Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN CHAPERONE PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
chaperone proteins
and to the use of these sequences in the diagnosis, treatment, and prevention
of reproductive, eye,
neuromuscular, metabolic, and autoimmune/inflammatory disorders, infectious
diseases, and cell
proliferative disorders including cancer.
BACKGROUND OF THE INVENTION
Molecular chaperones are proteins that interact with many other cellular
proteins. Chaperones
are involved in normal cellular functions, such as the folding of newly
synthesized polypepddes and the
assembly of multisubunit protein structures, in the transport of proteins
across membranes, and in the
stabilization of proteins in inactive configurations. This may give chaperones
a role in cell signaling, as
they can limit the access of proteins to their signaling partners, and in any
regulatory process dependent
on oligomerization or complex protein rearrangements. Chaperones are also
involved in cellular
responses to stresses such as toxicity and heat shock, and are therefore
called heat-shock proteins
(Hsp).
Chaperones are found in many cellular compartments. In the mitochondria, for
example,
chaperones on both sides of the membrane are involved in importing most of the
nuclear encoded
proteins necessary for oxidative phosphorylation. Chaperones may be divided
into several classes
named for their approximate molecular weights, including Hsp90, Hsp70,Hsp 60,
Hsp40 (also called
DnaJ), and the small Hsps (having molecular masses between 20 kD and 30 kD).
Mitochondrial
chaperones show a high degree of similarity to molecular chaperones in
bacteria, and in general
chaperones are ubiquitous and highly conserved, liom bacteria to humans
(Martinus, R.D. et al. (1995)
FASEB J. 9(5):371-378).
Molecular chaperone genes are activated by a variety of stresses, including
glucose deprivation,
ethanol, and heavy metals as well as heat shock, all of which affect protein
folding and aggregation.
Activation may be expected in any disorder that results in temperature
elevation. Molecular chaperones
have been suggested to play a role in the development of autoimmune conditions
and have been
implicated in a variety of metabolic and developmental disorders as well as in
response to trauma. In
addition, because many of the proteins that carry out the major mitochondrial
function, oxidative
phosphorylation, must be imported from the cytoplasm, any disorder affecting
metabolism may involve
mitochondrial import translocases.
Under normal or nonstressed conditions, constitutively expressed Hsps
facilitate proper protein
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folding and maturation, promote protein translocation across membranes, and
regulate hormone
receptor and protein kinase activity (Hightower, L.E. et al. (1991) Cell
66:191-197), antigen
presentation, protein degradation in the lysosome, and uncoating of clathrin-
coated vesicles. Hsps are
located in all major cellular compartments and function as monomers,
multimers, or in complexes with
other cellular proteins, which may determine the rate and specificity of Hsp
action. Different roles have
been ascribed to different classes of Hsps. Hsp20 proteins seem to form
heterooligomers that can
protect other proteins against heat-induced denaturation and aggregation.
Hsp40, homologous to the
bacterial DnaJ protein, and Hsp70, homologous to bacterial DnaK, act in
concert to aid in protein
folding and assembly of higher order protein complexes. Hsp60, along with
HsplO, binds misfolded
proteins and gives them the opportunity to refold correctly (Alberts, B. et
al. (1994) Molecular Bioloev
of the Cell Garland Publishing Co., New York, NY p. 608).
Hsp70 is a dimeric and ubiquitous protein which binds its substrates in an
extended
conformation through hydrophobic interactions. Hsp70 binds to newly
synthesized proteins and is
required for protein transport. The strength and specificity of Hsp70's
interaction with its substrates is
modified by binding and hydrolysis of ATP. Hsp70 has low protein affinity in
its ATP-bound state,
and increased protein affinity after ATP is hydrolyzed to ADP (Burston, S.G.
and Clarke, A.R. (1995)
Essays Biochem. 29:125-136). DnaJ chaperones work in concert with Hsp70. In
particular, DnaJ
interacts with the ATPase domain of Hsp70. The defining characteristic of the
DnaJ chaperone family
is an N-terminal, approximately 70 amino acid signature called the J domain,
which is required for
interactions with Hsp70. The tripeptide HPD seems to be particularly important
for this interaction
(Kelley, W. (1999) Curr. Biol. (1999) 9:8305-308). DnaJ stimulates ATP
hydrolysis, increasing the
affinity of Hsp70 for its protein substrate. GrpE, another co-chaperone,
promotes dissociation of ADP
from Hsp70, again modifying the Hsp70/substrate interaction and completing the
cycle (Burston and
Clarke, supra). Many eukaryotic DnaJ homologs have recently been described.
Growing evidence
suggests that specific DnaJ homologs interact with specific Hsp70 homologs to
form a chaperone
complex with affinity for specific substrates (Kelley, W. (1998) Trends in
Biochem. Sci. 23:222-227).
The DnaJ homolog Hsp40 was shown to co-localize with Hsp70 in the nuclei and
nucleoli of heat-
shocked HeLa cells (Ohtsuka, K. (1993) Biochem. Biophys. Res. Commun. 197:235-
240). Homologs
of GrpE have been identified in bovine, porcine, and rat tissues (Naylor, D.J.
et al. (1995) Biochim.
Biophys. Acta 1248:75-79).
The induction of heat shock proteins (Hsps), is a physiological and
biochemical response to
abrupt increases in temperature or exposure to a variety of other metabolic
insults including heavy
metals, amino acid analogs, toxins, and oxidative stress. This response occurs
in all prokaryotic and
eukaryotic cells and is characterized by repression of normal protein
synthesis and initiation of
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transcription of Hsp-encoding genes (Hightower, supra). During cellular
stress, Hsps form a complex
with proteins that misfold or unfold, either "rescuing" these proteins from
irreversible damage or
increasing their susceptibility to proteolytic attack. Overexpression of Hsps
in transgenic mice and rats,
or heat treatment of normal animals to induce Hsps, protects the heart muscle
from ischemic injury.
Both heat shock-induced and exogenous Hsps protect smooth muscle cells from
serum
deprivation-induced cell death. Overexpression of Hsps also protects murine
fibroblasts from both UV
light injury and proinflammatory cytokines released during UV exposure
(Marber, M.S. et al. (1995) J.
Clin. Invest. 95:1446-1456; Simon, M.M. et al. (1995) J. Clin. Invest. 95:926-
933). Specific Hsps
bind immunosuppressive drugs and may play a role in modulation of immune
responses. Hsps
expressed in cancer cells can protect the cancer cells from the cytotoxic
effects of drugs used in
anticancer therapies. Purified Hsps isolated from tumor cells and used as
antigens have been shown to
provide immunity to the tumors from which they are isolated (Udono, H. et al.
(1994) J. Immunol.
152:5398-5403; Young R.A. (1990) Annu. Rev. Immunol. 8:401-420; Marber, M.S.
et al. (1995) J.
Clin. Invest. 95:1446-1456; Simon, M.M. et al. (1995) J. Clin. Invest. 95:926-
933).
Chaperones are useful as markers of environmental stress and disease, and are
associated with
a variety of diseases and immune and drug responses. Several of the
constitutive Hsp genes are located
in the major histocompatibility complex on chromosome 6. Members of the Hsp
family have also been
shown to play roles in T-cell mediated regulation of inflammation and immune
recognition. For
example, Hsp90 binds to steroid hormone receptors, represses transcription in
the absence of the ligand,
and provides the proper folding of the ligand-binding domain in the presence
of the hormone (Burston
and Clarke, su ra). Heat shock treatment of B-cells enhances processing of
antigen and the assembly
and function of MHC class II molecules (Sargent, C.A. et al. (1989) Proc.
Natl. Acad. Sci. USA
86:1968-1972; Fang, Y. et al. (1996) J. Biol. Chem. 271:28697-28702; Hendrick,
J.P. et al. (1993)
Proc. Natl. Acad. Sci. USA 90:10216-10220). Abnormal transcription of Hsp70
has been associated
with major depression (Shimizu, S. et al. (1996) Biochem. Biophys. Res.
Commun. 219:745-752).
Hsp70 expression increases in response to tobacco smoke (Vayssier, M. et al.
(1998) Biochem.
Biophys. Res. Commun. 252:249-256). Hsp70 is involved in drug resistance in
breast cancer patients
treated with combination chemotherapies (Vargas-Roig, L.M. et al. (1998) Int.
J. Cancer 79:468-475).
Hsp70 variants are associated with clozapine-induced agranulocytosis, an
adverse drug reaction
(Turbay, D. et al. (1997) Blood 89:4167-4174). Knockout mice have provided
additional information
on the roles of Hsp70 in reproduction. For example, female homozygous knockout
mice for Hsp70 are
found to undergo normal meiosis and are fertile. In contrast, the homozygous
male knockout mice lack
postmeiotic spermatids and mature sperm, and are infertile (Dix, D.J. et al.
(1996) Proc. Natl. Acad.
Sci. U.S.A. 93:3264-3268). A DnaJ (Hsp40) homolog is essential for normal
placental development in
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mice (Hunter, P. et al. (1999) Development 126:1247-1258). Other DnaJ homologs
have been
implicated in viral DNA replication, secretion, tumor suppression, microtubule
formation in M phase,
and influenza virus infection (Kelley, 1998, supra).
The small Hsps are able to supress aggregation and heat inactivation of
various proteins,
including actin (Hickey, E. et al. (1986) Nucleic Acids Res. 14:4127-4145;
Miron, T. et al. (1991) J.
Cell Biol. 114:255-261 ). a-Crystallin, a protein abundant in the lens of the
eye, is an oligomer of two
subunits, aA and aB, which are 55% identical and belong to the small Hsp
family. a-Crystallin is
thought to be important for maintaining the transparency of the lens by
preventing denaturation and
aggregation of proteins. A missense mutation in the aA-crystallin gene is
associated with autosomal
dominant congenital cataracts (Litt, M. et al. (1998) Hum. Molec. Genet. 7:471-
474). However, the
functional role of a-crystallin is not confined to the eye. A missense
mutation of aB-crystallin has been
shown to cause a desmin-related myopathy (Vicart, P. et al. (1998) Nat. Genet.
20:92-95). Desmin-
related myopathies are inherited neuromuscular disorders.
The discovery of new human chaperone 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 reproductive, eye, neuromuscular, metabolic, and
autoimmuneJinflammatory disorders,
infectious diseases, and cell proliferative disorders including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human chaperone proteins,
referred to collectively
as "HCPN" and individually as "HCPN-1," "HCPN-2," "HCPN-3," "HCPN-4," "HCPN-
5," "HCPN-
6," "HCPN-7," "HCPN-8," "HCPN-9," "HCPN-10," and "HCPN-11." In one aspect, the
invention
provides an isolated 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:l-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:1-11.
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:1-11, 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
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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 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:l-11, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:l-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 polypepdde comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:l-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) culturing a cell under
conditions suitable for
expression of the polypeptide, wherein said cell is transformed with a
recombinant polynucleotide
comprising a promoter sequence operably linked to a polynucleotide encoding
the polypeptide, and b)
recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide 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:l-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 70% sequence
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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 70% 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 70% sequence
identity to a polynucleodde sequence selected from the group consisting of SEQ
ID N0:12-22, c) a
polynucleotide sequence complementary to a), d) a polynucleodde sequence
complementary to b), and e)
an RNA equivalent of a)-d). The method comprises a) amplifying said target
polynucleotide or
fragment thereof using polymerase chain reaction amplification, and b)
detecting the presence or
absence of said amplified target polynucleotide or fragment thereof, and,
optionally, if present, the
amount thereof.
The invention further provides a composition comprising an effective amount of
a polypepdde
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
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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 HCPN, comprising
adnunistering to a patient in need
of such treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO: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:l-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 HCPN, 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:l-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
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 HCPN, 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:l-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
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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:l-11,
b) a naturally
occurring amino acid sequence having at least 90°lo 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 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;
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
70% sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:12-
22, iii) a
polynucleodde 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 SEQ ID N0:12-22, ii) a naturally occurring polynucleotide
sequence having at least
70~'o sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
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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 polynucleodde
comprises a fragment of the above polynucleotide sequence; 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 HCPN.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of HCPN.
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 HCPN 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.
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
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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
"HCPN" refers to the amino acid sequences of substantially purified HCPN
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
HCPN. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of HCPN either by
directly interacting with
HCPN or by acting on components of the biological pathway in which HCPN
participates.
An "allelic variant" is an alternative form of the gene encoding HCPN. 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 HCPN include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as HCPN or a
polypeptide with at least one functional characteristic of HCPN. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding HCPN, and improper or unexpected hybridization to
allelic variants, with a
locus other than the normal chromosomal locus for the polynucleotide sequence
encoding HCPN. 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 HCPN. 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 HCPN 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
CA 02374743 2002-O1-25
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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
HCPN. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of HCPN either by
directly interacting with HCPN or by acting on components of the biological
pathway in which HCPN
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments thereof,
such as Fab, F(ab')z, and Fv fragments, which are capable of binding an
epitopic determinant.
Antibodies that bind HCPN polypeptides can be prepared using intact
polypeptides or using fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used
to immunize an animal (e.g., a mouse; a rat, or a rabbit) can be derived from
the translation of RNA, or
synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers
that are chemically coupled to peptides include bovine serum albumin,
thyroglobulin, and keyhole
limpet hemocyanin (KLH). The coupled peptide is then used to immunize the
animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies which
bind specifically to antigenic determinants (particular regions or three-
dimensional structures on the
protein). An antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to
elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
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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 HCPN, 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-S'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising a
given amino acid sequence" refer broadly to any composition containing the
given polynucleodde or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding HCPN or fragments of
HCPN may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
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
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
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Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide. Chemical
modifications of a polynucleotide sequence can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains
at least one biological or immunological function of the natural molecule. A
derivative polypeptide is
one modified by glycosylation, pegylation, or any similar process that retains
at least one biological or
immunological function of the polypeptide from which it was derived.
A "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 HCPN or the polynucleotide encoding HCPN
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 nucleodde/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
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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:l-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.
"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.
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For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and ''diagonals saved"=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
"percent similarity" between
aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn,'' that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10
Word Size: I1
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example, as
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 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. 1t is
understood that changes in
CA 02374743 2002-O1-25
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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
polynucleodde 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. SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
example, over the length of a fragment taken from a larger, defined polypepdde
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.
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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 complementarily..
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 ~g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (T,a 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 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 p~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.
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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 HCPN
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
HCPN 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 HCPN. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of HCPN.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
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preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an HCPN may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemizadon, proteolytic cleavage, and other
modifications known in the
S art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by
cell type depending on the enzymatic milieu of HCPN.
"Probe" refers to nucleic acid sequences encoding HCPN, 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"d
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
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
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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 selecaion
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.
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
CA 02374743 2002-O1-25
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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 HCPN, or fragments thereof, or HCPN 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
polypepdde comprising the epitope
A, or the presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell type
or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods well
known in the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences
into a prokaryotic or eukaryotic host cell. The method for transformation is
selected based on the type
of host cell being transformed and may include, but is not limited to,
bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
"transformed" cells
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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
reference molecule.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypepddes 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 polypepdde sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
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WO 01/09178 PCT/US00/21313
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 chaperone proteins
(HCPN), the
polynucleotides encoding HCPN, and the use of these compositions for the
diagnosis, treatment, or
prevention of reproductive, eye, neuromuscular, metabolic, and
autoimmune/inflammatory disorders,
infectious diseases, and cell proliferative disorders including cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
HCPN. 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 HCPN 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 HCPN 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 protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding HCPN. The first column of Table
3 lists the nucleotide
SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are
useful, for example, in hybridization or amplification technologies to
identify SEQ ID 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 HCPN as a fraction of total tissues expressing HCPN.
Column 4 lists
diseases, disorders, or conditions associated with those tissues expressing
HCPN as a fraction of total
tissues expressing HCPN. Column 5 lists the vectors used to subclone each cDNA
library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
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from which cDNA clones encoding HCPN 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 N0:14 maps to chromosome 10 within the interval from 46.2 to 46.8
centiMorgans.
This interval also contains a gene with homology to the murine leukemia viral
(bmi-1) oncogene.
SEQ ID NO:15 maps to chromosome 16 within the interval from 50.8 to 56.2
cendMorgans. This
interval also contains a gene associated with neuronal ceroid lipofuscinosis,
also known as Batten
disease. SEQ ID N0:22 maps to chromosome 1 within the interval from 78.3 to
84.2 centiMorgans,
to chromosome 6 within the interval liom 91.8 to 96.1 centiMorgans, to
chromosome 10 within the
interval from 93.8 to 96.9 centiMorgans, and to chromosome 12 within the
interval from 13.8 to 24.6
centiMorgans. The interval on chromosome 1 from 78.3 to 84.2 centiMorgans also
contains genes
and/or ESTs associated with myopathy, hypoketotic hypoglycemia and
hyperthyroxinemia. The
interval on chromosome 6 from 91.8 to 96.1 centiMorgans also contains a gene
and/or EST associated
with maple syrup urine disease. The interval on chromosome 12 from 13.8 to
24.6 centiMorgans
also contains genes and/or ESTs associated with T cell antigen T4 deficiency,
neonatal
adrenoleukodystrophy, and Zellweger syndrome.
The invention also encompasses HCPN variants. A preferred HCPN 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 HCPN amino acid sequence, and which contains at least one
functional or structural
characteristic of HCPN.
The invention also encompasses polynucleotides which encode HCPN. 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 HCPN. The
polynucleotide 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
HCPN. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at least
about 85%, or even at least about 95% polynucleotide sequence identity to the
polynucleotide sequence
encoding HCPN. 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 70%, or alternatively at least about 85%, or even at least about
95% polynucleotide
sequence identity to a nucleic acid sequence selected from the group
consisting of SEQ ID N0:12-22.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or structural characteristic of HCPN.
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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 HCPN, 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 HCPN, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode HCPN and its variants are generally
capable of
hybridizing to the nucleotide sequence of the naturally occurring HCPN under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding HCPN 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 HCPN 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 HCPN
and
HCPN 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 HCPN or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:12-22 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 polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (PE
Biosystems,
Foster City CA), thermostable T7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
CA 02374743 2002-O1-25
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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 Dynanucs,
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 Bioloev, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular
Biolo~y and Biotechnolo~y, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding HCPN 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 digesdons and
ligadons may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National Biosciences,
Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a
GC content of about 50% or more, and to anneal to the template at temperatures
of about 68°C to
72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
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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 HCPN may be cloned in recombinant DNA molecules that direct expression
of HCPN, 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 HCPN.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter HCPN-encoding sequences for a variety of
purposes including, but not
limited to, modification of the cloning, processing, and/or expression of the
gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction sites,
alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of HCPN, 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
27
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding HCPN 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,
HCPN 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 431 A peptide synthesizer (PE Biosystems). Additionally, the
amino acid sequence of
HCPN, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, su ra, pp. 28-53.)
In order to express a biologically active HCPN, the nucleotide sequences
encoding HCPN 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 HCPN. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
HCPN. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding HCPN 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
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vectors containing sequences encoding HCPN 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 Bioloey, 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 HCPN. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, su ra; Ausubel, 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.
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 HCPN. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding HCPN can be
achieved using a
nmltifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1 plasmid
(Life Technologies). Ligation of sequences encoding HCPN 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.
29
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264:5503-5509.) When large quantities of HCPN are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of HCPN 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 HCPN. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomvces 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, supra.)
Plant systems may also be used for expression of HCPN. Transcription of
sequences encoding
HCPN 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, supra; Brogue, supra; and Winter, supra.) 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 Technoloav (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 HCPN
may be ligated~into an
adenovirus transcription/translation complex consisting of the late promoter
and tripartite leader
sequence. Insertion in a non-essential El or E3 region of the viral genome may
be used to obtain
infective virus which expresses HCPN in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino polymers,
or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of
HCPN in cell lines is preferred. For example, sequences encoding HCPN 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
CA 02374743 2002-O1-25
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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 iiisD, which
alter cellular requirements
for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech),13
glucuronidase and its substrate B-glucuronide, or luciferase and its substrate
luciferin may be used.
These markers can be used not only to identify transformants, but also to
quantify the amount of
transient or stable protein expression attributable to a specific vector
system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding HCPN is inserted within a marker gene sequence, transformed
cells containing
sequences encoding HCPN can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding HCPN 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 HCPN
and that express
HCPN 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 HCPN 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
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activated cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering epitopes on HCPN is preferred, but
a competitive binding
assay may be employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. IV; Coligan, J.E.
et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and
Wiley-Interscience, New
York NY; and Pound, J.D. (1998) 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 HCPN
include oligolabeling,
nick translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the
sequences encoding HCPN, 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 HCPN 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 HCPN may be designed to contain signal sequences
which direct
secretion of HCPN 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
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sequences encoding HCPN 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
HCPN protein containing a
heterologous moiety that can be recognized by a commercially available
antibody may facilitate the
screening of peptide libraries for inhibitors of HCPN 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 HCPN encoding sequence and the heterologous.protein
sequence, so that HCPN
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 HCPN 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.
HCPN of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to HCPN. At least one and up to a plurality of test
compounds may be screened
for specific binding to HCPN. 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
HCPN, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, Coligan, J.E. et al. (1991) Current Protocols
in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which HCPN
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 HCPN,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing HCPN or cell membrane fractions which contain HCPN are
then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either HCPN or the
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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
HCPN, either in
solution or affixed to a solid support, and detecting the binding of HCPN to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
HCPN of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of HCPN. Such compounds may include agonists,
antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under conditions
permissive for HCPN
activity, wherein HCPN is combined with at least one test compound, and the
activity of HCPN in the
presence of a test compound is compared with the activity of HCPN in the
absence of the test
compound. A change in the activity of HCPN in the presence of the test
compound is indicative of a
compound that modulates the activity of HCPN. Alternatively, a test compound
is combined with an
in vitro or cell-free system comprising HCPN under conditions suitable for
HCPN activity, and the
assay is performed. In either of these assays, a test compound which modulates
the activity of HCPN
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 HCPN or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding
region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) 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
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therapeutic or toxic agents.
Polynucleotides encoding HCPN 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, hematopoiedc lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding HCPN 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 HCPN 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 HCPN, e.g., by secreting HCPN 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 HCPN and human chaperone proteins. In addition, the
expression of HCPN is
closely associated with reproductive disorders and with cancerous,
proliferating, inflamed, and
hematopoietic/immune tissues. Therefore, HCPN appears to play a role in
reproductive, eye,
neuromuscular, metabolic, and autoimmune/inllammatory disorders, infectious
diseases, and cell
proliferative disorders including cancer. In the treatment of disorders
associated with increased HCPN
expression or activity, it is desirable to decrease the expression or activity
of HCPN. In the treatment
of disorders associated with decreased HCPN expression or activity, it is
desirable to increase the
expression or activity of HCPN.
Therefore, in one embodiment, HCPN 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 HCPN. Examples of such disorders include, but are not limited to,
a reproductive disorder
such as a disorder of prolactin production, infertility, including tubal
disease, ovulatory defects, and
endometriosis, a disruption of the estrous cycle, a disruption of the
menstrual cycle, polycystic ovary
syndrome, ovarian hyperstimuladon syndrome, an endometrial or ovarian tumor, a
uterine fibroid,
autoimmune disorders, an ectopic pregnancy, and teratogenesis, cancer of the
breast, fibrocystic
breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal
sperm physiology, cancer
of the testis, cancer of the prostate, benign prostatic hyperplasia,
prostatitis, Peyronie's disease,
impotence, carcinoma of the male breast, and gynecomastia; a disorder of the
eye such as
conjunctivitis, keratoconjunctivitis sicca, keratitis, episcleritis, iritis,
posterior uveitis, glaucoma,
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amaurosis fugax, ischemic optic neuropathy, optic neuritis, Leber's hereditary
optic neuropathy, toxic
optic neuropathy, vitreous detachment, retinal detachment, cataract, macular
degeneration, central
serous chorioretinopathy, retinitis pigmentosa, melanoma of the choroid,
retrobulbar tumor, and
chiasmal tumor; a neuromuscular disorder such as a desmin-related myopathy; a
metabolic disorder
such as Zellweger syndrome, maple syrup urine disease, adrenoleukodystropy,
carnitine
palmitoyltransferase deficiency, Addison's disease, cerebrotendinous
xanthomatosis, congenital
adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty
hepatocirrhosis,
fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma,
glycogen storage diseases,
hereditary fructose intolerance, hyperadrenalism, hypoadrenalism,
hyperparathyroidism,
hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia,
hypothyroidism,
hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal
storage diseases,
mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria;
an
autoimmune/inflammatory disorder such as inflammation, actinic keratosis,
acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, bursitis,
cholecystitis, cirrhosis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
paroxysmal nocturnal
hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome,
episodic lymphopenia with
lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis, pancreatitis,
polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis, primary
thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal,
parasitic, protozoal, and helminthic infections, trauma, and hematopoietic
cancer including
lymphoma, leukemia, and myeloma; a viral infection, such as those caused by
adenoviruses (acute
respiratory disease, pneumonia), arenaviruses (lymphocytic choriomeningitis),
bunyaviruses
(Hantavirus), coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses
(hepatitis),
herpesviruses (herpes simplex virus, varicella-zoster virus, Epstein-Barr
virus, cytomegalovirus),
flaviviruses (yellow fever), orthomyxoviruses (influenza), papillomaviruses
(cancer),
paramyxoviruses (measles, mumps), picornoviruses (rhinovirus, poliovirus,
coxsackie-virus),
polyomaviruses (BK virus, JC virus), poxviruses (smallpox), reovirus (Colorado
tick fever),
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retroviruses (human immunodeficiency virus, human T lymphotropic virus),
rhabdoviruses (rabies),
rotaviruses (gastroenteritis), and togaviruses (encephalitis, rubella); and a
cell proliferative disorder
such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinur-ia,
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 HCPN 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 HCPN including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
HCPN 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 HCPN including,
but not limited to, those
provided above.
In still another embodiment, an agonist which modulates the activity of HCPN
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of HCPN including, but not limited to, those listed above.
In a further embodiment, an antagonist of HCPN may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of HCPN.
Examples of such
disorders include, but are not limited to, those reproductive, eye,
neuromuscular, metabolic, and
autoimmune/inflammatory disorders, infectious diseases, and cell proliferative
disorders, including
cancer, described above. In one aspect, an antibody which specifically binds
HCPN 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 HCPN.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HCPN may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of HCPN 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
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lower dosages of each agent, thus reducing the potential for adverse side
effects.
An antagonist of HCPN may be produced using methods which are generally known
in the art.
In particular, purified HCPN may be used to produce antibodies or to screen
libraries of
pharmaceutical agents to identify those which specifically bind HCPN.
Antibodies to HCPN 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 HCPN or with any fragment or
oligopepdde 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 HCPN
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 HCPN 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 HCPN may be prepared using any technique which
provides for the
production of antibody molecules by continuous cell lines in culture. These
include, but are not limited
to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-
hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
HCPN-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
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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 HCPN may also be
generated. For
example, such fragments include, but are not limited to, F(ab~z 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
HCPN and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two
non-interfering HCPN 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 HCPN. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of HCPN-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The ICa determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple
HCPN epitopes, represents the average affinity, or avidity, of the antibodies
for HCPN. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular HCPN
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
HCPN-antibody complex
must withstand rigorous manipulations. Low-affinity antibody preparations with
I~ ranging from
about 106 to 10' L/mole are preferred for use in immunopurification and
similar procedures which
ultimately require dissociation of HCPN, 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
39
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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 HCPN-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, su ra, and
Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding HCPN, 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 HCPN.
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 HCPN.
(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, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding HCPN may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked
inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe
combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-
242)), (ii)
CA 02374743 2002-O1-25
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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-I 1399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and Tar
panosoma cruzi). In the
case where a genetic deficiency in HCPN expression or regulation causes
disease, the expression of
HCPN 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 HCPN
are treated by constructing mammalian expression vectors encoding HCPN and
introducing these
vectors by mechanical means into HCPN-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 HCPN 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). HCPN may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, sera)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding HCPN 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
41
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of these
standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to HCPN expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding HCPN under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in an
appropriate vector producing cell line (VPCL) that expresses an envelope gene
with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant") discloses a
method for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells
(e.g., CD4+ T-cells), and the
return of transduced cells to a patient are procedures well known to persons
skilled in the art of gene
therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et
al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al.
(1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding HCPN to cells which have one or more genetic
abnormalities with respect to
the expression of HCPN. The construction and packaging of adenovirus-based
vectors are well known
to those with ordinary skill in the art. Replication defective adenovirus
vectors have proven to be
versatile for importing genes encoding immunoregulatory proteins into intact
islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia (1997) Nature 18:389:239-
242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
42
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
polynucleotides encoding HCPN to target cells which have one or more genetic
abnormalities with
respect to the expression of HCPN. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing HCPN 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 HCPN 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. Biotech. 9:464-
469). During alphavirus
RNA replication, a subgenomic RNA is generated that normally encodes the viral
capsid proteins. This
subgenomic RNA replicates to higher levels than the full-length genomic RNA,
resulting in the
overproduction of capsid proteins relative to the viral proteins with
enzymatic activity (e.g., protease
and polymerase). Similarly, inserting the coding sequence for HCPN into the
alphavirus genome in
place of the capsid-coding region results in the production of a large number
of HCPN-coding RNAs
and the synthesis of high levels of HCPN 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
HCPN 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
43
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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. Can,
Molecular and Immunoloeic 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 HCPN.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleoddes using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA sequences
encoding HCPN. 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
44
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
within the backbone of the molecule. This concept is inherent in the
production of PNAs and can be
extended in all of these molecules by the inclusion of nontraditional bases
such as inosine, queosine, and
wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms
of adenine, cytidine,
guanine, thymine, and uridine which are not as easily recognized by endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding HCPN. 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 HCPN
expression or activity, a compound which specifically inhibits expression of
the polynucleotide
encoding HCPN may be therapeutically useful, and in the treament of disorders
associated with
decreased HCPN expression or activity, a compound which specifically promotes
expression of the
polynucleotide encoding HCPN 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 HCPN is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
HCPN 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 HCPN. 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 Schizosaccharomvces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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 Remin~ton's
Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may
consist of HCPN,
antibodies to HCPN, and mimetics, agonists, antagonists, or inhibitors of
HCPN.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, infra-
arterial, intramedullary, intrathecal,
intraventricular, pulmonary, transdermal, subcutaneous, 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
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CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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 HCPN or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the
macromolecule. Alternatively, HCPN 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 HCPN
or fragments thereof, antibodies of HCPN, and agonists, antagonists or
inhibitors of HCPN, which
ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may
be determined by
standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSO (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity
of the disease state, the general health of the subject, the age, weight, and
gender of the subject, time
and frequency of adminisVation, drug combination(s), reaction sensitivities,
and response to therapy.
Long-acting compositions may be administered every 3 to 4 days, every week, or
biweekly depending
on the half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total
dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
47
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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 HCPN may be used for
the
diagnosis of disorders characterized by expression of HCPN, or in assays to
monitor patients being
treated with HCPN or agonists, antagonists, or inhibitors of HCPN. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
for HCPN include methods which utilize the antibody and a label to detect HCPN
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 HCPN, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
HCPN expression. Normal
or standard values for HCPN expression are established by combining body
fluids or cell extracts taken
from normal mammalian subjects, for example, human subjects, with antibody to
HCPN under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of HCPN
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 HCPN may
be used for
diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect and
quantify gene expression in biopsied tissues in which expression of HCPN may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
HCPN, and to monitor regulation of HCPN levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding HCPN or closely related
molecules may be used to
identify nucleic acid sequences which encode HCPN. 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 HCPN, allelic
variants, or related
sequences.
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Probes may also be used for the detection of related sequences, and may have
at least SO~Ir,
sequence identity to any of the HCPN 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 HCPN
gene.
Means for producing specific hybridization probes for DNAs encoding HCPN
include the
cloning of polynucleotide sequences encoding HCPN or HCPN derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety
of reporter groups, for example, by radionuclides such as 32P or 35S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding HCPN may be used for the diagnosis of
disorders
associated with expression of HCPN. Examples of such disorders include, but
are not limited to, a
reproductive disorder such as a disorder of prolactin production, infertility,
including tubal disease,
ovulatory defects, and endometriosis, a disruption of the estrous cycle, a
disruption of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an
endometrial or ovarian
tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and
teratogenesis, cancer of the
breast, fibrocystic breast disease, and galactorrhea, a disruption of
spermatogenesis, abnormal sperm
physiology, cancer of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis,
Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
a disorder of the eye
such as conjunctivitis, keratoconjunctivitis sicca, keratitis, episcleritis,
iritis, posterior uveitis,
glaucoma, amaurosis fugax, ischemic optic neuropathy, optic neuritis, Leber's
hereditary optic
neuropathy, toxic optic neuropathy, vitreous detachment, retinal detachment,
cataract, macular
degeneration, central serous chorioretinopathy, retinitis pigmentosa, melanoma
of the choroid,
retrobulbar tumor, and chiasmal tumor; a neuromuscular disorder such as a
desmin-related myopathy;
a metabolic disorder such as Zellweger syndrome, maple syrup urine disease,
adrenoleukodystropy,
carnidne palmitoyltransferase deficiency, Addison's disease, cerebrotendinous
xanthomatosis,
congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis,
diabetes, fatty hepatocirrhosis,
fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma,
glycogen storage diseases,
hereditary fructose intolerance, hyperadrenalism, hypoadrenalism,
hyperparathyroidism,
hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia,
hypothyroidism,
hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal
storage diseases,
mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria;
an
autoimmune/inflammatory disorder such as inflammation, actinic keratosis,
acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory
distress syndrome,
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allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroidids, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, bursitis,
cholecystitis, cirrhosis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's
syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
paroxysmal nocturnal
hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome,
episodic lymphopenia with
lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis,
myasthenia gravis,
myocardial or pericardial inflammation, myelofibrosis, osteoarthritis,
osteoporosis, pancreatitis,
polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis, primary
thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveids, Werner
syndrome,
complications of cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal,
parasitic, protozoal, and helminthic infections, trauma, and hematopoietic
cancer including
lymphoma, leukemia, and myeloma; a viral infection, such as those caused by
adenoviruses (acute
respiratory disease, pneumonia), arenaviruses (lymphocytic choriomeningitis),
bunyaviruses
(Hantavirus), coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses
(hepatitis),
herpesviruses (herpes simplex virus, varicella-zoster virus, Epstein-Barr
virus, cytomegalovirus),
flaviviruses (yellow fever), orthomyxoviruses (influenza), papillomaviruses
(cancer),
paramyxoviruses (measles, mumps), picornoviruses (rhinovirus, poliovirus,
coxsackie-virus),
polyomaviruses (BK virus, JC virus), poxviruses .(smallpox), reovirus
(Colorado tick fever),
retroviruses (human immunodeficiency virus, human T lymphotropic virus),
rhabdoviruses (rabies),
rotaviruses (gastroenteritis), and togaviruses (encephalitis, rubella); 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 . The
polynucleotide seduences
encoding HCPN 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 HCPN
expression. Such
qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding HCPN may be useful
in assays that
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detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding HCPN 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
HCPN 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 HCPN,
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 HCPN, under conditions suitable for hybridization
or amplification.
Standard hybridization may be quantified by comparing the values obtained from
normal subjects with
values from an experiment in which a known amount of a substantially purified
polynucleotide is used.
Standard values obtained in this manner may be compared with values obtained
from samples from
patients who are symptomatic for a disorder. Deviation from standard values is
used to establish the
presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the development
of the disease, or may provide a means for detecting the disease prior to the
appearance of actual
clinical symptoms. A more definitive diagnosis of this type may allow health
professionals to employ
preventative measures or aggressive treatment earlier thereby preventing the
development or further
progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding HCPN
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
HCPN, or a fragment of a polynucleotide complementary to the polynucleotide
encoding HCPN, and
will be employed under optimized conditions for identification of a specific
gene or condition.
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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 HCPN 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 HCPN are used to amplify
DNA using the
polymerise chain reaction (PCR). The DNA may be derived, for example, from
diseased or normal
tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause
differences in the secondary
and tertiary structures of PCR products in single-stranded form, and these
differences are detectable
using gel electrophoresis in non-denaturing gels. In fSCCP, the
oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in high-
throughput equipment such as
DNA sequencing machines. Additionally, sequence database analysis methods,
termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of
individual
overlapping DNA 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 HCPN include
radiolabeling or
biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C. et
al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives rapid
quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described 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
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progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for HCPN, or HCPN 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
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
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rest of the expression data. The normalization procedure is useful for
comparison of expression data
after treatment with different compounds. While the assignment of gene
function to elements of a
toxicant signature aids in interpretation of toxicity mechanisms, knowledge of
gene function is not
necessary for the statistical matching of signatures which leads to prediction
of toxicity. (See, for
example, Press Release 00-02 from the National Institute of Environmental
Health Sciences, released
February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.)
Therefore, it is
important and desirable in toxicological screening using toxicant signatures
to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological sample
containing nucleic acids with the test compound. Nucleic acids that are
expressed in the treated
biological sample are hybridized with one or more probes specific to the
polynucleotides of the
present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a proteome
can be subjected individually to further analysis. Proteome expression
patterns, or profiles, are
analyzed by quantifying the number of expressed proteins and their relative
abundance under given
conditions and at a given time. A profile of a cell's proteome may thus be
generated by separating and
analyzing the polypeptides of a particular tissue or cell type. In one
embodiment, the separation is
achieved using two-dimensional gel electrophoresis, in which proteins from a
sample are separated by
isoelectric focusing in the first dimension, and then according to molecular
weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner and
Anderson, supra). The proteins are
visualized in the gel as discrete and uniquely positioned spots, typically by
staining the gel with an agent
such as Coomassie Blue or silver or fluorescent stains. The optical density of
each protein spot 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.
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A proteomic profile may also be generated using antibodies specific for HCPN
to quantify the
levels of HCPN expression. In one embodiment, the antibodies are used as
elements on a microarray,
and protein expression levels are quantified by exposing the microarray to the
sample and detecting the
levels of protein bound to each array element (Lueking, A. et al. (1999) Anal.
Biochem. 270:103-111;
Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be
performed by a variety of
methods known in the art, for example, by reacting the proteins in the sample
with a thiol- or amino-
reactive fluorescent compound and detecting the amount of fluorescence bound
at each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and should
be analyzed in parallel with toxicant signatures at the transcript level.
There is a poor correlation
between transcript and protein abundances for some proteins in some tissues
(Anderson, N.L. and J.
Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures
may be useful in the
analysis of compounds which do not significantly affect the transcript image,
but which alter the
proteomic profile. In addition, the analysis of transcripts in body fluids is
difficult, due to rapid
degradation of mRNA, so proteomic profiling may be more reliable and
informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated biological
sample are separated so that the amount of each protein can be quantified. The
amount of each protein
is compared to.the amount of the corresponding protein in an untreated
biological sample. A difference
in the amount of protein between the two samples is indicative of a toxic
response to the test compound
in the treated sample. Individual proteins are identified by sequencing the
amino acid residues of the
individual proteins and comparing these partial sequences to the polypeptides
of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are incubated
with antibodies specific to the polypeptides of the present invention. The
amount of protein recognized
by the antibodies is quantified. The amount of protein in the treated
biological sample is compared with
the amount in an untreated biological sample. A difference in the amount of
protein between the 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.
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In another embodiment of the invention, nucleic acid sequences encoding HCPN
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 mufti-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 P1
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, e.g.,
Larder, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, sera, 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
HCPN 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 posidonal cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to l 1q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (See, e.g.,
Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, HCPN, 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
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between HCPN and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with HCPN, or
fragments thereof,
and washed. Bound HCPN is then detected by methods well known in the art.
Purified HCPN 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 HCPN specifically compete with a test compound
for binding HCPN. In
this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with HCPN.
In additional embodiments, the nucleotide sequences which encode HCPN 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.
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. 60/146,908 and U.S. Ser. No. 60/160,924, are hereby
expressly incorporated
by reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues
were homogenized and lysed in guanidinium isothiocyanate, while others were
homogenized and lysed
in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life
Technologies), a monophasic
solution of phenol and guanidine isothiocyanate. The resulting lysates were
centrifuged over CsCl
cushions or extracted with chloroform. RNA was precipitated from the lysates
with either isopropanol
or sodium acetate and ethanol, or by other routine methods.
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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), PSPORTl 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 XL1-Blue, XL1-
BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DHlOB from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo excision
using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were
purified using at least
one of the following: a Magic or WIZARD Minipreps DNA purification system
(Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8
Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid
purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1 ml of
distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in 384-
well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically using
PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence
scanner
(Labsystems Oy, Helsinki, Finland).
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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
S 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 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
Conned, and were screened
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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, su ra,
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 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
CA 02374743 2002-O1-25
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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 HCPN occurred. Analysis involved the categorization of
cDNA libraries by
organ/dssue 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 HESHP 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 G~nethon 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 N0:14, SEQ ID NO:15, and SEQ ID N0:22 are
described in The Invention as ranges, or intervals, of human chromosomes. More
than one map
location is reported for SEQ ID N0:22, indicating that previously mapped
sequences having
similarity, but not complete identity, to SEQ ID N0:22 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 centiMorgari (cM) is a unit of measurement based on
recombination
frequencies between chromosomal markers. On average, 1 cM is roughly
equivalent to 1 megabase
(Mb) of DNA in humans, although this can vary widely due to hot and cold spots
of recombination.)
The cM distances are based on genetic markers mapped by Genethon which provide
boundaries for
radiation hybrid markers whose sequences were included in each of the
clusters. Diseases associated
with the public and Incyte sequences located within the indicated intervals
are also reported in the
Invention where applicable.
VI. Extension of HCPN Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:12-22 were produced by
extension of an
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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)2S04,
and ~i-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 ~1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 gel to 10 ~1 aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose mini-gel to determine which reactions were successful in
extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%) agarose
gels, fragments were excised, and agar digested with Agar ACE (Promega).
Extended clones were
religated using T4 lipase (New England Biolabs, Beverly MA) into pUC 18 vector
(Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells were selected
on antibiotic-containing
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media, and individual colonies were picked and cultured overnight at
37°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham
Pharmacia Biotech) and Pfu DNA polymerise (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 oligonucleoddes, consisting of about
20 base pairs, is
specifically described, essentially the same procedure is used with larger
nucleotide fragments.
Oligonucleotides are designed using state-of-the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of ['y-
32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextrin bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or
Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
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photolithography, piezoelectric printing (ink-jet printing, See, e.g.,
Baldeschweiler, supra), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
technologies should be uniform and solid with a non-porous surface (Schena
(1999), s_upra). 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;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson
(1998) Nat. Biotechnol.
16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The array
elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element on
the microarray may be assessed. In one embodiment, microarray preparation and
usage is described in
detail below.
Tissue or Cell Sample 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/lil oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/Nl RNase inhibitor, 500 pM dATP, 500 i.~M dGTP, 500
pM dTTP, 40 i.~M
dCTP, 40 E1M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)' RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37°C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
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CA 02374743 2002-O1-25
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then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~.~1 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
fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/~.Q, 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 Erl of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 iM of 5X SSC in a corner of the chamber. The chamber containing the arrays
is incubated for
about 6.5 hours at 60°C. The arrays are washed for 10 min at 45
°C in a first wash buffer (1X SSC,
0.1% SDS), three times for 10 minutes each at 45 °C in a second wash
buffer (0.1X SSC), and dried.
Detection
CA 02374743 2002-O1-25
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Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
S 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 N.>] corresponding to the two
lluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the Iluorophores 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
Iluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are differentially
expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two
fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each spot
is centered in each element of the grid. The fluorescence signal within each
element is then integrated
to obtain a numerical value corresponding to the average intensity of the
signal. The software used
for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
IX. Complementary Polynucleotides
Sequences complementary to the HCPN-encoding sequences, or any parts thereof,
are used to
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detect, decrease, or inhibit expression of naturally occurring HCPN. 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 HCPN. 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 HCPN-encoding transcript.
X. Expression of HCPN
Expression and purification of HCPN is achieved using bacterial or virus-based
expression
systems. For expression of HCPN in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21 (DE3).
Antibiotic resistant bacteria express HCPN upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of HCPN in eukaryotic cells is
achieved by infecting insect
or mammalian cell lines with recombinant Auto~raphica californica nuclear
polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding HCPN 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 frueiperda (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, HCPN is synthesized as a fusion protein with,
e.g., glutathione S-
transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting
rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-kilodalton
enzyme from Schistosoma Lponicum, 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 HCPN 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
67
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, su ra,
ch. 10 and 16). Purified HCPN obtained by these methods can be used directly
in the assays shown in
Examples XI and XV.
XI. Demonstration of HCPN Activity
HCPN induction by heat or toxins may be demonstrated using primary cultures of
human
fibroblasts or human cell lines such as CCL-13, HEK293, or HEP G2 (ATCC). To
heat induce HCPN
expression, aliquots of cells are incubated at 42°C for 15, 30, or 60
minutes. Control aliquots are
incubated at 37 °C for the same time periods. To induce HCPN expression
by toxins, aliquots of cells
are treated with 100 pM arsenite or 20 mM azetidine-2-carboxylic acid for 0,
3, 6, or 12 hours. After
exposure to heat, arsenite, or the amino acid analogue, samples of the treated
cells are harvested and
cell lysates prepared for analysis by western blot.
Cells are lysed in lysis buffer containing 1 % Nonidet P-40, 0.15 M NaCl, 50
mM Tris-HCI, 5
mM EDTA, 2 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 1 mg/ml
leupepdn, and 1
m~ml pepstatin. Twenty micrograms of the cell lysate is separated on an 8% SDS-
PAGE gel and
transferred to a nitrocellulose membrane. After blocking with 5% nonfat dry
milk/phosphate-buffered
saline for 1 h, the membrane is incubated overnight at 4°C or at room
temperature for 2-4 hours with a
1:1000 dilution of anti-HCPN serum in 2% nonfat dry milk/phosphate-buffered
saline. The membrane
is then washed and incubated with a 1:1000 dilution of horseradish peroxidase-
conjugated goat
anti-rabbit IgG in 2% dry milk/phosphate-buffered saline. After washing with
0.1 % Tween 20 in
phosphate-buffered saline, the HCPN protein is detected and compared to
controls by using
chemiluminescence. Induction of HCPN under stress conditions is evidence of
HCPN activity.
Alternatively, HCPN activity can be determined by measuring the ability to
promote ATP
hydrolysis by Hsp70. Briefly, 1 ~tg Hsp70 protein is incubated with 1 nmol
unlabled ATP and 0.01
~Ci of a32P-ATP in ATPase buffer (50 mM HEPES, pH 7.4, 50 mM NaCI, 10 mM DTT,
and 2 mM
MgCl2) in a total volume of 20 itl at 30°C with or without HCPN. After
1 hr, 1 ~1 of the reaction is
spotted on polyethyleneimine cellulose TLC plates and developed in IM formic
acid with 0.5 M LiCI.
Plates are examined for conversion of a32P-ATP to a32P-ADP by phosphorimager
(Hunter, s, unra).
X1I. Functional Assays
HCPN function is assessed by expressing the sequences encoding HCPN 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
68
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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;
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 specif c 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 Cvtometry, Oxford, New York NY.
The influence of HCPN on gene expression can be assessed using highly purified
populations
of cells transfected with sequences encoding HCPN 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 HCPN and other genes of interest can be analyzed by northern
analysis or
microarray techniques.
XIII. Production of HCPN Specific Antibodies
HCPN 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 HCPN amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431 A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
69
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting andsera are tested for
antipeptide and anti-HCPN
activity by, for example, binding the peptide or HCPN to a substrate, blocking
with 1 % BSA, reacting
with rabbit antisera, washing, and reacting with radio-iodinated goat and-
rabbit IgG.
XIV. Purification of Naturally Occurring HCPN Using Specific Antibodies
Naturally occurring or recombinant HCPN is substantially purified by
immunoaffinity
chromatography using antibodies specific for HCPN. An immunoaffinity column is
constructed by
covalently coupling anti-HCPN 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 HCPN are passed over the immunoaffinity column, and the
column is washed
under conditions that allow the preferential absorbance of HCPN (e.g., high
ionic strength buffers in the
presence of detergent). The column is eluted under conditions that disrupt
antibody/HCPN 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 HCPN is collected.
XV. Identification of Molecules Which Interact with HCPN
HCPN, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a mull-well plate are incubated with the
labeled HCPN, washed, and
any wells with labeled HCPN complex are assayed. Data obtained using different
concentrations of
HCPN are used to calculate values for the number, affinity, and association of
HCPN with the
candidate molecules.
Alternatively, molecules interacting with HCPN 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).
HCPN 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
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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.
71
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
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CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
YUE, Henry
BANDMAN, Olga
TANG, Y. Tom
BAUGHN, Mariah R.
AZIMZAI, Yalda
LU, Dyung Aina M.
<120> HUMAN CHAPERONE PROTEINS
<130> PF-0728 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/146,908; 60/160,924
<151> 1999-08-03; 1999-10-22
<160> 22
<170> PERL Program
<210> 1
<211> 170
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 723593CD1
<400> 1
Met Ser His Arg Thr Ser Ser Thr Phe Arg Ala Glu Arg Ser Phe
1 5 10 15
His Ser Ser Ser Ser Ser Ser Ser Ser Ser Thr Ser Ser Ser Ala
20 25 30
Ser Arg Ala Leu Pro Ala Gln Asp Pro Pro Met Glu Lys Ala Leu
35 40 45
Ser Met Phe Ser Asp Asp Phe Gly Ser Phe Met Arg Pro His Ser
50 55 60
Glu Pro Leu Ala Phe Pro Ala Arg Pro Gly Gly Ala Gly Asn Ile
65 70 75
Lys Thr Leu Gly Asp Ala Tyr Glu Phe Ala Val Asp Val Arg Asp
80 85 90
Phe Ser Pro Glu Asp Ile Ile Val Thr Thr Ser Asn Asn His Ile
95 100 105
Glu Val Arg Ala Glu Lys Leu Ala Ala Asp Gly Thr Val Met Asn
110 115 120
Thr Phe Ala His Lys Cys Gln Leu Pro Glu Asp Val Asp Pro Thr
125 130 135
Ser Val Thr Ser Ala Leu Arg Glu Asp Gly Ser Leu Thr Ile Arg
140 145 150
Ala Arg Arg His Pro His Thr Glu His Val Gln Gln Thr Phe Arg
155 160 165
Thr Glu Ile Lys Ile
170
<210> 2
<211> 304
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1708350CD1
1/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
<400> 2
Met Ala Val Thr Lys Glu Leu Leu Gln Met Asp Leu Tyr Ala Leu
1 5 10 15
Leu Gly Ile Glu Glu Lys Ala Ala Asp Lys Glu Val Lys Lys Ala
20 25 30
Tyr Arg Gln Lys Ala Leu Ser Cys His Pro Asp Lys Asn Pro Asp
35 40 45
Asn Pro Arg Ala Ala Glu Leu Phe His Gln Leu Ser Gln Ala Leu
50 55 60
Glu Val Leu Thr Asp Ala Ala Ala Arg Ala Ala Tyr Asp Lys Val
65 70 75
Arg Lys Ala Lys Lys Gln Ala Ala Glu Arg Thr Gln Lys Leu Asp
80 85 90
Glu Lys Arg Lys Lys Val Lys Leu Asp Leu Glu Ala Arg Glu Arg
95 100 105
Gln Ala Gln Ala Gln Glu Ser Glu Glu Glu Glu Glu Ser Arg Ser
110 115 120
Thr Arg Thr Leu Glu Gln Glu Ile Glu Arg Leu Arg Glu Glu Gly
125 130 135
Ser Arg Gln Leu Glu Glu Gln Gln Arg Leu Ile Arg Glu Gln Ile
140 145 150
Arg Gln Glu Arg Asp Gln Arg Leu Arg Gly Lys Ala Glu Asn Thr
155 160 165
Glu Gly Gln Gly Thr Pro Lys Leu Lys Leu Lys Trp Lys Cys Lys
170 175 180
Lys Glu Asp Glu Ser Lys Gly Gly Tyr Ser Lys Asp Val Leu Leu
185 190 195
Arg Leu Leu Gln Lys Tyr Gly Glu Val Leu Asn Leu Val Leu Ser
200 205 210
Ser Lys Lys Pro Gly Thr Ala Val Val Glu Phe Ala Thr Val Lys
215 220 225
Ala Ala Glu Leu Ala Val'Gln Asn Glu Val Gly Leu Val Asp Asn
230 235 240
Pro Leu Lys Ile Ser Trp Leu Glu Gly Gln Pro Gln Asp Ala Val
245 250 255
Gly Arg Ser His Ser Gly Leu Ser Lys Gly Ser Val Leu Ser Glu
260 265 270
Arg Asp Tyr Glu Ser Leu Val Met Met Arg Met Arg Gln Ala Ala
275 280 285
Glu Arg Gln Gln Leu Ile Ala Arg Met Gln Gln Glu Asp Gln Glu
290 295 300
Gly Pro Pro Thr
<210> 3
<211> 483
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1742550CD1
<400> 3
Met Ala Lys Asp Ala Ser Ser Ala Asp Ile Arg Lys Ala Tyr Arg
1 5 10 15
Lys Leu Ser Leu Thr Leu His Pro Asp Lys Asn Lys Asp Glu Asn
20 25 30
Ala Glu Thr Gln Phe Arg Gln Leu Val Ala Ile Tyr Glu Val Leu
35 40 45
Lys Asp Asp Glu Arg Arg Gln Arg Tyr Asp Asp Ile Leu Ile Asn
50 55 60
Gly Leu Pro Asp Trp Arg Gln Pro Val Phe Tyr Tyr Arg Arg Val
65 70 75
Arg Lys Met Ser Asn Ala Glu Leu Ala Leu Leu Leu Phe Ile Ile
80 85 90
Leu Thr Val Gly His Tyr Ala Val Val Trp Ser Ile Tyr Leu Glu
95 100 105
2/ 16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
Lys Gln Leu Asp Glu Leu Leu Ser Arg Lys Lys Arg Glu Lys Lys
110 115 120
Lys Lys Thr Gly Ser Lys Ser Val Asp Val Ser Lys Leu Gly Ala
125 130 135
Ser Glu Lys Asn Glu Arg Leu Leu Met Lys Pro Gln Trp His Asp
140 145 150
Leu Leu Pro Cys Lys Leu Gly Ile Trp Phe Cys Leu Thr Leu Lys
155 160 165
Ala Leu Pro His Leu Ile Gln Asp Ala Gly Gln Phe Tyr Ala Lys
170 175 180
Tyr Lys Glu Thr Arg Leu Lys Glu Lys Glu Asp Ala Leu Thr Arg
185 190 195
Thr Glu Leu Glu Thr Leu Gln Lys Gln Lys Lys Val Lys Lys Pro
200 205 210
Lys Pro Glu Phe Pro Val Tyr Thr Pro Leu Glu Thr Thr Tyr Ile
215 220 225
Gln Ser Tyr Asp His Gly Thr Ser Ile Glu Glu Ile Glu Glu Gln
230 235 240
Met Asp Asp Trp Leu Glu Asn Arg Asn Arg Thr Gln Lys Lys Gln
245 250 255
Ala Pro Glu Trp Thr Glu Glu Asp Leu Ser Gln Leu Thr Arg Ser
260 265 270
Met Val Lys Phe Pro Gly Gly Thr Pro Gly Arg Trp Glu Lys Ile
275 280 285
Ala His Glu Leu Gly Arg Ser Val Thr Asp Val Thr Thr Lys Ala
290 295 300
Lys GTn Leu Lys Asp Ser Val Thr Cys Ser Pro Gly Met Val Arg
305 310 315
Leu Ser Glu Leu Lys Ser Thr Val Gln Asn Ser Arg Pro Ile Lys
320 325 330
Thr Ala Thr Thr Leu Pro Asp Asp Met Ile Thr Gln Arg Glu Asp
335 340 345
Ala Glu Gly Val Ala Ala Glu Glu Glu Gln Glu Gly Asp Ser Gly
350 355 360
Glu Gln Glu Thr Gly Ala Thr Asp Ala Arg Pro Arg Arg Arg Lys
365 370 375
Pro Ala Arg Leu Leu Glu Ala Thr Ala Lys Pro Glu Pro Glu Glu
380 385 390
Lys Ser Arg Ala Lys Arg Gln Lys Asp Phe Asp Ile Ala Glu Gln
395 400 405
Asn Glu Ser Ser Asp Glu Glu Ser Leu Arg Lys Glu Arg Ala Arg
410 415 420
Ser Ala Glu Glu Pro Trp Thr Gln Asn Gln Gln Lys Leu Leu Glu
425 430 435
Leu Ala Leu Gln Gln Tyr Pro Arg Gly Ser Ser Asp Arg Trp Asp
440 445 450
Lys Ile Ala Arg Cys Val Pro Ser Lys Ser Lys Glu Asp Cys Ile
455 460 465
Ala Arg Tyr Lys Leu Leu Val Glu Leu Val Gln Lys Lys Lys Gln
470 475 480
Ala Lys Ser
<210> 4
<211> 226
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1919301CD1
<400> 4
Met Ala Ala Met Arg Trp Arg Trp Trp Gln Arg Leu Leu Pro Trp
1 5 10 15
Arg Leu Leu Gln Ala Arg Gly Phe Pro Gln Asn Ser Ala Pro Ser
20 25 30
Leu Gly Leu Gly Ala Arg Thr Tyr Ser Gln Gly Asp Cys Ser Tyr
3/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
35 40 45
Ser Arg Thr Ala Leu Tyr Asp Leu Leu Gly Val Pro Ser Thr Ala
50 55 60
Thr Gln Ala Gln Ile Lys Ala Ala Tyr Tyr Arg Gln Cys Phe Leu
65 70 75
Tyr His Pro Asp Arg Asn Ser Gly Ser Ala Glu Ala Ala Glu Arg
80 85 90
Phe Thr Arg Ile Ser Gln Ala Tyr Val Val Leu Gly Ser Ala Thr
95 100 105
Leu Arg Arg Lys Tyr Asp Arg Gly Leu Leu Ser Asp Glu Asp Leu
110 115 120
Arg Gly Pro Gly Val Arg Pro Ser Arg Thr Pro Ala Pro Asp Pro
125 130 135
Gly Ser Pro Arg Thr Pro Pro Pro Thr Ser Arg Thr His Asp Gly
140 145 150
Ser Arg Ala Ser Pro Gly Ala Asn Arg Thr Met Phe Asn Phe Asp
155 160 165
Ala Phe Tyr Gln Ala His Tyr Gly Glu Gln Leu Glu Arg Glu Arg
170 175 180
Arg Leu Arg Ala Arg Arg Glu Ala Leu Arg Lys Arg Gln Glu Tyr
185 190 195
Arg Ser Met Lys Gly Leu Arg Trp Glu Asp Thr Arg Asp Thr Ala
200 205 210
Ala Ile Phe Leu Ile Phe Ser Ile Phe Ile Ile Ile Gly Phe Tyr
215 220 225
Ile
<210> 5
<211> 112
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2012055CD1
<400> 5
Met Met Ala Val Glu Gln Met Pro Lys Lys Asp Trp Tyr Ser Ile
1 5 10 15
Leu Gly Ala Asp Pro Ser Ala Asn Ile Ser Asp Leu Lys Gln Lys
20 25 30
Tyr Gln Lys Leu Ile Leu Met Tyr His Pro Asp Lys Gln Ser Thr
35 40 45
Asp Val Pro Ala Gly Thr Val Glu Glu Cys Val Gln Lys Phe Ile
50 55 60
Glu Ile Asp Gln Ala Trp Lys Ile Leu Gly Asn Glu Glu Thr Lys
65 70 75
Arg Glu Tyr Asp Leu Gln Arg Cys Glu Asp Asp Leu Arg Asn Val
80 85 90
Gly Pro Val Asp Ala Gln Val Tyr Leu Glu Glu Met Ser Trp Asn
95 100 105
Glu Val Thr Ser Gln Arg Gln
110
<210> 6
<211> 358
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2238062CD1
<400> 6
Met Ala Ala Thr Leu Gly Ser Gly Glu Arg Trp Thr Glu Ala Tyr
1 5 10 15
Ile Asp Ala Val Arg Arg Asn Lys Tyr Pro Glu Asp Thr Pro Pro
20 25 30
4/ 16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
Glu Ser His Asp Pro Cys Gly Cys Cys Asn Cys Met Lys Ala Gln
35 40 45
Lys Glu Lys Lys Ser Glu Asn Glu Trp Thr Gln Thr Arg Gln Gly
50 55 60
Glu Gly Asn Ser Thr Tyr Ser Glu Glu Gln Leu Leu Gly Val Gln
65 70 75
Arg Ile Lys Lys Cys Arg Asn Tyr Tyr Glu Ile Leu Gly Val Ser
80 85 90
Arg Asp Ala Ser Asp Glu Glu Leu Lys Lys Ala Tyr Arg Lys Leu
95 100 105
Ala Leu Lys Phe His Pro Asp Lys Asn Cys Ala Pro Gly Ala Thr
110 115 120
Asp Ala Phe Lys Ala Ile Gly Asn Ala Phe Ala Val Leu Ser Asn
125 130 135
Pro Asp Lys Arg Leu Arg Tyr Asp Glu Tyr Gly Asp Glu Gln Val
140 145 150
Thr Phe Thr Ala Pro Arg Ala Arg Pro Tyr Asn Tyr Tyr Arg Asp
155 160 165
Phe Glu Ala Asp Ile Thr Pro Glu Glu Leu Phe Asn Val Phe Phe
170 175 180
Gly Gly His Phe Pro Thr Gly Asn Ile His Met Phe Ser Asn Val
185 190 195
Thr Asp Asp Thr Tyr Tyr Tyr Arg Arg Arg His Arg His Glu Arg
200 205 210
Thr Gln Thr Gln Lys Glu Glu Glu Glu Glu Lys Pro Gln Thr Thr
215 220 225
Tyr Ser Ala Phe Ile Gln Leu Leu Pro Val Leu Val Ile Val Ile
230 235 240
Ile Ser Val Ile Thr Gln Leu Leu Ala Thr Asn Pro Pro Tyr Ser
245 250 255
Leu Phe Tyr Lys Ser Thr Leu Gly Tyr Thr Ile Ser Arg Glu Thr
260 265 270
Gln Asn Leu Gln Val Pro Tyr Phe Val Asp Lys Asn Phe Asp Lys
275 280 285
Ala Tyr Arg Gly Ala Ser Leu His Asp Leu Glu Lys Thr Ile Glu
290 295 300
Lys Asp Tyr Ile Asp Tyr Ile Gln Thr Ser Cys Trp Lys Glu Lys
305 310 315
Gln Gln Lys Ser Glu Leu Thr Asn Leu Ala Gly Leu Tyr Arg Asp
320 325 330
Glu Arg Leu Lys Gln Lys Ala Glu Ser Leu Lys Leu Glu Asn Cys
335 340 345
Glu Lys Leu Ser Lys Leu Ile Gly Leu Arg Arg Gly Gly
350 355
<210> 7
<211> 928
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1825012CD1
<400> 7
Met Gly Gly Ser Ala Ser Ser Gln Leu Asp Glu Gly Lys Cys Ala
1 5 10 15
Tyr Ile Arg Gly Lys Thr Glu Ala Ala Ile Lys Asn Phe Ser Pro
20 25 30
Tyr Tyr Ser Arg Gln Tyr Ser Val Ala Phe Cys Asn His Val Arg
35 40 45
Thr Glu Val Glu Gln Gln Arg Asp Leu Thr Ser Gln Phe Leu Lys
50 55 60
Thr Lys Pro Pro Leu Ala Pro Gly Thr Ile Leu Tyr Glu Ala Glu
65 70 75
Leu Ser Gln Phe Ser Glu Asp Ile Lys Lys Trp Lys Glu Arg Tyr
80 85 90
Val Val Val Lys Asn Asp Tyr Ala Val Glu Ser Tyr Glu Asn Lys
5/ 16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
95 100 105
Glu Ala Tyr Gln Arg Gly Ala Ala Pro Lys Cys Arg Ile Leu Pro
110 115 120
Ala Gly Gly Lys Val Leu Thr Ser Glu Asp Glu Tyr Asn Leu Leu
125 130 135
Ser Asp Arg His Phe Pro Asp Pro Leu Ala Ser Ser Glu Lys Glu
140 145 150
Asn Thr Gln Pro Phe Val Val Leu Pro Lys Glu Phe Pro Val Tyr
155 160 165
Leu Trp Gln Pro Phe Phe Arg His Gly Tyr Phe Cys Phe His Glu
170 175 180
Ala Ala Asp Gln Lys Arg Phe Ser Ala Leu Leu Ser Asp Cys Val
185 190 195
Arg His Leu Asn His Asp Tyr Met Lys Gln Met Thr Phe Glu Ala
200 205 210
Gln Ala Phe Leu Glu Ala Val Gln Phe Phe Arg Gln Glu Lys Gly
215 220 225
His Tyr Gly Ser Trp Glu Met Ile Thr Gly Asp Glu Ile Gln Ile
230 235 240
Leu Ser Asn Leu Val Met Glu Glu Leu Leu Pro Thr Leu Gln Thr
245 250 255
Asp Leu Leu Pro Lys Met Lys Gly Lys Lys Asn Asp Arg Lys Arg
260 265 270
Thr Trp Leu Gly Leu Leu Glu Glu Ala Tyr Thr Leu Val Gln His
275 280 285
Gln Val Ser Glu Gly Leu Ser Ala Leu Lys Glu Glu Cys Arg Ala
290 295 300
Leu Thr Lys Gly Leu Glu Gly Thr Ile Arg Ser Asp Met Asp Gln
305 310 315
Ile Val Asn Ser Lys Asn Tyr Leu Ile Gly Lys Ile Lys Ala Met
320 325 330
Val Ala Gln Pro Ala Glu Lys Ser Cys Leu Glu Ser Val Gln Pro
335 340 345
Phe Leu Ala Ser Ile Leu Glu Glu Leu Met Gly Pro Val Ser Ser
350 355 360
Gly Phe Ser Glu Val Arg Val Leu Phe Glu Lys Glu Val Asn Glu
365 370 375
Val Ser Gln Asn Phe Gln Thr Thr Lys Asp Ser Val Gln Leu Lys
380 385 390
Glu His Leu Asp Arg Leu Met Asn Leu Pro Leu His Ser Val Lys
395 400 405
Met Glu Pro Cys Tyr Thr Lys Val Asn Leu Leu His Glu Arg Leu
410 415 420
Gln Asp Leu Lys Ser Arg Phe Arg Phe Pro His Ile Asp Leu Val
425 430 435
Val Gln Arg Thr Gln Asn Tyr Met Gln Glu Leu Met Glu Asn Ala
440 445 450
Val Phe Thr Phe Glu Gln Leu Leu Ser Pro His Leu Gln Gly Glu
455 460 465
Ala Ser Lys Thr Ala Val Ala Ile Glu Lys Val Lys Leu Arg Val
470 475 480
Leu Lys Gln Tyr Asp Tyr Asp Ser Ser Thr Ile Arg Lys Lys Ile
485 490 495
Phe Gln Glu Ala Leu Val Gln Ile Thr Leu Pro Thr Val Gln Lys
500 505 510
Ala Leu Ala Ser Thr Cys Lys Pro Glu Leu Gln Lys Tyr Glu Gln
515 520 525
Phe Ile Phe Ala Asp His Thr Asn Met Ile His Val Glu Asn Val
530 535 540
Tyr Glu Glu Ile Leu His Gln Ile Leu Leu Asp Glu Thr Leu Lys
545 550 555
Val Ile Lys Glu Ala Ala Ile Leu Lys Lys His Asn Leu Phe Glu
560 565 570
Asp Asn Met Ala Leu Pro Ser Glu Ser Val Ser Ser Leu Thr Asp
575 580 585
Leu Lys Pro Pro Thr Gly Ser Asn Gln Ala Ser Pro Ala Arg Arg
590 595 600
6/ 16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
Ala Ser Ala Ile Leu Pro Gly Val Leu Gly Ser Glu Thr Leu Ser
605 610 615
Asn Glu Val Phe Gln Glu Ser Glu Glu Glu Lys Gln Pro Glu Val
620 625 630
Pro Ser Ser Leu Ala Lys Gly Glu Ser Leu Ser Leu Pro Gly Pro
635 640 645
Ser Pro Pro Pro Asp Gly Thr Glu Gln Val Ile Ile Ser Arg Val
650 655 660
Asp Asp Pro Val Val Asn Pro Val Ala Thr Glu Asp Thr Ala Gly
665 670 675
Leu Pro Gly Thr Cys Ser Ser Glu Leu Glu Phe Gly Gly Thr Leu
680 685 690
Glu Asp Glu Glu Pro Ala Gln Glu Glu Pro Glu Pro Ile Thr Ala
695 700 705
Ser Gly Ser Leu Lys Ala Leu Arg Lys Leu Leu Thr Ala Ser Val
710 715 720
Glu Val Pro Val Asp Ser Ala Pro Val Met Glu Glu Asp Thr Asn
725 730 735
Gly Glu Ser His Val Pro Gln Glu Asn Glu Glu Glu Glu Glu Lys
740 745 750
Glu Pro Ser Gln Ala Ala Ala Ile His Pro Asp Asn Cys Glu Glu
755 760 765
Ser Glu Val Ser Glu Arg Glu Ala Gln Pro Pro Cys Pro Glu Ala
770 775 780
His Gly Glu Glu Leu Gly Gly Phe Pro Glu Val Gly Ser Pro Ala
785 790 795
Ser Pro Pro Ala Ser Gly Gly Leu Thr Glu Glu Pro Leu Gly Pro
800 805 810
Met Glu Gly Glu Leu Pro Gly Glu Ala Cys Thr Leu Thr Ala His
815 820 825
Glu Gly Arg Gly Gly Lys Cys Thr Glu Glu Gly Asp Ala Ser Gln
830 835 840
Gln Glu Gly Cys Thr Leu Gly Ser Asp Pro Ile Cys Leu Ser Glu
845 850 855
Ser Gln Val Ser Glu Glu Gln Glu Glu Met Gly Gly Gln Ser Ser
860 865 870
Ala Ala Gln Ala Thr Ala Ser Val Asn Ala Glu Glu Ile Lys Val
875 880 885
Ala Arg Ile His Glu Cys Gln Trp Val Val Glu Asp Ala Pro Asn
890 895 900
Pro Asp Val Leu Leu Ser His Lys Asp Asp Val Lys Glu Gly Glu
905 910 915
Gly Gly Gln Glu Ser Phe Pro Glu Leu Pro Ser Glu Glu
920 925
<210> 8
<211> 159
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1906464CD1
<400> 8
Met Gln Arg Val Gly Asn Thr Phe Ser Asn Glu Ser Arg Val Ala
1 5 10 15
Ser Arg Cys Pro Ser Val Gly Leu Ala Glu Arg Asn Arg Val Ala
20 25 30
Thr Met Pro Val Arg Leu Leu Arg Asp Ser Pro Ala Ala Gln Glu
35 40 45
Asp Asn Asp His Ala Arg Asp Gly Phe Gln Met Lys Leu Asp Ala
50 55 60
His Gly Phe Ala Pro Glu Glu Leu Val Val Gln Val Asp Gly Gln
65 70 75
Trp Leu Met Val Thr Gly Gln Gln Gln Leu Asp Val Arg Asp Pro
80 85 90
Glu Arg Val Ser Tyr Arg Met Ser Gln Lys Val His Arg Lys Met
7/ 16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
95 100 105
Leu Pro Ser Asn Leu Ser Pro Thr Ala Met Thr Cys Cys Leu Thr
110 115 120
Pro Ser Gly Gln Leu Trp Val Arg Gly Gln Cys Val Ala Leu Ala
125 130 135
Leu Pro Glu Ala Gln Thr Gly Pro Ser Pro Arg Leu Gly Ser Leu
140 145 150
Gly Ser Lys Ala Ser Asn Leu Thr Arg
155
<210> 9
<211> 235
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1979146CD1
<400> 9
Met Trp Arg Gly Arg Ala Gly Ala Leu Leu Arg Val Trp Gly Phe
1 5 10 15
Trp Pro Thr Gly Val Pro Arg Arg Arg Pro Leu Ser Cys Asp Ala
20 25 30
Ala Ser Gln Ala Gly Ser Asn Tyr Pro Arg Cys Trp Asn Cys Gly
35 40 45
Gly Pro Trp Gly Pro Gly Arg Glu Asp Arg Phe Phe Cys Pro Gln
50 55 60
Cys Arg Ala Leu Gln Ala Pro Asp Pro Thr Arg Asp Tyr Phe Ser
65 70 75
Leu Met Asp Cys Asn Arg Ser Phe Arg Val Asp Thr Ala Asn Val
80 85 90
Gln His Arg Tyr Gln Gln Leu Gln Arg Leu Val His Pro Asp Phe
95 100 105
Phe Ser Gln Arg Ser Gln Thr Glu Lys Asp Phe Ser Glu Lys His
110 115 120
Ser Thr Leu Val Asn Asp Ala Tyr Lys Thr Leu Leu Ala Pro Leu
125 130 135
Ser Arg Gly Leu Tyr Leu Leu Lys Leu His Gly Ile Glu Ile Pro
140 145 150
Glu Arg Thr Asp Tyr Glu Met Asp Arg Gln Phe Leu Ile Glu Ile
155 160 165
Met Glu Ile Asn Glu Lys Leu Ala Glu Ala Glu Ser Glu Ala Ala
170 175 180
Met Lys Glu Ile Glu Ser Ile Val Lys Ala Lys Gln Lys Glu Phe
185 190 195
Thr Asp Asn Val Ser Ser Ala Phe Glu Gln Asp Asp Phe Glu Glu
200 205 210
Ala Lys Glu Ile Leu Thr Lys Met Arg Tyr Phe Ser Asn Ile Glu
215 220 225
Glu Lys Ile Lys Leu Lys Lys Ile Pro Leu
230 235
<210> 10
<211> 260
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5680480CD1
<400> 10
Met Gly Leu Leu Asp Leu Cys Glu Glu Val Phe Gly Thr Ala Asp
1 5 10 15
Leu Tyr Arg Val Leu Gly Val Arg Arg Glu Ala Ser Asp Gly Glu
20 25 30
Val Arg Arg Gly Tyr His Lys Val Ser Leu Gln Val His Pro Asp
35 40 45
8/ 16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
Arg Val Gly Glu Gly Asp Lys Glu Asp Ala Thr Arg Arg Phe Gln
50 55 60
Ile Leu Gly Lys Val Tyr Ser Val Leu Ser Asp Arg Glu Gln Arg
65 70 75
Ala Val Tyr Asp Glu Gln Gly Thr Val Asp Glu Asp Ser Pro Val
80 85 90
Leu Thr Gln Asp Arg Asp Trp Glu Ala Tyr Trp Arg Leu Leu Phe
95 100 105
Lys Lys Ile Ser Leu Glu Asp Ile Gln Ala Phe Glu Lys Thr Tyr
110 115 120
Lys Gly Ser Glu Glu Glu Leu Ala Asp Ile Lys Gln Ala Tyr Leu
125 130 135
Asp Phe Lys Gly Asp Met Asp Gln Ile Met Glu Ser Val Leu Cys
140 145 150
Val Gln Tyr Thr Glu Glu Pro Arg Ile Arg Asn Ile Ile Gln Gln
155 160 165
Ala Ile Asp Ala Gly Glu Val Pro Ser Tyr Asn Ala Phe Val Lys
170 175 180
Glu Ser Lys Gln Lys Met Asn Ala Arg Lys Arg Arg Ala Gln Glu
185 190 195
Glu Ala Lys Glu Ala Glu Met Ser Arg Lys Glu Leu Gly Leu Asp
200 205 210
Glu Gly Val Asp Ser Leu Lys Ala Ala Ile Gln Ser Arg Gln Lys
215 220 225
Asp Arg Gln Lys Glu Met Asp Asn Phe Leu Ala Gln Met Glu Ala
230 235 240
Lys Tyr Cys Lys Ser Ser Lys Gly Gly Gly Lys Lys Ser Ala Leu
245 250 255
Lys Lys Glu Lys Lys
260
<210> 11
<211> 269
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1459372CD1
<400> 11
Met Ala Gly Val Pro Glu Asp Glu Leu Asn Pro Phe His Val Leu
1 5 10 15
Gly Val Glu Ala Thr Ala Ser Asp Val Glu Leu Lys Lys Ala Tyr
20 25 30
Arg Gln Leu Ala Val Met Val His Pro Asp Lys Asn His His Pro
35 40 45
Arg Ala Glu Glu Ala Phe Lys Val Leu Arg Ala Ala Trp Asp Ile
50 55 60
Val Ser Asn Ala Glu Lys Arg Lys Glu Tyr Glu Met Lys Arg Met
65 70 75
Ala Glu Asn Glu Leu Ser Arg Ser Val Asn Glu Phe Leu Ser Lys
80 85 90
Leu Gln Asp Asp Leu Lys Glu Ala Met Asn Thr Met Met Cys Ser
95 100 105
Arg Cys Gln Gly Lys His Arg Arg Phe Glu Met Asp Arg Glu Pro
110 115 120
Lys Ser Ala Arg Tyr Cys Ala Glu Cys Asn Arg Leu His Pro Ala
125 130 135
Glu Glu Gly Asp Phe Trp Ala Glu Ser Ser Met Leu Gly Leu Lys
140 145 150
Ile Thr Tyr Phe Ala Leu Met Asp Gly Lys Val Tyr Asp Ile Thr
155 160 165
Glu Trp Ala Gly Cys Gln Arg Val Gly Ile Ser Pro Asp Thr His
170 175 180
Arg Val Pro Tyr His Ile Ser Phe Gly Ser Arg Ile Pro Gly Thr
185 190 195
Arg Gly Arg Gln Arg Ala Thr Pro Asp Ala Pro Pro Ala Asp Leu
9/ 16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
200 205 210
Gln Asp Phe Leu Ser Arg Ile Phe Gln Val Pro Pro Gly Gln Met
215 220 225
Pro Asn Gly Asn Phe Phe Ala Ala Pro Gln Pro Ala Pro Gly Ala
230 235 240
Ala Ala Ala Ser Lys Pro Asn Ser Thr Val Pro Lys Gly Glu Ala
245 250 255
Lys Pro Lys Arg Arg Lys Lys Val Arg Arg Pro Phe Gln Arg
260 265
<210> 12
<211> 1550
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 723593CB1
<400> 12
gtcggagcct ggcacgctcg cccagaggcc tgcgcccaca ccctctcctg tccagccctc 60
gcccgcctgg gcagggcccg gcgccgtccg tggatgagcc acagaacctc ttccaccttc 120
cgagcggaga gaagtttcca ttcctcttct tcttcctcct cctcttccac ctcctcctcg 180
gcctcccgtg ccctcccggc ccaggacccg cccatggaga aggccctgag catgttttcc 240
gatgactttg gcagcttcat gcggccccac tcggagcccc tggccttccc agcccgcccc 300
ggtggggcag gcaacatcaa gaccctagga gacgcctatg agtttgcggt ggacgtgaga 360
gacttctcac ctgaagacat cattgtcacc acctccaaca accacatcga ggtgcgggct 420
gagaagctgg cggctgacgg cactgtcatg aacaccttcg ctcacaagtg ccagctgccg 480
gaggacgtgg acccgacgtc ggtgacctcg gctctgcggg aggacggcag cctcactatc 540
cgggcacggc gtcacccgca tacagaacac gtccagcaga ccttccggac ggagatcaaa 600
atctgagtgc ctctcccttc cctttccctg tccccccgcc ccacgcctgc cagcaaagcc 660
tcgctaaccc cattacaaca gctccaggac atctcagccc aggttctagc ccccacgcac 720
cccagacccc aggtggacca tcctcccaaa ctagggccct ccactctatc cagggcaggc 780
cagggactcc ctggcctgac acatgatgcc cagatttcag atttggcctc cgtcacttaa 840
tccagagtac aggggctggg gtcagggaag gaagatctaa agaacccact gtgggtcagg 900
ggaatgggac cagcaggaca tatgggcaag ctctgcagga cagacagaca gacaaaccct 960
ctgatctatg aagtctctgc agggcaaggg gaccagggac ctggaaccct cttggccaag 1020
gggagtggga gggacagagg gaaggtcaca ggcaagggtg cctatctaag tggaactaat 1080
tgcccgaggg ctcagcaagg ccaagaggag acagccgtga cggtaaactt cccctctacc 1140
agcctccaag ccccacgcca gcgagcaggc tgcctgccca ccccgtgccc ccagccagct 1200
ggctgtgcca gggcagagcc atgccacatc tgtatataga tggggttttt ccaatacagc 1260
tggttcgtga taaactgcat gaaactcctg ccgtcctgcg cctgctgggg cctccaggca 1320
aggccacgtg gggttggggg tggggctggt ccttctccct cccacaggcc tgtgttcttg 1380
gggctgctcc catgcagaca ggatcaccta acagagatgg aagccagggc atggatgggg 1440
ctttgggtcc tcaaggttgg accccagctt cttgccacct tcccctccgg gcagtcagct 1500
ctccatccat ccccctcttt aatctatgaa tctataggct cggtgtgtgt 1550
<210> 13
<211> 1075
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1708350CB1
<400> 13
cagaacacaa ttcccagagg gctaggcgcc gctcggagcc tgcagtcctc acgcgcgctt 60
agactcttgg gagttgtagt acgaatccgt caggccggaa ccatggcagt gaccaaggag 120
ctcttacaga tggacctgta cgcgctgcta ggcattgagg agaaggcagc ggacaaagag 180
gtaaagaagg cgtataggca gaaggccctc tcctgccacc cagacaaaaa tccagataat 240
cccagagcag ctgaactctt ccaccagctt tctcaggcct tggaggtgct gaccgatgct 300
gcagccaggg ctgcatatga caaggtcagg aaagccaaga agcaagcagc agagaggacc 360
cagaaacttg atgagaaaag gaagaaagtg aagcttgacc tggaggcccg ggagcggcag 420
gcccaggccc aggagagtga ggaggaagag gagagccgga gcaccaggac actagagcaa 480
gagatcgaac gcctgagaga agagggttcc cggcagctgg aggaacagca gaggctcatc 540
cgggagcaga tacgccagga gcgtgaccag aggttgagag gaaaggcaga aaatactgaa 600
ggccaaggaa cccccaaact aaagctaaaa tggaagtgca agaaggagga tgagtcaaaa 660
10/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
ggtggctact ccaaagacgt cctcctacgg cttttgcaga agtatggtga ggttctcaac 720
ctggtgcttt ccagtaagaa gccaggcact gctgtggtgg agtttgcaac cgtcaaggca 780
gcggagctgg ctgtccagaa tgaagttggc ctggtggata accctctgaa gatttcctgg 840
ttggagggac agccccagga tgccgtgggc cgcagccact caggactgtc aaagggctca 900
gtgctgtcag agagggacta cgagagcctc gtcatgatgc gcatgcgcca ggcggccgag 960
cggcaacagc tgatcgcacg gatgcagcag gaagaccagg aggggccgcc tacgtagccc 1020
cagctccagc catccacccg tcagcccttt tcttcaacgt cgcgagataa attta 1075
<210> 14
<211> 1950
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1742550CB1
<400> 14
aagagagccc gaggcaggta gctgcagttc ccctccaaga cttctccaca cctgtttgac 60
caggtacaag atcaggcgcc ggggtcatct gttcactagg ccacggggtc aggacaagag 120
tcacccgcag ctctgaggcc agatggtaat tccaatcgcc tccccagttc agcagcgaac 180
ccagcaagac gaagataatt ttcgaaacat tcaggctcgg gagtagacgt cgcaatggag 240
tgctgtcctc gcggctttgg agccacgggg catggccaag gatgcatcat ctgcagacat 300
cagaaaagca tatcgtaagc tttcactaac tttacatcca gacaagaata aagatgaaaa 360
tgcagaaact cagtttagac aattggtggc catttatgaa gttttaaagg atgatgaacg 420
aaggcagagg tatgatgata ttctgatcaa tggacttcca gattggcgac agcctgtatt 480
ctactacagg cgggtgagaa aaatgagcaa tgctgagctg gcattactct tgttcattat 540
tctcacagtg ggtcattatg ctgtggtttg gtcaatctac ctggaaaaac aactggatga 600
actactaagt agaaaaaaga gagaaaagaa aaaaaagact ggcagcaaga gtgtggatgt 660
atcaaaactc ggtgcttcag aaaaaaatga aagattgctg atgaaaccac agtggcatga 720
tttgcttcca tgcaaactgg ggatttggtt ttgccttaca ctaaaagcat tacctcacct 780
catccaggat gctgggcagt tttatgctaa atataaagaa acaagattga aggaaaagga 840
agatgcactg actagaactg aacttgaaac acttcaaaaa cagaagaaag ttaaaaaacc 900
aaaacctgaa tttcctgtat acacaccttt agaaactaca tatattcagt cttatgatca 960
tggaacttcc atagaagaaa ttgaggaaca aatggatgat tggttggaaa acaggaaccg 1020
aacacagaaa aaacaggcac ctgaatggac agaagaggac ctcagccaac tgacaagaag 1080
tatggttaag ttcccaggag ggactccagg tcgatgggaa aagattgccc acgaattggg 1140
tcgatctgtg acagatgtga caaccaaagc caagcaactg aaggattcag tgacctgctc 1200
cccaggaatg gttagactct ccgaactcaa atcgacagtt cagaattcca ggcccatcaa 1260
aacggccacc accttgcccg atgacatgat cacccagcga gaggacgcag agggggtggc 1320
agcggaggag gagcaggagg gagactccgg tgagcaggag accggggcca ctgatgcccg 1380
gcctcggagg cggaagccag ccaggctgct ggaggctaca gcgaagccgg agccagagga 1440
gaagtccaga gccaagcggc agaaggactt tgacatagca gaacaaaacg agtccagcga 1500
cgaggagagc ctgagaaaag agagagctcg gtctgcagag gagccgtgga ctcaaaatca 1560
acagaaactt ctggaactgg cgttgcagca gtacccaagg ggatcctctg accgctggga 1620
caaaatagcc agatgtgtcc cgtccaagag caaggaagac tgtatcgcta ggtacaagtt 1680
gctggttgaa ctggtccaaa agaaaaaaca agctaaaagc tgaatattct gggagatgat 1740
gttcaccttc attttccaaa atgaatatct taaaaatctt atgcagaaat ttgcattttg 1800
tacctcaata tttctacgtc atgtgcctta gtaaaaaaaa ataataaata aataaaagat 1860
gagtgttgtg ctaaaaaaaa aaaaaaaaaa aaaaaactcg gtcgcaagct tattcccttt 1920
agtgagggtt aattttagct tgcactggcc 1950
<210> 15
<211> 1187
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1919301CB1
<400> 15
ctcttgcacc gcctgccgaa tcaattcaac atggcagcca tgcgctggcg atggtggcag 60
cggctgttac cttggaggtt gctgcaggcc cgtggctttc cacaaaattc tgcacccagc 120
ctgggcctag gagcgaggac ttattcccag ggcgactgct cgtattcgcg cacggcgctg 180
tatgatctgc tcggcgtccc ctccacagcc acgcaggccc aaatcaaggc ggcttactac 240
cgtcagtgct ttctctacca cccggaccgc aactccggga gcgcggaggc cgccgagcgc 300
11/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
ttcacgcgca tctcccaggc ctacgtggtg ctgggcagtg ccaccctccg tcgcaagtat 360
gatcgcggcc tactcagcga cgaggacctg cgcggacctg gcgtccggcc ctccaggacg 420
cccgcacccg accccggctc gccgcgtacc ccgccgccca cctctcggac ccacgacggt 480
tctcgggcct cccccggcgc caaccgcacg atgttcaact ttgacgcctt ctaccaggcc 540
cactatgggg aacaactgga gcgggaacgg cgcctgaggg cccggcggga ggcccttcgc 600
aaacggcagg agtatcggtc catgaaaggc ctccgctggg aggatacccg agacacggct 660
gccattttcc tcatcttttc aatcttcatc atcatcggct tttatattta atcggagaga 720
gaagggaagg ggagtgtccc cagccaaccc cccagaaacg gccttttttc ctgcctctga 780
acccttggcc gttgatagtc tacctttgct gggatccgaa ggaactgtac tccccctgcc 840
ctccccgacc cgcccagctt agccgatgac ctgcacatcg ctccactgtg gtccagaaaa 900
ggaggccttt cgatgtctga gaaagaggcc ccacgctgta gagtcccgaa agcccaggag 960
tgaagggggt tcctggagtc tctagggtgc ttcttccaga gtctgtcttc ttgcttccag 1020
atgtggtcaa cttctggaac actcgctgta gctttattgt ttagccccaa gcaagattta 1080
tctcctcctg ccccgcatgt gtatggtggg cctctgtaac cttgaaatgt gcaatgtgac 1140
caattgttga ctaccaaaag aaaaggtctg gggttgtaaa aaaaaaa 1187
<210> 16
<211> 740
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2012055CB1
<400> 16
cgaggagtgg gtagcagcgc ctatgtgaag ttagctaatc tgagaaggcc cacttctggt 60
tccatggatg atggcggttg agcagatgcc aaaaaaggat tggtacagca tcctgggagc 120
agacccatct gcaaatatat cagacctaaa acaaaaatat caaaaactca tattaatgta 180
tcatccagat aaacaaagta cagatgtacc agcaggaaca gtggaggaat gtgtacagaa 240
gttcatcgaa attgatcaag catggaaaat tctaggaaat gaagagacaa aaagagagta 300
tgacctgcag cggtgtgaag atgatctaag aaatgtagga ccagtagatg ctcaagtata 360
tcttgaagaa atgtcttgga atgaagttac ttctcagaga cagtaaaatg gaatgaccaa 420
tggatcagag attctttaag tcaaagggca caagcatttc aacttcccag gaaaatgaca 480
cacttaaaat ttccacgatc aggagcctaa gtattgcacc gtattgcctc ctttgggcat 540
ctcacttcag catcttgttg gttcatgtat catttgtaaa catcaaacac acacacacat 600
acccccatag atttaaaaaa acaacaacaa catggtgttg tgtttataga cttaagtcaa 660
gattcttgaa atagtgtgac actagaagag aaagtatcca gatgttgcat ttgataaata 720
gtctggcttt ctctaaagga 740
<210> 17
<211> 1361
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2238062CB1
<400> 17
tcgggcgcgg gggaggctcg gcggacctgc tgattgggaa ccgatatggc ggcgactctg 60
ggcagcgggg agcgctggac ggaagcttac attgacgcag ttagaagaaa caaataccca 120
gaagacacac ctcctgagag tcatgacccc tgtggctgct gtaactgcat gaaggcacaa 180
aaggaaaaga agtctgagaa tgagtggact cagacccggc agggtgaggg gaactccacg 240
tacagtgagg aacagctgct tggggtacaa aggatcaaga aatgcagaaa ttactatgaa 300
attctgggag tttctcgaga tgctagtgac gaagagctta agaaagctta cagaaaactc 360
gccctgaaat ttcaccctga caagaactgt gctcctggag caacagatgc tttcaaagca 420
ataggaaatg catttgcagt cctgagcaat cctgataaga gacttcgcta tgatgaatac 480
ggagatgaac aggtgacttt cactgcccct cgagccagac cttataatta ttacagggat 540
tttgaagctg acatcactcc agaagagctg ttcaacgtct tctttggagg acattttcct 600
acaggaaata ttcatatgtt ttcaaatgtg acagatgaca cttactatta ccgtcgacgg 660
caccgacatg agaggacaca gactcagaag gaggaggaag aagagaaacc tcagactaca 720
tattctgcat ttattcagct acttccagtt cttgtgattg tgattatatc tgtcattact 780
cagctgctgg ctactaatcc cccatatagt ctgttctata aatcgacctt gggctacacc 840
atttctagag aaactcagaa cctgcaggtg ccttactttg tggataaaaa ctttgacaag 900
gcctacagag gagcttctct gcatgacttg gagaaaacaa tagagaagga ttacattgat 960
tatatccaga ctagttgttg gaaggagaaa caacaaaagt cagagctgac aaatttggca 1020
12/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
ggattataca gagatgaacg attgaaacag aaagcagagt cgctgaaact tgaaaactgt 1080
gagaaacttt ccaaactcat tggcctacgc agaggtggct gagaggataa tggtcctacg 1140
cagggctggg gttttgctac ttgttcctat ttatgttcct gattccattt tataatacaa 1200
aactaggtaa tgatgaacac tttactattt gctaacttcg ttggttgggc agagtggcag 1260
gagcatgggc acgagagcca gatgtgtctt cacaggatcc ttcctgggga gtggctccag 1320
ggaccaggag tagttcatct aagttaaatt aatggcaagg c 1361
<210> 18
<211> 4475
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1825012CB1
<400> 18
cgcctctcga aggaagtttg ctcttaattt cagagccggg ttcgccgtcg gatcaacctc 60
caggagctag cagcgggcgc ggaccgggca gtttccgcgc tcagcacagg cagctcgcgg 120
tcatgggcgg ctcagcctcc agccagctgg acgagggcaa gtgcgcttac atccgaggga 180
aaactgaggc tgccatcaaa aacttcagtc cctactacag tcgtcagtac tctgtggctt 240
tctgcaatca cgtgcgcact gaagtagaac agcaaagaga tttaacgtca cagtttttga 300
agaccaagcc accattggcg cctggaacta ttttgtatga agcagagcta tcacaatttt 360
ctgaagacat aaagaagtgg aaggagagat acgttgtagt taaaaatgat tatgctgtgg 420
agagctatga gaataaagag gcctatcaga gaggagctgc tcctaaatgt cgaattcttc 480
cagccggtgg caaggtgtta acctcagaag atgaatataa tctgttgtct gacaggcatt 540
tcccagaccc tcttgcctcc agtgagaagg agaacactca gccctttgtg gtcctgccca 600
aggaattccc agtgtacctg tggcagccct tcttcagaca cggctacttc tgcttccacg 660
aggctgctga ccagaagagg tttagtgccc tcctgagtga ctgcgtcagg catctcaatc 720
atgattacat gaagcagatg acatttgaag cccaagcctt tttagaagct gtgcaattct 780
tccgacagga gaagggtcac tatggttcct gggaaatgat cactggggat gaaatccaga 840
tcctgagtaa cctggtgatg gaggagctcc tgcccactct tcagacagac ctgctgccta 900
agatgaaggg gaagaagaat gacagaaaga ggacgtggct tggtctcctc gaggaggcct 960
acaccctggt tcagcatcaa gtttcagaag gattaagtgc cttgaaggag gaatgcagag 1020
ctctgacaaa gggcctggaa ggaacgatcc gttctgacat ggatcagatt gtgaactcaa 1080
agaactattt aattggaaag atcaaagcga tggtggccca gccggcggag aaaagctgct 1140
tggagagtgt gcagccattc ctggcatcca tcctggagga gctcatggga ccagtgagct 1200
cgggattcag tgaagtacgt gtactctttg agaaagaggt gaatgaagtc agccagaact 1260
tccagaccac caaagacagt gtccagctaa aggagcatct agaccggctt atgaatcttc 1320'
cgctgcattc cgtgaagatg gaaccttgtt atactaaagt caacctgctt cacgagcgcc 1380
tgcaggatct caagagccgc ttcagattcc cccacattga tctggtggtt cagaggacac 1440
agaactacat gcaggagcta atggagaatg cagtgttcac ttttgagcag ttgctttccc 1500
cacatctcca aggagaggcc tccaaaactg cagttgccat tgagaaggtt aaactccgag 1560
tcttaaagca atatgattat gacagcagca ccatccgaaa gaagatattt caagaggcac 1620
tagttcaaat cacacttccc actgtgcaga aggcactggc gtccacatgc aaaccagagc 1680
ttcagaaata cgagcagttc atctttgcag atcataccaa tatgattcac gttgaaaatg 1740
tctatgagga gattttacat cagatcctgc ttgatgaaac tctgaaagtg ataaaggaag 1800
ctgctatctt gaagaaacac aacttatttg aagataacat ggccttgccc agtgaaagtg 1860
tgtccagctt aacagatcta aagcccccca cagggtcaaa ccaggccagc cctgccagga 1920
gagcttctgc cattctgcca ggagttctgg gtagtgagac cctcagtaac gaagtattcc 1980
aggagtcaga ggaagagaag cagcctgagg tccctagctc gttggccaaa ggagaaagcc 2040
tttctctccc tggcccaagc ccacccccag atgggactga gcaggtgatt atttcaagag 2100
tggatgaccc cgtggtgaat cctgtggcaa cagaggacac agcaggactc ccgggcacat 2160
gctcatcaga gctggagttt ggagggaccc ttgaggatga agaacccgcc caggaagagc 2220
cagaacccat cactgcctcg ggttctttga aggcgctcag aaagttgctg acagcgtccg 2280
tggaagtacc agtggactct gctccagtga tggaagaaga tacgaatggg gagagccacg 2340
ttccccaaga aaatgaagaa gaagaggaaa aagagcccag tcaggcagct gccatccacc 2400
ccgacaactg tgaagaaagt gaagtcagcg agagggaggc ccaacctccc tgtcccgagg 2460
cccatgggga ggagttgggg ggatttccag aggtaggcag cccagcctct ccgccagcca 2520
gtggagggct caccgaggag cccctggggc ccatggaggg ggagctccca ggagaggcct 2580
gcacactcac tgcccatgaa ggaagagggg gcaagtgtac cgaggaaggg gatgcctcac 2640
agcaagaggg ctgcacctta ggttctgacc ccatctgcct cagtgagagc caggtttctg 2700
aggaacaaga agagatggga gggcaaagca gcgcggccca ggccacggcc agtgtgaatg 2760
cagaggagat caaggtagcc cgtattcatg agtgtcagtg ggtggtggag gatgctccaa 2820
acccggatgt cctgctgtca cacaaagatg acgtgaagga gggagaaggt ggtcaggaga 2880
gtttcccaga gctgccctca gaggagtgaa agggacaatt tggctgaagt ctttctctga 2940
aaaaagccaa agggttatag gggtacactt aggggttgca tgcaagctgt taccaaaaaa 3000
13/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
tttttaagta ttttcttaat ttgaataata aaaccagagg aaatgcatac agggcatgag 3060
caactgaggc aaacctttgt ggacatgaat tgttctacga tgaatttttg ctttagtatt 3120
ttaataagaa ttacaaagac aatggcatac ttggggtgag agggagctga ggatgtctga 3180
ggagggaata gtattgcagg gaagactgag aaaacagtag gatgacagtt ttgagtatac 3240
tctgcacttt tcaattgtgc aatcttcttg tgcactttaa ggctttttaa ttttgtttga 3300
gaatgcaaat gtatactgta agtctacctt tactatctac tatgcctact tcaccatctc 3360
ttaaggactc ggcatttgtc cacagtcaga ctgcaagaga gggtaggtca tgaacagtca 3420
cccatgctgg ctgtagcccc cacagaggca atcatgccca atagattcaa gagaagctaa 3480
gcggaaatgg agggcggaag gtgtgatctg tgggactgtc tgggcctgtt actcatcctg 3540
ctatcaattt cttattaatt aatcttgatg attcttatta attaatcaca tttgcaggaa 3600
attcagatga ggcaagaaaa ttttattggc ctgggtaaga ctgaaagcat tccaaattag 3660
gcttagactg tgcaaagggc ttagctaagt tatcgagctt aaaacccgtc aattaaacaa 3720
acattatttg aacagttact gcatgccacg cactgtgttg ggcttagtaa taaaaaaaag 3780
aaaagataag tgcttgttct agcataaatt aaaaggtcca agggaattta atctggaaga 3840
gaacatatgc caatttttaa actatgacag cttttttttt tctctttcca ttcaaatagt 3900
cctggttcat tcccagaagg gcacaaaatg aatgaataaa taaataaatg aataaagaca 3960
aaagccaagg tgtatgctct caagttccaa agatgttatc aaaagctgaa atcatttgtt 4020
tggtcattca gcaagctaat tgagtctctg ttatatacca agcactgggg ataccatggc 4080
gaaaaacaac tttgttcctt cctcctagaa cttacatttt aatggaaata gacaaaacac 4140
atcttcttaa cggatggtga cctataacca ttaatgttga aaatggaaga gacttgcttc 4200
caaaagatta aaaggagttg ttcttttctc cttcagaaaa ataccagatc atttcctaaa 4260
atctccagtc ccaagtatta catcgtggtt tccctccccg actttttatt ttattttatt 4320
ctattttttt gagatggagt ctcactctgt cgccaaggct ggagtgcagt ggtgtgatct 4380
cggctcactg caacctccgc ctcctgggtt caggagtttc tcctctgtca gcgtcccaag 4440
tagctggaat tacagccatg cggcaccatt cccgg 4475
<210> 19
<211> 636
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1906464CB1
<400> 19
gaaccgagcg agcggagctg agctcgggta ggccgcgcga ggtccctcct ctccgggcgt 60
ccgtgcgcct agctctgcgc tgggagcctc gcgccctttg acagcagtta gttgctgact 120
cggatgcaga gagtcggtaa caccttctcc aacgagagcc gggtggcatc ccggtgtccc 180
agcgtgggcc ttgctgaacg gaaccgggtg gccacaatgc cggtgcggct gctcagggac 240
agtccagcgg ctcaggagga caatgaccat gccagagacg gtttccaaat gaagctggat 300
gcccacggct tcgccccgga ggaactggtg gtgcaggtgg atggccaatg gctgatggtg 360
accggacagc agcaactgga cgtcagggac ccggaaaggg tcagttaccg catgtcacag 420
aaggtgcacc ggaaaatgct cccgtccaac ctgagtccta ccgccatgac ctgctgcctg 480
accccctccg ggcagctgtg ggtcagaggc cagtgtgtgg cgctggccct ccctgaagcc 540
caaacaggac cgtccccgag actcgggagc ctcggctcta aggcttccaa cctgacccgg 600
taaacaaacg acgcgatgtg cagcaaaaaa aaaaaa 636
<210> 20
<211> 1090
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1979146CB1
<400> 20
gcttttcccc acgagtgacc acggctagat aggccgccgg ccagatgtgg cgggggagag 60
ccggggcttt gctccgggtg tgggggtttt ggccgacagg ggttcccaga aggagaccgc 120
taagctgcga tgctgcgtcg caggcgggaa gcaattatcc ccgctgttgg aactgcggcg 180
gcccatgggg ccccgggcgg gaggacaggt tcttctgccc acagtgccga gcgctgcagg 240
cacctgaccc cactcgagac tacttcagcc ttatggactg caaccgttcc ttcagagttg 300
atacagcgaa cgtccagcac aggtaccagc aactgcagcg tcttgtccac ccagatttct 360
tcagccagag gtctcagact gaaaaggact tctcagagaa gcattcgacc ctggtgaatg 420
atgcctataa gaccctcctg gcccccctga gcagaggact gtaccttcta aagctccatg 480
gaatagagat tcctgaaagg acagattatg aaatggacag gcaattcctc atagaaataa 540
14/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
tggaaatcaa tgaaaaactc gcagaagctg aaagtgaagc tgccatgaaa gagattgaat 600
ccattgtcaa agctaaacag aaagaattta ctgacaatgt gagcagtgct tttgaacaag 660
atgactttga agaagccaag gaaattttga caaagatgag atacttttca aatatagaag 720
aaaagatcaa gttaaagaag attccccttt aattgtggat agtttaaagt ttaaaaaata 780
aagttcttgc tgggcacagt ggctcacacc tgtaatccca gcactttggg aggctgaggt 840
gggtggatga caaggtcagg agttcaagac cagcttggcc aacatagtga aaccccgtct 900
ctgctgaaaa tacaaaaatt agccgggcat ggtggcgcgt gcctgtaatc ccagctactt 960
ggtaggccga ggcaggagaa tcgcttaaac ccgtgaggtg gaggttgcag tgagcagaga 1020
tcacgcaact gcactccagc ttgggcaaca gagtgagctt aatcttgaaa aataaataaa 1080
tgaaaatgat 1090
<210> 21
<211> 1447
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5680480CB1
<400> 21
cgaaaaagaa gcagtcctgg gttgtacccg gcgcacgtgg gagcggctgc ttcctccggg 60
gtcgtatctc cgcccggcat ggggctgctg gacctttgcg aggaagtgtt cggcaccgcc 120
gacctttacc gggtgctggg cgtgcgacgc gaggcctccg acggcgaggt ccgacgaggc 180
taccacaagg tgtccctgca ggtacacccg gaccgggtgg gtgagggcga caaggaggac 240
gccacccgcc gcttccagat cctgggaaaa gtctattccg ttctcagtga cagagaacag 300
agagcagtgt acgatgagca gggaacagtg gacgaggact ctcctgtgct cacccaagac 360
cgagactggg aggcgtattg gcggctactc tttaaaaaga tatctttaga ggacattcaa 420
gcttttgaaa agacatacaa aggttcggaa gaagagctgg ctgatattaa gcaggcctat 480
ctggacttca agggtgacat ggatcagatc atggagtctg tgctttgcgt gcagtacaca 540
gaggaaccca ggataaggaa tatcattcag caagctattg acgccggaga ggtcccatcc 600
tataatgcct ttgtcaaaga atcgaaacaa aagatgaatg caaggaaaag gagggctcag 660
gaagaggcca aagaagcaga aatgagcaga aaggagttgg ggcttgatga aggcgtggat 720
agcctgaagg cagccattca gagcagacaa aaggatcggc aaaaggaaat ggacaatttt 780
ctggctcaga tggaagcaaa gtactgcaaa tcttccaaag gaggagggaa aaaatctgct 840
ctcaagaaag aaaagaaata atggaatttt tctcttcaaa ggtccttagg tgtaaattga 900
tgccatcgta ggcaaggtgc aggcaggatt tgaaggcaaa agtcaattca gctcttgaga 960
aaaggtgtct ttccagcctg aatttttcag attgactaga ccaagcagaa tctctcaacc 1020
tgatcttagt atttcctaga aagcacttga cattgtgtga ggtctcacct gaaggaactt 1080
ggtggtgaca tttgggaggg tggagggagg cagtgtcctt cctgacagca cttgcctcca 1140
tggatcttct gtacacagaa ctcttatcta ggatgtggtt ctgttcatgc tgctttctgc 1200
gatgtgcgtg tctgttagaa taggctctct acccagctag aacaccttcc agacacttgc 1260
tggacagcta tcttccacat acttcccagt ttacatttgg tcttaatgat cttgaataga 1320
tcctctcttc attttactca gccaggtttt gtactgatgt acaggtgtta aattacttca 1380
agcatttttg taagaggtgt atataattca ataaaaaagg taaaacatga tgattaaaaa 1440
aaaaaaa 1447
<210> 22
<211> 1147
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1459372CB1
<400> 22
gccttgggtc aagcagaata ttaataggca ggggaatgca cctgtagcta gtgggcgcta 60
ctgccagcct gaagaggaag tggctcgact cttgaccatg gctggggttc ctgaggatga 120
gctaaaccct ttccatgtac tgggggttga ggccacagca tcagatgttg aactgaagaa 180
ggcctataga cagctggcag tgatggttca tcctgacaaa aatcatcatc cccgggctga 240
ggaggccttc aaggttttgc gagcagcttg ggacattgtc agcaatgctg aaaagcgaaa 300
ggagtatgag atgaaacgaa tggcagagaa tgagctgagc cggtcagtaa atgagtttct 360
gtccaagctg caagatgacc tcaaggaggc aatgaatact atgatgtgta gccgatgcca 420
aggaaagcat aggaggtttg aaatggaccg ggaacctaag agtgccagat actgtgctga 480
gtgtaatagg ctgcatcctg ctgaggaagg agacttttgg gcagagtcaa gcatgttggg 540
cctcaagatc acctactttg cactgatgga tggaaaggtg tatgacatca cagagtgggc 600
15/16
CA 02374743 2002-O1-25
WO 01/09178 PCT/US00/21313
tggatgccag cgtgtaggta tctccccaga tacccacaga gtcccctatc acatctcatt 660
tggttctcgg attccaggca ccagagggcg gcagagagcc accccagatg cccctcctgc 720
tgatcttcag gatttcttga gtcggatctt tcaagtaccc ccagggcaga tgcccaatgg 780
gaacttcttt gcagctcctc agcctgcccc tggagccgct gcagcctcta agcccaacag 840
cacagtaccc aagggagaag ccaaacctaa gcggcggaag aaagtgagga ggcccttcca 900
acgttgatgc cccttctctt tcctcaaatc aatgtcaggg agtcaaaagg gctgtagcac 960
aggatggagt ttgatttatc cctcctcccc caacacctag gaactgaatc tttttctttt 1020
tattttttga gatggagtct tgctctgttg cccagctgga gtgcagtggt gtgatctcag 1080
cttactgcaa cctctgtctc ccgggttcaa gcaattctcc catctcagcc tcctgagtag 1140
ctgggat 1147
16/16
