415
<212> DNA
<213> Homo Sapiens
54/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<220>
<221> misc_feature
<223> Incyte ID No: 1251534CB1
<400> 65
gcctcctcca cctcctcgtc tttctcccgg gaaccttgac gacgccttcc gcttggccct 60
gccttctgcc gcatccccgc cgccgcggcg ccttgaggag caggagaaga cgcagccggg 120
ccgccgccgt tagaggggtt cccggccgcc gctcgccccg tcggccgcca ccgcctccgg 180
ggtcagccct ctctctgggt ctccgCtttc tCCtgCCgCC agCgCCCC,JCt catcgccgcg 240
atggggctcc tggactcgga gccgggtagt gtcctaaacg tagtgtccac ggcactcaac 300
gacacggtag agttctaccg ctggacctgg tccatcgcag ataagcgtgt ggaaaattgg 360
cctctgatgc agtctccttg gcctacacta agtataagca ctctttatct cctgtttgtg 420
tggctgggtc caaaatggat gaaggaccga gaaccttttc agatgcgtct agtgctcatt 480
atctataatt ttgggatggt tttgcttaac ctctttatct tcagagagtt attcatggga 540
tcatataatg cgggatatag ctatatttgc cagagtgtgg attattctaa taatgttcat 600
gaagtcagga tagctgctgc tctgtggtgg tactttgtat ctaaaggagt tgagtatttg 660
gacacagtgt tttttattct gagaaagaaa aacaaccaag tttctttcct tcatgtgtat 720
catcactgta cgatgtttac cttgtggtgg attggaatta agtgggttgc aggaggacaa 780
gcattttttg gagcccagtt gaattccttt atccatgtga ttatgtactc atactatggg 840
ttaactgcat ttggcccatg gattcagaaa tatctttggt ggaaacgata cctgactatg 900
ttgcaactga ttcaattcca tgtgaccatt gggcacacgg Cactgtctct ttacactgac 960
tgccccttcc ccaaatggat gcactgggct ctaattgcct atgcaatcag cttcatattt 1020
ctctttctta acttctacat tcggacatac aaagagccta agaaaccaaa agctggaaaa 1080
acagccatga atggtatttc agcaaatggt gtgagcaaat cagaaaaaca actcatgata 1140
gaaaatggaa aaaagcagaa aaatggaaaa gcaaaaggag attaaattga actgggcctt 1200
aactgttgtt gacagtgagg aaaaactccc atatcatata aaatttcagg gaaaacagaa 1260
gcaaagg'aga gcttgggggt ggggagaaaa gacaaatgtg ctctatgtcc tagtaactct 1320
tagactgagt aaagtgttaa taccataccc agatgtttta tttatgaagt ttttatttta 1380
aacatttttt ttaaaaatta gccttgatat tctccagacc aaagcaatca ttaagtgact 1440
ttggggattc tccccctgtt cacatccagt tgtctaaagg atgagatttt tcatgtatct 1500
tatagtcact cattcttcgg tctgaatttt agacgatcac agaaacggtc tttatgaatt 1560
attttgataa attactaatt atcttatcta ctgactgaaa tcagtggtgt tacattttct 1620
tgtccaaagc tgaaaatgtg tatacactta aacttgcaca tttgaattca tttgctgacc 1680
ggaatggtca aatctctcca cctctagtca gagtataatt ttggttgtaa.ttaaattttt 1740
aaaatctgct gatctctgta gaatcttaga ggcttgatga tgatggtgtt ggtgaaaata 1800
agaaagaatt gcagtaaagt cttg°tctggt gacccagaga tcaccatgac ttgaggcaca 1860
aatcactgtg gggaaacaat tttttgtgat gaaaaggcag catttgaata ctcctgttag 1920
tagcagaaat atattatgaa aattaagatt attgtctgat tgaaacatga aacaactcat 1980
gtctttatta gtaacatcat aagatagtta catttatgtg ctgttagaat atgttgattt 2040
ttatcaggct ttccttgttt tgatttatgg ctgttcctga tttttcatat gtggaaatat 2100
acctacctct tccgttggaa agaacattta aaattaaata aattttaatt aaaaaatcaa 2160
ggagtcttct aatgtaaatt ttaatgttaa ctttcaaatc cactagtatt ttttttgctt 2220
ttatgacaaa tagcatacac caaacatttc tgtgaaacta tccttctctt tcaatgtgtt 2280
taattttgga gtaacgtttt ccttgtgact aagttgcaag atcttattta ttaactaggt 2340
atgaagtata aacccatttt ggtgcaatat tcttgactcc ttggtgctaa agattgttaa 2400
attcaatgct tgatgttaca aggtgttgtt aaaacacaaa atgaataaaa gtgagagtag 2460
tcagaactat aacattcaat ttgctattta caaatgaagt atttcatgta atataagtga 2520
acaactggaa ataaagtagg aaagaatttg tatcatgttt tactacatag gttaattttt 2580
taagggatgt tgcaaaggga ttactagaga aagacaaaat gtgaccaaaa aaaagcatga 2640
atatttctta agtatctcaa caacatgtca aagctgcatg tgtaggatgt atgctgtttg 2700
tacaaactat ttcagaatat tttgtaagct ataacatatt tattgtgcat taaaattaaa 2760
tactttttcc ccaaaggcat gcagtcatga gaattacaga aaatttgcaa catataaagt 2820
agtttgatct aagaggattc aacacctttg ttttgttgct cagtgtgtaa tgactgagat 2880
ttgtaaatct ttgtgaacat tctgtactgg ttcccaagag ctattcattc cctgctacct 2940
gatttcagca caataaatat acttctgctg tgggaaaaat atgatttttt tttgttaatc 3000
agtttattat aatgtaaact gtctgctaga tgtttatttg atttagttaa ttctaaattg 3060
ttttctgctg ctaatttctt atatagaaaa aatcaaacta gaacttgggt atctaccatt 3120
tctggtaaaa agcttatttt ctatagtagc atatttttac tttaaaaatt agctgctttt 3180
ggaagagctt ttcattcaga cataaaacat accattttct tctaaattat tcttaacata 3240
aatggggaag ttcttgtatt ctttcattta aatcattcag tccttactat aagtttcttt 3300
atagaagatg attataagat gtcataaata cagccttttc cagtagcttt cataatttct 3360
ctattatcac agatatgttg catgccttat atctagaaac tgatgaataa agatg 3415
55/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<210> 66
<211> 2289
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1402211CB1
<400> 66
ggacatgccg ggagttgcag taccctcagg aaggtagcgt cttgatctgc gtggcgtggt 60
tctgtgcctt gggaagagat gaatgggaag cggccagcgg agcccggccc agcccgggtg 120
ggaaaaaagg gaaagaagga ggtgatggcg gagttttcgg acgctgttac ggaagaaacc 180
ttgaaaaagc aggtggctga ggcctggagc cgcaggacgc cgttcagtca cgaagtcatt 240
gtcatggaca tggacccttt tcttcactgt gtgatcccaa acttcatcca aagccaagac 300
ttcttagaag ggcttcagaa ggaactgatg aacttggact tccatgagaa gtataatgat 360
ttatataagt tccagcagtc tgatgatttg aagaagagaa gagagcctca catctccact 420
ttaaggaaaa ttctgtttga agatttccgg tcctggcttt ctgatatttc taaaattgac 480
ctggaatcaa ccattgacat gtcctgtgct aaatatgaat tcactgatgc cctgctgtgc 540
catgatgatg agctggaagg gcgccggatt gccttcatcC tgtacctggt tcctccctgg 600
gacaggagca tgggtggtac cctggacctg tacagcattg atgaacactt tcagccgaag 660
cagattgtca agtctcttat cccttcgtgg aacaaactgg ttttctttga agtatctcct 720
gtgtcctttc accaggtgtc tgaagtgctg tctgaagaaa agtcacgttt gtctataagt 780
ggctggtttc atggtccatc attgactcgg cctcccaact actttgaacc ccccatacct 840
cggagccctc acatcccaca agatcatgag attttgtatg attggatcaa ccctacttat 900
ctggacatgg attaccaagt tcaaattcaa gaagagtttg aagaaagttc tgaaattctc 960
ctgaaggagt ttcttaagcc tgagaaattc acgaaagtct gtgaggcctt ggagcatgga 1020
catgtggaat ggagcagccg aggtccccct aacaaaaggt tttatgagaa agctgaggag 1080
agtaagcttc ctgagatatt gaaggagtgc atgaagttat ttcgctctga ggcactattc 1140
ttgctgctct ccaacttcaC aggcctgaag cttcatttct tggccccttc ggaagaagat 1200
gagatgaatg ataaaaaaga ggcagaaacc actgatatca ctgaagaagg gactagccat 1260
agtcctcctg agccagagaa taatcagatg gccatcagca acaacagcca acagagcaat 1320
gagcagacag acccagagcc agaggaaaat gaaacaaaga aagaatcaag tgttcccatg 1380
tgccaagggg aactgaggca ttggaagacc ggtcactaca ctttaattca tgaccatagc 1440
aaggctgaat ttgccctaga cttaattctg tactgtggct gtgaaggctg ggagccagaa 1500
.tatggcggtt ttacttctta cattgccaaa ggtgaagatg aagagctgct aacagtgaat 1560
ccagaaagca attctttggc attggtctac agagacagag agactctgaa atttgtcaag 1620
catattaacc accgaagcct ggaacaaaag aaaaccttcc caaacagaac aggtttctgg 1680
gacttttcat tcatctatta tgaatgacag cactgggcaa agctgaacaa aaatgtgacc 1740
cttcgtaatt actgggaagt ctgaaagagc taagcatgga gtcaaggaga actacatggt 1800
agcttgcctg acagtgttct taaaactggt tgtcttttac taggactcat aatgattgtc 1860
ctcaaccgag accttgagct tgcagctaag tacttatctc ttgattaaaa aaaaaaagtt 1920
ggcttttttt tttttaacat ttagtccttt ttccatattg gcttcttcag tgaattttta 1980
agttcaattt gtttttattg aggtaaaata tttataacat aaaactgacc agcttaccca 2040
tttttaaata tgcaattcag tggattaagt acattctcat tgttgtccag ccatcaccat 2100
catccatctc cagaagtttt ccatcttccc aaattctgtg cccattgaac aataactccc 2160
cacctcccct tcccctagca acagccatac cttttgtctc tatcatcaac ttcactactc 2220
atatttctca tgtaagtgga atcatacagt atttgtcctt ttgtgactgg tttcacttag 2280
cataaagtc 2289
<210> 67
<211> 4480
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1623474CB1
<400> 67
tacgggagcg tgcgggggcc gcgcgctgct tctctgaggc aggacggcac tgccgggagg 60
cggcggtgac aacgacggcg gtggtgacgg gcaccgggct cgcgggtgag acacagtaac 120
ctggttgaac tctgcatctg gaaagctgaa gactgaagaa agataagaga cattgactag 180
56/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tctggaaaca gggacatctt tggaacttcg ttttcatcca cagtaaactt ttgaagtgtc 240
atcaattgga attgatttct tcatcttatt ctgcctattg ggaagaacat ggcttcaagg 300
attttaagtt tccctttagt tttacatgaa ctttgtagga aacagagccc ttaaagggct 360
tgggaataac aagaagagat tgaagacaga gaagcttgcc ctgttttcct tgccccttca 420
aagaaaagga tttacagctc aaacttagaa cagctgttgt ccagctttag ccatcaagag 480
agaaataaat taaaccacca ttgccagact acaagccctg gtgaagtcag ggtgtgggag 540
tggtggcatt gagaagacta cctaaaagag acaaagactg cagtaaacaa agctcctctt 600
taaagttgga aggggcctca ggttccttct tggattgaaa tagaaataga aacacagggc 660
acacctcttt taggtgcagc tcacatttta tggaactgta gtcgtggagg tactatagta 720
tctcagaaga atttttcttt gcccaaagtt tttttgccat accctgatat tctctccttc 780
ttttgaagac ctgcctccat ccatgagctg tatcttgatc tgtctgactg tccatgtttt 840
ccacctgcaa ccatttgcat gtgtacagcc tactgtttgt ctccagtttt taaactgtac 900
aagttgtgtt tcttaatctt cccttctgcc ttgttctggg gaggtggtta ttcatcattt 960
ggaatcacct ttccccctcc catgtgcttt ccttcatttg agatcttttg acctttggct 1020
ttatttggga gggggaaggg tgataaagtt ttctgtttcc ctggttttct tttgtactcc 1080
tctctgttgc ttccctcctc ccattttctt gtctgttctg ccgctgtgtg ggcctgggct 1140
atgcggcagg gcagatttcc catcagagct ccaacatgcc cgcagagtct ggaaagagat 1200
tcaaacccag caagtatgtc ccggtctctg cagccgccat cttcctagtg ggagctacga 1260
cactcttctt tgcctttacg tgtccaggac taagcctgta tgtgtcacct gcagtgccca 1320
tctacaatgc aattatgttt ctctttgtgt tggccaactt cagcatggcc accttcatgg 1380
acccagggat tttccctcga gctgaggagg atgaggacaa ggaagatgat ttccgagctc 1440
ccctttacaa aacagtggag ataaagggca tccaggtgcg catgaaatgg tgtgccacct 1500
gCCgCtttta CCgtCCCCCt cgatgttccc actgcagtgt ctgtgacaac tgtgtggagg 1560
aatttgatca tcactgcccc tgggtgaata actgtattgg tcgccggaac taccgttatt 1620
ttttcctttt cctcctttcc ctgacagccc acattatggg tgtgtttggc tttggcctcc 1680
tttatgtcct ctaccacata gaggaactct caggggtccg cacggctgtc acaatggcag 1740
taatgtgtgt ggctggctta ttcttcatcc ctgtagctgg cctcacggga tttcacgtgg 1800
ttctggtggc caggggacgc acaaccaatg aacaggttac gggtaaattc cggggaggtg 1860
tgaacccctt caccaatggc tgctgtaaca atgtcagccg tgttctctgc agttctccag 1920
cacccaggta tttggggaga ccaaagaaag agaagacaat tgtaatcaga cctcccttcc 1980
ttcgaccaga agtttcagat gggcagataa ctgtgaagat catggataat ggcatccagg 2040
gagagctgag gagaacaaag tctaagggaa gcctggagat aacagagagc cagtctgcag 2100
atgctgaacc tccacctcct cctaagccag acctgagccg ttacacaggg ttgcgaacac 2160
acctcggcct ggctactaat gaggatagta gcttattggc caaggacagc cccccgacac 2220
ctaccatgta caagtatcgg ccgggttaca gtagcagcag tacgtcagct gccatgccgc 2280
attcctccag cgccaagttg agtcgtgggg acagcttgaa ggagccaacc tcaattgcag 2340
agagcagccg tcaccccagc taccgctcag agcccagctt ggaaccagag agcttccgtt 2400
ctcctacctt tggcaaaagt tttcacttcg atccactatc cagtggctca cgctcctcca 2460
gcctcaagtc agcccagggc acaggctttg agctgggcca gttgcaatcc attcgttcag 2520
agggcaccac ctccacctcc tataagagcc tggccaacca gacacgcaat ggaagcctat 2580
cttatgacag cttgctcaca ccttcagaca gccctgattt tgagtcagtg caggcagggc 2640
ctgagccaga cccaccttta ggctatacct ctcccttcct gtcagccagg ctggcccagc 2700
aacgggaagc tgagaggcac ccacgtttgg tgccaactgg cccaacacac cgagagccct 2760
caccagtccg ttacgacaat ctgtcgcgcc acattgtggc ctctctccag gaacgagaga 2820
agttgctgcg ccagtcaccc ccactcccgg gccgtgagga agaaccaggc ttgggggact 2880
caggcattca gtcaacacca ggctcgggcc atgcccctcg tactagttcc tcctcagatg 2940
attcaaagag atcacctttg ggcaagactc cactgggacg cccagctgtc ccccgttttg 3000
gcaagccaga tgggctaagg ggccggggag tagggtcccc tgaaccaggc ccaacagccc 3060
catacctggg ccgatcgatg tcttacagca gccaaaaagc ccaacctggt gtctctgaga 3120
cagaagaagt ggccttgcag ccattactga cacccaaaga tgaagtacag ctgaagacca 3180
cctacagcaa atccaacggg cagcccaaga gcttaggctc agcctcccct ggcccaggcc 3240
agccacctct cagtagcccc acgaggggag gagtcaagaa ggtgtcaggg gttggtggta 3300
ccacctatga gatttcggtg tgagccttcg gcacctcccc tccccaacgc ctctgcgcct 3360
acaccaaagg gccccaggtg gccaccttcc ttccctcaag gggctcccct cccgtgcatg 3420
gacatttttt aaaccaccga ttccaagagg atgaggagtg ttttctaaaa tgcagtaggc 3480
ttggggagtc ggagagttgg ggccctgaga ctggggtagc aaccccccct tttatctttt 3540
aagaccttcc cttccttgat ccctggacca gactcagtgg acatttgtgc aattgctcgc 3600
cctggaggga accagatcat ttttaaacca gaaataattt tttttattat tgttacggat 3660
tctatttttt tcctcttctg cgttaccagg tgtgtgtgta catataatat atatatatat 3720
atattataaa tatcaaagaa attatatatc tatcctggga tgggaaaatg agggagggat 3780
acatatacgg agggggatct tactcttccc attcctcaga ccagcaggaa aagaggggag 3840
acgtcagtct ttttcctgtg gttccctctc atttgtccca gttactaact acggaaatag 3900
catcctctgc tggtgctaag tgtgattagg aagaagcctg gggagaggtg agtctggaat 3960
57/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tttggtcaca agagggaagg acttggagag gagaattagt tttctaggct cattggcatt 4020
tagtttccct aggaaagggg tcaaaacttc aagacactgg tggtggtggg agatcaggaa 4080
aataacttgg cctagctcaa acaatattgg ataatcccct ccttggggga gagggattag 4140
agtgtgctcc tactggcccc ttggagcctc ccctagctta cacagttaac ttgattttaa 4200
aatccaaggc caggagagaa gaatccaaaa agcaatattt ttcatcacat gccaaaaacg 4260
ggggatagag agaaggagtg gcaggcctag gcccctccga ttgtcccttg ggggttaccc 4320
ctcagcccac ctcactatgg tgctgggtag aggggatacc tgggttctaa cctctaaata 4380
ggggagatcc cagcctccac aaagaggccc ttttattttt tattctgatt agccatttta 4440
aaccaacgag gaataaaaag aaatcctgat ctaaaaaaaa 4480
<210> 68
<211> 1568
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1706443CB1
<400> 68
ctgctcatgt gccccctccc actgaacagg cagccagcgg cctgtgcgag ctcctgtccg 60
tcaacagctg catgggccgt gtgaggcgca tctaccctca gctgctcctg gccctgctca 120
ttcaggtcca ttaccacatc ggcctcaacc tgcctggctg cgtggctcct cccaaggaca 180
ccaagaaggg tgcacagccc tctcccttcg tacctgtgcg ctgggtggtg aaagtggtga 240
aaaccctgct actgaggatg ggctgctctt atgagaccac gtttctggag gaccagggtg 300
gctgggagct catggagcag gtggagagcc accaccgcgg agtggccttg ctggcaaggg 360
ccatggtgca gtactcctgc caggagctgt gccgcatcct ctacctgctc atcccgctcc 420
tggagcgagg cgacgagaag cacaggatca cggccaccgc cttcttcgtg gagctcctcc 480
agatggagca ggtgcgccgg atccccgagg aatactctct ggggcggatg gcagaaggcc 540
tgagccacca cgaccccatc atgaaggtgc tgtccattcg aggcctggtc atcctggccc 600
gcaggtctga gaagaccgcc aaggtgaagg ccctcctgcc ctccatggtg aagggcctga 660
agaacatgga tgggatgctg gtggtggaag cggtccacaa cctcaaggct gtcttcaagg 720
ggcgggacca gaagctgatg gacagtgcgg tctatgtgga gatgctgcag atcctgctgc 780
cgcacttcag cgacgcacga gaggacgtgc gctcctcctg catcaacctg tatgggaagg 840
tggtccagaa gcttcgggca ccacgcactc aggccatgga ggagcagctg gtcagcacct 900
tggtgcccct actgctgacc atgcaggagg gcaactccaa ggtaagccag aagtgtgtga 960
agaccctgtt acgctgttct tacttcatgg cttgggagtt gccaaaaaga gcttatagcc 1020
ggaagccctg ggacaaccaa cagcagacag tggccaaaat ttgcaagtgc cttgtgaaca 1080
cccaccgaga cagcgccttc atattcctca gccagagcct ggagtatgcc aagaactcac 1140
gggcctccct ccggaagtgc tcagtcatgt tcatagggtc cctggtcccc tgcatggaga 1200
gcataatgac agaagatcgt ctgaatgaag tgaaagctgc tctggataac ttgagacatg 1260
acccagaagc atcagtgtgc atctacgcag cccaggtcca ggaccacatc ctggccagct 1320
gctggcagaa ctcctggctg ccgcacggga actcatgggt gtgttactca gccaccaccc 1380
accgctggag ccccagctgt gagaacctgc ccacttccca ccagcggcgc tcctggatca 1440
tgcaggcact gggctcctgg aagatgtcct tgaagaagtg acgtccctga gccccaaacc 1500
ctcctcaggg tggttgagtt ccagccatgc tccctataaa tgtcatgtgg cttaaaaaaa 1560
aaaaaaaa 1568
<210> 69
<211> 1887
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1748627CB1
<400> 69
acgaagtccc acgccccacc cggttctttg tttttttcaa aataaacata ggttatattt 60
tttcaaacat gagcattcat atattaaaga gcttttcaat ggccgaccat ggtggttcac 120
acctgcaatc ccagcacttt cggaggctga ggcaggtgga tcacttgagg tcaggagttc 180
gagaccagcc tggccagcat ggtgaaaccc tgtctctact aaaaatacaa aaattagtca 240
ggcatggtgg tgcgcacctg tagtcccagc tacttgggag gctgtggcat gagaattgct 300
58/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tgaatccagg aagcggaggt tgcaatgagc ccagatgaca ccactgcact ctagcttggg 360
cgacacagca agactctgtc gaggggcgga gtagcttccg gaaagggtac tgcatttccc 420
gtttctacct ccactgcacc cgcttattgc gtcttgctcc tgggtcacag agcctaaaac 480
gacacaccca acacgcccgc cggagttaca gctaaaggaa ggacagggga agcaatgaaa 540
tgccgagggc ggagccaaga gcgacactgg gggagcagga aaaggcgggg cttccgcttg 600
gggcatggag gctgtacctc ttacgtcact tccgtaaaca aacggagctg cggaggagcg 660
ggtcccggga tgtgaccggg gctctgcttg tggctgcggc ggtggcttct gaggctgtcg 720
ggtctttgcg ggttgcggaa gggggcccca atacccttct tcttcaggtc ttaagaagct 780
ggccgtggtg caataaggaa cttaaaacaa tggaagagcg gaaagtgaag aggaggagtc 840
ctaagtcttt tagtgcccac tgtactcagg ttgtcaatgc caaaaaaaat gccattccag 900
tgagtaaaag cacagggttt tcaaatcctg catcacagtc aacttcacag cgaccaaagt 960
taaaaagagt gatgaaagaa aagaccaaac ctcagggtgg agagggcaaa ggcgctcagt 1020
caactccgat ccagcactcc ttcctcactg atgtctcaga tgttcaggag atggagagag 1080
ggctgctcag tcttttgaat gatttccact ctggaaaact tcaagcattt ggaaatgaat 1140
gttccattga acagatggaa catgttcggg gaatgcagga gaaattagct cgcttgaatt 1200
tggagctcta tggggagtta gaggaacttc ctgaggataa gagaaaaaca gccagtgact 1260
ccaatctgga taggcttctg tcagatttag aagaattgaa ttcttccata caaaaactcc 1320
atttggcaga tgcacaagat gttccaaata cttctgctag ctaaaatgaa atgtagtttg 1380
ctttcttgtg atttgaagag aagcagcagt ctttactttt ccagccaaat ccagtagcag 1440
aatcatcttc cacaacaagg aattaaagta attaaagtgc aagttcaatg atcgttttca 1500
cttactgctt ttagtaaatg tgactcgctg taattcgcca taactggagg cctaggcctt 1560
gttgaaggct tcatcaggaa atgtgacgca gagattgtgc tgctgtgctt catattgttg 1620
ccttatggga ttatacttga aatgcattgt gctgatgttc ttgacagggc ggagggattt 1680
ctctttcctg agctcaccaa acttattcca gtgttgatcg caagctgttg atgcacaggc 1740
gtcttgtggc aagcccagct tcagtttatg ttagacaatc agtacacatg ctggtgtgtt 1800
ttccaatggc cagtgataag aaattaaata agtatttgtg agatttgcta ttttttaata 1860
aagtaattgt tttaaattaa aaaaaaa 1887
<210> 70
<211> 569
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> 2ncyte ID No: 1818332CB1
<400> 70
cggctcgagg gggtggggct gcgggaggcc ctggagcgcg gcggtgatgg cggggccggt 60
gaaggaccgc gaggccttcc agaggctcaa cttcctgtac caggccgccc attgtgtcct 120
tgcccaggac cccgagaacc aggcgctggc gaggttttac tgctacactg agaggaccat 180
tgcgaagcgg ctcgtcttgc ggcgagatcc~ctcggtgaag aggactctct gtcgaggctg 240
ctcttccctc ctcgtcccgg gcctcacctg cacccagcgc cagagacgct gcaggggaca 300
gcgctggacc gtacagacct gcctaacatg ccagcgcagc caacgcttcc tcaatgatcc 360
cgggcattta ctctggggag acaggcctga ggcccagctc gggagccaag cagattccaa 420
accactacaa cccttgccaa acacagccca ctccatttca gaccgccttc ctgaggagaa 480
aatgcagact cagggttcca gtaaccagtg atggattcac cccatctccc aaataaagtt 540
tacttgtttt acattccaaa aaaaaaaaa 569
<210> 71
<211> 2338
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1822832CB1
<400> 71
aacgtaggct gtcaggtctc cccctgtgca aatgggacat ccacttgaga gtcaagggtc 60
tgtttgggtg gcagggatag ccacttctga aggtagaaag aaaaataagc caccaaattg 120
gtatctttct gtgaaatgga catcgtgctt agagtctcca ttttccccac aacctggagg 180
aataagtatt gtcatctgca ttttatagct gaggaatctg actcaacaaa attaaattac 240
59/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tcacctaagc aattagcaat taaccaagtc tttctgattc agaaacccag ctgttgcctg 300
ttcatatcca gcccctgtat tgggatcaag atctgcccta ttctcagtgc agcaagatcc 360
aggcagatca cactggactc ccagcactga atctggctca aggggacatg aaatttgact 420
gggtcatggg gctcaggagc atcactctca aaaatagcag tacaggaaga ggcgatggcc 480.
ctaaacagca tttgcaggca gatcccatgt taatcataag ggccaggact ctctcactgt 540
ctgtctctct ctctgtctct cctctagggc tgaccccaca ttggacaccg ctgcatccat 600
gtccatcaca caacacagct gccgtttctt ctgcctgctt atgggaaagt cccctcttct 660
cctccgtttt cttctcttcc tgccctatca caccgtgcac ttctcccttt ccttaaagaa 720
ccaccatcaa ctttaggagg agggaaaggg gtggctctgg caggaaaagc cagaatcccc 78~
tctagccagc agagagagag aggaatggct gcatgttttc tcccccagtc caaggcactg 840
ggtcttggct gggttgaagg ttccaagctg ctctcctgct gtgtcggtga gttctggtca 900
acctgcaacc tcctgatatg gccattgcag ttcatcgagt cttcagggac tccccatggc 960
ctggagtact ttgccttgct tacacgggag aggagaatgg atttatagag aacatcatct 1020
aaatccaact tgaccattgt gtggccacac ttgctagatt gctatagtct aaatctagca 1080
ttgtagaaag acgggggagc ttggagctgc acaaacccag gtctggaact ggctccttac 1140
cttggaaggt gaatgatcct gccaggactc ttagcctccc tgggtctcaa tttctttatc 1200
tgtttcatgg gaatgaggat ctctgctggg tggttgggtg atgtgggggc tgtgtgaaaa 1260
cagcttgtca atacaagcca aaatagaaat atttctccac agagtatgaa ggtcaaatga 1320
gagaatacat ttaaattaaa tggaaaatta aaatggtaaa aaatgcaaag ctgtattgaa 1380
agttccgagc ttctctataa ggagcttttt gactatgtaa gaatcctgta ctcgttcccc 1440
ctaaatataa aaaaaagttg aaggaggcag aagggagagt gatgcacgat gggcaaggac 1500
ttcacctgct gttgctggct ttgaggatgg aggaaggagg ccacaaacct agaagctgga 1560
gcccctagaa gctagaaaag gcagggaccc gattctttcC ttgagcctcc agaagggaca 1620
cagccccgcc agcaccttga ctttagcctg gtgagatcct cttaggactt ttggcaatca 1680
gaactataag acagaaatgg aagccactga gtctgtagct gtttgttgca gcagcaatag 1740
aaaactaatg cagggcccaa gaaatcactg gtgatgagat cgggaaagtg ggctcaggag 1800
gtctggatct gtgatgagat ggggaaagtg ggctcaagag gtctggatct gtggtgagat 1860
gggggaagtg ggctcaggag gtctggatct gtgatgagat ggggaaagtg ggctcaggag 1920
gtctggatct gtgatgagat ggggaaagtg ggctcaggag gtctggatct gtgatgagat 1980
gggggaagtg ggctcaggag gtctggatct gtgatgagat gggggaagtg ggctcaggag 2040
gtctggatct gtgatgagat gggggaagtg ggctcaggag gtctggatct gtgatgagat 2100
ggggaaagtg ggctcaggag gtctggatct gtgatgagat gggggaagtg ggctcaggag 2160
gtctggatct ggggtgggga tctggagtgg aaggggaatt catttgttca ttgtctatcc 2220
ttttgtattg attgaatttt ttatatatat atgtgaattt tcacaataaa atttttttcc 2280
aaaataaaat aaacaaaagg ggctttttgc aacccaattc ctatctaaaa aaaaaaaa 2338
<210> 72
<211> 481
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1832219CB1
<400> 72
cctgggcggc gttggtccgg tgcgtcctgt tctacagcta tggccgggcc agctgcagct 60
ttccgccgct tgggcgcctt gtccggagct gcggccttag gcttcgcttc ctacggggcg 120
cacggcgccc aattcccaga tgcctacggg aaggagctgt ttgacaaggc caacaaacac 180
cacttcttac acagcctggc cctgttaggg gtgccccatt gcagaaagcc actctgggct 240
gggttattgc tagcttccgg aacgacctta ttctgcacca gcttttacta ccaggctctg 300
agtggagacc ccagcatcca gactttggcc cctgcgggag ggaccctgct actcttgggc 360
tggcttgcct tggctctttg agctcccttt tgcttaatta ctgggttttc tgggcagttt 420
tttttttaaa gagttggagt aagaagagga ttaaaaagga aaggcaaata aaaaaaaaaa 480
a 481
<210> 73
<211> 1255
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
60/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
<223> Incyte ID No: 1899010CB1
<400> 73
cggacggtgg gcggacgcgt gtgctgcggc gtcctagctg gcttacaggg cggcggcggg 60
gtgtgtgtcc tctgttaaga gtgctactcg cccggggttg atctgtgcat gccactcctg 120
ggtcagacgg tgaggtcggc gtctgcgagg acgcggcggt ggagtagaag ggcagccgga 180
gacaggcccg gcgccccttc cgaggctaga cggccccagc ttcgcgggga tcatggcatt 240
gctggtggac cgagtgcggg gccactggcg aatcgccgcc gggctcctgt tcaacctgct 300
ggtgtccatc tgcattgtgt tcctcaacaa atggatttat gtgtaccacg gcttccccaa 360
catgagcctg accctggtgc acttcgtggt cacctggctg ggcttgtata tctgccagaa 420
gctggacatc tttgccccca aaagtctgcc gccctccagg ctcctcctcc tggccctcag 480
cttctgtggc tttgtggtct tcactaacct ttctctgcag aacaacacca taggcaccta 540
tcagctggcc aaggccatga ccacgccggt gatcatagcc atccagacct tctgctacca 600
gaaaaccttc tccaccagaa tccagctcac gctgattcct ataactttag gtgtaatcct 660
aaattcttat tacgatgtga agtttaattt ccttggaatg gtgtttgctg ctcttggtgt 720
tttagttaca tccctttatc aagtgtgggt aggagccaaa cagcatgaat tacaagtgaa 780
ctcaatgcag ctgctgtact accaggctcc gatgtcatct gccatgttgc tggttgctgt 840
gcccttcttt gagccagtgt ttggagaagg aggaatattt ggtccctggt cagtttctgc 900
tttgcttatg gtgctgctat ctggagtaat agctttcatg gtgaacttat caatttattg 960
gatcattggg aacacttcac ctgtcaccta taacatgttc ggacacttca agttctgcat 1020
tactttattc ggaggatatg ttttatttaa ggatccactg tccattaatc aggcccttgg 1080
cattttatgt acattatttg gcattctcgc ctatacccac tttaagctca gtgaacagga 1140
aggaagtagg agtaaactgg cacaacgtcc ttaattgggt ttttgtggag aaaagaatgt 1200
tgtcccaaga agataaaaaa tattgttaag tgtgcaagtt attaaaaaaa aaaaa 1255
<210> 74
<211> 875
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2008768CB1
<400> 74
tagctgagca cgccctctga gccgctcggt ggacaccagg cactctagta ggcctggcct 60
acccagaaac agcaggagag agaagaaaca ggccagctgt gagaagccaa ggacaccgag 120
tcagtcatgg cacctaaggc ggcaaagggg gccaagccag agccagcacc agctccacct 18O
ccacccgggg ccaaacccga ggaagacaag aaggacggta aggagccatc ggacaaacct 240
caaaaggcgg tgcaggacca taaggagcca tcggacaaac ctcaaaaggc ggtgcagccc 300
aagcacgaag tgggcacgag gagggggtgt cgccgctacc ggtgggaatt aaaagacagc 360
aataaagagt tctggctctt ggggcacgct gagatcaaga ttcggagttt ggacctcttc 420
aacgacctga ttgcttgtgc gttccttgtg ggagccgtgg tctttgctgt gagaagtcgg 480
cgatccatga atctccacta cttacttgct gtgatcctta ttggtgcggc tggagttttt 540
gcttttatcg atgtgtgtct tcaaagaaac cacttcagag gcaagaaggc caaaaagcat 600
atgctggttc ctcctccagg aaaggaaaaa ggaccccagc agggcaaggg accagaaccc 660
gccaagccac cagaacctgg caagccacca gggccagcaa agggaaagaa atgacttgga 720
ggaggctcct ggtgtctgaa acggcagtgt attttacagc aatatgtttc cactctcttc 780
cttgtcttct ttctggaatg gttttctttt ccattttcat taccaccttt gcttggaaaa 840
gaatggatta atggattcta aaagcctaaa aaaaa 875
<210> 75
<211> 2188
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2070984CB1
<400> 75
cggcgacggc gacggcagcg gggacggcag cagtagcggg agcagcagcg tggacgcggc 60
tggcgctggc gccatgaacc cgctgtaagg cgcaggctgt gcagcacggg gtgcggggga 120
61/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
ggaggaggag gacgccgcgg tgaagttctc cgccatgaac ctgaggggcc tcttccagga 180
cttcaacccg agtaaattcc tcatctatgc ctgtctgctg ctgttctctg tgctgctggc 240
ccttcgtttg gatggcatca tacagtggag ttactgggct gtctttgctc caatatggct 300
gtggaagtta atggtcattg ttggagcctc agttggaact ggagtctggg cacgaaatcc 360
tcaatatcga gcagaaggag aaacgtgtgt ggagtttaaa gccatgttga ttgcagtggg 420
catccacttg ctcttgttga tgtttgaagt tctggtctgt gacagaatcg agagaggaag 480
ccatttctgg ctcctggtct tcatgccgct gttctttgtt tccccggtgt ctgttgcagc 540
ttgcgtttgg ggctttcgac atgacaggtc actagagtta gaaatcctgt gttctgtcaa 600
cattctccag tttatattca ttgccttaag actggacaag atcatccact ggccctggct 660
tgttgtgtgt gtcccgctgt ggattctcat gtcctttctg tgcctggtgg tcctctacta 720
cattgtgtgg tccgtcttgt tcttgcgctc tatggatgtg attgcggagc agcgcaggac 780
acacataacc atggccctga gctggatgac catcgtcgtg ccccttctta catttgagat 840
tctgctggtt cacaaactgg atggccacaa cgccttctcc tgcatcccga tctttgtccc 900
cctttggctc tcgttgatca cgctgatggc aaccacattt ggacagaagg gaggaaacca 960
ctggtggttt ggtatccgca aagatttctg tcagtttctg cttgaaatct tcccatttct 1020
acgagaatat ggaaacattt cctatgatct ccatcacgaa gataatgaag aaaccgaaga 1080
gaccccagtt ccggagcccc ctaaaatcgc acccatgttt cgaaagaagg ccagggtggt 1140
cattacccag agccctggga agtatgtgct cccacctccc aaattaaata tcgaaatgcc 1200
agattagatg ccacttccgg ggacagagct taagtggact gggacgcact ctctccgcct 1260
tcctctgccc cctcgttcac cccgcagacc agaaccagta ctggagctgg gtctccaggt 1320
acgtccatct catgccttgt ttgcatccag cgcctatcag ccactcacca cgacgggacg 1380
cggaagtggc aggtgacggg ggtgtgtgcc agcagatgcg gatgccagga agagtgtgag 1440
aacaggggtg ggattaccgt ctgtctggga ggggctccag gtacccctct tccccgtcag 1500
acccactggg agatggctgc ttgccaggcc cccagaagga acatctgtct atacggtgct 1560
gaaatcccaa tcaaaagtat tgtttagaaa tgtatttctc cacagggctg acctcctgca 1620
gctcgctgag cactcccagg tcctcagcac tcccaggtcg tggctggggc agtcagtagg 1680
aactgtaact atgtctctga tgcaccacgt gtttagacac agcacagtcc ttttttctgt 1740
tcctactgtg gaagtagttt ctctttgggc atgctgacag cagtttttca tagcctcacg 1800
gatgagccct ttctacggga gtgactccat gcttgtatac agagtattta tacaaatgtt 1860
ttagcatctt catatgcggt gttaacccct agttctgtac agcatattct gttcaagtat 1920
ttttttacaa gcttgtgctg taggcacatg ccttctgctg cagaagtgga cgcccgtggc 1980
acactccccc cccccccccg tggggtgcca cgccttcatg ggacattgcc acttctgccc 2040
tggaactcgt gcaggtacgt agtagctgct actgccacaa cggcaacacc aagcaagaga 2100
tggtccatgc ttttctgacg ttctcagaat agtggctagc ttcaaacctg acaagcgctg 2160
cttgaagccg gaacactaga gaatgttg 2188.
<210> 76
<211> 1561
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2193240CB1
<400> 76
tctggccatg ctcctgagga aagctggtgc catcacactc ccgtctgtta ccgtggccct 60
ggccaagcac tggacagcgg cgattgtgct atgaagggaa atcctccaaa gatatcctga 120
aaagagtagc tgcaaatgca ttgatgtcac tgctggctgt cagtagaaga gcacagaaac 180
atgctttgaa agccaatctt atagacaatt gcatggagca gatgaaacac ataaatgcac 240
aactgaacct agattctctg aggcctggga aagcagcatt gaaaaaaaag gaggatggtg 300
ttattaaaga gttaagcatt gccatgcagc tcctaagaaa ctgtetttat caaaatgagg 360
aatgtaaaga agcagctctt gaagctcacc ttgtccctgt cttgcactct ctctggcctt 420
ggattttgat ggatgattca ttgatgcaaa tttctctgca gctcctttgt gtctatactg 480
caaattttcc aaatggttgc agttctcttt gttggtcaag ttgtggacaa caccctgttc 540
aagctacaca tagaggagcc gtgagcaact ctctgatgct gtgtatccta aagttggctt 600
cccagatgcc actggagaac accacggttc agcagatggt ttttatgctt ctttcaaacc 660
tggccttgtc gcatgactgt aaaggagtaa ttcagaagag taacttctta cagaacttcc 720
tctctctagc attgccaaaa ggaggaaata aacatctaag taatctgact attctttggt 780
tgaagttact cctgaatata tcatctggag aagatgggca acaaatgatt ctgaggcttg 840
atggctgtct agacttacta acagagatga gcaaatacaa gcacaagagc agccctttat 900
tgcctcttct tatctttcat aatgtttgct tcagtcctgc aaataaaccc aagatcctgg 960
ctaatgaaaa agtcattact gtgcttgctg cctgtctgga aagtgagaat caaaatgctc 1020
62/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
agaggattgg agcagctgcc ctttgggctc tgatttacaa ttatcagaag gcaaaaacag 1080
ctttgaaaag cccatcagta aaaagaagag tggatgaagc atactcctta gcaaagaaaa 1140
ctttcccaaa ctcagaagca aaccctctaa atgcctatta tttgaaatgt cttgaaaacc 1200
tcgtgcagct ccttaattct tcctgagtgc catgggatgc tacaccttga agctgacagt 1260
catcaacagg ggagctaaag ttgaagccag ctgtgtgtag cagctgttac ctgaagacgt 1320
gctacctctc tacaaagtgt tgatcccctt ctttcccatg agagagagaa ctggtgatac 1380
tccaacaccg tccagttgtg gcagctctcc agaagtaata gcagctgaca actttctgtg 1440
ccttttcctt tctgttgaaa aggcatagaa agttctggga acataaacat ttttaccctt 1500
ttctatgcca tttattttgt aaaaatccta tttaacagtt atttaataaa acaagatttt 1560
g 1561
<210> 77
<211> 1777
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2235177CB1
<400> 77
ccaacatcag cgagggaggc agcttcgggg agctggggac catgggctcc aggatcaagc 60
agaatccaga gaccacattt gaagtatatg ttgaagtggc ctatcccagg acaggtggca 120
ctctttcaga tcctgaggtg cagaggcaat tcccggagga ctacagtgac caggaagttc 180
tacagacttt gaccaagttt tgtttcccct tctatgtgga cagcctcaca gttagccaag 240
ttggccagaa cttcacgttc gtgctcactg acattgacag caaacagaga ttcgggttct 300
gccgcttatc ttcaggagcg aagagctgct tctgtatctt aagctatctc ccctggttcg 360
aggtatttta taagctgctt aacatcctgg cagattacac gacaaaaaga caggaaaatc 420
agtggaatga gcttcttgaa actctgcaca aacttcccat ccctgaccca ggagtgtctg 480
tccatctcag cgtgcattct tattttactg tgcctgatac cagagaactt cccagcatac 540
ctgagaatag aaatctgaca gaatattttg tggctgtgga tgttaacaac atgttgcatc 600
tgtacgccag tatgctgtac gaacgccgga tactcatcat ttgcagcaaa ctcagcactc 660
tgactgcctg catccacggg tctgcggcga tgctctaccc catgtactgg cagcacgtgt 720
acatccccgt gctgccgccg catctgctgg actactgctg tgctcccatg ccctacctca 780
taggaatcca tttaagttta atggagaaag tcagaaacat ggccctggat gatgtcgtga 840
tcctgaatgt ggacaccaac accctggaaa cccccttcga tgacctccag agcctcccaa 900
acgacgtgat ctcttccctg aagaacaggc tgaaaaaggt ctccacaacc actggggatg 960
gtgtggccag agcgttcctc aaggcccagg ctgctttctt cggtagctac cgaaacgctc 1020
tgaaaatcga gccggaggag ccgatcactt tctgtgagga agccttcgtg tcccactacc 1080
gctccggagc catgaggcag ttcctgcaga acgccacaca gctgcagctc ttcaagcagt 1140
ttattgatgg tcgattagat cttctcaatt ccggcgaagg tttcagtgat gtttttgaag 1200
aggaaatcaa catgggcgag tacgctggca gtgacaaact gtaccatcag tggctctcca 1260
ctgtccggaa aggaagtgga gcaattctga atactgtaaa gaccaaagca aatccggcca 1320
tgaagactgt ctacaagttc gcaaaagatc atgcaaaaat gggaataaaa gaggtgaaaa 1380
accgcttgaa gcaaaaggac attgccgaga atggctgcgc ccccacccca gaagagcagc 1440
tgccaaagac tgcaccgtcc ccactggtgg aggccaagga ccccaagctc cgagaagacc 1500
ggcggccaat cacagtccac tttggacagg tgcgcccacc tcgtccacat gttgttaaga 1560
gaccaaagag caacatcgca gtggaaggcc ggaggacgtc tgtgccgagc cctgagcaaa 1620
acaccattgc aacaccagct acactccaca tcctacagaa aagcattacc cattttgcgg 1680
ccaagttccc gacgagaggc tggacctctt catcacattg acttacgccg ttgcttttcc 1740
agactgggca gaggggctga cttcgcagtg tgtgcca 1777
<210> 78
<211> 1841
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2416227CB1
<400> 78
cgaaagagaa acccggaggg cgccggggac tgggccgggg tctgcagggc tcagctgagc 60
63/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
ccatgagctc ccagagctaa cccctgaaca cccaggcggg caaagggctg atgtcggtag 120
tccccatcct ggaggggcag gctctgcgca tctgctcctg gcatggcgct gcggcacctc 180
gccctcctgg ctggccttct cgtgggagtc gccagcaagt ccatggagaa cacggcccag 240
ctgcccgagt gctgtgtgga tgtggtgggc gtcaacgcca gctgcccagg cgcaagtctg 300
tgtggtccag gctgttacag gcgctggaac gcggacggga gcgccagctg cgtccgctgt 360
gggaacggaa ccctcccagc ctacaacggc tccgagtgta gaagctttgc tggcccgggt 420
gcgccattcc ccatgaacag aagctcaggg acccccgggc ggccacatcc tggggctccg 480
cgcgtggccg cctccctctt cctgggcacg ttcttcatta gctccggcct catcctctcc 540
gtagctgggt tcttctacct caagcgctcc agtaaactcc ccagggcctg ctacagaaga 600
aacaaagctc cggccctgca gcctggcgaa gccgctgcaa tgatcccccc gccacagtcc 660
tcagtacgga agccgcgcta cgtcaggcgg gagcggcccc tggacagggc cacggatccc 720
gctgccttcc cgggggaggc ccgtatcagc aatgtctgac ctggaggccg agaccacgcc 780
acgcacttgg cggcagggac ccggaggccg aCCCCttggC gggaaccagc acaaagtgtt 840
ggcatcgccc ggcgcccggg acagtcctgg gcacagcctc ggctctgagt ccctccgcct 900
cccagcgacg gacgccaaag ggtcccgggc cgcctgaggc tCCtCCCCaC CaCagCCatC 960
tcgtttatcg gaccaggagc aggcatccat gagacctcag agcttcagat cgaggccttg 1020
gggggtccgg gcccccccag gaaacacggt gaggccccag cgcctgcagc caaagctggc 1080
acgatctatg gggcaggtgc cgctctgcct agaaaagcca ggggctctgc tgccgtgccc 1140
tccagagccc acagcgggca ggactcctcc agcaccacca cacccagtgg cccgagaccc 1200
ctctgagaac agtgaggctg gtcctcgtgc cgttccagcc ggtgcccggc cagtggggag 1260
gaoacagcct aggaaccagc tgcctgagac cagggtgcct ctgggctgtc ctcccgcgtg 1320
gcggagaccc caagcacgca gCCaCCCatt tCCggagCtg caggatagag cttcctcttg 1380
atctctgttt ttaagcagaa attcattgtg cagaaaagtc ctccagagct CtgtggCCCC 1440
gctcggatcc gCtggaCCCC CatgCCtggC tgatccctgc ccacgtgggg caggcccaca 1500
tctaaccccc acaagtcact gcctcactgc acctgccaag gctgccctgg cgctgagtcc 1560
tggggtccct cccggagttc ctgggagaaa ggcgccgtcg tggccgcctc ccgcacgcca 1620
ggcccgggct ccaccgtggg tctcagacgc cctgcggcac cggcaccgtc tgctttagca 1680
tgggaccccc atctgagggg tggcctggcc ttcggggtcc ccacgctcct ttgcgaagtc 1740
cactgtgggt gccatcatgg tctccgggac ctgggccagc gggaacgtgg gggcactggg 1800
tgtgctgata taaagtcggc attactcaaa aaaaaaaaaa a 1841
<210> 79
<211> 1616
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2461076CB1
<400> 79
agctggagac agagaggctg gccattgcgg gcataagcag aggcctagat caacagtgcc 60
aaggactggg atgagggtag tggtggtggg agtggagaac catgatgatg taagagacag 120
ttagtggctt ctgatccagg gaaggtggca gtgtagtgac agaaacctga aggggagtgg 180
atgggagtga tggccccact gcctgggggg agaggccagg ctggaggcgg gacctggagg 240
aatggaggcc agtgctaccc tcacactccc agcctcctgt cctggtggga tttggaccca 300
cagatcccca tggtgcatat gtcaagtctg gcctgttcca cctctttctc aaaggaaggt 360
tctgagcctc cctacccagt gtaggggtgg caacgatccc tcattcattc aagcatttat 420
gaaccaccct ccctaagtct agctcatgca gcaccaaatt tattcactta gctaatgatt 480
gacctactgc tgtgttctag gcaccaggga taccacatgg aggcgtgcgc cagagccctg 540
tcccctggag cccttgggtt caaatactgg ttctacctgg gagtgggcag tagttggcct 600
cccagccttg catcctcagc cattgaatta gttaacatgt gaagactctt agaacagcgt 660
aagcgctgca gaagaatttg ctgctgctgt tattattatt ccagacatta ttataaacag 720
aaagccgttc tgagctcaga agcgaactct cagcgggtcc ttctctaccc agaagaggtg 780
ttcataggaa cctgttagtc agagtgggaa cgggccaagg gtcactaggt cactgttttt 840
ttgttggttt ttttaagttt tataattaat ataaaaatgg ggtctcgcga tgttggccat 900
gttgttcttg aacttttggc ctcaagcagt cctcccgcct cagcctccca aagtgctagg .960
attacgggca cgagccacca catccagcca agctgggtca ctttgaactg aggccagggt 1020
ggattgcaga cgtggggcac atcccagccc ctgaattcag ggtccaccat cacacctgct 1080
cctgcctagc atcctgagag ggctgcccag aaaagagctt ttacaaacca gctgttgata 1140
agctcagaat tttagaaacc actgtcttca ttataagggc ataaggtaca aggatgaaac 1200
tccccctgtc tttactcttt cttagaactc tgggttttta catcccagtg aaaggggatc 1260
tgtcctctgg ttgtgaggac aaggcatgtc tttatgtgtt aaaaagagta accactgaca 1320
64/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
aggttttttt tgatcctttt aaaatttact ttcgacctgt aatcccagga ctttgggagg 1380
ccgaggcagg tggatcactt gggttaggag tttgagacca gcctggtcaa catggtgaaa 1440
ccccgtctct actaaaaaat acaaaaatta gctgggtgtg gtggtgtgca cctggaatct 1500
cagctattcg ggaggctaag acaggagaat cgcttgaacc caggaggcag aggttgcagt 1560
gagccgagat cacgccactg cactctagcc tgggtgacag agcgagactc catgtc 1616
<210> 80
<211> 1434
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1957517CB1
<400> 80
ccgccccgtc cgttgagggc ccgcgccgca tggaggccgg ctgaggagcg ccgctgcctc 60
gcctcggctc cccacaggtg caggaagccg ccgcccagcc atggacgggg aggagcagca 120
gccaccgcac gaggccaacg tggaacctgt tgtgccgtca gaggcttcag agccggtgcc 180
cagggtgctt tctggagacc cccagaacct gtccgacgtg gacgccttca acctgctcct 240
ggagatgaag ctgaagcggc ggcgtcagcg gcccaacctg ccgcgcactg tgacccagtt 300
ggtggctgag gacgggagca gggtgtacgt ggtggggaca gcccacttca gcgacgacag 360
caagagggac gttgtgaaga ccatccggga ggtgcagcct gacgtggtgg tcgtggagct 420
ctgccaatat cgtgtgtcca tgctgaagat ggacgagagc acgctgctgc gggaggccca 480
ggagctcagc ctggagaagc tgcagcaggc cgtgaggcag aacgggctca tgtcggggct 540
gatgcagatg ctgctgctga aggtgtctgc acacatcacc gagcagctgg gcatggcccc 600
aggtggcgag ttcagggagg ccttcaagga ggccagcaag gtgcctttct gcaagttcca 660
cctgggtgac cgacccatcc ccgtcacctt caagagggcc atcgcagcgc tctccttctg 720
gcagaaggtc aggctggctt ggggcctgtg cttcctgtca gaccccatca gcaaggatga 780
cgtggaacgc tgcaagcaga aggacctact ggagcagatg atggccgaga tgattggcga 840
gttcccagac ctgcaccgca ccatcgtctc ggagcgcgac gtctacctaa cctacatgct 900
gcgccaggcc gcgcggcgcc tcgagctgcc tcgggcctct gacgccgagc ccaggaagtg 960
cgtcccctcc gtggtcgtgg gcgtcgtggg catgggccac gtgcctggca tcgagaagaa 1020
ctggagcacc gacctcaaca tccaggagat catgaccgtg cccccgccgt ccgtctccgg 1080
cagagtgtct cggttggccg tgaaggccgc cttcttcggc ctgctgggct acagcctgta 1140
ctggatgggc cgccgcaccg cgagcctggt cctgtcgctg cccgccgcgc agtactgcct 1200
gcagagggtg accgaggccc ggcacaagta ggagactgct ccccgcccgc tcgggcccct 1260
gaggagccag tgcccccgcg gcacttctgg gtgccaggtg catcctagcc cgcccgaggc 1320
ccctgccacc ccccatgggg gtctgggccc ggcctcgcct gccctcctgg gccagtcacc 1380
cctcccccag cccacccaaa taaaggatta tttaactgtc tgaaaaaaaa aaaa 1434
<210> 81
<211> 2085
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 866038CB1
<400> 81
ccgcgcccgg ccccgccatg gtgtcctgga tcatctctcg cctggtggtg ggctgatgcg 60
gagctgggag ggagaggcca ctgcccttgg ctgagaggcc cagtaaccat gcctgcctcc 120
ctaggctcat ctttggcacc ctgtacccag cctattcttc ctacaaggcc gtgaagacaa 180
aaaacgtgaa ggaatatgtg aaatggatga tgtactggat cgtctttgcc ttcttcacca 240
cggccgagac gctcacggat atagtgctct cctggttccc cttctacttt gaactgaaga 300
tcgccttcgt gatatggctg ctgtcccctt acaccaaggg ctccagcgtg ctctaccgca 360
agttcgtgca cccaacgctg tccaacaagg agaaggagat cgacgagtac atcacgcagg 420
cccgagacaa gagctatgag accatgatga gggtgggcaa gaggggcctg aaccttgccg 480
ccaatgctgc agtcacagct gccgccaagg gccagggggt gctgtcagag aagctccgca 540
gcttcagcat gcaggacctg accctgatcc gggacgagga cgcactgccc ctgcagaggc 600
ctgacggccg cctccgaccc agccctggca gcctcctgga caccatcgag gacttaggag 660
atgaccctgc cctgagtcta aggtccagca caaacccggc agattcccgg acagaggctt 720
65/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
ctgaggatga catgggagac aaagctccca agagggccaa acccatcaaa aaagcgccca 780
aagctgagcc actggcttcc aagacactga agacccggcc caagaagaag acctctggcg 840
ggggcgactc agcttgagcc cctccacccc cgcaggctgc agagcaagga tgaagcctca 900
ggaggggcct cagacccagc ccctgctcca cactgtgcca gtagcctagg tgtctcaggc 960
ccctgggccc cgcagatggc catttccggt gcctgcccag tggccactct tctggaaggg 1020
gcttggaaaa gaggaaggag gcccagctgt gggggttgag ggtagagggt ggaccagagg 1080
ctgaggactg agccacccaa ggaggtgggg aCtgCtCggC CtCCaCCgCt gttCCJCtgC 1140
agccccgccc tgccccaccc acccagtgcc ttgctgaagc ccatagcaat ccgcttctca 1200
gaggtcctat cgtgtttcca ctgctcgcct tggtttggga gcagggaggg ggaagtccta 1260
gcccagatgg accaaggacg ggcctgaagg cacatggggg aaagggagca cacggggagg 1320
acgttgggga ccctgggtgg ggcctccagg tgcagctgtg gatggaagac agggattggc 1380
ctgtgcttca gcgaccagga tggccaggcc agagctgcag ctgggggctc ttttcctggt 1440
cattgggtgg ggctgagtgc cacatgttcc cacattaaaa aggggggtcc agggctgtgt 1500
gagtgtgtct ttctgggtct agggctcggg gtagtttggg tcaaggactg tccctccagc 1560
agtcgcctcc tCCCaCCCtg agccccacag tcatctggcc ctttccctgc tCaaCCCtCC 1620
atcctaggct ctgagcctca gaggacccag cccatgagag aacggggatc tggggggcct 1680
ctcacctgct cctatgacct tgctcccttt taggtcaccc cattgccacc gtgcccctgg 1740
gctggactcc cgtgctcctc agggcccacc cctgctctgt ctggtacagg cccctgctga 1800
gtgggcccct ctcctctgcc cctggggtcc atcccctctt gcccagggtc cccatcctgt 1860
accaagcaga ctgggcccta agacccctgg cagaacccag cctctgctca cacccgcccc 1920
agcttctgcc acggcttcag tcagggccag gaggaacacg tgaaggagaa agagaaatgc 1980
aggagccgcg gggctcccgg ttccttggga agagggtgcc cattggacct ttggcactgg 2040
atgagccaat aaaccaaact ctggcacctc aaaaaaaaaa aaaaa 2085
<210> 82
<211> 904
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ZD No: 3869704CB1
<400> 82
ggggagccca gctgtgctgt gggctcagga ggcagagctc tgggaatctc accatggcct 60
ggacccctct cctgctcccc ctcctcactt tctgcacagt ctctgaggcc tcctatgagc 120
tgacacagcc accctcggtg tcagtgtccc caggacaaac ggccaggatc acctgctctg 180
gagatgcatt gccaaaaaaa tatgcttatt ggtaccagca gaagtcaggc caggcccctg 240
tgctggtcat ctatgaggac aacaaacgac cctccgggat ccctgagaga ttctttggct 300
ccagctcagg gacaatggcc accttgacta tcagtggggc ccaggtggag gatgaagctg 360
actactactg ttactcaaca gacagcagtg gtaatgatag ggtgttcggc ggagggacca 420
agctgaccgt cctaggtcag cccaaggctg ccccctcggt cactctgttc ccaccctcct 480
ctgaggagct tcaagccaac aaggccacac tggtgtgtct cataagtgac ttctacccgg 540
gagccgtgac agtggcctgg aaggcagata gcagccccgt caaggcggga gtggagacca 600
ccacaccctc caaacaaagc aacaacaagt acgcggccag cagctacctg agcctgacgc 660
ctgagcagtg gaagtcccac aaaagctaca gctgccaggt cacgcatgaa gggagcaccg 720
tggagaagac agtggcccct acagaatgtt cataggttct catccctcac cccccaccac 780
gggagactag agctgcagga tcccagggga ggggtctctc ctcccacccc aaggcatcaa 840
gcccttctcc ctgcactcaa taaaccctca ataaatattc tcattgtcaa tcagaaaaaa 900
aaaa 904
<210> 83
<211> 1496
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte 2D No: 1415179CB1
<400> 83
gacgacagaa gtttgtacgg ttccaggagc tggctgtgca ggctcctcgg gccttctgct 60
acatgcctgc tggggacgcc cagctactcc tggcccccag cttcaaggga cagacgctgg 120
66/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
tgtatagaca cattgtggtg gatctcagtg cctagggggt gccgaggcct ctggtctcct 180
cagggtggcc actggaggat gggtgggggc ctctgtgagc accacgtatg cctgtgtgaa 240
gacaagcgac caacctgcat tcatgccctt gtatccgcac acgcccagtg tgtacaggcg 300
caccccgtgg actggcccag gaggacgtgc atcatcactg caatcagcat gaacaccagg 360
cacccctggg acacctaacg cccagttccc gtgaccccaa ctgcaggccc cccctactct 420
gcagcctttc ccatgctctg ccccctgtct catgcacgag tggtgcgggg tgctgggagc 480
gaggggggcc gCatCCtCCt gtCCttgtgC ttctccttct gcccctctgg tctttcctgt 540
tggtgctcca ggcattgttt acctgctctt gctccccgct gtagcccaca gccatatctg 600
tcatgcttcc ccggggccac tcacccctgc cccaccccgt ctgcatgctc acatggacgc 660
ggacggacac actcattaca cactcacacg ccccgactgc acccggtgtc catctataaa 720
catgtcaggg cacgtgtgca tacaagcaga ttcagcactg cctaccaggc tctcctgtta 780
ccttgcctct ctgcttggag ggggccccct ctgctcaccc ccagcgtgcc ccccccggaa 840
ctgataagga tgagaatggt ggtgccagct tctgagggcc tgcttggcct cctgggggca 900
aagcccctct gcccgaagca ataacaacag cagcagaagc aatcccgggg agctgccagg 960
ggtgtctgtc tctgttctcc ttggcgtttc cttcatggcc gtgaatccca gctcacccat 1020
ggacagaaga cctcccctca tctgaaagcc tttctgcctt ccttcctgta gctgtcgctt 1080
tctcaggctt ggactgtccc ctggttcacc ctgtctttgg gatgtggccc ttgagctggg 1140
cgggctcctt gggtggatta gagagctggc cgagctgcaa gcacctgagt tacctgcgag 1200
ggtgcccaga gtggagcttg gcctgagggc gtgttgcagc cctggcccca ccagtccaca 1260
gaggccccca ggagcactgc ctttcagtgg accgggtggg ggctggtggg aggggcccca 1320
ctagctgagc ttctgctgcc ctctgctgtc tgctgagatg ccaggctagt ggagagggca 1380
caccatgtgc ccaaatggtg tgatgctcct tgtagcagga taccagggta ctggggggca 1440
atgctatgat taacttgctt caaataaaaa gttcccgccc caaaaaaaaa aaaaaa 1496
<210> 84
<211> 2837
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1664792CB1
<400> 84
gctgcagctg cgccgccgcc ctggcgcagg CCdCgCCCga CCaCttCCaC atgCagtCgg 60
agtctctgat CgggCCCCtg atgcagacca tCtCCCaCCa gcactggaag gtccgtgtgg 120
ccgccattga agccacaggc gcagtgatcc attttggcaa cgggaagtcc gtggacgacg 180
tgctttccca ttttgctcag cgactgtttg atgacgtccc gcaggtccgg cgggcggtgg 240
cctccgtggt gggcggctgg ctgctgtgtc tgcgtgaccg ttactccttc ttccacaagc 300
tcatccctct gctgctcagt agcctcaacg acgaggtgcc tgaggtcagg cagctggctg 360
ccagcctctg ggaggacgtt ggcctgcagt ggcagaagga gaatgaggag gacctgaagg 420
acaagctgga ctttgcccct cccaccccac cccattaccc tccacatgag cgccgccctg 480
tgctgggctg ccgggagctc gtcttcagga acctctccaa gatcctccct gccctgtgcc 540
acgacatcac cgactgggtg gtggggaccc gagtgaagtc ggcacagctg ctcccagtgc 600
tgctgctgca tgccgaggac cacgccacgc agcacctgga ggtcgtcctc cggaccctgt 660
tccaggcctg caccgacgag gaggcagccg tggtccaaag ttgtaccaga tctgcagagc 720
tcgtcgggac gtttgtcagc cctgaggtgt ttctgaagct gatcttatcg acgctgaaga 780
agacgccctc tgcctccggc ctcctggtgc tggcctccgc catgcggggt tgcccccgag 840
aagccctcca gccgcacctg gcagccatcg ccacagagct ggcacaggcc cacatctgcc 900
aggcatctga aaacgacctc tacctggagc gcctgctgct gtgtgtgcag gctctggtgt 960
ctgtgtgtca tgaggactgt ggcgtggcca gcctgcagct cttggacgtg ctgctgacaa 1020
tagtggccct cgcaggtgct accggcctga gggacaaggc acaggagacg atggactcac 1080
tggccatggt ggagggtgtc agcagctgcc aggacctcta ccgcaagcac attggtcccc 1140
tcctggagcg ggtgaccgcg tcgcaccttg actggaccgc acactcgccg gagctcctgc 1200
agttcagtgt catcgtcgca cagtcaggcc ctgccctggg agaagccctg ccacacgtcg 1260
tgcccacgct gagggcctgt ctgcagccct cccaagaccc gcagatgcgc ctgaagctgt 1320
tCtCCatCCt gtCCdCCgtg ctgctcagag ccacggacac catcaactcc caggggcagt 1380
ttcccagcta cctcgagacg gtgacaaagg acatcctggc ccccaatctg cagtggcatg 1440
cggggaggac agccgcggcc atccgcacgg ctgccgtgtc ctgcctctgg gcgctcacca 1500
gcagcgaggt cctgtcggca gagcagatac gggacgtgca ggaaacactg atgccccagg 1560
tcctgaccac cctggaggag gattcgaaga tgacgcgact gatctcatgc cgtattatca 1620
acacgttctt aaaaacctcg ggcggcatga cggatccaga gaaactcatc aagatttatc 1680
ctgaactctt aaaacgccta gatgacgtgt ccaacgatgt gaggatggca gccgcctcca 1740
67/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
ccttggtcac ctggctgcag tgtgtcaagg gtgccaacgc aaaatcctac tatcagagca 1800
gtgtccagta cctgtaccga gagttgctgg ttcaccttga cgatccagag agggccatcc 1860
aggatgcaat tttagaggtc ctcaaagagg gcagcgggct gttcccagat ctcctggtga 1920
gggagacgga ggccgtcatc cacaagcacc gctcggccac ctactgcgag cagctcctgc 1980
agcatgtgca ggccgtgcca gccacacagt gaccacgctg gtttcagcca cggcacaccc 2040
ttgtccccac ctgagccaga gtttgtggcc tttaaatctc ataaacaagg cacctctgtg 2100
ccagcagtga gactgtgaca gcaagaatgt actcctcagg acacctgccc actctttccc 2160
tggaataaca gcctctgagt ggattctgca tgttatgtga tttgttctgt tcatcgagag 2220
ggctcccaaa catctgcagc tgatttgaaa ttaaaagtaa gtcgcagccg ctcctcccgc 2280
agccacttca gcagcatctt agattttaag cctcacgtgc gcagctggtt catgaactat 2340
tggctgcatc ctgcttaggt gcccaccaag aaggttttta cctacttaac aaaaaagaaa 2400
gaagccaaag tgattagaaa gaaatgaaat ctctttttgg gttctgtcta ctgaaattta 2460
atatctcagt gaacagacta aaaggaattt agaatcctaa caacttacca gatttctcct 2520
gttttaaata tactgggact ttaaaggtta tatgtccggt caccgtatgt tttaagtcgg 2580
tgttaatgct aacagtgttg aaaacaatat ttcatgagat ctaattgtgg ttgcccctat 2640
aggtagcagg aaagtaaagt tgcatttccc tctcgcacat tctacaccca agtgcctaaa 2700
agatctcatt gtaagtgggt agtgttaccg gaagccattg tgttcacacg ggggaaatgc 2760
Cgtatatatt tttcaacaaa tattaacgtt tatactttca tgtttgaaaa tttaattaag 2820
aatatttgtt ttaaaaa 2837
<210> 85
<211> 1123
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2079396CB1
<400> 85
taagcttgcg accgccattt tttttttttt tttttttttt tttttactcg tgttgcaatt 60
ttactgtaag tttgaaactg tcaatacaaa aagttacaaa gaattgttta taaagaagaa 120
gaagaaaaaa gcaaacccaa ccaagctcaa gcctagatcc gtatttggat tcagctcgtc 180
tcggggcgga gccaggcgtc acggcctccg gattaaagta tccccccggg gagtgtgctc 240
tgtgaatctg ggtggggagg gcgctcggtg ctgttcccag caacccacca ccctcctcct 300
agtgcttgca agaataggca gggaactcag ctgactgcat cagaacctga gaagctggag 360
gctgaagcca gacaccagcc tctcaggact tggggacact atgagcccac tcagccccac 420
aggtctgaat ctttggggag gggagggttc cagtctgcac tctgcccttg accatcaggg 480
cagggggatc acactggcta tcggcatcat ctctagcagt ttctccagcc ccagccctag 540
aatcaggcca agctctcagc actgcgtggg tttgatcttg aggattcttt accaccatcc 600
aggactgggg ggctgccgct catgggtttt gttgttaaga gacagggtct cgctctgtca 660
cccaggctgg agtgcagtgg catgatcacg gctcactgta gccttgacct cccgggctca 720
agcgatcctc cggcctcagc ctcccgagta gctgcgacca caggcctgtg ccagcactcc 780
tggcttgctc gtgctcatct gctatgacgt catgaatccc accagctacg acaacgtcct 840
catcaaggtg aggccggcct cctgggtggt ggccccacat ctgggcccgg ccgcacccct 900
gatcccgcct cctccctcca cagtggttcc ctgaggtcac gcatttctgc cgcgggatcc 960
ccatggtgct catcggctgc aagacagacc tggaatgttc cgccaagttt cgggagaatg 1020
tggaggacgt cttccgggag gccgccaagg tggctctcag cgctctgaag aaggcgcaac 1080
ggcagaagaa gcgccggctc tgcctgctgc tctgacccag ggc 1123
<210> 86
<211> 1549 .
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5390115CB1
<400> 86
ttccggggga gcggcgcggc ggcgcgggag tgccacctga ttcactgcac ttggaaccag 60
aagatgttgg gctctttcca gaagtcaaat ccttcctggg aggatgaaga tctgccagct 120
ctgaggattc tggctgggaa gaaaaagagc ctcaagcttt gaaggccatt cccatggtag 180
68/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
acagagctga tgtgaaaagt ggccatcctg ttgccccagg cacatgacct tctgaagtga 240
ccaggctgaa agggaagcaa tactcgtgtg atctctcacc ccgtcactca gggtggcgca 300
atcacgactc attggctcac tgcagcctag acctcccagc tggagcaatt ctcctgcctc 360
agccttctga gtagctggga ctacagttgg ttctaaagag tggtgagtca gaagagacgt 420
caggcagcaa gcgacttggg ccatggcctc tgacctagac ttctcacctc cggaggtgcc 480
cgagcccact ttcctggaga acctgctacg gtacggactc ttcctgggag ccatcttcca 540
gctcatctgt gtgctggcca tcatcgtacc cattcccaag tcccacgagg cggaggctga 600
accgtctgag cccagaagtg ctgaggtgac gaggaagccc aaggctgctg ttccttctgt 660
gaacaagagg cccaagaaag agactaagaa gaagcggtag aagaggaggc ctgaggagct 720
gggcgggcag ggagagggtc ttggggacag ccctcctggg aatctacatt gtgttccccc 780
gcattccagg ctcagggtct gaggaggctg tgacgcccta tgaccgcaga gatctagaca 840
gtcgtaacag tccccaggct ccagctgggc aatccaccac ttcctcttcc ttctgcttct 900
gtgacggttt agagtcaagg gggctgaaac acactgtgag catagactgt attaggtttg 960
ttcagaagcc gggtcagctc acagagtcac attttcttgc ttagtcatgt gtccctcctt 1020
gagttgcccc ctccttgtgg gtttacacta catttgggag tcattgtcta atgctgacaa 1080
gcacaccctc tcccattatt tgtgcactac agatctcctg ctgatcagtc acctttgttg 1140
ctgctgtgta gacagagcca ggcctcacct gtttgtttag gccaagatgc catggacatg 1200
cagcgttagt gatcccacta gctgcgacag ccaggcccag aaaatgcctg gcgtgagagc 1260
cagcagacag ccaggccggg gtaggcagtg cctgcttctg ctccatcagg tgcaggggat 1320
ttggctgaag gcgtgcatat ttcctgggca caaacttcct gagcctctga aatgggaggc 1380
tcgtcaattt cagaccaacc tcttttcaac ccatcatagc acgttcaagg tgtgcctttt 1440
acttctacct gtacatcccc catcccttca attctttcat tccctgacca gtgagagggt 1500
tcctggggga agtatggtga ataaactgac atgcatgctt caaaaaaaa 1549
<210> 87
<211> 4820
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1403326CB1
<400> 87
ctcacactct aacgcgtgta gaatagctgg gaccacagtt tcagctgtga gttcaacatg 60
gaggcaaatc agtgccccct ggttgtggaa ccatcttacc cagacctggt catcaatgta 120
ggagaagtga ctcttggaga agaaaacaga aaaaagctgc agaaaattca gagagaccaa 180
gagaaggaga gagttatgcg ggctgcatgt gctttattaa actcaggagg aggagtgatt 240
cgaatggcca agaaggttga gcatcccgtg gagatgggac tggatttaga acagtctttg 300
agagagctta ttcagtcttc agatctgcag gctttctttg agaccaagca acaaggaagg 360
tgtttttaca tttttgttaa atcttggagc agtggccctt tccctgaaga tcgctctttc 420
aagccccgcc tttgcagcct cagttcttca ttataccgta gatctgagac ctctgtgcgt 480
tccatggact caagagaggc attctgtttc ctgaagacca aaaggaagcc aaaaatcttg 540
gaagaaggac cttttcacaa aattcacaag ggtgtatacc aagagctccc taactcggat 600
cctgctgacc caaactcgga tcctgctgac ctaattttcc aaaaagacta tcttgaatat 660
ggtgaaatcc tgccttttcc tgagtctcag ttagtagagt ttaaacagtt ctctacaaaa 720
cacttccaag aatatgtaaa aaggacaatt ccagaatacg tccctgcatt tgcaaacact 780
ggaggaggct atctttttat tggagtggat gataagagta gggaagtcct gggatgtgca 840
aaagaaaatg ttgaccctga ctctttgaga aggaaaatag aacaagccat atacaaacta 900
ccttgtgttc atttttgcca accccaacgc ccgataacct tcacactcaa aattgtggat 960
gtgttaaaaa ggggagagct ctatggctat gcttgcatga tcagagtaaa tcccttctgc 1020
tgtgcagtgt tctcagaagc tcccaattca tggatagtgg aggacaagta cgtctgcagc 1080
ctgacaaccg agaaatgggt aggcatgatg acagacacag atccagatct tctacagttg 1140
tctgaagatt ttgaatgtca gctgagtcta tctagtgggc ctccccttag cagaccagtg 1200
tattctaaga aaggtctgga acacaaagct gatctacaac aacatttatt tccagttcca 1260
ccaggacatt tggaatgtac tccagagtcc ctctggaagg agctgtcttt acagcatgaa 1320
ggactaaagg agttaataca caagcaaatg cgacctttct cccagggaat tgtgatcctc 1380
tctagaagct gggctgtgga cctgaacttg caggagaagc caggagtcat ctgtgatgct 1440
ctgctgatag cacagaacag cacccccatt ctctacacca ttctcaggga gcaggatgca 1500
gagggccagg actactgcac tcgcaccgcc tttactttga agcagaagct agtgaacatg 1560
gggggctaca ccgggaaggt gtgtgtcagg gccaaggtcc tctgcctgag tcctgagagc 1620
agcgcagagg ccttggaggc tgcagtgtct ccgatggatt accctgcgtc ctatagcctt 1680
gcaggcaccc agcacatgga agccctgctg cagtccctcg tgattgtctt actcggcttc 1740
69/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
aggtctctct tgagtgacca gctcggctgt gaggttttaa atctgctcac agcccagcag 1800
tatgagatat tctccagaag cctccgcaag aacagagagt tgtttgtcca cggcttacct 1860
ggctcaggga agaccatcat ggccatgaag atcatggaga agatcaggaa tgtgtttcac 1920
tgtgaggcac acagaattct ctacgtttgt gaaaaccagc ctctgaggaa ctttatcagt 1980
gatagaaata tctgccgagc agagacccgg aaaactttcc taagagaaaa ctttgaacac 2040
attcaacaca tcgtcattga cgaagctcag aatttccgta ctgaagatgg ggactggtat 2100
gggaaggcaa aaagcatcac tcggagagca aagggtggcc caggaattct ctggatcttt 2160
ctggattact ttcagaccag ccacttggat tgcagtggcc tccctcctct ctcagaccaa 2220
tatccaagag aagagctcac cagaatagtt cgcaatgcag atccaatagc caagtactta 2280
caaaaagaaa tgcaagtaat tagaagtaat ccttcattta acatccccac tgggtgcctc 2340
gaggtatttc ctgaagccga atggtcccag ggtgttcagg gaaccttacg aattaagaaa 2400
tacttgactg tggagcaaat aatgacctgt gtggcagaca cgtgcaggcg cttctttgat 2460
aggggctatt ctccaaagga tgttgctgtg cttgtcagca ccgcaaaaga agtggagcac 2520
tataagtatg agctcttgaa agcaatgagg aagaaaaggg tggtgcagct cagtgatgca 2580
tgtgatatgt tgggtgatca cattgtgttg gacagtgttc ggcgattctc aggcctggaa 2640
aggagcatag tgtttgggat ccatccaagg acagctgacc cagctatctt acccaatgtt 2700
ctgatctgtc tggcttccag ggcaaaacaa cacctgtata tttttccgtg gggtggccat 2760
taggaagaac tccaaatcaa aatgctatgt aaatgtctat gggtgacagt ctgctgatgg 2820
tagaaacctt tctttttagt tcacaagtca gagatttgga cggagctgac acaaagagtt 2880
tggagctccc ccatttctgg ctctcctttc aggggttcct tccccaaccc ttttcagcag 2940
cggtggctgc cccccattct gacccctgac tcttccagcc agaaagatgg tggttttcta 3000
aaggaacttt agctgtcctg cacaatgccg atctgtgtct tgcattttgg gtaaaagcca 3060
taaaaataag aaactcagcc tgtggccttt ctttcttcca aggctgggct tcttttttta 3120
agtgacttca tgcagtttgt tgcttttaaa aatttgtcca gaatcgtttt ctgcagaagc 3180
atggtctgtt aggagcttac tggccgtagc agaagcaatt gtttcctgaa ttcttgacat 3240
ttatctttgc tgtattcatt tagggcttgg gagagtccga agataattca gt'cactgtca 3300
gattaataat tttgtcagga caaagaatac cgttatgatt atttaatcct ttaaaattgt 3360
ggtctccaga gcttgttctc agaatggccc agaccaagcc ttaattgtga tagtgaatat 3420
taatggtcac tttaaggaga aattataggc caagatgaaa tgaacataaa cctgtttgcc 3480
ctggctttca gtggaagatg atattagaga ccaaaatctg gttctgaagg tgtgtatcag 3540
ccctaaggtg aaccagactt gggaaagatt gtctttaaaa atcaatgagt ttatgtttta 3600
acttctcagc ttagttctat gcattgctct ataacacacc tagttaagtt ttatgttatt 3660
cttgaactgt gatttttttt ctatttactt tcatggtttg gtgggccatt gttatggact 3720
gaatgtttgt gtcccaccct tcacccccaa attcccgtgt tgaagcccca acctgcactg 3780
tggagctggg gctgctaagg aagtaattaa ggttacatga agtcatggtg gggctctgat 3840
ctgctaaggt tggtgtcctt atagggagag accccagaga gcttgttccc tccctccctg 3900
tgcatgcaaa caagagggca tgggagcaca cagagagatg gcagccacct acaagccaag 3960
aggagaagcc tcacaatcaa actctcgctg ctggcgagag tcttggactc tgtcttggac 4020
ttccagcctc cagactgtga gaaacaaatt tctgttgttt cagcttctca gtctctggtg 4080
ttttgttatt gcagcctgag aacacagctg tacgattatt tgtcaaacag aaaacactga 4140
tacttaacaa tgctaatgca attatttatt tgcttttcag tctctacaaa acgttctaaa 4200
acactaatct aaatattaac agtaaaatat ttgcataact aatggaaact aagaaatcat 4260
atgaccaata tttcacttat tggtaatctt actctactga tttcccccca gactgtgatt 4320
tttggaactt ccttgccttt ctcctgtctt tctgtgttta ttcatggaat tccagttatc 4380
tggggcttga aattgcaggc tctcctaact taagcaaaat ctgacagatc agcaaaatga 4440
gataaatgtt tcttttttct ttctgactgc attaaatcag atacaactca gcattaaaaa 4500
gctatctttg taaatgttgt tactaataaa ttagtcttat aagatccctg gactttggag 4560
ttgttgcaat gtctttgaga gtaattcttt aaaagtctaa tttcgactgg ttgtatctct 4620
ttatgattta ttgccccact aacaacattt gaaacaatat aatattttaa aatgtataaa 4680
taattatgaa tttttgttta gaacaaagag gattactgat atttgtttcc ctatgaatgg 4740
caaaaggttt agcttactac tgcatttctg ttttaaataa aaagttgaga gtttgtgtct 4800
cattaaactg gaaaaaaaaa 4820
<210> 88
<211> 3599
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7690129CB1
<400> 88
70/71
CA 02409778 2002-11-25
WO 01/98353 PCT/USO1/19862
ggggccccgt cctccagacc tggctgcagg acctgctgcg tcgtgggctg gtgcgggctg 60
cccagagcac aggagcctgg attgtcactg ggggtctgca cacgggcatc ggccggcatg 120
ttggtgtgcg ctgtacggga ccatcagatg gccagcactg ggggcaccaa ggtggtggcc 180
atgggtgtgg ccccctgggg tgtggtccgg aatagagaca ccctcatcaa ccccaagggc 240
tcgttccctg cgaggtaccg gtggcgcggt gacccggagg acggggtcca gtttcccctg 300
gactacaact actcggcctt cttcctggtg gacgacggca cacacggctg cctggggggc 360
gagaaccgct tccgcttgcg cctggagtcc tacatctcac agcagaagac gggcgtggga 420
gggactggaa ttgacatccc tgtcctgctc ctcctgattg atggtgatga gaagatgttg 480
acgcgaatag agaacgccac ccaggctcag ctcccatgtc tcctcgtggc tggctcaggg 540
ggagctgcgg actgcctggc ggagaccctg gaagacactc tggccccagg gagtggggga 600
gccaggcaag gcgaagcccg agatcgaatc aggcgtttct ttcccaaagg ggaccttgag 660
gtccttcagg cccaggtgga gaggattatg acccggaagg agctcctgac agtctattct 720
tctgaggatg ggtctgagga attcgagacc atagttttga aggcccttgt gaaggcctgt 780
gggagctcgg aggcctcagc ctacctggat gagctgcgtt tggctgtggc ttggaaccgc 840
gtggacattg cccagagtga actctttcgg ggggacatcc aatggcggtc cttccatctc 900
gaagcttccc tcatggacgc cctgctgaat gaccggcctg agttcgtgcg cttgctcatt 960
tcccacggcc tcagcctggg ccacttcctg accccgatgc gcctggccca actctacagc 1020
gcggcgccct ccaactcgct catccgcaac cttttggacc aggcgtccca cagcgcaggc 1080
accaaagccc cagccctaaa agggggagct gcggagctcc ggccccctga cgtggggcat 1140
gtgctgagga tgctgctggg gaagatgtgc gcgccgaggt acccctccgg gggcgcctgg 1200
gaccctcacc caggccaggg cttcggggag agcatgtatc tgctctcgga caaggccacc 1260
tcgccgctct cgctggatgc tggcctcggg caggccccct ggagcgacct gcttctttgg 1320
gcactgttgc tgaacagggc acagatggcc atgtacttct gggagatggg ttccaatgca 1380
gtttcctcag ctcttggggc ctgtttgctg ctccgggtga tggcacgcct ggagcctgac 1440
gctgaggagg cagcacggag gaaagacctg gcgttcaagt ttgaggggat gggcgttgac 1500
ctctttggcg agtgctatcg cagcagtgag gtgagggctg cccgcctcct cctccgtcgc 1560
tgcccgctct ggggggatgc cacttgcctc cagctggcca tgcaagctga cgcccgtgcc 1620
ttctttgccc aggatggggt acagtctctg ctgacacaga agtggtgggg agatatggcc 1680
agcactacac Ccatctgggc cctggttctc gccttctttt gccctccact catctacacc 1740
cgcctcatca ccttcaggaa atcagaagag gagcccacac gggaggagct agagtttgac 1800
atggatagtg tcattaatgg ggaagggcct gtcgggacgg cggacccagc cgagaagacg 1860
ccgctggggg tcccgcgcca gtcgggccgt ccgggttgct gcgggggccg ctgcgggggg 1920
cgccggtgcc tacgccgctg gttccacttc tggggcgcgc cggtgaccat cttcatgggc 1980
aacgtggtca gctacctgct gttcctgctg cttttctcgc gggtgctgct cgtggatttc 2040
cagccggcgc cgcccggctc cctggagctg ctgctctatt tctgggcttt cacgctgctg 2100
tgcgaggaac tgcgccaggg cctgagcgga ggcgggggca gcctcgccag cgggggcccc 2160
gggcctggcc atgcctcact gagccagcgc ctgcgcctct acctcgccga cagctggaac 2220
cagtgcgacc tagtggctct cacctgcttc ctcctgggcg tgggctgccg gctgaccccg 2280
ggtttgtacc acctgggccg cactgtcctc tgcatcgact tcatggtttt cacggtgcgg 2340
ctgcttcaca tcttcacggt caacaaacag ctggggccca agatcgtcat cgtgagcaag 2400
atgatgaagg acgtgttctt cttcctcttc ttcctcggcg tgtggctggt agcctatggc 2460
gtggccacgg aggggctcct gaggccacgg gacagtgact tcccaagtat cctgcgccgc 2520
gtcttctacc gtccctacct gcagatcttc gggcagattc cccaggagga catggacgtg 2580
gccctcatgg agcacagcaa ctgctcgtcg gagcccggct tctgggcaca ccctcctggg 2640
gcccaggcgg gcacctgcgt ctcccagtat gccaactggc tggtggtgct gctcctcgtc 2700
atcttcctgc tcgtggccaa catcctgctg gtcaacttgc tcattgccat gttcagttac 2760
acattcggca aagtacaggg caacagcgat ctctactgga aggcgcagcg ttaccgcctc 2820
atccgggaat tccactctcg gCCCgCgCtg gccccgccct ttatcgtcat ctcccacttg 2880
cgcctcctgc tcaggcaatt gtgcaggcga ccccggagcc cccagccgtc ctccccggcc 2940
ctcgagcatt tccgggttta cctttctaag gaagccgagc ggaagctgct aacgtgggaa 3000
tcggtgcata aggagaactt tctgctggca cgcgctaggg acaagcggga gagcgactcc 3060
gagcgtctga agcgcacgtc ccagaaggtg gacttggcac tgaaacagct gggacacatc 3120
cgcgagtacg aacagcgcct gaaagtgctg gagcgggagg tccagcagtg tagccgcgtc 3180
ctggggtggg tggccgaggc cctgagccgc tctgccttgc tgcccccagg tgggccgcca 3240
ccccctgacc tgcctgggtc caaagactga gccctgctgg cggacttcaa ggagaagccc 3300
ccacagggga ttttgctcct agagtaaggc tcatctgggc ctcggccccc gcacctggtg 3360
gccttgtcct tgaggtgagc cccatgtcca tctgggccac tgtcaggacc acctttggga 3420
gtgtcatcct tacaaaccac agcatgcccg gctcctccca gaaccagtcc cagcctggga 3480
ggatcaaggc ctggatcccg ggccgttatc catctggagg ctgcagggtc cttggggtaa 3540
cagggaccac agacccctca ccactcacag attcctcaca ctggggaaat aaaccattc 3599
71171
