Note: Descriptions are shown in the official language in which they were submitted.
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PROTEIN PHOSPHATASE AND KINASE PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of protein
phosphatase and
kinase proteins and to the use of these sequences in the diagnosis, treatment,
and prevention of
gastrointestinal disorders, immune system disorders, neurological disorders,
and cell proliferative
disorders, including cancer.
BACKGROUND OF THE INVENTION
Kinases and phosphatases are critical components of intracellular signal
transduction
mechanisms. Kinases catalyze the transfer of high energy phosphate groups from
adenosine
triphosphate (ATP) to hydroxyamino acids of various target proteins.
Phosphatases, in contrast,
remove phosphate groups from proteins. Reversible protein phosphorylation is
the main strategy for
regulating protein activity in eukaryotic cells. In general, proteins are
activated by phosphorylation in
response to extracellular signals such as hormones, neurotransmitters, and
growth and differentiation
factors. Protein dephosphorylation occurs when down-regulation of a signaling
pathway is required.
The combined activities of kinases and phosphatases regulate key cellular
processes such as
proliferation, differentiation, and cell cycle progression.
Protein Kinases
Kinases comprise the largest known enzyme superfamily and vary widely in their
target
proteins. Kinases may be categorized as protein tyrosine kinases (PTKs), which
phosphorylate
tyrosine residues, and protein serine/threonine kinases (STKs), which
phosphorylate serine and/or
threonine residues. Some kinases have dual specificity for both
serine/threonine and tyrosine
residues. Almost all kinases contain a conserved 250-300 amino acid catalytic
domain. This domain
can be further divided into 11 subdomains. N-terminal subdomains I-IV fold
into a two-lobed
structure which binds and orients the ATP donor molecule, and subdomain V
spans the two lobes. C-
terminal subdomains VI-XI bind the protein substrate and transfer the gamma
phosphate from ATP to
the hydroxyl group of a serine, threonine, or tyrosine residue. Each of the 11
subdomains contains
specific catalytic residues or amino acid motifs characteristic of that
subdomain. For example,
subdomain I contains an 8-amino acid glycine-rich ATP binding consensus motif,
subdomain II
contains a critical lysine residue required for maximal catalytic activity,
and subdomains VI through
IX comprise the highly conserved catalytic core. STKs and PTKs also contain
distinct sequence
motifs in subdomains VI and VIII which may confer hydroxyamino acid
specificity. Some STKs and
PTKs possess structural characteristics of both families. In addition, kinases
may also be classified
by additional amino acid sequences, generally between 5 and 100 residues,
which either flank or
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occur within the kinase domain. These additional amino acid sequences regulate
kinase activity and
determine substrate specificity. (Reviewed in Hardie, G. and Hanks, S. (1995)
The Protein Kinase
Facts Book, Vol I:7-20 Academic Press, San Diego, CA.)
The second messenger dependent protein kinases primarily mediate the effects
of second
messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate,
phosphatidylinositol,
3,4,5-triphosphate, cyclic ADPribose, arachidonic acid, diacylglycerol and
calcium-calmodulin. The
cyclic-AMP dependent protein kinases (PKA) are important members of the STK
family. Cyclic-
AMP is an intracellular mediator of hormone action in all animal cells that
have been studied.
Hormone-induced cellular responses include thyroid hormone secretion, cortisol
secretion,
progesterone secretion, glycogen breakdown, bone resorption, and regulation of
heart rate and force
of heart muscle contraction. PKA is found in all animal cells and is thought
to account for the effects
of cyclic-AMP in most of these cells. Altered PKA expression is implicated in
a variety of disorders
and diseases including cancer, thyroid disorders, diabetes, atherosclerosis,
and cardiovascular disease
(Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine,
McGraw-Hill, New York,
NY, pp. 416-431, 1887).
Calcium-calmodulin (CaM) dependent protein kinases are also members of the STK
family.
Calmodulin is a calcium receptor that mediates many calcium regulated
processes by binding to target
proteins. The principle target proteins in these processes are CaM-dependent
protein kinases (CaM
kinases). CaM kinases are involved in regulation of smooth muscle contraction,
glycogen breakdown
(phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase
II). CaM kinase I
phosphorylates a variety of substrates including the neurotransmitter-related
proteins synapsin I and
II, the gene transcription regulator, CREB, and the cystic fibrosis
conductance regulator protein,
CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:3679-3686). CaM kinase II
also phosphorylates
synapsin at different sites and controls the synthesis of catecholamines in
the brain through
phosphorylation and activation of tyrosine hydroxylase. Many CaM kinases are
activated not only by
CaM, but also by phosphorylation. For example, CaM kinase may
autophosphorylate itself or may be
phosphorylated by another kinase as part of a kinase cascade. The mRNA
encoding a calmodulin-
binding protein kinase-like protein was found to be enriched in mammalian
forebrain. This protein is
associated with vesicles in both axons and dendrites and accumulates largely
postnatally. The amino
acid sequence of this protein is similar to CaM-dependent STKs, and the
protein binds calmodulin in
the presence of calcium (Godbout, M. et al. (1994) J. Neurosci. 14:1-13).
The mitogen-activated protein kinases (MAP) are another STK family that
regulates
intracellular signaling pathways. The MAP kinases mediate signal transduction
from the cell surface
to the nucleus via phosphorylation cascades. Several subgroups have been
identified, and each
manifests different substrate specificities and responds to distinct
extracellular stimuli (Egan, S.E.
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and Weinberg, R.A. (1993) Nature 365:781-783). MAP kinase signaling pathways
are present in
mammalian cells as well as in yeast. The extracellular stimuli which activate
MAP kinase pathways
include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium,
heat shock,
endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as
tumor necrosis factor
(TNF) and interleukin-1 (IL-1). Altered MAP kinase expression is implicated in
a variety of disease
conditions including cancer, inflammation, immune disorders, and disorders
affecting growth and
development.
PTKs may be divided into transmembrane, receptor PTKs and nontransmembrane,
non
receptor PTKs. Transmembrane PTKs are receptors for most growth factors.
Binding of growth
factor to the receptor activates the transfer of a phosphate group from ATP to
selected intracellular
tyrosine side chains of the receptor and other intracellular signal
transduction proteins. Growth
factors (GF) which bind to receptor PTKs include epidermal GF, platelet-
derived GF, fibroblast GF,
hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial
GF, and macrophage
colony stimulating factor. Non-receptor PTKs lack transmembrane regions and,
instead, form
complexes with the intracellular regions of cell surface receptors. Receptors
that function through
non-receptor PTKs include those for cytokines and hormones (growth hormone and
prolactin), and
antigen-specific receptors on T and B lymphocytes.
Many PTKs were first identified as the products of mutant oncogenes in cancer
cells in which
their activation was no longer subject to normal cellular controls. In fact,
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 Tonks N.K.
(1992) Annu Rev Cell Biol 8:463-493). Regulation of PTK activity may therefore
be an important
strategy in controlling some types of cancer.
Protein Phosphatases
Phosphatases are characterized as either tyrosine-specific or serine/threonine-
specific based
on their preferred phospho-amino acid substrate. However, some phosphatases
exhibit dual
specificity for both phospho-tyrosine and phospho-serine/threonine residues.
Many serine/threonine-
specific phosphatases (STPs) have been biochemically purified and extensively
characterized. STPs
are generally comprised of two or more subunits and have broad and overlapping
protein substrate
specificities. STPs are found in the cytosol, nucleus, and mitochondria and in
association with
cytoskeletal and membranous structures. Some STPs require divalent cations,
such as Ca2+ or Mnz+,
for activity. STPs play important roles in glycogen metabolism, muscle
contraction, protein
synthesis, oocyte maturation, and hepatic metabolism. (Reviewed in Cohen, P.
(1989) Annu. Rev.
Biochem. 58:453-508.)
Protein phosphatase 2A (PP2A) is a well-characterized STP purified primarily
from
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mammalian tissues. A trimeric PP2A holoenzyme has been purified from rabbit
skeletal muscle.
(Hendrix, P. et al. (1993) J. Biol. Chem. 268:15267-15276.) PP2A holoenzyme
activity is stimulated
by polycationic macromolecules. PP2A holoenzyme consists of a single catalytic
subunit of 36
kilodaltons (kDa) and two regulatory subunits of 65 and 72 kDa. The regulatory
subunits appear to
determine the substrate-specificity, enzymatic activity, and subcellular
localization of the
holoenzyme. The 72 kDa subunit (PR72), in particular, increases the
stimulatory effect of
polycations and is phosphorylated by casein kinases I and II. The cDNA
encoding human PR72 has
been cloned, and PR72 gene transcripts have been detected exclusively in
skeletal muscle and heart,
suggesting that PR72 confers tissue-specificity to the PP2A holoenzyme. A 55
kDa regulatory
subunit was also purified from the trimeric form of protein phosphatase 2A
from rabbit skeletal
muscle. This PR55 subunit was found to be encoded by two genes, alpha and
beta. A high degree of
conservation was found in a comparison of the amino acid sequences of human
and rabbit PR55. The
human PR55 beta isoform was detected at high levels in a neuroblastoma-derived
cell line and at very
low levels in other human cell lines. This suggests the isoform is neuron-
specific (Mayer, R.E. et al.
(1991) Biochemistry 30:3589-3597).
In contrast to STPs, tyrosine-specific phosphatases (YPs) are generally
monomeric proteins
which function primarily in the transduction of signals across the plasma
membrane. YPs are
categorized as either transmembrane receptor-like proteins or soluble
nontransmembrane proteins.
YPs 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. YPs play important roles in lymphocyte activation
and cell adhesion. In
addition, the genes encoding at least eight YPs 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. (Reviewed in Charbonneau,
H. and Tonks, N.
K. (1992) Annu. Rev. Cell Biol. 8:463-493.)
The discovery of new protein phosphatase and kinase proteins and the
polynucleotides
encoding them satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of gastrointestinal disorders, immune
system disorders,
neurological disorders, and cell proliferative disorders, including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, protein phosphatase and kinase
proteins,
referred to collectively as "PPHKP" and individually as PPHKP-1," "PPHKP-2,"
"PPHKP-3,"
"PPHKP-4," "PPHKP-5," "PPHKP-6," "PPHKP-7," "PPHKP-8," "PPHKP-9," "PPHKP-10,"
and
"PPHKP-1 l." In one aspect, the invention provides an isolated polypeptide
comprising an amino acid
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sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-11, 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:1-11,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-11, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-11. In one alternative, the invention provides
an isolated
polypeptide comprising the amino acid sequence of SEQ ID NO:1-1 I.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-1 I, 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:1-11, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ )D NO:1-11, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-11. In one alternative, the
polynucleotide encodes a
polypeptide selected from the group consisting of SEQ ID NO:1-11. In another
alternative, the
polynucleotide is selected from the group consisting of SEQ ID N0:12-22.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-11, 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:1-11,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-11, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-11. In one alternative, the invention provides
a cell transformed
with the recombinant polynucleotide. In another alternative, the invention
provides a transgenic
organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-11, 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-11,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-11, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-11. 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
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polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11, 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:1-1 l, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:l-1 l, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:12-22, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:12-22, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). In one alternative,
the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:12-22, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:12-22, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the
probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:12-22, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:12-22, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). The method comprises
a) amplifying said
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target polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b)
detecting the presence or absence of said amplified target polynucleotide or
fragment thereof, and,
optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11, 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:1-11, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-11, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11, and a
pharmaceutically
acceptable excipient. In one embodiment, the composition comprises an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-11. The invention
additionally provides a
method of treating a disease or condition associated with decreased expression
of functional PPHKP,
comprising administering to a patient in need of such treatment the
composition.
IS The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-11,
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:1-11, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-11. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
agonist activity in the sample. In one alternative, the invention provides a
composition comprising an
agonist compound identified by the method and a pharmaceutically acceptable
excipient. In another
alternative, the invention provides a method of treating a disease or
condition associated with
decreased expression of functional PPHKP, comprising administering to a
patient in need of such
treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-
11, 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:1-11, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-1 l, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-11. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
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antagonist activity in the sample. In one alternative, the invention provides
a composition comprising
an antagonist compound identified by the method and a pharmaceutically
acceptable excipient. In
another alternative, the invention provides a method of treating a disease or
condition associated with
overexpression of functional PPHKP, comprising administering to a patient in
need of such treatment
the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-11, 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:1-11, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-11, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11. The method
comprises a)
combining the polypeptide with at least one test compound under suitable
conditions, and b)
detecting binding of the polypeptide to the test compound, thereby identifying
a compound that
specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-11,
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:1-11, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-11, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:I-11. The
method comprises a) combining the polypeptide with at least one test compound
under conditions
permissive for the activity of the polypeptide, b) assessing the activity of
the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of the
test compound with the activity of the polypeptide in the absence of the test
compound, wherein a
change in the activity of the polypeptide in the presence of the test compound
is indicative of a
compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:12-22, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
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b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:12-22, ii) a naturally occurring polynucleotide sequence having at least
90% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:12-
22, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of i) a polynucleotide sequence selected from the group consisting
of SEQ ID N0:12-22,
ii) a naturally occurnng polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:12-22,
iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide
comprises a fragment of
a polynucleotide sequence selected from the group consisting of i)-v) above;
c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the
treated biological sample with the amount of hybridization complex in an
untreated biological
sample, wherein a difference in the amount of hybridization complex in the
treated biological sample
is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 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 PPHKP.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of PPHKP.
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 PPHKP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
9
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DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"PPHKP" refers to the amino acid sequences of substantially purified PPHKP
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
PPHKP. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of PPHKP either by
directly interacting with
PPHKP or by acting on components of the biological pathway in which PPHKP
participates.
An "allelic variant" is an alternative form of the gene encoding PPHKP.
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 PPHKP include those sequences with
deletions,
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insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as PPHKP or a
polypeptide with at least one functional characteristic of PPHKP. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding PPHKP, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
PPHKP. 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 PPHKP. 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 PPHKP is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine. .
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a sequence of a
naturally occurring
protein molecule, "amino acid sequence" and like terms are not meant to limit
the amino acid
sequence to the complete native amino acid sequence associated with the
recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of PPHKP. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of PPHKP either by
directly interacting with PPHKP or by acting on components of the biological
pathway in which
PPHKP participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind PPHKP 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,
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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 o~ base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic PPHKP, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with specific
antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding PPHKP or fragments
of PPHKP 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.).
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"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(PE Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
S fragment assembly, such as the GELVIEW fragment assembly system (GCG,
Madison WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
IS Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide sequence can include, for example,
replacement of
hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative
polynucleotide encodes a
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polypeptide which retains at least one biological or immunological function of
the natural molecule.
A derivative polypeptide is one modified by glycosylation, pegylation, or any
similar process that
retains at least one biological or immunological function of the polypeptide
from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of PPHKP or the polynucleotide encoding PPHKP
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example,
a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide)
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ ID N0:12-22 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:12-22, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:12-22 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:12-22 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:12-22 and the region of SEQ ID N0:12-22 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
A fragment of SEQ ID NO:1-11 is encoded by a fragment of SEQ ID N0:12-22. A
fragment
of SEQ ID NO:1-11 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-11. For example, a fragment of SEQ ID NO:1-11 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-11.
The precise length of
a fragment of SEQ ID NO:1-11 and the region of SEQ ID NO:1-11 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full-length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full-length" polynucleotide sequence encodes a "full-length" polypeptide
sequence.
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"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCB)] Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Cap: 5 and Extension Gap: 2 penalties
Gap x drop-off. 50
Expect: 10
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Word Size: 1l
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (Apr-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: I penalties
Gap x drop-off: 50
Expect: 10
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
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 pg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.,
1989, Molecular Cloning: A Laboratory Manual, 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, sheared and denatured salmon sperm DNA at about 100-200
pg/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of PPHKP
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 PPHKP which is useful in any of the antibody production methods disclosed
herein or known in
the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of PPHKP. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of PPHKP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
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polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of a PPHKP may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of PPHKP.
"Probe" refers to nucleic acid sequences encoding PPHKP, 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 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. ( 1989) Molecular Cloning: A Laboratory Manual,
2°'' ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. (1990) PCR
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Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may 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, su ra. 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.
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Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins which control
transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding PPHKP, or fragments thereof, or PPHKP 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
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
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trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed" cells includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook, J. et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
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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 protein phosphatase and
kinase
proteins (PPHKP), the polynucleotides encoding PPHKP, and the use of these
compositions for the
diagnosis, treatment, or prevention of gastrointestinal disorders, immune
system disorders,
neurological disorders, and cell proliferative disorders, including cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
PPHKP. 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 PPHKP 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. In some cases, GenBank sequence identifiers are also
shown in column 5.
The Incyte clones and GenBank cDNA sequences, where indicated, in column 5
were used to
assemble the consensus nucleotide sequence of each PPHKP 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
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
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protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding PPHKP. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of
column 1. These
S fragments are useful, for example, in hybridization or amplification
technologies to identify SEQ ID
N0:12-22 and to distinguish between SEQ ID N0:12-22 and related polynucleotide
sequences. The
polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides. Column
3 lists tissue categories which express PPHKP as a fraction of total tissues
expressing PPHKP.
Column 4 lists diseases, disorders, or conditions associated with those
tissues expressing PPHKP as a
fraction of total tissues expressing PPHKP. Column 5 lists the vectors used to
subclone each cDNA
library. Of particular note is the tissue specific expression of SEQ ID N0:14,
SEQ ID N0:17, and
SEQ ID N0:22. About 75% of the cDNA libraries expressing SEQ ID N0:14 are
derived from
nervous system tissue, particularly brain and spinal cord tissue. About 88% of
the cDNA libraries
expressing SEQ ID N0:17 are derived from nervous system tissue, particularly
brain tissue. About
82% of the cDNA libraries expressing SEQ ID N0:22 are derived from
gastrointestinal tissue.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding PPHKP 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.
SEQ ID N0:12 maps to chromosome 7 within the interval from 84.40 to 90.30
centiMorgans,
to and to chromosome 11 within the interval from 89.80 to 90.70 centiMorgans .
The interval on
chromosome 7 from 84.40 to 90.30 centiMorgans also contains ESTs associated
with B-cell
CLL/lymphoma 7b. SEQ ID N0:16 maps to chromosome 20 within the interval from
the p-terminus
to 6.20 centiMorgans. This interval also contains ESTs associated with protein
tyrosine phosphatase,
non-receptor type substrate 1. SEQ ID N0:21 maps to chromosome 22 within the
interval from 0.0
to 40.2 centiMorgans. This interval also contains ESTs associated with
leukemia-associated
phosphoprotein p18, a cytosolic phosphoprotein found in increased levels in
the cells of various types
of human acute leukemia.
The invention also encompasses PPHKP variants. A preferred PPHKP 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 PPHKP amino acid sequence, and which contains at
least one functional or
structural characteristic of PPHKP.
The invention also encompasses polynucleotides which encode PPHKP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:12-22, which encodes PPHKP. The
polynucleotide
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WO 01/20004 PCT/US00/25515
sequences of SEQ ID N0:12-22, 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
PPHKP. 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 PPHKP. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:12-22 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 N0:12-22. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of PPHKP.
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 PPHKP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring PPHKP, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode PPHKP and its variants are
generally capable
of hybridizing to the nucleotide sequence of the naturally occurring PPHKP
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences encoding
PPHKP 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 PPHKP 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 PPHKP
and
PPHKP 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 PPHKP or any fragment thereof.
CA 02383927 2002-03-05
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Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:12-22 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(PE
Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia
Biotech,
Piscataway NJ), or combinations of polymerases and proofreading exonucleases
such as those found
in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably,
sequence preparation is automated with machines such as the MICROLAB 2200
liquid transfer
system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA)
and ABI
CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then carried out
using either the
ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA
sequencing
system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
The resulting
sequences are analyzed using a variety of algorithms which are well known in
the art. (See, e.g.,
Ausubel, F.M. (1997) Short Protocols in Molecular BioloQV, 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 PPHKP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR Methods
Applic. 2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. ( 1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
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CA 02383927 2002-03-05
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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, PE Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode PPHKP may be cloned in recombinant DNA molecules that direct
expression of
PPHKP, 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 PPHKP.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter PPHKP-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments. and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
27
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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 PPHKP, 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 PPHKP 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; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-
232.) Alternatively,
PPHKP itself or a fragment thereof may be synthesized using chemical methods.
For example,
peptide synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. ( 1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (PE Biosystems). Additionally, the
amino acid sequence of
PPHKP, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, sera, pp. 28-53.)
In order to express a biologically active PPHKP, the nucleotide sequences
encoding PPHKP
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding PPHKP. Such elements may vary in their
strength and
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WO 01/20004 PCT/US00/25515
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding PPHKP. Such signals include the ATG initiation codon and
adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding PPHKP 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 PPHKP 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 BioloQV, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding PPHKP. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945; Takamatsu,
N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
( 1984) Science 224:838-843; Winter, J. et al. ( 1991 ) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
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USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding PPHKP. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding PPHKP can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding PPHKP 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 PPHKP are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of PPHKP may be used.
For example, vectors
containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of PPHKP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, supra; and Scorer, su ra.)
Plant systems may also be used for expression of PPHKP. Transcription of
sequences
encoding PPHKP 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, su ra; Brogue, supra; and Winter,
su ra.) These
constructs can be introduced into plant cells by direct DNA transformation or
pathogen-mediated
transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~v (
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 PPHKP
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses PPHKP 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
CA 02383927 2002-03-05
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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 PPHKP in cell lines is preferred. For example, sequences encoding PPHKP can
be transformed
into cell lines using expression vectors which may contain viral origins of
replication and/or
endogenous expression elements and a selectable marker gene on the same or on
a separate vector.
Following the introduction of the vector, cells may be allowed to grow for
about 1 to 2 days in
enriched media before being switched to selective media. The purpose of the
selectable marker is to
confer resistance to a selective agent, and its presence allows growth and
recovery of cells which
successfully express the introduced sequences. Resistant clones of stably
transformed cells may be
propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. ( 1981 )
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), f3 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the 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 PPHKP is inserted within a marker gene sequence, transformed
cells containing
sequences encoding PPHKP can be identified by the absence of marker gene
function. Alternatively,
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a marker gene can be placed in tandem with a sequence encoding PPHKP 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 PPHKP
and that express
PPHKP 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 PPHKP
using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on PPHKP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. ( 1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
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 PPHKP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding PPHKP, 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 detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding PPHKP 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 PPHKP may be designed to contain
signal sequences which
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direct secretion of PPHKP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding PPHKP 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 PPHKP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of PPHKP
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 PPHKP encoding sequence and the
heterologous protein
sequence, so that PPHKP may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate
expression and purification of
fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled PPHKP 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.
PPHKP of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to PPHKP. At least one and up to a plurality of test
compounds may be
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screened for specific binding to PPHKP. Examples of test compounds include
antibodies,
oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
PPHKP, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. ( 1991 ) Current
Protocols in Immunoloey 1 (2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which PPHKP
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express PPHKP,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing PPHKP or cell membrane fractions which contain PPHKP
are then
contacted with a test compound and binding, stimulation, or inhibition of
activity of either PPHKP or
the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
PPHKP, either in
solution or affixed to a solid support, and detecting the binding of PPHKP to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carned out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
PPHKP of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of PPHKP. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
PPHKP activity, wherein PPHKP is combined with at least one test compound, and
the activity of
PPHKP in the presence of a test compound is compared with the activity of
PPHKP in the absence of
the test compound. A change in the activity of PPHKP in the presence of the
test compound is
indicative of a compound that modulates the activity of PPHKP. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising PPHKP under
conditions suitable for
PPHKP activity, and the assay is performed. In either of these assays, a test
compound which
modulates the activity of PPHKP may do so indirectly and need not come in
direct contact with the
test compound. At least one and up to a plurality of test compounds may be
screened.
In another embodiment, polynucleotides encoding PPHKP or their mammalian
homologs
may be "knocked out" in an animal model system using homologous recombination
in embryonic
stem (ES) cells. Such techniques are well known in the art and are useful for
the generation of animal
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models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding PPHKP may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding PPHKP can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding PPHKP is injected into animal ES cells,
and the injected
sequence integrates into the animal cell genome. Transformed cells are
injected into blastulae, and
the blastulae are implanted as described above. Transgenic progeny or inbred
lines are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress PPHKP, e.g., by secreting PPHKP
in its milk, may
also serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-
74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of PPHKP and protein phosphatase and kinase proteins. In
addition, the expression
of PPHKP is closely associated with gastrointestinal, inflamed, nervous,
proliferating and cancerous
tissue. Therefore, PPHKP appears to play a role in gastrointestinal disorders,
immune system
disorders, neurological disorders, and cell proliferative disorders, including
cancer. In the treatment
of disorders associated with increased PPHKP expression or activity, it is
desirable to decrease the
expression or activity of PPHKP. In the treatment of disorders associated with
decreased PPHKP
CA 02383927 2002-03-05
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expression or activity, it is desirable to increase the expression or activity
of PPHKP.
Therefore, in one embodiment, PPHKP 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 PPHKP. Examples of such disorders include, but are not limited to,
a gastrointestinal
disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal
stricture, esophageal
carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis,
gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis, ulcerative
colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic
obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea,
constipation, gastrointestinal
hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice,
hepatic
encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis,
Wilson's disease, alpha,-
antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver
infarction, portal vein
obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic
vein thrombosis, veno-
occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas; an
immune system disorder, such as acquired immunodeficiency syndrome (AIDS),
Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma; a
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
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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; and a cell proliferative disorder, such as actinic
keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and
cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and
uterus.
In another embodiment, a vector capable of expressing PPHKP 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 PPHKP including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
PPHKP 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 PPHKP
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of PPHKP
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of PPHKP including, but not limited to, those listed above.
In a further embodiment, an antagonist of PPHKP may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of PPHKP.
Examples of such
disorders include, but are not limited to, those gastrointestinal disorders,
immune system disorders,
neurological disorders, and cell proliferative disorders, including cancer
described above. In one
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aspect, an antibody which specifically binds PPHKP 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 PPHKP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding PPHKP may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of PPHKP 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 PPHKP may be produced using methods which are generally known
in the
art. In particular, purified PPHKP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind PPHKP.
Antibodies to PPHKP may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, humans,
and others may be immunized by injection with PPHKP or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
PPHKP have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of PPHKP 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 PPHKP 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
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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
PPHKP-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for PPHKP may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. ( 1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
PPHKP and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering PPHKP epitopes is generally used, but a
competitive binding assay
may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for PPHKP.
Affinity is expressed as an
association constant, K~, which is defined as the molar concentration of PPHKP-
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
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affinities for multiple PPHKP epitopes, represents the average affinity, or
avidity; of the antibodies
for PPHKP. The K~ determined for a preparation of monoclonal antibodies, which
are monospecific
for a particular PPHKP epitope, represents a true measure of affinity. High-
affinity antibody
preparations with K~ ranging from about 109 to 10'2 L/mole are preferred for
use in immunoassays in
which the PPHKP-antibody complex must withstand rigorous manipulations. Low-
affinity antibody
preparations with Ka ranging from about 106 to 10' L/mole are preferred for
use in
immunopurification and similar procedures which ultimately require
dissociation of PPHKP,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies,
Volume I: A Practical
Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A
Practical Guide to
Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of PPHKP-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding PPHKP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding PPHKP. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding PPHKP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
CA 02383927 2002-03-05
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al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997)
Nucleic Acids Res.
25( 14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding PPHKP may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X 1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Tr~panosoma cruzi). In the
case where a genetic deficiency in PPHKP expression or regulation causes
disease, the expression of
PPHKP from an appropriate population of transduced cells may alleviate the
clinical manifestations
caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
PPHKP are treated by constructing mammalian expression vectors encoding PPHKP
and introducing
these vectors by mechanical means into PPHKP-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson
(1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of PPHKP include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). PPHKP may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an
inducible promoter (e.g., the
41
CA 02383927 2002-03-05
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tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding PPHKP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. E6 ( 1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
( 1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to PPHKP expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding PPHKP under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
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In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding PPHKP to cells which have one or more genetic
abnormalities with respect
to the expression of PPHKP. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Crete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:51 I-544; and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding PPHKP to target cells which have one or more genetic
abnormalities with
respect to the expression of PPHKP. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing PPHKP to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye
Res.169:385-395). The construction of a HSV-1 virus vector has also been
disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. For HSV vectors, see also Goins, W.F, et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art. .
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding PPHKP to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full-length
genomic RNA,
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resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
PPHKP into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
PPHKP-coding RNAs and the synthesis of high levels of PPHKP in vector
transduced cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of PPHKP into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunoloy~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
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 PPHKP.
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 I S 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
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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 PPHKP. 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
1 S endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding PPHKP.
Compounds which may be effective in altering expression of a specific
polynucleotide may include,
but are not limited to, oligonucleotides, antisense oligonucleotides, triple
helix-forming
oligonucleotides, transcription factors and other polypeptide transcriptional
regulators, and non-
macromolecular chemical entities which are capable of interacting with
specific polynucleotide
sequences. Effective compounds may alter polynucleotide expression by acting
as either inhibitors or
promoters of polynucleotide expression. Thus, in the treatment of disorders
associated with increased
PPHKP expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding PPHKP may be therapeutically useful, and in the
treament of disorders
associated with decreased PPHKP expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding PPHKP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective in
altering polynucleotide expression; selection from an existing, commercially-
available or proprietary
library of naturally-occurring or non-natural chemical compounds; rational
design of a compound
based on chemical and/or structural properties of the target polynucleotide;
and selection from a
library of chemical compounds created combinatorially or randomly. A sample
comprising a
polynucleotide encoding PPHKP is exposed to at least one test compound thus
obtained. The sample
CA 02383927 2002-03-05
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may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
PPHKP are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding PPHKP. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of PPHKP, antibodies to PPHKP, and mimetics, agonists, antagonists, or
inhibitors of
PPHKP.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
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intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising PPHKP or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, PPHKP or a fragment thereof may be joined to
a short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
PPHKP or fragments thereof, antibodies of PPHKP, and agonists, antagonists or
inhibitors of
PPHKP, which ameliorates the symptoms or condition. Therapeutic efficacy and
toxicity may be
determined by standard pharmaceutical procedures in cell cultures or with
experimental animals, such
as by calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the
dose lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
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The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
S active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 /.cg to 100,000 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 PPHKP may be used
for the
diagnosis of disorders characterized by expression of PPHKP, or in assays to
monitor patients being
treated with PPHKP or agonists, antagonists, or inhibitors of PPHKP.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for PPHKP include methods which utilize the antibody and a
label to detect
PPHKP in human body fluids or in extracts of cells or tissues. The antibodies
may be used with or
without modification, and may be labeled by covalent or non-covalent
attachment of a reporter
molecule. A wide variety of reporter molecules, several of which are described
above, are known in
the art and may be used.
A variety of protocols for measuring PPHKP, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
PPHKP expression.
Normal or standard values for PPHKP expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibody to
PPHKP under conditions suitable for complex formation. The amount of standard
complex formation
may be quantitated by various methods, such as photometric means. Quantities
of PPHKP 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 PPHKP may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
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CA 02383927 2002-03-05
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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 PPHKP
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of PPHKP, and to monitor regulation of PPHKP levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding PPHKP or closely related
molecules may be used
to identify nucleic acid sequences which encode PPHKP. 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 PPHKP, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the PPHKP encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:12-22 or from
genomic sequences including promoters, enhancers, and introns of the PPHKP
gene.
Means for producing specific hybridization probes for DNAs encoding PPHKP
include the
cloning of polynucleotide sequences encoding PPHKP or PPHKP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are
cormnercially available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding PPHKP may be used for the diagnosis of
disorders
associated with expression of PPHKP. Examples of such disorders include, but
are not limited to, a
gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal
spasm, esophageal
stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric
carcinoma, anorexia, nausea,
emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,
gastroenteritis, intestinal
obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis,
pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis, ulcerative
colitis, ulcerative proctitis,
Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic
obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea,
constipation, gastrointestinal
hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice,
hepatic
encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis,
Wilson's disease, alpha,-
antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver
infarction, portal vein
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obstruction and thrombosis, centrilobular necrosis, peliosis hepatic, hepatic
vein thrombosis, veno-
occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy,
intrahepatic cholestasis of
pregnancy, and hepatic tumors including nodular hyperplasias; adenomas, and
carcinomas; an
immune system disorder, such as acquired immunodeficiency syndrome (AIDS),
Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema
nodosum, atrophic
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis,
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections, and trauma; a
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; and a cell proliferative disorder, such as actinic
keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue
disease (MCTD), myelofibrosis,
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paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and
cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder,
bone, bone marrow, brain,
breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis,
thymus, thyroid, and
uterus. The polynucleotide sequences encoding PPHKP 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 PPHKP expression. Such qualitative or quantitative methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding PPHKP may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding PPHKP 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 PPHKP 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
PPHKP, 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 PPHKP, 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
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development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
PPHKP 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 PPHKP, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
PPHKP, and will be employed under optimized conditions for identification of a
specific gene or
condition. Oligomers may also be employed under less stringent conditions for
detection or
quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding PPHKP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding
PPHKP are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of PPHKP include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. ( 1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
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rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to
monitor progression/regression of disease as a function of gene expression,
and to develop and
monitor the activities of therapeutic agents in the treatment of disease. In
particular, this information
may be used to develop a pharmacogenomic profile of a patient in order to
select the most appropriate
and effective treatment regimen for that patient. For example, therapeutic
agents which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for PPHKP, or PPHKP or fragments
thereof may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
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molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
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density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for PPHKP
to quantify
the levels of PPHKP expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed
by a variety of methods known in the art, for example, by reacting the
proteins in the sample with a
thiol- or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
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incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are
well known and thoroughly described in DNA Microarrays: A Practical Approach,
M. Schena, ed.
( 1999) Oxford University Press, London, hereby expressly incorporated by
reference.
In another embodiment of the invention, nucleic acid sequences encoding PPHKP
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial
chromosomes (HACs), yeast artificial chromosomes (PACs), bacterial artificial
chromosomes
(BACs), bacterial Pl constructions, or single chromosome cDNA libraries. (See,
e.g., Harrington, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of
the invention may be
used to develop genetic linkage maps, for example, which correlate the
inheritance of a disease state
with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, e.g., Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, su ra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding PPHKP on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
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may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, PPHKP, 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 PPHKP and the agent being tested may be measured.
I Another technique for drug screening provides for high throughput screening
of compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with PPHKP,
or fragments thereof,
and washed. Bound PPHKP is then detected by methods well known in the art.
Purified PPHKP 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 PPHKP specifically compete with a test compound
for binding PPHKP.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with PPHKP.
In additional embodiments, the nucleotide sequences which encode PPHKP 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
particular U.S. Ser. No. 60/154,141, are hereby expressly incorporated by
reference.
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EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XLl-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DH l OB, or ElectroMAX DH 1 OB from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
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ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out
using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373
or 377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base
calling software; or other sequence analysis systems known in the art. Reading
frames within the
cDNA sequences were identified using standard methods (reviewed in Ausubel,
1997, supra, unit
7.7). Some of the cDNA sequences were selected for extension using the
techniques disclosed in
Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
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
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sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:12-22. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel,
1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
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product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding PPHKP 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. Chromosomal Mapping of PPHKP Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:12-22 were compared
with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:12-22 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
The genetic map locations of SEQ ID N0:12, SEQ ID N0:16 and SEQ ID N0:21 are
described in The Invention as ranges, or intervals, of human chromosomes. More
than one map
location is reported for SEQ ID N0:12, indicating that previously mapped
sequences having
similarity, but not complete identity, to SEQ ID N0:12 were assembled into
their respective clusters.
The map position of an interval, in centiMorgans, is measured relative to the
terminus of the
chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on
recombination
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frequencies between chromosomal markers. On average, 1 cM is roughly
equivalent to 1 megabase
(Mb) of DNA in humans, although this can vary widely due to hot and cold spots
of recombination.)
The cM distances are based on genetic markers mapped by Genethon which provide
boundaries for
radiation hybrid markers whose sequences were included in each of the
clusters. Diseases associated
with the public and Incyte sequences located within the indicated intervals
are also reported in the
Invention where applicable.
VI. Extension of PPHKP Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:12-22 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
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and (3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1 X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~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.
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The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LBl2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:12-22 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:12-22 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 ,uCi of
[y-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
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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°1o sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena ( 1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (21 mer), 1 X
first strand buffer, 0.03 units/pl RNase inhibitor, 500 E.~M dATP, 500 pM
dGTP, 500 L~M dTTP, 40
NM dCTP, 40 L~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
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GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 p1 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60
°C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for 5 minutes and is aliquoted onto the
microarray surface and covered
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with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 p1 of SX SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60 °C. The arrays are washed for 10
min at 45 °C in a first wash
buffer ( 1 X SSC, 0.1 % SDS), three times for 10 minutes each at 45 °C
in a second wash buffer (0.1 X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines .
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
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emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
IX. Complementary Polynucleotides
Sequences complementary to the PPHKP-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring PPHKP.
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 PPHKP. 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
PPHKP-encoding
transcript.
X. Expression of PPHKP
Expression and purification of PPHKP is achieved using bacterial or virus-
based expression
systems. For expression of PPHKP 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 PPHKP upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of PPHKP in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autographica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding PPHKP by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, PPHKP is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
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affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
PPHKP 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,
sera, ch. 10 and 16). Purified PPHKP obtained by these methods can be used
directly in the assays
shown in Examples XI and XV.
XI. Demonstration of PPHKP Activity
Protein kinase activity is measured by quantifying the phosphorylation of a
protein substrate
by PPHKP in the presence of gamma-labeled 3ZP-ATP. PPHKP is incubated with the
protein
substrate, 3zP-ATP, and an appropriate kinase buffer. The 32P incorporated
into the substrate is
separated from free 3zP-ATP by electrophoresis and the incorporated 32P is
counted using a
radioisotope counter. The amount of incorporated 32P is proportional to the
activity of PPHKP. A
determination of the specific amino acid residue phosphorylated is made by
phosphoamino acid
analysis of the hydrolyzed protein.
Alternatively, protein phosphatase activity is measured by the hydrolysis of P-
nitrophenyl
phosphate (PNPP). PPHKP is incubated 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 of the reaction mixture 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 PPHKP (Diamond R.H. et al (1994) Mol Cell Biol 14:3752-62).
XII. Functional Assays
PPHKP function is assessed by expressing the sequences encoding PPHKP 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;
68
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated; laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of PPHICP on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding PPHKP 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 PPHI{P and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XIII. Production of PPHKP Specific Antibodies
PPHKP 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 PPHKP 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, sera, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431 A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are immunized with
the oligopeptide-
ICLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
PPHKP activity by, for example, binding the peptide or PPHKP to a substrate,
blocking with I %
BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated
goat anti-rabbit IgG.
69
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
XIV. Purification of Naturally Occurring PPHKP Using Specific Antibodies
Naturally occurring or recombinant PPHKP is substantially purified by
immunoaffinity
chromatography using antibodies specific for PPHKP. An immunoaffinity column
is constructed by
covalently coupling anti-PPHKP 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 PPHKP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of PPHKP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/PPHKP 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 PPHKP is collected.
XV. Identification of Molecules Which Interact with PPHKP
PPHKP, or biologically active fragments thereof, are labeled with'25I 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 PPHKP, washed,
and any wells with labeled PPHKP complex are assayed. Data obtained using
different
concentrations of PPHKP are used to calculate values for the number, affinity,
and association of
PPHKP with the candidate molecules.
Alternatively, molecules interacting with PPHKP 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).
PPHKP may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
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.
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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73
SUBSTITUTE SHEET (RULE 26)
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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74
SUBSTITUTE SHEET (RULE 26)
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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SUBSTITUTE SHEET (RULE 26)
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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76
SUBSTITUTE SHEET (RULE 26)
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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SUBSTITUTE SHEET (RULE 26)
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
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79
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
YUE, Henry
TANG, Y. Tom
BANDMAN, Olga
HILLMAN, Jennifer L.
BAUGHN, Mariah R.
AZIMZAI, Yalda
LU, Dyung Aina M.
<120> PROTEIN PHOSPHATASE AND KINASE PROTEINS
<130> PF-0742 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/154,141
<151> 1999-09-15
<160> 22
<170> PERL Program
<210> 1
<211> 329
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 365665CD1
<400> 1
Met Leu Asn Cys Ser Gln Asn Ser Ser Ser Ser Ser Val Trp Trp
1 5 10 15
Leu Lys Ser Pro Ala Phe Ser Ser Gly Ser Ser Glu Gly Asp Ser
20 25 30
Pro Trp Ser Tyr Leu Asn Ser Ser Gly Ser Ser Trp Val Ser Leu
35 40 45
Pro Gly Lys Met Arg Lys Glu Ile Leu Glu Ala Arg Thr Leu Gln
50 55 60
Pro Asp Asp Phe Glu Lys Leu Leu Ala Gly Val Arg His Asp Trp
65 70 75
Leu Phe Gln Arg Leu Glu Asn Thr Gly Val Phe Lys Pro Ser Gln
80 85 90
Leu His Arg Ala His Ser Ala Leu Leu Leu Lys Tyr Ser Lys Lys
95 100 105
Ser Glu Leu Trp Thr Ala Gln Glu Thr Ile Val Tyr Leu Gly Asp
110 115 120
Tyr Leu Thr Val Lys Lys Lys Gly Arg Gln Arg Asn Ala Phe Trp
125 130 135
Val His His Leu His Gln Glu Glu Ile Leu Gly Arg Tyr Val Gly
140 145 150
Lys Asp Tyr Lys Glu Gln Lys Gly Leu Trp His His Phe Thr Asp
155 160 165
Val Glu Arg Gln Met Thr Ala Gln His Tyr Val Thr Glu Phe Asn
170 175 180
Ljrs Arg Leu Tyr Glu Gln Asn Ile Pro Thr Gln Ile Phe Tyr Ile
1/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
185 190 195
Pro Ser Thr Ile Leu Leu Ile Leu Glu Asp Lys Thr Ile Lys Gly
200 205 . 210
Cys Ile Ser Val Glu Pro Tyr Ile Leu Gly Glu.Phe Val Lys Leu
215 220 225
Ser Asn Asn Thr Lys Val Val Lys Thr Glu Tyr Lys Ala Thr Glu
230 235 240
Tyr Gly Leu Ala Tyr Gly His Phe Ser Tyr Glu Phe Ser Asn His
245 250 255
Arg Asp Val Val Val Asp Leu Gln Gly Trp Val Thr Gly Asn Gly
260 265 270
Lys Gly Leu Ile Tyr Leu Thr Asp Pro Gln Ile His Ser Val Asp
275 280 285
Gln Lys Val Phe Thr Thr Asn Phe Gly Lys Arg Gly Ile Phe Tyr
290 295 300
Phe Phe Asn Asn Gln His Val Glu Cys Asn Glu Ile Cys His Arg
305 310 315
Leu Ser Leu Thr Arg Pro Ser Met Glu Lys Pro Cys Lys Ser
320 325
<210> 2
<211> 141
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID N0: 760934CD1
<400> 2
Met Leu Phe Thr Phe His Arg Glu Ser Leu Gln Met Ala Val Gly
1 5 10 15
Pro Phe Leu His Ile Leu Glu Ser Asn Leu Leu Lys Ala Met Asp
20 25 30
Ser Ala Thr Ala Pro Asp Lys Ile Arg Lys Leu Tyr Leu Tyr Ala
35 40 45
Ala His Asp Val Thr Phe Ile Pro Leu Leu Met Thr Leu Gly Ile
50 55 60
Phe Asp His Lys Trp Pro Pro Phe Ala Val Asp Leu Thr Met Glu
65 70 75
Leu Tyr Gln His Leu Glu Ser Lys Glu Trp Phe Val Gln Leu Tyr
80 85 90
Tyr His Gly Lys Glu Gln Val Pro Arg Gly Cys Pro Asp Gly Leu
95 100 105
Cys Pro Leu Asp Met Phe Leu Asn Ala Met Ser Val Tyr Thr Leu
110 115 120
Ser Pro Glu Lys Tyr His Ala Leu Cys Ser Gln Thr Gln Val Met
125 130 135
Glu Val Gly Asn Glu Glu
140
<210> 3
<211> 447
<212> PRT
<213> Homo Sapiens
<220>
2/ 19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
<221> misc_feature
<223> Incyte ID NO: 926043CD1
<400> 3
Met Gly Glu Asp Thr Asp Thr Arg Lys Ile Asn His Ser Phe Leu
1 5 10 15
Arg Asp His Ser Tyr Val Thr Glu Ala Asp Ile Phe Ser Thr Val
20 25 30
Glu Phe Asn His Thr Gly Glu Leu Leu Ala Thr Gly Asp Lys Gly
35 40 45
Gly Arg Val Val Ile Phe Gln Arg Glu Pro Glu Ser Lys Asn Ala
50 55 60
Pro His Ser Gln Gly Glu Tyr Asp Val Tyr Ser Thr Phe Gln Ser
65 70 75
His Glu Pro Glu Phe Asp Tyr Leu Lys Ser Leu Glu Ile Glu Glu
80 85 90
Lys Ile Asn Lys Ile Lys Trp Leu Pro Gln Gln Asn Ala Ala His
95 100 105
Ser Leu Leu Ser Thr Asn Asp Lys Thr Ile Lys Leu Trp Lys Ile
110 115 120
Thr Glu Arg Asp Lys Arg Pro Glu Gly Tyr Asn Leu Lys Asp Glu
125 130 ~ 135
Glu Gly Lys Leu Lys Asp Leu Ser Thr Val Thr Ser Leu Gln Val
140 145 150
Pro Val Leu Lys Pro Met Asp Leu Met Val Glu Val Ser Pro Arg
155 160 165
Arg Ile Phe Ala Asn Gly His Thr Tyr His Ile Asn Ser Ile Ser
170 175 180
Val Asn Ser Asp Cys Glu Thr Tyr Met Ser Ala Asp Asp Leu Arg
185 190 195
Ile Asn Leu Trp His Leu Ala Ile Thr Asp Arg Ser Phe Asn Ile
200 205 210
Val Asp Ile Lys Pro Ala Asn Met Glu Asp Leu Thr Glu Val Ile
215 220 225
Thr Ala Ser Glu Phe His Pro His His Cys Asn Leu Phe Val Tyr
230 235 240
Ser Ser Ser Lys Gly Ser Leu Arg Leu Cys Asp Met Arg Ala Ala
245 250 255
Ala Leu Cys Asp Lys His Ser Lys Leu Phe Glu Glu Pro Glu Asp
260 265 270
Pro Ser Asn Arg Ser Phe Phe Ser Glu Ile Ile Ser Ser Val Ser
275 280 285
Asp Val Lys Phe Ser His Ser Gly Arg Tyr Met Leu Thr Arg Asp
290 295 300
Tyr Leu Thr Val Lys Val Trp Asp Leu Asn Met Glu Ala Arg Pro
305 310 315
Ile Glu Thr Tyr Gln Val His Asp Tyr Leu Arg Ser Lys Leu Cys
320 325 330
Ser Leu Tyr Glu Asn Asp Cys Ile Phe Asp Lys Phe Glu Cys Ala
335 340 345
Trp Asn Gly Ser Asp Ser Val Ile Met Thr Gly Ala Tyr Asn Asn
350 355 360
Phe Phe Arg Met Phe Asp Arg Asn Thr Lys Arg Asp Val Thr Leu
365 370 375
Glu Ala Ser Arg Glu Ser Ser Lys Pro Arg Ala Val Leu Lys Pro
380 385 390
Arg Arg Val Cys Val Gly Gly Lys Arg Arg Arg Asp Asp Ile Ser
395 400 405
Val Asp Ser Leu Asp Phe Thr Lys Lys Ile Leu His Thr Ala Trp
410 415 420
His Pro Ala Glu Asn Ile Ile Ala Ile Ala Ala Thr Asn Asn Leu
425 430 435
Tyr Ile Phe Gln Asp Lys Val Asn Ser Asp Met His
3/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
440 445
<210> 4
<211> 666
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1234795CD1
<400> 4
Met Ala His Glu Met Ile Gly Thr Gln Ile Val Thr Glu Arg Leu
1 5 10 15
Val Ala Leu Leu Glu Ser Gly Thr Glu Lys Val Leu Leu Ile Asp
20 25 30
Ser Arg Pro Phe Val Glu Tyr Asn Thr Ser His Ile Leu Glu Ala
35 40 45
Ile Asn Ile Asn Cys Ser Lys Leu Met Lys Arg Arg Leu Gln Gln
50 55 60
Asp Lys Val Leu Ile Thr Glu Leu Ile Gln His Ser Ala Lys His
65 70 75
Lys Val Asp Ile Asp Cys Ser Gln Lys Val Val Val Tyr Asp Gln
80 85 90
Ser Ser Gln Asp Val Ala Ser Leu Ser Ser Asp Cys Phe Leu Thr
95 100 105
Val Leu Leu Gly Lys Leu Glu Lys Ser Phe Asn Ser Val His Leu
110 115 120
Leu Ala Gly Gly Phe Ala Glu Phe Ser Arg Cys Phe Pro Gly Leu
125 130 135
Cys Glu Gly Lys Ser Thr Leu Val Pro Thr Cys Ile Ser Gln Pro
140 145 150
Cys Leu Pro Val Ala Asn Ile Gly Pro Thr Arg Ile Leu Pro Asn
155 160 165
Leu Tyr Leu Gly Cys Gln Arg Asp Val Leu Asn Lys Glu Leu Met
170 175 180
Gln Gln Asn Gly Ile Gly Tyr Val Leu Asn Ala Ser Asn Thr Cys
185 190 195
Pro Lys Pro Asp Phe Ile Pro Glu Ser His Phe Leu Arg Val Pro
200 205 210
Val Asn Asp Ser Phe Cys Glu Lys Ile Leu Pro Trp Leu Asp Lys
215 220 225
Ser Val Asp Phe Ile Glu Lys Ala Lys Ala Ser Asn Gly Cys Val
230 235 240
Leu Val His Cys Leu Ala Gly Ile Ser Arg Ser Ala Thr Ile Ala
245 250 255
Ile Ala Tyr Ile Met Lys Arg Met Asp Met Ser Leu Asp Glu Ala
260 265 270
Tyr Arg Phe Val Lys Glu Lys Arg Pro Thr Ile Ser Pro Asn Phe
275 280 285
Asn Phe Leu Gly Gln Leu Leu Asp Tyr Glu Lys Lys Ile Lys Asn
290 295 300
Gln Thr Gly Ala Ser Gly Pro Lys Ser Lys Leu Lys Leu Leu His
305 310 315
Leu Glu Lys Pro Asn Glu Pro Val Pro Ala Val Ser Glu Gly Gly
320 325 330
Gln Lys Ser Glu Thr Pro Leu Ser Pro Pro Cys Ala Asp Ser Ala
335 340 345
Thr Ser Glu Ala Ala Gly Gln Arg Pro Val His Pro Ala Ser Val
350 355 360
4/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
Pro Ser Val Pro Ser Val Gln Pro Ser Leu Leu Glu Asp Ser Pro
365 370 375
Leu Val Gln Ala Leu Ser Gly Leu His Leu Ser Ala Asp Arg Leu
380 385 390
Glu Asp Ser Asn Lys Leu Lys Arg Ser Phe Ser Leu Asp Ile Lys
395 400 405
Ser Val Ser Tyr Ser Ala Ser Met Ala Ala Ser Leu His Gly Phe
410 415 420
Ser Ser Ser Glu Asp Ala Leu Glu Tyr Tyr Lys Pro Ser Thr Thr
425 430 435
Leu Asp Gly Thr Asn Lys Leu Cys Gln Phe Ser Pro Val Gln Glu
440 445 450
Leu Ser Glu Gln Thr Pro Glu Thr Ser Ser Leu Ile Arg Arg Lys
455 460 465
Pro Ala Ser Pro Arg Ser Cys Arg Pro Pro Gly Leu Gln Thr Ala
470 475 480
Arg Ala Ser Asp Cys Ile Arg Ser Glu Pro Ala Ala Val Ala Pro
485 490 495
Pro Arg Gly Pro Phe Tyr Leu His Cys Ile Glu Val Gly Ala Trp
500 505 510
Arg Thr Ile Thr Thr Pro Ala Ser Phe Ser Ala Phe Pro Pro Ala
515 520 525
Ser Ser Thr Ser Arg Ser Leu Leu Ala Trp Ala Leu Lys Gly Trp
530 535 540
His Ser Asp Ile Leu Ala Pro Gln Thr Ser Thr Pro Ser Leu Thr
545 550 555
Ser Ser Trp Tyr Phe Ala Thr Glu Ser Ser His Phe Tyr Ser Ala
560 565 570
Ser Ala Ile Tyr Gly Gly Ser Ala Ser Tyr Ser Ala Tyr Ser Cys
575 580 585
Ser Gln Leu Pro Thr Cys Gly Asp Gln Val Tyr Ser Val Arg Arg
590 595 600
Arg Gln Lys Pro Ser Asp Arg Ala Asp Ser Arg Arg Ser Trp His
605 610 615
Glu Glu Ser Pro Phe Glu Lys Gln Phe Lys Arg Arg Ser Cys Gln
620 625 630
Met Glu Phe Gly Glu Ser Ile Met Ser Glu Asn Arg Ser Arg Glu
635 640 645
Glu Leu Gly Lys Val Gly Ser Gln Ser Ser Phe Ser Gly Ser Met
650 655 660
Glu Ile Ile Glu Val Ser
665
<210> 5
<211> 358
<212> PRT
<213> Homo sapiens
<220>
<221> misC_feature
<223> Incyte ID NO: 1271505CD1
<400> 5
Met Arg Ala Thr Pro Leu Ala Ala Pro Ala Gly Ser Leu Ser Arg
1 5 10 15
Lys Lys Arg Leu Glu Leu Asp Asp Asn Leu Asp Thr Glu Arg Pro
20 25 30
Val Gln Lys Arg Ala Arg Ser Gly Pro Gln Pro Arg Leu Pro Pro
35 40 45
Cys Leu Leu Pro Leu Ser Pro Pro Thr Ala Pro Asp Arg Ala Thr
5/ 19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
50 55 60
Ala Val Ala Thr Ala Ser Arg Leu Gly Pro Tyr Val Leu Leu Glu
65 70 75
Pro Glu Glu Gly Gly Arg Ala Tyr Gln Ala Leu His Cys Pro Thr
80 85 90
Gly Thr Glu Tyr Thr Cys Lys Val Tyr Pro Val Gln Glu Ala Leu
95 100 105
Ala Val Leu Glu Pro Tyr Ala Arg Leu Pro Pro His Lys His Val
110 115 120
Ala Arg Pro Thr Glu Val Leu Ala Gly Thr Gln Leu Leu Tyr Ala
125 130 135
Phe Phe Thr Arg Thr His Gly Asp Met His Ser Leu Val Arg Thr
140 145 150
Arg His Arg Ile Pro Glu Pro Glu Ala Ala Val Leu Phe Arg Gln
155 160 165
Met Ala Thr Ala Leu Ala His Cys His Gln His Gly Leu Val Leu
170 175 180
Arg Asp Leu Lys Leu Cys Arg Phe Val Phe Ala Asp Arg Glu Arg
185 190 195
Lys Lys Leu Val Leu Glu Asn Leu Glu Asp Ser Cys Val Leu Thr
200 205 210
Gly Pro Asp Asp Ser Leu Trp Asp Lys His Ala Cys Pro Ala Tyr
215 220 225
Val Gly Pro Glu Ile Leu Ser Ser Arg Ala Ser Tyr Ser Gly Lys
230 235 240
Ala Ala Asp Val Trp Ser Leu Gly Val Ala Leu Phe Thr Met Leu
245 250 255
Ala Gly His Tyr Pro Phe Gln Asp Ser Glu Pro Val Leu Leu Phe
260 265 270
Gly Lys Ile Arg Arg Gly Ala Tyr Ala Leu Pro Ala Gly Leu Ser
275 280 285
Ala Pro Ala Arg Cys Leu Val Arg Cys Leu Leu Arg Arg Glu Pro
290 295 300
Ala Glu Arg Leu Thr Ala Thr Gly Ile Leu Leu His Pro Trp Leu
305 310 315
Arg Gln Asp Pro Met Pro Leu Ala Pro Thr Arg Ser His Leu Trp
320 325 330
Glu Ala Ala Gln Val Val Pro Asp Gly Leu Gly Leu Asp Glu Ala
335 340 345
Arg Glu Glu Glu Gly Asp Arg Glu Val Val Leu Tyr Gly
350 355
<210> 6
<211> 470
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1385073CD1
<400> 6
Met Pro Phe Gly Cys Val Thr Leu Gly Asp Lys Lys Asn Tyr Asn
1 5 10 15
Gln Pro Ser Glu Val Thr Asp Arg Tyr Asp Leu Gly Gln Val Ile
20 25 30
Lys Thr Glu Glu Phe Cys Glu Ile Phe Arg Ala Lys Asp Lys Thr
35 40 45
Thr Gly Lys Leu His Thr Cys Lys Lys Phe Gln Lys Arg Asp Gly
50 55 60
6/ 19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
Arg Lys Val Arg Lys Ala Ala Lys Asn Glu Ile Gly Ile Leu Lys
65 70 75
Met Val Lys His Pro Asn Ile Leu Gln Leu Val Asp Val Phe Val
80 85 90
Thr Arg Lys Glu Tyr Phe Ile Phe Leu Glu Leu Ala Thr Gly Arg
95 100 105
Glu Val Phe Asp Trp Ile Leu Asp Gln Gly Tyr Tyr Ser Glu Arg
110 115 120
Asp Thr Ser Asn Val Val Arg Gln Val Leu Glu Ala Val Ala Tyr
125 130 135
Leu His Ser Leu Lys Ile Val His Arg Asn Leu Lys Leu Glu Asn
140 145 150
Leu Val Tyr Tyr Asn Arg Leu Lys Asn Ser Lys Ile Val Ile Ser
155 160 ~ 165
Asp Phe His Leu Ala Lys Leu Glu Asn Gly Leu Ile Lys Glu Pro
170 175 180
Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Val Gly Arg Gln
185 190 195
Arg Tyr Gly Arg Pro Val Asp Cys Trp Ala Ile Gly Val Ile Met
200 205 210
Tyr Ile Leu Leu Ser Gly Asn Pro Pro Phe Tyr Glu Glu Val Glu
215 220 225
Glu Asp Asp Tyr Glu Asn His Asp Lys Asn Leu Phe Arg Lys Ile
230 235 240
Leu Ala Gly Asp Tyr Glu Phe Asp Ser Pro Tyr Trp Asp Asp Ile
245 250 255
Ser Gln Ala Ala Lys Asp Leu Val Thr Arg Leu Met Glu Val Glu
260 265 270
Gln Asp Gln Arg Ile Thr Ala Glu Glu Ala Ile Ser His Glu Trp
275 280 285
Ile Ser Gly Asn Ala Ala Ser Asp Lys Asn Ile Lys Asp Gly Val
290 295 300
Cys Ala Gln Ile Glu Lys Asn Phe Ala Arg Ala Lys Trp Lys Lys
305 310 315
Ala Val Arg Val Thr Thr Leu Met Lys Arg Leu Arg Ala Pro Glu
320 325 330
Gln Ser Ser Thr Ala Ala Ala Gln Ser Ala Ser Ala Thr Asp Thr
335 340 345
Ala Thr Pro Gly Ala Ala Asp Arg Ser Ala Thr Pro Ala Thr Asp
350 355 360
Gly Ser Ala Thr Pro Ala Thr Asp Gly Ser Val Thr Pro Ala Thr
365 370 375
Asp Gly Ser Ile Thr Pro Ala Thr Asp Gly Ser Val Thr Pro Ala
380 385 390
Thr Asp Arg Ser Ala Thr Pro Ala Thr Asp Gly Arg Ala Thr Pro
395 400 405
Ala Thr Glu Glu Ser Thr Val Pro Thr Thr Gln Ser Ser Ala Met
410 415 420
Leu Ala Thr Lys Ala Ala Ala Thr Pro Glu Pro Ala Met Ala Gln
425 430 435
Pro Asp Ser Thr Ala Pro Glu Gly Ala Thr Gly Gln Ala Pro Pro
440 445 450
Ser Ser Lys Gly Glu Glu Ala Ala Gly Tyr Ala Gln Glu Ser Gln
455 460 465
Arg Glu Glu Ala Ser
470
<210> 7
<211> 150
<212> PRT
7/ 19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1606974CD1
<400> 7
Met Gly Val Gln Pro Pro Asn Phe Ser Trp Val Leu Pro Gly Arg
1 5 10 15
Leu Ala Gly Leu Ala Leu Pro Arg Leu Pro Ala His Tyr Gln Phe
20 25 30
Leu Leu Asp Leu Gly Val Arg His Leu Val Ser Leu Thr Glu Arg
35 40 45
Gly Pro Pro His Ser Asp Ser Cys Pro Gly Leu Thr Leu His Arg
50 55 60
Leu Arg Ile Pro Asp Phe Cys Pro Pro Ala Pro Asp Gln Ile Asp
65 70 75
Arg Phe Val Gln Ile Val Asp Glu Ala Asn Ala Arg Gly Glu Ala
80 85 90
Val Gly Val His Cys Ala Leu Gly Phe Gly Arg Thr Gly Thr Met
95 100 105
Leu Ala Cys Tyr Leu Val Lys Glu Arg Gly Leu Ala Ala Gly Asp
110 115 120
Ala Ile Ala Glu Ile Arg Arg Leu Arg Pro Gly Ser Ile Glu Thr
125 130 135
Tyr Glu Gln Glu Lys Ala Val Phe Gln Phe Tyr Gln Arg Thr Lys
140 145 150
<210> 8
<211> 253
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1829744CD1
<400> 8
Met Ala Ala Ala Arg Ala Thr Thr Pro Ala Asp Gly Glu Glu Pro
1 5 10 15
Ala Pro Glu Ala Glu Ala Leu Ala Ala Ala Arg Glu Arg Ser Ser
20 25 30
Arg Phe Leu Ser Gly Leu Glu Leu Val Lys Gln Gly Ala Glu Ala
35 40 45
Arg Val Phe Arg Gly Arg Phe Gln Gly Arg Ala Ala Val Ile Lys
50 55 60
His Arg Phe Pro Lys Gly Tyr Arg His Pro Ala Leu Glu Ala Arg
65 70 75
Leu Gly Arg Arg Arg Thr Val Gln Glu Ala Arg Ala Leu Leu Arg
80 85 90
Cys Arg Arg Ala Gly Ile Ser Ala Pro Val Val Phe Phe Val Asp
95 100 105
Tyr Ala Ser Asn Cys Leu Tyr Met Glu Glu Ile Glu Gly Ser Val
110 115 120
Thr Val Arg Asp Tyr Ile Gln Ser Thr Met Glu Thr Glu Lys Thr
125 130 135
Pro Gln Gly Leu Ser Asn Leu Ala Lys Thr Ile Gly Gln Val Leu
140 145 150
Ala Arg Met His Asp Glu Asp Leu Ile His Gly Asp Leu Thr Thr
155 160 165
8/ 19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
Ser Asn Met Leu Leu Lys Pro Pro Leu Glu Gln Leu Asn Ile Val
170 175 180
Leu Ile Asp Phe Gly Leu Ser Phe Ile Ser Ala Leu Pro Glu Asp
185 190 195
Lys Gly Val Asp Leu Tyr Val Leu Glu Lys Ala Phe Leu Ser Thr
200 205 210
His Pro Asn Thr Glu Thr Val Phe Glu Ala Phe Leu Lys Ser Tyr
215 220 225
Ser Thr Ser Ser Lys Lys Ala Arg Pro Val Leu Lys Lys Leu Asp
230 235 240
Glu Val Arg Leu Arg Gly Arg Lys Arg Ser Met Val Gly
245 250
<210> 9
<211> 442
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 4030831CD1
<400> 9
Met Arg Arg Thr Arg Arg Pro Arg Phe Val Leu Met Asn Lys Met
1 5 10 15
Asp Asp Leu Asn Leu His Tyr Arg Phe Leu Asn Trp Arg Arg Arg
20 25 30
Ile Arg Glu Ile Arg Glu Val Arg Ala Phe Arg Tyr Gln Glu Arg
35 40 45
Phe Lys His Ile Leu Val Asp Gly Asp Thr Leu Ser Tyr His Gly
50 55 60
Asn Ser Gly Glu Val Gly Cys Tyr Val Ala Ser Arg Pro Leu Thr
65 70 75
Lys Asp Ser Asn Tyr Phe Glu Val Ser Ile Val Asp Ser Gly Val
80 85 90
Arg Gly Thr Ile Ala Val Gly Leu Val Pro Gln Tyr Tyr Ser Leu
95 100 105
Asp His Gln Pro Gly Trp Leu Pro Asp Ser Val Ala Tyr His Ala
110 115 120
Asp Asp Gly Lys Leu Tyr Asn Gly Arg Ala Lys Gly Arg Gln Phe
125 130 135
Gly Ser Lys Cys Asn Ser Gly Asp Arg Ile Gly Cys Gly Ile Glu
140 145 150
Pro Val Ser Phe Asp Val Gln Thr Ala Gln Ile Phe Phe Thr Lys
155 160 165
Asn Gly Lys Arg Val Gly Ser Thr Ile Met Pro Met Ser Pro Asp
170 175 180
Gly Leu Phe Pro Ala Val Gly Met His Ser Leu Gly Glu Glu Val
185 190 195
Arg Leu His Leu Asn Ala Glu Leu Gly Arg Glu Asp Asp Ser Val
200 205 210
Met Met Val Asp Ser Tyr Glu Asp Glu Trp Gly Arg Leu His Asp
215 220 225
Val Arg Val Cys Gly Thr Leu Leu Glu Tyr Leu Gly Lys Gly Lys
230 235 240
Ser Ile Val Asp Val Gly Leu Ala Gln Ala Arg His Pro Leu Ser
245 250 255
Thr Arg Ser His Tyr Phe Glu Val Glu Ile Val Asp Pro Gly Glu
260 265 270
Lys Cys Tyr Ile Ala Leu Gly Leu Ala Arg Lys Asp Tyr Pro Lys
9/ 19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
275 280 285
Asn Arg His Pro Gly Trp Ser Arg Gly Ser Val Ala Tyr His Ala
290 295 300
Asp Asp Gly Lys Ile Phe His Gly Ser Gly Val Gly Asp Pro Phe
305 310 315
Gly Pro Arg Cys Tyr Lys Gly Asp Ile Met Gly Cys Gly Ile Met
320 325 330
Phe Pro Arg Asp Tyr Ile Leu Asp Ser Glu Gly Asp Ser Asp Asp
335 340 345
Ser Cys Asp Thr Val Ile Leu Ser Pro Thr Ala Arg Ala Val Arg
350 355 360
Asn Val Arg Asn Val Met Tyr Leu His Gln Glu Gly Glu Glu Glu
365 370 375
Glu Glu Glu Glu Glu Glu Glu Glu Asp Gly Glu Glu Ile Glu Pro
380 385 390
Glu His Glu Gly Arg Lys Val Val Val Phe Phe Thr Arg Asn Gly
395 400 405
Lys Ile Ile Gly Lys Lys Asp Ala Val Val Pro Ser Gly Gly Phe
410 415 420
Phe Pro Thr Ile Gly Met Leu Ser Cys Gly Glu Lys Val Lys Val
425 430 435
Asp Leu His Pro Leu Ser Gly
440
<210> 10
<211> 659
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 5039718CD1
<400> 10
Met Ala Leu Val Thr Val Ser Arg Ser Pro Pro Gly Ser Gly Ala
1 5 10 15
Ser Thr Pro Val Gly Pro Trp Asp Gln Ala Val Gln Arg Arg Ser
20 25 30
Arg Leu Gln Arg Arg Gln Ser Phe Ala Val Leu Arg Gly Ala Val
35 40 45
Leu Gly Leu Gln Asp Gly Gly Asp Asn Asp Asp Ala Ala Glu Ala
50 55 60
Ser Ser Glu Pro Thr Glu Lys Ala Pro Ser Glu Glu Glu Leu His
65 70 75
Gly Asp Gln Thr Asp Phe Gly Gln Gly Ser Gln Ser Pro Gln Lys
80 85 90
Gln Glu Glu Gln Arg Gln His Leu His Leu Met Val Gln Leu Leu
95 100 105
Arg Pro Gln Asp Asp Ile Arg Leu Ala Ala Gln Leu Glu Ala Pro
110 115 120
Arg Pro Pro Arg Leu Arg Tyr Leu Leu Val Val Ser Thr Arg Glu
125 130 135
Gly Glu Gly Leu Ser Gln Asp Glu Thr Val Leu Leu Gly Val Asp
140 145 150
Phe Pro Asp Ser Ser Ser Pro Ser Cys Thr Leu Gly Leu Val Leu
155 160 165
Pro Leu Trp Ser Asp Thr Gln Val Tyr Leu Asp Gly Asp Gly Gly
170 175 180
Phe Ser Val Thr Ser Gly Gly Gln Ser Arg Ile Phe Lys Pro Ile
185 190 195
10/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
Ser Ile Gln Thr Met Trp Ala Thr Leu Gln Val Leu His Gln Ala
200 205 210
Cys Glu Ala Ala Leu Gly Ser Gly Leu Val Pro Gly Gly Ser Ala
215 220 225
Leu Thr Trp Ala Ser His Tyr Gln Glu Arg Leu Asn Ser Glu Gln
230 235 240
Ser Cys Leu Asn Glu Trp Thr Ala Met Ala Asp Leu Glu Ser Leu
245 250 255
Arg Pro Pro Ser Ala Glu Pro Gly Gly Ser Ser Glu Gln Glu Gln
260 265 270
Met Glu Gln Ala Ile Arg Ala Glu Leu Trp Lys Val Leu Asp Val
275 280 285
Ser Asp Leu Glu Ser Val Thr Ser Lys Glu Ile Arg Gln Ala Leu
290 295 300
Glu Leu Arg Leu Gly Leu Pro Leu Gln Gln Tyr Arg Asp Phe Ile
305 310 315
Asp Asn Gln Met Leu Leu Leu Val Ala Gln Arg Asp Arg Ala Ser
320 325 330
Arg Ile Phe Pro His Leu Tyr Leu Gly Ser Glu Trp Asn Ala Ala
335 340 345
Asn Leu Glu Glu Leu Gln Arg Asn Arg Val Thr His Ile Leu Asn
350 355 360
Met Ala Arg Glu Ile Asp Asn Phe Tyr Pro Glu Arg Phe Thr Tyr
365 370 375
His Asn Val Arg Leu Trp Asp Glu Glu Ser Ala Gln Leu Leu Pro
380 385 390
His Trp Lys Glu Thr His Arg Phe Ile Glu Ala Ala Arg Ala Gln
395 400 405
Gly Thr His Val Leu Val His Cys Lys Met Gly Val Ser Arg Ser
410 415 420
Ala Ala Thr Val Leu Ala Tyr Ala Met Lys Gln Tyr Glu Cys Ser
425 430 435
Leu Glu Gln Ala Leu Arg His Val Gln Glu Leu Arg Pro Ile Ala
440 445 450
Arg Pro Asn Pro Gly Phe Leu Arg Gln Leu Gln Ile Tyr Gln Gly
455 460 465
Ile Leu Thr Ala Ser Arg Gln Ser His Val Trp Glu Gln Lys Val
470 475 480
Gly Gly Val Ser Pro Glu Glu His Pro Ala Pro Glu Val Ser Thr
485 490 495
Pro Phe Pro Pro Leu Pro Pro Glu Pro Glu Gly Gly Gly Glu Glu
500 505 510
Lys Val Val Gly Met Glu Glu Ser Gln Ala Ala Pro Lys Glu Glu
515 520 525
Pro Gly Pro Arg Pro Arg Ile Asn Leu Arg Gly Val Met Arg Ser
530 535 540
Ile Ser Leu Leu Glu Pro Ser Leu Glu Leu Glu Ser Thr Ser Glu
545 550 555
Thr Ser Asp Met Pro Glu Val Phe Ser Ser His Glu Ser Ser His
560 565 570
Glu Glu Pro Leu Gln Pro Phe Pro Gln Leu Ala Arg Thr Lys Gly
575 580 585
Gly Gln Gln Val Asp Arg Gly Pro Gln Pro Ala Leu Lys Ser Arg
590 595 600
Gln Ser Val Val Thr Leu Gln Gly Ser Ala Val Val Ala Asn Arg
605 610 615
Thr Gln Ala Phe Gln Glu Gln Glu Gln Gly Gln~Gly Gln Gly Gln
620 625 630
Gly Glu Pro Cys Ile Ser Ser Thr Pro Arg Phe Arg Lys Val Val
635 640 645
Arg Gln Ala Ser Val His Asp Ser Gly Glu Glu Gly Glu Ala
650 655
11/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
<210> 11
<211> 145
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 5595281CD1
<400> 11
Met Leu Ser Ser Ser Pro Ala Ser Cys Thr Ser Pro Ser Pro Asp
1 5 10 15
Gly Glu Asn Pro Cys Lys Lys Val His Trp Ala Ser Gly Arg Arg
20 25 30
Arg Thr Ser Ser Thr Asp Ser Glu Ser Lys Ser His Pro Asp Ser
35 40 45
Ser Lys Ile Pro Arg Ser Arg Arg Pro Ser Arg Leu Thr Val Lys
50 55 60
Tyr Asp Arg Gly Gln Leu Gln Arg Trp Leu Glu Met Glu Gln Trp
65 70 75
Val Asp Ala Gln Val Gln Glu Leu Phe Gln Asp Gln Ala Thr Pro
80 85 90
Ser Glu Pro Glu Ile Asp Leu Glu Ala Leu Met Asp Leu Ser Thr
95 100 105
Glu Glu Gln Lys Thr Gln Leu Glu Ala Ile Leu Gly Asn Cys Pro
110 115 120
Arg Pro Thr Glu Ala Phe Ile Ser Glu Leu Leu Ser Gln Leu Lys
125 130 135
Lys Leu Arg Arg Leu Ser Arg Pro Gln Lys
140 145
<210> 12
<211> 1884
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 365665CB1
<400> 12
ctgcactacc acagaggaag gaaatcagcc tggaaacatg ctaaactgca gccagaactc 60
cagctcatcc tcagtgtggt ggctgaaatc acctgcattt tccagtggtt cttctgaggg 120
ggacagccct tggtcctatc tgaattccag tgggagttct tgggtttcat tgccgggaaa 180
gatgaggaaa gagatccttg aggctcgcac cttgcaacct gatgactttg aaaagctgtt 240
ggcaggagtg aggcatgatt ggctgtttca gagactagag aatacggggg tttttaagcc 300
cagtcaactc caccgagcac atagtgctct tttgttaaaa tattcaaaaa aatctgaact 360
gtggacggcc caggaaacta ttgtctattt gggggactac ttgactgtga agaaaaaagg.420
cagacaaaga aatgcttttt gggttcatca tcttcatcaa gaagaaattc tggggaggta 480
tgttgggaaa gactataagg agcagaaggg gctctggcac cacttcactg atgtggagcg 540
acagatgacc gcacagcact atgtgacaga atttaacaag agactctatg aacaaaacat 600
tcccacccag atattctaca tcccatccac aatactactg attttagagg acaagacaat 660
aaagggatgt atcagtgtgg agccttacat actgggagaa tttgtaaaat tgtcaaataa 720
cacgaaagtg gtgaaaacag aatacaaagc cacagaatat ggcttggcct atggccattt 780
ttcttatgag ttttctaatc atagagatgt tgtggtcgat ttacaaggtt gggtaaccgg 840
taatggaaaa ggactcatct acctcacaga tccccagatt cactccgttg atcagaaagt 900
tttcactacc aattttggaa agagaggaat tttttacttc tttaataacc agcatgtgga 960
atgtaatgaa atctgccatc gtctttcttt gactagacct tcaatggaga aaccatgtaa 1020
12/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
gtcataggct gtatggattg gtaatgtgac agaccttagt gatgctctca gatatctggt 1080
gatgctcgca aatatctggt tctctccttc caggcacata gaatacggca cagtctggtc 1140
ctttggggct tgggcagggc cgtgacacag gttctggcca atgatttgca agaggaattg 1200
atcagtatca ctttaagtcc tgcatttaat tggcagcaca agatcctgca gagcctcttt 1260
ccctctgcca cagttatcaa gaatgggtca ggagaccgct gcttctgggc ataagtcctg 1320
caaggaaagc aacatggaaa acagccccaa ctcacccatg agggatgaaa agcactcttg 1380
agaaaggcat gtgttgttta agccattgag attttagagc tttttgtcac tatctgtcaa 1440
gactgatact actggggctt ttcctattga tttgggagtt ctttacatat taaaaaaatg 1500
tgagcctttg tgatacgaat tcaatttgtt ttcctgtctt ttgacatttg actttgcata 1560
aaagtttatc tgtgcataat tttatatgta gttgaattca tcaatctttt attttgtatg 1620
gctttttggt tatgtataat acttagatcc tccttatact ctgagtttct ttctttttaa 1680
ttctcctgta tttccttcta gtataattaa atctgtaaaa agtaagatgg aagagtggta 1740
cagttttctt tatccagtct gtccttgatg ggcatttagg tagactggat aaagaaaatg 1800
tgatacatat acaccatgga acactatgtg tattaatcca ctctcacact gctatgaaga 1860
gatacctgag actgggtaat ttag 1884
<210> 13
<211> 784
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 760934CB1
<400> 13
cggacggtgg gcggacgcgt gggcggacgc gtgggcggac gcgtgggctc agcccctccc 60
atctcagttg tgttcacaac aggtctgtgt gctggtgcct gtgttgttgg gagtcttcat 120
tcacagagga ccccaagacc ggactcggtg ccatttctct ccttctggcc cgttgctggc 180
ccttatgcat tacttgcctt tttctcagct ctgtctggac tcatgaggtc ctgggggcag 240
aatcagtctg tgtcccctga atgctgagtg ccctctaggt gcttaaatgt tgttcacttt 300
tcacagggaa agtcttcaga tggcagtagg cccattcctc cacatcctag agagcaacct 360
gctgaaagcc atggactctg ccactgcccc cgacaagatc agaaagctgt atctctatgc 420
ggctcatgat gtgaccttca taccgctctt aatgaccctg gggatttttg accacaaatg 480
gccaccgttt gctgttgacc tgaccatgga actttaccag cacctggaat ctaaggagtg 540
gtttgtgcag ctctattacc acgggaagga gcaggtgccg agaggttgcc ctgatgggct 600
ctgcccgctg gacatgttct tgaatgccat gtcagtttat accttaagcc cagaaaaata 660
ccacgcactc tgctctcaaa ctcaggtgat ggaagttgga aatgaagagt aactgattta 720
taaaagcagg atgtgttgat tttaaaataa agtgccttta tacaatgaaa aaaaaaaaaa 780
aaaa 784
<210> 14
<211> 1657
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 926043CB1
<400> 14
ccagagcacc gggcacggcc ttcaatgggc gaggacacgg acacgcggaa aattaaccac 60
agcttcctgc gggaccacag ctatgtgact gaagctgaca tcttctctac cgttgagttc 120
aaccacacgg gagagctgct ggccacaggt gacaagggcg gccgggtcgt catcttccag 180
cgggaaccag agagtaaaaa tgcgccccac agccagggcg aatacgacgt gtacagcact 240
ttccagagcc acgagccgga gtttgactat ctcaagagcc tggagataga ggagaagatc 300
aacaagatca agtggctccc acagcagaac gccgcccact cactcctgtc caccaacgat 360
aaaactatca aattatggaa gattaccgaa cgagataaaa ggcccgaagg atacaacctg 420
aaggatgaag aggggaaact taaggacctg tccacggtga cgtcactgca ggtgccagtg 480
ctgaagccca tggatctgat ggtggaggtg agccctcgga ggatctttgc caatggccac 540
acctaccaca tcaactccat ctccgtcaac agtgactgcg agacctacat gtcggcggat 600
13/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
gacctgcgca tcaacctctg gcacctggcc atcaccgaca ggagcttcaa catcgtggac 660
atcaagccgg ccaacatgga ggaccttacg gaggtgatca cagcatctga gttccatccg 720
caccactgca acctcttcgt ctacagcagc agcaagggct ccctgcggct ctgcgacatg 780
cgggcagctg ccctgtgtga caagcattcc aagctctttg aagagcctga ggaccccagt 840
aaccgctcat tcttctcgga aatcatctcc tccgtgtccg acgtgaagtt cagccacagc 900
ggccgctaca tgctcacccg ggactacctt acagtcaagg tctgggacct gaacatggag 960
gcaagaccca tagagaccta ccaggtccat gactaccttc ggagcaagct ctgttccctg 1020
tacgagaacg actgcatttt cgacaagttt gaatgtgcct ggaacgggag cgacagcgtc 1080
atcatgaccg gggcctacaa caacttcttc cgcatgttcg atcggaacac caagcgggac 1140
gtgaccctgg aggcctcgag ggaaagcagc aagccccggg ctgtgctcaa gccacggcgc 1200
gtgtgcgtgg ggggcaagcg ccggcgtgat gacatcagtg tggacagctt ggacttcacc 1260
aagaagatcc tgcacacggc ctggcacccg gctgagaaca tcattgccat cgccgccacc 1320
aacaacctgt acatcttcca ggacaaggta aactctgaca tgcactaggt atgtgcagtt 1380
cccggcccct gccacccagc ctcatgcaag tcatccccga catgaccttc acgaccgcaa 1440
tgcaaggagg ggaagaaagt cacagcactg atgaggacag ctgcagaggt ggcagtgtgt 1500
ggacacagga agtttgggcc ccctccctgc cccagctttc ctaggccaga attgtgtttg 1560
gcagtaattg tctgtttaaa aaaataaaaa ggagaggaag cgttcaccgc cgcaaatcat 1620
aaaatggaca tgactgtgga gtcttacagt tcagggt 1657
<210> 15
<211> 2118
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1234795CB1
<400> 15
gggaaaagag gacttattgt tgtcatggcc catgagatga ttggaactca aattgttact 60
gagaggttgg tggctctgct ggaaagtgga acggaaaaag tgctgctaat tgatagccgg 120
ccatttgtgg aatacaatac atcccacatt ttggaagcca ttaatatcaa ctgctccaag 180
cttatgaagc gaaggttgca acaggacaaa gtgttaatta cagagctcat ccagcattca 240
gcgaaacata aggttgacat tgattgcagt cagaaggttg tagtttacga tcaaagctcc 300
caagatgttg cctctctctc ttcagactgt tttctcactg tacttctggg taaactggag 360
aagagcttca actctgttca cctgcttgca ggtgggtttg ctgagttctc tcgttgtttc 420
cctggcctct gtgaaggaaa atccactcta gtccctacct gcatttctca gccttgctta 480
cctgttgcca acattgggcc aacccgaatt cttcccaatc tttatcttgg ctgccagcga 540
gatgtcctca acaaggagct gatgcagcag aatgggattg gttatgtgtt aaatgccagc 600
aatacctgtc caaagcctga ctttatcccc gagtctcatt tcctgcgtgt gcctgtgaat 660
gacagctttt gtgagaaaat tttgccgtgg ttggacaaat cagtagattt cattgagaaa 720
gcaaaagcct ccaatggatg tgttctagtg cactgtttag ctgggatctc ccgctccgcc 780
accatcgcta tcgcctacat catgaagagg atggacatgt ctttagatga agcttacaga 840
tttgtgaaag aaaaaagacc tactatatct ccaaacttca attttctggg ccaactcctg 900
gactatgaga agaagattaa gaaccagact ggagcatcag ggccaaagag caaactcaag 960
ctgctgcacc tggagaagcc aaatgaacct gtccctgctg tctcagaggg tggacagaaa 1020
agcgagacgc ccctcagtcc accctgtgcc gactctgcta cctcagaggc agcaggacaa 1080
aggcccgtgc atcccgccag cgtgcccagc gtgcccagcg tgcagccgtc gctgttagag 1140
gacagcccgc tggtacaggc gctcagtggg ctgcacctgt ccgcagacag gctggaagac 1200
agcaataagc tcaagcgttc cttctctctg gatatcaaat cagtttcata ttcagccagc 1260
atggcagcat ccttacatgg cttctcctca tcagaagatg ctttggaata ctacaaacct 1320
tccactactc tggatgggac caacaagcta tgccagttct cccctgttca ggaactatcg 1380
gagcagactc ccgaaaccag ttccctgata aggaggaagc cagcatcccc aagaagctgc 1440
agaccgccag gccttcagac agccagagca agcgattgca ttcggtcaga accagcagca 1500
gtggcaccgc ccagaggtcc cttttatctc cactgcatcg aagtgggagc gtggaggaca 1560
attaccacac cagcttcctt ttcggccttt ccaccagcca gcagcacctc acgaagtctg 1620
ctggcctggg cccttaaggg ctggcactcg gatatcttgg ccccccagac ctctacccct 1680
tccctgacca gcagctggta ttttgccaca gagtcctcac acttctactc tgcctcagcc 1740
atctacggag gcagtgccag ttactctgcc tacagctgca gccagctgcc cacttgcgga 1800
gaccaagtct attctgtgcg caggcggcag aagccaagtg acagagctga ctcgcggcgg 1860
agctggcatg aagagagccc ctttgaaaag cagtttaaac gcagaagctg ccaaatggaa 1920
tttggagaga gcatcatgtc agagaacagg tcacgggaag agctggggaa agtgggcagt 1980
14/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
cagtctagct tttcgggcag catggaaatc attgaggtct cctgagaaga aagacacttg 2040
tgacttctat agacaatttt ttttttcttg ttcacaaaaa aattccctgt aaatctgaaa 2100
tatatatatg tacatacc 2118
<210> 16
<211> 2116
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1271505CB1
<400> 16
ggaggcggct ccgcgcgcgt ccgctgctag gacccgggca gggctggagc tgggctggga 60
tcccgagctc ggcagcagcg cacgggccgg cccacctgct ggtgccctgg aggctctgag 120
ccccggcggc gcccgggccc acgcggaacg acggggcgag atgcgagcca cccctctggc 180
tgctcctgcg ggttccctgt ccaggaagaa gcggttggag ttggatgaca acttagatac 240
cgagcgtccc gtccagaaac gagctcgaag tgggccccag cccagactgc ccccctgcct 300
gttgcccctg agcccaccta ctgctccaga tcgtgcaact gctgtggcca ctgcctcccg 360
tcttgggccc tatgtcctcc tggagcccga ggagggcggg cgggcctacc aggccctgca 420
ctgccctaca ggcactgagt atacctgcaa ggtgtacccc gtccaggaag ccctggccgt 480
gctggagccc tacgcgcggc tgcccccgca caagcatgtg gctcggccca ctgaggtcct 540
ggctggtacc cagctcctct acgccttttt cactcggacc catggggaca tgcacagcct 600
ggtgcgaacg cgccaccgta tccctgagcc tgaggctgcc gtgctcttcc gccagatggc 660
caccgccctg gcgcactgtc accagcacgg tctggtcctg cgtgatctca agctgtgtcg 720
ctttgtcttc gctgaccgtg agaggaagaa gctggtgctg gagaacctgg aggactcctg 780
cgtgctgact gggccagatg attccctgtg ggacaagcac gcgtgcccag cctacgtggg 840
acctgagata ctcagctcac gggcctcata ctcgggcaag gcagccgatg tctggagcct 900
gggcgtggcg Ctcttcacca tgctggccgg ccactacccc ttccaggact cggagcctgt 960
cctgctcttc ggcaagatcc gccgcggggc ctacgccttg cctgcaggcc tctcggcccc 1020
tgcccgctgt ctggttcgct gcctccttcg tcgggagcca gctgaacggc tcacagccac 1080
aggcatcctc ctgcacccct ggctgcgaca ggacccgatg cccttagccc caacccgatc 1140
ccatctctgg gaggctgccc aggtggtccc tgatggactg gggctggacg aagccaggga 1200
agaggaggga gacagagaag tggttctgta tggctaggac caccctacta cacgctcagc 1260
tgccaacagt ggattgagtt tgggggtagc tccaagcctt ctcctgcctc tgaactgagc 1320
caaaccttca gtgccttcca gaagggagaa aggcagaagc ctgtgtggag tgtgctgtgt 1380
acacatctgc tttgttccac acacatgcag ttcctgcttg ggtgcttatc aggtgccaag 1440
ccctgttctc ggtgctggga gtacagcagt gagcaaagga gacaatattc cctgctcaca 1500
gagatgacaa actggcatcc ttgagctgac aacacttttc catgaccata ggtcactgtc 1560
tacactgggt acactttgta ccagtgtcgg cctccactga tgctggtgct caggcacctc 1620
tgtccaagga caatcccttt cacaaacaaa ccagctgcct ttgtatcttg taccttttca 1680
gagaaaggga ggtatccctg tgccaaaggc tccaggcctc tcccctgcaa ctcaggaccc 1740
aagcccagct cactctggga actgtgttcc cagcatctct gtcctcttga ttaagagatt 1800
ctccttccag gcctaagcct gggatttggg ccagagataa gaatccaaac tatgaggcta 1860
gttcttgtct aactcaagac tgttctggaa tgagggtcca ggcctgtcaa ccatggggct 1920
tctgacctga gcaccaaggt tgagggacag gattaggcag ggtctgtcct gtggccacct 1980
ggaaagtccc aggtgggact cttctgggga cacttggggt ccacaatccc aggtccatac 2040
tctaggtttt ggataccatg agtatgtatg tttacctgtg cctaataaag gagaattatg 2100
aaataaaaaa aaaaaa 2116
<210> 17
<211> 2897
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1385073CB1
<400> 17
15/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
ctagccgaag cggctgcatc tggcgccgcg tctgccccgc gtgctcggag cggattctgc 60
ccgccgtccc cggagccctc ggcgccccgc tgagcccgcg,atcacttcct ccctgtgacc 120
aaccggcgct gcaggttaga gcctggcaat gccgtttggg tgtgtgactc tgggcgacaa 180
gaagaactat aaccagccat cggaggtgac tgacagatat gatttgggac aggtcatcaa 240
gactgaggag ttttgtgaaa tcttccgggc caaggacaag acgacaggca agctgcacac 300
ctgcaagaag ttccagaagc gggacggccg caaggtgcgg aaagctgcca agaacgagat 360
aggcatcctc aagatggtga agcatcccaa catcctacag ctggtggatg tgtttgtgac 420
ccgcaaggag tactttatct tcctggagct ggccacgggg agggaggtgt ttgactggat 480
cctggaccag ggctactact cggagcgaga cacaagcaac gtggtacggc aagtcctgga 540
ggccgtggcc tatttgcact cactcaagat cgtgcacagg aatctcaagc tggagaacct 600
ggtttactac aaccggctga agaactcgaa gattgtcatc agtgacttcc atctggctaa 660
gctagaaaat ggcctcatca aggagccctg tgggaccccc gagtatctgg ccccagaggt 720
ggtaggccgg cagcggtatg gacgccctgt ggactgctgg gccattggag tcatcatgta 780
catcctgctt tcaggcaacc cacctttcta tgaggaggtg gaagaagatg attatgagaa 840
ccatgataag aatctcttcc gcaagatcct ggctggtgac tatgagtttg actctccata 900
ttgggatgat atttcgcagg cagccaaaga cctggtcaca aggctgatgg aggtggagca 960
agaccagcgg atcactgcag aagaggccat ctcccatgag tggatttctg gcaatgctgc 1020
ttctgataag aacatcaagg atggtgtctg tgcccagatt gaaaagaact ttgccagggc 1080
caagtggaag aaggctgtcc gagtgaccac cctcatgaaa cggctccggg caccagagca 1140
gtccagcacg gctgcagccc agtcggcctc agccacagac actgccaccc ccggggctgc 1200
agaccgtagt gccaccccag ccacagatgg aagtgccacc ccagccactg atggcagtgt 1260
caccccagcc accgatggaa gcatcactcc agccactgat gggagtgtca ccccagccac 1320
tgacaggagc gctactccag ccactgatgg gagagccaca ccagccacag aagagagcac 1380
tgtgcccacc acccaaagca gtgccatgct ggccaccaag gcagctgcca cccctgagcc 1440
ggctatggcc cagccggaca gcacagcccc agagggcgcc acaggccagg ctccaccctc 1500
tagtaaaggg gaagaggctg ctggttatgc ccaggagtct caaagggagg aggccagctg 1560
agtaggcagc ctggtgaggg ggggcagggg atgggcagga gggtgggaga gtggatgagg 1620
ggcttctcac tgtacataga gtcactggca tgatgccctc gctcccccat gcccccacat 1680
cccagtgggg cataactagg ggtcacggga gagcagtctc gtctcctgtg tgtatgtgtg 1740
tgagtggtgg gcaggccagt ggcagggccg gccccagccc ctgcatggat tccttgtggc 1800
ttttctgtct tttgctagct tcaccagttt ctgttccttg tgggatgctg ctctagggat 1860
actcaggggg ctcctgctct ccttcccctt cccttcttgc ctcaccattc ccctaggcag 1920
gccctgcagg tcccacactc tcccaggccc taaacttggg cggccttgcc ctgagagctg 1980
gtcctccagc gaggccctgt cagcggtctt aggctcctgc acatgaaggt gtgtgcctgt 2040
ggtgtgtggg ctgctctagg agcagataca ggctggtata gaggatgcag aaaggtaggg 2100
cagtatgttt aagtccagac ttggcacatg gctagggata ctgctcacta gctgtggagg 2160
tcctcaggag tggagagaat gagtaggagg gcagaagctt ccatttttgt ccttcctaag 2220
accctgttat ttgtgttatt tcctgccttt ccgagtcctg cagtgggctg ccctgtaccc 2280
tgaacctcat gagcctctaa gggaaaggag gaacaattag gacgtggcaa tgagacctgg 2340
cagggcagag tacaagccca gcacccagtg tcccagcctt actgggtcct taccctgggc 2400
caaacaggga gggctgatac ctccttgctc ttcctagatg cccacctcct acaatctcag 2460
cccacaagtc ctctccaccc tagggggctt gctgcatggc aataactcat aatctgattt 2520
ggaggtttgc cctttacagg ggcagatttt ctgctcagtt caacaatgaa atgaagagga 2580
actccctctt tctacagctc acttctatca gaggcccagg tgcctcagag ccacattgag 2640
ttgctttttc tgggatgagg aagtagggtt aaactcccca gtttcctgag ggaggctcct 2700
gacaggtgcc ctttgtcaga ccctaccaca gcctggatag gcagccacat tggtcctcgc 2760
ccttgctcgg cactccgtgg tggtcctgcc cttctccctg catgcctgtg ggtctgctct 2820
ggtgtgtgaa ggtcggtggg ttaactgtgt gcctactgaa cctggcaaat aaacatcacc 2880
ctgcaaagcc aaaaaaa 2897
<210> 18
<211> 839
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1606974CB1
<400> 18
gtggtgagtt acttggctcg gagcgggcga ggggacgcgt gggcggagcg gggctggcca 60
gcctcggccc ccatgacccg ctgtcctgtg ccctttccca gcgatgggcg tgcagccccc 120
16/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
caacttctcc tgggtgcttc cgggccggct ggcgggactg gcgctgccgc ggctccccgc 180
ccactaccag ttcctgttgg acctgggcgt gcggcacctg gtgtccctga cggagcgcgg 240
gccccctcac agcgacagct gccccggcct caccctgcac cgcctgcgca tccccgactt 300
ctgcccgccg gcccccgacc agatcgaccg cttcgtgcag atcgtggacg aggccaacgc 360
acggggagag gctgtgggag tgcactgtgc tctgggcttt ggccgcactg gcaccatgct 420
ggcctgttac ctggtgaagg agcggggctt ggctgcagga gatgccattg ctgaaatccg 480
acgactacga cccggctcca tcgagaccta tgagcaggag aaagcagtct tccagttcta 540
ccagcgaacg aaataagggg ccttagtacc cttctaccag gccctcactc cccttcccca 600
tgttgtcgat ggggccagag atgaagggaa gtggactaaa gtattaaacc ctctagctcc 660
cattggctga agacactgaa gtagcccacc cctgcaggca ggtcctgatt gaaggggagg 720
cttgtactgc tttgttgaat aaatgagttt tacgaaccaa aaaaaaaaaa aaaaaaggcg 780
gccgcaagct tattcccttt agtgagggtt aattttagct tggcactggc cgtcgtata 839
<210> 19
<211> 1081
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 1829744CB1
<400> 19
gggaacctca ggcttcagag agccgaaaag ttgggaggcg taaccactta caggccggaa 60
gtgtccgggg tggacgcatt cgggtagccg aagaagtccc aggattgccg aagaagtccc 120
aggatttccg aagcgagccg aagcatcgcg acagttttca gagacagctg atcggttgga 180
gctgttgcgc cgagcagtca tggcggcggc cagagctact acgccggccg atggcgagga 240
gcccgccccg gaggctgagg ctctggccgc agcccgggag cggagcagcc gcttcttgag 300
cggcctggag ctggtgaagc agggtgccga ggcgcgcgtg ttccgtggcc gcttccaggg 360
ccgcgcggcg gtgatcaagc accgcttccc caagggctac cggcacccgg cgctggaggc 420
gcggcttggc agacggcgga cggtgcagga ggcccgggcg ctcctccgct gtcgccgcgc 480
tggaatatct gccccagttg tcttttttgt ggactatgct tccaactgct tatatatgga 540
agaaattgaa ggctcagtga ctgttcgaga ttatattcag tccactatgg agactgaaaa 600
aactccccag ggtctctcca acttagccaa gacaattggg caggttttgg ctcgaatgca 660
cgatgaagac ctcattcatg gtgatctcac cacctccaac atgctcctga aaccccccct 720
ggaacagctg aacattgtgc tcatagactt tgggctgagt ttcatttcag cacttccaga 780
ggataaggga gtagacctct atgtcctgga gaaggccttc ctcagtaccc atcccaacac 840
tgaaactgtg tttgaagcct ttctgaagag ctactccacc tcctccaaaa aggccaggcc 900
agtgctaaaa aaattagatg aagtgcgcct gagaggaaga aagaggtcca tggttgggta 960
gaagaatgtg tatgacaacc acacacagtg aagctctttt ttcaaagtaa atttgaagaa 1020
atgctacaag tatgagatga gatctaagta aaggtgttaa gatattttta aaaaaaaaaa 1080
a 1081
<210> 20
<211> 2924
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID N0: 4030831CB1
<400> 20
ggcggggacc cggatgtgtg tggtggcggc ggccgaagag cttgtgtgcg gagctgagag 60
gcctatggat gaggaggacg cggcggcccc ggtttgttct catgaacaag atggatgacc 120
tcaacctgca ctaccggttt ctgaattggc gccggcggat ccgggagatt cgagaggtcc 180
gagctttccg atatcaggag aggttcaaac atatccttgt agatggagat actttaagtt 240
atcatggaaa ctctggtgaa gttggctgct acgtggcttc tcgacccctg accaaggaca 300
gcaattattt tgaggtgtct attgtggaca gtggagtccg gggcaccatt gctgtggggc 360
tggtccctca gtactacagc ttggatcacc agcctggctg gttgcctgac tctgtagcct 420
accatgctga tgatggcaag ctgtacaatg gccgagccaa gggccgccag tttgggtcaa 480
17/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
agtgcaactc cggggaccgg attggctgtg gcattgagcc tgtgtccttt gatgtgcaga 540
ccgcccagat cttcttcacc aaaaatggga agcgggtggg ctctaccatc atgcccatgt 600
ccccagatgg actgttccca gcagtgggca tgcactccct gggtgaggag gtgcggctgc 660
acctcaacgc tgagctgggc cgtgaggacg acagcgtcat gatggtggac agttacgagg 720
atgaatgggg ccggctacat gatgtcagag tctgtgggac tctgctggag tacttaggga 780
agggcaaaag catcgtggat gtggggctgg cccaggcccg gcacccactc agcacccgca 840
gccactactt cgaggtggag atcgtggacc ctggagagaa atgctacatc gccctggggc 900
tggcacggaa ggactatccc aagaacaggc accctggctg gagcagaggg tctgtggctt 960
atcatgcaga cgatgggaag atcttccatg gcagtggtgt gggggacccc tttgggccac 1020
gctgttacaa aggggacatc atgggctgtg gaatcatgtt cccccgggac tacattttgg 1080
acagtgaggg ggacagtgat gacagttgtg acacagtgat cctgtctccg actgcccggg 1140
ccgtccggaa cgtgcggaat gtcatgtacc tgcaccagga aggggaagag gaagaggagg 1200
aagaggaaga ggaagaggat ggggaagaga tagagccgga gcatgagggc aggaaggtgg 1260
tggttttctt cactcggaat ggcaagatca ttgggaagaa ggatgctgtt gttccttctg 1320
gaggcttctt ccccaccatt ggaatgctga gctgcgggga gaaagtcaaa gtagatctgc 1380
accccttgag tggctagggc ctcccctcca gacctgctcc ttctccctgc tcaccctctg 1440
ctgggccagg cacccagttc ctgacttccc agaggcttcg tttacccagc aggcccctgg 1500
aggtgtgtag tcactctgcc cccactggct caggcccctg tcacgcttct ctgtgcccac 1560
gtttctgacc tggtgctgcc actgttgtca gtccctgggc ctgagtccct ggttggacag 1620
gaatggaccc aaagaatggt gttggtatgt gggtggtccc actcgctttg gtcagtgggc 1680
ttctgggtcc ccctttccct caccggccct gtgtgggtgg agaggcgtga gcaccctatc 1740
tcagctgcta ttcgggcatg atgctttgta gagggtagag tagacagccc cctcccctac 1800
tcaccatggt atttctcctt gaattcctct ttcttgtttt ctttcctggt tgtgtgaacc 1860
agttgctgct gtcatacccc tggcagggcc aggggacctc tctttggtca tctctgtcct 1920
ttcactggct gctgccccag gaagactcct ctaggctctc catctttccc ttgagagctg 1980
gctccccacc ccaacctgct caggcaccac agaggatcta ggtctctggc tccccatacc 2040
tggacccaca tgggtgggtg cctgttgcat gtttaagaga gaggggctgt gaggtgacag 2100
ggcactaggg ccttcactcc tttctcccct tccatccttt ctttaccagt gccacccatg 2160
tccctagctc ccgggtattg gggctgaggc tctggggcct gtctccctgc cagcgtgagg 2220
gcaagacccc agagccttag ctgagcaagc ccagaggggc agcgtggccc ctccctcccc 2280
ttttcctgcc ccgtcccatg cctcagcttg ctgcttgtgc cagttgcctg tttcgcttca 2340
gtgtttgatt ctagcactta catgtgtcct ccccaccaag ccctctatct ccttctaatc 2400
cttcaacccc tggccccctc cccgtaacag tgacttttcc agggaggaag aggcagcagg 2460
agctgttggc cttggtttgc acagagcggg tagggctgta gggaaagcgg gtgagctgtt 2520
gtgctgctgg gcctcccttt ggccctcgct tcccacccta cgatgtatga aatgtatgta 2580
cagaccagag atgtttatac agccgataaa gatggagttt ccgtatttat cagtatggcc 2640
ggaaccagga gcctttctag tccactgggc taggaacagg actgctggat gggggcagcc 2700
gaaggcagct tgctcatggg gagatgtgga ccaatgttgg gccagggatg ggaatcatat 2760
gttccatggg cctggctaca ggcctgagca cagatacgtc ccctgggaga tgaggctttg 2820
accttcctgt gaataagtgt tgactccaat ttcggctaaa gtttatagaa attctttatt 2880
attagacaaa aatagactct cttttttccc ctaaaaaaaa aaaa 2924
<210> 21
<211> 2781
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 5039718CB1
<400> 21
cggctcgagg cgggtccagg actgtccgcc gggttgaggg aaggggccgt gcccggtgcc 60
agcccaggtg ctcgcggcct ggctccatgg ccctggtcac agtgagccgt tcgcccccgg 120
gcagcggcgc ctccacgccc gtggggccct gggaccaggc ggtccagcga aggagtcgac 180
tccagcgaag gcagagcttt gcggtgctcc gtggggctgt cctgggactg caggatggag 240
gggacaatga tgatgcagca gaggccagtt ctgagccaac agagaaggcc ccgagtgagg 300
aggagctcca cggggaccag acagacttcg ggcaaggatc ccagagtccc cagaagcagg 360
aggagcagag gcagcacctg cacctcatgg tacagctgct gaggccgcag gatgacatcc 420
gcctggcagc ccagctggag gcaccccggc ctccccggct ccgctacctg ctggtagttt 480
ctacacgaga aggagaaggt ctgagccagg atgagacggt cctcctgggc gtggatttcc 540
ctgacagcag ctcccccagc tgcaccctgg gcctggtctt gcccctctgg agtgacaccc 600
18/19
CA 02383927 2002-03-05
WO 01/20004 PCT/US00/25515
aggtgtactt agatggagac gggggcttca gcgtgacgtc tggtgggcaa agccggatct 660
tcaagcccat ctccatccag accatgtggg ccacactcca ggtattgcac caagcatgtg 720
aggcagctct aggcagcggc cttgtaccgg gtggcagtgc cctcacctgg gccagccact 780
accaggagag actgaactcc gaacagagct gcctcaatga gtggacggct atggccgacc 840
tggagtctct gcggcctccc agtgccgagc ctggcgggtc ctcagaacag gagcagatgg 900
agcaggcgat ccgtgctgag ctgtggaaag tgttggatgt cagtgacctg gagagtgtca 960
cttccaaaga gatccgccag gctctggagc tgcgcctggg gctccccctc cagcagtacc 1020
gtgacttcat cgacaaccag atgctgctgc tggtggcaca gcgggaccga gcctcccgca 1080
tcttccccca cctctacctg ggctcagagt ggaacgcagc aaacctggag gagctgcaga 1140
ggaacagggt cacccacatc ttgaacatgg cccgggagat tgacaacttc taccctgagc 1200
gcttcaccta ccacaatgtg cgcctctggg atgaggagtc ggcccagctg ctgccgcact 1260
ggaaggagac gcaccgcttc attgaggctg caagagcaca gggcacccac gtgctggtcc 1320
actgcaagat gggcgtcagc cgctcagcgg ccacagtgct ggcctatgcc atgaagcagt 1380
acgaatgcag cctggagcag gccctgcgcc acgtgcagga gctccggccc atcgcccgcc 1440
ccaaccctgg cttcctgcgc cagctgcaga tctaccaggg catcctgacg gccagccgcc 1500
agagccatgt ctgggagcag aaagtgggtg gggtctcccc agaggagcac ccagcccctg 1560
aagtctctac accattccca cctcttccgc cagaacctga gggtggtggg gaggagaagg 1620
ttgtaggcat ggaagagagc caggcagccc cgaaagaaga gcctgggcca cggccacgta 1680
taaacctccg aggggtcatg aggtccatca gtcttctgga gccctccttg gagctggaga 1740
gcacctcaga gaccagtgac atgccagagg tcttctcttc ccacgagtct tcacatgaag 1800
agcctctgca gcccttccca cagcttgcaa ggaccaaggg aggccagcag gtggacaggg 1860
ggcctcagcc tgccctgaag tcccgccagt cagtggttac cctccagggc agtgccgtgg 1920
tggccaaccg gacccaggcc ttccaggagc aggagcaggg gcaggggcag gggcagggag 1980
agccctgcat ttcctctacg cccaggttcc ggaaggtggt gagacaggcc agcgtgcatg 2040
acagtggaga ggagggcgag gcctgagccc tcacacatgc ccacgctccc ctgacactga 2100
agaggatcca caactccttg gagaaacacc ctcacgtctg ttgccgcaca cattcctctc 2160
agctccgccc catacccgtc actacagcct cacctcccac ccctgtcact acggcctcac 2220
ctcccacccc tgtcactaca gcctcacctc ctacagcctt aagtcccagg cccatgtctg 2280
cctgtccaag ggctcaagac tttctaactg ggatgtggta gagggactga aggtaccttt 2340
gggggcaaca gcaccctagt ttcattctca actctagccc tgcacactca cctgtggcac 2400
ggaatgaaaa cagagcttcc cgtgcaaaaa gggtcacgcc tcccaccccc gccccctccc 2460
tgcacctcct gtcctctccc agttcattcc tggaaccagc caggccaggc aaccagtggc 2520
ccccaaaggc aggcaggatc ctcaggcccc agccgcggga ggctggaagg gctggcagat 2580
cgcttccctc atccacctcc accggtccag gtctttgctg ctgtccccag acctcctgtg 2640
acaccacgcc agatcacagg gcaccaggcc agagatagtc ttctttttgt cctttctggc 2700
ctctggctag tcagtttttc atagccttac agtatctggc tttgtactga gaaataaaac 2760
acattttcat aaaaaaaaaa a 2781
<210> 22
<211> 754
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID NO: 5595281CB1
<400> 22
gttctctgtc tcctcacttc cctctgtggc tctgctcaga actggcggtt tttcccagct 60
ccttgcccag accaatactt ccatgctgtc ttcaagccct gcttcctgca catctcccag 120
cccagatggg gagaacccat gtaagaaggt ccactgggct tctgggagga gaaggacatc 180
atccacagac tcagagtcca agtcccaccc ggactcctcc aagataccca ggtcccggag 240
acccagccgc ctgacagtga agtatgaccg gggccagctc cagcgctggc tggagatgga 300
gcaatgggtg gatgctcaag ttcaggagct cttccaggat caagcaaccc cttctgagcc 360
tgagattgac ctggaagctc tcatggatct atccacagag gagcagaaga ctcagctgga 420
ggccattctt gggaactgcc cccgccccac agaggctttt atctctgagc tgctcagtca 480
actcaagaaa ctccggagac tcagccggcc tcagaaataa gcctgagaga ccatctttag 540
cagcctcagc actgccaggc ctgccctgaa actccagatc ctggctaaga gggaaatagc 600
tccttgggac acaaacaaga aatgtggaca aggagggaca tttgcatact cctactgtct 660
gtgtggtcac agctagtttc tgtcagctgg gctctctggg agaaagctgg ctgttgtcca 720
atgccttcct tggcagccaa gtggataaaa cctt 754
19/19
