Note: Descriptions are shown in the official language in which they were submitted.
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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.
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-calmodulin, 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
serineJthreonine 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 residues. Almost all
kinases contain a conserved 250-300 amino acid catalytic domain containng
specific residues and
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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 II 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 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
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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. Cell
stimulation induces a signaling cascade leading to phosphorylation of MEK
(MAP/ERK 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; checlq~oint 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 serinelthreonine
protein 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.
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The CHI 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 isoforn~s of casein ,
kinase I from a variety of sources. There are at least five mammalian
isoforms, a, (3, 'y, 8, and E. 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 arid 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
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 catecholanunes and
seratonin, through
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phosphorylation/activation of tyrosine hydroxylase and tryptophan hydroxylase,
respectively (Fujisawa,
H. (1990) BioEssays 12:27-29). The mRNA encoding a calmodulin-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).
Mitogen-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
a
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 of the
microtubule organizing center, the centrosome, in animal cells (Fry, A.M., et
al. (1998) EMBO J.
17:470-481 ).
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
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induced. In the absence of such checkpoints, the damaged DNA is inherited by
aberrant 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 to
dephosphorylate and activate the cyclin-dependent kinase Cde2. Cdc2 activation
controls the entry of
cells into mitosis. (Peng, C-Y et al. (1997) Science 277:1501- 1505.) Thus,
activation of Chk1
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.
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
tissue and may be a proto-oncogene whose deregulated expression in normal
tissue leads to oncogenic
transformation.
5'-AMP-activated~rotein kinase
A ligand-activated STK protein kinase is 5'-AMP-activated protein kinase
(AMPK) (Gao, G. 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
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 leucine
zipper domain in addition to its N-terminal protein kinase domain. This C-
terminal domain appears to
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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. supra).
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.
supra). 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 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
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-
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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) su ra).
KINASES WITH NON-PROTEIN SUBSTRATES
Lipid 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) Curr. 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
membrane by inositol kinases, thus converting PI residues to the biphosphate
state (PIPZ). P1P2 is then
cleaved into inositol triphosphate (IP3) 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,
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
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type-2 diabetes (Shepard, supra).
An example of lipid kinase phosphorylation activity is the phosphoryladon 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
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
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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
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.
Pvrimidine Kinases
The pyrimidine 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
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.
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," "PKIN-7,"
"PKIN-8," "PKIN-9," "PKIN-10," "PKIN-1 l," and "PKIN-12." In one aspect, the
invention provides
an isolated polypeptide comprising an anuno acid sequence selected fiom the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from the
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group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:l-12, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12. In one
alternative, the invention
provides an isolated polypeptide comprising the amino acid sequence of SEQ ID
NO:1-12.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising an
amino acid sequence selected from the group consisting of a) an amino acid
sequence selected from the
group consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid
sequence having at least
90% sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
12, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12. In one alternative, the polynucleotide encodes a
polypeptide selected
from the group consisting of SEQ ID NO:l-12. In another alternative, the
polynucleotide is selected
from the group consisting of SEQ ID N0:13-24.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected fiom the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-12, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:l-12, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-12. In one alternative, the invention provides a
cell transformed with the
recombinant polynucleotide. In another alternative, the invention provides a
transgenic organism
comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an anuno acid sequence
selected from the group
consisting of SEQ ID NO:1-12, b) a naturally occurring amino acid sequence
having at least 90%
sequence identity to an amino acid sequence selected from the group consisting
of SEQ ID NO:1-12, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
N0:1-12, and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-12. 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
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polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:l-12, b) a naturally
occurring amino acid
sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12, c) a biologically active fragment of an amino
acid sequence selected
fiom the group consisting of SEQ ID N0:1-12, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12.
The invention further provides an isolated polynucleotide comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:13-24, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to 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 comprising a
polynucleotide sequence
selected from the group consiseing of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:13-24, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to 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 comprising a
polynucleotide sequence
selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:13-24, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:13-24, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). The method comprises a) amplifying said target
polynucleotide or
fragment thereof using polymerise chain reaction amplification, and b)
detecting the presence or
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absence of said amplified target polynucleotide or fragment thereof, and,
optionally, if present, the
amount thereof.
The invention further provides a composition comprising an effective amount of
a polypeptide
comprising an amino acid sequence selected from the group consisting of a) an
amino acid sequence
selected from the group consisting of SEQ ID N0:1-12, b) a naturally occurring
amino acid sequence
having at least 90% sequence identity to an amino acid sequence selected from
the group consisting of
SEQ ID NO:l-12, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:l-12, and a pharmaceutically acceptable
excipient. In one
embodiment, the composition comprises an amino acid sequence selected from the
group consisting of
SEQ 1D NO:1-12. The invention additionally 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 comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:l-12, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an amino acid
sequence selected from the
group consisting of SEQ ID N0:1-12, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12. 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 comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
12, b) a naturally
occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-12, c) a biologically active
tiagment of an amino
acid sequence selected from the group consisting of SEQ ID N0:1-12, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:1-
12. The method
comprises a) exposing a sample comprising the polypeptide to a compound, and
b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a composition comprising
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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 PKIN, 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 comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a
naturally occurring amino
acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the group
consisting of SEQ ID N0:1-12, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-12, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-12. 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 comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-12, b) a
naturally occurring
amino acid sequence having at least 90% sequence identity to an anuno acid
sequence selected from the
group consisting of SEQ ID NO:1-12, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-12, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID N0:1-12. The method
comprises a)
combining the polypeptide with at least one test compound under conditions
permissive for the
activity of the polypeptide, b) assessing the activity of the polypeptide in
the presence of the test
compound, and c) comparing the activity of the polypeptide in the presence of
the test compound with
the activity of the polypeptide in the absence of the test compound, wherein a
change in the activity of
the polypeptide in the presence of the test compound is indicative of a
compound that modulates the
activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:13-24, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The~invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample contaiW ng nucleic acids
with the test compound;
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b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:13-24, ii) a naturally occurring polynucleotide sequence having at least
90% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:13-
24, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to 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 comprising a polynucleotide sequence
selected liom the group
consisting of i) a polynucleotide sequence selected from the group consisting
of SEQ ID N0:13-24,
ii) a naturally occurring polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:13-24,
iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to 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 each polypeptide 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 each polypeptide sequence, including
predicted motifs and
domains, along with the methods, algorithms, and searchable databases used for
analysis of each
polypeptide.
Table 4 lists the cDNA and genomic DNA fragments which were used to assemble
each
polynucleotide sequence, along with selected fragments of the polynucleotide
sequences.
Table 5 shows the representative cDNA library for each polynucleotide 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.
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Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings
as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although
any machines, materials, and methods similar or equivalent to those described
herein can be used to
practice or test the present invention, the preferred machines, materials and
methods are now described.
All publications mentioned herein are cited for the purpose of describing and
disclosing the cell lines,
protocols, reagents and vectors which are reported in the publications and
which might be used in
connection with the invention. Nothing herein is to be construed as an
admission that the invention is
not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"PKIN" refers to the amino acid sequences of substantially purified PHIN
obtained from any
species, particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
PHIN. 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
PHIN or by acting on components of the biological pathway in which PHIN
participates.
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
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allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times in
a given sequence.
"Altered" nucleic acid sequences encoding 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
IS include aspartic acid and glutamic acid, and positively charged amino acids
may include lysine and
arginine. Amino acids with uncharged polar side chains having similar
hydrophilicity values may
include: asparagine and glutamine; and serine and threonine. Amino acids with
uncharged side chains
having similar hydrophilicity values may include: leucine, isoleucine, and
valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity of
PKIN. Antagonists may include proteins such as antibodies, 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.
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.
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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 "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
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
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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 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.
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A "deletion" refers to a change in the anuno 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. Chenucal
modifications of a polynucleotide can include, for example, replacement of
hydrogen by an alkyl, acyl,
hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide
which retains at least one
biological or immunological function of the natural molecule. A derivative
polypeptide is one modified
by glycosylation, pegylation, or any similar process that retains at least one
biological or immunological
function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
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
fiagment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:13-24 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:13-24, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:13-24 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:13-24 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:13-24 and the region of SEQ ID N0:13-24 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-12 is encoded by a fragment of SEQ ID N0:13-24. A
fragment
of SEQ ID NO:1-12 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:l-12. For example, a fragment of SEQ ID NO:l-12 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-12.
The precise length of
a fragment of SEQ ID NO:l-12 and the region of SEQ ID NO:1-12 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
CA 02395102 2002-06-19
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purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation colon
(e.g., methionine) followed by an open reading frame and a translation
termination colon. 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
Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn," that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
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Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: l0
Word Size: Il
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example, as
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
program (described and referenced above). For pairwise alignments of
polypeptide sequences using
CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3,
window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the "percent similarity"
between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(April-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
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Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: I penalties
Gap x drop-off:' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
a fragment of at least 15, at least 20, at least 30, at least 40, at least S0,
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 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 ~~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
23
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WO 01/46397 PCT/US00/35304
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,n and conditions
for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al. (1989) Molecular
Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor
Press, Plainview NY; specifically
see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present invention
include wash conditions of 68°C in the presence of about 0.2 x SSC and
about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or
42°C may be used. SSC concentration may
be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking
reagents are used to block non-specific hybridization. Such blocking reagents
include, for instance,
sheared and denatured salmon sperm DNA at about 100-200 pg/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid
sequence present in solution and another nucleic acid sequence immobilized on
a solid support (e.g.,
paper, membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells
or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
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.
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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,
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
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule. Typical
labels include radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are
short nucleic acids, usually DNA oligonucleotides, which may be annealed to a
target polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerise enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerise chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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. I-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
Protocols. A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
program (available to the public from the Whitehead InstitutelMIT Center for
Genome Research,
Cambridge MA) allows the user to input a "mispriming library," in which
sequences to avoid as primer
binding sites are user-specified. Primer3 is useful, in particular, for the
selection of oligonucleotides for
microarrays. (The source code for the latter two primer selection programs may
also be obtained from
their respective sources and modified to meet the user's specific needs.) The
PrimeGen program
(available to the public from the UK Human Genome Mapping Project Resource
Centre, Cambridge
UK) designs primers based on multiple sequence alignments, thereby allowing
selection of primers that
hybridize to either the most conserved or least conserved regions of aligned
nucleic acid sequences.
Hence, this program is useful for identification of both unique and conserved
oligonucleotides and
polynucleotide fragments. The oligonucleotides and polynucleotide fragments
identified by any of the
above selection methods are useful in hybridization technologies, for example,
as PCR or sequencing
primers, microarray elements, or specific probes to identify fully or
partially complementary
polynucleotides in a sample of nucleic acids. Methods of oligonucleotide
selection are not limited to
those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
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WO 01/46397 PCT/US00/35304
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
artifcial manipulation of isolated segments of nucleic acids, e.g., by genetic
engineering techniques
such as those described in Sambrook, supra. .The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing PKIN,
nucleic acids encoding PHIN, 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 tree 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
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WO 01/46397 PCT/US00/35304
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 cells"
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part of the host chromosome, as well as
transiently transformed
cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. ( 1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having at
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least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of the
nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or
at least 98% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an "allelic"
(as defined above), "splice," "species," or "polymorphic" variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. The corresponding
polypeptide may
possess additional functional domains or lack domains that are present in the
reference molecule.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides will generally have significant amino acid identity relative to
each other. A polymorphic
variant is a variation in the polynucleotide sequence of a particular gene
between individuals of a given
species. Polymorphic variants also may encompass "single nucleotide
polymorphisms" (SNPs) in
which the polynucleotide sequence varies by one nucleotide base. The presence
of SNPs may be
indicative of, for example, a certain population, a disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having at
least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human kinases (PKIN), the
polynucleotides
encoding PHIN, 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 ID NO:)
and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is
denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as
shown.
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Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2
show the
polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte
polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide 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 each of the polypeptides of the
invention. Columns
1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and
the corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer
Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains,
and motifs. Column 7
shows analytical methods for protein structurelfunction analysis and in some
cases, searchable
databases to which the analytical methods were applied.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from genomic
DNA, or any
combination of these two types of sequences. Columns 1 and 2 list the
polynucleotide sequence
identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide
consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide
of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column
4 lists fragments of
the polynucleotide sequences which are useful, for example, in hybridization
or amplification
technologies that identify SEQ ID N0:13-24 or that distinguish between SEQ ID
N0:13-24 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 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,
2287966H1 is the
identification number of an Incyte cDNA sequence, and BRA1NON01 is the cDNA
library from which
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
it is derived. Incyte cDNAs for which cDNA libraries are not indicated were
derived from pooled
cDNA libraries (e.g., 70166939V1). Alternatively, the identification numbers
in column 5 may refer to
GenBank cDNAs or ESTs (e.g., g2821547) which contributed to the assembly of
the full length
polynucleotide sequences. Alternatively, the identification numbers in column
5 may refer to coding
regions predicted by Genscan analysis of genomic DNA. For example,
g4454511.v113.gs_3.nt.edit is
the identification number of a Genscan-predicted coding sequence, with
g4454511 being the GenBank
identification number of the sequence to which Genscan was applied. The
Genscan-predicted coding
sequences may have been edited prior to assembly. (See Example IV.)
Alternatively, the identification
numbers in column 5 may refer to assemblages of both eDNA and Genscan-
predicted exons brought
together by an "exon stitching" algorithm. (See Example V.) Alternatively, the
identification numbers
in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon-stretching" algorithm. (See Example V.) In some cases, Incyte cDNA
coverage redundant
with the sequence coverage shown in column 5 was obtained to confirm the final
consensus
polynucleotide sequence, but the relevant Incyte cDNA identification numbers
are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide sequences
which were assembled using Incyte cDNA sequences. The representative cDNA
library is the Incyte
eDNA 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 PK1N 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 N0:13-24, which encodes PKIN. The
polynucleotide sequences of
SEQ 1D N0:13-24, as presented in the Sequence Listing, embrace the equivalent
RNA sequences,
wherein occurrences of the nitrogenous base thymine are replaced with uracil,
and the sugar backbone
is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
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 PK1N. A particular aspect of the invention encompasses a variant of a
polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:13-24 which has at
31
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
least about 70%, or alternatively at least about 85%, or even at least about
95% polynucleotide
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID N0:13-24.
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 colon 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 colon usage, e.g., inclusion
of non-naturally occurring
colons. Colons 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 colons
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.
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
N0:13-24 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 liagment of
32
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Applied
Biosystems), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA sequencing
system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system
(Molecular Dynamics,
Sunnyvale CA), or other systems known in the art. The resulting sequences are
analyzed using a
variety of 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 Biotechnolo~y, 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 in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
legations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991 ) Nucleic Acids
Res. 19:3055-3060).
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
33
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 PHIN.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter PHIN-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of 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
34
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 occurnng 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
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, supra, 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 a
suitable host. These elements include regulatory sequences, such as enhancers,
constitutive and
inducible promoters, and 5' and 3' untxanslated 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 by the
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 Biolo~y, John Wiley & Sons, New York NY, ch. 9,
13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding 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, supra; Ausubel, suQra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~v (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet..15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding 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 site
disrupts the LacZ gene, allowing a colorimetric screening procedure for
identification of transformed
36
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 antibodies,
vectors which direct high level expression of PHIN 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 PHIN. 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
nastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, su ra;
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 PHIN. Transcription of
sequences 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 liom TMV (Takamatsu, N. (1987) EMBO
J. 6:307-311).
Alternatively, plant promoters such as the small subunit of RUBISCO or heat
shock promoters may be
used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R. et
al. (1984) Science
224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-
105.) These constructs can
be introduced into plant cells by direct DNA transformation or pathogen-
mediated transfection. (See,
e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill,
New York NY, pp.
191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding PHIN
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.
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 artiricial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.)
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CA 02395102 2002-06-19
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For long term production of recombinant proteins in mammalian systems, stable
expression of
PKIN in cell lines is preferred. For example, sequences encoding PKIN can be
transformed into cell
lines using expression vectors which may contain viral origins of replication
and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk and apr cells, respectively. (See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or
herbicide resistance can be
used as the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers
resistance to the aminoglycosides neomycin and G-418; and als and pat confer
resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980)
Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and 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., anthoc;yanins, green fluorescent
proteins (GFP; Clontech),13
glucuronidase and its substrate 13-glucuronide, or luciferase and its
substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presencelabsence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding PHIN 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
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
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CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 Immunology, Greene Pub. Associates and
Wiley-Interscience, New
York NY; and Pound, J.D. (1998) Immunoc;hemical 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 polymerise
such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a variety of
commercially available
kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison
WI), and US
Biochemical. Suitable reporter molecules or labels which may be used for ease
of detection include
radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents,
as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding 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 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
39
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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), calmodulin binding peptide (CBP), 6-His, FLAG, c-
myc, and hemagglutinin
(HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate
fusion proteins on
immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-
chelate resins,
respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity
purification of fusion
proteins using commercially available monoclonal and polyclonal antibodies
that specifically recognize
these epitope tags. A fusion protein may also be engineered to contain a
proteolytic cleavage site
located between the PHIN encoding sequence and the heterologous protein
sequence, so that PKIN 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 that
specitlcally 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 identii-ied is closely related to the
natural ligand of
PKIN, e.g., a ligand or iiagment 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 Immunology 1 (2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which PKIN
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 polypepdde, 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 compounds) 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
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 Number 5,175,383 and U.S. Patent Number
5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse embryo
and grown in culture. The ES cells are transformed with a vector containing
the gene of interest
disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo;
Capecchi, M.R. (1989)
Science 244:1288-1292). The vector integrates into the corresponding region of
the host genome by
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homologous recombination. Alternatively, homologous recombination takes place
using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-
specific manner (Marth, J.D.
(1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids
Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell
blastocysts such as those from
the C57BL/6 mouse strain. The blastocysts are surgically transferred to
pseudopregnant dams, and the
resulting chimeric progeny are genotyped and bred to produce heterozygous or
homozygous strains.
Transgenic animals thus generated may be tested with potential therapeutic or
toxic agents.
Polynucleotides encoding 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 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 cancers, cell proliferation and cardiovascular diseases.
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, PHIN 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
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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-ec;todermal 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, Sjogrcn'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,
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
neurotibromatosis, 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, mural annular
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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 pulmonary disease, restrictive
pulmonary disease, chronic
obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial
asthma, bronchiectasis,
bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary
tuberculosis, diffuse
interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative
interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia
bronchiolitis
obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,
Goodpasture's syndromes,
idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular
disorders, pulmonary
alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural
effusions, pneumothorax,
pleural tumors, drug-induced lung disease, radiation-induced lung disease, and
complications of lung
transplantation; and a lipid disorder such as fatty liver, cholestasis,
primary biliary cirrhosis, carnitine
deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase
deficiency,
hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's
disease, Niemann-Pick's
disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2
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 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 including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
PKIN 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
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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 cancers, 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
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
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
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. For
example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin digestion
of the antibody molecule and Fab fragments generated by reducing the disulfide
bridges of the F(ab')2
fragments. Alternatively, Fab expression libraries may be constructed to allow
rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
PKIN and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
46
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
non-interfering PKIN epitopes is generally used, but a competitive binding
assay may also be employed
(Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay techniques
may be used to.assess the affinity of antibodies for 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 1~ determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple PHIN
epitopes, represents the average affinity, or avidity, of the antibodies for
PHIN. The Ka determined for
a preparation of monoclonal antibodies, which are monospecific for a
particular PHIN epitope,
represents a true measure of affinity. High-affinity antibody preparations
with Ka ranging from about
109 to 10'2 L/mole are preferred for use in immunoassays in which the 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
specirc 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 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.,
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CA 02395102 2002-06-19
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Slater, J.E. et al. (1998) J. Allergy Cli. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, su ra; 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.)
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 (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) 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,
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; Ivies,
Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin.
Biotechnol. 9:445-450).
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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 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 (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M. V.
and Blau, H.M. su ra)), 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 KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to 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.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses a
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method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al.
(1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding 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 Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-
242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding 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 Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant HSV
d92 which consists of a genome containing at least one exogenous gene to be
transferred to a cell under
the control of the appropriate promoter for purposes including human gene
therapy. Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al. (1994) Dev.
Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
known to those of ordinary
CA 02395102 2002-06-19
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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 on
the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA, resulting
in the overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g.,
protease and polymerase). Similarly, inserting the coding sequence for 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~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding PKIN.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
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scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA sequences
encoding 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' and/or 3' ends
of the molecule, or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine, and
wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms
of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding 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 PKIN
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding PKIN may be therapeutically useful, and in the treament of disorders
associated with
decreased PKIN 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 speciiuc polynucleotide. A test compound may be
obtained by any method
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commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurnng or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding 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 specif c
nucleotide is detected
by hybridization with a probe having a nucleotide sequence complementary to
the sequence of the
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 Schizosaccharomyces pombe gene
expression system (Atkins,
D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic
Acids Res. 28:E15) or a
human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem.
Biophys. Res. Commun.
268:8-13). A particular embodiment of the present invention involves screening
a combinatorial
library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides,
peptide nucleic acids, and
modified oligonucleotides) for antisense activity against a specific
polynucleotide sequence (Bruice,
T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
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 ReminQton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of PKIN,
antibodies to PKIN, and mimetics, agonists, antagonists, or inhibitors of
PKIN.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical,
sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the case
of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of fast-acting
formulations is well-known in the art. In the case of macromolecules (e.g.
larger peptides and proteins),
recent developments in the field of pulmonary delivery via the alveolar region
of the lung have enabled
the practical delivery of drugs such as insulin to blood circulation (see,
e.g., Patton. J.S. et al., U.S.
Patent No. 5,997,848). Pulmonary delivery has the advantage of administration
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
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
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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 ~g to 100,000 fig, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind PKIN may be used for
the diagnosis
of disorders characterized by expression of PKIN, or in assays to monitor
patients being treated with
PHIN 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 PHIN 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 or
standard values for PHIN expression are established by combining body fluids
or cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibodies
to PHIN under
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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 PHIN. 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
IS 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.
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 ID
N0:13-24 or liom
genomic sequences including promoters, enhancers, and introns of the PKIN
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,
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
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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, 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, 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, mural
annular calcification, mural
valve prolapse, rheumatic fever and rheumatic heart disease, infective
endocarditis, nonbacterial
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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 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 noninllammatory 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 palmitoyltransferase deficiency, myoadenylate deaminase
deficiency,
hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's
disease, Niemann-Pick's
disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2
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 PHIN 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
sequences encoding PK1N 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
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sample then the presence of altered levels of nucleotide sequences encoding
PK.IN 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 PHIN, 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 fiom 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
PHIN, and will be
employed under optimized conditions for identification of a specific gene or
condition. Oligomers may
also be employed under less stringent conditions for detection or
quantification of closely related DNA
or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding PHIN 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
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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
polymerise chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the secondary
and tertiary structures of PCR products in single-stranded form, and these
differences are detectable
using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA liagments 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 microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, PKIN, Iragments of PKIN, or antibodies specific for
PKIN may be
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used as elements on a microarray. The microarray may be used to monitor or
measure protein-protein
interactions, drug-target interactions, and gene expression profiles, as
described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at a
given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines, biopsies,
or other biological samples. The transcript image may thus reflect gene
expression in vivo, as in the
case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the
present invention
may also be used in conjunction with in vitro model systems and preclinical
evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and toxicity
(Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
If a test compound has a
signature similar to that of a compound with known toxicity, it is likely to
share those toxic properties.
These fingerprints or signatures are most useful and refined when they contain
expression information
from a large number of genes and gene families. Ideally, a genome-wide
measurement of expression
provides the highest quality signature. Even genes whose expression is 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
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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 pioteome 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, su ra). The proteins are
visualized in the gel as discrete and uniquely positioned spots, typically by
staining the gel with an agent
such as Coomassie Blue or silver or fluorescent stains. The optical density of
each protein spot is
generally proportional to the level of the protein in the sample. The optical
densities of equivalently
positioned protein spots from different samples, for example, from biological
samples either treated or
untreated with a test compound or therapeutic agent, are compared to identify
any changes in protein
spot density related to the treatment. The proteins in the spots are partially
sequenced using, for
example, standard methods employing chemical or enzymatic cleavage followed by
mass spectrometry.
The identity of the protein in a spot may be determined by comparing its
partial sequence, preferably of
at least 5 contiguous amino acid residues, to the polypeptide sequences of the
present invention. In
some cases, further sequence data may be obtained for definitive protein
identification.
A proteomic profile may also be generated using antibodies specific for 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 (Luelcing, 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
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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 tiom the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared with
the amount in an untreated biological sample. A difference in the amount of
protein between the two
samples is indicative of a toxic response to the test compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999)
Oxford University Press, London, hereby expressly incorporated by reference.
. In another embodiment of the invention, nucleic acid sequences encoding PHIN
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
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mapping. The sequences may be mapped to a particular chromosome, to a specific
region of a
chromosome, or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs),
yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs),
bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, for
example, 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-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.)
Examples of genetic map
data can be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM)
World Wide Web site. Correlation between the location of the gene encoding
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
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 PHIN 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.,
Geyser, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
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synthesized on a solid substrate. The test compounds are reacted with PKIN, or
tiagments thereof, and
washed. Bound PKIN is then detected by methods well known in the art. Purified
PKIN 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.
The disclosures of all patents, applications and publications, mentioned above
and below, in
particular U.S. Ser. No. 60/172,066, U.S. Ser. No. 60/176,107, U.S. Ser. No.
60/177,731, and U.S.
Ser. No. 60/178,573, are expressly incorporated by reference herein.
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. The Incyte
cDNA shown for SEQ
ID N0:13 was derived from a eDNA library constructed from musculoskeletal
tissue. The Incyte
cDNA shown for SEQ ID N0:14 was derived from cDNA libraries constructed from
prostate, brain
and ovarian tissues, including tissues associated with brain, prostate and
thyroid tumors. 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
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using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000
bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto
CA), or derivatives thereof. Recombinant plasmids were transformed into
competent E. coli cells
including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DHlOB, or
ElectroMAX
DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo excision
using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were
purified using at least
one of the following: a Magic or WIZARD Minipreps DNA purification system
(Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8
Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid
purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1 ml of
distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN I1 fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
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Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal cycler
(MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides were
carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the ABI
PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel, 1997,
supra, unit 7.7). Some of the cDNA sequences were selected for extension using
the techniques
disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing vector,
linker, and poly(A) sequences and by masking ambiguous bases, using algorithms
and programs based
on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The
Incyte cDNA
sequences or translations thereof were then queried against a selection of
public databases such as the
GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and
BLOCKS, PRINTS,
DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases
such as PFAM.
(HMM is a probabilistic approach which analyzes consensus primary structures
of gene families.
See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The
queries were
performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte
cDNA
sequences were assembled to produce full length polynucleotide sequences.
Alternatively, GenBank
cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-
predicted coding
sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages
to full length.
Assembly was performed using programs based on Phred, Phrap, and Conned, and
cDNA assemblages
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
polypeptide sequences. Alternatively, a polypeptide of the invention may begin
at any of the methionine
residues of the full length translated polypeptide. Full length polypeptide
sequences were subsequently
analyzed by querying against databases such as the GenBank protein databases
(genpept), SwissProt,
BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based
protein
family databases such as PFAM. Full length polynucleotide sequences are also
analyzed using
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MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA)
and
LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence
alignments are
generated using default parameters specified by the CLUSTAL algorithm as
incorporated into the
MEGALIGN multisequence alignment program (DNASTAR), which also calculates the
percent
identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly of
Incyte cDNA and full length sequences and provides applicable descriptions,
references, and threshold
parameters. The first column of Table 7 shows the tools, programs, and
algorithms used, the second
column provides brief descriptions thereof, the third column presents
appropriate references, all of
which are incorporated by reference herein in their entirety, and the fourth
column presents, where
applicable, the scores, probability values, and other parameters used to
evaluate the strength of a match
between two sequences (the higher the score or the lower the probability
value, the greater the identity
between two sequences).
The programs described above for the assembly and analysis of lull length
polynucleotide and
polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:13-24. 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
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 kinases. Potential human kinases were also identified by homology
to Incyte cDNA
sequences that had been annotated as 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 lncyte eDNA or public eDNA coverage of the Genscan-
predicted sequences,
thus providing evidence for transcription. When Incyte cDNA coverage was
available, this information
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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" Se9uences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification
program described in Example IV. Partial cDNAs assembled as described in
Example III were mapped
to genomic DNA and parsed into clusters containing related cDNAs and Genscan
exon predictions from
one or more genomic sequences. Each cluster was analyzed using an algorithm
based on graph theory
and dynamic programming to integrate cDNA and genomic information, generating
possible splice
variants that were subsequently confirmed, edited, or extended to create a
full length sequence.
Sequence intervals in which the entire length of the interval was present on
more than one sequence in
the cluster were identified, and intervals thus identified were considered to
be equivalent by transitivity.
For example, if an interval was present on a cDNA and two genomic sequences,
then all three intervals
were considered to be equivalent. This process allows unrelated but
consecutive genomic sequences to
be brought together, bridged by eDNA sequence. Intervals thus identified were
then "stitched" together
by the stitching algorithm in the order that they appear along their parent
sequences to generate the
longest possible sequence, as well as sequence variants. Linkages between
intervals which proceed
along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic
sequence) were
given preference over linkages which change parent type (cDNA to genomic
sequence). The resultant
stitched sequences were translated and compared by BLAST analysis to the
genpept and gbpri public
databases. Incorrect exons predicted by Genscan were corrected by comparison
to the top BLAST hit
from genpept. Sequences were further extended with additional cDNA sequences,
or by inspection of
genomic DNA, when necessary.
"Stretched" Seguences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
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 eDNA 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
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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 loom 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 N0:13-24 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID NO:l 3-24 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, or 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 G~nethon 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.nebi.nlm.nih.gov/genemap~, can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e.g., Sambrook, s-upra,
ch. 7; Ausubel (1995)
su ra, 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.
CA 02395102 2002-06-19
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The basis of the search is the product score, which is defined as:
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 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;
heroic 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 eDNA
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+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PC1 B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) P1COGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 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
(Amersham
Pharmacia Biotech), treated with Plu DNA polymerise (Stratagene) to till-in
restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells were selected
on antibiotic-containing
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media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
Garb 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 reamplilied
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 N0:13-24 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
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
X. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
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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
S 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;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a Iluorescent 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/i.~l oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/Nl RNase inhibitor, 500 ~M dATP, 500 ~M dGTP, 500 pM
dTTP, 40 NM
dCTP, 40 NM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction 'is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 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
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WO 01/46397 PCT/US00/35304
using 1 ml of glycogen (I 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 ~15X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element is
amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses
primers complementary to the vector sequences flanking the cDNA insert. Array
elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522 , incorporated herein by reference. 1 ~1 of the array
element DNA, at an average
concentration of 100 ng/~.xl, 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 Nl of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 ° C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 p1 of 5X SSC in a corner of the chamber. The chamber containing the arrays
is incubated for
about 6.5 hours at 60° C. The arrays are washed for 10 min at 45
° C in a first wash buffer (1X SSC,
0.1 % SDS), three times for 10 minutes each at 45 ° C in a second wash
buffer (0.1X SSC), and dried.
Detection
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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, Ine., 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 NJ7 corresponding to the two
Iluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fiuorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fiuorophore, 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
fiuorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
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XI. Complementary Polynucleotides
Sequences complementary to the PHIN-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
smaller or with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of PKIN. 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 PHIN 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 eDNA 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 Spodoptera
fru~iperda (Sf9) insect
cells in most cases, or human hepatocytes, in some cases. Infection of the
latter requires additional
genetic modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc.
Natl. Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
In most expression systems, 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,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma iaponicum, enables the purification of fusion proteins
on immobilized
glutathione under conditions that maintain protein activity and antigenicity
(Amersham Pharmacia
Biotech). Following purification, the GST moiety can be proteolytically
cleaved liom 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-
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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
Examples XVI, XVII, and XVIII where applicable.
XIII. Functional 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 ~sg 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 of
cells transfected with sequences encoding PKIN and either CD64 or CD64-GFP.
CD64 and CD64-
GFP are expressed on the surface of transfeeted 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.
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XIV. Production of PKIN 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.
S 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, supra, eh. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431 A
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-KL,H 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
immunoaflinity
chromatography using antibodies specific for PKIN. An immunoaftinity 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 immunoaftinity 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
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
PHIN is collected.
XVI. Identification of Molecules Which Interact with PKIN
PKIN, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled PKIN, washed, and
any wells with labeled PKIN complex are assayed. Data obtained using different
concentrations of
PHIN are used to calculate values for the number, affinity, and association of
PKIN with the candidate
molecules.
79
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 3zP-ATP. PKIN is incubated
with the protein
substrate, 32P-ATP, and an appropriate kinase buffer. The 32P incorporated
into the substrate is
separated from free 32P-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 32P-ATP. The reservoir of the centrifuged unit containing the 32P-
peptide product as retentate is
then counted in a scintillation counter. This procedure allows assay of any
type of protein kinase
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
H1 (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 PK1N is demonstrated in
vitro in an assay
containing PKIN, 501 of kinase buffer, lpg substrate, such as myelin basic
protein (MBP) or synthetic
peptide substrates, 1 mM DTT, 10 ~g ATP, and O.Sp.Ci [y-33P]ATP. The reaction
is incubated at
30°C for 30 minutes and stopped by pipetting onto P81 paper. The
unincorporated ['y-3~P]ATP is
removed by washing and the incorporated radioactivity is measured using a
radioactivity scintillation
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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 32P-labeled ATP as the phosphate
donor. The 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.
XVIII. Enhancement/Inhibition of Protein Kinase Activity
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 those skilled in molecular biology or related fields are intended to be
within the scope of the following
claims.
81
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
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CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
<110> INCYTE GENOMICS, INC.
YANG, Junming
BAUGHN, Mariah R.
BURFORD, Neil
AU-YOUNG, Janice
LU, Dyung Aina M.
REDDY, Roopa
YUE, Henry
YAO, Monique G.
LAL, Preeti
KAHN, Farrah A.
<120> HUMAN KINASES
<130> PI-0002 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/172,066; 60/176,107; 60/176,107; 60/177,731
<151> 1999-12-23; 2000-01-14; 2000-01-14; 2000-01-21
<160> 24
<170> PERL Program
<210> 1
<211> 466
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 058860CD1
<400> 1
Met Glu Asp Gly Thr Pro Asn Glu His Phe Tyr Thr Pro Thr Glu
1 5 10 15
Glu Arg Gly Ser Ala Tyr Glu Ile Trp Arg Ser Asp Ser Phe Gly
20 25 30
Thr Pro Asn Glu Ala Ile Glu Pro Lys Asp Asn Glu Met Pro Pro
35 40 45
Ser Phe Ile Glu Pro Leu Thr Lys Arg Lys Val Tyr Glu Asn Thr
50 55 60
Thr Leu Gly Phe Ile Val Glu Val Glu Gly Leu Pro Val Pro Gly
65 70 75
Val Lys Trp Tyr Arg Asn Lys Ser Leu Leu Glu Pro Asp Glu Arg
80 85 90
Ile Lys Met Glu Arg Val Gly Asn Val Cys Ser Leu Glu Ile Ser
95 100 105
Asn Ile Gln Lys Gly Glu Gly Gly Glu Tyr Met Cys His Ala Val
110 115 120
Asn Ile Ile Gly Glu Ala Lys Ser Phe Ala Asn Val Asp Ile Met
125 130 135
Pro Gln Glu Glu Arg Val Val Ala Leu Pro Pro Pro Val Thr His
140 145 150
Gln His Val Met Glu Phe Asp Leu Glu His Thr Thr Ser Ser Arg
155 160 165
Thr Pro Ser Pro Gln Glu Ile Val Leu Glu Val Glu Leu Ser Glu
170 175 180
Lys Asp Val Lys Glu Phe Glu Lys Gln Val Lys Ile Val Thr Val
185 190 195
Pro Glu Phe Thr Pro Asp His Lys Ser Met Ile Val Ser Leu Asp
200 205 210
Val Leu Pro Phe Asn Phe Val Asp Pro Asn Met Asp Ser Arg Glu
215 220 225
Gly Glu Asp Lys Glu Leu Lys Ile Asp Leu Glu Val Phe Glu Met
230 235 240
1/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Pro Pro Arg Phe Ile Met Pro Ile Cys Asp Phe Lys Ile Pro Glu
245 250 255
Asn Ser Asp Ala Val Phe Lys Cys Ser Val Ile Gly Ile Pro Thr
260 265 270
Pro Glu Val Lys Trp Tyr Lys Glu Tyr Met Cys Ile Glu Pro Asp
275 280 285
Asn Ile Lys Tyr Val Ile Ser Glu Glu Lys Gly Ser His Thr Leu
290 295 300
Lys Ile Arg Asn Val Cys Leu Ser Asp Ser Ala Thr Tyr Arg Cys
305 310 315
Arg Ala Val Asn Cys Val Gly Glu Ala Ile Cys Arg Gly Phe Leu
320 325 330
Thr Met Gly Asp Ser Glu Ile Phe Ala Val Ile Ala Lys Lys Ser
335 340 345
Lys Val Thr Leu Ser Ser Leu Met Glu Glu Leu Val Leu Lys Ser
350 355 360
Asn Tyr Thr Asp Ser Phe Phe Glu Phe Gln Val Val Glu Gly Pro
365 370 375
Pro Arg Phe Ile Lys Gly Ile Ser Asp Cys Tyr Ala Pro Ile Gly
380 385 390
Thr Ala Ala Tyr Phe Gln Cys Leu Val Arg Gly Ser Pro Arg Pro
395 400 405
Thr Val Tyr Trp Tyr Lys Asp Gly Lys Leu Val Gln Gly Arg Arg
410 415 420
Phe Thr Val Glu Glu Ser Gly Thr Gly Phe His Asn Leu Phe Ile
425 430 435
Thr Ser Leu Val Lys Ser Asp Glu Gly Glu Tyr Arg Cys Val Ala
440 445 450
Thr Asn Lys Ser Gly Met Ala Glu Ser Phe Ala Ala Leu Thr Leu
455 460 465
Thr
<210> 2
<211> 513
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2041716CD1
<400> 2
Met Glu Gly Gly Pro Ala Val Cys Cys Gln Asp Pro Arg Ala Glu
1 5 10 15
Leu Val Glu Arg Val Ala Ala Ile Asp Val Thr His Leu Glu Glu
20 25 30
Ala Asp Gly Gly Pro Glu Pro Thr Arg Asn Gly Val Asp Pro Pro
35 40 45
Pro Arg Ala Arg Ala Ala Ser Val Ile Pro Gly Ser Thr Ser Arg
50 55 60
Leu Leu Pro Ala Arg Pro Ser Leu Ser Ala Arg Lys Leu Ser Leu
65 70 75
Gln Glu Arg Pro Ala Gly Ser Tyr Leu Glu Ala Gln Ala Gly Pro
80 85 90
Tyr Ala Thr Gly Pro Ala Ser His Ile Ser Pro Arg Ala Trp Arg
95 100 105
Arg Pro Thr Ile Glu Ser His His Val Ala Ile Ser Asp Ala Glu
110 115 120
Asp Cys Val Gln Leu Asn Gln Tyr Lys Leu Gln Ser Glu Ile Gly
125 13 0 13 5
Lys Val Gly Leu Thr Asp Ala Tyr Leu Gln Gly Ala Tyr Gly Val
140 145 150
Val Arg Leu Ala Tyr Asn Glu Ser Glu Asp Arg His Tyr Ala Met
155 160 165
Lys Val Leu Ser Lys Lys Lys Leu Leu Lys Gln Tyr Gly Phe Pro
170 175 180
2/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Arg Arg Pro Pro Pro Arg Gly Ser Gln Ala Ala Gln Gly Gly Pro
185 190 195
Ala Lys Gln Leu Leu Pro Leu Glu Arg Val Tyr Gln Glu Ile Ala
200 205 210
Ile Leu Lys Lys Leu Asp His Val Asn Val Val Lys Leu Ile Glu
215 220 225
Val Leu Asp Asp Pro Ala Glu Asp Asn Leu Tyr Leu Val Asp Leu
230 235 240
Leu Arg Lys Gly Pro Val Met Glu Vah Pro Cys Asp Lys Pro Phe
245 250 255
Ser Glu Glu Gln Ala Arg Leu Tyr Leu Arg Asp Val Ile Leu Gly
260 265 270
Leu Glu Tyr Leu His Cys Gln Lys Ile Val His Arg Asp Ile Lys
275 280 285
Pro Ser Asn Leu Leu Leu Gly Asp Asp Gly His Val Lys Ile Ala
290 295 300
Asp Phe Gly Val Ser Asn Gln Phe Glu Gly Asn Asp Ala Gln Leu
305 310 315
Ser Ser Thr Ala Gly Thr Pro Ala Phe Met Ala Pro Glu Ala Ile
320 325 330
Ser Asp Ser Gly Gln Ser Phe Ser Gly Lys Ala Leu Asp Val Trp
335 340 345
Ala Thr Gly Val Thr Leu Tyr Cys Phe Val Tyr Gly Lys Cys Pro
350 355 360
Phe Ile Asp Asp Phe Ile Leu Ala Leu His Arg Lys Ile Lys Asn
365 370 375
Glu Pro Val Val Phe Pro Glu Glu Pro Glu Ile Ser Glu Glu Leu
380 385 390
Lys Asp Leu Ile Leu Lys Met Leu Asp Lys Asn Pro Glu Thr Arg
395 400 405
Ile Gly Val Pro Asp Ile Lys Leu His Pro Trp Val Thr Lys Asn
410 415 420
Gly Glu Glu Pro Leu Pro Ser Glu Glu Glu His Cys Ser Val Val
425 430 435
Glu Val Thr Glu Glu Glu Val Lys Asn Ser Val Arg Leu Ile Pro
440 445 450
Ser Trp Thr Thr Val Ile Leu Val Lys Ser Met Leu Arg Lys Arg
455 460 465
Ser Phe Gly Asn Pro Phe Glu Pro Gln Ala Arg Arg Glu Glu Arg
470 475 480
Ser Met Ser Ala Pro Gly Asn Leu Leu Val Lys Glu Gly Phe Gly
485 490 495
Glu Gly Gly Lys Ser Pro Glu Leu Pro Gly Val Gln Glu Asp Glu
500 505 510
Ala Ala Ser
<210> 3
<211> 1012
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472005CD1
<400> 3
Met Ala Pro Ala Arg Gly Arg Leu Pro Pro Ala Leu Trp Val Val
1 5 10 15
Thr Ala Ala Ala Ala Ala Ala Thr Cys Val Ser Ala Ala Arg Gly
20 25 30
Glu Val Asn Leu Leu Asp Thr Ser Thr Ile His Gly Asp Trp Gly
35 40 45
Trp Leu Thr Tyr Pro Ala His Gly Trp Asp Ser Ile Asn Glu Val
50 55 60
Asp Glu Ser Phe Gln Pro Ile His Thr Tyr Gln Val Cys Asn Val
65 70 75
3126
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Met Ser Pro Asn Gln Asn Asn Trp Leu Arg Thr Ser Trp Val Pro
80 85 90
Arg Asp Gly Ala Arg Arg Val Tyr Ala Glu Ile Lys Phe Thr Leu
95 100 105
Arg Asp Cys Asn Ser Met Pro Gly Val Leu Gly Thr Cys Lys Glu
110 115 120
Thr Phe Asn Leu Tyr Tyr Leu Glu Ser Asp Arg Asp Leu Gly Ala
125 130 135
Ser Thr Gln Glu Ser Gln Phe Leu Lys Ile Asp Thr Ile Ala Ala
140 145 150
Asp Glu Ser Phe Thr Gly Ala Asp Leu Gly Val Arg Arg Leu Lys
155 160 165
Leu Asn Thr Glu Val Arg Ser Val Gly Pro Leu Ser Lys Arg Gly
170 175 180
Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Leu Ala Ile Leu
185 190 195
Ser Leu Arg Ile Tyr Tyr Lys Lys Cys Pro Ala Met Val Arg Asn
200 205 210
Leu Ala Ala Phe Ser Glu Ala Val Thr Gly Ala Asp Ser Ser Ser
215 220 225
Leu Val Glu Val Arg Gly Gln Cys Val Arg His Ser Glu Glu Arg
230 235 240
Asp Thr Pro Lys Met Tyr Cys Ser Ala Glu Gly Glu Trp Leu Val
245 250 255
Pro Ile Gly Lys Cys Val Cys Ser Ala Gly Tyr Glu Glu Arg Arg
260 265 270
Asp Ala Cys Val Ala Cys Glu Leu Gly Phe Tyr Lys Ser Ala Pro
275 280 285
Gly Asp Gln Leu Cys Ala Arg Cys Pro Pro His Ser His Ser Ala
290 295 300
Ala Pro Ala Ala Gln Ala Cys His Cys Asp Leu Ser Tyr Tyr Arg
305 310 315
Ala Ala Leu Asp Pro Pro Ser Ser Ala Cys Thr Arg Pro Pro Ser
320 325 330
Ala Pro Val Asn Leu Ile Ser Ser Val Asn Gly Thr Ser Val Thr
335 340 345
Leu Glu Trp Ala Pro Pro Leu Asp Pro Gly Gly Arg Ser Asp Ile
350 355 360
Thr Tyr Asn Ala Val Cys Arg Arg Cys Pro Trp Ala Leu Ser Arg
365 370 375
Cys Glu Ala Cys Gly Ser Gly Thr Arg Phe Val Pro Gln Gln Thr
380 385 390
Ser Leu Val Gln Ala Ser Leu Leu Val Ala Asn Leu Leu Ala His
395 400 405
Met Asn Tyr Ser Phe Trp Ile Glu Ala Val Asn Gly Val Ser Asp
410 415 420
Leu Ser Pro Glu Pro Arg Arg Ala Ala Val Val Asn Ile Thr Thr
425 430 435
Asn Gln Ala Ala Pro Ser Gln Val Val Val Ile Arg Gln Glu Arg
440 445 450
Ala Gly Gln Thr Ser Val Ser Leu Leu Trp Gln Glu Pro Glu Gln
455 460 465
Pro Asn Gly Ile Ile Leu Glu Tyr Glu Ile Lys Tyr Tyr Glu Lys
470 475 480
Asp Lys Glu Met Gln Ser Tyr Ser Thr Leu Lys Ala Val Thr Thr
485 490 495
Arg Ala Thr Val Ser Gly Leu Lys Pro Gly Thr Arg Tyr Val Phe
500 505 510
Gln Val Arg Ala Arg Thr Ser Ala Gly Cys Gly Arg Phe Ser Gln
515 520 525
Ala Met Glu Val Glu Thr Gly Lys Pro Arg Pro Arg Tyr Asp Thr
530 535 540
Arg Thr Ile Val Trp Ile Cys Leu Thr Leu Ile Thr Gly Leu Val
545 550 555
Val Leu Leu Leu Leu Leu Ile Cys Lys Lys Arg His Cys Gly Tyr
560 565 570
Ser Lys Ala Phe Gln Asp Ser Asp Glu Glu Lys Met His Tyr Gln
4/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
575 580 585
Asn Gly Gln Ala Pro Pro Pro Val Phe Leu Pro Leu His His Pro
590 595 600
Pro Gly Lys Leu Pro Glu Pro Gln Phe Tyr Ala Glu Pro His Thr
605 610 615
Tyr Glu Glu Pro Gly Arg Ala Gly Arg Ser Phe Thr Arg Glu Ile
620 625 630
Glu Ala Ser Arg Ile His Ile Glu Lys Ile Ile Gly Ser Gly Asp
635 640 645
Ser Gly Glu Val Cys Tyr Gly Arg Leu Arg Val Pro Gly Gln Arg
650 655 660
Asp Val Pro Val Ala Ile Lys Ala Leu Lys Ala Gly Tyr Thr Glu
665 670 675
Arg Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser Ile Met Gly Gln
680 685 690
Phe Asp His Pro Asn Ile Ile Arg Leu Glu Gly Val Val Thr Arg
695 700 705
Gly Arg Leu Ala Met Ile Val Thr Glu Tyr Met Glu Asn Gly Ser
710 715 720
Leu Asp Thr Phe Leu Arg Thr His Asp Gly Gln Phe Thr Ile Met
725 730 735
Gln Leu Val Gly Met Leu Arg Gly Val Gly Ala Gly Met Arg Tyr
740 745 750
Leu Ser Asp Leu Gly Tyr Val His Arg Asp Leu Ala Ala Arg Asn
755 760 765
Val Leu Val Asp Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly
770 775 780
Leu Ser Arg Val Leu Glu Asp Asp Pro Asp Ala Ala Tyr Thr Thr
785 790 795
Thr Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile
800 805 810
Ala Phe Arg Thr Phe Ser Ser Ala Ser Asp Val Trp Ser Phe Gly
815 820 825
Val Val Met Trp Glu Val Leu Ala Tyr Gly Glu Arg Pro Tyr Trp
830 835 840
Asn Met Thr Asn Arg Asp Val Ser Ala Lys Pro Trp Gln Val Ile
845 850 855
Ser Ser Val Glu Glu Gly Tyr Arg Leu Pro Ala Pro Met Gly Cys
860 865 870
Pro His Ala Leu His Gln Leu Met Leu Asp Cys Trp His Lys Asp
875 880 885
Arg Ala Gln Arg Pro Arg Phe Ser Gln Ile Val Ser Val Leu Asp
890 895 900
Ala Leu Ile Arg Ser Pro Glu Ser Leu Arg Ala Thr Ala Thr Val
905 910 915
Ser Arg Cys Pro Pro Pro Ala Phe Val Arg Ser Cys Phe Asp Leu
920 925 930
Arg Gly Gly Ser Gly Gly Gly Gly Gly Leu Thr Val Gly Asp Trp
935 940 945
Leu Asp Ser Ile Arg Met Gly Arg Tyr Arg Asp His Phe Ala Ala
950 955 960
Gly Gly Tyr Ser Ser Leu Gly Met Val Leu Arg Met Asn Ala Gln
965 970 975
Asp Val Arg Ala Leu Gly Ile Thr Leu Met Gly His Gln Lys Lys
980 985 990
Ile Leu Gly Ser Ile Gln Thr Met Arg Ala Gln Leu Thr Ser Thr
995 1000 1005
Gln Gly Pro Arg Arg His Leu
1010
<210> 4
<211> 367
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
5/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
<223> Incyte ID No: 7472006CD1
<400> 4
Met Asp Asp Ala Ala Val Leu Lys Arg Arg Gly Tyr Leu Leu Gly
1 5 10 15
Ile Asn Leu Gly Glu Gly Ser Tyr Ala Lys Val Lys Ser Ala Tyr
20 25 30
Ser Glu Arg Leu Lys Phe Asn Val Ala ,Ile Lys Ile Ile Asp Arg
35 40 45
Lys Lys Ala Pro Ala Asp Phe Leu Glu Lys Phe Leu Pro Arg Glu
50 55 60
Ile Glu Ile Leu Ala Met Leu Asn His Cys Ser Ile Ile Lys Thr
65 70 75
Tyr Glu Ile Phe Glu Thr Ser His Gly Lys Val Tyr Ile Val Met
80 85 90
Glu Leu Ala Val Gln Gly Asp Leu Leu Glu Leu Ile Lys Thr Arg
95 100 105
Gly Ala Leu His Glu Asp Glu Ala Arg Lys Lys Phe His Gln Leu
110 115 120
Ser Leu Ala Ile Lys Tyr Cys His Asp Leu Asp Val Val His Arg
125 130 135
Asp Leu Lys Cys Asp Asn Leu Leu Leu Asp Lys Asp Phe Asn Ile
140 145 150
Lys Leu Ser Asp Phe Ser Phe Ser Lys Arg Cys Leu Arg Asp Asp
155 160 165
Ser Gly Arg Met Ala Leu Ser Lys Thr Phe Cys Gly Ser Pro Ala
170 175 ' 180
Tyr Ala Ala Pro Glu Val Leu Gln Gly Ile Pro Tyr Gln Pro Lys
185 190 195
Val Tyr Asp Ile Trp Ser Leu Gly Val Ile Leu Tyr Ile Met Val
200 205 210
Cys Gly Ser Met Pro Tyr Asp Asp Ser Asn Ile Lys Lys Met Leu
215 220 225
Arg Ile Gln Lys Glu His Arg Val Asn Phe Pro Arg Ser Lys His
230 235 240
Leu Thr Gly Glu Cys Lys Asp Leu Ile Tyr His Met Leu Gln Pro
245 250 255
Asp Val Asn Arg Arg Leu His Ile Asp Glu Ile Leu Ser His Cys
260 265 270
Trp Met Gln Pro Lys Ala Arg Gly Ser Pro Ser Val Ala Ile Asn
275 280 285
Lys Glu Gly Glu Ser Ser Arg Gly Thr Glu Pro Leu Trp Thr Pro
290 295 300
Glu Pro Gly Ser Asp Lys Lys Ser Ala Thr Lys Leu Glu Pro Glu
305 310 315
Gly Glu Ala Gln Pro Gln Ala Gln Pro Glu Thr Lys Pro Glu Gly
320 325 330
Thr Ala Met Gln Met Ser Arg Gln Ser Glu Ile Leu Gly Phe Pro
335 340 345
Ser Lys Pro Ser Thr Met Glu Thr Glu Glu Gly Pro Pro Gln Gln
350 355 360
Pro Pro Glu Thr Arg Ala Gln
365
<210> 5
<211> 798
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2902460CD1
<400> 5
Met Phe Glu Ala His Ile Gln Ala Gln Ser Ser Ala Ile Gln Ala
1 5 10 15
Pro Arg Ser Pro Arg Leu Gly Arg Ala Arg Ser Pro Ser Pro Cys
6/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
20 25 30
Pro Phe Arg Ser Ser Ser Gln Pro Pro Gly Arg Val Leu Val Gln
35 40 45
Gly Ala Arg Ser Glu Glu Arg Arg Thr Lys Ser Trp Gly Glu Gln
50 55 60
Cys Pro Glu Thr Ser Gly Thr Asp Ser Gly Arg Lys Gly Gly Pro
65 70 75
Ser Leu Cys Ser Ser Gln Val Lys Lys Gly Met Pro Pro Leu Pro
80 85 90
Gly Arg Ala Ala Pro Thr Gly Ser Glu Ala Gln Gly Pro Ser Ala
95 100 105
Phe Val Arg Met Glu Lys Gly Ile Pro Ala Ser Pro Arg Cys Gly
110 115 120
Ser Pro Thr Ala Met Glu Ile Asp Lys Arg Gly Ser Pro Thr Pro
125 130 135
Gly Thr Arg Ser Cys Leu Ala Pro Ser Leu Gly Leu Phe Gly Ala
140 145 150
Ser Leu Thr Met Ala Thr Glu Val Ala Ala Arg Val Thr Ser Thr
155 160 165
Gly Pro His Arg Pro Gln Asp Leu Ala Leu Thr Glu Pro Ser Gly
170 175 180
Arg Ala Arg Glu Leu Glu Asp Leu Gln Pro Pro Glu Ala Leu Val
185 190 195
Glu Arg Gln Gly Gln Phe Leu Gly Ser Glu Thr Ser Pro Ala Pro
200 205 210
Glu Arg Gly Gly Pro Arg Asp Gly Glu Pro Pro Gly Lys Met Gly
215 220 225
Lys Gly Tyr Leu Pro Cys Gly Met Pro Gly Ser Gly Glu Pro Glu
230 235 240
Val Gly Lys Arg Pro Glu Glu Thr Thr Val Ser Val Gln Ser Ala
245 250 255
Glu Ser Ser Asp Ala Leu Ser Trp Ser Arg Leu Pro Arg Ala Leu
260 265 270
Ala Ser Val Gly Pro Glu Glu Ala Arg Ser Gly Ala Pro Val Gly
275 280 285
Gly Gly Arg Trp Gln Leu Ser Asp Arg Val Glu Gly Gly Ser Pro
290 295 300
Thr Leu Gly Leu Leu Gly Gly Ser Pro Ser Ala Gln Pro Gly Thr
305 310 315
Gly Asn Val Glu Ala Gly Ile Pro Ser Gly Arg Met Leu Glu Pro
320 325 330
Leu Pro Cys Trp Asp Ala Ala Lys Asp Leu Lys Glu Pro Gln Cys
335 340 345
Pro Pro Gly Asp Arg Val Gly Val Gln Pro Gly Asn Ser Arg Val
350 355 360
Trp Gln Gly Thr Met Glu Lys Ala Gly Leu Ala Trp Thr Arg Gly
365 370 375
Thr Gly Val Glri Ser Glu Gly Thr Trp Glu Ser Gln Arg Gln Asp
380 385 390
Ser Asp Ala Leu Pro Ser Pro Glu Leu Leu Pro Gln Asp Gln Asp
395 400 405
Lys Pro Phe Leu Arg Lys Ala Cys Ser Pro Ser Asn Ile Pro Ala
410 415 420
Val Ile Ile Thr Asp Met Gly Thr Gln Glu Asp Gly Ala Leu Glu
425 430 435
Glu Thr Gln Gly Ser Pro Arg Gly Asn Leu Pro Leu Arg Lys Leu
440 445 450
Ser Ser Ser Ser Ala Ser Ser Thr Gly Phe Ser Ser Ser Tyr Glu
455 460 465
Asp Ser Glu Glu Asp Ile Ser Ser Asp Pro Glu Arg Thr Leu Asp
470 475 480
Pro Asn Ser Ala Phe Leu His Thr Leu Asp Gln Gln Lys Pro Arg
485 490 495
Val Ser Lys Ser Trp Arg Lys Ile Lys Asn Met Val His Trp Ser
500 505 510
Pro Phe Val Met Ser Phe Lys Lys Lys Tyr Pro Trp Ile Gln Leu
515 520 525
7/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Ala Gly His Ala Gly Ser Phe Lys Ala Ala Ala Asn Gly Arg Ile
530 535 540
Leu Lys Lys His Cys Glu Ser Glu Gln Arg Cys Leu Asp Arg Leu
545 550 555
Met Val Asp Val Leu Arg Pro Phe Val Pro Ala Tyr His Gly Asp
560 565 570
Val Val Lys Asp Gly Glu Arg Tyr Asn Gln Met Asp Asp Leu Leu
575 580 585
Ala Asp Phe Asp Ser Pro Cys Val Met Asp Cys Lys Met Gly Ile
590 595 600
Arg Thr Tyr Leu Glu Glu Glu Leu Thr Lys Ala Arg Lys Lys Pro
605 610 615
Ser Leu Arg Lys Asp Met Tyr Gln Lys Met Ile Glu Val Asp Pro
620 625 630
Glu Ala Pro Thr Glu Glu Glu Lys Ala Gln Arg Ala Val Thr Lys
635 640 645
Pro Arg Tyr Met Gln Trp Arg Glu Thr Ile Ser Ser Thr Ala Thr
650 655 660
Leu Gly Phe Arg Ile Glu Gly Ile Lys Lys Glu Asp Gly Thr Val
665 670 675
Asn Arg Asp Phe Lys Lys Thr Lys Thr Arg Glu Gln Val Thr Glu
680 685 690
Ala Phe Arg Glu Phe Thr Lys Gly Asn His Asn Ile Leu Ile Ala
695 700 705
Tyr Arg Asp Arg Leu Lys Ala Ile Arg Thr Thr Leu Glu Val Ser
710 715 720
Pro Phe Phe Lys Cys His Glu Val Ile Gly Ser Ser Leu Leu Phe
725 730 735
Ile His Asp Lys Lys Glu Gln Ala Lys Val Trp Met Ile Asp Phe
740 745 750
Gly Lys Thr Thr Pro Leu Pro Glu Gly Gln Thr Leu Gln His Asp
755 760 765
Val Pro Trp Gln Glu Gly Asn Arg Glu Asp Gly Tyr Leu Ser Gly
770 775 780
Leu Asn Asn Leu Val Asp Ile Leu Thr Glu Met Ser Gln Asp Ala
785 790 795
Pro Leu Ala
<210> 6
<211> 358
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6383934CD1
<400> 6
Met Asp Asp Ala Thr Val Leu Arg Lys Lys Gly Tyr Ile Val Gly
1 5 10 15
Ile Asn Leu Gly Lys Gly Ser Tyr Ala Lys Val Lys Ser Ala Tyr
20 25 30
Ser Glu Arg Leu Lys Phe Asn Val Ala Val Lys Ile Ile Asp Arg
35 40 45
Lys Lys Thr Pro Thr Asp Phe Val Glu Arg Phe Leu Pro Arg Glu
50 55 60
Met Asp Ile Leu Ala Thr Val Asn His Gly Ser Ile Ile Lys Thr
65 70 75
Tyr Glu Ile Phe Glu Thr Ser Asp Gly Arg Ile Tyr Ile Ile Met
80 85 90
Glu Leu Gly Val Gln Gly Asp Leu Leu Glu Phe Ile Lys Cys Gln
95 100 105
Gly Ala Leu His Glu Asp Val Ala Arg Lys Met Phe Arg Gln Leu
110 115 120
Ser Ser Ala Val Lys Tyr Cys His Asp Leu Asp Ile Val His Arg
125 130 135
8/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Asp Leu Lys Cys Glu Asn Leu Leu Leu Asp Lys Asp Phe Asn Ile
140 145 150
Lys Leu Ser Asp Phe Gly Phe Ser Lys Arg Cys Leu Arg Asp Ser
155 160 165
Asn Gly Arg Ile Ile Leu Ser Lys Thr Phe Cys Gly Ser Ala Ala
170 175 180
Tyr Ala Ala Pro Glu Val Leu Gln Ser Ile Pro Tyr Gln Pro Lys
185 190 195
Val Tyr Asp Ile Trp Ser Leu Gly Val Ile Leu Tyr Ile Met Val
200 205 210
Cys Gly Ser Met Pro Tyr Asp Asp Ser Asp Ile Lys Lys Met Leu
215 220 225
Arg Ile Gln Lys Glu His Arg Val Asn Phe Pro Arg Ser Lys His
230 235 240
Leu Thr Cys Glu Cys Lys Asp Leu Ile Tyr His Met Leu Gln Pro
245 250 255
Asp Val Ser Gln Arg Leu His Ile Asp Glu Ile Leu Ser His Ser
260 265 270
Trp Leu Gln Pro Pro Lys Pro Lys Ala Thr Ser Ser Ala Ser Phe
275 280 285
Lys Arg Glu Gly Glu Gly Lys Tyr Arg Ala Glu Cys Lys Leu Asp
290 295 300
Thr Lys Thr Gly Leu Arg Pro Asp His Arg Pro Asp His Lys Leu
305 310 315
Gly Ala Lys Thr Gln His Arg Leu Leu Val Val Pro Glu Asn Glu
320 325 330
Asn Arg Met Glu Asp Arg Leu Ala Glu Thr Ser Arg Ala Lys Asp
335 340 345
His His Ile Ser Gly Ala Glu Val Gly Lys Ala Ser Thr
350 355
<210> 7
<211> 1049
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3210906CD1
<400> 7
Met Pro Ala Gly Gly Arg Ala Gly Ser Leu Lys Asp Pro Asp Val
1 5 10 15
Ala Glu Leu Phe Phe Lys Asp Asp Pro Glu Lys Leu Phe Ser Asp
20 25 30
Leu Arg Glu Ile Gly His Gly Ser Phe Gly Ala Val Tyr Phe Ala
35 40 45
Arg Asp Val Arg Asn Ser Glu Val Val Ala Ile Lys Lys Met Ser
50 55 60
Tyr Ser Gly Lys Gln Ser Asn Glu Lys Trp Gln Asp Ile Ile Lys
65 70 75
Glu Val Arg Phe Leu Gln Lys Leu Arg His Pro Asn Thr Ile Gln
80 85 90
Tyr Arg Gly Cys Tyr Leu Arg Glu His Thr Ala Trp Leu Val Met
95 100 105
Glu Tyr Cys Leu Gly Ser Thr Ser Asp Leu Leu Glu Val His Lys
110 115 120
Lys Pro Leu Gln Glu Val Glu Ile Ala Ala Val Thr His Gly Ala
125 130 135
Leu Gln Gly Leu Ala Tyr Leu His Ser His Asn Met Ile His Arg
140 145 150
Asp Val Lys Ala Gly Asn Ile Leu Leu Ser Glu Pro Gly Leu Val
155 160 165
Lys Leu Gly Asp Phe Gly Ser Ala Ser Ile Met Ala Pro Ala Asn
170 175 180
Ser Phe Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Leu
185 190 195
9/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Ala Met Asp Glu Gly Gln Tyr Asp Gly Lys Val Asp Val Trp Ser
200 205 210
Leu Gly Ile Thr Cys Ile Glu Leu Ala Glu Arg Lys Pro Pro Leu
215 220 225
Phe Asn Met Asn Ala Met Ser Ala Leu Tyr His Ile Ala Gln Asn
230 235 240
Glu Ser Pro Val Leu Gln Ser Gly His Trp Ser Glu Tyr Phe Arg
245 250 255
Asn Phe Val Asp Ser Cys Leu Gln Lys Ile Pro Gln Asp Arg Pro
260 265 270
Thr Ser Glu Val Leu Leu Lys His Arg Phe Val Leu Arg Glu Arg
275 280 285
Pro Pro Thr Val Ile Met Asp Leu Ile Gln Arg Thr Lys Asp Ala
290 295 300
Val Arg Glu Leu Asp Ser Leu Gln Tyr Arg Lys Met Lys Lys Ile
305 310 315
Leu Phe Gln Glu Ala Pro Asn Gly Pro Gly Ala Glu Ala Pro Glu
320 325 330
Glu Glu Glu Glu Ala Glu Pro Tyr Met His Leu Ala Gly Thr Leu
335 340 345
Thr Ser Leu Glu Ser Ser His Ser Val Pro Ser Met Ser Ile Ser
350 355 360
Ala Ser Ser Gln Ser Ser Ser Val Asn Ser Leu Ala Asp Ala Ser
365 370 375
Asp Asn Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
380 385 390
Glu Glu Gly Pro Glu Ala Arg Glu Met Ala Met Met Gln Glu Gly
395 400 405
Glu His Thr Val Thr Ser His Ser Ser Ile Ile His Arg Leu Pro
410 415 420
Gly Ser Asp Asn Leu Tyr Asp Asp Pro Tyr Gln Pro Glu Ile Thr
425 ~ 430 435
Pro Ser Pro Leu Gln Pro Pro Ala Ala Pro Ala Pro Thr Ser Thr
440 445 450
Thr Ser Ser Ala Arg Arg Arg Ala Tyr Cys Arg Asn Arg Asp His
455 460 465
Phe Ala Thr Ile Arg Thr Ala Ser Leu Val Ser Arg Gln Ile Gln
470 475 480
Glu His Glu Gln Asp Ser Ala Leu Arg Glu Gln Leu Ser Gly Tyr
485 490 495
Lys Arg Met Arg Arg Gln His Gln Lys Gln Leu Leu Ala Leu Glu
500 505 510
Ser Arg Leu Arg Gly Glu Arg Glu Glu His Ser Ala Arg Leu Gln
515 520 525
Arg Glu Leu Glu Ala Gln Arg Ala Gly Phe Gly Ala Glu Ala Glu
530 535 540
Lys Leu Ala Arg Arg His Gln Ala Ile Gly Glu Lys Glu Ala Arg
545 550 555
Ala Ala Gln Ala Glu Glu Arg Lys Phe Gln Gln His Ile Leu Gly
560 565 570
Gln Gln Lys Lys Glu Leu Ala Ala Leu Leu Glu Ala Gln Lys Arg
575 580 585
Thr Tyr Lys Leu Arg Lys Glu Gln Leu Lys Glu Glu Leu Gln Glu
590 595 600
Asn Pro Ser Thr Pro Lys Arg Glu Lys Ala Glu Trp Leu Leu Arg
605 610 615
Gln Lys Glu Gln Leu Gln Gln Cys Gln Ala Glu Glu Glu Ala Gly
620 625 630
Leu Leu Arg Arg Gln Arg Gln Tyr Phe Glu Leu Gln Cys Arg Gln
635 640 645
Tyr Lys Arg Lys Met Leu Leu Ala Arg His Ser Leu Asp Gln Asp
650 655 660
Leu Leu Arg Glu Asp Leu Asn Lys Lys Gln Thr Gln Lys Asp Leu
665 670 675
Glu Cys Ala Leu Leu Leu Arg Gln His Glu Ala Thr Arg Glu Leu
680 685 690
Glu Leu Arg Gln Leu Gln Ala Val Gln Arg Thr Arg Ala Glu Leu
10/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
695 700 705
Thr Arg Leu Gln His Gln Thr Glu Leu Gly Asn Gln Leu Glu Tyr
710 715 720
Asn Lys Arg Arg Glu Gln Glu Leu Arg Gln Lys His Ala Ala Gln
725 730 735
Val Arg Gln Gln Pro Lys Ser Leu Lys Ser Lys Glu Leu Gln Ile
740 745 750
Lys Lys Gln Phe Gln Glu Thr Cys Lys Ile Gln Thr Arg Gln Tyr
755 760 765
Lys Ala Leu Arg Ala His Leu Leu Glu Thr Thr Pro Lys Ala Gln
770 775 780
His Lys Ser Leu Leu Lys Arg Leu Lys Glu Glu Gln Thr Arg Lys
785 790 795
Leu Ala Ile Leu Ala Glu Gln Tyr Asp Gln Ser Ile Ser Glu Met
800 805 810
Leu Ser Ser Gln Ala Leu Arg Leu Asp Glu Thr Gln Glu Ala Glu
815 820 825
Phe Gln Ala Leu Arg Gln Gln Leu Gln Gln Glu Leu Glu Leu Leu
830 835 840
Asn Ala Tyr Gln Ser Lys Ile Lys Ile Arg Thr Glu Ser Gln His
845 850 855
Glu Arg Glu Leu Arg Glu Leu Glu Gln Arg Val Ala Leu Arg Arg
860 865 870
Ala Leu Leu Glu Gln Arg Val Glu Glu Glu Leu Leu Ala Leu Gln
875 880 885
Thr Gly Arg Ser Glu Arg Ile Arg Ser Leu Leu Glu Arg Gln Ala
890 895 900
Arg Glu Ile Glu Ala Phe Asp Ala Glu Ser Met Arg Leu Gly Phe
905 910 915
Ser Ser Met Ala Leu Gly Gly Ile Pro Ala Glu Ala Ala Ala Gln
920 925 930
Gly Tyr Pro Ala Pro Pro Pro Ala Pro Ala Trp Pro Ser Arg Pro
935 940 945
Val Pro Arg Ser Gly Ala His Trp Ser His Gly Pro Pro Pro Pro
950 955 960
Gly Met Pro Pro Pro Ala Trp Arg Gln Pro Ser Leu Leu Ala Pro
965 970 975
Pro Gly Pro Pro Asn Trp Leu Gly Pro Pro Thr Gln Ser Gly Thr
980 985 990
Pro Arg Gly Gly Ala Leu Leu Leu Leu Arg Asn Ser Pro Gln Pro
995 1000 1005
Leu Arg Arg Ala Ala Ser Gly Gly Ser Gly Ser Glu Asn Val Gly
1010 1015 1020
Pro Pro Ala Ala Ala Val Pro Gly Pro Leu Ser Arg Ser Thr Ser
1025 1030 1035
Val Ala Ser His Ile Leu Asn Gly Ser Ser His Phe Tyr Ser
1040 1045
<210> 8
<211> 322
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3339024CD1
<400> 8
Met Pro Thr Phe Ser Ile Pro Gly Thr Leu Glu Ser Gly His Pro
1 5 10 15
Arg Asn Leu Thr Cys Ser Val Pro Trp Ala Cys Glu Gln Gly Thr
20 25 30
Pro Pro Thr Ile Thr Trp Met Gly Ala Ser Val Ser Ser Leu Asp
35 40 45
Pro Thr Ile Thr Arg Ser Ser Met Leu Ser Leu Ile Pro Gln Pro
50 55 60
Gln Asp His Gly Thr Ser Leu Thr Cys Gln Val Thr Leu Pro Gly
11 /26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
65 70 75
Ala Gly Val Thr Met Thr Arg Ala Val Arg Leu Asn Ile Ser Tyr
80 85 90
Pro Pro Gln Asn Leu Thr Met Thr Val Phe Gln Gly Asp Gly Thr
95 100 105
Ala Ser Thr Thr Leu Arg Asn Gly Ser Ala Leu Ser Val Leu Glu
110 115 120
Gly Gln Ser Leu His Leu Val Cys Ala Val Asp Ser Asn Pro Pro
125 130 135
Ala Arg Leu Ser Trp Thr Trp Gly Ser Leu Thr Leu Ser Pro Ser
140 145 150
Gln Ser Ser Asn Leu Gly Val Leu Glu Leu Pro Arg Val His Val
155 160 165
Lys Asp Glu Gly Glu Phe Thr Cys Arg Ala Gln Asn Pro Leu Gly
170 175 180
Ser Gln His Ile Ser Leu Ser Leu Ser Leu Gln Asn Glu Tyr Thr
185 190 195
Gly Lys Met Arg Pro Ile Ser Gly Val Thr Leu Gly Ala Phe Gly
200 205 210
Gly Ala Gly Ala Thr Ala Leu Val Phe Leu Tyr Phe Cys Ile Ile
215 220 225
Phe Val Val Val Arg Ser Cys Arg Lys Lys Ser Ala Arg Pro Ala
230 235 240
Val Gly Val Gly Asp Thr Gly Met Glu Asp Ala Asn Ala Val Trp
245 250 255
Gly Ser Ala Ser Gln Gly Pro Leu Ile Glu Ser Pro Ala Asp Asp
260 265 270
Ser Pro Pro His His Ala Pro Pro Ala Leu Ala Thr Pro Ser Pro
275 280 285
Glu Glu Gly Glu Ile Gln Tyr Ala Ser Leu Ser Phe His Lys Ala
290 295 300
Arg Pro Gln Tyr Pro Gln Glu Gln Glu Ala Ile Gly Tyr Glu Tyr
305 310 315
Ser Glu Ile Asn Ile Pro Lys
320
<210> 9
<211> 1212
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4436929CD1
<400> 9
Met Ala Asn Asp Ser Pro Ala Lys Ser Leu Val Asp Ile Asp Leu
1 5 10 15
Ser Ser Leu Arg Asp Pro Ala Gly Ile Phe Glu Leu Val Glu Val
20 25 30
Val Gly Asn Gly Thr Tyr Gly Gln Val Tyr Lys Gly Arg His Val
35 40 45
Lys Thr Gly Gln Leu Ala Ala Ile Lys Val Met Asp Val Thr Glu
50 55 60
Asp Glu Glu Glu Glu Ile Lys Leu Glu Ile Asn Met Leu Lys Lys
65 70 75
Tyr Ser His His Arg Asn Ile Ala Thr Tyr Tyr Gly Ala Phe Ile
80 85 90
Lys Lys Ser Pro Pro Gly His Asp Asp Gln Leu Trp Leu Val Met
95 100 105
Glu Phe Cys Gly Ala Gly Ser Ile Thr Asp Leu Val Lys Asn Thr
110 115 120
Lys Gly Asn Thr Leu Lys Glu Asp Trp Ile Ala Tyr Ile Ser Arg
125 130 135
Glu Ile Leu Arg Gly Leu Ala His Leu His Ile His His Val Ile
140 145 150
His Arg Asp Ile Lys Gly Gln Asn Val Leu Leu Thr Glu Asn Ala
12/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
155 160 165
Glu Val Lys Leu Val Asp Phe Gly Val Ser Ala Gln Leu Asp Arg
170 175 180
Thr Val Gly Arg Arg Asn Thr Phe Ile Gly Thr Pro Tyr Trp Met
185 190 195
Ala Pro Glu Val Ile Ala Cys Asp Glu Asn Pro Asp Ala Thr Tyr
200 205 210
Asp Tyr Arg Ser Asp Leu Trp Ser Cys Gly Ile Thr Ala Ile Glu
215 220 225
Met Ala Glu Gly Ala Pro Pro Leu Cys Asp Met His Pro Met Arg
230 235 240
Ala Leu Phe Leu Ile Pro Arg Asn Pro Pro Pro Arg Leu Lys Ser
245 250 255
Lys Lys Trp Ser Lys Lys Phe Phe Ser Phe Ile Glu Gly Cys Leu
260 265 270
Val Lys Asn Tyr Met Gln Arg Pro Ser Thr Glu Gln Leu Leu Lys
275 280 285
His Pro Phe Ile Arg Asp Gln Pro Asn Glu Arg Gln Val Arg Ile
290 295 300
Gln Leu Lys Asp His Ile Asp Arg Thr Arg Lys Lys Arg Gly Glu
305 310 315
Lys Asp Glu Thr Glu Tyr Glu Tyr Ser Gly Ser Glu Glu Glu Glu
320 325 330
Glu Glu Val Pro Glu Gln Glu Gly Glu Pro Ser Ser Ile Val Asn
335 340 345
Val Pro Gly Glu Ser Thr Leu Arg Arg Asp Phe Leu Arg Leu Gln
350 355 360
Gln Glu Asn Lys Glu Arg Ser Glu Ala Leu Arg Arg Gln Gln Leu
365 370 375
Leu Gln Glu Gln Gln Leu Arg Glu Gln Glu Glu Tyr Lys Arg Gln
380 385 390
Leu Leu Ala Glu Arg Gln Lys Arg Ile Glu Gln Gln Lys Glu Gln
395 400 405
Arg Arg Arg Leu Glu Glu Gln Gln Arg Arg Glu Arg Glu Ala Arg
410 415 420
Arg Gln Gln Glu Arg Glu Gln Arg Arg Arg Glu Gln Glu Glu Lys
425 430 435
Arg Arg Leu Glu Glu Leu Glu Arg Arg Arg Lys Glu Glu Glu Glu
440 445 450
Arg Arg Arg Ala Glu Glu Glu Lys Arg Arg Val Glu Arg Glu Gln
455 460 465
Glu Tyr Ile Arg Arg Gln Leu Glu Glu Glu Gln Arg His Leu Glu
470 475 480
Val Leu Gln Gln Gln Leu Leu Gln Glu Gln Ala Met Leu Leu His
485 490 495
Asp His Arg Arg Pro His Pro Gln His Ser Gln Gln Pro Pro Pro
500 505 510
Pro Gln Gln Glu Arg Ser Lys Pro Ser Phe His Ala Pro Glu Pro
515 520 525
Lys Ala His Tyr Glu Pro Ala Asp Arg Ala Arg Glu Val Glu Asp
530 535 540
Arg Phe Arg Lys Thr Asn His Ser Ser Pro Glu Ala Gln Ser Lys
545 550 555
Gln Thr Gly Arg Val Leu Glu Pro Pro Val Pro Ser Arg Ser Glu
560 565 570
Ser Phe Ser Asn Gly Asn Ser Glu Ser Val His Pro Ala Leu Gln
575 580 585
Arg Pro Ala Glu Pro Gln Val Pro Val Arg Thr Thr Ser Arg Ser
590 595 600
Pro Val Leu Ser Arg Arg Asp Ser Pro Leu Gln Gly Ser Gly Gln
605 610 615
Gln Asn Ser Gln Ala Gly Gln Arg Asn Ser Thr Ser Ser Ile Glu
620 625 630
Pro Arg Leu Leu Trp Glu Arg Val Glu Lys Leu Val Pro Arg Pro
635 640 645
Gly Ser Gly Ser Ser Ser Gly Ser Ser Asn Ser Gly Ser Gln Pro
650 655 660
13/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Gly Ser His Pro Gly Ser Gln Ser Gly Ser Gly Glu Arg Phe Arg
665 670 675
Val Arg Ser Ser Ser Lys Ser Glu Gly Ser Pro Ser Gln Arg Leu
680 685 690
Glu Asn Ala Val Lys Lys Pro Glu Asp Lys Lys Glu Val Phe Arg
695 700 705
Pro Leu Lys Pro Ala Gly Glu Val Asp Leu Thr Ala Leu Ala Lys
710 715 720
Glu Leu Arg Ala Val Glu Asp Val Arg Pro Pro His Lys Val Thr
725 730 735
Asp Tyr Ser Ser Ser Ser Glu Glu Ser Gly Thr Thr Asp Glu Glu
740 745 750
Asp Asp Asp Val Glu Gln Glu Gly Ala Asp Glu Ser Thr Ser Gly
755 760 765
Pro Glu Asp Thr Arg Ala Ala Ser Ser Leu Asn Leu Ser Asn Gly
770 775 780
Glu Thr Glu Ser Val Lys Thr Met Ile Val His Asp Asp Val Glu
785 790 795
Ser Glu Pro Ala Met Thr Pro Ser Lys Glu Gly Thr Leu Ile Val
800 805 810
Arg Gln Thr Gln Ser Ala Ser Ser Thr Leu Gln Lys His Lys Ser
815 820 825
Ser Ser Ser Phe Thr Pro Phe Ile Asp Pro Arg Leu Leu Gln Ile
830 835 840
Ser Pro Ser Ser Gly Thr Thr Val Thr Ser Val Val Gly Phe Ser
845 850 855
Cys Asp Gly Met Arg Pro Glu Ala Ile Arg Gln Asp Pro Thr Arg
860 865 870
Lys Gly Ser Val Val Asn Val Asn Pro Thr Asn Thr Arg Pro Gln
875 880 885
Ser Asp Thr Pro Glu Ile Arg Lys Tyr Lys Lys Arg Phe Asn Ser
890 895 900
Glu Ile Leu Cys Ala Ala Leu Trp Gly Val Asn Leu Leu Val Gly
905 910 915
Thr Glu Ser Gly Leu Met Leu Leu Asp Arg Ser Gly Gln Gly Lys
920 925 930
Val Tyr Pro Leu Ile Asn Arg Arg Arg Phe Gln Gln Met Asp Val
935 940 945
Leu Glu Gly Leu Asn Val Leu Val Thr Ile Ser Gly Lys Lys Asp
950 955 960
Lys Leu Arg Val Tyr Tyr Leu Ser Trp Leu Arg Asn Lys Ile Leu
965 970 975
His Asn Asp Pro Glu Val Glu Lys Lys Gln Gly Trp Thr Thr Val
980 985 990
Gly Asp Leu Glu Gly Cys Val His Tyr Lys Val Val Lys Tyr Glu
995 1000 1005
Arg Ile Lys Phe Leu Val Ile Ala Leu Lys Ser Ser Val Glu Val
1010 1015 1020
Tyr Ala Trp Ala Pro Lys Pro Tyr His Lys Phe Met Ala Phe Lys
1025 1030 1035
Ser Phe Gly Glu Leu Val His Lys Pro Leu Leu Val Asp Leu Thr
1040 1045 1050
Val Glu Glu Gly Gln Arg Leu Lys Val Ile Tyr Gly Ser Cys Ala
1055 1060 1065
Gly Phe His Ala Val Asp Val Asp Ser Gly Ser Val Tyr Asp Ile
1070 1075 1080
Tyr Leu Pro Thr His Ile Gln Cys Ser Ile Lys Pro His Ala Ile
1085 1090 1095
Ile Ile Leu Pro Asn Thr Asp Gly Met Glu Leu Leu Val Cys Tyr
1100 1105 1110
Glu Asp Glu Gly Val Tyr Val Asn Thr Tyr Gly Arg Ile Thr Lys
1115 1120 1125
Asp Val Val Leu Gln Trp Gly Glu Met Pro Thr Ser Val Ala Tyr
1130 1135 1140
Ile Arg Ser Asn Gln Thr Met Gly Trp Gly Glu Lys Ala Ile Glu
1145 1150 1155
Ile Arg Ser Val Glu Thr Gly His Leu Asp Gly Val Phe Met His
14/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
1160 1165 1170
Lys Arg Ala Gln Arg Leu Lys Phe Leu Cys Glu Arg Asn Asp Lys
1175 1180 1185
Val Phe Phe Ala Ser Val Arg Ser Gly Gly Ser Ser Gln Val Tyr
1190 1195 1200
Phe Met Thr Leu Gly Arg Thr Ser Leu Leu Ser Trp
1205 1210
<210> 10
<211> 280
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5046791CD1
<400> 10
Met Gln Pro Leu Arg Val Asn Ser Gln Pro Gly Pro Gln Lys Arg
1 5 10 15
Cys Leu Phe Val Cys Arg His Gly Glu Arg Met Asp Val Val Phe
20 25 30
Gly Lys Tyr Trp Leu Ser Gln Cys Phe Asp Ala Lys Gly Arg Tyr
35 40 45
Ile Arg Thr Asn Leu Asn Met Pro His Ser Leu Pro Gln Arg Ser
50 55 60
Gly Gly Phe Arg Asp Tyr Glu Lys Asp Ala Pro Ile Thr Val Phe
65 70 75
Gly Cys Met Gln Ala Arg Leu Val Gly Glu Ala Leu Leu Glu Ser
80 85 90
Asn Thr Ile Ile Asp His Val Tyr Cys Ser Pro Ser Leu Arg Cys
95 100 105
Val Gln Thr Ala His Asn Ile Leu Lys Gly Leu Gln Gln Glu Asn
110 115 120
His Leu Lys Ile Arg Val Glu Pro Gly Leu Phe Glu Trp Thr Lys
125 130 135
Trp Val Ala Gly Ser Thr Leu Pro Ala Trp Ile Pro Pro Ser Glu
140 145 150
Leu Ala Ala Ala Asn Leu Ser Val Asp Thr Thr Tyr Arg Pro His
155 160 165
Ile Pro Ile Ser Lys Leu Val Val Ser Glu Ser Tyr Asp Thr Tyr
170 175 180
Ile Ser Arg Ser Phe Gln Val Thr Lys Glu Ile Ile Ser Glu Cys
185 190 195
Lys Ser Lys Gly Asn Asn Ile Leu Ile Val Ala His Ala Ser Ser
200 205 210
Leu Glu Ala Cys Thr Cys Gln Leu Gln Gly Leu Ser Pro Gln Asn
215 220 225
Ser Lys Asp Phe Val Gln Met Val Arg Lys Ile Pro Tyr Leu Gly
230 235 240
Phe Cys Ser Cys Glu Glu Leu Gly Glu Thr Gly Ile Trp Gln Leu
245 250 255
Thr Asp Pro Pro Ile Leu Pro Leu Thr His Gly Pro Thr Gly Gly
260 265 270
Phe Asn Trp Arg Glu Thr Leu Leu Gln Glu
275 280
<210> 11
<211> 114
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1416174CD1
<400> 11
15/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Met Leu Ala Ile Ser Pro Ser His Leu Gly Ala Asp Leu Val Ala
1 5 10 15
Ala Pro His Ala Arg Phe Asp Asp Gly Leu Val His Leu Cys Trp
20 25 30
Val Arg Thr Gly Ile Ser Arg Ala Ala Leu Leu Arg Leu Phe Leu
35 40 45
Ala Met Glu Arg Gly Ser His Phe Ser Leu Gly Cys Pro Gln Leu
50 55 60
Gly Tyr Ala Ala Ala Arg Ala Phe Arg Leu Glu Pro Leu Thr Pro
65 70 75
Arg Gly Val Leu Thr Val Asp Gly Glu Gln Val Glu Tyr Gly Pro
80 85 90
Leu Gln Ala Gln Met His Pro Gly Ile Gly Thr Leu Leu Thr Gly
95 100 105
Pro Pro Gly Cys Pro Gly Arg Glu Pro
110
<210> 12
<211> 375
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3244919CD1
<400> 12
Met Gly Ser Ser Met Ser Ala Ala Thr Ala Arg Arg Pro Val Phe
1 5 10 15
Asp Asp Lys Glu Asp Val Asn Phe Asp His Phe Gln Ile Leu Arg
20 25 30
Ala Ile Gly Lys Gly Ser Phe Gly Lys Val Cys Ile Val Gln Lys
35 40 45
Arg Asp Thr Glu Lys Met Tyr Ala Met Lys Tyr Met Asn Lys Gln
50 55 60
Gln Cys Ile Glu Arg Asp Glu Val Arg Asn Val Phe Arg Glu Leu
65 70 75
Glu Ile Leu Gln Glu Ile Glu His Val Phe Leu Val Asn Leu Trp
80 85 90
Tyr Ser Phe Gln Asp Glu Glu Asp Met Phe Met Val Val Asp Leu
95 100 105
Leu Leu Gly Gly Asp Leu Arg Tyr His Leu Gln Gln Asn Val Gln
110 115 120
Phe Ser Glu Asp Thr Val Arg Leu Tyr Ile Cys Glu Met Ala Leu
125 130 135
Ala Leu Asp Tyr Leu Arg Gly Gln His Ile Ile His Arg Asp Val
140 145 150
Lys Pro Asp Asn Ile Leu Leu Asp Glu Arg Gly His Ala His Leu
155 160 165
Thr Asp Phe Asn Ile Ala Thr Ile Ile Lys Asp Gly Glu Arg Ala
170 175 180
Thr Ala Leu Ala Gly Thr Lys Pro Tyr Met Ala Pro Glu Ile Phe
185 190 195
His Ser Phe Val Asn Gly Gly Thr Gly Tyr Ser Phe Glu Val Asp
200 205 210
Trp Trp Ser Val Gly Val Met Ala Tyr Glu Leu Leu Arg Gly Trp
215 220 225
Arg Pro Tyr Asp Ile His Ser Ser Asn Ala Val Glu Ser Leu Val
230 235 240
Gln Leu Phe Ser Thr Val Ser Val Gln Tyr Val Pro Thr Trp Ser
245 250 255
Lys Glu Met Val Ala Leu Leu Arg Lys Leu Leu Thr Val Asn Pro
260 265 270
Glu His Arg Leu Ser Ser Leu Gln Asp Val Gln Ala Ala Pro Ala
275 280 285
Leu Ala Gly Val Leu Trp Asp His Leu Ser Glu Lys Arg Val Glu
290 295 300
16/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
Pro Gly Phe Val Pro Asn Lys Gly Arg Leu His Cys Asp Pro Thr
305 310 315
Phe Glu Leu Glu Glu Met Ile Leu Glu Ser Arg Pro Leu His Lys
320 325 330
Lys Lys Lys Arg Leu Ala Lys Asn Lys Ser Arg Asp Asn Ser Arg
335 340 345
Asp Ser Ser Gln Ser Ala Pro Arg Ser Lys Ser Lys Pro Ser Thr
350 355 360
Gln Arg Gln Gly Ser Trp Ala Leu Ala Ser Ser Gly Leu Gly Glu
365 370 375
<210> 13
<211> 1859
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 058860CB1
<400> 13
gagagatact ccacaccccc aggagagact ctagagagat attccacacc cccaggagag 60
actctggaga gatactccac acccccagga gagactctag agcgatattc cacaccccca 120
ggggaggcac tagagagata ttctattcct actggaggac caaaccccac tggtactttt 180
aaaacatatc catcaaaaat agaaatggaa gacggtacac caaatgagca tttctacaca 240
cctacagaag agaggggttc agcttatgaa atatggcgtt ccgattcatt tggtacaccc 300
aatgaagcca ttgagccaaa agacaatgaa atgcctccat cttttattga acctctgacc 360
aaaaggaagg tatatgaaaa cacaacacta ggcttcattg ttgaagttga aggtcttcca 420
gttcctggtg tgaaatggta tcgaaataaa tctttactag agccagatga aagaatcaaa 480
atggaaagag tgggtaatgt gtgttcactg gaaatttcta acattcaaaa aggagaaggg 540
ggagagtaca tgtgtcatgc tgtaaacatc ataggggaag caaagagctt tgcaaatgta 600
gacataatgc cccaggaaga aagagtggtg gcactaccac ctccagtaac acatcagcat 660
gtcatggagt ttgatttgga acacaccaca tcatcaagaa caccttctcc tcaagaaatt 720
gtcctggaag ttgaattaag tgaaaaagac gttaaagaat ttgagaagca ggtgaaaata 780
gtgacagttc ccgaatttac tcctgaccat aaaagtatga ttgtgagtct agatgttctt 840
ccatttaatt ttgtagatcc aaatatggat tcaagggagg gagaagacaa agaactaaaa 900
attgatttag aagtatttga aatgcctcct cgctttataa tgcctatttg tgattttaaa 960
attccagaaa attcagatgc tgtattcaaa tgttcagtca tagggatccc gactcccgaa 1020
gttaagtggt ataaagaata tatgtgtatt gagccagata atattaaata cgtgattagc 1080
gaggagaagg gaagtcacac tcttaaaatt cgaaatgtct gtctttctga tagtgcaaca 1140
tacaggtgca gagctgtgaa ttgtgtagga gaggctatct gtcggggatt cctcaccatg 1200
ggagattctg aaatatttgc tgtgatagca aagaaaagca aagtgacttt aagcagttta 1260
atggaagaat tggtcttaaa gagcaactac acagacagtt tttttgaatt tcaggtggtg 1320
gaagggcctc ccaggtttat caaaggtatt tctgactgtt atgcaccaat aggtacagca 1380
gcatattttc agtgcttagt tcgtggctct ccaagaccca cggtttactg gtacaaagat 1440
ggaaaattag tccaaggaag aaggttcact gttgaggaaa gtggcacagg gttccataac 1500
ctgtttataa caagcttagt aaagagtgat gaaggagagt ataggtgtgt agctacaaac 1560
aaatcaggaa tggctgagag ctttgcagca ctcaccttaa cttaaaatgt aatgttttag 1620
tgcctcagta attattagca ttgatctgag tgctttcata ttttccaaat tatgtggatc 1680
taataaactt ccaaacaggt ccaccatatt tgaattcatt accttggaga cccctaaaga 1740
aataatctct atgtagaaat ctcatctttg taatacatgt aaatattttg ttatctgaac 1800
tgtggaatca tcacttgtgt caatcatgct gtgtaatatc aaacacaatt aaatctctc 1859
<210> 14
<211> 3501
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2041716CB1
<400> 14
gtggtgtggc tgcagtggag agttcccaac aaggctacgc agaagaaccc ccttgactga 60
agcaatggag gggggtccag ctgtctgctg ccaggatcct cgggcagagc tggtagaacg 120
ggtggcagcc atcgatgtga ctcacttgga ggaggcagat ggtggcccag agcctactag 180
17/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
aaacggtgtg gaccccccac cacgggccag agctgcctct gtgatccctg gcagtacttc 240
aagactgctc ccagcccggc ctagcctctc agccaggaag ctttccctac aggagcggcc 300
agcaggaagc tatctggagg cgcaggctgg gccttatgcc acggggcctg ccagccacat 360
ctccccccgg gcctggcgga ggcccaccat cgagtcccac cacgtggcca tctcagatgc 420
agaggactgc gtgcagctga accagtacaa gctgcagagt gagattggca aggtggggct 480
gactgatgcc tatctgcagg gtgcctacgg tgtggtgagg ctggcctaca acgaaagtga 540
agacagacac tatgcaatga aagtcctttc caaaaagaag ttactgaagc agtatggctt 600
tccacgtcgc cctcccccga gagggtccca ggctgcccag,ggaggaccag ccaagcagct 660
gctgcccctg gagcgggtgt accaggagat tgccatcctg aagaagctgg accacgtgaa 720
tgtggtcaaa ctgatcgagg tcctggatga cccagctgag gacaacctct atttggttga 780
cctcctgaga aaggggcccg tcatggaagt gccctgtgac aagcccttct cggaggagca 840
agctcgcctc tacctgcggg acgtcatcct gggcctcgag tacttgcact gccagaagat 900
cgtccacagg gacatcaagc catccaacct gctcctgggg gatgatgggc acgtgaagat 960
cgccgacttt ggcgtcagca accagtttga ggggaacgac gctcagctgt ccagcacggc 1020
gggaacccca gcattcatgg cccccgaggc catttctgat tccggccaga gcttcagtgg 1080
gaaggccttg gatgtatggg ccactggcgt cacgttgtac tgctttgtct atgggaagtg 1140
cccgttcatc gacgatttca tcctggccct ccacaggaag atcaagaatg agcccgtggt 1200
gtttcctgag gagccagaaa tcagcgagga gctcaaggac ctgatcctga agatgttaga 1260
caagaatccc gagacgagaa ttggggtgcc agacatcaag ttgcaccctt gggtgaccaa 1320
gaacggggag gagccccttc cttcggagga ggagcactgc agcgtggtgg aggtgacaga 1380
ggaggaggtt aagaactcag tcaggctcat ccccagctgg accacggtga tcctggtgaa 1440
gtccatgctg aggaagcgtt cctttgggaa cccgtttgag ccccaagcac ggagggaaga 1500
gcgatccatg tctgctccag gaaacctact ggtgaaagaa gggtttggtg aagggggcaa 1560
gagcccagag ctccccggcg tccaggaaga cgaggctgca tcctgagccc ctgcatgcac 1620
ccagggccac ccggcagcac actcatcccg cgcctccaga ggcccacccc tcatgcaaca 1680
gccgcccccg caggcagggg gctggggact gcagccccac tcccgcccct cccccatcgt 1740
gctgcatgac ctccacgcac gcacgtccag ggacagactg gaatgtatgt catttggggt 1800
cttgggggca gggctcccac gaggccatcc tcctcttctt ggacctcctt ggcctgaccc 1860
attctgtggg gaaaccgggt gcccatggag cctcagaaat gccacccggc tggttggcat 1920
ggcctggggc aggaggcaga ggcaggagac caagatggca ggtggaggcc aggcttacca 1980
caacggaaga gacctcccgc tggggccggg caggcctggc tcagctgcca caggcatatg 2040
gtggagaggg gggtaccctg cccaccttgg ggtggtggca ccagagctct tgtctattca 2100
gacgctggta tgggggctcg gacccctcac tggggacagg gccagtgttg gagaattctg 2160
attccttttt tgttgtcttt tacttttgtt tttaacctgg gggttcgggg agaggccctg 2220
cttgggaaca tctcacgagc tttcctacat cttccgtggt tcccagcaca gcccaagatt 2280
atttggcagc caagtggatg gaactaactt tcctggactg tgtttcgcat tcggcgttat 2340
ctggaaagtg gactgaacgg aatcaagctc tgagcagagg cctgaagcgg aagcaccaca 2400
tcgtccctgc ccatctcact ctctcccttg atgatgcccc tagagctgag gctggagaag 2460.
acaccagggc tgactttgac cgagggccat ggacgcgaca ggcctgtggc cctgcgcatg 2520
ctgaaataac tggaacccag cctctcctcc tacaccggcc tacccatctg ggcccaagag 2580
ctgcactcac actcctacaa cgaaggacaa actgtccagg tcggagggat cacgagacac 2640
agaacctgga ggggtgtgca cgctggcagg tggcctctgc ggcaattgcc tcaccctgag 2700
gacatcagca gtcagcctgc tcagagcggg ggtgctggag cgcgtgcaga cacagctctt 2760
ccggagcagc cttcaccttc tctctgggat cagtgtccgg ctggccgacg tggcatttgc 2820
tgaccgaatg ctcatagagg ttgaccccca cagggtcacg caggactcgg acactgccct 2880
ggaaacatgg atggacaagg gcttttggcc acaggtgtgg gtgtcctgtt ggaggagggc 2940
ttgtttggag aagggaggct ggctggggga gaaacccgga tcccgctgca tctccgcgcc 3000
tgtgggtgca tgtcgcgtgc tcatctgttg cacacagctc actcgtatgt cctgcactgg 3060
tacatgcatc tgtaatacag tttctacgtc tatttaaggc taggagccga atgtgcccca 3120
ttgtcagtgg gtccacgttt ctccccggct cctctgggct aaggcagtgt ggcccgaggc 3180
ttaaaaagtt actcggtact gtttttaaga acacttttat agagttagtg gaaggcaagt 3240
taagagccaa tcactgatcc ccaagtgttt cttgagcatc tggtctgggg ggaccacttt 3300
gatcggaccc acccttggaa agctcagggg taggcccagg tgggatgctc accctgtcac 3360
tgagggtttt ggttggcatc gttgtttttg aatgtagcac aagcgatgag caaactctat 3420
aagagtgttt taaaaattaa cttcccagga agtgagttaa aaacaataaa agccctttct 3480
tgagttaaaa agaaaaaaaa a 3501
<210> 15
<211> 3039
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472005CB1
<400> 15
18/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
atggcccccg cccggggccg cctgccccct gcgctctggg tcgtcacggc cgcggcggcg 60
gcggccacct gcgtgtccgc ggcgcgcggc gaagtgaatt tgctggacac gtcgaccatc 120
cacggggact ggggctggct cacgtatccg gctcatgggt gggactccat caacgaggtg 180
gacgagtcct tccagcccat ccacacgtac caggtttgca acgtcatgag ccccaaccag 240
aacaactggc tgcgcacgag ctgggtcccc cgagacggcg cccggcgcgt ctatgctgag 300
atcaagttta ccctgcgcga ctgcaacagc atgcctggtg tgctgggcac ctgcaaggag 360
accttcaacc tctactacct ggagtcggac cgcgacctgg gggccagcac acaagaaagc 420
cagttcctca aaatcgacac cattgcggcc gacgagagct tcacaggtgc cgaccttggt 480
gtgcggcgtc tcaagctcaa cacggaggtg cgcagtgtgg gtcccctcag caagcgcggc 540
ttctacctgg ccttccagga cataggtgcc tgcctggcca tcctctctct ccgcatctac 600
tataagaagt gccctgccat ggtgcgcaat ctggctgcct tctcggaggc agtgacgggg 660
gccgactcgt cctcactggt ggaggtgagg ggccagtgcg tgcggcactc agaggagcgg 720
gacacaccca agatgtactg cagcgcggag ggcgagtggc tcgtgcccat cggcaaatgc 780
gtgtgcagtg ccggctacga ggagcggcgg gatgcctgtg tggcctgtga gctgggcttc 840
tacaagtcag cccctgggga ccagctgtgt gcccgctgcc ctccccacag ccactccgca 900
gctccagccg cccaagcctg ccactgtgac ctcagctact accgtgcagc cctggacccg 960
ccgtcctcag cctgcacccg gccaccctcg gcaccagtga acctgatctc cagtgtgaat 1020
gggacatcag tgactctgga gtgggcccct cccctggacc caggtggccg cagtgacatc 1080
acctacaatg ccgtgtgccg ccgctgcccc tgggcactga gccgctgcga ggcatgtggg 1140
agcggcaccc gctttgtgcc ccagcagaca agcctggtgc aggccagcct gctggtggcc 1200
aacctgctgg cccacatgaa ctactccttc tggatcgagg ccgtcaatgg cgtgtccgac 1260
ctgagccccg agccccgccg ggccgctgtg gtcaacatca ccacgaacca ggcagccccg 1320
tcccaggtgg tggtgatccg tcaagagcgg gcggggcaga ccagcgtctc gctgctgtgg 1380
caggagcccg agcagccgaa cggcatcatc ctggagtatg agatcaagta ctacgagaag 1440
gacaaggaga tgcagagcta ctccaccctc aaggccgtca ccaccagagc caccgtctcc 1500
ggcctcaagc cgggcacccg ctacgtgttc caggtccgag cccgcacctc agcaggctgt 1560
ggccgcttca gccaggccat ggaggtggag accgggaaac cccggccccg ctatgacacc 1620
aggaccattg tctggatctg cctgacgctc atcacgggcc tggtggtgct tctgctcctg 1680
ctcatctgca agaagaggca ctgtggctac agcaaggcct tccaggactc ggacgaggag 1740
aagatgcact atcagaatgg acaggcaccc ccacctgtct tcctgcctct gcatcacccc 1800
ccgggaaagc tcccagagcc ccagttctat gcggaacccc acacctacga ggagccaggc 1860
cgggcgggcc gcagtttcac tcgggagatc gaggcctcta ggatccacat cgagaaaatc 1920
atcggctctg gagactccgg ggaagtctgc tacgggaggc tgcgggtgcc agggcagcgg 1980
gatgtgcccg tggccatcaa ggccctcaaa gccggctaca cggagagaca gaggcgggac 2040
ttcctgagcg aggcgtccat catggggcaa ttcgaccatc ccaacatcat ccgcctcgag 2100
ggtgtcgtca cccgtggccg cctggcaatg attgtgactg agtacatgga gaacggctct 2160
ctggacacct tcctgaggac ccacgacggg cagttcacca tcatgcagct ggtgggcatg 2220
ctgagaggag tgggtgccgg catgcgctac ctctcagacc tgggctatgt ccaccgagac 2280
ctggccgccc gcaacgtcct ggttgacagc aacctggtct gcaaggtgtc tgacttcggg 2340
ctctcacggg tgctggagga cgacccggat gctgcctaca ccaccacggg cgggaagatc 2400
cccatccgct ggacggcccc agaggccatc gccttccgca ccttctcctc ggccagcgac 2460
gtgtggagct tcggcgtggt catgtgggag gtgctggcct atggggagcg gccctactgg 2520
aacatgacca accgggatgt gagtgccaag ccctggcagg tcatcagctc tgtggaggag 2580
gggtaccgcc tgcccgcacc catgggctgc ccccacgccc tgcaccagct catgctcgac 2640
tgttggcaca aggaccgggc gcagcggcct cgcttctccc agattgtcag tgtcctcgat 2700
gcgctcatcc gcagccctga gagtctcagg gccaccgcca cagtcagcag gtgcccaccc 2760
cctgccttcg tccggagctg ctttgacctc cgagggggca gcggtggcgg tgggggcctc 2820
accgtggggg actggctgga ctccatccgc atgggccggt accgagacca cttcgctgcg 2880
ggcggatact cctctctggg catggtgcta cgcatgaacg cccaggacgt gcgcgccctg 2940
ggcatcaccc tcatgggcca ccagaagaag atcctgggca gcattcagac catgcgggcc 3000
cagctgacca gcacccaggg gccccgccgg cacctctga 3039
<210> 16
<211> 1104
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 7472006CB1
<400> 16
atggatgacg ctgctgtcct caagcgacga ggctacctcc tggggataaa tttaggagag 60
ggctcctatg caaaagtaaa atctgcttac tctgagcgcc tgaagttcaa tgtggcgatc 120
aagatcatcg accgcaagaa ggcccccgca gacttcttgg agaaattcct tccccgggaa 180
attgagattc tggccatgtt aaaccactgc tccatcatta agacctacga gatctttgag 240
acatcacatg gcaaggtcta catcgtcatg gagctcgcag tccagggcga cctcctcgag 300
19/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
ttaatcaaaa cccggggagc cctgcatgag gacgaagctc gcaagaagtt ccaccagctt 360
tccttggcca tcaagtactg ccacgacctg gacgtcgtcc accgggacct caagtgtgac 420
aaccttctcc ttgacaagga cttcaacatc aagctgtccg acttcagctt ctccaagcgc 480
tgcctgcggg atgacagtgg tcgaatggcc ttaagcaaga ccttctgtgg gtcaccagcg 540
tatgcggccc cagaggtgct gcagggcatt ccctaccagc ccaaggtgta cgacatctgg 600
agcctaggcg tgatcctcta catcatggtc tgcggctcca tgccctacga cgactccaac 660
atcaagaaga tgctgcgtat ccagaaggag caccgcgtca acttcccacg ctccaagcac 720
ctgacaggcg agtgcaagga cctcatctac cacatgctgc ag.cccgacgt caaccggcgg 780
ctccacatcg acgagatcct cagccactgc tggatgcagc ccaaggcacg gggatctccc 840
tctgtggcca tcaacaagga gggggagagt tcccggggaa ctgaaccctt gtggaccccc 900
gaacctggct ctgacaagaa gtctgccacc aagctggagc ctgagggaga ggcacagccc 960
caggcacagc ctgagacaaa acccgagggg acagcaatgc aaatgtccag gcagtcggag 1020
atcctgggtt tccccagcaa gccgtcgact atggagacag aggaagggcc cccccaacag 1080
cctccagaga cgcgggccca gtga 1104
<210> 17
<211> 3939
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2902460CB1
<400> 17
ccgcagtgtg ctggaaaggc agctgcggca gtagcgtgag cagcccaagt tgggctggtc 60
gcctgcgagg ggaccggcag caggtggtgg cagccggtac cctctccccg ccaggccgga 120
ggaggccaag aggaagctgc ggatcttgca gcgcgagttg cagaacgtgc aggtgaacca 180
gaaagtgggc atgtttgagg cgcacatcca ggcacagagc tccgccattc aagcgccccg 240
cagcccgcgt ttgggcaggg ctcgctcgcc ctccccgtgc cccttccgca gcagcagtca 300
gccccctgga agggtcctgg ttcagggcgc ccggagcgag gaacggagga caaagtcctg 360
gggggagcaa tgtccagaga cttcaggaac cgactccggg aggaaaggag ggcccagcct 420
atgctcctcg caggtgaaga aaggaatgcc acctcttccc ggccgggctg cccctacagg 480
atcagaggct cagggtccat ccgcttttgt aaggatggag aagggtatcc ctgccagtcc 540
ccgctgtggc tcacccacag ctatggaaat tgacaaaagg ggctctccta ccccgggaac 600
tcggagctgc ctagctccct cattggggct gttcggagct agcttaacga tggccacgga 660
agtggcagcg agagttacat ccactgggcc acaccgtcca caggatcttg ccctcactga 720
gccgtctggg agagcccgtg agcttgagga cctgcagccc ccagaggccc tggtggagag 780
gcaggggcag tttctgggca gtgagacaag cccagcccca gaaaggggcg ggccccgcga 840
tggagaaccc cctgggaaga tggggaaagg atatctgccc tgtggcatgc cgggctctgg 900
ggagcctgaa gtgggcaaaa ggccagagga gacgactgtg agcgtgcaaa gcgcagagtc 960
ctctgatgcc ctgagctggt ccaggctgcc cagggccctg gcctccgtag gccctgagga 1020
ggcccgaagt ggggcccccg tgggcggggg gcgttggcag ctctccgaca gagtggaggg 1080
agggtcccca acgctgggct tgcttggggg cagcccctca gcacagccgg ggaccgggaa 1140
tgtggaggcg ggaattcctt ctggcagaat gctggagcct ttgccctgtt gggacgctgc 1200
gaaagatctg aaagaacctc agtgccctcc tggggacagg gtgggtgtgc agcctgggaa 1260
ctccagggtt tggcagggca ccatggagaa agccggtttg gcttggacgc gtggcacagg 1320
ggtgcaatca gaggggactt gggaaagcca gcggcaggac agtgatgccc tcccaagtcc 1380
ggagctgcta ccccaagatc aggacaagcc tttcctgagg aaggcctgca gccccagcaa 1440
catacctgct gtcatcatta cagacatggg cacccaggag gatggggcct tggaggagac 1500
gcagggaagc cctcggggca acctgcccct gaggaaactg tcctcttcct cggcctcctc 1560
cacgggcttc tcctcatcct acgaagactc agaggaggac atctccagtg accctgagcg 1620
caccctggac cccaactcag ccttcctgca taccctggac cagcagaaac ctagagtgag 1680
caaatcatgg aggaagataa aaaacatggt gcactggtct cccttcgtca tgtccttcaa 1740
gaagaagtac ccctggatcc agctggcagg acacgcaggg agtttcaagg cagctgccaa 1800
tggcaggatc ctgaagaagc actgtgagtc agagcagcgc tgcctggacc ggctgatggt 1860
ggatgtgctg aggcccttcg tacctgccta ccatggggat gtggtgaagg acggggagcg 1920
ctacaaccag atggacgacc tgctggccga cttcgactcg ccctgtgtga tggactgcaa 1980
gatgggaatc aggacctacc tggaggagga gctcacgaag gcccggaaga agcccagcct 2040
gcggaaggac atgtaccaga agatgatcga ggtggacccc gaggccccca ccgaggagga 2100
aaaagcacag cgggctgtga ccaagccacg gtacatgcag tggcgggaga ccatcagctc 2160
cacggccacc ctggggttca ggatcgaggg aatcaagaaa gaagacggca ccgtgaaccg 2220
ggacttcaag aagaccaaaa cgagggagca ggtcaccgag gccttcagag agttcactaa 2280
aggaaaccat aacatcctga tcgcctatcg ggaccggctg aaggccattc gaaccactct 2340
agaagtttct cccttcttca agtgccacga ggtcattggc agctccctcc tcttcatcca 2400
cgacaagaag gaacaggcca aagtgtggat gatcgacttt gggaaaacca cgcccctgcc 2460
tgagggccag accctgcagc atgacgtccc ctggcaggag gggaaccggg aggatggcta 2520
20/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
cctctcgggg ctcaataacc tcgtcgacat cctgaccgag atgtcccagg atgccccact 2580
cgcctgagct gcccacgccc tccctggccc ccgcctgggc ctcctttcct cctcctgtgc 2640
ttcctttctc gttcctaact tttccttcac ttacacctga ctgaccctcc tgaactgcac 2700
tacaagacac tttgtagaag aggagatgag agtttctagt cattttccta acttcagggc 2760
ttggaggtgg tgtttgcact gctttttgta gagagggtca cctactagaa gagaaatgcc 2820
cagtcttaga ggtgggtcag gtgtagagct ggagggggtc cctggctgct gaggggaccc 2880
taccagatga gccctgcctc tgggagcccc ctaggaagca ccagcctgga cctaccacct 2940
gcggaggcct gctgccccct ggcggccagt gctgttagag tgctgccaag cacagcctta 3000
tttctgccgg ggcctcccca ccggagagcc cagggggccg gccgggttcc tggtccctgg 3060
ctgggagcag ggctttctgg tagttggggc acaaaaccat cggggaacca catgttgact 3120
gtgagcaaag tgtcttccga ttagcagcct cagggatgcc ctggtggcct ctccagggct 3180
gctcaggcaa ggccccccac ccatctggta tggaaacctg ccggctccag gccagaccca 3240
ggagccaaga gaaggctgaa gccagcttgg ctgtgttctc tgatctaggc cttcccagag 3300
gaggcgagca gaagctgtgc cacttggaat tgcaacccat gagttcagaa ggcacactct 3360
gccatgctga gctccaaggg tgctaccagg ggaagatggg atctatagag tctctgggcc 3420
ctggccccag ggaggagcac atttttcttg accctcacct acctggtgct agttggtcaa 3480
ccctgcctgc atacatgggc tcctgtcatg gggcccagag tcccttgcag atatagaaat 3540
aggggaggag ctcaggtctg cgccaggcag gaagaaggca ggcttctggc ttccagaggt 3600
gccgcggtgg cctcctggca tcatttgtta ttgcctctga aacaagcctt actgcctgga 3660
gggcttagat tcctgcttct ccaatgtagt gtgggtatct tgtagggtat gtggtggatg 3720
ccagggcgtg ctccaggcac ctcttcctga agtctctgca tttggagatt cgtggagaac 3780
ctatttaagc ccaattttaa ctgaaagcca gtgagtctga tatggaaggg aatgtaaaat 3840
ttgcctgact tcttaagaac aaaaccccca gctctgtgcc ccatgctcct tggggcttgc 3900
cacccactcc tttgctgtca gaggtacagg agctgggag 3939
<210> 18
<211> 1381
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 6383934CB1
<400> 18
atgaggacaa tgcctgctgg cccacatgac ggggggatgt agacggcagc ggcgccagtc 60
gctcctggca ccatggacga tgccacagtc ctaaggaaga agggttacat cgtaggcatc 120
aatcttggca agggttccta cgcaaaagtc aaatctgcct actctgagcg cctcaagttc 180
aatgtggctg tcaagatcat cgaccgcaag aaaacaccta ctgactttgt ggagagattc 240
cttcctcggg agatggacat cctggcaact gtcaaccacg gctccatcat caagacttac 300
gagatctttg agacctctga cggacggatc tacatcatca tggagcttgg cgtccagggc 360
gacctcctcg agttcatcaa gtgccaggga gccctgcatg aggacgtggc acgcaagatg 420
ttccgacagc tctcctccgc cgtcaagtac tgccacgacc tggacatcgt ccaccgggac 480
ctcaagtgcg agaaccttct cctcgacaag gacttcaaca tcaagctgtc tgactttggc 540
ttctccaagc gctgcctgcg ggacagcaat gggcgcatca tcctcagcaa gaccttctgc 600
gggtcggcag catatgcagc ccccgaggtg ctgcagagca tcccctacca gcccaaggtg 660
tatgacatct ggagcctagg cgtgatcctc tacatcatgg tctgcggctc catgccctac 720
gacgactccg acatcaagaa gatgctgcgt atccagaagg agcaccgcgt caacttccca 780
cgctccaagc acctgacctg cgagtgcaag gacctcatct accacatgct gcagcccgac 840
gtcagccagc ggctccacat cgatgagatc ctcagccact cgtggctgca gccccccaag 900
cccaaagcca cgtcttctgc ctccttcaag agggaggggg agggcaagta ccgcgctgag 960
tgcaaactgg acaccaagac aggcttgagg cccgaccacc ggcccgacca caagcttgga 1020
gccaaaaccc agcaccggct gctggtggtg cccgagaacg agaacaggat ggaggacagg 1080
ctggccgaga cctccagggc caaagaccat cacatctccg gagctgaggt ggggaaagca 1140
agcacctagc atgacaatgg ccccgttgtg tgtggtgggg gtcggggttg gggggcatgg 1200
tgcagtcggc cttcacgtaa actaagtagg caggtaggat ctgaagaagg cacaggtgca 1260
agtaaaattc gtcaattaaa ccactatttt gattacgttc cattagcttt cttccactta 1320
gcagcaaaga cgttccttac tgaccaccaa ataaaccaca gggtgtgtgc aagcatcaaa 1380
a 1381
<210> 19
<211> 3904
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
21 /26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
<223> Incyte ID No: 3210906CB1
<400> 19
tattcggggt tcagacccca caatcagaaa tccggaattc ggcagctgtc gccctcgacg 60
agggggagga ctggaccgcg aggtcagatt aggttgtcac cccctcccct ccaggggagg 120
cttcccgggc ccgcccctca ggaagggcga aagccgagga agaggtggca aggggaaagg 180
tctccttgcc cctctccctg acttggcaga gccgctggag gaccccaggc ggaagcggag 240
gcgctggggc accatagtga,cccctaccag gccaggcccc actctcaggg cccccagggg 300
ccaccatgcc agctgggggc cgggccggga gcctgaagga cccagatgtg gctgagctct 360
tcttcaagga tgacccagaa aagctcttct ctgacctccg ggaaattggc catggcagct 420
ttggagccgt atactttgcc cgggatgtcc ggaatagtga ggtggtggcc atcaagaaga 480
tgtcctacag tgggaagcag tccaatgaga aatggcaaga catcatcaag gaggtgcggt 540
tcttacagaa gctccggcat cccaacacca ttcagtaccg gggctgttac ctgagggagc 600
acacggcttg gctggtaatg gagtattgcc tgggctcaac ttctgacctt ctagaagtgc 660
acaagaaacc ccttcaggag gtagagatcg cagctgtgac ccacggggcg cttcagggcc 720
tggcatatct gcactcccac aacatgatcc atagggatgt gaaggctgga aacatcctgc 780
tgtcagagcc agggttagtg aagctagggg actttggttc tgcgtccatc atggcacctg 840
ccaactcctt cgtgggcacc ccatactgga tggcacccga ggtgatcctg gccatggatg 900
aggggcagta cgatggcaaa gtggacgtct ggtccttggg gataacctgc atcgagctgg 960
ctgaacggaa accaccgctc tttaacatga atgcgatgag tgccttatac cacattgcac 1020
agaacgaatc ccccgtgctc cagtcaggac actggtctga gtacttccgg aattttgtcg 1080
actcctgtct tcagaaaatc cctcaagaca gaccaacctc agaggttctc ctgaagcacc 1140
gctttgtgct ccgggagcgg ccacccacag tcatcatgga cctgatccag aggaccaagg 1200
atgccgtgcg ggagctggac agcctgcagt accgcaagat gaagaagatc ctgttccaag 1260
aggcacccaa cggccctggt gccgaggccc cagaggagga agaggaggcc gagccctaca 1320
tgcacctggc cgggactctg accagcctcg agagtagcca ctcagtgccc agcatgtcca 1380
tcagcgcctc cagccagagc agctccgtca acagcctagc agatgcctca gacaacgagg 1440
aagaggagga ggaggaggag gaagaggagg aggaggaaga aggccctgaa gcccgggaga 1500
tggccatgat gcaggagggg gagcacacag tcacctctca cagctccatt atccaccggc 1560
tgccgggctc tgacaaccta tatgatgacc cctaccagcc agagataacc cccagccctc 1620
tccagccgcc tgcagcccca gctcccactt ccaccacctc ttccgcccgc cgccgggcct 1680
actgccgtaa ccgagaccac tttgccacca tccgaaccgc ctccctggtc agccgtcaga 1740
tccaggagca tgagcaggac tctgcgctgc gggagcagct gagcggctat aagcggatgc 1800
gacgacagca ccagaagcag ctgctggccc tggagtcacg gctgaggggt gaacgggagg 1860
agcacagtgc acggctgcag cgggagcttg aggcgcagcg ggctggcttt ggggcagagg 1920
cagaaaagct ggcccggcgg caccaggcca taggtgagaa ggaggcacga gctgcccagg 1980
ccgaggagcg gaagttccag cagcacatcc ttgggcagca gaagaaggag ctggctgccc 2040
tgctggaggc acagaagcgg acctacaaac ttcgcaagga acagctgaag gaggagctcc 2100.
aggagaaccc cagcactccc aagcgggaga aggccgagtg gctgctgcgg cagaaggagc 2160
agctccagca gtgccaggcg gaggaggaag cagggctgct gcggcggcag cgccagtact 2220
ttgagctgca gtgtcgccag tacaagcgca agatgttgct ggctcggcac agcctggacc 2280
aggacctgct gcgggaggac ctgaacaaga agcagaccca gaaggacttg gagtgtgcac 2340
tgctgcttcg gcagcacgag gccacgcggg agctggagct gcggcagctc caggccgtgc 2400
agcgcacgcg ggctgagctc acccgcctgc agcaccagac ggagctgggc aaccagctgg 2460
agtacaacaa gcggcgtgag caagagttgc ggcagaagca tgcggcccag gttcgccagc 2520
agcccaagag cctcaaatct aaggagctgc agatcaagaa gcagttccag gagacgtgta 2580
agatccagac tcggcagtac aaggctctgc gagcacactt gctggagacc acgcccaaag 2640
ctcagcacaa gagcctcctt aagcggctca aggaagagca gacccgcaag ctggcgatct 2700
tggcggagca gtatgaccag tccatctcag agatgctcag ctcacaggcg ctgcggcttg 2760
atgagaccca ggaggcagag ttccaggccc ttcggcagca gcttcaacag gagctggagc 2820
tgctcaacgc ttaccagagc aagatcaaga tccgcacaga gagccagcac gagagggagc 2880
tgcgggagct ggagcagagg gtcgcgctgc ggcgggcact gctggagcag cgggtggaag 2940
aggagctgct ggccctgcag acaggacgct ccgagcgaat ccgcagtctg cttgagcggc 3000
aggcccgtga gatcgaggcc ttcgatgcgg aaagcatgag gctgggcttc tccagcatgg 3060
ctctgggggg catcccggct gaagctgctg cccagggcta tcctgctcca ccccctgccc 3120
cagcctggcc ctcccgtccc gttccccgtt ctggggcaca ctggagccat ggccctcctc 3180
caccaggcat gccccctcca gcctggcgtc agccgtctct gctggctccc ccaggccccc 3240
caaactggct ggggcccccc acacaaagtg ggacaccccg tggcggagcc ctgctgctgc 3300
taagaaacag cccccagccc ctgcggcggg cagcctcggg gggcagtggc agtgagaatg 3360
tgggcccccc tgctgccgcg gtgcccgggc ccctgagccg cagcaccagt gtcgcttccc 3420
acatcctcaa tggttcttcc cacttctatt cctgaggtgc agcggggagg agcagatgag 3480
ctgggcaggg caggggtggg tggagcctga ccctggaggg cactgagctg gaggcccctg 3540
caagggtagg ggacaagatg taggctccag ctcccctcag acctcctcat ctcatgagct 3600
tcttggggct ggccagtggc ccagggccag cttggcgata gatgcctcaa ggctgcctgg 3660
gagccccgcc tccctaccat ggtgccaggg gtctccctcc gccacctagg aaaggaggga 3720
gatgtgcgtg tcaaatattc atctagtccc ctgggggagg ggaagggtgg gtctagacat 3780
actatattca gagaactata ctaccctcac agtgaggccc tcagacctgc cacagggcag 3840
22/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
agcaggtctg gggcctgagg cagggagaat gagaggccac ttactggcag gaaggatcag 3900
gatg 3904
<210> 20
<211> 1987
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3339024CB1
<400> 20
gaagaaccct gaggaacaga cttacctcag caaccctggc acctccaacc cgacacatgc 60
tactgctgct gctactgctg ccacccctgc tctgtgggag agtgggggct aaggaacaga 120
aggattacct gctgacaatg tagaagtccg tgacggtgca ggagggcctg tgtgtctctg 180
tgctttgctc cttctcctac ccccaaaatg gctggactgc ctccgatcca gttcatggct 240
actggttccg ggcaggggac catgtaagcc ggaacattcc agtggccaca aacaacccag 300
ctcgagcagt gcaggaggag actcgggacc gattccacct ccttggggac ccacagaaca 360
aggattgtac cctgagcatc agagacacca gagagagtga tgcagggaca tacgtctttt 420
gtgtagagag aggaaatatg aaatggaatt ataaatatga ccagctctct gtgaatgtga 480
cagcgtccca ggacctactg tcaagataca ggctggaggt gccagagtcg gtgactgtgc 540
aggagggtct gtgtgtctct gtgccctgca gtgtccttta cccccattac aactggactg 600
cctctagccc tgtttatgga tcctggttca aggaaggggc cgatatacca tgggatattc 660
cagtggccac aaacacccca agtggaaaag tgcaagagga tacccacggt cgattcctcc 720
tccttgggga cccacagacc aacaactgct ccctgagcat cagagatgcc aggaaggggg 780
attcagggaa gtactacttc caggtggaga gaggaagcag gaaatggaac tacatatatg 840
acaagctctc tgtgcatgtg acagccctga ctcacatgcc caccttctcc atcccgggga 900
ccctggagtc tggccacccc aggaacctga cctgctctgt gccctgggcc tgtgaacagg 960
ggacgccccc cacgatcacc tggatggggg cctccgtgtc ctccctggac cccactatca 1020
ctcgctcctc gatgctcagc ctcatcccac agccccagga ccatggcacc agcctcacct 1080
gtcaggtgac cttgcctggg gccggcgtga ccatgaccag ggctgtccga ctcaacatat 1140
cctatcctcc tcagaacttg accatgactg tcttccaagg agatggcaca gcatccacaa 1200
ccttgaggaa tggctcggcc ctttcagtcc tggagggcca gtccctgcac cttgtctgtg 1260
ctgtcgacag caatccccct gccaggctga gctggacctg ggggagcctg accctgagcc 1320
cctcacagtc ctcgaacctt ggggtgctgg agctgcctcg agtgcatgtg aaggatgaag 1380
gggaattcac ctgccgagct cagaaccctc taggctccca gcacatttcc ctgagcctct 1440
ccctgcaaaa cgagtacaca ggcaaaatga ggcctatatc aggagtgacg ctaggggcat 1500
tcgggggagc tggagccaca gccctggtct tcctgtactt ctgcatcatc ttcgttgtag 1560
tgaggtcctg caggaagaaa tcggcaaggc cagcagtggg cgtgggggat acaggcatgg 1620
aggacgcaaa cgctgtctgg ggctcagcct ctcagggacc cctgattgaa tccccggcag 1680
atgacagccc cccacaccat gctccgccag ccctggccac cccctcccca gaggaaggag 1740
agatccagta tgcatccctc agcttccaca aagcgaggcc tcagtaccca caggaacagg 1800
aggccatcgg ctatgagtac tccgagatca acatccccaa gtgagaaact gcagagactc 1860
aggcctgttt gagggctcac gacccctcca gcaaagaagc ccgagactga ttcctttaga 1920
attaaaagcc ctccatgctg tgcaacgggg gatccactag ttaagagcgg cgcacccgcg 1980
tgcccct 1987
<210> 21
<211> 3925
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4436929CB1
<400> 21
ccgtcctcga ggcgaggaga gtaccgggcc ggcccggctg ccgcgcgagg agcgcggtcg 60
gcggcctggt ctgcggctga gatacacaga gcgacagaga catttattgt tatttgtttt 120
ttggtggcaa aaagggaaaa tggcgaacga ctcccctgca aaaagtctgg tggacatcga 180
cctctcctcc ctgcgggatc ctgctgggat ttttgagctg gtggaagtgg ttggaaatgg 240
cacctatgga caagtctata agggtcgaca tgttaaaacg ggtcagttgg cagccatcaa 300
agttatggat gtcactgagg atgaagagga agaaatcaaa ctggagataa atatgctaaa 360
gaaatactct catcacagaa acattgcaac atattatggt gctttcatca aaaagagccc 420
tccaggacat gatgaccaac tctggcttgt tatggagttc tgtggggctg ggtccattac 480
agaccttgtg aagaacacca aagggaacac actcaaagaa gactggatcg cttacatctc 540
23/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
cagagaaatc ctgaggggac tggcacatct tcacattcat catgtgattc accgggatat 600
caagggccag aatgtgttgc tgactgagaa tgcagaggtg aaacttgttg actttggtgt 660
gagtgctcag ctggacagga ctgtggggcg gagaaatacg ttcataggca ctccctactg 720
gatggctcct gaggtcatcg cctgtgatga gaacccagat gccacctatg attacagaag 780
tgatctttgg tcttgtggca ttacagccat tgagatggca gaaggtgctc cccctctctg 840
tgacatgcat ccaatgagag cactgtttct cattcccaga aaccctcctc cccggctgaa 900
gtcaaaaaaa tggtcgaaga agttttttag ttttatagaa gggtgcctgg tgaagaatta 960
catgcagcgg ccc,tctacag agcagctttt gaaacatcct tttataaggg atcagccaaa 1020
tgaaaggcaa gttagaatcc agcttaagga tcatatagat cgtaccagga agaagagagg 1080
cgagaaagat gaaactgagt atgagtacag tgggagtgag gaagaagagg aggaagtgcc 1140
tgaacaggaa ggagagccaa gttccattgt gaacgtgcct ggtgagtcta ctcttcgccg 1200
agatttcctg agactgcagc aggagaacaa ggaacgttcc gaggctcttc ggagacaaca 1260
gttactacag gagcaacagc tccgggagca ggaagaatat aaaaggcaac tgctggcaga 1320
gagacagaag cggattgagc agcagaaaga acagaggcga cggctagaag agcaacaaag 1380
gagagagcgg gaagctagaa ggcagcagga acgtgaacag cgaaggagag aacaagaaga 1440
aaagaggcgt ctagaggagt tggagagaag gcgcaaagaa gaagaggaga ggagacgggc 1500
agaagaagaa aagaggagag ttgaaagaga acaggagtat atcaggcgac agctagaaga 1560
ggagcagcgg cacttggaag tccttcagca gcagctgctc caggagcagg ccatgttact 1620
gcatgaccat aggaggccgc acccgcagca ctcgcagcag ccgccaccac cgcagcagga 1680
aaggagcaag ccaagcttcc atgctcccga gcccaaagcc cactacgagc ctgctgaccg 1740
agcgcgagag gtggaagata gatttaggaa aactaaccac agctcccctg aagcccagtc 1800
taagcagaca ggcagagtat tggagccacc agtgccttcc cgatcagagt ctttttccaa 1860
tggcaactcc gagtctgtgc atcccgccct gcagagacca gcggagccac aggttcctgt 1920
gagaacaaca tctcgctccc ctgttctgtc ccgtcgagat tccccactgc agggcagtgg 1980
gcagcagaat agccaggcag gacagagaaa ctccaccagc agtattgagc ccaggcttct 2040
gtgggagaga gtggagaagc tggtgcccag acctggcagt ggcagctcct cagggtccag 2100
caactcagga tcccagcccg ggtctcaccc tgggtctcag agtggctccg gggaacgctt 2160
cagagtgaga tcatcatcca agtctgaagg ctctccatct cagcgcctgg aaaatgcagt 2220
gaaaaaacct gaagataaaa aggaagtttt cagacccctc aagcctgctg gcgaagtgga 2280
tctgaccgca ctggccaaag agcttcgagc agtggaagat gtacggccac ctcacaaagt 2340
aacggactac tcctcatcca gtgaggagtc ggggacgacg gatgaggagg acgacgatgt 2400
ggagcaggaa ggggctgacg agtccacctc aggaccagag gacaccagag cagcgtcatc 2460
tctgaatttg agcaatggtg aaacggaatc tgtgaaaacc atgattgtcc atgatgatgt 2520
agaaagtgag ccggccatga ccccatccaa ggagggcact ctaatcgtcc gccagactca 2580
gtccgctagt agcacactcc agaaacacaa atcttcctcc tcctttacac cttttataga 2640
ccccagatta ctacagattt ctccatctag cggaacaaca gtgacatctg tggtgggatt 2700
ttcctgtgat gggatgagac cagaagccat aaggcaagat cctacccgga aaggctcagt 2760
ggtcaatgtg aatcctacca acactaggcc acagagtgac accccggaga ttcgtaaata 2820
caagaagagg tttaactctg agattctgtg tgctgcctta tggggagtga atttgctagt 2880
gggtacagag agtggcctga tgctgctgga cagaagtggc caagggaagg tctatcctct 2940
tatcaaccga agacgatttc aacaaatgga cgtacttgag ggcttgaatg tcttggtgac 3000
aatatctggc aaaaaggata agttacgtgt ctactatttg tcctggttaa gaaataaaat 3060
acttcacaat gatccagaag ttgagaagaa gcagggatgg acaaccgtag gggatttgga 3120
aggatgtgta cattataaag ttgtaaaata tgaaagaatc aaatttctgg tgattgcttt 3180
gaagagttct gtggaagtct atgcgtgggc accaaagcca tatcacaaat ttatggcctt 3240
taagtcattt ggagaattgg tacataagcc attactggtg gatctcactg ttgaggaagg 3300
ccagaggttg aaagtgatct atggatcctg tgctggattc catgctgttg atgtggattc 3360
aggatcagtc tatgacattt atctaccaac acatatccag tgtagcatca aaccccatgc 3420
aatcatcatc ctccccaata cagatggaat ggagcttctg gtgtgctatg aagatgaggg 3480
ggtttatgta aacacatatg gaaggatcac caaggatgta gttctacagt ggggagagat 3540
gcctacatca gtagcatata ttcgatccaa tcagacaatg ggctggggag agaaggccat 3600
agagatccga tctgtggaaa ctggtcactt ggatggtgtg ttcatgcaca aaagggctca 3660
aagactaaaa ttcttgtgtg aacgcaatga caaggtgttc tttgcctctg ttcggtctgg 3720
tggcagcagt caggtttatt tcatgacctt aggcaggact tctcttctga gctggtagaa 3780
gcagtgtgat ccagggatta ctggcctcca gagtcttcaa gatcctgaga acttggaatt 3840
ccttgtaact ggagctcgga gctgcaccga gggcaaccag gacagctgtg tgtgcagacc 3900
tcatgtgttg ggttctctcc cctcc 3925
<210> 22
<211> 1210
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5046791CB1
24/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
<400> 22
ttacaggtca tctaccccta taccccacaa aatgacgatg agctggagct ggtccccggg 60
gacttcatct tcatgtctcc aatggagcag accagcacca gcgagggttg gatctatggc 120
acgtccttaa ccaccggctg ctctggactc ctcctgagaa ttacattacc aaggctgatg 180
aatgcagcac ctggatattt catggttctt attcaatctt aaatacatcg tcatccaact 240
ctctcacgtt tggggatgga gtattggaga ggcggcctta tgaggaccag gggctcgggg 300
agacgactcc tcttactatc atctgccagc ccatgcagcc gctgagggtc aacagccagc 360
ccggccccca, gaagcgatgc ctttttgtgt gtcggcatgg tgagaggatg,gatgttgtgt 420
ttgggaagta ctggctgtcc cagtgcttcg atgccaaagg ccgctacata cgcaccaacc 480
tgaacatgcc tcatagttta cctcagcgga gtggtggttt ccgagattac gagaaagatg 540
ctcccatcac tgtgtttgga tgcatgcaag caagactagt gggtgaagcc ttattagaga 600
gcaataccat tatcgatcat gtctattgct ccccgtccct tcgctgcgtt cagactgcac 660
acaatatctt gaaaggttta caacaagaaa atcacttgaa gatccgtgta gagcccggct 720
tatttgagtg gacaaaatgg gttgctggga gcacattacc tgcatggata cctccatcag 780
agttagctgc agccaacctg agtgttgata caacctacag acctcacatt ccaatcagca 840
aattagttgt ttcagaatcc tatgatactt atatcagtag aagtttccaa gtaacaaaag 900
aaataataag tgaatgtaaa agtaaaggaa ataacatcct gattgtggcc cacgcatctt 960
cccttgaagc gtgtacctgc caacttcagg gcctgtcacc tcagaactcc aaggacttcg 1020
tacaaatggt ccgaaagatc ccatatctgg gattttgttc ctgtgaagaa ttaggagaaa 1080
ctggaatatg gcagctgaca gatccaccaa tccttcctct tacccatgga ccaactgggg 1140
gcttcaactg gagagagacc ttgcttcaag aataaaccat accagtgaac aagaaggaaa 1200
aaaaaaaaaa 1210
<210> 23
<211> 1521
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1416174CB1
<400> 23
ggcacggtgc tgggcctcgc cacactgcac acctaccgcg gacgcctctc ctacctcccc 60
gccactgtgg aacctgcctc gcccacccct gcccatagcc tgcctcgtgc caagtcggag 120
ctgaccctaa ccccagaccc agccccgccc atggcccact cacccctgca tcgttctgtg 180
tctgacctgc ctcttcccct gccccagcct gccctggcct ctcctggctc gccagaaccc 240
ctgcccatcc tgtccctcaa cggtgggggc ccagagctgg ctggggactg gggtggggct 300
ggggatgctc cgctgtcccc ggacccactg ctgtcttcac ctcctggctc tcccaaggca 360
gctctacact cacccgtctc cgaagggccc ccgtaattcc cccatcctct gggctcccac 420
ttcccacccc tgatgcccgg gtaggggcct ccacctgcgg cccgcccgac cacctgctgc 480
ctccgctggg caccccgctg cccccagact gggtgacgct ggagggggac tttgtgctca 540
tgttggccat ctcgcccagc cacctaggcg ctgacctggt ggcagctccg catgcgcgct 600
tcgacgacgg cctggtgcac ctgtgctggg tgcgtacggg catctcgcgg gctgcgctgc 660
tgcgcctttt cttggccatg gagcgtggta gccacttcag cctgggctgt ccgcagctgg 720
gctacgccgc ggcccgtgcc ttccgcctag agccgctcac accacgcggc gtgctcacag 780
tggacgggga gcaggtggag tatgggccgc tacaggcaca gatgcaccct ggcatcggta 840
cactgctcac tgggcctcct ggctgcccgg ggcgggagcc ctgaaactaa acaagcttgg 900
tacccgccgg gggcggggcc tacattccaa tggggcggag ctgagctagg gggtgtggcc 960
tggctgctag agttgtggtg gcaggggccc tggccccgtc tcaggattgc gctcgctttc 1020
atgggaccag acgtgatgct ggaaggtggg cgtcgtcacg gttaaagaga aatgggctcg 1080
tcccgagggt agtgcctgat caatgagggc ggggcctggc gtctgatctg gggccgccct 1140
tacggggcag ggctcagtcc tgacgcttgc cacctgctcc tacccggcca ggatggctga 1200
gggcggagtc tattttacgc gtcgcccaat gacaggacct ggaatgtact ggctggggta 1260
ggcctcagtg agtcggccgg tcagggcccg cagcctcgcc ccatccactc cggtgcctcc 1320
atttagctgg ccaatcagcc caggaggggc aggttccccg gggccggcgc taggatttgc 1380
actaatgttc ctctccccgc gggtgggggc ggggaaattc atatcccctg ttcgtctcat 1440
gcgcgtcctc cgtccccaat ctaaaaagca attgaaaagg tctatgcaat aaaggcagtc 1500
gcttcattcc tctcaaaaaa a 1521
<210> 24
<211> 1640
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
25/26
CA 02395102 2002-06-19
WO 01/46397 PCT/US00/35304
<223> Incyte ID No: 3244919CB1
<400> 24
gcagcgccgc ggcgtccccg ggctcgccgc cccccggccg cgcgcgcccc gccggctccg 60
acgcgccctc ggccctgccg ccgcccgctg ctggccagcc ccgggcccgg gactcgggcg 120
atgtccgctc gcagccgcgc cccctgtttc agtggagcaa gtggaagaag aggatgggct 180
cgtccatgtc ggcggccacc gcgcggaggc cggtgtttga cgacaaggag gacgtgaact 240
tcgaccactt ccagatcctt cgggccattg ggaagggcag ctttggcaag gtgtgcattg 300
tgcagaagcg ggacacggag aagatgtacg ccatgaagta catgaacaag cagcagtgca 360
tcgagcgcga cgaggtccgc aacgtcttcc gggagctgga gatcctgcag gagatcgagc 420
acgtcttcct ggtgaacctc tggtactcct tccaggacga ggaggacatg ttcatggtcg 480
tggacctgct actgggcggg gacctgcgct accacctgca gcagaacgtg cagttctccg 540
aggacacggt gaggctgtac atctgcgaga tggcactggc tctggactac ctgcgcggcc 600
agcacatcat ccacagagat gtcaagcctg acaacattct cctggatgag agaggacatg 660
cacacctgac cgacttcaac attgccacca tcatcaagga cggggagcgg gcgacggcat 720
tagcaggcac caagccgtac atggctccgg agatcttcca ctcttttgtc aacggcggga 780
ccggctactc cttcgaggtg gactggtggt cggtgggggt gatggcctat gagctgctgc 840
gaggatggag gccctatgac atccactcca gcaacgccgt ggagtccctg gtgcagctgt 900
tcagcaccgt gagcgtccag tatgtcccca cgtggtccaa ggagatggtg gccttgctgc 960
ggaagctcct cactgtgaac cccgagcacc ggctctccag cctccaggac gtgcaggcag 1020
ccccggcgct ggccggcgtg ctgtgggacc acctgagcga gaagagggtg gagccgggct 1080
tcgtgcccaa caaaggccgt ctgcactgcg accccacctt tgagctggag gagatgatcc 1140
tggagtccag gcccctgcac aagaagaaga agcgcctggc caagaacaag tcccgggaca 1200
acagcaggga cagctcccag tccgccccac ggagcaagtc caagccatcc acccagaggc 1260
aagggagctg ggccttggca tcctcgggct tgggagaatg actatcttca agactgcctc 1320
gatgccatcc agcaagactt cgtgattttt aacagagaaa agctgaagag gagccaggac 1380
ctcccgaggg agcctctccc cgcccctgag tccagggatg ctgcggagcc tgtggaggac 1440
gaggcggaac gctccgccct gcccatgtgc ggccccattt gcccctcggc cgggagcggc 1500
taggccggga cgcccgtggt cctcacccct tgagctgctt tggagactcg gctgccagag 1560
ggagggccat gggccgaggc ctggcattca cgttcccacc cagcctggct ggcggtgccc 1620
acagtgcccc ggacacattt 1640
26/26
