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FULL-LENGTH MOLECULES EXPRESSED IN HUMAN TISSUES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of full-length
molecules
expressed in human tissues and to the use of these sequences in the analysis
of human gene expression,
genetic linkage, and genetic variability, and in the diagnosis, treatment, and
prevention of
developmental, cell proliferative, and immunological disorders.
BACKGROUND OF THE INVENTION
It is estimated that only 2'70 of mammalian DNA encodes proteins, and only a
small fraction
of the genes that encode proteins is actually expressed in a particular cell
at any time. The various
types of cells in a multicellular organism differ dramatically both in
structure and function, and the
identity of a particular cell is conferred by its unique pattern of gene
expression. In addition, different
cell types express overlapping but distinctive sets of genes throughout
development. Cell growth and
1~ proliferation, cell differentiation, the immune response, apoptosis, and
other processes that contribute
to organism development and survival are governed by regulation of gene
expression. Appropriate
gene regulation also ensures that cells function efficiently by expressing
only those genes whose
functions are required at a given time. Factors that influence gene expression
include extracellular
signals that mediate cell-cell communication and coordinate the activities of
different cell types.
Gene expression is regulated at the level of DNA and RNA transcription, and at
the level of mRNA
translation.
Aberrant expression or mutations in genes and their products may cause, or
increase
susceptibility to, a variety of human diseases such as cancer and other cell
proliferative disorders.
The identification of these genes and their products is the basis of an ever-
expanding effort to finding
markers for early detection of diseases and targets for their prevention and
treatment. For example,
cancer represents a type of cell proliferative disorder that affects nearly
every tissue in the body. The
development of cancer, or oncogenesis, is often correlated with the conversion
of a normal gene into a
cancer-causing gene, or oncogene, through abnormal expression or mutation.
Oncoproteins, the
products of oncogenes, include a variety of molecules that influence cell
proliferation, such as growth
factors, growth factor receptors, intracellular signal transducers, nuclear
transcription factors, and
cell-cycle control proteins. In contrast, tumor-suppressor genes are involved
in inhibiting cell
proliferation. Mutations which reduce or abrogate the function of tumor-
suppressor genes result in
aberrant cell proliferation and cancer. Thus a wide variety of genes and their
products have been
found that are associated with cell proliferative disorders such as cancer,
but many more may exist
that are yet to be discovered.
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DNA-based arrays can provide an efficient, high-throughput method to examine
gene
expression and genetic variability. For example, SNPs, or single nucleotide
polymorphisms, are the
most common type of human genetic variation. DNA-based arrays can dramatically
accelerate the
discovery of SNPs in hundreds and even thousands of genes. Likewise, such
arrays can be used for
SNP genotyping in which DNA samples from individuals or populations are
assayed for the presence
of selected SNPs. These approaches will ultimately lead to the systematic
identification of all genetic
variations in the human genome and the correlation of certain genetic
variations with disease
susceptibility, responsiveness to drug treatments. and other medically
relevant information. (See, for
example, Wang, D.G. et al. (1998) Science 280:1077-1082.)
DNA-based array technology is especially important for the rapid analysis of
global gene
expression patterns. For example, genetic predisposition, disease, or
therapeutic treatment may
directly or indirectly affect the expression of a large number of genes in a
given tissue. In this case, it
is useful to develop a profile, or transcript image, of all the genes that are
expressed and the levels at
which they are expressed in that particular tissue. A profile generated from
an individual or
population affected with a certain disease or undergoing a particular therapy
may be compared with a
profile likewise generated from a control individual or population. Such
analysis does not require
knowledge of gene function, as the expression profiles can subjected to
mathematical analyses which
simply treat each gene as a marker. Furthermore, gene expression profiles may
help dissect biological
pathways by identifying all the genes expressed, for example, at a certain
developmental stage, in a
particular tissue, or in response to disease or treatment. (See, for example,
Lander, E.S. et al. (1996)
Science 274:536-539.)
The discovery of new full-length molecules expressed in human tissues and the
polynucleotides
encoding them satisfies a need in the art by providing new compositions which
are useful in the analysis
of human gene expression, genetic linkage, and genetic variability, and in the
diagnosis, prevention, and
treatment of developmental, cell proliferative, and immunological disorders.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, full-length molecules expressed
in human tissues,
referred to collectively as ''FLEXHT" and individually as "FLEXHT-1,'' "FLEXHT-
2," "FLEXHT-3,"
"FLEXHT-4," "FLEXHT-5," "FLEXHT-6," ''FLEXHT-7," "FLEXHT-8,'' "FLEXHT-9,''
''FLEXHT-10," "FLEXHT-11," "FLEXHT-12," "FLEXHT-13," "FLEXHT-14," "FLEXHT-15,"
"FLEXHT-16," ''FLEXHT-17," ''FLEXHT-18,'' "FLEXHT-19," ''FLEXHT-20," "FLEXHT-
21,''
''FLEXHT-22," "FLEXHT-23," "FLEXHT-24,'' "FLEXHT-25,'' "FLEXHT-26," "FLEXHT-
27,"
''FLEXHT-28,'' "FLEXHT-29," "FLEXHT-30," ''FLEXHT-31,'' ''FLEXHT-32," "FLEXHT-
33,''
"FLEXHT-34," "FLEXHT-35," "FLEXHT-36," ''FLEXHT-37,'' "FLEXHT-38," ''FLEXHT-
39,"
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"FLEXHT-40," "FLEXHT-41," "FLEXHT-42." ''FLEXHT-43," ''FLEXHT-44," "FLEXHT-
45,"
''FLEXHT-46," ''FLEXHT-47," "FLEXHT-48," ''FLEXHT-49," "FLEXHT-50," "FLEXHT-
51,"
"FLEXHT-52," ''FLEXHT-53," ''FLEXHT-54," and ''FLEXHT-55." 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-
55, 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:1-55, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-55, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:1-
55. In one alternative,
the invention provides an isolated polypeptide comprising the amino acid
sequence of SEQ ID NO:1-55.
The invention further provides an isolated polynucleotide encoding 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:1-55, 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-
55, c) a biologically active fragment of an amino acid sequence selected from
the group consisting of
SEQ ID NO:1-55, and d) an immunogenic fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-55. In one alternative, the polynucleotide encodes a
polypeptide selected
from the group consisting of SEQ ID NO:1-55. In another alternative, the
polynucleotide is selected
from the group consisting of SEQ ID N0:56-110.
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:1-55, 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:1-55, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
NO:1-5~, and d) an immunogenic fiagment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-55. In one alternative, the invention provides a
cell transformed with the
recombinant polynucleotide. In another alternative, the invention provides a
transgenic organism
comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:l-55, 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-55, c)
a biologically active fragment of an amino acid sequence selected from the
group consisting of SEQ ID
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NO:1-~~. and d) an immunogenic fragment of an amino acid sequence selected
from the group
consisting of SEQ ID NO:l-55. 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:1-55, 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-55, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-55, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-55.
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 N0:56-110, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:56-110, 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 N0:56-110, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:56-110, 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
4
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selected from the group consisting of a) a polynucleotide sequence selected
from the group consisting of
SEQ ID N0:56-110, b) a naturally occurring polynucleotide sequence having at
least 90% sequence
identity to a polynucleotide sequence selected from the group consisting of
SEQ ID N0:56-110, c) a
polynucleotide sequence complementary to a), d) a polynucleotide sequence
complementary to b), and e)
an RNA equivalent of a)-d). The method comprises a) amplifying said target
polynucleotide or
fragment thereof using 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 pharmaceutical composition comprising an
effective amount
of a polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:l-55, b) a
naturally occurring amino
acid sequence having at least 90'70 sequence identity to an amino acid
sequence selected from the group
consisting of SEQ ID NO:1-55, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-55, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-55, and a
pharmaceutically acceptable
excipient. In one embodiment, the pharmaceutical composition comprises an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-55. The invention
additionally provides a method
of treating a disease or condition associated with decreased expression of
functional FLEXHT,
comprising administering to a patient in need of such treatment the
pharmaceutical composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID NO:1-55, 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:1-55, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:l-55, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:l-55. The method
comprises a)
exposing a sample comprising the polypeptide to a compound, and b) detecting
agonist activity in the
sample. In one alternative, the invention provides a pharmaceutical
composition comprising an agonist
compound identified by the method and a pharmaceutically acceptable excipient.
In another
alternative, the invention provides a method of treating a disease or
condition associated with
decreased expression of functional FT.EXHT, comprising administering to a
patient in need of such
treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising 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-
55, b) a naturally
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occurring amino acid sequence having at least 90% sequence identity to an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-55, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-55, and d) an
immunogenic fragment
of an amino acid sequence selected from the group consisting of SEQ ID NO:I-
55. The method
comprises a) exposing a sample comprising the polypeptide to a compound, and
b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a pharmaceutical
composition comprising an antagonist compound identified by the method and a
pharmaceutically
acceptable excipient. In another alternative, the invention provides a method
of treating a disease or
condition associated with overexpression of functional FLEXHT, comprising
administering to a
patient in need of such treatment the pharmaceutical 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:1-55, 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-55, c) a biologically active fragment of an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-55, and d) an immunogenic fragment of
an amino acid
sequence selected from the group consisting of SEQ ID NO:1-55. The method
comprises a) combining
the polypeptide with at least one test compound under suitable conditions, and
b) detecting binding
of the polypeptide to the test compound, thereby identifying a compound that
specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-55,
b) a naturally
occurnng amino acid sequence having at least 90% sequence identity to an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-55, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-55, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-55. 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
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sequence selected from the group consisting of SEQ ID N0:56-I 10, 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 an isolated polynucleotide comprising at least
20 contiguous
nucleotides of a polynucleotide comprising a polynucleotide sequence selected
from the group consisting
of a) a polynucleotide sequence which is complementary to a polynucleotide
sequence selected from the
group consisting of SEQ ID N0:56-110, and b) an RNA equivalent of a). The
invention also provides
a composition comprising at least one of these isolated polynucleotides and a
detectable label, said
composition being useful for the detection of altered expression of human
FLEXHT.
The invention further provides a microarray wherein at least one element of
the microarray is
an isolated polynucleotide comprising at least 20 contiguous nucleotides of a
polynucleotide comprising
a polynucleotide sequence selected from the group consisting of a) a
polynucleotide sequence which is
complementary to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:56-110,
and b) an RNA equivalent of a). The invention also provides a method of using
the microarray for
generating a transcript image of a sample which contains polynucleotides. The
method comprises a)
labeling the polynucleotides of the sample, b) contacting the elements of the
microarray with the labeled
polynucleotides of the sample under conditions suitable for the formation of a
hybridization complex,
and c) quantifying the expression of the polynucleotides in the sample.
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 FLEXHT.
Table 2 shows features of each polypeptide sequence, including protein
phosphorylation and
glycosylation sites.
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 FLEXHT were isolated.
Table 5 shows features of selected polypeptide sequences, including the
identity of the
polypeptide, potential motifs, homologous sequences, and methods, algorithms,
and searchable
databases used for analysis of FLEXHT.
Table 6 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold parameters.
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DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
~ may vary. It is also to be understood that the terminology used herein is
for the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which will
be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms ''a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell'' includes a plurality of such host cells, and a
reference to ''an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same meanings
as commonly understood by one of ordinary skill in the art to which this
invention belongs. Although
any machines, materials, and methods similar or equivalent to those described
herein can be used to
practice or test the present invention, the preferred machines, materials and
methods are now described.
All publications mentioned herein are cited for the purpose of describing and
disclosing the cell lines,
protocols, reagents and vectors which are reported in the publications and
which might be used in
connection with the invention. Nothing herein is to be construed as an
admission that the invention is
not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"FLEXHT'' refers to the amino acid sequences of substantially purified FLEXHT
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
FLEXHT. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of FLEXHT either by
directly interacting with
FLEXHT or by acting on components of the biological pathway in which FLEXHT
participates.
An "allelic variant'' is an alternative form of the gene encoding FLEXHT.
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
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a given sequence.
''Altered" nucleic acid sequences encoding FLEXHT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as FLEXHT or a
polypeptide with at least one functional characteristic of FLEXHT. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe of
the polynucleotide encoding FLEXHT, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
FLEXHT. 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 FLEXHT. 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 FLEXHT is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence'' refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or synthetic
molecules. Where ''amino acid sequence'' is recited to refer to a sequence of
a naturally occurring
protein molecule, ''amino acid sequence" and like terms are not meant to limit
the amino acid sequence
to the complete native amino acid sequence associated with the recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well known
in the art.
The term "antagonist' refers to a molecule which inhibits or attenuates the
biological activity of
FLEXHT. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of FLEXHT either by
directly interacting with FLEXHT or by acting on components of the biological
pathway in which
FLEXHT participates.
The term "antibody ' refers to intact immunoglobulin molecules as well as to
fragments thereof,
such as Fab, F(ab')~, and Fv fragments, which are capable of binding an
epitopic determinant.
Antibodies that bind FLEXHT 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
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translation of RNA, or synthesized chemically. and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies which
bind specifically to antigenic determinants (particular regions or three-
dimensional structures on the
protein). An antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to
elicit the immune response) for binding to an antibody.
The term "antisense'' refers to any composition capable of base-pairing with
the ''sense '
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA; RNA:
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages
such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus'' can refer to the antisense
strand, and the
designation ''positive'' or "plus" can refer to the sense strand of a
reference DNA molecule.
The term ''biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise. "immunologically
active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic FLEXHT, or
of any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with specific
antibodies.
''Complementary'' describes the relationship between two single-stranded
nucleic acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A ''composition comprising a given polynucleotide sequence" and a
''composition comprising a
given amino acid sequence'' refer broadly to any composition containing the
given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding FLEXHT or fragments
of FLEXHT may
be employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
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associated with a stabilizing went such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCl), detergents
(e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
"Consensus sequence'' refers to a nucleic acid sequence which has been
subjected to repeated
~ DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR
kit (Perkin-Elmer,
Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has
been assembled from one
or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for fragment
assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or
Phrap (University
of Washington, Seattle WA). Some sequences have been both extended and
assembled to produce the
consensus sequence.
"Conservative amino acid substitutions'' are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of the
protein is conserved and not significantly changed by such substitutions. The
table below shows amino
acids which may be substituted for an original amino acid in a protein and
which are regarded as
conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of the
side chain.
A "deletion'' refers to a change in the amino acid or nucleotide sequence that
results in the
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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 FLEXHT or the polynucleotide encoding
FLEXHT
which is identical in sequence to but shorter in length than the parent
sequence. A fragment may
comprise up to the entire length of the defined sequence, minus one
nucleotide/amino acid residue.
For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or
amino acid residues.
A fragment used as a probe, primer, antigen, therapeutic molecule, or for
other purposes, may be at
least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500
contiguous nucleotides or
amino acid residues in length. Fragments may be preferentially selected from
certain regions of a
molecule. For example, a polypeptide fragment may comprise a certain length of
contiguous amino
acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a
polypeptide) as shown
in a certain defined sequence. Clearly these lengths are exemplary, and any
length that is supported
by the specification, including the Sequence Listing, tables, and figures, may
be encompassed by the
present embodiments.
A fragment of SEQ ID N0:56-110 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:56-110, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:56-110 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:56-110 from related polynucleotide sequences. The precise length of
a fragment of SEQ
ID N0:56-110 and the region of SEQ ID N0:56-110 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:1-55 is encoded by a fragment of SEQ ID N0:56-110. A
fragment of SEQ ID NO:1-55 comprises a region of unique amino acid sequence
that specifically
identities SEQ ID NO:1-55. For example, a fragment of SEQ ID NO:1-55 is useful
as an
immunogenic peptide for the development of antibodies that specifically
recognize SEQ ID NO:l-55.
The precise length of a fragment of SEQ ID NO:1-55 and the region of SEQ ID
NO:1-55 to which the
fragment corresponds are routinely determinable by one of ordinary skill in
the art based on the
intended purpose for the fragment.
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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.
1~ and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters
are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved''=4. The "weighted"
residue weight table is
selected as the default. Percent identity is reported by CLUSTAL V as the
''percent similarity" between
aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms is
provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment Search
Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which
is available from several
sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence analysis
programs including "blastn,'' that is used to align a known polynucleotide
sequence with other
polynucleotide sequences from a variety of databases. Also available is a tool
called "BLAST 2
Sequences'' that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The
"BLAST 2 Sequences'' tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSL1M62
Reward for match: 1
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Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off:' S0
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example, as
defined by a particular SEQ ID number, or may be measured over a shorter
length, for example, over
the length of a fragment taken from a larger, defined sequence, for instance,
a fragment of at least 20, at
least 30, at least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such
lengths are exemplary only, and it is understood that any fragment length
supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be used to
describe a length over which
percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes in
a nucleic acid sequence can be made using this degeneracy to produce multiple
nucleic acid sequences
that all encode substantially the same protein.
The phrases ''percent identity" and ''% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some alignment
methods take into account conservative amino acid substitutions. Such
conservative substitutions,
explained in more detail above, generally preserve the charge and
hydrophobicity at the site of
substitution, thus preserving the structure (and therefore function) of the
polypeptide.
Percent identity between polypeptide sequences may be determined using the
default parameters
of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e
sequence alignment
program (described and referenced above). For pairwise alignments of
polypeptide sequences using
CLUSTAL V, the default parameters are set as follows: Ktuple-=1, gap
penalty=3, window=5, and
"diagonals saved''=5. The PAM250 matrix is selected as the default residue
weight table. As with
polynucleotide alignments, the percent identity is reported by CLUSTAL V as
the ''percent similarity'
between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12
(Apr-21-2000) with blastp set at default parameters. Such default parameters
may be, for example:
Matrix: BLOSUM62
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Open Gap: ll and Extension Gap: I penalties
Gap x drop-off:' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence, for
example, as defined by a particular SEQ ID number, or may be measured over a
shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for instance,
a fragment of at least 15, at least 20, at least 30, at least 40, at least 50,
at least 70 or at least 150
contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment length
supported by the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
"Human artificial chromosomes'' (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term ''humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the stringency
of the hybridization process, with more stringent conditions allowing less non-
specific binding, i.e.,
binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by one of
ordinary skill in the art and
may be consistent among hybridization experiments, whereas wash conditions may
be varied among
experiments to achieve the desired stringency, and therefore hybridization
specificity. Permissive
annealing conditions occur, for example, at 68°C in the presence of
about 6 x SSC, about 190 (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~ for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 5090 of the
CA 02373191 2001-11-05
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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, 2nd 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 qg/ml. Organic
solvent, such as
formamide at a concentration of about 35-50% v/v, may also be used under
particular circumstances,
such as for RNA:DNA hybridizations. Useful variations on these wash conditions
will be readily
apparent to those of ordinary skill in the art. Hybridization, particularly
under high stringency
conditions, may be suggestive of evolutionary similarity between the
nucleotides. Such similarity is
strongly indicative of a similar role for the nucleotides and their encoded
polypeptides.
The term "hybridization complex'' refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A hybridization
complex may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid
sequence present in solution and another nucleic acid sequence immobilized on
a solid support (e.g.,
paper, membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells
or their nucleic acids have been fixed).
The words "insertion" and "addition'' refer to changes in an amino acid or
nucleotide sequence
resulting in the addition of one or more amino acid residues or nucleotides,
respectively.
"Immune response'' can refer to conditions associated with inflammation,
trauma, immune
2~ 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 FLEXHT
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
FLEXHT which is useful in any of the antibody production methods disclosed
herein or known in the
art.
The term "microarray'' refers to an arrangement of a plurality of
polynucleotides, polypeptides,
or other chemical compounds on a substrate.
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The terms ''element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of FLEXHT. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other biological,
functional, or immunological properties of FLEXHT.
The phrases ''nucleic acid" and ''nucleic acid sequence" refer to a
nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer to DNA or
RNA of genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the sense or the
antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-
like material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and stop
transcript elongation, and
may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an FLEXHT may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in the
art. These processes may occur synthetically or biochemically. Biochemical
modifications will vary by
cell type depending on the enzymatic milieu of FLEXHT.
"Probe" refers to nucleic acid sequences encoding FLEXHT, their complements,
or fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule. Typical
labels include radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers'' are
short nucleic acids, usually DNA oligonucleotides, which may be annealed to a
target polynucleotide by
complementary base-pairing. The primer may then be extended along the target
DNA strand by a DNA
polymerise enzyme. Primer pairs can be used for amplification (and
identification) of a nucleic acid
sequence, e.g., by the polymerise chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may
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be considerably loner 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 a1.,1987, Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al., 1990, PCR
Protocols. A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to 5,000
nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection
programs have incorporated additional features for expanded capabilities. For
example, the PrimOU
primer selection program (available to the public from the Genome Center at
University of Texas South
West Medical Center, Dallas TX) is capable of choosing specific primers from
megabase sequences
and is thus useful for designing primers on a genome-wide scope. The Primer3
primer selection
program (available to the public from the Whitehead Institute/MIT Center for
Genome Research,
Cambridge MA) allows the user to input a ''nuspriming 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.
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This artificial combination is often accomplished by chenucal synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for
example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control
transcription, translation, or RNA
stability.
''Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample'' is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding FLEXHT, or fragments thereof, or FLEXHT itself, may comprise a
bodily fluid; an
extract fiom a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic DNA,
RNA. or cDNA, in solution or bound to a substrate: a tissue; a tissue print;
etc.
The terms "specific binding'' and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular structure
of the protein, e.g., the antigenic determinant or epitope, recognized by the
binding molecule. For
example, if an antibody is specific for epitope ''A," the presence of a
polypeptide comprising the epitope
A, or the presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will
reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60~7o free,
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preferably at least 75% free, and most preferably at least 90% free from other
components with which
they are naturally associated.
A "substitution'' refers to the replacement of one or more amino acid residues
or nucleotides by
different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image' refers to the collective pattern of gene expression by a
particular cell type
or tissue under given conditions at a given time.
''Transformation'' describes a process by which exogenous DNA is introduced
into a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods well
known in the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences
into a prokaryotic or eukaryotic host cell. The method for transformation is
selected based on the type
of host cell being transformed and may include, but is not limited to,
bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment. The term
''transformed" cells
includes stably transformed cells in which the inserted DNA is capable of
replication either as an
autonomously replicating plasmid or as part of the host chromosome, as well as
transiently transformed
cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook et al. (1989),
supra.
A "variant'' of a particular nucleic acid sequence is defined as a nucleic
acid sequence having at
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)
CA 02373191 2001-11-05
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set at default parameters. Such a pair of nucleic acids may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or
at least 98% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an ''allelic"
(as defined above), "splice,'' "species," or "polymorphic'' variant. A splice
variant may have significant
identity to a reference molecule, but will generally have a greater or lesser
number of polynucleotides
due to alternative splicing of exons during mRNA processing. The corresponding
polypeptide may
possess additional functional domains or lack domains that are present in the
reference molecule.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides generally will have significant amino acid identity relative to
each other. A polymorphic
variant is a variation in the polynucleotide sequence of a particular gene
between individuals of a given
species. Polymorphic variants also may encompass ''single nucleotide
polymorphisms'' (SNPs) in
which the polynucleotide sequence varies by one nucleotide base. The presence
of SNPs may be
indicative of, for example, a certain population, a disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having at
least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of the
polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version
2Ø9 (May-07-1999)
set at default parameters. Such a pair of polypeptides may show, for example,
at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new full-length molecules expressed
in human
tissues (FLEXHT), the polynucleotides encoding FLEXHT, and the use of these
compositions for the
analysis of human gene expression, genetic linkage, and genetic variability,
and for the diagnosis,
treatment, or prevention of developmental, cell proliferative, and
immunological disorders.
2~ Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
FLEXHT. 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 FLEXHT were identified, and column 4
shows the cDNA libraries
from which these clones were isolated. Column 5 shows Incyte clones and their
corresponding cDNA
libraries. Clones for which cDNA libraries are not indicated were derived from
pooled cDNA libraries.
The Incyte clones in column 5 were used to assemble the consensus nucleotide
sequence of each
FLEXHT 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
3~ polypeptide; column 3 shows potential phosphorylation sites: and column 4
shows potential
21
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
glycosylation sites.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding FLEXHT. The Iirst 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
N0:56-110 and to distinguish between SEQ ID N0:56-110 and related
polynucleotide sequences.
The polypeptides encoded by the indicated fragments of SEQ ID N0:56, SEQ ID
N0:58-69, SEQ ID
N0:71-72, SEQ ID N0:74-76, SEQ ID N0:79-85, SEQ ID N0:87, SEQ ID N0:90-98, SEQ
ID
NO:100-103, and SEQ ID NO:105-110 are useful, for example, as immunogenic
peptides. Column 3
lists tissue categories which express FLEXHT as a fraction of total tissues
expressing FLEXHT.
Column 4 lists diseases, disorders, or conditions associated with those
tissues expressing FLEXHT as a
fraction of total tissues expressing FLEXHT. Column 5 lists the vectors used
to subclone each cDNA
library.
The columns of Table 4 show descriptions of the tissues used to construct the
cDNA libraries
from which cDNA clones encoding FLEXHT were isolated. Column 1 references the
nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
The columns of Table 5 show various properties of polypeptides of the
invention: column 1
references the SEQ ID NO; column 2 shows the identity of the polypeptide along
with relevant
citations, all of which are expressly incorporated by reference herein in
their entirety; column 3 shows
the amino acid residues comprising signature sequences, domains, and motifs,
and homologous
sequences as identified by BLAST analysis; and column 4 shows analytical
methods and in some cases,
searchable databases to which the analytical methods were applied. The methods
of column 4 were
used to characterize each polypeptide through sequence homology and protein
motifs.
The invention incorporates the nucleic acid sequences disclosed in the
Sequence Listing and the
use of these sequences in the diagnosis and treatment of disease states
characterized by altered
expression of FLEXHT genes or defects in FLEXHT protein function. The
invention further utilizes
these sequences in hybridization and amplification technologies, and in
particular, in technologies which
comprehensively assess gene expression patterns correlated with specific cells
or tissues and their
responses in vivo or in vitro to pharmaceutical agents, toxins. and other
treatments. In this manner, the
sequences of the present invention are used to develop a transcript image for
a particular cell or tissue.
The invention also encompasses FLEXHT variants. A preferred FLEXHT 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 FLEXHT amino acid sequence, and which contains at
least one functional or
22
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
structural characteristic of FLEXHT.
The invention also encompasses polynucleotides which encode FLEXHT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected from
the group consisting of SEQ ID N0:56-110, which encodes FLEXHT. The
polynucleotide sequences
~ of SEQ ID N0:56-110, 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
FLEXHT. 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 FLEXHT. A particular aspect of the invention encompasses a variant of
a polynucleotide
sequence comprising a sequence selected from the group consisting of SEQ ID
N0:56-110 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 N0:56-110.
Any one of the polynucleotide variants described above can encode an amino
acid sequence which
contains at least one functional or structural characteristic of FLEXHT.
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 FLEXHT, 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 FLEXHT, and all such variations are to be considered as being
specifically disclosed.
Although nucleotide sequences which encode FLEXHT and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring FLEXHT under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding FLEXHT
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 FLEXHT 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 FLEXHT
and
23
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
FLEXHT 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 FLEXHT or any fragment thereof.
Hybridization
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
N0:56-110 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.) For example, stringent salt concentration will ordinarily be
less than about 750 mM
NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and
50 mM trisodium
citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium
citrate. Low
stringency hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while high
stringency hybridization can be obtained in the presence of at least about 35%
formamide, and most
preferably at least about 50% formamide. Stringent temperature conditions will
ordinarily include
temperatures of at least about 30°C, more preferably of at least about
37°C, and most preferably of at
least about 42°C. Varying additional parameters, such as hybridization
time, the concentration of
detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion
of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these
various conditions as needed. In a preferred embodiment, hybridization will
occur at 30°C in 750 mM
NaCl, 75 mM trisodium citrate, and 1 % SDS. In a more preferred embodiment,
hybridization will
occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35%
formamide, and 100 ~c~ml
denatured salmon sperm DNA (ssDNA). In a most preferred embodiment,
hybridization will occur at
42°C in 250 mM NaCl, 25 mM trisodium citrate, 1 % SDS, 50 % formamide,
and 200 ~g/ml ssDNA.
Useful variations on these conditions will be readily apparent to those
skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency
conditions can be defined by salt concentration and by temperature. As above,
wash stringency can be
increased by decreasing salt concentration or by increasing temperature. For
example, stringent salt
concentration for the wash steps will preferably be less than about 30 mM NaCl
and 3 mM trisodium
citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium
citrate. Stringent
temperature conditions for the wash steps will ordinarily include temperature
of at least about 25°C,
more preferably of at least about 42°C, and most preferably of at least
about 68°C. In a preferred
embodiment, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium
citrate, and 0.1% SDS.
In a more preferred embodiment, wash steps will occur at 42°C in 15 mM
NaCl, 1.5 mM trisodium
24
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
citrate, and 0.1 % SDS. In a most preferred embodiment, wash steps will occur
at 68°C in 15 mM
NaCI, 1.5 mM trisodium citrate, and 0.1 % SDS. Additional variations on these
conditions will be
readily apparent to those skilled in the art. Further examples of
hybridization conditions, including
annealing and wash conditions, are described in ''Definitions."
~ cDNA Sequencing
Methods for DNA sequencing are well known in the art and may be used to
practice any of the
embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment of
DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(Perkin-Elmer),
thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway NJ), or
combinations of
polymerises and proofreading exonucleases such as those found in the ELONGASE
amplification
system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation
is automated with
machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV),
PTC200 thermal
cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Perkin-
Elmer).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing
system (Perkin-
Elmer), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale
CA), or
other systems known in the art. The resulting sequences are analyzed using a
variety of algorithms
which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short
Protocols in Molecular
Biolo~y, 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 FLEXHT may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising a
known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al.
(1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent
to known sequences in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom, M. et al.
(1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction
enzyme digestions and
legations may be used to insert an engineered double-stranded sequence into a
region of unknown
sequence before performing PCR. Other methods which may be used to retrieve
unknown sequences
are known in the art. (See, e.g.. Parker, J.D. et al. (1991) Nucleic Acids
Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries
(Clontech, Palo
2~
CA 02373191 2001-11-05
WO 00/70047 PCT/LTS00/13299
Alto CA) to walk genomic DNA. This procedure avoids the need to screen
libraries and is useful in
fording intron/exon junctions. For all PCR-based methods, primers may be
designed using
commercially available software, such as OLIGO 4.06 Primer Analysis software
(National Biosciences,
Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a
GC content of about 50% or more, and to anneal to the template at temperatures
of about 68°C to
72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence into 5'
non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze the
size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Outputllight intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the
entire process
from loading of samples to computer analysis and electronic data display may
be computer controlled.
Capillary electrophoresis is especially preferable for sequencing small DNA
fragments which may be
present in limited amounts in a particular sample.
cDNA Expression
In another embodiment of the invention, polynucleotide sequences or fragments
thereof which
encode FLEXHT may be cloned in recombinant DNA molecules that direct
expression of FLEXHT, 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 FLEXHT.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter FLEXHT-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
26
CA 02373191 2001-11-05
WO 00170047 PCT/US00/13299
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 FLEXHT, such as its biological or
enzymatic activity or its
~ ability to bind to other molecules or compounds. DNA shuffling is a process
by which a library of
gene variants is produced using PCR-mediated recombination of gene fragments.
The library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding FLEXHT may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively,
FLEXHT itself or a fragment thereof may be synthesized using chemical methods.
For example,
peptide synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431A peptide synthesizer (Perkin-Elmer). Additionally. the amino
acid sequence of
FLEXHT, or any part thereof, may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide or
a polypeptide having a
sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.)
In order to express a biologically active FLEXHT, the nucleotide sequences
encoding FLEXHT
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
27
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
polynucleotide sequences encoding FLEXHT. Such elements may vary in their
strength and specificity.
Specific initiation signals may also be used to achieve more efficient
translation of sequences encoding
FLEXHT. Such signals include the ATG initiation codon and adjacent sequences,
e.g. the Kozak
sequence. In cases where sequences encoding FLEXHT and its initiation codon
and upstream
regulatory sequences are inserted into the appropriate expression vector, no
additional transcriptional or
translational control signals may be needed. However, in cases where only
coding sequence, or a
fragment thereof, is inserted, exogenous translational control signals
including an in-frame ATG
initiation codon should be provided by the vector. Exogenous translational
elements and initiation
codons may be of various origins, both natural and synthetic. The efficiency
of expression may be
enhanced by the inclusion of enhancers appropriate for the particular host
cell system used. (See, e.g.,
Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding FLEXHT and appropriate transcriptional
and translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic techniques,
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding FLEXHT. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or
tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra: Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods
Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-
1945: Takamatsu,
N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue. R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105: The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J, and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
3~ delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
28
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
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 FLEXHT. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding FLEXHT can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1 plasmid
(Life Technologies). Ligation of sequences encoding FLEXHT into the vector's
multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure for
identification of transformed
bacteria containing recombinant molecules. In addition, these vectors may be
useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper phage, and
creation of nested
deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster
(1989) J. Biol. Chem.
264:5503-5509.) When large quantities of FLEXHT are needed, e.g. for the
production of antibodies,
vectors which direct high level expression of FLEXHT may be used. For example,
vectors containing
the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of FLEXHT. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable integration
of foreign sequences into the host genome for stable propagation. (See. e.g.,
Ausubel, 1995, supra;
Bitter, su ra; and Scorer, supra.)
Plant systems may also be used for expression of FLEXHT. Transcription of
sequences
encoding FLEXHT 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 Technoloey
(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 FLEXHT
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
29
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
infective virus which expresses FLEXHT in host cells. (See, e.g., Logan, J.
and T. Shenk (1984) Prcx;.
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
FLEXHT in cell lines is preferred. For example. sequences encoding FLEXHT can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media before
being switched to selective media. The purpose of the selectable marker is to
confer resistance to a
selective agent, and its presence allows growth and recovery of cells which
successfully express the
introduced sequences. Resistant clones of stably transformed cells may be
propagated using tissue
culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include,
but are not limited to, the herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase
genes, for use in tk- and apr cells, respectively. (See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or
herbicide resistance can be
used as the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers
resistance to the aminoglycosides neomycin and G-418; and als and pat confer
resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980)
Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and hisD, which
alter cellular requirements
for metabolites. (See, e. g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins
(GFP; Clontech), li
glucuronidase and its substrate li-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
CA 02373191 2001-11-05
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sequence encoding FLEXHT is inserted within a marker gene sequence,
transformed cells containing
sequences encoding FLEXHT can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding FLEXHT 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 FLEXHT
and that express
FLEXHT 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 FLEXHT
using either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and
fluorescence
activated cell sorting (FACS). A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering epitopes on FLEXHT is preferred,
but a competitive binding
assay may be employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et
al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN,
Sect. IV; Coligan, J.E.
et al. (1997) Current Protocols in Immunolo~y> Greene Pub. Associates and
Wiley-Interscience, New
York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa
NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization
or PCR probes for detecting sequences related to polynucleotides encoding
FLEXHT include
oligolabeling, nick translation, end-labeling, or PCR amplification using a
labeled nucleotide.
Alternatively, the sequences encoding FLEXHT, 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 FLEXHT 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
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and/or the vector used. As will be understood by those of skill in the art,
expression vectors containing
polynucleotides which encode FLEXHT may be designed to contain signal
sequences which direct
secretion of FLEXHT 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, Mantissas VA) and may be chosen to ensure the correct
modification and processing
of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding FLEXHT may be ligated to a heterologous sequence resulting
in translation of a
1~ fusion protein in any of the aforementioned host systems. For example, a
chimeric FLEXHT protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of FLEXHT
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
immunoaflinity purification of
fusion proteins using commercially available monoclonal and polyclonal
antibodies that specifically
2~ recognize these epitope tags. A fusion protein may also be engineered to
contain a proteolytic cleavage
site located between the FLEXHT encoding sequence and the heterologous protein
sequence, so that
FLEXHT 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 FLEXHT 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, 3'S-methionine.
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Fragments of FLEXHT may be produced not only by recombinant production, but
also by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
supra, pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation. Automated
synthesis may be
achieved, for example, using the ABI 431A peptide synthesizer (Perkin-Elmer).
Various fragments of
FLEXHT may be synthesized separately and then combined to produce the full
length molecule.
Screening Assays
FLEXHT of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to FLEXHT. At least one and up to a plurality of test
compounds may be
screened for specific binding to FLEXHT. 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
FLEXHT, 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 FLEXHT
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 FLEXHT,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or E.
coli. Cells expressing FLEXHT or cell membrane fractions which contain FLEXHT
are then
contacted with a test compound and binding, stimulation, or inhibition of
activity of either FLEXHT
or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
FLEXHT, either in
solution or affixed to a solid support, and detecting the binding of FLEXHT 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.
FLEXHT of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of FLEXHT. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
FLEXHT activity, wherein FLEXHT is combined with at least one test compound,
and the activity of
FLEXHT in the presence of a test compound is compared with the activity of
FLEXHT in the absence
of the test compound. A change in the activity of FLEXHT in the presence of
the test compound is
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indicative of a compound that modulates the activity of FLEXHT. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising FLEXHT under
conditions suitable for
FLEXHT activity. and the assay is performed. In either of these assays, a test
compound which
modulates the activity of FLEXHT 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.
Preferably, an ELISA assay using, e.g., a monoclonal or polyclonal antibody,
can measure
polypeptide level in a sample. The antibody can measure polypeptide level by
either binding, directly
or indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
All of the above assays can be used in a diagnostic or prognostic context. The
molecules
discovered using these assays can be used to treat disease or to bring about a
particular result in a
patient (e.g., blood vessel growth) by activating or inhibiting the
polypeptide/molecule. Moreover,
the assays can discover agents which may inhibit or enhance the production of
the polypeptide from
suitably manipulated cells or tissues.
Transcript Ima~in~
Another embodiment relates to the use of polynucleotide sequences encoding
FLEXGEM to
develop a transcript image of a tissue or cell type. A transcript image is the
collective pattern of gene
expression by a particular tissue or cell type under given conditions and at a
given time. This pattern
of gene expression is defined by the number of expressed genes and their
abundance. Thus the
polynucleotide sequences of the present invention may be used to develop a
transcript image of a
tissue or cell type by hybridizing, preferably in a microarray format, the
polynucleotide sequences of
the present invention to the totality of transcripts or reverse transcripts of
a tissue or cell type. The
resultant transcript image would provide a profile of FLEXGEM gene activity.
Transcript images which profile FLEXGEM gene expression may be generated using
transcripts isolated from tissues, cell lines, biopsies, or other biological
samples. The transcript
image may thus reflect FLEXGEM 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 may be
used to profile FLEXGEM
gene expression in distinct tissue types. This process can be used to
determine FLEXGEM gene
activity in a particular tissue type relative to this activity in a different
tissue type. Transcript images
may be used to generate a profile of FLEXGEM gene expression characteristic of
diseased tissue.
Transcript images of tissues before and after treatment may be used for
diagnostic purposes, to
monitor the progression of disease, and to monitor the efficacy of drug
treatments for diseases which
affect the activity of genes encoding FLEXGEM.
Transcript images which profile FLEXGEM gene expression may also be used in
conjunction
with in vitro model systems and preclinical evaluation of pharmaceuticals.
Transcript images of cell
lines can be used to assess FLEXGEM activity and/or to identify cell lines
that lack or misregulate
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CA 02373191 2001-11-05
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this activity. Such cell lines may then be treated with pharmaceutical agents,
and a transcript image
following treatment may indicate the efficacy of these agents in restoring
desired levels of this
activity. A similar approach may be used to assess the toxicity of
pharmaceutical agents as reflected
by undesirable changes in FLEXGEM activity. Candidate pharmaceutical agents
may be evaluated
by comparing their associated transcript images with those of pharmaceutical
agents of known
effectiveness.
Trans~enic Animals
In another embodiment, polynucleotides encoding FLEXHT 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; Capecclii, 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-IoxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (March, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding FLEXHT may also be manipulated in vitro in ES cells
derived
from human blastocysts. Human ES cells have the potential to differentiate
into at least eight separate
cell lineages including endoderm, mesoderm, and ectodermal cell types. These
cell lineages
differentiate into, for example, neural cells, hematopoietic lineages, and
cardiomyocytes (Thomson,
J.A. et al. (1998) Science 282:1145-1147).
Polynucleotides encoding FLEXHT 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 FLEXHT 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 FLEXHT, e.g., by secreting
FLEXHT in its nulk, may
3~
CA 02373191 2001-11-05
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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 FLEXHT and full-length molecules expressed in human
tissues. In addition, the
expression of FLEXHT is closely associated with cell proliferation and the
immune response.
Therefore, FLEXHT appears to play a role in developmental, cell proliferative,
and immunological
disorders. In the treatment of disorders associated with increased FLEXHT
expression or activity, it
is desirable to decrease the expression or activity of FLEXHT. In the
treatment of disorders
associated with decreased FLEXHT expression or activity, it is desirable to
increase the expression or
activity of FLEXHT.
Therefore, in one embodiment, FLEXHT 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 FLEXHT. Examples of such disorders include, but are not limited
to, a developmental
disorder such as renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; 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, a cancer of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; and an immunological
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, 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, paroxysmal nocturnal hemoglobinuria, hepatitis,
hypereosinophilia, irritable bowel
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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,
systenuc lupus
erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic
purpura, ulcerative
colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal
circulation, trauma, and hematopoietic cancer including lymphoma, leukemia,
and myeloma.
In another embodiment, a vector capable of expressing FLEXHT 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 FLEXHT including, but not limited to, those
described above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
FLEXHT 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 FLEXHT including, but
not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of FLEXHT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or activity
of FLEXHT including, but not limited to, those listed above.
In a further embodiment, an antagonist of FLEXHT may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
FLEXHT. Examples of such
disorders include, but are not limited to, those developmental, cell
proliferative, and immunological
disorders described above. In one aspect, an antibody which specifically binds
FLEXHT 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 FLEXHT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding FLEXHT may be administered to a subject to treat or prevent a
disorder associated with
increased expression or activity of FLEXHT including, but not limited to,
those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made by
one of ordinary skill in the art, according to conventional pharmaceutical
principles. The combination
of therapeutic agents may act synergistically to effect the treatment or
prevention of the various
disorders described above. Using this approach, one may be able to achieve
therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse side
effects.
An antagonist of FLEXHT may be produced using methods which are generally
known in the
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art. In particular, purified FLEXHT may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind FLEXHT.
Antibodies to FLEXHT 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 FLEXHT or with any fiagment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants
used in humans. BCG
(bacilli Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
FLEXHT 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
FLEXHT 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 FLEXHT 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
FLEXHT-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g., Burton.
D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
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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 FLEXHT may also be
generated.
For example, such fragments include, but are not limited to, F(ab~~ fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of the
F(ab~2 fragments. Alternatively, Fab expression libraries may be constructed
to allow rapid and easy
identification of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specilicities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
FLEXHT and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive
to two non-interfering FLEXHT 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 FLEXHT. Affinity is
expressed as an association
constant, Ka, which is defined as the molar concentration of FLEXHT-antibody
complex divided by the
molar concentrations of free antigen and free antibody under equilibrium
conditions. The Ka determined
for a preparation of polyclonal antibodies, which are heterogeneous in their
affinities for multiple
FLEXHT epitopes, represents the average affinity, or avidity, of the
antibodies for FLEXHT. The Ka
determined for a preparation of monoclonal antibodies, which are monospecific
for a particular
FLEXHT epitope, represents a true measure of affinity. High-affinity antibody
preparations with I~
ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in
which the FLEXHT-
antibody complex must withstand rigorous manipulations. Low-affinity antibody
preparations with I~
ranging from about 10~ to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of FLEXHT, preferably in
active form. from the
antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to determine
the quality and suitability of such preparations for certain downstream
applications. For example, a
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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 FLEXHT-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 FLEXHT,
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
FLEXHT. 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
FLEXHT. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana
Press Inc., Totawa
NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A.D. (1990) Blood
76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
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 FLEXHT 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 Somia, N. (1997) Nature
389:239-242)), (ii)
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites (e.g.,
against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D. (1988)
Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in FLEXHT expression or regulation causes
disease, the expression of
FLEXHT 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
FLEXHT are treated by constructing mammalian expression vectors encoding
FLEXHT and
introducing these vectors by mechanical means into FLEXHT-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 FLEXHT 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). FLEXHT 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. U.S.A.
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, su ra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding FLEXHT 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
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
(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 manunalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to FLEXHT expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding FLEXHT 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. U.S.A. 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. U.S.A. 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 FLEXHT to cells which have one or more genetic
abnormalities with respect
to the expression of FLEXHT. The construction and packagTing 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
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
polynucleoddes encoding FLEXHT to target cells which have one or more genetic
abnormalities with
respect to the expression of FLEXHT. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing FLEXHT 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
1~ 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 FLEXHT to target cells. The biology of the
prototypic alphavirus,
Senlliki 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 FLEXHT into the
alphavirus genome in
place of the capsid-coding region results in the production of a large number
of FLEXHT-coding RNAs
and the synthesis of high levels of FLEXHT 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
FLEXHT 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 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
of alphaviruses, performing alphavirus cDNA and RNA transfections, and
performing alphavirus
infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions -10
and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can
~ be achieved using triple helix base-pairing methodology. Triple helix
pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently for the
binding of polymerases,
transcription factors, or regulatory molecules. Recent therapeutic advances
using triplex DNA have
been described in the literature. (See, e.g., Gee. J.E. et al. (1994) in
Huber, B.E. and B.I. Carr,
Molecular and Immunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-
177.) A
complementary sequence or antisense molecule may also be designed to block
translation of mRNA by
preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding FLEXHT.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by
any method known in the art for the synthesis of nucleic acid molecules. These
include techniques for
chemically synthesizing oligonucleotides such as solid phase phosphoramidite
chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA sequences
encoding FLEXHT. 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 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
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 FT.EXHT.
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
FLEXHT expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding FLEXHT may be therapeutically useful, and in the
treament of disorders
associated with decreased FLEXHT expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding FLEXHT 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 FLEXHT 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
FLEXHT 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 FLEXHT. 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.
4~
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
5.932,435: Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human
cell line such as HeLa
cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A
particular
embodiment of the present invention involves screening a combinatorial library
of oligonucleotides
(such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and
modified oligonucleotides)
for antisense activity against a specific polynucleotide sequence (Bruice,
T.W. et al. (1997) U.S.
Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022.691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable for
use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells taken
from the patient and clonally propagated for autologous transplant back into
that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15 :462-466. )
Any of the therapeutic methods described above may be applied to any subject
in need of such
therapy, including, for example, mammals such as humans, dogs, cats, cows,
horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
pharmaceutical
composition which generally comprises an active ingredient formulated with a
pharmaceutically
acceptable excipient. Excipients may include, for example, sugars, starches,
celluloses, gums, and
proteins. Various formulations are commonly known and are thoroughly discussed
in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
pharmaceutical
compositions may consist of FLEXHT, antibodies to FLEXHT, and nlimetics,
agonists, antagonists, or
inhibitors of FLEXHT.
The pharmaceutical compositions utilized in this invention may be administered
by any number
of routes including, but not limited to, oral, intravenous, intramuscular,
intra-arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasah enteral,
topical, sublingual, or rectal means.
Pharmaceutical 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.
Pharmaceutical compositions suitable for use in the invention include
compositions wherein the
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active ingredients are contained in an effective amount to achieve the
intended purpose. The
determination of an effective dose is well within the capability of those
skilled in the art.
Specialized forms of pharmaceutical compositions may be prepared for direct
intracellular
delivery of macromolecules comprising FLEXHT or fragments thereof. For
example, liposome
preparations containing a cell-impermeable macromolecule may promote cell
fusion and intracellular
delivery of the macromolecule. Alternatively. FLEXHT 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
FLEXHT or fragments thereof, antibodies of FLEXHT, and agonists, antagonists
or inhibitors of
FLEXHT, 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 EDS~ (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/ED;o ratio.
Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained from cell
culture assays and animal
studies are used to formulate a range of dosage for human use. The dosage
contained in such
compositions is preferably within a range of circulating concentrations that
includes the ED;o with little
or no toxicity. The dosage varies within this range depending upon the dosage
form employed, the
sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the subject
requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the active
moiety or to maintain the desired effect. Factors which may be taken into
account include the severity
of the disease state, the general health of the subject, the age, weight, and
gender of the subject, time
and frequency of administration, drug combination(s), reaction sensitivities,
and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3 to 4 days,
every week, or
biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total
dose of
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind FLEXHT may be used
for the
diagnosis of disorders characterized by expression of FLEXHT, or in assays to
monitor patients being
treated with FLEXHT or agonists, antagonists, or inhibitors of FLEXHT.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for FLEXHT include methods which utilize the antibody and a
label to detect
FLEXHT 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 FLEXHT, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of FLEXHT expression.
Normal or standard values for FLEXHT expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibody to
FLEXHT under conditions suitable for complex formation. The amount of standard
complex formation
may be quantitated by various methods, such as photometric means. Quantities
of FLEXHT 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 FLEXHT
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 FLEXHT may
be correlated with
disease. The diagnostic assay may be used to determine absence, presence, and
excess expression of
FLEXHT, and to monitor regulation of FLEXHT levels during therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding FLEXHT or closely related
molecules may be used
to identify nucleic acid sequences which encode FLEXHT. 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
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
probe identities only naturally occurring sequences encoding FLEXHT, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the FLEXHT encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:56-110 or from
genomic sequences including promoters, enhancers, and introns of the FLEXHT
gene.
Means for producing specific hybridization probes for DNAs encoding FLEXHT
include the
cloning of polynucleotide sequences encoding FLEXHT or FLEXHT 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 ~ZP or 3'S, or by
enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin coupling systems,
and the like.
Polynucleotide sequences encoding FLEXHT may be used for the diagnosis of
disorders
associated with expression of FLEXHT. Examples of such disorders include, but
are not limited to, a
developmental disorder such as renal tubular acidosis, anemia, Cushing's
syndrome, achondroplastic
dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome
(Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation),
Smith-Magenis
syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia,
hereditary keratodermas,
hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis, hypothyroidism,
hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; a cell
proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal
nocturnal
2~ hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma.
teratocarcinoma, and, in
particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas, parathyroid,
penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and
uterus; and an immunological
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, bronchitis, bursitis, cholecystitis, cirrhosis, contact
dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis
fetalis, erythema nodosum,
49
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atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'
disease, Hashimoto's
thyroiditis, 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, trauma, and hematopoietic cancer including lymphoma, leukemia,
and myeloma. The
polynucleotide sequences encoding FLEXHT 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 FLEXHT
expression. Such qualitative or quantitative methods are well known in the
art.
In a particular aspect, the nucleotide sequences encoding FLEXHT may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding FLEXHT 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 FLEXHT 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
FLEXHT, 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 FLEXHT, 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
CA 02373191 2001-11-05
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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 FLEXHT
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
FLEXHT, or a fragment of a polynucleotide complementary to the polynucleotide
encoding FLEXHT,
and will be employed under optimized conditions for identification of a
specific gene or condition.
Oligomers may also be employed under less stringent conditions for detection
or quantification of
closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding FLEXHT 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 FLEXHT 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).
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Methods which may also be used to quantify the expression of FLEXHT include
radiolabeling
or biotinyladng 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.
The polynucleotides are also useful for identifying individuals on the basis
of minute
biological samples, for example, by matching the restriction fragment length
polymorphism (RFLP)
pattern of a sample's DNA to that of an individual's DNA. The polynucleotides
of the present
invention can also be used to determine the actual base-by-base DNA sequence
of selected portions of
an individual's genome. These sequences can be used to prepare PCR primers for
amplifying and
isolating such selected DNA, which can then be sequenced. Using this
technique, an individual can
be identified through a unique set of DNA sequences. Once a unique ID database
is established for an
1~ individual, positive identification of that individual can be made from
extremely small tissue samples.
DNA-based identification techniques are critical in forensic technology. DNA
sequences
taken from very small biological samples such as tissues, e.g., hair, skin, or
body fluids (e.g., blood,
saliva, semen, etc.), can be amplified using, e.g., PCR, to identify
individuals. (See, e.g., Erlich, H.
(1992) PCR Technoloey> Freeman and Co., New York NY). Similarly,
polynucleotides of the
present invention can be used as polymorphic markers.
There is also a need for reagents capable of identifying the source of a
particular tissue.
Appropriate reagents can comprise, for example, DNA probes or primers prepared
from the sequences
of the present invention that are specific for particular tissues. Panels of
such reagents can identify
tissue by species and/or by organ type. In a similar fashion, these reagents
can be used to screen
2~ tissue cultures for contamination.
The polynucleotides of the present invention can also be used as molecular
weight markers on
nucleic acid gels or Southern blots, as diagnostic probes for the presence of
a specific mRNA in a
particular cell type, in the creation of subtracted cDNA libraries which aid
in the discovery of novel
polynucleotides, in selection and synthesis of oligomers for attachment to an
array or other support,
and as an antigen to elicit an immune response.
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
3~ Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
J7
CA 02373191 2001-11-05
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used to identify genetic variants. mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to monitor
progression/regression of disease as a function of gene expression. and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and effective
treatment regimen for that patient. For example, therapeutic agents which are
highly effective and
display the fewest side effects may be selected for a patient based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for FLEXHT, or FLEXHT 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.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-
2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types
of microarrays are well
known and thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999)
Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding FLEXHT
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial Pl
constructions, or single chromosome cDNA libraries. (See, e.g., Harrington,
J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be
used to develop genetic
linkage maps, for example, which correlate the inheritance of a disease state
with the inheritance of a
particular chromosome region or restriction fragment length polymorphism
(RFLP). (See, e.g.,
Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.)
Examples of genetic map
data can be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM)
World Wide Web site. Correlation between the location of the gene encoding
FLEXHT on a physical
53
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
map and a specific disorder, or a predisposition to a specific disorder, may
help define the region of
DNA associated with that disorder and thus may further positional cloning
efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse, may
reveal associated markers even if the exact chromosomal locus is not known.
This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely localized
by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia
to l 1q22-23, any sequences
mapping to that area may represent associated or regulatory genes for further
investigation. (See, e.g.,
Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the
instant invention may
also be used to detect differences in the chromosomal location due to
translocation, inversion, etc.,
among normal, carrier, or affected individuals.
In another embodiment of the invention, FLEXHT, its catalytic or immunogenic
fragments, or
1~ oligopeptides thereof can be used for screening libraries of compounds in
any of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between FLEXHT 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 FLEXHT,
or fragments thereof,
and washed. Bound FLEXHT is then detected by methods well known in the art.
Purified FLEXHT
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 FLEXHT specifically compete with a test compound
for binding
FLEXHT. In this manner, antibodies can be used to detect the presence of any
peptide which shares
one or more antigenic determinants with FLEXHT.
In additional embodiments, the nucleotide sequences which encode FLEXHT 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.
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Without iiuther 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. 09/311,894, U.S. Ser. No. 09/311,940, and U.S. Ser.
No. 09/311.937, 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.
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), PSPORTI 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
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
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).
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 (Perkin-Elmer) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out
using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or
377 sequencing system (Perkin-Elmer) in conjunction with standard ABI
protocols and base calling
software: or other sequence analysis systems known in the art. Reading frames
within the cDNA
sequences were identified using standard methods (reviewed in Ausubel, 1997,
supra, unit 7.7). Some
of the cDNA sequences were selected for extension using the techniques
disclosed in Example V.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 6 summarizes the tools, programs, and algorithms used and provides
applicable descriptions,
references, and threshold parameters. The first colunm of Table 6 shows the
tools, programs, and
algorithms used, the second column provides brief descriptions thereof, the
third column presents
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WO 00/70047 PCT/US00/13299
appropriate references, all of which are incorporated by reference herein in
their entirety, and the fourth
column presents, where applicable, the scores, probability values, and other
parameters used to evaluate
the strength of a match between two sequences (the higher the score, the
greater the homology between
two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software
Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and
polypeptide sequence alignments were generated using the default parameters
specified by the clustal
algorithm as incorporated into the MEGALIGN multisequence alignment program
(DNASTAR), which
also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA sequences
and by masking ambiguous bases, using algorithms and programs based on BLAST,
dynamic
programing, and dinucleotide nearest neighbor analysis. The sequences were
then queried against a
selection of public databases such as the GenBank primate, rodent, mammalian,
vertebrate, and
eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation
using programs based on BLAST. FASTA, and BLIMPS. The sequences were assembled
into full
length polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened
for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length
polynucleotide sequences were translated to derive the corresponding full
length amino acid sequences,
and these full length sequences were subsequently analyzed by querying against
databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM,
Prosite,
and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM
is a
probabilistic approach which analyzes consensus primary structures of gene
families. (See, e.g.,
Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide and
amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:56-110. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a gene
and involves the hybridization of a labeled nucleotide sequence to a membrane
on which RNAs from a
particular cell type or tissue have been bound. (See, e. g., Sambrook, supra,
ch. 7; Ausubel, 1995,
supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
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
x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the length
of the sequence match. The product score is a normalized value between 0 and
100, and is calculated
as follows: the BLAST score is multiplied by the percent nucleotide identity
and the product is divided
by (5 times the length of the shorter of the two sequences). The BLAST score
is calculated by
assigning a score of +5 for every base that matches in a high-scoring segment
pair (HSP), and -4 for
every mismatch. Two sequences may share more than one HSP (separated by gaps).
If there is more
than one HSP, then the pair with the highest BLAST score is used to calculate
the product score. The
product score represents a balance between fractional overlap and quality in a
BLAST alignment. For
example, a product score of 100 is produced only for 100% identity over the
entire length of the shorter
of the two sequences being compared. A product score of 70 is produced either
by 100% identity and
70% overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79% identity
and 100% overlap.
The results of northern analyses are reported as a percentage distribution of
libraries in which
the transcript encoding FLEXHT occurred. Analysis involved the categorization
of cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoiedc/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in Table
3.
V. Extension of FLEXHT Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:56-110 were produced by
extension of an
appropriate fragment of the full length molecule using oligonucleotide primers
designed from this
fragment. One primer was synthesized to initiate 5' extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using OLIGO
4.06 software (National Biosciences), or another appropriate program, to be
about 22 to 30 nucleotides
in length, to have a GC content of about 50% or more, and to anneal to the
target sequence at
58
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
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)~SO4,
and ~-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.
1~ The concentration of DNA in each well was determined by dispensing 100 pl
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 ~l aliquot of the reaction mixture was
analyzed by electrophoresis
on a 1 % agarose mini-gel to determine which reactions were successful in
extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to 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 polymerase (Stratagene) to fill-in
restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells were selected
on antibiotic-containing
media, and individual colonies were picked and cultured overnight at 37
°C in 384-well plates in LB/2x
carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following parameters:
Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C,
1 min; Step 4: 72°C, 2 min; Step 5: steps 2,
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at
4°C. DNA was quantified by
PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were
reamplified using the same conditions as described above. Samples were diluted
with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer
sequencing primers and
~ the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the polynucleotide sequences of SEQ ID N0:56-110 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:56-110 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide fragments.
Oligonucleotides are designed using state-of-the-art software such as OLIGO
4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~cCi of [y-
32P] adenosine
triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase
(DuPont NEN, Boston
MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size
exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot
containing 10' counts per
minute of the labeled probe is used in a typical membrane-based hybridization
analysis of human
genomic DNA digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or
Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VII. Transcript Image Analysis
Transcript images are generated as described in Seilhamer, J.J. et al.,
"Comparative Gene
Transcript Analysis," U.S. Patent No. 5,840,484. incorporated herein by
reference.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink-jet printing, See, e.g.,
Baldeschweiler, supra), mechanical
microspotting technologies, and derivatives thereof. The substrate in each of
the aforementioned
CA 02373191 2001-11-05
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technologies should be uniform and solid with a non-porous surface (Schena
(1999), supra). Suggested
substrates include silicon, silica, glass slides, glass chips, and silicon
wafers. Alternatively, a procedure
analogous to a dot or slot blot may also be used to arrange and link elements
to the surface of a
substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be
produced using available methods and machines well known to those of ordinary
skill in the art and may
contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
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/~.M oligo-(dT)
primer (2lmer), 1X first
strand buffer, 0.03 units/ql RNase inhibitor, 500 NM dATP, 500 ~M dGTP, 500 ~M
dTTP, 40 EiM
dCTP, 40 ~M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro transcription
from non-coding yeast genomic DNA. After incubation at 37 ° C for 2 hr,
each reaction sample (one
with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium
hydroxide and
incubated for 20 minutes at 85 °C to the stop the reaction and degrade
the RNA. Samples are purified
using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are
ethanol precipitated
using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is
then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook
NY) and
resuspended in 14 ~.~1 SX SSC/0.2% SDS.
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Microarrav Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water, and
coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are
cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 E~l of the array
element DNA, at an average
concentration of 100 ng/E.il, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 nl 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 nunutes at 60
°C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 E.il of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65 °C for 5 minutes and is aliquoted onto the
microarray surface and covered with
an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly
larger than a microscope slide. The chamber is kept at 100% humidity
internally by the addition of
140 Eil 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 (O.1X SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for
CyS. Each array is
typically scanned twice, one scan per fluorophore using the appropriate
filters at the laser source,
although the apparatus is capable of recording the spectra from both
fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are differentially
expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two
fluorophores and adding identical amounts of each to the hybridization
mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
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 FLEXHT-encoding sequences, or any parts
thereof, are used
to detect, decrease, or inhibit expression of naturally occurring FLEXHT.
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
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
using OLIGO 4.06 software (National Biosciences) and the coding sequence of
FLEXHT. 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 FLEXHT-
encoding transcript.
X. Expression of FLEXHT
Expression and purification of FLEXHT is achieved using bacterial or virus-
based expression
systems. For expression of FLEXHT 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 FLEXHT upon induction with isopropyl
beta-D-
thiogalactopyranoside (IPTG). Expression of FLEXHT 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 FLEXHT by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K. et
al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther.
7:1937-1945.)
In most expression systems, FLEXHT 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 j~aponicum, 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 FLEXHT at
specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification
using commercially available monoclonal and polyclonal anti-FLAG antibodies
(Eastman Kodak). 6-
His, a stretch of six consecutive histidine residues, enables purification on
metal-chelate resins
(QIAGEN). Methods for protein expression and purification are discussed in
Ausubel (1995, su ra,
ch. 10 and 16). Purified FLEXHT obtained by these methods can be used directly
in the assays in
Examples XII and XIV.
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
XI. Functional Assays
FLEXHT function is assessed by expressing the sequences encoding FLEXHT at
physiologically elevated levels in mammalian cell culture systems. cDNA is
subcloned into a
mammalian expression vector containing a strong promoter that drives high
levels of cDNA expression.
Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1
plasmid
(Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ~g of
recombinant vector are
transiently transfected into a human cell line, for example, an endothelial or
hematopoietic cell line,
using either liposome formulations or electroporation. 1-2 ~g of an additional
plasmid containing
sequences encoding a marker protein are co-transfected. Expression of a marker
protein provides a
means to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA
expression from the recombinant vector. Marker proteins of choice include,
e.g., Green Fluorescent
Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an
automated, laser optics-based technique, is used to identify transfected cells
expressing GFP or CD64-
GFP and to evaluate the apoptotic state of the cells and other cellular
properties. FCM detects and
quantifies the uptake of fluorescent molecules that diagnose events preceding
or coincident with cell
death. These events include changes in nuclear DNA content as measured by
staining of DNA with
propidium iodide; changes in cell size and granularity as measured by forward
light scatter and 90
degree side light scatter; down-regulation of DNA synthesis as measured by
decrease in
bromodeoxyuridine uptake; alterations in expression of cell surface and
intracellular proteins as
measured by reactivity with specific antibodies; and alterations in plasma
membrane composition as
measured by the binding of fluorescein-conjugated Annexin V protein to the
cell surface. Methods in
flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford,
New York NY.
The influence of FLEXHT on gene expression can be assessed using highly
purified
populations of cells transfected with sequences encoding FLEXHT 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 FLEXHT and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XII. Production of FLEXHT Specific Antibodies
FLEXHT 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.
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Alternatively, the FLEXHT 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 (Perkin-Elmer) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-FLEXHT
activity by, for example, binding the peptide or FLEXHT to a substrate,
blocking with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring FLEXHT Using Specific Antibodies
Naturally occurring or recombinant FLEXHT is substantially purified by
immunoaffinity
chromatography using antibodies specific for FLEXHT. An immunoaffinity column
is constructed by
covalently coupling anti-FLEXHT 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 FLEXHT are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of FLEXHT
(e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/FLEXHT 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 FLEXHT is collected.
XIV. Identification of Molecules Which Interact with FLEXHT
FLEXHT, or biologically active fragments thereof, are labeled with'z5I 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 mufti-well plate are incubated with the
labeled FLEXHT, washed,
and any wells with labeled FLEXHT complex are assayed. Data obtained using
different
concentrations of FLEXHT are used to calculate values for the number,
affinity, and association of
FLEXHT with the candidate molecules.
Alternatively, molecules interacting with FLEXHT 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).
FLEXHT may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
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CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent
No. 6.057,101).
Various modifications and variations of the described methods and systems of
the invention will
be apparent to those skilled in the art without departing from the scope and
spirit of the invention.
Although the invention has been described in connection with certain
embodiments, it should be
understood that the invention as claimed should not be unduly limited to such
specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention which are obvious
to those skilled in molecular biology or related fields are intended to be
within the scope of the following
claims.
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CA 02373191 2001-11-05
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CA 02373191 2001-11-05
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SEQUENCE LISING
<110> INCYTE GENOMICS, INC.
YUE, Henry
TANG, Y. Tom
LAL, Preeti
REDDY, Ro~pa
BATRA, Sajeev
BAUGHN, Mariah R.
YANG, Junming
AZIMZAI, Yalda
LU, Dyung Aina M.
AU-YOUNG, Janice
SHIH, Leo L.
<120> FULL-LENGTH MOLECULES EXPRESSED IN HUMAN TISSUES
<130> PF-0695 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/311,894; 09/311,940; 09/311,937
<151> 1999-05-14; 1999-05-14; 1999-05-14
<160> 110
<170> PERL Program
<210> 1
<211> 349
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1841446CD1
<400> 1
Met Ala Lys Ala Gly Asp Lys Ser Ser Ser Ser Gly Lys Lys Ser
1 5 10 15
Leu Lys Arg Lys Ala Ala Ala Glu Glu Leu Gln Glu Ala Ala Gly
20 25 30
Ala Gly Asp Gly Ala Thr Glu Asn Gly Val Gln Pro Pro Lys Ala
35 40 45
Ala Ala Phe Pro Pro Gly Phe Ser Ile Ser Glu Ile Lys Asn Lys
50 55 60
Gln Arg Arg His Leu Met Phe Thr Arg Trp Lys Gln Gln Gln Arg
65 70 75
Lys Glu Lys Leu Ala Ala Lys Lys Lys Leu Lys Lys Glu Arg Glu
80 85 90
Ala Leu Gly Asp Lys Ala Pro Pro Lys Pro Val Pro Lys Thr Ile
95 100 105
Asp Asn Gln Arg Val Tyr Asp Glu Thr Thr Val Asp Pro Asn Asp
110 115 120
Glu Glu Val Ala Tyr Asp Glu Ala Thr Asp Glu Phe Ala Ser Tyr
125 130 135
Phe Asn Lys Gln Thr Ser Pro Lys Ile Leu Ile Thr Thr Ser Asp
140 145 150
Arg Pro His Gly Arg Thr Val Arg Leu Cys Glu Gln Leu Ser Thr
155 160 165
Val Ile Pro Asn Ser His Val Tyr Tyr Arg Arg Gly Leu Ala Leu
170 175 180
Lys Lys Ile Ile Pro Gln Cys Ile Ala Arg Asp Phe Thr Asp Leu
185 190 195
Ile Val Ile Asn Glu Asp Arg Lys Thr Pro Asn Gly Leu Ile Leu
1/72
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200 205 210
Ser His Leu Pro Asn Gly Pro Thr Ala His Phe Lys Met Ser Ser
215 220 225
Val Arg Leu Arg Lys Glu Ile Lys Arg Arg Gly Lys Asp Pro Thr
230 235 240
Glu His Ile Pro Glu Ile Ile Leu Asn Asn Phe Thr Thr Arg Leu
245 250 255
Gly His Ser Ile Gly Arg Met Phe Ala Ser Leu Phe Pro His Asn
260 265 270
Pro Gln Phe Ile Gly Arg Gln Val Ala Thr Phe His Asn Gln Arg
275 280 285
Asp Tyr Ile Phe Phe Arg Phe His Arg Tyr Ile Phe Arg Ser Glu
290 295 300
Lys Lys Val Gly Ile Gln Glu Leu Gly Pro Arg Phe Thr Leu Lys
305 310 315
Leu Arg Ser Leu Gln Lys Gly Thr Phe Asp Ser Lys Tyr Gly Glu
320 325 330
Tyr Glu Trp Val His Lys Pro Arg Glu Met Asp Thr Ser Arg Arg
335 340 345
Lys Phe His Leu
<210> 2
<211> 169
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1850310CD1
<400> 2
Met Gln Cys Leu Leu Pro Tyr Gln Ser Lys Glu Pro Ser Cys Leu
1 5 10 15
Pro Pro Leu Pro Leu Asn Leu Pro Leu Pro Pro Cys Leu Cys Pro
20 25 30
Leu Leu Gln Leu Asn Ala Ala Met Thr Arg Lys Glu Lys Thr Lys
35 40 45
Glu Gly Gln Arg Ala Ala Gln Phe Ser Ala Gly Ala Asp Ala Gly
50 55 60
Ser Gly Gly Gly Leu Ser Arg Gln Lys Asp Thr Lys Arg Pro Met
65 70 75
Leu Leu Val Ile His Asp Val Val Leu Glu Leu Leu Thr Ser Ser
80 85 90
Asp Cys His Ala Asn Pro Arg Lys Tyr Pro Thr Cys Gln Lys Ser
95 100 105
Glu Val Leu Gly Val Ser Ile Tyr Val Ser Ile Cys Pro Ser Thr
110 115 120
Arg Pro Arg Asp Lys Asn Lys Thr Lys Lys Arg Cys Gln Val Leu
125 13 0 13 5
Glu Ala Val Leu Val Ser Lys Pro Ser Gly Ser Cys His Gln Gly
140 145 150
Ser Phe Glu Ile Val Pro His Val Lys Gly Asn Leu Ala Phe Thr
155 160 165
Ser Ser Asn His
<210> 3
<211> 316
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1887020CD1
<400> 3
2/72
CA 02373191 2001-11-05
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Met Glu Ser Asn Val Lys Val Gln Arg Gln Glu Gly Ala Lys Val
1 5 10 15
Ser Leu Met Ser Pro Asp Gln Leu Arg Asn Lys Phe Pro Trp Ile
20 25 30
Asn Thr Glu Gly Val Ala Leu Ala Ser Tyr Gly Met Glu Asp Glu
35 40 45
Gly Trp Phe Asp Pro Trp Cys Leu Leu Gln Gly Leu Arg Arg Lys
50 55 60
Val Gln Ser Leu Gly Val Leu Phe Cys Gln Gly Glu Val Thr Arg
65 70 75
Phe Val Ser Ser Ser Gln Arg Met Leu Thr Thr Asp Asp Lys Ala
80 85 90
Val Val Leu Lys Arg Ile His Glu Val His Va1 Lys Met Asp Arg
95 100 105
Ser Leu Glu Tyr Gln Pro Val Glu Cys Ala Ile Val Ile Asn Ala
110 115 120
Ala Gly Ala Trp Ser Ala Gln Ile Ala Ala Leu Ala Gly Val Gly
125 130 135
Glu Gly Pro Pro Gly Thr Leu Gln Gly Thr Lys Leu Pro Val Glu
140 145 150
Pro Arg Lys Arg Tyr Val Tyr Val Trp His Cys Pro Gln Gly Pro
155 160 165
Gly Leu Glu Thr Pro Leu Val Ala Asp Thr Ser Gly Ala Tyr Phe
170 175 180
Arg Arg Glu Gly Leu Gly Ser Asn Tyr Leu Gly Gly Arg Ser Pro
185 190 195
Thr Glu Gln Glu Glu Pro Asp Pro Ala Asn Leu Glu Val Asp His
200 205 210
Asp Phe Phe Gln Asp Lys Val Trp Pro His Leu Ala Leu Arg Val
215 220 225
Pro Ala Phe Glu Thr Leu Lys Val Gln Ser Ala Trp Ala Gly Tyr
230 235 240
Tyr Asp Tyr Asn Thr Phe Asp Gln Asn Gly Val Val Gly Pro His
245 250 255
Pro Leu Val Val Asn Met Tyr Phe Ala Thr Gly Phe Ser Gly His
260 265 270
Gly Leu Gln Gln Ala Pro Gly Ile Gly Arg Ala Val Ala Glu Met
275 280 285
Val Leu Lys Gly Arg Phe Gln Thr Ile Asp Leu Ser Pro Phe Leu
290 295 300
Phe Thr Arg Phe Tyr Leu Gly Glu Lys Ile Gln Glu Asn Asn Ile
305 310 315
Ile
<210> 4
<211> 220
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1911421CD1
<400> 4
Met Lys Ser Val Ile Tyr His Ala Leu Ser Gln Lys Glu Ala Asn
1 5 10 15
Asp Ser Asp Val Gln Pro Ser Gly Ala Gln Arg Ala Glu Ala Phe
20 25 30
Val Arg Ala Phe Leu Lys Arg Ser Thr Pro Arg Met Ser Pro Gln
35 40 45
Ala Arg Glu Asp Gln Leu Gln Arg Lys Ala Val Val Leu Glu Tyr
50 55 60
Phe Thr Arg His Lys Arg Lys Glu Lys Lys Lys Lys Ala Lys Gly
65 70 75
Leu Ser Ala Arg Gln Arg Arg Glu Leu Arg Leu Phe Asp Ile Lys
80 85 90
3/72
CA 02373191 2001-11-05
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Pro Glu Gln Gln Arg Tyr Ser Leu Phe Leu Pro Leu His Glu Leu
95 100 105
Trp Lys Gln Tyr Ile Arg Asp Leu Cys Ser Gly Leu Lys Pro Asp
110 115 120
Thr Gln Pro Gln Met Ile Gln Ala Lys Leu Leu Lys Ala Asp Leu
125 130 135
His Gly Ala Ile Ile Ser Val Thr Lys Ser Lys Cys Pro Ser Tyr
140 145 150
Val Gly Ile Thr Gly Ile Leu Leu Gln Glu Thr Lys His Ile Phe
155 160 165
Lys Ile Ile Thr Lys Glu Asp Arg Leu Lys Val Ile Pro Lys Leu
170 175 180
Asn Cys Val Phe Thr Val Glu Thr Asp Gly Phe Ile Ser Tyr Ile
185 190 195
Tyr Gly Ser Lys Phe Gln Leu Arg Ser Ser Glu Arg Ser Ala Lys
200 205 210
Lys Phe Lys Ala Lys Gly Thr Ile Asp Leu
215 220
<210> 5
<211> 235
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1911910CD1
<400> 5
Met Gly Ser Thr Glu Ser Ser Glu Gly Arg Arg Val Ser Phe Gly
1 5 10 15
Val Asp Glu Glu Glu Arg Val Arg Val Leu Gln Gly Val Arg Leu
20 25 30
Ser Glu Asn Val Val Asn Arg Met Lys Glu Pro Ser Ser Pro Pro
35 40 45
Pro Ala Pro Thr Ser Ser Thr Phe Gly Leu Gln Asp Gly Asn Leu
50 55 60
Arg Ala Pro His Lys Glu Ser Thr Leu Pro Arg Ser Gly Ser Ser
65 70 75
Gly Gly Gln Gln Pro Ser Gly Met Lys Glu Gly Val Lys Arg Tyr
80 85 90
Glu Gln Glu His Ala Ala Ile Gln Asp Lys Leu Phe Gln Val Ala
95 100 105
Lys Arg Glu Arg Glu Ala Ala Thr Lys His Ser Lys Ala Ser Leu
110 115 120
Pro Thr Gly Glu Gly Ser Ile Ser His Glu Glu Gln Lys Ser Val
125 130 135
Arg Leu Ala Arg Glu Leu Glu Ser Arg Glu Ala Glu Leu Arg Arg
140 145 150
Arg Asp Thr Phe Tyr Lys Glu Gln Leu Glu Arg Ile Glu Arg Lys
155 160 165
Asn Ala Glu Met Tyr Lys Leu Ser Ser Glu Gln Phe His Glu Ala
170 175 180
Ala Ser Lys Met Glu Ser Thr Ile Lys Pro Arg Arg Val Glu Pro
185 190 195
Val Cys Ser Gly Leu Gln Ala Gln Ile Leu His Cys Tyr Arg Asp
200 205 210
Arg Pro His Glu Val Leu Leu Cys Ser Asp Leu Val Lys Ala Tyr
215 220 225
Gln Arg Cys Val Ser Ala Ala His Lys Gly
230 235
<210> 6
<211> 487
<212> PRT
4/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1928920CD1
<400> 6
Met Ala Ser Ser Ala Glu Gly Asp Glu Gly Thr Val Val Ala Leu
1 5 10 15
Ala Gly Val Leu Gln Ser Gly Phe Gln Glu Leu Ser Leu Asn Lys
20 25 30
Leu Ala Thr Ser Leu Gly Ala Ser Glu Gln Ala Leu Arg Leu Ile
35 40 45
Ile Ser Ile Phe Leu Gly Tyr Pro Phe Ala Leu Phe Tyr Arg His
50 55 60
Tyr Leu Phe Tyr Lys Glu Thr Tyr Leu Ile His Leu Phe His Thr
65 70 75
Phe Thr Gly Leu Ser Ile Ala Tyr Phe Asn Phe Gly Asn Gln Leu
80 85 90
Tyr His Ser Leu Leu Cys Ile Val Leu Gln Phe Leu Ile Leu Arg
95 100 105
Leu Met Gly Arg Thr Ile Thr Ala Val Leu Thr Thr Phe Cys Phe
110 115 120
Gln Met Ala Tyr Leu Leu Ala Gly Tyr Tyr Tyr Thr Ala Thr Gly
125 130 135
Asn Tyr Asp Ile Lys Trp Thr Met Pro His Cys Val Leu Thr Leu
140 145 150
Lys Leu Ile Gly Leu Ala Val Asp Tyr Phe Asp Gly Gly Lys Asp
155 160 165
Gln Asn Ser Leu Ser Ser Glu Gln Gln Lys Tyr Ala Ile Arg Gly
170 175 180
Val Pro Ser Leu Leu Glu Val Ala Gly Phe Ser Tyr Phe Tyr Gly
185 190 195
Ala Phe Leu Val Gly Pro Gln Phe Ser Met Asn His Tyr Met Lys
200 205 210
Leu Val Gln Gly Glu Leu Ile Asp Ile Pro Gly Lys Ile Pro Asn
215 220 225
Ser Ile Ile Pro Ala Leu Lys Arg Leu Ser Leu Gly Leu Phe Tyr
230 235 240
Leu Val Gly Tyr Thr Leu Leu Ser Pro His Ile Thr Glu Asp Tyr
245 250 255
Leu Leu Thr Glu Asp Tyr Asp Asn His Pro Phe Trp Phe Arg Cys
260 265 270
Met Tyr Met Leu Ile Trp Gly Lys Phe Val Leu Tyr Lys Tyr Val
275 280 285
Thr Cys Trp Leu Val Thr Glu Gly Val Cys Ile Leu Thr Gly Leu
290 295 300
Gly Phe Asn Gly Phe Glu Glu Lys Gly Lys Ala Lys Trp Asp Ala
305 310 315
Cys Ala Asn Met Lys Val Trp Leu Phe Glu Thr Asn Pro Arg Phe
320 325 330
Thr Gly Thr Ile Ala Ser Phe Asn Ile Asn Thr Asn Ala Trp Val
335 340 345
Ala Arg Tyr Ile Phe Lys Arg Leu Lys Phe Leu Gly Asn Lys Glu
350 355 360
Leu Ser Gln Gly Leu Ser Leu Leu Phe Leu Ala Leu Trp His Gly
365 370 375
Leu His Ser Gly Tyr Leu Val Cys Phe Gln Met Glu Phe Leu Ile
380 385 390
Val Ile Val Glu Arg Gln Ala Ala Arg Leu Ile Gln Glu Ser Pro
395 400 405
Thr Leu Ser Lys Leu Ala Ala Ile Thr Val Leu Gln Pro Phe Tyr
410 415 420
Tyr Leu Val Gln Gln Thr Ile His Trp Leu Phe Met Gly Tyr Ser
425 430 435
Met Thr Ala Phe Cys Leu Phe Thr Trp Asp Lys Trp Leu Lys Val
440 445 450
5/72
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Tyr Lys Ser Ile Tyr Phe Leu Gly His Ile Phe Phe Leu Ser Leu
455 460 465
Leu Phe Ile Leu Pro Tyr Ile His Lys Ala Met Val Pro Arg Lys
470 475 480
Glu Lys Leu Lys Lys Met Glu
485
<210> 7
<211> 212
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2170846CD1
<400> 7
Met Ala Ala Pro Pro Gln Leu Arg Ala Leu Leu Val Val Val Asn
1 5 10 15
Ala Leu Leu Arg Lys Arg Arg Tyr His Ala Ala Leu Ala Val Leu
20 25 30
Lys Gly Phe Arg Asn Gly Ala Val Tyr Gly Ala Lys Ile Arg Ala
35 40 45
Pro His Ala Leu Val Met Thr Phe Leu Phe Arg Asn Gly Ser Leu
5p 55 60
Gln Glu Lys Leu Trp Ala Ile Leu Gln Ala Thr Tyr Ile His Ser
65 70 75
Trp Asn Leu Ala Arg Phe Val Phe Thr Tyr Lys Gly Leu Arg Ala
80 85 90
Leu Gln Ser Tyr Ile Gln Gly Lys Thr Tyr Pro Ala His Ala Phe
95 100 105
Leu Ala Ala Phe Leu Gly Gly Ile Leu Val Phe Gly Glu Asn Asn
110 115 120
Asn Ile Asn Ser Gln Ile Asn Met Tyr Leu Leu Ser Arg Val Leu
125 130 135
Phe Ala Leu Ser Arg Leu Ala Val Glu Lys Gly Tyr Ile Pro Glu
140 145 150
Pro Arg Trp Asp Pro Phe Pro Leu Leu Thr Ala Val Val Trp Gly
155 160 165
Leu Val Leu Trp Leu Phe Glu Tyr His Arg Ser Thr Leu Gln Pro
170 175 180
Ser Leu Gln Ser Ser Met Thr Tyr Leu Tyr Glu Asp Ser Asn Val
185 190 195
Trp His Asp Ile Ser Asp Phe Leu Ile Tyr Asn Lys Ser Arg Pro
200 205 210
Ser Asn
<210> 8
<211> 241
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2176361CD1
<400> 8
Met Ala Pro Val Arg Arg Ser Ala Lys Trp Arg Pro Gly Gly Ile
1 5 10 15
Glu Ala Arg Gly Glu Gly Val Ser Thr Val Gly Tyr Arg Asn Lys
20 25 30
Asn Val Arg Gln Lys Thr Trp Arg Pro Asn His Pro Gln Ala Phe
35 40 45
Val Gly Ser Val Arg Glu Gly Gln Gly Phe Ala Phe Arg Arg Lys
50 55 60
6/72
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Leu Lys Ile Gln Gln Ser Tyr Lys Lys Leu Leu Arg Lys Glu Lys
65 70 75
Lys Ala Gln Thr Ser Leu Glu Ser Gln Phe Thr Asp Arg Tyr Pro
80 85 90
Asp Asn Leu Lys His Leu Tyr Leu Ala Glu Glu Glu Arg His Arg
95 100 105
Lys Gln Ala Arg Lys Val Asp His Pro Leu Ser Glu Gln Val His
110 115 120
Gln Pro Leu Leu Glu Glu Gln Cys Ser Ile Asp Glu Pro Leu Phe
125 130 135
Glu Asp Gln Cys Ser Phe Asp Gln Pro Gln Pro Glu Glu Gln Cys
140 145 150
Ile Lys Thr Val Asn Ser Phe Thr Ile Pro Lys Lys Asn Lys Lys
155 160 165
Lys Thr Ser Asn Gln Lys Ala Gln Glu Glu Tyr Glu Gln Ile Gln
170 175 180
Ala Lys Arg Ala Ala Lys Lys Gln Glu Phe Glu Arg Arg Lys Gln
185 190 195
Glu Arg Glu Glu Ala Gln Arg Gln Tyr Lys Lys Lys Lys Met Glu
200 205 210
Val Phe Lys Ile Leu Asn Lys Lys Thr Lys Lys Gly Gln Pro Asn
215 220 225
Leu Asn Val Gln Met Glu Tyr Leu Leu Gln Lys Ile Gln Glu Lys
230 235 240
Cys
<210> 9
<211> 375
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2212732CD1
<400> 9
Met Pro Gln Glu Leu Pro Gln Ser Pro Arg Thr Arg Gln Pro Glu
1 5 10 15
Pro Asp Phe Tyr Cys Val Lys Trp Ile Pro Trp Lys Gly Glu Gln
20 25 30
Thr Pro Ile Ile Thr Gln Ser Thr Asn Gly Pro Cys Pro Leu Leu
35 40 45
Ala Ile Met Asn Ile Leu Phe Leu Gln Trp Lys Val Lys Leu Pro
50 55 60
Pro Gln Lys Glu Val Ile Thr Ser Asp Glu Leu Met Ala His Leu
65 70 75
Gly Asn Cys Leu Leu Ser Ile Lys Pro Gln Glu Lys Ser Glu Gly
80 85 90
Leu Gln Leu Asn Phe Gln Gln Asn Val Asp Asp Ala Met Thr Val
95 100 105
Leu Pro Lys Leu Ala Thr Gly Leu Asp Val Asn Val Arg Phe Thr
110 115 120
Gly Val Ser Asp Phe Glu Tyr Thr Pro Glu Cys Ser Val Phe Asp
125 130 135
Leu Leu Gly Ile Pro Leu Tyr His Gly Trp Leu Val Asp Pro Gln
140 145 150
Gln Ser Pro Glu Ala Val Arg Ala Val Gly Lys Leu Ser Tyr Asn
155 160 165
Gln Leu Val Glu Arg Ile Ile Thr Cys Lys His Ser Ser Asp Thr
170 175 180
Asn Leu Val Thr Glu Gly Leu Ile Ala Glu Gln Phe Leu Glu Thr
1g5 190 195
Thr Ala Ala Gln Leu Thr Tyr His Gly Leu Cys Glu Leu Thr Ala
200 205 210
Ala Ala Lys Glu Gly Glu Leu Ser Va1 Phe Phe Arg Asn Asn His
215 220 225
?/72
CA 02373191 2001-11-05
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Phe Ser Thr Met Thr Lys His Lys Ser His Leu Tyr Leu Leu Val
230 235 240
Thr Asp Gln Gly Phe Leu Gln Glu Glu Gln Val Val Trp Glu Ser
245 250 255 .
Leu His Asn Val Asp Gly Asp Ser Cys Phe Cys Asp Ser Asp Phe
260 265 270
His Leu Ser His Ser Leu Gly Lys Gly Pro Gly Ala Glu Gly Gly
275 280 285
Ser Gly Ser Pro Glu Lys Gln Leu Gln Val Asp Gln Asp Tyr Leu
290 295 300
Ile Ala Leu Ser Leu Gln Gln Gln Gln Pro Arg Gly Pro Leu Gly
305 310 315
Leu Thr Asp Leu Glu Leu Ala Gln Gln Leu Gln Gln Glu Glu Tyr
320 325 330
Gln Gln Gln Gln Ala Ala Gln Pro Val Arg Met Arg Thr Arg Val
335 340 345
Leu Ser Leu Gln Gly Arg Gly Ala Thr Ser Gly Arg Pro Ala Gly
350 355 360
Glu Arg Arg Gln Arg Pro Lys His Glu Ser Asp Cys Ile Leu Leu
365 370 375
<210> 10
<211> 429
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2303457CD1
<400> 10
Met Ser Asn Arg Asn Asn Asn Lys Leu Pro Ser Asn Leu Pro Gln
1 5 10 15
Leu Gln Asn Leu Ile Lys Arg Asp Pro Pro Ala Tyr Ile Glu Glu
20 25 30
Phe Leu Gln Gln Tyr Asn His Tyr Lys Ser Asn Val Glu Ile Phe
35 40 45
Lys Leu Gln Pro Asn Lys Pro Ser Lys Glu Leu Ala Glu Leu Val
50 55 60
Met Phe Met Ala Gln Ile Ser His Cys Tyr Pro Glu Tyr Leu Ser
65 70 75
Asn Phe Pro Gln Glu Val Lys Asp Leu Leu Ser Cys Asn His Thr
80 85 90
Val Leu Asp Pro Asp Leu Arg Met Thr Phe Cys Lys Ala Leu Ile
95 100 105
Leu Leu Arg Asn Lys Asn Leu Ile Asn Pro Ser Ser Leu Leu Glu
110 115 120
Leu Phe Phe Glu Leu Phe Arg Cys His Asp Lys Leu Leu Arg Lys
125 130 135
Thr Leu Tyr Thr His Ile Val Thr Asp Ile Lys Asn Ile Asn Ala
140 145 150
Lys His Lys Asn Asn Lys Val Asn Val Val Leu Gln Asn Phe Met
155 160 165
Tyr Thr Met Leu Arg Asp Ser Asn Ala Thr Ala Ala Lys Met Ser
170 175 180
Leu Asp Val Met Ile Glu Leu Tyr Arg Arg Asn Ile Trp Asn Asp
185 190 195
Ala Lys Thr Val Asn Val Ile Thr Thr Ala Cys Phe Ser Lys Val
200 205 210
Thr Lys Ile Leu Val Ala Ala Leu Thr Phe Phe Leu Gly Lys Asp
215 220 225
Glu Asp Glu Lys Gln Asp Ser Asp Ser Glu Ser Glu Asp Asp Gly
230 235 240
Pro Thr Ala Arg Asp Leu Leu Val Gln Tyr Ala Thr Gly Lys Lys
245 250 255
Ser Ser Lys Asn Lys Lys Lys Leu Glu Lys Ala Met Lys Val Leu
8/72
CA 02373191 2001-11-05
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260 265 270
Lys Lys Gln Lys Lys Lys Lys Lys Pro Glu Val Phe Asn Phe Ser
275 280 285
Ala Ile His Leu Ile His Asp Pro Gln Asp Phe Ala Glu Lys Leu
290 295 300
Leu Lys Gln Leu Glu Cys Cys Lys Glu Arg Phe Glu Val Lys Met
305 310 315
Met Leu Met Asn Leu Ile Ser Arg Leu Val G1y Ile His Glu L~eu
320 325 330
Phe Leu Phe Asn Phe Tyr Pro Phe Leu Lys Arg Phe Leu Lys Pro
335 340 345
His Gln Arg Glu Val Thr Lys Ile Leu Leu Phe Val Glu Lys Asp
350 355 360
Ser His His Leu Val Pro Gln Gly Phe Phe Asn Ser Trp Leu Met
365 370 375
Leu Gly Glu Lys Ile Phe Phe Asn Gly Lys Lys Ser Gly Lys Met
380 385 390
Leu Met Thr Val Gly Asn Leu Met Val Lys Arg Gly Val Tyr Lys
395 400 405
Arg Ser Lys Val Phe Leu Gly Gly Asn Ser Val Gly Arg Asn Phe
410 415 420
Phe Gln Lys Asn Pro Gly Gly Ser Ser
425
<210> 11
<211> 329
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2317552CD1
<400> 11
Met Glu Val Ala Glu Pro 5er Ser Pro Thr Glu Glu Glu Glu Glu
1 5 10 15
Glu Glu Glu His Ser Ala Glu Pro Arg Pro Arg Thr Arg Ser Asn
20 25 30
Pro Glu Gly Ala Glu Asp Arg Ala Val Gly Ala Gln Ala Ser Val
35 40 45
Gly Ser Arg Ser Glu Gly Glu Gly Glu Ala Ala Ser Ala Asp Asp
50 55 60
Gly Ser Leu Asn Thr Ser Gly Ala Gly Pro Lys Ser Trp Gln Val
65 70 75
Pro Pro Pro Ala Pro Glu Val Gln Ile Arg Thr Pro Arg Val Asn
80 85 90
Cys Pro Glu Lys Val Ile Ile Cys Leu Asp Leu Ser Glu Glu Met
95 100 105
Ser Leu Pro Lys Leu Glu Ser Phe Asn Gly Ser Lys Thr Asn Ala
110 115 120
Leu Asn Val Ser Gln Lys Met Ile Glu Met Phe Val Arg Thr Lys
125 130 135
His Lys Ile Asp Lys Ser His Glu Phe Ala Leu Val Val Val Asn
140 145 150
Asp Asp Thr Ala Trp Leu Ser Gly Leu Thr Ser Asp Pro Arg Glu
155 160 165
Leu Cys Ser Cys Leu Tyr Asp Leu Glu Thr Ala Ser Cys Ser Thr
170 175 180
Phe Asn Leu Glu Gly Leu Phe Ser Leu Ile Gln Gln Lys Thr Glu
185 190 195
Leu Pro Val Thr Glu Asn Val Gln Thr Ile Pro Pro Pro Tyr Val
200 205 210
Val Arg Thr Ile Leu Val Tyr Ser Arg Pro Pro Cys Gln Pro Gln
215 220 225
Phe Ser Leu Thr Glu Pro Met Lys Lys Met Phe Gln Cys Pro Tyr
230 235 240
9/72
CA 02373191 2001-11-05
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Phe Phe Phe Asp Val Val Tyr Ile His Asn Gly Thr Glu Glu Lys
245 250 255
Glu Glu Glu Met Ser Trp Lys Asp Met Phe Ala Phe Met Gly Ser
260 265 270
Leu Asp Thr Lys Gly Thr Ser Tyr Lys Tyr Glu Val Ala Leu Ala
275 280 285
Gly Pro Ala Leu Glu Leu His Asn Cys Met Ala Lys Leu Leu Ala
290 295 300
His Pro Leu Gln Arg Pro Cys Gln Ser His Ala Ser Tyr Ser Leu
305 310 315
Leu Glu Glu Glu Asp Glu Ala Ile Glu Val Glu Ala Thr Val
320 325
<210> 12
<211> 476
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2416366CD1
<400> 12
Met Gln Asn Asp Ser Phe His Ser Asp Ser His Met Asp Arg Lys
10 15
Lys Phe His Ser Ser Asp Ser Glu Glu Glu Glu His Lys Lys Gln
20 25 30
Lys Met Asp Ser Asp Glu Asp Glu Lys Glu Gly Glu Glu Glu Lys
35 40 45
Val Ala Lys Arg Lys Ala Ala Val Leu Ser Asp Ser Glu Asp Glu
50 55 60
Glu Lys Ala Ser Ala Lys Lys Ser Arg Val Val Ser Asp Ala Asp
65 70 75
Asp Ser Asp Ser Asp Ala Val Ser Asp Lys Ser Gly Lys Arg Glu
80 85 90
Lys Thr Ile Ala Ser Asp Ser Glu Glu Glu Ala Gly Lys Glu Leu
g5 100 105
Ser Asp Lys Lys Asn Glu Glu Lys Asp Leu Phe Gly Ser Asp Ser
110 115 120
Glu Ser Gly Asn Glu Glu Glu Asn Leu Ile Ala Asp Ile Phe Gly
125 130 135
Glu Ser Gly Asp Glu Glu Glu Glu Glu Phe Thr Gly Phe Asn Gln
140 145 150
Glu Asp Leu Glu Glu Glu Lys Gly Glu Thr Gln Val Lys Glu Ala
155 160 165
Glu Asp Ser Asp Ser Asp Asp Asn Ile Lys Arg Gly Lys His Met
170 175 180
Asp Phe Leu Ser Asp Phe Glu Met Met Leu Gln Arg Lys Lys Ser
185 190 195
Met Ser Gly Lys Arg Arg Arg Asn Arg Asp Gly Gly Thr Phe Ile
200 205 210
Ser Asp Ala Asp Asp Val Val Ser Ala Met Ile Val Lys Met Asn
215 220 225
Glu Ala Ala Glu Glu Asp Arg Gln Leu Asn Asn Gln Lys Lys Pro
230 235 240
Ala Leu Lys Lys Leu Thr Leu Leu Pro Ala Val Val Met His Leu
245 250 255
Lys Lys Gln Asp Leu Lys Glu Thr Phe Ile Asp Ser Gly Val Met
260 265 270
Ser Ala Ile Lys Glu Trp Leu Ser Pro Leu Pro Asp Arg Ser Leu
275 280 285
Pro Ala Leu Lys Ile Arg Glu Glu Leu Leu Lys Ile Leu Gln Glu
290 295 300
Leu Pro Ser Val Ser Gln Glu Thr Leu Lys His Ser Gly Ile Gly
305 310 315
Arg Ala Val Met Tyr Leu Tyr Lys His Pro Lys Glu Ser Arg Ser
10/72
CA 02373191 2001-11-05
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320 325 330
Asn Lys Asp Met Ala Gly Lys Leu Ile Asn Glu Trp Ser Arg Pro
335 340 345
Ile Phe Gly Leu Thr Ser Asn Tyr Lys Gly Met Thr Arg Glu Glu
350 355 360
Arg Glu Gln Arg Asp Leu Glu Gln Met Pro Gln Arg Arg Arg Met
365 370 375
Asn Ser Thr Gly Gly Gln Thr Pro Arg Arg Asp Leu Glu Lys Val
380 385 390
Leu Thr Gly Glu Glu Lys Ala Leu Arg Pro Gly Asp Pro Gly Phe
395 400 405
Cys Ala Arg Ala Arg Val Pro Met Pro Ser Asn Lys Asp Tyr Val
410 415 420
Val Arg Pro Lys Trp Asn Val Glu Met Glu Ser Ser Arg Phe Gln
425 430 435
Ala Thr Ser Lys Lys Gly Ile Ser Arg Leu Asp Lys Gln Met Arg
440 445 450
Lys Phe Thr Asp Ile Arg Lys Lys Ser Arg Ser Ala His Ala Val
455 460 465
Lys Ile Ser Ile Glu Gly Asn Lys Met Pro Leu
470 475
<210> 13
<211> 366
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2472980CD1
<400> 13
Met Ala Ala Ala Tyr Phe Pro Asp Cys Ile Val Arg Pro Phe Gly
1 5 10 15
Ser Ser Val Asn Thr Phe Gly Lys Leu Gly Cys Asp Leu Asp Met
20 25 30
Phe Leu Asp Leu Asp Glu Thr Arg Asn Leu Ser Ala His Lys Ile
35 40 45
Ser Gly Asn Phe Leu Met Glu Phe Gln Val Lys Asn Val Pro Ser
50 55 60
Glu Arg Ile Ala Thr Gln Lys Ile Leu Ser Val Leu Gly Glu Cys
65 70 75
Leu Asp His Phe Gly Pro Gly Cys Val Gly Val Gln Lys Ile Leu
80 85 90
Asn Ala Arg Cys Pro Leu Val Arg Phe Ser His Gln Ala Ser Gly
95 100 105
Phe Gln Cys Asp Leu Thr Thr Asn Asn Arg Ile Ala Leu Thr Ser
110 115 120
Ser Glu Leu Leu Tyr Ile Tyr Gly Ala Leu Asp Ser Arg Val Arg
125 130 135
Ala Leu Val Phe Ser Val Arg Cys Trp Ala Arg Ala His Ser Leu
140 145 150
Thr Ser Ser Ile Pro Gly Ala Trp Ile Thr Asn Phe Ser Leu Thr
155 160 165
Met Met Val Ile Phe Phe Leu Gln Arg Arg Ser Pro Pro Ile Leu
170 175 180
Pro Thr Leu Asp Ser Leu Lys Thr Leu Ala Asp Ala Glu Asp Lys
185 190 195
Cys Val Ile Glu Gly Asn Asn Cys Thr Phe Val Arg Asp Leu Ser
200 205 210
Arg Ile Lys Pro Ser Gln Asn Thr Glu Thr Leu Glu Leu Leu Leu
215 220 225
Lys Glu Phe Phe Glu Tyr Phe Gly Asn Phe Ala Phe Asp Lys Asn
230 235 240
Ser Ile Asn Ile Arg Gln Gly Arg Glu Gln Asn Lys Pro Asp Ser
245 250 255
11 /72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Ser Pro Leu Tyr Ile Gln Asn Pro Phe Glu Thr Ser Leu Asn Ile
260 265 270
Ser Lys Asn Val Ser Gln Ser Gln Leu Gln Lys Phe Val Asp Leu
275 280 285
Ala Arg Glu Ser Ala Trp Ile Leu Gln Gln Glu Asp Thr Asp Arg
290 295 300
Pro Ser Ile Ser Ser Asn Arg Pro Trp Gly Leu Val Ser Leu Leu
305 310 315
Leu Pro Ser Ala Pro Asn Arg Lys Ser Phe Thr Lys Lys Lys Ser
320 325 330
Asn Lys Phe Ala Ile Glu Thr Val Lys Asn Leu Leu Glu Ser Leu
335 340 345
Lys Gly Asn Arg Thr Glu Asn Phe Thr Lys Thr Ser Glv_ Lys Arg
350 355 360
Thr Ile Ser Thr Gln Thr
365
<210> 14
<211> 152
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2541640CD1
<400> 14
Met Gly Gly Val Gly Val Ala Glu Ala Ala Arg Pro Leu Leu Ser
1 5 10 15
Trp Pro Thr Ile Ser Leu Thr Ile Phe Thr Ala Val Asn Ser Ser
20 25 30
Gln Gly Gly Gly Leu Val Gln Arg Gln Leu Arg Phe His Asn Ser
35 40 45
His Arg Val Leu Cys Arg Arg Cys Pro Cys Pro Pro Thr Pro Ala
50 55 60
Trp Trp Glu Cys Asp Ala Arg Leu Leu Pro Pro Pro Trp Pro Pro
65 70 75
Val Pro Pro Ala Ser Thr Ser Pro Glu Ile Leu Pro Thr Pro His
80 85 90
Leu His Arg Ser Pro His Ala Pro Gly Ala Pro Lys Pro Pro Pro
95 100 105
Asn Pro Thr His Pro Gly Ala Gly Thr Gly Val Ser Glu Leu Ser
110 115 120
Gln Gly Pro Trp Glu Val Ala Gly Thr Gly Ala Ser Cys Ser Leu
125 130 135
Phe His Phe Pro Phe Arg Ile Trp Pro Gly Trp Arg Thr Gly Gln
140 145 150
Asp Gly
<210> 15
<211> 233
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2695204CD1
<400> 15
Met Gly Arg Arg Leu Lys Gly Ala Arg Arg Leu Lys Leu Ser Pro
1 5 10 15
Leu Arg Ser Leu Arg Lys Gly Pro Gly Leu Leu Ser Pro Pro Ser
20 25 30
Ala Ser Pro Val Pro Thr Pro Ala Val Ser Arg Thr Leu Leu Gly
35 40 45
12/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Asn Phe Glu Glu Ser Leu Leu Arg Gly Arg Phe Ala Pro Ser Gly
50 55 60
His Ile Glu Gly Phe Thr Ala Glu Ile Gly Ala Ser Gly Ser Tyr
65 70 75
Cys Pro Gln His Val Thr Leu Pro Val Thr Val Thr Phe Phe Asp
80 85 90
Val Ser Glu Gln Asn Ala Pro Ala Pro Phe Leu Gly Ile Val Asp
95 100 105
Leu Asn Pro Leu Gly Arg Lys Gly Tyr Ser Val Pro Lys Val Gly
110 115 120
Thr Val Gln Val Thr Leu Phe Asn Pro Asn Gln Thr Val Val Lys
125 130 135
Met Phe Leu Val Thr Phe Asp Phe Ser Asp Met Pro Ala Ala His
140 145 150
Met Thr Phe Leu Arg His Arg Leu Phe Leu Val Pro Val Gly Glu
155 160 165
Glu Gly Asn Ala Asn Pro Thr His Arg Leu Leu Cys Tyr Leu Leu
170 175 180
His Leu Arg Phe Arg Ser Ser Arg Ser Gly Arg Leu Ser Leu His
185 190 195
Gly Asp Ile Arg Leu Leu Phe Ser Arg Arg Ser Leu Glu Leu Asp
200 205 210
Thr Gly Leu Pro Tyr Glu Leu Gln Ala Val Thr Glu Ala Pro His
215 220 225
Asn Pro Arg Tyr Ser Pro Leu Pro
230
<210> 16
<211> 357
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2805526CD1
<400> 16
Met Glu Val Leu Arg Pro Gln Leu Ile Arg Ile Asp Gly Arg Asn
1 5 10 15
Tyr Arg Lys Asn Pro Val Gln Glu Gln Thr Tyr Gln His Glu Glu
20 25 30
Asp Glu Glu Asp Phe Tyr Gln Gly Ser Met Glu Cys Ala Asp Glu
35 40 45
Pro Cys Asp Ala Tyr Glu Val Glu Gln Thr Pro Gln Gly Phe Arg
50 55 60
Ser Thr Leu Arg Ala Pro Ser Leu Leu Tyr Lys His Ile Val Gly
65 70 75
Lys Arg Gly Asp Thr Arg Lys Lys Ile Glu Met Glu Thr Lys Thr
80 85 90
Ser Ile Ser Ile Pro Lys Pro Gly Gln Asp Gly Glu Ile Val Ile
95 100 105
Thr Gly Gln His Arg Asn Gly Val Ile Ser Ala Arg Thr Arg Ile
110 115 120
Asp Val Leu Leu Asp Thr Phe Arg Arg Lys Gln Pro Phe Thr His
125 130 135
Phe Leu Ala Phe Phe Leu Asn Glu Val Glu Val Gln Glu Gly Phe
140 145 150
Leu Arg Phe Gln Glu Glu Val Leu Ala Lys Cys Ser Met Asp His
155 160 165
Gly Val Asp Ser Ser Ile Phe Gln Asn Pro Lys Lys Leu His Leu
170 175 180
Thr Ile Gly Met Leu Val Leu Leu Ser Glu Glu Glu Ile Gln Gln
185 190 195
Thr Cys Glu Met Leu Gln Gln Cys Lys Glu Glu Phe Ile Asn Asp
200 205 210
Ile Ser Gly Gly Lys Pro Leu Glu Val Glu Met Ala Gly Ile Glu
13/72
CA 02373191 2001-11-05
WO 00/70047 PCT/iJS00/13299
215 220 225
Tyr Met Asn Asp Asp Pro Gly Met Val Asp Val Leu Tyr Ala Lys
230 235 240
Val His Met Lys Asp Gly Ser Asn Arg Leu Gln Glu Leu Val Asp
245 250 255
Arg Val Leu Glu Arg Phe Gln Ala Ser Gly Leu Ile Val Lys Glu
260 265 270
Trp Asn Ser Val Lys Leu His Ala Thr Val Met Asn Thr Leu Phe
275 280 285
Arg Lys Asp Pro Asn Ala Glu Gly Arg Tyr Asn Leu Tyr Thr Ala
290 295 300
Glu Gly Lys Tyr Ile Phe Lys Glu Arg Glu Ser Phe Asp Gly Arg
305 310 315
Asn Ile Leu Lys Leu Phe Glu Asn Phe Tyr Phe Gly Ser Leu Lys
320 325 330
Leu Asn Ser Ile His Ile Ser Gln Arg Phe Thr Val Asp Ser Phe
335 340 345
Gly Asn Tyr Ala Ser Cys Gly Gln Ile Asp Phe Ser
350 355
<210> 17
<211> 251
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2850382CD1
<400> 17
Met Glu Pro Gly Glu Glu Leu Glu Glu Glu Gly Ser Pro Gly Gly
1 5 10 15
Arg Glu Asp Gly Phe Thr Ala Glu His Leu Ala Ala Glu Ala Met
20 25 30
Ala Ala Asp Met Asp Pro Trp Leu Val Phe Asp Ala Arg Thr Thr
35 40 45
Pro Ala Thr Glu Leu Asp Ala Trp Leu Ala Lys Tyr Pro Pro Ser
50 55 60
Gln Val Thr Arg Tyr Gly Asp Pro Gly Ser Pro Asn Ser Glu Pro
65 70 75
Val Gly Trp Ile Ala Val Tyr Gly Gln Gly Tyr Ser Pro Asn Ser
80 85 90
Gly Asp Val Gln Gly Leu Gln Ala Ala Trp Glu Ala Leu Gln Thr
95 100 105
Ser Gly Arg Pro Ile Thr Pro Gly Thr Leu Arg Gln Leu Ala Ile
110 115 120
Thr His His Val Leu Ser Gly Lys Trp Leu Met His Leu Ala Pro
125 130 135
Gly Phe Lys Leu Asp His Ala Trp Ala Gly Ile Ala Arg Ala Val
140 145 150
Val Glu Gly Arg Leu Gln Val Ala Lys Val Ser Pro Arg Ala Lys
155 160 165
Glu Gly Gly Arg Gln Val Ile Cys Val Tyr Thr Asp Asp Phe Thr
170 175 180
Asp Arg Leu Gly Val Leu Glu Ala Asp Ser Ala Ile Arg Ala Ala
185 190 195
Gly Ile Lys Cys Leu Leu Thr Tyr Lys Pro Asp Val Tyr Thr Tyr
200 205 210
Leu Gly Ile Tyr Arg Ala Asn Arg Trp His Leu Cys Pro Thr Leu
215 220 ;225
Tyr Glu Ser Arg Phe Gln Leu Gly Gly Ser Ala Arg Gly Ser Arg
230 235 240
Val Leu Asp Arg Ala Asn Asn Val Glu Leu Thr
245 250
14/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<210> 18
<211> 105
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2929276CD1
<400> 18
Met Ser Ile Tyr Phe Pro Ile His Cys Pro Asp Tyr Leu Arg Ser
1 5 10 15
Ala Lys Met Thr Glu Val Met Met Asn Thr Gln Pro Met Glu Glu
20 25 30
Ile Gly Leu Ser Pro Arg Lys Asp Gly Leu Ser Tyr Gln Ile Phe
35 40 45
Pro Asp Pro Ser Asp Phe Asp Arg Cys Cys Lys Leu Lys Asp Arg
50 55 60
Leu Pro Ser Ile Val Val Glu Pro Thr Glu Gly Glu Val Glu Ser
65 70 75
Gly Glu Leu Arg Trp Pro Pro Glu Glu Phe Leu Val Gln Glu Asp
80 85 90
Glu Gln Asp Asn Cys Glu Glu Thr Ala Lys Glu Asn Lys Glu Gln
95 100 105
<210> 19
<211> 876
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3033039CD1
<400> 19
Met Thr Met Asp Ala Leu Leu Ala Arg Leu Lys Leu Leu Asn Pro
1 5 10 15
Asp Asp Leu Arg Glu Glu Ile Val Lys Ala Gly Leu Lys Cys Gly
20 25 30
Pro Ile Thr Ser Thr Thr Arg Phe Ile Phe Glu Lys Lys Leu Ala
35 40 45
Gln Ala Leu Leu Glu Gln Gly Gly Arg Leu Ser Ser Phe Tyr His
50 55 60
His Glu Ala Gly Val Thr Ala Leu Ser Gln Asp Pro Gln Arg Ile
65 70 75
Leu Lys Pro Ala Glu Gly Asn Pro Thr Asp Gln Ala Gly Phe Ser
80 85 90
Glu Asp Arg Asp Phe Gly Tyr Ser Val Gly Leu Asn Pro Pro Glu
95 100 105
Glu Glu Ala Val Thr Ser Lys Thr Cys Ser Val Pro Pro Ser Asp
110 115 120
Thr Asp Thr Tyr Arg Ala Gly Ala Thr Ala Ser Lys Glu Pro Pro
125 130 135
Leu Tyr Tyr Gly Val Cys Pro Val Tyr Glu Asp Val Pro Ala Arg
140 145 150
Asn Glu Arg Ile Tyr Val Tyr Glu Asn Lys Lys Glu Ala Leu Gln
155 160 165
Ala Val Lys Met Ile Lys Gly Ser Arg Phe Lys Ala Phe Ser Thr
170 175 180
Arg Glu Asp Ala Glu Lys Phe Ala Arg Gly Ile Cys Asp Tyr Phe
185 190 195
Pro Ser Pro Ser Lys Thr Ser Leu Pro Leu Ser Pro Val Lys Thr
200 205 210
Ala Pro Leu Phe Ser Asn Asp Arg Leu Lys Asp Gly Leu Cys Leu
215 220 225
Ser Glu Ser Glu Thr Val Asn Lys Glu Arg Ala Asn Ser Tyr Lys
15/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
230 235 240
Asn Pro Arg Thr Gln Asp Leu Thr Ala Lys Leu Arg Lys Al.a Val
245 250 255
Glu Lys Gly Glu Glu Asp Thr Phe Ser Asp Leu Ile Trp Ser Asn
260 265 270
Pro Arg Tyr Leu Ile Gly Ser Gly Asp Asn Pro Thr Ile Val Gln
275 280 285
Glu Gly Cys Arg Tyr Asn Val Met His Val Ala Ala Lys Glu Asn
290 295 300
Gln Ala Ser Ile Cys Gln Leu Thr Leu Asp Val Leu Glu Asn Pro
305 310 315
Asp Phe Met Arg Leu Met Tyr Pro Asp Asp Asp Glu Ala Met Leu
320 325 330
Gln Lys Arg Ile Arg Tyr Val Val Asp Leu Tyr Leu Asn Thr Pro
335 340 345
Asp Lys Met Gly Tyr Asp Thr Pro Leu His Phe Ala Cys Lys Phe
350 355 360
Gly Asn Ala Asp Val Val Asn Val Leu Ser Ser His His Leu Ile
365 370 375
Val Lys Asn Ser Arg Asn Lys Tyr Asp Lys Thr Pro Glu Asp Val
380 385 390
Ile Cys Glu Arg Ser Lys Asn Lys Ser Val Glu Leu Lys Glu Arg
395 400 405
Ile Arg Glu Tyr Leu Lys Gly His Tyr Tyr Val Pro Leu Leu Arg
410 415 420
Ala Glu Glu Thr Ser Ser Pro Val Ile Gly Glu Leu Trp Ser Pro
425 430 435
Asp Gln Thr Ala Glu Ala Ser His Val Ser Arg Tyr Gly Gly Ser
440 445 450
Pro Arg Asp Pro Val Leu Thr Leu Arg Ala Phe Ala Gly Pro Leu
455 460 465
Ser Pro Ala Lys Ala Glu Asp Phe Arg Lys Leu Trp Lys Thr Pro
470 475 480
Pro Arg Glu Lys Ala Gly Phe Leu His His Val Lys Lys Ser Asp
485 490 495
Pro Glu Arg Gly Phe Glu Arg Val Gly Arg Glu Leu Ala His Glu
500 505 510
Leu Gly Tyr Pro Trp Val Glu Tyr Trp Glu Phe Leu Gly Cys Phe
515 520 525
Val Asp Leu Ser Ser Gln Glu Gly Leu Gln Arg Leu Glu Glu Tyr
530 535 540
Leu Thr Gln Gln Glu Ile Gly Lys Lys Ala Gln Gln Glu Thr Gly
545 550 555
Glu Arg Glu Ala Ser Cys Arg Asp Lys Ala Thr Thr Ser Gly Ser
560 565 570
Asn Ser Ile Ser Val Arg Ala Phe Leu Asp Glu Asp Asp Met Ser
575 580 585
Leu Glu Glu Ile Lys Asn Arg Gln Asn Ala Ala Arg Asn Asn Ser
590 595 600
Pro Pro Thr Val Gly Ala Phe Gly His Thr Arg Cys Ser Ala Phe
605 610 615
Pro Leu Glu Gln Glu Ala Asp Leu Ile Glu Ala Ala Glu Pro Gly
620 625 630
Gly Pro His Ser Ser Arg Asn Gly Leu Cys His Pro Leu Asn His
635 640 645
Ser Arg Thr Leu Ala Gly Lys Arg Pro Lys Ala Pro Arg Gly Glu
650 655 660
Glu Ala His Leu Pro Pro Val Ser Asp Leu Thr Val Glu Phe Asp
665 670 675
Lys Leu Asn Leu Gln Asn Ile Gly Arg Ser Val Ser Lys Thr Pro
680 685 690
Asp Glu Ser Thr Lys Thr Lys Asp Gln Ile Leu Thr Ser Arg Ile
695 700 705
Asn Ala Val Glu Arg Asp Leu Leu Glu Pro Ser Pro Ala Asp Gln
710 715 720
Leu Gly Asn Gly His Arg Arg Thr Glu Ser Glu Met Ser Ala Arg
725 730 735
16/?2
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Ile Ala Lys Met Ser Leu Ser Pro Ser Ser Pro Arg His Glu Asp
740 745 750
Gln Leu Glu Val Thr Arg Glu Pro Ala Arg Arg Leu Phe Leu Phe
755 760 765
Gly Glu Glu Pro Ser Lys Leu Asp Gln Asp Val Leu Ala Ala Leu
770 775 780
Glu Cys Ala Asp Val Asp Pro His Gln Phe Pro Ala Val His Arg
785 790 795
Trp Lys Ser Ala Val Leu Cys Tyr Ser Pro Ser Asp Arg Gln Ser
800 805 810
Trp Pro Ser Pro Ala Val Lys Gly Arg Phe Lys Ser Gln Leu Pro
g15 820 825
Asp Leu Ser Gly Pro His Ser Tyr Ser Pro Gly Arg Asn Ser Val
830 835 840
Ala Gly Ser Asn Pro Ala Lys Pro Gly Leu Gly Ser Pro Gly Arg
845 850 855
Tyr Ser Pro Val His Gly Ser Gln Leu Arg Arg Met Ala Arg Leu
860 865 870
Ala Glu Leu Ala Ala Leu
875
<210> 20
<211> 254
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3213216CD1
<400> 20
Met Leu Glu His Lys Ile Phe Lys Arg Phe Trp Leu Val Ile Asn
10 15
Gln Glu Gly Asn Met Val Thr Ala Arg Gln Glu Pro Arg Leu Val
20 25 30
Leu Ile Ser Leu Thr Cys Asp Gly Asp Thr Leu Thr Leu Ser Ala
35 40 45
Ala Tyr Thr Lys Asp Leu Leu Leu Pro Ile Lys Thr Pro Thr Thr
50 55 60
Asn Ala Val His Lys Cys Arg Val His Gly Leu Glu Ile Glu Gly
65 70 75
Arg Asp Cys Gly Glu Ala Ala Ala Gln Trp Ile Thr Ser Phe Leu
80 85 90
Lys Ser Gln Pro Tyr Arg Leu Val His Phe Glu Pro His Met Arg
95 100 105
Pro Arg Arg Pro His Gln Ile Ala Asp Leu Phe Arg Pro Lys Asp
110 115 120
Gln Ile Ala Tyr Ser Asp Thr Ser Pro Phe Leu Ile Leu Ser Glu
125 130 135
Ala Ser Leu Ala Asp Leu Asn Ser Arg Leu Glu Lys Lys Val Lys
140 145 150
Ala Thr Asn Phe Arg Pro Asn Ile Val Ile Ser Gly Cys Asp Val
155 160 165
Tyr Ala Glu Asp Ser Trp Asp Glu Leu Leu Ile Gly Asp Val Glu
170 175 180
Leu Lys Arg Val Met Ala Cys Ser Arg Cys Ile Leu Thr Thr Val
185 190 195
Asp Pro Asp Thr Gly Val Met Ser Arg Lys Glu Pro Leu Glu Thr
200 205 210
Leu Lys Ser Tyr Arg Gln Cys Asp Pro Ser Glu Arg Lys Leu Tyr
215 220 225
Gly Lys Ser Pro Leu Phe Gly Gln Tyr Phe Val Leu Glu Asn Pro
230 235 240
Gly Thr Ile Lys Val Gly Asp Pro Val Tyr Leu Leu Gly Gln
245 250
I 7/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<210> 21
<211> 259
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3220944CD1
<400> 21
Met Lys Met Val Thr Gly Ala Val Ala Ser Val Leu Glu Asp Glu
1 5 10 15
Ala Thr Asp Thr ser Asp Ser Glu Gly Ser Cys Gly Ser Glu Lys
20 25 30
Asp His Phe Tyr Ser Asp Asp Asp Ala Ile Glu Ala Asp Ser Glu
35 40 45
Gly Asp Ala Glu Pro Cys Asp Lys Glu Asn Glu Asn Asp Gly Glu
5p 55 60
Ser Ser Val Gly Thr Asn Met Gly Trp Ala Asp Ala Met Ala Lys
65 70 75
Val Leu Asn Lys Lys Thr Pro Glu Ser Lys Pro Thr Ile Leu Val
80 85 90
Lys Asn Lys Lys Leu Glu Lys Glu Lys Glu Lys Leu Lys Gln Glu
g5 100 105
Arg Leu Glu Lys Ile Lys Gln Arg Asp Lys Arg Leu Glu Trp Glu
110 115 120
Met Met Cys Arg Val Lys Pro Asp Val Val Gln Asp Lys Glu Thr
125 130 135
Glu Arg Asn Leu Gln Arg Ile Ala Thr Arg Gly Val Val Gln Leu
140 145 150
Phe Asn Ala Val Gln Lys His Gln Lys Asn Val Asp Glu Lys Val
155 160 165
Lys Glu Ala Gly Ser Ser Met Arg Lys Arg Ala Lys Leu Ile Ser
170 175 180
Thr Val Ser Lys Lys Asp Phe Ile Ser Val Leu Arg Gly Met Asp
185 190 195
Gly Ser Thr Asn Glu Thr Ala Ser Ser Arg Lys Lys Pro Lys Ala
200 205 210
Lys Gln Thr Glu Val Lys Ser Glu Glu Gly Pro Gly Trp Thr Ile
215 220 225
Leu Arg Asp Asp Phe Met Met Gly Ala Ser Met Lys Asp Trp Asp
230 235 240
Lys Glu Ser Asp Gly Pro Asp Asp Ser Arg Pro Glu Ser Ala Ser
245 250 255
Asp Ser Asp Thr
<210> 22
<211> 65
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3224631CD1
<400> 22
Met Asp Tyr Gly Val Gln Phe Thr Ala Ser Arg Lys Phe Leu Thr
1 5 10 15
Ile Thr Pro Ile Val Leu Tyr Phe Leu Thr Ser Phe Tyr Thr Lys
20 25 30
Tyr Asp Gln Ile His Phe Val Leu Asn Thr Val Ser Leu Met Ser
35 40 45
Val Leu Ile Pro Lys Leu Leu Gln Leu His Gly Val Arg Ile Phe
50 55 60
Gly Ile Asn Lys Tyr
18/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<210> 23
<211> 163
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3242839CD1
<400> 23
Met Val Gly Gly Gly Gly Val Gly Gly Gly Leu Leu Glu Asn Ala
1 5 10 15
Asn Pro Leu Ile Tyr Gln Arg Ser Gly Glu Arg Pro Val Thr Ala
20 25 30
G1y Glu Glu Asp Glu Gln Val Pro Asp Ser Ile Asp Ala Arg Glu
35 40 45
Ile Phe Asp Leu Ile Arg Ser Ile Asn Asp Pro Glu His Pro Leu
50 55 60
Thr Leu Glu Glu Leu Asn Val Val Glu Gln Val Arg Val Gln Val
65 70 75
Ser Asp Pro Glu Ser Thr Val Ala Val Ala Phe Thr Pro Thr Ile
80 85 90
Pro His Cys Ser Met Ala Thr Leu Ile Gly Leu Ser Ile Lys Val
95 100 105
Lys Leu Leu Arg Ser Leu Pro Gln Arg Phe Lys Met Asp Val His
110 115 120
Ile Thr Pro Gly Thr His Ala Ser Glu His Ala Val Asn Lys Gln
125 130 135
Leu Ala Asp Lys Glu Arg Val Ala Ala Ala Leu Glu Asn Thr His
140 145 150
Leu Leu Glu Val Val Asn Gln Cys Leu Ser Ala Arg Ser
155 160
<210> 24
<211> 199
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3274451CD1
<400> 24
Met Thr Pro Leu Ala Pro Trp Asp Pro Lys Tyr Glu Ala Lys Ala
1 5 10 15
Gly Pro Arg Pro Val Trp Gly Ala Asn Cys Ser Ser Gly Ala Ser
20 25 30
Phe Ser Gly Arg Thr Leu Cys His Pro Ser Phe Trp Pro Leu Tyr
35 40 45
Glu Ala Ala Ser Gly Arg Gly Leu Arg Pro Val Ala Pro Ala Thr
50 55 60
Gly His Trp Asn Gly Gln Gln Ala Pro Pro Asp Ala Gly Phe Pro
65 70 75
Val Val Cjrs Cys Glu Asp Val Phe Leu Ser Asp Pro Leu Leu Pro
80 85 90
Arg Gly Gln Arg Val Pro Leu Tyr Leu Ser Lys Ala Pro Gln Gln
95 100 105
Met Met Gly Ser Leu Lys Leu Leu Pro Pro Pro Pro Ile Met Ser
110 115 120
Ala Arg Val Leu Pro Arg Pro Ser Pro Ser Arg Gly Pro Ser Thr
125 130 135
Ala Trp Leu Ser Gly Pro Glu Leu Ile Ala Leu Thr Gly Leu Leu
140 145 150
Gln Met Ser Gln Gly Glu Pro Arg Pro Ser Ser Ser Ala Val Gly
155 160 165
19/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Pro Pro Asp His Thr Ser Asp Pro Pro Ser Pro Cys Gly Ser Pro
170 175 180
Ser Ser Ser Gln Gly Ala Asp Leu Ser Leu Pro Gln Thr Pro Asp
185 190 195
Thr His Cys Pro
<210> 25
<211> 231
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3284256CD1
<400> 25
Met Ala Gly Met Val Asp Phe Gln Asp Glu Glu Gln Val Lys Ser
1 5 10 15
Phe Leu Glu Asn Met Glu Val Glu Cys Asn Tyr His Cys Tyr His
20 25 30
Glu Lys Asp Pro Asp Gly Cys Tyr Arg Leu Val Asp Tyr Leu Glu
35 40 45
Gly Ile Arg Lys Asn Phe Asp Glu Ala Ala Lys Val Leu Lys Phe
50 55 60
Asn Cys Glu Glu Asn Gln His Ser Asp Ser Cys Tyr Lys Leu Gly
65 70 75
Ala Tyr Tyr Val Thr Gly Lys Gly Gly Leu Thr Gln Asp Leu Lys
80 85 90
Ala Ala Ala Arg Cys Phe Leu Met Ala Cys Glu Lys Pro Gly Lys
95 100 105
Lys Ser Ile Ala Ala Cys His Asn Val Gly Leu Leu Ala His Asp
110 115 120
Gly Gln Val Asn Glu Asp Gly Gln Pro Asp Leu Gly Lys Ala Arg
125 130 135
Asp Tyr Tyr Thr Arg Ala Cys Asp Gly Gly Tyr Thr Ser Ser Cys
140 145 150
Phe Asn Leu Ser Ala Met Phe Leu Gln Gly Ala Pro Gly Phe Pro
155 160 165
Lys Asp Met Asp Leu Ala Cys Lys Tyr Ser Met Lys Ala Cys Asp
170 175 180
Leu Gly His Ile Trp Ala Cys Ala Asn Ala Ser Arg Met Tyr Lys
185 190 195
Leu Gly Asp Gly Val Asp Lys Asp Glu Ala Lys Ala Glu Val Leu
200 205 210
Lys Asn Arg Ala Gln Gln Leu His Lys Glu Gln Gln Lys Gly Val
215 220 225
Gln Pro Leu Thr Phe Gly
230
<210> 26
<211> 412
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3507004CD1
<400> 26
Met Ala Pro Val Glu His Val Val Ala Asp Ala Gly Ala Phe Leu
1 5 10 15
Arg His Ala Ala Leu Gln Asp Ile Gly Lys Asn Ile Tyr Thr Ile
20 25 30
Arg Glu Val Val Thr Glu Ile Arg Asp Lys Ala Thr Arg Arg Arg
35 40 45
20/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Leu Ala Val Leu Pro Tyr Glu Leu Arg Phe Lys Glu Pro Leu Pro
50 55 60
Glu Tyr Val Arg Leu Val Thr Glu Phe Ser Lys Lys Thr Gly Asp
65 70 75
Tyr Pro Ser Leu Ser Ala Thr Asp Ile Gln Val Leu Ala Leu Thr
80 85 90
Tyr Gln Leu Glu Ala Glu Phe Val Gly Val Ser His Leu Lys Gln
95 100 105
Glu Pro Gln Lys Val Lys Val Ser Ser Ser Ile Gln His Pro Glu
110 115 120
Thr Pro Leu His Ile Ser Gly Phe His Leu Pro Tyr Lys Pro Lys
125 130 135
Pro Pro ;~ln Glu Thr Glu Lys Gly His Ser Ala Cys Glu Pro Glu
140 145 150
Asn Leu Glu Phe Ser Ser Phe Met Phe Trp Arg Asn Pro Leu Pro
155 160 165
Asn Ile Asp His Glu Leu Gln Glu Leu Leu Ile Asp Arg Gly Glu
170 175 180
Asp Val Pro Ser Glu Glu Glu Glu Glu Glu Glu Asn Gly Phe Glu
185 190 195
Asp Arg Lys Asp Asp Ser Asp Asp Asp Gly Gly Gly Trp Ile Thr
200 205 210
Pro Ser Asn Ile Lys Gln Ile Gln Gln Glu Leu Glu Gln Cys Asp
215 220 225
Val Pro Glu Asp Val Arg Val Gly Cys Leu Thr Thr Asp Phe Ala
230 235 240
Met Gln Asn Val Leu Leu Gln Met Gly Leu His Val Leu Ala Val
245 250 255
Asn Gly Met Leu Ile Arg Glu Ala Arg Ser Tyr Ile Leu Arg Cys
260 265 270
His Gly Cys Phe Lys Thr Thr Ser Asp Met Ser Arg Val Phe Cys
275 280 285
Ser His Cys Gly Asn Lys Thr Leu Lys Lys Val Ser Val Thr Val
290 295 300
Ser Asp Asp Gly Thr Leu His Met His Phe Ser Arg Asn Pro Lys
305 310 315
Val Leu Asn Pro Arg Gly Leu Arg Tyr Ser Leu Pro Thr Pro Lys
320 325 330
Gly Gly Lys Tyr Ala Ile Asn Pro His Leu Thr Glu Asp Gln Arg
335 340 345
Phe Pro Gln Leu Arg Leu Ser Gln Lys Ala Arg Gln Lys Thr Asn
350 355 360
Val Phe Ala Pro Asp Tyr Ile Ala Gly Val Ser Pro Phe Val Glu
365 370 375
Asn Asp Ile Ser Ser Arg Ser Ala Thr Leu Gln Val Arg Asp Ser
380 385 390
Thr Leu Gly Ala Gly Arg Arg Arg Leu Asn Pro Asn Ala Ser Arg
395 400 405
Lys Lys Phe Val Lys Lys Arg
410
<210> 27
<211> 272
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3585823CD1
<400> 27
Met Thr Pro Ile Leu Glu Gly Ser His Arg Ala His Ser Leu Leu
1 5 10 15
Phe Glu Asn Ser Asp Ser Phe Ser Glu Asp Ser Ser Thr Leu Gly
20 25 30
Arg Thr Arg Ser Leu Pro Ile Thr Ile Glu Met Leu Lys Val Pro
21/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
35 40 45
Asp Asp Glu Glu Glu Glu Glu Gln Thr Cys Pro Ser Thr Phe Ser
50 55 60
Glu Glu Met Thr Pro Thr Ser Val Ile Pro Lys Leu Pro Gln Cys
65 70 75
Leu Arg Glu Glu Glu Glu Lys Glu Ser Asp Ser Asp Ser Glu Gly
80 85 90
Pro Ile Gln Tyr Arg Asp Glu Glu Asp Glu Asp Glu Ser Tyr Gln
95 100 105
Ser Ala Leu Ala Asn Lys Val Lys Arg Lys Asp Thr Leu Ala Met
110 115 120
Lys Leu Asn His Arg Pro Ser Glu Pro Glu Leu Asn Leu Asn Ser
125 130 135
Trp Pro Cys Lys Ser Lys Glu Glu Trp Asn Glu Ile Arg His Gln
140 145 150
Ile Gly Asn Thr Leu Ile Arg Arg Leu Ser Gln Arg Pro Thr Pro
155 160 165
Glu Glu Leu Glu Gln Arg Asn Ile Leu Gln Pro Lys Asn Glu Ala
170 175 180
Asp Arg Gln Ala Glu Lys Arg Glu Ile Lys Arg Arg Leu Thr Arg
185 190 195
Lys Leu Ser Gln Arg Pro Thr Val Ala Glu Leu Leu Ala Arg Lys
200 205 210
Ile Leu Arg Phe Asn Glu Tyr Val Glu Val Thr Asp Ala Gln Asp
215 220 225
Tyr Asp Arg Arg Ala Asp Lys Pro Trp Thr Lys Leu Thr Pro Ala
230 235 240
Asp Lys Ala Ala Ile Arg Lys Glu Leu Asn Glu Phe Lys Ser Ser
245 250 255
Glu Met Glu Val His Glu Glu Ser Lys His Phe Thr Arg Tyr His
260 265 270
Arg Pro
<210> 28
<211> 242
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3743822CD1
<400> 28
Met Ala Glu Ala Val Glu Arg Thr Asp Glu Leu Val Arg Glu Tyr
1 5 10 15
Leu Leu Phe Arg Gly Phe Thr His Thr Leu Arg Gln Leu Asp Ala
20 25 30
Glu Ile Lys Ala Asp Lys Glu Lys Gly Phe Arg Val Asp Lys Ile
35 40 45
Val Asp Gln Leu Gln Gln Leu Met Gln Val Tyr Asp Leu Ala Ala
50 55 60
Leu Arg Asp Tyr Trp Ser Tyr Leu Glu Arg Arg Leu Phe Ser Arg
65 70 75
Leu Glu Asp Ile Tyr Arg Pro Thr Ile His Lys Leu Lys Thr Ser
80 85 90
Leu Phe Arg Phe Tyr Leu Val Tyr Thr Ile Gln Thr Asn Arg Asn
95 100 105
Asp Lys Ala Gln Glu Phe Phe Ala Lys Gln Ala Thr Glu Leu Gln
110 115 120
Asn Gln Ala Glu Trp Lys Asp Trp Phe Val Leu Pro Phe Leu Pro
125 130 135
Ser Pro Asp Thr Asn Pro Thr Phe Ala Thr Tyr Phe Ser Arg Gln
140 145 150
Trp Ala Asp Thr Phe Ile Val Ser Leu His Asn Phe Leu Ser Val
155 160 165
Leu Phe Gln Cys Met Pro Val Pro Val Ile Leu Asn Phe Asp Ala
22/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
170 175 180
Glu Cys Gln Arg Thr Asn Gln Val Gln Glu Glu Asn Glu Val Leu
185 190 195
Arg Gln Lys Leu Phe Ala Leu Gln Ala Glu Ile His Arg Leu Lys
200 205 210
Lys Glu Glu Gln Gln Pro Glu Glu Glu Glu Ala Leu Val Gln His
215 220 225
Lys Leu Pro Pro Tyr Val Ser Asn Met Asp Arg Leu Gly Asp Ser
230 235 240
Glu Leu
<210> 29
<211> 285
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3808027CD1
<400> 29
Met Thr Thr Leu Thr His Arg Ala Arg Arg Thr Glu Ile Ser Lys
1 5 10 15
Asn Ser Glu Lys Lys Met Glu Ser Glu Glu Asp Ser Asn Trp Glu
20 25 30
Lys Ser Pro Asp Asn Glu Asp Ser Gly Asp Ser Lys Asp Tle Arg
35 40 45
Leu Thr Leu Met Glu Glu Val Leu Leu Leu Gly Leu Lys Asp Lys
50 55 60
Glu Gly Tyr Thr Ser Phe Trp Asn Asp Cys Ile Ser Ser Gly Leu
65 70 75
Arg Gly Gly Ile Leu Ile Glu Leu Ala Met Arg Gly Arg Ile Tyr
80 85 90
Leu Glu Pro Pro Thr Met Arg Lys Lys Arg Leu Leu Asp Arg Lys
95 100 105
Val Leu Leu Lys Ser Asp Ser Pro Thr Gly Asp Val Leu Leu Asp
110 115 120
Glu Thr Leu Lys His Ile Lys Ala Thr Glu Pro Thr Glu Thr Val
125 130 135
Gln Thr Trp Ile Glu Leu Leu Thr Gly Glu Thr Trp Asn Pro Phe
140 145 150
Lys Leu Gln Tyr Gln Leu Arg Asn Val Arg Glu Arg Ile Ala Lys
155 160 165
Asn Leu Val Glu Lys Gly Ile Leu Thr Thr Glu Lys Gln Asn Phe
170 175 180
Leu Leu Phe Asp Met Thr Thr His Pro Val Thr Asn Thr Thr Glu
185 190 195
Lys Gln Arg Leu Val Lys Lys Leu Gln Asp Ser Val Leu Glu Arg
200 205 210
Trp Val Asn Asp Pro Gln Arg Met Asp Lys Arg Thr Leu Ala Leu
215 220 225
Leu Val Leu Ala His Ser Ser Asp Val Leu Glu Asn Val Phe Ser
230 235 240
Ser Leu Thr Asp Asp Lys Tyr Asp Val Ala Met Asn Arg Ala Lys
245 250 255
Asp Leu Val Glu Leu Asp Pro Glu Val Glu Gly Thr Lys Pro Ser
260 265 270
Ala Thr Glu Met Ile Trp Ala Val Leu Ala Ala Phe Asn Lys Ser
275 280 285
<210> 30
<211> 89
<212> PRT
<213> Homo Sapiens
23/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<220>
<221> misc_feature
<223> Incyte ID No: 4016220CD1
<400> 30
Met Gly Thr Lys Arg Glu Ala Ile Leu Lys Val Leu Glu Asn Leu
1 5 10 15
Thr Pro Glu Glu Leu Lys Lys Phe Lys Met Lys Leu Gly Thr Val
20 25 30
Pro Leu Arg Glu Gly Phe Glu Arg Ile Pro Arg Gly Ala Leu Gly
35 40 45
Gln Leu Asp Ile Val Asp Leu Thr Asp Lys Leu Val Ala Ser Tyr
50 55 60
Tyr Glu Asp Tyr Ala Ala Glu Leu Val Val Ala Val Leu Arg Asp
65 70 75
Met Arg Met Leu Glu Glu Ala Ala Arg Leu Gln Arg Ala Ala
80 85
<210> 31
<211> 210
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4093555CD1
<400> 31
Met Ala Thr Glu Thr Val Glu Leu His Lys Leu Lys Leu Ala Glu
1 5 10 15
Leu Lys Gln Glu Cys Leu Ala Arg Gly Leu Glu Thr Lys Gly Ile
20 25 30
Lys Gln Asp Leu Ile His Arg Leu Gln Ala Tyr Leu Glu Glu His
35 40 45
Ala Glu Glu Glu Ala Asn Glu Glu Asp Val Leu Gly Asp Glu Thr
50 55 60
Glu Glu Glu Glu Thr Lys Pro Ile Glu Leu Pro Val Lys Glu Glu
65 70 75
Glu Pro Pro Glu Lys Thr Val Asp Val Ala Ala Glu Lys Lys Val
80 85 90
Val Lys Ile Thr Ser Glu Ile Pro Gln Thr Glu Arg Met Gln Lys
95 100 105
Arg Ala Glu Arg Phe Asn Val Pro Val Ser Leu Glu Ser Lys Lys
110 115 120
Ala Ala Arg Ala Ala Arg Phe Gly Ile Ser Ser Val Pro Thr Lys
125 130 135
Gly Leu Ser Ser Asp Asn Lys Pro Met Val Asn Leu Asp Lys Leu
140 145 150
Lys Glu Arg Ala Gln Arg Phe Gly Leu Asn Val Ser Ser Ile Ser
155 160 165
Arg Lys Ser Glu Asp Asp Glu Lys Leu Lys Lys Arg Lys Glu Arg
170 175 180
Phe Gly Ile Val Thr Ser Ser Ala Gly Thr Gly Thr Thr Glu Asp
185 190 195
Thr Glu Ala Lys Lys Arg Lys Arg Ala Glu Arg Phe Gly Ile Ala
200 205 210
<210> 32
<211> 271
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4829366CD1
24; 72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<400> 32
Met Ala Ala Glu Glu Pro Gln Gln Gln Lys Gln Glu Pro Leu Gly
1 5 10 15
Ser Asp Ser Glu Gly Val Asn Cys Leu Ala Tyr Asp Glu Ala Ile
20 25 30
Met Ala Gln Gln Asp Arg Ile Gln Gln Glu Ile Ala Val Gln Asn
35 40 45
Pro Leu Val Ser Glu Arg Leu Glu Leu Ser Val Leu Tyr Lys Glu
50 55 60
Tyr Ala Glu Asp Asp Asn Ile Tyr Gln Gln Lys Ile Lys Asp Leu
65 70 75
His Lys Lys Tyr Ser Tyr Ile Arg Lys Thr Arg Pro Asp Gly Asn
80 85 90
Cys Phe Tyr Arg Ala Phe Gly Phe Ser His Leu Glu Ala Leu Leu
95 100 105
Asp Asp Ser Lys Glu Leu Gln Arg Phe Lys Ala Val Ser Ala Lys
110 115 120
Ser Lys Glu Asp Leu Val Ser Gln Gly Phe Thr Glu Phe Thr Ile
125 130 135
Glu Asp Phe His Asn Thr Phe Met Asp Leu Ile Glu Gln Val Glu
140 145 150
Lys Gln Thr Ser Val Ala Asp Leu Leu Ala Ser Phe Asn Asp Gln
155 160 165
Ser Thr Ser Asp Tyr Leu Val Val Tyr Leu Arg Leu Leu Thr Ser
170 175 180
Gly Tyr Leu Gln Arg Glu Ser Lys Phe Phe Glu His Phe Ile Glu
185 190 195
Gly Gly Arg Thr Val Lys Glu Phe Cys Gln Gln Glu Val Glu Pro
200 205 210
Met Cys Lys Glu Ser Asp His Ile His Ile Ile Ala Leu Ala Gln
215 220 225
Ala Leu Ser Val Ser Ile Gln Val Glu Tyr Met Asp Arg Gly Glu
230 235 240
Gly Gly Thr Thr Asn Pro His Ile Phe Pro Glu Gly Ser Glu Pro
245 250 255
Lys Val Tyr Leu Leu Tyr Arg Pro Gly His Tyr Asp Ile Leu Tyr
260 265 270
Lys
<210> 33
<211> 389
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4830720CD1
<400> 33
Met Glu Ala Leu Gly Lys Leu Lys Gln Phe Asp Ala Tyr Pro Lys
1 5 10 15
Thr Leu Glu Asp Phe Arg Val Lys Thr Cys Gly Gly Ala Thr Val
20 25 30
Thr Ile Val Ser Gly Leu Leu Met Leu Leu Leu Phe Leu Ser Glu
35 40 45
Leu Gln Tyr Tyr Leu Thr Thr Glu Val His Pro Glu Leu Tyr Val
50 55 60
Asp Lys Ser Arg Gly Asp Lys Leu Lys Ile Asn Ile Asp Val Leu
65 70 75
Phe Pro His Met Pro Cys Ala Tyr Leu Ser Ile Asp Ala Met Asp
80 85 90
Val Ala Gly Glu Gln Gln Leu Asp Val Glu His Asn Leu Leu Lys
95 100 105
Gln Arg Leu Asp Lys Asp Gly Ile Pro Val Ser Ser Glu Ala Glu
110 115 120
Arg His Glu Leu Gly Lys Val Glu Val Thr Val Phe Asp Pro Asp
25/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
125 130 135
Ser Leu Asp Pro Asp Arg Cys Glu Ser Cys Tyr Gly Ala Glu Ala
140 145 150
Glu Asp Ile Lys Cys Cys Asn Thr Cys Glu Asp Val Arg Glu Ala
155 160 165
Tyr Arg Arg Arg Gly Trp Ala Phe Lys Asn Pro Asp Thr Ile Glu
170 175 180
Gln Cys Arg Arg Giu Gly Phe Ser Gln Lys Met Gln Glu Gln Lys
185 190 195
Asn Glu Gly Cys Gln Val Tyr Gly Phe Leu Glu Val Asn Lys Val
200 205 210
Ala Gly Asn Phe His Phe Ala Pro Gly Lys Ser Phe Gln Gln Ser
215 220 225
His Val His Val His Asp Leu Gln Ser Phe Gly Leu Asp Asn Ile
230 235 240
Asn Met Thr His Tyr Ile Gln His Leu Ser Phe Gly Glu Asp Tyr
245 250 255
Pro Gly Ile Val Asn Pro Leu Asp His Thr Asn Val Thr Ala Pro
260 265 270
Gln Ala Ser Met Met Phe Gln Tyr Phe Val Lys Val Val Pro Thr
275 280 285
Val Tyr Met Lys Val Asp Gly Glu Ala Pro Leu Pro Pro Gln Val
290 295 300
Leu Arg Thr Asn Gln Phe Ser Val Thr Arg His Glu Lys Val Ala
305 310 315
Asn Gly Leu Leu Gly Asp Gln Gly Leu Pro Gly Val Phe Val Leu
320 325 330
Tyr Glu Leu Ser Pro Met Met Val Lys Leu Thr Glu Lys His Arg
335 340 345
Ser Phe Thr His Phe Leu Thr Gly Val Cys Ala Ile Ile Gly Gly
350 355 360
Met Phe Thr Val Ala Gly Leu Ile Asp Ser Leu Ile Tyr His Ser
365 370 375
Ala Arg Ala Ile Gln Lys Lys Ile Asp Leu Gly Lys Thr Thr
380 385
<210> 34
<211> 228
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5389730CD1
<400> 34
Met Ser Tyr Ala Glu Lys Pro Asp Glu Ile Thr Lys Asp Glu Trp
1 5 10 15
Met Glu Lys Leu Asn Asn Leu His Val Gln Arg Ala Asp Met Asn
20 25 30
Arg Leu Ile Met Asn Tyr Leu Val Thr Glu Gly Phe Lys Glu Ala
35 40 45
Ala Glu Lys Phe Arg Met Glu Ser Gly Ile Glu Pro Ser Val Asp
50 55 60
Leu Glu Thr Leu Asp Glu Arg Ile Lys Ile Arg Glu Met Ile Leu
65 70 75
Lys Gly Gln Ile Gln Glu Ala Ile Ala Leu Ile Asn Ser Leu His
80 85 90
Pro Glu Leu Leu Asp Thr Asn Arg Tyr Leu Tyr Phe His Leu Gln
95 100 105
Gln Gln His Leu Ile Glu Leu Ile Arg Gln Arg Glu Thr Glu Ala
110 115 120
Ala Leu Glu Phe Ala Gln Thr Gln Leu Ala Glu Gln Gly Glu Glu
125 130 135
Ser Arg Glu Cys Leu Thr Glu Met Glu Arg Thr Leu Ala Leu Leu
140 145 150
26/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Ala Phe Asp Ser Pro Glu Glu Ser Pro Phe Gly Asp Leu Leu His
155 160 165
Thr Met Gln Arg Gln Lys Val Trp Ser Glu Val Asn Gln Ala Val
170 175 180
Leu Asp Tyr Glu Asn Arg Glu Ser Thr Pro Lys Leu Ala Lys Leu
185 190 195
Leu Lys Leu Leu Leu Trp Ala Gln Asn Glu Leu Asp Gln Lys Lys
200 205 210
Val Lys Tyr Pro Lys Met Thr Asp Leu Ser Lys Gly Val Ile Glu
215 220 225
Glu Pro Lys
<210> 35
<211> 330
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5397088CD1
<400> 35
Met Ala Pro Glu Glu Asn Ala Gly Thr Glu Leu Leu Leu Gln Ser
1 5 10 15
Phe Glu Arg Arg Phe Leu Ala Ala Arg Thr Leu Arg Ser Phe Pro
20 25 30
Trp Gln Ser Leu Glu Ala Lys Leu Arg Asp Ser Ser Asp Ser Glu
35 40 45
Leu Leu Arg Asp Ile Leu His Lys Thr Val Lys His Pro Val Cys
50 55 60
Val Lys His Pro Pro Ser Val Lys Tyr Ala Arg Cys Phe Leu Ser
65 70 75
Glu Leu Ile Lys Lys His Glu Ala Val His Thr Glu Pro Leu Asp
80 85 90
Glu Leu Tyr Glu Ala Leu Ala Glu Thr Leu Met Ala Lys Glu Ser
95 100 105
Thr Gln Gly His Arg Ser Tyr Leu Leu Pro Ser Gly Gly Ser Val
110 115 120
Thr Leu Ser Glu Ser Thr Ala Ile Ile Ser Tyr Gly Thr Thr Gly
125 13 0 135
Leu Val Thr Trp Asp Ala Ala Leu Tyr Leu Ala Glu Trp Ala Ile
140 145 150
Glu Asn Pro Ala Val Phe Thr Asn Arg Thr Val Leu Glu Leu Gly
155 160 165
Ser Gly Ala Gly Leu Thr Gly Leu Ala Ile Cys Lys Met Cys Arg
170 175 180
Pro Arg Ala Tyr Ile Phe Ser Asp Cys His Ser Arg Val Leu Glu
185 190 195
Gln Leu Arg Gly Asn Val Leu Leu Asn Gly Leu Ser Leu Glu Ala
200 205 210
Asp Ile Thr Ala Lys Leu Asp Ser Pro Arg Val Thr Val Ala Gln
215 220 225
Leu Asp Trp Asp Val Ala Thr Val His Gln Leu Ser Ala Phe Gln
230 235 240
Pro Asp Val Val Ile Ala Ala Asp Val Leu Tyr Cys Pro Glu Ala
245 250 255
Ile Met Ser Leu Val Gly Val Leu Arg Arg Leu Ala Ala Cys Arg
260 265 270
Glu Asp Gln Arg Ala Pro Glu Val Tyr Val Ala Phe Thr Val Arg
275 280 285
Asn Pro Glu Thr Cys Gln Leu Phe Thr Thr Glu Leu Gly Arg Ala
290 295 300
Gly Ile Arg Trp Glu Val Glu Pro Arg His Glu Gln Lys Leu Phe
305 310 315
Pro Tyr Glu Glu His Leu Glu Met Ala Met Leu Asn Leu Thr Leu
320 325 330
27/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<210> 36
<211> 307
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5425521CD1
<400> 36
Met Ala Ala Leu Ala Pro Leu Pro Pro Leu Pro Ala Gln Phe Lys
1 5 10 15
Ser Ile Gln His His Leu Arg Thr Ala Gln Glu His Asp Lys Arg
20 25 30
Asp Pro Val Val Ala Tyr Tyr Cys Arg Leu Tyr Ala Met Gln Thr
35 40 45
Gly Met Lys Ile Asp Ser Lys Thr Pro Glu Cys Arg Lys Phe Leu
50 55 60
Ser Lys Leu Met Asp Gln Leu Glu Ala Leu Lys Lys Gln Leu Gly
65 70 75
Asp Asn Glu Ala Ile Thr Gln Glu Ile Val Gly Cys Ala His Leu
80 85 90
Glu Asn Tyr Ala Leu Lys Met Phe Leu Tyr Ala Asp Asn Glu Asp
95 100 105
Arg Ala Gly Arg Phe His Lys Asn Met Ile Lys Ser Phe Tyr Thr
110 115 120
Ala Ser Leu Leu Ile Asp Val Ile Thr Val Phe Gly Glu Leu Thr
125 13 0 13 5
Asp Glu Asn Val Lys His Arg Lys Tyr Ala Arg Trp Lys Ala Thr
140 145 150
Tyr Ile His Asn Cys Leu Lys Asn Gly Glu Thr Pro Gln Ala Gly
155 160 165
Pro Val Gly Ile Glu Glu Asp Asn Asp Ile Glu Glu Asn Glu Asp
170 175 180
Ala Gly Ala Ala Ser Leu Pro Thr Gln Pro Thr Gln Pro Ser Ser
185 190 195
Ser Ser Thr Tyr Asp Pro Ser Asn Met Pro Ser Gly Asn Tyr Thr
200 205 210
Gly Ile Gln Ile Pro Pro Gly Ala His Ala Pro Ala Asn Thr Pro
215 220 225
Ala Glu Val Pro His Ser Thr Gly Val Ala Ser Asn Thr Ile Gln
230 235 240
Pro Thr Pro Gln Thr Ile Pro Ala Ile Asp Pro Ala Leu Phe Asn
245 250 255
Thr Ile Ser Gln Gly Asp Val Arg Leu Thr Pro Glu Asp Phe Ala
260 265 270
Arg Ala Gln Lys Tyr Cys Lys Tyr Ala Gly Ser Ala Leu Gln Tyr
275 280 285
Glu Asp Val Ser Thr Ala Val Gln Asn Leu Gln Lys Ala Leu Lys
290 295 300
Leu Leu Thr Thr Gly Arg Glu
305
<210> 37
<211> 163
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5495427CD1
<400> 37
Met Ala Trp Ala Val Ser His Phe Arg Pro Gly Pro Glu Val Trp
1 5 10 15
28/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Asp Thr Ala Ser Met Ala Ala Ser Lys Val Lys Gln Asp Met Pro
20 25 30
Pro Pro Gly Gly Tyr Gly Pro Ile Asp Tyr Lys Arg Asn Leu Pro
35 40 45
Arg Arg Gly Leu Ser Gly Tyr Ser Met Leu Ala Ile Gly Ile Gly
50 55 60
Thr Leu Ile Tyr Gly His Trp Ser Ile Met Lys Trp Asn Arg Glu
65 70 75
Arg Arg Arg Leu Gln Ile Glu Asp Phe Glu Ala Arg Ile Ala Leu
80 85 90
Leu Pro Leu Leu Gln Ala Glu Thr Asp Arg Arg Thr Leu Gln Met
95 100 105
Leu Arg Glu Asn Leu Glu Glu Glu Ala Ile Ile Met Lys Asp Val
110 115 120
Pro Asp Trp Lys Val Gly Glu Ser Val Phe His Thr Thr Arg Trp
125 130 135
Val Pro Pro Leu Ile Gly Glu Leu Tyr Gly Leu Arg Thr Thr Glu
140 145 150
Glu Ala Leu His Ala Ser His Gly Phe Met Trp Tyr Thr
155 160
<210> 38
<211> 202
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 003908CD1
<400> 38
Met Ala Val Pro Ala Ala Leu Ile Leu Arg Glu Ser Pro Ser Met
1 5 10 15
Lys Lys Ala Val Ser Leu Ile Asn Ala Ile Asp Thr Gly Arg Phe
20 25 30
Pro Arg Leu Leu Thr Arg Ile Leu Gln Lys Leu His Leu Lys Ala
35 40 45
Glu Ser Ser Phe Ser Glu Glu Glu Glu Glu Lys Leu Gln Ala Ala
50 55 60
Phe Ser Leu Glu Lys Gln Asp Leu His Leu Val Leu Glu Thr Ile
65 70 75
Ser Phe Ile Leu Glu Gln Ala Val Tyr His Asn Val Lys Pro Ala
80 85 90
Ala Leu Gln Gln Gln Leu Glu Asn Ile His Leu Arg Gln Asp Lys
95 100 105
Ala Glu Ala Phe Val Asn Thr Trp Ser Ser Met Gly Gln Glu Thr
110 115 120
Val Glu Lys Phe Arg Gln Arg Ile Leu Ala Pro Cys Lys Leu Glu
125 130 135
Thr Val Gly Trp Gln Leu Asn Leu Gln Met Ala His Ser Ala Gln
140 145 150
Ala Lys Leu Lys Ser Pro Gln Ala Val Leu Gln Leu Gly Val Asn
155 160 165
Asn Glu Asp Ser Lys Ser Leu Glu Lys Val Leu Val Glu Phe Ser
170 175 180
His Lys Glu Leu Phe Asp Phe Tyr Asn Lys Leu Glu Thr Ile Gln
185 190 195
Ala Gln Leu Asp Ser Leu Thr
200
<210> 39
<211> 209
<212> PRT
<213> Homo sapiens
29/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<220>
<221> misc_feature
<223> Incyte ID No: 085915CD1
<400> 39
Met Ile Asp Asn Phe Phe Ser Glu Pro Thr Thr Lys Ser Trp Glu
1 5 10 15
Ile Ile Thr Val Glu Glu Ala Lys Arg Arg Lys Ser Thr Cys Ser
20 25 30
Tyr Tyr Glu Asp Glu Asp Glu Glu Val Leu Pro Val Leu Arg Pro
35 40 45
His Ser Ala Leu Leu Glu Asn Met His Ile Glu Gln Leu Ala Arg
50 55 60
Arg Leu Pro Ala Arg Val Gln Gly Tyr Pro Trp Arg Leu Ala Tyr
65 70 75
Ser Thr Leu Glu His Gly Thr Ser Leu Lys Thr Leu Tyr Arg Lys
80 85 90
Ser Ala Ser Leu Asp Ser Pro Val Leu Leu Val Ile Lys Asp Met
95 100 105
Asp Asn Gln Ile Phe Gly Ala Tyr Ala Thr His Pro Phe Lys Phe
110 115 120
Ser Asp His Tyr Tyr Gly Thr Gly Glu Thr Phe Leu Tyr Thr Phe
125 130 135
Ser Pro His Phe Lys Val Phe Lys Trp Ser Gly Glu Asn Ser Tyr
140 145 150
Phe Ile Asn Gly Asp Ile Ser Ser Leu Glu Leu Gly Gly Gly Gly
155 160 165
Gly Arg Phe Gly Leu Trp Leu Asp Ala Asp Leu Tyr His Gly Arg
170 175 180
Ser Asn Ser Cys Ser Thr Phe Asn Asn Asp Ile Leu Ser Lys Lys
185 190 195
Glu Asp Phe Ile Val Gln Asp Leu Glu Val Trp Ala Phe Asp
200 205
<210> 40
<211> 314
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 478615CD1
<400> 40
Met Pro Gln Met Arg Gln Thr Pro Thr Asp Lys Pro Leu Cys Pro
1 5 10 15
Ser Arg Thr His Lys Val Leu Pro Ile Leu Glu Ile Leu Tyr His
20 25 30
Val Glu Glu Arg Asn Ser His His Val Tyr Met Ala Leu Ile Ile
35 40 45
Leu Leu Ile Leu Thr Glu Asp Asp Gly Phe Asn Arg Ser Ile His
50 55 60
Glu Val Ile Leu Lys Asn Ile Thr Trp Tyr Ser Glu Arg Val Leu
65 70 75
Thr Glu Ile Ser Leu Gly Ser Leu Leu Ile Leu Val Val Ile Arg
80 85 90
Thr Ile Gln Tyr Asn Met Thr Arg Thr Arg Asp Lys Tyr Leu His
95 100 105
Thr Asn Cys Leu Ala Ala Leu Ala Asn Met Ser Ala Gln Phe Arg
110 115 120
Ser Leu His Gln Tyr Ala Ala Gln Arg Ile Ile Ser Leu Phe Ser
125 130 135
Leu Leu Ser Lys Lys His Asn Lys Val Leu Glu Gln Ala Thr Gln
140 145 150
Ser Leu Arg Gly Ser Leu Ser Ser Asn Asp Val Pro Leu Pro Asp
155 160 165
30/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Tyr Ala Gln Asp Leu Asn Val Ile Glu Glu Val Ile Arg Met Met
170 175 180
Leu Glu Ile Ile Asn Ser Cys Leu Thr Asn Ser Leu His His Asn
185 190 195
Pro Asn Leu Val Tyr Ala Leu Leu Tyr Lys Arg Asp Leu Phe Glu
200 205 210
Gln Phe Arg Thr His Pro Ser Phe Gln Asp Ile Met Gln Asn Ile
215 220 225
Asp Leu Val Ile Ser Phe Phe Ser Ser Arg Leu Leu Gln Ala Gly
230 235 240
Ala Glu Leu Ser Val Glu Arg Val Leu Glu Ile Ile Lys Gln Gly
245 250 255
Val Val Ala Leu Pro Lys Asp Arg Leu Lys Lys Phe Pro G1u Leu
260 265 270
Lys Phe Lys Tyr Val Glu Glu Glu Gln Pro Glu Glu Phe Phe Ile
275 280 285
Pro Tyr Val Trp Ser Leu Val Tyr Asn Ser Ala Val Gly Leu Tyr
290 295 300
Trp Asn Pro Gln Asp Ile Gln Leu Phe Thr Met Asp Ser Asp
305 310
<210> 41
<211> 137
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 924880CD1
<400> 41
Met Glu Val Leu Glu Met Arg Ala Lys Asn Pro Val Pro Gln Leu
1 5 10 15
Arg Lys Phe Lys Thr Asn Val Leu Pro Phe Arg Gln Asn Asp Ser
20 25 30
Ser Ser His Cys Gln Lys Ser Gly Ser Pro Ile Ser Ser Glu Glu
35 40 45
Arg Arg Arg Arg Asp Lys Gln His Leu Asp Asp Ile Thr Ala Ala
50 55 60
Arg Leu Leu Pro Leu His His Met Pro Thr Gln Leu Leu Ser Ile
65 70 75
Glu Glu Ser Leu Ala Leu Gln Lys Gln Gln Lys Gln Asn Tyr Glu
80 85 90
Glu Met Gln Ala Lys Leu Ala Ala Gln Lys Leu Ala Glu Arg Leu
95 100 105
Asn Ile Lys Met Arg Ser Tyr Asn Pro Glu Gly Glu Ser Ser Gly
110 115 120
Arg Tyr Arg Glu Val Arg Asp Glu Asp Asp Asp Trp Ser Ser Asp
125 130 135
Glu Phe
<210> 42
<211> 245
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 955431CD1
<400> 42
Met Ser Asp Glu Phe Ser Leu Ala Asp Ala Leu Pro Glu His Ser
1 5 10 15
Pro Ala Lys Thr Ser Ala Val Ser Asn Thr Lys Pro Gly Gln Pro
20 25 30
31/?2
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Pro Gln Gly Trp Pro Gly Ser Asn Pro Trp Asn Asn Pro Ser Ala
35 40 45
Pro Ser Ser Val Pro Ser Gly Leu Pro Pro Ser Ala Thr Pro Ser
50 55 60
Thr Val Pro Phe Gly Pro Ala Pro Thr Gly Met Tyr Pro Ser Val
65 70 75
Pro Pro Thr Gly Pro Pro Pro Gly Pro Pro Ala Pro Phe Pro Pro
30 85 90
Ser Gly Pro Ser Cys Pro Pro Pro Gly Gly Pro Tyr Pro Ala Pro
95 100 105
Thr Val Pro Gly Pro Gly Pro Thr Gly Pro Tyr Pro Thr Pro Asn
110 115 120
Met Pro Phe Pro Glu Leu Pro Arg Pro Tyr Gly Ala Pro Thr Asp
125 130 135
Pro Ala Ala Ala Gly Pro Leu Gly Pro Trp Gly Ser Met Ser Ser
140 145 150
Gly Pro Trp Ala Pro Gly Met Gly Gly Gln Tyr Pro Thr Pro Asn
155 160 165
Met Pro Tyr Pro Ser Pro Gly Pro Tyr Pro Ala Pro Pro Pro Pro
170 175 180
Gln Ala Pro Gly Ala Ala Pro Pro Val Pro Trp Gly Thr Val Pro
185 190 195
Pro Gly Ala Trp Gly Pro Pro Ala Pro Tyr Pro Ala Pro Thr Gly
200 205 210
Ser Tyr Pro Thr Pro Gly Leu Tyr Pro Thr Pro Ser Asn Pro Phe
215 220 225
Gln Val Pro Ser Gly Pro Ser Gly Ala Pro Pro Met Pro Gly Gly
230 235 240
Pro His Ser Tyr His
245
<210> 43
<211> 179
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1275918CD1
<400> 43
Met Glu Thr Lys Asp Gln Lys Lys Gln Arg Lys Lys Asn Ser Gly
1 5 10 15
Pro Lys Ala Ala Lys Lys Lys Lys Arg His Leu Gln Asp Leu Gln
20 25 30
Leu Gly Asp Glu Glu Asp Ala Trp Lys Arg Asn Pro Lys Ala Phe
35 40 45
Ala Phe Gln Ser Ala Val Trp Met Ala Arg Ser Phe His Arg Thr
50 55 60
Gln Asp Leu Lys Thr Lys Lys His His Ile Pro Val Val Asp Arg
65 70 75
Thr Pro Leu Glu Pro Pro Pro Ile Val Val Val Val Met Gly Pro
80 85 90
Pro Lys Val Gly Lys Ser Thr Leu Ile Gln Cys Leu Ile Arg Asn
95 100 105
Phe Thr Arg Gln Lys Leu Thr Lys Ile Arg Gly Pro Val Met Ile
110 115 120
Val Ser Gly Lys Lys Leu Arg Leu Thr Ile Ile Glu Cys Gly Cys
125 13 0 13 5
Asp Ile Asn Met Met Ile Asp Leu Ala Glu Val Ala Asp Leu Val
140 145 150
Ser Glu Gln Gly Gln Pro Gly Val Leu Met Glu Thr Tyr Ser Ile
155 160 165
Val Ile Gly Tyr Leu Pro Arg Asp Glu Gly Asn Arg Val Leu
170 175
32/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<210> 44
<211> 165
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1290896CD1
<400> 44
Met Leu Ser Leu Cys Ala Gln Asp Leu Thr Gln Met Leu Ala Leu
1 5 10 15
Ser Arg His Ser Leu Leu S2r Pro Leu Leu Ser Val Thr Ser Phe
20 25 30
Arg Arg Phe Tyr Arg Gly Asp Ser Pro Thr Asp Ser Gln Lys Asp
35 40 45
Met Ile Glu Ile Pro Leu Pro Pro Trp Gln Glu Arg Thr Asp Glu
50 55 60
Ser Ile Glu Thr Lys Arg Ala Arg Leu Leu Tyr Glu Ser Arg Lys
65 70 75
Arg Gly Met Leu Glu Asn Cys Ile Leu Leu Ser Leu Phe Ala Lys
80 85 90
Glu His Leu Gln His Met Thr Glu Lys Gln Leu Asn Leu Tyr Asp
95 100 105
Arg Leu Ile Asn Glu Pro Ser Asn Asp Trp Asp Ile Tyr Tyr Trp
110 115 120
Ala Thr Glu Ala Lys Pro Ala Pro Glu Ile Phe Glu Asn Glu Val
125 130 135
Met Ala Leu Leu Arg Asp Phe Ala Lys Asn Lys Asn Lys Glu Gln
140 145 150
Arg Leu Arg Ala Pro Asp Leu Glu Tyr Leu Phe Glu Lys Pro Arg
155 160 165
<210> 45
<211> 177
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1342736CD1
<400> 45
Met Gly Val Ser Val Asp Val His Gln Val Tyr Lys Tyr Pro Phe
1 5 10 15
Glu Gln Val Val Ala Ser Phe Leu Arg Lys Tyr Pro Asn Pro Met
20 25 30
Asp Lys Asn Val Ile Ser Val Lys Ile Met Glu Glu Lys Arg Asp
35 40 45
Glu Ser Thr Gly Val Ile Tyr Arg Lys Arg Ile Ala Ile Cys Gln
50 55 60
Asn Val Val Pro Glu Ile Leu Arg Lys Val Ser Ile Leu Lys Val
65 70 75
Pro Asn Ile Gln Leu Glu Glu Glu Ser Trp Leu Asn Pro Arg Glu
80 85 90
Arg Asn Met Ala Ile Arg Ser His Cys Leu Thr Trp Thr Gln Tyr
95 100 105
Ala Ser Met Lys Glu Glu Ser Val Phe Arg Glu Ser Met Glu Asn
110 115 120
Pro Asn Trp Thr Glu Phe Ile Gln Arg Gly Arg Ile Ser Ile Thr
125 13 0 135
Gly Val Gly Phe Leu Asn Cys Val Leu Glu Thr Phe Ala Ser Thr
140 145 150
Phe Leu Arg Gln Gly Ala Gln Lys Gly Ile Arg Ile Met Glu Met
155 160 165
Leu Leu Lys Glu Gln Cys Gly Ala Pro Leu Ala Glu
33/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
170 175
<210> 46
<211> 215
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1394209CD1
<400w 46
Met Ser Ile Leu Glu Thr Ile Thr Ser Leu Asn Gln Glu Ala Ser
1 5 10 15
Ala Ala Arg Ala Ser Thr Glu Thr Ser Asn Ala Lys Thr Ser Glu
20 25 30
Arg Ala Ser Lys Lys Leu Pro Ser Gln Pro Thr Thr Asp Thr Ser
35 40 45
Thr Asp Lys Glu Arg Thr Ser Glu Asp Met Ala Asp Lys Glu Lys
50 55 60
Ser Thr Ala Asp Ser Gly Gly Glu Gly Leu Glu Thr Ala Pro Lys
65 70 75
Ser Glu Glu Phe Ser Asp Leu Pro Cys Pro Val Glu Glu Ile Lys
80 85 90
Asn Tyr Thr Lys Glu His Asn Asn Leu Ile Leu Leu Asn Lys Asp
95 100 105
Val Gln Gln Glu Ser Ser Glu Gln Lys Asn Lys Ser Thr Asp Lys
110 115 120
Gly Glu Lys Lys Pro Asp Ser Asn Glu Lys Gly Glu Arg Lys Lys
125 13 0 13 5
Glu Lys Lys Glu Lys Thr Glu Lys Lys Phe Asp His Ser Lys Lys
140 145 150
Ser Glu Asp Thr Gln Lys Val Lys Asp Glu Lys Gln Ala Lys Glu
155 160 165
Lys Glu Val Glu Ser Leu Lys Leu Pro Ser Glu Lys Asn Ser Asn
170 175 180
Lys Ala Lys Thr Val Glu Gly Thr Lys Glu Glu Glu Asn Lys Thr
185 190 195
Gln Lys Lys Lys Ser Ser Tyr Tyr Lys Asn Ile Leu Arg Ala Gln
200 205 210
Leu Leu Asn Tyr Thr
215
<210> 47
<211> 133
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1394647CD1
<400> 47
Met Leu Ser Pro Glu Ala Glu Arg Val Leu Arg Tyr Leu Val Glu
1 5 10 15
Val Glu Glu Leu Ala Glu Glu Val Leu Ala Asp Lys Arg Gln Ile
20 25 30
Val Asp Leu Asp Thr Lys Arg Asn Gln Asn Arg Glu Gly Leu Arg
35 40 45
Ala Leu Gln Lys Asp Leu Ser Leu Ser Glu Asp Val Met Val Cys
50 55 60
Phe Gly Asn Met Phe Ile Lys Met Pro His Pro Glu Thr Lys Glu
65 70 75
Met Ile Glu Lys Asp Gln Asp His Leu Asp Lys Glu Ile Glu Lys
80 85 90
34/?2
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Leu Arg Lys Gln Leu Lys Val Lys Val Asn Arg Leu Phe Glu Ala
95 100 105
Gln Gly Lys Pro Glu Leu Lys Gly Phe Asn Leu Asn Pro Leu Asn
110 115 120
Gln Asp Glu Leu Lys Ala Leu Lys Val Ile Leu Lys Gly
125 130
<210> 48
<211> 579
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1436854CD1
<400> 48
Met Ser Ala Ser Ser Leu Leu Glu Gln Arg Pro Lys Gly Gln Gly
1 5 10 15
Asn Lys Val Gln Asn Gly Ser Val His Gln Lys Asp Gly Leu Asn
20 25 30
Asp Asp Asp Phe Glu Pro Tyr Leu Ser Pro Gln Ala Arg Pro Asn
35 40 45
Asn Ala Tyr Thr Ala Met Ser Asp Ser Tyr Leu Pro Ser Tyr Tyr
50 55 60
Ser Pro Ser Ile Gly Phe Ser Tyr Ser Leu Gly Glu Ala Ala Trp
65 70 75
Ser Thr Gly Gly Asp Thr Ala Met Pro Tyr Leu Thr Ser Tyr Gly
80 85 90
Gln Leu Ser Asn Gly Glu Pro His Phe Leu Pro Asp Ala Met Phe
95 100 105
Gly Gln Pro Gly Ala Leu Gly Ser Thr Pro Phe Leu Gly Gln His
110 115 120
Gly Phe Asn Phe Phe Pro Ser Gly Ile Asp Phe Ser Ala Trp Gly
125 130 135
Asn Asn Ser Ser Gln Gly Gln Ser Thr Gln Ser Ser Gly Tyr Ser
140 145 150
Ser Asn Tyr Ala Tyr Ala Pro Ser Ser Leu Gly Gly Ala Met Ile
155 160 165
Asp Gly Gln Ser Ala Phe Ala Asn Glu Thr Leu Asn Lys Ala Pro
170 175 180
Gly Met Asn Thr Ile Asp Gln Gly Met Ala Ala Leu Lys Leu Gly
185 190 195
Ser Thr Glu Val Ala Ser Asn Val Pro Lys Val Val Gly Ser Ala
200 205 210
Val Gly Ser Gly Ser Ile Thr Ser Asn Ile Val Ala Ser Asn Ser
215 220 225
Leu Pro Pro Ala Thr Ile Ala Pro Pro Lys Pro Ala Ser Trp Ala
230 235 240
Asp Ile Ala Ser Lys Pro Ala Lys Gln Gln Pro Lys Leu Lys Thr
245 250 255
Lys Asn Gly Ile Ala Gly Ser Ser Leu Pro Pro Pro Pro Ile Lys
260 265 270
His Asn Met Asp Ile Gly Thr Trp Asp Asn Lys Gly Pro Val Ala
275 280 285
Lys Ala Pro Ser Gln Ala Leu Val Gln Asn Ile Gly Gln Pro Thr
290 295 300
Gln Gly Ser Pro Gln Pro Val Gly Gln Gln Ala Asn Asn Ser Pro
305 310 315
Pro Val Ala Gln Ala Ser Val Gly Gln Gln Thr Gln Pro Leu Pro
320 325 330
Pro Pro Pro Pro Gln Pro Ala Gln Leu Ser Val Gln Gln Gln Ala
335 340 345
Ala Gln Pro Thr Arg Trp Val Ala Pro Arg Asn Arg Gly Ser Gly
350 355 360
Phe Gly His Asn Gly Val Asp Gly Asn Gly Val Gly Gln Ser Gln
35/?2
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
365 370 375
Ala Gly Ser Gly Ser Thr Pro Ser Glu Pro His Pro Val Leu Glu
380 385 390
Lys Leu Arg Ser Ile Asn Asn Tyr Asn Pro Lys Asp Phe Asp Trp
395 400 405
Asn Leu Lys His Gly Arg Val Phe Ile Ile Lys Ser Tyr Ser Glu
410 415 420
Asp Asp Ile His Arg Ser Ile Lys Tyr Asn Ile Trp Cys Ser Thr
425 430 435
Glu His Gly Asn Lys Arg Leu Asp Ala Ala Tyr Arg Ser Met Asn
440 445 450
Gly Lys Gly Pro Val Tyr Leu Leu Phe Ser Val Asn Gly Ser Gly
455 460 465
His Phe Cys Gly Val Ala Glu Met Lys Ser Ala Val Asp Tyr Asn
470 475 480
Thr Cys Ala Gly Val Trp Ser Gln Asp Lys Trp Lys Gly Arg Phe
485 490 495
Asp Val Arg Trp Ile Phe Val Lys Asp Val Pro Asn Ser Gln Leu
500 505 510
Arg His Ile Arg Leu Glu Asn Asn Glu Asn Lys Pro Val Thr Asn
515 520 525
Ser Arg Asp Thr Gln Glu Val Pro Leu Glu Lys Ala Lys Gln Val
530 535 540
Leu Lys Ile Ile Ala Ser Tyr Lys His Thr Thr Ser Ile Phe Asp
545 550 555
Asp Phe Ser His Tyr Glu Lys Arg Gln Glu Glu Glu Glu Ser Val
560 565 570
Lys Lys Glu Arg Gln Gly Arg Gly Lys
575
<210> 49
<211> 139
<212> PRT .
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1447955CD1
<400> 49
Met Glu Thr Asp Cys Asn Pro Met Glu Leu Ser Ser Met Ser Gly
1 5 10 15
Phe Glu Glu Gly Ser Glu Leu Asn Gly Phe Glu Gly Thr Asp Met
20 25 30
Lys Asp Met Arg Leu Glu Ala Glu Ala Val Val Asn Asp Val Leu
35 40 45
Phe Ala Val Asn Asn Met Phe Val Ser Lys Ser Leu Arg Cys Ala
50 55 60
Asp Asp Val Ala Tyr Ile Asn Val Glu Thr Lys Glu Arg Asn Arg
65 70 75
Tyr Cys Leu Glu Leu Thr Glu Ala Gly Leu Lys Val Val Gly Tyr
80 85 90
Ala Phe Asp Gln Val Asp Asp His Leu Gln Thr Pro Tyr His Glu
95 100 105
Thr Val Tyr Ser Leu Leu Asp Thr Leu Ser Pro Ala Tyr Arg Glu
110 115 120
Ala Phe Gly Asn Ala Leu Leu Gln Arg Leu Glu Ala Leu Lys Arg
125 130 135
Asp Gly Gln Ser
<210> 50
<211> 314
<212> PRT
<213> Homo Sapiens
36/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<220>
<221> misc_feature
<223> Incyte ID No: 1454689CD1
<400> 50
Met Ser Ala Ala Glu Ala Gly Gly Val Phe His Arg Ala Arg Gly
1 5 10 15
Arg Thr Leu Ala Ala Phe Pro Ala Glu Lys Glu Ser Glu Trp Lys
20 25 30
Gly Pro Phe Tyr Phe Ile Leu Gly Ala Asp Pro Gln Phe Gly Leu
35 40 45
Ile Lys Ala Trp Ser Thr Gly Asp Cys Asp Asn Gly Gly Asp Glu
50 55 60
Trp Glu Gln Glu Ile Arg Leu Thr Glu Gln Ala Val Gln Ala Ile
65 70 75
Asn Lys Leu Asn Pro Lys Pro Lys Phe Phe Va1 Leu Cys Gly Asp
80 85 90
Leu Ile His Ala Met Pro Gly Lys Pro Trp Arg Thr Glu Gln Thr
95 100 105
Glu Asp Leu Lys Arg Val Leu Arg Ala Val Asp Arg Ala Ile Pro
110 115 120
Leu Val Leu Val Ser Gly Asn His Asp Ile Gly Asn Thr Pro Thr
125 130 135
Ala Glu Thr Val Glu Glu Phe Cys Arg Thr Trp Gly Asp Asp Tyr
140 145 150
Phe Ser Phe Trp Val Gly Gly Val Leu Phe Leu Val Leu Asn Ser
155 160 165
Gln Phe Tyr Glu Asn Pro Ser Lys Cys Pro Ser Leu Lys Gln Ala
170 175 180
Gln Asp Gln Trp Leu Asp Glu Gln Leu Ser Ile Ala Arg Gln Arg
185 190 195
His Cys Gln His Ala Ile Val Leu Gln His Ile Pro Leu Phe Leu
200 205 210
Glu Ser Ile Asp Glu Asp Asp Asp Tyr Tyr Phe Asn Leu Ser Lys
215 220 225
Ser Thr Arg Lys Lys Leu Ala Asp Lys Phe Ile His Ala Gly Val
230 235 240
Arg Val Val Phe Ser Gly His Tyr His Arg Asn Ala Gly Gly Thr
245 250 255
Tyr Gln Asn Leu Asp Met Val Val Ser Ser Ala Ile Gly Cys Gln
260 265 270
Leu Gly Arg Asp Pro His Gly Leu Arg Val Val Val Val Thr Ala
275 280 285
Glu Lys Ile Val His Arg Tyr Tyr Ser Leu Asp Glu Leu Ser Glu
290 295 300
Lys Gly Ile Glu Asp Asp Leu Met Asp Leu Ile Lys Lys Lys
305 310
<210> 51
<211> 355
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1568009CD1
<400> 51
Met Pro Phe Leu Gly Gln Asp Trp Arg Ser Pro Gly Gln Asn Trp
1 5 10 15
Val Lys Thr Ala Asp Gly Trp Lys Arg Phe Leu Asp Glu Lys Ser
20 25 30
Gly Ser Phe Val Ser Asp Leu Ser Ser Tyr Cys Asn Lys Glu Val
35 40 45
Tyr Asn Lys Glu Asn Leu Phe Asn Ser Leu Asn Tyr Asp Val Ala
50 55 60
3'/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Ala Lys Lys Arg Lys Lys Asp Met Leu Asn Ser Lys Thr Lys Thr
65 70 75
Gln Tyr Phe His Gln Glu Lys Trp Ile Tyr Val His Lys Gly Ser
80 85 90
Thr Lys Glu Arg His Gly Tyr Cys Thr Leu Gly Glu Ala Phe Asn
95 100 105
Arg Leu Asp Phe Ser Thr Ala Ile Leu Asp Ser Arg Arg Phe Asn
110 115 120
Tyr Val Val Arg Leu Leu Glu Leu Ile Ala Lys Ser Gln Leu Thr
125 130 135
Ser Leu Ser Gly Ile Ala Gln Lys Asn Phe Met Asn Ile Leu Glu
140 145 150
Lys Val Val Leu Lys Val Leu Glu Asp Gln Gln Asn Ile Arg Leu
155 160 165
Ile Arg Glu Leu Leu Gln Thr Leu Tyr Thr Ser Leu Cys Thr Leu
170 175 180
Val Gln Arg Val Gly Lys Ser Val Leu Val Gly Asn Ile Asn Met
185 190 195
Trp Val Tyr Arg Met Glu Thr Ile Leu His Trp Gln Gln Gln Leu
200 205 210
Asn Asn Ile Gln Ile Thr Arg Pro Ala Phe Lys Gly Leu Thr Phe
215 220 225
Thr Asp Leu Pro Leu Cys Leu Gln Leu Asn Ile Met Gln Arg Leu
230 235 240
Ser Asp Gly Arg Asp Leu Val Ser Leu G1y Gln Ala Ala Pro Asp
245 250 255
Leu His Val Leu Ser Glu Asp Arg Leu Leu Trp Lys Lys Leu Cys
260 265 270
Gln Tyr His Phe Ser Glu Arg Gln Ile Arg Lys Arg Leu Ile Leu
275 280 285
Ser Asp Lys Gly Gln Leu Asp Trp Lys Lys Met Tyr Phe Lys Leu
290 295 300
Val Arg Cys Tyr Pro Arg Lys Glu Gln Tyr Gly Asp Thr Leu Gln
305 310 315
Leu Cys Lys His Cys His Ile Leu Ser Trp Lys Gly Thr Asp His
320 325 330
Pro Cys Thr Ala Asn Asn Pro Glu Ser Cys Ser Val Ser Leu Ser
335 340 345
Pro Gln Asp Phe Ile Asn Leu Phe Lys Phe
350 355
<210> 52
<211> 179
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1677811CD1
<400> 52
Met Gln Arg Gln Asn Phe Arg Pro Pro Thr Pro Pro Tyr Pro Gly
1 5 10 15
Pro Gly Gly Gly Gly Trp Gly Ser Gly Ser Ser Phe Arg Gly Thr
20 25 30
Pro Gly Gly Gly Gly Pro Arg Pro Pro Ser Pro Arg Asp Gly Tyr
35 40 45
Gly Ser Pro His His Thr Pro Pro Tyr Gly Pro Arg Ser Arg Pro
50 55 60
Tyr Gly Ser Ser His Ser Pro Arg His Gly Gly Ser Phe Pro Gly
65 70 75
Gly Arg Phe Gly Ser Pro Ser Pro Gly Gly Tyr Pro Gly Ser Tyr
80 85 90
Ser Arg Ser Pro Ala Gly Ser Gln Gln Gln Phe Gly Tyr Ser Pro
95 100 105
Gly Gln Gln Gln Thr His Pro Gln Gly Ser Pro Arg Thr Ser Thr
38/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
110 115 120
Pro Phe Gly Ser Gly Arg Val Arg Glu Lys Arg Met Ser Asn Glu
125 130 135
Leu Glu Asn Tyr Phe Lys Pro Ser Met Leu Glu Asp Pro Trp Ala
140 145 150
Gly Leu Glu Pro Val Ser Val Val Asp Ile Ser Gln Gln Tyr Ser
155 160 165
Asn Thr Gln Thr Phe Thr Gly Lys Lys Gly Arg Tyr Phe Cys
170 175
<210> 53
<211> 403
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1756951CD1
<400> 53
Met Ala Ala Phe Leu Lys Met Ser Val Ser Val Asn Phe Phe Arg
1 5 10 15
Pro Phe Thr Arg Phe Leu Val Pro Phe Thr Leu His Arg Lys Arg
20 25 30
Asn Asn Leu Thr Ile Leu Gln Arg Tyr Met Ser Ser Lys Ile Pro
35 40 45
Ala Val Thr Tyr Pro Lys Asn Glu Ser Thr Pro Pro Ser Glu Glu
50 55 60
Leu Glu Leu Asp Lys Trp Lys Thr Thr Met Lys Ser Ser Val Gln
65 70 75
Glu Glu Cys Val Ser Thr Ile Ser Ser Ser Lys Asp Glu Asp Pro
80 85 90
Leu Ala Ala Thr Arg Glu Phe Ile Glu Met Trp Arg Leu Leu Gly
95 100 105
Arg Glu Val Pro Glu His Ile Thr Glu Glu Glu Leu Lys Thr Leu
110 115 120
Met Glu Cys Val Ser Asn Thr Ala Lys Lys Lys Tyr Leu Lys Tyr
125 130 135
Leu Tyr Thr Lys Glu Lys Val Lys Lys Ala Arg Gln Ile Lys Lys
140 145 150
Glu Met Lys Ala Ala Ala Arg Glu Glu Ala Lys Asn Ile Lys Leu
155 160 165
Leu Glu Thr Thr Glu Glu Asp Lys Gln Lys Asn Phe Leu Phe Leu
170 175 180
Arg Leu Trp Asp Arg Asn Met Asp Ile Ala Met Gly Trp Lys Gly
185 190 195
Ala Gln Ala Met Gln Phe Gly Gln Pro Leu Val Phe Asp Met Ala
200 205 210
Tyr Glu Asn Tyr Met Lys Arg Lys Glu Leu Gln Asn Thr Val Ser
215 220 225
Gln Leu Leu Glu Ser Glu Gly Trp Asn Arg Arg Asn Val Asp Pro
230 235 240
Phe His Ile Tyr Phe Cys Asn Leu Lys Ile Asp Gly Ala Leu His
245 250 255
Arg Glu Leu Val Lys Arg Tyr Gln Glu Lys Trp Asp Lys Leu Leu
260 265 270
Leu Thr Ser Thr Glu Lys Ser His Val Asp Leu Phe Pro Lys Asp
275 280 285
Ser Ile Ile Tyr Leu Thr Ala Asp Ser Pro Asn Val Met Thr Thr
290 295 300
Phe Arg His Asp Lys Val Tyr Val Ile Gly Ser Phe Val Asp Lys
305 310 315
Ser Met Gln Pro Gly Thr Ser Leu Ala Lys Ala Lys Arg Leu Asn
320 325 330
Leu Ala Thr Glu Cys Leu Pro Leu Asp Lys Tyr Leu Gln Trp Glu
335 340 345
39/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
Ile Gly Asn Lys Asn Leu Thr Leu Asp Gln Met Ile Arg Ile Leu
350 355 360
Leu Cys Leu Lys Asn Asn Gly Asn Trp Gln Glu Ala Leu Gln Phe
365 370 375
Val Pro Lys Arg Lys His Thr Gly Phe Leu Glu Ile Ser Gln His
380 385 390
Ser Gln Glu Phe Ile Asn Arg Leu Lys Lys Ala Lys Thr
395 400
<210> 54
<211> 163
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1833547CD1
<400> 54
Met Asp Val Arg Ser Arg Val Asp Ser Lys Thr Leu Thr Arg Asn
1 5 10 15
Thr Arg Ile Ile Ala Glu Ala Leu Thr Arg Val Ile Tyr Asn Leu
20 25 30
Thr Glu Lys Gly Thr Pro Pro Asp Met Pro Val Phe Thr Glu Gln
35 40 45
Met Ile Gln Gln Glu Gln Leu Asp Ser Val Met Asp Trp Leu Thr
50 55 60
Asn Gln Pro Arg Ala Ala Gln Leu Val Asp Lys Asp Ser Thr Phe
65 70 75
Leu Ser Thr Leu Glu His His Leu Ser Arg Tyr Leu Lys Asp Val
80 85 90
Lys Gln His His Val Lys Ala Asp Lys Arg Asp Pro Glu Phe Val
95 100 105
Phe Tyr Asp Gln Leu Lys Gln Val Met Asn Ala Tyr Arg Val Lys
110 115 120
Pro Ala Val Phe Asp Leu Leu Leu Ala Val Gly Ile Ala Ala Tyr
125 130 135
Leu Gly Met Ala Tyr Val Ala Val Gln His Phe Ser Leu Leu Tyr
140 145 150
Lys Thr Val Gln Arg Leu Leu Val Lys Ala Lys Thr Gln
155 160
<210> 55
<211> 110
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1833723CD1
<400> 55
Met Gln Ala Ala Leu Glu Val Thr Ala Arg Tyr Cys Gly Arg Glu
1 5 10 15
Leu Glu Gln Tyr Gly Gln Cys Val Ala Ala Lys Pro Glu Ser Trp
20 25 30
Gln Arg Asp Cys His Tyr Leu Lys Met Ser Ile Ala Gln Cys Thr
35 40 45
Ser Ser His Pro Ile Ile Arg Gln Ile Arg Gln Ala Cys Ala Gln
50 55 60
Pro Phe Glu Ala Phe Glu Glu Cys Leu Arg Gln Asn Glu Ala Ala
65 70 75
Val Gly Asn Cys Ala Glu His Met Arg Arg Phe Leu Gln Cys Ala
80 85 90
Glu Gln Val Gln Pro Pro Arg Ser Pro Ala Thr Val Glu Ala Gln
40/72
CA 02373191 2001-11-05
WO 00170047 PCT/US00/13299
95 100 105
Pro Leu Pro Ala Ser
110
<210> 56
<211> 1929
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1841446CB1
<400> 56
cggaagcaaa ggagccaaga ccatggcgaa agccggggat aagagcagca gcagcgggaa 60
gaaaagtcta aaacggaaag ccgctgccga agaacttcag gaggctgcag gcgctgggga 120
tggggcgacg gaaaacgggg tccaaccccc gaaagcggct gcctttccgc caggctttag 180
catttcggag attaaaaaca aacagcggcg acacttaatg ttcacgcggt ggaaacagca 240
gcagcggaag gaaaagttgg cagctaagaa aaaacttaaa aaagaaagag aggctcttgg 300
cgataaggct ccaccaaagc ctgtacccaa gaccattgac aaccagcgag tgtatgatga 360
aaccacagta gaccctaatg atgaagaggt cgcttatgat gaagctacag atgaatttgc 420
ttcttacttc aacaaacaga cttctcccaa gattctcatc acaacatcag atagacctca 480
tgggagaaca gtacgactct gtgaacagct ctccacagtt ataccaaact cacatgttta 540
ttacagaaga ggactggctc tgaaaaaaat tattccacag tgcatcgcaa gagatttcac 600
agacctgatt gttattaatg aagatcgtaa aaccccaaat ggacttattt tgagtcactt 660
gccaaatggc ccaactgctc attttaaaat gagcagtgtt cgtcttcgta aagaaattaa 720
gagaagaggc aaggacccca cagaacacat acctgaaata attctgaata attttacaac 780
acggctgggt cattcaattg gacgtatgtt tgcatctctc tttcctcata atcctcaatt 840
tatcggaagg caggttgcca cattccacaa tcaacgggat tacatattct tcagatttca 900
cagatacata ttcaggagtg aaaagaaagt gggaattcag gaacttggac cacgttttac 960
cttaaaatta aggtctcttc agaaaggaac ctttgattct aaatatggag agtatgaatg 1020
ggtccataag ccccgggaaa tggatacaag tagaagaaaa ttccatttat aaagtactga 1080
gagaatgata ttggattttg ctgaacaggc ctatcttgaa ctttggtaaa ttatttttga 1140
cagaatactc ttttcaaaat ggcatttgct gatttcataa acctttcacg tctggacgaa 1200
ttaccaaatg ccatgaattg ccactgtgtg tttatgtaga aaatacaaat aaaagttatt 1260
ttgatggctt aggtttcctt aaacttagtt ctcttgtttt tgggtaactg tgaataatta 1320
agttggaatc aagattcaga ttaactttcc tatttgcata gaacacatga gaggaataaa 1380
atggttggta aatattggct aacccttgat ttttatacca gattaacctt ggattcccag 1440
tgtctggcac agttttaata gcttaaatgg aggccaggtt tctggatgtt ttaacattct 1500
cttaagcctt cagaagggta aaaaatttaa agcaaaatga tctaccaggg tttaaagcaa 1560
agttgcaaat tactgaagct aatctttgct tcctgatttt gaggtttttg gttttttgtg 1620
cccacgttgt ggggagctct tttttacctc attacatggt gctgtagtac tccattcagg 1680
cactgaaaca aagttaaccc tataagtaac tcatggatgg aaacccgtag aacttaacag 1740
cctcctcctg accttaaaag aataaaggtt cacagtttac ctttaattcc ctagcagtct 1800
tgccagatgt atggcataaa gtcatgtgag aagagtaggt ggaaaaaact gtacaaactt 1860
aaccccttca ggtgttcaga acagattaat ataccatgta tttaatacca ataataatgc 1920
aaaataaag 1929
<210> 57
<211> 2113
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1850310CB1
<400> 57
ctgccacgcc actgtgtcat gaaggaaagt gaaagggaac gaggaagtag gaatgcccac 60
gctcgttgac tccgtgggtg aatacagcag ttaggacata cacaccatca cctttgaaag 120
tgcttgtttg ggggagggaa ggacatacgg gtaactagaa ctacccagcg agtcgtccag 180
aggagaggat caggtttgag tcaggaggct ccctgtactg gagtcgtccc actattcctc 240
aagaaatctt agaaccagct tgtgaggaaa aacatttttt aatgtaataa aaatatgcca 300
ttattctttg aaatgccaaa tgatataaat attttgccta atacatattt attgtagatg 360
aaatgcactc ttctcgatga ggcctcgatt tgaatcaatg gggtgggcca caggaaatgt 420
cagaggaacc agaactcaga actcttcctc ctggaccttt cttcccttcc cttggaggta 480
41/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
tccctttgaa tcaggcctct ctcttctcat cagtctgtag cttcccccct tgtataacct 540
gctttccttt ttacatttat taaaagtgga ttttgtaaaa gcatttcatt gacacgcgac 600
ctatcacaga caatggaatt cgtcagtggt ggtaagactg aaatcctgat gcttttcaca 660
cttcttgtct cttgctatgt atttctgcct ctagccttgc catgttttgc cttttttttt 720
tctttttggc caattccttt ttatatgtgc ccacaacaga ggtggggaga cacggagcac 780
cctgggtcct tcccagcgct gctgggcagg ccccgtctcc aggccccagc tgttgaaact 840
ttgaagggca acaaacaacc atccacactg ccggacccta ggctgttcag ggaggcagct 900
catttcca~ccggccccag gacacccagc ctgtgcccca caaggatctc tctaaatggg 960
agggattgag gctacttttc tgccaagccc tattaagtag taatgtgggg aaacccactg 1020
tgtcagtgca ggaagcccta gacaaatgtt ttcaaataaa tttcactgcc cagcctgcac 1080
agatttccat ttgaagtact tcccatccac cctgacaccc aaaggggttt ttttgttttg 1140
ttttgttttt gagacagggt cttgctttgt tgcccaggct ggagtgcagt gacgtggtca 1200
tagctcactg cagcctcaac ctcctgggct caagtgaccc tcctgcctca gcctcccaaa 1260
gttctgagat gataggcatg agccattgtg cctagcctat tttgattttt ttcttagagt 1320
caaggtcttg ctctgttgcc caggctgatc ttggacttgc gagccaccat gcctggctgg 1380
gtttttttaa aaatagaatc tcactgatag cctgcaagaa acagatgcag tgcctgcttc 1440
cgtatcagtc caaggagccc tcgtgtttgc cacctttacc tttgaacctc cccctgcctc 1500
cctgcctgtg tccgcttttg cagctcaatg cagccatgac aaggaaagaa aagacaaagg 1560
aaggccagag agccgcgcag ttctctgcag gtgcagatgc aggcagtgga ggtggcctga 1620
gcaggcagaa ggacaccaag cgccctatgt tgcttgtcat tcatgacgtg gtcttggagc 1680
ttctgactag ttcagactgc cacgccaacc ccagaaaata ccccacatgc cagaaaagtg 1740
aagtcctagg tgtttccatc tatgtttcaa tctgtccatc taccaggcct cgcgataaaa 1800
acaaaacaaa aaaacgctgc caggttttag aagcagttct ggtctcaaaa ccatcaggat 1860
cctgccacca gggttctttt gaaatagtac cacatgtaaa agggaatttg gctttcactt 1920
catctaatca ctgaattgtc aggctttgat tgataattgt agaaataagt agccttctgt 1980
tgtgggaata agttataatc agtattcatc tctttgtttt ttgtcactct tttctctcta 2040
attgtgtcat ttgtactgtt tgaaaaatat ttcttctata aaattaaact aacctgcctt 2100
aagaaaaaaa aaa 2113
<210> 58
<211> 1652
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1887020CB1
<400> 58
gacgaggtgc tattcgagtg ctagtggtgg aacgggacca cacgtattca caggcctcca 60
ctgggctctc agtaggtggg atttgtcagc agttctcatt gcctgagaac atccagctct 120
ccctcttttc agccagcttt ctacggaaca tcaatgagta cctggccgta gtcgatgctc 180
ctcccctgga cctccggttc aacccctcgg gctacctctt gctggcttca gaaaaggatg 240
ctgcagccat ggagagcaac gtgaaagtgc agaggcagga gggagccaaa gtttctctga 300
tgtctcctga tcagcttcgg aacaagtttc cctggataaa cacagaggga gtggctttgg 360
cgtcttatgg gatggaggac gaaggttggt ttgacccctg gtgtctgctc caggggcttc 420
ggcgaaaggt ccagtccttg ggagtccttt tctgccaggg agaggtgaca cgttttgtct 480
cttcatctca acgcatgttg accacagatg acaaagcggt ggtcttgaaa aggatccatg 540
aagtccatgt gaagatggac cgcagcctgg agtaccagcc tgtggaatgc gccattgtga 600
tcaacgcagc cggagcctgg tctgcgcaaa tcgcagcact ggctggtgtt ggagaggggc 660
cgcctggcac cctgcagggc accaagctac ctgtggagcc gaggaaaagg tatgtgtatg 720
tgtggcactg cccccaggga ccaggcctag agactccgct tgttgcagac accagtggag 780
cctattttcg ccgggaagga ttaggtagca actacctagg tggtcgtagc cccactgagc 840
aggaagaacc ggacccggcg aacctggaag tggaccatga tttcttccag gacaaggtgt 900
ggccccattt ggccctgagg gtcccagctt ttgagactct gaaggttcag agcgcctggg 960
ccggctatta cgactacaac acctttgacc agaatggcgt ggtgggcccc cacccgctag 1020
ttgtcaacat gtactttgct actggcttca gtggtcacgg gctccagcag gcccctggca 1080
ttgggcgagc tgtagcagag atggtactga agggcaggtt ccagaccatc gacctgagcc 1140
ccttcctctt tacccgcttt tacttgggag agaagatcca ggagaacaac atcatctgag 1200
catgtgtgct ctgcactggc tccactggct tgcatcctgg ctgtgttcac agccttgttt 1260
gctgcttcca tcttccccag tactgtgcca ggccttctcc ccctccccag tgtcctctcc 1320
tctcaggcag gccattgcac ccatatggct gggcaggcac aggcagtgag gccgaggcca 1380
atagcgagtg atgagcggga tcctaggact gatctgtagc ccatgctgat gtcacccacc 1440
agggcaatcc atctggaggc ctgagcaccc tggcccagga ctggcttcat cctggcactg 1500
accaggaaag actgcctctg accctcttag cagacagagc ccaggcatgg gagcactctg 1560
gggcagcctg gctcaggttt attgattttc gtctgtttac cctatccatt aatcaataca 1620
tgtaattaac tccttccaaa aaaaaaaaaa as 1652
42/72
CA 02373191 2001-11-05
WO 00170047 PCT/US00/13299
<210> 59
<211> 1120
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1911421CB1
<400> 59
agagcgccgg aagcggtccg agaatgaaga gtgtgatcta ccatgcattg tctcagaaag 60
aggcgaatga ctccgatgtc cagccttcag gagcacagcg ggccgaggcc ttcgtgaggg 120
ccttcctgaa gcgcagcacg ,cccgcatga gcccgcaggc ccgcgaggac cagctgcagc 180
gcaaggcggt ggtcctggag tacttcaccc gccacaagcg caaggagaag aagaagaaag 240
ccaaaggcct ctctgccagg caaaggaggg agctgcggct ctttgacatt aaaccagagc 300
agcagagata cagccttttc ctccctctcc atgaactctg gaaacagtac atcagggacc 360
tgtgcagtgg gctcaagcca gacacgcagc cacagatgat tcaggccaag ctcttaaagg 420
cagatcttca cggggctatt atttcagtga caaaatccaa atgcccctct tatgtgggta 480
ttacaggaat ccttctacag gaaacaaagc acattttcaa aattatcacc aaagaagacc 540
gcctgaaagt tatccccaag ctaaactgcg tgttcactgt ggaaaccgat ggctttattt 600
cctacattta cgggagcaaa ttccagcttc ggtcaagtga acggtctgcg aagaagttca 660
aagcgaaggg aacgattgac ctgtgaattc tttgccgtct aaggcagttg tttatgacag 720
ctgaaaactg gacactccct aaatgtccac ctttcagtga agagatagtt aagccaattc 780
catttataga ccacctccag ccagtgacgc tccgagttga ggatgttgaa caacatggga 840
aggtcgcagc gtactaagtg aagaagtcag aggacagagg aatttctctt tctaggagat 900
tttcattttg tgtgactccc atggggagga acagactggc aggaagcaca ccggggttaa 960
cactggttga cttgaatagg attattcgat ttttaaaaat acttttccat gttttctgag 1020
tgctctatga taaatcagtt gcatctgtga taatacagta catatgtgga cataaacagg 1080
gatcaaataa aggaggtatt gctgcaaaaa aaaaaaaaaa 1120
<210> 60
<211> 1000
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1911910CB1
<400> 60
agtcctggaa agcgttgttg gcccggttgc tctggagccg ggtctcgggt ctggtggctg 60
ccggccctgc ggcatctcgc catggggagc acggagagca gcgagggccg cagggtgtcc 120
ttcggagtgg acgaggagga gcgggtccgg gtgctgcagg gtgtccggct gtctgaaaac 180
gtggtgaacc gcatgaagga gcccagctct ccaccccctg ctcccacatc ttctaccttt 240
ggccttcaag atggcaactt gagagcccct cacaaagaat ccacactgcc caggtcgggg 300
agcagtggtg gccagcagcc ctcagggatg aaggagggtg tcaagaggta tgaacaggag 360
catgctgcta tccaggataa gctcttccag gtggcaaaga gggaaagaga ggctgccacc 420
aagcactcca aggcatccct gcccacgggc gaaggcagca tcagccatga ggagcagaag 480
tcagtccggc tggccaggga gctggagagc agagaggcag agctaagacg ccgtgacacc 540
ttctacaagg agcagctgga gcgtattgag aggaagaatg ctgagatgta taaactgtct 600
tcagagcaat tccatgaggc agcctcaaag atggagagca caataaagcc ccgcagggtg 660
gagcccgtct gctcagggtt gcaggcccag attctccact gctaccgaga tcgcccgcat 720
gaggtgctgc tgtgctcgga cctggtcaag gcataccagc gctgcgtgag cgccgcccac 780
aagggctgag gagcagacat cattccctgc cctggcagtg acttggagcc ctgaagaagg 840
gaccaatcat gggaccacag ccactgtgcc ctgccgtttc ctgctgggcc cctgcatatg 900
cccctgagcc tggggctgcc acgtgtttag gaaacaaagt atgcgctact gtctgaaaac 960
aaataaagca gatgcctttg ttttcaaaaa aaaaaaaaaa 1000
<210> 61
<211> 2273
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1928920CB1
43/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<400> 61
gggggtgaag cgatacgttt tgcccgcatt cggggcgcgc ggactggggg ggtccctgtg 60
gggctcccgg agttaagatg gcgtcctcag cggaggggga cgaggggact gtggtggcgc 120
tggcgggggt tctgcagtcg ggtttccagg agctgagcct taacaagttg gcgacgtccc 180
tgggcgcgtc agaacaggcg ctgcggctga tcatctccat cttcctgggt tacccctttg 240
ctttgtttta tcggcattac cttttctaca aggagaccta cctcatccac ctcttccata 300
cctttacagg cctctcaatt gcttatttta actttggaaa ccagctctac cactccctgc 360
tgtgtattgt gctteagttc ctcatc~ttc gactaatggg ccgcaccatc actgccgtcc 420
tcactacctt ttgcttccag atggcctacc ttctggctgg atactattac actgccaccg 480
gcaactacga tatcaagtgg acaatgccac attgtgttct gactttgaag ctgattggtt 540
tggctgttga ctactttgac ggagggaaag atcagaattc cttgtcctct gagcaacaga 600
aatatgccat acgtggtgtt ccttccctgc tggaagttgc tggtttctcc tacttctatg 660
gggccttctt ggtagggccc cagttctcaa tgaatcacta catgaagctg gtgcagggag 720
agctgattga cataccagga aagataccaa acagcatcat tcctgctctc aagcgcctga 780
gtctgggcct tttctaccta gtgggctaca cactgctcag cccccacatc acagaagact 840
atctcctcac tgaagactat gacaaccacc ccttctggtt ccgctgcatg tacatgctga 900
tctggggcaa gtttgtgctg tacaaatatg tcacctgttg gctggtcaca gaaggagtat 960
gcattttgac gggcctgggc ttcaatggct ttgaagaaaa gggcaaggca aagtgggatg 1020
cctgtgccaa catgaaggtg tggctctttg aaacaaaccc ccgcttcact ggcaccattg 1080
cctcattcaa catcaacacc aacgcctggg tggcccgcta catcttcaaa cgactcaagt 1140
tccttggaaa taaagaactc tctcagggtc tctcgttgct attcctggcc ctctggcacg 1200
gcctgcactc aggatacctg gtctgcttcc agatggaatt cctcattgtt attgtggaaa 1260
gacaggctgc caggctcatt caagagagcc ccaccctgag caagctggcc gccattactg 1320
tcctccagcc cttctactat ttggtgcaac agaccatcca ctggctcttc atgggttact 1380
ccatgactgc cttctgcctc ttcacgtggg acaaatggct taaggtgtat aaatccatct 1440
atttccttgg ccacatcttc ttcctgagcc tactattcat attgccttat attcacaaag 1500
caatggtgcc aaggaaagag aagttaaaga agatggaata atccatttcc ctggtggcct 1560
gtgcgggact ggtgcagaaa ctactcgtct cccttttcac agcactcctt tgccccagag 1620
cagagaatgg aaaagccagg gaggtggaag atcgatgctt ccagctgtgc ctctgctgcc 1680
agccaagtct tcatttgggg ccaaagggga aacttttttt tggagaaggc gtcttgcttt 1740
gtcacccacg ctggaatgca gtggcgggat ctcagctcac cgcaacctcc acctcctggg 1800
ttcaagtgat tttcctgcct cagcctccca agtagctggg aatacaggca cgccaccatg 1860
cccagctaat ttttgtattt tcagtagaaa cgggatttca ccacgttggc caggctggtc 1920
tcgaactcct gaccgcaagt gatccacccg cctccgcctc ccaaagtgct gggattacag 1980
gcgtgagcca ccgtgcccgg cccaaagggg aaactcttgt gggaggagca gaggggctca 2040
catctcccct ctgattcccc catgcacatt gccttatctc tccccatcta gccaggaatc 2100
tattgtgttt ttcttctgcc aatttactat gattgtgtat gtgccgctac caccaccccc 2160
cccatggggg ggtggagagg ggtgcaaggc cctgcctgct ccactttttc taccttggaa 2220
ctgtattaga taaaatcact tctgtttgtt cagtttttca aaaaaaaaaa aaa 2273
<210> 62
<211> 925
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2170846CB1
<400> 62
cgcagaaaca gcctatagac gccacgagtc ggcggcgcta ccgaggggct gtgggcgcgc 60
agctggaacc tccggctgtc agtgcgctta cagttcctaa ccccgaccct gcgcgcaccc 120
gcactatggc agccccgccg cagctaaggg ctctgctcgt agtcgtcaac gcactgctgc 180
gcaagcgccg ctaccacgct gcgttggccg tgcttaaggg cttccggaac ggggctgtct 240
atggagccaa aatccgggcc cctcacgcgc tggtcatgac ctttctcttc cggaatggca 300
gcctccagga gaagctgtgg gccatactgc aggccacata tatccactcc tggaacctgg 360
cacggtttgt gttcacctac aagggtctcc gtgccctgca gtcctacata caaggcaaga 420
cctacccagc acacgcattc ctggcggcct tcctcggggg tatcctggtg tttggagaaa 480
acaataacat caacagccag atcaacatgt acctgttgtc acgcgtcctg tttgccctga 540
gccgcctggc tgtagagaag ggctacatcc ctgaacccag gtgggacccg ttcccgctgc 600
tcactgcggt ggtgtggggg ctggtgctgt ggctctttga gtatcaccga tccaccctgc 660
agccctcgct gcagtcctcc atgacctacc tctatgagga cagcaatgta tggcacgaca 720
tctcagactt cctcatctat aacaagagcc gtccctccaa ttaatgcagc cctgaggtgt 780
ctggctgtgg ctcaagattt ggccccatgc agaccctccc aaaggatact gccttctcaa 840
gatcataggc ctcagactcc aactggtgtt atcccagggt tctgtttgct gaagtaaaaa 900
cactgatttt aaaaaaaaaa aaaaa 925
X4/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<210> 63
<211> 1570
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2176361CB1
<400> 63
gggcctccgt ctccgacgct gacttcctca gcgcaccagc actgtctccg gtggcaagtc 60
gaccagctcc tcccacacgg attctgtgta gaaatccacg gtatgtgcat tggaaatgga 120
cagggcatcc ctcaggaact gcagcagtcc ctgcaacttg gcacgcagcg tgggcaggtc 180
cggggtcacc gggagagggc aagaagccgc catgacgctc accctccgag gctgtaggtg 240
gcgcaaacat ggccgtgagg cgccagctcg gcccaaatta gcacaggcag gctcagtaga 300
gctccgagcc ggattccttc tgagcgattg attgcttcgg cttggtgacg tattttgtgg 360
gcgtccgtcg tggtcttctg atgacgcatt agtcggctgc aatggcgccg gtgaggcggt 420
ccgcgaagtg gcggcctggt ggtattgagg cgcgtggtga aggggtttcc actgtcgggt 480
acaggaataa gaatgtgaga cagaagacat ggcggcctaa ccacccgcaa gccttcgtgg 540
ggagcgttcg cgagggacaa ggctttgctt ttcgaagaaa actgaaaata cagcaaagtt 600
acaagaaatt gctacggaag gaaaagaagg ctcaaacgtc actggaatct caattcacag 660
atcgataccc agataatctg aaacatctct atttagctga agaggaaaga cataggaagc 720
aagcaagaaa agtcgaccat cctttgtcag aacaagttca ccagccgttg cttgaagaac 780
agtgtagcat tgacgagcct ttatttgaag atcagtgtag ctttgaccag cctcagccag 840
aagaacaatg tattaaaaca gtaaactcct ttacaattcc aaagaaaaat aaaaagaaaa 900
catcaaatca aaaagcacaa gaagaatatg aacagataca agccaaacgt gctgctaaga 960
aacaagaatt cgagaggaga aaacaggaga gagaagaagc ccaaaggcag tacaaaaaga 1020
agaaaatgga agtgtttaaa atactgaaca aaaagactaa aaagggccaa ccaaacttga 1080
atgtacaaat ggagtacctt cttcaaaaaa tacaagaaaa atgttaaaca ttttgttcct 1140
acaggttaaa atatctgctg cctattaggt tcttctgtga catgtgcctc ccagcagtga 1200
actaaatttg tcgacataaa ctggattgct aaactatgct aaatataaga tgttcacata 1260
tttttattat ggtaaaaaat tttctaaata tgttctacat gtttcttatt tatttgcctc 1320
tgaaggaagg ttggcctgaa gaactgaaag aacctcttat tttgcaagac aggcccaagc 1380
atgtaatact tttgtaccat atgagattta tatgaaataa attttttaaa aataaggaat 1440
cagagctatc aatgaagcat ttcaatgaaa tatttcaatt tagtaaacag ttacgttgtt 1500
ttaaaattta ttttaatgat gagggaggca aagactcctc ttggactttt tatttatttt 1560
aagtacatgt 1570
<210> 64
<211> 1868
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2212732CB1
<400> 64
tcaccatgag gatccagccc ttggtaaggc cgggactgca gaagcagtca tccctgaaaa 60
ccatgaggtt ctggcaggcc cagatgagca ccctcaggac acagatgcaa gagatgctga 120
tggggaggct agagatcggg agccagcaga ccaagctttg ctgcttagcc agtgtgggga 180
taaccttgag tcccctctgc ctgaagctag ctcagctcca ccggggccaa cccttgggac 240
actgcctgaa gtagagacaa taagggcatg ctccatgccc caggagcttc ctcagtcccc 300
caggacccga cagcctgagc cagatttcta ctgtgtcaag tggatccctt ggaaaggaga 360
acagacaccc atcatcaccc agagcactaa cggcccttgc cctctccttg ccatcatgaa 420
catcctcttt cttcagtgga aggtgaagct ccccccgcag aaggaagtga tcacatcgga 480
tgagctcatg gcccatcttg gaaactgcct cctgtccatc aagccccagg agaagtcaga 540
gggacttcag cttaattttc agcagaatgt ggatgatgca atgacagtgc tgcctaaact 600
ggccacaggt ctggatgtca atgtgcgatt cacaggcgtc tctgattttg agtatacacc 660
cgagtgcagt gtctttgacc tgctaggcat acctctgtac catggctggc ttgttgatcc 720
acagcagagt cctgaggctg tgcgtgcagt tgggaaactg agttacaacc agctggtgga 780
gaggatcatc acctgcaaac actccagtga caccaacctc gtgacagaag gcctgattgc 840
agagcagttc ctggagacca ccgcggccca gctgacctac cacggactgt gtgagctgac 900
agcagctgct aaggagggtg aacttagcgt ctttttccga aacaaccact ttagcaccat 960
gactaagcat aagagtcact tatacctact ggtcactgac cagggctttc tacaggagga 1020
gcaagtcgta tgggagagcc tgcacaatgt ggatggagac agctgctttt gtgactctga 1080
ctttcacctg agtcattccc tgggcaaggg gcctggagca gaaggtggga gtggctcccc 1140
45/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
agaaaagcag ctgcaggtag accaggacta cctgattgct ctgtccctgc agcagcaaca 1200
gccacgaggc ccgctggggc ttaccgactt ggagctggcc cagcagcttc agcaagagga 1260
gtatcaacag cagcaggcag cgcagccagt gcggatgcgg acgcgggtcc tgtcactgca 1320
ggggagagga gccacatctg gacgcccagc cggggagcgt cggcagaggc cgaagcacga 1380
gtcagactgc attctgctgt agctctgccc cagtgccagg ctggcctgcc ccttcttcca 1440
gaggctatgg ctagttggct tgctcccccg cctccacccc tgagatgtgc tggataactt 1500
atttatggac tgttggggat gagagcaggc aacaaatgcc aaggtcagac ttggtaatgt 1560
ccttgacctc acgtgctgct gccttctctg cctcccaccc agggcaacac taggattggt 1620
gggtttctgg ttctcaactc ccggtccctg aatagtcaca cgtatgtaca gactgaggct 1680
ctggggtgag gtccctatcc agaatgcatc tcttctgctt cccatccctg ctgcctggat 1740
gctcctgatc acctaggcag gcctgtctcc agttgtttca gagcttaatt tgggtttcta 1800
tctcttattt gtaatgcctt cctggggttt ggaaataaaa cttctggccg ggcaaaaaaa 1860
aaaaaaa~ 1868
<210> 65
<211> 1401
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2303457CB1
<400> 65
ccaagctgca gctggcaggg attgcggggt gccggccgtc tgagtttttt taaaactgct 60
cgccgcgaag tctgtctgca gccaaaatgt ccaacagaaa caacaacaag cttcccagca 120
acctgccgca gttacagaat ctaatcaagc gagacccgcc ggcctacatc gaggagtttc 180
tacagcagta taatcactac aaatccaatg tggagatttt caaattgcaa ccaaataaac 240
ccagcaaaga actagcagag ctggtgatgt ttatggcaca gattagtcac tgctacccag 300
agtacctaag taattttcct caagaggtga aagatcttct ctcctgcaat cataccgtat 360
tggatccaga tctgcgaatg acattttgca aagctttgat cttgctgaga aataagaatc 420
tcatcaatcc atcaagcctg ctagaactct tctttgaact ttttcgttgc catgataaac 480
ttctgcgaaa gactttatac acacatattg tgactgatat caagaatata aatgcaaaac 540
acaagaacaa taaagtgaat gtagtattgc aaaatttcat gtacaccatg ttaagagata 600
gcaatgcaac cgcagccaag atgtctttag atgtaatgat tgaactctac agaaggaaca 660
tctggaatga tgcaaaaact gtcaatgtta tcacaactgc atgtttctct aaggtcacca 720
agatattagt tgccgctttg acattctttc ttgggaaaga tgaagatgaa aaacaggaca 780
gtgactccga atctgaggat gatggaccaa cagcaagaga cctgctagta caatatgcta 840
cagggaagaa aagttccaaa aacaagaaaa agttggaaaa ggcaatgaaa gtgctcaaga 900
aacaaaaaaa gaagaaaaaa ccagaggtgt ttaacttttc agccattcac ttgattcatg 960
atccccaaga ttttgcggaa aaactactaa agcagcttga gtgctgtaag gagaggtttg 1020
aagtgaagat gatgctcatg aaccttatct ccagattggt gggaattcat gagcttttcc 1080
tcttcaattt ctatcccttt ttgaaaaggt ttctgaagcc ccaccaaagg gaggtaacca 1140
agatccttct gtttgttgaa aaagattctc atcacttagt accccaaggg ttttttaatt 1200
catggttaat gcttggggaa aaaatttttt ttaacggaaa aaaatctggg aaaatgttaa 1260
tgacagttgg gaatttaatg gttaaaagag gggtatataa acgctccaaa gtgttccttg 1320
ggggaaatag tgttggaaga aatttttttc aaaaaaatcc agggggctca agttaaaaaa 1380
accccccagg cgggtgtgta t 1401
<210> 66
<211> 1409
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2317552CB1
<400> 66
gtcggggctg cgccgtacaa cttccggctg taaagatggc ggcttcctag tgagtcggcg 60
gctgatttag aaggaggttc aggctacggt gagccgaagc cacacaggag ccatggaagt 120
ggcagagccc agcagcccca ctgaagagga ggaggaggaa gaggagcact cggcagagcc 180
tcggccccgc actcgctcca atcctgaagg ggctgaggac cgggcagtag gggcacaggc 240
cagcgtgggc agccgcagcg agggtgaggg tgaggccgcc agtgctgatg atgggagcct 300
caacacttca ggagccggcc ctaagtcctg gcaggtgccc ccgccagccc ctgaggtcca 360
aattcggaca ccaagggtca actgtccaga gaaagtgatt atctgcctgg acctgtcaga 420
ggaaatgtca ctgccaaagc tggagtcgtt caacggctcc aaaaccaacg ccctcaatgt 480
46/72
CA 02373191 2001-11-05
WO 00170047 PCT/US00/13299
ctcccagaag atgattgaga tgttcgtgcg gacaaaacac aagatcgaca aaagccacga 540
gtttgcactg gtggtggtga acgatgacac ggcctggctg tctggcctga cctccgaccc 600
ccgcgagctc tgtagctgcc tctatgatct ggagacggcc tcctgttcca ccttcaatct 660
ggaaggactt ttcagcctca tccagcagaa aactgagctt ccggtcacag agaacgtgca 720
gacgattccc ccgccatatg tggtccgcac catccttgtc tacagccgtc caccttgcca 780
gccccagttc tccttgacgg agcccatgaa gaaaatgttc cagtgcccat atttcttctt 840
tgacgttgtt tacatccaca atggcactga ggagaaggag gaggagatga gttggaagga 900
tatgtttg~cc ttcatgggca gcctggatac caagggtacc agctacaaat atgaggtggc 960
actggctggg ccagccctgg agttgcacaa ctgcatggcg aaactgttgg cccaccccct 1020
gcagcggcct tgccagagcc atgcttccta cagcctgctg gaggaggagg atgaagccat 1080
tgaggttgag gccactgtct gaaccatccc tgtacatctg caccttcttg tgcaaggaag 1140
tccttggcct aaagccttgg ttctcaaact gggttccttg ggacctccgg ggtggggggg 1200
ttcc:aggagg cacgtagggt accttgcagg gtcctaggag ggaaacccag gattccagga 1260
gggatcccag gaactgtggg cacccatttt ctgtgtctcc cagcccattt ccactcctag 1320
tttgtcatgg ataatttttg ttcttccctg tgtgattttt gccatcaaaa taaaaatttg 1380
agactcgtta accgaaaaaa aaaaaaaaa 1409
<210> 67
<211> 1888
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2416366CB1
<400> 67
tgtgattcgg agagtgagga tccctcaagg aaccaggcta gtgattcggg aaatgaggag 60
ctactcaaac cccgagtcag tgactctgag agtgaggggc ctcagaaggg gcctgccagt 120
gactcagaaa ctgaggatgc gtccagacac aaacagaagc cagagtcaga tgatgacagc 180
gacagggaga ataagggaga ggatacagaa atgcagaatg actccttcca ttcagacagc 240
catatggaca gaaaaaagtt tcacagttct gatagtgagg aggaagaaca caaaaagcaa 300
aaaatggaca gtgatgaaga tgaaaaagag ggtgaggagg agaaagtagc gaagagaaaa 360
gctgctgtgc tttctgatag tgaagatgaa gagaaagcat cagcaaagaa gagtcgtgtt 420
gtctctgatg cagatgactc tgacagtgat gctgtatcag acaagtcagg caaaagagag 480
aagaccatag catctgacag tgaggaagaa gctgggaaag aattgtctga taagaaaaat 540
gaagagaagg atctgtttgg gagtgacagt gagtcaggca atgaagaaga aaatcttatt 600
gcagacatat ttggagaatc tggtgatgaa gaggaagaag aatttacagg ttttaaccaa 660
gaagatctgg aagaagaaaa aggtgaaaca caggtaaaag aagcagaaga ttcagattct 720
gatgataaca taaagagagg aaaacatatg gactttctgt cagattttga gatgatgttg 780
cagcgaaaaa agagcatgag tggcaagcgc agacggaacc gcgatggtgg cacctttatt 840
agtgatgcag acgacgtcgt gagtgccatg atcgtcaaga tgaatgaagc tgctgaggaa 900
gacagacagt tgaacaatca aaaaaagcca gcactgaaaa aattaacttt actgcctgct 960
gtagttatgc accttaagaa gcaggacctt aaagaaacat tcattgacag tggtgtgatg 1020
tctgccatca aagaatggct ctcacctcta ccagatagga gtttgcctgc actcaagatc 1080
cgggaggagc tgctgaagat cctgcaagag ctgcctagtg tgagccagga gaccctgaag 1140
catagtggga ttggacgagc agtgatgtat ctctataaac accccaagga gtcaaggtct 1200
aacaaggaca tggcagggaa attaatcaat gagtggtcta ggcctatatt tggtcttacc 1260
tcaaactaca aaggaatgac aagagaagaa agggagcaga gagatctaga acagatgcct 1320
caacgacgaa gaatgaacag cactggtggt cagacaccca gaagagacct ggaaaaggtg 1380
ctgacaggag aggagaaggc tcttagacct ggagatcctg gattctgtgc ccgtgcaagg 1440
gtcccaatgc cttcaaacaa ggactatgtt gtcaggccca aatggaatgt ggaaatggag 1500
tcatccaggt ttcaggcgac ctccaagaag ggtatcagtc gactggataa acagatgaga 1560
aagttcacag atataaggaa aaaaagcaga tctgcacacg cagtgaaaat cagcattgag 1620
ggcaacaaaa tgccattgtg accttgcctg gaatgtgtcc ccatctctac tctaagaaat 1680
gcgcaatgga ctctttggag aaagaagata ttttaaaaca tttttagtgt gtctgtaaat 1740
ggttcagcgt gtatcagatg ttgtcatagg actcacattt ctctcagtta tatttaaaac 1800
cgttgtgtac tttgtacaaa ggaatactag tcatacttct ataaacttta cacaataaaa 1860
tttcattctg gttaaaaaaa aaaaaaaa 1888
<210> 68
<211> 1897
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
X7/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<222> 1892
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 2472980CB1
<400> 68
ttattgaaga catggccgcc gcgtattttc cagactgcat agtcagaccc tttggctcct 60
cagtcaacac ttttgggaag ttaggatgtg atttggacat gtttttggat ctagatgaaa 120
ccagaaacct cagcgctcac aagatctcag gaaattttct gatggaattt caagtgaaaa 180
atgttccttc agaaagaatt gcaactcaga agatcctgtc tgtgttagga gagtgccttg 240
accactttgg ccctggctgt gtgggtgtgc aaaaaatatt aaatgcccgg tgtccgctcg 300
tgaggttctc acaccaggcc tccggatttc agtgtgattt gactacgaac aataggattg 360
ccttgacaag ttccgaactc ctttatatat atggtgccct agactcaaga gtgagagcct 420
tggtgttcag tgtacggtgc tgggctcgag cacattcact aacaagtagt attcctggtg 480
catggattac aaatttctcc cttacaatga tggtcatctt ttttctccag agaagatcac 540
cccctattct tccaacacta gattccttaa aaaccctagc agatgcagaa gataaatgtg 600
taatagaagg caacaactgc acatttgttc gtgacttgag tagaattaaa ccttcacaga 660
acacagaaac attagaatta ctactgaagg aattttttga gtattttggc aattttgctt 720
tcgataaaaa ttccataaat attcgacagg gaagggagca aaacaaacct gattcttctc 780
ctctgtacat tcagaatcca tttgaaactt ctctcaacat aagcaaaaat gtaagtcaaa 840
gccagctgca aaaatttgta gatttggccc gagaaagtgc ctggatttta caacaggaag 900
atacagatcg accttccata tcaagtaatc ggccctgggg gctggtatcc ctattgctac 960
catctgctcc aaacagaaag tcctttacca agaagaaaag caataagttt gcaattgaaa 1020
cagtcaaaaa cttgctagaa tctttaaaag gtaacagaac agaaaatttc acaaaaacca 1080
gtgggaagag aacaattagt actcagacat gatggctgct acattgtgta aagaactggg 1140
cttagcctat caaatggtct gtggacttac ttggaaaaac tgatttgaaa ctttcacaga 1200
tctcagcttt catctgatgt cacttttcat gatcttctca ttggccccct taacctggtc 1260
tgaagttctg ggatgttttc agtttgatca gtctgatact cagtggcact ttattaaaac 1320
atcagctgtg gagtgtggcg gtgcacacct gtagtcccag ctgctcagga ggctgaggca 1380
ggaggatctc ttgagcccag gattttgaat ccatcgtgga caacatagca agattccatc 1440
tctaaaaaaa atgaaaataa acataagcca caaggaatgg gtgaaagatt attgtaatgt 1500
gctttaacta aataggtaaa tatactaaac aaatgctaaa actcagtttt aggatgaaac 1560
cattgttgat atccacatca gtccctgttt agaaaacatt taaaatgact tttagttatg 1620
tacagtacgt tggcaatgaa tacattaagc ttcaaaattt ggtagtgctc tcgaatatgt 1680
atatttgtat ttttcaagcg aagttctctt attcacatat aaattaaagt gggttggtac 1740
tgatatcaaa aaatgtttat gtttttagaa cagacatttc agtcactgca ttcttaggta 1800
ttccaaacca aatatgatga catcaataga ttgcatttta aaaatattgt ttgatttttc 1860
tatggtcaaa aataaaattg tgttctactt tnctggt 1897
<210> 69
<211> 1132
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2541640CB1
<400> 69
gtggagcctc gaacccgaga cgctagaccc aatttggtgc ttatgggagg agtgggagtg 60
gcagaggcag cgagaccact cctttcttgg cccaccattt ctctcaccat ttttactgca 120
gtgaactcct ctcagggtgg ggggctggta cagaggcaac tccgctttca taactctcac 180
agggtccttt gtcggaggtg tccctgccct cctaccccag cgtggtggga gtgcgacgcc 240
aggcttctcc ctcctccctg gcctcctgtg cctccagcct ccacatctcc tgagatcctc 300
cccaccccac acttacacag gtccccacac gcccctggtg cccctaaacc gccacccaac 360
cccactcacc ctggggccgg gactggggtg tcagagctga gccagggacc ttgggaggtg 420
gctgggaccg gcgcctcctg ctccctcttt cattttccct ttcggatttg gccaggatgg 480
aggacaggac aggatggctg agaggggcta gcgctccccc ttctcttaaa ggaacagggt 540
gaccctgctg gctggctccg cccttgggaa caggcacgga ccacgcccct tgctgggctc 600
tgtggccctc actctttagc taaatttcca tctggaaatg ggggaggggg gtaaccagag 660
ccacatcctt tatcgaggaa gctgggggat tcctagaagc ccttcccaga cagaccctca 720
gtctggagcc tagatccact acgcactaac catgccctgt gaagatggtc ttcggggaca 780
gagggaggtt tggtgctgga gtttaagggg cagcaaacat ctaacagctt ctggttccag 840
ctctgccacc gctcaccttg gacaaatcat tcggcggtac tgagccaggc tttcctgcct 900
tgtcaacaaa ggcggccagt ccagctgacc cccaagtttc cttccagctt tacatctttt 960
48/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
ttggaacggg gtctctctac atagcccagg tcgatcttga actcctggct tcaagtgatc 1020
ctcacacctt agcctcctga gtagctggga ttctctactt cttaatattg aattcccaac 1080
catattacag agaaccaaaa aaacccttgt gcctgcaaat aaataaaaaa as 1132
<210> 70
<211> 1763
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 1612, 1613, 1737, 1745
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 2695204CB1
<400> 70
aggctacagc tagggctgct cagtaactgc cccaaccact accacgtccc caagtgcaac 60
gggacccacg gaactacagg gtcctgctga gctcaggagc tgcacggaat ggtggcagtc 120
accttgccag tgcagcaggc atggatcaga ctgcaagcta ggggcacgag gcatcagttg 180
ggaagagggg acgcacagct aggcttgagg cctcttcatc gggatgtccc caggcccccc 240
agcccaggcc cagcataaag gccgtgttgg ggggcccccc tgacccaagg ggggcttcat 300
gcgccacgtg caggcggagc gtagtccatc ctcagagccg gacgtggccc ttcacagcct 360
ccagtcaggc agggggccct ccagggtggc ctgctcatgg gctacagccc agcagggggg 420
gcgacatccc ccggggtcta ccaggcccct gccccaccaa gcgaaggctg cttcctgctg 480
gagaagcccc agatgtcagc tctgaggaag aggggccagc ccctcggagg cgccggggat 540
ccctgggcca ccctactgct gccaacagtt ctgatgccaa agccacaccc ttctggagcc 600
acctgctgcc tgggcccaaa gagcctgttt tggacccaac agactgcggt cccatggggc 660
ggaggctgaa aggagcccgt cgcctgaagc tgagccccct tcgaagcctc cggaaggggc 720
caggcctgct gagccccccc agtgcctccc ctgttcctac ccctgctgtc agccgtaccc 780
tgctgggcaa ctttgaggaa tcattgctgc gaggacgttt tgcaccatct ggccacattg 840
agggcttcac agcagaaatt ggagctagtg ggtcatactg cccccagcac gtcacgctgc 900
ctgtcactgt cacattcttt gatgtttctg agcaaaatgc cccggctccc ttcctgggca 960
tcgtggatct gaaccccttg gggaggaagg gttacagcgt gcccaaggtg ggcaccgtcc 1020
aagtgacctt atttaacccc aaccagactg tggtaaagat gttccttgtg acctttgact 1080
tctcggacat gcctgctgcc cacatgacct tcctgcgcca tcgcctcttt ttggtgcctg 1140
tgggtgagga gggaaatgct aaccccaccc accgcctcct ctgctacttg ctgcacctca 1200
ggttccggag ctcccgctca ggccgcttaa gcctgcatgg agatatccgc ctgctttttt 1260
cccgccggag cctggagctg gacacagggc tcccctacga actgcaggct gtgaccgagg 1320
cccctcataa tccacgttat tcacctttgc cctgattgcc agcactctga acccatgcgg 1380
gctaatgacc tgcccatcct gctccatctt agagaacata tatggagaga cagcaagaga 1440
cccttcaggc ttgaattaaa gccctcacca tgctcacgcc caaatggatt atttgggtgt 1500
ttaaagcttc tgattcttac tacaccctgc cctacttcgg gtactccatg tgcctgtccc 1560
ctcccttggg tttcccagga cagcttaggt agtagggagg aactggagct annctgagat 1620
tttctcaagt tcccaggcaa gttatgccag cgttgcctcc ctgtcctggg caggggccac 1680
ttgttatttt atttattttt aatttataat ttattcaatt ggattgcctt ggtaaantcc 1740
cacanctgat aattggcatc act 1763
<210> 71
<211> 2110
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2805526CB1
<400> 71
cagagaacgc gctcgcgtag aggaattccg ggtcctcctc ctcgccctac cttggtttac 60
ctgcagccgc ctagtcctct tctccttctc tttgatgatt cagggcctcg gcttccctca 120
cgatattgca gaaagacaca gctttcctct tcctctccaa accacctcga agcaggggca 180
taattggaat atcatttgga gaaagtgtca tggaagttct gcgtccacag cttataagaa 240
ttgatggccg gaattacagg aagaatccag tccaagaaca gacctatcaa catgaagaag 300
atgaagagga cttctatcaa ggctccatgg agtgtgctga tgagccctgt gatgcctacg 360
aggtggagca gaccccacaa ggattccggt ctactttgag ggcccccagc ttgctctata 420
49/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
agcatatagt tggaaagaga ggggacacta ggaagaaaat agaaatggag accaaaactt 480
ctattagcat tcctaaacct ggacaagacg gggaaattgt aatcactggc cagcatcgaa 540
atggtgtaat ttcagcccga acacggattg atgttctttt ggacactttt cgaagaaagc 600
agcccttcac tcacttcctt gcctttttcc tcaatgaagt tgaggttcag gaaggattcc 660
tgagattcca ggaggaagta ctggcgaagt gctccatgga tcatggggtt gacagcagca 720
ttttccagaa tcctaaaaag cttcatctaa ctattgggat gttggtgctt ttgagtgagg 780
aagagatcca gcagacatgt gagatgctac agcagtgtaa agaggaattc attaatgata 840
tttctggggg taaaccc~ta gaagtggaga tggcagggat agaatacatg aatgatgatc 900
ctggcatggt ggatgttctt tacgccaaag tccatatgaa agatggctcc aacaggctac 960
aagaattagt tgatcgagtg ctggaacgtt ttcaggcatc tggactaata gtgaaagagt 1020
ggaatagtgt gaaactgcat gctacagtta tgaatacact attcaggaaa gaccccaatg 1080
ctgaaggcag gtacaatctc tacacagcgg aaggcaaata tatcttcaag gaaagagaat 1140
catttgatgg ccgaaatatt ttaaagttgt ttgagaactt ctactttggc tccctaaagc 1200
tgaattcaat tcacatctct cagaggttca ccgtagacag ctttggaaac tacgcttcct 1260
gtggacaaat tgacttctcc tgaggtggat cttggaaagc actagaaact aaacatcttc 1320
accaggtgct gaagaaaagt gtcttcgttt taattgccaa gcagggatgt ggacatttgg 1380
atggtgactt tcctgggtgg ttccccatag attcaccatt gcctctaatg gtgtctacac 1440
ccgtcatact accagctgag atggtggtgg gcataaggag aatttgtgcc tataaccctt 1500
agtgtgttct ggtttttttt cttttaattt ttaaattgtc gtaaaatact cataaaacat 1560
actgtcttca ccatttttaa gtgcacagtt cagtaacgtt aactgttaat acattcataa 1620
tgctgtgtgg ccgtcaccgc cgtccatctc cataggcttc tcagcttgta aaatggaaac 1680
tgtacccatt aaacagtaat tcccactcct cccagccccc gcagccacca ttctgctttc 1740
tgtctctctg gttttgacta ttctcagtat ctcatataag tggaatcata cagtgacttg 1800
tctttttgtg actggcttat ttcacttagc ataatgtcct caaggttcat ccatgttgtg 1860
tcaggtgaca ggatttcctt cctgtattat acatataatg gaatgttccg ttacatgtgc 1920
gtaccacact ctgttcctcc atcagtgaac acttggcttg cttcctcttg actattggga 1980
gtagtgctaa tacagacacg ggggtgcaaa tatctctttg agactctgcc tttaattcct 2040
ttaccaccca gagttttatc tttagcaaaa taaccattaa agttgtttgc cttttaaaaa 2100
aaaaaaaaaa 2110
<210> 72
<211> 1493
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2850382CB1
<400> 72
tggcggtggc gcggaccacg gcaggagcgg accgggcccg gggctgggcc ggcgtcgaac 60
gcagcgaagg ccggaggatg gaaccaggtg aggagctgga agaggagggc tctccaggtg 120
gccgtgagga tggcttcacc gccgagcacc tggctgcaga ggccatggca gctgacatgg 180
acccctggct agtgtttgat gcccgcacaa cgcctgccac tgagctggat gcctggctgg 240
ccaagtaccc accatcccaa gttacccgct atggggaccc cggttcaccc aactcagagc 300
ctgtgggctg gattgcagtg tatgggcagg gctacagccc caactccggg gacgtgcagg 360
gcctgcaggc agcctgggaa gctctgcaga ccagtgggcg gcccatcaca ccgggtaccc 420
tgcgccagct cgccatcacc caccacgtgc tctcgggcaa gtggcttatg catctggcac 480
cgggcttcaa gctggaccac gcctgggctg gcattgcccg ggccgtggtt gaaggccggc 540
ttcaggtggc caaggtgagc ccacgtgcca aggagggtgg gcgccaggtc atctgtgttt 600
acacggacga cttcacggac cgcttgggtg tactggaggc ggattcagcc atccgtgcag 660
cgggcattaa gtgcctgctc acctacaagc ctgatgtcta cacctacctg ggcatctacc 720
gggccaatcg ctggcacctc tgccccactc tctatgagag ccgtttccag cttgggggta 780
gtgcccgtgg ctcccgagtg cttgaccgtg ccaacaacgt ggaactgacc tagaggggcc 840
aaattgggga gactgcccac tcccccctcc atgctggggt tggatcctcc tgtcttcctc 900
cttgtcccat gaaggccaca ccccccagct ctggggactc ctaggtcact tgggaactac 960
ctgcatcttc agtccccttg aacttctgcc ctctgttcag ggctgacaca agccccacag 1020
gctggggggc tccggttccc tgagggatga gccttcagcc tccctttgta atgctgctcc 1080
tctccactgc ccagcaccat gagttgggtg cagacaccta gaaggagaga cttcttggaa 1140
cgctcatccc ccgctatacc tccccttcct cctgcatctc cccttctttc cttccccctc 1200
aggagagaga aaacttagtg cttccagccc ttcttggagc cttcatggtc caggggtagg 1260
ggccccactg gcctgagcat gccattttga ggggagggta gttgtgccta cttatcccct 1320
ggcagagggg atgccaggac catggacatg aggcttgccc atccctgcca acttacacag 1380
cctgtaccac tgtcccccct tccttggcta ctttgacatg tgcctgctcc tggcatttca 1440
ataaaacccg gcttgggtct gtgtcttgag tgttttttaa aaaaaaaaaa aaa 1493
<210> 73
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<211> 2930
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2929276CB1
<400> 73
tggagagcgg ggagttgtgt ccaccttgcc gacgtcgcta gccgtggggc tgtcctggga 60
aggcggacgg cgagcgcccg gtgtccgcac tcggccgcct gccgtgcccg tctgcgcccg 120
tgtcatcctc actcgggacg cagggaccgt ttttaaatca caggggcgtg tgtcagcctg 180
ccctaggact tcatgtctat atatttcccc attcactgcc ccgactatct gagatcggcc 240
aagatgactg aggtgatgat gaacacccag cccatggagg agatcggcct cagcccccgc 300
aaggatggcc tttcctacca gatcttccca gacccgtcag attttgaccg ctgctgcaaa 360
ctgaaggacc gtctgccctc catagtggtg gaacccacag aaggggaggt ggagagcggg 420
gagctccggt ggccccctga ggagttcctg gtccaggagg atgagcaaga taactgcgaa 480
gagacagcga aagaaaataa agagcagtag agtccctgtg gactcccatg ggtcatacca 540
gccagcatct gttcctgaac tgtgtttttc ccatcatgac ggaagaagag agtgagccgc 600
aattgttctg aaaatgtcaa acgaggcttc tgttttgcac ctgcagatca ccgagttggt 660
tttcttttct tttcttgcct tttttttttt gaaatttgcc gagcagtgga gccctctgac 720
aatttgcaag gccctctgag aaaggaagct gcttagagcc agggggttag tgggtgaggg 780
gagcgagtgc tgtttttgag atcattatct gaactcaggc agcctagtag aggcagtggt 840
gggattccaa tgggtcttgg tgggtgggag gtggggcatg tgcaaagcaa gcaaggaaca 900
tttggggtaa gaaaacaaac atgaggcaaa agaaaaaata catgttttta agaaaacatt 960
gagcagagaa ctgcagccag gatgcgctca gcagacattc actctggctg ctgggacatc 1020
agaaaacaaa gtcttcatct ctctctccag tttcacccac cccacccttt gctttcattt 1080
caggtgtgtt ggtctatatg acagggagga gagtaaagga gagcaggagc aattggctgc 1140
ctgcaaagcc agctggaggt gaagtgcagg aaaggaaagg tcaccccatt ctactccatg 1200
gcctctctgc tcccagctgt ggtaggctca catagccagt gtgatcggtt tttaagaggc 1260
agtgcttttc agcttttctc cctgatatat ccattttgct tcccagcact ttttaggagt 1320
agtgagagca cttcctgccc ttgttggaag ccccagggtg gacactcagc acgaaggtct 1380
ctcccttaac tgctgccctt ccaagacttg ctcccgagat ggagtgggcg tggtcttcca 1440
ggctggccct tccttctcct caccgccacc ttccctgccc cagccccagc agccatgggt 1500
acatgggtcc ccagctcacc tatggattcc cgccagtctg cccagctgca gtactcacgc 1560
cccatggggg atcttggtct gtttttcttg tgggagccta gtggagagca gacgtggctt 1620
tttatgtgtc ttgttgggga ggtgacttgc atggtgggga caaggctgtc gtggcaacct 1680
tgggatcgag tttgagacta aaggatgtca tgagatccct ggcttctccc catgttgttc 1740
ccggacaagg gcagaaggga ggcatggcaa gggacctctg ctgtccttac tcaacagtgg 1800
tcctcatccc tccccacctc ccactgcttc ctgcaagggc accagttgta tgagaaagtt 1860
ggcctttgga cttaggattt cttattgtag ctaagagcca tctgaagcag caggttgcag 1920
gacaaatgct tcagtccgcc gagagcagta ccgtgtggcc aagaggtgga ctcagagcct 1980
tccttgagct aaactcggcc aaccaaggca cgcagcatgt cccctcaggt ctccagtcag 2040
tccaggttga ccctcagttc tggacgtgtg tatatagctg tatttaatac ctcaaggtca 2100
ttgtggctct ggggatgcca gggcaggagg acgagggtgc gctgtggaca cagcagtccg 2160
cggaattccg ttctgggaag ccaatggtcg ccggcacccc ttgcttcctc cctctgttgt 2220
ctgcctgtgt gacacacatc aatggcaata acttcttcca actcctcgca gaagtgggag 2280
aggccggcag cctgcaccga gaggggcttt cctctctctt gctccccgct tcgttctgtt 2340
ttggctgcag agagtggttc atccatactc tcattccctc gcctcccctt gtggacgggg 2400
gtcttgcctt ttcaattcct gtgttttggt gtcttccctt atctgctacc ctgaatcacc 2460
tgtcctggtc ttgctgtgtg atgggaacat gcttgtaaac tgcgtaacaa atctactttg 2520
tgtatgtgtc tgtttatggg ggtggtttat tatttttgct ggtccctaga ccactttgta 2580
tgaccgtttg cagtctgagc aggccagggg ctgacagcta atgtcaggac cctcagcggt 2640
ggagcctgct ggggggaccc agctgctctt ggacaagtgg ctgagctcct atctggcctc 2700
ctcttttttt tttttttcaa gtaatttgtg tgtatttcta actgattgta ttgaaaaaat 2760
tcctagtatt r_cagtaaaaa tgcctgttgt gagatgaacc tcctgtaact tctatctgtt 2820
cttttttgag gctcagggag aaactagcat tttttttttt ccaaactact ttttgtcact 2880
gtgacagttg taaataaagt ttgaaaatgc tttccaaaaa aaaaaaaaaa 2930
<210> 74
<211> 4204
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3033039CB1
Sl/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<400> 74
atgacaatgg atgctctgtt ggctcgattg aaacttctga atccagatga ccttagagaa 60
gaaatcgtca aagccggatt gaaatgtgga cccattacat caactacaag gttcattttt 120
gagaaaaaat tggctcaggc tttactggag caaggaggaa ggctgtcttc tttctaccac 180
catgaggcag gtgtcacagc tctcagccag gacccacaaa ggattttgaa gccagctgaa 240
gggaacccaa ctgatcaggc tggtttttct gaagacagag attttggtta cagtgtgggc 300
ctgaatcctc cagaggagga agctgtgaca tccaagacct gctcggtgcc ccctagtgac 360
accgacacct acagagctgg agcgactgcg tctaaggagc cgcccctgta ctatggggtg 420
tgtccagtgt atgaggacgt cccagcgaga aatgaaagga tctatgttta tgaaaataaa 480
aaggaagcat tgcaagctgt caagatgatc aaagggtccc gatttaaagc tttttctacc 540
agagaagacg ctgagaaatt tgctagagga atttgtgatt atttcccttc tccaagcaaa 600
acgtccttac cactgtctcc tgtgaaaaca gctccactct ttagcaatga caggttgaaa 660
gatggtttgt gcttgtcgga atcagaaaca gtcaacaaag agcgagcgaa cagttacaaa 720
aatccccgca cgcaggacct caccgccaag cttcggaaag ctgtggagaa gggagaggag 780
gacacctttt ctgaccttat ctggagcaac ccccggtatc tgataggctc aggagacaac 840
cccactatcg tgcaggaagg gtgcaggtac aacgtgatgc atgttgctgc caaagagaac 900
caggcttcca tctgccagct gactctggac gtcctggaga accctgactt catgaggctg 960
atgtaccctg atgacgacga ggccatgctg cagaagcgta tccgttacgt ggtggacctg 1020
tacctcaaca cccccgacaa gatgggctat gacacaccgt tgcattttgc ttgtaagttt 1080
ggaaatgcag atgtagtcaa cgtgctttcg tcacaccatt tgattgtaaa aaactcaagg 1140
aataaatatg ataaaacacc tgaagatgta atttgtgaaa gaagcaaaaa taaatctgtg 1200
gaactgaagg agcggatcag agagtattta aagggccact actacgtgcc cctcctgaga 1260
gcggaagaga cttcttctcc agtcatcggg gagctgtggt ccccagacca gacggctgag 1320
gcctctcacg tcagccgcta tggaggcagc cccagagacc cggtactgac cctgagagcc 1380
ttcgcagggc ccctgagtcc agccaaggca gaagattttc gcaagctctg gaaaactcca 1440
cctcgagaga aagcaggctt ccttcaccac gtcaagaagt cggacccgga aagaggcttt 1500
gagagagtgg gaagggagct agctcatgag ctggggtatc cctgggttga atactgggaa 1560
tttctgggct gttttgttga tctgtcttcc caggaaggcc tgcaaagact agaagaatat 1620
ctcacacagc aggaaatagg caaaaaggct caacaagaaa caggagaacg ggaagcctcc 1680
tgccgagata aagccaccac gtctggcagc aattccattt ccgtgagggc gtttctagat 1740
gaagatgaca tgagcttgga agaaataaaa aatcggcaaa atgcagctcg aaataacagc 1800
ccgcccacag tcggtgcttt tggacatacg aggtgcagcg ccttcccctt ggagcaggag 1860
gcagacctca tagaagccgc cgagccggga ggtccacaca gcagcagaaa tgggctctgc 1920
catcctctga atcacagcag gaccctggcg ggcaagagac caaaggcccc ccgtggggag 1980
gaagcccatc tgccacctgt ctcggatttg actgttgagt ttgataaact gaatttgcaa 2040
aatataggac gtagcgtttc caagacacca gatgaaagta caaaaactaa agatcagatc 2100
ctgacttcaa gaatcaatgc agtagaaaga gacttgttag agccttctcc cgcagaccaa 2160
ctcgggaatg gccacaggag gacagaaagt gaaatgtcag ccaggatcgc taaaatgtcc 2220
ttgagtccca gcagccccag gcacgaggat cagctcgagg tcaccaggga accggccagg 2280
cggctcttcc tttttggaga ggagccatca aaactcgatc aggatgtttt ggccgctctt 2340
gaatgtgcag acgtcgaccc ccatcagttc ccggccgtgc acagatggaa gagtgctgtc 2400
ctgtgctact caccctcgga cagacagagt tggcccagtc ccgcggtgaa aggaaggttc 2460
aagtctcagc tgccagatct cagtggccct cacagctaca gtccggggag aaacagcgtg 2520
gctggaagca accccgcaaa gccaggcctg ggcagtcctg ggcgctacag ccccgtgcac 2580
gggagccagc tccgcaggat ggcgcgcctg gctgagcttg ccgccctgta ggcttggcgc 2640
tgggctctcg gtttgttctt catttttaaa gaaggaaggg tcatatgttt attgctaaac 2700
tgtcaaaaag gaatatattc tgattaaatt attactcctc actttgaggg tgtgagaatt 2760
ttagaagatt taaatgttct atataacact tagatttctg atattttgga agaagttaga 2820
agttaatgaa agcaaactca gttaccaatt ttctggaaaa tatccatgtg gtaatggtag 2880
actttttagg tggcaatttc taggtctgaa atatagcaga ggaaagggcg ctgaggcagt 2940
tgcaggcagg cagccctgta cttaccctgt actcacctca tccgacagac gctgtggatg 3000
aggaggggct tggcggaggc gtgagcaccg atgtcccttt gataacctgc actcaccaag 3060
atgaactatt tgccgccctg tcttttcctg ggttgggggg tggcatctga tggtggcaga 3120
gtgcctgttg gttcgcccgt gggtctcatg gttcagacag agggaggtgg acggcaggga 3180
tcagggagcc aggagcgcgc ctcagacttg cagcaaccat tgtgatttgg gttgttcgga 3240
atatttaaat tactgatcag aagatgaaag tagcttttct cttgggaagt cttgcagccc 3300
gtgggagtga taccaggagc aacacagagc tcagcagcgg cgccaaggtg ttccctgttt 3360
cctcagcacg tgagccttca ccgcctgctt cattcaggag ccagtgcagc agtaatacag 3420
tctatacatt gttctgtttt caaatttatc ctgaggcttt gttgagcata aatgattata 3480
cgataaaggt atccgttatt ttggaactca tttcagttgg gatctcctgt atgcagagtg 3540
ttgcatttag aggtttgagt cccatcttgg tttcttgccg tgctgactgt agccttcacc 3600
ttgacttgaa tgaaggtctg tggttggaat gtgtgaggag ccgctgaggt gttcaggagg 3660
tgctgcctgg aggtcggttt cttcctgggt gttacgggca actgctcaca cagttgtttc 3720
tctgtgaaca tttccagtgt ttaatccaaa atgaaaaccc accaatgctt ttgctaactt 3780
cagtgccttt tataaatcat ttttaaattt cctgaacttg ctttttgagg atatacaggg 3840
atattaagta gacgcaggat tgtttttgtt tgtaaaaatt ctgaattgaa actttgtttt 3900
aaaaaaaggc ttctttcttt catatgacaa gagataggtc aggaatattg gaatcaagat 3960
52/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
ttaaatgtta aaattcgatt ttgttacaca gggtgtgttc atttgttttg tagcagacaa 4020
gatctagatc ccagacagaa acaacacatg ctattctaaa aagccgcatt ttaaaaggca 4080
ccttggttct caaaagaaat cagaatatgg atattcgtag tgatgatctg ttttctctaa 4140
aatcttacca tattgtctgt atatggttgt aaattcaaat ggaaagtaaa acgttttggc 4200
cctg 4204
<210> 75
<211> 2149
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3213216CB1
<400> 75
ctccgcttag tgattttcca ttcactgacc ccgaggaact tcgctccttg gctatgaatc 60
ctgagctgtc tttgctgtaa tacagagttg agcctaatct ctttccccta ttgtgatgcc 120
cctgttacag tagccttgaa tagtcttcct taccttttta acaagcgtct gagggaattt 180
ttttccttta atagcttggt acatcaagca ggagcctgac tcctaaacca cgctgttcgg 240
ggtatgctga cattgttgtc tggaatataa cctgatttgg aagtgacaag tgactgagga 300
agtacctgta gaccaggaat actgggccag aagaaaaaaa tactgtctag tttagcaaat 360
tgcagaatgg acagcactga atgttggaac ataaaatttt taaaaggttt tggcttgtga 420
tcaaccagga gggaaacatg gttactgctc gccaggaacc tcgcctggtc ctgatttccc 480
tgacctgcga tggtgacacc ctgactctca gtgcagccta cacaaaggac ctactactgc 540
ctatcaaaac gcccaccaca aatgcagtgc acaagtgcag agtgcacggc ctggagatag 600
agggcaggga ctgtggcgag gccgccgccc agtggataac cagcttcctg aagtcacagc 660
cctaccgcct ggtgcacttc gagcctcaca tgcgaccgag acgtcctcat caaatagcag 720
acttgttccg acccaaggac cagattgctt actcagacac cagcccattc ttgatccttt 780
ctgaggcgtc gctggcggat ctcaactcca ggctagagaa gaaagttaaa gcaaccaact 840
tcaggcccaa tattgtaatt tcaggatgcg atgtctatgc agaggattct tgggatgagc 900
ttcttattgg tgacgtggaa ctgaaaaggg tgatggcttg ttccagatgc attttaacca 960
cagtggaccc agacaccggt gtcatgagca ggaaggaacc gctggaaac~, ctgaagagtt 1020
atcgccagtg tgacccttca gaacgaaagt tatatggaaa atcaccactc tttgggcagt 1080
attttgtgct ggaaaaccca gggaccatca aagtgggaga ccctgtgtac ctgctgggcc 1140
agtaatggga accgtatgtc ctggaatatt agatgccttt taaaaatgtt ctcaaaaatg 1200
acaacacttg aagcatggtg tttcagaact gagacctcta cattttcttt aaatttgtga 1260
ttttcacatt tttcgtcttt tggacttctg gtgtctcaat gcttcaatgt cccagtgcaa 1320
aaagtaaaga aatatagtct caataactta gtaggacttc agtaagtcac ttaaatgaca 1380
agacaggatt ctgaaaactc cccgtttaac tgattatgga atagttcttt ctcctgcttc 1440
tccgtttatc taccaagagc gcagacttgc atcctgtcac taccactcgt tagagaaaga 1500
gaagaagaga aagaggaaga gtgggtgggc tggaagaata tcctagaatg tgttattgcc 1560
cctgttcatg aggtacgcaa tgaaaattaa attgcacccc aaatatggct ggaatgccac 1620
ttcccttttc ttctcaagcc ccgggctagc ttttgaaatg gcataaagac tgaggtgacc 1680
ttcaggaagc actgcagata ttaattttcc atagatctgg atctggccct gctgcttctc 1740
agacagcatt ggatttccta aaggtgctca ggaggatggt tgtgtagtca tggacgaccc 1800
ctggatcctt gccattcccc tcagctaatg acggagtgct ccttctccag ttcggggtga 1860
aaaagttctg aattctgtgg aggagaagaa aagtgattca gtgatttcag atagactact 1920
gaaaaccttt aaagggggaa aaggaaagca tatgtcagtt gtttaaaacc caatatctat 1980
tttttaactg attgtataac tctaagatct gatgaagtat attttttatt gccattttgt 2040
cctttgatta tattgggaag ttgactaaac ttgaaaaatg tttttaaaac tgtgaataaa 2100
tggaagctac tttgactagt ttcaaaaaac aaacaaaaaa aaaaggaaa 2149
<210> 76
<211> 1348
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3220944CB1
<400> 76
gcagccgccg ctccggactc acgtgtgagt gaggaagaaa acctgaaaaa gaccccaaag 60
aagaagatga aaatggtaac tggagccgta gcgtcggtgc tggaagacga ggccacagac 120
acttctgata gtgaaggaag ctgtggatcg gaaaaggacc acttttattc tgatgatgac 180
gcaatagaag ctgacagtga gggtgatgct gagccctgtg acaaagaaaa tgaaaatgat 240
53/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
ggagaatcaa gtgttgggac taatatgggc tgggcagatg ctatggctaa agtcctcaac 300
aagaaaactc ctgaaagtaa acctactatt ctggtcaaaa ataagaagct ggaaaaggaa 360
aaagaaaagt taaagcaaga aagactagag aaaataaaac agcgtgataa gaggctggag 420
tgggaaatga tgtgcagagt aaagccagat gttgtccaag acaaagagac agagagaaat 480
cttcagagaa ttgcaacaag gggtgtggtg caattattta atgctgttca gaaacatcaa 540
aagaatgttg atgaaaaggt taaggaagct ggaagttcta tgagaaagcg tgctaagttg 600
atatcaactg tttccaagaa agatttcatc agtgttttga gagggatgga tggaagtaca 660
aatgagactg-cttcaagcag gaagaaacca aaagccaaac agactgaagt gaaatcagaa 720
gaaggcccag gttggacgat cctacgtgat gatttcatga tgggagcatc tatgaaagac 780
tgggacaagg aaagtgatgg gccagatgac agcagaccag aatctgcaag tgactctgat 840
acataaagca tcataggaaa tacaattgca gtcgttttat tttttctaga aaaatatgtc 900
atcctctgat agttggggaa ttataaggat accatttgta agaaagccaa aagacttttg 960
ccagatttca tatttcccct tttcatgtac actttatata tacttcatta aaattatatt 1020
ttaaaccctt gtataatttt aagcattgtt cctcagaaca tttgtaaaag gatatatttc 1080
tgcttgacca gcgagatgtg cattttgcca ggatcatatt ggtcatgtct attggtgtat 1140
tatttcagta tcaccaatgt tttcagaaat acagtactaa ttcatcatta aactctttga 1200
agttaatatt tttctgcctt ctaacttata gactcaacta tgtatctgta gtttttggga 1260
atggttggtg ttttttgctt tgtgttggga agttattgag aaaacctata taataaaatt 1320
taaaattata gttaaaaaaa aaaaaaaa 1348
<210> 77
<211> 662
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3224631CB1
<400> 77
ggagctgcca gccgggaggc tgcagccgcg ggttgttaca gctgctggag cagcagcggc 60
ccccgctccc gggaaccgtt cccgggccgt tgatcttcgg ccccacacga acagcagaga 120
ggggcagcag gatgaatgtg ggcacagcgc acagcgaggt gaaccccaac acgcgggtga 180
tgaacagccg tggcatctgg ctctcctacg tgctggccat cggtctcctc cacatcgtgc 240
tgctgagcat cccgtttgtg agtgtccctg tcgtctggac cctcaccaac ctcattcaca 300
acatgggcat gtatatcttc ctgcacacgg tgaaggggac accctttgag acccggacca 360
gggcaaggcg aggctgctaa cccactggga gcagatggat tatggggtcc agttcacggc 420
ctctcggaag ttcttgacca tcacacccat cgtgctgtac ttcctcacca gcttctacac 480
taagtacgac cagatccatt ttgtgctcaa caccgtgtca ttgatgagcg tgcttatccc 540
caagctgctc cagctccacg gagtccggat ttttgggatc aataagtact gagagtgcag 600
caccttcaca tgtccagggt ggtaagggag gggtagggta aaaggcatgt gctgcaacac 660
to 662
<210> 78
<211> 679
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3242839CB1
<400> 78
gcctgctagg atcagcggtg gtggttccgc gatggtaggc ggcggcgggg tcggcggcgg 60
cctcctggag aatgccaacc ccctcatcta ccagcgctct ggggagcggc ctgtgacggc 120
aggcgaggag gacgagcagg ttcccgacag catcgacgca cgcgagatct tcgatctgat 180
tcgctccatc aatgacccgg agcatccact gacgctagag gagttgaacg tagtagagca 240
ggtgcgggtt caggttagcg accccgagag tacagtggct gtggctttca caccaaccat 300
tccgcactgc agcatggcca cccttattgg tctgtccatc aaggtcaagc ttctgcgctc 360
ccttcctcag cgtttcaaga tggacgtgca cattactccg gggacccatg cctcagagca 420
tgcagtgaac aagcaacttg cagataagga gcgggtggca gctgccctgg agaacaccca 480
cctcttggag gttgtgaatc agtgcctgtc agcccgctcc tgagcctggc ctttgacccc 540
tcagcctgca tactggtatc ctggtcccag ctcctgccag ggctgttacc gttgttttct 600
tgaatcactc acaatgagaa actaacattt tgctttttgt aataaagtta atttatattc 660
agttaaaaaa aaaaaaaaa 679
<210> 79
54/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<211> 1160
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3274451CB1
<400> 79
cggaatgttg ccctggtcgc cgccgcggtg gggcgcgggc ccggaggagg cgcccgaggg 60
accaggcctt ccggcgggca aggaccaggg cgagccctgg agcgccggag cggacgcccc 120
cagggagctg gggacacccc catgggacag gtgaggggct cggacggagc cgtgggggga 180
cagcgactgg gggcctccat gccttcacca cgcgctctac ggaggcttct ccattgtccc 240
tgctggagat tgggtccctc ggtctccctt gcaccaggca cccttgggct ccaggggagg 300
gtgcgtgggg gcggtgtggc tgcagcctca gtccctgccc ctccagccca cagcccccat 360
cccgtagccc ttcctggatc tggagagagc agctgctgct gccccaccac ctgccaggcc 420
cggccactga gcagcccccc ggagccccag atccccgttc cccccaggtg gcctgggagg 480
tggccccctc gaggatgact ccactagcgc cctgggaccc caagtatgaa gccaaagcag 540
gacctcggcc ggtgtggggg gccaactgta gctcaggagc ctcgttctca ggccggacgc 600
tgtgtcaccc ctcattctgg ccgctgtatg aagcagcctc gggcaggggt ctcaggcccg 660
tggcccctgc cacagggcac tggaatggac agcaggcgcc cccagatgca gggttcccgg 720
tggtgtgctg tgaagatgtc ttcctctcgg accctctgct gccccggggg cagcgtgttc 780
ccctgtacct gtccaaggcc ccccagcaga tgatgggctc cctgaaactg ctgccgccgc 840
cccccatcat gtctgccagg gtgctccccc gcccatcacc ctcccggggc ccctccactg 900
cctggctcag cgggccggag ctgatcgctc tcactggcct gctgcagatg agccaggggg 960
agcctaggcc cagctcctcc gcggttggcc ccccagacca tacctctgac ccacccagcc 1020
cctgtggtag ccccagcagt tctcagggtg ctgacctctc tctcccacag accccagaca 1080
cccattgtcc atagccttct cagggcagag tgggctggtt gtgttgacaa taaaacagtg 1140
ttggtttgca aaaaaaaaaa 1160
<210> 80
<211> 949
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3284256CB1
<400> 80
gcgcgaacca tggccggcat ggtggacttc caggatgagg agcaggtcaa gtcctttttg 60
gagaacatgg aggtggagtg caactaccac tgctaccacg agaaggaccc ggacggttgc 120
tatcggctgg tggactattt ggaagggatc cggaagaatt ttgatgaggc tgccaaggtg 180
ttgaagttta actgtgaaga gaaccagcac agtgatagct gctacaaact gggggcctac 240
tatgtgactg gaaaaggtgg tctgacccag gacctgaaag ctgccgccag gtgctttttg 300
atggcgtgtg agaagcctgg aaagaagtca atagcagcat gtcacaacgt tggcctcctg 360
gcacatgatg gacaggttaa tgaggatggc cagcctgact tgggaaaggc cagggactac 420
tacacaaggg cctgtgatgg tggctatact tccagttgct tcaacctcag tgccatgttc 480
ctgcagggtg ccccaggctt tcccaaggac atggacctgg catgtaaata ctccatgaaa 540
gcctgtgacc tgggtcatat ctgggcctgt gccaatgcca gtcgcatgta caagctgggg 600
gatggtgttg ataaggatga ggccaaggcc gaggtgctaa aaaatcgagc ccagcagcta 660
cacaaagaac agcagaaagg tgtccaaccc ttaacatttg ggtaatgtgg tccaccctcc 720
ctcccaggca acaaactgct tgaggctggc agctcctgtt tctgaagact gatgcagccc 780
ttgaaggtca acctgctgga gcaaaaaaac ttgggacttg aatttgtagc tccatttaca 840
tggatccatt gccccagcta ctggagtata gcctacaatg tttatttcag tcaatattcc 900
tttatctggg tgttctgtac aatgtgagat tacagtcaat atgccgttg 949
<210> 81
<211> 1517
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3507004CB1
<400> 81
55/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
cgctcccctc tcacgcagcc aacatggctc cagtggagca cgttgtggcg gatgctgggg 60
ctttcctgcg gcatgcggct ctgcaggaca tcgggaagaa catttacacc atccgggagg 120
tggtcactga gattcgggac aaggccacac gcaggcggct cgctgtcctg ccctacgagc 180
tgcggttcaa ggagccctta ccggaatacg tgcggctggt gactgagttt tcaaagaaaa 240
caggagacta ccccagcctc tctgccacgg acatccaagt gcttgcactc acataccagt 300
tggaagcaga gtttgttggg gtgtctcacc taaaacaaga accacagaag gttaaggtga 360
gctcatcgat tcagcaccca gaaacacctc tgcacatttc tggtttccat ctgccctaca 420
agcctaaa~c cccacaagaa acagaaaaag- gaca~tcagc ttgtgagcct gagaacctgg 480
aatttagttc cttcatgttc tggagaaacc ctttgcccaa catcgatcat gaactgcagg 540
agctgctgat tgacagaggt gaggacgttc caagtgagga ggaggaggag gaagaaaacg 600
ggtttgaaga cagaaaagat gacagcgatg acgacggggg tggctggata acccccagta 660
acatcaagca gatccagcag gagctggagc agtgtgacgt ccccgaggac gtgcgggttg 720
gctgcctgac cacagacttc gccatgcaga atgttctgct gcagatgggg ctgcacgtgc 780
tggcggtgaa cggcatgctg attcgtgagg cccggagcta catcttgcgc tgccatggct 840
gtttcaagac aacgtctgac atgagccgag tgttctgctc acactgtggg aacaagaccc 900
tgaagaaagt gtccgtgacc gtcagcgacg acggcaccct gcacatgcac ttctcccgca 960
accccaaggt gctgaacccc cgcggcctcc ggtactcgct tcccactccc aaagggggca 1020
aatacgccat caacccccat ctcaccgagg atcagcgctt ccctcagctg cgactctccc 1080
aaaaggccag gcagaaaacc aacgtgttcg cccctgacta catcgccggg gtgtcaccct 1140
ttgtcgagaa tgacatctcc agccgctcag ctaccctgca ggtccgggac agcaccttgg 1200
gagctgggcg gagacgctta aatcccaacg cttccagaaa gaagtttgtg aagaaaaggt 1260
gaagagcgag ttcccgcagg caaattggat gggcgtctgg ccgccgtgga gttccggtga 1320
cccatttccc cagccgtgtc gtctccagga ccacccgatg gaaataacag gcgggcttca 1380
cggtgcggct ctgtccgccc atgccccgct gggtctgcag ggaactggac tgtcccatgg 1440
cctgtgagca ccggagcgcc tggctgcctg ccaaggaagt gcaattgcat aaaaacagaa 1500
agaacaacgc cctggag 1517
<210> 82
<211> 1991
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3585823CB1
<400> 82
cagaaattga gtttccacca tccttagatc tacaccagga gattccccag caggaagatc 60
agaaaaagga agtccccaag aggatactgg accagaactt tggggagccc catataacct 120
ctaggctgcc tccactccca ctgcatattc gaatccagca ggccctcacc agcccacttc 180
ccatgactcc tattctggag ggttctcaca gagctcattc gttgcttttt gaaaacagtg 240
acagcttttc tgaggacagc agtacgctgg gtcggaccag gtctcttccc atcactattg 300
aaatgctaaa agttccagac gatgaagaag aagaggagca aacctgtcca tccacattca 360
gtgaagaaat gacacctacc tcagtcattc ctaaattacc acagtgtcta cgggaggaag 420
aagagaagga gagcgactct gattcagaag gtcccattca gtaccgagat gaagaagatg 480
aagatgaaag ctatcagagt gctctcgcca acaaagtgaa gaggaaagac acactggcaa 540
tgaagttgaa ccacagaccc agtgaaccag agttgaacct gaattcttgg ccttgtaaaa 600
gcaaggagga gtggaatgaa atacggcacc agattggaaa cacactgatc cggcgactga 660
gtcaaagacc aacaccagaa gaactagaac aacgcaatat attgcaacct aaaaatgaag 720
ctgatcgtca ggcagaaaaa cgagaaatta aacgtcggct cactagaaag ctcagtcaaa 780
ggccaactgt cgctgaactc cttgccagga agattctgag gtttaatgaa tatgtagagg 840
taacagatgc tcaagattat gaccggcgag ccgacaaacc ttggaccaaa ctgacccctg 900
ctgacaaggc tgccataaga aaagaattaa atgaatttaa aagctccgag atggaggttc 960
atgaagagag caaacatttt acacgctacc atcgtccatg atgccaaagg ttgagagagg 1020
aatcaacatg gctgctttgc tgcttccttc tccaaagtga catatggagg gaactttagc 1080
acttcccagc acagccagaa ttgcatcctc tgggatcttc tgaggtggac agcactttga 1140
atgtagcatt tcactggaac agagtcttat gtgctgcacc gggggcaaaa caacactttg 1200
tcagtgcttt tgaacctttc aatattgtag catgcttgag gagtttttcc cttactggcc 1260
accaaagttc tgaaccactt gcaggttcca ggttttactg gctgcaccac accccttccc 1320
ctagatgact gcctgtgcag agacacagtt tgcaccatta gccttacctg ccctgccctg 1380
attgtgagac ccaaatgtgt aggctctaaa ttccagccat caaatccaat tcctggtggg 1440
gaaaaccttc tggagacccc caaccttctg ataaaagagt ctctacctcc agggaaagcc 1500
ttcttaccac actggcatat cagatgaaag cattgcactg tacctctcgt aacacagcaa 1560
tacagtcctc ttgaggcact caagcctgag aggaagctca ggatctgaca tgttcttcct 1620
tttcctcaca agtcatcatg attttttatt ttaaaataat ctggaagtaa tgggaactta 1680
gtttttcctg aactccaacc agaatccaaa ttggttagat gaggccaggc gcggtggctc 1740
acgcctgtaa tcccagcact ttgggaggcc gaggtgggtg gatcacctga ggtcaggagt 1800
56/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
tcaagaccag cctggccaac atggtgaaac cccatctcta ctaaaaatac aaaaattagc 1860
caggtgtggt ggcgcctggt tgaggcatga gaatcgcttg aacccaggag gtggaggttg 1920
cagtgagcca agatcatgcc tactgcactc cagactggca acaaagtggg actctgtctt 1980
aaaaaaaaaa a 1991
<210> 83
<211> 1238
< 212 > DFdA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3743822CB1
<400> 83
acgctagcgc tgcgatggcg gaggccgtgg agcgcactga cgagctggtc cgggagtacc 60
tgctcttccg cgggttcacg cacacactgc ggcagctgga cgccgagatc aaggcggaca 120
aggagaaggg gttccgggtg gataagattg tggaccagct gcagcagtta atgcaggtgt 180
atgacttggc tgcccttcgg gattattgga gctacttgga gcgtcggctc ttcagccgct 240
tggaggatat atacagaccc acaatccaca agctgaaaac cagcctgttt cgattttatc 300
ttgtctacac aatccagaca aacagaaatg acaaggctca ggagttcttt gcaaagcagg 360
ccacggaact ccagaaccag gctgagtgga aggattggtt tgtcctgccc ttcctgccat 420
ccccggacac caaccccacc tttgctacct acttttctcg acagtgggct gacaccttca 480
ttgtgtccct gcacaacttc ctgagcgtcc tgtttcagtg catgccagtc cctgtgatcc 540
tgaactttga tgcggagtgt cagaggacta accaggttca agaagaaaat gaagttctgc 600
gtcagaagct ttttgcattg caagctgaaa tccaccgact gaagaaagag gagcaacagc 660
cagaagagga agaggccttg gtccaacaca aattgcctcc ttatgtctcc aacatggacc 720
gcctggggga ctcggaactg tgagtgtgtg tgacggccct ttccttttct ttgccctctg 780
tgctctcagc cccacagttc cctctgatct tgccttgcct gcagtcctct gtgcctgctg 840
aaggagaatt gcggaagagc ctcctggcac acactgctct tgcttgagag ctttattttt 900
atttttagag atggagtctc gctctgtcgc ccaggctaga gtgccgtggc acaatcttgg 960
ctcaaagcaa cttctgcctc ctgggttcag gagaaccact tgagcccagg aggcggaggt 1020
tgcagtgagc cgagatcacg ccactgggag aacccaactc aaaaaaaaaa aaaaattttt 1080
gggttatgga cctttttgga tttgggacct tggggttaag gcgctcaacc tgtacctttt 1140
ccatttggtg ggggtcggag cccgaattcg gaaccatgta aaagctgttt cctggttaaa 1200
attggtaacc ggccaaaatt cccacaaaca ttggggcg 1238
<210> 84
<211> 1448
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3808027CB1
<400> 84
gactacgtct gcatcgaagg atcaggaagt ttgtgctctc tgcgtggcta agtttttcac 60
ctactaggac gggggtgggg tggggagaac aggtgtcctt ctaaaataca gcacaagcta 120
cagcctgcgt ccagccataa cccaggagta acatcagaaa caggtgagaa tgaccacttt 180
aactcaccgg gcccgtcgca ctgaaataag caagaactct gaaaagaaga tggaaagtga 240
ggaagacagt aattgggaga aaagtccaga caatgaagat tctggagact ctaaggatat 300
ccgccttact cttatggaag aagtattgct tctgggacta aaagataaag aggggtacac 360
atctttctgg aatgactgca tatcatcagg cctgcgaggg ggcatcctga tagagctggc 420
catgcggggt cgaatctatc tggaaccccc gaccatgcgt aagaagcgac tactagacag 480
aaaggtactg ctaaagtcag acagcccaac aggtgatgtt ttactggatg aaactctgaa 540
acacatcaaa gcaactgaac ccacagaaac tgtccaaaca tggatagagc tactcactgg 600
tgagacctgg aaccccttca aattacagta ccagctgaga aatgtacgag agcgcatcgc 660
aaagaaccta gtagagaaag gtattctaac cactgagaag cagaatttcc tgctatttga 720
catgactact catccagtga ccaatacaac agagaaacag cgactagtga aaaaacttca 780
agatagtgta ctagagcggt gggtaaatga ccctcagcgt atggacaagc gaacactagc 840
actcctggtg ctagcccact cctctgatgt gctagagaat gtcttctcct ctctgacaga 900
tgacaagtat gatgtggcaa tgaatcgagc caaggactta gtagaactgg accctgaagt 960
ggaagggaca aagcctagtg ccacagaaat gatctgggct gtgctggcag ccttcaataa 1020
atcttaaagc cggcaggtgg atttctcctt ttcccctgct ggctggtgac tgtcagagac 1080
cccatcactg agttttgtgt gatgagatgt tttcatcatt tttgtttcct tttcttgaat 1140
cagacttgtg atttggggag agcaacttca gggcttctcc atagaccttg accattataa 1200
57/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
gcagacctta tttctcccta cacttccact ccataggtaa ataaactggg tgaagccaca 1260
cacacccaat gaacattttc tcccatttgg tggtttagtt tcacccagtg tgcttttgga 1320
taagcagaaa tatctaaatg ggattttgag ttctccagct atagaatgtg aatgagataa 1380
aaagtgaact atctattata tttttctgag tttgttttct atcataaata catgttttca 1440
cagccttt 1448
<210> 85
<211> 807
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4016220CB1
<400> 85
cggacctggg gcctgaccat ggaatcagtt ccattcctgc ctcaatgact tgcaacctgt 60
ttcctcctct gcagattggg aacagcaatc cccacatcaa caatttggcg cccccactct 120
cccaggggaa actgggggac cctctaggcg gtgtttgcag agtcccctcc cagcaggagc 180
cacagggttg aataggagct gccagatcag tctccttccc gggacatccc cgggggcggc 240
agctccacct gctgcaggaa aggtaggacc tgatggctcc aggccctcgg aggctcagag 300
gcgcagggct gagccatggg aacgaagcgc gaggccatcc tgaaggtgct ggagaacctg 360
acaccggagg agctcaagaa gttcaagatg aagctgggga cggtgccgct gcgcgagggc 420
tttgagcgca tcccgcgggg cgcgctcggg cagctagata tcgtggacct caccgacaag 480
ctggtcgcct cctactacga ggactacgca gccgagctcg tcgtggccgt gctgcgcgac 540
atgcgcatgt tggaggaggc cgcacggctg cagcgggctg cgtgagggcc actctgagct 600
ggaggcgggt caaggcaaaa ggagtaatgc aacgcctgtg aagccagccc cacgcgcgca 660
ccagtcgcgt aagacaacag cagtgtctcc acctcgggga ccaaggacgc ctccgtgcct 720
ccagaccccg cctcctccag cccctgcacc tgtcatttat tcttccactg cccaataaat 780
attcatggca gactttaaaa aaaaaaa 807
<210> 86
<211> 1071
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4093555CB1
<400> 86
ggtggggcaa gctgcctaga gaagtgagag cgacgtcagc tattgaccaa tgggaagagc 60
tgatggtatg gcgtgggagc aagagtgaca acgattggtc agccttgcat ctctacgcct 120
aaggcgggag ctcctggagg cggaggccgc gggtgggggc ctagtggagt gaggggtaac 180
aagatggcga ccgagacggt ggagctccat aagctaaagc ttgccgaact aaagcaagaa 240
tgtcttgctc gtggtttgga gaccaaggga ataaagcaag atcttatcca cagactccag 300
gcatatcttg aagaacatgc tgaagaggag gcaaatgaag aagatgtact gggagatgaa 360
acagaggaag aagaaacaaa gcccattgag ctccctgtca aagaggaaga accccctgaa 420
aaaactgttg atgtggcagc agagaagaaa gtggtgaaaa ttacatctga aataccacag 480
actgagagaa tgcagaagag ggctgaacga ttcaatgtac ctgtgagctt ggagagtaag 540
aaagctgctc gggcagctag gtttgggatt tcttcagttc caacaaaagg tctgtcatct 600
gataacaaac ctatggttaa cttggataag ctgaaggaaa gagctcaaag atttggtttg 660
aatgtctctt caatctccag aaagtctgaa gatgatgaga aactgaaaaa gaggaaggag 720
cgatttggga ttgtcacaag ttcagctgga actggaacca cagaggatac agaggcaaag 780
aagaggaaaa gagcagagcg ctttgggatt gcctgatgaa aagttcctga tactttctgt 840
tctccagtgt tttccatttc tctccttctt cttggtcaca tatatgccta aatgcacagt 900
catgtgccta cgtcctgcct cgcaatgagg gagcatgtac cccaggtaca tccatgaact 960
gcggcagcag tttgacttat tgctgtttca gctttaaggt tgttgtgttt ttgtttttga 1020
ttatgttgct tgttaataaa aaaaaataga aaagagaaaa aaaaaaaaaa a 1071
<210> 87
<211> 988
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
58;?2
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<223> Incyte ID No: 4829366CB1
<400> 87
tcggtagtgc ggcgctgttt aaagatggcg gcggaggaac ctcagcagca gaagcaggag 60
ccgctgggca gcgactccga aggtgttaac tgtctggcct atgatgaagc catcatggct 120
cagcaggacc gaattcagca agagattgct gtgcagaacc ctctggtgtc agagcggctg 180
gagctctcgg tcctatacaa ggagtatgct gaagatgaca acatctatca acagaagatc 240
aaggacctcc acaaaaagta ctcgtacatc cgcaagacca ggcctgacgg caactgtttc 300
tatcgggctt tcggattctc ccacttggag gcactgctgg atgacagcaa ggagttgcag 360
cggttcaagg ctgtgtctgc caagagcaag gaagacctgg tgtcccaggg cttcactgaa 420
ttcacaattg aggatttcca caacacgttc atggacctga ttgagcaggt ggagaagcag 480
acctctgtcg ccgacctgct ggcctccttc aatgaccaga gcacctccga ctaccttgtg 540
gtctacctgc ggctgctcac ctcgggctac ctgcagcgcg agagcaagtt cttcgagcac 600
ttcatcgagg gtggacggac tgtcaaggag ttctgccagc aggaggtgga gcccatgtgc 660
aaggagagcg accacatcca catcattgcg ctggcccagg ccctcagcgt gtccatccag 720
gtggagtaca tggaccgcgg cgagggcggc accaccaatc cgcacatctt ccctgagggc 780
tccgagccca aggtctacct tctctaccgg cctggacact acgatatcct ctacaaatag 840
ggctggctcc agcccgctgc tgccctgctg cccccctctg ccaggcgcta gacatgtaca 900
gaggtttttc tgtggttgta aatggtccta tttcaccccc ttcttcctgt cacatgaccc 960
ccccccatgt tttattaaag ggggtgca 988
<210> 88
<211> 1385
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4830720CB1
<400> 88
cccacgcgtc cggccggaag agggagtctg taggggcggg ccggctggcg tcccctttcc 60
ggccggtccc catggaggcg ctggggaagc tgaagcagtt cgatgcctac cccaagactt 120
tggaggactt ccgggtcaag acctgcgggg gcgccaccgt gaccattgtc agtggccttc 180
tcatgctgct actgttcctg tccgagctgc agtattacct caccacggag gtgcatcctg 240
agctctacgt ggacaagtcg cggggagata aactgaagat caacatcgat gtactttttc 300
cgcacatgcc ttgtgcctat ctgagtattg atgccatgga tgtggccgga gaacagcagc 360
tggatgtgga acacaacctg ctcaagcaac gactagataa agatggcatc cccgtgagct 420
cagaggctga gcggcatgag cttgggaaag tcgaggtgac ggtgtttgac cctgactccc 480
tggaccctga tcgctgtgag agctgctatg gtgctgaggc agaagatatc aagtgctgta 540
acacctgtga agatgtgcgg gaggcatatc gccgtagagg ctgggccttc aagaacccag 600
atactattga gcagtgccgg cgagagggct tcagccagaa gatgcaggag cagaagaatg 660
aaggctgcca ggtgtatggc ttcttggaag tcaataaggt ggccggaaac ttccactttg 720
cccctgggaa gagcttccag cagtcccatg tgcacgtcca tgacttgcag agctttggcc 780
ttgacaacat caacatgacc cactacatcc agcacctgtc atttggggag gactatccag 840
gcattgtgaa ccccctggac cacaccaatg tcactgcgcc ccaagcctcc atgatgttcc 900
agtactttgt gaaggtggtg cccactgtgt acatgaaggt ggacggagag gctcccctcc 960
caccccaggt actgaggaca aatcagttct ctgtgaccag acatgagaag gttgccaatg 1020
ggctgttggg cgaccaaggc cttcccggag tcttcgtcct ctatgagctc tcgcccatga 1080
tggtgaagct gacggagaag cacaggtcct tcacccactt cctgacaggt gtgtgcgcca 1140
tcattggggg catgttcaca gtggctggac tcatcgattc gctcatctac cactcagcac 1200
gagccatcca gaagaaaatt gatctaggga agacaacgta gtcaccctcg gtgcttcctc 1260
tgtctcctct ttctccctgg cctgtggttg tcccccagcc tctgccaccc tccacctcct 1320
cggtcagccc cagccccagg ttgataaatc tattgattga ttgtgatagt aaaaaaaaaa 1380
aaaaa 1385
<210> 89
<211> 1524
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5389730CB1
<400> 89
gccgccacct ctccccagcc caggagaggc tgcggagccg cagccgccca gaccgcgcag 60
59/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
cgcgggaggc aggttccgca cgaaataaat cagaatgagt tatgcagaaa aacccgatga 120
aatcacgaaa gatgagtgga tggaaaagct caataacttg catgtccaga gagcagacat 180
gaaccgcctc atcatgaact acctggtcac agagggcttt aaggaagcag cggagaagtt 240
tcgaatggaa tctggaatcg aacctagtgt ggatctggaa acacttgatg aacgaatcaa 300
gatccgggag atgatactga aaggtcagat tcaggaggcc atcgccttga tcaacagcct 360
ccacccagag ctcttggaca caaaccggta tctttacttc catttgcagc aacagcattt 420
gatcgagctg atccgccagc gggagacaga ggcggcgctg gagtttgcac agactcagct 480
ggcggagcag gg~gaggaga gccgagagtg cctcacagag atggagcgta ccctggcact 540
gctggccttt gacagtcccg aggagtcgcc cttcggagac ctcctccaca ccatgcagag 600
gcagaaggtg tggagtgaag ttaaccaagc tgtgctagat tatgaaaatc gcgagtcaac 660
acccaaactg gcaaaattac tgaaactact actttgggct cagaacgagc tggaccagaa 720
gaaagtaaaa tatcccaaaa tgacagacct cagcaagggt gtgattgagg agcccaagta 780
gcgcctgcgc ttgcgtggtg gatccaacac cagccctgcg tcgtgggact tgcctcagat 840
cagcctgcga ctgcaagatt cttactgcag tagagaactc tttttctccc ttgtactttt 900
ttttgacctg gcatcttttt atagggaaaa atggcctttg taggcagtgg aaaacttgca 960
aggaaagctg ccgtctcttt ggcagtctga tgcagagcct gcactctggc actcgctgaa 1020
gaatctggaa ggttgcggtt tgctcttcca gtgttcgggg gcctctggct gctgaaggat 1080
tcggtctacc acggagggct gtgctgttag gctgcatccc actcaaaata caggaaaagc 1140
acgaatcatg attctgcttt ctgttagctt aggcagacat tgggccttca cctacaagtt 1200
tttccttacc cctgtggttt ttgtgttttt ttttttttct ttttccatag gaaagaatat 1260
ataaatttgt aaatcctaat tcaaagatgg ctcatgtgtg agggcattga gtttgatttg 1320
ttttcccttt ggtctgggtt gtgtggcttt tgggggatgc gtgtgagggg gctatgtgtt 1380
ttttaatttt ttaaatatat attttggtgc tgtgtgtggt aagagacttg ttcctagtgg 1440
atcaatgaac catctcttct gggcagtttt gttgaaaata aaggtttctc tttgatttca 1500
agaatgacca aaaaaaaaaa aaaa 1524
<210> 90
<211> 1288
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5397088CB1
<400> 90
ccgcccacgt catggcgccc gaggagaacg cggggaccga actcttgctg cagagtttcg 60
agcgccgctt cctggcggca cgcacactgc gctccttccc ctggcagagc ttagaagcaa 120
agttaagaga ctcatcagat tctgagctgc tgcgggatat tttgcacaag actgtgaagc 180
atcctgtgtg tgtgaagcac ccgccgtccg tcaaatatgc ccggtgcttt ctctcagaac 240
tcatcaaaaa gcacgaggct gtccacacag agcctttgga cgagctgtat gaagcgctgg 300
cggagaccct gatggccaag gagtccaccc agggccaccg gagctatttg ctgccctcgg 360
gaggctcggt cacactctcc gagagcacgg ccatcatctc ctacggtacc acaggcctgg 420
tcacatggga cgccgccctc taccttgcag aatgggccat cgagaacccg gcagtcttca 480
ctaacaggac tgtcctagag cttggcagtg gtgctggcct cacaggcctg gccatctgca 540
agatgtgccg cccccgggca tacatcttca gcgactgtca cagccgggtc cttgagcagc 600
tccgagggaa tgtccttctc aatggcctct cattagaggc agacatcact gccaagttag 660
acagccccag ggtgacagtg gcccagctgg actgggacgt cgcgacggtc catcagctct 720
ctgccttcca gccagatgtt gtcattgcag cagatgtgct gtattgccca gaagccatca 780
tgtcgctggt cggcgtcctg cggaggctgg ctgcctgccg ggaggaccag cgggctcctg 840
aggtctacgt ggcctttacc gtccgcaacc cagagacgtg ccagctgttc accaccgagc 900
taggccgggc cgggatcaga tgggaagtgg aacctcgtca tgagcagaaa ctgtttccct 960
acgaagagca cttggagatg gcaatgctga atctcaccct gtaggactca cacacgactc 1020
caacgggatt gtgagaatca agtcactctc atgggaagaa tttttatatg ggaaagcgga 1080
taaaactttc attggactgg aatgtttgga gaatgttaat ttccaaatca ggaaccacaa 1140
actgccctct aataagacat cggctatcta agcgtgtggg tgcccccttt ctgccagcag 1200
ttgtggttct taagaaaatc accgataaat cagacatgaa aattctggct ccaaaaacag 1260
cattgtctgt gtgcgagtgg gagcgtgt 1288
<210> 91
<211> 1161
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5425521CB1
60/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<400> 91
gccggtggag cgcgagtagg aagtggtgag ttcggagtag agatggccgc gcttgcaccg 60
ctgcccccgc tccccgcaca gttcaagagc atacagcatc atctgaggac ggctcaggag 120
catgacaagc gagaccctgt ggtggcttat tactgtcgtt tatacgcaat gcagactgga 180
atgaagatcg atagtaaaac tcctgaatgt cgcaaatttt tatcaaagtt aatggatcag 240
ttagaagctc taaagaagca gttgggtgat aatgaagcta ttactcaaga aatagtgggc 300
tgtgcccatt tggagaatta tgctttgaaa atgtttttgt atgcagacaa tgaagatcgt 360
gctggacgat ttcacaaaaa catgatcaag tccttctata ctgcaagtct tttgatagat 420
gtcataacag tatttggaga actcactgat gaaaatgtga aacacaggaa gtatgccaga 480
tggaaggcaa catacatcca taattgttta aagaatgggg agactcctca agcaggccct 540
gttggaattg aagaagataa tgatattgaa gaaaatgaag atgctggagc agcctctctg 600
cccactcagc caactcagcc atcatcatct tcaacttatg acccaagcaa catgccatca 660
ggcaactata ctggaataca gattcctccg ggtgcacacg ctccagctaa tacaccagca 720
gaagtgcctc acagcacagg tgtagcaagt aatactatcc aacctactcc acagactata 780
cctgccattg atcccgcact tttcaataca atttcccagg gggatgttcg tctaacccca 840
gaagactttg ctagagctca gaagtactgc aaatatgctg gcagtgcttt gcagtatgaa 900
gatgtaagca ctgctgtcca gaatctacaa aaggctctca agttactgac gacaggcaga 960
gaatgaagcc tttgtatgac agacccatgt atttttggca tgaggaacta acagtccatt 1020
actctatctt cagcctatca ggatcacagt tttaaggaag acttggtttt gttgaatatg 1080
acaatgaaat ctgtgtgtat cagattttta ttgaagcatt catcagcagc ctcagccagt 1140
tttcattgtc catttactag a 1161
<210> 92
<211> 614
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5495427CB1
<400> 92
gccgcgacca tctcaagtgt ttcgccacac tcatcatggc gtgggcagta agtcacttcc 6i)
gcccgggacc ggaagtgtgg gatactgcga gtatggcggc gtcaaaggtg aagcaggaca 120
tgcctccgcc ggggggctat gggcccatcg actacaaacg gaacttgccg cgtcgaggac 180
tgtcgggcta cagcatgctg gccataggga ttggaaccct gatctacggg cactggagca 240
taatgaagtg gaaccgtgag cgcaggcgcc tacaaatcga ggacttcgag gctcgcatcg 300
cgctgttgcc actgttacag gcagaaaccg accggaggac cttgcagatg cttcgggaga 360
acctggagga ggag.gccatc atcatgaagg acgtgcccga ctggaaggtg ggggagtctg 420
tgttccacac aacccgctgg gtgcccccct tgatcgggga gctgtacggg ctgcgcacca 480
cagaggaggc tctccatgcc agccacggct tcatgtggta cacgtaggcc ctgtgccctc 540
cggccacctg gatccctgcc cctccccact gggacggaat aaatgctctg cagacctgga 600
aaaaaaaaaa aaaa 614
<210> 93
<211> 1462
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 003908CB1
<400> 93
gttcggcgca gctaacagac ggcggcagtg cgagaaagcc gaagatggcg gtccccgcgg 60
cgctgatcct acgggagagc cccagcatga agaaagcagt gtcactgata aatgcaatag 120
atacaggaag atttccacgg ttgctcactc ggattcttca aaaacttcac ctgaaggctg 180
agagcagttt cagtgaagaa gaggaagaaa aacttcaagc ggcattttct ctagagaaac 240
aagatcttca cctagttctt gaaacaatat catttatttt agaacaggca gtgtatcaca 300
atgtgaagcc agcagctttg cagcagcaat tagagaacat tcatcttaga caagacaaag 360
ctgaagcatt tgtcaatact tggtcttcta tgggtcaaga aacagttgaa aagttccggc 420
agagaattct ggctccctgt aagctagaga ctgttggatg gcagcttaac cttcagatgg 480
ctcactctgc tcaagcaaaa ctaaaatctc ctcaagctgt gttacaactc ggagtgaaca 540
atgaagattc aaagagcctg gagaaagttc ttgtggaatt cagtcacaag gagttgtttg 600
atttctataa caagctagag actatacaag cacagctgga ttcccttaca tgatgttttc 660
gaagactgtt tttttcatca cgctcctgcc acctcattat tttgcattga agatacattg 720
ccaggttgtg ttttctgaag gattcagtga cttgctttct gtaaattata tggcttatca 780
61/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
cttcttagac aaataacaac caatagagat cattgttaag aatactgagg ttctaatata 840
ctttctttag ttctgtgagc caacagtaat tattaagaac actttccctt taaaggaaac 900
acaagtgaat accatattgt ttttactgtc atagtgttgc tttcttgcct gtcctgctta 960
gtttttactt gctggatgat accataatgt atcaaggagc gtccatggat acaagataag 1020
atgtgtacct tagtagaata cagagctttg gtaattacat gaataaaatt aagaaaatag 1080
ccatatacaa tcaaatacac tatggcattt ttatttgaat atgatgagta tattttgctt 1140
cggaaataat ataggaagga aatgtaaaat agtgagtagt atggtatcag ttaattccag 1200
tctgagcttc tctgtcaact tcagtttctc tctcagttta atgatttaat aatagtccag 1260
gtttttgtgt gtttttcttt atactgcaaa ttaataatga ttcactttat agtttgggag 1320
acagaatcag gtcttgaata aaataattgt aatgagtgct aaatgggcac cattattcga 1380
atcagatacc ttttatattc tctttccata aatacgttga tttctgtcaa taaaattttt 1440
gtgtcttaaa aaaaaaaaaa as 1462
<210> 94
<211> 1828
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 085915CB1
<400> 94
gcacaaatgg ataataaatc tgaagttcag ttgtggctgt taaagagaat tcaggtaccc 60
attgaagata tacttccttc aaaagaagaa aaaagcaaga ccccacccat gttcctgtgc 120
atcaaagtgg gaaaaccaat gagaaaatcc tttgccactc acactgcagc catggtccag 180
cagtacggca aacggagaaa gcagccagag tactggtttg ctgttcctcg ggagagggtg 240
gatcatttgt acacattctt tgttcagtgg tctcccgatg tctatggaaa agatgccaaa 300
gagcaaggct ttgtggtggt ggagaaggaa gaactgactc atgattgaca acttcttcag 360
tgagccaaca accaagagct gggagatcat cactgttgaa gaggcaaagc gcaggaagag 420
cacatgcagc tactatgaag acgaggacga agaggtgctg cctgtcctac ggccccacag 480
cgcgctcctg gagaatatgc acatcgagca gctggcccga cgccttcctg caagggtgca 540
agggtatcca tggagactgg cctatagcac gttagagcac gggaccagct taaagacgct 600
ctaccggaaa tcggcatcac tagacagtcc tgtcctattg gtcatcaaag atatggataa 660
tcagattttt ggagcatatg caactcatcc tttcaagttc agtgaccact attatggcac 720
aggcgaaact tttctctaca cattcagccc tcattttaag gtctttaagt ggagtggaga 780
aaattcatac tttatcaatg gagacataag ttctttagaa cttggtggtg gagggggacg 840
atttggttta tggctagatg ctgatttata ccacggacga agcaactctt gcagcacttt 900
caataatgat attctttcca aaaaggaaga cttcatagtt caggatctgg aggtgtgggc 960
atttgattga aattcagact gccttaaaat ataacattaa aaagactggg ttcgatcagc 1020
cctcctaaag ctggctggaa aaagaagccc cagcccagcc tgcctcatcc caccccaatg 1080
cttcctttct gccatcatct cagagcatga tcacattgca gaaagattct ggaaggtcca 1140
tgtagagggc agacattgaa gaaagaaact taaaatccag gttgttgaaa agactttgta 1200
ctcccacttc ctccaaatcc atacagtgag gaatcagagt gtttatagat atatgagttg 1260
aatgcaattt ttatttttgg taactgtgaa aaataagata ctgtggatat atacatgcct 1320
tgtgtattta tcagcataat tttcattacc aaaatgtaca gctattattt gccatggaaa 1380
aggttagtct catttagaaa aatcgaaagt gcacagcact taaagggaat atatgaggtt 1440
ttttttttaa aaaaaaaaaa cttttattta ttatttgtag tatattgtct gaaatgtgtc 1500
ggcagttttt tttcttttaa tgtgtcaaat cttgaaatat taaatgtata cattttgtgc 1560
tatgttttgg gaacaaatct gtttgattta tatagtttta tattgaattt tttttgccct 1620
gattgtttag ggtgataggt cttaagcagc atatatctat atatctgtat gtgggtatat 1680
agagatatat gtgtgtgtgt atgtatatgt acatatacat atatatgagt gtataattct 1740
aaatttctaa aaactcatta tgaatgttca tcaatttgac tattataggc cagctttcca 1800
tttagtcaat aaaagcgtac atttttag 1828
<210> 95
<211> 1365
<212> DNA _
<213> Homo Sapiens
<220>
<221> unsure
<222> 87, 124
<223> a or g or c or t, unknown, or other
<220>
<221> misc feature
62/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<223> Incyte ID No: 478615CB1
<400> 95
aggactgctt tatggacttg catcaggagt agcaacagga ctctggactg tcttcacact 60
aggtggtgtg ggcagcaaag cggctgnctc tccagagctt tcttcccctc tggccaacca 120
gagnctcctg cttctgctgg tgttggccaa tctgacagat gcctcaaatg cgccaaaccc 180
ctacagacaa gccattatgt ccttcaagaa cacacaaggt tttaccaatt cttgagattc 240
tgtatcatgt tgaagaaagg aattcacacc atgtgtatat ggcccttata atattgttga 300
tccttacgga agatgatggc ttcaacagat ccattcatga agtgatacta aaaaatatta 360
cttggtattc agaacgagtt ttaactgaaa tctccttggg gagtctcctg atcctggtgg 420
taataagaac cattcaatac aacatgacta ggacacgaga caagtacctt cacacaaatt 480
gtttggcagc tttagcaaat atgtcggcac agtttcgttc tctccatcag tatgctgccc 540
agaggatcat cagtttattt tctttgctgt ctaaaaaaca caacaaagtt ctggaacaag 600
ccacacagtc cttgagaggt tcgctgagtt ctaatgatgt tcctctacca gattatgcac 660
aagacctaaa tgtcattgaa gaagtgattc gaatgatgtt agagatcatc aactcctgcc 720
tgacaaattc ccttcaccac aacccaaact tggtatacgc cctgctttac aaacgcgatc 780
tctttgaaca atttcgaact catccttcat ttcaggatat aatgcaaaat attgatctgg 840
tgatctcctt ctttagctca aggttgctgc aagctggagc tgagctgtca gtggaacggg 900
tcctggaaat cattaagcaa ggcgtcgttg cgctgcccaa agacagactg aagaaatttc 960
cagaattgaa attcaaatat gtggaagagg agcagcccga ggagtttttt atcccctatg 1020
tctggtctct tgtctacaac tcagcagtcg gcctgtactg gaatccacag gacatccagc 1080
tgttcaccat ggattccgac tgagggcagg atgctctccc acccggaccc ctccagccaa 1140
gcagcccttc aagttctttt atttctgggt aacagaagta gacagacagg ttacttggtg 1200
tatcttctgt taaagaggat tgcacgagtg tgttttcctc acacactttg atttggagaa 1260
ttggtgctag ttggcaatag ataactcagc gtagatagta ttgcaaaaag gggaggaaat 1320
acacaacaat aataaatgta aaaacctgct attcaaaaaa aaaaa 1365
<210> 96
<211> 1631
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 924880CB1
<400> 96
ggatcaccat tgcggaccaa ggtgaacaac agtcagaaga aaacgcaagt actaagaact 60
tgacaggcct ttccagtggg actgagaaga aacctcatta catggaagtg ctagaaatgc 120
gagccaaaaa cccagtgccc cagctgcgta aatttaaaac caatgtgtta ccttttcgac 180
aaaatgattc atctagtcat tgccagaaga gtgggtctcc tatttcctca gaagagcggc 240
ggcgcaggga taagcagcat cttgatgaca tcacagcagc tcggcttcta ccacttcacc 300
atatgcccac gcagctgctc tccatagaag aatccttggc acttcagaaa cagcagaaac 360
agaattatga ggagatgcaa gcaaagctcg cagcgcaaaa attagctgaa agactgaata 420
ttaaaatgcg gagttataat ccagaagggg agtcttcagg gagataccga gaagtaaggg 480
atgaagatga cgattggtcc tctgatgaat tctgaagata atctcctaaa tcactgacgt 540
tgagatgtca tcatcttaca tcagactttc taactagtat caagatcagt gtcagatatt 600
gttgagggaa gtaattttat aaagttacac aaaggtagtt ataaaaaaag cccagtttgt 660
ctttcagaag gtgactttca tgtgcttgaa aagtttaata tttgaatatt gtgtttaacc 720
acatggtatt aaaattttgc aatatattgt gtattggtct gatattttag tatatagtag 780
aacatacttt tttttcttta agccaaatga aaagaggtaa ctttgctttt ttcctttttc 840
ttacctatca aatagcattt attacatgtc tttcagtgaa atacttagtt gttccaggca 900
cctaaaatca attaggaaga catagttccc ttcttttttg ggtaatgaag ggagcagtct 960
aaagaattgt atgcatgttt gcatggggtt attgagacat tggatgtgaa atacctagga 1020
agacatgtat ggagaatgct taacaaatgc tcttctcctt tttctgtctt cccttaggaa 1080
gaacgttatc tctcttctga ttaggaagga cttccctttt gagtagatgc tggaaagtgg 1140
aggttttttt tgttggtttg tttgtttggt tttgtgtttt tttttgagat ggggtcttgc 1200
tctgtcgccc aggctggagt gcagtggcag gatctcggct cactgcaatc taggtggagg 1260
tctttttaaa taacctcatg ttacttcagg caaatctggt acaggagaag cccaagctaa 1320
ctgagctggg tgaaggcttg tgcatcacct ggcttgagtt ccttgctgtc acagatgata 1380
ggttcatgta cacaacatta atgtttgata aggaaagcat tttttttttg ttaaaattga 1440
attgtcagta ctttactttt ttccccccaa ataggtatat atagactgca gaagcttcag 1500
tcccatatat aaacgttcat gtcattttag aggattatat ggttgtctcc agagaaatta 1560
acttacatta tcaaaaaatt ctttgtctaa tatattggag tctttaaaag tgttgaagaa 1620
taaaatatta t 1631
<210> 97
63/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<211> 2533
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 955431CB1
<400> 97
gggggagaac gggcggaggc ggtgcgctcg gcgcttcctg ttccggcgcc aggaggagcc 60
gcgcgctgct ggtgctgttg ccgccgctgc tctagctgcc gtcagtcagg ctgcgcccgc 120
gtcttcaggg cccagtccct cggacccatc gccgcttcta gaccctactg cggtctcgga 180
tattgccggg aaaatgtctg atgaattttc gttggcagat gcactacctg aacactcccc 240
tgccaaaacc tctgctgtga gcaatacaaa acctggccaa cctcctcaag gctggccagg 300
ctccaaccct tggaataatc cgagtgctcc atcttcagtg ccatctggac tcccaccaag 360
tgcaacaccc tccactgtgc cttttggacc agcaccaaca ggaatgtatc cctccgtgcc 420
tcccaccgga ccacctccag gacccccagc accctttcct ccttccggac catcatgtcc 480
cccacctggt ggtccttatc cagccccaac tgtgccgggc cctggcccca cagggccata 540
tcctacacca aatatgccct ttccagagct acccagacca tatggtgcac ccacagatcc 600
agctgcagct ggtcctttag gtccatgggg atccatgtct tctggacctt gggcgccagg 660
aatgggaggg cagtatccta cccctaatat gccatatcca tctccaggcc catatcccgc 720
tcctcctcct ccccaagccc ctggggcagc accacctgtt ccatggggca ccgttccacc 780
aggagcctgg ggaccaccag caccatatcc tgcccctaca ggatcgtatc ccacaccagg 840
actctatcct actcccagta atcctttcca agtgccttca ggaccttctg gtgctccacc 900
aatgcctggt ggcccccatt cttaccatta agttaacaat ggacgaagag atgacgcttt 960
gctttttgaa gtacatgtat atgcacatga atgcatatat aaaaattgct ggtttcacta 1020
ttagagggca ttcatgaaag aacaactctt gcacctctca gagaagataa ctgcctcttg 1080
tacttggatg cgtagtacat catatgtata caatcagata aaagcataga agtaaatcat 1140
tcggatgtga tttttatttg gttttcatgg aaagttaaag tgataaagta tattgaatag 1200
ttctttgaca gaatttgttt aaactatgaa actacacact taaaaatcta agatgtggat 1260
tattgttaga atctgcaact tcattggcaa attatttcaa gtatttttct ataatcactt 1320
tccccttcta aataaataaa cttcgagaat aacccatcat aatccaaaca aatgatgcct 1380
caacattttg agctgctctg tcggacaaat aaacctggtc ctcttgaggt tatattttgg 1440
atatacattt ttaaactgtc agtaattatt gtcagatgtg gagttcaata gccagccagt 1500
gttcattttt atccttgagc ttttagtaaa aacttcctgg ttttattttt agtcattggg 1560
tcatacagca ctaaagtctg ctatttatgg aaactaactt ttttgttttt aatccaggcc 1620
aacatgtatg taaattaaat ttttagataa ttgattatct ctttgtacta cttgagattt 1680
gattatgaga tgtgcatatt gctttgggaa gagctcgagg aaggaaataa ttctctcctt 1740
tgttttgaac ctcaaactag ataaacccta ggaattgctt aactgcaaca agtaattttc 1800
attcccacaa aaacctgagg cagctctttt gcccagagcg ttccctgtag ccacccccac 1860
cccacttgcc cttggttctt tagaaggagc acacacatcc cttgattcct ccctgatgtg 1920
gtaaactggc acactccagg ggtctaaaac ataaaacagt tgtgtttagg gaaccttaag 1980
tcatgcagac atgactgttc tctttgtaca agtgtgaatc aaaatatgta tctctttttc 2040
agagtctggt taagctatgt cattgtctac tgcatagttt cctgagtctg tttgtaaagt 2100
gcttatggct aacagttcag ttctgtattt gttgacaggt aaataagtgg agttgagtgc 2160
catctttgaa aaaattaccc tctagctcta acactgaaaa taataataaa ttgtagatct 2220
ctgcaactaa gtttaaagca gtgtgactgt gttgcttaaa tatcaagtat tgtttataac 2280
caccaaaaaa aaaaaagccc tggtagtttt ttggcacctt atgtttaaat cagattctta 2340
gatttggagt agacctgacc ttgttattta ttagataaca ttttgaatgt atccattgga 2400
tttctaaaat gtattgtgaa tttctcagac aaacaggatt tatgctggag ctctgttttg 2460
cttagaaata aaatatttag tagtttattt ctgctctaat taaaatgtca agaatgccaa 2520
aaaaaaaaaa aaa 2533
<210> 98
<211> 1266
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1275918CB1
<400> 98
tgcgacctga ggagtcacag ttgtgagcaa gttcggtgcc gcgggctggg attagaatta 60
cttgttattg gtaaatagcc actatggaga ctaaggacca gaagaaacaa agaaagaaaa 120
atagtggacc caaagctgca aagaaaaaga agcggcatct gcaggatctc cagctaggag 180
acgaagaaga tgcctggaag agaaatccca aagcttttgc atttcagtct gctgtgtgga 240
54/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
tggctcgatc ctttcacagg actcaggatt tgaagacaaa aaagcatcat attccagtgg 300
ttgatcgaac tccactagag cccccaccaa tagtggtagt ggtgatgggg cctccaaaag 360
ttggaaagag cactttgata caatgcctca ttcggaactt cacccggcag aagttgacca 420
agatcagagg ccctgtgatg atcgtgtcag gtaaaaagct ccgactcacc attattgaat 480
gtgggtgtga cattaacatg atgattgatc tggctgaagt agcagatctg gtaagtgagc 540
aggggcagcc tggggtgctg atggagactt acagcattgt gataggttat ttaccccgtg 600
atgaagggaa tagagtttta tgattattaa aggaatcatg gtcatcatca aggatacaat 660
agatgtgatt gaattgatga taataataat agtaataaat gttgttatat taataataca 720
aatggaatgt gtacaataaa atgtattcca aaatctaaaa gcaaatcaca ggatagagaa 780
aacctttaat gaacacagct aataagaact cattaacatg catacatata tacctacaaa 840
tatatcatag taagtaccat ttcttcttgg acccttcctg gcactgtgct aactgctttc 900
tacaaattta gcttacataa acccttgata cacccttgag gtgagtaggt attatctgta 960
gtttacataa gatgaaatag agcctcccag cagttaagta acttgtgtga agatgggacc 1020
cttgttccta atggttctag aacctttatc cttagtgata ataagtaagt acatgaattg 1080
cctgaagaag tggtcagagt ttatcataga aaatctagat agtactcagt gtgtggaaaa 1140
atgtacacgc tcttgcattt atggaaatga taatgttcac tatttctgat attctattaa 1200
ctatcttttt tttaaactat cttttaaatt aactaaaata aaatacaaat taccaaaaaa 1260
aaaaaa 1266
<210> 99
<211> 1423
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1290896CB1
<400> 99
ccggaagtgc ccgcgcaccg gtttccggtg caggtgggga aaatggcggt gtctacagtg 60
ttctcgactt cgtcgctggg aattggtttg tttgttgctg ttggctcagt gtctccatcg 120
gggaaggagg atctggggtt tcctactcca ccatcttcac ctcttgctga ggtcaccacc 180
ttgacatcat atcttgataa gttgtcaact tctctggatt gggagccttt gggggattag 240
ggatcatgct gtctctgtgt gctcaggatt tgacacagat gcttgctctg tcaaggcaca 300
gcctattgtc tcctttgctc agtgtgacat cattcagacg cttctacaga ggtgacagcc 360
caacagattc ccaaaaggac atgattgaaa tccctttgcc tccatggcag gagagaactg 420
atgaatccat agaaaccaaa agagcccgcc tgctctatga gagcagaaag aggggaatgt 480
tggaaaactg cattcttctt agtctttttg ctaaagaaca tctgcagcac atgacagaaa 540
agcagctgaa cctctatgac cgcctgatta acgagcctag taatgactgg gatatttact 600
actgggccac agaagctaaa ccagccccag aaatatttga aaatgaagtc atggccctgc 660
tgagagactt tgctaaaaac aaaaacaaag agcagagact gcgtgcccca gatcttgagt 720
acctctttga aaagccacgt tgagctgtgc tccacggcct ggcatggggg ttcagtctgt 780
ggatggtaac tacttatgat ggacgttagc cttgcttccg gcttcttaga tgcccagctg 840
ccctacccca gaccactggt cctgcctcaa gtgatggaca taaccctctc ctaggacata 900
catgtaaatg cacaatgtga ctcattctca tacttttttg ttcagctctg agcctcaaaa 960
gtgatttgtc agcagcgctg gcatgcaggc tttgcctggc tgctatctct agaggcaagt 1020
ctgccacgag agggcactgc aggcgaagca gttgtgcttg ctcattgcct cagcccaagt 1080
gtactcaaag aaaagaggca gccagctgtg cgtgctgtac ggaaagcctc ccgccctccc 1140
tgcagctccc cgccctcagt ggcccagggc tttgttgaag tggaactccc cacttccaac 1200
ctggtatggc tccttctgcg aagggaagct gatcccagcc tcctgagctg aatccttcaa 1260
aggcacagca ggcagaatga tggccacagg caggagtggt gtgggcacac tgcttaggag 1320
tcaacatctt catcattggg ctttctttaa aacgtgcact ttgtaatttg aaagagaatt 1380
attaaagcat actgaaaaaa ggaaatttaa aaaaaaaaaa aaa 1423
<210> 100
<211> 911
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1342736CB1
<400> 100
ctgtccgagt gtgggcgcct ccgggcctct gcggaccctg ggcagctctg agctcgcgga 60
ggcgtggccg gtgcgcgggg cccgcggcgc gcggggatgg gggtctcggt ggatgtgcac 120
caggtgtaca agtacccctt cgagcaggtg gtcgccagct ttctccgaaa gtaccccaac 180
65/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
cccatggata aaaatgtcat ctcagtaaaa atcatggagg aaaaaagaga tgaatcaaca 240
ggggtcatct acagaaagag gattgcaatc tgtcagaacg tggttccaga aattttaagg 300
aaggtgagca ttttgaaagt acctaatatc caattagaag aggagtcatg gctcaatcct 360
cgggaaagaa acatggccat acggagtcac tgccttacgt ggacacagta tgcatccatg 420
aaggaagagt ctgtcttccg ggaaagtatg gaaaacccaa attggacaga gttcattcaa 480
agaggcagga tttcaatcac aggggttgga tttctcaact gtgttttaga aacttttgcc 540
agcacattct tacgacaggg agcccagaag ggaattagaa tcatggagat gctgctaaag 600
gaacagtgtg gtgccecctt agctgaataa agaatcatca gagagctgta tccatgtcta 660
cttttttatt tatttatttt ttttttgaga atcacttctt ggccacagca catcacagac 720
acagtttctg gaatacaggt ttgaggatac agtgttggca gcttcaagaa gagaagacct 780
tcttgccagg acataaaatg ataccctcct ctgggagcct gcttcgaata gtgggactca 840
gggagataag accttcttgc tggattttta tgacacaatc tctttataat ttaacaaata 900
aaggaaaggg a 911
<210> 101
<211> 1231
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1394209CB1
<400> 101
aatcacaaag aggaaggaag tggcaacaca gctcccgatg atgagaaacc agacacttcc 60
cttattacac aaggtgttcc tactcctggg cccagtgcta atgtagccaa tgatgccatg 120
tcgatattgg aaaccataac ttctcttaac caagaagcca gtgctgctag ggcttcaaca 180
gaaacatcaa atgccaagac cagtgagaga gcgtcaaaaa aacttccatc tcagccaacc 240
actgatacta gtactgacaa agaaagaact tcagaggaca tggctgataa agaaaaatct 300
acagctgact ctggaggtga aggactggaa acagccccaa agtctgaaga gttcagcgac 360
ctcccctgtc cagtcgaaga aattaaaaat tacacaaaag agcataataa tttaattctg 420
ctaaataagg atgttcaaca ggaaagcagt gagcaaaaaa ataaatcaac agacaaaggt 480
gaaaagaagc cagacagcaa tgagaaagga gaaagaaaga aagaaaagaa ggaaaagact 540
gaaaagaaat ttgatcactc aaaaaagagt gaagatacac agaaagttaa agatgaaaaa 600
caagcaaagg aaaaagaagt agagagttta aaacttcctt cagaaaagaa cagtaataaa 660
gctaaaactg ttgaagggac aaaagaagag gaaaataaaa cacagaaaaa aaaatcatct 720
tattataaga acatactcag agctcaatta ctaaattaca cttaaaattt tctgctttaa 780
atacgtgtca aactatcaaa caaggcagag tggtttccag aaaatacaaa ggcaaagtag 840
acttctgtaa aaataactaa actattaatt tataatgtat gatataataa taaaatgaat 900
tttaagtaac tgaagtattt actacctaat aaaaattagg atatccaact atgaactaag 960
ttgcttaaaa taatgtatgt gaataaatta taagacaaat tataaattaa taacttctgt 1020
caatttactc tgtgaacaga tatatgagta cacaaaaata caaatattta ttgctttttt 1080
tcttaagcaa aggggatata agtctccaaa agtattaaat aaacttatta aaatatgaag 1140
gcaaatttga tacttccctg atcccaaaac actctgttct ttaaactttg taggatgttg 1200
ccagttttta tataccctcc tttggcccct c 1231
<210> 102
<211> 1600
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1394647CB1
<400> 102
cccacgcgtc cggattttgt tgtgttcact gctgcatcct aagcacataa aactgcttgg 60
cacataataa gtgctcaaga agcatgaacc gagggagtaa cgggtcagag gcagcccgag 120
gactgagctc tctgagcctg cagagggcgc tccagccccg cccccaaagc taggccccgc 180
ctctcgcagc cggctggcgc actagtgtta gagcctgcgc accaccgttc ttttaactgc 240
gcaggcgcgc cggaagcacc tagagagcgg cgcgtgcgca gcgggagtcg aagcggagat 300
cccggggtcg cgcgagagcc gcaagcggag ttggtgggcg ctatgctatc acccgaggca 360
gagcgagtgc tgcggtacct tgtagaagtg gaggagctcg ccgaggaggt gctggcggac 420
aagcggcaga ttgtggacct ggacactaaa aggaatcaga atcgagaggg cctgagggcc 480
ctgcagaagg atctcagcct ctctgaagat gtgatggttt gcttcgggaa catgtttatc 540
aagatgcctc accctgagac aaaggaaatg attgaaaaag atcaagatca tctggataaa 600
gaaatagaaa aactgcggaa gcaacttaaa gtgaaggtca accgcctttt tgaggcccaa 660
66/72
CA 02373191 2001-11-05
WO 00/70047 PCT/LJS00/13299
ggcaaaccgg agctgaaggg ttttaacttg aaccccctca accaggatga gcttaaagct 720
ctcaaggtca tcttgaaagg atgagactca agaaccaaga tgggggacca gcaacccccc 780
agggtcatgg aggacccagg accctccaac cttgacacct gtaaggacag gatctgccct 840
gtaaggggcc agccgtcagg aatctggcca tgaaaacctc tttgtagtgc ttggctactc 900
tgtgatggca ggagggaacc ttcagcctgt ctggctgctg gacctggaca ccagggctcg 960
gtggacacaa gatctattga cgggccttgg tagccaccag tgggtgtgtg gggcagtggc 1020
tgtgggggtg taagaatgac tgcaacaggc acttcccaac aatggcctgc tgttcacatg 1080
gaccctgagc aaggaaggag ggagggaggg gcagagtgga gtgtcattcc agcattcctc 1140
tcagaaggga gagaggtttt caggctggtg ccatgcgatt ggaataaagc aggaggctca 1200
tgggtggttg ctgaatgaag aacagaatct tggtgctttg tggctcacca cagccatctg 1260
tggggcaggc acacacacct cccgccagct ccaattttgc actttttccc tgcttgattc 1320
caagagtagg tgctgcctag cagcccttcg tggccactct ttactcagga gggccttgca 1380
gagtcctgca ccaggcctgg gtgagtggat gcgcctctta ccatatgaca cgtgtcaaga 1440
tgcccttccg ccccctctga aagtggggcc cggccagcac tgctcgttac tgtctgcctt 1500
cagtggtctg aggtcccagt atgaactgcc gtgaagtcaa aactcttatg tgttcattaa 1560
gggctcaata aatgttagct gaatgaaaaa aaaaaaaaaa 1600
<210> 103
<211> 2750
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1436854CB1
<400> 103
gccgagtcgg agccggagcc tgagccgcgc gctgtgtctc cgctgcgtcc gccgaggccc 60
ccgagtgtca gggacaaaag cctccgcctg ctcccgcagc cggggctcat ctgccgccgc 120
cgccgcgctg aggagagttc gccgccgtcg ccgcccgtga ggatctgaga gccatgtcgg 180
ccagcagcct cttggagcag agaccaaaag gtcaaggaaa caaagtacaa aatggatctg 240
tacatcaaaa ggatggatta aacgatgatg attttgaacc ttacttgagt ccacaggcaa 300
ggcccaataa tgcatatact gccatgtcag attcctactt acccagttac tacagtccct 360
ccattggctt ctcctattct ttgggtgaag ctgcttggtc tacggggggt gacacagcca 420
tgccctactt aacttcttat ggacagctga gcaacggaga gccccacttc ctaccagatg 480
caatgtttgg gcaaccagga gccctaggta gcactccatt tcttggtcag catggtttta 540
atttctttcc cagtgggatt gacttctcag catggggaaa taacagttct cagggacagt 600
ctactcagag ctctggatat agtagcaatt atgcttatgc acctagctcc ttaggtggag 660
ccatgattga tggacagtca gcttttgcca atgagaccct caataaggct cctggcatga 720
atactataga ccaagggatg gcagcactga agttgggtag cacagaagtt gcaagcaatg 780
ttccaaaagt tgtaggttct gctgttggta gcgggtccat tactagtaac atcgtggctt 840
ccaatagttt gcctccagcc accattgctc ctccaaaacc agcatcttgg gctgatattg 900
ctagcaagcc tgcaaaacag caacctaaac tgaagaccaa gaatggcatt gcagggtcaa 960
gtcttccgcc acccccgata aagcataaca tggatattgg aacttgggat aacaagggtc 1020
ccgttgcaaa agccccctca caggctttgg ttcagaatat aggtcagcca acccaggggt 1080
ctcctcagcc tgtaggtcag caggctaaca atagcccacc agtggctcag gcatcagtag 1140
ggcaacagac acagccattg cctccacctc caccacagcc tgcccagctt tcagtccagc 1200
aacaggcagc tcagccaacc cgctgggtag cacctcggaa ccgtggcagt gggttcggtc 1260
ataatggggt ggatggtaat ggagtaggac agtctcaggc tggttctgga tctactcctt 1320
cagaacccca cccagtgttg gagaagcttc ggtccattaa taactataac cccaaagatt 1380
ttgactggaa tctgaaacat ggccgggttt tcatcattaa gagctactct gaggacgata 1440
ttcaccgttc cattaagtat aatatttggt gcagcacaga gcatggtaac aagagactgg 1500
atgctgctta tcgttccatg aacgggaaag gccccgttta cttacttttc agtgtcaacg 1560
gcagtggaca cttctgtggc gtggcagaaa tgaaatctgc tgtggactac aacacatgtg 1620
caggtgtgtg gtcccaggac aaatggaagg gtcgttttga tgtcaggtgg atttttgtga 1680
aggacgttcc caatagccaa ctgcgacaca ttcgcctaga gaacaacgag aataaaccag 1740
tgaccaactc tagggacact caggaagtgc ctctggaaaa ggctaagcag gtgttgaaaa 1800
ttatagccag ctacaagcac accacttcca tttttgatga cttctcacac tatgagaaac 1860
gccaagagga agaagaaagt gttaaaaagg aacgtcaagg tcgtgggaaa taaaaggcag 1920
ttctacacag actgcagcaa cggttgcatc tgcatatcct aagaggaaaa aatgaccttc 1980
aagagaatta ggactttttt cttaatttca ctgacttcag agacgattgc agacttgcag 2040
tttaagtatt ggaatttcac aaaagacata ggacttaact ggaaaatgaa aaaaaaaaga 2100
aaaagaaaaa actaaacaaa aaatccctct aggtagttta ggtgaaaaat gtccctttta 2160
ttttggcttt ggttgtgatt tcagagcata atgctatgtt tttttgtctt tttactatgt 2220
ttttcggatt tttaagtccg taagtgcata cagttttctc taatttttaa accctttcct 2280
cctcccattt tgacatttgc acttggagaa cacttgagtt gtgaaggttt tgggcatcca 2340
ccccagaaag tgggaatttg attttatcct tccgaactgg aagaacattt ttatgaagaa 2490
67/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
tttttgtcta ggagaatata acagtgttac ccaaggttgt gtctttaagg gtggttcatt 2460
ttctctgacc ttttgttact caaagtaaag tactaggagt cctaagaaat gttctgttct 2520
tgtacattat actgattaag tcaggattaa tttgatttca aagctgagaa cagtggtaaa 2580
aactcgttta cagaaatgca ttttggaaga gaaaaatact gtaaaacgtg tcgtgaatgt 2640
ttcttcagtt tcttgttcag ccaatgagga aagggcattg cctttctttt taccattaat 2700
cacttctcaa taaacgtgag atcctgttga gcatcaaaaa aaaaaaaaaa 2750
<210> 104
<211> 2685
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1447955CB1
<400> 104
ggggcggggc ggaagaaggc ggggagggag ggggcggcac cgcggtcggc tccagtcgcc 60
tccggaggaa ggaagaagag gacgccgggc gcgcagctcg gtgctgatta tcacaactgt 120
ttggtgacct acttcactga cctgtgagga ttccttccct tcaagtactg gattcttgat 180
ctttctgcat catcaagaat ggaaacagac tgtaatccca tggagctaag cagtatgtca 240
ggatttgaag aaggttcaga gctgaacggt tttgaaggaa ctgacatgaa agacatgagg 300
ctcgaagctg aagcagttgt aaatgatgtt ctctttgctg ttaacaacat gtttgtctcg 360
aaaagcctgc ggtgtgcgga tgatgtggcc tatatcaatg tggaaacaaa ggaaagaaac 420
agatattgcc tagaactcac tgaagcaggg ctcaaggtgg taggctatgc ttttgaccag 480
gtagatgatc atttacagac tccctaccat gaaacagtct actccttgtt ggatacactc 540
agccccgcct accgagaagc atttggaaac gcactgcttc aaagactgga agctttgaaa 600
agagatggac agtcatgact acactttttc ctttcagagg ggctggtgct ggtacagaat 660
gttgatataa agcttaaaat tcttgcatat ggtcatagaa aatgcatctt tggttttgtg 720
tttttatcac ttgcttccaa cttaggcttt tggctcagaa gattattgaa taatgatttg 780
tcttagtttc tgtttcagta agggaattct gaggccgttg ctatgatacc atcattaaga 840
cattcacatg tcttcatata atatctcttc atttcaaatc ctaatcacta tttcatacta 900
ttacagggct ttgatgctgc cagcactgtc ttttacatag gaaattctag atttgcacag 960
taatagagga attagaagta cctaactata cactttgatt cagcctgcta aatcaggggt 1020
tcaatactag cttggacaaa ctttgtagta attaattgct accagcctta ttggaaacaa 1080
attatcaact agtttcccct gcacaaattt tgaaattcac tgcttcactt aatctattta 1140
tattactaat aatggattaa taaagatgaa ttaattatat attacttaac tagtattaaa 1200
tgaaaaacag ggactgaaat agttctgtat tccgtgtttg caacagccag ccaactaagc 1260
agaggataaa ccgttagcaa atgaatgtaa taattactca tttccaagat atctaagcac 1320
ataagcaaat acaggaacag acttcattct ttttcttaac aaaaaagcat cttcagtgtg 1380
tgatttaaaa gaaagaagat tctggtttcc tagaaaacaa tattttggcc tgtgttgatt 1440
cttattctga atgtgtgttt acataatgta cagtatatat tcagaaagta tttttgcttc 1500
aacgtttact ttctatgatg tagtgctttg gtattcctac agcaccccac cttccccaac 1560
agatgtacag tgttctgtct ccattcgaaa tctacaatgt aatatgagtg cattgtatgg 1620
gtttgaaacc aaaggatgaa tgaagcattc agagacttaa tatttgaaaa aggaatagtc 1680
agtattttat attttattac aggtactgat atttataaat ttaataaact gtaccatgct 1740
gctgcatgtt ttcaagtaca tgttgaacag taaggattgg ggagttgttt tttaatggtc 1800
acctaaagca gctgctatag aaatgttgaa ctaaaatttt gcatctggtc ataccttcat 1860
gcatttatca tttgcagata tttttccatc attattaaaa aacaggaact tttaggctct 1920
gaagatcatg tggaccagag caaattaaag ttcagtttgt gtcacaattc attgccagac 1980
ttcattggaa tgctttgttt gatgatgtat gttcattctc agctttattt tcagatgctt 2040
aactgggcaa tgaagtctaa cttcaggttg aactttctca tgtttaatct caggctaaat 2100
gtaaatgata tttgtaaagt ttgaataaaa ttctgtttac tcattttgag ttagtatgaa 2160
aaaaagtgat tgtatgttta agaattgaaa ttgttcattt tgtgataaat gattaattcc 2220
aagagcttgt ttccattttt taattctgca ttgttacatt tggatttgaa atgactgaag 2280
tgagctttct cttgttacta ctatgaaaga gcaatgagga acaagaggag aggacattaa 2340
taaaactaca cagatataca tgagaatcta gatattaaaa cctccatttt acctggtctg 2400
tttaaagaac ttaaccagca gataggccag gtgtcgtggc tcacacttgc aattccagtg 2460
gtttgggagg cccaggcagg aggatcactt gaggccagga gtgcaagaca gcctgtgcaa 2520
catagatccc atttctacaa aaaattataa aagcatggcc tggtgacaca tgcctgcagt 2580
cctagctatt aggaaagctg aagcatgaag attgcttgag cccaggaatt ggagactgca 2640
gtgagctaca atcgtgctcg tcctccagcc tggatgcaga cagag 2685
<210> 105
<211> 2439
<212> DNA
<213> Homo Sapiens
68/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
<220>
<221> misc_feature
<223> Incyte ID No: 1454689CB1
<400> 105
caaagcggga caggtccagc ggttgttctg tgtgaagtcg cgcggcttcc acccacgcag 60
tgttctaagt gaaggccaga aactcgctcg ccatgtcggc tgcagaggcg gggggtgttt 120
tccacagagc caggggcagg accctggccg cgtttcccgc agaaaaggaa agcgaatgga 180
aaggcccatt ctacttcatc ctgggcgcag acccacagtt tgggctgatc aaggcctggt 240
ccactgggga ctgtgacaat ggcggtgacg aatgggaaca ggagatccgt ctaactgagc 300
aagccgtcca ggccatcaac aagctgaacc ccaaacccaa attcttcgtt ctgtgcggcg 360
acctcatcca cgccatgcca gggaagccgt ggcggacgga gcagacggag gacctgaagc 420
gagtgcttag ggcagtggac agggccatcc cactggtcct tgtcagcggc aaccatgaca 480
ttggcaacac ccccacggcc gagaccgtcg aggagttctg ccggacttgg ggagatgact 540
acttcagctt ctgggtcggg ggcgtcctgt tcctggtcct caactcccag ttctacgaga 600
acccctccaa atgccccagc ctgaagcagg ctcaggacca gtggctggac gagcagctga 660
gcatcgcgag gcagcggcac tgccagcatg ccatcgtcct ccagcacatc ccgctgttcc 720
tggagagcat cgacgaggac gacgactact acttcaacct cagcaagtcc actcggaaga 780
agttggcaga caagttcatc cacgcaggtg tcagagtcgt gttctcaggc cactaccaca 840
ggaatgccgg gggtacctac cagaacctcg acatggtggt gtcatctgcc attggatgcc 900
agctgggcag agacccccac gggctccgag tcgtggtggt caccgccgag aaaattgttc 960
accgatacta cagtctagat gagctgagtg agaaaggaat agaagacgat ctcatggatt 1020
tgatcaagaa aaaatgacgc tccttcccgt tcccgttcac ttttcacttg cactattttt 1080
ttattttgcc agaaacagca gctgcacaca acctcttgct gaaatataaa aatagcccag 1140
gcaggtttgt gaatttatgt ccttttgcat aaatcagaga gctggggccc tgtcctgaat 1200
tatattcaaa atagaactgc atttccttta aaatggagcg aatgatcctg gaaatcagtt 1260
tttaaaatat tagactatct ctgcatggat gtttgaataa attctcctaa tgctgtgttt 1320
ctcaactcca cttttgaatt ggattgtatt ctggttagca tatggtcaaa gtctgcctat 1380
cgacctcaat tccaatctga cagccaagta tgatctttga taattcctta aacaaactac 1440
acctgattgt attaaattgt ggtcacgatt cccaaaacgc taggcaaaac aaagttcatc 1500
tcctttcctt tccagcggaa cgaacagctg tctcccttgt tgtgagagcc atgcagactt 1560
attttgacaa ggagccaagt aggcaccatt tactgttcaa ttctgagact tctgatgata 1620
ggaacagaat gcagttgata cagctcggag cttaaatttg ccttcctggg tgcctagaaa 1680
agtcaccttt gcacataaat tggccatttc tgaaaatctc ctccaagaga ctgaaaccaa 1740
agctctaaaa cgccgcttct tcagtctctg gggagcacgt tgagttggtt cacatccaca 1800
ttctggctgt gtagccactc cacagatggg tggtctcttg ggatcagaat gcccctccac 1860
tcatgagact cttcattttg tccactttga caggaaaagt gggaatgtat gcagagctct 1920
caaaagaaac aaaaaaggcc aaaacggtgc cttcagccac atcctctgaa ttggccctga 1980
cttggactaa atgcactaat gcaaaatccc ttgacaaaag cgcataggtt atttcaaacc 2040
agcattgttt tttatgtaac ctgttttacc gcatcttctc agcagcttct gaccactgct 2100
caattttctc ctttacagcc attgttctgg tggacaaata acctaggtac tccaaatcct 2160
ggcaggaaaa atatacagca ttatgaaaca gcactcagta atcctaaaat gatttccaaa 2220
gctggttaca catgacctgc aaagtctatt aaattaaaag actttctcat tacagagttt 2280
agtcaacgta gcaaaaccat gggaaattaa atggaaagat aattaaatat gtaaattcat 2340
aaggaacaaa agacgagaag ttaattatac aataggctgg tgacaccaga gttcattgtt 2400
atgtgccaca agaaaattaa attttttaaa aaaaaaaaa 2439
<210> 106
<211> 1507
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1568009CB1
<400> 106
caacgcttga gatcctctcc gctcccgcca ccccgcaggt gccccgcgcc gttcccgccg 60
ccccgccgcc cccgtcgcgg gcccctgcac cccgagcatc cgccccgggt ggcacgtccc 120
cgagcccacc aggccggccc cgtctcccca tccgtctagt ccgctcgcgg tgccatgcca 180
ttcctcgggc aggactggcg gtcccccggg cagaactggg tgaagacggc cgacggctgg 240
aagcgcttcc tggatgagaa gagcggcagt ttcgtgagcg acctcagcag ttactgcaac 300
aaggaggtat acaataagga gaatcttttc aacagcctga actatgatgt tgcagccaag 360
aagagaaaga aggacatgct gaatagcaaa accaaaactc agtatttcca ccaagaaaaa 420
tggatctatg ttcacaaagg aagtactaaa gagcgccatg gatattgcac cctgggggaa 480
gctttcaaca gactggactt ctcaactgcc attctggatt ccagaagatt taactacgtg 540
gtccggctgt tggagctgat agcaaagtca cagctcacat ccctgagtgg catcgcccaa 600
69/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
aagaacttca tgaatatttt ggaaaaagtg gtactgaaag tccttgaaga ccagcaaaac 660
attagactaa taagggaact actccagacc ctctacacat ccttatgtac ac.tggtccaa 720
agagtcggca agtctgtgct ggtcgggaac attaacatgt gggtgtatcg gatggagacg 780
attctccact ggcagcagca gctgaacaac attcagatca ccaggcctgc cttcaaaggc 840
ctcaccttca ctgacctgcc tttgtgccta caactgaaca tcatgcagag gctgagcgac 900
gggcgggacc tggtcagcct gggccaggct gcccccgacc tgcacgtgct cagcgaagac 960
cggctgctgt ggaagaaact ctgccagtac cacttctccg agcggcagat ccgcaaacga 1020
ttaattctgt cagacaaagg gcagctggat tggaagaaga tgtatttcaa acttgtccga 1080
tgttacccaa ggaaagagca gtatggagat acccttcagc tctgcaaaca ctgtcacatc 1140
ctttcctgga agggcactga ccatccgtgc actgccaata acccagagag ctgctccgtt 1200
tcactttcac cccaggactt tatcaacttg ttcaagttct gaatcccagc acatgacaac 1260
acttcagaag ggtccccctg ctgactggag agctgggaat atggcatttg gacacttcat 1320
ttgt-~aatag tgtacatttt aaacattggc tcgaaacttc agagataagt catggagagg 1380
acattggagg ggagaaatgc agttgctgac tgggaattta agaatgtgaa cttctcacta 1440
gaattggtat ggaaaagcaa aatactgtaa ataaactttt tttctaacaa ttaaaaaaaa 1500
aaaaaaa 1507
<210> 107
<211> 1132
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1677811CB1
<400> 107
tgctgggctt gggtgtccgt cctttcctgt ctccttttct tcctcttttc acttaaatcc 60
actgagtctc tcgccaagtc agtgatttag gtaatcccgc cgcccctggc ggaacctttg 120
tgacgccgga actgatgtgc cgtatggtgg ctgcggtcgg aggactgttt tggtaaagga 180
ctagcagttc tctgcggagg gccggttgat acagttccgg tgggagaacg cggctgcgag 240
gttttcggct ttggctcctg atatgcagcg acagaatttt cggcccccaa ctcctcctta 300
ccctggtccg ggtggaggag gttggggtag cggaagcagc ttccggggaa ccccgggcgg 360
gggcggacca cggccgccct cccctcgaga cgggtacggg agtccgcacc acacgccgcc 420
gtacgggccc cggtctaggc cgtacgggag cagtcactct ccgcgacacg gcggcagctt 480
cccggggggc cggttcgggt ctccgtcccc tggcggctac cctggctcct actccaggtc 540
ccccgcgggg tcccagcagc aattcggcta ctccccaggg cagcagcaga cccaccccca 600
gggttctcca aggacatcta caccatttgg atcagggcgt gttagagaaa aaagaatgtc 660
taatgagttg gaaaattatt tcaagccttc aatgcttgaa gatccttggg ctggcctaga 720
accagtatct gtagtggata taagccaaca atacagcaat actcaaacat tcacaggcaa 780
aaaaggaaga tacttttgtt aacatttctg aaattcaact ggaagcttca tgtgtcagga 840
acatcttgga caaaacttta agttgtgttg atataaattt acccaaagat gatgactttg 900
attggataat tagtaaggtc tttttgttat ttttcatcgt atcaggtatt gttgatatta 960
gagaaaaaag taggataact tgcaacattt agctctggaa gtacctacca cattttagag 1020
atttaccgtt tccatatatt taacattcct ggttacataa tggacatttg tcttttaatg 1080
ttttttcaat gttttaaaat aaaacatttt gtcttctaaa aaaaaaaaaa as 1132
<210> 108
<211> 1822
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1756951CB1
<400> 108
ggctgtgtag actgttgggt aggctgcgtg ctagcttcgg cgcggatccc tgggcgtccg 60
tacgtcggag tccttcgtcc tccagggtcc ctgttctttg cgccagcggg aaccactatc 120
tctgcactcc tggggttttg ttacatggct gctttcctca aaatgagtgt tagtgtcaat 180
ttcttcagac ctttcaccag gtttttggtg ccatttaccc ttcataggaa gagaaataac 240
ttaacaattt tgcagagata catgtcttcc aaaataccag ctgttactta tcctaaaaat 300
gagagtacac ccccttctga agagctagag ttggataagt ggaaaactac catgaaatct 360
agtgtgcaag aagaatgtgt ttcaacaatc tcaagcagta aggatgaaga tcctctagct 420
gccaccagag agttcattga gatgtggaga ttgcttggca gagaagtacc agaacacatc 480
actgaagaag agctcaaaac ccttatggaa tgtgtttcta acacagcaaa aaaaaaatat 540
ttaaaatatt tatatacgaa ggaaaaagtg aaaaaagcta ggcaaataaa aaaggaaatg 600
70/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
aaagcagcag caagggaaga agcaaaaaat atcaagctgc tagaaaccac tgaggaagat 660
aaacagaaaa actttctatt tttacgactt tgggatagga atatggacat agcaatgggc 720
tggaagggtg cccaggccat gcagtttgga caacctttgg tttttgacat ggcttacgaa 780
aattatatga aacgaaaaga attgcagaat actgtttccc agcttttaga aagtgaagga 840
tggaacagaa gaaatgttga tcctttccat atttatttct gcaatctaaa aatagatggt 900
gctttgcaca gagagttagt taaacggtat caagaaaaat gggacaaatt gcttttaaca 960
tcaacagaaa agtctcatgt agatttattt ccaaaggaca gtattatcta tttaactgca 1020
gattctccca atgttatgac tactttcagg catgacaaag tttatgtaat tgggtctttt 1080
gttgataaga gtatgcagcc aggcacatcc ctagccaagg caaaacggct gaacctggca 1140
actgaatgcc ttccattaga taaatattta caatgggaaa ttggtaacaa aaatctcacc 1200
ttagatcaaa tgatacgtat tttgttatgt ctgaaaaaca atggtaattg gcaagaggct 1260
ctgcaattcg ttcccaagag aaaacatact ggttttctgg agatttctca gcattctcaa 1320
gagtttatca acagactaaa gaaggcaaag acttaattca ttttcaaaag gttctctgaa 1380
tgtgcacaga acacgtggct caaatgagaa catttgatgg cttaaaaagt aaatgcgtta 1440
gaaatacagt tctgttaatg tatttcttcc caaacaattc atttttctct tctaaaggta 1500
gtctttccca actgactgta gggttgtgtc ttttcccaat taaatatctg cagaactttg 1560
ggattatact ttgtttactg tagaaagata ataaaaagag ttgtccaaga ttgttgaaca 1620
gaataatctt tatcccagtt aaatagttgt accattggta gactttttta tggaggttcc 1680
tagagggtgg tgccctgggg tgggcttgga agctctgcac cccttccccc atagctttcc 1740
ccgtgcatct ctttgtctgt atgttttgta atatctttta cagtaaactg gtaaatgtgt 1800
ttccttcaaa aaaaaaaaaa as 1822
<210> 109
<211> 933
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1833547CB1
<400> 109
cacgagcgct tcgccatccg ccgactgccc gccttcacgc tgtcccacct ggagagccac 60
cgtgacggcc agcgcagcag catcatggac gtgcggtccc gggtggattc taagaccctg 120
acccgtaaca cgaggatcat tgcagaggcc ctgactcgag tcatctacaa cctgacagag 180
aaggggacac ccccagacat gccggtgttc acagagcaga tgatccagca ggagcagctg 240
gactcggtga tggactggct caccaaccag ccgcgggccg cgcagctggt ggacaaggac 300
agcaccttcc tcagcacgct ggagcaccac ctgagccgct acctgaagga cgtgaagcag 360
caccacgtca aggctgacaa gcgggaccca gagtttgtct tctacgacca gctgaagcaa 420
gtgatgaatg cgtacagagt caagccggcc gtctttgacc tgctcctggc tgttggcatt 480
gctgcctacc tcggcatggc ctacgtggct gtccagcact tcagcctcct ctacaagacc 540
gtccagaggc tgctcgtgaa ggccaagaca cagtgacaca gccaccccca cagccggagc 600
ccccgccgct ccacagtccc tggggccgag cacgagtgag tggacactgc cccgccgcgg 660
gcggccctgc agggacaggg gccctctccc tccccggcgg tggttggaac actgaattac 720
agagcttttt tctgttgctc tccgagactg gggggggatt gtttcttctt ttccttgtct 780
ttgaacttcc ttggaggaga gcttgggaga cgtcccgggg ccaggctacg gacttgcgga 840
cgagcccccc agtcctggga gccggccgcc ctcggtctgg tgtaagcaca catgcacgat 900
taaagaggag acgccgggaa aaaaaaaaaa aaa 933
<210> 110
<211> 681
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1833723CB1
<400> 110
cgagccgggc cggaagaaga ggtagggcgc cgccgtgaca gattagtcct aaagggaacg 60
gggttgttag ttcaattggc taccggaaaa aaccaggctg ggctgggcgc cgccatgaca 120
accgataccg gaaaaggcgg gtcgttcccc ccggacagcc ctacgccggc aaaggtctcg 180
agatgcaggc ggccctagag gtcaccgctc gctactgtgg ccgggagctg gagcagtatg 240
gccagtgtgt ggcggccaag ccggaatcct ggcagcggga ctgtcactac cttaagatga 300
gcattgccca gtgcacatcc tcccacccaa tcatccgcca gatccgccag gcctgtgctc 360
agccttttga ggccttcgag gagtgtcttc gacagaacga ggcagctgtg ggcaactgtg 420
cagagcatat gcgccgcttc ctgcagtgcg ctgagcaggt gcagccgcca cgctcacctg 480
71/72
CA 02373191 2001-11-05
WO 00/70047 PCT/US00/13299
caactgtgga ggcacagcca cttcctgcct cctgaggact cctctgacgg caggaaaact 540
ggacatgaat gactgccccc ccgcccctcc cctgcagagt ggccagatgg agtcctgagc 600
cctggacatg ggcccggctt tcctggatat caggacttcc aataaataaa gactctgtat 660
actgggaaaa aaaaaaaaaa a 681
72/72
