Note: Descriptions are shown in the official language in which they were submitted.
WO 00/55332 CA 02364739 2001-08-20 pCT/US00/07277
REGULATORS OF INTRACELLULAR PHOSPHORYLATION
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of regulators
of intracellular
phosphorylation and to the use of these sequences in the diagnosis, treatment,
and prevention of
neurological, cell proliferative, and autoimmune/inflammatory disorders.
BACKGROUND OF THE INVENTION
Reversible protein phosphorylation is the main strategy for controlling the
activities of
eukaryotic cells. Kinases and phosphatases regulate reversible phosphorylation
reactions, and are
thus critical components of intracellular signal transduction pathways.
Protein kinases transfer the
high energy phosphate from adenosine triphosphate (ATP) to specific protein
targets in response to
extracellular signals (such as hormones, neurotransmitters, and growth and
differentiation factors),
cell cycle checkpoints (for example, signals associated with mitotic events),
and environmental or
nutritional stresses. Protein phosphatases mediate kinase effects by removing
phosphate groups from
proteins.
It is estimated that more than 1000 of the 10,000 proteins active in a typical
mammalian cell
are phosphorylated. In general, protein activity is stimulated by
phosphorylation, and this is
analogous to turning on a molecular switch. When the switch is turned on, the
appropriate protein
kinase activates a metabolic enzyme, regulatory protein, receptor,
cytoskeletal protein, ion channel or
pump, or transcription factor. Protein activity is repressed by
dephosphorylation when down-
regulation of a signaling pathway is required. The coordinate activities of
kinases and phosphatases
regulate key processes such as cell proliferation, cell differentiation, cell-
cell communication, and
cell cycle progression. Uncontrolled signaling has been implicated in a
variety of disease conditions
including inflammation, cancer, arteriosclerosis, and psoriasis.
Protein kinases phosphorylate protein acceptor molecules on hydroxylated amino
acids.
These kinases comprise the largest known protein group, a superfamily of
enzymes with widely
varied functions and specificities. Protein kinases are usually named after
substrates, regulatory
molecules, or some aspect of a mutant phenotype. With regard to substrates,
protein kinases may be
roughly divided into two groups: those that phosphorylate tyrosine residues
(protein tyrosine kinases
(PTKs)), and those that phosphorylate serine or threonine residues
(serine/threonine kinases (STKs)).
A few protein kinases have dual specificity and phosphorylate serine,
threonine, and tyrosine
residues. Some STKs and PTKs possess structural characteristics of both
families (Hardie, G. and S.
Hanks (1995) The Protein Kinase Facts Book, Vol. I:7-20, Academic Press, San
Diego CA).
Almost all kinases contain a conserved 250-300 amino acid kinase domain that
folds into a
WO 00/55332 CA 02364739 2001-08-20 PCT/US00/07277
two-lobed structure. The primary structure of the kinase domain is conserved
and can be further
subdivided into 11 subdomains. The smaller N-terminal lobe of the kinase
domain, which contains
subdomains I through IV, is primarily involved in the binding and orientation
of the ATP (or GTP)
donor molecule. The larger C terminal lobe, which contains subdomains VI
through XI, binds the
protein substrate and carries out transfer of the gamma phosphate from ATP to
the hydroxyl group of
a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.
Each of the 11
subdomains contains specific amino acid residues and motifs that are
characteristic of the particular
subdomain and are highly conserved (Hardie, G. and S. Hanks (1995) The Protein
Kinase Facts
Book, Vol. I:7-20, 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 (subdomain II), and the second containing an aspartate
residue important for
catalytic activity (subdomain VI). Kinases may also be categorized into
families by the different
amino acid sequences (generally between 5 and 100 residues) located on either
side of, or inserted
into, the kinase domain. These additional amino acid sequences are involved in
the regulation of
each kinase as the kinase recognizes and interacts with its target protein.
PTKs may be classified as either transmembrane or non-transmembrane proteins.
Transmembrane PTKs function as receptors for most growth factors. Growth
factors bind to the
receptor tyrosine kinase (RTK), causing the RTK to phosphorylate itself
(autophosphorylation) and
specific intracellular second messenger proteins. Growth factors that bind
RTKs include epidermal
growth factor, platelet-derived growth factor, fibroblast growth factor,
hepatocyte growth factor,
insulin and insulin-like growth factors, nerve growth factor, vascular
endothelial growth factor, and
macrophage colony stimulating factor.
Non-transmembrane PTKs form signaling complexes with the cytosolic domains of
plasma
membrane receptors. Receptors that signal through non-transmembrane PTKs
include receptors for
cytokines and hormones (e.g., growth hormone and proiactin), and antigen-
specific receptors on T
and B lymphocytes. Many non-transmembrane PTKs were first identified as the
products of mutant
oncogenes in cancer cells in which PTK activation was no longer subject to
normal cellular controls.
About one third of the known oncogenes encode PTKs, and it is well known that
cellular
transformation (oncogenesis) is often accompanied by increased tyrosine
phosphorylation activity
(Charbonneau H and N.K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-493).
Regulation of PTK
activity may therefore be an important strategy in controlling some types of
cancer.
STKs are non-transmembrane proteins. STKs include second messenger-dependent
protein
kinases, which primarily mediate the effects of second messengers such as
cyclic AMP, cyclic GMP,
inositol triphosphate, phosphatidylinositol 3,4,5-triphosphate, cyclic ADP
ribose, arachidonic acid,
diacylglycerol, and calcium-calmodulin (CaM). STKs include cyclic AMP
dependent protein kinases
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(PKAs), which are involved in mediating hormone-induced cellular responses;
CaM-dependent
protein kinases, which are involved in regulation of smooth muscle
contraction, glycogen breakdown,
and neurotransmission; mitogen-activated protein (MAP) kinases, which mediate
signal transduction
from the cell surface to the nucleus via phosphorylation cascades; and
diacylglycerol-activated
protein kinase C (PKC), which mediates glycogen breakdown and activation of
various transcription
factors. PKC mu is a novel member of the PKC family that, like other PKCs,
contains a
zinc-finger-like, cysteine-rich motif in the N-terminal region necessary for
phorbol ester binding, and
is also capable of phorbol ester-independent kinase activity (Johannes, F.J.
et al. (1994) J. Biol.
Chem. 269:6140-6148).
The PKAs are activated by cAMP produced within the cell in response to hormone
stimulation. 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, 1987).
CaM dependent protein kinases are activated by calmodulin, an intracellular
calcium receptor, in
response to the concentration of free calcium in the cell. CaM kinase I and
CaM kinase II play
important roles in the regulation of neurotransmission, and kinases have been
associated with
neurological disorders such as Alzheimer's disease and with cognitive effects
of aging. (See, for
example, Lynch, M.A. (1998) Prog. Neurobiol. 56:571-589 and Bonkale, W.L. et
al. (1999) Brain
Res. 818:383-396.) CASK is a neuronal cell surface protein (neurexin) that
includes a calmodulin-
dependent protein kinase domain and is present in relatively high
concentrations in brain synaptic
plasma membranes (Hata, Y. et al. (1996) J. Neurosci. 16:2488-2494). CASK
forms part of a
complex capable of binding the amyloid precursor protein (APP) implicated in
Alzheimer's Disease,
and may play an important role in APP processing (Borg, J.P. et al. (1998) J.
Biol. Chem. 273:31633-
31636).
The cyclin-dependent protein kinases (CDKs) are STKs that control the
progression of cells
through the cell cycle. 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, CDK activation
requires the phosphorylation of a specific threonine residue and the
dephosphorylation of a specific
tyrosine residue. 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).
The MAP kinases comprise another STK family that regulates intracellular
signaling
pathways. The MAP kinases mediate signal transduction from the cell surface to
the nucleus via
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phosphorylation cascades. The extracellular stimuli which activate MAP kinase
pathways include
epidermal growth factor, ultraviolet light, hyperosmolar medium, heat shock,
endotoxic
lipopolysaccharide, and pro-inflammatory cytokines such as tumor necrosis
factor and interleukin-I .
Altered MAP kinase expression is implicated in a variety of disease conditions
including cancer,
inflammation, immune disorders, and disorders affecting growth and
development.
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 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). DRAKI 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. These types of kinases, ZIP, DAP, and DRAK, 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 apoptotic 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 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
apoptotic pathway (Inohara et al., su ra .
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Protein phosphatases regulate the effects of protein kinases by removing
phosphate groups
from molecules activated by kinases. Phosphatases are characterized as either
tyrosine-specific or
serine/threonine-specific based on their preferred phospho-amino acid
substrate, although some
protein phosphatases have dual specificity for both serine/threonine and
tyrosine groups.
Serine/threonine phosphatases dephosphorylate phosphoserine and
phosphothreonine residues, and
are important regulators of many cAMP-mediated hormone responses (Cohen, P.
(1989) Annu. Rev.
Biochem. 58:453-508). Serine/threonine phosphatases generally comprise two or
more subunits and
have broad and overlapping protein substrate specificities.
Tyrosine phosphatases are generally monomeric proteins which function
primarily in the
transduction of signals across the plasma membrane, and are categorized as
either transmembrane
receptor-like proteins or soluble non-transmembrane proteins. Tyrosine
phosphatases reverse the
effects of PTKs, removing phosphate groups from tyrosine residues of
phosphorylated proteins, and
play a significant role in cell cycle and cell signaling processes, lymphocyte
activation, and cell
adhesion (Charbonneau, H. and N.K. Tonks (1992) Annu. Rev. Cell Biol. 8:463-
493). In the process
of cell division, for example, a specific tyrosine phosphatase called M-phase
inducer phosphatase
plays a key role in the induction of mitosis by dephosphorylating and
activating CDC2, a cell
division-specific PTK (Krishna, S. et al. ( 1990) Proc. Natl. Acad. Sci. USA
87:5139-5143).
Tyrosine phosphatases share a conserved catalytic domain of about 250 amino
acids which
contains the active site. The active site consensus sequence consists of 13
amino acids, including a
cysteine residue that is essential for phosphatase activity. In addition, the
genes encoding at least
eight tyrosine phosphatases have been mapped to chromosomal regions that are
translocated or
rearranged in various neoplastic conditions, including lymphoma, leukemia,
small cell lung
carcinoma, adenocarcinoma, and neuroblastoma (Charbonneau, H. and N.K. Tonks (
1992) Annu.
Rev. Cell Biol. 8:463-493). As previously noted, many PTKs are encoded by
oncogenes, and
oncogenesis is often accompanied by increased tyrosine phosphorylation
activity. It is therefore
possible that tyrosine phosphatases may prevent or reverse cell transformation
and the growth of
various cancers by controlling the levels of tyrosine phosphorylation in
cells. This hypothesis is
supported by studies showing that overexpression of tyrosine phosphatases can
suppress
transformation in cells, and that specific inhibition of tyrosine phosphatases
can enhance cell
transformation (Charbonneau and Tonks, su ra).
In addition to protein phosphorylation, lipid phosphorylation also plays a
role in certain
signaling pathways. The phosphorylation of phosphatidylinositol is involved in
activation of the
PKC signaling pathway. Recently, a sphingolipid metabolite, sphingosine-1-
phosphate (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
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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
PKC, 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).
The discovery of new regulators of intracellular phosphorylation 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 neurological, cell proliferative, and
autoimmune/inflammatory
disorders.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, regulators of intracellular
phosphorylation,
referred to collectively as "HRIP" and individually as "HRIP-I," "HRIP-2,"
"HRIP-3," "HRIP-4,"
"HRIP-5," "HRIP-6," "HRIP-7," "HRIP-8," "HRIP-9," "HRIP-10," "HRIP-11," "HRIP-
12," "HRIP-
13," and "HRIP-14." In one aspect, the invention provides an isolated
polypeptide comprising.a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-14, 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:1-14, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-14, or d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:I-14. In one
alternative, the
invention provides an isolated polypeptide comprising the amino acid sequence
of SEQ ID NO:I-14.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
a) an amino acid sequence selected from the group consisting of SEQ ID NO:I-
14, 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:1-14, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-14, or d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:I-14. In one
alternative, the polynucleotide is selected from the group consisting of SEQ
ID NO:IS-28.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
a) an amino acid
sequence selected from the group consisting of SEQ ID N0:1-14, 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:1-14, c) a biologically active fragment of an amino
acid sequence selected
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WO 00/55332 PCT/US00/07277
from the group consisting of SEQ ID NO:1-14, or d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-14. In one
alternative, the invention
provides a cell transformed with the recombinant polynucleotide. In another
alternative, the invention
provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising a)
an amino
acid sequence selected from the group consisting of SEQ ID NO:1-14, 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:1-14, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-14, or d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-14. 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
IS polypeptide comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-14, 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:1-14, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-14, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-14.
The invention further provides an isolated polynucleotide comprising a) a
polynucleotide
sequence selected from the group consisting of SEQ ID NO:15-28, b) a naturally
occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected
from the group consisting of SEQ ID NO:15-28, c) a polynucleotide sequence
complementary to a),
or d) a polynucleotide sequence complementary to b). In one alternative, the
polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a) a
polynucleotide sequence selected from the group consisting of SEQ ID NO:15-28,
b) a naturally
occurring polynucleotide sequence having at least 90% sequence identity to a
polynucleotide
sequence selected from the group consisting of SEQ ID NO:15-28, c) a
polynucleotide sequence
complementary to a), or d) a polynucleotide sequence complementary to b). The
method comprises a)
hybridizing the sample with a probe comprising at least 16 contiguous
nucleotides comprising a
sequence complementary to said target polynucleotide in the sample, and which
probe specifically
hybridizes to said target polynucleotide, under conditions whereby a
hybridization complex is formed
WO 00/$$332 CA 02364739 2001-08-20 PCT/US00/07277
between said probe and said target polynucleotide, and b) detecting the
presence or absence of said
hybridization complex, and optionally, if present, the amount thereof. In one
alternative, the probe
comprises at least 30 contiguous nucleotides. In another alternative, the
probe comprises at least 60
contiguous nucleotides.
The invention further. provides a pharmaceutical composition comprising an
effective amount
of a polypeptide comprising a) an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-14, 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:I-14, c) a
biologically active
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-14, or d) an
immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ ID
NO:1-14, and a pharmaceutically acceptable excipient. The invention
additionally provides a method
of treating a disease or condition associated with decreased expression of
functional HRIP,
comprising administering to a patient in need of such treatment the
pharmaceutical composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising a) an amino acid sequence selected from
the group consisting of
SEQ ID NO: I-14, 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:I-
14, c) a biologically
active fragment of an amino acid sequence selected from the group consisting
of SEQ ID NO:1-14, or
d) an immunogenic fragment of an amino acid sequence selected from the group
consisting of SEQ
ID NO:1-14. 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 pharmaceutical composition comprising an agonist compound identified by the
method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional HRIP, comprising
administering to a patient in need of such treatment the pharmaceutical
composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising a) an amino acid sequence selected
from the group
consisting of SEQ ID NO:I-14, 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:I-14,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID N0:1-14, or d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:I-14. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a pharmaceutical composition comprising an antagonist
compound identified by
the method and a pharmaceutically acceptable excipient. In another
alternative, the invention
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provides a method of treating a disease or condition associated with
overexpression of functional
HRIP, comprising administering to a patient in need of such treatment the
pharmaceutical
composition.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID NO:15-28, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding HRIP.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
IS sequences, and methods, algorithms, and searchable databases used for
analysis of HRIP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-
specific
expression patterns of each nucleic acid sequence as determined by northern
analysis; diseases,
disorders, or conditions associated with these tissues; and the vector into
which each cDNA was
cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which
cDNA clones
encoding HRIP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze HRIP, along
with
applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
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Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"HRIP" refers to the amino acid sequences of substantially purified HRIP
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
HRIP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of HRIP either by
directly interacting with
HRIP or by acting on components of the biological pathway in which HRIP
participates.
An "allelic variant" is an alternative form of the gene encoding HRIP. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding HRIP include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as HRIP or a
polypeptide with at least one functional characteristic of HRIP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding HRIP, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
HRIP. 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 HRIP. 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 HRIP is
retained, For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
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amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurnng or synthetic
molecules. Where "amino acid sequence" is recited to refer to an amino acid
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 polymerise chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of HRIP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of HRIP either by
directly interacting with HRIP or by acting on components of the biological
pathway in which HRIP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind HRIP polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived
from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly
used carriers that are chemically coupled to peptides include bovine serum
albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
strand of a specific nucleic acid sequence. Antisense compositions may include
DNA; RNA; peptide
nucleic acid (PNA); oligonucleotides having modified backbone linkages such as
phosphorothioates,
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methylphosphonates, or benzylphosphonates; oligonucleotides having modified
sugar groups such as
2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having
modified bases such
as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2 =deoxyguanosine. Antisense
molecules may be
produced by any method including chemical synthesis or transcription. Once
introduced into a cell,
the complementary antisense .molecule base-pairs with a naturally occurring
nucleic acid sequence
produced by the cell to form duplexes which block either transcription or
translation. The
designation "negative" or "minus" can refer to the antisense strand, and the
designation "positive" or
"plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" refers to the
capability of the natural, recombinant, or synthetic HRIP, or of any
oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to bind with
specific antibodies.
The terms "complementary" and "complementarity" refer to the natural binding
of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A 5'." Complementarity between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarity exists between the single stranded molecules. The degree
of complementarity
between nucleic acid strands has significant effects on the efficiency and
strength of the hybridization
between the nucleic acid strands. This is of particular importance in
amplification reactions, which
depend upon binding between nucleic acid strands, and in the design and use of
peptide nucleic acid
(PNA) molecules.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding HR1P or fragments of
HRIP may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk
CT) in the 5' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
one or more Incyte Clones and, in some cases, one or more public domain ESTs,
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI). Some sequences have been both extended and assembled to produce the
consensus sequence.
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"Conservative amino acid substitutions" are those substitutions that, when
made, least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
A derivative
polynucleotide encodes a polypeptide which retains at least one biological or
immunological function
of the natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or
any similar process that retains at least one biological or immunological
function of the polypeptide
from which it was derived.
A "fragment" is a unique portion of HRIP or the polynucleotide encoding HRIP
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,
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a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID NO:15-28 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID NO:15-28, for example, as distinct from any
other sequence in the
same genome. A fragment of SEQ ID NO:15-28 is useful, for example, in
hybridization and
amplification technologies and in analogous methods that distinguish SEQ ID
NO:15-28 from related
polynucleotide sequences. The precise length of a fragment of SEQ ID NO:15-28
and the region of
SEQ ID NO:15-28 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:1-14 is encoded by a fragment of SEQ ID NO:I S-28. A
fragment
of SEQ ID NO:1-14 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:I-14. For example, a fragment of SEQ ID NO:l-14 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-14.
The precise length of
a fragment of SEQ ID NO:1-14 and the region of SEQ ID N0:1-14 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
The term "similarity" refers to a degree of complementarity. There may be
partial similarity
or complete similarity. The word "identity" may substitute for the word
"similarity." A partially
complementary sequence that at least partially inhibits an identical sequence
from hybridizing to a
target nucleic acid is referred to as "substantially similar." The inhibition
of hybridization of the
completely complementary sequence to the target sequence may be examined using
a hybridization
assay (Southern or northern blot, solution hybridization, and the like) under
conditions of reduced
stringency. A substantially similar sequence or hybridization probe will
compete for and inhibit the
binding of a completely similar (identical) sequence to the target sequence
under conditions of
reduced stringency. This is not to say that conditions of reduced stringency
are such that non-specific
binding is permitted, as reduced stringency conditions require that the
binding of two sequences to
one another be a specific (i.e., a selective) interaction. The absence of non-
specific binding may be
tested by the use of a second target sequence which lacks even a partial
degree of complementarity
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(e.g., less than about 30% similarity or identity). In the absence of non-
specific binding, the
substantially similar sequence or probe will not hybridize to the second non-
complementary target
sequence.
The phrases "percent identity" and "°Io identity," as applied to
polynucleotide sequences,
refer to the percentage of residue matches between at least two polynucleotide
sequences aligned
using a standardized algorithm. Such an algorithm may insert, in a
standardized and reproducible
way, gaps in the sequences being compared in order to optimize alignment
between two sequences,
and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequence pairs.
Alternatively, a suite of commonly used and Freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø9 (May-07-1999) set at default parameters. Such default parameters may be,
for example:
Matrix: BLOSUM62
Reward for match: l
Penalty for mismatch: -2
Open Gap: S and Extension Gap: 2 penalties
Gap x drop-off:' S0
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Expect: 10
Word Size: 11
Filter: on -
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known.. Sorne
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the
hydrophobicity and acidity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø9
(May-07-1999) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
Open Gap: II and Extension Gap: I penalties
Gap x drop-off:' S0
Expect: JO
Word Size: 3
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Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of identity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 pg/ml denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Generally, such wash temperatures
are selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring
Harbor Press, Plainview NY;
specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
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invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1 % SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, denatured salmon sperm DNA at about 100-200 ~g/ml.
Organic solvent, such
as formamide at a concentration of about 35-50% v/v, may also be used under
particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Itot 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 m an amino acid or
nucleotide
sequence resulting in the addition of one or more arr~ino 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 HRIP
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 HRIP which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
The terms "element'' and "array element" in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HRIP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of HRIP.
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
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synthetic origin which may be single-stranded or double.-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with the second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if.the promoter affects the transcription or
expression of the coding
sequence. Generally, operably linked DNA sequences may be in close proximity
or contiguous and,
where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Probe" refers to nucleic acid sequences encoding HRIP, 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 polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least I50 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook et al., 1989, Molecular Cloning~A Laborator~Manual, 2"d ed.,
vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel et a1.,1987, Current Protocols in
Molecular Biology,
Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis et al., 1990,
PCR Protocols, A
Guide to Methods and Applications, Academic Press, San Diego CA. PCR primer
pairs can be
derived from a known sequence, for example, by using computer programs
intended for that purpose
such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the
art fox such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
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100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
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instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding HRIP, or fragments thereof, or HRIP itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occur under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign
nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is
selected based on the type of host cell being transformed and may include, but
is not limited to, viral
infection, electroporation, heat shock, 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
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PCT/US00/07277
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, and plants and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
suQra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides generally will have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least
98% or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human regulators of
intracellular
phosphorylation (HRIF'), the polynucleotides encoding HRIP, and the use of
these compositions for
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WO 00/55332 PCT/US00/07277
the diagnosis, treatment, or prevention of neurological, cell proliferative,
and
autoimmune/inflammatory disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
HRIP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of
the polypeptide
and nucleotide sequences, respectively. Column 3 shows the clone IDs of the
Incyte clones in which
nucleic acids encoding each HRIP were identified, and column 4 shows the cDNA
libraries from
which these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA
libraries. The Incyte clones in column 5 were used to assemble the consensus
nucleotide sequence of
each HRIP and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
IS shows homologous sequences as identified by BLAST analysis; and column 7
shows analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding HRIP. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments
are useful, for example, in hybridization or amplification technologies to
identify SEQ ID NO:15-28
and to distinguish between SEQ ID NO:15-28 and related polynucleotide
sequences. For SEQ ID
NO:15-27, the polypeptides encoded by these fragments are useful, for example,
as immunogenic
peptides. Column 3 lists tissue categories which express HRIP as a fraction of
total tissues
expressing HRIP. Column 4 lists diseases, disorders, or conditions associated
with those tissues
expressing HRIP as a fraction of total tissues expressing HRIP. Column 5 lists
the vectors used to
subclone each cDNA library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding HRIP were isolated. Column 1 references the
nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated,
and column 3 shows
the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
The invention also encompasses HRIP variants. A preferred HRIP 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 HRIP amino acid sequence, and which contains at least one
functional or structural
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characteristic of HRIP.
The invention also encompasses polynucleotides which encode HRIP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID NO: IS-28, which encodes HRIP. The
polynucleotide sequences
of SEQ ID NO:15-28, 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
HRIP. In
particular, such a variant polynucleotide sequence will have at least about
80%, or alternatively at
least about 90%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding HRIP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
NO:15-28 which has at least about 80%, or alternatively at least about 90%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
of SEQ ID NO:IS-28. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of HRIP.
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 HRIP, some
bearing rninimal
similarity to the polynucleotide sequences of any known and naturally occurnng
gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring HRIP, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode HRIP and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring HRIP under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding HRIP or
its derivatives possessing a substantially different codon usage, e.g.,
inclusion of non-naturally
occurring codons. Codons may be selected to increase the rate at which
expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance with the
frequency with which
particular codons are utilized by the host. Other reasons for substantially
altering the nucleotide
sequence encoding HRIP 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 HRIP
and
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PCT/US00/07277
HRIP 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 HRIP 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
NO:15-28 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Perkin-
Elmer), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway
NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (M3 Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377
DNA sequencing
system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics,
Sunnyvale CA), or other systems known in the art. The 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 Biology, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. (1995)
Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding HRIP 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
CA 02364739 2001-08-20
WO 00/55332 PCT/LTS00/07277
digestioris 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
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the
entire process
from loading of samples to computer analysis and electronic data display may
be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode HRIP may be cloned in recombinant DNA molecules that direct
expression of HRIP, 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 HRIP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter HRIP-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
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WO 00/55332 PCT/US00/07277
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 HRIP, such as its biological or enzymatic
activity or its ability to
bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding HRIP 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, HRIP itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. ( 1995) Science 269:202-204.) Automated synthesis may be
achieved using the
ABI 431A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of HRIP, or
any part thereof, may be altered during direct synthesis and/or combined with
sequences from other
proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, T. ( 1984) Proteins, Structures and
Molecular Pro erties, WH
Freeman, New York NY.)
In order to express a biologically active HRIP, the nucleotide sequences
encoding HRIP 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
27
W~ 00/55332 CA 02364739 2001-08-20 PCT/US00/07277
inducible promoters, and 5' and 3' untranslated regions in the vector and in
polynucleotide sequences
encoding HRIP. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
HRIP. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding HRIP and its initiation codon and upstream regulatory
sequences are inserted into
the appropriate expression vector, no additional transcriptional or
translational control signals may be
needed. However, in cases where only coding sequence, or a fragment thereof,
is inserted, exogenous
translational control signals including an in-frame ATG initiation codon
should be provided by the
vector. Exogenous translational elements and initiation codons may be of
various origins, both
natural and synthetic. The efficiency of expression may be enhanced by the
inclusion of enhancers
appropriate for the particular host cell system used. (See, e.g., Scharf, D.
et al. (1994) Results Probl.
Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding HRIP 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 maybe utilized to contain and
express sequences
encoding HRIP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding HRIP. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding HRIP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORTI
plasmid (Life Technologies). Ligation of sequences encoding HRIP into the
vector's multiple cloning
site disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of HRIP are needed, e.g. for the
production of
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WO 00/55332 PCT/US00/07277
antibodies, vectors which direct high level expression of HRIP may be used.
For example, vectors
containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HRIl'. 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~astoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. ( 1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. ( 1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of HRIP. Transcription of
sequences encoding
HRIP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV
used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO
J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; 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 Y earbook 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 HRIP
may be ligated into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential EI or E3 region of the viral genome may
be used to obtain
infective virus which expresses HRIP in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of HRIF' in cell lines is preferred. For example, sequences encoding HRIP 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
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WO 00/55332 CA 02364739 2001-08-20 PCT/US00/07277
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., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate B-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding HRIP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding HRIP can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding HRIP 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 HRIP
and that express
HRIP may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of HRIP 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
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on HRIP 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 Laborator~Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (.1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. ( 1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
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 HRIP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding HRIP, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
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 detectian 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 HRIP 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 HRIP may be designed to contain signal
sequences which
direct secretion of HRIP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
31
WO 00155332 CA 02364739 2001-08-20
PCT/US00l07277
sequences encoding HRIl' 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 HRIP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of HRIP 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 HRIP encoding sequence and the
heterologous protein
sequence, so that HRIP may be cleaved away from the heterologous moiety
following purification.
IS 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 HRIP may
be achieved in
vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
Fragments of HRIP may be produced not only by recombinant means, but also by
direct
peptide synthesis using solid-phase techniques. (See, e.g., Creighton, su ra
pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation. Automated
synthesis may be
achieved, for example, using the ABI 431A peptide synthesizer (Perkin-Elmer).
Various fragments of
HRIP may be synthesized separately and then combined to produce the full
length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of HRIP and regulators of intracellular phosphorylation. In
addition, the expression
of HRIP is closely associated with neurological tissue, with cancer and other
cell proliferative
disorders, and with inflammation and the immune response. Therefore, HRIP
appears to play a role
in neurological, cell proliferative, and autoimmuneiinflammatory disorders. In
the treatment of
disorders associated with increased HRIP expression or activity, it is
desirable to decrease the
expression or activity of HRIP. In the treatment of disorders associated with
decreased HRIP
32
WO ~~/55332 CA 02364739 2001-08-20 PCZ'/LTS~O/07277
expression or activity, it is desirable to increase the expression or activity
of HRIP.
Therefore, in one embodiment, HRIP 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 HRIP.
Examples of such disorders include, but are not limited to, a neurological
disorder such as epilepsy,
ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive neural
muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating
diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural abscess,
suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease; prion diseases
including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal
familial insomnia, nutritional and metabolic diseases of the nervous system,
neurofibromatosis,
tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central nervous system,
cerebral palsy,
neuroskeletal disorders, autonomic nervous system disorders, cranial nerve
disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders, peripheral
nervous system
disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine,
and toxic myopathies;
myasthenia gravis, periodic paralysis; mental disorders including mood,
anxiety, and schizophrenic
disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia,
diabetic neuropathy,
tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia,
Tourette's disorder,
progressive supranuclear palsy, corticobasal degeneration, and familial
frontotemporal dementia; a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and a
cancer including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; and an
autoimmunelinflammatory 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
33
WO 00155332 CA 02364739 2001-08-20 pCTlUS00/07277
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.
In another embodiment, a vector capable of expressing HRIP 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 HRIP including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
HRIP 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 HRIP
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of HRIP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HRIP including, but not limited to, those listed above.
In a further embodiment, an antagonist of HRIP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of HRIP.
Examples of such
disorders include, but are not limited to, those neurological, cell
proliferative, and
autoimmune/inflammatory disorders described above. In one aspect, an antibody
which specifically
binds HRIP 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 HRIP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HRIP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of HRIP 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 HRIP may be produced using methods which are generally known
in the art.
In particular, purified HRIF' may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind HRIP.
Antibodies to HRIP may also
34
WO 00/55332 CA 02364739 2001-08-20 PCT/US00/07277
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 HRIP 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 Cor~nebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
HRIP have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein and contain the
entire amino acid sequence of a small, naturally occurring molecule. Short
stretches of HRIP 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 HRIP 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
HRIP-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
CA 02364739 2001-08-20
WO 00155332 PCT/US00/07277
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 foi HRIP may also be
generated.
For example, such fragments include, but are not limited to, F(ab~., 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
HRIP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering HRIP epitopes is generally used, but a
competitive binding assay may
also be employed (Pound, su ra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for HRIP. Affinity
is expressed as an
association constant, K~, which is defined as the molar concentration of HRIP-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple HRIP epitopes, represents the average affinity, or
avidity, of the antibodies for
HRIP. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular HRIP epitope, represents a true measure of affinity. High-affinity
antibody preparations
with K~ ranging from about 109 to 10''' Llmole are preferred for use in
immunoassays in which the
HRIP-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with K~ ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of HRIP, preferably in active
form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, DC;
Liddell, J.E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of HRIP-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
36
CA 02364739 2001-08-20
WO 00/55332 PCT/LTS00/07277
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding HRIP, or
any fragment
or complement thereof, may be used for therapeutic purposes. In one aspect,
the complement of the
polynucleotide encoding HRIP may be used in situations in which it would be
desirable to block the
transcription of the mRNA. In particular, cells may be transformed with
sequences complementary to
polynucleotides encoding HRIP. Thus, complementary molecules or fragments may
be used to
modulate HRIP activity, or to achieve regulation of gene function. Such
technology is now well
known in the art, and sense or antisense oligonucleotides or larger fragments
can be designed from
various locations along the coding or control regions of sequences encoding
HRIP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses, or
from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the targeted
organ, tissue, or cell population. Methods which are well known to those
skilled in the art can be
used to construct vectors to express nucleic acid sequences complementary to
the polynucleotides
encoding HRIP. (See, e.g., Sambrook, supra; Ausubel, 1995, supra.)
Genes encoding HRIP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof,
encoding HRIP. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in
the absence of integration into the DNA, such vectors may continue to
transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient expression may last
for a month or more
with a non-replicating vector, and may last even longer if appropriate
replication elements are part of
the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or
regulatory regions of the gene encoding HRIP. Oligonucleotides derived from
the transcription
initiation site, e.g., between about positions -10 and +10 from the start
site, may be employed.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix
pairing is useful because it causes inhibition of the ability of the double
helix to open sufficiently for
the binding of polymerases, transcription factors, or regulatory molecules.
Recent therapeutic
advances using triplex DNA have been described in the literature. (See, e.g.,
Gee, J.E. et al. (1994) in
Huber, B.E. and B.I. Carr, Molecular and Immunolo ig c 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
37
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
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 HRIP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding HRIP. 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.
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.
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CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
An additional embodiment of the invention relates to the administration of a
pharmaceutical
or sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any of the
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of HRIP,
antibodies to HRIP, and mimetics, agonists, antagonists, or inhibitors of
HRIP. The compositions
may be administered alone or in combination with at least one other agent,
such as a stabilizing
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier including,
but not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered
to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing
of the active compounds into preparations which can be used pharmaceutically.
Further details on
techniques for formulation and administration may be found in the latest
edition of Remin ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral administration.
Such carriers enable the pharmaceutical compositions to be formulated as
tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose, mannitol,
and sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums,
including arabic and
tragacanth; and proteins, such as gelatin and collagen. If desired,
disintegrating or solubilizing agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and
alginic acid or a salt thereof,
such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated sugar
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone,
carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
product identification or to
characterize the quantity of active compound, i.e., dosage.
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WO 00/55332 CA 02364739 2001-08-20
PCT/CTS00/07277
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders, such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules,
the active compounds may be.dissolved or suspended in suitable liquids, such
as fatty oils, liquid, or
liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution, Ringer's
solution, or physiologically buffered saline. Aqueous injection suspensions
may contain substances
which increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or
dextran. Additionally, suspensions of the active compounds may be prepared as
appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil,
or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or
liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the suspension may
also contain suitable
stabilizers or agents to increase the solubility of the compounds and allow
for the preparation of
highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
bawier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a manner
that is known in the art, e.g., by means of conventional mixing, dissolving,
granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and succinic
acids. Salts tend to be more soluble in aqueous or other protonic solvents
than are the corresponding
free base forms. In other cases, the preparation may be a lyophilized powder
which may contain any
or all of the following: I mM to 50 mM histidine, 0. I % to 2% sucrose, and
2°~o to 7% mannitol, at a
pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an
appropriate
container and labeled for treatment of an indicated condition. For
administration of HRIP, such
labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein
the active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs, or pigs.
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
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 HRIP
or fragments thereof, antibodies of HRIP, and agonists, antagonists or
inhibitors of HRIP, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LD59 (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio.
Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDSO with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be deter~rrrined by the practitioner, in light of
factors related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4
days, every week, or biweekly depending on the half life and clearance rate of
the particular
formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 fig, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind HRIP may be used for
the
diagnosis of disorders characterized by expression of HRIP, or in assays to
monitor patients being
treated with HRIP or agonists, antagonists, or inhibitors of HRIP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for HRIP include methods which utilize the antibody and a label to detect HRIP
in human body fluids
41
VVO 00/5532 CA 02364739 2001-08-20
PCT/US00/07277
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 HRIP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
HRIP expression. Normal
or standard values for HRIP expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to HRIP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of HRIP
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 HRIP 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 HRIP
may be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
HRIP.; and to monitor regulation of HRIP levels during therapeutic
intervention.
In one aspect; hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HRIP or closely related
molecules may be used to
identify nucleic acid sequences which encode HRIP. The specificity of the
probe, whether it is made
from a highly specific region, e.g., the 5'regulatory region, or from a less
specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding HRIP, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least SO~Io
sequence identity to any of the HRIP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:
I S-28 or from
genomic sequences including promoters, enhancers, and introns of the HRIP
gene.
Means for producing specific hybridization probes for DNAs encoding HRIP
include the
cloning of polynucleotide sequences encoding HRIP or HRIP 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.
42
WO 00155332 CA 02364739 2001-08-20 PCTlUS00/07277
Polynucleotide sequences encoding HRIP may be used for the diagnosis of
disorders
associated with expression of HRIP. Examples of such disorders include, but
are not limited to, a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis;
inherited, metabolic,
endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis; mental
disorders including
mood, anxiety, and schizophrenic disorders; seasonal affective disorder (SAD);
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia; a cell proliferative disorder such as actinic
keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and a
cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and
uterus; and an autoimmune/inflammatory disorder such as acquired
immunodeficiency syndrome
(AIDS), Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
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PCT/US00/07277
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. The polynucleotide sequences encoding HRIP 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 HRIP expression. Such qualitative or quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding HRIP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HR1P may be labeled by standard methods and added to a
fluid or tissue sample
from a patient under conditions suitable for the formation of hybridization
complexes. After a
suitable incubation period, the sample is washed and the signal is quantified
and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding HRIP 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 HRIP,
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 HRIP, 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, ar may provide a means for detecting the disease
prior to the appearance
44
CA 02364739 2001-08-20
wo ooiss33z rcTrosooio~2~~
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding HRIP
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 HRIP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
HRIP, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions far
detection or
quantification of closely related DNA or RNA sequences.
Methods which may also be used to quantify the expression of HRIP include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer of interest is
presented in various dilutions and a spectrophotometric or colorimetric
response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The microarray
can be used to monitor the expression level of large numbers of genes
simultaneously and to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, and to develop and
monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
( 1995) PCT application W095/35505; Heller, R.A. et al. ( 1997) Proc. Natl.
Acad. Sci. USA 94:2150
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
In another embodiment of the invention, nucleic acid sequences encoding HRIP
may be used
to generate hybridization probes useful in mapping the naturally occurring
genomic sequence. The
sequences may be mapped to a particular chromosome, to a specific region of a
chromosome, or to
artificial chromosome constructions, e.g., human artificial chromosomes
(HACs), yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single
chromosome cDNA libraries. (See, e.g., Harrington, J.J, et al. ( 1997) Nat.
Genet. 15:345-355; Price,
C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-
154.)
WO 00/55332 CA 02364739 2001-08-20
PCT/US00l07277
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome
mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (
1995) in Meyers, supra,
pp. 965-968.) Examples of genetic map data can be found in various scientific
journals or at the
Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation
between the
location of the gene encoding.HRIP on a physical chromosomal map and a
specific disorder, or a
predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the number or arm of a particular human
chromosome is not
known. New sequences can be assigned to chromosomal arms by physical mapping.
This provides
valuable information to investigators searching for disease genes using
positional cloning or other
gene discovery techniques. Once the disease or syndrome has been crudely
localized by genetic
linkage to a particular genomic region, e.g., ataxia-telangiectasia to I 1 q22-
23, any sequences mapping
to that area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti,
R.A. et al. ( 1988) Nature 336:577-580.) The nucleotide sequence of the
subject invention may also
be used to detect differences in the chromosomal location due to
translocation, inversion, etc., among
normal, carrier, or affected individuals.
In another embodiment of the invention, HRIP, 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 HRIP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. ( I 984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with HRIP, or
fragments thereof,
and washed. Bound HRIP is then detected by methods well known in the art.
Purified HRIP 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 HRIP specifically compete with a test compound
for binding HRIP. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
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WO 00/55332 PCT/US00/07277
antigenic determinants with HRIP.
In additional embodiments, the nucleotide sequences which encode HRIP may be
used in any
molecular biology techniques that have yet to be developed, provided the new
techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
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
particularU.S. Ser. No. 60/125,593, U.S. Ser. No. 60/135,049, and U.S. Ser.
No. 60/143,188, are
hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN.
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, s_~ra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
47
WU 00/$$332 CA 02364739 2001-08-20 PCT/US00/07277
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.l plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Pharmaceuticals, Palo Alto
CA). Recombinant
plasmids were transformed into competent E. coli cells including XLI-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DHIOB, or ElectroMAX DHIOB from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system
(Stratagene) or by cell lysis. Plasmids were purified using at least one of
the following: a Magic or
WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep
purification kit (Edge
Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
QIAWELL 8
Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid
purification kit from QIAGEN.
Following precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or
without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
cDNA sequencing reactions were processed using standard methods or high-
throughput
instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cycler or
the PTC-200
thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser
(Robbins Scientific)
or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing
reactions were
prepared using reagents provided by Amersham Pharmacia Biotech or supplied in
ABI sequencing
kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction
kit (Perkin-Elmer).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) in conjunction with
standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, s_ unra, unit 7.7). Some of the cDNA sequences were selected for
extension using the techniques
disclosed in Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
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WO 00/55332 PCT/US00/07277
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
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, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. ( 1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
NO:15-28. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
sera, ch. 7; Ausubel,
49
WO 00/55332 CA 02364739 2001-08-20 PC'fi[jS00/07277
1995, s_ unra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte
Pharmaceuticals). This
analysis is much faster than multiple membrane-based hybridizations. In
addition, the sensitivity of
the computer search can be modified to determine whether any particular match
is categorized as
exact or similar. The basis of the search is the product score, which is
defined as:
% sequence identity x % maximum BLAST score
100
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a 1 % to 2% error, and, with a product score of 70, the match will be
exact. Similar molecules
are usually identified by selecting those which show product scores between 15
and 40, although
lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding HRIP occurred. Analysis involved the categorization of
cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Extension of HRIP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID NO:15-28 were produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg'-+, (NH4),S04,
and (3-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and4 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 girl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~tl 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 ~l to 10 ~l aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Phar~rrracia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, individual colonies were picked and cultured
overnight at 37°C in 384-
well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Phar7nacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, IS sec; Step 3:
60°C, I min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
51
CA 02364739 2001-08-20
WO 00!55332 PCT/US00/07277
In like manner, the nucleotide sequences of SEQ ID N0:15-28 are used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension, and an
appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:15-28 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
[y-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 ane of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, s-upra.)
An array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, UV, chemical, or mechanical bonding procedures. A typical array may
be produced by hand
or using available methods and machines and contain any appropriate number of
elements. After
hybridization, nonhybridized probes are removed and a scanner used to
determine the levels and
patterns of fluorescence. The degree of complementarity and the relative
abundance of each probe
which hybridizes to an element on the microarray may be assessed through
analysis of the scanned
images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise
the elements of the microarray. Fragments suitable for hybridization can be
selected using software
well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs,
ESTs, or
fragments thereof corresponding to one of the nucleotide sequences of the
present invention, or
52
WO 00/55332 CA 02364739 2001-08-20
PCTIUS00107277
selected at random from a cDNA library relevant to the present invention, are
arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide
using, e.g., UV cross-linking
followed by thermal and chemical treatments and subsequent drying. (See, e.g.,
Schena, M. et al.
( 1995) Science 270:467-470; Shalom D. et al. ( 1996) Genome Res. 6:639-645.)
Fluorescent probes
are prepared and used for hybridization to the elements on the substrate. The
substrate is analyzed by
procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the HRIP-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring HR1P. 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 HRIP. 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 HR1P-encoding transcript.
IX. Expression of HRIP
Expression and purification of HRIP is achieved using bacterial or virus-based
expression
systems. For expression of HR1P in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express HRIP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of HRIP in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Auto~raphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovin.~s is
replaced with cDNA encoding HRIP 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 frug_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: I 937-1945.)
In most expression systems, HRIP 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,
53
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosomalaponicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
HRIP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel ( 1995,
supra, ch. 10 and 16). Purified HRIP obtained by these methods can be used
directly in the following
activity assay.
X. Demonstration of HRIP Activity
Kinase activity of HRIP is measured by the phosphorylation of appropriate
substrates using
gamma-labeled 3zP-ATP and quantitation of the incorporated radioactivity using
a beta radioisotope
counter. HRIP is incubated with the protein substrate, 32P-ATP, and an
appropriate kinase buffer.
The 3zP incorporated into the product is separated from free 32P-ATP by
electrophoresis and the
incorporated 32P is counted. The amount of 32P recovered is proportional to
the kinase activity of
HRIP in the assay. A determination of the specific amino acid residue
phosphorylated by protein
kinase activity is made by phosphoamino acid analysis of the hydrolyzed
protein.
Alternatively, protein phosphatase activity of HRIP is measured by the
hydrolysis of P-
nitrophenyl phosphate (PNPP). HRIP is incubated together with PNPP in HEPES
buffer pH 7.5, in
the presence of 0.1 % b-mercaptoethanol at 37 °C for 60 min. The
reaction is stopped by the addition
of 6 ml of 10 N NaOH and the increase in light absorbance at 410 nm resulting
from the hydrolysis of
PNPP is measured using a spectrophotometer. The increase in light absorbance
is proportional to the
activity of HRIP in the assay (Diamond, R.H. et al. (1994) Mol. Cell Biol.
14:3752-3762).
XI. Functional Assays
HRIP function is assessed by expressing the sequences encoding HRIP at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~.g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
54
CA 02364739 2001-08-20
WO 00!55332 PCTIUS00/07277
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 Cvtometrv, Oxford, New York NY.
The influence of HRIP on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding HRIP and either CD64 or CD64-GFP.
CD64 and CD64-
GFP are expressed on the surface of transfected cells and bind to conserved
regions of human
immunoglobulin G (IgG). Transfected cells are efficiently separated from
nontransfected cells using
magnetic beads coated with either human IgG or antibody against CD64 (DYNAL,
Lake Success
NY). mRNA can be purified from the cells using methods well known by those of
skill in the art.
Expression of mRNA encoding HRIP and other genes of interest can be analyzed
by northern analysis
or microarray techniques.
XII. Production of HRIP Specific Antibodies
HRIP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. ( 1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the HR1P amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, s-bra.) Rabbits are immunized with
the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
HRIP activity by, for example, binding the peptide or HRIP to a substrate,
blocking with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
WO 00/55332 CA 02364739 2001-08-20
PCT/US00/07277
XIII. Purification of Naturally Occurring HRIP Using Specific Antibodies
Naturally occurnng or recombinant HRIP is substantially purified by
immunoaffinity
chromatography using antibodies specific for HRIP. An immunoaffinity column is
constructed by
covalently coupling anti-HRIP 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 HRIP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of HRIP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/HRIP 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 HRIP is collected.
XIV. Identification of Molecules Which Interact with HRIP
HRIP, 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 HRIP, washed, and
any wells with labeled HRIP complex are assayed. Data obtained using different
concentrations of
HRIP are used to calculate values for the number, affinity; and association of
HRIP with the
candidate molecules.
Alternatively, molecules interacting with HRIP are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song ( 1989, Nature 340:245-246), or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
56
WO 00/5$332 CA 02364739 2001-08-20
PCT/US00/07277
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67
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
BANDMAN, Olga
TANG, Y. Tom
YUE,Henry
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
AZIMZAI, Yalda
LU, Dyung Aina M.
AU-YOUNG, Janice
<120> REGULATORS OF INTRACELLULAR PHOSPHORYLATION
<130> PF-0683 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/125,593; 60/135,049; 60/143,188
<151> 1999-03-18; 1999-05-20; 1999-07-09
<160> 28
<170> PERL Program
<210> 1
<211> 482
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 480457CD1
<400> 1
Met Pro Pro Ser Pro Leu Asp Asp Arg Val Val Val Ala Leu Ser
1 5 10 15
Arg Pro Va1 Arg Pro G1n Asp Leu Asn Leu Cys Leu Asp Ser Ser
20 25 30
Tyr Leu Gly Ser Ala Asn Pro Gly Ser Asn Ser His Pro Pro Val
35 40 45
Ile Ala Thr Thr Val Val Ser Leu Lys Ala Ala Asn Leu Thr Tyr
50 55 60
Met Pro Ser Ser Ser Gly Ser Ala Arg Ser Leu Asn Cys Gly Cys
65 70 75
Ser Ser Ala Ser Cys Cys Thr Val Ala Thr Tyr Asp Lys Asp Asn
80 85 90
Gln Ala Gln Thr Gln Ala Ile Ala Ala Gly Thr Thr Thr Thr Ala
95 100 105
Ile Gly Thr Ser Thr Thr Cys Pro Ala Asn Gln Met Val Asn Asn
110 115 120
Asn Glu Asn Thr Gly Ser Leu Ser Pro Ser Ser Gly Val Gly Ser
125 130 135
Pro Val Ser Gly Thr Pro Lys Gln Leu Ala Ser Ile Lys Ile Ile
140 145 150
Tyr Pro Asn Asp Leu Ala Lys Lys Met Thr Lys Cys Ser Lys Ser
155 160 165
His Leu Pro Ser Gln Gly Pro Val Ile Ile Asp Cys Arg Pro Phe
170 175 180
1/24
W~ 00/$5332 CA 02364739 2001-08-20
PCT/US00/07277
PF-0683 PCT
Met Glu Tyr Asn Lys Ser His Ile Gln Gly Ala Val His Ile Asn
185 190 195
Cys Ala Asp Lys Ile Ser Arg Arg Arg Leu Gln Gln Gly Lys Ile
200 205 210
Thr Val Leu Asp Leu Ile Ser Cys Arg Glu Gly Lys Asp Ser Phe
215 220 225
Lys Arg Ile Phe Ser Lys Glu Ile Ile Val Tyr Asp Glu Asn Thr
230 235 240
Asn Glu Pro Ser Arg Val Met Pro Ser Gln Pro Leu His Ile Val
245 250 255
Leu Glu Ser Leu Lys Arg Glu Gly Lys Glu Pro Leu Val Leu Lys
260 265 270
Gly Gly Leu Ser Ser Phe Lys Gln Asn His Glu Asn Leu Cys Asp
275 280 285
Asn Ser Leu Gln Leu Gln Glu Cys Arg Glu Val Gly Gly Gly Ala
290 295 300
Ser Ala Ala Ser Ser Leu Leu Pro Gln Pro Ile Pro Thr Thr Pro
305 310 315
Asp Ile Glu Asn Ala Glu Leu Thr Pro Ile Leu Pro Phe Leu Phe
320 325 330
Leu Gly Asn Glu Gln Asp Ala Gln Asp Leu Asp Thr Met Gln Arg
335 340 345
Leu Asn Ile Gly Tyr Val Ile Asn Val Thr Thr His Leu Pro Leu
350 355 360
Tyr His Tyr Glu Lys Gly Leu Phe Asn Tyr Lys Arg Leu Pro Ala
365 370 375
Thr Asp Ser Asn Lys Gln Asn Leu Arg Gln Tyr Phe Glu Glu Ala
380 385 390
Phe Glu Phe Ile G1u Glu Ala His Gln Cys Gly Lys Gly Leu Leu
395 400 405
Ile His Cys Gln Ala Gly Val Ser Arg Ser Ala Thr Ile Val Ile
410 415 420
Ala Tyr Leu Met Lys His Thr Arg Met Thr Met Thr Asp Ala Tyr
425 430 435
Lys Phe Val Lys Gly Lys Arg Pro Ile Ile Ser Pro Asn Leu Asn
440 445 450
Phe Met Gly Gln Leu Leu Glu Phe Glu Glu Asp Leu Asn Asn Gly
455 460 465
Val Thr Pro Arg Ile Leu Thr Pro Lys Leu Met Gly Val Glu Thr
470 475 480
Val Val
<210> 2
<211> 190
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 563663CD1
<400> 2
Met Ser Arg Arg Arg Phe Asp Cys Arg Ser Ile Ser Gly Leu Leu
1 5 10 15
Thr Thr Thr Pro Gln Ile Pro I1e Lys Met Glu Asn Phe Asn Asn
2C 25 30
Phe Tyr Ile Leu Thr Ser Lys G1u Leu Gly Arg Gly Lys Phe Ala
35 40 45
Va1 Val Arg Gln Cys Tle Ser Lys Ser Thr Gly Gln Glu Tyr Ala
50 55 60
2/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
Ala Lys Phe Leu Lys Lys Arg Arg Arg Gly Gln Asp Cys Arg Ala
65 70 75
Glu Ile Leu His Glu Ile Ala Val Leu Glu Leu Ala Lys Ser Cys
80 85 90
Pro Arg Val Ile Asn Leu His Glu Val Tyr Glu Asn Thr Ser Glu
95 100 105
Ile Ile Leu Ile Leu Glu Tyr Ala Ala Gly Gly Glu Ile Phe Ser
110 115 120
Leu Cys Leu Pro Glu Leu Ala Glu Met Val Ser Glu Asn Asp Val
125 130 135
Ile Arg Leu Ile Lys Gln Ile Leu Glu Gly Val Tyr Tyr Leu His
140 145 150
Gln Asn Asn Ile Val His Leu Asp Leu Lys Pro Gln Asn Ile Leu
155 160 165
Leu Ser Ser Ile Tyr Pro Leu Gly Asp Ile Lys Ile Val Asp Gly
170 175 180
Gly Met Ser Arg Lys Ile Gly Gln Cys Val
185 190
<210> 3
<211> 455
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1425842CD1
<400> 3
Met Ser Ser Leu Gly Ala Ser Phe Val Gln Ile Lys Phe Asp Asp
1 5 10 15
Leu Gln Phe Phe Glu Asn Cys Gly Gly Gly Ser Phe Gly Ser Val
20 25 30
Tyr Arg Ala Lys Trp Ile Ser Gln Asp Lys Glu Val Ala Val Lys
35 40 45
Lys Leu Leu Lys Ile Glu Lys Glu Ala Glu Ile Leu Ser Val Leu
50 55 60
Ser His Arg Asn Ile Ile Gln Phe Tyr Gly Val Ile Leu Glu Pro
65 70 75
Pro Asn Tyr Gly Ile Val Thr Glu Tyr Ala Ser Leu Gly Ser Leu
80 85 90
Tyr Asp Tyr Ile Asn Ser Asn Arg Ser Glu Glu Met Asp Met Asp
95 100 105
His Ile Met Thr Trp Ala Thr Asp Val Ala Lys Gly Met His Tyr
110 115 120
Leu His Met Glu Ala Pro Val Lys Val Ile His Arg Asp Leu Lys
125 130 135
Ser Arg Asn Val Val Ile Ala Ala Asp Gly Val Leu Lys Ile Cys
140 145 150
Asp Phe Gly Ala Ser Arg Phe His Asn His Thr Thr His Met Ser
155 160 165
Leu Val Gly Thr Phe Pro Trp Met Ala Pro Glu Val Ile Gln Ser
170 175 180
Leu Pro Val Ser Glu Thr Cys Asp Thr Tyr Ser Tyr Gly Val Val
185 190 195
Leu Trp Glu Met Leu Thr Arg Glu Val Pro Phe Lys Gly Leu Glu
200 205 210
Gly Leu Gln Val Ala Trp Leu Val Val Glu Lys Asn Glu Arg Leu
215 220 225
Thr Ile Pro Ser Ser Cys Pro Arg Ser Phe Ala Glu Leu Leu His
3/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-06$3 PCT
230 235 240
G1n Cys Trp Glu Ala Asp Ala Lys Lys Arg Pro Ser Phe-Lys Gln
245 250 255
Ile Ile Ser Ile Leu Glu Ser Met Ser Asn Asp Thr Ser Leu Pro
260 265 270
Asp Lys Cys Asn Ser Phe Leu His Asn Lys Ala Glu Trp Arg Cys
275 280 285
Glu Ile Glu Ala Thr Leu Glu Arg Leu Lys Lys Leu Glu Arg Asp
290 295 300
Leu Ser Phe Lys Glu Gln Glu Leu Lys G1u Arg Glu Arg Arg Leu
305 310 315
Lys Met Trp Glu Gln Lys Leu Thr Glu Gln Ser Asn Thr Pro Leu
320 325 330
Leu Leu Pro Leu Ala Ala Arg Met Ser Glu Glu Ser Tyr Phe Glu
335 340 345
Ser Lys Thr Glu Glu Ser Asn Ser Ala Glu Met Ser Cys Gln Ile
350 355 360
Thr Ala Thr Ser Asn Gly Glu Gly His Gly Met Asn Pro Ser Leu
365 370 375
Gln Ala Met Met Leu Met Gly Phe Gly Asp Ile Phe Ser Met Asn
380 385 390
Lys Ala Gly Ala Val Met His Ser Gly Met Gln Ile Asn Met Gln
395 400 405
Ala Lys Gln Asn Ser Ser Lys Thr Thr Ser Lys Arg Arg Gly Lys
410 415 420
Lys Val Asn Met Ala Leu Gly Phe Ser Asp Phe Asp Leu Ser Glu
425 430 435
Gly Asp Asp Asp Asp Asp Asp Asp Gly Glu G1u Glu Asp Asn Asp
440 445 450
Met Asp Asn Ser Glu
455
<210> 4
<211> 485
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2349047CD1
<400> 4
Met Ala Lys Gly Gly Ile Phe Pro Arg Pro Arg Cys Asp Ser Ser
1 5 10 15
Ser Leu Leu Glu Cys Arg Lys Ala Ile Ser Arg Glu Va1 Lys Ala
20 25 30
Met Ala Ser Leu Asp Asn Glu Phe Val Leu Arg Leu Glu Gly Val
35 40 45
Ile Glu Lys Val Asn Trp Asp Gln Asp Pro Lys Pro Ala Leu Val
50 55 60
Thr Lys Phe Met Glu Asn Gly Ser Leu Ser Gly Leu Leu Gln Ser
65 70 75
Gln Cys Pro Arg Pro Trp Pro Leu Leu Cys Arg Leu Leu Lys Glu
80 85 90
Val Val Leu Gly Met Phe Tyr Leu His Asp Gln Asn Pro Val Leu
95 100 105
Leu His Arg Asp Leu Lys Pro Ser Asn Val Leu Leu Asp Pro Glu
110 115 120
Leu His Val Lys Leu Ala Asp Phe Gly Leu Ser Thr Phe Gln Gly
125 130 135
4/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
Gly Ser Gln Ser Gly Thr Gly Ser Gly Glu Pro Gly Gly Thr Leu
140 145 150
Gly Tyr Leu Ala Pro Glu Leu Phe Val Asn Val Asn Arg Lys Ala
155 160 165
Ser Thr Ala Ser Asp Val Tyr Ser Phe Gly Ile Leu Met Trp Ala
170 175 180
Val Leu Ala Gly Arg Glu Val Glu Leu Pro Thr Glu Pro Ser Leu
185 190 195
Val Tyr Glu Ala Val Cys Asn Arg Gln Asn Arg Pro Ser Leu Ala
200 205 210
Glu Leu Pro Gln Ala Gly Pro Glu Thr Pro Gly Leu Glu Gly Leu
215 220 225
Lys Glu Leu Met Gln Leu Cys Trp Ser Ser Glu Pro Lys Asp Arg
230 235 240
Pro Ser Phe Gln Glu Cys Leu Pro Lys Thr Asp Glu Val Phe Gln
245 250 255
Met Val Glu Asn Asn Met Asn Ala Ala Val Ser Thr Val Lys Asp
260 265 270
Phe Leu Ser Gln Leu Arg Ser Ser Asn Arg Arg Phe Ser Ile Pro
275 280 285
Glu Ser Gly Gln Gly Gly Thr Glu Met Asp Gly Phe Arg Arg Thr
290 295 300
Ile Glu Asn Gln His Ser Arg Asn Asp Val Met Val Ser Glu Trp
305 310 315
Leu Asn Lys Leu Asn Leu Glu Glu Pro Pro Ser Ser Val Pro Lys
320 325 330
Lys Cys Pro Ser Leu Thr Lys Arg Ser Arg Ala Gln Glu Glu Gln
335 340 345
Val Pro Gln Ala Trp Thr Ala Gly Thr Ser Ser Asp Ser Met Ala
350 355 360
Gln Pro Pro Gln Thr Pro Glu Thr Ser Thr Phe Arg Asn Gln Met
365 370 375
Pro Ser Pro Thr Ser Thr Gly Thr Pro Ser Pro Gly Pro Arg Gly
380 385 390
Asn Gln Gly Ala Glu Arg Gln Gly Met Asn Trp Ser Cys Arg Thr
395 400 405
Pro Glu Pro Asn Pro Val Thr Gly Arg Pro Leu Val Asn Ile Tyr
410 415 420
Asn Cys Ser Gly Val Gln Val Gly Asp Asn Asn Tyr Leu Thr Met
425 430 435
Gln Gln Thr Thr Ala Leu Pro Thr Trp Gly Leu Ala Pro Ser Gly
440 445 450
Lys Gly Arg Gly Leu Gln His Pro Pro Pro Val Gly Ser Gln Glu
455 460 465
Gly Pro Lys Asp Pro Glu Ala Trp Ser Arg Pro Gln Gly Trp Tyr
470 475 480
Asn His Ser Gly Lys
485
<210> 5
<211> 384
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2415617CD1
<400> 5
Met Asp Pro Ala Gly Gly Pro Arg Gly Val Leu Pro Arg Pro Cys
5/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
1 5 10 15
Arg Val Leu Val Leu Leu Asn Pro Arg Gly Gly Lys Gly Lys Ala
20 25 30
Leu Gln Leu Phe Arg Ser His Val Gln Pro Leu Leu Ala Glu Ala
35 40 45
Glu Ile Ser Phe Thr Leu Met Leu Thr Glu Arg Arg Asn His Ala
50 55 60
Arg Glu Leu Val Arg Ser Glu Glu Leu Gly Arg Trp Asp Ala Leu
65 70 75
Val Val Met Ser Gly Asp Gly Leu Met His Glu Val Val Asn Gly
80 85 90
Leu Met Glu Arg Pro Asp Trp Glu Thr Ala Ile Gln Lys Pro Leu
95 100 105
Cys Ser Leu Pro Ala Gly Ser Gly Asn Ala Leu Ala Ala Ser Leu
110 115 120
Asn His Tyr Ala Gly Tyr Glu Gln Val Thr Asn Glu Asp Leu Leu
125 130 135
Thr Asn Cys Thr Leu Leu Leu Cys Arg Arg Leu Leu Ser Pro Met
140 145 150
Asn Leu Leu Ser Leu His Thr Ala Ser Gly Leu Arg Leu Phe Ser
155 160 165
Val Leu Ser Leu Ala Trp Gly Phe Ile Ala Asp Val Asp Leu Glu
170 175 180
Ser Glu Lys Tyr Arg Arg Leu Gly Glu Met Arg Phe Thr Leu Gly
185 190 195
Thr Phe Leu Arg Leu Ala Ala Leu Arg Thr Tyr Arg Gly Arg Leu
200 205 210
Ala Tyr Leu Pro Val Gly Arg Val Gly Ser Lys Thr Pro Ala Ser
215 220 225
Pro Val Val Val Gln Gln Gly Pro Val Asp Ala His Leu Val Pro
230 235 240
Leu Glu Glu Pro Val Pro Ser His Trp Thr Val Val Pro Asp Glu
245 250 255
Asp Phe Val Leu Val Leu Ala Leu Leu His Ser His Leu Gly Ser
260 265 270
Glu Met Phe Ala Ala Pro Met Gly Arg Cys Ala Ala Gly Val Met
275 280 285
His Leu Phe Tyr Val Arg Ala Gly Val Ser Arg Ala Met Leu Leu
290 295 300
Arg Leu Phe Leu Ala Met Glu Lys Gly Arg His Met Glu Tyr Glu
305 310 315
Cys Pro Tyr Leu Val Tyr Val Pro Val Val Ala Phe Arg Leu Glu
320 325 330
Pro Lys Asp Gly Lys Gly Val Phe Ala Val Asp Gly Glu Leu Met
335 340 345
Val Ser Glu Ala Val Gln Gly Gln Val His Pro Asn Tyr Phe Trp
350 355 360
Met Val Ser Gly Cys Val Glu Pro Pro Pro Ser Trp Lys Pro Gln
365 370 375
Gln Met Pro Pro Pro Glu Glu Pro Leu
380
<210> 6
<211> 81
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3815186CD1
6/24
CA 02364739 2001-08-20
WO 00/55332 PCTiUS00i07277
PF-0683 PCT
<400> 6
Met Arg Trp Tyr Gln Pro Pro Asn Asp Trp Arg Ile Leu Val Leu
1 5 10 15
Cys Leu Ser Ser Tyr Ala Val Leu Met Cys Leu Leu Ser Ile Trp
20 25 30
Gln Arg Asp Lys Arg Asp Thr Ser Asn Phe Asp Lys Glu Phe Thr
35 40 45
Arg Gln Pro Val Glu Leu Thr Pro Thr Asp Lys Leu Phe Ile Met
50 55 60
Asn Leu Asp Gln Asn Glu Phe Ala Gly Phe Ser Tyr Thr Asn Pro
65 70 75
Glu Phe Val Ile Asn Val
<210> 7
<211> 721
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5504544CD1
<400> 7
Met Leu Phe Gly Leu Val Arg Gln Gly Leu Lys Cys Asp Gly Cys
1 5 10 15
Gly Leu Asn Tyr His Lys Arg Cys Ala Phe Ser Ile Pro Asn Asn
20 25 30
Cys Ser Gly Ala Arg Lys Arg Arg Leu Ser Ser Thr Ser Leu Ala
35 40 45
Ser Gly His Ser Val Arg Leu Gly Thr Ser Glu Ser Leu Pro Cys
50 55 60
Thr Ala Glu Glu Leu Ser Arg Ser Thr Thr Glu Leu Leu Pro Arg
65 70 75
Arg Pro Pro Ser Ser Ser Ser Ser Ser Ser Ala Ser Ser Tyr Thr
80 85 90
Gly Arg Pro Ile Glu Leu Asp Lys Met Leu Leu Ser Lys Val Lys
95 100 105
Val Pro His Thr Phe Leu Ile His Ser Tyr Thr Arg Pro Thr Val
110 115 120
Cys Gln Ala Cys Lys Lys Leu Leu Lys Gly Leu Phe Arg Gln Gly
125 130 135
Leu Gln Cys Lys Asp Cys Lys Phe Asn Cys His Lys Arg Cys Ala
140 145 150
Thr Arg Val Pro Asn Asp Cys Leu Gly Glu Ala Leu Ile Asn Gly
155 160 165
Asp Val Pro Met Glu Glu Ala Thr Asp Phe Ser Glu Ala Asp Lys
170 175 180
Ser Ala Leu Met Asp Glu Ser Glu Asp Ser Gly Val Ile Pro Gly
185 190 195
Ser His Ser Glu Asn Ala Leu His Ala Ser Glu Glu Glu Glu Gly
200 205 210
Glu Gly Gly Lys Ala Gln Ser Ser Leu Gly Tyr Ile Pro Leu Met
215 220 225
Arg Val Val Gln Ser Val Arg His Thr Thr Arg Lys Ser Ser Thr
230 235 240
Thr Leu Arg Glu Gly Trp Val Val His Tyr Ser Asn Lys Asp Thr
245 250 255
Leu Arg Lys Arg His Tyr Trp Arg Leu Asp Cys Lys Cys Ile Thr
260 265 270
7/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
Leu Phe Gln Asn Asn Thr Thr Asn Arg Tyr Tyr Lys Glu Ile Pro
275 280 - 285
Leu Ser Glu Ile Leu Thr Val Glu Ser Ala Gln Asn Phe Ser Leu
290 295 300
Val Pro Pro Gly Thr Asn Pro His Cys Phe Glu Ile Val Thr Ala
305 310 315
Asn Ala Thr Tyr Phe Val Gly Glu Met Pro Gly Gly Thr Pro Gly
320 325 330
Gly Pro Ser Gly Gln Gly Ala Glu Ala Ala Arg Gly Trp Glu Thr
335 340 345
Ala Ile Arg Gln Ala Leu Met Pro Val Ile Leu Gln Asp Ala Pro
350 355 360
Ser Ala Pro Gly His Ala Pro His Arg Gln Ala Ser Leu Ser Ile
365 370 375
Ser Val Ser Asn Ser Gln Ile Gln Glu Asn Val Asp Ile Ala Thr
380 385 390
Val Tyr Gln Ile Phe Pro Asp Glu Val Leu Gly Ser Gly Gln Phe
395 400 405
Gly Val Val Tyr Gly Gly Lys His Arg Lys Thr Gly Arg Asp Val
410 415 420
Ala Val Lys Val Ile Asp Lys Leu Arg Phe Pro Thr Lys Gln Glu
425 430 435
Ser Gln Leu Arg Asn Glu Val Ala Ile Leu Gln Ser Leu Arg His
440 445 450
Pro Gly Ile Val Asn Leu Glu Cys Met Phe Glu Thr Pro Glu Lys
455 460 465
Val Phe Val Val Met Glu Lys Leu His Gly Asp Met Leu Glu Met
470 475 480
Ile Leu Ser Ser Glu Lys Gly Arg Leu Pro Glu Arg Leu Thr Lys
485 490 495
Phe Leu Ile Thr Gln Ile Leu Val Ala Leu Arg His Leu His Phe
500 505 510
Lys Asn Ile Val His Cys Asp Leu Lys Pro Glu Asn Val Leu Leu
515 520 525
Ala Ser Ala Asp Pro Phe Pro Gln Val Lys Leu Cys Asp Phe Gly
530 535 540
Phe Ala Arg Ile Ile Gly Glu Lys Ser Phe Arg Arg Ser Val Val
545 550 555
Gly Thr Pro Ala Tyr Leu Ala Pro Glu Val Leu Leu Asn Gln Gly
560 565 570
Tyr Asn Arg Ser Leu Asp Met Trp Ser Val Gly Val Ile Met Tyr
575 580 585
Val Ser Leu Ser Gly Thr Phe Pro Phe Asn Glu Asp Glu Asp I1e
590 595 600
Asn Asp Gln Ile Gln Asn Ala Ala Phe Met Tyr Pro Ala Ser Pro
605 610 615
Trp Ser His Ile Ser Ala Gly Ala Ile Asp Leu Ile Asn Asn Leu
620 625 630
Leu Gln Val Lys Met Arg Lys Arg Tyr Ser Val Asp Lys Ser Leu
635 640 645
Ser His Pro Trp Leu Gln Glu Tyr Gln Thr Trp Leu Asp Leu Arg
650 655 660
Glu Leu Glu Gly Lys Met Gly Glu Arg Tyr Ile Thr His Glu Ser
665 670 675
Asp Asp Ala Arg Trp Glu Gln Phe Ala Ala Glu His Pro Leu Pro
680 685 690
Gly Ser Gly Leu Pro Thr Asp Arg Asp Leu Gly Gly Ala Cys Pro
695 700 705
Pro Gln Asp His Asp Met Gln Gly Leu Ala Glu Arg Ile Ser Val
710 715 720
Leu
8/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
<210> 8
<211> 249
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1511326CD1
<400> 8
Met Ala Ser Ser Asp Glu Asp Gly Thr Asn Gly Gly Ala Ser Glu
1 5 10 15
Ala Gly Glu Asp Arg Glu Ala Pro Gly Gln Arg Arg Arg Leu Gly
20 25 30
Phe Leu Ala Thr Ala Trp Leu Thr Phe Tyr Asp Ile Ala Met Thr
35 40 45
Ala Gly Trp Leu Val Leu Ala Ile Ala Met Val Arg Phe Tyr Met
50 55 60
G1u Lys Gly Thr His Arg Gly Leu Tyr Lys Ser Ile Gln Lys Thr
65 70 75
Leu Lys Phe Phe Gln Thr Phe Ala Leu Leu Glu Ile Val His Cys
80 85 90
Leu Ile Gly Ile Val Pro Thr Ser Val Ile Val Thr Gly Val Gln
95 100 105
Val Ser Ser Arg Ile Phe Met Val Trp Leu Ile Thr His Ser Ile
110 115 120
Lys Pro Ile Gln Asn Glu Glu Ser Val Va1 Leu Phe Leu Va1 Ala
125 130 135
Trp Thr Val Thr Glu Ile Thr Arg Tyr Ser Phe Tyr Thr Phe Ser
140 145 150
Leu Leu Asp His Leu Pro Tyr Phe Ile Lys Trp Ala Arg Tyr Asn
155 160 165
Phe Phe Ile Ile Leu Tyr Pro Val Gly Val Ala Gly Glu Leu Leu
170 175 180
Thr Ile Tyr Ala A1a Leu Pro His Val Lys Lys Thr Gly Met Phe
185 190 195
Ser Ile Arg Leu Pro Asn Lys Tyr Asn Val Ser Phe Asp Tyr Tyr
200 205 210
Tyr Phe Leu Leu Ile Thr Met Ala Ser Tyr Ile Pro Leu Phe Pro
215 220 225
Gln Leu Tyr Phe His Met Leu Arg Gln Arg Arg Lys Val Leu His
230 235 240
Gly Glu Val Ile Val Glu Lys Asp Asp
245
<210> 9
<211> 146
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1519120CD1
<400> 9
Met Ala Asp Asp Asp Val Leu Phe Glu Asp Val Tyr Glu Leu Cys
1 5 10 15
Glu Val Ile Gly Lys Gly Pro Phe Ser Val Val Arg Arg Cys Ile
20 25 30
9/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
Asn Arg Glu Thr G1y Gln Gln Phe Ala Val Lys Ile Val Asp Val
35 40 45
Ala Lys Phe Thr Ser Ser Pro Gly Leu Ser Thr Glu Gly Lys Arg
50 55 60
Trp Ile Ser Asn Leu Lys Arg Glu Ala Ser Ile Cys His Met Leu
65 70 75
Lys His Pro His Ile Val Glu Leu Leu Glu Thr Tyr Ser Ser Asp
80 85 90
Gly Met Leu Tyr Met Val Phe Glu Phe Met Asp Gly Ala Asp Leu
95 100 105
Cys Phe Glu Ile Val Lys Arg Ala Asp Ala Gly Phe Val Tyr Ser
110 115 120
Glu Ala Val Ala Ser Ile Leu Asp Lys His Ser Trp Lys Gln Leu
125 130 135
Gly Asp His Leu Asn Thr Ala Leu Ser Ser Ala
140 145
<210> 10
<211> 524
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1673761CD1
<400> 10
Met Asn Ile Ala Asn Arg Lys Gln Glu Glu Met Lys Asp Met Ile
1 5 10 15
Val Glu Thr Leu Asn Thr Met Lys Glu Glu Leu Leu Asp Asp Ala
20 25 30
Thr Asn Met Glu Phe Lys Asp Val Ile Val Pro Glu Asn Gly Glu
35 40 45
Pro Val Gly Thr Arg Glu Ile Lys Cys Cys Ile Arg Gln Ile Gln
50 55 60
Glu Leu Ile Ile Ser Arg Leu Asn Gln Ala Val Ala Asn Lys Leu
65 70 75
Ile Ser Ser Val Asp Tyr Leu Arg Glu Ser Phe Val Gly Thr Leu
80 85 90
Glu Arg Cys Leu Gln Ser Leu Glu Lys Ser Gln Asp Val Ser Val
95 100 105
His Ile Thr Ser Asn Tyr Leu Lys Gln Ile Leu Asn Ala Ala Tyr
110 115 120
His Val Glu Val Thr Phe His Ser Gly Ser Ser Val Thr Arg Met
125 130 135
Leu Trp Glu Gln Ile Lys Gln Ile Ile Gln Arg Ile Thr Trp Val
140 145 150
Ser Pro Pro Ala Ile Thr Leu Glu Trp Lys Arg Lys Val Ala Gln
155 160 165
Glu Ala Ile Glu Ser Leu Ser Ala Ser Lys Leu Ala Lys Ser Ile
170 175 180
Cys Ser Gln Phe Arg Thr Arg Leu Asn Ser Ser His Glu Ala Phe
185 190 195
Ala Ala Ser Leu Arg Gln Leu Glu Ala Gly His Ser Gly Arg Leu
200 205 210
Glu Lys Thr Glu Asp Leu Trp Leu Arg Val Arg Lys Asp His Ala
215 220 225
Pro Arg Leu Ala Arg Leu Ser Leu Glu Ser Cys Ser Leu Gln Asp
230 235 240
Val Leu Leu His Arg Lys Pro Lys Leu Gly Gln Glu Leu Gly Arg
10/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
245 250 255
Gly Gln Tyr Gly Val Val Tyr Leu Cys Asp Asn Trp Gly Gly His
260 265 270
Phe Pro Cys Ala Leu Lys Ser Val Val Pro Pro Asp Glu Lys His
275 280 285
Trp Asn Asp Leu Ala Leu Glu Phe His Tyr Met Arg Ser Leu Pro
290 295 300
Lys His Glu Arg Leu Val Asp Leu His Gly Ser Val Ile Asp Tyr
305 310 315
Asn Tyr Gly Gly Gly Ser Ser Ile Ala Val Leu Leu Ile Met Glu
320 325 330
Arg Leu His Arg Asp Leu Tyr Thr Gly Leu Lys Ala Gly Leu Thr
335 340 345
Leu Glu Thr Arg Leu Gln Ile Ala Leu Asp Val Val G1u Gly Ile
350 355 360
Arg Phe Leu His Ser Gln Gly Leu Val His Arg Asp Ile Lys Leu
365 370 375
Lys Asn Val Leu Leu Asp Lys Gln Asn Arg Ala Lys Ile Thr Asp
380 385 390
Leu Gly Phe Cys Lys Pro Glu Ala Met Met Ser Gly Ser Ile Val
395 400 405
Gly Thr Pro Ile His Met Ala Pro Glu Leu Phe Thr Gly Lys Tyr
410 415 420
Asp Asn Ser Val Asp Val Tyr Ala Phe Gly Ile Leu Phe Trp Tyr
425 430 435
Ile Cys Ser Gly Ser Val Lys Leu Pro Glu Ala Phe Glu Arg Cys
440 445 450
A1a Ser Lys Asp His Leu Trp Asn Asn Val Arg Arg Gly Ala Arg
455 460 465
Pro Glu Arg Leu Pro Va1 Phe Asp Glu Glu Cys Trp Gln Leu Met
470 475 480
Glu Ala Cys Trp Asp Gly Asp Pro Leu Lys Arg Pro Leu Leu Gly
485 490 495
Ile Val Gln Pro Met Leu Gln Gly Ile Met Asn Arg Leu Cys Lys
500 505 510
Ser Asn Ser Glu Gln Pro Asn Arg Gly Leu Asp Asp Ser Thr
515 520
<210> 11
<211> 509
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1270442CD1
<400> 11
Met Arg Leu Arg Glu Arg Ser Leu Arg Gln Asp Pro Asp Leu Arg
1 5 10 15
Gln Glu Leu Ala Ser Leu Ala Arg Gly Cys Asp Phe Val Leu Pro
20 25 30
Ser Arg Phe Lys Lys Arg Leu Lys Ala Phe Gln Gln Val Gln Thr
35 40 45
Arg Lys Glu Glu Pro Leu Pro Pro Ala Thr Ser Gln Ser Ile Pro
50 55 60
Thr Phe Tyr Phe Pro Arg Gly Arg Pro Gln Asp Ser Val Asn Va1
65 70 75
Asp Ala Val Ile Ser Lys Ile Glu Ser Thr Phe Ala Arg Phe Pro
80 85 90
11/24
CA 02364739 2001-08-20
WO 00/55332 PCT/CTS00/07277
PF-0683 PCT
His Glu Arg Ala Thr Met Asp Asp Met Gly Leu Val Ala Lys Ala
95 100 - 105
Cys Gly Cys Pro Leu Tyr Trp Lys Gly Pro Leu Phe Tyr Gly Ala
110 115 120
Gly Gly Glu Arg Thr Gly Ser Val Ser Val His Lys Phe Val Ala
125 130 135
Met Trp Arg Lys Ile Leu Gln Asn Cys His Asp Asp Ala Ala Lys
140 145 150
Phe Val His Leu Leu Met Ser Pro Gly Cys Asn Tyr Leu Val Gln
155 160 165
Glu Asp Phe Val Pro Phe Leu Gln Asp Val Val Asn Thr His Pro
170 175 180
Gly Leu Ser Phe Leu Lys Glu Ala Ser Glu Phe His Ser Arg Tyr
185 190 195
I1e Thr Thr Val Ile G1n Arg Ile Phe Tyr A1a Va1 Asn Arg Ser
200 205 210
Trp Ser Gly Arg Ile Thr Cys Ala Glu Leu Arg Arg Ser Ser Phe
215 220 225
Leu Gln Asn Val Ala Leu Leu Glu Glu Glu Ala Asp Ile Asn Gln
230 235 240
Leu Thr Glu Phe Phe Ser Tyr Glu His Phe Tyr Val Ile Tyr Cys
245 250 255
Lys Phe Trp Glu Leu Asp Thr Asp His Asp Leu Leu Ile Asp Ala
260 265 270
Asp Asp Leu Ala Arg His Asn Asp His Ala Leu Ser Thr Lys Met
275 280 285
Ile Asp Arg Ile Phe Ser Gly Ala Val Thr Arg Gly Arg Lys Val
290 295 300
Gln Lys Glu Gly Lys Ile Ser Tyr Ala Asp Phe Val Trp Phe Leu
305 310 315
Ile Ser Glu Glu Asp Lys Lys Thr Pro Thr Ser Ile Glu Tyr Trp
320 325 330
Phe Arg Cys Met Asp Leu Asp Gly Asp Gly Ala Leu Ser Met Phe
335 340 345
Glu Leu Glu Tyr Phe Tyr Glu Glu Gln Cys Arg Ser Val Asp Ser
350 355 360
Met Ala Ile Glu Ala Leu Pro Phe Gln Asp Cys Leu Cys Gln Met
365 370 375
Leu Asp Leu Val Lys Pro Arg Thr Glu Gly Lys Ile Thr Leu Gln
380 385 390
Asp Leu Lys Arg Cys Lys Leu Ala Asn Val Phe Phe Asp Thr Phe
395 400 405
Phe Asn Ile Glu Lys Tyr Leu Asp His Glu Gln Lys Glu Gln Ile
410 415 420
Ser Leu Leu Arg Asp Gly Asp Ser Gly Gly Pro Glu Leu Ser Asp
425 430 435
Trp Glu Lys Tyr Ala Ala Glu Glu Tyr Asp Ile Leu Val Ala Glu
440 445 450
Glu Thr Ala Gly Glu Pro Trp Glu Asp Gly Phe Glu Ala Glu Leu
455 460 465
Ser Pro Val Glu Gln Lys Leu Ser Ala Leu Arg Ser Pro Leu Ala
470 475 480
Gln Arg Pro Phe Phe Glu Ala Pro Ser Pro Leu Gly Ala Val Asp
485 490 495
Leu Tyr Glu Tyr Ala Cys Gly Asp Glu Asp Leu Glu Pro Leu
500 505
<210> 12
<211> 142
<212> PRT
12/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1877133CD1
<400> 12
Met Ile Ser Thr Ala Arg Val Pro Ala Asp Lys Pro Val Arg Ile
1 5 10 15
Ala Phe Ser Leu Asn Asp Ala Ser Asp Asp Thr Pro Pro Glu Asp
20 25 30
Ser Ile Pro Leu Val Phe Pro Glu Leu Asp Gln Gln Leu Gln Pro
35 40 45
Leu Pro Pro Cys His Asp Ser Glu Glu Ser Met Glu Val Phe Lys
50 55 60
Gln His Cys Gln Ile Ala Glu Glu Tyr His Glu Val Lys Lys Glu
65 70 75
Ile Thr Leu Leu Glu Gln Arg Lys Lys Glu Leu Ile Ala Lys Leu
80 85~ 90
Asp Gln Ala Glu Lys Glu Lys Val Asp Ala Ala Glu Leu Val Arg
95 100 105
Glu Phe Glu Ala Leu Thr Glu Glu Asn Arg Thr Leu Arg Leu Ala
110 115 120
Gln Ser Gln Cys Val Glu Gln Leu Glu Lys Leu Arg Ile Gln Tyr
125 130 135
Gln Lys Arg Gln Gly Ser Ser
140
<210> 13
<211> 221
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2636759CD1
<400> 13
Met Thr Ser Gly G1u Val Lys Thr Ser Leu Lys Asn Ala Tyr Ser
1 5 10 15
Ser Ala Lys Arg Leu Ser Pro Lys Met Glu Glu Glu Gly Glu Glu
20 25 30
G1u Asp Tyr Cys Thr Pro Gly Ala Phe Glu Leu Glu Arg Leu Phe
35 40 45
Trp Lys Gly Ser Pro Gln Tyr Thr His Val Asn Glu Val Trp Pro
50 55 60
Lys Leu Tyr Ile Gly Asp Glu Ala Thr Ala Leu Asp Arg Tyr Arg
65 70 75
Leu Gln Lys Ala Gly Phe Thr His Val Leu Asn Ala Ala His Gly
80 85 90
Arg Trp Asn Val Asp Thr Gly Pro Arg Leu Leu Pro Arg His Gly
95 100 105
His Pro Val Pro Arg Arg Gly Gly Pro Thr Thr Cys Pro Pro Phe
110 115 120
Asp Leu Ser Val Phe Phe Tyr Pro Ala Ala Ala Phe Ile Asp Arg
125 130 135
Ala Leu Ser Asp Asp His Ser Lys Ile Leu Val His Cys Val Met
140 145 150
Gly Arg Ser Arg Ser Ala Thr Leu Val Leu Ala Tyr Leu Met Ile
155 160 165
13/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
His Lys Asp Met Thr Leu Val Asp Ala Ile Gln Gln Val Ala Lys
170 175 180
Asn Arg Cys Val Leu Pro Asn Arg Gly Phe Leu Lys Gln Leu Arg
185 190 195
Glu Leu Asp Lys Gln Leu Val Gln Gln Arg Arg Arg Ser Gln Arg
200 205 210
Gln Asp Gly Glu Glu Glu Asp Asp Arg Glu Leu
215 220
<210> 14
<211> 462
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2716815CD1
<400> 14
Met Ser Ile Ala Gly Val Ala Ala Gln Glu Ile Arg Val Pro Leu
1 5 10 15
Lys Thr Gly Phe Leu His Asn G1y Arg Ala Met Gly Asn Met Arg
20 25 30
Lys Thr Tyr Trp Ser Ser Arg Ser Glu Phe Lys Asn Asn Phe Leu
35 40 45
Asn Ile Asp Pro Ile Thr Met Ala Tyr Ser Leu Asn Ser Ser A1a
50 55 60
Gln Glu Arg Leu Ile Pro Leu Gly His Ala Ser Lys Ser Ala Pro
65 70 75
Met Asn Gly His Cys Phe Ala Glu Asn Gly Pro Ser Gln Lys Ser
80 85 90
Ser Leu Pro Pro Leu Leu Ile Pro Pro Ser Glu Asn Leu Gly Pro
95 100 105
His Glu Glu Asp Gln Val Val Cys Gly Phe Lys Lys Leu Thr Val
110 115 120
Asn Gly Val Cys Ala Ser Thr Pro Pro Leu Thr Pro Ile Lys Asn
125 130 135
Ser Pro Ser Leu Phe Pro Cys Ala Pro Leu Cys Glu Arg Gly Ser
140 145 150
Arg Pro Leu Pro Pro Leu Pro Ile Ser Glu Ala Leu Ser Leu Asp
155 160 165
Asp Thr Asp Cys Glu Val Glu Phe Leu Thr Ser Ser Asp Thr Asp
170 175 180
Phe Leu Leu Glu Asp Ser Thr Leu Ser Asp Phe Lys Tyr Asp Val
185 190 195
Pro Gly Arg Arg Ser Phe Arg Gly Cys Gly Gln Ile Asn Tyr Ala
200 205 210
Tyr Phe Asp Thr Pro Ala Val Ser Ala Ala Asp Leu Ser Tyr Va1
215 220 225
Ser Asp Gln Asn Gly Gly Val Pro Asp Pro Asn Pro Pro Pro Pro
230 235 240
Gln Thr His Arg Arg Leu Arg Arg Ser His Ser Gly Pro Ala Gly
245 250 255
Ser Phe Asn Lys Pro Ala Ile Arg Ile Ser Asn Cys Cys Ile His
260 265 270
Arg Ala Ser Pro Asn Ser Asp Glu Asp Lys Pro Glu Val Pro Pro
275 280 285
Arg Val Pro Ile Pro Pro Arg Pro Val Lys Pro Asp Tyr Arg Arg
290 295 300
Trp Ser Ala Glu Val Thr Ser Ser Thr Tyr Ser Asp Glu Asp Arg
14/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
305 310 315
Pro Pro Lys Val Pro Pro Arg Glu Pro Leu Ser Pro Ser Asn Ser
320 325 330
Arg Thr Pro Ser Pro Lys Ser Leu Pro Ser Tyr Leu Asn Gly Val
335 340 345
Met Pro Pro Thr Gln Ser Phe Ala Pro Asp Pro Lys Tyr Val Ser
350 355 360
Ser Lys Ala Leu Gln Arg Gln Asn Ser Glu Gly Ser Ala Ser Lys
365 370 375
Val Pro Cys Ile Leu Pro Ile Ile Glu Asn Gly Lys Lys Val Ser
380 385 390
Ser Thr His Tyr Tyr Leu Leu Pro Glu Arg Pro Pro Tyr Leu Asp
395 400 405
Lys Tyr Glu Lys Phe Phe Arg Glu Ala Glu Glu Thr Asn Gly Gly
410 415 420
Ala Gln Ile Gln Pro Leu Pro Ala Asp Cys Gly Ile Ser Ser Ala
425 430 435
Thr Glu Lys Pro Asp Ser Lys Thr Lys Met Asp Leu Gly Gly His
440 445 450
Val Lys Arg Lys His Leu Ser Tyr Val Val Ser Pro
455 460
<210> 15
<211> 2192
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 480457CB1
<400> 15
gatcaatgaa gccgagtgaa tgggggctga atgtgcgagt ccatagctga agaggagcgc 60
cagatggtgg aggaatacac ttatttatga aactgtcttg agttcttctt gaattgccag 120
ttttcagcct cctcatgcct ccgtctcctt tagacgacag ggtagtagtg gcactatcta 180
ggcccgtccg acctcaggat ctcaaccttt gtttagactc tagttacctt ggctctgcca 240
acccaggcag taacagccac cctcctgtca tcgccaccac cgttgtgtcc ctcaaggctg 300
cgaatctgac gtatatgccc tcatccagcg gctctgcccg ctcgctgaat tgtggatgca 360
gcagtgccag ctgctgcact gtggcaacct acgacaagga caatcaggcc caaacccaag 420
ccattgccgc tggcaccacc accactgcca tcggaacctc taccacctgc cctgctaacc 480
agatggtcaa caataatgag aatacaggct ctctaagtcc atcaagtggg gtgggcagcc 540
ctgtgtcagg gacccccaag cagctagcca gcatcaaaat aatctacccc aatgacttgg 600
caaagaagat gaccaaatgc agcaagagtc acctgccgag tcagggccct gtcatcattg 660
actgcaggcc cttcatggag tacaacaaga gtcacatcca aggagctgtc cacattaact 720
gtgccgataa gatcagccgg cggagactgc agcagggcaa gatcactgtc ctagacttga 780
tttcctgtag ggaaggcaag gactctttca agaggatctt ttccaaagaa attatagttt 840
atgatgagaa taccaatgag ccaagccgag tgatgccctc ccagccactt cacatagtcc 900
tcgagtccct gaagagagaa ggcaaagaac ctctggtgtt gaaaggtgga cttagtagtt 960
ttaagcagaa ccatgaaaac ctctgtgaca actccctcca gctccaagag tgccgggagg 1020
tggggggcgg cgcatccgcg gcctcgagct tgctacctca gcccatcccc accacccctg 1080
acatcgagaa cgctgagctc acccccatct tgcccttcct gttccttggc aatgagcagg 1140
atgctcagga cctggacacc atgcagcggc tgaacatcgg ctacgtcatc aacgtcacca 1200
ctcatcttcc cctctaccac tatgagaaag gcctgttcaa ctacaagcgg ctgccagcca 1260
ctgacagcaa caagcagaac ctgcggcagt actttgaaga ggcttttgag ttcattgagg 1320
aagctcacca gtgtgggaag gggcttctca tccactgcca ggctggggtg tcccgctccg 1380
ccaccatcgt catcgcttac ttgatgaagc acactcggat gaccatgact gatgcttata 1440
aatttgtcaa aggcaaacga ccaattatct ccccaaacct taacttcatg gggcagttgc 1500
tagagttcga ggaagaccta aacaacggtg tgacaccgag aatccttaca ccaaagctga 1560
tgggcgtgga gacggttgtg tgacaatggt ctggatggaa aggattgctg ctctccatta 1620
ggagacaatg aggaaggagg atggattctg gttttttttc tttctttttt tttttgtagt 1680
15124
WO 00/55332 CA 02364739 2001-08-20
PCT/US00l07277
PF-0683 PCT
tgggagtaag tttgtgaatg gaaacaaact tgtttaaaca ctttattttt aacaagtgta 1740
agaagactat aacttttgat gccattgaga ttcacctccc acaaactgac aaattaagga 1800
ggttaaagaa gtaatttttt taagccaaca ataaaaatat aatacaactt gtttctcccc 1860
cttttccttt taagctattt gtagagttta tgactaaata gtctgtgcag gttcatagac 1920
cgaagatact acacacttta aaccaattaa aaagaaccaa aagtaaatag aaaagacatt 1980
gaatcaccaa ggcctgggat caacctgggc tgtccacaca gaaaacaaaa acccaaccaa 2040
accaagccct gttgtgctca ctggtgcaaa gagaagatca gggcagctta agtggtctaa 2100
gaatccttca ggcattcttt aaggagaaaa aggatacctt tgattttgtg tgtttcatgc 2160
tctggatttt tttttttttc cttctctggg tt 2192
<210> 16
<211> 1338
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 959, 1029, 1159
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 563663CB1
<400> 16
ccttgtctgg aaaagtaaaa gtggatcctg ccacgttcgg agctccctgg cgcctcgccc 60
ggctggagct agagaactcg tcctgtggcg gcccccggcg tggggcggga cagcggcccc 120
ctggaggggg cagtcccggg agaacctgcg gcggccggag cggtaaaaat aagtgactaa 180
agaagcagac ctgggaatca cctaacatgt cgaggaggag atttgattgc cgaagtattt 240
caggcctact aactacaact cctcaaattc caataaaaat ggaaaacttt aataatttct 300
atatacttac atctaaagag ctagggagag gaaaatttgc tgtggttaga caatgtatat 360
caaaatctac tggccaagaa tatgctgcaa aatttctaaa aaagagaaga agaggacagg 420
attgtcgggc agaaatttta cacgagattg ctgtgcttga attggcaaag tcttgtcccc 480
gtgttattaa tcttcatgag gtctatgaaa atacaagtga aatcattttg atattggaat 540
atgctgcagg tggagaaatt ttcagcctgt gtttacctga gttggctgaa atggtttctg 600
aaaatgatgt tatcagactc attaaacaaa tacttgaagg agtttattat ctacatcaga 660
ataacattgt acaccttgat ttaaagccac agaatatatt actgagcagc atataccctc 720
tcggggacat taaaatagta gatgggggaa tgtctcgaaa aatagggcaa tgcgtgtgac 780
cttcgggaaa tcatgggaac accagaatat ttagctccag aaatcctgaa ctatgatccc 840
attaccacag aaacagatat gtggaatatt ggtataatag catatatgtc gctaactcac 900
acatcaccat ttgtgggaga agataatcaa gaaacatacc tcaatctctc tcaagttant 960
gtagattatt cggaagaaac tttcccatca gtttcacagc tggccacaga ctttattcag 1020
agcccttcng taacacctcc agagaaacga caccagcaga gatctgcctc cctcattctc 1080
gcctacagca gcgggacttt gcaacattgt ctcacctgaa cgaacctcca gttcccctca 1140
aactcaggat caatctgtna ggtctctaag acaagactct taatcccctc gcatggaact 1200
cgggtgaaga gaagtacgag atctcccgcg gttgcagact gctcaaacca ttcgtctgat 1260
atctatcccc cccccccacc tgtccaattg gttttacatc tttcttgctc aggcttcctc 1320
gacttgcttc ccccctac 1338
<210> 17
<211> 1706
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1425842CB1
<400> 17
16/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
cgtgggagcc gcgcgggccg ctgtcgtccc aacccccgcc gccctcgtcg cgcgcggggc 60
ctccgcgccc ccggctgctg ctcacgcccc gcccgggagc cagattttgt ggaagtataa 120
tactttgtca ttatgagatg tcgtctctcg gtgcctcctt tgtgcaaatt aaatttgatg 180
acttgcagtt ttttgaaaac tgcggtggag gaagttttgg gagtgtttat cgagccaaat 240
ggatatcaca ggacaaggag gtggctgtaa agaagctcct caaaatagag aaagaggcag 300
aaatactcag tgtcctcagt cacagaaaca tcatccagtt ttatggagta attcttgaac 360
ctcccaacta tggcattgtc acagaatatg cttctctggg atcactctat gattacatta 420
acagtaacag aagtgaggag atggatatgg atcacattat gacctgggcc actgatgtag 480
ccaaaggaat gcattattta catatggagg ctcctgtcaa ggtgattcac agagacctca 540
agtcaagaaa cgttgttata gctgctgatg gagtattgaa gatctgtgac tttggtgcct 600
ctcggttcca taaccataca acacacatgt ccttggttgg aactttccca tggatggctc 660
cagaagttat ccagagtctc cctgtgtcag aaacttgtga cacatattcc tatggtgtgg 720
ttctctggga gatgctaaca agggaggtcc cctttaaagg tttggaagga ttacaagtag 780
cttggcttgt agtggaaaaa aacgagagat taaccattcc aagcagttgc cccagaagtt 840
ttgctgaact gttacatcag tgttgggaag ctgatgccaa gaaacggcca tcattcaagc 900
aaatcatttc aatcctggag tccatgtcaa atgacacgag ccttcctgac aagtgtaact 960
cattcctaca caacaaggcg gagtggaggt gcgaaattga ggcaactctt gagaggctaa 1020
agaaactaga gcgtgatctc agctttaagg agcaggagct taaagaacga gaaagacgtt 1080
taaagatgtg ggagcaaaag ctgacagagc agtccaacac cccgcttctc ttgcctcttg 1140
ctgcaagaat gtctgaggag tcttactttg aatctaaaac agaggagtca aacagtgcag 1200
agatgtcatg tcagatcaca gcaacaagta acggggaggg ccatggcatg aacccaagtc 1260
tgcaggccat gatgctgatg ggctttgggg atatcttctc aatgaacaaa gcaggagctg 1320
tgatgcattc tgggatgcag ataaacatgc aagccaagca gaattcttcc aaaaccacat 1380
ctaagagaag ggggaagaaa gtcaacatgg ctctggggtt cagtgatttt gacttgtcag 1440
.aaggtgacga tgatgatgat gatgacggtg aggaggagga taatgacatg gataatagtg 1500
aatgaaagca gaaagcaaag taataaaatc acaaatgttt ggaaaacaca aaagtaactt 1560
gtttatctca gtctgtacaa aaacagtaag gaggcagaaa gccaagcact gcatttttag 1620
ggcaatcaca tttacatgac cgtatttctt atcattctac tttattctgc tacagaaaac 1680
gcgggagatt agccaagagt tattat 1706
<210> 18
<211> 2140
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2349047CB1
<400> 18
aaaaagggta acaacccgga aagtagactc accgtcttgg tctagagact gacccctgca 60
cagacagacc ccttcccctc tctgcgaaag gaccaagccc cagaagtcac tccatctcct 120
acggctcgca atttccagag gccccctggc accttccagc ctgatgtcgt gcgtcaagtt 180
atggcccagc ggtgcccccg cccccttggt gtccatcgag gaactggaga accaggagct 240
cgtcggcaaa ggcgggttcg gcacagtgtt ccgggcgcaa cataggaagt ggggctacga 300
tgtggcggtc aagatcgtaa actcgtgagt gaccccggtt gaccccagcc gcaatctggc 360
caaaaaggtg gagccactgt gctctggggc ctgaatggcg aagggaggga ttttcccacg 420
accccggtgc gactccagct ctctcctgga gtgtaggaag gcgatatcca gggaggtcaa 480
ggccatggca agtctggata acgaattcgt gctgcgccta gaaggggtta tcgagaaggt 540
gaactgggac caagatccca agccggctct ggtgactaaa ttcatggaga acggctcctt 600
gtcggggctg ctgcagtccc agtgccctcg gccctggccg ctcctttgcc gcctgctgaa 660
agaagtggtg cttgggatgt tttacctgca cgaccagaac ccggtgctcc tgcaccggga 720
cctcaagcca tccaacgtcc tgctggaccc agagctgcac gtcaagctgg cagattttgg 780
cctgtccaca tttcagggag gctcacagtc agggacaggg tccggggagc cagggggcac 840
cctgggctac ttggccccag aactgtttgt taacgtaaac cggaaggcct ccacagccag 900
tgacgtctac agcttcggga tcctaatgtg ggcagtgctt gctggaagag aagttgagtt 960
gccaaccgaa ccatcactcg tgtacgaagc agtgtgcaac aggcagaacc ggccttcatt 1020
ggctgagctg ccccaagccg ggcctgagac tcccggctta gaaggactga aggagctaat 1080
gcagctctgc tggagcagtg agcccaagga cagaccctcc ttccaggaat gcctaccaaa 1140
aactgatgaa gtcttccaga tggtggagaa caatatgaat gctgctgtct ccacggtaaa 1200
17/24
WO 00155332 CA 02364739 2001-08-20 PCTJ[JSO~/07277
PF-0683 PCT
ggatttcctg tctcagctca ggagcagcaa taggagattt tctatcccag agtcaggcca 1260
aggagggaca gaaatggatg gctttaggag aaccatagaa aaccagcact ctcgtaatga 1320
tgtcatggtt tctgagtggc taaacaaact gaatctagag gagcctccca gctctgttcc 1380
taaaaaatgc ccgagcctta ccaagaggag cagggcacaa gaggagcagg ttccacaagc 1440
ctggacagca ggcacatctt cagattcgat ggcccaacct ccccagactc cagagacctc 1500
aactttcaga aaccagatgc ccagccctac ctcaactgga acaccaagtc ctggaccccg 1560
agggaatcag ggggctgaga gacaaggcat gaactggtcc tgcaggaccc cggagccaaa 1620
tccagtaaca gggcgaccgc tcgttaacat atacaactgc tctggggtgc aagttggaga 1680
caacaactac ttgactatgc aacagacaac tgccttgccc acatggggct tggcaccttc 1740
gggcaagggg aggggcttgc agcacccccc accagtaggt tcgcaagaag gccctaaaga 1800
tcctgaagcc tggagcaggc cacagggttg gtataatcat agcgggaaat aaagcacctt 1860
ccaagcttgc ctccaagagt tacgagttaa ggaagagtgc caccccttga ggcccctgac 1920
ttccttctag ggcagtctgg cctgcccaca aactgacttt gtgacctgtc ccccaggagt 1980
caataaacat gatggaaata aagcaccttc caagcttgcc tccaagagtt acgagttaag 2040
gaagagtgcc accccttgag gcccctgact tccttctagg gcagtctggc ctgcccacaa 2100
actgactttg tgacctgtcc cccaggagtc aataaacatg 2140
<210> 19
<211> 1573
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2415617CB1
<400> 19
gccccacagc cggccctgcg acgcccgcct gggcagcacc gataaggagc tgaaggcagg 60
agccgccgcc acgggcagcg ccccacagcg ccagggaccc cctggcagcg ggagccgcgg 120
gtcgaggtta tggatccagc gggcggcccc cggggcgtgc tcccgcggcc ctgccgcgtg 180
ctggtgctgc tgaacccgcg cggcggcaag ggcaaggcct tgcagctctt ccggagtcac 240
gtgcagcccc ttttggctga ggctgaaatc tccttcacgc tgatgctcac tgagcggcgg 300
aaccacgcgc gggagctggt gcggtcggag gagctgggcc gctgggacgc tctggtggtc 360
atgtctggag acgggctgat gcacgaggtg gtgaacgggc tcatggagcg gcctgactgg 420
gagaccgcca tccagaagcc cctgtgtagc ctcccagcag gctctggcaa cgcgctggca 480
gcttccttga accattatgc tggctatgag caggtcacca atgaagacct cctgaccaac 540
tgcacgctat tgctgtgccg ccggctgctg tcacccatga acctgctgtc tctgcacacg 600
gcttcggggc tgcgcctctt ctctgtgctc agcctggcct ggggcttcat tgctgatgtg 660
gacctagaga gtgagaagta tcggcgtctg ggggagatgc gcttcactct gggcaccttc 720
ctgcgtctgg cagccctgcg cacctaccgc ggccgactgg cctacctccc tgtaggaaga 780
gtgggttcca agacacctgc ctcccccgtt gtggtccagc agggcccggt agatgcacac 840
cttgtgccac tggaggagcc agtgccctct cactggacag tggtgcccga cgaggacttt 900
gtgctagtcc tggcactgct gcactcgcac ctgggcagtg agatgtttgc tgcacccatg 960
ggccgctgtg cagctggcgt catgcatctg ttctacgtgc gggcgggagt gtctcgtgcc 1020
atgctgctgc gcctcttcct ggccatggag aagggcaggc atatggagta tgaatgcccc 1080
tacttggtat atgtgcccgt ggtcgccttc cgcttggagc ccaaggatgg gaaaggtgtg 1140
tttgcagtgg atggggaatt gatggttagc gaggccgtgc agggccaggt gcacccaaac 1200
tacttctgga tggtcagcgg ttgcgtggag cccccgccca gctggaagcc ccagcagatg 1260
ccaccgccag aagagccctt atgacccctg ggccgcgctg tgccttagtg tctacttgca 1320
ggacccttcc tccttcccta gggctgcagg gcctgtccac agctcctgtg ggggtggagg 1380
agactcctct ggagaagggt gagaaggtgg aggctatgct ttggggggac aggccagaat 1440
gaagtcctgg gtcaggagcc cagctggctg ggcccagctg cctatgtaag gccttctagt 1500
ttgttctgag acccccaccc cacgaaccaa atccaaataa agtgacattc ccagcctgaa 1560
aaaaaaaaaa aaa 1573
<210> 20
<211> 862
<212> DNA
<213> Homo sapiens
18/24
WO 00/55332 CA 02364739 2001-08-20
PCTlUS00/07277
PF-0683 PCT
<220>
<221> misc_feature
<223> Incyte ID No: 3815186CB1
<400> 20
aacccagaga gtagggtggt catgattctg tggtggtgca ggcatgtttt cttagcatta 60
tttaccatca tattacgtat aagaaagctg acagtggaga gagattaaaa gcaggactat 120
taggggatag agagtaattg gaggcctttt tataatgtgc agattcgtga tttttgaaaa 180
gcatagcttt gattctgctt tccttttttt aacttggttt tttaaaattt gcacacacac 240
accactttat ggcaattctt aactgacatt caatgactta cttcttttct tagaaaattt 300
ccaccacatt tctatcccca agccaacata caatgtgaaa tgaaagccag tgcgtggagt 360
gcagctgcta aaaattttca gcacagggct ctttctgact ctgctcatga gatggtatca 420
gccacccaat gactggcgta tcttggtcct gtgtctttct tcttacgctg tgttaatgtg 480
tttactttcc atttggcaga gagacaagag agacacctcc aacttcgaca aagagttcac 540
cagacagcct gtggaactga cccccactga taaactcttc atcatgaact tggaccaaaa 600
tgaatttgct ggcttctcgt atactaaccc agagtttgtc attaatgtgt aggtgaatgc 660
agattccatc gctgagcctg tgtgtaaggc tgcaggctga atgtctatta tcaattccag 720
tcttccagga ttcatggtgc ctctgttggc atccgtcatg tggagagctt gtcttagagg 780
gcttttcttt gtatgtatag cttgctagtt tgttttctac atttcaaaat gtttagttta 840
gaataagtgc attgcccact ga 862
<210> 21
<211> 2744
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5504544CB1
<400> 21
ccggccccgg gttccggggt ctcctttcac atccagatcg ggctgacccg cgagttcgtg 60
ctgttgcccg ccgcctccga gctggctcat gtgaagcagc tggcctgttc catcgtggac 120
cagaagttcc ctgagtgtgg cttctacggc ctttacgaca agatcctgct tttcaaacat 180
gaccccacgt cggccaacct cctgcagctg gtgcgctcgt ccggagacat ccaggagggc 240
gacctggtgg aggtggtgct gtcggcctcg gccaccttcg aggacttcca gatccgcccg 300
cacgccctca cggtgcactc ctatcgggcg cctgccttct gtgatcactg cggggagatg 360
ctcttcggcc tagtgcgcca gggcctcaag tgcgatggct gcgggctgaa ctaccacaag 420
cgctgtgcct tcagcatccc caacaactgt agtggggccc gcaaacggcg cctgtcatcc 480
acgtctctgg ccagtggcca ctcggtgcgc ctcggcacct ccgagtccct gccctgcacg 540
gctgaagagc tgagccgtag caccaccgaa ctcctgcctc gccgtccccc gtcatcctct 600
tcctcctctt ctgcctcatc gtatacgggc cgccccattg agctggacaa gatgctgctc 660
tccaaggtca aggtgccgca caccttcctc atccacagct atacacggcc caccgtttgc 720
caggcttgca agaaactcct caagggcctc ttccggcagg gcctgcaatg caaagactgc 780
aagtttaact gtcacaaacg ctgcgccacc cgcgtcccta atgactgcct gggggaggcc 840
cttatcaatg gagatgtgcc gatggaggag gccaccgatt tcagcgaggc tgacaagagc 900
gccctcatgg atgagtcaga ggactccggt gtcatccctg gctcccactc agagaatgcg 960
ctccacgcca gtgaggagga ggaaggcgag ggaggcaagg cccagagctc cctggggtac 1020
atccccctaa tgagggtggt gcaatcggtg cgacacacga cgcggaaatc cagcaccacg 1080
ctgcgggagg gttgggtggt tcattacagc aacaaggaca cgctgagaaa gcggcactat 1140
tggcgcctgg actgcaagtg tatcacgctc ttccagaaca acacgaccaa cagatactat 1200
aaggaaattc cgctgtcaga aatcctcacg gtggagtccg cccagaactt cagccttgtg 1260
ccgccgggca ccaacccaca ctgctttgag atcgtcactg ccaatgccac ctacttcgtg 1320
ggcgagatgc ctggcgggac tccgggtggg ccaagtgggc agggggctga ggccgcccgg 1380
ggctgggaga cagccatccg ccaggccctg atgcccgtca tccttcagga cgcacccagc 1440
gccccaggcc acgcgcccca cagacaagct tctctgagca tctctgtgtc caacagtcag 1500
atccaagaga atgtggacat tgccactgtc taccagatct tccctgacga agtgctgggc 1560
tcagggcagt ttggagtggt ctatggagga aaacaccgga agacaggccg ggacgtggca 1620
gttaaggtca ttgacaaact gcgcttccct accaagcagg agagccagct ccggaatgaa 1680
gtggccattc tgcagagcct gcggcatccc gggatcgtga acctggagtg catgttcgag 1740
19/24
WO 00/55332 CA 02364739 2001-08-20 PCTlLTS00/07277
PF-0683 PCT
acgcctgaga aagtgtttgt ggtgatggag aagctgcatg gggacatgtt ggagatgatc 1800
ctgtccagtg agaagggccg gctgcctgag cgcctcacca agttcctcat cacccagatc 1860
ctggtggctt tgagacacct tcacttcaag aacattgtcc actgtgactt gaaaccagaa 1920
aacgtgttgc tggcatcagc agacccattt cctcaggtga agctgtgtga ctttggcttt 1980
gctcgcatca tcggcgagaa gtcgttccgc cgctcagtgg tgggcacgcc ggcctacctg 2040
gcacccgagg tgctgctcaa ccagggctac aaccgctcgc tggacatgtg gtcagtgggc 2100
gtgatcatgt acgtcagcct cagcggcacc ttccctttca acgaggatga ggacatcaat 2160
gaccagatcc agaacgccgc cttcatgtac ccggccagcc cctggagcca catctcagct 2220
ggagccattg acctcatcaa caacctgctg caggtgaaga tgcgcaaacg ctacagcgtg 2280
gacaaatctc tcagccaccc ctggttacag gagtaccaga cgtggctgga cctccgagag 2340
ctggagggga agatgggaga gcgatacatc acgcatgaga gtgacgacgc gcgctgggag 2400
cagtttgcag cagagcatcc gctgcctggg tctgggctgc ccacggacag ggatctcggt 2460
ggggcctgtc caccacagga ccacgacatg caggggctgg cggagcgcat cagtgttctc 2520
tgaggtcctg tgccctcgtc cagctgctgc cctccacagc ggttcttcac aggatcccag 2580
caatgaactg ttctagggaa agtggcttcc tgcccaaact ggatgggaca cgtggggagt 2640
ggggtggggg gagctatttc caaggcccct ccctgtttcc ccagcaatta aaacggactc 2700
atctctggcc ccatggcctt gatctcagca aaaaaaaaaa aaaa 2744
<210> 22
<211> 1266
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1511326CB1
<400> 22
gggcgcctga cggaagcggc ggcagcgggc agcggctctc gggctgcagg ctgggcaggg 60
tcccctccca cgctcctgcc gctgtctccc acgtccccca ggtgcgcggc caccatggcg 120
tccagcgacg aggacggcac caacggcggc gcctcggagg ccggcgagga ccgggaggct 180
cccggccagc ggaggcgcct ggggttcttg gccaccgcct ggctcacctt ctacgacatc 240
gccatgaccg cggggtggtt ggttctagct attgccatgg tacgttttta tatggaaaaa 300
ggaacacaca gaggtttata taaaagtatt cagaagacac ttaaattttt ccagacattt 360
gccttgcttg agatagttca ctgtttaatt ggaattgtac ctacttctgt gattgtgact 420
ggggtccaag tgagttcaag aatctttatg gtgtggctca ttactcacag tataaaacca 480
atccagaatg aagagagtgt ggtgcttttt ctggtcgcgt ggactgtgac agagatcact 540
cgctattcct tctacacatt cagccttctt gaccacttgc catacttcat taaatgggcc 600
agatataatt tttttatcat cttatatcct gttggagttg ctggtgaact tcttacaata 660
tacgctgcct tgccgcatgt gaagaaaaca ggaatgtttt caataagact tcctaacaaa 720
tacaatgtct cttttgacta ctattatttt cttcttataa ccatggcatc atatatacct 780
ttgtttccac aactctattt tcatatgtta cgtcaaagaa gaaaggtgct tcatggagag 840
gtgattgtag aaaaggatga ttaaatgatc tctgcaaaca aggtgctttt tccagaataa 900
ccaagattac ctgagtccaa gttttaataa caagaataaa caactttgtg aaatatcatg 960
gattgtatgg tttcttaaaa tataacttga gacacgtggt atttgccagt atttgtgttc 1020
ctcttgtgcc agatctattt tttacaagaa ctgtgcaaat atcagtaact tttgggtagg 1080
tattgattat taggaaaata attaggtgta ttatctgggg gaaaaaaaaa cttttgctaa 1140
gttttttttg aaacatgctc aaagcttttt aaatcaatat ttagaaatta gtttaacgat 1200
ttactattat acctgctagt gatatttatg tgatatttat aaatgaaaat aaatgcaaaa 1260
ttatat 1266
<210> 23
<211> 594
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1519120CB1
20/24
CA 02364739 2001-08-20
WO 00/55332 PCT/US00/07277
PF-0683 PCT
<400> 23
gcggccgcta tcccctccgg accatggccg acgacgacgt gctgttcgag gatgtgtacg 60
agctgtgcga ggtgatcgga aagggtccct tcagtgttgt acgacgatgt atcaacagag 120
aaactgggca acaatttgct gtaaaaattg ttgatgtagc caagttcaca tcaagtccag 180
ggttaagtac agaaggtaag agatggattt caaatctaaa gcgggaagcc agtatctgtc 240
atatgctgaa acatccacac attgtagagt tattggagac atatagctca gatggaatgc 300
tttacatggt tttcgaattt atggatggag cagatctgtg ttttgaaatc gtaaagcgag 360
ctgacgctgg ttttgtgtac agtgaagctg tagccagcat cttagacaaa catagctgga 420
aacaacttgg agaccacctt aatacagctt tgagctcagc atgacttgaa tgtcttcaaa 480
ttcattgccc tgactccttc tggctgaaga gcaagggacc tagtctcagg gatggagcag 540
ccatggctat ggcacaaaat aaagaatatg ctcaatgaaa aaaaaaaaaa aaaa 594
<210> 24
<211> 2280
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1673761CB1
<400> 24
cccaaacgtc tggaatatac tcgaaaaaag gagaatgagt tgtatgaatc attgatgaat 60
attgccaacc gaaagcagga ggaaatgaag gatatgattg ttgagacact taataccatg 120
aaggaggaac ttctggatga tgctactaac atggagttta aagacgtcat tgtccctgag 180
aatggagaac cagtaggcac cagagagatc aaatgctgca tccgacagat ccaggaactc 240
atcatctccc gacttaatca ggcagtggct aataagctga tcagctcagt ggattacctg 300
agggaaagct tcgtcggaac cctggaacga tgtctgcaga gcctggagaa gtctcaggat 360
gtctcagttc acatcaccag taattatctc aaacagatct taaatgctgc ctatcatgtt 420
gaagtcacgt ttcactcagg gtcgtcagtt acaaggatgc tatgggagca aatcaaacag 480
atcatccagc gcatcacatg ggtgagccca cctgccatca ctctggaatg gaagaggaag 540
gtggcccagg aagccattga gagcctcagc gcctccaaat tggctaagag catttgcagc 600
caattccgga ctcggctcaa tagttcccac gaggcttttg cagcctcctt gcggcagctg 660
gaagctggcc actcaggccg gttagagaaa acggaagatc tatggctgag ggttcggaaa 720
gatcatgctc cccgcctggc ccgcctttct ctggaaagct gttctttaca ggatgtcttg 780
cttcatcgta aacctaaact gggacaggaa ctgggccggg gccagtatgg tgtggtatac 840
ctgtgtgaca actggggagg acacttccct tgtgccctca aatcagttgt Ccctccagat 900
gagaagcact ggaatgatct ggctttggaa tttcactata tgaggtctct gccgaagcat 960
gagcgattgg tggatctcca tggttcagtc attgactaca actatggtgg tggctccagc 1020
attgctgtgc tcctcattat ggagcggcta caccgggatc tctacacagg gctgaaggct 1080
gggctgaccc tggagacacg tttgcagata gcactagatg tggtggaggg aatccgcttc 1140
ctgcacagcc agggacttgt ccatcgtgat atcaaactga aaaatgtgct gctggataag 1200
cagaaccgtg ccaagatcac tgacttagga ttctgcaagc cagaggccat gatgtcaggc 1260
agcattgtgg ggacaccaat ccatatggcc cctgaacttt tcacagggaa gtacgataat 1320
tccgtggatg tctacgcttt tggaattctt ttctggtata tctgctcagg ctctgtcaag 1380
ctccctgagg catttgagag gtgtgctagc aaagaccatc tctggaacaa tgtgcggagg 1440
ggggctcgcc cagaacgtct tcctgtgttt gatgaggagt gctggcagtt gatggaagcc 1500
tgttgggatg gcgacccctt gaagaggcct ctcttgggca ttgtccagcc catgctccag 1560
ggcatcatga atcggctctg caagtccaat tctgagcagc caaacagagg actagatgat 1620
tctacttgaa agcaaagacc tttctctttc actctctagt tatttccttc cccctcacct 1680
tttggccatg gggagaattt gacatttatt cactatagga cacactccca agggaactgg 1740
tgcttgctgg gaaacttgga accttcccag gcagggatga ctcctggaca gtgaagagtt 1800
gaatgactga gcatattcag cagctcactg aagcgccaag ctatcccttt agcaaaaaag 1860
tgtctcagat gtgtaaaagc tgaggaatgt ggtgttctgg cttcacaaat gaaaaggagg 1920
cagatgttac cattgtcttt tcactgtata tacttctaag acagcaagcg ggacactgca 1980
gtggcaatag tgttaaaaaa tctcattctc atgatttttg gctctagcta ggaatagttt 2040
agtcaggact cagaaaattt gagcttagtt ctgggattat gaaaacatgg ggacaaaaac 2100
aataacttgt ggatgtctgg ttcctgctgt ctgcagccag gtatctcagg ttgcaatggt 2160
cagaagtccc agtgagggag ctagaaacag agctatctgt tcctcatagt gccagtgtgt 2220
ttacatttat aggaccacat atccctggtt tctgggggag gtttcactgt acacagccaa 2280
21/24
WO 00/55332 CA 02364739 2001-08-20 PCT/US00/07277
PF-0683 PCT
<210> 25
<211> 1679
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1270442CB1
<400> 25
gcgcgccggc ggcatgcgac tccgcgagcg ctcgctgcgc caggaccccg acctgcgcca 60
ggagctggcc tcactggccc gcggctgcga cttcgtgctg ccctctcggt tcaagaagcg 120
gctgaaggcc ttccagcagg ttcagacacg gaaagaagag cctctgcccc cggccacgag 180
ccaaagcatt ccgaccttct acttccccag aggacgcccg caggactccg tcaacgtgga 240
tgccgtcatc agcaagatcg agagcacctt cgcccggttc ccccacgaga gggccaccat 300
ggacgacatg ggcctggtgg ccaaggcctg cggctgcccc ctctactgga aggggccgct 360
cttctatggc gccggcgggg agcgcacggg ctccgtgtcc gtccacaagt tcgtcgccat 420
gtggagaaaa atcctccaga actgccacga cgacgcggcc aagttcgtcc atctgctcat 480
gagccccggc tgcaactacc tggtgcagga ggactttgtc cccttcttgc aggacgtggt 540
gaacacgcac ccggggctgt cgttcctgaa ggaggcgtcc gagttccact cgcgctacat 600
caccacggtc atccagcgga tcttctacgc cgtgaaccgg tcctggtccg gcaggatcac 660
ctgcgccgag ctgcggagga gctccttcct gcagaatgtg gcgctgctgg aggaggaggc 720
ggacatcaac cagctgaccg aattcttctc gtacgagcat ttctacgtca tctactgcaa 780
gttctgggag ctggacacgg accacgacct gctcatcgac gcggacgacc tggcgcggca 840
caatgaccac gccctttcta ccaagatgat agacaggatc ttctcaggag cagtcacacg 900
aggcagaaaa gtgcagaagg aagggaagat cagctatgcc gactttgtct ggtttttgat 960
ctctgaggaa gacaaaaaaa caccgaccag catcgagtac tggttccgct gcatggacct 1020
ggacggggac ggcgccctgt ccatgttcga gctcgagtac ttctacgagg agcagtgccg 1080
aagcgtggac agcatggcca tcgaggccct gcccttccag gactgcctct gccagatgct 1140
ggacctggtc aagccgagga ctgaagggaa gatcacgctg caggacctga agcgctgcaa 1200
gctggccaac gtcttcttcg acaccttctt caacatcgag aagtacctcg accacgagca 1260
gaaagagcag atctccctgc tcagggacgg tgacagcggc ggccccgagc tctcggactg 1320
ggagaagtac gcggccgagg agtacgacat cctggtggcc gaggagaccg cgggagagcc 1380
ctgggaggac gggttcgagg ccgagctcag ccctgtggag cagaagctga gtgcgctgcg 1440
ctccccgctg gcccagaggc ccttcttcga ggcgccctca ccgctgggcg ccgtggacct 1500
gtacgagtac gcgtgcgggg acgaggacct ggagccgctg tgacgccgcc cgcgagaacg 1560
ccgccgcggg gccgctcccc acgtgccacc accgggccac cgcggctcgt gtaaaaactg 1620
ttgtggaaaa tgagtgcgtt tgtacggaat gataaacttt tatttattca aaaaaaaaa 1679
<210> 26
<211> 1053
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1877133CB1
<400> 26
aaggaaagga gagaagggtt gtgaagggaa gcggaatgga agggaaggga ggtcccgtgg 60
gacgctgggg tctggggcag agcaggtagc agcgtgctgc cctgacagct gtctccgctc 120
ctcagattgt cagtggctgc tatgcagcag gtgcagcctg gtctctcact gagtctctac 180
tccacaaagg caacgactgg ccaaggcagt ggctggctct gggttacaca agtgcagaca 240
ctcaactaag tgagctggaa gacccaggag aaggcggagg ctcaggtgcc cacatgatca 300
gcacagccag ggtacctgct gacaagcctg tacgcatcgc ctttagcctc aatgacgcct 360
cagatgatac accccctgaa gactccattc ctttggtctt tccagaatta gaccagcagc 420
tacagcccct gccgccttgt catgactccg aggaatccat ggaggtgttc aaacagcact 480
gccaaatagc agaagaatac catgaggtca aaaaggaaat caccctgctt gagcaaagga 540
agaaggagct cattgccaag ttagatcagg cagaaaagga gaaggtggat gctgctgagc 600
22/24
CA 02364739 2001-08-20
WO 00/55332 PCTiUS00/07277
PF-0683 PCT
tggttcggga attcgaggct ctgacggagg agaatcggac gttgaggttg gcccagtctc 660
aatgtgtgga acaactggag aaacttcgaa tacagtatca gaagaggcag ggctcgtcct 720
aactttaaat ttttcagtgt gagcatacga ggctgatgac tgccctgtgc tggccaaaag 780'
atttttattt taaatgaata gtgagtcaga tctattgctt ctctgtatta cccacatgac 840
aactgtctat aatgagttta ctgcttgcca gcttctagct tgagagaagg gatattttaa 900
atgagatcat taacgtgaaa ctattactag tatatgtttt tggagatcag aattcttttc 960
caaagatata tgtttttttc ttttttagga agatatgatc atgctgtaca acagggtaga 1020
aaatgataaa aatagactta ttgctgaccc cag 1053
<210> 27
<211> 1212
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2636759CB1
<400> 27
gacggcacgc aaggtcagcg tcacagatct gaccctaaaa ataggcctct gttgccagtc 60
ggggtggctg ggcgtgcggc tgctacatgc cccacggacc agaacctccc gacgcggcca 120
ggccccggca cacccagctg cagaaaggag agaaaatccc ttggctctaa aatgacatct 180
ggagaagtga agacaagcct caagaatgcc tactcatctg ccaagaggct gtcgccgaag 240
atggaggagg aaggggagga ggaggactac tgcacccctg gagcctttga gctggagcgg 300
ctcttctgga agggcagtcc ccagtacacc cacgtcaacg aggtctggcc caagctctac 360
attggcgatg aggcgacggc gctggaccgc tataggctgc agaaggcggg gttcacgcac 420
gtgctgaacg cggcccacgg ccgttggaac gtggacactg ggccccgact actaccgcga 480
catggacatc cagtaccacg gcgtggaggc ccgacgacct gcccaccttt cgacctcagt 540
gtcttcttct acccggcggc agccttcatc gacagagcgc taagcgacga ccacagtaag 600
atcctggttc actgcgtcat gggccgcagc cggtcagcca ccctggtcct ggcctacctg 660
atgatccaca aggacatgac cctggtggac gccatccagc aagtggccaa gaaccgctgc 720
gtcctcccga accggggctt tttgaagcag ctccgggagc tggacaagca gctggtgcag 780
cagaggcgac ggtcccagcg ccaggacggt gaggaggagg atgacaggga gctgtaggcc 840
cgactcacag ggccagcaga ggcacttggg gacagagggg agaggcagaa catagccctg 900
gcctaggact ccagagaagg gatggtgaaa ccgaagctcg actcttccaa accatcttgt 960
tcaacttccc catgtgtgct ggggacaggg aggacccaga gctgcccccg ggcagagctg 1020
agcgctcagc ctctcagcaa aatgggaggg acgggctccc cggctctggg tcacagagga 1080
gcatgccacg ctgcaccaag tcccctgctt tggttttgtt tttttggtga gaaggaagag 1140
ggaaaatttt ttaaattgtg gggcattatt tttgtaaata tccttcgggc tttgtttaaa 1200
aacaaaaaaa as 1212
<210> 28
<211> 3093
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2716815CB1
<400> 28
gcagagtgct agcaggagcg cgagccagca agaggcgcct gcgcgatgtc cgggcccctg 60
agccgcggcg ctgagccagc cgggacggac atgcgcggga gggcgccgcg gggcagccgc 120
cgctcctccg ggggaatgaa agctactggt tgattttaaa gtgcctgggc ctcacaggtt 180
tggagatgtc ccagaataag gcacaatgtc aatagcagga gttgctgctc aggagatcag 240
agtcccatta aaaactggat ttctacataa tggccgagcc atggggaata tgaggaagac 300
ctactggagc agtcgcagtg agtttaaaaa caacttttta aatattgacc cgataaccat 360
ggcctacagt ctgaactctt ctgctcagga gcgcctaata ccacttgggc atgcttccaa 420
atctgctccg atgaatggcc actgctttgc agaaaatggt ccatctcaaa agtccagctt 480
23/24
WO 0~i55332 CA 02364739 2001-08-20 PCT/US00/07277
PF-0683 PCT
gccccctctt cttattcccc caagtgaaaa cttgggacca catgaagagg atcaagttgt 540
atgtggtttt aagaaactca cagtgaatgg ggtttgtgct tccacccctc cactgacacc 600
cataaaaaac tccccttccc ttttcccctg tgcccctctt tgtgaacggg gttctaggcc 660
tcttccaccg ttgccaatct ctgaagccct ctctctggat gacacagact gtgaggtgga 720
attcctaact agctcagata cagacttcct tttagaagac tctacacttt ctgatttcaa 780
atatgatgtt cctggcaggc gaagcttccg tgggtgtgga caaatcaact atgcatattt 840
tgatacccca gctgtttctg cagcagatct cagctatgtg tctgaccaaa atggaggtgt 900
cccagatcca aatcctcctc cacctcagac ccaccgaaga ttaagaaggt ctcattcggg 960
accagctggc tcctttaaca agccagccat aaggatatcc aactgttgta tacacagagc 1020
ttctcctaac tccgatgaag acaaacctga ggttcccccc agagttccca tacctcctag 1080
accagtaaag ccagattata gaagatggtc agcagaagtt acttcgagca cctatagtga 1140
tgaagacagg cctcccaaag taccgccaag agaacctttg tcaccgagta actcgcgcac 1200
accgagtccc aaaagccttc cgtcttacct caatggggtc atgcccccga cacagagctt 1260
tgcccctgat cccaagtatg tcagcagcaa agcactgcaa agacagaaca gcgaaggatc 1320
tgccagtaag gttccttgca ttctgcccat tattgaaaat gggaagaagg ttagttcaac 1380
acattattac ctactacctg aacgaccacc atacctggac aaatatgaaa aattttttag 1440
ggaagcagaa gaaacaaatg gaggcgccca aatccagcca ttacctgctg actgcggtat 1500
atcttcagcc acagaaaagc cagactcaaa aacaaaaatg gatctgggtg gccacgtgaa 1560
gcgtaaacat ttatcctatg tggtttctcc ttagaccttg gggtcatggt tcagcagagg 1620
ttacatagga gcaaatggtt ctcaattttc cagtttgatt gaagtgcaga gaaaaatccc 1680
ttagattgca aaataaaata gttgaactct ctgtcttcat gtggaaggtt tagagcagtt 1740
gtgagatgct gttatgctga gaaaccctga ctttgttagt gttggaaaaa agtcttacaa 1800
gtctataatt taaagatgtg atggtgggga ggggaggatg gggaagcttt ttatatatgc 1860
atacattaca tacctatata taaacttgtg gtataaccat agaccatagc tgcaggttaa 1920
ccaattagtt actatcgtag agtaatatat attcagaata ataaactcaa gctggagaaa 1980
tgagtcctga tagactgaaa attgagcaaa tggaagaaga tacagtattg tttagatcag 2040
aatcattaaa aaatattttt gtttagtaag tttgaagatt tctggctttt aggccttttc 2100
tattttgttc catttatttt tgcaggcaat cttttccatg gagggcaggg tatccattct 2160
ttaccatggg tgtacctgct taggttaaaa atcataccaa ggcctcatac ttccaggttt 2220
catgttgcgt cttgttgagg gagggagagc aggttacttg gcaaccatat tgtcacctgt 2280
acctgtcaca catcttgaaa aataaaacga taatagaact agtgactaat tttcccttac 2340
agttcctgct tggtcccacc cactgaagta gctcatcgta gtgcgggccg tattagaggc 2400
agtggggtac gttagactca gatggaaaag tattctaggt gccagtgtta ggatgtcagt 2460
tttacaaaat aatgaagcaa ttagctatgt gattgagagt tattgtttgg ggatgtgtgt 2520
tgtggttttg cttttttttt ttagactgta ttaataaaca tacaacacaa gctggccttg 2580
tgttgctggt tcctattcag tatttcctgg ggattgtttg ctttttaagt aaaacacttc 2640
tgacccatag ctcagtatgt ctgaattcca gaggtcacat cagcatcttt ctgctttgaa 2700
aactctcaca gctgtggctg cttcacttag atgcagtgag acacatagtt ggtgttccga 2760
ttttcacatc cttccatgta tttatcttga agagataagc acagaagaga aggtgctcac 2820
taacagaggt acattactgc aatgttctct taacagttaa acaagctgtt tacagtttaa 2880
actgctgaat attatttgag ctatttaaag cttattatat tttagtatga actaaatgaa 2940
ggttaaaaca tgcttaagaa aaatgcactg atttctgcat tatgtgtaca gtattggaca 3000
aaggatttta ttcattttgt tgcattattt tgaatattgt cttttcattt taataaagtt 3060
ataatactta tttatgataa aaaaaaaaaa aaa 3093
24/24
