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HUMAN KINASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
kinases and to the
use of these sequences in the diagnosis, treatment, and prevention of cancer,
immune disorders,
disorders affecting growth and development, cardiovascular diseases, and lipid
disorders, and in the
assessment of the effects of exogenous compounds on the expression of nucleic
acid and amino acid
sequences of human kinases.
1o BACKGROUND OF THE INVENTION
Kinases comprise the largest known enzyme superfamily and vary widely in their
target
molecules. Kinases catalyze the transfer of high energy phosphate groups from
a phosphate donor to
a phosphate acceptor. Nucleotides usually serve as the phosphate donor in
these reactions, with most
kinases utilizing adenosine triphosphate (ATP). The phosphate acceptor can be
any of a variety of
molecules, including nucleosides, nucleotides, lipids, carbohydrates, and
proteins. Proteins are
phosphorylated on hydroxyamino acids. Addition of a phosphate group alters the
local charge on the
acceptor molecule, causing internal conformational changes and potentially
influencing intermolecular
contacts. Reversible protein phosphorylation is the primary method for
regulating protein activity in
eukaryotic cells. In general, proteins are activated by phosphorylation in
response to extracellular
signals such as hormones, neurotransmitters, and growth and differentiation
factors. The activated
proteins initiate the cell's intracellular response by way of intracellular
signaling pathways and second
messenger molecules such as cyclic nucleotides, calcium-calinodulin, inositol,
and various mitogens,
that regulate protein phosphorylation.
Kinases are involved in all aspects of a cell's function, from basic metabolic
processes, such
as glycolysis, to cell-cycle regulation, differentiation, and communication
with the extracellular
environment through signal transduction cascades. Inappropriate
phosphorylation of proteins in cells
has been linked to changes in cell cycle progression and cell differentiation.
Changes in the cell cycle
have been linked to induction of apoptosis or cancer. Changes in cell
differentiation have been linked
to diseases and disorders of the reproductive system, immune system, and
skeletal muscle.
There are two classes of protein kinases. One class, protein tyrosine kinases
(PTKs),
phosphorylates tyrosine residues, and the other class, protein
serine/threonine kinases (STKs),
phosphorylates serine and threonine residues. Some PTKs and STKs possess
structural
characteristics of both families and have dual specificity for both tyrosine
and serine/threonine
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residues. Alinost all kinases contain a conserved 250-300 amino acid catalytic
domain containing
specific residues and sequence motifs characteristic of the kinase family. The
protein kinase catalytic
domain can be further divided into 11 subdomains. N-terminal subdomains I-IV
fold into a two-lobed
structure which binds and orients the ATP donor molecule, and subdomain V
spans the two lobes. C-
terminal subdomains VI-XI bind the protein substrate and transfer the gamma
phosphate from ATP to
the hydroxyl group of a tyrosine, serine, or threonine residue. Each of the 11
subdomains contains
specific catalytic residues or amino acid motifs characteristic of that
subdomain. For example,
subdomain I contains an 8-amino acid glycine-rich ATP binding consensus motif,
subdomain 1I
contains a critical lysine residue required for maximal catalytic activity,
and subdomains VI through IX
comprise the highly conserved catalytic core. PTKs and STKs also contain
distinct sequence motifs
in subdomains VI and VIII which may confer hydroxyamino acid specificity.
In addition, kinases may also be classified by additional amino acid
sequences, generally
between 5 and 100 residues, which either flank or occur within the kinase
domain. These additional
amino acid sequences regulate kinase activity and determine substrate
specificity. (Reviewed in
Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book, Vol I p.p. 17-
20 Academic Press,
San Diego, CA.). In particular, two protein kinase signature sequences have
been identified in the
kinase domain, the first containing an active site lysine residue involved in
ATP binding, and the
second containing an aspartate residue important for catalytic activity. If a
protein analyzed includes
the two protein kinase signatures, the probability of that protein being a
protein kinase is close to 100%
(PROSITE: PDOC00100, November 1995).
Protein Tyrosine Kinases
Protein tyrosine kinases (PTKs) may be classified as either transmembrane,
receptor PTKs
or nontransmembrane, nonreceptor PTK proteins. Transmembrane tyrosine kinases
function as
receptors for most growth factors. Growth factors bind to the receptor
tyrosine ~kinase (RTK), which
causes the receptor to phosphorylate itself (autophosphorylation) and specific
intracellular second
messenger proteins. Growth factors (GF) that associate with receptor PTKs
include epidermal GF,
platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like
GFs, nerve GF, vascular
endothelial GF, and macrophage colony stimulating factor.
Nontransmembrane, nonreceptor PTKs lack transmembrane regions and, instead,
form
signaling complexes with the cytosolic domains of plasma membrane receptors.
Receptors that
function through non-receptor PTKs include those for cytokines and hormones
(growth hormone and
prolactin), and antigen-specific receptors on T and B lymphocytes.
Many PTKs were first identified as oncogene products in cancer cells in which
PTK
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activation was no longer subject to normal cellular controls. In fact, about
one third of the known
oncogenes encode PTKs. Furthermore, cellular transformation (oncogenesis) is
often accompanied
by increased tyrosine phosphorylation activity (Charbonneau, H. and Tonks, N.
K. (1992) Annu. Rev.
Cell Biol. 8:463-93). Regulation of PTK activity may therefore be an important
strategy in controlling
some types of cancer.
Protein Serine/Threonine Kinases
Protein serine/threonine kinases (STKs) are nontransmembrane proteins. A
subclass of
STKs are known as ERKs (extracellular signal regulated kinases) or MAPS
(mitogen-activated
protein kinases) and are activated after cell stimulation by a variety of
hormones and growth factors.
i0 Cell stimulation induces a signaling cascade leading to phosphorylation of
MEK (MAP/BRK kinase)
which, in turn, activates ERK via serine and threonine phosphorylation. A
varied number of proteins
represent the downstream effectors for the active ERK and implicate it in the
control of cell
proliferation and differentiation, as well as regulation of the cytoskeleton.
Activation of ERK is
normally transient, and cells possess dual specificity phosphatases that are
responsible for its down-
regulation. Also, numerous studies have shown that elevated ERK activity is
associated with some
cancers. Other STKs include the second messenger dependent protein kinases
such as the
cyclic-AMP dependent protein kinases (PKA), calcium-calmodulin (CaM) dependent
protein kinases,
and the mitogen-activated protein kinases (MAP); the cyclin-dependent protein
kinases; checkpoint
and cell cycle kinases; proliferation-related kinases; 5'-AMP-activated
protein kinases; and kinases
involved in apoptosis.
The second messenger dependent protein kinases primarily mediate the effects
of second
messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate,
phosphatidylinositol,
3,4,5-triphosphate, cyclic ADPribose, arachidonic acid, diacylglycerol and
calcium-calmodulin. The
PKAs are involved in mediating hormone-induced cellular responses and are
activated by cAMP
produced within the cell in response to hormone stimulation. cAMP is an
intracellular mediator of
hormone action in all animal cells that have been studied. Hormone-induced
cellular responses include
thyroid hormone secretion, cortisol secretion, progesterone secretion,
glycogen breakdown, bone
resorption, and regulation of heart rate and force of heart muscle
contraction. PKA is found in all
animal cells and is thought to account for the effects of cAMP in most of
these cells. Altered PKA
expression is implicated in a variety of disorders and diseases including
cancer, thyroid disorders,
diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K.J. et
al. (1994) Harrison's
Principles of Internal Medicine, McGraw-Hill, New York, NY, pp. 416-431,
1887).
The casein kinase I (CKI) gene family is another subfamily of serine/threonine
protein
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kinases. This continuously expanding group of kinases have been implicated in
the regulation of
numerous cytoplasmic and nuclear processes, including cell metabolism, and DNA
replication and
repair. CKI enzymes are present in the membranes, nucleus, cytoplasm and
cytoskeleton of
eukaryotic cells, and on the mitotic spindles of mammalian cells (Fish, K.J.
et al., (1995) J. Biol. Chem.
270:14875-14883.
The CKI family members all have a short amino-terminal domain of 9-76 amino
acids, a highly
conserved kinase domain of 284 amino acids, and a variable carboxyl-terminal
domain that ranges
from 24 to over 200 amino acids in length (Cegielska, A. et al., (1998) J.
Biol. Chem. 273:1357-1364.)
The CKI family is comprised of highly related proteins, as seen by the
identification of isoforms of
casein kinase I from a variety of sources. There are at least five mammalian
isoforms, a, ~3, y, 8, and
s. Fish et al., identified CKI-epsilon from a human placenta cDNA library. It
is a basic protein of 416
amino acids and is closest to CKI-delta. Through recombinant expression, it
was determined to
phosphorylate known CKI substrates and was inhibited by the CKI-specific
inhibitor CKI-7. The
human gene for CKI-epsilon was able to rescue yeast with a slow-growth
phenotype caused by
deletion of the yeast CKI locus, HRR250 (Fish et al, supra.)
The mammalian circadian mutation tau was found to be a semidominant autosomal
allele of
CKI-epsilon that markedly shortens period length of circadian rhythms in
Syrian hamsters. The tau
locus is encoded by casein kinase I-epsilon, which is also a homolog of the
Drosophila circadian gene
double-time. Studies of both the wildtype and tau mutant CKI-epsilon enzyme
indicated that the
mutant enzyme has a noticeable reduction in the maximum velocity and
autophosphorylation state.
Further, in vitro, CKI-epsilon is able to interact with mammalian PERIOD
proteins, while the mutant
enzyme is deficient in its ability to phosphorylate PERIOD. Lowrey et al.,
have proposed that CKI-
epsilon plays a major role in delaying the negative feedback signal within the
transcription-translation-
based autoregulatory loop that composes the core of the circadian mechanism.
Therefore the CKI-
epsilon enzyme is an ideal target for pharmaceutical compounds influencing
circadian rhythms, jet-lag
and sleep, in addition to other physiologic and metabolic processes under
circadian regulation (Lowrey,
P.L. et al., (2000) Science 288:483-491.)
Calcium-Calmodulin Dependent Protein Kinases
Calcium-calmodulin dependent (CaM) kinases are involved in regulation of
smooth muscle
contraction, glycogen breakdown (phosphorylase kinase), and neurotransmission
(CaM kinase I and
CaM kinase II). CaM dependent protein kinases are activated by calmodulin, an
intracellular calcium
receptor, in response to the concentration of free calcium in the cell. Many
CaM kinases are also
activated by phosphorylation. Some CaM kinases are also activated by
autophosphorylation or by
4
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other regulatory kinases. CaM kinase I phosphorylates a variety of substrates
including the
neurotransmitter-related proteins synapsin I and II, the gene transcription
regulator, CREB, and the
cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al.
(1995) EMBO Journal
14:3679-3686). CaM kinase II also phosphorylates synapsin at different sites
and controls the
synthesis of catecholamines in the brain through phosphorylation and
activation of tyrosine
hydroxylase. CaM kinase II controls the synthesis of catecholamines and
seratonin, through
phosphorylation/activation of tyrosine hydroxylase and tryptophan hydroxylase,
respectively (Fujisawa,
H. (1990) BioEssays 12:27-29). The mRNA encoding a calinodulin-binding protein
kinase-like protein
was found to be enriched in mammalian forebrain. This protein is associated
with vesicles in both
axons and dendrites and accumulates largely postnatally. The amino acid
sequence of this protein is
similar to CaM-dependent STKs, and the protein binds calmodulin in the
presence of calcium
(Godbout, M. et al. (1994) J. Neurosci. 14:1-13).
Mito~en-Activated Protein Kinases
The mitogen-activated protein kinases (MAP) which mediate signal transduction
from the cell
surface to the nucleus via phosphorylation cascades are another STK family
that regulates
intracellular signaling pathways. Several subgroups have been identified, and
each manifests different
substrate specificities and responds to distinct extracellular stimuli (Egan,
S.E. and Weinberg, R.A.
(1993) Nature 365:781-783). MAP kinase signaling pathways are present in
mammalian cells as well
as in yeast. The extracellular stimuli which activate MAP kinase pathways
include epidermal growth
factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic
lipopolysaccharide (LPS),
and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and
interleukin-1 (IL-1).
Altered MAP kinase expression is implicated in a variety of disease conditions
including cancer,
inflammation, immune disorders, and disorders affecting growth and
development.
Cyclin-Dependent Protein Kinases
The cyclin-dependent protein kinases (CDKs) are STKs that control the
progression of cells
through the cell cycle. The entry and exit of a cell from mitosis are
regulated by the synthesis and
destruction of a family of activating proteins called cyclins. Cyclins are
small regulatory proteins that
bind to and activate CDKs, which then phosphorylate and activate selected
proteins involved in the
mitotic process. CDKs are unique in that they require multiple inputs to
become activated. In addition
to cyclin binding, CDK activation requires the phosphorylation of a specific
threonine residue and the
dephosphorylation of a specific tyrosine residue on the CDK.
Another family of STKs associated with the cell cycle are the NIMA (never in
mitosis)-
related kinases (Neks). Both CDKs and Neks are involved in duplication,
maturation, and separation
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of the microtubule organizing center, the centrosome, in animal cells (Fry,
A.M., et al. (1998) EMBO
J. 17:470-481). The NIM-related kinases also include NIK1 histidine kinases,
which function in signal
transmission (Yamada-Okabe, T. et al. (1999) J. Bacteriol. 181:7243-7247).
Checkpoint and Cell Cycle Kinases
In the process of cell division, the order and timing of cell cycle
transitions are under control of
cell cycle checkpoints, which ensure that critical events such as DNA
replication and chromosome
segregation are carried out with precision. If DNA is damaged, e.g. by
radiation, a checkpoint
pathway is activated that arrests the cell cycle to provide time for repair.
If the damage is extensive,
apoptosis is induced. In the absence of such checkpoints, the damaged DNA is
inherited by aberrant
to cells which may cause proliferative disorders such as cancer. Protein
kinases play an important role
in this process. For example, a specific kinase, checkpoint kinase 1 (Chkl),
has been identified in
yeast and mammals, and is activated by DNA damage in yeast. Activation of Chkl
leads to the arrest
of the cell at the G2/M transition. (Sanchez, Y. et al. (1997) Science
277:1497-1501.) Specifically,
Chkl phosphorylates the cell division cycle phosphatase CDC25, inhibiting its
normal function which is
15 to dephosphorylate and activate the cyclin-dependent kinase Cdc2. Cdc2
activation controls the entry
of cells into mitosis. (Peng, C-Y et al. (1997) Science 277:1501- 1505.) Thus,
activation of Chkl
prevents the damaged cell from entering mitosis. A similar deficiency in a
checkpoint kinase, such as
Chkl, may also contribute to cancer by failure to arrest cells with damaged
DNA at other checkpoints
such as G2/M.
20 Proliferation-Related Kinases
Proliferation-related kinase is a serum/cytokine inducible STK that is
involved in regulation of
the cell cycle and cell proliferation in human megakarocytic cells (Li, B. et
al. (1996) J. Biol. Chem.
271:19402-8). Proliferation-related kinase is related to the polo (derived
from Drosophila polo gene)
family of STKs implicated in cell division. Proliferation-related kinase is
downregulated in lung tumor
25 tissue and may be a proto-oncogene whose deregulated expression in normal
tissue leads to oncogenic
transformation.
The RET (rearranged during transfection) proto-oncogene encodes a tyrosine
kinase receptor
involved in both multiple endocrine neoplasia type 2, an inherited cancer
syndrome, and Hirschsprung
disease, a developmental defect of enteric neurons. RET and its functional
ligand, glial cell line-
3o derived neurotrophic factor, play key roles in the development of the human
enteric nervous system
(Pachnis, V. et al. (1998) Am. J. Physiol. 275:6183-6186).
=AMP-activated protein kinase
A ligand-activated STK protein kinase is 5 =AMP-activated protein kinase
(AMPK) (Gao, G.
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et al. (1996) J. Biol Chem. 271:8675-8681). Mammalian AMPK is a regulator of
fatty acid and sterol
synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and
hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways
to cellular stresses
such as heat shock and depletion of glucose and ATP. AMPK is a heterotrimeric
complex comprised
of a catalytic alpha subunit and two non-catalytic beta and gamma subunits
that are believed to
regulate the activity of the alpha subunit. Subunits of AMPK have a much wider
distribution in
non-lipogenic tissues such as brain, heart, spleen, and lung than expected.
This distribution suggests
that its role may extend beyond regulation of lipid metabolism alone.
Kinases in Apoptosis
l0 Apoptosis is a highly regulated signaling pathway leading to cell death
that plays a crucial role
in tissue development and homeostasis. Deregulation of this process is
associated with the
pathogenesis of a number of diseases including autoimmune disease,
neurodegenerative disorders, and
cancer. Various STKs play key roles in this process. ZIP kinase is an STK
containing a C-terminal
leuciue zipper domain in addition to its N-terminal protein kinase domain.
This C-terminal domain
appears to mediate homodimerization and activation of the kinase as well as
interactions with
transcription factors such as activating transcription factor, ATF4, a member
of the cyclic-AMP
responsive element binding protein (ATF/CREB) family of transcriptional
factors (Sanjo, H. et al.
(1998) J. Biol. Chem, 273:29066-29071). DRAKl and DRAK2 are STKs that share
homology with
the death-associated protein kinases (DAP kinases), known to function in
interferon-y induced
apoptosis (Sanjo et al. su ra). Like ZIP kinase, DAP kinases contain a C-
terminal protein-protein
interaction domain, in the form of ankyrin repeats, in addition to the N-
terminal kinase domain. ZIP,
DAP, and DRAK kinases induce morphological changes associated with apoptosis
when transfected
into NIH3T3 cells (Sanjo et al. su ra). However, deletion of either the N-
terminal kinase catalytic
domain or the C-terminal domain of these proteins abolishes apoptosis
activity, indicating that in
addition to the kinase activity, activity in the C-terminal domain is also
necessary for apoptosis,
possibly as an interacting domain with a regulator or a specific substrate.
RICK is another STK recently identified as mediating a specific apoptotic
pathway involving
the death receptor, CD95 (Inohara, N. et al. (1998) J. Biol. Chem. 273:12296-
12300). CD95 is a
member of the tumor necrosis factor receptor superfamily and plays a critical
role in the regulation
and homeostasis of the immune system (Nagata, S. (1997) Cell 88:355-365). The
CD95 receptor
signaling pathway involves recruitment of various intracellular molecules to a
receptor complex
following ligand binding. This process includes recruitment of the cysteine
protease caspase-8 which,
in turn, activates a caspase cascade leading to cell death. RICK is composed
of an N-terminal kinase
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catalytic domain and a C-terminal "caspase-recruitment" domain that interacts
with caspase-like
domains, indicating that RICK plays a role in the recruitment of caspase-8.
This interpretation is
supported by the fact that the expression of RICK in human 293T cells promotes
activation of
caspase-8 and potentiates the induction of apoptosis by various proteins
involved in the CD95
apoptosis pathway (Inohara et al. su ra .
Mitochondrial Protein Kinases
A novel class of eukaryotic kinases, related by sequence to prokaryotic
histidine protein
kinases, are the mitochondrial protein kinases (MPKs) which seem to have no
sequence similarity with
other eukaryotic protein kinases. These protein kinases are located
exclusively in the mitochondrial
l0 matrix space and may have evolved from genes originally present in
respiration-dependent bacteria
which were endocytosed by primitive eukaryotic cells. MPKs are responsible for
phosphorylation and
inactivation of the branched-chain alpha-ketoacid dehydrogenase and pyruvate
dehydrogenase
complexes (Harris, R.A. et al. (1995) Adv. Enzyme Regul. 34:147-162). Five
MPKs have been
identified. Four members correspond to pyruvate dehydrogenase kinase isozymes,
regulating the
activity of the pyruvate dehydrogenase complex, which is an important
regulatory enzyme at the
interface between glycolysis and the citric acid cycle. The fifth member
corresponds to a branched-
chain alpha-ketoacid dehydrogenase kinase, important in the regulation of the
pathway for the disposal
of branched-chain amino acids. (Harris, R.A. et al. (1997) Adv. Enzyme Regul.
37:271-293). Both
starvation and the diabetic state are known to result in a great increase in
the activity of the pyruvate
dehydrogenase kinase in the liver, heart and muscle of the rat. This increase
contributes in both
disease states to the phosphorylation and inactivation of the pyruvate
dehydrogenase complex and
conservation of pyruvate and lactate for gluconeogenesis (Harris (1995)
supra).
KINASES WITH NON-PROTEIN SUBSTRATES
Liyid and Inositol kinases
Lipid kinases phosphorylate hydroxyl residues on lipid head groups. A family
of kinases
involved in phosphorylation of phosphatidylinositol (PI) has been described,
each member
phosphorylating a specific carbon on the inositol ring (Leevers, S.J. et al.
(1999) C~rr. Opin. Cell. Biol.
11:219-225). The phosphorylation of phosphatidylinositol is involved in
activation of the protein kinase
C signaling pathway. The inositol phospholipids (phosphoinositides)
intracellular signaling pathway
begins with binding of a signaling molecule to a G-protein linked receptor in
the plasma membrane.
This leads to the phosphorylation of phosphatidylinositol (PI) residues on the
inner side of the plasma .
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membrane by inositol kinases, thus converting PI residues to the biphosphate
state (PIPZ). PIPZ is
then cleaved into inositol triphosphate (1P3) and diacylglycerol. These two
products act as mediators
for separate signaling pathways. Cellular responses that are mediated by these
pathways are
glycogen breakdown in the liver in response to vasopressin, smooth muscle
contraction in response to
acetylcholine, and thrombin-induced platelet aggregation.
PI 3-kinase (PI3K), which phosphorylates the D3 position of PI and its
derivatives, has a
central role in growth factor signal cascades involved in cell growth,
differentiation, and metabolism.
PI3K is a heterodimer consisting of an adapter subunit and a catalytic
subunit. The adapter subunit
acts as a scaffolding protein, interacting with specific tyrosine-
phosphorylated proteins, lipid moieties,
to and other cytosolic factors. When the adapter subunit binds tyrosine
phosphorylated targets, such as
the insulin responsive substrate (IRS)-1, the catalytic subunit is activated
and converts PI (4,5)
bisphosphate (PIPZ) to PI (3,4,5) P3 (PIPS). PIPS then activates a number of
other proteins, including
PKA, protein kinase B (PKB), protein kinase C (PKC), glycogen synthase kinase
(GSK)-3, and p70
ribosomal s6 kinase. PI3K also interacts directly with the cytoskeletal
organizing proteins, Rac, rho,
and cdc42 (Shepherd, P.R., et al. (1998) Biochem. J. 333:471-490). Animal
models for diabetes, such
as obese and fat mice, have altered PI3K adapter subunit levels. Specific
mutations in the adapter
subunit have also been found in an insulin-resistant Danish population,
suggesting a role for PI3K in
type-2 diabetes (Shepard, su ra).
PKC is also activated by diacylglycerol (DAG). Phorbol esters (PE) are analogs
of DAG and
tumor promoters that cause a variety of physiological changes when
administered to cells and tissues.
PE and DAG bind to the N-terminal region of PKC. This region contains one or
more copies of a
cysteine-rich domain about 50 amino-acid residues long and essential for
DAG/PE-binding.
Diacylglycerol kinase (DGK), the enzyme that converts DAG into phosphatidate,
contains two copies
of the DAG/PE-binding domain in its N-terminal section (Azzi, A. et al. (
1992) Eur. J. Biochem.
208:547-557).
An example of lipid kinase phosphorylation activity is the phosphorylation of
D-erythro-sphingosine to the sphingolipid metabolite, sphingosine-1-phosphate
(SPP). SPP has
emerged as a novel lipid second-messenger with both extracellular and
intracellular actions (Kohama,
T. et al. (1998) J. Biol. Chem. 273:23722-23728). Extracellularly, SPP is a
ligand for the G-protein
coupled receptor EDG-1 (endothelial-derived, G-protein coupled receptor).
Intracellularly, SPP
regulates cell growth, survival, motility, and cytoskeletal changes. SPP
levels are regulated by
sphingosine kinases that specifically phosphorylate D-erythro-sphingosine to
SPP. The importance of
sphingosine kinase in cell signaling is indicated by the fact that various
stimuli, including
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platelet-derived growth factor (PDGF), nerve growth factor, and activation of
protein kinase C,
increase cellular levels of SPP by activation of sphingosine kinase, and the
fact that competitive
inhibitors of the enzyme selectively inhibit cell proliferation induced by
PDGF (Kohama et al. su ra).
Purine Nucleotide Kinases
The purine nucleotide kinases, adenylate kinase (ATP:AMP phosphotransferase,
or AdK) and
guanylate kinase ( ATP:GMP phosphotransferase, or GuK) play a key role in
nucleotide metabolism
and are crucial to the synthesis and regulation of cellular levels of ATP and
GTP, respectively. These
two molecules are precursors in DNA and RNA synthesis in growing cells and
provide the primary
source of biochemical energy in cells (ATP), and signal transduction pathways
(GTP). Inhibition of
various steps in the synthesis of these two molecules has been the basis of
many antiproliferative
drugs for cancer and antiviral therapy (Pillwein, K. et al. (1990) Cancer Res.
50:1576-1579).
AdK is found in almost all cell types and is especially abundant in cells
having high rates of
ATP synthesis and utilization such as skeletal muscle. In these cells AdK is
physically associated with
mitochondria and myofibrils, the subcellular structures that are involved in
energy production and
utilization, respectively. Recent studies have demonstrated a major function
for AdK in transferring
high energy phosphoryls from metabolic processes generating ATP to cellular
components consuming
ATP ( Zeleznikar, R.J. et al. (1995) J. Biol. Chem. 270:7311-7319). Thus AdK
may have a pivotal
role in maintaining energy production in cells, particularly those having a
high rate of growth or
metabolism such as cancer cells, and may provide a target for suppression of
its activity to treat
certain cancers. Alternatively, reduced AdK activity may be a source of
various metabolic,
muscle-energy disorders that can result in cardiac or respiratory failure and
may be treatable by
increasing AdK activity.
GuK, in addition to providing a key step in the synthesis of GTP for RNA and
DNA synthesis,
also fulfills an essential function in signal transduction pathways of cells
through the regulation of GDP
and GTP. Specifically, GTP binding to membrane associated G proteins mediates
the activation of cell
receptors, subsequent intracellular activation of adenyl cyclase, and
production of the second
messenger, cyclic AMP. GDP binding to G proteins inhibits these processes. GDP
and GTP levels
also control the activity of certain oncogenic proteins such as p21'~ known to
be involved in control of
cell proliferation and oncogenesis (Bos, J.L. (1989) Cancer Res. 49:4682-
4689). High ratios of
GTP:GDP caused by suppression of GuK cause activation of p21'~ and promote
oncogenesis.
Increasing GuK activity to increase levels of GDP and reduce the GTP:GDP ratio
may provide a
therapeutic strategy to reverse oncogenesis.
GuK is an important enzyme in the phosphorylation and activation of certain
antiviral drugs
l0
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useful in the treatment of herpes virus infections. These drugs include the
guanine homologs acyclovir
and buciclovir (Miller, W.H. and Miller R.L. (1980) J. Biol. Chem. 255:7204-
7207; Stenberg, K. et al.
(1986) J. Biol. Chem. 261:2134-2139). Increasing GuK activity in infected
cells may provide a
therapeutic strategy for augmenting the effectiveness of these drugs and
possibly for reducing the
necessary dosages of the drugs.
Pyrimidine Kinases
The pyrirnidine kinases are deoxycytidine kinase and thymidine kinase 1 and 2.
Deoxycytidine
kinase is located in the nucleus, and thymidine kinase 1 and 2 are found in
the cytosol (Johansson, M.
et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:11941-11945). Phosphorylation
of
to deoxyribonucleosides by pyrimidine kinases provides an alternative pathway
for de novo synthesis of
DNA precursors. The role of pyrimidine kinases, like purine kinases, in
phosphorylation is critical to
the activation of several chemotherapeutically important nucleoside analogues
(Arner E.S. and
Eriksson, S. (1995) Pharmacol. Ther. 67:155-186).
The discovery of new human kinases, and the polynucleotides encoding them,
satisfies a need
in the art by providing new compositions which are useful in the diagnosis,
prevention, and treatment
of cancer, immune disorders, disorders affecting growth and development,
cardiovascular diseases,
and lipid disorders, and in the assessment of the effects of exogenous
compounds on the expression of
nucleic acid and amino acid sequences of human kinases.
2o SUMMARY OF THE INVENTION
The invention features purified polypeptides, human kinases, referred to
collectively as
"PKIN" and individually as "PKIN-1," "PKIN-2," "PKIN-3," "PKIN-4," "PKIN-5,"
"PKIN-6,'>
«P~-7~» «P~-8~» «P~-9~» «P~-10~» «P~-11~» «P~-12,» «P~-13~» «P~-14~»
«P~-15 » «P~_16 » «P~-17 » «P~-18 » «P~-19 » «P~-20 » «P~-21 " and "PKIN-
> > > > > > >
22." 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:I-22, b) a polypeptide comprising a naturally occurring amino acid sequence
at least 90% identical
to an amino acid sequence selected from the group consisting of SEQ )D NO:1-
22, c) a biologically
active fragment of a polypeptide having an amino acid sequence selected from
the group consisting of
3o SEQ 117 NO:1-22, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-22. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-22.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected from
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the group consisting of a) a polypeptide comprising an amino acid sequence
selected from the group
consisting of SEQ 1D N0:1-22, b) a polypeptide comprising a naturally
occurring amino acid sequence
at least 90% identical to an amino acid sequence selected from the group
consisting of SEQ 1D N0:1-
22, c) a biologically active fragment of a polypeptide having an amino acid
sequence selected from the
group consisting of SEQ 1D N0:1-22, and d) an immunogenic fragment of a
polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID NO:1-22. In
one alternative, the
polynucleotide encodes a polypeptide selected from the group consisting of SEQ
1D NO:1-22. In
another alternative, the polynucleotide is selected from the group consisting
of SEQ ID N0:23-44.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ 1D NO:1-22, b) a polypeptide comprising a naturally occurnng amino acid
sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID N0:1-22, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ 1D N0:1-22, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ D7 NO:1-22. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative, the
invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ ID NO:1-22, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ ID N0:1-22, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ )D NO:1-22, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-22. The method
comprises a)
culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid sequence
selected from the group consisting of SEQ 1D NO:1-22, b) a polypeptide
comprising a naturally
occurring amino acid sequence at least 90% identical to an amino acid sequence
selected from the
group consisting of SEQ 1D NO:1-22, c) a biologically active fragment of a
polypeptide having an
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amino acid sequence selected from the group consisting of SEQ ID N0:1-22, and
d) an immunogenic
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-22.
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 N0:23-44, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ )17 N0:23-44,
c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide complementary to
the polynucleotide of b), and e) an RNA equivalent of a)-d). In one
alternative, the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting of
a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of SEQ
ID N0:23-44, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:23-44,
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 )D
N0:23-44, b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90%
identical to a polynucleotide sequence selected from the group consisting of
SEQ )D N0:23-44, c) a
polynucleotide complementary to the polynucleotide of a), d) a polynucleotide
complementary to the
polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
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The invention further provides a composition comprising an effective amount of
a polypeptide
selected from the group consisting of a) a polypeptide comprising an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-22, b) a polypeptide comprising a
naturally occurring
amino acid sequence at least 90% identical to an amino acid sequence selected
from the group
consisting of SEQ )17 NO:1-22, c) a biologically active fragment of a
polypeptide having an amino acid
sequence selected from the group consisting of SEQ )D N0:1-22, and d) an
irntnunogenic fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ ID NO:1-22,
and a pharmaceutically acceptable excipient. In one embodiment, the
composition comprises an amino
acid sequence selected from the group consisting of SEQ D7 NO:1-22. The
invention additionally
to provides a method of treating a disease or condition associated with
decreased expression of
functional PKIN, comprising administering to a patient in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ >D NO:1-22, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-22, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-22, and d) an
immunogenic fragment of a polypeptide having an. amino acid sequence selected
from the group
consisting of SEQ )D NO:1-22. 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 PKIN, 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 )D NO:1-22, b) a
polypeptide
comprising a naturally occurring amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ 117 NO:1-22, c) a
biologically active fragment of
3o a polypeptide having an amino acid sequence selected from the group
consisting of SEQ )D N0:1-22,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ ID N0:1-22. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
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invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
PK1N, 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 1D NO:1-22, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ll7 N0:1-22, c) a biologically active
fragment of a polypeptide
l0 having an amino acid sequence selected from the group consisting of SEQ )D
NO:1-22, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ 117 NO:1-22. The method comprises a) combining the
polypeptide with at least one
test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-22, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ )D NO:1-22, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ 1D
NO:l-22, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )D N0:1-22. 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
polynucleotide sequence selected from the group consisting of SEQ )D N0:23-44,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, and b) detecting
altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
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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
>D N0:23-44, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ )D N0:23-44,
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 N0:23-
44, ii) a
polynucleotide comprising a naturally occurring polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:23-44,
iii} a polynucleotide
complementary to the polynucleotide of i), iv) a polynucleotide complementary
to the polynucleotide of
ii), and v) an RNA equivalent of i)-iv). Alternatively, the target
polynucleotide comprises a fragment
of a polynucleotide sequence selected from the group consisting of i)-v)
above; c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the treated
biological sample with the amount of hybridization complex in an untreated
biological sample, wherein
a difference in the amount of hybridization complex in the treated biological
sample is indicative of
toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
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.
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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.
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.
2o 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
"PKIN" refers to the amino acid sequences of substantially purified PKIN
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.
3o The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
PKIN. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of PKIN either by
directly interacting with
PKIN or by acting on components of the biological pathway in which PKIN
participates.
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An "allelic variant" is an alternative form of the gene encoding PKIN. 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 PKIN include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as PKIN or a
polypeptide with at least one functional characteristic of PKIN. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding PKIN, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding PKIN. 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 PKIN. 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 PKIN is retained. For example, negatively charged
amino acids may
include aspartic acid and glutarnic acid, and positively charged amino acids
may include lysine and
2o 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 phenylalauine 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 PKIN. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
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molecules, or any other compound or composition which modulates the activity
of PKIN either by
directly interacting with PKIN or by acting on components of the biological
pathway in which PKIN
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind PKIN polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from
the translation of RNA,
or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly used
carriers that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and
keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen used -
to elicit the immune response) for binding to an antibody:
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163 ), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules. The
nucleotide components of an aptamer may have modified sugar groups (e.g., the
2'-OH group of a
ribonucleotide may be replaced by 2'-F or 2'-NH2), which may improve a desired
property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels
in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci.
USA 96:3606-3610).
The term "spiegeliner" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
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handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
l0 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 PKIN, 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 PKIN or fragments of
PKIN 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' and/or the 3' direction, and
resequenced, or which has been
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assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
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
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one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of PKIN or the polynucleotide encoding PKIN
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example, a
fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10, 15,
16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
2o example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ m N0:23-44 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ >l7 N0:23-44, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:23-44 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish SEQ
B7 N0:23-44 from related polynucleotide sequences. The precise length of a
fragment of SEQ ID
N0:23-44 and the region of SEQ ID N0:23-44 to which the fragment corresponds
are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ )I7 NO:1-22 is encoded by a fragment of SEQ m N0:23-44. A
fragment of SEQ ID NO:1-22 comprises a region of unique amino acid sequence
that specifically
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identifies SEQ ID NO:1-22. For example, a fragment of SEQ ID NO:1-22 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:1-22.
The precise length of a fragment of SEQ >I7 NO:1-22 and the region of SEQ 117
N0:1-22 to which
the fragment corresponds are routinely determinable by one of ordinary skill
in the art based on the
intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation codon
(e.g., methionine) followed by an open reading frame and a translation
termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between two
or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer to
the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps in
the sequences being compared in order to optimize alignment between two
sequences, and therefore
achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
2o Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G.
et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available from
several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlin.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
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"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
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off:' S0
l0 Expect: l0
Word Size: I1
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 S0, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length supported
by the sequences shown herein, in the tables, figures, or Sequence Listing,
may be used to describe a
length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the 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
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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: I1 and Extension Gap: 1 penalties
Gap x drop-off. 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ )D number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
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
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conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill in
the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1% (w/v) SDS, and about 100 pg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T"~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating T", and
conditions for nucleic acid hybridization are well known and can be found in
Sarnbrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C maybe used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 p,g/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
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disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of PKIN
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
PKIN 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 PKIN. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of PKIN.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an PKIN may involve lipidation,
glycosylation,
3o 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 PKIN.
"Probe" refers to nucleic acid sequences encoding PKIN, their complements, or
fragments
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thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed
to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
to 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
Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas
South West Medical Center, Dallas TX) is capable of choosing specific primers
from megabase
sequences and is thus useful for designing primers on a genome-wide scope. The
Primer3 primer
selection program (available to the public from the Whitehead Institute/MIT
Center for Genome
Research, Cambridge MA) allows the user to input a "mispriming library," in
which sequences to
avoid as primer binding sites are user-specified. Primer3 is useful, in
particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter two primer
selection programs may
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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 S' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
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.
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The term "sample" is used in its broadest sense. A sample suspected of
containing PKIN,
nucleic acids encoding PKIN, 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% 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 viral
infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed
3o 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
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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.
l0 Techniques for transferring the DNA of the present invention into such
organisms are widely known
and provided in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to each
other. A polymorphic variant is a variation in the polynucleotide sequence of
a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
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
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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 human kinases (PKIN), the
polynucleotides encoding PKIN, and the use of these compositions for the
diagnosis, treatment, or
prevention of cancer, immune disorders, disorders affecting growth and
development, cardiovascular
diseases, and lipid 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 117 NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide 117) 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 117) 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 m) 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 MOT1FS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structure/function analysis and in some
cases, searchable
databases to which the analytical methods were applied.
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Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and these
properties establish that the claimed polypeptides are human kinases.
For example, SEQ ID N0:1 is 91 % identical to human casein kinase I-alpha
(GenBank ID
g852055) as determined by the Basic Local Alignment Search Tool (BLAST). (See
Table 2.) The
BLAST probability score is 2.9e-167, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. SEQ ID N0:1 also contains a
eukaryotic protein kinase
domain as determined by searching for statistically significant matches in the
hidden Markov model
(IEVVIM)-based PFAM database of conserved protein family domains. (See Table
3.) Data from
BLIIVVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative
evidence that SEQ
l0 ID N0:1 is a protein kinase.
For example, SEQ B7 NO:10 is 91% identical to Mus musculus FYVE finger-
containing
phosphoinositide kinase (GenBank ID g4200446) 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 NO:10
also contains a
phosphatidyl inositol 4-phosphate S-kinase domain as determined by searching
for statistically
significant matches in the hidden Markov model (HIVIM)-based PFAM database of
conserved protein
family domains. (See Table 3.) Data from PRODOM analysis provides further
corroborative
evidence that SEQ ID N0:10 is a phosphoinositide kinase.
For example, SEQ ID N0:12 is 71 % identical to human serine/threonine protein
kinase
(GenBank D7 g7160989) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 1.7e-148, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ D7 N0:12 also contains
a eukaryotic
protein kinase domain as determined by searching for statistically significant
matches in the hidden
Markov model (HIVIM)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from BLIIVVIPS and MOTIFS analyses provide further corroborative evidence
that SEQ ID
N0:12 is protein kinase.
For example, SEQ ID N0:13 is 86% identical to murine pantothenate kinase 1
beta (GenBank
117 g6690020) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.)
The BLAST probability score is 1.6e-129, which indicates the probability of
obtaining the observed
polypeptide sequence alignment by chance. Pantothenate kinase (PanK) is
proposed to be the master
regulator of CoA biosynthesis in mammalian cells, by controlling flux through
the CoA biosynthetic
pathway. Changes in the level of tissue PanK activitiy is reflected by the
concurrent changes in the
levels of CoA as seen in various metabolic states. Alterations in CoA levels
and PanK activity are
33
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seen during starvation/feeding, pathological states such as diabetes and by
treatment with
hypolipidemic drugs (Rock, C.O. et al., (2000) J. Biol. Chem. 275:1377-1383.)
For example, SEQ 1D N0:16 is 68% identical to Mus musculus Nck-interacting
kinase-like
embryo specific kinase (GenBank ID 86472874) 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 1D N0:16
also contains a
eukaryotic protein kinase domain as determined by searching for statistically
significant matches in the
hidden Markov model (HIVIM)-based PFAM database of conserved protein family
domains. (See
Table 3.) Data from BLIIVVIPS, MOTIFS, and PROFILESCAN analyses provide
further
corroborative evidence that SEQ D7 N0:16 is a protein kinase.
For example, SEQ ID N0:19 is 99% identical to human RET tyrosine kinase
receptor
(GenBank 1D 85419753) 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 N0:19 also contains
a eukaryotic
protein kinase domain as determined by searching for statistically significant
matches in the hidden
Markov model (HIVIM)-based PFAM database of conserved protein family domains.
(See Table 3.)
Data from BLIIVVIPS, MOTIFS, and PROFILESCAN analyses provide further
corroborative evidence
that SEQ 1D N0:19 is a tyrosine kinase.
For example, SEQ 1D N0:22 is 33% identical to Gallus gallus smooth muscle
myosin light
2o chain kinase precursor (GenBank 1D 8212661) as determined by the Basic
Local Alignment Search
Tool (BLAST). (See Table 2.) The BLAST probability score is 1.2 e-60, which
indicates the
probability of obtaining the observed polypeptide sequence alignment by
chance. SEQ ID N0:22 also
contains two eukaryotic protein kinase domains as determined by searching for
statistically significant
matches in the hidden Markov model (HIVIM)-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 N0:22 is a protein kinase.
SEQ >D N0:2-9, SEQ 1D N0:11, SEQ 1D N0:14-15, SEQ 1D N0:17-18, and SEQ 1D
N0:20-21 were analyzed and annotated in a similar manner. The algorithms and
parameters for the
analysis of SEQ ID NO:1-22 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
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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 N0:23-44 or that distinguish between SEQ D7
N0:23-44 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,
18381287 is the
identification number of an Incyte cDNA sequence, and CARDNOTO1 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., 71583296V1). Alternatively, the identification
numbers in column 5 may
refer to GenBank cDNAs or ESTs which contributed to the assembly of the full
length polynucleotide
sequences. In addition, the identification numbers in column 5 may identify
sequences derived from
the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the
designation "ENST"). Alternatively, the identification numbers in column 5 may
be derived from the
2o NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences
including the
designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e.,
those sequences
including the designation "NP"). Alternatively, the identification numbers in
column 5 may refer to
assemblages of both cDNA and Genscan-predicted exons brought together by an
"exon stitching"
algorithm. For example, FL_~c:~~~XXX Nl Nz_YYYYY N3 NQ represents a "stitched"
sequence in
which ~'~~~XXX is the identification number of the cluster of sequences to
which the algorithm was
applied, and YYYYY is the number of the prediction generated by the algorithm,
and N,,2,3..., if present,
represent specific exons that may have been manually edited during analysis
(See Example V).
Alternatively, the identification numbers in column 5 may refer to assemblages
of exons brought
together by an "exon-stretching" algorithm. For example,
FLXX~:XXX~IAAAA~BBBBB_1 N is the
identification number of a "stretched" sequence, with I~:~~~XXX being the
Incyte project identification
number, gAAAAA being the GenBank identification number of the human genomic
sequence to which
the "exon-stretching" algorithm was applied, gBBBBB being the GenBank
identification number or
NCBI RefSeq identification number of the nearest GenBank protein homolog, and
N referring to
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specific exons (See Example V). In instances where a RefSeq sequence was used
as a protein
homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by
"NM," "NP," or "NT")
may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
to ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK)
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences (see
Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
' In some cases, Incyte cDNA coverage redundant with the sequence coverage
shown in
column 5 was obtained to confirm the final consensus polynucleotide sequence,
but the relevant Incyte
cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library
is the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences
which were used to assemble and confirm the above polynucleotide sequences.
The tissues and
vectors which were used to construct the cDNA libraries shown in Table 5 are
described in Table 6.
The invention also encompasses PKIN variants. A preferred PKIN 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 PKIN amino acid sequence, and which contains at least
one functional or
structural characteristic of PKIN.
The invention also encompasses polynucleotides which encode PKIN. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:23-44, which encodes PKIN. The
polynucleotide sequences
of SEQ ID N0:23-44, as presented in the Sequence Listing, embrace the
equivalent RNA sequences,
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WO 02/33099 PCT/USO1/47728
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
PKIN. 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 PKIN. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence corrlprising a sequence selected from the group
consisting of SEQ )D N0:23-
44 which has at least about 70%, or alternatively at least about 85%, or even
at least about 95%
polynucleotide sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ
l0 ID N0:23-44. Any one of the polynucleotide variants described above can
encode an amino acid
sequence which contains at least one functional or structural characteristic
of PKIN.
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 PKIN, some
bearing minimal similarity
to the polynucleotide sequences of any known and naturally occurring gene, may
be produced. Thus,
the invention contemplates each and every possible variation of polynucleotide
sequence that could be
made by selecting combinations based on possible codon choices. These
combinations are made in
accordance with the standard triplet genetic code as applied to the
polynucleotide sequence of
naturally occurring PKIN, and all such variations are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode PKIN and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring PKIN under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding PKIN 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 PKIN 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 PKIN
and
PKIN 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 PKIN or any fragment thereof.
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Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:23-44 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
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 polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
to combinations of polymerases and proofreading exonucleases such as those
found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carned out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and BiotechnoloQV, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding PKIN 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 inhuman 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).
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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
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 PKIN may be cloned in recombinant DNA molecules that direct expression
of PKIN, 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 PKIN.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter PKIN-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.
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WO 02/33099 PCT/USO1/47728
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or improve
the biological properties of PKIN, 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 PKIN may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
PKIN itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide.
2o 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 PKIN, 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, su ra, pp. 28-53.)
In order to express a biologically active PKIN, the nucleotide sequences
encoding PKIN 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
CA 02425963 2003-04-15
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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 PKIN. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
PKIN. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding PKIN 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
l0 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 PKIN 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 Biology, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding PKIN. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, su ra; Ausubel, su ra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New
York 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,
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M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc.
Natl. Acad. Sci. USA
90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994)
Mol. I_m_m__unol. 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 PKIN. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding PKIN can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding PKIN into the
vector's multiple cloning
l0 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 PKIN are needed, e.g. for the
production of
15 antibodies, vectors which direct high level expression of PKIN 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 PKIN. 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
20 vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra;
Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of PKIN. Transcription of
sequences
25 encoding PKIN may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.) These
30 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
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where an adenovirus is used as an expression vector, sequences encoding PKIN
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 PKIN in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
S 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
to 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
PKIN in cell lines is preferred. For example, sequences encoding PK1N can be
transformed into cell
15 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
20 introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and Apr. cells, respectively.
(See, e.g., Wigler, M. et
25 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)
30 J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), B glucuronidase and its substrate B-glucuronide, or
luciferase and its substrate
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luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest
is also present, the presence and expression of the gene may need to be
confirmed. For example, if
the sequence encoding PKIN is inserted within a marker gene sequence,
transformed cells containing
sequences encoding PKIN can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding PKIN under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
l0 expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding PKIN
and that express
PKIN 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 PKIN 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 PKIN 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 I-m_m__unoloay, 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
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 PKIN
include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding PKIN, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
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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 PKIN 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 PKIN may be designed to contain signal sequences
which direct
to secretion of PKIN 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 PKIN 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 PKIN protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of PKIN 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), calinodulin binding peptide (CBP), 6-
His, FLAG, c-myc, and
hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion
proteins on immobilized glutathione, maltose, phenylarsine oxide, calinodulin,
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 PKIN encoding sequence and the heterologous protein
sequence, so that PKIN
CA 02425963 2003-04-15
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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 PKIN 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.
PKIN of the present invention or fragments thereof may be used to screen for
compounds
l0 that specifically bind to PKIN. At least one and up to a plurality of test
compounds may be screened
for specific binding to PKIN. 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
PKIN, 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 PKIN
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 PKIN,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing PKIN or cell membrane fractions which contain PKIN are
then contacted with
a test compound and binding, stimulation, or inhibition of activity of either
PKIN 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
PKIN, either in solution
or affixed to a solid support, and detecting the binding of PKIN to the
compound. Alternatively, the
assay may detect or measure binding of a test compound in the presence of a
labeled competitor.
Additionally, the assay may be carried out using cell-free preparations,
chemical libraries, or natural
product mixtures, and the test cornpound(s) may be free in solution or affixed
to a solid support.
PKIN of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of PKIN. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for PKIN
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activity, wherein PKIN is combined with at least one test compound, and the
activity of PKIN in the
presence of a test compound is compared with the activity of PKIN in the
absence of the test
compound. A change in the activity of PKIN in the presence of the test
compound is indicative of a
compound that modulates the activity of PKIN. Alternatively, a test compound
is combined with an in
vitro or cell-free system comprising PKIN under conditions suitable for PKIN
activity, and the assay
is performed. In either of these assays, a test compound which modulates the
activity of PKIN 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 PKIN or their mammalian
homologs may be
"knocked out" in an animal model system using homologous recombination in
embryonic stem (ES)
cells. Such techniques are well known in the art and are useful for the
generation of animal models of
human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337.) For example,
mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and
grown in culture. The ES cells are transformed with a vector containing the
gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi,
M.R. (1989) Science
244:1288-1292). The vector integrates into the corresponding region of the
host genome by
homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Clip. 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 PKIN 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 PKIN can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a region
of a polynucleotide encoding PKIN 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
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potential pharmaceutical agents to obtain information on treatment of a human
disease. Alternatively,
a mammal inbred to overexpress PKIN, e.g., by secreting PKIN 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 PKIN and human kinases. In addition, the expression of PKIN is
closely associated with
brain, breast tumor, cardiovascular, digestive, fallopian tube tumor, fetal
stomach, nervous, ovarian
tumor, pancreatic tumor, peritoneal tumor, pituitary gland, placental,
prostate tumor, neural, spinal cord,
and testicular tissues, and with umbilical cord blood dendritic cells.
Therefore, PKIN appears to play a
role in cancer, immune disorders, disorders affecting growth and development,
cardiovascular
diseases, and lipid disorders. In the treatment of disorders associated with
increased PKIN expression
or activity, it is desirable to decrease the expression or activity of PKIN.
In the treatment of disorders
associated with decreased PKIN expression or activity, it is desirable to
increase the expression or
activity of PKIN.
Therefore, in one embodiment, PKIN 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 PKIN.
Examples of such disorders include, but are not limited to, a cancer, such as
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus, leukemias
such as multiple myeloma,
and lymphomas such as Hodgkin's disease; an immune 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,
3o 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,
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bacterial, fungal, parasitic, protozoal, and helininthic infections, and
trauma; a growth and
developmental disorder, such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus,
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; a cardiovascular disease, such as 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,
congestive heart failure,
ischemic heart disease, angina pectoris, myocardial infarction, hypertensive
heart disease,
degenerative valvular heart disease, calcific aortic valve stenosis,
congenitally bicuspid aortic valve,
mitral annular calcification, mitral valve prolapse, rheumatic fever and
rheumatic heart disease,
infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of
systemic lupus
erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis,
pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation, congenital lung
anomalies, atelectasis, pulmonary congestion and edema, pulinonary 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
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noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced
lung disease, radiation-
induced lung disease, and complications of lung transplantation; and a lipid
disorder such as fatty liver,
cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine
paltnitoyltransferase deficiency,
myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage
disorders such Fabry's
disease, Gaucher's disease, Niemann-Pick's disease, metachromatic
leukodystrophy,
adrenoleukodystrophy, GMZ gangliosidosis, and ceroid lipofuscinosis,
abetalipoproteinemia, Tangier
disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses,
acute panniculitis,
disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia,
minimal change disease,
lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with
hypertriglyceridemia,
primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver
disease, lecithin:cholesterol
acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia,
hypocholesterolemia, Tay-
Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid
myopathies, and obesity.
In another embodiment, a vector capable of expressing PK1N 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 PKIN including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
PI~NN 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 PKIN including,
but not limited to, those
provided above.
In still another~embodiment, an agonist which modulates the activity of PKIN
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of PKIN including, but not limited to, those listed above.
In a further embodiment, an antagonist of PKIN may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of PKIN.
Examples of such
disorders include, but are not limited to, those cancer, immune disorders,
disorders affecting growth
and development, cardiovascular diseases, and lipid disorders described above.
In one aspect, an
antibody which specifically binds PKIN 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
PKIN.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PKIN may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of PKIN including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
CA 02425963 2003-04-15
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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 PKIN may be produced using methods which are generally known
in the art.
In particular, purified PKIN may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind PKIN.
Antibodies to PKIN 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 PKIN 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 PKIN
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 PKIN
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 PKIN 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
3o 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
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splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
PKIN-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 PKIN may also be
generated.
Forexample, 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
PKIN and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering PKIN epitopes is generally used, but a competitive
binding assay may also be
employed (Pound, su ra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to assess the affinity of antibodies for PKIN. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of PKIN-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ke
determined for a preparation of polyclonal antibodies, which are heterogeneous
in their affinities for
multiple PKIN epitopes, represents the average affinity, or avidity, of the
antibodies for PKIN. The
Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a particular
PKIN epitope, represents a true measure of affinity. High-affinity antibody
preparations with Ka
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ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in
which the PKIN-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with Ka
ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of PKIN, preferably in active
form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press,
Washington DC; Liddell,
J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John
Wiley & Sons, New York
NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
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 PKIN-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. su ra.)
In another embodiment of the invention, the polynucleotides encoding PKIN, 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
PKIN. Such technology is well known in the art, and antisense oligonucleotides
or larger fragments
2o can be designed from various locations along the coding or control regions
of sequences encoding
PKIN. (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 Clip. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
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In another embodiment of the invention, polynucleotides encoding PKIN 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 (SC>D)-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),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
l0 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 PKIN expression or regulation causes
disease, the expression of
PKIN 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
PKIN are treated by constructing mammalian expression vectors encoding PKIN
and introducing
these vectors by mechanical means into PKIN-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 PKIN include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA);
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
PKIN
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
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(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) C~rr. 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 PKIN from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, 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 PKIN expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding PKIN 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.5. Patent No. 5,910,434 to Rigg ("Method
for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses
a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ 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)
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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 PKIN to cells which have one or more genetic
abnormalities with respect to
the expression of PKIN. 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 No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"), hereby
incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et
al. (1999) Annu. Rev.
to 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 PKIN to target cells which have one or more genetic
abnormalities with
respect to the expression of PKIN. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing PKIN to cells of the central nervous
system, for which HSV has a
tropism. The construction and packaging of herpes-based vectors are well known
to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92
which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by
this patent are the construction and use of recombinant HSV strains deleted
for ICP4, ICP27 and
ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-
532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding PKIN to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
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on the SFV genome (Garoff, H. and K.-J. Li (1998) C~.irr. 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
PKIN into the alphavirus
genome in place of the capsid-coding region results in the production of a
large number of PKIN-
coding RNAs and the synthesis of high levels of PKIN in vector transduced
cells. While alphavirus
infection is typically associated with cell lysis within a few days, the
ability to establish a persistent
infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis
virus (SIN) indicates that
the lytic replication of alphaviruses can be altered to suit the needs of the
gene therapy application
(Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range'of
alphaviruses will allow the
introduction of PKIN into a variety of cell types. The specific transduction
of a subset of cells in a
population may require the sorting of cells prior to transduction. The methods
of manipulating
infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and
performing alphavirus infections, are well known to those with ordinary skill
in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
be achieved using triple helix base-pairing methodology. Triple helix pairing
is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo 'sac Approaches, Futura Publishing, Mt. Kisco NY, pp.
163-177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA
by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding PKIN.
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
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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 PKIN. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines, cells,
or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' andlor 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-, thin-, 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
2o compound which is effective in altering expression of a polynucleotide
encoding PKIN. 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 PI~NN
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding PKIN may be therapeutically useful, and in the treatment of disorders
associated with
decreased PK1N expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding PKIN 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
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altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection. from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding PKIN 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
PKIN 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
to polynucleotide encoding PKIN. The amount of hybridization may be
quantified, thus forming the
basis for a comparison of the expression of the polynucleotide both with and
without exposure to one
or more test compounds. Detection of a change in the expression of a
polynucleotide exposed to a
test compound indicates that the test compound is effective in altering the
expression of the
polynucleotide. A screen for a compound effective in altering expression of a
specific polynucleotide
can be carried out, for example, using a Schizosaccharom~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.
Biotechnol. 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
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formulations are commonly known and are thoroughly discussed in the latest
edition of Remin tg on's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of PKIN,
antibodies to PKIN, and mimetics, agonists, antagonists, or inhibitors of
PK1N.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intxaperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-
acting formulations is well-known in the art. In the case of macromolecules
(e.g. larger peptides and
proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the lung
have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of
administration without
needle injection, and obviates the need for potentially toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising PKIN or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, PKIN 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 PKIN
or fragments thereof, antibodies of PKIN, and agonists, antagonists or
inhibitors of PKIN, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
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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.
Normal dosage amounts may vary from about 0.1 ~cg to 100,000 ~.cg, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind PKIN may be used for
the
diagnosis of disorders characterized by expression of PKIN, or in assays to
monitor patients being
treated with PKIN or agonists, antagonists, or inhibitors of PKIN. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for PKIN include methods which utilize the antibody and a label to
detect PKIN 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 PKIN, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
PKIN expression. Normal
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or standard values for PKIN expression are established by combining body
fluids or cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibodies
to PKIN under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of PKIN
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 PKIN 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 PKIN
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
PKIN, and to monitor regulation of PKIN levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding PKIN or closely related
molecules may be used to
identify nucleic acid sequences which encode PKIN. 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 PKIN, allelic
variants, or related
sequences.
2o Probes may also be used for the detection of related sequences, and may
have at least 50%
sequence identity to any of the PKIN encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ 1D
N0:23-44 or from
genomic sequences including promoters, enhancers, and introns of the PK1N
gene.
Means for producing specific hybridization probes for DNAs encoding PKIN
include the
cloning of polynucleotide sequences encoding PKIN or PKIN derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding PKIN may be used for the diagnosis of
disorders
associated with expression of PKIN. Examples of such disorders include, but
are not limited to, a
cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
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teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and uterus,
leukemias such as multiple myeloma and lymphomas such as Hodgkin's disease; an
immune disorder,
such as acquired imrnunodeficiency syndrome (A)DS), 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 growth and developmental disorder, such as actinic
keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and
cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and uterus,
renal tubular acidosis, anemia, C~shing'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; a cardiovascular
disease, such as 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
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artery bypass graft surgery, congestive heart failure, ischemic heart disease,
angina pectoris,
myocardial infarction, hypertensive heart disease, degenerative valvular heart
disease, calcific aortic
valve stenosis, congenitally bicuspid aortic valve, mural annular
calcification, mitral valve prolapse,
rheumatic fever and rheumatic heart disease, infective endocarditis,
nonbacterial thrombotic
endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart
disease, cardiomyopathy,
myocarditis, pericarditis, neoplastic heart disease, congenital heart disease,
and complications of
cardiac transplantation, congenital lung anomalies, atelectasis, pulmonary
congestion and edema,
pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary
hypertension, vascular
sclerosis, obstructive pulinonary 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; and a
lipid disorder such as fatty liver, cholestasis, primary biliary cirrhosis,
carnitine deficiency, carnitine
palinitoyltransferase deficiency, myoadenylate deaminase deficiency,
hypertriglyceridemia, lipid
2o storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's
disease, metachromatic
leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and ceroid
lipofuscinosis,
abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes
mellitus, lipodystrophy,
lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa,
lipoid adrenal hyperplasia,
minimal change disease, lipomas, atherosclerosis, hypercholesterolemia,
hypercholesterolemia with
hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal
disease, liver disease,
lecithin:cholesterol acyltransferase deficiency, cerebrotendinous
xanthomatosis, sitosterolemia,
hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia,
hyperlipemia, lipid
myopathies, and obesity. The polynucleotide sequences encoding PKIN 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 PKIN expression. Such qualitative or quantitative methods are
well known in the art.
In a particular aspect, the nucleotide sequences encoding PKIN may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
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sequences encoding PKIN 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
PKIN 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 PKIN,
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 PKIN, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects with
values from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation from standard values is
used to establish the
presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding PKIN
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 PKIN, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
PKIN, and will be employed under optimized conditions for identification of a
specific gene or
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condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding PKIN 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 PKIN are used to amplify
DNA using the
polymerase chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
to 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 PKIN include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
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 rnicroarray may also
be used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
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progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic
profile.
In another embodiment, PKIN, fragments of PKIN, or antibodies specific for
PKIN 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.
to A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
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provides the highest quality signature. Even genes whose expression is not
altered by any tested
compounds are important as well, as the levels of expression of these genes
are used to normalize the
rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the present
invention, so that transcript levels corresponding to the polynucleotides of
the present invention may be
quantified. The transcript levels in the treated biological sample are
compared with levels in an
untreated biological sample. Differences in the transcript levels between the
two samples are
indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating
and analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins
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
3o 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
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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 PKIN
to quantify the
levels of PKIN 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 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
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two samples is indicative of a toxic response to the test compound in the
treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D. et
al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding PKIN
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. Either
coding or noncoding sequences may be used, and in some instances, noncoding
sequences may be
preferable over coding sequences. For example, conservation of a coding
sequence among members
of a multi-gene family may potentially cause undesired cross hybridization
during chromosomal
mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (PACs), bacterial artificial chromosomes (BACs),
bacterial Pl
constructions, or single chromosome cDNA libraries. (See, e.g., 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
2o 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, Larder, 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-Uliich, et al. (1995) in Meyers, su ra, 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 PKIN on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another 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
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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, PKIN, 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 PKIN 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.,
Geysers, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with PKIN, or
fragments thereof,
and washed. Bound PKIN is then detected by methods well known in the art.
Purified PK1N 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 PKIN specifically compete with a test compound
for binding PKIN. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with PKIN.
In additional embodiments, the nucleotide sequences which encode PKIN 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 embodiments are, therefore,
to be construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way
whatsoever.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
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embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No. 60/242,410, U.S. Ser. No. 60/244,068, U.S. Ser. No.
60/245,708, U.S. Ser.
No. 60/247,672, U.S. Ser. No. 60/249,565, U.S. Ser. No. 60/252,730, and U.S.
Ser. No. 60/250,807,
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 CsCl
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
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, su ra,
units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies),
PCDNA2.1 plasmid
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(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or
pINCY (Incyte
Genomics), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli
cells including XL1-Blue, XI,1-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
l0 AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and
QIAWELL 8 Plasmid,
QIAWELL 8 Plus Plasmid, Q1AWELL 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).
2o 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 Phartnacia 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
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techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HIvIM)-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.)
l0 The queries were performed using programs based on BLAST, FASTA, BLIIVVIPS,
and HIVIMER.
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 (HIVIM)-based protein family databases such as PFAM. Full length
polynucleotide
sequences are also analyzed using MACDNASIS PRO software (Hitachi Software
Engineering,
South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and
polypeptide
sequence alignments are generated using default parameters specified by the
CLUSTAL algorithm as
incorporated into the MEGALIGN multisequence alignment program (DNASTAR),
which also
calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a
match between two sequences (the higher the score or the lower the probability
value, the greater the
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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 )D
N0:23-44. 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 human kinases were initially identified by running the Genscan gene
identification
program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-
purpose gene identification program which analyzes genomic DNA sequences from
a variety of
l0 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 human kinases, the encoded polypeptides were analyzed by
querying against
PFAM models for human kinases. Potential human kinases were also identified by
homology to
Incyte cDNA sequences that had been annotated as human kinases. 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
)II 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
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based on graph theory and dynamic programming to integrate cDNA and genomic
information,
generating possible splice variants that were subsequently confirmed, edited,
or extended to create a
full length sequence. Sequence intervals in which the entire length of the
interval was present on
more than one sequence in the cluster were identified, and intervals thus
identified were considered to
be equivalent by transitivity. For example, if an interval was present on a
cDNA and two genomic
sequences, then all three intervals were considered to be equivalent. This
process allows unrelated
but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals
thus identified were then "stitched" together by the stitching algorithm in
the order that they appear
along their parent sequences to generate the longest possible sequence, as
well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence
(cDNA to cDNA or
genomic sequence to genomic sequence) were given preference over linkages
which change parent
type (cDNA to genomic sequence). The resultant stitched sequences were
translated and compared
by BLAST analysis to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan
were corrected by comparison to the top BLAST hit from genpept. Sequences were
further extended
with additional cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
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 PKIN Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:23-44 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 )D N0:23-44 were assembled into clusters of contiguous and overlapping
sequences using
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assembly algorithms such as 1?hrap (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 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.nlin.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 N0:29 was mapped to chromosome 1 within the interval
from 199.20
to 203.00 centiMorgans, to chromosome 13 within the interval from 105.20
centiMorgans to the q
terminus, and to chromosome 6 within the interval from 59.60 to 72.20
centiMorgans. More than one
map location is reported for SEQ ID N0:29, 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.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
search can be modified to determine whether any particular match is
categorized as exact or similar.
The basis of the search is the product score, which is defined as:
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BLAST Score x Percent Identity
x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
5 length of the sequence match. The product score is a normalized value
between 0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding PKIN are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female; genitalia,
male; germ cells; hemic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or
urinary tract. The number of libraries in each category is counted and divided
by the total number of
libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding PKIN. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of PKIN Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
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fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68 °C to about 72 °C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
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+, (NH~)2S04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE
enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters
for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 60°C, 1 min;
Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step
6: 68 °C, 5 min; Step 7: storage
at 4°C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C,
3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4:
68°C, 2 min; Step 5: Steps 2, 3, and 4
repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
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(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
recoveries were reamplified using the same conditions as described above.
Samples were diluted with
20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure or
are used to obtain 5'regulatory sequences using the above procedure along with
oligonucleotides
designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:23-44 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~.cCi of
[Y 32P] 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
carned out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
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compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), su ra).
Suggested substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a
procedure analogous to a dot or slot blot may also be used to arrange and link
elements to the surface
of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
A typical array may
be produced using available methods and machines well known to those of
ordinary skill in the art and
may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science
270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and
J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described
in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)'
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/~1 RNase inhibitor, 500 p,M dATP, 500 p,M dGTP, 500
pM dTTP, 40 ~.M
dCTP, 40 pM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
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with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM 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 p.1 SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
to is amplified from bacterial cells containing vectors with cloned cDNA
inserts. PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5 pg.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 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 p1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
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larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of 140
p,1 of SX SSC in a corner of the chamber. The chamber containing the arrays is
incubated for about
6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in
a first wash buffer (1X SSC, 0.1
SDS), three times for 10 minutes each at 45° C in a second wash buffer
(0.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 rim for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that location
to be correlated with a weight ratio of hybridizing species of 1:100,000. When
two samples from
different sources (e.g., representing test and control cells), each labeled
with a different fluorophore,
are hybridized to a single array for the purpose of identifying genes that are
differentially expressed,
the calibration is done by labeling samples of the calibrating cDNA with the
two fluorophores and
adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping emission
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spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides
Sequences complementary to the PKIN-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring PKIN. Although
use of oligonucleotides
comprising from about 15 to 30 base pairs is described, essentially the same
procedure is used with
to smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of PK1N. 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 PKIN-encoding transcript.
XII. Expression of PKIN
Expression and purification of PKIN is achieved using bacterial or virus-based
expression
systems. For expression of PKIN in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express PKIN upon induction with isopropyl beta-
D-thiogalactopyranoside
(IPTG). Expression of PKIN in eukaryotic cells is achieved by infecting insect
or mammalian cell
lines with recombinant Autographica californica nuclear polyhedrosis virus
(AcMNPV), commonly
known as baculovirus. The nonessential polyhedrin gene of baculovirus is
replaced with cDNA
encoding PKIN 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 S~odoptera
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, PKIN 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,
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CA 02425963 2003-04-15
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affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma Lponicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Phartnacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved from PKIN 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 PKIN obtained by these methods can be used directly
in the assays shown in
to Examples XVI, XVII, and XVI>I, where applicable.
XIII. ~nctional Assays
PKIN function is assessed by expressing the sequences encoding PKIN at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 Ecg 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 PKIN on gene expression can be assessed using highly purified
populations
CA 02425963 2003-04-15
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of cells transfected with sequences encoding PKIN 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 PKIN and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIV. Production of PHIN Specific Antibodies
PKIN 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 PKIN 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, su ra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-PKIN activity by, for example, binding the peptide or
PKIN 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 PKIN Using Specific Antibodies
Naturally occurring or recombinant PKIN is substantially purified by
immunoaffinity
chromatography using antibodies specific for PKIN. An immunoaffinity column is
constructed by
covalently coupling anti-PKIN 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 PKIN are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of PKIN (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
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CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
antibody/PKIN 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 PKIN is collected.
XVI. Identification of Molecules Which Interact with PKIN
PKIN, or biologically active fragments thereof, are labeled with 1~I 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 PKIN, washed, and
any wells with labeled PKIN complex are assayed. Data obtained using different
concentrations of
PKIN are used to calculate values for the number, affinity, and association of
PKIN with the
candidate molecules.
Alternatively, molecules interacting with PKIN 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).
PKIN 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 PKIN Activity
Generally, protein kinase activity is measured by quantifying the
phosphorylation of a protein
substrate by PKIN in the presence of gamma-labeled 32P-ATP. PKIN is incubated
with the protein
substrate, 3zP-ATP, and an appropriate kinase buffer. The 32P incorporated
into the substrate is
separated from free'zP-ATP by electrophoresis and the incorporated 32P is
counted using a
radioisotope counter. The amount of incorporated 32P is proportional to the
activity of PKIN. A
determination of the specific amino acid residue phosphorylated is made by
phosphoamino acid
analysis of the hydrolyzed protein.
In one alternative, protein kinase activity is measured by quantifying the
transfer of gamma
phosphate from adenosine triphosphate (ATP) to a serine, threonine or tyrosine
residue in a protein
substrate. The reaction occurs between a protein kinase sample with a
biotinylated peptide substrate
and gamma 32P-ATP. Following the reaction, free avidin in solution is added
for binding to the
biotinylated 32P-peptide product. The binding sample then undergoes a
centrifugal ultrafiltration
process with a membrane which will retain the product-avidin complex and allow
passage of free
gamma 3zP-ATP. The reservoir of the centrifuged unit containing the'zP-peptide
product as retentate
is then counted in a scintillation counter. This procedure allows assay of any
type of protein kinase
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CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
sample, depending on the peptide substrate and kinase reaction buffer
selected. This assay is provided
in kit form (ASUA, Affinity Ultrafiltration Separation Assay, Transbio
Corporation, Baltimore MD,
U.S. Patent No. 5,869,275). Suggested substrates and their respective enzymes
are as follows:
Histone Hl (Sigma) and p34°a~2kinase, Annexin I, Angiotensin (Sigma)
and EGF receptor kinase,
Annexin II and src kinase, ERK1 & ERK2 substrates and MEK, and myelin basic
protein and ERK
(Pearson, J.D. et al. (1991) Methods in Enzymology 200:62-81).
In another alternative, protein kinase activity of PKIN is demonstrated in
vitro in an assay
containing PKIN, SOwI of kinase buffer, leg substrate, such as myelin basic
protein (MBP) or
synthetic peptide substrates, 1 mM DTT, 10 ~g ATP, and O.S~Ci ['y-33P]ATP. The
reaction is
to incubated at 30°C for 30 minutes and stopped by pipetting onto P81
paper. The unincorporated ['y-
33P~ATP is removed by washing and the incorporated radioactivity is measured
using a radioactivity
scintillation counter. Alternatively, the reaction is stopped by heating to
100 °C in the presence of SDS
loading buffer and visualized on a 12% SDS polyacrylamide gel by
autoradiography. Incorporated
radioactivity is corrected for reactions carried out in the absence of PKIN or
in the presence of the
inactive kinase, K38A.
In yet another alternative, adenylate kinase or guanylate kinase activity may
be measured by
the incorporation of 32P from gamma-labeled 32P -ATP into ADP or GDP using a
gamma radioisotope
counter. The enzyme, in a kinase buffer, is incubated together with the
appropriate nucleotide
mono-phosphate substrate (AMP or GMP) and 3zP-labeled ATP as the phosphate
donor. The
2o reaction is incubated at 37°C and terminated by addition of
trichloroacetic acid. The acid extract is
neutralized and subjected to gel electrophoresis to separate the mono-, di-,
and triphosphonucleotide
fractions. The diphosphonucleotide fraction is cut out and counted. The
radioactivity recovered is
proportional to the enzyme activity.
In yet another alternative, other assays for PKIN include scintillation
proximity assays (SPA),
scintillation plate technology and filter binding assays. Useful substrates
include recombinant proteins
tagged with glutathione transferase, or synthetic peptide substrates tagged
with biotin. Inhibitors of
PKIN activity, such as small organic molecules, proteins or peptides, may be
identified by such assays.
Kinase activity of PKIN may be determined by its ability to convert
polyphosphate substrate
(Polyp) to ATP in the presence of ADP. PKIN and Poly P are incubated at
37°C for 40 minutes and
3o then at 90°C for 2 minutes in a buffer containing 50 mM Tris-HCl, pH
7.4, 40 mM ammonium sulfate,
4 mM MgClz, and 5 ~M ADP. The reaction mixture is diluted 1:100 in 100 mM Tris-
HCl (pH 8.0), 4
mM EDTA, which is then diluted 1:1 in luciferase reaction mixture (ATP
Bioluminescence Assay Kit
CLS II; Boehringer Mannheim). The ATP generated is then quantitated using a
luminometer
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CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
(Kornberg, A. et al. (1999) Annu. Rev. Biochem. 68:89-125; Ault-Riche, D. et
al. (1998) J. Bacteriol.
180:1841-1847).
Kinase activity of PKIN, as measured by phosphorylation of substrate, may be
determined
using an immune complex kinase assay well known in the art. COS7 cells are
transfected with an
expression plasmid constructed from a FLAG tag expression vector (pMEl8S-FLAG)
containing
PKIN DNA. A control transfection using vector alone without the PKIN DNA
insert is done in
parallel. After 48 hours, the cells are lysed in buffer A (20 mM HEPES-NaOH,
pH 7.5, 3 mM
MgCl2, 100 mM NaCl2, 1 mM dithiothreitol, 1 mM phenylinethanesulfonyl
fluoride, 1 ~g/ml leupeptin, l
mM EGTA, 1 mM Na3Vo4, 10 mM NaF, 20 mM ~i-glycerophosphate, and 0.5% Triton X-
100) and
to centrifuged at 14,000 rpm. Supernatants are incubated with anti-FLAG
antibody (M2 monoclonal
antibody; Eastman Kodak Co.) in a SO% slurry of protein A-Sepharose (Amersham
Pharmacia
Biotech) for 1.5 hours at 4°C. Immune complexes are precipitated and
washed twice in buffer A and
twice in buffer B (20 mM HEPES-NaOH, pH 7.5, 1 mM dithiothreitol, 10 wM
Na3Vo4, 2 mM (3-
glycerophosphate, 0.1 mM phenylmethanesulfonyl fluoride, 0.1 ~g/ml leupeptin,
0.1 mM EGTA.)
Precipitates are incubated in buffer B containing 0.17 mg/ml myelin basic
protein (MBP) (Sigma), 20
p,M ATP, and 5 ~Ci of ['y-'ZP]ATP (NEN Life Science Products) at 30°C
for 20 minutes. The
reaction is stopped by the addition of 4X Laemmli sample buffer (SO mM Tris-
HCI, pH 6.8, 2% SDS,
30 mM dithiothreitol, and 10% glycerol) and heated at 95°C for 5
minutes. Proteins are separated by
SDS-polyacrylamide gel electrophoresis and radioactivity incorporated into MBP
is detected by
2o autoradiography (Nakano, K. et al. (2000) J. Biol. Chem. 275:20533-20539.)
In yet another alternative, an assay for PanK activity of PKIN includes the
enzyme
preparation method as described in Vallari, D.S. et al., (1987) J. Biol. Chem.
262:2468-247.
Pantothenate kinase-specific activities in cell lysates are calculated as a
function of protein
concentration with the assay being linear with respect to both time and
protein input. Protein
concentrations are measured using the Bradford assay using bovine 7-globulin
as a standard.
Standard assays contain D-[1-14C]pantothenate (45.5 p.M; specific activity SS
mCi/mmol), ATP (2.5
mM, pH 7.0), MgCl2 (2.5 mM), Tris-HCl (0.1 M, pH 7.5), and l5pg of protein
from a soluble cell
extract in a total volume of 40 p1. The mixture is incubated for 10 min. at 37
°C, and the reaction is
stopped by depositing a 30-~,1 aliquot onto a Whatman DE81 ion-exchange filter
disc which is then
washed in three changes of 1 % acetic acid in 95 % ethanol (25 ml/disc) to
remove unreacted
pantothenate. 4'-Phosphopantothenate is quantitated by counting the dried disc
in 3 ml of scintillation
solution (Rock, su ra).
XVIII. Enhancement/Inhibition of Protein Kinase Activity '
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CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Agonists or antagonists of PKIN activation or inhibition may be tested using
assays described
in section XVII. Agonists cause an increase in PKIN activity and antagonists
cause a decrease in
PKIN activity.
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 to those skilled in molecular biology or related fields are intended to be
within the scope of the
following claims.
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
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CA 02425963 2003-04-15
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CA 02425963 2003-04-15
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Table 5
PolynucleotideIncyte Representative Library
SEQ ID Project
NO: ID:
24 7483046CB COLCTUT03
1
25 71636374CBCARDNOTO1
1
26 7480597CB DRGLNOTO1
1
27 3227248CB COTRNOTO1
1
28 4207273CB TESTNOCO1
1
29 7483334CB ADRENOT03
1
30 7483337CB UTRSTMR02
1
31 6035509CB PITUNOT06
1
32 7373485CB MCLDTXT02
1
33 5734965CB PROSTUS23
1
34 7473788CB1BRAINOT19
35 3107989CB STOMFET02
1
37 2963414CB SCORNOT04
1
38 7477139CB1PLACFER06
39 55009053CB1SINITME01
41 7483053CB1BRAYDIN03
42 7483117CB ADREFECO1
1
43 7484498CB BRAITDR03
1
44 7638121CB1MUSLTDR02
116
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<110> INCYTE GENOMICS, INC.
GURURAJAN, Rajagopal
BAUGHN, Mariah R.
WALIA, Narinder K.
ELLIOTT, Vicki S.
XU, Yuming
ARVIZU, Chandra
YAO, Monique G.
RAMKUMAR, Jayalaxmi
DING, Li
TANG, Y. Tom
HAFALIA, April J.A.
NGUYEN, Danniel B.
GANDHI, Ameena R.
LU, Yan
YUE, Henry
BURFORD, Neil
BANDMAN, Olga
TRIBOULEY, Catherine
LAL, Preeti G.
RECIPON, Shirley A.
LU, Dyung Aina M.
BOROWSKY, Mark L.
THORNTON, Michael
SWARNAKER Anita
THANGAVELU, Kavitha
KHAN, Farrah A.
ISON, Craig H.
<120> HUMAN KINASES
<130> PI-0262 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/242,410; 60/244,068; 60/245,708; 60/247,672; 60/249,565;
60/252,730; 60/250,807
<151> 2000-10-20; 2000-10-27; 2000-11-03; 2000-11-09; 2000-11-16
2000-11-22; 2000-12-O1
<160> 44
<170> PERL Program
<210> 1
<211> 337
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7482896CD1
<400> 1
Met Thr Asn Asn Ser Gly Ser Lys Ala Glu Leu Val Val Gly Gly
1 5 10 15
1/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Val Tyr Leu Gly Ile Thr Thr Thr Asn Gly Glu Asp Val Ala Val
35 40 45
Lys Leu Glu Ser Gln Lys Val Lys His Pro Gln Leu Leu Tyr Glu
50 55 60
Ser Lys Leu Tyr Thr Ile Leu Gln Gly Gly Val Gly Ile Pro His
65 70 75
Met His Trp Tyr Gly Gln Glu.Lys Asp Asn Asn Val Leu Val Met
80 85 90
Asp Leu Leu Gly Pro Ser Leu Glu Asp Leu Phe Asn Phe Cys Ser
95 100 105
Arg Arg Phe Thr Met Lys Thr Val Leu Met Leu Ala Asp Gln Met
110 115 120
Ile Ser Arg Ile Glu Tyr Val His Thr Lys Asn Phe Leu His Arg
125 130 135
Asp Ile Lys Pro Asp Asn Phe Leu Met Gly Thr Gly Arg His Cys
140 145 150
Asn Lys Leu Phe Leu Ile Asp Phe Gly Leu Ala Lys Lys Tyr Arg
155 160 165
Asp Asn Arg Thr Arg Gln His Ile Pro Tyr Arg Glu Asp Lys His
170 175 180
Leu Ile Gly Thr Val Arg Tyr Ala Ser Ile Asn Ala His Leu Gly
185 190 195
Ile Glu Gln Ser Arg Arg Asp Asp Met Glu Ser Leu Gly Tyr Val
200 205 210
Phe Met Tyr Phe Asn Arg Thr Ser Leu Pro Trp Gln Gly Leu Arg
215 220 225
Ala Met Thr Lys Lys Gln Lys Tyr Glu Lys Ile Ser Glu Lys Lys
230 235 240
Met Ser Thr Pro Val Glu Val Leu Cys Lys Gly Phe Pro Ala Glu
245 250 255
Phe Ala Met Tyr Leu Asn Tyr Cys Arg Gly Leu Arg Phe Glu Glu
260 265 270
Val Pro Asp Tyr Met Tyr Leu Arg Gln Leu Phe Arg Ile Leu Phe
275 280 285
Arg Thr Leu Asn His Gln Tyr Asp Tyr Thr Phe Asp Trp Thr Met
290 . 295 300
Leu Lys Gln Lys Ala Ala Gln Gln Ala Ala Ser Ser Ser Gly Gln
305 310 315
Gly Gln Gln Ala Gln Thr Gln Thr Gly Lys Gln Thr Glu.Lys Asn
320 325 330
Lys Asn Asn Val Lys Asp Asn
335
<210> 2
<211> 475
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7483046CD1
<400> 2
Met Glu Arg Arg Leu Arg Ala Leu Glu Gln Leu Ala Arg Gly Glu
1 5 10 15
Ala Gly Gly Cys Pro Gly Leu Asp Gly Leu Leu Asp Leu Leu Leu
2/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
20 25 30
Ala Leu His His Glu Leu Ser Ser Gly Pro Leu Arg Arg Glu Arg
35 40 45
Ser Val Ala Gln Phe Leu Ser Trp Ala Ser Pro Phe Val Ser Lys
50 55 60
Val Lys Glu Leu Arg Leu Gln Arg Asp Asp Phe Glu Ile Leu Lys
65 70 75
Val Ile Gly Arg Gly Ala Phe Gly Glu Val Thr Val Val Arg Gln
80 85 90
Arg Asp Thr Gly Gln Ile Phe Ala Met Lys Met Leu His Lys Trp
95 100 105
Glu Met Leu Lys Arg Ala Glu Thr Ala Cys Phe Arg Glu Glu Arg
110 115 120
Asp Val Leu Val Lys Gly Asp Ser Arg Trp Val Thr Thr Leu His
125 130 135
Tyr Ala Phe Gln Asp Glu Glu Tyr Leu Tyr Leu Val Met Asp Tyr
140 145 150
Tyr Ala Gly Gly Asp Leu Leu Thr Leu Leu Ser Arg Phe Glu Asp
155 160 165
Arg Leu Pro Pro Glu Leu Ala Gln Phe Tyr Leu Ala Glu Met Val
170 175 180
Leu Ala Ile His Ser Leu His Gln Leu Gly Tyr Val His Arg Asp
185 190 195
Val Lys Pro Asp Asn Val Leu Leu Asp Val Asn Gly His Ile Arg
200 205 210
Leu Ala Asp Phe Gly Ser Cys Leu Arg Leu Asn Thr Asn Gly Met
215 220 225
Val Asp Ser Ser Val Ala Val Gly Thr Pro Asp Tyr Ile Ser Pro
230 235 240
Glu Ile Leu Gln Ala Met Glu Glu Gly Lys Gly His Tyr Gly Pro
245 250 255
Gln Cys Asp Trp Trp Ser Leu Gly Val Cys Ala Tyr Glu Leu Leu
260 265 270
Phe Gly Glu Thr Pro Phe Tyr Ala Glu Ser Leu Val Glu Thr Tyr
275 280 285
Gly Lys Ile Met Asn His Glu Asp His Leu Gln Phe Pro Pro Asp
290 295 300
Val Pro Asp Val Pro Ala Ser Ala Gln Asp Leu Ile Arg Gln Leu
305 310 315
Leu Cys Arg Gln Glu Glu Arg Leu Gly Arg Gly Gly Leu Asp Asp
320 325 330
Phe Arg Asn His Pro Phe Phe Glu Gly Val Asp Trp Glu Arg Leu
335 340 345
Ala Ser Ser Thr Ala Pro Tyr Ile Pro Glu Leu Arg Gly Pro Met
350 355 360
Asp Thr Ser Asn Phe Asp Val Asp Asp Asp Thr Leu Asn His Pro
365 370 375
Gly Thr Leu Pro Pro Pro Ser His Gly Ala Phe Ser Gly His His
380 385 390
Leu Pro Phe Val Gly Phe Thr Tyr Thr Ser Gly Ser His Ser Pro
395 400 405
Glu Ser Ser Ser Glu Ala Trp Ala Ala Leu Glu Arg Lys Leu Gln
410 415 420
Cys Leu Glu Gln Glu Lys Val Glu Leu Ser Arg Lys His Gln Glu
425 430 435
Ala Leu His Ala Pro Thr Asp His Arg Glu Leu Glu Gln Leu Arg
3/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
440 445 450
Lys Glu Val Gln Thr Leu Arg Asp Arg Leu Pro Gly Ile Pro Ser
455 460 465
Ala His Pro His Pro Leu Leu Glu Phe Leu
470 475
<210> 3
<211> 675
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 71636374CD1
<400> 3
Met Thr Thr Ser His Met Asn Gly His Val Thr Glu Glu Ser Asp
1 5 10 15
Ser Glu Val Lys Asn Val Asp Leu Ala Ser Pro Glu Glu His Gln
20 25 30
Lys His Arg Glu Met Ala Val Asp Cys Pro Gly Asp Leu Gly Thr
35 40 45
Arg Met Met Pro Ile Arg Arg Ser Ala Gln Leu Glu Arg Ile Arg
50 55 60
Gln Gln Gln Glu Asp Met Arg Arg Arg Arg Glu Glu Glu Gly Lys
65 70 75
Lys Gln Glu Leu Asp Leu Asn Ser Ser Met Arg Leu Lys Lys Leu
80 85 90
Ala Gln Ile Pro Pro Lys Thr Gly Ile Asp Asn Pro Met Phe Asp
95 100 105
Thr Glu Glu Gly Ile Val Leu Glu Ser Pro His Tyr Ala Val Lys
110 115 120
Ile Leu Glu Ile Glu Asp Leu Phe Ser Ser Leu Lys His Ile Gln
125 130 135
His Thr Leu Val Asp Ser Gln Ser Gln Glu Asp Ile Ser Leu Leu
140 145 150
Leu Gln Leu Val Gln Asn Lys Asp Phe Gln Asn Ala Phe Lys Ile
155 160 165
His Asn Ala Ile Thr Val His Met Asn Lys Ala Ser Pro Pro Phe
170 175 180
Pro Leu Ile Ser Asn Ala Gln Asp Leu Ala Gln Glu Val Gln Thr
185 190 195
Val Leu Lys Pro Val His His Lys Glu Gly Gln Glu Leu Thr Ala
200 205 210
Leu Leu Asn Thr Pro His Ile Gln Ala Leu Leu Leu Ala His Asp
215 220 225
Lys Val Ala Glu Gln Glu Met Gln Leu Glu Pro Ile Thr Asp Glu
230 235 240
Arg Val Tyr Glu Ser Ile Gly Gln Tyr Gly Gly Glu Thr Val Lys
245 250 255
Ile Val Arg Ile Glu Lys Ala Arg Asp Ile Pro Leu Gly Ala Thr
260 265 270
Val Arg Asn Glu Met Asp Ser Val Ile Ile Ser Arg Ile Val Lys
275 280 285
Gly Gly Ala Ala Glu Lys Ser Gly Leu Leu His Glu Gly Asp Glu
290 295 300
4/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Val Leu Glu Ile Asn Gly Ile Glu Ile Arg Gly Lys Asp Val Asn
305 310 315
Glu Val Phe Asp Leu Leu Ser Asp Met His Gly Thr Leu Thr Phe
320 325 330
Val Leu Ile Pro Ser Gln Gln Ile Lys Pro Pro Pro Ala Lys Glu
335 340 345
Thr Val Ile His Val Lys Ala His Phe Asp Tyr Asp Pro Ser Asp
350 355 360
Asp Pro Tyr Val Pro Cys Arg Glu Leu Gly Leu Ser Phe Gln Lys
365 370 375
Gly Asp Ile Leu His Val Ile Ser Gln Glu Asp Pro Asn Trp Trp
380 385 390
Gln Ala Tyr Arg Glu Gly Asp Glu Asp Asn Gln Pro Leu Ala Gly
395 400 405
Leu Val Pro Gly Lys Ser Phe Gln Gln Gln Arg Glu Ala Met Lys
410 415 420
Gln Thr Ile Glu Glu Asp Lys Glu Pro,Glu Lys Ser Gly Lys Leu
425 430 435
Trp Cys Ala Lys Lys Asn Lys Lys Lys Arg Lys Lys Val Leu Tyr
440 445 450
Asn Ala Asn Lys Asn Asp Asp Tyr Asp Asn Glu Glu Ile Leu Thr
455 460 465
Tyr Glu Glu Met Ser Leu Tyr His Gln Pro Ala Asn Arg Lys Arg
470 475 480
Pro Ile Ile Leu Ile Gly Pro Gln Asn Cys Gly Gln Asn Glu Leu
485 490 495
Arg Gln Arg Leu Met Asn Lys Glu Lys Asp Arg Phe Ala Ser Ala
500 505 510
Val Pro His Thr Thr Arg Ser Arg Arg Asp Gln Glu Val Ala Gly
515 520 525
Arg Asp Tyr His Phe Val Ser Arg Gln Ala Phe Glu Ala Asp Ile
530 535 540
Ala Ala Gly Lys Phe Ile Glu His Gly Glu Phe Glu Lys Asn Leu
545 550 555
Tyr Gly Thr Ser Ile Asp Ser Val Arg Gln Val Ile Asn Ser Gly
560 565 570
Lys Ile Cys Leu Leu Ser Leu Arg Thr Gln Ser Leu Lys Thr Leu
575 580 585
Arg Asn Ser Asp Leu Lys Pro Tyr Ile Ile Phe Ile Ala Pro Pro
590 595 600
Ser Gln Glu Arg Leu Arg Ala Leu Leu Ala Lys Glu Gly Lys Asn
605 610 615
Pro Lys Pro Glu Glu Leu Arg Glu Ile Ile Glu Lys Thr Arg Glu
620 625 630
Met Glu Gln Asn Asn Gly His Tyr Phe Asp Thr Ala Ile Val Asn
635 640 645
Ser Asp Leu Asp Lys Ala Tyr Gln Glu Leu Leu Arg Leu Ile Asn
650 655 660
Lys Leu Asp Thr Glu Pro Gln Trp Val Pro Ser Thr Trp Leu Arg
665 670 675
<210> 4
<211> 835
<212> PRT
<213> Homo Sapiens
5/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
<220>
<221> misc_feature
<223> Incyte ID No: 7480597CD1
<400> 4
Met Ala Glu Gly Lys Glu Gly Gln Val Pro Ser Tyr Met Asp Gly
1 5 10 15
Ser Arg Gln Arg Glu Asn Glu Glu Asp Ala Lys Ala Glu Thr Pro
20 25 30
Asp Val Thr Ile Arg Ser Tyr Glu Ile Tyr Ser Leu Pro Trp Asn
35 40 45
Arg Gln Gln Gly Leu Cys Asp His Ser Leu Lys Tyr Leu Ser Ser
50 55 60
Arg Ile Thr Glu Arg Lys Leu Gln Gly Ser Trp Leu Pro Ala Ser
65 70 75
Arg Gly A~sn Leu Glu Lys Pro Phe Leu Gly Pro Arg Gly Pro Val
80 85 90
Val Pro Leu Phe Cys Pro Arg Asn Gly Leu His Ser Ala His Pro
95 100 105
Glu Asn Ser Pro Leu Lys Pro Arg Val Val Thr Val Val Lys Leu
110 115 120
Gly Gly Gln Arg Pro Arg Lys Ile Thr Leu Leu Leu Asn Arg Arg
125 130 135
Ser Val Gln Thr Phe Glu Gln Leu Leu Ala Asp Ile Ser Glu Ala
140 145 150
Leu Gly Ser Pro Arg Trp Lys Asn Asp Arg Val Arg Lys Leu Phe
155 160 165
Asn Leu Lys Gly Arg Glu Ile Arg Ser Val Ser Asp Phe Phe Arg
170 175 180
Glu Gly Asp Ala Phe Ile Ala Met Gly Lys Glu Pro Leu Thr Leu
185 190 195
Lys Ser Ile Gln Val Ala Val Glu Glu Leu Tyr Pro Asn Lys Ala
200 205 210
Arg Ala Leu Thr Leu Ala Gln His Ser Arg Ala Pro Ser Pro Arg
215 220 225
Leu Arg Ser Arg Leu Phe Ser Lys Ala Leu Lys Gly Asp His Arg
230 235 240
Cys Gly Glu Thr Glu Thr Pro Lys Ser Cys Ser Glu Val Ala Gly
245 250 255
Cys Lys Ala Ala Met Arg His Gln Gly Lys Ile Pro Glu Glu Leu
260 265 270
Ser Leu Asp Asp Arg Ala Arg Thr Gln Lys Lys Trp Gly Arg Gly
275 280 285
Lys Trp Glu Pro Glu Pro Ser Ser Lys Pro Pro Arg Glu Ala Thr
290 295 300
Leu Glu Glu Arg His Ala Arg Gly Glu Lys His Leu Gly Val Glu
305 310 315
Ile Glu Lys Thr Ser Gly Glu Ile Ile Arg Cys Glu Lys Cys Lys
320 325 330
Arg Glu Arg Glu Leu Gln Gln Ser Leu Glu Arg Glu Arg Leu Ser
335 340 345
Leu Gly Thr Ser Glu Leu Asp Met Gly Lys Gly Pro Met Tyr Asp
350 355 360
Val Glu Lys Leu Val Arg Thr Arg Ser Cys Arg Arg Ser Pro Glu
365 370 375
Ala Asn Pro Ala Ser Gly Glu Glu Gly Trp Lys Gly Asp Ser His
6/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
380 385 390
Arg Ser Ser Pro Arg Asn Pro Thr Gln Glu Leu Arg Arg Pro Ser
395 400 405
Lys Ser Met Asp Lys Lys Glu Asp Arg Gly Pro Glu Asp Gln Glu
410 415 420
Ser His Ala Gln Gly Ala Ala Lys Ala Lys Lys Asp Leu Val Glu
425 430 435
Val Leu Pro Val Thr Glu Glu Gly Leu Arg Glu Val Lys Lys Asp
440 445 450
Thr Arg Pro Met Ser Arg Ser Lys His Gly Gly Trp Leu Leu Arg
455 460 465
Glu His Gln Ala Gly Phe Glu Lys Leu Arg Arg Thr Arg Gly Glu
470 475 480
Glu Lys Glu Ala Glu Lys Glu Lys Lys Pro Cys Met Ser Gly Gly
485 490 495
Arg Arg Met Thr Leu Arg Asp Asp Gln Pro Ala Lys Leu Glu Lys
500 505 510
Glu Pro Lys Thr Arg Pro Glu Glu Asn Lys Pro Glu Arg Pro Ser
515 520 525
Gly Arg Lys Pro Arg Pro Met Gly Ile Ile Ala Ala Asn Val Glu
530 535 540
Lys His Tyr Glu Thr Gly Arg Val Ile Gly Asp Gly Asn Phe Ala
545 550 555
Val Val Lys Glu Cys Arg His Arg Glu Thr Arg Gln Ala Tyr Ala
560 565 570
Met Lys Ile Ile Asp Lys Ser Arg Leu Lys Gly Lys Glu Asp Met
575 580 585
Val Asp Ser Glu Ile Leu Ile Ile Gln Ser Leu Ser His Pro Asn
590 595 600
Ile Val Lys Leu His Glu Val Tyr Glu Thr Asp Met Glu Ile Tyr
605 610 615
Leu Ile Leu Glu Tyr Val Gln Gly Gly Asp Leu Phe Asp Ala Ile
620 625 630
Ile Glu Ser Val Lys Phe Pro Glu Pro Asp Ala Ala Leu Met Ile
635 640 645
Met Asp Leu Cys Lys Ala Leu Val His Met His Asp Lys Ser Ile
650 655 660
Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Val Gln Arg Asn
665 670 675
Glu Asp Lys Ser Thr Thr Leu Lys Leu Ala Asp Phe Gly Leu Ala
680 685 690
Lys His Val Val Arg Pro Ile Phe Thr Val Cys Gly Thr Pro Thr
695 700 705
Tyr Val Ala Pro Glu Ile Leu Ser Glu Lys Gly Tyr Gly Leu Glu
710 715 720
Val Asp Met Trp Ala Ala Gly Val Ile Leu Tyr Ile Leu Leu Cys
725 730 735
Gly Phe Pro Pro Phe Arg Ser Pro Glu Arg Asp Gln Asp Glu Leu
740 745 750
Phe Asn Ile Ile Gln Leu Gly His Phe Glu Phe Leu Pro Pro Tyr
755 760 765
Trp Asp Asn Ile Ser Asp Ala Ala Lys Asp Leu Val Ser Arg Leu
770 775 780
Leu Val Val Asp Pro Lys Lys Arg Tyr Thr Ala His Gln Val Leu
785 790 795
Gln His Pro Trp Ile Glu Thr Ala Gly Lys Thr Asn Thr Val Lys
7/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
800 805 810
Arg Gln Lys Gln Val Ser Pro Ser Ser Glu Gly His Phe Arg Ser
815 820 825
Gln His Lys Arg Val Val Glu Gln Val Ser
830 835
<210> 5
<211> 373
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3227248CD1
<400> 5
Met Lys Leu Ile Asn Gly Lys Lys Gln Thr Phe Pro Trp Phe Gly
1 5 10 15
Met Asp Ile Gly Gly Thr Leu Val Lys Leu Val Tyr Phe Glu Pro
20 25 30
Lys Asp Ile Thr Ala Glu Glu Glu Gln Glu Glu Val Glu Asn Leu
35 40 45
Lys Ser Ile Arg Lys Tyr Leu Thr Ser Asn Thr Ala Tyr Gly Lys
50 55 60
Thr Gly Ile Arg Asp Val His Leu Glu Leu Lys Asn Leu Thr Met
65 70 75
Cys Gly Arg Lys Gly Asn Leu His Phe Ile Arg Phe Pro Ser Cys
80 85 90
Ala Met His Arg Phe Ile Gln Met Gly Ser Glu Lys Asn Phe Ser
95 100 105
Ser Leu His Thr Thr Leu Cys Ala Thr Gly Gly Gly Ala Phe Lys
110 115 120
Phe Glu Glu Asp Phe Arg Met Ile Ala Asp Leu Gln Leu His Lys
125 130 135
Leu Asp Glu Leu Asp Cys Leu Ile Gln Gly Leu Leu Tyr Val Asp
140 145 150
Ser Val Gly Phe Asn Gly Lys Pro Glu Cys Tyr Tyr Phe Glu Asn
155 160 165
Pro Thr Asn Pro Glu Leu Cys Gln Lys Lys Pro Tyr Cys Leu Asp
170 175 180
Asn Pro Tyr Pro Met Leu Leu Val Asn Met Gly Ser Gly Val Ser
185 190 195
Ile Leu Ala Val Tyr Ser Lys Asp Asn Tyr Lys Arg Val Thr Gly
200 205 210
Thr Ser Leu Gly Gly Gly Thr Phe Leu Gly Leu Cys Cys Leu Leu
215 220 225
Thr Gly Cys Glu Thr Phe Glu Glu Ala Leu Glu Met Ala Ala Lys
230 235 240
Gly Asp Ser Thr Asn Val Asp Lys Leu Val Lys Asp Ile Tyr Gly
245 250 255
Gly Asp Tyr Glu Arg Phe Gly Leu Gln Gly Ser Ala Val Ala Ser
260 265 270
Ser Phe Gly Asn Met Met Ser Lys Glu Lys Arg Asp Ser Ile Ser
275 280 285
Lys Glu Asp Leu Ala Arg Ala Thr Leu Val Thr Ile Thr Asn Asn
290 295 300
8/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Ile Gly Ser Ile Ala Arg Met Cys Ala Leu Asn Glu Asn Ile Asp
305 310 315
Arg Val Val Phe Val Gly Asn Phe Leu Arg Ile Asn Met Val Ser
320 325 330
Met Lys Leu Leu Ala Tyr Ala Met Asp Phe Trp Ser Lys Gly Gln
335 340 345
Leu Lys Ala Leu Phe Leu Glu His Glu Gly Tyr Phe Gly Ala Val
350 355 360
Gly Ala Leu Leu Glu Leu Phe Lys Met Thr Asp Asp Lys
365 370
<210> 6
<211> 735
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4207273CD1
<400> 6
Met Pro Gln Ile Ala Lys Lys Gln Ser Thr His Arg Thr Gln Lys
1 5 10 15
Pro Lys Lys Gln Ser Phe Pro Cys Ile Cys Lys Asn Pro Gly Thr
20 25 30
Gln Lys Ser Cys Val Pro Leu Ser Val Gln Pro Thr Glu Pro Arg
35 40 45
Leu Asn Tyr Leu Asp Leu Lys Tyr Ser Asp Met Phe Lys Glu Ile
50 55 60
Asn Ser Thr Ala Asn Gly Pro Gly Ile Tyr Glu Met Phe Gly Thr
65 70 75
Pro Val Tyr Cys His Val Arg Glu Thr Glu Arg Asp Glu Asn Thr
80 85 90
Tyr Tyr Arg Glu Ile Cys Ser Ala Pro Ser Gly Arg Arg Ile Thr
95 100 105
Asn Lys Cys Arg Ser Ser His Ser Glu Arg Lys Ser Asn Ile Arg
110 115 120
Thr Arg Leu Ser Gln Lys Lys Thr His Met Lys Cys Pro Lys Thr
125 130 135
Ser Phe Gly Ile Lys Gln Glu His Lys Val Leu Ile Ser Lys Glu
140 145 150
Lys Ser Ser Lys Ala Val His Ser Asn Leu His Asp Ile Glu Asn
155 160 165
Gly Asp Gly Ile Ser Glu Pro Asp Trp Gln Ile Lys Ser Ser Gly
170 175 180
Asn Glu Phe Leu Ser Ser Lys Asp Glu Ile His Pro Met Asn Leu
185 190 195
Ala Gln Thr Pro Glu Gln Ser Met Lys Gln Asn Glu Phe Pro Pro
200 205 210
Val Ser Asp Leu Ser Ile Val Glu Glu Val Ser Met Glu Glu Ser
215 220 225
Thr Gly Asp Arg Asp Ile Ser Asn Asn Gln Ile Leu Thr Thr Ser
230 235 240
Leu Arg Asp Leu Gln Glu Leu Glu Glu Leu His His Gln Ile Pro
245 250 255
Phe Ile Pro Ser Glu Asp Ser Trp Ala Val Pro Ser Glu Lys Asn
9/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
260 265 270
Ser Asn Lys Tyr Val Gln Gln Glu Lys Gln Asn Thr Ala Ser Leu
275 280 285
Ser Lys Val Asn Ala Ser Arg Ile Leu Thr Asn Asp Leu Glu Phe
290 295 300
Asp Ser Val Ser Asp His Ser Lys Thr Leu Thr Asn Phe Ser Phe
305 310 315
Gln Ala Lys Gln Glu Ser Ala Ser Ser Gln Thr Tyr Gln Tyr Trp
320 325 330
Val His Tyr Leu Asp His Asp Ser Leu Ala Asn Lys Ser Ile Thr
335 340 345
Tyr Gln Met Phe Gly Lys Thr Leu Ser Gly Thr Asn Ser Ile Ser
350 355 360
Gln Glu Ile Met Asp Ser Val Asn Asn Glu Glu Leu Thr Asp Glu
365 370 375
Leu Leu Gly Cys Leu Ala Ala Glu Leu Leu Ala Leu Asp Glu Lys
380 385 390
Asp Asn Asn Ser Cys Gln Lys Met Ala Asn Glu Thr Asp Pro Glu
395 400 405
Asn Leu Asn Leu Val Leu Arg Trp Arg Gly Ser Thr Pro Lys Glu
410 415 420
Met Gly Arg Glu Thr Thr Lys Val Lys Ile Gln Arg His Ser Ser
425 430 435
Gly Leu Arg Ile Tyr Asp Arg Glu Glu Lys Phe Leu Ile Ser Asn
440 445 450
Glu Lys Lys Ile Phe Ser Glu Asn Ser Leu Lys Ser Glu Glu Pro
455 460 465
Ile Leu Trp Thr Lys Gly Glu Ile Leu Gly Lys Gly Ala Tyr Gly
470 475 480
Thr Val Tyr Cys Gly Leu Thr Ser Gln Gly Gln Leu Ile Ala Val
485 490 495
Lys Gln Val Ala Leu Asp Thr Ser Asn Lys Leu Ala Ala Glu Lys
500 505 510
Glu Tyr Arg Lys Leu Gln Glu Glu Val Asp Leu Leu Lys Ala Leu
515 520 525
Lys His Val Asn Ile Val Ala Tyr Leu Gly Thr Cys Leu Gln Glu
530 535 540
Asn Thr Val Ser Ile Phe Met Glu Phe Val Pro Gly Gly Ser Ile
545 550 555
Ser Ser Ile Ile Asn Arg Phe Gly Pro Leu Pro Glu Met Val Phe
560 565 570
Cys Lys Tyr Thr Lys Gln Ile Leu Gln Gly Val Ala Tyr Leu His
575 580 585
Glu Asn Cys Val Val His Arg Asp Ile Lys Gly Asn Asn Val Met
590 595 600
Leu Met Pro Thr Gly Ile Ile Lys Leu Ile Asp Phe Gly Cys Ala
605 610 615
Arg Arg Leu Ala Trp Ala Gly Leu Asn Gly Thr His Ser Asp Met
620 625 630
Leu Lys Ser Met His Gly Thr Pro Tyr Trp Met Ala Pro Glu Val
635 640 645
Ile Asn Glu Ser Gly Tyr Gly Arg Lys Ser Asp Ile Trp Ser Ile
650 655 660
Gly Cys Thr Val Phe Glu Met Ala Thr Gly Lys Pro Pro Leu Ala
665 670 675
Ser Met Asp Arg Met Ala Ala Met Phe Tyr Ile Gly Ala His Arg
10/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
680 685 690
Gly Leu Met Pro Pro Leu Pro Asp His Phe Ser Glu Asn Ala Ala
695 700 705
Asp Phe Val Arg Met Cys Leu Thr Arg Asp Gln His Glu Arg Pro
710 715 720
Ser Ala Leu Gln Leu Leu Lys His Ser Phe Leu Glu Arg Ser His
725 730 735
<210> 7
<211> 506
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483334CD1
<400> 7
Met Asp Asp Tyr Met Val Leu Arg Met Ile Gly Glu Gly Ser Phe
1 5 10 15
Gly Arg Ala Leu Leu Val Gln Leu Glu Ser Ser Asn Gln Met Phe
20 25 30
Ala Met Lys Glu Ile Arg Leu Pro Lys Ser Phe Ser Asn Thr Gln
35 40 45
Asn Ser Arg Lys Glu Ala Val Leu Leu Ala Lys Met Lys His Pro
50 55 60
Asn Ile Val Ala Phe Lys Glu Ser Phe Glu Ala Glu Gly His Leu
65 70 75
Tyr Ile Val Met Glu Tyr Cys Asp Gly Gly Asp Leu Met Gln Lys
80 85- 90
Ile Lys Gln Gln Lys Gly Lys Leu Phe Pro Glu Asp Met Ile Leu
95 100 105
Asn Trp Phe Thr Gln Met Cys Leu Gly Val Asn His Ile His Lys
110 115 120
Lys Arg Val Leu His Arg Asp Ile Lys Ser Lys Asn Ile Phe Leu
125 130 135
Thr Gln Asn Gly Lys Val Lys Leu Gly Asp Phe Gly Ser Ala Arg
140 145 150
Leu Leu Ser Asn Pro Met Ala Phe Ala Cys Thr Tyr Val Gly Thr
155 160 165
Pro Tyr Tyr Val Pro Pro Glu Ile Trp Glu Asn Leu Pro Tyr Asn
170 175 180
Asn Lys Ser Asp Ile Trp Ser Leu Gly Cys Ile Leu Tyr Glu Leu
185 190 195
Cys Thr Leu Lys His Pro Phe Gln Ala Asn Ser Trp Lys Asn Leu
200 205 210
Ile Leu Lys Val Cys Gln Gly Cys Ile Ser Pro Leu Pro Ser His
215 220 225
Tyr Ser Tyr Glu Leu Gln Phe Leu Val Lys Gln Met Phe Lys Arg
230 235 240
Asn Pro Ser His Arg Pro Ser Ala Thr Thr Leu Leu Ser Arg Gly
245 250 255
Ile Val Ala Arg Leu Val Gln Lys Cys Leu Pro Pro Glu Ile Ile
260 265 270
Met Glu Tyr Gly Glu Glu Val Leu Glu Glu Ile Lys Asn Ser Lys
11/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
275 280 285
His Asn Thr Pro Arg Lys Lys Thr Asn Pro Ser Arg Ile Arg Ile
290 295 300
Ala Leu Gly Asn Glu Ala Ser Thr Val Gln Glu Glu Glu Gln Asp
305 310 315
Arg Lys Gly Ser His Thr Asp Leu Glu Ser Ile Asn Glu Asn Leu
320 325 330
Val Glu Ser Ala Leu Arg Arg Val Asn Arg Glu Glu Lys Gly Asn
335 340 345
Lys Ser Val His Leu Arg Lys Ala Ser Ser Pro Asn Leu His Arg
350 355 360
Arg Gln Trp Glu Lys Asn Val Pro Asn Thr Ala Leu Thr Ala Leu
365 370 375
Glu Asn Ala Ser Ile Leu Thr Ser Ser Leu Thr Ala Glu Asp Asp
380 385 390
Arg Gly Gly Ser Val Ile Lys Tyr Ser Lys Asn Thr Thr Arg Lys
395 400 405
Gln Trp Leu Lys Glu Thr Pro Asp Thr Leu Leu Asn Ile Leu Lys
410 415 420
Asn Ala Asp Leu Ser Leu Ala Phe Gln Thr Tyr Thr Ile Tyr Arg
425 430 435
Pro Gly Ser Glu Gly Phe Leu Lys Gly. Pro Leu Ser Glu Glu Thr
440 445 450
Glu Ala Ser Asp Ser Val Asp Gly Gly His Asp Ser Val Ile Leu
455 460 465
Asp Pro Glu Arg Leu Glu Pro Gly Leu Asp Glu Glu Asp Thr Asp
470 475 480
Phe Glu Glu Glu Asp Asp Asn Pro Asp Trp Val Ser Glu Leu Lys
485 490 495
Lys Arg Ala Gly Trp Gln Gly Leu Cys Asp Arg
500 505
<210> 8
<211> 2014
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483337CD1
<400> 8
Met Glu Thr Leu Asn Gly Ala Gly Asp Thr Gly Gly Lys Pro Ser
1 5 10 15
Thr Arg Gly Gly Asp Pro Ala Ala Arg Ser Arg Arg Thr Glu Gly
20 25 30
Ile Arg Ala Ala Tyr Arg Arg Gly Asp Arg Gly Gly Ala Arg Asp
35 40 45
Leu Leu Glu Glu Ala Cys Asp Gln Cys Ala Ser Gln Leu Glu Lys
50 55 60
Gly Gln Leu Leu Ser Ile Pro Ala Ala Tyr Gly Asp Leu Glu Met
65 70 75
Val Arg Tyr Leu Leu Ser Lys Arg Leu Val Glu Leu Pro Thr Glu
80 85 90
Pro Thr Asp Asp Asn Pro Ala Val Val Ala Ala Tyr Phe Gly His
95 100 105
12/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Thr Ala Val Val Gln Asn Thr Leu Pro Thr Glu Pro Thr Asp Asp
110 115 120
Asn Pro Ala Val Val Ala Ala Tyr Phe Gly His Thr Ala Val Val
125 130 135
Gln Glu Leu Leu Glu Ser Leu Pro Gly Pro Cys Ser Pro Gln Arg
140 145 150
Leu Leu Asn Trp Met Leu Ala Leu Ala Cys Gln Arg Gly His Leu
155 160 165
Gly Val Val Lys Leu Leu Val Leu Thr His Gly Ala Asp Pro Glu
170 175 180
Ser Tyr Ala Val Arg Lys Asn Glu Phe Pro Val Ile Val Arg Leu
185 190 195
Pro Leu Tyr Ala Ala Ile Lys Ser Gly Asn Glu Asp Ile Ala Ile
200 205 210
Phe Leu Leu Arg His Gly Ala Tyr Phe Cys Ser Tyr Ile Leu Leu
215 220 225
Asp Ser Pro Asp Pro Ser Lys His Leu Leu Arg Lys Tyr Phe Ile
230 235 240
Glu Ala Ser Pro Leu Pro Ser Ser Tyr Pro Gly Lys Thr Ala Leu
245 250 255
Arg Val Lys Trp Ser His Leu Arg Leu Pro Trp Val Asp Leu Asp
260 265 270
Trp Leu Ile Asp Ile Ser Cys Gln Ile Thr Glu Leu Asp Leu Ser
275 280 285
Ala Asn Cys Leu Ala Thr Leu Pro Ser Val Ile Pro Trp Gly Leu
290 295 300
Ile Asn Leu Arg Lys Leu Asn Leu Ser Asp Asn His Leu Gly Glu
305 310 315
Leu Pro Gly Val Gln Ser Ser Asp Glu Ile Ile Cys Ser Arg Leu
320 325 330
Leu Glu Ile Asp Ile Ser Ser Asn Lys Leu Ser His Leu Pro Pro
335 340 345
Gly Phe Leu His Leu Ser Lys Leu Gln Lys Leu Thr Ala Ser Lys
350 355 360
Asn Cys Leu Glu Lys Leu Phe Glu Glu Glu Asn Ala Thr Asn Trp
365 370 375
Ile Gly Leu Arg Lys Leu Gln Glu Leu Asp Ile Ser Asp Asn Lys
380 385 390
Leu Thr Glu Leu Pro Ala Leu Phe Leu His Ser Phe Lys Ser Leu
395 400 405
Asn Ser Leu Asn Val Ser Arg Asn Asn Leu Lys Val Phe Pro Asp
410 415 420
Pro Trp Ala Cys Pro Leu Lys Cys Cys Lys Ala Ser Arg Asn Ala
425 430 435
Leu Glu Cys Leu Pro Asp Lys Met Ala Val Phe Trp Lys Asn His
440 445 450
Leu Lys Asp Val Asp Phe Ser Glu Asn Ala Leu Lys Glu Val Pro
455 460 465
Leu Gly Leu Phe Gln Leu Asp Ala Leu Met Phe Leu Arg Leu Gln
470 475 480
Gly Asn Gln Leu Ala Ala Leu Pro Pro Gln Glu Lys Trp Thr Cys
485 490 495
Arg Gln Leu Lys Thr Leu Asp Leu Ser Arg Asn Gln Leu Gly Lys
500 505 510
Asn Glu Asp Gly Leu Lys Thr Lys Arg Ile Ala Phe Phe Thr Thr
515 520 525
13/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Arg Gly Arg Gln Arg Ser Gly Thr Glu Ala Glu Thr Thr Met Glu
530 535 540
Phe Ser Ala Ser Leu Val Thr Ile Val Phe Leu Ser Asn Asn Cys
545 550 555
Asn Leu Cys Ala Tyr Thr Cys Ala Ala Ser Val Leu Glu Phe Pro
560 565 570
Ala Phe Leu Ser Glu Ser Leu Glu Val Leu Cys Leu Asn Asp Asn
575 580 585
His Leu Asp Thr Val Pro Pro Ser Val Cys Leu Leu Lys Ser Leu
590 595 600
Ser Glu Leu Tyr Leu Gly Asn Asn Pro Gly Leu Arg Glu Leu Pro
605 610 615
Pro Glu Leu Gly Gln Leu Gly Asn Leu Trp Gln Leu Asp Thr Glu
620 625 630
Asp Leu Thr Ile Ser Asn Val Pro Ala Glu Ile Gln Lys Glu Gly
635 640 645
Pro Lys Ala Met Leu Ser Tyr Leu Arg Ala Gln Leu Arg Lys Ala
650 655 660
Glu Lys Cys Lys Leu Met Lys Met Ile Ile Val Gly Pro Pro Arg
665 670 675
Gln Gly Lys Ser Thr Leu Leu Glu Ile Leu Gln Thr Gly Arg Ala
680 685 690
Pro Gln Val Val His Gly Glu Ala Thr Ile Arg Thr Thr Lys Trp
695 700 705
Glu Leu Gln Arg Pro Ala Gly Ser Arg Ala Lys Val Lys Asp Gly
710 715 720
Leu Arg Ala Glu Ser Leu Trp Val Glu Ser Val Glu Phe Asn Val
725 730 735
Trp Asp Ile Gly Gly Pro Ala Ser Met Ala Thr Val Asn Gln Cys
740 745 750
Phe Phe Thr Asp Lys Ala Leu Tyr Val Val.Va1 Trp Asn Leu Ala
755 760 765
Leu Gly Glu Glu Ala Val Ala Asn Leu Gln Phe Trp Leu Leu Asn
770 775 780
Ile Glu Ala Lys Ala Pro Asn Ala Val Val Leu Val Val Gly Thr
785 790 795
His Leu Asp Leu Ile Glu Ala Lys Phe Arg Val Glu Arg Ile Ala
800 805 810
Thr Leu Arg Ala Tyr Val Leu Ala Leu Cys Arg Ser Pro Ser Gly
815 820 825
Ser Arg Ala Thr Gly Phe Pro Asp Ile Thr Phe Lys His Leu His
830 835 840
Glu Ile Ser Cys Lys Ser Leu Glu Gly Gln Glu Gly Leu Arg Gln
845 850 855
Leu Ile Phe His Val Thr Cys Ser Met Lys Asp Val Gly Ser Thr
860 865 870
Ile Gly Cys Gln Arg Leu Ala Gly Arg Leu Ile Pro Arg Ser Tyr
875 880 885
Leu Ser Leu Gln Glu Ala Val Leu Ala Glu Gln Gln Arg Arg Ser
890 895 900
Arg Asp Asp Asp Val Gln Tyr Leu Thr Asp Arg Gln Leu Glu Gln
905 910 915
Leu Val Glu Gln Thr Pro Asp Asn Asp Ile Lys Asp Tyr Glu Asp
920 925 930
Leu Gln Ser Ala Ile Ser Phe Leu Ile Glu Thr Gly Thr Leu Leu
935 940 945
14/75
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His Phe Pro Asp Thr Ser His Gly Leu Arg Asn Leu Tyr Phe Leu
950 955 960
Asp Pro Ile Trp Leu Ser Glu Cys Leu Gln Arg Ile Phe Asn Ile
965 970 975
Lys Gly Ser Arg Ser Val Ala Lys Asn Gly Val Ile Arg Ala Glu
980 985 990
Asp Leu Arg Met Leu Leu Val Gly Thr Gly Phe Thr Gln Gln Thr
995 1000 1005
Glu Glu Gln Tyr Phe Gln Phe Leu Ala Lys Phe Glu Ile Ala Leu
1010 1015 1020
Pro Val Ala Asn Asp Ser Tyr Leu Leu Pro His Leu Leu Pro Ser
1025 1030 1035
Lys Pro Gly Leu Asp Thr His Gly Met Arg His Pro Thr Ala Asn
1040 1045 1050
Thr Ile Gln Arg Val Phe Lys Met Ser Phe Val Pro Val Gly Phe
1055 1060 1065
Trp Gln Arg Phe Ile Ala Arg Met Leu Ile Ser Leu Ala Glu Met
1070 1075 1080
Asp Leu Gln Leu Phe Glu Asn Lys Lys Asn Thr Lys Ser Arg Asn
1085 1090 1095
Arg Lys Val Thr Ile Tyr Ser Phe Thr Gly Asn Gln Arg Asn Arg
1100 110.5 1110
Cys Ser Thr Phe Arg Val Lys Arg Asn Gln Thr Ile Tyr Trp Gln
1115 1120 1125
Glu Gly Leu Leu Val Thr Phe Asp Gly Gly Tyr Leu Ser Val Glu
1130 1135 1140
Ser Ser Asp Val Asn Trp Lys Lys Lys Lys Ser Gly Gly Met Lys
1145 1150 1155
Ile Val Cys Gln Ser Glu Val Arg Asp Phe Ser Ala Met Ala Phe
1160 1165 1170
Ile Thr Asp His Val Asn Ser Leu Ile Asp Gln Trp Phe Pro Ala
1175 1180 1185
Leu Thr Ala Thr Glu Ser Asp Gly Thr Pro Leu Met Glu Gln Tyr
1190 1195 1200
Val Pro Cys Pro Val Cys Glu Thr Ala Trp Ala Gln His Thr Asp
1205 1210 1215
Pro Ser Glu Lys Ser Glu Asp Val Gln Tyr Phe Asp Met Glu Asp
1220 1225 1230
Cys Val Leu Thr Ala Ile Glu Arg Asp Phe Ile Ser Cys Pro Arg
1235 1240 1245
His Pro Asp Leu Pro Val Pro Leu Gln Glu Leu Val Pro Glu Leu
1250 1255 1260
Phe Met Thr Asp Phe Pro Ala Arg Leu Phe Leu Glu Asn Ser Lys
1265 1270 1275
Leu Glu His Ser Glu Asp Glu Gly Ser Val Leu Gly Gln Gly Gly
1280 1285 1290
Ser Gly Thr Val Ile Tyr Arg Ala Arg Tyr Gln Gly Gln Pro Val
1295 1300 1305
Ala Val Lys Arg Phe His Ile Lys Lys Phe Lys Asn Phe Ala Asn
1310 1315 1320
Val Pro Ala Asp Thr Met Leu Arg His Leu Arg Ala Thr Asp Ala
1325 1330 1335
Met Lys Asn Phe Ser Glu Phe Arg Gln Glu Ala Ser Met Leu His
1340 1345 1350
Ala Leu Gln His Pro Cys Ile Val Ala Leu Ile Gly Ile Ser Ile
1355 1360 1365
15/75
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His Pro Leu Cys Phe Ala Leu Glu Leu Ala Pro Leu Ser Ser Leu
1370 1375 1380
Asn Thr Val Leu Ser Glu Asn Ala Arg Asp Ser Ser Phe Ile Pro
1385 1390 1395
Leu Gly His Met Leu Thr Gln Lys Ile Ala Tyr Gln Ile Ala Ser
1400 1405 1410
Gly Leu Ala Tyr Leu His Lys Lys Asn Ile Ile Phe Cys Asp Leu
1415 1420 1425
Lys Ser Asp Asn Ile Leu Val Trp Ser Leu Asp Val Lys Glu His
1430 1435 1440
Ile Asn Ile Lys Leu Ser Asp Tyr Gly Ile Ser Arg Gln Ser Phe
1445 1450 1455
His Glu Gly Ala Leu Gly Val Glu Gly Thr Pro Gly Tyr Gln Ala
1460 1465 1470
Pro Glu Ile Arg Pro Arg Ile Val Tyr Asp Glu Lys Val Asp Met
1475 1480 1485
Phe Ser Tyr Gly Met Val Leu Tyr Glu Leu Leu Ser Gly Gln Arg
1490 1495 1500
Pro Ala Leu Gly His His Gln Leu Gln Ile Ala Lys Lys Leu Ser
1505 1510 1515
Lys Gly Ile Arg Pro Val Leu Gly Gln Pro Glu Glu Val Gln Phe
1520 1525 1530
Arg Arg Leu Gln Ala Leu Met Met Glu Cys Trp Asp Thr Lys Pro
1535 1540 1545
Glu Lys Arg Pro Leu Ala Leu Ser Val Val Ser Gln Met Lys Asp
1550 1555 1560
Pro Thr Phe Ala Thr Phe Met Tyr Glu Leu Cys Cys Gly Lys Gln
1565 1570 1575
Thr Ala Phe Phe Ser Ser Gln Gly Gln Glu Tyr Thr Val Val Phe
1580 1585 1590
Trp Asp Gly Lys Glu Glu Ser Arg Asn Tyr Thr Val Val Asn Thr
1595 1600 1605
Glu Lys Gly Leu Met Glu Val Gln Arg Met Cys Cys Pro Gly Met
1610 1615 1620
Lys Val Ser Cys Gln Leu Gln Val Gln Arg Ser Leu Trp Thr Ala
1625 1630 1635
Thr Glu Asn Ser Tyr Leu Val Leu Ala Gly Leu Ala Asp Gly Leu
1640 1645 1650
Val Ala Val Phe Pro Val Val Arg Gly Thr Pro Lys Asp Ser Cys
1655 1660 1665
Ser Tyr Leu Cys Ser His Thr Ala Asn Arg Ser Lys Phe Ser Ile
1670 1675 1680
Ala Asp Glu Asp Ala Arg Gln Asn Pro Tyr Pro Val Lys Ala Met
1685 1690 1695
Glu Val Val Asn Ser Gly Ser Glu Val Trp Tyr Ser Asn Gly Pro
1700 1705 1710
Gly Leu Leu Val Ile Asp Cys Ala Ser Leu Glu Ile Cys Arg Arg
1715 1720 1725
Leu Glu Pro Tyr Met Ala Pro Ser Met Val Thr Ser Val Val Cys
1730 1735 1740
Ser Ser Glu Gly Arg Gly Glu Glu Val Val Trp Cys Leu Asp Asp
1745 1750 1755
Lys Ala Asn Ser Leu Val Met Tyr His Ser Thr Thr Tyr Gln Leu
1760 1765 1770
Cys Ala Arg Tyr Phe Cys Gly Val Pro Ser Pro Leu Arg Asp Met
1775 1780 1785
16/75
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Phe Pro Val Arg Pro Leu Asp Thr Glu Pro Pro Ala Ala Ser His
1790 1795 1800
Thr Ala Asn Pro Lys Val Pro Glu Gly Asp Ser Ile Ala Asp Val
1805 1810 1815
Ser Ile Met Tyr Ser Glu Glu Leu Gly Thr Gln Ile Leu Ile His
1820 1825 1830
Gln Glu Ser Leu Thr Asp Tyr Cys Ser Met Ser Ser Tyr Ser Ser
1835 1840 1845
Ser Pro Pro Arg Gln Ala Ala Arg Ser Pro Ser Ser Leu Pro Ser
1850 1855 1860
Ser Pro Ala Ser Ser Ser Ser Val Pro Phe Ser Thr Asp Cys Glu
1865 1870 1875
Asp Ser Asp Met Leu His Thr Pro Gly Ala Ala Ser Asp Arg Ser
1880 1885 1890
Glu His Asp Leu Thr Pro Met Asp Gly Glu Thr Phe Ser Gln His
1895 1900 1905
Leu Gln Ala Val Lys Ile Leu Ala Val Arg Asp Leu Ile Trp Val
1910 1915 1920
Pro Arg Arg Gly Gly Asp Val Ile Val Ile Gly Leu Glu Lys Asp
1925 1930 1935
Ser Gly Ala Gln Arg Gly Arg Val Ile Ala Val Leu Lys Ala Arg
1940 1945 1950
Glu Leu Thr Pro His Gly Val Leu Val Asp Ala Ala Val Val Ala
1955 1960 1965
Lys Asp Thr Val Val Cys Thr Phe Glu Asn Glu Asn Thr Glu Trp
1970 1975 1980
Cys Leu Ala Val Trp Arg Gly Trp Gly Ala Arg Glu Phe Asp Ile
1985 1990 1995
Phe Tyr Gln Ser Tyr Glu Glu Leu Gly Arg Leu Glu Ala Cys Thr
2000 2005 2010
Arg Lys Arg Arg
<210> 9
<211> 348
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6035509CD1
<400> 9 -
Met Met Leu Gly Leu Glu Ser Leu Pro Asp Pro Thr Asp Thr Trp
1 5 10 15
Glu Ile Ile Glu Thr Ile Gly Lys Gly Thr Tyr Gly Lys Val Tyr
20 25 30
Lys Val Thr Asn Lys Arg Asp Gly Ser Leu Ala Ala Val Lys Ile
35 40 45
Leu Asp Pro Val Ser Asp Met Asp Glu Glu Ile Glu Ala Glu Tyr
50 55 60
Asn Ile Leu Gln Phe Leu Pro Asn His Pro Asn Val Val Lys Phe
65 70 75
Tyr Gly Met Phe Tyr Lys Ala Asp His Cys Val Gly Gly Gln Leu
80 85 90
Trp Leu Val Leu Glu Leu Cys Asn Gly Gly Ser Val Thr Glu Leu
17/75
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95 100 105
Val Lys Gly Leu Leu Arg Cys Gly Gln Arg Leu Asp Glu Ala Met
110 115 120
Ile Ser Tyr Ile Leu Tyr Gly Ala Leu Leu Gly Leu Gln His Leu
125 130 135
His Asn Asn Arg Ile Ile His Arg Asp Val Lys Gly Asn Asn Ile
140 145 150
Leu Leu Thr Thr Glu Gly Gly Val Lys Leu Val Asp Phe Gly Val
155 160 165
Ser Ala Gln Leu Thr Ser Thr Arg Leu Arg Arg Asn Thr Ser Val
170 175 180
Gly Thr Pro Phe Trp Met Ala Pro Glu Val Ile Ala Cys Glu Gln
185 190 195
Gln Tyr Asp Ser Ser Tyr Asp Ala Arg Cys Asp Val Trp Ser Leu
200 205 210
Gly Ile Thr Ala Ile Glu Leu Gly Asp Gly Asp Pro Pro Leu Phe
215 220 225
Asp Met His Pro Val Lys Thr Leu Phe Lys Ile Pro Arg Asn Pro
230 235 240
Pro Pro Thr Leu Leu His Pro Glu Lys Trp Cys Glu Glu Phe Asn
245 250 255
His Phe Ile Ser Gln Cys Leu Ile Lys Asp Phe Glu Arg Arg Pro
260 265 270
Ser Val Thr His Leu Leu Asp His Pro Phe Ile Lys Gly Val His
275 280 285
Gly Lys Val Leu Phe Leu Gln Lys Gln Leu Ala Lys Val Leu Gln
290 295 300
Asp Gln Lys His Gln Asn Pro Val Ala Lys Thr Arg His Glu Arg
305 310 315
Met His Thr Arg Arg Pro Tyr His Val Glu Asp Ala Glu Lys Tyr
320 325 330
Cys Leu Glu Asp Asp Leu Val Asn Leu Glu Val Leu Asp Glu Val
335 340 345
Leu Asn Ile
<210> 10
<211> 2042
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7373485CD1
<400> 10
Met Ala Thr Asp Asp Lys Thr Ser Pro Thr Leu Asp Ser Ala Asn
1 5 10 15
Asp Leu Pro Arg Ser Pro Thr Ser Pro Ser His Leu Thr His Phe
20 25 30
Lys Pro Leu Thr Pro Asp Gln Asp Glu Pro Pro Phe Lys Ser Ala
35 40 45
Tyr Ser Ser Phe Val Asn Leu Phe Arg Phe Asn Lys Glu Arg Ala
~ 50 55 60
Glu Gly Gly Gln Gly Glu Gln Gln Pro Leu Ser Gly Ser Trp Thr
65 70 75
18/75
CA 02425963 2003-04-15
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Ser Pro Gln Leu Pro Ser Arg Thr Gln Ser Val Arg Ser Pro Thr
80 85 90
Pro Tyr Lys Lys Gln Leu Asn Glu Glu Leu Gln Arg Arg Ser Ser
95 100 105
Ala Leu Asp Thr Arg Arg Lys Ala Glu Pro Thr Phe Gly Gly His
110 115 120
Asp Pro Arg Thr Ala Val Gln Leu Arg Ser Leu Ser Thr Val Leu
125 130 135
Lys Arg Leu Lys Glu Ile Met Glu Gly Lys Ser Gln Asp Ser Asp
140 145 150
Leu Lys Gln Tyr Trp Met Pro Asp Ser Gln Cys Lys Glu Cys Tyr
155 160 165
Asp Cys Ser Glu Lys Phe Thr Thr Phe Arg Arg Arg His His Cys
170 175 180
Arg Leu Cys Gly Gln Ile Phe Cys Ser Arg Cys Cys Asn Gln Glu
185 190 195
Ile Pro Gly Lys Phe Met Gly Tyr Thr Gly Asp Leu Arg Ala Cys
200 205 210
Thr Tyr Cys Arg Lys Ile Ala Leu Ser Tyr Ala His Ser Thr Asp
215 220 225
Ser Asn Ser Ile Gly Glu Asp Leu Asn Ala Leu Ser Asp Ser Ala
230 235 240
Cys Ser Val Ser Val Leu Asp Pro Ser Glu Pro Arg Thr Pro Val
245 250 255
Gly Ser Arg Lys Ala Ser Arg Asn Ile Phe Leu Glu Asp Asp Leu
260 265 270
Ala Trp Gln Ser Leu Ile His Pro Asp Ser Ser Asn Thr Pro Leu
275 280 285
Ser Thr Arg Leu Val Ser Val Gln Glu Asp Ala Gly Lys Ser Pro
290 295 300
Ala Arg Asn Arg Ser Ala Ser Ile Thr Asn Leu Ser Leu Asp Arg
305 310 315
Ser Gly Ser Pro Met Val Pro Ser Tyr Glu Thr Ser Val Ser Pro
320 325 330
Gln Ala Asn Arg Thr Tyr Val Arg Thr Glu Thr Thr Glu Asp Glu
335 340 345
Arg Lys Ile Leu Leu Asp Ser Val Gln Leu Lys Asp Leu Trp Lys
350 355 360
Lys Ile Cys His His Ser Ser Gly Met Glu Phe Gln Asp His Arg
365 370 375
Tyr Trp Leu Arg Thr His Pro Asn Cys Ile Val Gly Lys Glu Leu
380 385 390
Val Asn Trp Leu Ile Arg Asn Gly His Ile Ala Thr Arg Ala Gln
395 400 405
Ala Ile Ala Ile Gly Gln Ala Met Val Asp Gly Arg Trp Leu Asp
410 415 420
Cys Val Ser His His Asp Gln Leu Phe Arg Asp Glu Tyr Ala Leu
425 430 435
Tyr Arg Pro Leu Gln Ser Thr Glu Phe Ser Glu Thr Pro Ser Pro
440 445 450
Asp Ser Asp Ser Val Asn Ser Val Glu Gly His Ser Glu Pro Ser
455 460 465
Trp Phe Lys Asp Ile Lys Phe Asp Asp Ser Asp Thr Glu Gln Ile
470 475 480
Ala Glu Glu Gly Asp Asp Asn Leu Ala Lys Tyr Leu Ile Ser Asp
485 490 495
19/75
CA 02425963 2003-04-15
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Thr Gly Gly Gln Gln Leu Ser Ile Ser Asp Ala Phe Ile Lys Glu
500 505 510
Ser Leu Phe Asn Arg Arg Val Glu Glu Lys Ser Lys Glu Leu Pro
515 520 525
Phe Thr Pro Leu Gly Trp His His Asn Asn Leu Glu Leu Leu Arg
530 535 540
Glu Glu Asn Gly Glu Lys Gln Ala Met Glu Arg Leu Leu Ser Ala
545 550 555
Asn His Asn His Met Met Ala Leu Leu Gln Gln Leu Leu His Ser
560 565 570
Asp Ser Leu Ser Ser Ser Trp Arg Asp Ile Ile Val Ser Leu Val
575 580 585
Cys Gln Val Val Gln Thr Val Arg Pro Asp Val Lys Asn Gln Asp
590 595 600
Asp Asp Met Asp Ile Arg Gln Phe Val His Ile Lys Lys Ile Pro
605 610 615
Gly Gly Lys Lys Phe Asp Ser Val Val Val Asn Gly Phe Val Cys
620 625 630
Thr Lys Asn Ile Ala His Lys Lys Met Asn Ser Cys Ile Lys Asn
635 640 645
Pro Lys Ile Leu Leu Leu Lys Cys Ser Ile Glu Tyr Leu Tyr Arg
650 ' 655 660
Glu Glu Thr Lys Phe Thr Cys Ile Asp Pro Ile Val Leu Gln Glu
665 670 675
Arg Glu Phe Leu Lys Asn Tyr Val Gln Arg Ile Val Asp Val Arg
680 685 690
Pro Thr Leu Val Leu Val Glu Lys Thr Val Ser Arg Ile Ala Gln
695 700 705
Asp Met Leu Leu Glu His Gly Ile Thr Leu Val Ile Asn Val Lys
710 715 720
Ser Gln Val Leu Glu Arg Ile Ser Arg Met Thr Gln Gly Asp Leu
725 730 735
Val Met Ser Met Asp Gln Leu Leu Thr Lys Pro Arg Leu Gly Thr
740 745 750
Cys His Lys Phe Tyr Met Gln Ile Phe Gln Leu Pro Asn Glu Gln
755 760 765
Thr Lys Thr Leu Met Phe Phe Glu Gly Cys Pro Gln His Leu Gly
770 775 780
Cys Thr Ile Lys Leu Arg Gly Gly Ser Asp Tyr Glu Leu Ala Arg
785 790 795
Val Lys Glu Ile Leu Ile Phe Met Ile Cys Val Ala Tyr His Ser
800 805 810
Gln Leu Glu Ile Ser Phe Leu Met Asp Glu Phe Ala Met Pro Pro
815 820 825
Thr Leu Met Gln Asn Pro Ser Phe His Ser Leu Ile Glu Gly Arg
830 835 840
Gly His Glu Gly Ala Val Gln Glu Gln Tyr Gly Gly Gly Ser Ile
845 850 855
Pro Trp Asp Pro Asp Ile Pro Pro Glu Ser Leu Pro Cys Asp Asp
860 865 870
Ser Ser Leu Leu Glu Ser Arg Ile Val Phe Glu Lys Gly Glu Gln
875 880 885
Glu Asn Lys Asn Leu Pro Gln Ala Val Ala Ser Val Lys His Gln
890 895 900
Glu His Ser Thr Thr Ala Cys Pro Ala Gly Leu Pro Cys Ala Phe
905 910 915
20/75
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Phe Ala Pro Val Pro Glu Ser Leu Leu Pro Leu Pro Val Asp Asp
920 925 930
Gln Gln Asp Ala Leu Gly Ser Glu Leu Pro Glu Ser Leu Gln Gln
935 940 945
Thr Val Val Leu Gln Asp Pro Lys Ser Gln Ile Arg Ala Phe Arg
950 955 960
Asp Pro Leu Gln Asp Asp Thr Gly Leu Tyr Val Thr Glu Glu Val
965 970 975
Thr Ser Ser Glu Asp Lys Arg Lys Thr Tyr Ser Leu Ala Phe Lys
980 985 990
Gln Glu Leu Lys Asp Val Lle Leu Cys Ile Ser Pro Val Ile Thr
995 1000 1005
Phe Arg Glu Pro Phe Leu Leu Thr Glu Lys Gly Met Arg Cys Ser
1010 1015 102 0
Thr Arg Asp Tyr Phe Ala Glu Gln Val Tyr Trp Ser Pro Leu Leu
1025 1030 1035
Asn Lys Glu Phe Lys Glu Met Glu Asn Arg Arg Lys Lys Gln Leu
1040 1045 1050
Leu Arg Asp Leu Ser Gly Leu Gln Gly Met Asn Gly Ser Ile Gln
1055 1060 1065
Ala Lys Ser Ile Gln Val Leu Pro Ser His Glu Leu Val Ser Thr
1070 1075 1080
Arg Ile Ala Glu His Leu Gly Asp Ser Gln Ser Leu Gly Arg Met
1085 1090 1095
Leu Ala Asp Tyr Arg Ala Arg Gly Gly Arg Ile Gln Pro Lys Asn
1100 1105 1110
Ser Asp Pro Phe Ala His Ser Lys Asp Ala Ser Ser Thr Ser Ser
1115 1120 1125
Gly Lys Ser Gly Ser Lys Asn Glu Gly Asp Glu Glu Arg Gly Leu
1130 1135 1140
Ile Leu Ser Asp Ala Val Trp Ser Thr Lys Val Asp Cys Leu Asn
1145 1150 1155
Pro Ile Asn His Gln Arg Leu Cys Val Leu Phe Ser Ser Ser Ser
1160 1165 1170
Ala Gln Ser Ser Asn Ala Pro Ser Ala Cys Val Ser Pro Trp Ile
1175 1180 1185
Val Thr Met Glu Phe Tyr Gly Lys Asn Asp Leu Thr Leu Gly Ile
1190 1195 1200
Phe Leu Glu Arg Tyr Cys Phe Arg Pro Ser Tyr Gln Cys Pro Ser
1205 1210 1215
Met Phe Cys Asp Thr Pro Met Val His His Ile Arg Arg Phe Val
1220 1225 1230
His Gly Gln Gly Cys Val Gln Ile Ile Leu Lys Glu Leu Asp Ser
1235 1240 1245
Pro Val Pro Gly Tyr Gln His Thr Ile Leu Thr Tyr Ser Trp Cys
1250 1255 1260
Arg Ile Cys Lys Gln Val Thr Pro Val Val Ala Leu Ser Asn Glu
1265 1270 1275
Ser Trp Ser Met Ser Phe Ala Lys Tyr Leu Glu Leu Arg Phe Tyr
1280 1285 1290
Gly His Gln Tyr Thr Arg Arg Ala Asn Ala Glu Pro Cys Gly His
1295 1300 1305
Ser Ile His His Asp Tyr.His Gln Tyr Phe Ser Tyr Asn Gln Met
1310 1315 1320
Val Ala Ser Phe Ser Tyr Ser Pro Ile Arg Leu Leu Glu Val Cys
1325 1330 1335
21/75
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Val Pro Leu Pro Lys Ile Phe Ile Lys Arg Gln Ala Pro Leu Lys
1340 1345 1350
Val Ser Leu Leu Gln Asp Leu Lys Asp Phe Phe Gln Lys Val Ser
1355 1360 1365
Gln Val Tyr Val Ala Ile Asp Glu Arg Leu Ala Ser Leu Lys Thr
1370 1375 1380
Asp Thr Phe Ser Lys Thr Arg Glu Glu Lys Met Glu Asp Ile Phe
1385 1390 1395
Ala Gln Lys Glu Met Glu Glu Gly Glu Phe Lys Asn Trp Ile Glu
1400 1405 1410
Lys Met Gln Ala Arg Leu Met Ser Ser Ser Val Asp Thr Pro Gln
1415 1420 1425
Gln Leu Gln Ser Val Phe Glu Ser Leu Ile Ala Lys Lys Gln Ser
1430 1435 1440
Leu Cys Glu Val Leu Gln Ala Trp Asn Asn Arg Leu Gln Asp Leu
1445 1450 1455
Phe Gln Gln Glu Lys Gly Arg Lys Arg Pro Ser Val Pro Pro Ser
1460 1465 1470
Pro Gly Arg Leu Arg Gln Gly Glu Glu Ser Lys Ile Ser Ala Met
1475 1480 1485
Asp Ala Ser Pro Arg Asn Ile Ser Pro Gly Leu Gln Asn Gly Glu
1490 1495 1500
Lys Glu Asp Arg Phe Leu Thr Thr Leu Ser Ser Gln Ser Ser Thr
1505 1510 1515
Ser Ser Thr His Leu Gln Leu Pro Thr Pro Pro Glu Val Met Ser
1520 1525 1530
Glu Gln Ser Val Gly Gly Pro Pro Glu Leu Asp Thr Ala Ser Ser
1535 1540 1545
Ser Glu Asp Val Phe Asp Gly His Leu Leu Gly Ser Thr Asp Ser
1550 1555 1560
Gln Val Lys Glu Lys Ser Thr Met Lys Ala Ile Phe Ala Asn Leu
1565 1570 1575
Leu Pro Gly Asn Ser Tyr Asn Pro Ile Pro Phe Pro Phe Asp Pro
1580 1585 1590
Asp Lys His Tyr Leu Met Tyr Glu His Glu Arg Val Pro Ile Ala
1595 1600 1605
Val Cys Glu Lys Glu Pro Ser Ser Ile Ile Ala Phe Ala Leu Ser
1610 1615 1620
Cys Lys Glu Tyr Arg Asn Ala Leu Glu Glu Leu Ser Lys Ala Thr
1625 1630 1635
Gln Trp Asn Ser Ala Glu Glu Gly Leu Pro Thr Asn Ser Thr Ser
1640 1645 1650
Asp Ser Arg Pro Lys Ser Ser Ser Pro Ile Arg Leu Pro Glu Met
1655 1660 1665
Ser Gly Gly Gln Thr Asn Arg Thr Thr Glu Thr Glu Pro Gln Pro
1670 1675 1680
Thr Lys Lys Ala Ser Gly Met Leu Ser Phe Phe Arg Gly Thr Ala
1685 1690 1695
Gly Lys Ser Pro Asp Leu Ser Ser Gln Lys Arg Glu Thr Leu Arg
1700 1705 1710
Gly Ala Asp Ser Ala Tyr Tyr Gln Val Gly Gln Thr Gly Lys Glu
1715 1720 1725
Gly Thr Glu Asn Gln Gly Val Glu Pro Gln Asp Glu Val Asp Gly
1730 1735 1740
Gly Asp Thr Gln Lys Lys Gln Leu Ile Asn Pro His Val Glu Leu
1745 1750 1755
22/75
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Gln Phe Ser Asp Ala Asn Ala Lys Phe Tyr Cys Arg Leu Tyr Tyr
1760 1765 1770
Ala Gly Glu Phe His Lys Met Arg Glu Val Ile Leu Asp Ser Ser
1775 1780 1785
Glu Glu Asp Phe Ile Arg Ser Leu Ser His Ser Ser Pro Trp Gln
1790 1795 1800
Ala Arg Gly Gly Lys Ser Gly Ala Ala Phe Tyr Ala Thr Glu Asp
1805 1810 1815
Asp Arg Phe Ile Leu Lys Gln Met Pro Arg Leu Glu Val Gln Ser
1820 1825 1830
Phe Leu Asp Phe Ala Pro His Tyr Phe Asn Tyr Ile Thr Asn Ala
1835 1840 1845
Val Gln Gln Lys Arg Pro Thr Ala Leu Ala Lys Ile Leu Gly Val
1850 1855 1860
Tyr Arg Ile Gly Tyr Lys Asn Ser Gln Asn Asn Thr Glu Lys Lys
1865 1870 1875
Leu Asp Leu Leu Val Met Glu Asn Leu Phe Tyr Gly Arg Lys Met
1880 1885 1890
Ala Gln Val Phe Asp Leu Lys Gly Ser Leu Arg Asn Arg Asn Val
1895 1900 1905
Lys Thr Asp Thr Gly Lys Glu Ser Cys Asp Val Val Leu Leu Asp
1910 1915 1920
Glu Asn Leu Leu Lys Met Val Arg Asp Asn Pro Leu Tyr Ile Arg
1925 1930 1935
Ser His Ser Lys Ala Val Leu Arg Thr Ser Ile His Ser Asp Ser
1940 1945 1950
His Phe Leu Ser Ser His Leu Ile Ile Asp Tyr Ser Leu Leu Val
1955 1960 1965
Gly Arg Asp Asp Thr Ser Asn Glu Leu Val Val Gly Ile Ile Asp
1970 1975 1980
Tyr Ile Arg Thr Phe Thr Trp Asp Lys Lys Leu Glu Met Val Val
1985 1990 1995
Lys Ser Thr Gly Ile Leu Gly Gly Gln Gly Lys Met Pro Thr Val
2000 2005 2010
Val Ser Pro Glu Leu Tyr Arg Thr Arg Phe Cys Glu Ala Met Asp
2015 2020 2025
Lys Tyr Phe Leu Met Val Pro Asp His Trp Thr Gly Leu Gly Leu
2030 2035 2040
Asn Cys
<210> 11
<211> 551
<212> PRT
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 5734965CD1
<400> 11
Met Ser Gly Gly Glu Gln Lys Pro Glu Arg Tyr Tyr Val Gly Val
1 5 10 15
Asp Val Gly Thr Gly Ser Val Arg Ala Ala Leu Val Asp Gln Ser
20 25 30
Gly Val Leu Leu Ala Phe Ala Asp Gln Pro Ile Lys Asn Trp Glu
23/75
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35 40 45
Pro Gln Phe Asn His His Glu Gln Ser Ser Glu Asp Ile Trp Ala
50 55 60
Ala Cys Cys Val Val Thr Lys Lys Val Val Gln Gly Ile Asp Leu
65 70 75
Asn Gln Ile Arg Gly Leu Gly Phe Asp Ala Thr Cys Ser Leu Val
80 85 90
Val Leu Asp Lys Gln Phe His Pro Leu Pro Val Asn Gln Glu Gly
95 100 105
Asp Ser His Arg Asn Val Ile Met Trp Leu Asp His Arg Ala Val
110 115 120
Ser Gln Val Asn Arg Ile Asn Glu Thr Lys His Ser Val Leu Gln
125 130 135
Tyr Val Gly Gly Val Met Ser Val Glu Met Gln Ala Pro Lys Leu
140 145 150
Leu Trp Leu Lys Glu Asn Leu Arg Glu Ile Cys Trp Asp Lys Ala
155 160 165
Gly His Phe Phe Asp Leu Pro Asp Phe Leu Ser Trp Lys Ala Thr
170 175 180
Gly Val Thr Ala Arg Ser Leu Cys Ser Leu Val Cys Lys Trp Thr
185 190 195
Tyr Ser Ala Glu Lys Gly Trp Asp Asp Ser Phe Trp Lys Met Ile
200 205 210
Gly Leu Glu Asp Phe Val Ala Asp Asn Tyr Ser Lys Ile Gly Asn
215 220 225
Gln Val Leu Pro Pro Gly Ala Ser Leu Gly Asn Gly Leu Thr Pro
230 235 240
Glu.Ala Ala Arg Asp Leu Gly Leu Leu Pro Gly Ile Ala Val Ala
245 250 255
Ala Ser Leu Ile Asp Ala His Ala Gly Gly Leu Gly Val Ile Gly
260 265 270
Ala Asp Val Arg Gly His Gly Leu Ile Cys Glu Gly Gln Pro Val
275 280 285
Thr Ser Arg Leu Ala Val Ile Cys Gly Thr Ser Ser Cys His Met
290 295 300
Gly Ile Ser Lys Asp Pro Ile Phe Val Pro Gly Val Trp Gly Pro
305 310 315
Tyr Phe Ser Ala Met Val Pro Gly Phe Trp Leu Asn Glu Gly Gly
320 325 330
Gln Ser Val Thr Gly Lys Leu Ile Asp His Met Val Gln Gly His
335 340 345
Ala Ala Phe Pro Glu Leu Gln Val Lys Ala Thr Ala Arg Cys Gln
350 355 360
Ser Ile Tyr Ala Tyr Leu Asn Ser His Leu Asp Leu Ile Lys Lys
365 370 375
Ala Gln Pro Val Gly Phe Leu Thr Val Asp Leu His Val Trp Pro
380 385 390
Asp Phe His Gly Asn Arg Ser Pro Leu Ala Asp Leu Thr Leu Lys
395 400 405
Gly Met Val Thr Gly Leu Lys Leu Ser Gln Asp Leu Asp Asp Leu
410 415 420
Ala Ile Leu Tyr Leu Ala Thr Val Gln Ala Ile Ala Leu Gly Thr
425 430 435
Arg Phe Ile Ile Glu Ala Met Glu Ala Ala Gly His Ser Ile Ser
440 445 450
Thr Leu Phe Leu Cys Gly Gly Leu Ser Lys Asn Pro Leu Phe Val
24/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
455 460 465
Gln Met His Ala Asp Ile Thr Gly Met Pro Val Val Leu Ser Gln
470 475 480
Glu Val Glu Ser Val Leu Val Gly Ala Ala Val Leu Gly Ala Cys
485 490 495
Ala Ser Gly Asp Phe Ala Ser Val Gln Glu Ala Met Ala Lys Met
500 505 510
Ser Lys Val Gly Lys Val Val Phe Pro Arg Leu Gln Asp Lys Lys
515 520 525
Tyr Tyr Asp Lys Lys Tyr Gln Val Phe Leu Lys Leu Val Glu His
530 535 540
Gln Lys Glu Tyr Leu Ala Ile Met Asn Asp Asp
545 550
<210> 12
<211> 485
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473788CD1
<400> 12
Met Arg Ser Gly Ala Glu Arg Arg Gly Ser Ser Ala Ala Ala Ser
1 5 10 15
Pro Gly Ser Pro Pro Pro Gly Arg Ala Arg Pro Ala Gly Ser Asp
20 25 30
Ala Pro Ser Ala Leu Pro Pro Pro Ala Ala Gly Gln Pro Arg Ala
35 40 45
Arg Asp Ser Gly Asp Val Arg Ser Gln Pro Arg Pro Leu Phe Gln
50 55 60
Trp Ser Lys Trp Lys Lys Arg Met Gly Ser Ser Met Ser Ala Ala
65 70 75
Thr Ala Arg Arg Pro Val Phe Asp Asp Lys Glu Asp Val Asn Phe
80 85 90
Asp His Phe Gln Ile Leu Arg Ala Ile Gly Lys Gly Ser Phe Gly
95 100 105
Lys Val Cys Ile Val Gln Lys Arg Asp Thr Glu Lys Met Tyr Ala
110 115 120
Met Lys Tyr Met Asn Lys Gln Gln Cys Ile Glu Arg Asp Glu Val
125 130 135
Arg Asn Val Phe Arg Glu Leu Glu Ile Leu Gln Glu Ile Glu His
140 145 150
Val Phe Leu Val Asn Leu Trp Tyr Ser Phe Gln Asp Glu Glu Asp
155 160 165
Met Phe Met Val Val Asp Leu Leu Leu Gly Gly Asp Leu Arg Tyr
170 175 180
His Leu Gln Gln Asn Val Gln Phe Ser Glu Asp Thr Val Arg Leu
185 190 195
Tyr Ile Cys Glu Met Ala Leu Ala Leu Asp Tyr Leu Arg Gly Gln
200 205 210
His Ile Ile His Arg Asp Val Lys Pro Asp Asn Ile Leu Leu Asp
215 220 225
Glu Arg Gly His Ala His Leu Thr Asp Phe Asn Ile Ala Thr Ile
230 235 240
25/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Ile Lys Asp Gly Glu Arg Ala Thr Ala Leu Ala Gly Thr Lys Pro
245 250 255
Tyr Met Ala Pro Glu Ile Phe His Ser Phe Val Asn Gly Gly Thr
260 265 270
Gly Tyr Ser Phe Glu Val Asp Trp Trp Ser Val Gly Val Met Ala
275 280 285
Tyr Glu Leu Leu Arg Gly Trp Arg Pro Tyr Asp Ile His Ser Ser
290 295 300
Asn Ala Val Glu Ser Leu Val Gln Leu Phe Ser Thr Val Ser Val
305 310 315
Gln Tyr Val Pro Thr Trp Ser Lys Glu Met Val Ala Leu Leu Arg
320 325 330
Lys Leu Leu Thr Val Asn Pro Glu His Arg Leu Ser Ser Leu Gln
335 340 345
Asp Val Gln Ala Ala Pro Ala Leu Ala Gly Val Leu Trp Asp His
350 355 360
Leu Ser Glu Lys Arg Val Glu Pro Gly Phe Val Pro Asn Lys Gly
365 370 375
Arg Leu His Cys Asp Pro Thr Phe Glu Leu Glu Glu Met Ile Leu
380 385 390
Glu Ser Arg Pro Leu His Lys Lys Lys Lys Arg Leu Ala Lys Asn
395 400 405
Lys Ser Arg Asp Asn Ser Arg Asp Ser Ser Gln Ser Glu Asn Asp
410 415 420
Tyr Leu Gln Asp Cys Leu Asp Ala Ile Gln Gln Asp Phe Val Ile
425 430 435
Phe Asn Arg Glu Lys Leu Lys Arg Ser Gln Asp Leu Pro Arg Glu
440 445 450
Pro Leu Pro Ala Leu Ser Pro Gly Met Leu Arg Ser Leu Trp Arg
455 460 465
Thr Arg Arg Thr Leu Arg Leu Pro Met Cys Gly Pro Ile Cys Pro
470 475 480
Ser Ala Gly Ser Gly
485
<210> 13
<211> 282
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3107989CD1
<400> 13
Met Pro Ala Phe Ile Gln Met Gly Arg Asp Lys Asn Phe Ser Ser
1 5 10 15
Leu His Thr Val Phe Cys Ala Thr Gly Gly Gly Ala Tyr Lys Phe
20 25 30
Glu Gln Asp Phe Leu Thr Ile Gly Asp Leu Gln Leu Cys Lys Leu
35 40 45
Asp Glu Leu Asp Cys Leu Ile Lys Gly Ile Leu Tyr Ile Asp Ser
50 55 60
Val Gly Phe Asn Gly Arg Ser Gln Cys Tyr Tyr Phe Glu Asn Pro
65 70 75
Ala Asp Ser Glu Lys Cys Gln Lys Leu Pro Phe Asp Leu Lys Asn
26/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
80 85 90
Pro Tyr Pro Leu Leu Leu Val Asn Ile Gly Ser Gly Val Ser Ile
95 100 105
Leu Ala Val Tyr Ser Lys Asp Asn Tyr Lys Arg Val Thr Gly Thr
110 115 120
Ser Leu Gly Gly Gly Thr Phe Phe Gly Leu Cys Cys Leu Leu Thr
125 130 135
Gly Cys Thr Thr Phe Glu Glu Ala Leu Glu Met Ala Ser Arg Gly
140 145 150
Asp Ser Thr Lys Val Asp Lys Leu Val Arg Asp Ile Tyr Gly Gly
155 160 165
Asp Tyr Glu Arg Phe Gly Leu Pro Gly Trp Ala Val Ala Ser Ser
170 175 180
Phe Gly Asn Met Met Ser Lys Glu Lys Arg Asp Ser Ile Ser Lys
185 190 195
Glu Asp Leu Ala Arg Ala Thr Leu Val Thr Ile Thr Asn Asn Ile
200 205 210
Gly Ser Ile Ala Arg Met Cys Ala Leu Asn Glu Asn Ile Asp Arg
215 220 225
Val Val Phe Val Gly Asn Phe Leu Arg Ile Asn Met Val Ser Met
230 235 240
Lys Leu Leu Ala Tyr Ala Met Asp Phe Trp Ser Lys Gly Gln Leu
245 250 255
Lys Ala Leu Phe Leu Glu His Glu Gly Tyr Phe Gly Ala Val Gly
260 265 270
Ala Leu Leu Glu Leu Phe Lys Met Thr Asp Asp Lys
275 280
<210> 14
<211> 151
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7482887CD1
<400> 14
Met Ala Asn Thr Glu Ser Ile Ile Ile Asn Pro Ser Ala Val Gln
1 5 10 15
His Ser Leu Val Gly Glu Ile Ile Lys Tyr Ser Glu Gln Lys Gly
20 25 30
Phe Tyr Leu Val Thr Met Lys Phe Leu Arg Ala Ser Glu Lys Pro
35 40 45
Leu Lys Pro His Tyr Thr Asn Leu Lys Asp His Pro Phe Phe Pro
50 55 60
Asp Leu Val Lys Tyr Met Asn Ser Gly Gln Val Val Ala Met Val
65 70 75
Leu Glu Gly Leu Asn Val Ala Lys Thr Gly Leu Arg Met Leu Gly
80 85 90
Glu Thr Asn Ser Leu Gly Ser Met Leu Glu Thr Ile Ile Arg Arg
95 100 105
Asp Phe Cys Ala Lys Ile Gly Gly Asn Val Ile Gly Gly Ser Asp
110 115 120
Ser Leu Gln Ser Ala Glu Lys Glu Ile Ser Leu Trp Phe Lys Pro
125 130 135
27/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Lys Glu Pro Val Asp Tyr Arg Ser Cys Ala Tyr Asp Trp Val Tyr
140 145 150
Ala
<210> 15
<211> 410
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2963414CD1
<400> 15
Met Val Val Gln Asn Ser Ala Asp Ala Gly Asp Met Arg Ala Gly
1 5 10 15
Val Gln Leu Glu Pro Phe Leu His Gln Val Gly Gly His Met Ser
20 25 30
Val Met Lys Tyr Asp Glu His Thr Val Cys Lys Pro Leu Val Ser
35 40 45
Arg Glu Gln Arg Phe Tyr Glu Ser Leu Pro Leu Ala Met Lys Arg
50 55 60
Phe Thr Pro Gln Tyr Lys Gly Thr Val Thr Val His Leu Trp Lys
65 70 75
Asp Ser Thr Gly His Leu Ser Leu Val Ala Asn Pro Val Lys Glu
80 85 90
Ser Gln Glu Pro Phe Lys Val Ser Thr Glu Ser Ala Ala Val Ala
95 100 105
Ile Trp Gln Thr Leu Gln Gln Thr Thr Gly Ser Asn Gly Ser Asp
110 115 120
Cys Thr Leu Ala Gln Trp Pro His Ala Gln Leu Ala Arg Ser Pro
125 130 135
Lys Glu Ser Pro Ala Lys Ala Leu Leu Arg Ser Glu Pro His Leu
140 145 150
Asn Thr Pro Ala Phe Ser Leu Val Glu Asp Thr Asn Gly Asn Gln
155 160 165
Val Glu Arg Lys Ser Phe Asn Pro Trp Gly Leu Gln Cys His Gln
170 175 180
Ala His Leu Thr Arg Leu Cys Ser Glu Tyr Pro Glu Asn Lys Arg
185 190 195
His Arg Phe Leu Leu Leu Glu Asn Val Val Ser Gln Tyr Thr His
200 205 210
Pro Cys Val Leu Asp Leu Lys Met Gly Thr Arg Gln His Gly Asp
215 220 225
Asp Ala Ser Glu Glu Lys Lys Ala Arg His Met Arg Lys Cys Ala
230 235 240
Gln Ser Thr Ser Ala Cys Leu Gly Val Arg Ile Cys Gly Met Gln
245 250 255
Val Tyr Gln Thr Asp Lys Lys Tyr Phe Leu Cys Lys Asp Lys Tyr
260 265 270
Tyr Gly Arg Lys Leu Ser Val Glu Gly Phe Arg Gln Ala Leu Tyr
275 280 285
Gln Phe Leu His Asn Gly Ser His Leu Arg Arg Glu Leu Leu Glu
290 295 300
Pro Ile Leu His Gln Leu Arg Ala Leu Leu Ser Ile Ile Arg Ser
28/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
305 310 315
Gln Ser Ser Tyr Arg Phe Tyr Ser Ser Ser Leu Leu Val Ile Tyr
320 325 330
Asp Gly Gln Glu Pro Pro Glu Arg Ala Pro Gly Ser Pro His Pro
335 340 345
His Glu Ala Pro Gln Ala Ala His Gly Ser Ser Pro Gly Gly Leu
350 355 360
Thr Lys Val Asp Ile Arg Met Ile Asp Phe Ala His Thr Thr Tyr
365 370 375
Lys Gly Tyr Trp Asn Glu His Thr Thr Tyr Asp Gly Pro Asp Pro
380 385 390
Gly Tyr Ile Phe Gly Leu Glu Asn Leu Ile Arg Ile Leu Gln Asp
395 400 405
Ile Gln Glu Gly Glu
410
<210> 16
<211> 1581
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477139CD1
<400> 16
Met Ala Gly Pro Gly Gly Trp Arg Asp Arg Glu Val Thr Asp Leu
1 5 10 15
Gly His Leu Pro Asp Pro Thr Gly Ile Phe Ser Leu Asp Lys Thr
20 25 30
Ile Gly Leu Gly Thr Tyr Gly Arg Ile Tyr Leu Gly Leu His Glu
35 40 45
Lys Thr Gly Ala Phe Thr Ala Val Lys Val Met Asn Ala Arg Lys
50 55 60
Thr Pro Leu Pro Glu Ile Gly Arg Arg Val Arg Val Asn Lys Tyr
65 70 75
Gln Lys Ser Val Gly Trp Arg Tyr Ser Asp Glu Glu Glu Asp Leu
80 85 90
Arg Thr Glu Leu Asn Leu Leu Arg Lys Tyr Ser Phe His Lys Asn
95 100 105
Ile Val Ser Phe Tyr Gly Ala Phe Phe Lys Leu Ser Pro Pro Gly
110 115 120
Gln Arg His Gln Leu Trp Met Val Met Glu Leu Cys Ala Ala Gly
125 130 135
Ser Val Thr Asp Val Val Arg Met Thr Ser Asn Gln Ser Leu Lys
140 145 150
Glu Asp Trp Ile Ala Tyr Ile Cys Arg Glu Ile Leu Gln Gly Leu
155 160 165
Ala His Leu His Ala His Arg Val Ile His Arg Asp Ile Lys Gly
170 175 180
Gln Asn Val Leu Leu Thr His Asn Ala Glu Val Lys Leu Val Asp
185 190 195
Phe Gly Val Ser Ala Gln Val Ser Arg Thr Asn Gly Arg Arg Asn
200 205 210
Ser Phe Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Asp
215 220 225
29/75
CA 02425963 2003-04-15
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Cys Asp Glu Asp Pro Arg Arg Ser Tyr Asp Tyr Arg Ser Asp Val
230 235 240
Trp Ser Val Gly Ile Thr Ala Ile Glu Met Ala Glu Gly Ala Pro
245 250 255
Pro Leu Cys Asn Leu Gln Pro Leu Glu Ala Leu Phe Val Ile Leu
260 265 270
Arg Glu Ser Ala Pro Thr Val Lys Ser Ser Gly Trp Ser Arg Lys
275 280 285
Phe His Asn Phe Met Glu Lys Cys Thr Ile Lys Asn Phe Leu Phe
290 295 300
Arg Pro Thr Ser Ala Asn Met Leu Gln His Pro Phe Val Arg Asp
305 310 315
Ile Lys Asn Glu Arg His Val Val Glu Ser Leu Thr Arg His Leu
320 325 330
Thr Gly Ile Ile Lys Lys Arg Gln Lys Lys Gly Ile Pro Leu Ile
335 340 345
Phe Glu Arg Glu Glu Ala Ile Lys Glu Gln Tyr Thr Val Arg Arg
350 355 360
Phe Arg Gly Pro Ser Cys Thr His Glu Leu Leu Arg Leu Pro Thr
365 370 375
Ser Ser Arg Cys Arg Pro Leu Arg Val Leu His Gly Glu Pro Ser
380 385 390
Gln Pro Arg Trp Leu Pro Asp Arg Glu Glu Pro Gln Val Gln Ala
395 400 405
Leu Gln Gln Leu Gln Gly Ala Ala Arg Val Phe Met Pro Leu Gln
410 415 420
Ala Leu Asp Ser Ala Pro Lys Pro Leu Lys Gly Gln Ala Gln Ala
425 430 435
Pro Gln Arg Leu Gln Gly Ala Ala Arg Val Phe Met Pro Leu Gln
440 445 450
Ala Gln Val Lys Ala Lys Ala Ser Lys Pro Leu Gln Met Gln Ile
455 460 465
Lys Ala Pro Pro Arg Leu Arg Arg Ala Ala Arg Val Leu Met Pro
470 475 480
Leu Gln Ala Gln Val Arg Ala Pro Arg Leu Leu Gln Val Gln Ser
485 490 495
Gln Val Ser Lys Lys Gln Gln Ala Gln Thr Gln Thr Ser Glu Pro
500 505 510
Gln Asp Leu Asp Gln Val Pro Glu Glu Phe Gln Gly Gln Asp Gln
515 520 525
Val Pro Glu Gln Gln Arg Gln Gly Gln Ala Pro Glu Gln Gln Gln
530 535 540
Arg His Asn Gln Val Pro Glu Gln Glu Leu Glu Gln Asn Gln Ala
545 550 555
Pro Glu Gln Pro Glu Val Gln Glu Gln Ala Ala Glu Pro Ala Gln
560 565 570
Ala Glu Thr Glu Ala Glu Glu Pro Glu Ser Leu Arg.Val Asn Ala
575 580 585
Gln Val Phe Leu Pro Leu Leu Ser Gln Asp His His Val Leu Leu
590 595 600
Pro Leu His Leu Asp Thr Gln Val Leu Ile Pro Val Glu Gly Gln
605 610 615
Thr Glu Gly Ser Pro Gln Ala Gln Ala Trp Thr Leu Glu Pro Pro
620 625 630
Gln Ala Ile Gly~Ser Val Gln Ala Leu Ile Glu Gly Leu Ser Arg
635 640 645
30/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Asp Leu Leu Arg Ala Pro Asn Ser Asn Asn Ser Lys Pro Leu Gly
650 655 660
Pro Leu Gln Thr Leu Met Glu Asn Leu Ser Ser Asn Arg Phe Tyr
665 670 675
Ser Gln Pro Glu Gln Ala Arg Glu Lys Lys Ser Lys Val Ser Thr
680 685 690
Leu Arg Gln Ala Leu Ala Lys Arg Leu Ser Pro Lys Arg Phe Arg
695 700 705
Ala Lys Ser Ser Trp Arg Pro Glu Lys Leu Glu Leu Ser Asp Leu
710 715 720
Glu Ala Arg Arg Gln Arg Arg Gln Arg Arg Trp Glu Asp Ile Phe
725 730 735
Asn Gln His Glu Glu Glu Leu Arg Gln Val Asp Lys Thr Ser Trp
740 745 750
Arg Gln Trp Gly Pro Ser Asp Gln Leu Ile Asp Asn Ser Phe Thr
755 760 765
Gly Met Gln Asp Leu Lys Lys Tyr Leu Lys Gly Lys Thr Thr Phe
770 775 780
His Asn Val Gln Val Val Ile Tyr Arg Ala Val Lys Gly Asn Asp
785 790 795
Asp Val Ala Thr Arg Ser Thr Val Pro Gln Arg Ser Leu Leu Glu
800 805 810
Gln Ala Gln Lys Pro Ile Asp Ile Arg Gln Arg Ser Ser Gln Asn
815 820 825
Arg Gln Asn Trp Leu Ala Ala Ser Gly Asp Ser Lys His Lys Ile
830 835 840
Leu Ala Gly Lys Thr Gln Ser Tyr Cys Leu Thr Ile Tyr Ile Ser
845 850 855
Glu Val Lys Lys Glu Glu Phe Gln Glu Gly Met Asn Gln Lys Cys
860 865 870
,Gln Gly Ala Gln Val Gly Leu Gly Pro Glu Gly His Cys Ile Trp
875 880 885
Gln Leu Gly Glu Ser Ser Ser Glu Glu Glu Ser Pro Val Thr Gly
890 895 900
Arg Arg Ser Gln Ser Ser Pro Pro Tyr Ser Thr Ile Asp Gln Lys
905 910 915
Leu Leu Val Asp Ile His Val Pro Asp Gly Phe Lys Val Gly Lys
920 925 930
Ile Ser Pro Pro Val Tyr Leu Thr Asn Glu Trp Val Gly Tyr Asn
935 940 945
Ala Leu Ser Glu Ile Phe Arg Asn Asp Trp Leu Thr Pro Ala Pro
950 955 960
Val Ile Gln Pro Pro Glu Glu Asp Gly Asp Tyr Val Glu Leu Tyr
965 970 975
Asp Ala Ser Ala Asp Thr Asp Gly Asp Asp Asp Asp Glu Ser Asn
980 985 990
Asp Thr Phe Glu Asp Thr Tyr Asp His Ala Asn Gly Asn Asp Asp
995 1000 1005
Leu Asp Asn Gln Val Asp Gln Ala Asn Asp Val Cys Lys Asp His
1010 1015 1020
Asp Asp Asp Asn Asn Lys Phe Val Asp Asp Val Asn Asn Asn Tyr
1025 1030 1035
Tyr Glu Ala Pro Ser Cys Pro Ser Leu Leu Ser Gly Gln Ala Met
1040 1045 1050
Ala Glu Met Glu Ala Ala Ser Lys Met Val Met Met Glu Val Val
1055 1060 .1065
31/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Glu Lys Arg Lys Pro Thr Glu Ala Met Glu Ala Ile Gln Pro Ile
1070 1075 1080
Glu Ala Met Glu Glu Val Gln Pro Val Arg Asp Asn Ala Ala Ile
1085 1090 1095
Gly Asp Gln Glu Glu His Ala Ala Asn Ile Gly Ser Glu Arg Arg
1100 1105 1110
Gly Ser Glu Gly Asp Gly Gly Lys Gly Val Val Arg Thr Ser Glu
1115 1120 1125
Glu Ser Gly Ala Leu Gly Leu Asn Gly Glu Glu Asn Cys Ser Glu
1130 1135 1140
Thr Asp Gly Pro Gly Leu Lys Arg Pro Ala Ser Gln Asp Phe Glu
1145 1150 1155
Tyr Leu Gln Glu Glu Pro Gly Gly Gly Asn Glu Ala Ser Asn Ala
1160 1165 1170
Ile Asp Ser Gly Ala Ala Pro Ser Ala Pro Asp His Glu Ser Asp
1175 1180 1185
Asn Lys Asp Ile Ser Glu Ser Ser Thr Gln Ser Asp Phe Ser Ala
1190 1195 1200
Asn His Ser Ser Pro Ser Lys Gly Ser Gly Met Ser Ala Asp Ala
1205 1210 1215
Asn Phe Ala Ser Ala Ile Leu Tyr Ala Gly Phe Val Glu Val Pro
1220 1225 1230
Glu Glu Ser Pro Lys Gln Pro Ser Glu Val Asn Val Asn Pro Leu
1235 1240 1245
Tyr Val Ser Pro Ala Cys Lys Lys Pro Leu Ile His Met Tyr Glu
1250 1255 1260
Lys Glu Phe Thr Ser Glu Ile Cys Cys Gly Ser Leu Trp Gly Val
1265 1270 1275
Asn Leu Leu Leu Gly Thr Arg Ser Asn Leu Tyr Leu Met Asp Arg
1280 1285 1290
Ser Gly Lys Ala Asp Ile Thr Lys Leu Ile Arg Arg Arg Pro Phe
1295 1300 1305
Arg Gln Ile Gln Val Leu Glu Pro Leu Asn Leu Leu Ile Thr Ile
1310 1315 1320
Ser Gly His Lys Asn Arg Leu Arg Val Tyr His Leu Thr Trp Leu
1325 1330 1335
Arg Asn Lys Ile Leu Asn Asn Asp Pro Glu Ser Lys Arg Arg Gln
1340 1345 1350
Glu Glu Met Leu Lys Thr Glu Glu Ala Cys Lys Ala Ile Asp Lys
1355 1360 1365
Leu Thr Gly Cys Glu His Phe Ser Val Leu Gln His Glu Glu Thr
1370 1375 1380
Thr Tyr Ile Ala Ile Ala Leu Lys Ser Ser Ile His Leu Tyr Ala
1385 1390 1395
Trp Ala Pro Lys Ser Phe Asp Glu Ser Thr Ala Ile Lys Val Phe
1400 1405 1410
Pro Thr Leu Asp His Lys Pro Val Thr Val Asp Leu Ala Ile Gly
1415 1420 1425
Ser Glu Lys Arg Leu Lys Ile Phe Phe Ser Ser Ala Asp Gly Tyr
1430 1435 1440
His Leu Ile Asp Ala Glu Ser Glu Val Met Ser Asp Val Thr Leu
1445 1450 1455
Pro Lys Asn Asn Ile Ile Ile Leu Pro Asp Cys Leu Gly Ile Gly
1460 1465 1470
Met Met Leu Thr Phe Asn Ala Glu Ala Leu Ser Val Glu Ala Asn
1475 1480 1485
32/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Glu Gln Leu Phe Lys Lys Ile Leu Glu Met Trp Lys Asp Ile Pro
1490 1495 1500
Ser Ser Ile Ala Phe Glu Cys Thr Gln Arg Thr Thr Gly Trp Gly
1505 1510 1515
Gln Lys Ala Ile Glu Val Arg Ser Leu Gln Ser Arg Val Leu Glu
1520 1525 1530
Ser Glu Leu Lys Arg Arg Ser Ile Lys Lys Leu Arg Phe Leu Cys
1535 1540 1545
Thr Arg Gly Asp Lys Leu Phe Phe Thr Ser Thr Leu Arg Asn His
1550 1555 1560
His Ser Arg Val Tyr Phe Met Thr Leu Gly Lys Leu Glu Glu Leu
1565 1570 1575
Gln Ser Asn Tyr Asp Val
1580
<210> 17
<211> 1084
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55009053CD1
<400> 17
Met Glu Thr Gln Ala Val Ala Thr Ser Pro Asp Gly Arg Tyr Leu
1 5 10 15
Lys Phe Asp Ile Glu Ile Gly Arg Gly Ser Phe Lys Thr Val Tyr
20 25 30
Arg Gly Leu Asp Thr Asp Thr Thr Val Glu Val Ala Trp Cys Glu
35 40 45
Leu Gln Thr Arg Lys Leu Ser Arg Ala Glu Arg Gln Arg Phe Ser
50 55 60
Glu Glu Val Glu Met Leu Lys Gly Leu Gln His Pro Asn Ile Val
65 70 75
Arg Phe Tyr Asp Ser Trp Lys Ser Val Leu Arg Gly Gln Val Cys
80 85 90
Ile Val Leu Val Thr Glu Leu Met Thr Ser Gly Thr Leu Lys Thr
95 100 105
Tyr Leu Arg Arg Phe Arg Glu Met Lys Pro Arg Val Leu Gln Arg
110 115 120
Trp Ser Arg Gln Ile Leu Arg Gly Leu His Phe Leu His Ser Arg
125 130 135
Val Pro Pro Ile Leu His Arg Asp Leu Lys Cys Asp Asn Val Phe
140 145 150
Ile Thr Gly Pro Ser Gly Ser Val Lys Ile Gly Asp Leu Gly Leu
155 160 165
Ala Thr Leu Lys Arg Ala Ser Phe Ala Lys Ser Val Ile Gly Thr
170 175 180
Pro Glu Phe Met Ala Pro Glu Met Tyr Glu Glu Lys Tyr Asp Glu
185 190 195
Ala Val Asp Val Tyr Ala Phe Gly Met Cys Met Leu Glu Met Ala
200 205 210
Thr Ser Glu Tyr Pro Tyr Ser Glu Cys Gln Asn Ala Ala Gln Ile
215 220 225
Tyr Arg Lys Val Thr Ser Gly Arg Lys Pro Asn Ser Phe His Lys
33/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
230 235 240
Val Lys Ile Pro Glu Val Lys Glu Ile Ile Glu Gly Cys Ile Arg
245 250 255
Thr Asp Lys Asn Glu Arg Phe Thr Ile Gln Asp Leu Leu Ala His
260 265 270
Ala Phe Phe Arg Glu Glu Arg Gly Val His Val Glu Leu Ala Glu
275 280 285
Glu Asp Asp Gly Glu Lys Pro Gly Leu Lys Leu Trp Leu Arg Met
290 295 300
Glu Asp Ala Arg Arg Gly Gly Arg Pro Arg Asp Asn Gln Ala Ile
305 310 315
Glu Phe Leu Phe Gln Leu Gly Arg Asp Ala Ala Glu Glu Val Ala
320 325 330
Gln Glu Met Val Ala Leu Gly Leu Val Cys Glu Ala Asp Tyr Gln
335 340 345
Pro Val Ala Arg Ala Val Arg Glu Arg Val Ala Ala Ile Gln Arg
350 355 360
Lys Arg Glu Lys Leu Arg Lys Ala Arg Glu Leu Glu Ala Leu Pro
365 370 375
Pro Glu Pro Gly Pro Pro Pro Ala Thr Val Pro Met Ala Pro Gly
380 385 390
Pro Pro Ser Val Phe Pro Pro Glu Pro Glu Glu Pro Glu Ala Asp
395 400 405
Gln His Gln Pro Phe Leu Phe Arg His Ala Ser Tyr Ser Ser Thr
410 415 420
Thr Ser Asp Cys Glu Thr Asp Gly Tyr Leu Ser Ser Ser Gly Phe
425 430 435
Leu Asp Ala Ser Asp Pro Ala Leu Gln Pro Pro Gly Gly Val Pro
440 445 450
Ser Ser Leu Ala Glu Ser His Leu Cys Leu Pro Ser Ala Phe Ala
455 460 465
Leu Ser Ile Pro Arg Ser Gly Pro Gly Ser Asp Phe Ser Pro Gly
470 475 480
Asp Ser Tyr Ala Ser Asp Ala Ala Ser Gly Leu Ser Asp Val Gly
485 490 495
Glu Gly Met Gly Gln Met Arg Arg Pro Pro Gly Arg Asn Leu Arg
500 505 510
Arg Arg Pro Arg Ser Arg Leu Arg Val Thr Ser Val Ser Asp Gln
515 520 525
Asn Asp Arg Val Val Glu Cys Gln Leu Gln Thr His Asn Ser Lys
530 535 540
Met Val Thr Phe Arg Phe Asp Leu Asp Gly Asp Ser Pro Glu Glu
545 550 555
Ile Ala Ala Ala Met Val Tyr Asn Glu Phe Ile Leu Pro Ser Glu
560 565 570
Arg Asp Gly Phe Leu Arg Arg Ile Arg Glu Ile Ile Gln Arg Val
575 580 585
Glu Thr Leu Leu Lys Arg Asp Thr Gly Pro Met Glu Ala Ala Glu
590 595 600
Asp Thr Leu Ser Pro Gln Glu Glu Pro Ala Pro Leu Pro Ala Leu
605 610 615
Pro Val Pro Leu Pro Asp Pro Ser Asn Glu Glu Leu Gln Ser Ser
620 625 630
Thr Ser Leu Glu His Arg Ser Trp Thr Ala Phe Ser Thr Ser Ser
635 640 645
Ser 5er Pro Gly Thr Pro Leu Ser Pro Gly Asn Pro Phe Ser Pro
34/75
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WO 02/33099 PCT/USO1/47728
650 655 660
Gly Thr Pro Ile Ser Pro Gly Pro Ile Phe Pro Ile Thr Ser Pro
665 670 675
Pro Cys His Pro Ser Pro Ser Pro Phe Ser Pro Ile Ser Ser Gln
680 685 690
Val Ser Ser Asn Pro Ser Pro His Pro Thr Ser Ser Pro Leu Pro
695 700 705
Phe Ser Ser Ser Thr Pro Glu Phe Pro Val Pro Leu Ser Gln Cys
710 715 720
Pro Trp Ser Ser Leu Pro Thr Thr Ser Pro Pro Thr Phe Ser Pro
725 730 735
Thr Cys Ser Gln Val Thr Leu Ser Ser Pro Phe Phe Pro Pro Cys
740 745 750
Pro Ser Thr Ser Ser Phe Pro Ser Thr Thr Ala Ala Pro Leu Leu
755 760 765
Ser Leu Ala Ser Ala Phe Ser Leu Ala Val Met Thr Val Ala Gln
770 775 780
Ser Leu Leu Ser Pro Ser Pro Gly Leu Leu Ser Gln Ser Pro Pro
785 790 795
Ala Pro Pro Ser Pro Leu Pro Ser Leu Pro Leu Pro Pro Pro Val
800 805 810
Ala Pro Gly Gly Gln Glu Ser Pro Ser Pro His Thr Ala Glu Val
815 820 825
Glu Ser Glu Ala Ser Pro Pro Pro Ala Arg Pro Leu Pro Gly Glu
830 835 840
Ala Arg Leu Ala Pro Ile Ser Glu Glu Gly Lys Pro Gln Leu Val
845 850 855
Gly Arg Phe Gln Val Thr Ser Ser Lys Glu Pro Ala Glu Pro Leu
860 865 870
Pro Leu Gln Pro Thr Ser Pro Thr Leu Ser Gly Ser Pro Lys Pro
875 880 885
Ser Thr Pro Gln Leu Thr Ser Glu Ser Ser Asp Thr Glu Asp Ser
890 895 900
Ala Gly Gly Gly Pro Glu Thr Arg Glu Ala Leu Ala Glu Ser Asp
905 910 915
Arg Ala Ala Glu Gly Leu Gly Ala Gly Val Glu Glu Glu Gly Asp
920 925 930
Asp Gly Lys Glu Pro Gln Val Gly Gly Ser Pro Gln Pro Leu Ser
935 940 945
His Pro Ser Pro Val Trp Met Asn Tyr Ser Tyr Ser Ser Leu Cys
950 955 960
Leu Ser Ser Glu Glu Ser Glu Ser Ser Gly Glu Asp Glu Glu Phe
965 970 975
Trp Ala Glu Leu Gln Ser Leu Arg Gln Lys His Leu Ser Glu Val
980 985 990
Glu Thr Leu Gln Thr Leu Gln Lys Lys Glu Ile Glu Asp Leu Tyr
995 1000 1005
Ser Arg Leu Gly Lys Gln Pro Pro Pro Gly Ile Val Ala Pro Ala
1010 1015 1020
Ala Met Leu Ser Ser Arg Gln Arg Arg Leu Ser Lys Gly Ser Phe
1025 1030 1035
Pro Thr Ser Arg Arg Asn Ser Leu Gln Arg Ser Glu Pro Pro Gly
1040 1045 1050
Pro Gly Ile Met Arg Arg Asn Ser Leu Ser Gly Ser Ser Thr Gly
1055 1060 1065
Ser Gln Glu Gln Arg Ala Ser Lys Gly Val Thr Phe Ala Gly Asp
35/75
CA 02425963 2003-04-15
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Val Gly Arg Met
1070 1075 1080
<210> 18
<211> 600
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474648CD1
<400> 18
Met Gly Glu Ser Gly Asn His His Phe Gln Gln Thr Asn Thr Gly
1 5 10 15
Thr Glu Asn Gln Thr Ala His Val Leu Thr His Lys Trp Glu Leu
20 25 30
Asp Asn Glu Asn Ile Trp Ala Gln Gly Gly Glu His His Lys Leu
35 40 45
Gly Pro Val Met Gly Trp Lys Ala Arg Ser Gly Lys Thr Leu Gly
50 55 60
Glu Ile Pro Asn Val Gly Thr Leu Thr Leu Leu Thr Gly Tyr Gly
65 70 75
Gly Cys Gln Leu Pro Cys Cys Lys Asp Thr Gln Ala Ala Tyr Gly
80 85 90
Glu Thr His Val Val Arg Ser Gly Gly Leu Leu Pro Thr Ala Ser
95 100 105
Trp Glu Leu Arg Pro Ala Asp Ser His Thr Val Thr Ser Asp Asp
110 115 120
Pro Gly Val Ser Val Val Ser Gly Tyr Pro Gly Gly Cys Leu Pro
125 130 135
Asp His Asp Pro Pro Val Gly Phe Leu Ser Glu Gly Pro Ala Pro
140 145 150
Arg Ser Cys Ser Leu Ile Lys Gly Gly Gly Thr Gly Leu Ala Ala
155 160 165
Ser Arg Val Pro Arg Ser Arg Glu Arg Arg Ala Cys Cys Gly Tyr
170 175 180
Gly Val Arg Arg Gln Gln Glu Gly Gly Pro Gly Ala Thr Ser Ala
185 190 195
Gly Leu Gly Gln Ala Arg Arg Ser Lys Pro Ser Arg Arg Arg Arg
200 205 210
Arg Gly Ala Trp Ala Arg Gly Gly Gly Pro Gly Gly Ala Glu Asp
215 220 225
Thr Gly Gly Ser Leu Pro Ser Gln Val Arg Pro Pro Gly Pro Cys
230 235 240
Gln Cys Pro Val Gln Phe Leu Phe Asp Ile Ser Glu Gln Gly Val
245 250 255
Gln Arg Met Gly Lys Lys Arg Ala Gly Ala Ala Ala Asn Lys Gly
260 265 270
Arg Asn Ser Tyr Leu Arg Arg Tyr Asp Ile Lys Ala Leu Ile Gly
275 280 285
Thr Gly Ser Phe Ser Arg Val Val Arg Val Glu Gln Lys Thr Thr
290 295 300
Lys Lys Pro Phe Ala Ile Lys Val Met Glu Thr Arg Glu Arg Glu
305 310 315
36/75
CA 02425963 2003-04-15
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Gly Arg Glu Ala Cys Val Ser Glu Leu Ser Val Leu Arg Arg Val
320 325 330
Ser His Arg Tyr Ile Val Gln Leu Met Glu Ile Phe Glu Thr Glu
335 340 345
Asp Gln Val Tyr Met Val Met Glu Leu Ala Thr Gly Gly Glu Leu
350 355 360
Phe Asp Arg Leu Ile Ala Gln Gly Ser Phe Thr Glu Arg Asp Ala
365 370 375
Val Arg Ile Leu Gln Met Val Ala Asp Gly Ile Arg Tyr Leu His
380 385 390
Ala Leu Gln Ile Thr His Arg Asn Leu Lys Pro Glu Asn Leu Leu
395 400 405
Tyr Tyr His Pro Gly Glu Glu Ser Lys Ile Leu Ile Thr Asp Phe
410 415 420
Gly Leu Ala Tyr Ser Gly Lys Lys Ser Gly Asp Trp Thr Met Lys
425 430 435
Thr Leu Cys Gly Thr Pro Glu Tyr Ile Ala Pro Glu Val Leu Leu
440 445 450
Arg Lys Pro Tyr Thr Ser Ala Val Asp Met Trp Ala Leu Gly Val
455 460 465
Ile Thr Tyr Ala Leu Leu Ser Gly Phe Leu Pro Phe Asp Asp Glu
470 475 480
Ser Gln Thr Arg Leu Tyr Arg Lys Ile Leu Lys Gly Lys Tyr Asn
485 490 495
Tyr Thr Gly Glu Pro Trp Pro Ser Ile Ser His Leu Ala Lys Asp
500 505 510
Phe Ile Asp Lys Leu Leu Ile Leu Glu Ala Gly His Arg Met Ser
515 520 525
Ala Gly Gln Ala Leu Asp His Pro Trp Val Ile Thr Met Ala Ala
530 535 540
Gly Ser Ser Met Lys Asn Leu Gln Arg Ala Ile Ser Arg Asn Leu
545 550 555
Met Gln Arg Ala Ser Pro His Ser Gln Ser Pro Gly Ser Ala Gln
560 565 570
Ser Ser Lys Ser His Tyr Ser His Lys Ser Arg His Met Trp Ser
575 580 585
Lys Arg Asn Leu Arg Ile Val Glu Ser Pro Leu Ser Ala Leu Leu
590 595 600
<210> 19
<211> 1114
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483053CD1
<400> 19
Met Ala Lys Ala Thr Ser Gly Ala Ala Gly Leu Arg Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Pro Leu Leu Gly Lys Val Ala Leu Gly Leu Tyr
20 25 ~ 30
Phe Ser Arg Asp Ala Tyr Trp Glu Lys Leu Tyr Val Asp Gln Ala
35 40 45
37/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Ala Gly Thr Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro
50 55 60
Glu Glu Val Pro Ser Phe Arg Leu Gly Gln His Leu Tyr Gly Thr
65 70 75
Tyr Arg Thr Arg Leu His Glu Asn Asn Trp Ile Cys Ile Gln Glu
80 85 90
Asp Thr Gly Leu Leu Tyr Leu Asn Arg Ser Leu Asp His Ser Ser
95 100 105
Trp Glu Lys Leu Ser Val Arg Asn Arg Gly Phe Pro Leu Leu Thr
110 115 120
Val Tyr Leu Lys Val Phe Leu Ser Pro Thr Ser Leu Arg Glu Gly
125 130 135
Glu Cys Gln Trp Pro Gly Cys Ala Arg Val Tyr Phe Ser Phe Phe
140 145 150
Asn Thr Ser Phe Pro Ala Cys Ser Ser Leu Lys Pro Arg Glu Leu
155 160 165
Cys Phe Pro Glu Thr Arg Pro Ser Phe Arg Ile Arg Glu Asn Arg
170 175 180
Pro Pro Gly Thr Phe His Gln Phe Arg Leu Leu Pro Val Gln Phe
185 190 195
Leu Cys Pro Asn Ile Ser Val Ala Tyr Arg Leu Leu Glu Gly Glu
200 205 210
Gly Leu Pro Phe Arg Cys Ala Pro Asp Ser Leu Glu Val Ser Thr
215 220 225
Arg Trp Ala Leu Asp Arg Glu Gln Arg Glu Lys Tyr Glu Leu Val
230 235 240
Ala Val Cys Thr Val His Ala Gly Ala Arg Glu Glu Val Val Met
245 250 255
Val Pro Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser Ala Pro
260 265 270
Thr Phe Pro Ala Gly Val Asp Thr Ala Ser Ala Val Val Glu Phe
275 280 285
Lys Arg Lys Glu Asp Thr Val Val Ala Thr Leu Arg Val Phe Asp
290 295 300
Ala Asp Val Val Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr
305 310 315
Ser Thr Leu Leu Pro Gly Asp Thr Trp Ala Gln Gln Thr Phe Arg
320 325 330
Val Glu His Trp Pro Asn Glu Thr Ser Val Gln Ala Asn Gly Ser
335 340 345
Phe Val Arg Ala Thr Val His Asp Tyr Arg Leu Val Leu Asn Arg
350 355 360
Asn Leu Ser Ile Ser Glu Asn Arg Thr Met Gln Leu Ala Val Leu
365 370 375
Val Asn Asp Ser Asp Phe Gln Gly Pro Gly Ala Gly Val Leu Leu
380 385 390
Leu His Phe Asn Val Ser Val Leu Pro Val Ser Leu His Leu Pro
395 400 405
Ser Thr Tyr Ser Leu Ser Val Ser Arg Arg Ala Arg Arg Phe Ala
410 415 420
Gln Ile Gly Lys Val Cys Val Glu Asn Cys Gln Ala Phe Ser Gly
425 430 435
Ile Asn Val Gln Tyr Lys Leu His Ser Ser Gly Ala Asn Cys Ser
440 445 450
Thr Leu Gly Val Val Thr Ser Ala Glu Asp Thr Ser Gly Ile Leu
455 460 465
38/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Phe Val Asn Asp Thr Lys Ala Leu Arg Arg Pro Lys Cys Ala Glu
470 475 480
Leu His Tyr Met Val Val Ala Thr Asp Gln Gln Thr Ser Arg Gln
485 490 495
Ala Gln Ala Gln Leu Leu Val Thr Val Glu Gly Ser Tyr Val Ala
500 505 510
Glu Glu Ala Gly Cys Pro Leu Ser Cys Ala Val Ser Lys Arg Arg
515 520 525
Leu Glu Cys Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg
530 535 540
Cys Glu Trp Arg Gln Gly Asp Gly Lys Gly Ile Thr Arg Asn Phe
545 550 555
Ser Thr Cys Ser Pro Ser Thr Lys Thr Cys Pro Asp Gly His Cys
560 565 570
Asp Val Val Glu Thr Gln Asp Ile Asn Ile Cys Pro Gln Asp Cys
575 580 585
Leu Arg Gly Ser Ile Val Gly Gly His Glu Pro Gly Glu Pro Arg
590 595 600
Gly Ile Lys Ala Gly Tyr Gly Thr Cps Asn Cys Phe Pro Glu Glu
605 610 615
Glu Lys Cys Phe Cys Glu Pro Glu Asp Ile Gln Asp Pro Leu Cys
620 625 630
Asp Glu Leu Cys Arg Thr Val Ile Ala Ala Ala Val Leu Phe Ser
635 640 645
Phe Ile Val Ser Val Leu Leu Ser Ala Phe Cys Ile His Cys Tyr
650 655 660
His Lys Phe Ala His Lys Pro Pro Ile Ser Ser Ala Glu Met Thr
665 670 675
Phe Arg Arg Pro Ala Gln Ala Phe Pro Val Ser Tyr Ser Ser Ser
680 685 690
Ser Ala Arg Arg Pro Ser Leu Asp Ser Met Glu Asn Gln Val Ser
695 700 705
Val Asp Ala Phe Lys Ile Leu Glu Asp Pro Lys Trp Glu Phe Pro
710 715 720
Arg Lys Asn Leu Val Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe
725 730 735
Gly Lys Val Val Lys Ala Thr Ala Phe His Leu Lys Gly Arg Ala
740 745 750
Gly Tyr Thr Thr Val Ala Val Lys Met Leu Lys Glu Asn Ala Ser
755 760 765
Pro Ser Glu Leu Arg Asp Leu Leu Ser Glu Phe Asn Val Leu Lys
770 775 780
Gln Val Asn His Pro His Val Ile Lys Leu Tyr Gly Ala Cys Ser
785 790 795
Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr Gly
800 805 810
Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg Lys Val Gly Pro Gly
815 820 825
Tyr Leu Gly Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His
830 835 840
Pro Asp Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe Ala
845 850 855
Trp Gln Ile Ser Gln Gly Met Gln Tyr Leu Ala Glu Met Lys Leu
860 865 870
Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly
875 880 885
39/75
CA 02425963 2003-04-15
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Arg Lys Met Lys Ile Ser Asp Phe Gly Leu Ser Arg Asp Val Tyr
890 895 900
Glu Glu Asp Ser Tyr Val Lys Arg Ser Gln Gly Arg Ile Pro Val
905 910 915
Lys Trp Met Ala Ile Glu Ser Leu Phe Asp His Ile Tyr Thr Thr
920 925 930
Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Val
935 940 945
Thr Leu Gly Gly Asn Pro Tyr Pro Gly Ile Pro Pro Glu Arg Leu
950 955 960
Phe Asn Leu Leu Lys Thr Gly His Arg Met Glu Arg Pro Asp Asn
965 970 975
Cys Ser Glu Glu Met Tyr Arg Leu Met Leu Gln Cys Trp Lys Gln
980 985 990
Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile Ser Lys Asp Leu
995 1000 1005
Glu Lys Met Met Val Lys Arg Arg Asp Tyr Leu Asp Leu Ala Ala
1010 1015 1020
Ser Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly Leu Ser Glu
1025 1030 1035
Glu Glu Thr Pro Leu Val Asp Cys Asn Asn Ala Pro Leu Pro Arg
1040 1045 1050
Ala Leu Pro Ser Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser
1055 1060 1065
Asp Pro Asn Trp Pro Gly Glu Ser Pro Val Pro Leu Thr Arg Ala
1070 1075 1080
Asp Gly Thr Asn Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val
1085 1090 1095
Tyr Ala Asn Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met Asp
1100 1105 1110
Thr Phe Asp Ser
<210> 20
<211> 567
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483117CD1
<400> 20
Met Asp Asp Lys Asp Ile Asp Lys Glu Leu Arg Gln Lys Leu Asn
1 5 10 15
Phe Ser Tyr Cys Glu Glu Thr Glu Ile Glu Gly Gln Lys Lys Val
20 25 30
Glu Glu Ser Arg Glu Ala Ser Ser Gln Thr Pro Glu Lys Gly Glu
35 40 45
Val Gln Asp Ser Glu Ala Lys Gly Thr Pro Pro Trp Thr Pro Leu
50 55 60
Ser Asn Val His Glu Leu Asp Thr Ser Ser Glu Lys Asp Lys Glu
65 70 75
Ser Pro Asp Gln Ile Leu Arg Thr Pro Val Ser His Pro Leu Lys
80 85 90
Cys Pro Glu Thr Pro Ala Gln Pro Asp Ser Arg Ser Lys Leu Leu
40/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
95 100 105
Pro Ser Asp Ser Pro Ser Thr Pro Lys Thr Met Leu Ser Arg Leu
110 115 120
Val Ile Ser Pro Thr Gly Lys Leu Pro Ser Arg Gly Pro Lys His
125 130 135
Leu Lys Leu Thr Pro Ala Pro Leu Lys Asp Glu Met Thr Ser Leu
140 145 150
Ala Leu Val Asn Ile Asn Pro Phe Thr Pro Glu Ser Tyr Lys Lys
155 160 165
Leu Phe Leu Gln Ser Gly Gly Lys Arg Lys Ile Arg Gly Asp Leu
170 175 180
Glu Glu Ala Gly Pro Glu Glu Gly Lys Gly Gly Leu Pro Ala Lys
185 190 195
Arg Cys Val Leu Arg Glu Thr Asn Met Ala Ser Arg Tyr Glu Lys
200 205 210
Glu Phe Leu Glu Val Glu Lys Ile Gly Val Gly Glu Phe Gly Thr
215 220 225
Val Tyr Lys Cys Ile Lys Arg Leu Asp Gly Cys Val Tyr Ala Ile
230 235 240
Lys Arg Ser Met Lys Thr Phe Thr Glu Leu Ser Asn Glu Asn Ser
245 250 255
Ala Leu His Glu Val Tyr Ala His Ala Val Leu Gly His His Pro
260 265 270
His Val Val Arg Tyr Tyr Ser Ser Trp Ala Glu Asp Asp His Met
275 280 285
Ile Ile Gln Asn Glu Tyr Cys Asn Gly Gly Ser Leu Gln Ala Ala
290 295 300
Ile Ser Glu Asn Thr Lys Ser Gly Asn His Phe Glu Glu Pro Lys
305 310 315
Leu Lys Asp Ile Leu Leu Gln Ile Ser Leu Gly Leu Asn Tyr Ile
320 325 330
His Asn Ser Ser Met Val His Leu Asp Ile Lys Pro Ser Asn Ile
335 340 345
Phe Ile Cys His Lys Met Gln Ser Glu Ser Ser Gly Val Ile Glu
350 355 360
Glu Val Glu Asn Glu Ala Asp Trp Phe Leu Ser Ala Asn Val Met
365 370 375
Tyr Lys Ile Gly Asp Leu Gly His Ala Thr Ser Ile Asn Lys Pro
380 385 390
Lys Val Glu Glu Gly Asp Ser Arg Phe Leu Ala Asn Glu Ile Leu
395 400 405
Gln Glu Asp Tyr Arg His Leu Pro Lys Ala Asp Ile Phe Ala Leu
410 415 420
Gly Leu Thr Ile Ala Val Ala Ala Gly Ala Glu Ser Leu Pro Thr
425 430 435
Asn Gly Ala Ala Trp His His Ile Arg Lys Gly Asn Phe Pro Asp
440 445 450
Val Pro Gln Glu Leu Ser Glu Ser Phe Ser Ser Leu Leu Lys Asn
455 460 465
Met Ile Gln Pro Asp Ala Glu Gln Arg Pro Ser Ala Ala Ala Leu
470 475 480
Ala Arg Asn Thr Val Leu Arg Pro Ser Leu Gly Lys Thr Glu Glu
485 490 495
Leu Gln Gln Gln Leu Asn Leu Glu Lys Phe Lys Thr Ala Thr Leu
500 505 510
Glu Arg Glu Leu Arg Glu Ala Gln Gln Ala Gln Ser Pro Gln Gly
41/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
515 520 525
Tyr Thr His His Gly Asp Thr Gly Val Ser Gly Thr His Thr Gly
530 535 540
Ser Arg Ser Thr Lys Arg Leu Val Gly Gly Lys Ser Ala Arg Ser
545 550 555
Ser Ser Phe Thr Ser Gly Glu Arg Glu Pro Leu His
560 565
<210> 21
<211> 2054
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7484498CD1
<400> 21
Met Leu Lys Phe Lys Tyr Gly Ala Arg Asn Pro Leu Asp Ala Gly
1 5 10 15
Ala Ala Glu Pro Ile Ala Ser Arg Ala Ser Arg Leu Asn Leu Phe
20 25 30
Phe Gln Gly Lys Pro Pro Phe Met Thr Gln Gln Gln Met Ser Pro
35 40 45
Leu Ser Arg Glu Gly Ile Leu Asp Ala Leu Phe Val Leu Phe Glu
50 55 60
Glu Cys Ser Gln Pro Ala Leu Met Lys Ile Lys His Val Ser Asn
65 70 75
Phe Val Arg Lys Tyr Ser Asp Thr Ile Ala Glu Leu Gln Glu Leu
80 85 90
Gln Pro Ser Ala Lys Asp Phe Glu Val Arg Ser Leu Val Gly Cys
95 100 105
Gly His Phe Ala Glu Val Gln Val Val Arg Glu Lys Ala Thr Gly
110 115 120
Asp Ile Tyr Ala Met Lys Val Met Lys Lys Lys Ala Leu Leu Ala
125 130 135
Gln Glu Gln Val Ser Phe Phe Glu Glu Glu Arg Asn Ile Leu Ser
140 145 150
Arg Ser Thr Ser Pro Trp Ile Pro Gln Leu Gln Tyr Ala Phe Gln
155 160 165
Asp Lys Asn His Leu Tyr Leu Val Met Glu Tyr Gln Pro Gly Gly
170 175 180
Asp Leu Leu Ser Leu Leu Asn Arg Tyr Glu Asp Gln Leu Asp Glu
185 190 195
Asn Leu Ile Gln Phe Tyr Leu Ala Glu Leu Ile Leu Ala Val His
200 205 210
Ser Val His Leu Met Gly Tyr Val His Arg Asp Ile Lys Pro Glu
215 220 225
Asn Ile Leu Val Asp Arg Thr Gly His Ile Lys Leu Val Asp Phe
230 235 240
Gly Ser Ala Ala Lys Met Asn Ser Asn Lys Met Val Asn Ala Lys
245 250 255
Leu Pro Ile Gly Thr Pro Asp Tyr Met Ala Pro Glu Val Leu Thr
260 265 270
Val Met Asn Gly Asp Gly Lys Gly Thr Tyr Gly Leu Asp Cys Asp
275 280 285
42/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Trp Trp Ser Val Gly Val Ile Ala Tyr Glu Met Ile Tyr Gly Arg
290 295 300
Ser Pro Phe Ala Glu Gly Thr Ser Ala Arg Thr Phe Asn Asn Ile
305 310 315
Met Asn Phe Gln Arg Phe Leu Lys Phe Pro Asp Asp Pro Lys Val
320 325 330
Ser Ser Asp Phe Leu Asp Leu Ile Gln Ser Leu Leu Cys Gly Gln
335 340 345
Lys Glu Arg Leu Lys Phe Glu Gly Leu Cys Cys His Pro Phe Phe
350 355 360
Ser Lys Ile Asp Trp Asn Asn Ile Arg Asn Ser Pro Pro Pro Phe
365 370 375
Val Pro Thr Leu Lys Ser Asp Asp Asp Thr Ser Asn Phe Asp Glu
380 385 390
Pro Glu Lys Asn Ser Trp Val Ser Ser Ser Pro Cys Gln Leu Ser
395 400 405
Pro Ser Gly Phe Ser Gly Glu Glu Leu Pro Phe Val Gly Phe Ser
410 415 420
Tyr Ser Lys Ala Leu Gly Ile Leu Gly Arg Ser Glu Ser Val Val
425 430 435
Ser Gly Leu Asp Ser Pro Ala Lys Thr Ser Ser Met Glu Lys Lys
440 445 450
Leu Leu Ile Lys Ser Lys Glu Leu Gln Asp Ser Gln Asp Lys Cys
455 460 465
His Lys Met Glu Gln Glu Met Thr Arg Leu His Arg Arg Val Ser
470 475 480
Glu Val Glu'Ala Val Leu Ser Gln Lys Glu Val Glu Leu Lys Ala
485 490 495
Ser Glu Thr Gln Arg Ser Leu Leu Glu Gln Asp Leu Ala Thr Tyr
500 505 510
Ile Thr Glu Cys Ser Ser Leu Lys Arg Ser Leu Glu Gln Ala Arg
515 520 525
Met Glu Val Ser Gln Glu Asp Asp Lys Ala Leu Gln Leu Leu His
530 535 540
Asp Ile Arg Glu Gln Ser Arg Lys Leu Gln Glu Ile Lys Glu Gln
545 550 555
Glu Tyr Gln Ala Gln Val Glu Glu Met Arg Leu Met Met Asn Gln
560 565 570
Leu Glu Glu Asp Leu Val Ser Ala Arg Arg Arg Ser Asp Leu Tyr
575 580 585
Glu Ser Glu Leu Arg Glu Ser Arg Leu Ala Ala Glu Glu Phe Lys
590 595 600
Arg Lys Ala Thr Glu Cys Gln His Lys Leu Leu Lys Ala Lys Asp
605 610 615
Gln Gly Lys Pro Glu Val Gly Glu Tyr Ala Lys Leu Glu Lys Ile
620 625 630
Asn Ala Glu Gln Gln Leu Lys Ile Gln Glu Leu Gln Glu Lys Leu
635 640 645
Glu Lys Ala Val Lys Ala Ser Thr Glu Ala Thr Glu Leu Leu Gln
650 655 660
Asn Ile Arg Gln Ala Lys Glu Arg Ala Glu Arg Glu Leu Glu Lys
665 670 675
Leu Gln Asn Arg Glu Asp Ser Ser Glu Gly Ile Arg Lys Lys Leu
680 685 690
Val Glu Ala Glu Glu Arg Arg His Ser Leu Glu Asn Lys Val Lys
695 700 705
43/75
CA 02425963 2003-04-15
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Arg Leu Glu Thr Met Glu Arg Arg Glu Asn Arg Leu Lys Asp Asp
710 715 720
Ile Gln Thr Lys Ser Gln Gln Ile Gln Gln Met Ala Asp Lys Ile
725 730 735
Leu Glu Leu Glu Glu Lys His Arg Glu Ala Gln Val Ser Ala Gln
740 745 750
His Leu Glu Val His Leu Lys Gln Lys Glu Gln His Tyr Glu Glu
755 760 765
Lys Ile Lys Val Leu Asp Asn Gln Ile Lys Lys Asp Leu Ala Asp
770 775 780
Lys Glu Thr Leu Glu Asn Met Met Gln Arg His Glu Glu Glu Ala
785 790 795
His Glu Lys Gly Lys Ile Leu Ser Glu Gln Lys Ala Met Ile Asn
800 805 810
Ala Met Asp Ser Lys Ile Arg Ser Leu Glu Gln Arg Ile Val Glu
815 820 825
Leu Ser Glu Ala Asn Lys Leu Ala Ala Asn Ser Ser Leu Phe Thr
830 835 840
Gln Arg Asn Met Lys Ala Gln Glu Glu Met Ile Ser Glu Leu Arg
845 850 855
Gln Gln Lys Phe Tyr Leu Glu Thr Gln Ala Gly Lys Leu Glu Ala
860 865 870
Gln Asn Arg Lys Leu Glu Glu Gln Leu Glu Lys Ile Ser His Gln
875 880 885
Asp His Ser Asp Lys Asn Arg Leu Leu Glu Leu Glu Thr Arg Leu
890 895 900
Arg Glu Val Ser Leu Glu His Glu Glu Gln Lys Leu Glu Leu Lys
905 910 915
Arg Gln Leu Thr Glu Leu Gln Leu Ser Leu Gln Glu Arg Glu Ser
920 925 930
Gln Leu Thr Ala Leu Gln Ala Ala Arg Ala Ala Leu Glu Ser Gln
935 940 945
Leu Arg Gln Ala Lys Thr Glu Leu Glu Glu Thr Thr Ala Glu Ala
950 955 960
Glu Glu Glu Ile Gln Ala Leu Thr Ala His Arg Asp Glu Ile Gln
965 970 975
Arg Lys Phe Asp Ala Leu Arg Asn Ser Cys Thr Val Ile Thr Asp
980 985 990
Leu Glu Glu Gln Leu Asn Gln Leu Thr Glu Asp Asn Ala Glu Leu
995 1000 1005
Asn Asn Gln Asn Phe Tyr Leu Ser Lys Gln Leu Asp Glu Ala Ser
1010 1015 1020
Gly Ala Asn Asp Glu Ile Val Gln Leu Arg Ser Glu Val Asp His
1025 1030 1035
Leu Arg Arg Glu Ile Thr Glu Arg Glu Met Gln Leu Thr Ser Gln
1040 1045 1050
Lys Gln Thr Met Glu Ala Leu Lys Thr Thr Cys Thr Met Leu Glu
1055 1060 1065
Glu Gln Val Met Asp Leu Glu Ala Leu Asn Asp Glu Leu Leu Glu
1070 1075 1080
Lys Glu Arg Gln Trp Glu Ala Trp Arg Ser Val Leu Gly Asp Glu
1085 1090 1095
Lys Ser Gln Phe Glu Cys Arg Val Arg Glu Leu Gln Arg Met Leu
1100 1105 1110
Asp Thr Glu Lys Gln Ser Arg Ala Arg Ala Asp Gln Arg Ile Thr
1115 1120 1125
44/75
CA 02425963 2003-04-15
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Glu Ser Arg Gln Val Val Glu Leu Ala Val Lys Glu His Lys Ala
1130 1135 1140
Glu Ile Leu Ala Leu Gln Gln Ala Leu Lys Glu Gln Lys Leu Lys
1145 1150 1155
Ala Glu Ser Leu Ser Asp Lys Leu Asn Asp Leu Glu Lys Lys His
1160 1165 1170
Ala Met Leu Glu Met Asn Ala Arg Ser Leu Gln Gln Lys Leu Glu
1175 1180 1185
Thr Glu Arg Glu Leu Lys Gln Arg Leu Leu Glu Glu Gln Ala Lys
1190 1195 1200
Leu Gln Gln Gln Met Asp Leu Gln Lys Asn His Ile Phe Arg Leu
1205 1210 1215
Thr Gln Gly Leu Gln Glu Ala Leu Asp Arg Ala Asp Leu Leu Lys
1220 1225 1230
Thr Glu Arg Ser Asp Leu Glu Tyr Gln Leu Glu Asn Ile Gln Val
1235 1240 1245
Leu Tyr Ser His Glu Lys Val Lys Met Glu Gly Thr Ile Ser Gln
1250 1255 1260
Gln Thr Lys Leu Ile Asp Phe Leu Gln Ala Lys Met Asp Gln Pro
1265 1270 1275
Ala Lys Lys Lys Lys Val Pro Leu Gln Tyr Asn Glu Leu Lys Leu
1280 1285 1290
Ala Leu Glu Lys Glu Lys Ala Arg Cys Ala Glu Leu Glu Glu Ala
1295 1300 1305
Leu Gln Lys Thr Arg Ile Glu Leu Arg Ser Ala Arg Glu Glu Ala
1310 1315 1320
Ala His Arg Lys Ala Thr Asp His Pro His Pro Ser Thr Pro Ala
1325 1330 1335
Thr Ala Arg Gln Gln Ile Ala Met Ser Ala Ile Val Arg Ser Pro
1340 1345 1350
Glu His Gln Pro Ser Ala Met Ser Leu Leu Ala Pro Pro Ser Ser
1355 1360 1365
Arg Arg Lys Glu Ser Ser Thr Pro Glu Glu Phe Ser Arg Arg Leu
1370 1375 1380
Lys Glu Arg Met His His Asn Ile Pro His Arg Phe Asn Val Gly
1385 1390 1395
Leu Asn Met Arg Ala Thr Lys Cys Ala Val Cys Leu Asp Thr Val
1400 1405 1410
His Phe Gly Arg Gln Ala Ser Lys Cys Leu Glu Cys Gln Val Met
1415 1420 1425
Cys His Pro Lys Cys Ser Thr Cys Leu Pro Ala Thr Cys Gly Leu
1430 1435 1440
Pro Ala Glu Tyr Ala Thr His Phe Thr Glu Ala Phe Cys Arg Asp
1445 1450 1455
Lys Met Asn Ser Pro Gly Leu Gln Thr Lys Glu Pro Ser Ser Ser
1460 1465 1470
Leu His Leu Glu Gly Trp Met Lys Val Pro Arg Asn Asn Lys Arg
1475 1480 1485
Gly Gln Gln Gly Trp Asp Arg Lys Tyr Ile Val Leu Glu Gly Ser
1490 1495 1500
Lys Val Leu Ile Tyr Asp Asn Glu Ala Arg Glu Ala Gly Gln Arg
1505 1510 1515
Pro Val Glu Glu Phe Glu Leu Cys Leu Pro Asp Gly Asp Val Ser
1520 1525 1530
Ile His Gly Ala Val Gly Ala Ser Glu Leu Ala Asn Thr Ala Lys
1535 1540 1545
45/75
CA 02425963 2003-04-15
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Ala Asp Val Pro Tyr Ile Leu Lys Met Glu Ser His Pro His Thr
1550 1555 1560
Thr Cys Trp Pro Gly Arg Thr Leu Tyr Leu Leu Ala Pro Ser Phe
1565 1570 1575
Pro Asp Lys Gln Arg Trp Val Thr Ala Leu Glu Ser Val Val Ala
1580 1585 1590
Gly Gly Arg Val Ser Arg Glu Lys Ala Glu Ala Asp Ala Lys Leu
1595 1600 1605
Leu Gly Asn Ser Leu Leu Lys Leu Glu Gly Asp Asp Arg Leu Asp
1610 1615 1620
Met Asn Cys Thr Leu Pro Phe Ser Asp Gln Val Val Leu Val Gly
1625 1630 1635
Thr Glu Glu Gly Leu Tyr Ala Leu Asn Val Leu Lys Asn Ser Leu
1640 1645 1650
Thr His Val Pro Gly Ile Gly Ala Val Phe Gln Ile Tyr Ile Ile
1655 1660 1665
Lys Asp Leu Glu Lys Leu Leu Met Ile Ala Gly Glu Glu Arg Ala
1670 1675 1680
Leu Cys Leu Val Asp Val Lys Lys Val Lys Gln Ser Leu Ala Gln
1685 1690 1695
Ser His Leu Pro Ala Gln Pro Asp Ile Ser Pro Asn Ile Phe Glu
170p 1705 1710
Ala Val Lys Gly Cys His Leu Phe Gly Ala Gly Lys Ile Glu Asn
1715 1720 1725
Gly Leu Cys Ile Cys Ala Ala Met Pro Ser Lys Val Val Ile Leu
1730 1735 1740
Arg Tyr Asn Glu Asn Leu Ser Lys Tyr Cys Ile Arg Lys Glu Ile
1745 1750 1755
Glu Thr Ser Glu Pro Cys Ser Cys Ile His Phe Thr Asn Tyr Ser
1760 1765 1770
Ile Leu Ile Gly Thr Asn Lys Phe Tyr Glu Ile Asp Met Lys Gln
1775 1780 1785
Tyr Thr Leu Glu Glu Phe Leu Asp Lys Asn Asp His Ser Leu Ala
1790 1795 1800
Pro Ala Val Phe Ala Ala Ser Ser Asn Ser Phe Pro Val Ser Ile
1805 1810 1815
Val Gln Val Asn Ser Ala Gly Gln Arg Glu Glu Tyr Leu Leu Cys
1820 1825 1830
Phe His Glu Phe Gly Val Phe Val Asp Ser Tyr Gly Arg Arg Ser
1835 1840 1845
Arg Thr Asp Asp Leu Lys Trp Ser Arg Leu Pro Leu Ala Phe Ala
1850 1855 1860
Tyr Arg Glu Pro Tyr Leu Phe Val Thr His Phe Asn Ser Leu Glu
1865 1870 1875
Val Ile Glu Ile Gln Ala Arg Ser Ser Ala Gly Thr Pro Ala Arg
1880 1885 1890
Ala Tyr Leu Asp Ile Pro Asn Pro Arg Tyr Leu Gly Pro Ala Ile
1895 1900 1905
Ser Ser Gly Ala Ile Tyr Leu Ala Ser Ser Tyr Gln Asp Lys Leu
1910 1915 1920
Arg Val Ile Cys Cys Lys Gly Asn Leu Val Lys Glu Ser Gly Thr
1925 1930 1935
Glu His His Arg Gly Pro Ser Thr Ser Arg Ser Ser Pro Asn Lys
1940 1945 1950
Arg Gly Pro Pro Thr Tyr Asn Glu His Ile Thr Lys Arg Val Ala
1'955 1960 1965
46/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
Ser Ser Pro Ala Pro Pro Glu Gly Pro Ser His Pro Arg Glu Pro
1970 1975 1980
Ser Thr Pro His Arg Tyr Arg Glu Gly Arg Thr Glu Leu Arg Arg
1985 1990 1995
Asp Lys Ser Pro Gly Arg Pro Leu Glu Arg Glu Lys Ser Pro Gly
2000 2005 2010
Arg Met Leu Ser Thr Arg Arg Glu Arg Ser Pro Gly Arg Leu Phe
2015 2020 2025
Glu Asp Ser Ser Arg Gly Arg Leu Pro Ala Gly Ala Val Arg Thr
2030 2035 2040
Pro Leu Ser Gln Val Asn Lys Val Trp Asp Gln Ser Ser Val
2045 2050
<210> 22
<211> 1665
<212> PRT
<213> Homo sapiens
<220>
<221> mist feature
<223> Incyte ID No: 7638121CD1
<400> 22
Met Gly Cys Cys Arg Leu Gly Cys Gly Gly Cys Ser Val Ala His
1 5 10 15
Ser Val Ser Gln Gly Leu Thr Asn His Pro Ser Met Val Gly Cys
20 25 30
Gly Trp His Pro Gly Leu Cys Gly Trp Gly Gly Gly Leu His Ser
35 40 45
Ser Leu Pro Ala Leu Pro Gly Pro Pro Ser Met Gln Val Thr Ile
50 55 60
Glu Asp Val Gln Ala Gln Thr Gly Gly Thr Ala Gln Phe Glu Ala
65 70 75
Ile Ile Glu Gly Asp Pro Gln Pro Ser Val Thr Trp Tyr Lys Asp
80 . 85 90
Ser Val Gln Leu Val Asp Ser Thr Arg Leu Ser Gln Gln Gln Glu
95 100 105
Gly Thr Thr Tyr Ser Leu Val Leu Arg His Met Ala Ser Lys Asp
110 115 120
Ala Gly Val Tyr Thr Cys Leu Ala Gln Asn Thr Gly Gly Gln Val
125 130 135
Leu Cys Lys Ala Glu Leu Leu Val Leu Gly Gly Asp Asn Glu Pro
140 145 150
Asp Ser Glu Lys Gln Ser His Arg Arg Lys Leu His Ser Phe Tyr
155 160 165
Glu Val Lys Glu Glu Ile Gly Arg Gly Val Phe Gly Phe Val Lys
170 175 180
Arg Val Gln His Lys Gly Asn Lys Ile Leu Cys Ala Ala Lys Phe
185 190 195
Ile Pro Leu Arg Ser Arg Thr Arg Ala Gln Ala Tyr Arg Glu Arg
200 205 210
Asp Ile Leu Ala Ala Leu Ser His Pro Leu Val Thr Gly Leu Leu
215 220 225
Asp Gln Phe Glu Thr Arg Lys Thr Leu Ile Leu Ile Leu Glu Leu
230 235 240
Cys Ser Ser Glu Glu Leu Leu Asp Arg Leu Tyr Arg Lys Gly Val
47/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
245 250 255
Val Thr Glu Ala Glu Val Lys Val Tyr Ile Gln Gln Leu Val Glu
260 265 270
Gly Leu His Tyr Leu His Ser His Gly Val Leu His Leu Asp Ile
275 280 285
Lys Pro Ser Asn Ile Leu Met Val His Pro Ala Arg Glu Asp Ile
290 295 300
Lys Ile Cys Asp Phe Gly Phe Ala Gln Asn Ile Thr Pro Ala Glu
305 310 315
Leu Gln Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val Ser Pro Glu
320 325 330
Ile Ile Gln Gln Asn Pro Val Ser Glu Ala Ser Asp Ile Trp Ala
335 340 345
Met Gly Val Ile Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro Phe
350 355 360
Ala Gly Glu Ser Asp Arg Ala Thr Leu Leu Asn Val Leu Glu Gly
365 370 375
Arg Val Ser Trp Ser Ser Pro Met Ala Ala His Leu Ser Glu Asp
380 385 390
Ala Lys Asp Phe Ile Lys Ala Thr Leu Gln Arg Ala Pro Gln Ala
395 400 405
Arg Pro Ser Ala Ala Gln Cys Leu Ser His Pro Trp Phe Leu Lys
410 415 420
Ser Met Pro Ala Glu Glu Ala His Phe Ile Asn Thr Lys Gln Leu
425 430 435
Lys Phe Leu Leu Ala Arg Ser Arg Trp Gln Arg Ser Leu Met Ser
440 445 450
Tyr Lys Ser Ile Leu Val Met Arg Ser Ile Pro Glu Leu Leu Arg
455 460 465
Gly Pro Pro Asp Ser Pro Ser Leu Gly Val Ala Arg His Leu Cys
470 475 480
Arg Asp Thr Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Asp Asn
485 490 495
Glu Leu Ala Pro Phe Ala Arg Ala Lys Ser Leu Pro Pro Ser Pro
500 505 510
Val Thr His Ser Pro Leu Leu His Pro Arg Gly Phe Leu Arg Pro
515 520 525
Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala Ser Glu Arg Ser Thr
530 535 540
Glu Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly Ala Gly Pro Pro
545 550 555
Ala Ala Gln Gly Cys Val Pro Arg His Ser Val Ile Arg Ser Leu
560 565 570
Phe Tyr His Gln Ala Gly Glu Ser Pro Glu His Gly Ala Leu Ala
575 580 585
Pro Gly Ser Arg Arg His Pro Ala Arg Arg Arg His Leu Leu Lys
590 595 600
Gly Gly Tyr Ile Ala Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu
605 610 615
Met Glu His Arg Val Leu Glu Glu Glu Ala Ala Arg Glu Glu Gln
620 625 630
Ala Thr Leu Leu Ala Lys Ala Pro Ser Phe Glu Thr Ala Leu Arg
635 640 645
Leu Pro Ala Ser Gly Thr His Leu Ala Pro Gly His Ser His Ser
650 655 660
Leu Glu His Asp Ser Pro Ser Thr Pro Arg Pro Ser Ser Glu Ala
48/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
665 670 675
Cys Gly Glu Ala Gln Arg Leu Pro Ser Ala Pro Ser Gly Gly Ala
680 685 690
Pro Ile Arg Asp Met Gly His Pro Gln Gly Ser Lys Gln Leu Pro
695 700 705
Ser Thr Gly Gly His Pro Gly Thr Ala Gln Pro Glu Arg Pro Ser
710 715 720
Pro Asp Ser Pro Trp Gly Gln Pro Ala Pro Phe Cys His Pro Lys
725 730 735
Gln Gly Ser Ala Pro Gln Glu Gly Cys Ser Pro His Pro Ala Val
740 745 750
Ala Pro Cys Pro Pro Gly Ser Phe Pro Pro Gly Ser Cys Lys Glu
755 760 765
Ala Pro Leu Val Pro Ser Ser Pro Phe Leu Gly Gln Pro Gln Ala
770 775 780
Pro Leu Ala Pro Ala Lys Ala Ser Pro Pro Leu Asp Ser Lys Met
785 790 795
Gly Pro Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro
800 805 810
Cys Ser Ser Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val
815 820 825
Ser Ser Leu Arg Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly
830 835 840
Pro Ser Leu Asp Ala Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu
845 850 855
Ser Asp Ser Thr Pro Thr Leu Gln Arg Pro Gln Glu Gln Val Thr
860 865 870
Met Arg Lys Phe Ser Leu Gly Gly Arg Gly Gly Tyr Ala Gly Val
875 880 885
Ala Gly Tyr Gly Thr Phe Ala Phe Gly Gly Asp Ala Gly Gly Met
890 895 900
Leu Gly Gln Gly Pro Met Trp Ala Arg Ile Ala Trp Ala Val Ser
905 910 915
Gln Ser Glu Glu Glu Glu Gln Glu Glu Ala Arg Ala Glu Ser Gln
920 925 930
Ser Glu Glu Gln Gln Glu Ala Arg Ala Glu Ser Pro Leu Pro Gln
935 940 945
Val Ser Ala Arg Pro Val Pro Glu Val Gly Arg Ala Pro Thr Arg
950 955 960
Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp Ile Gly Gln Val Ser
965 970 975
Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala Glu Ala Ala Asp
980 985 990
Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala Tyr Leu Asn
995 1000 1005
Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu Phe Met
1010 1015 1020
Ile Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro Ser
1025 1030 1035
Pro Met Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp
1040 1045 1050
Pro Trp Pro Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr
1055 1060 1065
Glu Glu Ser Glu Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val
1070 1075 1080
Gly Arg Lys Arg Lys Trp Ser Ser Pro Ser Arg Ser Leu Phe His
49/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
1085 1090 1095
Phe Pro Gly Arg His Leu Pro Leu Asp Glu Pro Ala Glu Leu Gly
1100 1105 1110
Leu Arg Glu Arg Val Lys Ala Ser Val Glu His Ile Ser Arg Ile
1115 1120 1125
Leu Lys Gly Arg Pro Glu Gly Leu Glu Lys Glu Gly Pro Pro Arg
1130 1135 1140
Lys Lys Pro Gly Leu Ala Ser Phe Arg Leu Ser Gly Leu Lys Ser
1145 1150 1155
Trp Asp Arg Ala Pro Thr Phe Leu Arg Glu Leu Ser Asp Glu Thr
1160 1165 1170
Val Val Leu Gly Gln Ser Val Thr Leu Ala Cys Gln Val Ser Ala
1175 1180 1185
Gln Pro Ala Ala Gln Ala Thr Trp Ser Lys Asp Gly Ala Pro Leu
1190 1195 1200
Glu Ser Ser Ser Arg Val Leu Ile Ser Ala Thr Leu Lys Asn Phe
1205 1210 1215
Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu Asp Leu Gly Val
1220 1225 1230
Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val Thr Thr Thr
1235 1240 1245
Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro Cys Pro
1250 1255 1260
Asp Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp Lys
1265 1270 1275
Pro Val Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cps Ser
1280 1285 1290
Leu Glu Gly Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp
1295 1300 1305
Cys Cys Tyr Leu Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr
1310 1315 1320
Phe Arg Thr Ala Cys Val Ser Lys Ala Gly Met Gly Pro Tyr Ser
1325 1330 1335
Ser Pro Ser Glu Gln Val Leu Leu Gly Gly Pro Ser His Leu Ala
1340 1345 1350
Ser Glu Glu Glu Ser Gln Gly Arg Ser Ala Gln Pro Leu Pro Ser
1355 1360 1365
Thr Lys Thr Phe Ala Phe Gln Thr Gln Ile Gln Arg Gly Arg Phe
1370 1375 1380
Ser Val Val Arg Gln Cys Trp Glu Lys Ala Ser Gly Arg Ala Leu
1385 1390 1395
Ala Ala Lys Ile Ile Pro Tyr His Pro Lys Asp Lys Thr Ala Val
1400 1405 1410
Leu Arg Glu Tyr Glu Ala Leu Lys Gly Leu Arg His Pro His Leu
1415 1420 1425
Ala Gln Leu His Ala Ala Tyr Leu Ser Pro Arg His Leu Val Leu
1430 1435 1440
Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu Leu Pro Cys Leu Ala
1445 1450 1455
Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys Asp Tyr Leu Trp
1460 1465 1470
Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln His Ile Leu
1475 1480 1485
His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu Tyr Asn
1490 1495 1500
Leu Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser Gln
50/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
1505 1510 1515
Glu Lys Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr
1520 1525 1530
Met Ala Pro Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr
1535 1540 1545
Asp Ile Trp Ala Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala
1550 1555 1560
Glu Tyr Pro Val Ser Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly
1565 1570 1575
Leu Arg Lys Gly Leu Val Arg Leu Ser Arg Cys Tyr Ala Gly Leu
1580 1585 1590
Ser Gly Gly Ala Val Ala Phe Leu Arg Ser Thr Leu Cys Ala Gln
1595 1600 1605
Pro Trp Gly Arg Pro Cys Ala Ser Ser Cys Leu Gln Cys Pro Trp
1610 1615 1620
Leu Thr Glu Glu Gly Pro Ala Cys Ser Arg Pro Ala Pro Val Thr
1625 1630 1635
Phe Pro Thr Ala Arg Leu Arg Val Phe Val Arg Asn Arg Glu Lys
1640 1645 1650
Arg Arg Ala Leu Leu Tyr Lys Arg His Asn Leu Ala Gln Val Arg
1655 1660 1665
<210> 23
<211> 1014
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7482896CB1
<400> 23
atgacaaaca acagcggctc caaagccgaa ctcgttgtgg gagggaaata caaactggtg 60
cggaagatcg ggtctggctc ctttggagac gtttatctgg gcatcaccac caccaacggc 120
gaggacgtag cagtgaagct ggaatctcag aaggtcaagc acccccagtt gctgtatgag 180
agcaaactct acacgattct tcaaggtggg gttggcatcc cccacatgca ctggtatggt 240
caggaaaaag acaacaatgt gctagtcatg gaccttctgg gacccagcct cgaagacctc 300
tttaatttct gttcaagaag gttcaccatg aaaactgtac ttatgttagc cgaccagatg 360
atcagcagaa ttgaatacgt gcatacaaag aattttctac accgagacat taaaccagat 420
aacttcctga tgggtactgg gcgtcactgt aataagttgt tccttattga ttttggtttg 480
gccaaaaagt acagagacaa caggaccagg caacacatac cgtacagaga agataaacac 540
ctcattggca ctgtccgata tgccagcatc aatgcacatc ttggtattga gcagagccgc 600
cgagatgaca tggaatcctt aggctacgtt ttcatgtatt ttaatagaac cagcctgccg 660
tggcaaggac taagggctat gacaaaaaaa caaaaatatg aaaagattag tgagaagaag 720
atgtccaccc ctgttgaagt tttatgtaag gggtttcctg cagaattcgc catgtacttg 780
aactactgtc gtgggctgcg ctttgaggaa gtcccagatt acatgtatct gaggcagcta 840
ttccgcattc ttttcaggac cctgaaccac caatatgact acacatttga ttggacgatg 900
ttaaagcaga aagcagcaca gcaggcagcc tcttccagtg ggcagggtca gcaggcccaa 960
acccagacag gcaagcaaac tgaaaaaaac aagaataatg tgaaagataa ctaa 1014
<210> 24
<211> 1530
<212> DNA
<213> Homo Sapiens
51/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
<220>
<221> misc feature
<223> Incyte ID No: 7483046CB1
<400> 24
cggcctgaca ggcgggcatg cgggcggcca gactgtagcc gagcagcgag gctccggccg 60
cagccatgga gcggcggctg cgcgcgctgg agcagctggc gcggggcgag gccggcggct 120
gcccggggct cgacggcctc ctagatctgc tgctggcgct gcaccacgag ctcagcagcg 180
gccccctacg gcgggagcgc agcgtggcgc agttcctgag ctgggccagc cccttcgtat 240
caaaggtgaa agaactgcgt ctgcagagag atgactttga gatcttgaag gtgatcggcc 300
gaggagcctt tggggaggtc accgtggtga ggcagaggga cactgggcag atttttgcca 360
tgaaaatgct gcacaagtgg gagatgctga agagggctga gacagcctgt ttccgggagg 420
agcgggatgt gctcgtgaaa ggggacagcc gttgggtgac cactctgcac tatgccttcc 480
aagacgagga gtacctgtac cttgtgatgg actactatgc tggtggggac ctcctgacgc 540
tgctgagccg cttcgaggac cgtctcccgc ccgagctggc ccagttctac ctggctgaga 600
tggtgctggc catccactcg ctgcaccagc tgggttatgt ccacagggat gtcaagccag 660
acaacgtcct gctggatgtg aacgggcaca ttcgcctggc tgacttcggc tcctgcctgc 720
gtctcaacac caacggcatg gtggattcat cagtggcagt agggacgccg gactatatct 780
cccctgagat cctgcaggcc atggaggagg gcaagggcca ctacggccca cagtgtgact 840
ggtggtcgct tggagtctgc gcctatgagc tgctctttgg ggagacgccc ttctatgctg 900
agtccttggt ggaaacctac ggcaagatca tgaaccacga ggaccacctg cagttccccc 960
cggacgtgcc tgacgtgcca gccagcgccc aagacctgat ccgccagctg ctgtgtcgcc 1020
aggaagagcg gctaggccgt ggtgggctgg atgacttccg gaaccatcct ttcttcgaag 1080
gcgtggactg ggagcggctg gcgagcagca cggcccccta tattcctgag ctgcgggggc 1140
ccatggacac ctccaacttt gatgtggatg acgacaccct caaccatcca gggaccctgc 1200
caccgccctc ccacggggcc ttctccggcc atcacctgcc attcgtgggc ttcacctaca 1260
cctcaggcag tcacagtcct gagagcagct ctgaggcttg ggctgccctg gagcggaagc 1320
tccagtgtct ggagcaggag aaggtggagc tgagcaggaa gcaccaagag gccctgcacg 1380
cccccacaga ccatcgggag ctggagcagc tacggaagga agtgcagact ctgcgggaca 1440
ggctgccagg tatcccttcc gcccaccccc accctctcct tgagtttctg tgaattaaaa 1500
tatttgcaaa tccaaaaaaa aaaaaaaagg 1530
<210> 25
<211> 3150
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 71636374CB1
<400> 25
attggcttat aggaaaaatt gatttataaa aagtggtaca ggttttcata gataaccatg 60
acaacatccc atatgaatgg gcatgttaca gaggaatcag acagcgaagt aaaaaatgtt 120
gatcttgcat caccagagga acatcagaag caccgagaga tggctgttga ctgccctgga 180
gatttgggca ccaggatgat gccaatacgt cgaagtgcac agttggagcg tattcggcaa 240
caacaggagg acatgaggcg taggagagag gaagaaggga aaaagcaaga acttgacctt 300
aattcttcca tgagacttaa gaaactagcc caaattcctc caaagaccgg aatagataac 360
cctatgtttg atacagagga aggaattgtc ttagaaagtc ctcattatgc tgtgaaaata 420
ttagaaatag aagacttgtt ttcttcactt aaacatatcc aacatacttt ggtagattct 480
cagagccagg aggatatttc actgctttta caacttgttc aaaataagga tttccagaat 540
gcatttaaga tacacaatgc catcacagta cacatgaaca aggccagtcc tccatttcct 600
cttatctcca acgcacaaga tcttgctcaa gaggtacaaa ctgttttgaa gccagttcat 660
cataaggaag gacaagaact aactgctttg ctgaatactc cacatattca ggcactttta 720
ctggcccacg ataaggttgc tgagcaggaa atgcagctag agcccattac agatgagaga 780
gtttatgaaa gtattggcca gtatggagga gaaactgtaa aaatagttcg tatagaaaag 840
52/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
gctcgtgata ttccgttggg tgctacagtt cgtaatgaaa tggactctgt catcattagc 900
cggatagtaa aagggggtgc tgcagagaaa agtggtctgt tgcatgaagg agatgaagtt 960
ctagagatta atggcattga aattcggggg aaagatgtca atgaggtttt tgacttgttg 1020
tctgatatgc atggtacttt gacttttgtc ctgattccca gtcaacagat caagccgcct 1080
cctgccaagg aaacagtaat ccatgtaaaa gctcattttg actatgaccc ctcagatgac 1140
ccttatgttc catgtcgaga gttaggtctg tcttttcaaa aaggtgatat acttcatgtg 1200
atcagtcaag aagatccaaa ctggtggcag gcctacaggg aaggggacga agataatcaa 1260
cctctagccg ggcttgttcc agggaaaagc tttcagcagc aaagggaagc catgaaacaa 1320
accatagaag aagataagga gccagaaaaa tcaggaaaac tgtggtgtgc aaagaagaat 1380
aaaaagaaga ggaaaaaggt tttatataat gccaataaaa atgatgatta tgacaacgag 1440
gagatcttaa cctatgagga aatgtcactt tatcatcagc cagcaaatag gaagagacct 1500
atcatcttga ttggtccaca gaactgtggc cagaatgaat tgcgtcagag gctcatgaac 1560
aaagaaaagg accgctttgc atctgcagtt cctcatacaa cccggagtag gcgagaccaa 1620
gaagtagccg gtagagatta ccactttgtt tcgcggcaag cattcgaggc agacatagca 1680
gctggaaagt tcattgagca tggtgaattt gagaagaatt tgtatggaac tagcatagat 1740
tctgtacggc aagtgatcaa ctctggcaaa atatgtcttt taagtcttcg tacacagtca 1800
ttgaagactc tccggaattc agatttgaaa ccatatatta tcttcattgc acccccttca 1860
caagaaagac ttcgggcatt attggccaaa gaaggcaaga atccaaagcc tgaagagttg 1920
agagaaatca ttgagaagac aagagagatg gagcagaaca atggccacta ctttgatacg 1980
gcaattgtga attccgatct tgataaagcc tatcaggaat tgcttaggtt aattaacaaa 2040
cttgatactg aacctcagtg ggtaccatcc acttggctga ggtgaaagaa acatccattc 2100
tgtggcatgt tggacttgat ctggcaaaaa ctgccaatag gaggactgcc cgacactgca 2160
gcaagattga ggataagatg gaaggcagca gtataagctg tagatctgtt cttagatctc 2220
ttgaattagt gagacgacag ttcccttagg cagtttgtgc atggcatcct ttattctcta 2280
tacatggctt tagcggttct tgcctcattt tgggattcta aatggaagct ttcaacagag 2340
cattccattt tgtcctgtta aaaccttttg ttttcaccta aaccctttct gcttagttgt 2400
atctctgtga aaaacttgta tacacaagcg tccatgtctc acacaaatat tgatgtgatt 2460
attcttaagt gttaaatcat taacacttaa atgacttcat tgggaatatt gagcagaggg 2520
actgtgcttc tatgcactgg gcaaggcagt atttgcttag gaaactaatt tagtcatcag 2580
agatactttc ctaaaaagga aaaataaaaa acaaaatggt gccactttgg gttgaagcta 2640
ctttgttagg cttgaattca tttatatgtc ttttgattct taaaaaaaca aaaaacattc 2700
cattagaagc accagttttt ttgctcagac tttgtggatc agactctaca ctcaacacac 2760
tctaatctac ttaaaggtat acaaaatatg ctgatctttt ttaaattatg atttcctgaa 2820
tttttttctt aagtcgtctc aactgattta ctcacttagc ttcccttccc tcatcagcat 2880
agtataatag aatgtatgtt acatttttat gaatggcagg tgttcattat aatctgtatt 2940
gacttaaaaa gtttcttcct catgatgcta atagtttttt gtatacatgg gaggatagca 3000
catttgacag tttttgcatt tttatgtatg agcacagtat cctatgactg tgctacgtat 3060
atataggtaa taaactggaa ttctgttgat gaatatagct gctgtactgt atattaatat 3120
ttaatagatc aacaaatggt cattgaaaac 3150
<210> 26
<211> 2901
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7480597CB1
<400> 26
atggcggaag gcaaggaagg gcaagtccca tcttacatgg atggcagcag gcaaagagag 60
aatgaggaag atgcaaaagc ggaaacccct gatgtaacca tcagatctta tgagatttat 120
tcactaccat ggaacagaca gcaaggccta tgtgaccatt ctctaaaata tttaagctcg 180
agaatcacag agcggaagct gcaaggctcc tggctgcctg ccagccgagg gaatctggag 240
aaaccattcc tggggccgcg tggccccgtc gtgcccttgt tctgccctcg gaatggcctt 300
cactcagcac atcctgagaa cagccctctg aagcccaggg tcgtgaccgt agtgaagctg 360
53/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
ggtgggcagc gcccccgaaa gatcactctg ctcctcaaca ggcgatcagt gcagacgttc 420
gagcagctct tagctgacat ctcagaagcc ttgggctctc ccagatggaa gaatgaccgt 480
gtgaggaaac tgtttaacct caagggcagg gaaatcagga gcgtctctga tttcttcagg 540
gaaggggatg ctttcatagc tatgggcaaa gaaccactga cactgaagag cattcaggtg 600
gctgtagaag aactgtaccc caacaaagcc cgggccctga cactggccca gcacagccgt 660
gccccttctc caaggctgag gagcaggctg tttagcaagg ctctgaaagg agaccaccgc 720
tgtggggaga ccgagacccc caagagctgc agcgaagttg caggatgcaa ggcagccatg 780
aggcaccagg ggaagatccc cgaggagctt tcactagatg acagagcgag gacccagaag 840
aagtggggga gggggaaatg ggagccagaa cccagtagca agccccccag ggaagccact 900
ctggaagaga ggcacgcaag gggagagaag catcttgggg tggagattga aaagacctcg 960
ggtgaaatta tcagatgcga gaagtgcaag agagagaggg agctccagca gagcctggag 1020
cgtgagaggc tttctctggg gaccagtgag ctggatatgg ggaagggccc aatgtatgat 1080
gtggagaagc tggtgaggac cagaagctgc aggaggtctc ccgaggcaaa tcctgcaagt 1140
ggggaggaag ggtggaaggg tgacagccac aggagcagcc ccaggaatcc cactcaagag 1200
ctgaggagac ccagcaagag catggacaag aaagaggaca gaggcccaga ggatcaagaa 1260
agccatgctc agggagcagc caaggccaag aaggaccttg tggaagttct tcctgtcaca 1320
gaggaggggc tgagggaggt gaagaaggac accaggccca tgagcaggag caaacatggt 1380
ggctggctcc tgagagagca ccaggcgggc tttgagaagc tccgcaggac ccgaggagaa 1440
gagaaggagg cagagaagga gaaaaagcca tgtatgtctg gaggcagaag gatgactctc 1500
agagatgacc aacctgcaaa gctagaaaag gagcccaaga cgaggccaga agagaacaag 1560
ccagagcggc ccagcggtcg gaagccacgg cccatgggca tcattgccgc caatgtggaa 1620
aagcattatg agactggccg ggtcattggg gatgggaact ttgctgtcgt gaaggagtgc 1680
agacaccgcg agaccaggca ggcctatgcg atgaagatca ttgacaagtc cagactcaag 1740
ggcaaggagg acatggtgga cagtgagatc ttgatcatcc agagcctctc tcaccccaac 1800
atcgtgaaat tgcatgaagt ctacgaaaca gacatggaaa tctacctgat cctggagtac 1860
gtgcagggag gagacctttt tgacgccatc atagaaagtg tgaagttccc ggagcccgat 1920
gctgccctca tgatcatgga cttatgcaaa gccctcgtcc acatgcacga caagagcatt 1980
gtccaccggg acctcaagcc ggaaaacctt ttggttcagc gaaatgagga caaatctact 2040
accttgaaat tggctgattt tggacttgca aagcatgtgg tgagacctat atttactgtg 2100
tgtgggaccc caacttacgt agctcccgaa attctttctg agaaaggtta tggactggag 2160
gtggacatgt gggctgctgg cgtgatcctc tatatcctgc tgtgtggctt tcccccattc 2220
cgcagccctg agagggacca ggacgagctc tttaacatca tccagctggg ccactttgag 2280
ttcctccccc cttactggga caatatctct gatgctgcta aagatctggt gagccggttg 2340
ctggtggtag accccaaaaa gcgctacaca gctcatcagg ttcttcagca cccctggatc 2400
gaaacagctg gcaagaccaa tacagtgaaa cgacagaagc aggtgtcccc cagcagcgag 2460
ggtcacttcc ggagccagca caagagggtt gtggagcagg tatcatagtc accaccttgg 2520
gaatctgtcc agcccccagt tctgctcaag gacagagaaa aggatagaag tttgagagaa 2580
aaacaatgaa agaggcttct tcacataatt ggtgaatcag agggagagac actgagtata 2640
ttttaaagca tattaaaaaa attaagtcaa tgttaaatgt cacaacatat ttttagattt 2700
gtatatttaa agcctttaat acatttttgg ggggtaagca ttgtcatcag tgaggaattt 2760
tggtaataat gatgtgtttt gcttcccctt tgtaaccaag tttattctgt actacaggag 2820
tggtgcttac cagggtctaa actccccctg tgagattaat aaggtgcatt gtggtctttc 2880
tgtgttaata aaatgtggtc c 2901
<210> 27
<211> 1671
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3227248CB1
<400> 27
atgaagctta taaatggcaa aaagcaaaca ttcccatggt ttggcatgga catcggtgga 60
acgctggtta aattggtgta tttcgagccg aaggatatta cagccgaaga ggagcaagag 120
54/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
gaagtggaga acctgaagag catccggaag tatttgactt ctaatactgc ttatgggaaa 180
actgggatcc gagacgtcca cctggaactg aaaaacctga ccatgtgtgg acgcaaaggg 240
aacctgcact tcatccgctt tcccagctgt gctatgcaca ggttcattca gatgggcagc 300
gagaagaact tctctagcct tcacaccacc ctctgtgcca caggaggcgg ggctttcaaa 360
ttcgaagagg acttcagaat gattgctgac ctgcagctgc ataaactgga tgaactggac 420
tgtctgattc agggcctgct ttatgtcgac tctgttggct tcaacggcaa gccagaatgt 480
tactattttg aaaatcccac aaatcctgaa ttgtgtcaaa aaaagccgta ctgccttgat 540
aacccatacc ctatgttgct ggttaacatg ggctcaggtg tcagcattct agccgtgtac 600
tccaaggaca actataaaag agttacaggg accagtcttg gaggtggaac attcctaggc 660
ctatgttgct tgctgactgg ttgtgagacc tttgaagaag ctctggaaat ggcagctaaa 720
ggcgacagca ccaatgttga taaactggtg aaggacattt acggaggaga ctatgaacga 780
tttggccttc aaggatctgc tgtagcatca agctttggca acatgatgag taaagaaaag 840
cgagattcca tcagcaagga agacctcgcc cgggccacat tggtcaccat caccaacaac 900
attggctcca ttgctcggat gtgtgcgttg aatgagaaca tagacagagt tgtgtttgtt 960
ggaaattttc tcagaatcaa tatggtctcc atgaagctgc tggcatatgc catggatttt 1020
tggtccaaag gacaactgaa agctctgttt ttggaacatg agggttattt tggagccgtt 1080
ggggcactgt tggaactgtt caaaatgact gatgataagt agagacgagc agtggaggaa 1140
acagcctccc aaaaggacag agaactaaaa aattgctgct ggagaaggtg aaagtcgctt 1200
tgggacggaa gccaagccat tatggcagat gaacctgctg gatttgtaaa taatttaaaa 1260
tccttccaga tgatctttta ctcttaggtt ttgagctaat gattcaaaac gggggaatat 1320
aaaaggtttt ttttctgtat actgtatttt tttaaaaaaa tggtgcagcg tggccaaacc 1380
taccaattgt atgcattaac tttgaaaagt tgtttgatgt ttaagaagga cctgatatgt 1440
aagcgctggt catttttctt ctggggttta ctgatcagtg tggtgatttt aacttcattt 1500
agtaattact ctaggagatt ttaccttgac ttatattttt catgacgttt catgatttgc 1560
tgttggtttc aaatgaaact acaaatctgg catgttttac tgtgaacact tttgttattt 1620
gttttgtacc ctttttgtct tgtttttctg ttttagttgt cttctgaaaa a 1671
<210> 28
<211> 2577
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4207273CB1
<400> 28
atgccacaga tagcaaagaa gcaatcaact caccggactc agaaacctaa aaagcaatca 60
tttccttgca tctgtaaaaa tccaggaaca cagaagtcat gtgttcctct ctctgttcaa 120
ccgacagagc caagactaaa ttacctagat cttaagtata gtgatatgtt caaagaaatc 180
aattcaactg ctaatggacc tggaatctat gaaatgtttg ggacccctgt ttattgtcat 240
gtgcgagaga ctgaaaggga tgaaaacacg tattaccgtg agatatgttc ggctccatca 300
ggcagacgta tcaccaataa atgtcgatct tcacacagtg agaggaagag caatatcaga 360
acaagacttt ctcagaaaaa aacacatatg aaatgcccaa agacttcatt tggcattaaa 420
caagagcaca aagtcttaat ttctaaagaa aagagttcca aggctgtaca tagcaaccta 480
catgacattg aaaatggtga tggtatttca gaaccagact ggcagataaa gtcttcagga 540
aatgagtttc tatcttccaa agatgaaatt catcccatga acttggctca gacacctgag 600
cagtccatga aacagaatga attccctcct gtctcagatt tatccattgt tgaagaagtt 660
tctatggaag agtctactgg tgatagagac atttctaaca atcaaatact caccacaagc 720
ctcagagatc tgcaagaact tgaagagcta catcaccaga tcccatttat cccttcagaa 780
gacagctggg cagtgcccag tgagaagaat tctaacaagt atgtacagca agaaaagcag 840
aatacagcat ctcttagtaa agtaaatgcc agccgaattt taactaatga tctagagttt 900
gatagtgttt cagatcactc taaaacactt acaaatttct ctttccaagc aaaacaagaa 960
agtgcatctt cccagacata tcaatattgg gtacattatt tggatcatga tagtttagca 1020
aataagtcaa tcacatatca aatgtttgga aaaaccttaa gtggcacaaa ttcaatttcc 1080
caagaaatta tggactctgt aaataatgaa gaattgacag atgaactatt aggttgtcta 1140
55/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
gctgcagaat tattagctct tgatgagaaa gataacaact cttgccaaaa aatggcaaat 1200
gaaacagatc ctgaaaacct aaatcttgtc ctcagatgga gaggaagtac cccaaaagaa 1260
atgggcagag agacaacaaa agtcaaaata cagaggcata gtagtgggct caggatatat 1320
gacagggagg agaaatttct catctcaaat gaaaagaaga tattttctga aaatagttta 1380
aagtctgaag aacctatcct atggaccaag ggtgagattc ttggaaaggg agcctacggc 1440
acagtatact gtggtctcac tagtcaagga cagctaatag ctgtaaaaca ggtggctttg 1500
gatacctcta ataaattagc tgctgaaaag gaataccgga aactacagga agaagtagat 1560
ttgctcaaag cactgaaaca tgtcaacatt gtggcctatt tggggacatg cttgcaagag 1620
aacactgtga gcattttcat ggagtttgtt cctggtggct caatctctag tattataaac 1680
cgttttgggc cattgcctga gatggtgttc tgtaaatata cgaaacaaat acttcaaggt 1740
gttgcttatc tccatgagaa ctgtgtggta catcgcgata tcaaaggaaa taatgttatg 1800
ctcatgccaa ctggaataat aaagctgatt gactttggct gtgccaggcg tttggcctgg 1860
gcaggtttaa atggcaccca cagtgacatg cttaagtcca tgcatgggac tccatattgg 1920
atggccccag aagtcatcaa tgagtctggc tatggacgga aatcagatat ctggagcatt 1980
ggttgtactg tgtttgagat ggctacaggg aagcctccac tggcttccat ggacaggatg 2040
gccgccatgt tttacatcgg agcacaccga gggctgatgc ctcctttacc agaccacttc 2100
tcagaaaatg cagcagactt tgtgcgcatg tgcctgacca gggaccagca tgagcgacct 2160
tctgctctcc agctcctgaa gcactccttc ttggagagaa gtcactgaat atacatcaag 2220
actttcttcc cagttccact gcagatgctc ccttgcttaa ttgtggggaa tgatggctaa 2280
gggatctttg tttccccact gaaaattcag tctaacccag tttaagcaga tcctatggag 2340
tcattaactg aaagttgcag ttacatatta gcctcctcaa gtgtcagaca ttattactca 2400
tagtatcaga aaacatgttc ttaataacaa caaaaaacta tttcagtgtt tacagttttg 2460
attgtccagg aactacattc tctagtgttt tatatgacat ttctttttat ttttggcctg 2520
tcctgtcaat tttaatgttg ttagtttaaa ataaattgta aaaacaaaaa aaaaaaa 2577
<210> 29
<211> 2110
<212> DNA
<213> Homo sapiens
<220>
<221> mist feature
<223> Incyte ID No: 7483334CB1
<400> 29
ctagggtcgc cggggaagcg gtttgggaga gcccatggtg actgcgtgag tggagcccag 60
ctgtgtggat gccccagcat ggatgactac atggtcctga gaatgattgg ggagggctcc 120
ttcggcagag ctcttttggt tcagcttgaa agcagtaatc agatgtttgc catgaaagaa 180
ataaggcttc ccaagtcttt ctctaataca cagaattcta ggaaggaggc tgttctttta 240
gccaaaatga aacaccctaa tattgttgcc ttcaaagaat catttgaagc tgaaggacac 300
ttgtatattg tgatggaata ctgtgatgga ggggatctaa tgcaaaagat taaacagcag 360
aaaggaaagt tatttcctga agacatgata cttaattggt ttacccaaat gtgccttgga 420
gtaaatcaca ttcacaagaa acgtgtgcta cacagagata tcaagtccaa gaatatcttc 480
ctcactcaga atggaaaagt gaaattggga gactttggat ctgcccgtct tctctccaat 540
ccgatggcat ttgcttgtac ctatgtggga actccttatt atgtgcctcc agaaatttgg 600
gaaaacctgc cttataacaa taaaagtgac atctggtcct tgggttgcat cctgtatgaa 660
ctctgtaccc ttaagcatcc atttcaggca aatagttgga aaaatcttat cctcaaagta 720
tgtcaagggt gcatcagtcc actgccgtct cattactcct atgaacttca gttcctagtc 780
aagcagatgt ttaaaaggaa tccctcacat cgcccctcgg ctacaacgct tctctctcga 840
ggcatcgtag ctcggcttgt ccagaagtgc ttaccccccg agatcatcat ggaatatggt 900
gaggaagtat tagaagaaat aaaaaattcg aagcataaca caccaagaaa aaaaacaaac 960
cccagcagaa tcaggatagc tttgggaaat gaagcaagca cagtgcaaga ggaagaacaa 1020
gatagaaagg gtagccatac tgatttggaa agcattaatg aaaatttagt tgaaagtgca 1080
ttgagaagag taaacagaga agaaaaaggt aataagtcag tccatctgag gaaagccagt 1140
tcaccaaatc ttcatagacg acagtgggag aaaaatgtac ccaatacagc tcttacagct 1200
ttggaaaatg catccatact cacctccagt ttaacagcag aggacgatag aggtggttct 1260
56/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
gtaataaagt acagcaaaaa tactactcgt aagcagtggc tcaaagagac ccctgacact 1320
ttgttgaaca tccttaagaa tgctgatctc agcttggctt ttcaaacata cacaatatat 1380
agaccaggtt cagaagggtt cttgaaaggc cccctgtctg aagaaacaga agcatcggac 1440
agtgttgatg gaggtcacga ttctgtcatt ttggatccag agcgacttga gcctgggcta 1500
gatgaggagg acacggactt tgaggaggaa gatgacaacc ccgactgggt gtcagagctg 1560
aagaagcgag ctggatggca aggcctgtgc gacagataat gcctgaggaa atgttcctga 1620
gtcacgctga ggagagcctt cactcaggag ttcatgctga gatgatcatg agttcatgcg 1680
acgtatattt tcctttggaa acagaatgaa gcagaggaaa ctcttaatac ttaaaatcgt 1740
tcttgattag tatcgtgagt ttgaaaagtc tagaactcct gtaagttttt gaactcaagg 1800
gagaaggtat agtggaatga gtgtgagcat cgggctttgc agtcccatag aacagaaatg 1860
ggatgctagc gtgccactac ctacttgtgt gattgtggga aattacttaa cctcttcaag 1920
ccccaatttc ctcaaccata aaatgaagat aataatgcct acctcagagg gatgctgacc 1980
acagaccttt atagcagccc gtatgatatt attcacatta tgatatgtgt ttattattat 2040
gtgactcttt ttacatttcc taaaggtttg agaattaaat atatttaatt atgatttaaa 2100
aaaaaaaaaa 2110
<210> 30
<211> 7093
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7483337CB1
<400> 30
cgaggggacg cctcgcgacg gttcctggga gagctggcgg cggccttgct ctgcgcgctc 60
ttcgcgccgc cctccccgcc cgcccgcctc aggattgagg aagtgcgtct gggcccggcc 120
ccggcgcggg gggcagacgg cggtgggacg gccaggcccc ggccccgcca gtgtgtccgc 180
ccggccccgc gtcccggagg agtcagctgt gtgtccagaa cgtgccatgg agacgcttaa 240
cggtgccggg gacacgggcg gcaagccgtc cacgcggggc ggtgaccctg cagcgcggtc 300
ccgcaggacg gaaggcatcc gcgccgcgta caggcgggga gaccgcggcg gcgcccggga 360
cctgctggag gaggcctgcg accagtgcgc gtcccagctg gaaaagggcc agcttctgag 420
catcccggca gcctatgggg atctggagat ggtccgctac ctactcagca agagactggt 480
ggagctgccc accgagccca cggatgacaa cccagccgtg gtggcagcgt attttggaca 540
cacggcagtt gtgcaaaata cgctgcccac cgagcccacg gatgacaacc cagccgtggt 600
ggcagcgtat tttggacaca cggcagttgt gcaggaattg cttgagtcct taccaggtcc 660
ctgcagtccc cagcggcttc tgaactggat gctggccttg gcttgccagc gagggcacct 720
gggggttgtg aagctcctgg tcctgacgca cggggctgac ccggagagct acgctgtcag 780
gaagaatgag ttccctgtca-tcgtgcgctt gcccctgtat gcggccatca agtcagggaa 840
tgaagacatt gcaatattcc tgcttcggca tggggcctat ttctgttcct acatcttgct 900
ggatagtcct gaccccagca aacatctgct gagaaagtac ttcattgaag ccagtccctt 960
gcccagcagt tatccgggaa aaacagctct ccgtgtgaaa tggtcccatc tcagactgcc 1020
ctgggtagac ctagactggc tcatagacat ctcctgccag atcacggagc tcgacctttc 1080
tgccaactgc ctggcgaccc tcccctcggt tatcccctgg ggcctcatca atctccggaa 1140
gctgaacctc tccgacaacc acctggggga gctgcctggc gtgcagtcat cggacgaaat 1200
catctgttcc aggctacttg aaattgacat ttccagcaac aagttgtccc acctccctcc 1260
tggattcttg cacctctcaa aacttcaaaa actgacagct tcaaaaaatt gtttagaaaa 1320
attgttcgaa gaagaaaatg ccactaactg gataggttta cggaagctac aggaacttga 1380
tatatctgac aataaattga cagaactccc tgccctgttc cttcactctt tcaagtccct 1440
caattctctg aatgtctcca gaaacaacct gaaggtgttt ccagatccct gggcctgccc 1500
tttgaaatgt tgtaaagctt ccagaaatgc cctggaatgt ctgccagaca aaatggctgt 1560
cttttggaaa aatcacctga aggatgtgga tttctcagaa aacgcactca aagaagttcc 1620
cctgggactt ttccagcttg atgccctcat gttcttgagg ttacagggga accagctggc 1680
ggcacttcca cctcaagaga agtggacctg caggcagctc aaaaccctgg atctctccag 1740
aaaccaactt ggcaaaaatg aagatggact gaaaacgaag cgtattgcct ttttcaccac 1800
57/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
cagaggtcgc cagcgctccg ggactgaggc agagacaact atggagttca gtgcatctct 1860
ggtaaccatt gtgttcctgt ctaacaactg taacctctgt gcatacacat gtgcagcaag 1920
tgtgctggaa tttccggcct tcctaagtga gtctttggaa gtcctttgcc tgaacgacaa 1980
ccacctcgac acagtccctc cctcggtttg cctactgaag agcttatcag agctctactt 2040
gggaaacaac cctggcctcc gggagctccc tcctgagctg gggcagctgg gcaacctctg 2100
gcagctggac actgaagacc tgaccatcag caatgtgcct gcagaaatcc aaaaagaagg 2160
ccccaaagca atgctgtctt acctgcgtgc tcagctgcgg aaagcggaaa agtgcaagct 2220
gatgaagatg atcatcgtgg gtcccccgcg ccagggcaag tccaccctcc tggagatctt 2280
acagacgggg agggcccccc aggtggtgca tggagaggcc accatcagga ccaccaagtg 2340
ggagctccag aggccggctg gctcgagagc caaggtcaag gatggtctgc gtgcagagtc 2400
cctgtgggtt gagtccgtgg agttcaacgt ctgggacatc gggggaccgg ccagcatggc 2460
cactgtcaac cagtgcttct tcacggacaa ggccctgtac gtggtggtct ggaacctggc 2520
gctgggggag gaggccgtgg ccaacctcca gttctggctg ctcaacatcg aggccaaggc 2580
cccaaacgcc gtggtgctgg tggtcgggac gcacctggat ttaattgaag ccaagttccg 2640
tgtggaaagg attgcaacgc tgcgtgccta tgtgctggca ctctgccgct ccccctccgg 2700
ctccagggcc acaggcttcc cagacatcac cttcaaacac ttacatgaga tttcctgcaa 2760
gagcctggaa ggtcaggaag ggctgcgaca gctgattttc cacgtcacgt gcagcatgaa 2820
ggacgtcggc agcaccatcg gctgccagcg actggcaggg cggctgatcc ccaggagcta 2880
cctgagcctg caggaggccg tgctggcaga gcagcagcgc cgcagccggg acgacgacgt 2940
gcagtacctg acggacaggc agctggagca gctggtggag cagacgcccg acaacgacat 3000
caaggactac gaggacctgc agtcagccat cagcttcctc atagaaaccg gcaccctgct 3060
ccatttcccg gacaccagcc acggcctgag gaacctctac ttcctcgacc ctatttggct 3120
ctccgaatgt ctgcagagga tctttaatat taagggctct cggtcagtgg ccaagaatgg 3180
ggtgatcaga gcagaagacc tcaggatgct gctggtgggg actggcttca cgcagcagac 3240
ggaagagcag tacttccagt tcctggccaa gtttgagatc gccctgcccg tcgccaatga 3300
cagctacctc ctgccccatc tccttccatc taaacctggc ctggacaccc acggtatgcg 3360
gcaccccaca gccaacacca ttcagagggt atttaagatg agcttcgttc ccgttggctt 3420
ctggcaaagg tttatagcac ggatgctgat cagcctggcg gagatggacc tgcagctttt 3480
tgaaaacaag aagaatacta aaagcaggaa caggaaagtc accatttaca gttttacagg 3540
aaaccagaga aatcgctgta gcacattcag agtgaaaaga aatcagacca tctattggca 3600
ggaagggctc ctggtcactt ttgatggggg ctacctcagt gtggaatctt ccgacgtgaa 3660
ctggaaaaag aagaaaagcg gaggaatgaa aattgtttgc caatcagaag tgagggactt 3720
ctcagccatg gctttcatca cggaccacgt caattccttg attgatcagt ggtttcccgc 3780
cctgacagcc acagagagcg acgggacgcc actcatggag cagtacgtgc cctgcccggt 3840
ctgcgagaca gcctgggccc agcacacgga ccccagtgag aaatcagagg atgtgcagta 3900
cttcgacatg gaagactgtg tcctgacggc catcgagcgg gacttcatct cctgccccag 3960
acacccggac ctccccgtgc cgctgcagga gctggtccct gaactgttca tgaccgactt 4020
cccggccagg ctcttcctgg agaacagcaa gctggagcac agcgaggacg agggcagcgt 4080
cctgggccag ggcggcagtg gcaccgtcat ctaccgggcc cggtaccagg gccagcctgt 4140
ggccgtcaag cgcttccaca tcaaaaaatt caagaacttt gctaacgtac cggcagacac 4200
catgctgagg cacctgcggg ccaccgatgc catgaagaac ttctccgagt tccggcagga 4260
ggccagcatg ctgcacgcgc tgcagcaccc ctgcatcgtg gcgctcatcg gcatcagcat 4320
ccacccgctc tgcttcgccc tggagctcgc gccgctcagc agcctcaaca ccgtgctgtc 4380
cgagaacgcc agagattctt cctttatacc cctgggacac atgctcaccc aaaaaatagc 4440
ctaccagatc gcctcgggcc tggcctacct gcacaagaaa aacatcatct tctgtgacct 4500
gaagtcggac aacattctgg tgtggtccct tgacgtcaag gagcacatca acatcaagct 4560
atctgactac gggatttcga ggcagtcatt ccatgagggc gccctaggcg tggagggcac 4620
tcctggctac caggccccag agatcaggcc tcgcattgta tatgatgaga aggtagatat 4680
gttctcctat ggaatggtgc tctacgagtt gctgtcagga cagcgccctg cactgggcca 4740
ccaccagctc cagattgcca agaagctgtc caagggcatc cgcccggttc tggggcagcc 4800
ggaggaagtg cagttccggc gactgcaggc gctcatgatg gagtgctggg acactaagcc 4860
agagaagcga ccgctggccc tgtcggtggt gagccagatg aaggacccga cttttgccac 4920
cttcatgtat gaactgtgct gtgggaagca gacagccttc ttctcatccc agggccagga 4980
gtacaccgtg gtgttttggg atggaaaaga ggagtccagg aactacacgg tggtgaacac 5040
agagaagggc ctcatggagg tgcagaggat gtgctgccct gggatgaagg tgagctgcca 5100
gctccaggtc cagagatccc tgtggacagc caccgagaat tcctacctgg tcttagcggg 5160
58/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
cctcgccgat gggcttgtgg ctgtgtttcc cgtggtgcgg ggcaccccaa aggacagctg 5220
ctcctacctg tgctcacaca cagccaacag gtccaagttc agcatcgcgg atgaagacgc 5280
acggcagaac ccctacccag tgaaggccat ggaggtggtc aacagcggct ctgaggtctg 5340
gtacagcaat gggccgggcc tccttgtcat cgactgtgcc tccctggaga tctgcaggcg 5400
gctggagccc tacatggccc cctccatggt tacgtcagtc gtgtgcagct ctgagggcag 5460
aggggaggag gtcgtctggt gcctggatga caaggccaac tccttggtga tgtaccactc 5520
caccacctac cagctgtgtg cccggtactt ctgcggggtc cccagccccc tcagggacat 5580
gtttcccgtg cggcccttgg acacggaacc cccggcagcc agccacacgg ccaacccaaa 5640
ggtgcctgag ggggactcca tcgcggacgt gagcatcatg tacagtgagg agctgggcac 5700
gcagatcctg atccaccagg aatcactcac tgactactgc tccatgtcct cctactcctc 5760
atccccaccc cgccaggctg ccaggtcccc ctcaagcctc cccagctccc cagcaagttc 5820
ttccagtgtg cctttctcca ccgactgcga ggactcagac atgctacata cgcccggtgc 5880
tgcctccgac aggtctgagc atgacctgac ccccatggac ggggagacct tcagccagca 5940
cctgcaggcc gtgaagatcc tcgccgtcag agacctcatt tgggtcccca ggcgcggtgg 6000
agatgttatc gtcattggcc tggagaagga ttctggcgcc cagcggggcc gagtcattgc 6060
cgtcttaaaa gcccgagagc tgactccgca tggggtgctg gtggatgctg ccgtggtggc 6120
aaaggacact gttgtgtgca cctttgaaaa tgaaaacaca gagtggtgcc tggccgtctg 6180
gaggggctgg ggcgccaggg agttcgacat tttctaccag tcctacgagg agctgggccg 6240
gctggaggct tgcactcgca agagaaggta attcctgtgg aatgactgtc acacatcaga 6300
gctggctggc ccggggctgc agcctgactc ctctgccatc ggcctctagt tctccaagga 6360
cctagaagac agatggagtt ctcccctgaa ctccttgctg ctaagaagtg ctgagaagtt 6420
actcgcctgg cggtggctcc agggttctct ggttctctgg agcagagttc tctgaatacc 6480
ccatccccca actgctgatt ttacagcccc agggaagaca gtggtatcag gctgggagcg 6540
gcctcctctg gcctccccca tcagtttgca ggagcagggg tgcaggatcc tgttctgagc 6600
tgggtcaaac aaagcagggc cgggccttcc tgccatcccc aggtctcaga tggaattaca 6660
ctagaggccc tccgctggga agcacttgag gtagggcagg aggggggctg tgacccctgc 6720
cctttccccg ccagagacct caggctctca gcacattcca caggctcctg agtccccgag 6780
gcctgggcca gcttgggcaa gccaagatca gatgtctctg tgttcgggaa ggtctccgtg 6840
tgggaaagcc cttgggggat cccgggtgag gagtgttgcc ccatccagag aatgaatgag 6900
ttcctttaag tgccaccgcc agcaagccca gaggcacaca ttctgagtgc acccgcttag 6960
cctttacatt cctctccacc gacaaaagga aggggaaact caatcagcag gacttcagaa 7020
agggccttgt gtttatagct ttgtcaagta aatttggacg cagctggaaa cacaggcctg 7080
tttgttgcac ata 7093
<210> 31
<211> 1800
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6035509CB1
<400> 31
gctgcagagt gctttacttt caacaagatg gagtcttgct ctgtttccca gcctgtagtg 60
cagtgacaca gtcttggctc actgtaacct ctgcctcctg ggttcaagtg attctcctgc 120
ctcagcctcc tgagtagctg ggattacagg aaacatctgt atggattatt tcactataat 180
cctatgatgc ttggacttga atcacttcca gatcccacag acacctggga aattata,gag 240
accattggta aaggcaccta tggcaaagtc tacaaggtaa ctaacaagag agatgggagc 300
ctggctgcag tgaaaattct ggatccagtc agtgatatgg atgaagaaat tgaggcagaa 360
tacaacattt tgcagttcct tcctaatcat cccaatgttg taaagtttta tgggatgttt 420
tacaaagcgg atcactgtgt agggggacag ctgtggctgg tcctggagct gtgtaatggg 480
ggctcagtca ctgagcttgt caaaggtcta ctcagatgtg gccagcggtt ggatgaagca 540
atgatctcat acatcttgta cggggccctc ttgggccttc agcatttgca caacaaccga 600
atcatccacc gtgatgtgaa ggggaataac attcttctga caacagaagg aggagttaag 660
ctcgttgact ttggtgtttc agctcaactc accagtacac gtctgcggag aaacacatct 720
59/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
gttggcaccc cgttctggat ggcccctgag gtcattgcct gtgagcagca gtatgactct 780
tcctatgacg ctcgctgtga cgtctggtcc ttggggatca cagctattga actgggggat 840
ggagaccctc ccctctttga catgcatcct gtgaaaacac tctttaagat tccaagaaat 900
cctccaccta ctttacttca tccagaaaaa tggtgtgaag aattcaacca ctttatttca 960
cagtgtctta ttaaggattt tgaaaggcga ccttccgtca cacatctcct tgaccaccca 1020
tttattaaag gagtacatgg aaaagttctg tttctgcaaa aacagctggc caaggttctc 1080
caagaccaga agcatcaaaa tcctgttgct aaaaccaggc atgagaggat gcataccaga 1140
agaccttatc atgtggaaga tgctgaaaaa tactgccttg aggatgattt ggtcaaccta 1200
gaggttctgg atgaggtact aaatatttag tagacaattc tcattgaaga catttgtttc 1260
atgtgaatgg tctgaacttt ctgttgtaga ccatgtcctc ctaaggtcat ttgaaaattt 1320
aattgtttgt gtagctatgg gatgaagttc agggagcatt cagttgctgt gactatgatc 1380
ctgtgctgtg tttatttaga tagcccctag aatgatgaag agaaaaggat ttggattttt 1440
gcaataaagc tctttatatt gtagccttaa tgatggatta tatcagctga aaatattttg 1500
tttgataaaa tttgataaaa tatttcaatt aacccttaag aagttgtttg ttcttcataa 1560
gaaagagctt catttaggga aatagtgaag ttaatatagc ttgaattcta aatttgaagt 1620
ctgtgataat ccccatttaa aatatgcatg tttaatagag ctgttaattg cactggacct 1680
gtttatgctg agtctaactc tggggattgt taccttcaat gtctaaatca ctaaagtgta 1740
atacaaagtg gttaattctg tatttatgcc acctaggttt taagtgcagt gctttgagaa 1800
<210> 32
<211> 6347 .
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7373485CB1
<400> 32
ggaagcgaga agccgcatca accatgtaag cagcttcgct tcctgccgca accgtccgcg 60
gcctgaggag cccaccgccg ctctcggggg ccgacttccg ggggctgagc cgttgaagcg 120
gaggctgggg cggggggcag ccggcgcggc cggggcagga ggcgcagact catgaaatgg 180
ccacagatga taagacgtcc ccaacactgg actctgctaa tgatttgcct cgatctccta 240
ctagtccttc tcatctcaca cactttaaac ctttgactcc tgatcaagat gagccccctt 300
ttaaatcagc ttatagttct tttgtaaatc tctttcgttt taacaaagag agagcagaag 360
gaggccaggg agaacagcag cctttgagtg gaagttggac cagccctcag ctcccttcga 420
ggacacagtc tgttaggtca cccacacctt ataaaaagca gcttaatgag gaactccagc 480
ggcgctcttc agcattagac acaagaagga aagcagaacc tacctttgga ggtcatgacc 540
ctcgtacagc tgttcagctt cgaagcctca gcacagtatt aaaacgcctc aaggaaatca 600
tggaggggaa aagccaggat agtgacctga aacaatactg gatgccagat agccaatgta 660
aagagtgcta tgactgtagt gagaaattta caacctttag gcgcagacac cattgccgac 720
tatgtgggca gattttctgc agtcgttgct gtaatcaaga aatccctgga aaatttatgg 780
gctatacagg agacctccga gcttgcacat attgtagaaa aatagcctta agttatgctc 840
attccacaga cagtaattct attggggaag acttgaatgc tctttcagat tctgcttgct 900
ctgtgtctgt gcttgatcca agtgaacccc gaacacctgt tgggagtagg aaagccagcc 960
gtaacatatt tttagaggat gatttggcct ggcaaagttt gattcatcca gattcctcaa 1020
atactcctct ttcaacaaga cttgtatctg tgcaagagga tgctgggaaa tctcctgctc'1080
gaaatagatc agccagcatt actaacctgt cactggatag atctggttct cctatggtac 1140
cttcatatga gacatctgtc agtccccagg ctaaccgaac atatgttagg acagagacca 1200
ctgaggatga acgcaaaatt cttctggaca gtgtgcagtt aaaagacctg tggaaaaaaa 1260
tctgccatca cagcagtgga atggagtttc aggatcaccg ctactggttg agaacgcatc 1320
ccaactgcat tgtaggaaag gaattagtca actggctaat ccgaaatggg catattgcca 1380
caagggcaca agctatagca attggacaag caatggttga tggacgttgg ctggattgtg 1440
ttagtcatca cgaccagctt ttcagagatg agtatgcgct gtatagacca ctgcagagta 1500
cagaattttc tgagacgcct tctcccgaca gtgactcagt gaactccgtg gaaggacact 1560
ctgagccatc ctggtttaaa gacataaagt ttgatgacag tgacacagaa cagatagctg 1620
60/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
aagaaggtga cgataatttg gctaagtatt tgatttctga cactggagga caacagctct 1680
caataagtga cgctttcatc aaagaatcct tatttaatcg ccgagtagag gaaaaatcca 1740
aagagctgcc tttcacacct ttgggctggc atcataacaa cctggagctc ctgagggagg 1800
agaatgggga gaaacaagcc atggagaggt tgctttcagc taatcataac cacatgatgg 1860
cactactcca gcagttgctc catagtgact cactgtcatc atcttggagg gacatcatcg 1920
tgtcattggt ctgccaggtt gttcagacag tccgacctga tgtcaagaac caggatgatg 1980
acatggatat ccgtcagttt gtccacatca aaaaaatccc aggtggaaag aagtttgatt 2040
ctgtggttgt caatggcttt gtttgtacca agaacattgc acataaaaag atgaattctt 2100
gtattaaaaa ccctaaaatt cttctgttga agtgttccat tgagtatctc tacagagaag 2160
aaactaagtt tacttgcatt gatcctattg tgcttcagga aagggaattc ttgaagaatt 2220
atgtccagcg aatagttgat gttcgaccca ccttggttct tgttgagaaa acagtgtctc 2280
ggattgccca ggacatgtta ttggaacatg gcattacttt ggtcattaat gtaaagtcac 2340
aagttttgga acgaatcagt cgaatgaccc aaggtgattt agtgatgtca atggaccagc 2400
tgcttacgaa accacgcctg ggcacttgtc acaaatttta tatgcagata tttcagttgc 2460
ctaatgaaca aaccaagaca ctgatgtttt ttgaaggttg tccacagcac ctaggctgta 2520
caatcaagct aagaggaggc tctgattatg agctggctcg agttaaggag atcctaatat 2580
ttatgatctg tgttgcttat cattctcaac tagaaatatc ctttctcatg gatgaatttg 2640
ctatgcctcc cacattaatg caaaaccctt cattccattc cctgattgag ggacgagggc 2700
atgagggggc tgtccaagag cagtacggtg gaggttccat cccctgggat cctgacatcc 2760
ctcctgagtc tctgccctgt gatgatagca gtttgctgga atcgaggatt gtgtttgaga 2820
agggtgagca ggaaaataaa aatcttccgc aggctgttgc ctctgtgaag catcaagaac 2880
atagcacaac agcttgcccg gcgggtctcc cttgtgcttt ctttgcacct gtaccggaat 2940
cattgttgcc actccctgtg gatgaccaac aagatgcttt aggcagcgag ctgccagaga 3000
gtttgcagca aacagttgtg ctgcaggatc ccaaaagcca gataagagcc tttagagacc 3060
ctctacagga tgacactgga ttatatgtta ctgaggaagt cacctcctct gaagataaac 3120
gaaagactta ttctttggcc tttaagcagg aattaaaaga tgtgatcctc tgtatctccc 3180
cagtaatcac attccgagaa ccctttcttt taactgaaaa ggggatgaga tgctctaccc 3240
gagattattt tgcagagcag gtttactggt ctcctctcct caataaagaa ttcaaagaaa 3300
tggagaacag gaggaagaaa cagctgctca gggatctctc tggacttcag ggcatgaatg 3360
gaagtattca ggccaagtct attcaagtct taccctcaca tgagctagtg agcactagaa 3420
ttgctgagca tctgggcgat agccagagct tgggtagaat gctggccgat tatcgagcca 3480
gaggaggaag aattcagccc aaaaattcag acccttttgc tcattcaaag gatgcatcaa 3540
gtacttcaag tggcaaatca ggaagcaaaa acgagggtga tgaagagaga gggcttattc 3600
tgagtgatgc tgtgtggtca acaaaggtgg actgtctgaa tcccattaat caccagagac 3660
tttgtgtgct cttcagcagc tcttctgccc agtccagcaa tgctcctagt gcctgtgtca 3720
gtccttggat tgtaacaatg gaattttatg gaaagaatga tcttacatta ggaatatttt 3780
tagagagata ctgtttcagg ccttcttatc agtgtccaag catgttctgt gataccccca 3840
tggtacatca tattcggcgc tttgttcatg gccaaggctg tgtgcagata atcctgaagg 3900
agttggattc tccagtacct ggatatcagc atacaattct tacatattcc tggtgtagaa 3960
tctgcaaaca ggtaacacca gttgttgctc tttccaatga gtcctggtct atgtcatttg 4020
caaaatacct tgaacttagg ttttatgggc accagtatac tcgcagagcc aacgctgagc 4080
cctgtggtca ctccatccat catgattatc accagtattt ctcctataac cagatggtgg 4140
cgtctttcag ttattctccc attcggcttc ttgaagtatg tgttccactc cccaaaatat 4200
tcattaagcg tcaggcccca ttaaaagtgt cccttcttca ggatctgaag gacttctttc 4260
aaaaagtttc acaggtatat gttgccattg atgaaagact tgcatctttg aaaactgata 4320
catttagtaa aacaagagag gaaaaaatgg aagatatttt tgcacagaaa gagatggaag 4380
aaggtgagtt caagaactgg attgagaaga tgcaagcaag gctcatgtct tcctctgtag 4440
atacccctca gcaactgcag tcggtctttg agtcactcat tgccaagaaa caaagtctct 4500
gtgaagtgct gcaagcttgg aataacaggt tgcaggacct tttccaacag gaaaagggta 4560
gaaagagacc ttcagttcct ccaagtcctg gaagactgag acaaggggaa gaaagcaaga 4620
taagtgcgat ggatgcatct ccacggaata tttctccagg acttcagaat ggagaaaaag 4680
aggatcgctt cttaacaact ttgtccagcc agagctccac cagttctact catctccaat 4740
tgcctacgcc acctgaagtc atgtctgaac agtcagtggg agggccccct gagctagata 4800
cagccagcag ttccgaagat gtgtttgatg ggcatttgct gggatccaca gacagccaag 4860
tgaaggaaaa gtcaaccatg aaagccatct ttgcaaattt gcttccagga aatagctata 4920
atcctattcc atttcctttt gatccagata aacactactt aatgtatgaa catgaacgag 4980
61/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
tgcccattgc agtctgcgag aaggaaccca gctccatcat tgcttttgct ctcagttgta 5040,
aagaataccg aaatgcctta gaggaattgt ctaaagcgac tcagtggaac agtgccgaag 5100
aagggcttcc aacaaatagt acttcagata gcagaccaaa gagtagcagc cctatcagat 5160
tacctgaaat gagtggagga cagacaaatc gtacaacaga aacagaacca caaccaacca 5220
aaaaggcttc tggaatgctg tccttcttca gagggacagc agggaaaagc cccgatctct 5280
cttcccagaa gagagagacc ttacgtggag cagatagtgc ttactaccag gttgggcaga 5340
caggcaagga ggggaccgag aatcaaggcg ttgagcctca agatgaagta gatggaggag 5400
atacgcaaaa gaagcaactc ataaatcctc atgtggaact tcaattttca gatgctaatg 5460
ccaagtttta ctgtcggctc tactatgcgg gagagtttca taagatgcgt gaagtgattc 5520
tggacagcag tgaggaagat ttcattcgtt ccctctccca ctcatcaccc tggcaggccc 5580
ggggaggcaa atcaggagct gccttctatg caactgagga tgatagattt attttgaagc 5640
aaatgcctcg tctggaagtc cagtccttcc tcgactttgc accacattac ttcaattata 5700
ttacaaatgc tgttcaacaa aagaggccca cggcgttggc caaaattctt ggagtttaca 5760
gaattggtta taagaactct cagaacaaca ctgagaagaa gttagatctc cttgtcatgg 5820
aaaatctttt ctacgggaga aagatggcac aggtttttga tttgaagggc tctcttagga 5880
atcggaatgt aaaaactgac actggaaaag agagttgtga tgtggtcctg ctagatgaaa 5940
atctcctaaa gatggttcga gacaaccctc tatatattcg ttctcattcc aaagctgtgc 6000
tgagaacctc gatccatagt gactcccatt tcctttctag ccacctcatt atagattatt 6060
ctttgctggt tgggcgagat gatactagca atgagctagt agttggaatt atagattata 6120
ttcgaacatt tacatgggac aaaaagcttg agatggttgt gaaatcaaca ggaattttag 6180
gtggacaagg taaaatgcca acagtggtgt ctccggagtt gtacaggact aggttttgtg 6240
aggcaatgga caagtatttc ctaatggtac cagaccactg gacaggcttg ggtctgaatt 6300
gctgaaatca agacatattt gaaatggact gtgaggaaaa ggggaac 6347
<210> 33
<211> 1876
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5734965CB1
<400> 33
tggggttcgg cgcggctacg tgcagaatcc gtctagctaa aatgtaattt cagattggac 60
aagtactgtg gaggaactgc aatgtctggt ggagaacaga aaccagagag gtactatgtg 120
ggtgtggacg ttggaacagg cagtgtccgt gcagctctgg tggaccagag tggggtcctg 180
ttggcttttg cagaccagcc aattaagaat tgggagcccc agttcaacca ccatgagcag 240
tcctccgagg acatctgggc tgcgtgctgt gttgtcacaa agaaagttgt acaagggatt 300
gatttaaacc aaattcgagg acttgggttt gatgccacgt gttctctggt tgttttggat 36U
aagcagtttc acccattacc agtcaaccag gaaggggatt cccatcgaaa cgtcatcatg 420
tggctggacc atcgagcagt cagtcaagtt aacaggatca atgagaccaa gcacagtgtc 480
ctccagtacg tcgggggggt gatgtctgtg gaaatgcagg ccccgaaact tctgtggctg 540
aaagagaact tgagagagat ttgctgggat aaggcgggac atttctttga tctcccggac 600
ttcttatcgt ggaaggcaac aggtgtcaca gcacggtctc tctgctccct ggtgtgtaag 660
tggacatatt cagcagagaa aggctgggac gacagtttct ggaaaatgat tggtttggaa 720
gactttgttg cagataatta cagcaaaata ggaaaccaag tgctacctcc tggagcttct 780
cttggaaatg ggctcacacc agaggcagca agagaccttg gccttctccc tgggattgcg 840
gtcgcagctt cactcattga tgcccatgca ggaggactag gagtgattgg ggcagatgtg 900
agagggcacg gcctcatctg tgaggggcag ccagtgacgt cacggctggc tgtcatctgt 960
ggaacgtctt cttgtcacat ggggatcagc aaagacccga tttttgtacc aggcgtctgg 1020
gggccttatt tctcagccat ggtacctggg ttctggctga atgaaggtgg tcagagcgtt 1080
actggaaaat tgatagacca catggtacaa ggccatgctg cttttccaga actacaagta 1140
aaggccacag ccagatgcca gagtatatat gcatatttga acagtcacct ggatctgatt 1200
aagaaggctc agcctgtggg tttccttact gttgatttac atgtttggcc agatttccat 1260
ggcaaccggt ctcccttagc agatctgaca ctaaagggca tggtcaccgg attgaaactg 1320
62/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
tctcaggacc ttgatgatct tgccattctc tacctggcca cagttcaagc cattgctttg 1380
gggactcgct tcattataga agccatggag gcagcagggc actcaatcag tactcttttc 1440
ctatgtggag gcctcagcaa gaatcccctt tttgtgcaaa tgcatgcgga cattactggc 1500
atgcctgtgg tcctgtcgca agaggtggag tccgttcttg tgggtgctgc tgttctgggt 1560
gcctgtgcct caggggattt cgcttctgta caggaagcaa tggcaaaaat gagcaaagtt 1620
gggaaagttg tgttcccgag actacaggat aaaaaatact atgataagaa ataccaagta 1680
ttcctgaagc tggttgaaca ccagaaggag tatttggcga tcatgaatga tgactgaaca 1740
gggcttgcag gtgctgatgc cagaagcttc tgtgccattg cattaaagac ttctgtcatt 1800
tgatccatgt tcaagaccct tgaggtattg tttcatcatt tctgtattgt ctttcaataa 1860
agaatacaaa catgtg 1876
<210> 34
<211> 1487
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7473788CB1
<400> 34
atgaggagtg gcgccgaacg caggggcagc agcgccgcgg cgtccccggg ctcgccgccc 60
cccggccgcg cgcgccccgc cggctccgac gcgccctcgg ccctgccgcc gcccgctgct 120
ggccagcccc gggcccggga ctcgggcgat gtccgctcgc agccgcgccc cctgtttcag 180
tggagcaagt ggaagaagag gatgggctcg tccatgtcgg cggccaccgc gcggaggccg 240
gtgtttgacg acaaggagga cgtgaacttc gaccacttcc agatccttcg ggccattggg 300
aagggcagct ttggcaaggt gtgcattgtg cagaagcggg acacggagaa gatgtacgcc 360
atgaagtaca tgaacaagca gcagtgcatc gagcgcgacg aggtccgcaa cgtcttccgg 420
gagctggaga tcctgcagga gatcgagcac gtcttcctgg tgaacctctg gtactccttc 480
caggacgagg aggacatgtt catggtcgtg gacctgctac tgggcgggga cctgcgctac 540
cacctgcagc agaacgtgca gttctccgag gacacggtga ggctgtacat ctgcgagatg 600
gcactggctc tggactacct gcgcggccag cacatcatcc acagagatgt caagcctgac 660
aacattctcc tggatgagag aggacatgca cacctgaccg acttcaacat tgccaccatc 720
atcaaggacg gggagcgggc gacggcatta gcaggcacca agccgtacat ggctccggag 780
atcttccact cttttgtcaa cggcgggacc ggctactcct tcgaggtgga ctggtggtcg 840
gtgggggtga tggcctatga gctgctgcga ggatggaggc cctatgacat ccactccagc 900
aacgccgtgg agtccctggt gcagctgttc agcaccgtga gcgtccagta tgtccccacg 960
tggtccaagg agatggtggc cttgctgcgg aagctcctca ctgtgaaccc cgagcaccgg 1020
ctctccagcc tccaggacgt gcaggcagcc ccggcgctgg ccggcgtgct gtgggaccac 1080
ctgagcgaga agagggtgga gccgggcttc gtgcccaaca aaggccgtct gcactgcgac 1140
cccacctttg agctggagga gatgatcctg gagtccaggc ccctgcacaa gaagaagaag 1200
cgcctggcca agaacaagtc ccgggacaac agcagggaca gctcccagtc cgagaatgac 1260
tatcttcaag actgcctcga tgccatccag caagacttcg tgatttttaa cagagaaaag 1320
ctgaagagga gccaggacct cccgagggag cctctccccg ccctgagtcc agggatgctg 1380
cggagcctgt ggaggacgag gcggacgctc cgcctgccca tgtgcggccc catttgcccc 1440
tcggccggga gcggctaggc cgggacgccc gtggtcctca ccccttg 1487
<210> 35
<211> 1884
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3107989CB1
63/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
<400> 35
gaggtgacca attttctctc caaaagagaa aggaagttga ttaaaaaaag aatccatgct 60
ccaaagcggc agccaaatcc atctatggcc cccaatgcat cacccagaaa ggggttccag 120
actctcctgc aaaaggccaa ctctacttcc cggctcccac ttcccctcct tcgccacagg 180
agggtggcga aggatttata acccacctct ttctttcagt tgccatggag acaagcccca 240
gtcctttcat tccttctggt acctctctct ccaacgcagg cggaaaggag gcggcttagc 300
ccaaacatgc tgggggaggg gctggcggcc tcgacggcag ctgcggaact aggccgaggg 360
acaaaggcta agtttttcca tggtttggac tggatatcgg tggaactctg gtcaagctgg 420
tatattttga acccaaagac atcactgctg aagaagaaga ggaagaagtg gaaagtctta 480
aaagcattcg gaagtacctg acctccaatg tggcttatgg gtctacaggc attcgggacg 540
tgcacctcga gctgaaggac ctgactctgt gtggacgcaa aggcaatctg cactttatac 600
gctttcccac tcatgacatg cctgctttta ttcaaatggg cagagataaa aacttctcga 660
gtctccacac tgtcttttgt gccactggag gtggagcgta caaatttgag caggattttc 720
tcacaatagg tgatcttcag ctttgcaaac tggatgaact agattgcttg atcaaaggaa 780
ttttatacat tgactcagtc ggattcaatg gacggtcaca gtgctattac tttgaaaacc 840
ctgctgattc tgaaaagtgt cagaagttac catttgattt gaaaaatccg tatcctctgc 900
ttctggtgaa cattggctca ggggttagca tcttagcagt atattccaaa gataattaca 960
aacgggtcac aggtactagt cttggaggag gaactttttt tggtctctgc tgtcttctta 1020
ctggctgtac cacttttgaa gaagctcttg aaatggcatc tcgtggagat agcaccaaag 1080
tggataaact agtacgagat atttatggag gggactatga gaggtttgga ctgccaggct 1140
gggctgtggc ttcaagcttt ggaaacatga tgagcaagga aaagcgagat tccatcagca 1200
aggaagacct cgcccgggcc acattggtca ccatcaccaa caacattggc tccattgctc 1260
ggatgtgtgc gttgaatgag aacatagaca gagttgtgtt tgttggaaat tttctcagaa 1320
tcaatatggt ctccatgaag ctgctggcat atgccatgga tttttggtcc aaaggacaac 1380
tgaaagctct gtttttggaa catgagggtt attttggagc cgttggggca ctgttggaac 1440
tgttcaaaat gactgatgac aagtagagac gagcagtgga ggaaacagcc tcccaaaagg 1500
acagagaact aaaaaattgc tgctggagaa ggtgaaagtc gctttgggac ggaagccaag 1560
ccattatggc agatgaacct gctggatttg taaataattt aaaatccttc cagatgatct 1620
tttactctta ggttttgagc taatgattca aaacggggga atataaaagg ttttttttct 1680
gtatactgta tttttttaaa aaaatggtgc agcgtggcca aacctaccaa ttgtatgcat 1740
taactttgaa aagttgtttg atgtttaaga aggacctgat atgtaagcgc tggtcatttt 1800
tcttctgggg tttactgatc agtgtggtga ttttaacttc atttagtaat tactctagga 1860
gattttacct tgacttatat tttc 1884
<210> 36
<211> 1070
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7482887CB1
<400> 36
gcaaatcaca cagcatggca gctcccagtc ctcctgcctc ttctgcattc cagacctgct 60
ctttaaaaac ctgggcattc cctccacaaa ttgaagagtg gaattttttt tcacctgctc 120
ttcctcttgc tggcacagat cataaagtct tgctctcttt ctatcacatc tcattattat 180
tttggcttct ttctacaagc aaggagcagc aggccctttt acattaccat tagtgaaggc 240
acttgagtta aatccgcaca acgaatctta ctcttgcctg taatcccagc actttggaaa 300
gccaaggcgg gtggatcacc tgaggtcagg agttcgagac cagcctggcc aatgtggtaa 360
aacctatctc tactaaaaat acaaaaaatt ggccaggtgt ggtggtgggg gcctgtaatc 420
tcagctactt gggaggctaa ggcaggagaa ttgcttgaat ctgggagaca gaggttgcgg 480
tgggccaaga tagcgccact gcactccagc cttagcaaca agagcacaac tccatctcaa 540
aataataata ataatttctt ggctccaagt ctcagctccc gcaccacctg acactgtcag 600
atcctcaggc catggccaac actgagagca tcattatcaa tccgagtgct gttcagcaca 660
gcctggtggg tgaaatcatc aaatactctg agcagaaggg attctacctg gtgaccatga 720
64/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
agttccttcg ggcctctgag aaacccctga agccgcacta cactaacctg aaagaccacc 780
cattcttccc ggaccttgtg aagtacatga actcagggca ggttgtggcc atggtcctgg 840
aggggctgaa tgtggcaaag acagggctaa ggatgcttgg ggagaccaat tcattgggct 900
ctatgctaga gactattatt cgcagggact tctgcgctaa aataggcggg aacgtcattg 960
gtggcagtga ttcattacaa agtgctgaaa aagaaatcag cctatggttt aagcccaaag 1020
aaccagttga ctacagatct tgtgcttatg actgggtcta tgcatgatag 1070
<210> 37
<211> 2890
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2963414CB1
<400> 37
gtgacccttc cctccccagg ccacggcagc ccggccctcc cgggcagacc tcccgcacca 60
gggctctggt gaacagcaaa tgctccacgc tgggacgggc cattgcctga tgcctgtaca 120
tggtgggcac tgagagacaa gattcctggg ccctgccttc catacactcc ccacgatctc 180
ggaggaagct ctgaggaccc cgctgagaac ccacagacag gaggacaact gcgctatgac 240
agcaataaag gccaagaagg agaaagttga ggaccgctga cagccccgtg tgctgttggg 300
agctgccctt tctacttcaa accttcctct agcagactgt gcagggaccc cccaccacca 360
ccatctgccg ccatggttgt gcaaaacagc gcagacgccg gggacatgag ggcaggcgtg 420
cagctggagc ccttcctgca ccaggtcggg gggcacatga gcgtgatgaa gtatgacgag 480
catacggtgt gcaagcccct cgtctcccgg gagcagaggt tctatgaatc cctgccgctg 540
gccatgaagc ggttcacccc acagtacaaa ggtaccgtca cagtgcacct ctggaaagac 600
agcacaggcc atctcagctt ggttgccaac ccagtgaagg agagccagga gcccttcaag 660
gtctccacag agtcggcggc ggtggccata tggcagacgc tccagcagac caccggcagc 720
aatggcagcg actgcaccct tgcccagtgg ccgcabgccc agctggcacg ctcacccaag 780
gagagcccgg ccaaggctct tctgaggtcc gagccccacc tcaacactcc agccttctcg 840
ctggtggaag acaccaacgg aaaccaggtt gagaggaaga gcttcaaccc gtggggcctg 900
caatgccacc aggcccacct gacccgcctg tgctccgagt acccagagaa caagcggcat 960
cggttcttgt tgctggaaaa tgtagtgtca cagtacacgc atccctgtgt cctggatctg 1020
aagatgggga cccggcagca cggcgatgat gcatcggagg agaagaaggc ccgccacatg 1080
aggaagtgtg cgcagagcac ctcagcctgc ctgggtgtgc gcatctgcgg catgcaggtt 1140
tatcaaacag ataagaagta ctttctctgc aaagacaagt actatggaag aaaactctca 1200
gtggaggggt tcagacaagc cctctatcag ttcctacata atggaagcca cctccggagg 1260
gagctcctgg agcccatcct gcaccagctc cgggccctcc tctccatcat taggagccag 1320
agttcatacc gcttctattc cagctctctc cttgtcatct atgatgggca ggaaccacca 1380
gaaagagccc caggcagccc gcatcctcac gaggctcccc aggcagccca cggtagctct 1440
cccggtggtc tcaccaaggt tgacatccgc atgattgact ttgctcatac cacatacaag 1500
ggctactgga atgagcacac cacctacgat ggaccagacc ctggctatat ttttggcctg 1560
gaaaacctca tcaggatcct gcaggatatc caagagggag aatgaaactt cctgggctta 1620
tctggattct tctgggctat agatctcaaa tagagacctg ttggttgcta gggtagtcca 1680
gacacccctt agatgtcttc ataatagtcc tatctacctt caaaaaccat ctctatatat 1740
ggcagactat attaacagct gctgaacaaa tcagctctgg aggtgattcc acatcccctg 1800
gcattatgct ctaatgctgc tcatcggaga acagacagcc aggataaagt ggcaccttct 1860
ggagtacact ggagggggca gcccaagtta gaggccagca ttgctgacat tctggaatat 1920
ttgcatctaa aaatgtttac tcgttgccat gctgcagtcc gcacaagctg tgaggcagaa 1980
aacttgactt gaagcagcct tgaagagtga gttcatgagc tcatggtttt tctccttgta 2040
tggactgctc gctccaaggg caggcagagc tcatgaatgc ctcttatctt cctaagcgga 2100
gttttaggtg acacaggatg aagcagaaga gatctaccca tctcacctgc tctgcaccca 2160
gcttctaagt ggacaaagcc aagcccaggc atgagctctg gcaaagcaag accccagatt 2220
ctccattttt gcctgtggaa aggagggtcc ctttacaggc ttttttttcc tttttttccc 2280
ccaaaatctc ttaaaatgag gaatctctta gcagactttg gagttcccca ttctgccaca 2340
65/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
ttctgaccat gagacgcggc ttgcagtggg ggtgaacgca cataaaaagg gaccactgac 2400
gtcctgctct actctctgct ttctatttat ttattttggg ggtgggttgg ggagtcagaa 2460
gaacctggag gacggaggaa accaggggca atgtttacaa gactggtgga caagtgtaaa 2520
tatggaataa gaacaaacag ttctaattaa ttccttcttc tgcagtacgg aaacctatta 2580
caatgccctt gagtcaagca ctgagatacg ttacccaatt agggaaataa atttgttaat 2640
aaaattgctg aggtcaccag tgattattgg tgtgccttat taccctttcc atttgtttat 2700
tctgatcaca ctgtgtggta gttccaattt atgagcgact agcatatacc acaagaacag 2760
ttcactgatt tcctacaatc cttcagggaa ctcgggtgga aatggtggct aataaaatat 2820
ttgcatgtat ctgcaaggga ggcaccagac ctgagaagtg gtccttttat ttgaatctca 2880
tacaatgtac 2890
<210> 38
<211> 5198
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7477139CB1
<400> 38
cgacacggag cacccttcta gcttcttcgt ctccaggact gacgctcagg ctcctctctc 60
gccttagccc aacttgcttt cccgcctcgc aaactccggt ttccctccac tcccaactct 120
tttcactaca cgtttcccct cctctatctc ccacgccacg aaccccgatc cccagactcc 180
tctctcccgc cctcctcctt cctctctcct cccttcaact cttcatccgc ttccacctca 240
gactctgcgc gcacccaatt cagtcgcccg ctcccgttcg gctcctcgaa gccatggcgg 300
gacctggggg ctggagggac agggaggtca cggatctggg ccacctgccg gatccaactg 360
gaatattctc actagataaa accattggcc ttggtactta tggcagaatc tatttgggac 420
ttcatgagaa gactggtgca tttacagctg ttaaagtgat gaacgctcgt aagacccctt 480
tacctgaaat aggaaggcga gtgagagtga ataaatatca aaaatctgtt gggtggagat 540
acagtgatga ggaagaggat ctcaggactg aactcaacct tctgaggaag tactctttcc 600
acaaaaacat tgtgtccttc tatggagcat ttttcaagct gagtccccct ggtcagcggc 660
accaactttg gatggtgatg gagttatgtg cagcaggttc ggtcactgat gtagtgagaa 720
tgaccagtaa tcagagttta aaagaagatt ggattgctta tatctgccga gaaatccttc 780
agggcttagc tcaccttcac gcacaccgag taattcaccg ggacatcaaa ggtcagaatg 840
tgctgctgac tcataatgct gaagtaaaac tggttgattt tggagtgagt gcccaggtga 900
gcagaactaa tggaagaagg aatagtttca ttgggacacc atactggatg gcacctgagg 960
tgattgactg tgatgaggac ccaagacgct cctatgatta cagaagtgat gtgtggtctg 1020
tgggaattac tgccattgaa atggctgaag gagcccctcc tctgtgtaac cttcaaccct 1080
tggaagctct cttcgttatt ttgcgggaat ctgctcccac agtcaaatcc agcggatggt 1140
cccgtaagtt ccacaatttc atggaaaagt gtacgataaa aaatttcctg tttcgtccta 1200
cttctgcaaa catgcttcaa cacccatttg ttcgggatat aaaaaatgaa cgacatgttg 1260
ttgagtcatt aacaaggcat cttactggaa tcattaaaaa aagacagaaa aaaggaatac 1320
ctttgatctt tgaaagagaa gaagctatta aggaacagta caccgtgaga agattcagag 1380
gaccctcttg cactcacgag cttctgagat tgccaaccag cagcagatgc agaccactta 1440
gagtcctgca tggggaaccc tctcagccaa ggtggctacc tgatcgagaa gagccacagg 1500
tccaggcact tcagcagcta cagggagcag ccagggtatt catgccactg caggctctgg 1560
acagtgcacc taagcctcta aaggggcagg ctcaggcacc tcaacgacta caaggggcag 1620
ctcgggtgtt catgccacta caggctcagg tgaaggctaa agcctctaaa cctctacaaa 1680
tgcagattaa ggcacctcca cgactacgga gggcagccag ggtgctcatg ccactacagg 1740
cacaggttag ggcacctagg cttctgcagg tacagtccca ggtatccaaa aagcagcagg 1800
cccagaccca gacatcagaa ccacaagatt tggaccaggt accagaggaa tttcagggtc 1860
aagatcaggt acccgaacaa caaaggcagg gccaggcccc tgaacaacag cagaggcaca 1920
accaggtgcc tgaacaagag ctggagcaga accaggcacc tgaacagcca gaggtacagg 1980
aacaggctgc cgagcctgca caggcagaga ctgaggcaga ggaacctgag tcattacgag 2040
taaatgccca ggtatttctg cccctgctat cacaagatca ccatgtgctg ttgccactac 2100
66/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
atttggatac tcaggtgctc attccagtag aggggcaaac tgaaggatca cctcaggcac 2160
aggcttggac actagaaccc ccacaggcaa ttggctcagt tcaagcactg atagagggac 2220
tatcaagaga cttgcttcgg gcaccaaact caaataactc aaagccactt ggtccgttgc 2280
aaaccctgat ggaaaatctg tcatcaaata ggttttactc acaaccagaa caggcacggg 2340
agaaaaaatc aaaagtttct actctgaggc aagcactggc aaaaagacta tcaccaaaga 2400
ggttcagggc aaagtcatca tggagacctg aaaagcttga actctcggat ttagaagccc 2460
gcaggcaaag gcgccaacgc agatgggaag atatctttaa tcagcatgag gaagaattga 2520
gacaagttga taaaaccagt tggcgtcagt ggggtccttc agaccagttg attgacaata 2580
gtttcactgg tatgcaagac ctgaagaaat atctcaaagg aaaaacaaca tttcataatg 2640
ttcaagttgt tatctacaga gcagttaagg ggaatgatga tgttgcaaca aggtctaccg 2700
ttcctcagcg gtctcttttg gaacaagctc agaagcccat tgacatcaga caaaggagtt 2760
cgcaaaatcg tcaaaattgg ctggcagcat caggtgattc aaagcacaaa attttagcag 2820
gcaaaacaca gagctactgt ttaacaattt atatttcaga agtcaagaaa gaagaatttc 2880
aagaaggaat gaatcaaaag tgtcagggag cccaagtagg attaggacct gaaggccatt 2940
gtatttggca attgggtgaa tcttcttctg aggaagaaag tcctgtgact ggaaggaggt 3000
ctcagtcatc accaccttat tctactattg atcagaagtt gctggttgac atccatgttc 3060
cagatggatt taaagtagga aaaatatcac cccctgtata cttgacaaac gaatgggtag 3120
gctataatgc actctctgaa atcttccgga atgattggtt aactccggca cctgtcattc 3180
agccacctga agaggatggt gattatgttg aactctatga tgccagtgct gatactgatg 3240
gtgatgatga tgatgagtct aatgatactt ttgaagatac ctatgatcat gccaatggca 3300
atgatgactt ggataaccag gttgatcagg ctaatgatgt ttgtaaagac catgatgatg 3360
acaacaataa gtttgttgat gatgtaaata ataattatta tgaggcgcct agttgtccaa 3420
gcttgttgtc agggcaagct atggcagaga tggaagctgc aagcaagatg gttatgatgg 3480
aagtcgtgga aaagaggaag cctacagagg ctatggaagc catacagcca atagaagcca 3540
tggaggaagt gcagccagtg agggacaatg cagccattgg agatcaggaa gaacatgcag 3600
ccaatatagg cagtgaaaga agaggcagtg agggtgatgg aggtaaggga gtcgttcgaa 3660
ccagtgaaga gagtggagcc cttggactca atggagaaga aaattgctca gagacagatg 3720
gtccaggatt gaagagacct gcgtctcagg actttgaata tctacaggag gagccaggtg 3780
gtggaaatga ggcctcaaat gccattgact caggtgctgc accgtcagca cctgatcatg 3840
agagtgacaa taaggacata tcagaatcat caacacaatc agatttttct gccaatcact 3900
catctccttc caaaggttct gggatgtctg ctgatgctaa ctttgccagt gccatcttat 3960
acgctggatt cgtagaagta cctgaggaat cacctaagca accctctgaa gtcaatgtta 4020
acccactcta tgtctctcct gcatgtaaaa aaccactaat ccacatgtat gaaaaggagt 4080
tcacttctga gatctgctgt ggttctttgt ggggagtcaa tttgctgttg ggaacccgat 4140
ctaatctata tctgatggac agaagtggaa aggctgacat tactaaactt ataaggcgaa 4200
gaccattccg ccagattcaa gtcttagagc cactcaattt gctgattacc atctcaggtc 4260
ataagaacag acttcgggtg tatcatctga cctggttgag gaacaagatt ttgaataatg 4320
atccagaaag taaaagaagg caagaagaaa tgctgaagac agaggaagcc tgcaaagcta 4380
ttgataagtt aacaggctgt gaacacttca gtgtcctcca acatgaagaa acaacatata 4440
ttgcaattgc tttgaaatca tcaattcacc tttatgcatg ggcaccaaag tcctttgatg 4500
aaagcactgc tattaaagta tttccaacac ttgatcataa gccagtgaca gttgacctgg 4560
ctattggttc tgaaaaaaga ctaaagattt tcttcagctc agcagatgga tatcacctca 4620
tcgatgcaga atctgaggtt atgtctgatg tgaccctgcc aaagaataat atcatcattt 4680
tacctgattg cttgggaatt ggcatgatgc tcaccttcaa tgctgaagcc ctctctgtgg 4740
aagcaaatga acaactcttc aagaagatcc ttgaaatgtg gaaagacata ccatcttcta 4800
tagcttttga atgtacacag cgaaccacag gatggggcca aaaggccatt gaagtgcgct 4860
ctttgcaatc cagggttctg gaaagtgagc tgaagcgcag gtcaattaag aagctgagat 4920
tcctgtgcac ccggggtgac aagctgttct ttacctctac cctgcgcaat caccacagcc 4980
gggtttactt catgacactt ggaaaacttg aagagctcca aagcaattat gatgtctaaa 5040
agtttccagt gatttattac cacattataa acatcatgta taggcagtct gcatcttcag 5100
atttcagaga ttaaatgagt attcagtttt atttttagta aagattaaat ccaaaacttt 5160
acttttaatg tagcacagaa tagttttaat gagaaatg 5198
<210> 39
<211> 3969
<212> DNA
67/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 55009053CB1
<400> 39
cttttttcct ttcagtgtgc ttcaaatgtc acgacacagg ttagctcagt cgacttgggg 60
ctgctgagct ctggtccctg ccagcctcac cgctcggacc cccccgatcc tccggactcc 120
gctggtcctg gccacgcgag gagcccacgc tagctccaaa gaatcccccg agggcacgtg 180
gaccgaggga gcccctgtga aggctgcgga agactccgcg cgtcccgagc tcccggactc 240
tgcagtgggc ccggggtcca gggagccgct aagggtccct gaagctgtgg ccctagagcg 300
gcggcgggag caggaagaaa aggaggacat ggagacccag gctgtggcaa cgtcccccga 360
tggccgatac ctcaagtttg acatcgagat tggacgtggc tccttcaaga cggtgtatcg 420
agggctagac accgacacca cagtggaggt ggcctggtgt gagctgcaga ctcggaaact 480
gtctagagct gagcggcagc gcttctcaga ggaggtggag atgctcaagg ggctgcagca 540
ccccaacatc gtccgcttct atgattcgtg gaagtcggtg ctgaggggcc aggtttgcat 600
cgtgctggtc accgaactca tgacctcggg cacgctcaag acgtacctga ggcggttccg 660
ggagatgaag ccgcgggtcc ttcagcgctg gagccgccaa atcctgcggg gacttcattt 720
cctacactcc cgggttcctc ccatcctgca ccgggatctc aagtgcgaca atgtctttat 780
cacgggacct tctggctctg tcaaaatcgg ggacctgggc ctggccacgc tcaagcgcgc 840
ctcctttgcc aagagtgtca tcgggacccc ggaattcatg gcccccgaga tgtacgagga 900
aaagtacgat gaggccgtgg acgtgtacgc gttcggcatg tgcatgctgg agatggccac 960
ctctgagtac ccgtactccg agtgccagaa tgccgcgcaa atctaccgca aggtcacttc 1020
gggcagaaag ccgaacagct tccacaaggt gaagataccc gaggtgaagg agatcattga 1080
aggctgcatc cgcacggata agaacgagag gttcaccatc caggacctcc tggcccacgc 1140
cttcttccgc gaggagcgcg gtgtgcacgt ggaactagcg gaggaggacg acggcgagaa 1200
gccgggcctc aagctctggc tgcgcatgga ggacgcgcgg cgcggggggc gcccacggga 1260
caaccaggcc atcgagttcc tgttccagct gggccgggac gcggccgagg aggtggcaca 1320
ggagatggtg gctctgggct tggtctgtga agccgattac cagccagtgg cccgtgcagt 1380
acgtgaacgg gttgctgcca tccagcgaaa gcgtgagaag ctgcgtaaag caagggaatt 1440
ggaggcactc ccaccagagc caggacctcc accagcaact gtgcccatgg cccccggtcc 1500
ccccagtgtc ttcccccctg agcctgagga gccagaggca gaccagcacc agcccttcct 1560
tttccgccac gccagctact catctaccac ttcggattgc gagactgatg gctacctcag 1620
ctcctccggc ttcctggatg cctcagaccc tgcccttcag ccccctgggg gggtgccatc 1680
cagcctggct gagtcccatc tctgcctgcc ctcggctttt gccctatcca ttccacgttc 1740
tggccctgga agtgactttt cccccgggga cagctatgcc tcagatgcag cttcaggcct 1800
tagcgatgtg ggagaaggga tgggacaaat gaggagaccc ccagggagga atctccggcg 1860
cagaccccga tcccggctgc gggtcactag tgtctcagac cagaatgaca gagtggttga 1920
gtgccagcta cagacccata acagcaagat ggtgaccttc cgatttgatc tggatgggga 1980
cagcccggaa gagattgcag ctgccatggt atataacgag ttcattctgc cttcggagcg 2040
agatggattt ctcagacgga ttcgggagat tatccagcga gtggagaccc tgttgaagag 2100
agacactggc cccatggagg ctgctgaaga caccctaagc ccccaggagg agccagcacc 2160
attacctgcc ctgcccgtcc ccctcccaga cccatccaat gaagagctcc agagcagcac 2220
ctccctggag cacaggagct ggacagcctt ctccacctcc tcatcttctc ctggaactcc 2280
tttgtctcct ggaaacccat tttcccctgg aacccccatt tccccaggtc ccatcttccc 2340
catcacttct cccccatgtc atcccagccc ctccccattc tcccccattt cttcccaggt 2400
ctcctcaaat ccctctccac accccaccag ctctccactt ccattctcct ccagcacacc 2460
cgagtttccg gtcccactct ctcagtgtcc ctggagttct ctccccacga cttctccacc 2520
tacgttctct cccacttgtt ctcaggtcac tcttagttcc cctttctttc ctccgtgccc 2580
ctccacttct tccttcccct ccaccacagc agcccctctc ctttctctgg ctagtgcctt 2640
ctcactggct gtgatgactg tggcccagtc cctgctgtcc ccctcacctg ggctcctttc 2700
ccagtctcct ccagcccctc ctagtcccct ccctagcctg ccccttcccc ctcccgttgc 2760
tcctggtggc caggaaagcc cttcacccca cacagctgag gtggagagtg aggcctcacc 2820
acctcctgct cggcccctcc caggggaagc caggctggcg cccatctctg aagagggaaa 2880
gccgcagctt gttgggcgtt tccaagtgac ttcatccaag gaaccggctg agcctcttcc 2940
68/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
cttgcagcca acatccccca ctctctc,tgg ttctccaaaa ccttcaaccc ctcagctcac 3000
ttcagagagc tcagatacag aggacagtgc tggaggcggg ccagagacca gggaagctct 3060
ggctgagagc gaccgtgcag ctgagggtct gggggctgga gttgaggagg aaggagatga 3120
tgggaaggaa ccccaagttg ggggcagccc ccaacccctg agccatccca gcccagtgtg 3180
gatgaactac tcctacagca gcctgtgttt gagcagcgag gagtcagaaa gcagtgggga 3240
agatgaggag ttctgggctg agctgcagag tcttcggcag aagcacttgt cagaggtgga 3300
aacactacag acactacaga aaaaagaaat tgaagatttg tacagccggc tggggaagca 3360
gcccccaccg ggtattgtgg ccccagctgc tatgctgtcc agccgccagc gccgcctctc 3420
caagggcagc ttccccacct cccgccgcaa cagcctacag cgctctgagc ccccaggccc 3480
tggcatcatg cgaaggaact ctctgagtgg cagcagcacc ggctcccagg agcagcgggc 3540
aagcaagggg gtgacattcg ccggggatgt tggcaggatg tgaattcaga acagaagcca 3600
tgtatctccc ccacaccagg gcccaccatg gagcttgtgt tctcagaatc tgatgctttc 3660
tgatcaacaa aactgagcaa ggaagatccc aacactgaag gggtagaagg ccaggggggc 3720
atggagagtg cagctccatt atagtgaaga gccaaacata tgtgaactgt ttgctgtgtg 3780
gaggtgttag ttctgctgcc taccatcttc atctctagca cctcccctgc caagagtcaa 3840
ccactaagca atcccaccca agcctggatg cttctagagg ggcccactcc cagctgggag 3900
agtgtagggg atatgctcac accacattag cagcaaccaa taaaaatgct ggaaacaaga 3960
aaaaaaaaa 3969
<210> 40
<211> 1803
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7474648CB1
<400> 40
atgggtgaaa gtggaaacca tcattttcag caaactaaca caggaacaga aaaccaaaca 60
gcacatgttc tcactcataa gtgggagttg gacaatgaaa acatatgggc acagggaggg 120
gaacatcaca aactgggacc tgtcatgggt tggaaggcta ggagtgggaa aacattagga 180
gaaataccta acgtaggcac actcacactc ctcactggct atgggggatg ccagctgcca 240
tgctgcaagg acactcaggc agcctatgga gaaacccacg tggtgcggag tggaggcctt 300
ctgccaacag ccagctggga actgaggcct gctgacagtc acacggtgac cagcgatgat 360
ccaggcgtct cggtcgttag cgggtatcct gggggctgtc tccctgacca cgacccccca 420
gtggggtttc tttccgaggg tcccgcccct cgcagctgct ctttgataaa gggcggagga 480
acggggctgg ctgcttcccg agtccccagg tcccgcgagc ggcgggcgtg ttgcgggtat 540
ggggtgcggc gccagcagga aggtggtccc ggggccacca gcgctggctt gggccaagca 600
cgaaggtcaa aaccaagccg gcgtcggagg cgcggggcct gggcccgagg cggcggccca 660
ggcggcgcag aggatacagg tggctcgctt ccgagccaag ttcgaccccc gggtccttgc 720
cagtgcccag tacaatttct ctttgacatc tctgaacagg gagttcagag gatgggaaaa 780
aagagagcag gagcagcagc aaacaaggga aggaattcct atcttcggag atatgacatc 840
aaagctctta ttgggacagg cagtttcagc agggttgtca gggtagagca gaagaccacc 900
aagaaacctt ttgcaataaa agtgatggaa accagagaga gggaaggtag agaagcgtgc 960
gtgtctgagc tgagcgtcct gcggcgggtt agccatcgtt acattgtcca gctcatggag 1020
atctttgaga ctgaggatca agtttacatg gtaatggagc tggctaccgg aggggagctc 1080
tttgatcgac tcattgctca gggatccttt acagagcggg atgccgtcag gatcctccag 1140
atggttgctg atgggattag gtatttgcat gcgctgcaga taactcatag gaatctaaag 1200
cctgaaaacc tcttatacta tcatccaggt gaagagtcga aaattttaat tacagatttt 1260
ggtttggcat actccgggaa aaaaagtggt gactggacaa tgaagacact ctgtgggacc 1320
ccagagtaca tagctcctga ggttttgcta aggaagcctt ataccagtgc agtggacatg 1380
tgggctcttg gtgtgatcac atatgcttta cttagcggat tcctgccttt tgatgatgaa 1440
agccagacaa ggctttacag gaagattctg aaaggcaaat ataattatac aggagagcct 1500
tggccaagca tttcccactt ggcgaaggac tttatagaca aactactgat tttggaggct 1560
ggtcatcgca tgtcagctgg ccaggccctg gaccatccct gggtgatcac catggctgca 1620
69/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
gggtcttcca tgaagaatct ccagagggcc atatcccgaa acctcatgca gagggcctct 1680
ccccactctc agagtcctgg atctgcacag tcttctaagt cacattattc tcacaaatcc 1740
aggcatatgt ggagcaagag aaacttaagg atagtagaat cgccactgtc tgcgcttttg 1800
taa 1803
<210> 41
<211> 3472
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483053CB1
<400> 41
atggcgaagg cgacgtccgg tgccgcgggg ctgcgtctgc tgttgctgct gctgctgccg 60
ctgctaggca aagtggcatt gggcctctac ttctcgaggg atgcttactg ggagaagctg 120
tatgtggacc aggcagccgg cacgcccttg ctgtacgtcc atgccctgcg ggacgcccct 180
gaggaggtgc ccagcttccg cctgggccag catctctacg gcacgtaccg aacacggctg 240
catgagaaca actggatctg catccaggag gacaccggcc tcctctacct taaccggagc 300
ctggaccata gctcctggga gaagctcagt gtccgcaacc gcggctttcc cctgctcacc 360
gtctacctca aggtcttcct gtcacccaca tcccttcgtg agggcgagtg ccagtggcca 420
ggctgtgccc gcgtatactt ctccttcttc aacacctcct ttccagcctg cagctccctc 480
aagccccggg agctctgctt cccagagaca aggccctcct tccgcattcg ggagaaccga 540
cccccaggca ccttccacca gttccgcctg ctgcctgtgc agttcttgtg ccccaacatc 600
agcgtggcct acaggctcct ggagggtgag ggtctgccct tccgctgcgc cccggacagc 660
ctggaggtga gcacgcgctg ggccctggac cgcgagcagc gggagaagta cgagctggtg 720
gccgtgtgca ccgtgcacgc cggcgcgcgc gaggaggtgg tgatggtgcc cttcccggtg 780
accgtgtacg acgaggacga ctcggcgccc accttccccg cgggcgtcga caccgccagc 840
gccgtggtgg agttcaagcg gaaggaggac accgtggtgg ccacgctgcg tgtcttcgat 900
gcagacgtgg tacctgcatc aggggagctg gtgaggcggt acacaagcac gctgctcccc 960
ggggacacct gggcccagca gaccttccgg gtggaacact ggcccaacga gacctcggtc 1020
caggccaacg gcagcttcgt gcgggcgacc gtacatgact ataggctggt tctcaaccgg 1080
aacctctcca tctcggagaa ccgcaccatg cagctggcgg tgctggtcaa tgactcagac 1140
ttccagggcc caggagcggg cgtcctcttg ctccacttca acgtgtcggt gctgccggtc 1200
agcctgcacc tgcccagtac ctactccctc tccgtgagca ggagggctcg ccgatttgcc 1260
cagatcggga aagtctgtgt ggaaaactgc caggcgttca gtggcatcaa cgtccagtac 1320
aagctgcatt cctctggtgc caactgcagc acgctagggg tggtcacctc agccgaggac 1380
acctcgggga tcctgtttgt gaatgacacc aaggccctgc ggcggcccaa gtgtgccgaa 1440
cttcactaca tggtggtggc caccgaccag cagacctcta ggcaggccca ggcccagctg 1500
cttgtaacag tggaggggtc atatgtggcc gaggaggcgg gctgccccct gtcctgtgca 1560
gtcagcaaga gacggctgga gtgtgaggag tgtggcggcc tgggctcccc aacaggcagg 1620
tgtgagtgga ggcaaggaga tggcaaaggg atcaccagga acttctccac ctgctctccc 1680
agcaccaaga cctgccccga cggccactgc gatgttgtgg agacccaaga catcaacatt 1740
tgccctcagg actgcctccg gggcagcatt gttgggggac acgagcctgg ggagccccgg 1800
gggattaaag ctggctatgg cacctgcaac tgcttccctg aggaggagaa gtgcttctgc 1860
gagcccgaag acatccagga tccactgtgc gacgagctgt gccgcacggt gatcgcagcc 1920
gctgtcctct tctccttcat cgtctcggtg ctgctgtctg ccttctgcat ccactgctac 1980
cacaagtttg cccacaagcc acccatctcc tcagctgaga tgaccttccg gaggcccgcc 2040
caggccttcc cggtcagcta ctcctcttcc agtgcccgcc ggccctcgct ggactccatg 2100
gagaaccagg tctccgtgga tgccttcaag atcctggagg atccaaagtg ggaattccct 2160
cggaagaact tggttcttgg aaaaactcta ggagaaggcg aatttggaaa agtggtcaag 2220
gcaacggcct tccatctgaa aggcagagca gggtacacca cggtggccgt gaagatgctg 2280
aaagagaacg cctccccgag tgagcttcga gacctgctgt cagagttcaa cgtcctgaag 2340
caggtcaacc acccacatgt catcaaattg tatggggcct gcagccagga tggcccgctc 2400
ctcctcatcg tggagtacgc caaatacggc tccctgcggg gcttcctccg cgagagccgc 2460
70/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
aaagtggggc ctggctacct gggcagtgga ggcagccgca actccagctc cctggaccac 2520
ccggatgagc gggccctcac catgggcgac ctcatctcat ttgcctggca gatctcacag 2580
gggatgcagt atctggccga gatgaagctc gttcatcggg acttggcagc cagaaacatc 2640
ctggtagctg aggggcggaa gatgaagatt tcggatttcg gcttgtcccg agatgtttat 2700
gaagaggatt cgtacgtgaa gaggagccag ggtcggattc cagttaaatg gatggcaatt 2760
gaatcccttt ttgatcatat ctacaccacg caaagtgatg tatggtcttt tggtgtcctg 2820
ctgtgggaga tcgtgaccct agggggaaac ccctatcctg ggattcctcc tgagcggctc 2880
ttcaaccttc tgaagaccgg ccaccggatg gagaggccag acaactgcag cgaggagatg 2940
taccgcctga tgctgcaatg ctggaagcag gagccggaca aaaggccggt gtttgcggac 3000
atcagcaaag acctggagaa gatgatggtt aagaggagag actacttgga ccttgcggcg 3060
tccactccat ctgactccct gatttatgac gacggcctct cagaggagga gacaccgctg 3120
gtggactgta ataatgcccc cctccctcga gccctccctt ccacatggat tgaaaacaaa 3180
ctctatggca tgtcagaccc gaactggcct ggagagagtc ctgtaccact cacgagagct 3240
gatggcacta acactgggtt tccaagatat ccaaatgata gtgtatatgc taactggatg 3300
ctttcaccct cagcggcaaa attaatggac acgtttgata gttaacattt ctttgtgaaa 3360
ggtaatggac tcacaagggg aagaaacatg ctgagaatgg aaagtctacc ggccctttct 3420
ttgtgaacgt cacattggcc gagccgtgtt cagttcccag gtggcagact cg 3472
<210> 42
<211> 1704
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7483117CB1
<400> 42
atggatgaca aagatattga caaagaacta aggcagaaat taaacttttc ctattgtgag 60
gagactgaga ttgaagggca gaagaaagta gaagaaagca gggaggcttc gagccaaacc 120
ccagagaagg gtgaagtgca ggattcagag gcaaagggta caccaccttg gactcccctt 180
agcaacgtgc atgagctcga cacatcttcg gaaaaagaca aagaaagtcc agatcagatt 240
ttgaggactc cagtgtcaca ccctctcaaa tgtcctgaga caccagccca accagacagc 300
aggagcaagc tgctgcccag tgacagcccc tctactccca aaaccatgct gagccggttg 360
gtgatttctc caacagggaa gcttccttcc agaggcccta agcatttgaa gctcacacct 420
gctcccctca aggatgagat gacctcattg gctctggtca atattaatcc cttcactcca 480
gagtcctata aaaaattatt tcttcaatct ggtggcaaga ggaaaataag aggagatctt 540
gaggaagctg gtccagagga aggcaaggga gggctgcctg ccaagagatg tgttttacga 600
gaaaccaaca tggcttcccg ctatgaaaaa gaattcttgg aggttgaaaa aattggggtt 660
ggcgaatttg gtacagtcta caagtgcatt aagaggctgg atggatgtgt ttatgcaata 720
aagcgctcta tgaaaacttt tacagaatta tcaaatgaga attcggcttt gcatgaagtt 780
tatgctcacg cagtgcttgg gcatcacccc catgtggtac gttactattc ctcatgggca 840
gaagatgacc acatgatcat tcagaatgaa tactgcaatg gtgggagttt gcaagctgct 900
atatctgaaa acactaagtc tggcaatcat tttgaagagc caaaactcaa ggacatcctt 960
ctacagattt cccttggcct taattacatc cacaactcta gcatggtaca cctggacatc 1020
aaacctagta atatattcat ttgtcacaag atgcaaagtg aatcctctgg agtcatagaa 1080
gaagttgaaa atgaagctga ttggtttctc tctgccaatg tgatgtataa aattggtgac 1140
ctgggccacg caacatcaat aaacaaaccc aaagtggaag aaggagatag tcgcttcctg 1200
gctaatgaga ttttgcaaga ggattaccgg caccttccca aagcagacat atttgccttg 1260
ggattaacaa ttgcagtggc tgcaggagca gagtcattgc ccaccaatgg tgctgcatgg 1320
caccatatcc gcaagggtaa ctttccggac gttcctcagg agctctcaga aagcttttcc 1380
agtctgctca agaacatgat ccaacctgat gccgaacaga gaccttctgc agcagctctg 1440
gccagaaata cagttctccg gccttccctg ggaaaaacag aagagctcca acagcagctg 1500
aatttggaaa agttcaagac tgccacactg gaaagggaac tgagagaagc ccagcaggcc 1560
cagtcacccc agggatatac ccatcatggt gacactgggg tctctgggac ccacacagga 1620
tcaagaagca caaaacgcct ggtgggagga aagagtgcaa ggtcttcaag ctttacctca 1680
71/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
ggagagcgtg agcctctgca ttaa 1704
<210> 43
<211> 6298
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7484498CB1
<400> 43
cgcggggcgg aacagatcgc agacctgggg gttcgcagag ccgccagtgg ggagatgttg 60
aagttcaaat atggagcgcg gaatcctttg gatgctggtg ctgctgaacc cattgccagc 120
cgggcctcca ggctgaatct gttcttccag gggaaaccac cctttatgac tcaacagcag 180
atgtctcctc tttcccgaga agggatatta gatgccctct ttgttctctt tgaagaatgc 240
agtcagcctg ctctgatgaa gattaagcac gtgagcaact ttgtccggaa gtattccgac 300
accatagctg agttacagga gctccagcct tcggcaaagg acttcgaagt cagaagtctt 360
gtaggttgtg gtcactttgc tgaagtgcag gtggtaagag agaaagcaac cggggacatc 420
tatgctatga aagtgatgaa gaagaaggct ttattggccc aggagcaggt ttcatttttt 480
gaggaagagc ggaacatatt atctcgaagc acaagcccgt ggatccccca attacagtat 540
gcctttcagg acaaaaatca cctttatctg gtcatggaat atcagcctgg aggggacttg 600
ctgtcacttt tgaatagata tgaggaccag ttagatgaaa acctgataca gttttaccta 660
gctgagctga ttttggctgt tcacagcgtt catctgatgg gatacgtgca tcgagacatc 720
aagcctgaga acattctcgt tgaccgcaca ggacacatca agctggtgga ttttggatct 780
gccgcgaaaa tgaattcaaa caagatggtg aatgccaaac tcccgattgg gaccccagat 840
tacatggctc ctgaagtgct gactgtgatg aacggggatg gaaaaggcac ctacggcctg 900
gactgtgact ggtggtcagt gggcgtgatt gcctatgaga tgatttatgg gagatccccc 960
ttcgcagagg gaacctctgc cagaaccttc aataacatta tgaatttcca gcggtttttg 1020
aaatttccag atgaccccaa agtgagcagt gactttcttg atctgattca aagcttgttg 1080
tgcggccaga aagagagact gaagtttgaa ggtctttgct gccatccttt cttctctaaa 1140
attgactgga acaacattcg taactctcct ccccccttcg ttcccaccct caagtctgac 1200
gatgacacct ccaattttga tgaaccagag aagaattcgt gggtttcatc ctctccgtgc 1260
cagctgagcc cctcaggctt ctcgggtgaa gaactgccgt ttgtggggtt ttcgtacagc 1320
aaggcactgg ggattcttgg tagatctgag tctgttgtgt cgggtctgga ctcccctgcc 1380
aagactagct ccatggaaaa gaaacttctc atcaaaagca aagagctaca agactctcag 1440
gacaagtgtc acaagatgga gcaggaaatg acccggttac atcggagagt gtcagaggtg 1500
gaggctgtgc ttagtcagaa ggaggtggag ctgaaggcct ctgagactca gagatccctc 1560
ctggagcagg accttgctac ctacatcaca gaatgcagta gcttaaagcg aagtttggag 1620
caagcacgga tggaggtgtc ccaggaggat gacaaagcac tgcagcttct ccatgatatc 1680
agagagcaga gccggaagct ccaagaaatc aaagagcagg agtaccaggc tcaagtggaa 1740
gaaatgaggt tgatgatgaa tcagttggaa gaggatcttg tctcagcaag aagacggagt 1800
gatctctacg aatctgagct gagagagtct cggcttgctg ctgaagaatt caagcggaaa 1860
gcgacagaat gtcagcataa actgttgaag gctaaggatc aagggaagcc tgaagtggga 1920
gaatatgcga aactggagaa gatcaatgct gagcagcagc tcaaaattca ggagctccaa 1980
gagaaactgg agaaggctgt aaaagccagc acggaggcca ccgagctgct gcagaatatc 2040
cgccaggcaa aggagcgagc cgagagggag ctggagaagc tgcagaaccg agaggattct 2100
tctgaaggca tcagaaagaa gctggtggaa gctgaggaac gccgccattc tctggagaac 2160
aaggtaaaga gactagagac catggagcgt agagaaaaca gactgaagga tgacatccag 2220
acaaaatccc aacagatcca gcagatggct gataaaattc tggagctcga agagaaacat 2280
cgggaggccc aagtctcagc ccagcaccta gaagtgcacc tgaaacagaa agagcagcac 2340
tatgaggaaa agattaaagt gttggacaat cagataaaga aagacctggc tgacaaggag 2400
acactggaga acatgatgca gagacacgag gaggaggccc atgagaaggg caaaattctc 2460
agcgaacaga aggcgatgat caatgctatg gattccaaga tcagatccct ggaacagagg 2520
attgtggaac tgtctgaagc caataaactt gcagcaaata gcagtctttt tacccaaagg 2580
aacatgaagg cccaagaaga gatgatttct gaactcaggc aacagaaatt ttacctggag 2640
72/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
acacaggctg ggaagttgga ggcccagaac cgaaaactgg aggagcagct ggagaagatc 2700
agccaccaag accacagtga caagaatcgg ctgctggaac tggagacaag attgcgggag 2760
gtcagtctag agcacgagga gcagaaactg gagctcaagc gccagctcac agagctacag 2820
ctctccctgc aggagcgcga gtcacagttg acagccctgc aggctgcacg ggcggccctg 2880
gagagccagc ttcgccaggc gaagacagag ctggaagaga ccacagcaga agctgaagag 2940
gagatccagg cactcacggc acatagagat gaaatccagc gcaaatttga tgctcttcgt 3000
aacagctgta ctgtaatcac agacctggag gagcagctaa accagctgac cgaggacaac 3060
gctgaactca acaaccaaaa cttctacttg tccaaacaac tcgatgaggc ttctggcgcc 3120
aacgacgaga ttgtacaact gcgaagtgaa gtggaccatc tccgccggga gatcacggaa 3180
cgagagatgc agcttaccag ccagaagcaa acgatggagg ctctgaagac cacgtgcacc 3240
atgctggagg aacaggtcat ggatttggag gccctaaacg atgagctgct agaaaaagag 3300
cggcagtggg aggcctggag gagcgtcctg ggtgatgaga aatcccagtt tgagtgtcgg 3360
gttcgagagc tgcagaggat gctggacacc gagaaacaga gcagggcgag agccgatcag 3420
cggatcaccg agtctcgcca ggtggtggag ctggcagtga aggagcacaa ggctgagatt 3480
ctcgctctgc agcaggctct caaagagcag aagctgaagg ccgagagcct ctctgacaag 3540
ctcaatgacc tggagaagaa gcatgctatg cttgaaatga atgcccgaag cttacagcag 3600
aagctggaga ctgaacgaga gctcaaacag aggcttctgg aagagcaagc caaattacag 3660
cagcagatgg acctgcagaa aaatcacatt ttccgtctga ctcaaggact gcaagaagct 3720
ctagatcggg ctgatctact gaagacagaa agaagtgact tggagtatca gctggaaaac 3780
attcaggttc tctattctca tgaaaaggtg aaaatggaag gcactatttc tcaacaaacc 3840
aaactcattg attttctgca agccaaaatg gaccaacctg ctaaaaagaa aaaggttcct 3900
ctgcagtaca atgagctgaa gctggccctg gagaaggaga aagctcgctg tgcagagcta 3960
gaggaagccc ttcagaagac ccgcatcgag ctccggtccg cccgggagga agctgcccac 4020
cgcaaagcaa cggaccaccc acacccatcc acgccagcca ccgcgaggca gcagatcgcc 4080
atgtccgcca tcgtgcggtc gccagagcac cagcccagtg ccatgagcct gctggccccg 4140
ccatccagcc gcagaaagga gtcttcaact ccagaggaat ttagtcggcg tcttaaggaa 4200
cgcatgcacc acaatattcc tcaccgattc aacgtaggac tgaacatgcg agccacaaag 4260
tgtgctgtgt gtctggatac cgtgcacttt ggacgccagg catccaaatg tctcgaatgt 4320
caggtgatgt gtcaccccaa gtgctccacg tgcttgccag ccacctgcgg cttgcctgct 4380
gaatatgcca cacacttcac cgaggccttc tgccgtgaca aaatgaactc cccaggtctc 4440
cagaccaagg agcccagcag cagcttgcac ctggaagggt ggatgaaggt gcccaggaat 4500
aacaaacgag gacagcaagg ctgggacagg aagtacattg tcctggaggg atcaaaagtc 4560
ctcatttatg acaatgaagc cagagaagct ggacagaggc cggtggaaga atttgagctg 4620
tgccttcccg acggggatgt atctattcat ggtgccgttg gtgcttccga actcgcaaat 4680
acagccaaag cagatgtccc atacatactg aagatggaat ctcacccgca caccacctgc 4740
tggcccggga gaaccctcta cttgctagct cccagcttcc ctgacaaaca gcgctgggtc 4800
accgccttag aatcagttgt cgcaggtggg agagtttcta gggaaaaagc agaagctgat 4860
gctaaactgc ttggaaactc cctgctgaaa ctggaaggtg atgaccgtct agacatgaac 4920
tgcacgctgc ccttcagtga ccaggtggtg ttggtgggca ccgaggaagg gctctacgcc 4980
ctgaatgtct tgaaaaactc cctaacccat gtcccaggaa ttggagcagt cttccaaatt 5040
tatattatca aggacctgga gaagctactc atgatagcag gagaagagcg ggcactgtgt 5100
cttgtggacg tgaagaaagt gaaacagtcc ctggcccagt cccacctgcc tgcccagccc 5160
gacatctcac ccaacatttt tgaagctgtc aagggctgcc acttgtttgg ggcaggcaag 5220
attgagaacg ggctctgcat ctgtgcagcc atgcccagca aagtcgtcat tctccgctac 5280
aacgaaaacc tcagcaaata ctgcatccgg aaagagatag agacctcaga gccctgcagc 5340
tgtatccact tcaccaatta cagtatcctc attggaacca ataaattcta cgaaatcgac 5400
atgaagcagt acacgctcga ggaattcctg gataagaatg accattcctt ggcacctgct 5460
gtgtttgccg cctcttccaa cagcttccct gtctcaatcg tgcaggtgaa cagcgcaggg 5520
cagcgagagg agtacttgct gtgtttccac gaatttggag tgttcgtgga ttcttacgga 5580
agacgtagcc gcacagacga tctcaagtgg agtcgcttac ctttggcctt tgcctacaga 5640
gaaccctatc tgtttgtgac ccacttcaac tcactcgaag taattgagat ccaggcacgc 5700
tcctcagcag ggacccctgc ccgagcgtac ctggacatcc cgaacccgcg ctacctgggc 5760
cctgccattt cctcaggagc gatttacttg gcgtcctcat accaggataa attaagggtc 5820
atttgctgca agggaaacct cgtgaaggag tccggcactg aacaccaccg gggcccgtcc 5880
acctcccgca gcagccccaa caagcgaggc ccacccacgt acaacgagca catcaccaag 5940
cgcgtggcct ccagcccagc gccgcccgaa ggccccagcc acccgcgaga gccaagcaca 6000
73/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
ccccaccgct accgcgaggg gcggaccgag ctgcgcaggg acaagtctcc tggccgcccc 6060
ctggagcgag agaagtcccc cggccggatg ctcagcacgc ggagagagcg gtcccccggg 6120
aggctgtttg aagacagcag caggggccgg ctgcctgcgg gagccgtgag gaccccgctg 6180
tcccaggtga acaaggtctg ggaccagtct tcagtataaa tctcagccag aaaaaccaac 6240
tcctcatctt gatctgcagg aaaacaccaa acacactatg gaactctgct gatgggga 6298
<210> 44
<211> 5454
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte ID No: 7638121CB1
<400> 44
cacgcacacc gcacgtacgg ggttgggccc agctgggtta taagcgtgat ccccatgccc 60
cctgcccagg ctggggggca tttgcacatc tgcaaaggcc tcccagcctg tcccagccct 120
gccccagcct gggaccccca cattctactc accgtgtctc ctcagagggg ccagaaccct 180
ccactgggga gaggcaagtg gcggtgaact tggtgtccat aggaccctgt ccctgagagc 240
gacagctgag ttagtgagct ccactggccc caccaactcc ttctgatcac ctggccagct 300
gaggtcagag tgggagaggc agtggttcca ttgaaggagt actcctaact gtcagaagcc 360
tgggcggtca ggatggggtg ctgtcgcttg ggctgcgggg ggtgttcagt tgcccacagt 420
gtatctcagg gtctcaccaa ccatccaagc atggtaggct gtggctggca cccagggttg 480
tgtggctggg gaggtggtct ccacagttcc ctccctgccc tcccagggcc cccatccatg 540
caggtaacca tcgaggatgt gcaggcacag acaggcggaa cggcccaatt cgaggctatc 600
attgagggcg acccacagcc ctcggtgacc tggtacaagg acagcgtcca gctggtggac 660
agcacccggc ttagccagca gcaagaaggc accacatact ccctggtgct gaggcatatg 720
gcctcgaagg atgccggcgt ttacacctgc ctggcccaaa acactggtgg ccaggtgctc 780
tgcaaggcag agctgctggt gcttgggggg gacaatgagc cggactcaga gaagcaaagc 840
caccggagga agctgcactc cttctatgag gtcaaggagg agattggaag gggcgtgttt 900
ggcttcgtaa aaagagtgca gcacaaagga aacaagatct tgtgcgctgc caagttcatc 960
cccctacgga gcagaactcg ggcccaggca tacagggagc gagacatcct ggccgcgctg 1020
agccacccgc tggtcacggg gctgctggac cagtttgaga cccgcaagac cctcatcctc 1080
atcctggagc tgtgctcatc cgaggagctg ctggaccgcc tgtacaggaa gggcgtggtg 1140
acggaggccg aggtcaaggt ctacatccag cagctggtgg aggggctgca ctacctgcac 1200
agccatggcg ttctccacct ggacataaag ccctctaaca tcctgatggt gcatcctgcc 1260
cgggaagaca ttaaaatctg cgactttggc tttgcccaga acatcacccc agcagagctg 1320
cagttcagcc agtacggctc ccctgagttc gtctcccccg agatcatcca gcagaaccct 1380
gtgagcgaag cctccgacat ttgggccatg ggtgtcatct cctacctcag cctgacctgc 1440
tcatccccat ttgccggcga gagtgaccgt gccaccctcc tgaacgtcct ggaggggcgc 1500
gtgtcatgga gcagccccat ggctgcccac ctcagcgaag acgccaaaga cttcatcaag 1560
gctacgctgc agagagcccc tcaggcccgg cctagtgcgg cccagtgcct ctcccacccc 1620
tggttcctga aatccatgcc tgcggaggag gcccacttca tcaacaccaa gcagctcaag 1680
ttcctcctgg cccgaagtcg ctggcagcgt tccctgatga gctacaagtc catcctggtg 1740
atgcgctcca tccctgagct gctgcggggc ccacccgaca gcccctccct cggcgtagcc 1800
cggcacctct gcagggacac tggtggctcc tccagttcct cctcctcctc tgacaacgag 1860
ctcgccccat ttgcccgggc taagtcactg ccaccctccc cggtgacaca ctcaccactg 1920
ctgcaccccc ggggcttcct gcggccctcg gccagcctgc ctgaggaagc cgaggccagt 1980
gagcgctcca ccgaggcccc agctccgcct gcatctcccg agggtgccgg gccaccggcc 2040
gcccagggct gcgtgccccg gcacagcgtc atccgcagcc tgttctacca ccaggcgggt 2100
gagagccctg agcacggggc cctggccccg gggagcaggc ggcacccggc ccggcggcgg 2160
cacctgctga agggcgggta cattgcgggg gcgctgccag gcctgcgcga gccactgatg 2220
gagcaccgcg tgctggagga ggaggccgcc agggaggagc aggccaccct cctggccaaa 2280
gccccctcat tcgagactgc cctccggctg cctgcctctg gcacccactt ggcccctggc 2340
cacagccact ccctggaaca tgactctccg agcacccccc gcccctcctc ggaggcctgc 2400
74/75
CA 02425963 2003-04-15
WO 02/33099 PCT/USO1/47728
ggtgaggcac agcgactgcc ttcagccccc tccggggggg cccctatcag ggacatgggg 2460
caccctcagg gctccaagca gcttccatcc actggtggcc acccaggcac tgctcagcca 2520
gagaggccat ccccggacag cccttggggg cagccagccc ctttctgcca ccccaagcag 2580
ggttctgccc cccaggaggg ctgcagcccc cacccagcag ttgccccatg ccctcctggc 2640
tccttccctc caggatcttg caaagaggcc cccttagtac cctcaagccc cttcttggga 2700
cagccccagg caccccttgc ccctgccaaa gcaagccccc cattggactc taagatgggg 2760
cctggagaca tctctcttcc tgggaggcca aaacccggcc cctgcagttc cccagggtca 2820
gcctcccagg cgagctcttc ccaagtgagc tccctcaggg tgggctcctc ccaggtgggc 2880
acagagcctg gcccctccct ggatgcggag ggctggaccc aggaggctga ggatctgtcc 2940
gactccacac ccaccttgca gcggcctcag gaacaggtga ccatgcgcaa gttctccctg 3000
ggtggtcgcg ggggctacgc aggcgtggct ggctatggca cctttgcctt tggtggagat 3060
gcagggggca tgctggggca ggggcccatg tgggccagga tagcctgggc tgtgtcccag 3120
tcggaggagg aggagcagga ggaggccagg gctgagtccc agtcggagga gcagcaggag 3180
gccagggctg agagcccact gccccaggtc agtgcaaggc ctgtgcctga ggtcggcagg 3240
gctcccacca ggagctctcc agagcccacc ccatgggagg acatcgggca ggtctccctg 3300
gtgcagatcc gggacctgtc aggtgatgcg gaggcggccg acacaatatc cctggacatt 3360
tccgaggtgg accccgccta cctcaacctc tcagacctgt acgatatcaa gtacctccca 3420
ttcgagttta tgatcttcag gaaagtcccc aagtccgctc agccagagcc gccctccccc 3480
atggctgagg aggagctggc cgagttcccg gagcccacgt ggccctggcc aggtgaactg 3540
ggcccccacg caggcctgga gatcacagag gagtcagagg atgtggacgc gctgctggca 3600
gaggctgccg tgggcaggaa gcgcaagtgg tcctcgccgt cacgcagcct cttccacttc 3660
cctgggaggc acctgccgct ggacgagcct gcagagctgg ggctgcgtga gagagtgaag 3720
gcctccgtgg agcacatctc ccggatcctg aagggcaggc cggaaggtct ggagaaggag 3780
gggcccccca ggaagaagcc aggccttgct tccttccggc tctcaggtct gaagagctgg 3840
gaccgagcgc cgacattcct aagggagctc tcagatgaga ctgtggtcct gggccagtca 3900
gtgacactgg cctgccaggt gtcagcccag ccagctgccc aggccacctg gagcaaagac 3960
ggagcccccc tggagagcag cagccgtgtc ctcatctctg ccaccctcaa gaacttccag 4020
cttctgacca tcctggtggt ggtggctgag gacctgggtg tgtacacctg cagcgtgagc 4080
aatgcgctgg ggacagtgac caccacgggc gtcctccgga aggcagagcg cccctcatct 4140
tcgccatgcc cggatatcgg ggaggtgtac gcggatgggg tgctgctggt ctggaagccc 4200
gtggaatcct acggccctgt gacctacatt gtgcagtgca gcctagaagg cggcagctgg 4260
accacactgg cctccgacat ctttgactgc tgctacctga ccagcaagct ctcccggggt 4320
ggcacctaca ccttccgcac ggcatgtgtc agcaaggcag gaatgggtcc ctacagcagc 4380
ccctcggagc aagtcctcct gggagggccc agccacctgg cctctgagga ggagagccag 4440
gggcggtcag cccaacccct gcccagcaca aagaccttcg cattccagac acagatccag 4500
aggggccgct tcagcgtggt gcggcaatgc tgggagaagg ccagcgggcg ggcgctggcc 4560
gccaagatca tcccctacca ccccaaggac aagacagcag tgctgcgcga atacgaggcc 4620
ctcaagggcc tgcgccaccc gcacctggcc cagctgcacg cagcctacct cagcccccgg 4680
cacctggtgc tcatcttgga gctgtgctct gggcccgagc tgctcccctg cctggccgag 4740
agggcctcct actcagaatc cgaggtgaag gactacctgt ggcagatgtt gagtgccacc 4800
cagtacctgc acaaccagca catcctgcac ctggacctga ggtccgagaa catgatcatc 4860
accgaataca acctgctcaa ggtcgtggac ctgggcaatg cacagagcct cagccaggag 4920
aaggtgctgc cctcagacaa gttcaaggac tacctagaga ccatggctcc agagctcctg 4980
gagggccagg gggctgttcc acagacagac atctgggcca tcggtgtgac agccttcatc 5040
atgctgagcg ccgagtaccc ggtgagcagc gagggtgcac gcgacctgca gagaggactg 5100
cgcaaggggc tggtccggct gagccgctgc tacgcggggc tgtccggggg cgccgtggcc 5160
ttcctgcgca gcactctgtg cgcccagccc tggggccggc cctgcgcgtc cagctgcctg 5220
cagtgcccgt ggctaacaga ggagggcccg gcctgttcgc ggcccgcgcc cgtgaccttc 5280
cctaccgcgc ggctgcgcgt cttcgtgcgc aatcgcgaga agagacgcgc gctgctgtac 5340
aagaggcaca acctggccca ggtgcgctga gggtcgcccc ggccacaccc ttggtctccc 5400
cgctgggggt cgctgcagac gcgccaataa aaacgcacag ccgggcgaga aaaa 5454
75/75
