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HUMAN TRANSCRIPTIONAL REGULATOR PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human
transcriptional
regulator proteins and to the use of these sequences in the diagnosis,
treatment, and prevention of cell
proliferative, autoimmune/inflammatory, and developmental disorders.
BACKGROUND OF THE INVENTION
Multicellular organisms are comprised of diverse cell types that differ
dramatically both in
structure and function. The identity of a cell is determined by its
characteristic pattern of gene
expression, and different cell types express overlapping but distinctive sets
of genes throughout
development. Spatial and temporal regulation of gene expression is critical
for the control of cell
proliferation, cell differentiation, apoptosis, and other processes that
contribute to organismal
development. Furthermore, gene expression is regulated in response to
extracellular signals that
mediate cell-cell communication and coordinate the activities of different
cell types. Appropriate
gene regulation also ensures that cells function efficiently by expressing
only those genes whose
functions are required at a given time.
Transcription Factors
Transcriptional regulatory proteins are essential for the control of gene
expression. Some of
these proteins function as transcription factors that initiate, activate,
repress, or terminate gene
transcription. Transcription factors generally bind to the promoter, enhancer,
and upstream
regulatory regions of a gene in a sequence-specific manner, although some
factors bind regulatory
elements within or downstream of a gene's coding region. Transcription factors
may bind to a
specific region of DNA singly or as a complex with other accessory factors.
(Reviewed in Lewin, B.
( 1990) Genes IV, Oxford University Press, New York, NY, and Cell Press,
Cambridge, MA, pp. 554-
570.)
The double helix structure and repeated sequences of DNA create topological
and chemical
features which can be recognized by transcription factors. These features are
hydrogen bond donor
and acceptor groups, hydrophobic patches, major and minor grooves, and
regular, repeated stretches
of sequence which induce distinct bends in the helix. Typically, transcription
factors recognize
specific DNA sequence motifs of about 20 nucleotides in length. Multiple,
adjacent transcription
factor-binding motifs may be required for gene regulation.
Many transcription factors incorporate DNA-binding structural motifs which
comprise either
a helices or Q sheets that bind to the major groove of DNA. Four well-
characterized structural motifs
are the helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix.
Proteins containing these
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motifs may act alone as monomers, or they may form homo- or heterodimers that
interact with DNA.
The helix-turn-helix motif consists of two a helices connected at a fixed
angle by a short
chain of amino acids. One of the helices binds to the major groove. Helix-turn-
helix motifs are
exemplified by the homeobox motif which is present in homeodomain proteins.
These proteins are
critical for specifying the anterior-posterior body axis during development
and are conserved
throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of
Drosophila
melano ag ster are prototypical homeodomain proteins (Pabo, C.O. and R.T.
Sauer (1992) Ann. Rev.
Biochem. 61:1053-1095).
The zinc finger motif, which binds zinc ions, generally contains tandem
repeats of about 30
amino acids consisting of periodically spaced cysteine and histidine residues.
Examples of this
sequence pattern include the C2H2-type, C4-type, and C3HC4-type ("RING"
finger) zinc fingers, and
the PHD domain (Lewin, supra ; Aasland, R. et al. (1995) Trends Biochem. Sci
20:56 - 59). Zinc
forger proteins each contain an a helix and an antiparallel Q sheet whose
proximity and conformation
are maintained by the zinc ion. Contact with DNA is made by the arginine
preceding the a helix and
by the second, third, and sixth residues of the a helix. The zinc finger motif
may be repeated in a
tandem array within a protein, such that the a helix of each zinc finger in
the protein makes contact
with the major groove of the DNA double helix. This repeated contact between
the protein and the
DNA produces a strong and specific DNA-protein interaction. The strength and
specificity of the
interaction can be regulated by the number of zinc finger motifs within the
protein.
The leucine zipper motif comprises a stretch of amino acids rich in leucine
which can form
an amphipathic a helix. This structure provides the basis for dimerization of
two leucine zipper
proteins. The region adjacent to the leucine zipper is usually basic, and upon
protein dimerization, is
optimally positioned for binding to the major groove. Proteins containing such
motifs are generally
referred to as bZIP transcription factors. The leucine zipper motif is found
in the proto-oncogenes
Fos and Jun, which comprise the heterodimeric transcription factor AP1,
involved in cell growth and
the determination of cell lineage (Papavassiliou, A. G. (1995) N. Engl. J.
Med. 332:45-47).
The helix-loop-helix motif (HLH) consists of a short a helix connected by a
loop to a longer
a helix. The loop is flexible and allows the two helices to fold back against
each other and to bind to
DNA. The oncogene Myc, a transcription factor that activates genes required
for cellular
proliferation, contains a prototypical HLH motif.
Most transcription factors contain characteristic DNA binding motifs, and
variations on the
above motifs and new motifs have been and are currently being characterized
(Faisst, S. and S. Meyer
(1992) Nucl. Acids Res. 20:3-26). These include the forkhead motif, found in
transcription factors
involved in development and oncogenesis (Hacker, U. et al. (1995) EMBO J.
14:5306-5317).
Chromatin Associated Proteins
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In the nucleus, DNA is packaged into chromatin, the compact organization of
which limits
the accessibility of DNA to transcription factors and plays a key role in gene
regulation (Lewin,
supra, pp. 409-410). The compact structure of chromatin is determined and
influenced by chromatin-
associated proteins such as the histones, the high mobility group (HMG)
proteins, helicases, and the
chromodomain proteins. There are five classes of histones, H1, H2A, H2B, H3,
and H4, all of which
are highly basic, low molecular weight proteins. The fundamental unit of
chromatin, the nucleosome,
consists of 200 base pairs of DNA associated with two copies each of H2A, H2B,
H3, and H4. H1
links adjacent nucleosomes. HMG proteins are low molecular weight, non-histone
proteins that may
play a role in unwinding DNA and stabilizing single-stranded DNA. Helicases,
which are DNA-
dependent ATPases, unwind DNA, allowing access for transcription factors.
Chromodomain proteins
play a key role in the formation of highly compacted heterochromatin, which
is. transcriptionally
silent.
Diseases and disorders related to , e~guulation
Many neoplastic disorders in humans can be attributed to inappropriate gene
expression.
Malignant cell growth may result from either excessive expression of tumor
promoting genes or
insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer
Surv. 15:89-104).
The zinc finger-type transcriptional regulator WT1 is a tumor-suppressor
protein that is inactivated in
children with Wilm's tumor. The oncogene bcl-6, which plays an important role
in large-cell
lymphoma, is also a zinc-finger protein (Papavassiliou, supra). Chromosomal
translocations may
also produce chimeric loci which fuse the coding sequence of a transcriptional
regulator with the
regulatory regions of a second unrelated gene. In Burkitt's lymphoma, for
example, the transcription
factor Myc is translocated to the immunoglobulin heavy chain locus, greatly
enhancing Myc
expression and resulting in rapid cell growth leading to leukemia (Latchman,
D. S. (1996) N. Engl. J.
Med. 334:28-33).
In addition, the immune system responds to infection or trauma by activating a
cascade of
events that coordinate the progressive selection, amplification, and
mobilization of cellular defense
mechanisms. A complex and balanced program of gene activation and repression
is involved in this
process. However, hyperactivity of the immune system as a result of improper
or insufficient
regulation of gene expression may result in considerable tissue or organ
damage. This damage is
well documented in immunological responses associated with arthritis,
allergens, heart attack, stroke,
and infections (Isselbacher et al. Harrison's Principles of Internal Medicine,
13/e, McGraw Hill, Inc.
and Teton Data Systems Software, 1996). The causative gene for autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED) was recently isolated and found to
encode a protein
with two PHD-type zinc finger motifs (Bjorses, P. et al. (1998) Hum. Mol.
Genet. 7:1547-1553).
Furthermore, the generation of multicellular organisms is based upon the
induction and
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coordination of cell differentiation at the appropriate stages of development.
Central to this process
is differential gene expression, which confers the distinct identities of
cells and tissues throughout the
body. Failure to regulate gene expression during development can result in
developmental disorders.
Human developmental disorders caused by mutations in zinc forger-type
transcriptional regulators
include: urogenital developmental abnormalities associated with WT1; Greig
cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type
A (GLI3), and
Townes-Brocks syndrome, characterized by anal, renal, limb, and ear
abnormalities (SALL1)
(Engelkamp, D. and V. van Heyningen (1996) Curr. Opin. Genet. Dev. 6:334-342;
Kohlhase, J. et al.
(1999) Am. J. Hum. Genet. 64:435-445).
The discovery of new human transcriptional regulator proteins and the
polynucleotides
encoding them satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of cell proliferative,
autoimmune/inflammatory, and
developmental disorders.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, human transcriptional regulator
proteins,
referred to collectively as "TXREG" and individually as "TXREG-1," "TXREG-2,"
"TXREG-3,"
"TXREG-4," "TXREG-5," "TXREG-6," "TXREG-7," "TXREG-8," "TXREG-9," "TXREG-10,"
"TXREG-11," "TXREG-12," "TXREG-13," "TXREG-14," "TXREG-15," "TXREG-16," "TXREG-
17," "TXREG-18," "TXREG-19," "TXREG-20," "TXREG-21," "TXREG-22," "TXREG-23,"
"TXREG-24," "TXREG-25," "TXREG-26," "TXREG-27," "TXREG-28," "TXREG-29," "TXREG-
30," "TXREG-31," and "TXREG-32." 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 )D NO:1-32, 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:1-32, c) a biologically active fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-32, and d) an immunogenic fragment of an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-32. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ )D NO:1-32.
The invention further provides an isolated polynucleotide encoding a
polypeptide comprising
an amino acid sequence selected from the group consisting of a) an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-32, 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:1-32, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ )D NO:1-32, and d) an immunogenic fragment of an amino acid
sequence selected
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from the group consisting of SEQ >D NO:1-32. In one alternative, the
polynucleotide encodes a
polypeptide selected from the group consisting of SEQ )D NO:1-32. In another
alternative, the
polynucleotide is selected from the group consisting of SEQ )D N0:33-64.
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 >l7 NO:1-32, 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 )D NO:1-32,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ )D NO:1-32, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ >D NO:1-32. 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 >T7 NO:1-32, 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:1-32,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ )D NO:1-32, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ )D NO:1-32. 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 )D NO:1-32, 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 1D NO:1-32, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:I-32, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ 1D NO:1-32.
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 >D N0:33-64, b) a naturally occurnng polynucleotide sequence
having at least
70% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:33-64, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
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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 N0:33-64, b) a naturally occurring polynucleotide
sequence having at least
70% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ >D
N0:33-64, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). The method comprises
a) hybridizing the
sample with a probe comprising at least 20 contiguous nucleotides comprising a
sequence
complementary to said target polynucleotide in the sample, and which probe
specifically hybridizes to
said target polynucleotide, under conditions whereby a hybridization complex
is formed between said
probe and said target polynucleotide or fragments thereof, and b) detecting
the presence or absence of
said hybridization complex, and optionally, if present, the amount thereof. In
one alternative, the
probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a} a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:33-64, b) a naturally occurring polynucleotide
sequence having at least
70% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ 1D
N0:33-64, 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 )D NO:1-32, 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 B7 NO:1-32, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-32, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ m NO:1-32, and a
pharmaceutically
acceptable excipient. In one embodiment, the pharmaceutical composition
comprises an amino acid
sequence selected from the group consisting of SEQ >D NO:1-32. The invention
additionally
provides a method of treating a disease or condition associated with decreased
expression of
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functional TXREG, 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 )D NO:1-32,
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 I1.7 NO:1-32, c) a biologically
active fragment of an amino
acid sequence selected from the group consisting of SEQ )D NO:1-32, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
>D NO:1-32. 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 TXREG, 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 m NO:1-
32, 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 )D NO:1-32, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ 1D NO:1-32, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
)D NO:1-32. 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 TXREG, 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:1-32, 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 )D NO:1-32, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ II7 NO:1-32, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ )D NO:1-32. The method
comprises a)
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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:1-32,
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:1-32, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-32, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-32. The
method comprises a) combining the polypeptide with at least one test compound
under conditions
permissive for the activity of the polypeptide, b) assessing the activity of
the polypeptide in the
presence of the test compound, and c) comparing the activity of the
polypeptide in the presence of the
test compound with the activity of the polypeptide in the absence of the test
compound, wherein a
change in the activity of the polypeptide in the presence of the test compound
is indicative of a
compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
sequence selected from the group consisting of SEQ ID N0:33-64, the method
comprising a).
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding TXREG.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of TXREG.
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 TXREG were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
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polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"TXREG" refers to the amino acid sequences of substantially purified TXREG
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
TXREG. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of TXREG either by
directly interacting with
TXREG or by acting on components of the biological pathway in which TXREG
participates.
An "allelic variant" is an alternative form of the gene encoding TXREG.
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.
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Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding TXREG include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as TXREG or
a polypeptide with at least one functional characteristic of TXREG. Included
within this definition
are polymorphisms which may or may not be readily detectable using a
particular oligonucleotide
probe of the polynucleotide encoding TXREG, and improper or unexpected
hybridization to allelic
variants, with a locus other than the normal chromosomal locus for the
polynucleotide sequence
encoding TXREG. 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 TXREG. 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 TXREG 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 TXREG. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of TXREG either by
directly interacting with TXREG or by acting on components of the biological
pathway in which
TXREG participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic determinant.
Antibodies that bind TXREG polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
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oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurnng
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 TXREG, 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 TXREG or fragments
of TXREG 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 agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(PE Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
Wn 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
IS as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
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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 TXREG or the polynucleotide encoding TXREG
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 N0:33-64 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:33-64, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:33-64 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:33-64 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:33-64 and the region of SEQ ID N0:33-64 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:1-32 is encoded by a fragment of SEQ ID N0:33-64. A
fragment
of SEQ ID NO:1-32 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-32. For example, a fragment of SEQ ID NO:1-32 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-32.
The precise length of
a fragment of SEQ ID NO:1-32 and the region of SEQ ID NO:1-32 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 Wn. CLUSTAL V is
described in
IS Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G.
et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBn Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: I
Penalty for mismatch: -2
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Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
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 )D number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (Apr-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: 71 and Extension Gap: 1 penalties
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WO 00/78954 PCT/US00/16766
Gap x drop-off:' SO
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ )D number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions
and remain hybridized
after the "washing" step(s). The washing steps) is particularly important in
determining the
stringency of the hybridization process, with more stringent conditions
allowing less non-specific
binding, i.e., binding between pairs of nucleic acid strands that are not
perfectly matched. Permissive
conditions for annealing of nucleic acid sequences are routinely determinable
by one of ordinary skill
in the art and may be consistent among hybridization experiments, whereas wash
conditions may be
varied among experiments to achieve the desired stringency, and therefore
hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the
presence of about 6 x SSC, about
1 % (w/v) SDS, and about 100 pg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5.°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.,
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WO 00/78954 PCT/US00/16766
1989, Molecular Clonine: A Laboratory Manual, 2"~ ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
pg/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of TXREG
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 TXREG which is useful in any of the antibody production methods disclosed
herein or known in
the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of TXREG. For example,
modulation
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WO 00/78954 PCT/US00/16766
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of TXREG.
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 TXREG 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 TXREG.
"Probe" refers to nucleic acid sequences encoding TXREG, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
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example Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, 2'~
ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et a1.,1987, Current
Protocols in Molecular
BioloQV, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al., 1990, PCR
Protocols, A Guide to Methods and Anulications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurnng or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
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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 S' 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 TXREG, or fragments thereof, or TXREG itself, may comprise a
bodily fluid; an
extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic DNA,
RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print;
etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 7S% 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.
CA 02375414 2001-11-28
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"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) 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
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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 human transcriptional regulator
proteins
(TXREG), the polynucleotides encoding TXREG, and the use of these compositions
for the
diagnosis, treatment, or prevention of cell proliferative,
autoimmune/inflammatory, and
developmental disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
TXREG. Columns 1 and 2 show the sequence identification numbers (SEQ )D NOs)
of the
polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
)Ds of the Incyte
clones in which nucleic acids encoding each TXREG 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 TXREG and are useful as fragments in hybridization
technologies.
The columns of Table 2 show various properties of each of the polypeptides of
the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid
residues in each
polypeptide; column 3 shows potential phosphorylation sites; column 4 shows
potential glycosylation
sites; column 5 shows the amino acid residues comprising signature sequences
and motifs; column 6
shows homologous sequences as identified by BLAST analysis; and column 7 shows
analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
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WO 00/78954 PCT/US00/16766
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding TXREG. The first column of Table
3 lists the
nucleotide SEQ 117 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
N0:33-64 and to distinguish between SEQ ID N0:33-64 and related polynucleotide
sequences. The
polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides. Column
3 lists tissue categories which express TXREG as a fraction of total tissues
expressing TXREG.
Column 4 lists diseases, disorders, or conditions associated with those
tissues expressing TXREG as a
fraction of total tissues expressing TXREG. 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 TXREG were isolated. Column 1 references the
nucleotide SEQ
)D NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
SEQ ID N0:33 maps to chromosome 1 within the interval from 199.2 to 203.0
centiMorgans,
to chromosome 6 within the interval from 59.6 to 73.9 centiMorgans, and to
chromosome 13 within
the interval from 112.8 to 117.5 centiMorgans. The interval on chromosome 6
from 59.6 to 73.9
centiMorgans also contains genes associated with methylmalonic CoA mutase
deficiency and retinal
degeneration. The interval on chromosome 13 from 112.8 to 117.5 centiMorgans
also contains genes
associated with Oguchi disease (night blindness) and Factor X deficiency. SEQ
ID N0:34 maps to
chromosome 13 within the interval from 112.8 to 117.5 centiMorgans. This
interval also contains
genes associated with Oguchi disease (night blindness) and Factor X
deficiency. SEQ ID N0:35
maps to chromosome 12 within the interval from 113.3 to 126.1 centiMorgans.
This interval also
contains genes associated with spinocerebellar ataxia, mevalonate kinase
deficiency, alcohol
intolerance, and myocardial hypertrophy. SEQ ID N0:36 maps to chromosome 1
within the interval
from 155.2 to 157.4 centiMorgans, and to chromosome 16 within the interval
from 83.7 to 86.6
centiMorgans. The interval on chromosome 1 from 155.2 to 157.4 centiMorgans
also contains genes
associated with leukemia and adrenal hyperplasia. The interval on chromosome
16 from 83.7 to 86.6
centiMorgans also contains a gene associated with cortisol 11-beta-keto
reductase deficiency. SEQ
ID N0:38 maps to chromosome 9 within the interval from 59.9 to 64.5
centiMorgans. SEQ ID
N0:40 maps to chromosome 18 within the interval from 61.2 to 63.2
centiMorgans. SEQ ID N0:44
maps to chromosome 2 within the interval from 180.6 to 188.2 centiMorgans.
This interval also
contains a gene associated with glutamate decarboxylase deficiency. SEQ ID
N0:45 maps to
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WO 00/78954 PCT/US00/16766
chromosome 13 within the interval from 112.8 to 117.5 centiMorgans. This
interval also contains
genes associated with Oguchi disease (night blindness) and Factor X
deficiency. SEQ ID N0:47
maps to chromosome 8 within the interval from 75.0 to 90.2 centiMorgans. This
interval also
contains genes associated with branchiootorenal dysplasia and Zellweger
syndrome. SEQ ID N0:61
maps to chromosome 5 within the interval from 63.9 to 69.6 centiMorgans. SEQ
ID N0:62 maps to
chromosome 7 within the interval from 120.7 to 123.9 centiMorgans. This
interval also contains
genes associated with lipoamide dehydrogenase deficiency, neonatal cutis laxa,
and tumor
suppression. SEQ ID N0:64 maps to chromosome 1 within the interval from 157.4
to 186.4
centiMorgans, to chromosome 5 within the interval from 175.3 to 182.4
centiMorgans, and to
chromosome 14 within the interval from 7.5 to 21.9 centiMorgans. The interval
on chromosome 1
from 157.4'to 186.4 centiMorgans also contains genes associated with
autoimmune diseases,
leukemia, and Gaucher disease. The interval on chromosome 14 from 7.5 to 21.9
centiMorgans also
contains genes associated with apoptosis, hypertrophic cardiomopathy, and
oculopharyngeal
muscular dystrophy.
The invention also encompasses TXREG variants. A preferred TXREG 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 TXREG amino acid sequence, and which contains at
least one functional or
structural characteristic of TXREG.
The invention also encompasses polynucleotides which encode TXREG. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:33-64, which encodes TXREG. The
polynucleotide
sequences of SEQ >D N0:33-64, 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
TXREG. 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 TXREG. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ )D
N0:33-64 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 N0:33-64. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of TXREG.
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 TXREG, some
bearing minimal
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WO 00/78954 PCT/US00/16766
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 TXREG, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode TXREG and its variants are
generally capable
of hybridizing to the nucleotide sequence of the naturally occurring TXREG
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences encoding
TXREG 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 TXREG and its derivatives without altering the
encoded amino acid
IS 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 TXREG
and
TXREG 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 TXREG or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ ID
N0:33-64 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(PE
Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia
Biotech,
Piscataway NJ), or combinations of polymerases and proofreading exonucleases
such as those found
in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably,
sequence preparation is automated with machines such as the MICROLAB 2200
liquid transfer
system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA)
and ABI
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then carried out
using either the
ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA
sequencing
system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
The resulting
sequences are analyzed using a variety of algorithms which are well known in
the art. (See, e.g.,
Ausubel, F.M. (1997) Short Protocols in Molecular BioloQV, John Wiley & Sons,
New York NY, unit
7.7; Meyers, R.A. (1995) Molecular Biology and BiotechnoloQV, Wiley VCH, New
York NY, pp.
856-853.)
The nucleic acid sequences encoding TXREG may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR Methods
Applic. 2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
digestions and ligations may be used to insert an engineered double-stranded
sequence into a region
of unknown sequence before performing PCR. Other methods which may be used to
retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 Primer Analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
length, to have a GC content of about 50% or more, and to anneal to the
template at temperatures of
about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of sequence
into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
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WO 00/78954 PCT/US00/16766
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide-
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Output/light intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode TXREG may be cloned in recombinant DNA molecules that direct
expression of
TXREG, 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 TXREG.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter TXREG-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
Number
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of TXREG, 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.
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WO 00/78954 PCT/US00/16766
In another embodiment, sequences encoding TXREG 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, TXREG itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. ( 1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (PE Biosystems).
Additionally, the amino
acid sequence of TXREG, 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 TXREG, the nucleotide sequences
encoding TXREG
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding TXREG. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding TXREG. Such signals include the ATG initiation codon and
adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding TXREG 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 TXREG 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
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Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-
17; Ausubel, F.M. et
al. (1995) Current Protocols in Molecular BioloQV, John Wiley & Sons, New York
NY, ch. 9, 13, and
16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding TXREG. 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;
Broglie, 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
Harnngton, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding TXREG. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding TXREG 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 TXREG 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 TXREG are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of TXREG may be used.
For example, vectors
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WO 00/78954 PCT/US00/16766
containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of TXREG. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, supra; and Scorer, supra.)
Plant systems may also be used for expression of TXREG. Transcription of
sequences
encoding TXREG may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used
alone or in comhination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, su ra; Broglie, 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 Technolo~y
(1992) McGraw
Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding TXREG
may be ligated into
an adenovirus transcriptionltranslation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses TXREG in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. SV40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of TXREG in cell lines is preferred. For example, sequences encoding TXREG 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
CA 02375414 2001-11-28
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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 Q-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding TXREG is inserted within a marker gene sequence, transformed
cells containing
sequences encoding TXREG can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding TXREG 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 TXREG
and that
express TXREG 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 TXREG
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 TXREG is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
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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 ImmunoloQV, 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 TXREG
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding TXREG, 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 Wn, 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 TXREG may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode TXREG may be designed to contain
signal sequences
which direct secretion of TXREG through a prokaryotic or eukaryotic cell
membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding TXREG may be ligated~to a heterologous sequence resulting
in translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric TXREG protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
32
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
facilitate the screening of peptide libraries for inhibitors of TXREG
activity. Heterologous protein
and peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffinity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the TXREG encoding sequence and the
heterologous
protein sequence, so that TXREG 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 TXREG may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
TXREG of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to TXREG. At least one and up to a plurality of test
compounds may be
screened for specific binding to TXREG. 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
TXREG, 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 TXREG
binds, onto 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 TXREG,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing TXREG or cell membrane fractions which contain TXREG
are then
contacted with a test compound and binding, stimulation, or inhibition of
activity of either TXREG or
the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
33
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WO 00/78954 PCT/US00/16766
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
TXREG, either in
solution or affixed to a solid support, and detecting the binding of TXREG 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.
TXREG of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of TXREG. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
TXREG activity, wherein TXREG is combined with at least one test compound, and
the activity of
TXREG in the presence of a test compound is compared with the activity of
TXREG in the absence
of the test compound. A change in the activity of TXREG in the presence of the
test compound is
indicative of a compound that modulates the activity of TXREG. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising TXREG under
conditions suitable for
TXREG activity, and the assay is performed. In either of these assays, a test
compound which
modulates the activity of TXREG may do so indirectly and need not come in
direct contact with the
test compound. At least one and up to a plurality of test compounds may be
screened.
In another embodiment, polynucleotides encoding TXREG or their mammalian
homologs
may be "knocked out" in an animal model system using homologous recombination
in embryonic
stem (ES) cells. Such techniques are well known in the art and are useful for
the generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding TXREG may also be manipulated in vitro in ES cells
derived from
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WO 00/78954 PCT/US00/16766
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 TXREG 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 TXREG 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 TXREG, e.g., by secreting TXREG
in its milk, may
also serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-
74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of TXREG and human transcriptional regulator proteins. In
addition, the expression
of TXREG is closely associated with cell proliferation and inflammation.
Therefore, TXREG
appears to play a role in cell proliferative, autoimmune/inflammatory, and
developmental disorders.
In the treatment of disorders associated with increased TXREG expression or
activity, it is desirable
to decrease the expression or activity of TXREG. In the treatment of disorders
associated with
decreased TXREG expression or activity, it is desirable to increase the
expression or activity of
TXREG.
Therefore, in one embodiment, TXREG 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 TXREG. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cinrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an
autoimmune/inflammatory
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease,
adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma; and 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, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss.
In another embodiment, a vector capable of expressing TXREG 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 TXREG including, but not limited to, those described
above.
In a further embodiment, a pharmaceutical composition comprising a
substantially purified
TXREG 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 TXREG including, but
not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of TXREG
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of TXREG including, but not limited to, those listed above.
In a further embodiment, an antagonist of TXREG may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of
TXREG. Examples of such
disorders include, but are not limited to, those cell proliferative,
autoimmune/inflammatory, and
developmental disorders described above. In one aspect, an antibody which
specifically binds
TXREG 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 TXREG.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding TXREG may be administered to a subject to treat or prevent a disorder
associated with
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WO 00/78954 PCT/US00/16766
increased expression or activity of TXREG 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 TXREG may be produced using methods which are generally known
in the
art. In particular, purified TXREG may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind TXREG.
Antibodies to TXREG 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 TXREG or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium narvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
TXREG 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 TXREG 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 TXREG 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
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CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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
TXREG-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for TXREG may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
TXREG and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering TXREG 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 TXREG.
Affinity is expressed as an
association constant, Ka, which is defined as the molar concentration of TXREG-
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 TXREG epitopes, represents the average affinity, or
avidity, of the antibodies
for TXREG. The Ka determined for a preparation of monoclonal antibodies, which
are monospecific
for a particular TXREG epitope, represents a true measure of affinity. High-
affinity antibody
preparations with Ka ranging from about 109 to 10'z L/mole are preferred for
use in immunoassays in
which the TXREG-antibody complex must withstand rigorous manipulations. Low-
affinity antibody
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CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
preparations with Ka ranging from about 106 to 10' L/mole are preferred for
use in
immunopurification and similar procedures which ultimately require
dissociation of TXREG,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies,
Volume I: A Practical
A~nroach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A
Practical Guide to
Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of TXREG-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity,
and guidelines for antibody quality and usage in various applications, are
generally available. (See,
e.g., Catty, supra, and Coligan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding TXREG, 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 TXREG. 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 TXREG. (See, e.g., Agrawal, S., ed. ( 1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of viral
vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g.,
Miller, A:D. (1990) Blood
76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other
gene delivery mechanisms include liposome-derived systems, artificial viral
envelopes, and other
systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et
al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997)
Nucleic Acids Res.
25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding TXREG 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 (SC)D)-X1 disease
characterized by X
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WO 00/78954 PCT/US00/16766
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)
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 falc~arum and
Trypanosoma cruzi). In the
case where a genetic deficiency in TXREG expression or regulation causes
disease, the expression of
TXREG 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
TXREG are treated by constructing mammalian expression vectors encoding TXREG
and introducing
these vectors by mechanical means into TXREG-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 TXREG include,
but are not
limited to, the PCDNA 3.1, EPTTAG, 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). TXREG may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or ~i-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.
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding TXREG from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to TXREG expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding TXREG 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 TXREG to cells which have one or more genetic
abnormalities with respect
to the expression of TXREG. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
41
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. ( 1999)
Annu. Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding TXREG to target cells which have one or more genetic
abnormalities with
respect to the expression of TXREG. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing TXREG to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye
Res.169:385-395). The construction of a HSV-1 virus vector has also been
disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is
hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use
of recombinant
HSV d92 which consists of a genome containing at least one exogenous gene to
be transferred to a
cell under the control of the appropriate promoter for purposes including
human gene therapy. Also
taught by this patent are the construction and use of recombinant HSV strains
deleted for ICP4, ICP27
and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The
manipulation of cloned
herpesvirus sequences, the generation of recombinant virus following the
transfection of multiple
plasmids containing different segments of the large herpesvirus genomes, the
growth and propagation
of herpesvirus, and the infection of cells with herpesvirus are techniques
well known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding TXREG to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotech. 9:464-
469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full-length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
TXREG into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
TXREG-coding RNAs and the synthesis of high levels of TXREG in vector
transduced cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
42
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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 TXREG into a variety of cell types. The specific
transduction of a subset of
cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunoloeic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163- ,
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding TXREG.
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 TXREG. 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
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CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
constructs that synthesize complementary RNA, constitutively or inducibly, can
be introduced into
cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding TXREG.
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
TXREG expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding TXREG may be therapeutically useful, and in the
treament of disorders
associated with decreased TXREG expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding TXREG 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 TXREG 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
TXREG 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 TXREG. The amount of hybridization may be
quantified, thus
44
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5.932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691 ).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
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 TXREG, antibodies to TXREG, and
mimetics, agonists,
antagonists, or inhibitors of TXREG.
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,
intranasal, 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
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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 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 TXREG or fragments thereof. For example,
liposome
preparations containing a cell-impermeable macromolecule may promote cell
fusion and intracellular
delivery of the macromolecule. Alternatively, TXREG 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
TXREG or fragments thereof, antibodies of TXREG, and agonists, antagonists or
inhibitors of
TXREG, which ameliorates the symptoms or condition. Therapeutic efficacy and
toxicity may be
determined by standard pharmaceutical procedures in cell cultures or with
experimental animals, such
as by calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDso (the
dose lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDso ratio.
Pharmaceutical compositions
which exhibit large therapeutic indices are preferred. The data obtained from
cell culture assays and
animal studies are used to formulate a range of dosage for human use. The
dosage contained in such
compositions is preferably within a range of circulating concentrations that
includes the EDso with
little or no toxicity. The dosage varies within this range depending upon the
dosage form employed,
the sensitivity of the patient, and the route of administration.
The exact dosage will be 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
46
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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 ~cg to 100,000 fig, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind TXREG may be used
for the
diagnosis of disorders characterized by expression of TXREG, or in assays to
monitor patients being
treated with TXREG or agonists, antagonists, or inhibitors of TXREG.
Antibodies useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for TXREG include methods which utilize the antibody and a
label to detect
TXREG 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 TXREG, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of TXREG expression.
Normal or standard values for TXREG expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibody to
TXREG under conditions suitable for complex formation. The amount of standard
complex
formation may be quantitated by various methods, such as photometric means.
Quantities of TXREG
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 TXREG 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 TXREG
may be correlated
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CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of TXREG, and to monitor regulation of TXREG levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding TXREG or closely related
molecules may be used
to identify nucleic acid sequences which encode TXREG. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurnng sequences encoding TXREG, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50°l0
sequence identity to any of the TXREG encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:33-64 or from
genomic sequences including promoters, enhancers, and introns of the TXREG
gene.
Means for producing specific hybridization probes for DNAs encoding TXREG
include the
, cloning of polynucleotide sequences encoding TXREG or TXREG derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or'SS,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding TXREG may be used for the diagnosis of
disorders
associated with expression of TXREG. Examples of such disorders include, but
are not limited to, a
cell proliferative disorder, such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus; an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis,
anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis,
autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis,
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis,
dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
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CA 02375414 2001-11-28
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erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; and a developmental disorder, such as renal tubular acidosis, anemia,
Cushing's syndrome,
achondroplastic dwa~sm, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation),
Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as
Syndenham's chorea
and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss. The polynucleotide sequences encoding TXREG 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 TXREG expression. Such qualitative or quantitative
methods are well
known in the art.
In a particular aspect, the nucleotide sequences encoding TXREG may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding TXREG 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 TXREG 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
TXREG, 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 TXREG, 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
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polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
TXREG 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 TXREG, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
TXREG, 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 TXREG 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
TXREG are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
CA 02375414 2001-11-28
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sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of TXREG include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described in Seilhamer, J.J. et al.,
"Comparative Gene Transcript
Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The
microarray may also be
used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to
monitor 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 TXREG, or TXREG 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 TXREG
may be
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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 P1 constructions, or single chromosome cDNA libraries. (See,
e.g., Harrington, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of
the invention may be
used to develop genetic linkage maps, for example, which correlate the
inheritance of a disease state
with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, e.g., 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 TXREG on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information is
valuable to investigators searching for disease genes using positional cloning
or other gene discovery
techniques. Once the gene or genes responsible for a disease or syndrome have
been crudely
localized by genetic linkage to a particular genomic region, e.g., ataxia-
telangiectasia to l 1q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The
nucleotide sequence of
the instant invention may also be used to detect differences in the
chromosomal location due to
translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, TXREG, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between TXREG and the agent being tested may be measured.
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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 TXREG,
or fragments thereof,
and washed. Bound TXREG is then detected by methods well known in the art.
Purified TXREG
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 TXREG specifically compete with a test compound
for binding
TXREG. In this manner, antibodies can be used to detect the presence of any
peptide which shares
one or more antigenic determinants with TXREG.
In additional embodiments, the nucleotide sequences which encode TXREG may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following preferred specific
embodiments are, therefore, to be construed as merely illustrative, and not
limitative of the remainder
of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below, in
particular U.S. Ser. No.60/140,109 are hereby expressly incorporated by
reference.
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some
tissues were homogenized and lysed in guanidinium isothiocyanate, while others
were homogenized
and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a
monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
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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, su ra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
pcDNA2.1 plasmid
(Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant
plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-
BIueMRF, or
SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life
Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega); an
AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96
plasmid purification kit from QIAGEN. Following precipitation, plasmids were
resuspended in 0.1
ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSICAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
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2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carned out
using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373
or 377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base
calling software; or other sequence analysis systems known in the art. Reading
frames within the
cDNA sequences were identified using standard methods (reviewed in Ausubel,
1997, supra, unit
7.7). Some of the cDNA sequences were selected for extension using the
techniques disclosed in
Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
sequences.
The polynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programing, and dinucleotide nearest neighbor analysis. The sequences
were then queried
against a selection of public databases such as the GenBank primate, rodent,
mammalian, vertebrate,
and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and 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,
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WO 00/78954 PCT/US00/16766
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 )D
N0:33-64. 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,
s_ unra, ch. 7; Ausubel,
1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 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 TXREG occurred. Analysis involved the categorization
of cDNA libraries by
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organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Chromosomal Mapping of TXREG Encoding Polynucleotides
The cDNA sequences which were used to assemble SEQ ID N0:33-64 were compared
with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ ID N0:33-64 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
The genetic map locations of SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID
N0:36, SEQ ID N0:38, SEQ ID N0:40, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:47,
SEQ ID
N0:61, SEQ ID N0:62, and SEQ ID N0:64 are described in The Invention as
ranges, or intervals, of
human chromosomes. More than one map location is reported for SEQ ID N0:33,
SEQ ID N0:36,
and SEQ ID N0:64, indicating that previously mapped sequences having
similarity, but not complete
identity, to SEQ ID N0:33, SEQ ID N0:36, and SEQ ID N0:64 were assembled into
their respective .
clusters. The map position of an interval, in centiMorgans, is measured
relative to the terminus of the
chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on
recombination
frequencies between chromosomal markers. On average, 1 cM is roughly
equivalent to I megabase
(Mb) of DNA in humans, although this can vary widely due to hot and cold spots
of recombination.)
The cM distances are based on genetic markers mapped by Genethon which provide
boundaries for
radiation hybrid markers whose sequences were included in each of the
clusters. Diseases associated
with the public and Incyte sequences located within the indicated intervals
are also reported in the
Invention where applicable.
VI. Extension of TXREG Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:33-64 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
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WO 00/78954 PCT/US00/16766
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known ~in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2', (NH4)zS04,
and ~i-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~1 to 10 /.d 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 Wn, and
sonicated or sheared prior to relegation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
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WO 00/78954 PCT/US00/16766
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:33-64 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ 1D N0:33-64 are employed to screen
cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
[y-3zP] 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
carned out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena ( 1999),
59
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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, W, 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 Pr~aration
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/lrl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/lil RNase inhibitor, 500 E,~M dATP, 500 liM
dGTP, 500 l~M dTTP, 40
pM dCTP, 40 E.~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 E~l SX SSC/0.2% SDS.
Microarray Preparation
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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 I-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.
Anray elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/ld, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60
°C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX 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 ~.il of SX SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60 °C. The arrays are washed for 10
min at 45 °C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45 °C in
a second wash buffer (0.1X
SSC), and dried.
Detection
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
61
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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 TXREG-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring TXREG.
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
62
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of TXREG.
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
TXREG-encoding
transcript.
X. Expression of TXREG
Expression and purification of TXREG is achieved using bacterial or virus-
based expression
systems. For expression of TXREG 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 TXREG upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of TXREG in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant AutoQraphica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding TXREG by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, TXREG 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 janonicum, 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
TXREG at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues, enables
purification on metal-chelate
resins (QIAGEN). Methods for protein expression and purification are discussed
in Ausubel (1995,
supra, ch. 10 and 16). Purified TXREG obtained by these methods can be used
directly in the assays
shown in Examples XI and XV.
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WO 00/78954 PCT/US00/16766
XI. Demonstration of TXREG Activity
TXREG activity is measured by its ability to stimulate transcription of a
reporter gene (Liu,
H.Y. et al. (1997) EMBO J. 16(17):5289-5298). The assay employs a well
characterized reporter
gene construct, LexAoP LacZ, that consists of LexA DNA transcriptional control
elements (LexAoP)
fused to sequences encoding the E. coli LacZ enzyme. The methods for
constructing and expressing
fusions genes, introducing them into cells, and measuring LacZ enzyme
activity, are well known to
those skilled in the art. Sequences encoding TXREG are cloned into a plasmid
that directs the
synthesis of a fusion protein, LexA-TXREG, consisting of TXREG and a DNA
binding domain
derived from the LexA transcription factor. The resulting plasmid, encoding a
LexA-TXREG fusion
protein, is introduced into yeast cells along with a plasmid containing the
LexAoP LacZ reporter gene.
The amount of LacZ enzyme activity associated with LexA-TXREG transfected
cells, relative to
control cells, is proportional to the amount of transcription stimulated by
the TXREG.
XII. Functional Assays
TXREG function is assessed by expressing the sequences encoding TXREG 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
,ug 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 TXREG on gene expression can be assessed using highly
purified
64
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
populations of cells transfected with sequences encoding TXREG 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 TXREG and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XIII. Production of TXREG Specific Antibodies
TXREG substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harnngton, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the TXREG amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to ICL,H
(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-
Ki,H complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
TXREG activity by, for example, binding the peptide or TXREG to a substrate,
blocking with~.1 %
BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated
goat anti-rabbit IgG.
XIV. Purification of Naturally Occurring TXItEG Using Specific Antibodies
Naturally occurnng or recombinant TXREG is substantially purified by
immunoaffinity
chromatography using antibodies specific for TXREG. An immunoaffinity column
is constructed by
covalently coupling anti-TXREG 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 TXREG are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of TXREG (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/TXREG 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 TXREG is collected.
XV. Identification of Molecules Which Interact with TX)REG
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
TXREG, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled TXREG, washed,
and any wells with_labeled TXREG complex are assayed. Data obtained using
different
concentrations of TXREG are used to calculate values for the number, affinity,
and association of
TXREG with the candidate molecules.
Alternatively, molecules interacting with TXREG 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).
TXREG may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101 ).
I5. 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.
66
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
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07
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
LAL, Preeti
YUE, Henry
TANG, Y. Tom
BAUGHN, Mariah R.
AZIMZAI, Yalda
TRAM, Bao
<120> HUMAN TRANSCRIPTIONAL REGULATOR PROTEINS
<130> PF-0713 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/140,109
<151> 1999-06-18
<160> 64
<170> PERL Program
<210> 1
<211> 192
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 091502CD1
<400> 1
Met Ala Ser Lys Gly Pro Ser Ala Ser Ala Ser Pro Glu Asn Ser
1 5 10 15
Ser Ala Gly Gly Pro Ser Gly Ser Ser Asn Gly Ala Gly Glu Ser
20 25 30
Gly Gly Gln Asp Ser Thr Phe Glu Cys Asn Ile Cys Leu Asp Thr
35 40 45
Ala Lys Asp Ala Val Ile Ser Leu Cys Gly His Leu Phe Cys Trp
50 55 60
Pro Cys Leu His Gln Trp Leu Glu Thr Arg Pro Asn Arg Gln Val
65 70 75
Cys Pro Val Cys Lys Ala Gly Ile Ser Arg Asp Lys Val Ile Pro
80 85 90
Leu Tyr Gly Arg Gly Ser Thr Gly Gln Gln Asp Pro Arg Glu Lys
95 100 105
Thr Pro Pro Arg Pro Gln Gly Gln Arg Pro Glu Pro Glu Asn Arg
110 115 120
Gly Gly Phe Gln Gly Phe Gly Phe Gly Asp Gly Gly Phe Gln Met
125 130 135
Ser Phe Gly Ile Gly Ala Phe Pro Phe Gly Ile Phe Ala Thr Ala
140 145 150
Phe Asn Ile Asn Asp Gly Arg Pro Pro Pro Ala Val Pro Gly Thr
155 160 165
Pro Gln Tyr Val Asp Glu Gln Phe Leu Ser Arg Leu Phe Leu Phe
170 175 180
Val Ala Leu Val Ile Met Phe Trp Leu Leu Ile Ala
185 190
<210> 2
<211> 169
<212> PRT
<213> Homo Sapiens
1 /50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<220>
<221> misc_feature
<223> Incyte ID No: 763816CD1
<400> 2
Met Asp Ile Lys Gly Gln Glu Ser Ser Ser Asp Gln Glu Gln Val
1 5 10 15
Asp Val Glu Ser Ile Asp Phe Ser Lys Glu Asn Lys Met Asp Met
20 25 30
Thr Ser Pro Glu Gln Ser Arg Asn Val Leu Gln Phe Thr Glu Glu
35 40 45
Lys Glu Ala Phe Ile Ser Glu Glu Glu Ile Ala Lys Tyr Met Lys
50 55 60
Arg Gly Lys Gly Lys Tyr Tyr Cys Lys Ile Cys Cys Cys Arg Ala
65 70 75
Met Lys Lys Gly Ala Val Leu His His Leu Val Asn Lys His Asn
80 85 90
Val His Ser Pro Tyr Lys Cys Thr Ile Cys Gly Lys Ala Phe Leu
95 100 105
Leu Glu Ser Leu Leu Lys Asn His Val Ala Ala His Gly Gln Ser
110 115 120
Leu Leu Lys Cys Pro Arg Cys Asn Phe Glu Ser Asn Phe Pro Arg
125 130 135
Gly Phe Lys Lys His Leu Thr His Cys Gln Ser Arg His Asn Glu
140 145 150
Glu Ala Asn Lys Lys Leu Met Glu Ala Leu Glu Pro Pro Leu Glu
155 160 165
Glu Gln Gln Ile
<210> 3
<211> 498
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 961184CD1
<400> 3
Met Pro Phe Leu Ser Leu His Arg Ser Pro His Gly Pro Ser Lys
1 5 10 15
Leu Cys Asp Asp Pro Gln Ala Ser Leu Val Pro Glu Pro Val Pro
20 25 30
Gly Gly Cys Gln Glu Pro Glu Glu Met Ser Trp Pro Pro Ser Gly
35 40 45
Glu Ile Ala Ser Pro Pro Glu Leu Pro Ser Ser Pro Pro Pro Gly
50 55 60
Leu Pro Glu Val Ala Pro Asp Ala Thr Ser Thr Gly Leu Pro Asp
65 70 75
Thr Pro Ala Ala Pro Glu Thr Ser Thr Asn Tyr Pro Val Glu Cys
80 85 90
Thr Glu Gly Ser Ala Gly Pro Gln Ser Leu Pro Leu Pro Ile Leu
95 100 105
Glu Pro Val Lys Asn Pro Cys Ser Val Lys Asp Gln Thr Pro Leu
110 115 120
Gln Leu Ser Val Glu Asp Thr Thr Ser Pro Asn Thr Lys Pro Cys
125 130 135
Pro Pro Thr Pro Thr Thr Pro Glu Thr Ser Pro Pro Pro Pro Pro
140 145 150
Pro Pro Pro Ser Ser Thr Pro Cys Ser Ala His Leu Thr Pro Ser
155 160 165
Ser Leu Phe Pro Ser Ser Leu Glu Ser Ser Ser Glu Gln Lys Phe
170 175 180
Tyr Asn Phe Val Ile Leu His Ala Arg Ala Asp Glu His Ile Ala
185 190 195
Leu Arg Val Arg Glu Lys Leu Glu Ala Leu Gly Val Pro Asp Gly
200 205 210
2/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
Ala Thr Phe Cys Glu Asp Phe Gln Val Pro Gly Arg Gly Glu Leu
215 220 225
Ser Cys Leu Gln Asp Ala Ile Asp His Ser Ala Phe Ile Ile Leu
230 235 240
Leu Leu Thr Ser Asn Phe Asp Cys Arg Leu Ser Leu His Gln Val
245 250 255
Asn Gln Ala Met Met Ser Asn Leu Thr Arg Gln Gly Ser Pro Asp
260 265 270
Cys Val Ile Pro Phe Leu Pro Leu Glu Ser Ser Pro Ala Gln Leu
275 280 285
Ser Ser Asp Thr Ala Ser Leu Leu Ser Gly Leu Val Arg Leu Asp
290 295 300
Glu His Ser Gln Ile Phe Ala Arg Lys Val Ala Asn Thr Phe Lys
305 310 315
Pro His Arg Leu Gln Ala Arg Lys Ala Met Trp Arg Lys Glu Gln
320 325 330
Asp Thr Arg Ala Leu Arg Glu Gln Ser Gln His Leu Asp Gly Glu
335 340 345
Arg Met Gln Ala Ala Ala Leu Asn Ala Ala Tyr Ser Ala Tyr Leu
350 355 360
Gln Ser Tyr Leu Ser Tyr Gln Ala Gln Met Glu Gln Leu Gln Val
365 370 375
Ala Phe Gly Ser His Met Ser Phe Gly Thr Gly Ala Pro Tyr Gly
380 385 390
Ala Arg Met Pro Phe Gly Gly Gln Val Pro Leu Gly Ala Pro Pro
395 400 405
Pro Phe Pro Thr Trp Pro Gly Cys Pro Gln Pro Pro Pro Leu His
410 415 420
Ala Trp Gln Ala Gly Thr Pro Pro Pro Pro Ser Pro Gln Pro Ala
425 430 435
Ala Phe Pro Gln Ser Leu Pro Phe Pro Gln Ser Pro Ala Phe Pro
440 445 450
Thr Ala Ser Pro Ala Pro Pro Gln Ser Pro Gly Leu Gln Pro Leu
455 460 465
Ile Ile His His Ala Gln Met Val Gln Leu Gly Leu Asn Asn His
470 475 480
Met Trp Asn Gln Arg Gly Ser Gln Ala Pro Glu Asp Lys Thr Gln
485 490 495
Glu Ala Glu
<210> 4
<211> 615
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1255525CD1
<400> 4
Met Leu Leu Gly Phe Leu Lys Leu Gln Gln Lys Trp Ser Tyr Glu
1 5 10 15
Asp Lys Asp Gln Arg Pro Leu Asn Gly Phe Lys Asp Gln Leu Cys
20 25 30
Ser Leu Val Phe Met Ala Leu Thr Asp Pro Ser Thr Gln Leu Gln
35 40 45
Leu Val Gly Ile Arg Thr Leu Thr Val Leu Gly Ala Gln Pro Asp
50 55 60
Leu Leu Ser Tyr Glu Asp Leu Glu Leu Ala Val Gly His Leu Tyr
65 70 75
Arg Leu Ser Phe Leu Lys Glu Asp Ser Gln Ser Cys Arg Val Ala
80 85 90
Ala Leu Glu Ala Ser Gly Thr Leu Ala Ala Leu Tyr Pro Val Ala
95 100 105
Phe Ser Ser His Leu Val Pro Lys Leu Ala Glu Glu Leu Arg Val
110 115 120
Gly Glu Ser Asn Leu Thr Asn Gly Asp Glu Pro Thr Gln Cys Ser
3/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
125 130 135
Arg His Leu Cys Cys Leu Gln Ala Leu Ser Ala Val Ser Thr His
140 145 150
Pro Ser Ile Val Lys Glu Thr Leu Pro Leu Leu Leu Gln His Leu
155 160 165
Trp Gln Val Asn Arg Gly Asn Met Val Ala Gln Ser Ser Asp Val
170 175 ~ 180
Ile Ala Val Cys Gln Ser Leu Arg Gln Met Ala Glu Lys Cys Gln
185 190 195
Gln Asp Pro Glu Ser Cys Trp Tyr Phe His Gln Thr Ala Ile Pro
200 205 210
Cys Leu Leu Ala Leu Ala Val Gln Ala Ser Met Pro Glu Lys Glu
215 220 225
Pro Ser Val Leu Arg Lys Val Leu Leu Glu Asp Glu Val Leu Ala
230 235 240
Ala Met Val Ser Val Ile Gly Thr Ala Thr Thr His Leu Ser Pro
245 250 255
Glu Leu Ala Ala Gln Ser Val Thr His Ile Val Pro Leu Phe Leu
260 265 270
Asp Gly Asn Val Ser Phe Leu Pro Glu Asn Ser Phe Pro Ser Arg
275 280 285
Phe Gln Pro Phe Gln Asp Gly Ser Ser Gly Gln Arg Arg Leu Ile
290 295 300
Ala Leu Leu Met Ala Phe Val Cys Ser Leu Pro Arg Asn Val Glu
305 310 315
Ile Pro Gln Leu Asn Gln Leu Met Arg Glu Leu Leu Glu Leu Ser
320 325 330
Cys Cys His Ser Cys Pro Phe Ser Ser Thr Ala Ala Ala Lys Cys
335 340 345
Phe Ala Gly Leu Leu Asn Lys His Pro Ala Gly Gln Gln Leu Asp
350 355 360
Glu Phe Leu Gln Leu Ala Val Asp Lys Val Glu Ala Gly Leu Gly
365 370 375
Ser Gly Pro Cys Arg Ser Gln Ala Phe Thr Leu Leu Leu Trp Val
380 385 390
Thr Lys Ala Leu Val Leu Arg Tyr His Pro Leu Ser Ser Cys Leu
395 400 405
Thr Ala Arg Leu Met Gly Leu Leu Ser Asp Pro Glu Leu Gly Pro
410 415 420
Ala Ala Ala Asp Gly Phe Ser Leu Leu Met Ser Asp Cys Thr Asp
425 430 435
Val Leu Thr Arg Ala Gly His Ala Glu Val Arg Ile Met Phe Arg
440 445 450
Gln Arg Phe Phe Thr Asp Asn Val Pro Ala Leu Val Gln Gly Phe
455 460 465
His Ala Ala Pro Gln Asp Val Lys Pro Asn Tyr Leu Lys Gly Leu
470 475 480
Ser His Val Leu Asn Arg Leu Pro Lys Pro Val Leu Leu Pro Glu
485 490 495
Leu Pro Thr Leu Leu Ser Leu Leu Leu Glu Ala Leu Ser Cys Pro
500 505 510
Asp Cys Val Val Gln Leu Ser Thr Leu Ser Cys Leu Gln Pro Leu
515 520 525
Leu Leu Glu Ala Pro Gln Val Met Ser Leu His Val Asp Thr Leu
530 535 540
Val Thr Lys Phe Leu Asn Leu Ser Ser Ser Pro Ser Met Ala Val
545 550 555
Arg Ile Ala Ala Leu Gln Cys Met His Ala Leu Thr Arg Leu Pro
560 565 570
Thr Pro Val Leu Leu Pro Tyr Lys Pro Gln Val Ile Arg Ala Leu
575 580 585
Ala Lys Pro Leu Asp Asp Lys Lys Arg Leu Val Arg Lys Glu Ala
590 595 600
Val Ser Ala Arg Gly Glu Trp Phe Leu Leu Gly Ser Pro Gly Ser
605 610 615
<210> 5
4/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<211> 120
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1297447CD1
<400> 5
Met Ile Thr Ser Gln Gly Ser Val Ser Phe Arg Asp Val Thr Val
1 5 10 15
Gly Phe Thr Gln Glu Glu Trp Gln His Leu Asp Pro Ala Gln Arg
20 25 30
Thr Leu Tyr Arg Asp Val Met Leu Glu Asn Tyr Ser His Leu Val
35 40 45
Ser Val Gly Tyr Cys Ile Pro Lys Pro Glu Val Ile Leu Lys Leu
50 55 60
Glu Lys Gly Glu Glu Pro Trp Ile Leu Glu Glu Lys Phe Pro Ser
65 70 75
Gln Ser His Leu Gly Glu Leu Val Cys Ala Arg Trp Asn Leu Lys
80 85 90
Glu Gly Arg Ser Gln Arg Val Ser Leu Asp Asn Lys Thr Ile Glu
95 100 105
Met Phe Phe Arg Asn His Val Leu Glu Ala Pro Asp Leu Trp Lys
110 115 120
<210> 6
<211> 543
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1441094CD1
<400> 6
Met Met Phe Gly Gly Tyr Glu Thr Ile Glu Ala Tyr Glu Asp Asp
1 5 10 15
Leu Tyr Arg Asp Glu Ser Ser Ser Glu Leu Ser Val Asp Ser Glu
20 25 30
Val Glu Phe Gln Leu Tyr Ser Gln Ile His Tyr Ala Gln Asp Leu
35 40 45
Asp Asp Val Ile Arg Glu Glu Glu His Glu Glu Lys Asn Ser Gly
50 55 60
Asn Ser Glu Ser Ser Ser Ser Lys Pro Asn Gln Lys Lys Leu Ile
65 70 75
Val Leu Ser Asp Ser Glu Val Ile Gln Leu Ser Asp Gly Ser Glu
80 85 90
Val Ile Thr Leu Ser Asp Glu Asp Ser Ile Tyr Arg Cys Lys Gly
95 100 105
Lys Asn Val Arg Val Gln Ala Gln Glu Asn Ala His Gly Leu Ser
110 115 120
Ser Ser Leu Gln Ser Asn Glu Leu Val Asp Lys Lys Cys Lys Ser
125 130 135
Asp Ile Glu Lys Pro Lys Ser Glu Glu Arg Ser Gly Val Ile Arg
140 145 150
Glu Val Met Ile Ile Glu Val Ser Ser Ser Glu Glu Glu Glu Ser
155 160 165
Thr Ile Ser Glu Gly Asp Asn Val Glu Ser Trp Met Leu Leu Gly
170 175 180
Cys Glu Val Asp Asp Lys Asp Asp Asp Ile Leu Leu Asn Leu Val
185 190 195
Gly Cys Glu Asn Ser Val Thr Glu Gly Glu Asp Gly Ile Asn Trp
200 205 210
Ser Ile Ser Asp Lys Asp Ile Glu Ala Gln Ile Ala Asn Asn Arg
215 220 225
Thr Pro Gly Arg Trp Thr Gln Arg Tyr Tyr Ser Ala Asn Lys Asn
5/5~
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
230 235 240
Ile Ile Cys Arg Asn Cys Asp Lys Arg Gly His Leu Ser Lys Asn
245 250 255
Cys Pro Leu Pro Arg Lys Val Arg Arg Cys Phe Leu Cys Ser Arg
260 265 270
Arg Gly His Leu Leu Tyr Ser Cys Pro Ala Pro Leu Cys Glu Tyr
275 280 285
Cys Pro Val Pro Lys Met Leu Asp His Ser Cys Leu Phe Arg His
290 295 300
Ser Trp Asp Lys Gln Cys Asp Arg Cys His Met Leu Gly His Tyr
305 310 315
Thr Asp Ala Cys Thr Glu Ile Trp Arg Gln Tyr His Leu Thr Thr
320 325 330
Lys Pro Gly Pro Pro Lys Lys Pro Lys Thr Pro Ser Arg Pro Ser
335 340 345
Ala Leu Ala Tyr Cys Tyr His Cys Ala Gln Lys Gly His Tyr Gly
350 355 360
His Glu Cys Pro Glu Arg Glu Val Tyr Asp Pro Ser Pro Val Ser
365 370 375
Pro Phe Ile Cys Tyr Tyr Asp Asp Lys Tyr Glu Ile Gln Glu Arg
380 385 390
Glu Lys Arg Leu Lys Gln Lys Ile Lys Val Leu Lys Lys Asn Gly
395 400 405
Val Ile Pro Glu Pro Ser Lys Leu Pro Tyr Ile Lys Ala Ala Asn
410 415 420
Glu Asn Pro His His Asp Ile Arg Lys Gly Arg Ala Ser Trp Lys
425 430 435
Ser Asn Arg Trp Pro Gln Glu Asn Lys Glu Thr Gln Lys Glu Met
440 445 450
Lys Asn Lys Asn Arg Asn Trp Glu Lys His Arg Lys Ala Asp Arg
455 460 465
His Arg Glu Val Asp Glu Asp Phe Pro Arg Gly Pro Lys Thr Tyr
470 475 480
Ser Ser Pro Gly Ser Phe Lys Thr Gln Lys Pro Ser Lys Pro Phe
485 490 495
His Arg Ser Ser His Tyr His Thr Ser Arg Glu Asp Lys Ser Pro
500 505 510
Lys Glu Gly Lys Arg Gly Lys Gln Lys Lys Lys Glu Arg Cys Trp
515 520 525
Glu Asp Asp Asp Asn Asp Asn Leu Phe Leu Ile Lys Gln Arg Lys
530 535 540
Lys Lys Ser
<210> 7
<211> 633
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1479382CD1
<400> 7
Met Tyr Ser Thr Arg Lys Asn Cys Ala Gln Leu Trp Leu Gly Pro
1 5 10 15
Ala Ala Phe Ile Asn His Asp Cys Arg Pro Asn Cys Lys Phe Val
20 25 30
Ser Thr Gly Arg Asp Thr Ala Cys Val Lys Ala Leu Arg Asp Ile
35 40 45
Glu Pro Gly Glu Glu Ile Ser Cys Tyr Tyr Gly Asp Gly Phe Phe
50 55 60
Gly Glu Asn Asn Glu Phe Cys Glu Cys Tyr Thr Cys Glu Arg Arg
65 70 75
Gly Thr Gly Ala Phe Lys Ser Arg Val Gly Leu Pro Ala Pro Ala
80 85 90
Pro Val Ile Asn Ser Lys Tyr Gly Leu Arg Glu Thr Asp Lys Arg
95 100 105
6/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
Leu Asn Arg Leu Lys Lys Leu Gly Asp Ser Ser Lys Asn Ser Asp
110 115 120
Ser Gln Ser Val Ser Ser Asn Thr Asp Ala Asp Thr Thr Gln Glu
125 130 135
Lys Asn Asn Ala Thr Ser Asn Arg Lys Ser Ser Val Gly Val Lys
140 145 150
Lys Asn Ser Lys Ser Arg Thr Leu Thr Arg Gln Ser Met Ser Arg
155 160 165
Ile Pro Ala Ser Ser Asn Ser Thr Ser Ser Lys Leu Thr His Ile
170 175 180
Asn Asn Ser Arg Val Pro Lys Lys Leu Lys Lys Pro Ala Lys Pro
185 190 195
Leu Leu Ser Lys Ile Lys Leu Arg Asn His Cys Lys Arg Leu Glu
200 205 210
Gln Lys Asn Ala Ser Arg Lys Leu Glu Met Gly Asn Leu Val Leu
215 220 225
Lys Glu Pro Lys Val Val Leu Tyr Lys Asn Leu Pro Ile Lys Lys
230 235 240
Asp Lys Glu Pro Glu Gly Pro Ala Gln Ala Ala Val Ala Ser Gly
245 250 255
Cys Leu Thr Arg His Ala Ala Arg Glu His Arg Gln Asn Pro Val
260 265 270
Arg Gly Ala His Ser Gln Gly Glu Ser Ser Pro Cys Thr Tyr Ile
275 280 285
Thr Arg Arg Ser Val Arg Thr Arg Thr Asn Leu Lys Glu Ala Ser
290 295 300
Asp Ile Lys Leu Glu Pro Asn Thr Leu Asn Gly Tyr Lys Ser Ser
305 310 315
Val Thr Glu Pro Cys Pro Asp Ser Gly Glu Gln Leu Gln Pro Ala
320 325 330
Pro Val Leu Gln Glu Glu Glu Leu Ala His Glu Thr Ala Gln Lys
335 340 345
Gly Glu Ala Lys Cys His Lys Ser Asp Thr Gly Met Ser Lys Lys
350 355 360
Lys Ser Arg Gln Gly Lys Leu Val Lys Gln Phe Ala Lys Ile Glu
365 370 375
Glu Ser Thr Pro Val His Asp Ser Pro Gly Lys Asp Asp Ala Val
380 385 390
Pro Asp Leu Met Gly Pro His Ser Asp Gln Gly Glu His Ser Gly
395 400 405
Thr Val Gly Val Pro Val Ser Tyr Thr Asp Cys Ala Pro Ser Pro
410 415 420
Val Gly Cys Ser Val Val Thr Ser Asp Ser Phe Lys Thr Lys Asp
425 430 435
Ser Phe Arg Thr Ala Lys Ser Lys Lys Lys Arg Arg Ile Thr Arg
440 445 450
Tyr Asp Ala Gln Leu Ile Leu Glu Asn Asn Ser Gly Ile Pro Lys
455 460 465
Leu Thr Leu Arg Arg Arg His Asp Ser Ser Ser Lys Thr Asn Asp
470 475 480
Gln Glu Asn Asp Gly Met Asn Ser Ser Lys Ile Ser Ile Lys Leu
485 490 495
Ser Lys Asp His Asp Asn Asp Asn Asn Leu Tyr Val Ala Lys Leu
500 505 510
Asn Asn Gly Phe Asn Ser Gly Ser Gly Ser Ser Ser Thr Lys Leu
515 520 525
Lys Ile Gln Leu Lys Arg Asp Glu Glu Asn Arg Gly Ser Tyr Thr
530 535 540
Glu Gly Leu His Glu Asn Gly Val Cys Cys Ser Asp Pro Leu Ser
545 550 555
Leu Leu Glu Ser Arg Met Glu Val Asp Asp Tyr Ser Gln Tyr Glu
560 565 570
Glu Glu Ser Thr Asp Asp Ser Ser Ser Ser Glu Gly Asp Glu Glu
575 580 585
Glu Asp Asp Tyr Asp Asp Asp Phe Glu Asp Asp Phe Ile Pro Leu
590 595 600
Pro Pro Ala Lys Arg Leu Arg Leu Ile Val Gly Lys Asp Ser Ile
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
605 610 615
Asp Ile Asp Ile Ser Ser Arg Arg Arg Glu Asp Gln Ser Leu Arg
620 625 630
Leu Asn Ala
<210> 8
<211> 312
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1503131CD1
<400> 8
Met Ser Ala Phe Ser Glu Ala Ala Leu Glu Lys Lys Leu Ser Glu
1 5 10 15
Leu Ser Asn Ser Gln Gln Ser Val Gln Thr Leu Ser Leu Trp Leu
20 25 30
Ile His His Arg Lys His Ser Arg Pro Ile Val Thr Val Trp Glu
35 40 45
Arg Glu Leu Arg Lys Ala Lys Pro Asn Arg Lys Leu Thr Phe Leu
50 55 60
Tyr Leu Ala Asn Asp Val Ile Gln Asn Ser Lys Arg Lys Gly Pro
65 70 75
Glu Phe Thr Lys Asp Phe Ala Pro Val Ile Val Glu Ala Phe Lys
80 85 90
His Val Ser Ser Glu Thr Asp Glu Ser Cys Lys Lys His Leu Gly
95 100 105
Arg Val Leu Ser Ile Trp Glu Glu Arg Ser Val Tyr Glu Asn Asp
110 115 120
Val Leu Glu Gln Leu Lys Gln Ala Leu Tyr Gly Asp Lys Lys Pro
125 130 135
Arg Lys Arg Thr Tyr Glu Gln Ile Lys Val Asp Glu Asn Glu Asn
140 145 150
Cys Ser Ser Leu Gly Ser Pro Ser Glu Pro Pro Gln Thr Leu Asp
155 160 165
Leu Val Arg Ala Leu Gln Asp Leu Glu Asn Ala Ala Ser Gly Asp
170 175 180
Ala Ala Val His Gln Arg Ile Ala Ser Leu Pro Val Glu Val Gln
185 190 195
Glu Val Ser Leu Leu Asp Lys Ile Thr Asp Lys Glu Ser Gly Glu
200 205 210
Arg Leu Ser Lys Met Val Glu Asp Ala Cys Met Leu Leu Ala Asp
215 220 225
Tyr Asn Gly Arg Leu Ala Ala Glu Ile Asp Asp Arg Lys Gln Leu
230 235 240
Thr Arg Met Leu Ala Asp Phe Leu Arg Cys Gln Lys Glu Ala Leu
245 250 255
Ala Glu Lys Glu His Lys Leu Glu Glu Tyr Lys Arg Lys Leu Ala
260 265 270
Arg Val Ser Leu Val Arg Lys Glu Leu Arg Ser Arg Ile Gln Ser
275 280 285
Leu Pro Asp Leu Ser Arg Leu Pro Asn Val Thr Gly Ser His Met
290 295 300
His Leu Pro Phe Ala Gly Asp Ile Tyr Ser Glu Asp
305 310
<210> 9
<211> 377
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1594803CD1
8/S~
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<400> 9
Met Phe Asn Gly Gly Met Ala Thr Thr Ser Thr Glu Ile Glu Leu
1 5 10 15
Pro Asp Val Glu Pro Ala Ala Phe Leu Ala Leu Leu Lys Phe Leu
20 25 30
Tyr Ser Asp Glu Val Gln Ile Gly Pro Glu Thr Val Met Thr Thr
35 40 45
Leu Tyr Thr Ala Lys Lys Tyr Ala Val Pro Ala Leu Glu Ala His
50 55 60
Cys Val Glu Phe Leu Lys Lys Asn Leu Arg Ala Asp Asn Ala Phe
65 70 75
Met Leu Leu Thr Gln Ala Arg Leu Phe Asp Glu Pro Gln Leu Ala
80 85 90
Ser Leu Cys Leu Glu Asn Ile Asp Lys Asn Thr Ala Asp Ala Ile
95 100 105
Thr Ala Glu Gly Phe Thr Asp Ile Asp Leu Asp Thr Leu Val Ala
110 115 120
Val Leu Glu Arg Asp Thr Leu Gly Ile Arg Glu Val Arg Leu Phe
125 130 135
Asn Ala Val Val Arg Trp Ser Glu Ala Glu Cys Gln Arg Gln Gln
140 145 150
Leu Gln Val Thr Pro Glu Asn Arg Arg Lys Val Leu Gly Lys Ala
155 160 165
Leu Gly Leu Ile Arg Phe Pro Leu Met Thr Ile Glu Glu Phe Ala
170 175 180
Ala Gly Pro Ala Gln Ser Gly Ile Leu Val Asp Arg Glu Val Val
185 190 195
Ser Leu Phe Leu His Phe Thr Val Asn Pro Lys Pro Arg Val Glu
200 205 210
Phe Ile Asp Arg Pro Arg Cys Cys Leu Arg Gly Lys Glu Cys Ser
215 220 225
Ile Asn Arg Phe Gln Gln Val Glu Ser Arg Trp Gly Tyr Ser Gly
230 235 240
Thr Ser Asp Arg Ile Arg Phe Ser Val Asn Lys Arg Ile Phe Val
245 250 255
Val Gly Phe Gly Leu Tyr Gly Ser Ile His Gly Pro Thr Asp Tyr
260 265 270
Gln Val Asn Ile Gln Ile Ile His Thr Asp Ser Asn Thr Val Leu
275 280 285
Gly Gln Asn Asp Thr Gly Phe Ser Cys Asp Gly Ser Ala Ser Thr
290 295 300
Phe Arg Val Met Phe Lys Glu Pro Val Glu Val Leu Pro Asn Val
305 310 315
Asn Tyr Thr Ala Cys Ala Thr Leu Lys Gly Pro Asp Ser His Tyr
320 325 330
Gly Thr Lys Gly Leu Arg Lys Val Thr His Glu Ser Pro Thr Thr
335 340 345
Gly Ala Lys Thr Cys Phe Thr Phe Cys Tyr Ala Ala Gly Asn Asn
350 355 360
Asn Gly Thr Ser Val Glu Asp Gly Gln Ile Pro Glu Val Ile Phe
365 370 375
Tyr Thr
<210> 10
<211> 170
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1736129CD1
<400>
Met Glu Pro Pro Glu Glu Asn Lys Gln Gln Asp
Asp Asn Met Ser
1 5 10 15
Pro Lys Arg Pro Gln Ser Pro Gly Asn Ile Cys
Glu Ser Gly His
20 25 30
9/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
Leu Gly Ala Pro Lys Cys Thr Arg Cys Leu Ile Thr Phe Ala Asp
35 40 45
Ser Lys Phe Gln Glu Arg His Met Lys Arg Glu His Pro Ala Asp
50 55 60
Phe Val Ala Gln Lys Leu Gln Gly Val Leu Phe Ile Cys Phe Thr
65 70 75
Cys Ala Arg Ser Phe Pro Ser Ser Lys Ala Leu Ile Thr His Gln
80 85 90
Arg Ser His Gly Pro Ala Ala Lys Pro Thr Leu Pro Val Ala Thr
95 100 105
Thr Thr Ala Gln Pro Thr Phe Pro Cys Pro Asp Cys Gly Lys Thr
110 115 120
Phe Gly Gln Ala Val Ser Leu Arg Arg His Arg Gln Met His Glu
125 130 135
Val Arg Ala Pro Pro Gly Thr Phe Ala Cys Thr Glu Cys Gly Gln
140 145 150
Asp Phe Ala Gln Glu Ala Gly Leu His Gln His Tyr Ile Arg His
155 160 165
Ala Arg Gly Glu Leu
170
<210> 11
<211> 160
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1874312CD1
<400> 11
Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys
1 5 10 15
Ala Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala
20 25 30
Pro Ala Thr Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly
35 40 45
Thr Val Ala Leu Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu
50 55 60
Leu Leu Ile Arg Lys Leu Pro Phe Gln Arg Leu Val Arg Glu Ile
65 70 75
Ala Gln Asp Phe Lys Thr Asp Leu Arg Phe Gln Ser Ser Ala Val
80 85 90
Met Ala Leu Gln Glu Ala Cys Glu Ala Tyr Leu Val Gly Leu Phe
95 100 105
Glu Asp Thr Asn Leu Cys Gly Ile Gln Arg Gln Ala Arg His Tyr
110 115 120
His Ala Gln Gly His Pro Thr His Pro Pro Ala Ser Ala Glu Glu
125 130 135
Arg Ala Val Ile Thr Val Gly Leu Ser Cys Arg Ser Lys Gln Arg
140 145 150
Val Phe Phe Arg Ala Thr Thr Phe Ser Lys
155 160.
<210> 12
<211> 219
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1969301CD1
<400> 12
Met Asn Arg Leu Phe Gly Lys Ala Lys Pro Lys Ala Pro Pro Pro
1 5 10 15
Ser Leu Thr Asp Cys Ile Gly Thr Val Asp Ser Arg Ala Glu Ser
10/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
20 25 30
Ile Asp Lys Lys Ile Ser Arg Leu Asp Ala Glu Leu Val Lys Tyr
35 40 45
Lys Asp Gln Ile Lys Lys Met Arg Glu Gly Pro Ala Lys Asn Met
50 55 60
Val Lys Gln Lys Ala Leu Arg Val Leu Lys Gln Lys Arg Met Tyr
65 70 75
Glu Gln Gln Arg Asp Asn Leu Ala Gln Gln Ser Phe Asn Met Glu
80 85 90
Gln Ala Asn Tyr Thr Ile Gln Ser Leu Lys Asp Thr Lys Thr Thr
95 100 105
Val Asp Ala Met Lys Leu Gly Val Lys Glu Met Lys Lys Ala Tyr
110 115 120
Lys Gln Val Lys Ile Asp Gln Ile Glu Asp Leu Gln Asp Gln Leu
125 130 135
Glu Asp Met Met Glu Asp Ala Asn Glu Ile Gln Glu Ala Leu Ser
140 145 150
Arg Ser Tyr Gly Thr Pro Glu Leu Asp Glu Asp Asp Leu Glu Ala
155 160 165
Glu Leu Asp Ala Leu Gly Asp Glu Leu Leu Ala Asp Glu Asp Ser
170 175 180
Ser Tyr Leu Asp Glu Ala Ala Ser Ala Pro Ala Ile Pro Glu Gly
185 190 195
Val Pro Thr Asp Thr Lys Asn Lys Asp Gly Val Leu Val Asp Glu
200 205 210
Phe Gly Leu Pro Gln Ile Pro Ala Ser
215
<210> 13
<211> 142
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1986873CD1
<400> 13
Met Asp Met Thr Ser Pro Glu Gln Ser Arg Asn Val Leu Gln Phe
1 5 10 15
Thr Glu Glu Lys Glu Ala Phe Ile Ser Glu Glu Glu Ile Ala Lys
20 25 30
Tyr Met Lys Arg Gly Lys Gly Lys Tyr Tyr Cys Lys Ile Cys Cys
35 40 45
Cys Arg Ala Met Lys Lys Gly Ala Val Leu His His Leu Val Asn
50 55 60
Lys His Asn Val His Ser Pro Tyr Lys Cys Thr Ile Cys Gly Lys
65 70 75
Ala Phe Leu Leu Glu Ser Leu Leu Lys Asn His Val Ala Ala His
80 85 90
Gly Gln Ser Leu Leu Lys Cys Pro Arg Cys Asn Phe Glu Ser Asn
95 100 105
Phe Pro Arg Gly Phe Lys Lys His Leu Thr His Cys Gln Ser Arg
110 115 120
His Asn Glu Glu Ala Asn Lys Lys Leu Met Glu Ala Leu Glu Pro
125 130 135
Pro Leu Glu Glu Gln Gln Ile
140
<210> 14
<211> 524
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2010820CD1
11 /50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<400> 14
Met Ala Glu Ile Lys Val Lys Leu Ile Glu Ala Lys Glu Ala Leu
1 5 10 15
Glu Asn Cys Ile Thr Leu Gln Asp Phe Asn Arg Ala Ser Glu Leu
20 25 30
Lys Glu Glu Ile Lys Ala Leu Glu Asp Ala Arg Ile Asn Leu Leu
35 40 45
Lys Glu Thr Glu Gln Leu Glu Ile Lys Glu Val His Ile Glu Lys
50 55 60
Asn Asp Ala Glu Thr Leu Gln Lys Cys Leu Ile Leu Cys Tyr Glu
65 70 75
Leu Leu Lys Gln Met Ser Ile Ser Thr Gly Leu Ser Ala Thr Met
80 85 90
Asn Gly Ile Ile Glu Ser Leu Ile Leu Pro Gly Ile Ile Ser Ile
95 100 105
His Pro Val Val Arg Asn Leu Ala Val Leu Cys Leu Gly Cys Cys
110 115 120
Gly Leu Gln Asn Gln Asp Phe Ala Arg Lys His Phe Val Leu Leu
125 130 135
Leu Gln Val Leu Gln Ile Asp Asp Val Thr Ile Lys Ile Ser Ala
140 145 150
Leu Lys Ala Ile Phe Asp Gln Leu Met Thr Phe Gly Ile Glu Pro
155 160 165
Phe Lys Thr Lys Lys Ile Lys Thr Leu His Cys Glu Gly Thr Glu
170 175 180
Ile Asn Ser Asp Asp Glu Gln Glu Ser Lys Glu Val Glu Glu Thr
185 190 195
Ala Thr Ala Lys Asn Val Leu Lys Leu Leu Ser Asp Phe Leu Asp
200 205 210
Ser Glu Val Ser Glu Leu Arg Thr Gly Ala Ala Glu Gly Leu Ala
215 220 225
Lys Leu Met Phe Ser Gly Leu Leu Val Ser Ser Arg Ile Leu Ser
230 235 240
Arg Leu Ile Leu Leu Trp Tyr Asn Pro Val Thr Glu Glu Asp Val
245 250 255
Gln Leu Arg His Cys Leu Gly Val Phe Phe Pro Val Phe Ala Tyr
260 265 270
Ala Ser Arg Thr Asn Gln Glu Cys Phe Glu Glu Ala Phe Leu Pro
275 280 285
Thr Leu Gln Thr Leu Ala Asn Ala Pro Ala Ser Ser Pro Leu Ala
290 295 300
Glu Ile Asp Ile Thr Asn Val Ala Glu Leu Leu Val Asp Leu Thr
305 310 315
Arg Pro Ser Gly Leu Asn Pro Gln Ala Lys Thr Ser Gln Asp Tyr
320 325 330
Gln Ala Leu Thr Val His Asp Asn Leu Ala Met Lys Ile Cys Asn
335 340 345
Glu Ile Leu Thr Ser Pro Cys Ser Pro Glu Ile Arg Val Tyr Thr
350 355 360
Lys Ala Leu Ser Ser Leu Glu Leu Ser Ser His Leu Ala Lys Asp
365 370 375
Leu Leu Val Leu Leu Asn Glu Ile Leu Glu Gln Val Lys Asp Arg
380 385 390
Thr Cys Leu Arg Ala Leu Glu Lys Ile Lys Ile Gln Leu Glu Lys
395 400 405
Gly Asn Lys Glu Phe Gly Asp Gln Ala Glu Ala Ala Gln Asp Ala
410 415 420
Thr Leu Thr Thr Thr Thr Phe Gln Asn Glu Asp Glu Lys Asn Lys
425 430 435
Glu Val Tyr Met Thr Pro Leu Arg Gly Val Lys Ala Thr Gln Ala
440 445 450
Ser Lys Ser Thr Gln Leu Lys Thr Asn Arg Gly Gln Arg Lys Val
455 460 465
Thr Val Ser Ala Arg Thr Asn Arg Arg Cys Gln Thr Ala Glu Ala
470 475 480
Asp Ser Glu Ser Asp His Glu Val Pro Glu Pro Glu Ser Glu Met
485 490 495
12/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
Lys Leu Arg Arg Ala Lys Thr Ala Ala Leu Glu Lys
Met Pro
Arg
500 505 510
Ser Asn Ala Gln Phe Leu Asn Glu Asp Leu Ser
Lys Leu
Leu
515 520
<210>15
<211>500
<212>PRT
<213>Homo Sapiens
<220>
<221>misc_feature
<223>Incyte 2013818CD1
ID
No:
<400> 15
Met Pro Gly Gln Ser Val Arg Lys Lys Thr Arg Lys Ala Lys Glu
1 5 10 15
Ile Ser Glu Ala Ser Glu Asn Ile Tyr Ser Asp Val Arg Gly Leu
20 25 30
Ser Gln Asn Gln Gln Ile Pro Gln Asn Ser Val Thr Pro Arg Arg
35 40 45
Gly Arg Arg Lys Lys Glu Val Asn Gln Asp Ile Leu Glu Asn Thr
50 55 60
Ser Ser Val Glu Gln Glu Leu Gln Ile Thr Thr Gly Arg Glu Ser
65 70 75
Lys Arg Leu Lys Ser Ser Gln Leu Leu Glu Pro Ala Val Glu Glu
80 85 90
Thr Thr Lys Lys Glu Val Lys Val Ser Ser Val Thr Lys Arg Thr
95 100 105
Pro Arg Arg Ile Lys Arg Ser Val Glu Asn Gln Glu Ser Val Glu
110 115 120
Ile Ile Asn Asp Leu Lys Val Ser Thr Val Thr Ser Pro Ser Arg
125 130 135
Met Ile Arg Lys Leu Arg Ser Thr Asn Leu Asp Ala Ser Glu Asn
140 145 150
Thr Gly Asn Lys Gln Asp Asp Lys Ser Ser Asp Lys Gln Leu Arg
155 160 165
Ile Lys His Val Arg Arg Val Arg Gly Arg Glu Val Ser Pro Ser
170 175 180
Asp Val Arg Glu Asp Ser Asn Leu Glu Ser Ser Gln Leu Thr Val
185 190 195
Gln Ala Glu Phe Asp Met Ser Ala Ile Pro Arg Lys Arg Gly Arg
200 205 210
Pro Arg Lys Ile Asn Pro Ser Glu Asp Val Gly Ser Lys Ala Val
215 220 225
Lys Glu Glu Arg Ser Pro Lys Lys Lys Glu Ala Pro Ser Ile Arg
230 235 240
Arg Arg Ser Thr Arg Asn Thr Pro Ala Lys Ser Glu Asn Val Asp
245 250 255
Val Gly Lys Pro Ala Leu Gly Lys Ser Ile Leu Val Pro Asn Glu
260 265 270
Glu Leu Ser Met Val Met Ser Ser Lys Lys Lys Leu Thr Lys Lys
275 280 285
Thr Glu Ser Gln Ser Gln Lys Arg Ser Leu His Ser Val Ser Glu
290 295 300
Glu Arg Thr Asp Glu Met Thr His Lys Glu Thr Asn Glu Gln Glu
305 310 315
Glu Arg Leu Leu Ala Thr Ala Ser Phe Thr Lys Ser Ser Arg Ser
320 325 330
Ser Arg Thr Arg Ser Ser Lys Ala Ile Leu Leu Pro Asp Leu Ser
335 340 345
Glu Pro Asn Asn Glu Pro Leu Phe Ser Pro Ala Ser Glu Val Pro
350 355 360
Arg Lys Ala Lys Ala Lys Lys Ile Glu Val Pro Ala Gln Leu Lys
365 370 375
Glu Leu Val Ser Asp Leu Ser Ser Gln Phe Val Ile Ser Pro Pro
380 385 390
13/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
Ala Leu Arg Ser Arg Gln Lys Asn Thr Ser Asn Lys Asn Lys Leu
395 400 405
Glu Asp Glu Leu Lys Asp Asp Ala Gln Ser Val Glu Thr Leu Gly
410 415 420
Lys Pro Lys Ala Lys Arg Ile Arg Thr Ser Lys Thr Lys Gln Ala
425 430 435
Ser Lys Asn Thr Glu Lys Glu Ser Ala Trp Ser Pro Pro Pro Ile
440 445 450
Glu Ile Arg Leu Ile Ser Pro Leu Ala Ser Pro Ala Asp Gly Val
455 460 465
Lys Ser Lys Pro Arg Lys Thr Thr Glu Val Thr Gly Thr Gly Leu
470 475 480
Gly Arg Asn Arg Lys Lys Leu Ser Ser Tyr Pro Lys Gln Ile Leu
485 490 495
Arg Arg Lys Met Leu
500
<210> 16
<211> 119
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2302032CD1
<400> 16
Met Asn Ala Ser Ser Glu Gly Glu Ser Phe Ala Gly Ser Val Gln
1 5 10 15
Ile Pro Gly Gly Thr Thr Val Leu Val Glu Leu Thr Pro Asp Ile
20 25 30
His Ile Cys Gly Ile Cys Lys Gln Gln Phe Asn Asn Leu Asp Ala
35 40 45
Phe Val Ala His Lys Gln Ser Gly Cys Gln Leu Thr Gly Thr Ser
50 55 60
Ala Ala Ala Pro Ser Thr Val Gln Phe Val Ser Glu Glu Thr Val
65 70 75
Pro Ala Thr Gln Thr Gln Thr Thr Thr Arg Thr Ile Thr Ser Glu
80 85 90
Thr Gln Thr Ile Thr Gly Thr Ala Gly Ala Trp Gly Ser Arg Pro
95 100 105
Glu Leu Ala Trp Leu Cys Leu Lys His Val His Gly Thr Cys
110 115
<210> 17
<211> 544
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2326109CD1
<400> 17
Met Ile His Val Arg Arg His Glu Thr Arg Arg Asn Ser Lys Ser
1 5 10 15
His Val Pro Glu Gln Lys Ser Arg Val Asp Trp Arg Arg Thr Lys
20 25 30
Arg Ser Ser Ile Ser Gln Leu Leu Asp Ser Asp Glu Glu Leu Asp
35 40 45
Ser Glu Glu Phe Asp Ser Asp Glu Glu Leu Asp Ser Asp Glu Ser
50 55 60
Phe Glu Asn Asp Glu Glu Leu Asp Ser Asn Lys Gly Pro Asp Cys
65 70 75
Asn Lys Thr Pro Gly Ser Glu Arg Glu Leu Asn Leu Ser Lys Ile
80 85 90
Gln Ser Glu Gly Asn Asp Ser Lys Cys Leu Ile Asn Ser Gly Asn
14/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
95 100 105
Gly Ser Thr Tyr Glu Glu Glu Thr Asn Lys Ile Lys His Arg Asn
110 115 120
Ile Asp Leu Gln Asp Gln Glu Lys His Leu Ser Gln Glu Asp Asn
125 130 135
Asp Leu Asn Lys Gln Thr Gly Gln Ile Ile Glu Asp Asp Gln Glu
140 145 150
Lys His Leu Ser Gln Glu Asp Asn Asp Leu Asn Lys Gln Thr Gly
155 160 165
Gln Ile Ile Glu Asp Asp Leu Glu Glu Glu Asp Ile Lys Arg Gly
170 175 180
Lys Arg Lys Arg Leu Ser Ser Val Met Cys Asp Ser Asp Glu Ser
185 190 195
Asp Asp Ser Asp Ile Leu Val Arg Lys Val Gly Val Lys Arg Pro
200 205 210
Arg Arg Val Val Glu Asp Glu Gly Ser Ser Val Glu Met Glu Gln
215 220 225
Lys Thr Pro Glu Lys Thr Leu Ala Ala Gln Lys Arg Glu Lys Leu
230 235 240
Gln Lys Leu Lys Glu Leu Ser Lys Gln Arg Ser Arg Gln Arg Arg
245 250 255
Ser Ser Gly Arg Asp Phe Glu Asp Ser Glu Lys Glu Ser Cys Pro
260 265 270
Ser Ser Asp Glu Val Asp Glu Glu Glu Glu Glu Asp Asn Tyr Glu
275 280 285
Ser Asp Glu Asp Gly Asp Asp Tyr Ile Ile Asp Asp Phe Val Val
290 295 300
Gln Asp Glu Glu Gly Asp Glu Glu Asn Lys Asn Gln Gln Gly Glu
305 310 315
Lys Leu Thr Thr Ser Gln Leu Lys Leu Val Lys Arg Asn Ser Leu
320 325 330
Tyr Ser Phe Ser Asp His Tyr Thr His Phe Glu Arg Val Val Lys
335 340 345
Ala Leu Leu Ile Asn Ala Leu Asp Glu Ser Phe Leu Gly Thr Leu
350 355 360
Tyr Asp Gly Thr Arg Gln Lys Ser Tyr Ala Lys Asp Met Leu Thr
365 370 375
Ser Leu His Tyr Leu Asp Asn Arg Phe Val Gln Pro Arg Leu Glu
380 385 390
Ser Leu Val Ser Arg Ser Arg Trp Lys Glu Gln Tyr Lys Glu Arg
395 400 405
Val Glu Asn Tyr Ser Asn Val Ser Ile His Leu Lys Asn Pro Glu
410 415 420
Asn Cys Ser Cys Gln Ala Cys Gly Leu His Arg Tyr Cys Lys Tyr
425 430 435
Ser Val His Leu Ser Gly Glu Leu Tyr Asn Thr Arg Thr Met Gln
440 445 450
Ile Asp Asn Phe Met Ser His Asp Lys Gln Val Phe Thr Val Gly
455 460 465
Arg Ile Cys Ala Ser Arg Thr Arg Ile Tyr His Lys Leu Lys His
470 475 480
Phe Lys Phe Lys Leu Tyr Gln Glu Cys Cys Thr Ile Ala Met Thr
485 490 495
Glu Glu Val Glu Asp Glu Gln Val Lys Glu Thr Val Glu Arg Ile
500 505 510
Phe Arg Arg Ser Lys Glu Asn Gly Trp Ile Lys Glu Lys Tyr Gly
515 520 525
Gln Leu Glu Glu Tyr Leu Asn Phe Ala Asp Tyr Phe Gln Glu Glu
530 535 540
Lys Phe Glu Leu
<210> 18
<211> 869
<212> PRT
<213> Homo sapiens
<220>
15/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<221> misc_feature
<223> Incyte ID No: 2354751CD1
<400> 18
Met Arg Trp Gly His His Leu Pro Arg Ala Ser Trp Gly Ser Gly
1 5 10 15
Phe Arg Arg Ala Leu Gln Arg Pro Asp Asp Arg Ile Pro Phe Leu
20 25 30
Ile His Trp Ser Trp Pro Leu Gln Gly Glu Arg Pro Phe Gly Pro
35 40 45
Pro Arg Ala Phe Ile Arg His His Gly Ser Ser Val Asp Ser Ala
50 55 60
Pro Pro Pro Gly Arg His Gly Arg Leu Phe Pro Ser Ala Ser Ala
65 70 75
Thr Glu Ala Ile Gln Arg His Arg Arg Asn Leu Ala Glu Trp Phe
80 85 90
Ser Arg Leu Pro Arg Glu Glu Arg Gln Phe Gly Pro Thr Phe Ala
95 100 105
Leu Asp Thr Val His Val Asp Pro Val Ile Arg Glu Ser Thr Pro
110 115 120
Asp Glu Leu Leu Arg Pro Pro Ala Glu Leu Ala Leu Glu His Gln
125 130 135
Pro Pro Gln Ala Gly Leu Pro Pro Leu Ala Leu Ser Gln Leu Phe
140 145 150
Asn Pro Asp Ala Cys Gly Arg Arg Val Gln Thr Val Val Leu Tyr
155 160 165
Gly Thr Val Gly Thr Gly Lys Ser Thr Leu Val Arg Lys Met Val
170 175 180
Leu Asp Trp Cys Tyr Gly Arg Leu Pro Ala Phe Glu Leu Leu Ile
185 190 195
Pro Phe Ser Cys Glu Asp Leu Ser Ser Leu Gly Pro Ala Pro Ala
200 205 210
Ser Leu Cys Gln Leu Val Ala Gln Arg Tyr Thr Pro Leu Lys Glu
215 220 225
Val Leu Pro Leu Met Ala Ala Ala Gly Ser His Leu Leu Phe Val
230 235 240
Leu His Gly Leu Glu His Leu Asn Leu Asp Phe Arg Leu Ala Gly
245 250 255
Thr Gly Leu Cys Ser Asp Pro Glu Glu Pro Gln Glu Pro Ala Ala
260 265 270
Ile Ile Val Asn Leu Leu Arg Lys Tyr Met Leu Pro Gln Ala Ser
275 280 285
Ile Leu Val Thr Thr Arg Pro Ser Ala Ile Gly Arg Ile Pro Ser
290 295 300
Lys Tyr Val Gly Arg Tyr Gly Glu Ile Cys Gly Phe Ser Asp Thr
305 310 315
Asn Leu Gln Lys Leu Tyr Phe Gln Leu Arg Leu Asn Gln Pro Tyr
320 325 330
Cys Gly Tyr Ala Val Gly Gly Ser Gly Val Ser Ala Thr Pro Ala
335 340 345
Gln Arg Asp His Leu Val Gln Met Leu Ser Arg Asn Leu Glu Gly
350 355 360
His His Gln Ile Ala Ala Ala Cys Phe Leu Pro Ser Tyr Cys Trp
365 370 375
Leu Val Cys Ala Thr Leu His Phe Leu His Ala Pro Thr Pro Ala
380 385 390
Gly Gln Thr Leu Thr Ser Ile Tyr Thr Ser Phe Leu Arg Leu Asn
395 400 405
Phe Ser Gly Glu Thr Leu Asp Ser Thr Asp Pro Ser Asn Leu Ser
410 415 420
Leu Met Ala Tyr Ala Ala Arg Thr Met Gly Lys Leu Ala Tyr Glu
425 430 435
Gly Val Ser Ser Arg Lys Thr Tyr Phe Ser Glu Glu Asp Val Cys
440 445 450
Gly Cys Leu Glu Ala Gly Ile Arg Thr Glu Glu Glu Phe Gln Leu
455 460 465
Leu His Ile Phe Arg Arg Asp Ala Leu Arg Phe Phe Leu Ala Pro
16/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
470 475 480
Cys Val Glu Pro Gly Arg Ala Gly Thr Phe Val Phe Thr Val Pro
485 490 495
Ala Met Gln Glu Tyr Leu Ala Ala Leu Tyr Ile Val Leu Gly Leu
500 505 510
Arg Lys Thr Thr Leu Gln Lys Val Gly Lys Glu Val Ala Glu Leu
515 520 525
Val Gly Arg Val Gly Glu Asp Val Ser Leu Val Leu Gly Ile Met
530 535 540
Ala Lys Leu Leu Pro Leu Arg Ala Leu Pro Leu Leu Phe Asn Leu
545 550 555
Ile Lys Val Val Pro Arg Val Phe Gly Arg Met Val Gly Lys Ser
560 565 570
Arg Glu Ala Val Ala Gln Ala Met Val Leu Glu Met Phe Arg Glu
575 580 585
Glu Asp Tyr Tyr Asn Asp Asp Val Leu Asp Gln Met Gly Ala Ser
590 595 600
Ile Leu Gly Val Glu Gly Pro Arg Arg His Pro Asp Glu Pro Pro
605 610 615
Glu Asp Glu Val Phe Glu Leu Phe Pro Met Phe Met Gly Gly Leu
620 625 630
Leu Ser Ala His Asn Arg Ala Val Leu Ala Gln Leu Gly Cys Pro
635 640 645
Ile Lys Asn Leu Asp Ala Leu Glu Asn Ala Gln Ala Ile Lys Lys
650 655 660
Lys Leu Gly Lys Leu Gly Arg Gln Val Leu Pro Pro Ser Glu Leu
665 670 675
Leu Asp His Leu Phe Phe His Tyr Glu Phe Gln Asn Gln Arg Phe
680 685 690
Ser Ala Glu Val Leu Ser Ser Leu Arg Gln Leu Asn Leu Ala Gly
695 700 705
Val Arg Met Thr Pro Val Lys Cys Thr Val Val Ala Ala Val Leu
710 715 720
Gly Ser Gly Arg His Ala Leu Asp Glu Val Asn Leu Ala Ser Cys
725 730 735
Gln Leu Asp Pro Ala Gly Leu Arg Thr Leu Leu Pro Val Phe Leu
740 745 750
Arg Ala Arg Lys Leu Gly Leu Gln Leu Asn Ser Leu Gly Pro Glu
755 760 765
Ala Cys Lys Asp Leu Arg Asp Leu Leu Leu His Asp Gln Cys Gln
770 775 780
Ile Thr Thr Leu Arg Leu Ser Asn Asn Pro Leu Thr Glu Ala Gly
785 790 795
Val Ala Val Leu Met Glu Gly Leu Ala Gly Asn Thr Ser Val Thr
800 805 810
His Leu Ser Leu Leu His Thr Gly Leu Gly Asp Glu Gly Leu Glu
815 820 825
Leu Leu Ala Ala Gln Leu Asp Arg Asn Arg Gln Leu Gln Glu Leu
830 835 840
Asn Val Ala Tyr Asn Gly Ala Gly Asp Thr Ala Ala Leu Ala Leu
845 850 855
Ala Arg Ala Ala Arg Glu His Pro Ser Leu Glu Leu Leu Gln
860 865
<210> 19
<211> 128
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2378058CD1
<400> 19
Met Met Thr Ala Val Ser Leu Thr Thr Arg Pro Gln Glu Ser Val
1 5 10 15
Ala Phe Glu Asp Val Ala Val Tyr Phe Thr Thr Lys Glu Trp Ala
17/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
20 25 30
Ile Met Val Pro Ala Glu Arg Ala Leu Tyr Arg Asp Val Met Leu
35 40 45
Glu Asn Tyr Glu Ala Val Ala Phe Val Val Pro Pro Thr Ser Lys
50 55 60
Pro Ala Leu Val Ser His Leu Glu Gln Gly Lys Glu Ser Cys Phe
65 70 75
Thr Gln Pro Gln Gly Val Leu Ser Arg Asn Asp Trp Arg Ala Gly
80 85 90
Trp Ile Gly Tyr Leu Glu Leu Arg Arg Tyr Thr Tyr Leu Ala Lys
95 100 105
Ala Val Leu Arg Arg Ile Val Ser Lys Ile Phe Arg Asn Arg Gln
110 115 120
Cys Trp Glu Asp Arg Arg Lys Ala
125
<210> 20
<211> 301
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2595747CD1
<400> 20
Met Leu Leu Thr Gln Ala Arg Leu Phe Asp Glu Pro Gln Leu Ala
1 5 10 15
Ser Leu Cys Leu Asp Thr Ile Asp Lys Ser Thr Met Asp Ala Ile
20 25 30
Ser Ala Glu Gly Phe Thr Asp Ile Asp Ile Asp Thr Leu Cys Ala
35 40 45
Val Leu Glu Arg Asp Thr Leu Ser Ile Arg Glu Ser Arg Leu Phe
50 55 60
Gly Ala Val Val Arg Trp Ala Glu Ala Glu Cys Gln Arg Gln Gln
65 70 75
Leu Pro Val Thr Phe Gly Asn Lys Gln Lys Val Leu Gly Lys Ala
80 85 90
Leu Ser Leu Ile Arg Phe Pro Leu Met Thr Ile Glu Glu Phe Ala
95 100 105
Ala Gly Pro Ala Gln Ser Gly Ile Leu Ser Asp Arg Glu Val Val
110 115 120
Asn Leu Phe Leu His Phe Thr Val Asn Pro Lys Pro Arg Val Glu
125 130 135
Tyr Ile Asp Arg Pro Arg Cys Cys Leu Arg Gly Lys Glu Cys Cys
140 145 150
Ile Asn Arg Phe Gln Gln Val Glu Ser Arg Trp Gly Tyr Ser Gly
155 160 165
Thr Ser Asp Arg Ile Arg Phe Thr Val Asn Arg Arg Ile Ser Ile
170 175 180
Val Gly Phe Gly Leu Tyr Gly Ser Ile His Gly Pro Thr Asp Tyr
185 190 195
Gln Val Asn Ile Gln Ile Ile Glu Tyr Glu Lys Lys Gln Thr Leu
200 205 210
Gly Gln Asn Asp Thr Gly Phe Ser Cys Asp Gly Thr Ala Asn Thr
215 220 225
Phe Arg Val Met Phe Lys Glu Pro Ile Glu Ile Leu Pro Asn Val
230 235 240
Cys Tyr Thr Ala Cys Ala Thr Leu Lys Gly Pro Asp Ser His Tyr
245 250 255
Gly Thr Lys Gly Leu Lys Lys Val Val His Glu Thr Pro Ala Ala
260 265 270
Ser Lys Thr Val Phe Phe Phe Phe Ser Ser Pro Gly Asn Asn Asn
275 280 285
Gly Thr Ser Ile Glu Asp Gly Gln Ile Pro Glu Ile Ile Phe Tyr
290 295 300
Thr
18/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<210> 21
<211> 402
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2634391CD1
<400> 21
Met Ile Asp Gln Ala Ser Leu Tyr Gln Tyr Ser Pro Gln Asn Gln
1 5 10 15
His Val Glu Gln Gln Pro His Tyr Thr His Lys Pro Thr Leu Glu
20 25 30
Tyr Ser Pro Phe Pro Ile Pro Pro Gln Ser Pro Ala Tyr Glu Pro
35 40 45
Asn Leu Phe Asp Gly Pro Glu Ser Gln Phe Cys Pro Asn Gln Ser
50 55 60
Leu Val Ser Leu Leu Gly Asp Gln Arg Glu Ser Glu Asn Ile Ala
65 70 75
Asn Pro Met Gln Thr Ser Ser Ser Val Gln Gln Gln Asn Asp Ala
80 85 90
His Leu His Ser Phe Ser Met Met Pro Ser Ser Ala Cys Glu Ala
95 100 105
Met Val Gly His Glu Met Ala Ser Asp Ser Ser Asn Thr Ser Leu
110 115 120
Pro Phe Ser Asn Met Gly Asn Pro Met Asn Thr Thr Gln Leu Gly
125 130 135
Lys Ser Leu Phe Gln Trp Gln Val Glu Gln Glu Glu Ser Lys Leu
140 145 150
Ala Asn Ile Ser Gln Asp Gln Phe Leu Ser Lys Asp Ala Asp Gly
155 160 165
Asp Thr Phe Leu His Ile Ala Val Ala Gln Gly Arg Arg Ala Leu
170 175 180
Ser Tyr Val Leu Ala Arg Lys Met Asn Ala Leu His Met Leu Asp
185 190 195
Ile Lys Glu His Asn Gly Gln Ser Ala Phe Gln Val Ala Val Ala
200 205 210
Ala Asn Gln His Leu Ile Val Gln Asp Leu Val Asn Ile Gly Ala
215 220 225
Gln Val Asn Thr Thr Asp Cys Trp Gly Arg Thr Pro Leu His Val
230 235 240
Cys Ala Glu Lys Gly His Ser Gln Val Leu Gln Ala Ile Gln Lys
245 250 255
Gly Ala Val Gly Ser Asn Gln Phe Val Asp Leu Glu Ala Thr Asn
260 265 270
Tyr Asp Gly Leu Thr Pro Leu His Cys Ala Val Ile Ala His Asn
275 280 285
Ala Val Val His Glu Leu Gln Arg Asn Gln Gln Pro His Ser Pro
290 295 300
Glu Val Gln Glu Leu Leu Leu Lys Asn Lys Ser Leu Val Asp Thr
305 310 315
Ile Lys Cys Leu Ile Gln Met Gly Ala Ala Val Glu Ala Lys Ala
320 325 330
Tyr Asn Gly Asn Thr Ala Leu His Val Ala Ala Ser Leu Gln Tyr
335 340 345
Arg Leu Thr Gln Leu Asp Ala Val Arg Leu Leu Met Arg Lys Gly
350 355 360
Ala Asp Pro Ser Thr Arg Asn Leu Glu Asn Glu Gln Pro Val His
365 370 375
Leu Val Pro Asp Gly Pro Val Gly Glu Gln Ile Arg Arg Ile Leu
380 385 390
Lys Gly Lys Ser Ile Gln Gln Arg Ala Pro Pro Tyr
395 400
<210> 22
<211> 254
19/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2637522CD1
<400> 22
Met Asn Phe Thr Val Gly Phe Lys Pro Leu Leu Gly Asp Ala His
1 5 10 15
Ser Met Asp Asn Leu Glu Lys Gln Leu Ile Cys Pro Ile Cys Leu
20 25 30
Glu Met Phe Ser Lys Pro Val Val Ile Leu Pro Cys Gln His Asn
35 40 45
Leu Cys Arg Lys Cys Ala Asn Asp Val Phe Gln Ala Ser Asn Pro
50 55 60
Leu Trp Gln Ser Arg Gly Ser Thr Thr Val Ser Ser Gly Gly Arg
65 70 75
Phe Arg Cys Pro Ser Cys Arg His Glu Val Val Leu Asp Arg His
80 85 90
Gly Val Tyr Gly Leu Gln Arg Asn Leu Leu Val Glu Asn Ile Ile
95 100 105
Asp Ile Tyr Lys Gln Glu Ser Ser Arg Pro Leu His Ser Lys Ala
110 115 120
Glu Gln His Leu Met Cys Glu Glu His Glu Glu Glu Lys Ile Asn
125 130 135
Ile Tyr Cys Leu Ser Cys Glu Val Pro Thr Cys Ser Leu Cys Lys
140 145 150
Val Phe Gly Ala His Lys Asp Cys Glu Val Ala Pro Leu Pro Thr
155 160 165
Ile Tyr Lys Arg Gln Lys Ser Glu Leu Ser Asp Gly Ile Ala Met
170 175 180
Leu Val Ala Gly Asn Asp Arg Val Gln Ala Val Ile Thr Gln Met
185 190 195
Glu Glu Val Cys Gln Thr Ile Glu Asp Asn Ser Arg Arg Gln Lys
200 205 210
Gln Leu Leu Asn Gln Arg Phe Glu Ser Leu Cys Ala Val Leu Glu
215 220 225
Glu Arg Asn Gly Glu Leu Leu Gln Ala Leu Ala Arg Glu Gln Ala
230 235 240
Gly Gln Ala Ser Thr Arg Ser Asp Gly Thr His Ser Gly Gln
245 250
<210> 23
<211> 553
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2650980CD1
<400> 23
Met Ala Thr Asp Thr Ser Gln Gly Glu Leu Val His Pro Lys Ala
1 5 10 15
Leu Pro Leu Ile Val Gly Ala Gln Leu Ile His Ala Asp Lys Leu
20 25 30
Gly Glu Lys Val Glu Asp Ser Thr Met Pro Ile Arg Arg Thr Val
35 40 45
Asn Ser Thr Arg Glu Thr Pro Pro Lys Ser Lys Leu Ala Glu Gly
50 55 60
Glu Glu Glu Lys Pro Glu Pro Asp Ile Ser Ser Glu Glu Ser Val
65 70 75
Ser Thr Val Glu Glu Gln Glu Asn Glu Thr Pro Pro Ala Thr Ser
80 85 90
Ser Glu Ala Glu Gln Pro Lys Gly Glu Pro Glu Asn Glu Glu Lys
95 100 105
2~/SO
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
Glu Glu Asn Lys Ser Ser Glu Glu Thr Lys Lys Asp Glu Lys Asp
110 115 120
Gln Ser Lys Glu Lys Glu Lys Lys Val Lys Lys Thr Ile Pro Ser
125 130 135
Trp Ala Thr Leu Ser Ala Ser Gln Leu Ala Arg Ala Gln Lys Gln
140 145 150
Thr Pro Met Ala Ser Ser Pro Arg Pro Lys Met Asp Ala Ile Leu
155 160 165
Thr Glu Ala Ile Lys Ala Cys Phe Gln Lys Ser Gly Ala Ser Val
170 175 180
Val Ala Ile Arg Lys Tyr Ile Ile His Lys Tyr Pro Ser Leu Glu
185 190 195
Leu Glu Arg Arg Gly Tyr Leu Leu Lys Gln Ala Leu Lys Arg Glu
200 205 210
Leu Asn Arg Gly Val Ile Lys Gln Val Lys Gly Lys Gly Ala Ser
215 220 225
Gly Ser Phe Val Val Val Gln Lys Ser Arg Lys Thr Pro Gln Lys
230 235 240
Ser Arg Asn Arg Lys Asn Arg Ser Ser Ala Val Asp Pro Glu Pro
245 250 255
Gln Val Lys Leu Glu Asp Val Leu Pro Leu Ala Phe Thr Arg Leu
260 265 270
Cys Glu Pro Lys Glu Ala Ser Tyr Ser Leu Ile Arg Lys Tyr Val
275 280 285
Ser Gln Tyr Tyr Pro Lys Leu Arg Val Asp Ile Arg Pro Gln Leu
290 295 300
Leu Lys Asn Ala Leu Gln Arg Ala Val Glu Arg Gly Gln Leu Glu
305 310 315
Gln Ile Thr Gly Lys Gly Ala Ser Gly Thr Phe Gln Leu Lys Lys
320 325 330
Ser Gly Glu Lys Pro Leu Leu Gly Gly Ser Leu Met Glu Tyr Ala
335 340 345
Ile Leu Ser Ala Ile Ala Ala Met Asn Glu Pro Lys Thr Cys Ser
350 355 360
Thr Thr Ala Leu Lys Lys Tyr Val Leu Glu Asn His Pro Gly Thr
365 370 375
Asn Ser Asn Tyr Gln Met His Leu Leu Lys Lys Thr Leu Gln Lys
380 385 390
Cys Glu Lys Asn Gly Trp Met Glu Gln Ile Ser Gly Lys Gly Phe
395 400 405
Ser Gly Thr Phe Gln Leu Cys Phe Pro Tyr Tyr Pro Ser Pro Gly
410 415 420
Val Leu Phe Pro Lys Lys Glu Pro Asp Asp Ser Arg Asp Glu Asp
425 430 435
Glu Asp Glu Asp Glu Ser Ser Glu Glu Asp Ser Glu Asp Glu Glu
440 445 450
Pro Pro Pro Lys Arg Arg Leu Gln Lys Lys Thr Pro Ala Lys Ser
455 460 465
Pro Gly Lys Ala Ala Ser Val Lys Gln Arg Gly Ser Lys Pro Ala
470 475 480
Pro Lys Val Ser Ala Ala Gln Arg Gly Lys Ala Arg Pro Leu Pro
485 490 495
Lys Lys Ala Pro Pro Lys Ala Lys Thr Pro Ala Lys Lys Thr Arg
500 505 510
Pro Ser Ser Thr Val Ile Lys Lys Pro Ser Gly Gly Ser Ser Lys
515 520 525
Lys Pro Ala Thr Ser Ala Arg Lys Glu Val Lys Leu Pro Gly Lys
530 535 540
Gly Lys Ser Thr Met Lys Lys Ser Phe Arg Val Lys Lys
545 550
<210> 24
<211> 461
<212> PRT
<213> Homo Sapiens
<220>
21 /50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<221> misc_feature
<223> Incyte ID No: 2939607CD1
<400> 24
Met Asp Ala Lys Ser Leu Thr Ala Trp Ser Arg Thr Leu Val Thr
1 5 10 15
Phe Lys Asp Val Phe Val Asp Phe Thr Arg Glu Glu Trp Lys Leu
20 25 30
Leu Asp Thr Ala Gln Gln Ile Val Tyr Arg Asn Val Met Leu Glu
35 40 45
Asn Tyr Lys Asn Leu Val Ser Leu Gly Tyr Gln Leu Thr Lys Pro
50 55 60
Asp Val Ile Leu Arg Leu Glu Lys Gly Glu Glu Pro Trp Leu Val
65 70 75
Glu Arg Glu Ile His Gln Glu Thr His Pro Asp Ser Glu Thr Ala
80 85 90
Phe Glu Ile Lys Ser Ser Val Ser Ser Arg Ser Ile Phe Lys Asp
95 100 105
Lys Gln Ser Cys Asp Ile Lys Met Glu Gly Met Ala Arg Asn Asp
110 115 120
Leu Trp Tyr Leu Ser Leu Glu Glu Val Trp Lys Cys Arg Asp Gln
125 130 135
Leu Asp Lys Tyr Gln Glu Asn Pro Glu Arg His Leu Arg Gln Val
140 145 150
Ala Phe Thr Gln Lys Lys Val Leu Thr Gln Glu Arg Val Ser Glu
155 160 165
Ser Gly Lys Tyr Gly Gly Asn Cys Leu Leu Pro Ala Gln Leu Val
170 175 180
Leu Arg Glu Tyr Phe His Lys Arg Asp Ser His Thr Lys Ser Leu
185 190 195
Lys His Asp Leu Val Leu Asn Gly His Gln Asp Ser Cys Ala Ser
200 205 210
Asn Ser Asn Glu Cys Gly Gln Thr Phe Cys Gln Asn Ile His Leu
215 220 225
Ile Gln Phe Ala Arg Thr His Thr Gly Asp Lys Ser Tyr Lys Cys
230 235 240
Pro Asp Asn Asp Asn Ser Leu Thr His Gly Ser Ser Leu Gly Ile
245 250 255
Ser Lys Gly Ile His Arg Glu Lys Pro Tyr Glu Cys Lys Glu Cys
260 265 270
Gly Lys Phe Phe Ser Trp Arg Ser Asn Leu Thr Arg His Gln Leu
275 280 285
Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys
290 295 300
Ser Tyr Ser Gln Arg Ser His Leu Val Val His His Arg Ile His
305 310 315
Thr Gly Leu Lys Pro Phe Glu Cys Lys Asp Cys Gly Lys Cys Phe
320 325 330
Ser Arg Ser Ser His Leu Tyr Ser His Gln Arg Thr His Thr Gly
335 340 345
Glu Lys Pro Tyr Glu Cys His Asp Cys Gly Lys Ser Phe Ser Gln
350 355 360
Ser Ser Ala Leu Ile Val His Gln Arg Ile His Thr Gly Glu Lys
365 370 375
Pro Tyr Glu Cys Cys Gln Cys Gly Lys Ala Phe Ile Arg Lys Asn
380 385 390
Asp Leu Ile Lys His Gln Arg Ile His Val Gly Glu Glu Thr Tyr
395 400 405
Lys Cys Asn Gln Cys Gly Ile Ile Phe Ser Gln Asn Ser Pro Phe
410 415 420
Ile Val His Gln Ile Ala His Thr Gly Glu Gln Phe Leu Thr Cys
425 430 435
Asn Gln Cys Gly Thr Ala Leu Val Asn Thr Ser Asn Leu Ile Gly
440 445 450
Tyr Gln Thr Asn His Ile Arg Glu Lys Ala Tyr
455 460
22/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<210> 25
<211> 159
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3098421CD1
<400> 25
Met Asp Lys Pro Arg Lys Glu Asn Glu Glu Glu Pro Gln Ser Ala
1 5 10 15
Pro Lys Thr Asp Glu Glu Arg Pro Pro Val Glu His Ser Pro Glu
20 25 30
Lys Gln Ser Pro Glu Glu Gln Ser Ser Glu Glu Gln Ser Ser Glu
35 40 45
Glu Glu Phe Phe Pro Glu Glu Leu Leu Pro Glu Leu Leu Pro Glu
50 55 60
Met Leu Leu Ser Glu Glu Arg Pro Pro Gln Glu Gly Leu Ser Arg
65 70 75
Lys Asp Leu Phe Glu Gly Arg Pro Pro Met Glu Gln Pro Pro Cys
80 85 90
Gly Val Gly Lys His Lys Leu Glu Glu Gly Ser Phe Lys Glu Arg
95 100 105
Leu Ala Arg Ser Arg Pro Gln Phe Arg Gly Asp Ile His Gly Arg
110 115 120
Asn Leu Ser Asn Glu Glu Met Ile Gln Ala Ala Asp Glu Leu Glu
125 130 135
Glu Met Lys Arg Val Arg Asn Lys Leu Met Ile Met His Trp Lys
140 145 150
Ala Lys Arg Ser Arg Pro Tyr Pro Ile
155
<210> 26
<211> 373
<212> PRT
<213> Homo sapiens
<220>
<221> unsure
<222> 368
<223> unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 3296650CD1
<400> 26
Met Lys Ala Leu Phe Lys His Glu Ser Leu Gly Ser Gln Pro Leu
1 5 10 15
His Asp Arg Val Leu Gln Val Pro Gly Leu Ala Gln Gly Gly Cys
20 25 30
Cys Arg Glu Asp Ala Met Val Ala Ser Arg Leu Thr Pro Gly Ser
35 40 45
Gln Gly Leu Leu Lys Met Glu Asp Val Ala Leu Thr Leu Thr Pro
50 55 60
Gly Trp Thr Gln Leu Asp Ser Ser Gln Val Asn Leu Tyr Arg Asp
65 70 75
Glu Lys Gln Glu Asn His Ser Ser Leu Val Ser Leu Gly Gly Glu
80 85 90
Ile Gln Thr Lys Ser Arg Asp Leu Pro Pro Val Lys Lys Leu Pro
95 100 105
Glu Lys Glu His Gly Lys Ile Cys His Leu Arg Glu Asp Ile Ala
110 115 120
Gln Ile Pro Thr His Ala Glu Ala Gly Glu Gln Glu Gly Arg Leu
125 130 135
Gln Arg Lys Gln Lys Asn Ala Ile Gly Ser Arg Arg His Tyr Cys
23/50
Tyr Gln Thr Asn His Ile Arg Glu Lys Ala Tyr
455 460
22/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
140 145 150
His Glu Cys Gly Lys Ser Phe Ala Gln Ser Ser Gly Leu Thr Lys
155 160 165
His Arg Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Glu Asp
170 175 180
Cys Gly Lys Thr Phe Ile Gly Ser Ser Ala Leu Val Ile His Gln
185 190 195
Arg Val His Thr Gly Glu Lys Pro Tyr Glu Cys Glu Glu Cys Gly
200 205 210
Lys Val Phe Ser His Ser Ser Asn Leu Ile Lys His Gln Arg Thr
215 220 225
His Thr Gly Glu Lys Pro Tyr Glu Cys Asp Asp Cys Gly Lys Thr
230 235 240
Phe Ser Gln Ser Cys Ser Leu Leu Glu His His Lys Ile His Thr
245 250 255
Gly Glu Lys Pro Tyr Gln Cys Asn Met Cys Gly Lys Ala Phe Arg
260 265 270
Arg Asn Ser His Leu Leu Arg His Gln Arg Ile His Gly Asp Lys
275 280 285
Asn Val Gln Asn Pro Glu His Gly Glu Ser Trp Glu Ser Gln Gly
290 295 300
Arg Thr Glu Ser Gln Trp Glu Asn Thr Glu Ala Pro Val Ser Tyr
305 310 315
Lys Cys Asn Glu Cys Glu Arg Ser Phe Thr Arg Asn Arg Ser Leu
320 325 330
Ile Glu His Gln Lys Ile His Thr Gly Asp Lys Pro Tyr Gln Cys
335 340 345
Asp Thr Cys Gly Lys Gly Phe Thr Arg Thr Ser Tyr Leu Val Gln
350 355 360
His Gln Arg Ser His Val Gly Xaa Lys Thr Leu Ser Gln
365 370
<210> 27
<211> 330
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3687719CD1
<400> 27
Met Gln Gln Gln Pro Leu Pro Gly Pro Gly Ala Pro Thr Thr Glu
1 5 10 15
Pro Thr Lys Pro Pro Tyr Ser Tyr Ile Ala Leu Ile Ala Met Ala
20 25 30
Ile Gln Ser Ser Pro Gly Gln Arg Ala Thr Leu Ser Gly Ile Tyr
35 40 45
Arg Tyr Ile Met Gly Arg Phe Ala Phe Tyr Arg His Asn Arg Pro
50 55 60
Gly Trp Gln Asn Ser Ile Arg His Asn Leu Ser Leu Asn Glu Cys
65 70 75
Phe Val Lys Val Pro Arg Asp Asp Arg Lys Pro Gly Lys Gly Ser
80 85 90
Tyr Trp Thr Leu Asp Pro Asp Cys His Asp Met Phe Glu His Gly
95 100 105
Ser Phe Leu Arg Arg Arg Arg Arg Phe Thr Arg Gln Thr Gly Ala
110 115 120
Glu Gly Thr Arg Gly Pro Ala Lys Ala Arg Arg Gly Pro Leu Arg
125 130 135
Ala Thr Ser Gln Asp Pro Gly Val Pro Asn Ala Thr Thr Gly Arg
140 145 150
Gln Cys Ser Phe Pro Pro Glu Leu Pro Asp Pro Lys Gly Leu Ser
155 160 165
Phe Gly Gly Leu Val Gly Ala Met Pro Ala Ser Met Cys Pro Ala
170 175 180
Thr Thr Asp Gly Arg Pro Arg Pro Pro Met Glu Pro Lys Glu Ile
24/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
185 190 195
Ser Thr Pro Lys Pro Ala Cys Pro Gly Glu Leu Pro Val Ala Thr
200 205 210
Ser Ser Ser Ser Cys Pro Ala Phe Gly Phe Pro Ala Gly Phe Ser
215 220 225
Glu Ala Glu Ser Phe Asn Lys Ala Pro Thr Pro Val Leu Ser Pro
230 235 240
Glu Ser Gly Ile Gly Ser Ser Tyr Gln Cys Arg Leu Gln Ala Leu
245 250 255
Asn Phe Cys Met Gly Ala Asp Pro Gly Leu Glu His Leu Leu Ala
260 265 270
Ser Ala Ala Pro Ser Pro Ala Pro Pro Thr Pro Pro Gly Ser Leu
275 280 285
Arg Ala Pro Leu Pro Leu Pro Thr Asp His Lys Glu Pro Trp Val
290 295 300
Ala Gly Gly Phe Pro Val Gln Gly Gly Ser Gly Tyr Pro Leu Gly
305 310 315
Leu Thr Pro Cys Leu Tyr Arg Thr Pro Gly Met Phe Phe Phe Glu
320 325 330
<210> 28
<211> 396
<212> PRT
<213> Homo sapiens
<220>
<221> misC_feature
<223> Incyte ID No: 3774188CD1
<400> 28
Met Lys Ala Val Lys Ser Glu Arg Glu Arg Gly Ser Arg Arg Arg
1 5 10 15
His Arg Asp Gly Asp Val Val Leu Pro Ala Gly Val Val Val Lys
20 25 30
Gln Glu Arg Leu Ser Pro Glu Val Ala Pro Pro Ala His Arg Arg
35 40 45
Pro Asp His Ser Gly Gly Ser Pro Ser Pro Pro Thr Ser Glu Pro
50 55 60
Ala Arg Ser Gly His Arg Gly Asn Arg Ala Arg Gly Val Ser Arg
65 70 75
Ser Pro Pro Lys Lys Lys Asn Lys Ala Ser Gly Arg Arg Ser Lys
80 85 90
Ser Pro Arg Ser Lys Arg Asn Arg Ser Pro His His Ser Thr Val
95 100 105
Lys Val Lys Gln Glu Arg Glu Asp His Pro Arg Arg Gly Arg Glu
110 115 120
Asp Arg Gln His Arg Glu Pro Ser Glu Gln Glu His Arg Arg Ala
125 130 135
Arg Asn Ser Asp Arg Asp Arg His Arg Gly His Ser His Gln Arg
140 145 150
Arg Thr Ser Asn Glu Arg Pro Gly Ser Gly Gln Gly Gln Gly Arg
155 160 165
Asp Arg Asp Thr Gln Asn Leu Gln Ala Gln Glu Glu Glu Arg Glu
170 175 180
Phe Tyr Asn Ala Arg Arg Arg Glu His Arg Gln Arg Asn Asp Val
185 190 195
Gly Gly Gly Gly Ser Glu Ser Gln Glu Leu Val Pro Arg Pro Gly
200 205 210
Gly Asn Asn Lys Glu Lys Glu Val Pro Ala Lys Glu Lys Pro Ser
215 220 225
Phe Glu Leu Ser Gly Ala Leu Leu Glu Asp Thr Asn Thr Phe Arg
230 235 240
Gly Val Val Ile Lys Tyr Ser Glu Pro Pro Glu Ala Arg Ile Pro
245 250 255
Lys Lys Arg Trp Arg Leu Tyr Pro Phe Lys Asn Asp Glu Val Leu
260 265 270
Pro Val Met Tyr Ile His Arg Gln Ser Ala Tyr Leu Leu Gly Arg
25/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
275 280 285
His Arg Arg Ile Ala Asp Ile Pro Ile Asp His Pro Ser Cys Ser
290 295 300
Lys Gln His Ala Val Phe Gln Tyr Arg Leu Val Glu Tyr Thr Arg
305 310 315
Ala Asp Gly Thr Val Gly Arg Arg Val Lys Pro Tyr Ile Ile Asp
320 325 330
Leu Gly Ser Gly Asn Gly Thr Phe Leu Asn Asn Lys Arg Ile Glu
335 340 345
Pro Gln Arg Tyr Tyr Glu Leu Lys Glu Lys Asp Val Leu Lys Phe
350 355 360
Gly Phe Ser Ser Arg Glu Tyr Val Leu Leu His Glu Ser Ser Asp
365 370 375
Thr Ser Glu Ile Asp Arg Lys Asp Asp Glu Asp Glu Glu Glu Glu
380 385 390
Glu Glu Val Ser Asp Ser
395
<210> 29
<211> 126
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4349106CD1
<400> 29
Met Gly Leu Leu Thr Phe Arg Asp Val Ala Ile Glu Phe Ser Arg
1 5 10 15
Glu Glu Trp Glu His Leu Asp Ser Asp Gln Lys Leu Leu Tyr Gly
20 25 30
Asp Val Met Leu Glu Asn Tyr Gly Asn Leu Val Ser Leu Gly Leu
35 40 45
Ala Val Ser Lys Pro Asp Leu Ile Thr Phe Leu Glu Gln Arg Lys
50 55 60
Glu Pro Trp Asn Val Lys Ser Ala Glu Thr Val Ala Ile Gln Pro
65 70 75
Asp Ile Phe Ser His Asp Thr Gln Gly Leu Leu Arg Lys Lys Leu
80 85 90
Ile Glu Ala Ser Phe Gln Lys Val Ile Leu Asp Gly Tyr Gly Ser
95 100 105
Cys Gly Pro Gln Asn Leu Asn Leu Arg Lys Glu Trp Glu Ser Glu
110 115 120
Gly Lys Ile Ile Leu Trp
125
<210> 30
<211> 519
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4834217CD1
<400> 30
Met Ala Ala Glu Ala Ala Asp Leu Gly Leu Gly Ala Ala Val Pro
1 5 10 15
Val Glu Leu Arg Arg Glu Arg Arg Met Val Cys Val Glu Tyr Pro
20 25 30
Gly Val Val Arg Asp Val Ala Lys Met Leu Pro Thr Leu Gly Gly
35 40 45
Glu Glu Gly Val Ser Arg Ile Tyr Ala Asp Pro Thr Lys Arg Leu
50 55 60
Glu Leu Tyr Phe Arg Pro Lys Asp Pro Tyr Cys His Pro Val Cys
65 70 75
26/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
Ala Asn Arg Phe Ser Thr Ser Ser Leu Leu Leu Arg Ile Arg Lys
80 85 90
Arg Thr Arg Arg Gln Lys Gly Val Leu Gly Thr Glu Ala His Ser
95 100 105
Glu Val Thr Phe Asp Met Glu Ile Leu Gly Ile Ile Ser Thr Ile
110 115 120
Tyr Lys Phe Gln Gly Met Ser Asp Phe Gln Tyr Leu Ala Val His
125 130 135
Thr Glu Ala Gly Gly Lys His Thr Ser Met Tyr Asp Lys Val Leu
140 145 150
Met Leu Arg Pro Glu Lys Glu Ala Phe Phe His Gln Glu Leu Pro
155 160 165
Leu Tyr Ile Pro Pro Pro Ile Phe Ser Arg Leu Asp Ala Pro Val
170 175 180
Asp Tyr Phe Tyr Arg Pro Glu Thr Gln His Arg Glu Gly Tyr Asn
185 190 195
Asn Pro Pro Ile Ser Gly Glu Asn Leu Ile Gly Leu Ser Arg Ala
200 205 210
Arg Arg Pro His Asn Ala Ile Phe Val Asn Phe Glu Asp Glu Glu
215 220 225
Val Pro Lys Gln Pro Leu Glu Ala Ala Ala Gln Thr Trp Arg Arg
230 235 240
Val Cys Thr Asn Pro Val Asp Arg Lys Val Glu Glu Glu Leu Arg
245 250 255
Lys Leu Phe Asp Ile Arg Pro Ile Trp Ser Arg Asn Ala Val Lys
260 265 270
Ala Asn Ile Ser Val His Pro Asp Lys Leu Lys Val Leu Leu Pro
275 280 285
Phe Ile Ala Tyr Tyr Met Ile Thr Gly Pro Trp Arg Ser Leu Trp
290 295 300
Ile Arg Phe Gly Tyr Asp Pro Arg Lys Asn Pro Asp Ala Lys Ile
305 310 315
Tyr Gln Val Leu Asp Phe Arg Ile Arg Cys Gly Met Lys His Gly
320 325 330
Tyr Ala Pro Ser Asp Leu Pro Val Lys Ala Lys Arg Ser Thr Tyr
335 340 345
Asn Tyr Ser Leu Pro Ile Thr Val Lys Lys Thr Ser Ser Gln Leu
350 355 360
Val Thr Met His Asp Leu Lys Gln Gly Leu Gly Pro Ser Gly Thr
365 370 375
Ser Gly Ala Arg Lys Pro Ala Ser Ser Lys Tyr Lys Leu Lys Asp
380 385 390
Ser Val Tyr Ile Phe Arg Glu Gly Ala Leu Pro Pro Tyr Arg Gln
395 400 405
Met Phe Tyr Gln Leu Cys Asp Leu Asn Val Glu Glu Leu Gln Lys
410 415 420
Ile Ile His Arg Asn Asp Gly Ala Glu Asn Ser Cys Thr Glu Arg
425 430 435
Asp Gly Trp Cys Leu Pro Lys Thr Ser Asp Glu Leu Arg Asp Thr
440 445 450
Met Ser Leu Met Ile Arg Gln Thr Ile Arg Ser Lys Arg Pro Ala
455 460 465
Leu Phe Ser Ser Ser Ala Lys Ala Asp Gly Gly Lys Glu Gln Leu
470 475 480
Thr Tyr Glu Ser Gly Glu Asp Glu Glu Asp Glu Glu Glu Glu Glu
485 490 495
Glu Glu Glu Glu Asp Phe Lys Pro Ser Asp Gly Ser Glu Asn Glu
500 505 510
Met Glu Thr Glu Ile Leu Asp Tyr Val
515
<210> 31
<211> 493
<212> PRT
<213> Homo sapiens
<220>
27/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<221> misc_feature
<223> Incyte ID No: 5156094CD1
<400> 31
Met Ile Leu Asn Ser Leu Ser Leu Cys Tyr His Asn Lys Leu Ile
1 5 10 15
Leu Ala Pro Met Val Arg Val Gly Thr Leu Pro Met Arg L'eu Leu
20 25 30
Ala Leu Asp Tyr Gly Ala Asp Ile Val Tyr Cys Glu Glu Leu Ile
35 40 45
Asp Leu Lys Met Ile Gln Cys Lys Arg Val Val Asn Glu Val Leu
50 55 60
Ser Thr Val Asp Phe Val Ala Pro Asp Asp Arg Val Val Phe Arg
65 70 75
Thr Cys Glu Arg Glu Gln Asn Arg Val Val Phe Gln Met Gly Thr
80 85 90
Ser Asp Ala Glu Arg Ala Leu Ala Val Ala Arg Leu Val Glu Asn
95 100 105
Asp Val Ala Gly Ile Asp Val Asn Met Gly Cys Pro Lys Gln Tyr
110 115 120
Ser Thr Lys Gly Gly Met Gly Ala Ala Leu Leu Ser Asp Pro Asp
125 130 135
Lys Ile Glu Lys Ile Leu Ser Thr Leu Val Lys Gly Thr Arg Arg
140 145 150
Pro Val Thr Cys Lys Ile Arg Ile Leu Pro Ser Leu Glu Asp Thr
155 160 165
Leu Ser Leu Val Lys Arg Ile Glu Arg Thr Gly Ile Ala Ala Ile
170 175 180
Ala Val His Gly Arg Lys Arg Glu Glu Arg Pro Gln His Pro Val
185 190 195
Ser Cys Glu Val Ile Lys Ala Ile Ala Asp Thr Leu Ser Ile Pro
200 205 210
Val Ile Ala Asn Gly Gly Ser His Asp His Ile Gln Gln Tyr Ser
215 220 225
Asp Ile Glu Asp Phe Arg Gln Ala Thr Ala Ala Ser Ser Val Met
230 235 240
Val Ala Arg Ala Ala Met Trp Asn Pro Ser Ile Phe Leu Lys Glu
245 250 255
Gly Leu Arg Pro Leu Glu Glu Val Met Gln Lys Tyr Ile Arg Tyr
260 265 270
Ala Val Gln Tyr Asp Asn His Tyr Thr Asn Thr Lys Tyr Cys Leu
275 280 285
Cys Gln Met Leu Arg Glu Gln Leu Glu Ser Pro Gln Gly Arg Leu
290 295 300
Leu His Ala Ala Gln Ser Ser Arg Glu Ile Cys Glu Ala Phe Gly
305 310 315
Leu Gly Ala Phe Tyr Glu Glu Thr Thr Gln Glu Leu Asp Ala Gln
320 325 330
Gln Ala Arg Leu Ser Ala Lys Thr Ser Glu Gln Thr Gly Glu Pro
335 340 345
Ala Glu Asp Thr Ser Gly Val Ile Lys Met Ala Val Lys Phe Asp
350 355 360
Arg Arg Ala Tyr Pro Ala Gln Ile Thr Pro Lys Met Cys Leu Leu
365 370 375
Glu Trp Cys Arg Arg Glu Lys Leu Ala Gln Pro Val Tyr Glu Thr
380 385 390
Val Gln Arg Pro Leu Asp Arg Leu Phe Ser Ser Ile Val Thr Val
395 400 405
Ala Glu Gln Lys Tyr Gln Ser Thr Leu Trp Asp Lys Ser Lys Lys
410 415 420
Leu Ala Glu Gln Ala Ala Ala Ile Val Cys Leu Arg Ser Gln Gly
425 430 435
Leu Pro Glu Gly Arg Leu Gly Glu Glu Ser Pro Ser Leu His Lys
440 445 450
Arg Lys Arg Glu Ala Pro Asp Gln Asp Pro Gly Gly Pro Arg Ala
455 460 465
Gln Glu Leu Ala Gln Pro Gly Asp Leu Cys Lys Lys Pro Phe Val
28/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
470 475 480
Ala Leu Gly Ser Gly Glu Glu Ser Pro Leu Glu Gly Trp
485 490
<210> 32
<211> 516
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5665139CD1
<400> 32
Met His Gly Arg Lys Asp Asp Ala Gln Lys Gln Pro Val Lys Asn
1 5 10 15
Gln Leu Gly Leu Asn Pro Gln Ser His Leu Pro Glu Leu Gln Leu
20 25 30
Phe Gln Ala Glu Gly Lys Ile Tyr Lys Tyr Asp His Met Glu Lys
35 40 45
Ser Val Asn Ser Ser Ser Leu Val Ser Pro Pro Gln Arg Ile Ser
50 55 60
Ser Thr Val Lys Thr His Ile Ser His Thr Tyr Glu Cys Asn Phe
65 70 75
Val Asp Ser Leu Phe Thr Gln Lys Glu Lys Ala Asn Ile Gly Thr
80 85 90
Glu His Tyr Lys Cys Asn Glu Arg Gly Lys Ala Phe His Gln Gly
95 100 105
Leu His Phe Thr Ile His Gln Ile Ile His Thr Lys Glu Thr Gln
110 115 120
Phe Lys Cys Asp Ile Cys Gly Lys Ile Phe Asn Lys Lys Ser Asn
125 130 135
Leu Ala Ser His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys
140 145 150
Cys Asn Glu Cys Gly Lys Val Phe His Asn Met Ser His Leu Ala
155 160 165
Gln His Arg Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asn
170 175 180
Glu Cys Gly Lys Val Phe Asn Gln Ile Ser His Leu Ala Gln His
185 190 195
Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys
200 205 210
Gly Lys Val Phe His Gln Ile Ser His Leu Ala Gln His Arg Thr
215 220 225
Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Lys Cys Gly Lys
230 235 240
Val Phe Ser Arg Asn Ser Tyr Leu Val Gln His Leu Ile Ile His
245 250 255
Thr Gly Glu Lys Pro Tyr Arg Cys Asn Val Cys Gly Lys Val Phe
260 265 270
His His Ile Ser His Leu Ala Gln His Gln Arg Ile His Thr Gly
275 280 285
Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Val Phe Ser His
290 295 300
Lys Ser Ser Leu Val Asn His Trp Arg Ile His Thr Gly Glu Lys
305 310 315
Pro Tyr Lys Cys Asn Glu Cys Gly Lys Val Phe Ser His Lys Ser
320 325 330
Ser Leu Val Asn His Trp Arg Ile His Thr Gly Glu Lys Pro Tyr
335 340 345
Lys Cys Asn Glu Cys Gly Lys Val Phe Ser Arg Asn Ser Tyr Leu
350 355 360
Ala Gln His Leu Ile Ile His Ala Gly Glu Lys Pro Tyr Lys Cys
365 370 375
Asp Glu Cys Asp Lys Ala Phe Ser Gln Asn Ser His Leu Val Gln
380 385 390
His His Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asp Glu
29/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
395 400 405
Cys Gly Lys Val Phe Ser Gln Asn Ser Tyr Leu Ala Tyr His Trp
410 415 420
Arg Ile His Thr Gly Glu Lys Ala Tyr Lys Cys Asn Glu Cys Gly
425 430 435
Lys Val Phe Gly Leu Asn Ser Ser Leu Ala His His Arg Lys Ile
440 445 450
His Thr Gly Glu Lys Pro Phe Lys Cys Asn Glu Cys Gly Lys Ala
455 460 465
Phe Ser Met Arg Ser Ser Leu Thr Asn His His Ala Ile His Thr
470 475 480
Gly Glu Lys His Phe Lys Cys Asn Glu Cys Gly Lys Leu Phe Arg
485 490 495
Asp Asn Ser Tyr Leu Val Arg His Gln Arg Phe His Ala Gly Lys
500 505 510
Lys Ser Asn Thr Cys Asn
515
<210> 33
<211> 3163
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 091502CB1
<400> 33
gtcggaggtc ttacccaaca gattgacgcg gcgttagtat tggccgtgta cccgaaaaac 60
tgattgactg ggctggcgtt aactgtgcgg aggcaggatt tccctgggga aagtcttcac 120
tcagagcttc gctgacagcc aaggatggca agcaaggggc cctcggcctc tgcatctcct 180
gagaactcca gtgcaggggg gcccagtggg agcagcaatg gcgctggcga gagcggaggg 240
caggacagca ctttcgagtg caacatctgc ttggacacag ccaaggatgc cgtcatcagc 300
ctgtgtggcc acctcttctg ttggccgtgt ttacatcagt ggttggagac cagacctaac 360
agacaggtgt gtcctgtttg caaagctggc atcagccgag acaaggtcat ccccctctat 420
ggaaggggca gcactgggca acaggacccc agagagaaga cccctcctcg tcctcaagga 480
cagaggccag agccggagaa tagaggggga tttcaaggat ttggatttgg agatggtggc 540
ttccagatgt cttttggaat tggggcattt ccctttggga tatttgccac agcatttaat 600
ataaatgatg ggcggcctcc tccagctgtc cctgggacac cccagtatgt ggacgagcag 660
ttcctgtcac gcctcttcct atttgtggcc ctggtgatca tgttctggct cctgattgcc 720
taatgctggg ctcctgccta catccgtggc agggctctgg actggtgacg tgccacccca 780
actcctggtg tttggcttcc tggctaatct tgactcctgg aatcagtggg atcagtaaca 840
catcaaggag tcttgtttct tcatcagagc tttggaactc gagaccagtt ggcgatgacc 900
cctgaatatc gccaccgctg taaacactct ataacttcag gccttggcat tgagtcatct 960
ctcatgggtg acaccatgaa atcttgtttc agccagttct gcaggtcctg actctgcaga 1020
gggaagaggc agaaagagag aaactgtcag agtataattt cacctgagtt taatattaca 1080
gaaacaaagg gatgcaccaa atggtatttc tggaaatttt catgtcttta aatacccctt 1140
ggtaagttgc ttctgaagcc agtgggggct cctcagatag agaggttccc ctttcaaatc 1200
ccagtgccgc tctgttctct ttccttcccc tcccactccc cctcttcttc ctctgtagag 1260
atgcaagaaa ttgctgtccc ataaaaatca taattgcagt agctaaagct ggggtcactt 1320
cgtgaattca ccagagactc aaagatcttt tattggctct gggctgtgct cagtgtcttt 1380
ggcctcagag aacaacttga atgacttcct ggtttcctgg cataaattat tcctggtgag 1440
acatgtggct taactcacag gtttcccatc agctttctcc ctaaaactat gttcatctgc 1500
ctctctctgc cagagaacat acagccgaga atactgccga agctgagact gactactgtg 1560
cattaggaaa gacctggagt caggactttg gtgggatttg gagctccgag gcagtaataa 1620
ctgaacaagc agccctgtcc cctaggctgc agaagcttga atgcatcctc tcccagaacc 1680
tgccacagga aactgggggc tttgtcaggt cagcccaact gcatgcaaaa gaccaccatc 1740
ctcagaagcc aagttgtctt ttatgaagag gcaaggaaag gggaaaccca catgtgaccc 1800
tgattttggt atggcttgat agagttccct gaaaactcct tgtatgtgtg ctaaaaccag 1860
ggaagcatgt gactgccaag caggcaaccc ctgatgattt gtaaagccag gtggcagggc 1920
cttggggagc cccagcacaa tgatattgtg tggtcttccc tcctgtggaa tcgaggggaa 1980
attattcttc ccaatacctt gatttgattt tcagtttcat aagcttcttc ctctgaatct 2040
tattgaggga ctatggtacc aagcaggtag gactgttcac ctggtggaac agttcttgct 2100
ctgccttcta ggcttcatcc cagaaatcca gcctctttct ggagacccca aagctggagg 2160
gagatgggct ttcctctggg cctctctcct actttgccat ccacactgct cctggctaac 2220
cccagcaata accaacaaat ggtaggaagc cccatctatt gctttttctc aattatgact 2280
gcatagttta tggaaacaaa gatcttgagg aagatgaggg aagccctccc ctcttcacag 2340
30/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
tccccatctc ttctcctttg tacctgtcaa caccagagtt cagtgtttaa acagacaaaa 2400
tataaagtat tgagtaggtg gttcatatgc cgaatccact tggtaggaag aaaccaccag 2460
ctttatagtg tgcctgatag attttaaaca ttcctgggca cccactcaag agtgctcttt 2520
ttatcacctt ctggaaatcc gcaaagttgc aggggcctct ggagtgtctc ttctctagag 2580
agaattggtg ggacccccct cagtgcagtg gccccaacta gtggagggag agaggactta 2640
agtcagatgg actcaacaga aatgggtttc cagaagaata atgaaaagtt gtgggtagga 2700
aaatgaatca tttggactct tcaatgaaat ggagtgagcc caggagagct cagccaacag 2760
aggcactctg ggaacctgtt agtaaagcca ggctggccaa atgccatttg attttgaacc 2820
tcgtaggtcc ccactcaccc tctgccagga gctaagtaag gcaggagagc tgacttggga 2880
ctcctggctc ggccccaaca gggagccccc ttcccaccat ccctcggcaa gctcaccacc 2940
tcatccttct gccaaggcag ctttcctttc ttttgtgtgt tttctgtgtt cttagcctcc 3000
accctcctcc tgccaccctt gtggactagg accaggtcct gaccccagtc agaaaatgat 3060
gatatgtaca gtggcacacc ttaaccagtc actaattttc actgttgtga aagtgatttg 3120
atttagaatt aaacaaatgg ttttacatta ctatgaaaaa aaa 3163
<210> 34
<211> 2185
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 763816CB1
<400> 34
ggatctccag agctcagaaa accctcaggg tcaccagatc tttggaagct ttctcctgat 60
cagcggaaaa cttctcctgc ttcacttgat ttccctgagt cccagaaaag ttcccgtggt 120
ggttctcctg atctctggaa gtcttccttt tttattgagc ctcagaaacc tgtcttccct 180
gagagccgaa aaccatgtcc ttatgggcca tctgaggccc caaaagcagc ctcagatatc 240
tggaagctgt tctctctatc gatactgagc ctagaaaacc tgccctgttt cccgagcctg 300
ccaaaacagc ccctcctgct tctccagaag cacgcaaacg tgcccttttt ccagagcccc 360
ggaagcatgc ccttttccct gaactcccca aatctgctct attctcagaa tcacagaagg 420
ctgttgagct tggtgatgaa ctacaaatag atgccataga tgatcaaaaa tgtgatattt 480
tggttcagga agaacttcta gcttcaccta agaaactctt agaagatact ttatttcctt 540
cctcaaagaa gctcaagaaa gacaaccaag agagctcaga cgctgagctt agtagtagtg 600
agtacataaa aacagatttg gatgcgatgg atattaaggg ccaggaatca agcagtgatc 660
aagagcaggt tgatgtggaa tccattgatt ttagcaaaga gaacaaaatg gacatgacta 720
gtccagagca gtctagaaat gtgctacagt ttactgaaga aaaagaagct tttatctctg 780
aagaggagat tgcaaaatac atgaagcgtg gaaaaggaaa gtattattgc aaaatttgtt 840
gctgtcgtgc tatgaaaaaa ggtgctgttt tgcatcattt ggttaataag cataatgttc 900
atagccctta caaatgcaca atctgtggaa aggcttttct tttggaatct ctccttaaaa 960
atcatgtagc agcccatggg caaagtttac ttaaatgtcc acgttgtaat tttgaatcaa 1020
atttcccaag aggttttaag aaacatttaa ctcattgtca aagccggcat aatgaagagg 1080
caaataaaaa gctaatggaa gctcttgaac cgccactgga ggagcagcaa atttgataac 1140
acagtgtgaa tatttgttct acaaaggtgt ttgttggaac cattctttgt aagtatagct 1200
tatcagatag catagttgga tcagtagatg acatgtatgg tgtaccgtgt ttcactgtct 1260
cagttgtgtt actaagaatg agcatttgat catttttttc tggtctctgt ctatgtgact 1320
atcttgtaag tcaataaatt tctgtatagt ccagatggat taaacttctc atttctttta 1380
aatatgtatg aataataata caaggaagta ggcattccat ttaataatca agagcaagtt 1440
gtactcaaag cattcagtta aagtgtatct gtgtgtggaa ctaatttcag acaatagaaa 1500
atattagttg aaatgtttaa gaattaggca tgaaaaataa atttgagaaa ttttgtttcc 1560
ttacatgtat ttttaaatca taagagttat tttctatctg atgtaaaatt agtttataaa 1620
tcttaatcag cttctagatg tttattagct tttatgtcat gaaatgttgg agtctcaggg 1680
ttgctgattt tctgctaatg ggaaaaattg actaagtctt taaaatagtt tgcagccttc 1740
tcccacagga gacaagtgaa agataagtgt gattttagat ctttcttgtc catagttgtt 1800
ttcagtggag tcttccattc tgtatcttac cctaagatct ggttcttccc tccccatccc 1860
caccccccac ccaccgcctg ccagctcaca ctaatagatg attcttaatt gccaaatgtg 1920
ttagagtttg tatatcctac tcctgggcct tacatgtcgc ctgttggggc ttaagaccag 1980
gttgataagt aggaactgaa agtcttccag attcacagta gaaaatttta tagacatttc 2040
tgttaaagaa atatatcgat tttatgtttt tcaattatgt tactgtaaat accttgtacc 2100
tgttcatgga ttattttatt ctaaaatatt ttgtcaaatg tgtatcaacc aaattaaaaa 2160
gaaaggtttt catgtcaaaa aaaaa 2185
<210> 35
<211> 2408
<212> DNA
<213> Homo sapiens
31 /50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<220>
<221> unsure
<222> 2243
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 961184CB1
<400> 35
ggagcatccg gacgctccag tccaatctgg gctgcctccc accatcctcg gctttgccct 60
ctgggaccag gagcctccca cgccccattg acggtgtttc ggactggagc caagggtgct 120
ccctgcgatc cactggcagc cctgcctccc tggccagcaa cttggaaatc agccagtccc 180
ctaccatgcc cttcctcagc ctgcaccgca gcccacatgg gcccagcaag ctctgtgacg 240
acccccaggc cagcttggtg cccgagcctg tccccggtgg ctgccaggag cctgaggaga 300
tgagctggcc gccatcgggg gagattgcca gcccaccaga gctgccaagc agcccacctc 360
ctgggcttcc cgaagtggcc ccagatgcaa cctccactgg cctccctgat acccccgcag 420
ctccagaaac cagcaccaac tacccagtgg agtgcaccga ggggtctgca ggcccccagt 480
ctctcccctt gcctattctg gagccggtca aaaacccctg ctctgtcaaa gaccagacgc 540
cactccaact ttctgtagaa gataccacct ctccaaatac caagccgtgc ccacctactc 600
ccaccacccc agaaacatcc cctcctcctc ctcctcctcc tccttcatct actccttgtt 660
cagctcacct gaccccctcc tccctgttcc cttcctccct ggaatcatca tcggaacaga 720
aattctataa ctttgtgatc ctccacgcca gggcagacga acacatcgcc ctgcgggttc 780
gggagaagct ggaggccctt ggcgtgcccg acggggccac cttctgcgag gatttccagg 840
tgccggggcg cggggagctg agctgcctgc aggacgccat agaccactca gctttcatca 900
tcctacttct cacctccaac ttcgactgtc gcctgagcct gcaccaggtg aaccaagcca 960
tgatgagcaa cctcacgcga caggggtcgc cagactgtgt catccccttc ctgcccctgg 1020
agagctcccc ggcccagctc agctccgaca cggccagcct gctctccggg ctggtgcggc 1080
tggacgaaca ctcccagatc ttcgccagga aggtggccaa caccttcaag ccccacaggc 1140
ttcaggcccg aaaggccatg tggaggaagg aacaggacac ccgagccctg cgggaacaga 1200
gccaacacct ggacggtgag cggatgcagg cggcggcact gaacgcagcc tactcagcct 1260
acctccagag ctacttgtcc taccaggcac agatggagca gctccaggtg gcttttggga 1320
gccacatgtc atttgggact ggggcgccct atggggctcg aatgcccttt gggggccagg 1380
tgcccctggg agccccgcca ccctttccca cttggccggg gtgcccgcag ccgccacccc 1440
tgcacgcatg gcaggctggc acccccccac cgccctcccc acagccagca gcctttccac 1500
agtcactgcc cttcccgcag tccccagcct tccctacggc ctcacccgca ccccctcaga 1560
gcccagggct gcaacccctc attatccacc acgcacagat ggtacagctg gggctgaaca 1620
accacatgtg gaaccagaga gggtcccagg cgcccgagga caagacgcag gaggcagaat 1680
gaccgcgtgt ccttgcctga ccacctgggg aacacccctg gacccaggca tcggccagga 1740
ccccatagag caccccggtc tgccctgtgc cctgtggaca gtggaagatg aggtcatctg 1800
ccactttcag gacattgtcc gggagccctt catttaggac aaaacgggcg cgatgatgcc 1860
ctggctttca gggtggtcag aactggatac ggtgtttaca attccaatct ctctatttct 1920
gggtgaaggg tcttggtggt gggggtattg ctacggtctt ttaattataa taaatattta 1980
ttgaatgcta aaccatatca aacacttccc aaaatttaca agcaagagag agttaaaatt 2040
aggaaatata tctgtgtaaa taacaatctg cctatacatt gtcttataaa ttaatttctc 2100
tttacatagt tttgagtaGt attttaagat tgcatctttt ccagtgttcc ttttgtcaaa 2160
aaatgttttc atgaggaagg agaatgttgt gtgtttacag agccaatgta tattaaattg 2220
agagaaactg atggggaagg ggnatcgact ttataggcag taaagtattt acaaaccatg 2280
actgcctatt ttaaaaccgt aagccaaatt tgtattgtag ttaatccaaa ttggtcctgt 2340
taagtgaaat tggttagaat ctgggtttaa aaacttttta attttataaa ataaccaggg 2400
aggatttt 2408
<210> 36
<211> 2564
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No. 1255525CB1
<400> 36
ccaacctcat tttttctctg ttgccacagg accctttgtg gaggagttgt ttgaagtgac 60
atcctgttat ttccctatcg attttacccc tccacctaat gatccccatg gtatccagag 120
agaagacctc atcctgagtc ttcgcgctgt gctggcttct acaccacgat ttgctgagtt 180
tctgctgccc ctgttgattg agaaagtgga ttctgaggtt ctgagtgcca agttggattc 240
tctacagact ctgaatgctt gctgtgctgt gtatggacag aaggaactga aggacttcct 300
32/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
ccccagcctt tgggcttcta tccgcagaga gagcagccag cggcggacaa tccttgaaat 360
gctcctgggt ttcttgaagc tgcagcagaa atggagctat gaagacaaag atcaaaggcc 420
tctgaatggc ttcaaggacc agctgtgctc actggtattc atggctctaa cagaccccag 480
cacccagctt cagcttgttg gcatccgtac actgacagtc ttgggtgccc agccagatct 540
cctatcttat gaggacttgg agctggcagt gggtcacctg tacagactga gcttcctgaa 600
ggaggattcc cagagttgca gggtggcagc actggaagca tcaggaaccc tggctgctct 660
ctaccctgtg gccttcagca gccacctcgt acccaagctc gctgaggagc tgcgtgtagg 720
ggagtcaaat ttgactaacg gagatgagcc cacccaatgc tcccggcatc tgtgctgtct 780
gcaagccttg tcagctgtat caacacatcc cagcatcgtc aaggagacac tgcctctgct 840
gctgcagcat ctctggcaag tgaacagagg gaatatggtt gcacaatcca gtgacgttat 900
tgctgtctgt cagagcctca gacagatggc agaaaaatgt cagcaggacc ctgagagttg 960
ctggtatttc caccagacag ctataccttg cctgcttgcc ttggctgtgc aggcctctat 1020
gccagagaag gagccctcag ttctgagaaa agtactattg gaggatgagg tgttggctgc 1080
catggtgtct gtcattggca ctgctacaac ccacctgagc cctgagttag ctgcccagag 1140
tgtgacacac attgtgcccc tcttcttgga tggcaacgtg tcctttctgc ctgaaaacag 1200
cttcccgagc agattccagc cattccagga tggctcctca gggcagaggc ggctgattgc 1260
actgcttatg gcctttgtct gctccctgcc tcgaaatgtg gaaatccctc agctgaacca 1320
actcatgcgg gagcttttgg aactgagctg ctgccacagc tgcccctttt cttccaccgc 1380
tgctgccaag tgctttgcag gactcctcaa caagcaccct gcagggcagc agctggatga 1440
attcctacag ctagctgtgg acaaagtgga ggctggcctg ggctctgggc cctgtcgtag 1500
tcaggccttc actcttcttc tctgggtaac aaaggcccta gtgctcagat accatcctct 1560
cagctcctgc cttacagccc ggctcatggg cctcctgagt gacccagaat taggtccagc 1620
agcagctgat ggcttctctc tgctcatgtc tgactgcact gatgtgctga ctcgtgctgg 1680
ccatgccgaa gtgcggatca tgttccgcca gcggttcttc acagataatg tgcctgcttt 1740
ggtccagggc ttccatgctg ctccccaaga tgtgaagcca aactacttga agggtctttc 1800
tcatgtactt aacaggctgc ccaagcctgt actcttgcca gagctgccca cgcttctttc 1860
cttgctgctg gaggccctgt cctgccctga ctgtgtggtg cagctctcca ccctcagctg 1920
ccttcagcct cttctactgg aagcacccca agtcatgagt cttcacgtgg acaccctcgt 1980
caccaagttt ctgaacctca gctctagccc ttccatggct gtccggatcg ccgcactgca 2040
gtgcatgcat gctctcactc gcctgcccac ccctgtgctg ctgccgtaca aaccacaggt 2100
gattcgggcc ttagccaaac ccctggatga caagaagaga ctggtgcgca aggaagcagt 2160
gtcagccaga ggggagtggt ttctgttggg gagccctggc agctgagccc tcagtcctgg 2220
cctagactgt tctgacaatc taacctggga ttactaactg ttgagccatc ttccccaaag 2280
cagggaaacc actggtctct gactgccttt cccacagaca cagcacaaat gctaggcctc 2340
tgttgcatgg ctgtacaaag aacataagag tccatatttc tagtggattt gtaaaataag 2400
tgtgtgtgag acacttgcgt ttgaagaaag atctagggtc ctgggtctct tgcatttata 2460
tgtcagaaaa ggggcgatat gctgctgagg ggtgagtgca tatgagtgtg gccctgagga 2520
ccagggctgg cagatgttgt ctacctgctg aagaataaag atcg 2564
<210> 37
<211> 957
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1297447CB1
<400> 37
ccttctgggc atgtctgcca tatggctcca ggtttgtttt tctccccggc actctgacgg 60
ggagggctcc cggcatctcc tggcgtccgg gtagaggacg cggaggatgc tgagctgctg 120
gcgcactgca gcacaactag agatgtacgg atgcccccat cttgatctta cagaatcaga 180
ggtacagccg cgagaaagag tcaagaacag acagagtcgc ttgaggactc aggagggtgt 240
ttgctgcgtt gacaacagac tacaccctca cagtttgctc tgctcttcca acaccagtgg 300
aagatgatca catcccaggg atcagtgtcg tttagggatg tgactgtggg cttcactcaa 360
gaggagtggc agcatctgga ccctgctcag aggaccctgt acagggatgt gatgctggag 420
aactacagcc accttgtctc agtagggtat tgcattccta aaccagaagt gattctcaag 480
ttggagaaag gcgaggagcc atggatatta gaggaaaaat ttccaagcca gagtcatctg 540
ggtgagttag tatgtgccag atggaattta aaggaaggta gatcacaaag ggtaagtttg 600
gataataaga ccattgaaat gttctttagg aatcatgttt tagaggctcc agacctttgg 660
aagtaacatg ttgacgtgac ccaaatatgc agtgactctg aatcacccag catctcccag 720
tatcactttc atacacacat cttgttgttt tttttaaatt gtgatataaa ctatatagtt 780
tagccagaga ttaataaaat tttatatata tatgtaacac tcatgtaccc atcattgaga 840
tgaatatata aagtgatttt tcaacacccc cagaaagctc cctgttgtac tcttcccagt 900
aaagagcatc cctccttttg taaaattaaa tatatttttc tataaaaaaa aaaaaaa 957
<210> 38
33/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<211> 2701
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1441094CB1
<400> 38
gcggcagtga gtatgtgtgt gacggacccc cgccgcccgc ggctcgggac ccctgcctac 60
cctccttctt gcccgccggg ctgtggaact agcgcgtgcc ccctcgccgg cctcgacgtc 120
tcccgtccgc ccctcactcg tggcagggcg cagctcctgc ttcaaggtta ctgacttttt 180
atgatgtttg gtggctatga gactatagaa gcatacgaag atgatcttta tcgagatgag 240
tcatctagtg aactgagtgt tgatagtgag gtggaatttc aactctatag ccaaattcat 300
tatgcccaag atcttgatga tgtcatcagg gaggaagagc atgaagaaaa gaactctggg 360
aattcggaat cttcgagtag taaaccaaat cagaagaagc taatcgtcct ttcagatagt 420
gaggtcatcc agctgtcaga tgggtcagag gtcatcactt tgtctgatga agacagtatt 480
tatagatgta aaggaaagaa tgttagagtt caagcacaag aaaatgccca tggtctttct 540
tcttctcttc aatctaatga gctggttgat aagaaatgca agagtgatat tgagaagcct 600
aaatctgaag agagatcagg tgtaatccga gaggtcatga ttatagaggt cagttcaagt 660
gaagaggaag agagcaccat ttcagaaggt gataatgtgg aaagctggat gctactggga 720
tgtgaagtag atgataaaga tgatgatatc cttctcaacc ttgtgggatg tgaaaactct 780
gttactgaag gagaagatgg tataaactgg tccatcagtg acaaagacat tgaggcccag 840
atagctaata accgaacacc tggaagatgg acccagcggt actattcagc caacaaaaac 900
attatctgta gaaattgtga caaacgtggt catttatcaa aaaactgccc cttaccacga 960
aaagttcgtc gctgcttcct gtgctccagg agaggacatc tcctgtattc ctgtccagcc 1020
cccctttgtg aatactgtcc tgtgcctaag atgttggacc actcatgtct tttcagacat 1080
tcctgggata aacagtgtga ccgatgtcat atgctaggcc actatacaga tgcttgcaca 1140
gaaatctgga ggcagtatca cctaacgacc aaacctggac cacccaaaaa gccgaagacc 1200
ccttcaagac catcagcctt agcatattgc tatcactgcg cgcaaaaagg ccattatgga 1260
cacgaatgtc cagaaagaga agtgtatgac ccgtctccag tatctccatt catctgctac 1320
tatgatgaca aatatgaaat tcaggagaga gaaaagagac taaaacaaaa aataaaagta 1380
ctcaagaaaa atggggttat cccagagcca tccaagctac cttatataaa agcagcaaat 1440
gagaaccccc accatgatat aaggaagggc cgtgcctcat ggaaaagcaa caggtggcct 1500
caagaaaata aagaaacaca aaaagaaatg aagaacaaga atagaaactg ggagaagcac 1560
aggaaggctg acagacatcg tgaagtggat gaggattttc ccaggggccc caaaacctac 1620
tcttctcctg gcagttttaa aacccagaag ccttctaagc cctttcaccg ttcatcacat 1680
taccacacgt caagagaaga caagtctccc aaggaaggca agaggggcaa gcagaagaaa 1740
aaggagaggt gctgggaaga tgatgacaat gataacttat ttcttattaa gcagagaaaa 1800
aaaaagtctt aagccgtcag gcagcctctg atgtggcttt tcattgttca tttggccttt 1860
gtgtctataa ccttctggca ctgtgtttat tatctatgat taaataaagt gagtttttgg 1920
ttttgttttt ttaatttcag ccattcctag agttactgaa tatccatgga gatctcaatt 1980
ctctgtgtcc aacaggatat taggtaagaa agtacaaaga taaacctgga cttctcctat 2040
tccaatatgt catctttact cagaatccta gggataggta gaagaattca tcttttcaag 2100
aaagtgtttt aaaaatactt tgggaaaaaa actgcatcaa aggtaattta tcctcaaatt 2160
aaatccttgc aggaagagaa attaacacta agaaaaagtc atcaatattt ttcaactttt 2220
tttttttttt tttttacttt ggaaaggaca ataactataa acctatatcc agattttctt 2280
tctgctgaag ctgttgtcag aatcttcctt tggacaaaac atcactagct gactataaaa 2340
acaaaagtgt catcattgaa gccctgaaga ggcagggaat tgagcttcag caaaatacag 2400
gaaaagaact atccagtata aatgtcagaa gacagatttc ctaaacaagt aaaagagaca 2460
tcaaaaattt taacataatc acaatgaaat cattttttac cacttttaca gcggtgtttc 2520
aagcggactg tcactcagat ctgcagagat gaatattact caaaaaattt ttttgttctc 2580
ttgcattttt ttcaactacc gcaaagctat acagattttt ttgtacttgt ggatcttttg 2640
tacttctcat aacctaatgt cagactaaga aaaataaaat atctgagagt aaaaaaaaaa 2700
a 2701
<210> 39
<211> 2517
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1479382CB1
<400> 39
ggagaaaacg acttcagtgt catgtactcc acaaggaaaa actgtgctca actctggctg 60
34/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
ggtcctgctg cgtttataaa ccatgattgc agacctaatt gtaagtttgt gtcaactggt 120
cgagatacag catgtgtgaa ggctctaaga gacattgaac ctggagaaga aatttcttgt 180
tattatggag atgggttctt tggagaaaat aatgagttct gcgagtgtta cacttgcgaa 240
agacggggca ctggtgcttt taaatccaga gtgggactgc ctgcgcctgc tcctgttatc 300
aatagcaaat atggactcag agaaacagat aaacgtttaa ataggcttaa aaagttaggt 360
gacagcagca aaaattcaga cagtcaatct gtcagctcta acactgatgc agataccact 420
caggaaaaaa acaatgcaac ttctaaccga aaatcttcag ttggcgtaaa aaagaatagc 480
aagagcagaa cgttaacgag gcaatctatg tcaagaattc cagcttcttc caactctacc 540
tcatctaagc taactcatat aaataattcc agggtaccaa agaaactgaa gaagcctgca 600
aagcctttac tttcaaagat aaaattgaga aatcattgca agcggctgga gcaaaagaat 660
gcttcaagaa aactcgaaat gggaaactta gtactgaaag agcctaaagt agttctgtat 720
aaaaatttgc ccattaaaaa agataaggag ccagagggac cagcccaagc cgcagttgcc 780
agcgggtgct tgactagaca cgcggcgaga gaacacagac agaatcctgt gagaggtgct 840
cattcgcagg gggagagctc gccctgcacc tacataactc ggcggtcagt gaggacaaga 900
acaaatctga aggaggcctc tgacatcaag cttgaaccaa atacgttgaa tggctataaa 960
agcagtgtga cggaaccttg ccccgacagt ggtgaacagc tgcagccagc tcctgtgctg 1020
caggaggaag aactggctca tgagactgca caaaaagggg aggcaaagtg tcataagagt 1080
gacacaggca tgtccaaaaa gaagtcacga caaggaaaac ttgtgaaaca gtttgcaaaa 1140
atagaggaat ctactccagt gcacgattct cctggaaaag acgacgcggt accagatttg 1200
atgggtcccc attctgacca gggtgagcac agtggcactg tgggcgtgcc tgtgagctac 1260
acagactgtg ctccttcacc cgtcggttgt tcagttgtga catcagatag cttcaaaaca 1320
aaagacagct ttagaactgc aaaaagtaaa aagaagaggc gaatcacaag gtatgatgca 1380
cagttaatcc tagaaaataa ctctgggatt cccaaattga ctcttcgtag gcgtcatgat 1440
agcagcagca aaacaaatga ccaagagaat gatggaatga actcttccaa aataagcatc 1500
aagttaagca aagaccatga caacgataac aatctctatg tagcaaagct taataatgga 1560
tttaactcag gatcaggcag tagttctaca aaattaaaaa tccagctaaa acgagatgag 1620
gaaaataggg ggtcttatac agaggggctt catgaaaatg gggtgtgctg cagtgatcct 1680
ctttctctct tggagtctcg aatggaggtg gatgactata gtcagtatga ggaagaaagt 1740
acagatgatt cctcctcttc tgagggcgat gaagaggagg atgactatga tgatgacttt 1800
gaagacgatt ttattcctct tcctccagct aagcgcttga ggttaatagt tggaaaagac 1860
tctatagata ttgacatttc ttcaaggaga agagaagatc agtctttaag gcttaatgcc 1920
taagctcttg gtcttaactt gacctgggat aactacttta aagaaataaa aaattccagt 1980
caattattcc tcaactgaaa gtttagtggc agcacttcta ttgtcccttc acttatcagc 2040
atactattgt agaaagtgta cagcatactg actcaattct taagtctgat ttgtgcaaat 2100
ttttatcgta ctttttaaat agccttctta cgtgcaattc tgagttagag gtaaagccct 2160
gttgtaaaat aaaggctcaa gcaaaattgt acagtgatag caactttcca cacaggacgt 2220
tgaaaacagt aatgtggcta cacagttttt ttaactgtaa gagcatcagc tggctcttta 2280
atatatgact aaacaataat ttaaaacaaa tcatagtagc agcatattaa gggtttctag 2340
tatgctaata tcaccagcaa tgatctttgg ctttttgatt tatttgctag atgtttcccc 2400
cttggagttt tgtcagtttc acactgtttg ctggcccagg tgtactgttt gtggcctttg 2460
ttaatatcgc aaaccattgg ttgggagtca gattggtttc ttaaaaaaaa aaaaaaa 2517
<210> 40
<211> 1698
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1503131CB1
<400> 40
gactacgctt gctggtttgc ggccggtctt ggatgaagcg gcggccgtgg tgagagcgtg 60
gggaagggtg gggtgagggg gcgaggccgc agctagggcg gcgaaactct cctcccctcg 120
gccccaccgc gtgggacggc gtgaacgtgg tgtcggaggg atgtcagcct tctctgaggc 180
ggcgctggag aagaagctgt cggagttgag caactcgcag cagagcgtgc agaccttgtc 240
cctgtggctc attcaccacc gtaaacactc gcggcccatc gtcaccgtgt gggagcggga 300
gctgcggaaa gccaaaccaa acaggaagct tacttttctc tacctagcca atgatgtcat 360
acagaacagc aagaggaagg ggccagagtt tacaaaagat tttgcaccag ttatagtgga 420
ggcttttaag catgtttcaa gtgaaactga tgaaagttgt aagaagcacc ttggaagagt 480
gttatctatt tgggaagaaa ggtctgttta tgaaaatgat gtattagaac aacttaaaca 540
agctctgtat ggtgataaga agcctaggaa gcgaacttat gaacagataa aggtggatga 600
aaatgaaaac tgttcctctc tgggatctcc aagtgaacca ccacagactc tagatctcgt 660
tagagcatta caagatctgg aaaatgcagc ctcaggtgat gcagcagttc atcagaggat 720
agcttcttta cctgttgaag tccaagaagt atctctatta gataaaataa cagataaaga 780
atctggagaa aggctttcca aaatggtaga ggatgcgtgt atgttgctgg cagattacaa 840
tggcagattg gcggcagaaa tagatgatag aaagcaactc actcgaatgt tagcagattt 900
35/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
tcttcgttgt caaaaggaag cccttgcaga gaaagagcat aaattggaag agtacaagcg 960
caagctagcc agagtttccc tggtgcgcaa agaactcagg tcccggatcc agagcctgcc 1020
agacttatct cgattgccca atgtcactgg cagccacatg cacctgccct ttgcgggaga 1080
catctacagt gaagattgat ggaccagcct ctttccaggt cccaggactt tgcaagagat 1140
ggagacaggt taggtggata gtcctgtagt gttatttttg tatattgttg agaaagaaac 1200
actaacaaaa ggaacaacga gtaatttata aaattgttta aaatgttggt ttgtttacta 1260
ttttattggt aaattaacat gacttaattt gaacaaagtg atgatttgtg tgtattaata 1320
agctcacaaa tagtgattat tttcaaaaga tctttttgta aatttttttt tttgatccaa 1380
ggccttgtga gaaatctctt gtttgtattt cttcaggtat tttcaattgt ttttctttat 1440
tttcttcagc atttaatcac tgtatactat gtaattcctg aaggggaaga actaattagg 1500
agttgattga gactttatta tggtatagtt aggacttgat tgtagaaggg actaaatgtt 1560
ccaacataat ctttaccctt ctccccaaac aaaatatcaa ttctgtgtct ctacatgcct 1620
ttgattcctt agaggtcggt aatttaacat aaaacaggga tgtcactttg aggggcagat 1680
gaaagtctag agacctgt 1698
<210> 41
<211> 2250
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1594803CB1
<400> 41
gctcgcagcg catccccgcg cacaggttcg tgctggccgt gggcagcgcg tctttgatgc 60
catgttcaac gggggaatgg ccacaacatc cacggagatt gagctgcccg acgtggaacc 120
cgctgccttc ctcgcactgc tcaagtttct ctactcggac gaggtgcaga ttggcccgga 180
gacggtgatg accacgctat acaccgccaa gaagtacgcg gtgccagcgc tcgaggccca 240
ttgcgtggag ttcctgaaga agaacctgcg agccgacaac gccttcatgc tgctcacgca 300
ggcgcgactc ttcgatgaac cgcagctggc cagcctgtgc ctggagaaca tcgacaaaaa 360
cactgcagac gccatcaccg cggagggctt caccgacatt gacctggaca cgctggtggc 420
tgtcctggag cgcgacacac tgggcatccg tgaggtgcgg ctgttcaatg ccgttgtccg 480
ctggtccgag gccgagtgtc agcggcagca gctgcaggtg acgccagaga acaggcggaa 540
ggttctgggc aaggccctgg gcctcattcg cttcccgctc atgaccatcg aggagttcgc 600
tgcaggtccc gcacagtcgg gcatcctggt ggaccgcgag gtggtcagcc tcttcctgca 660
cttcaccgtc aaccccaagc cacgagtgga gttcattgac cggccccgct gctgcctgcg 720
tgggaaggag tgcagcatca accgcttcca gcaggtggag agtcgctggg gctacagcgg 780
gaccagtgac cgcatcaggt tctcagtcaa caagcgcatc ttcgtggtgg gatttgggct 840
gtatggatcc atccacgggc ccaccgacta ccaagtgaac atccagatta ttcacaccga 900
tagcaacacc gtcttgggcc agaacgacac gggcttcagc tgcgacggct cagccagcac 960
cttccgcgtc atgttcaagg agccggtgga ggtgctgccc aacgtcaact acacggcctg 1020
tgccacgctc aagggcccag actcccacta cggcaccaaa ggcctgcgca aggtgacaca 1080
cgagtcgccc accacgggcg ccaagacctg cttcaccttt tgctacgcgg ccgggaacaa 1140
caatggcaca tccgtggagg acggccagat ccccgaggtc atcttctaca cctaggctgc 1200
ccgacaccga caccgccctc cctccgtggg gatagccgca gccccaggcc atcatctgct 1260
gctggggccc ccccaccacg cggtgccagg cccagtgtcc cccaggccgt ctgtccactc 1320
catgccacct ttctcagcat caggacgggg ttgccctgtg ttcaccacga gtgtggctgc 1380
tggatcaggg cagccgggga ggtggccagg ccagtggcca ggccctgtgg agacaatccc 1440
tcaggactag ggacagggct gtgccggcct gggccagggc ccacggaccc gcagctcagg 1500
gcgcctgccc acgtcgtctg ccggcggtgc gccgcgggcg tccctcgcgt ctcttcactg 1560
cacattgcaa tgcatttgcg attcccattt ctctgctagg agccagcctg ggtggcgctg 1620
ctcccagagc cgtgggtccc agaccttgcg ttccttttgt tcctgtccgt ttatcaggac 1680
acgggcccca cctgtcacgt gcccgaggcc acccaagccc agcctgcggg gcgttcccac 1740
tgcctggatg ccggcttgag ttctgcgcac gcaggattca gtgtggggac ggcccctgcc 1800
ggataggcct agccctggcc caggtggtga gcggtttgca gtgtccgttc tcatccacct 1860
gatgggccca gataaaggcc cccgctgtcc agcctccctg gacggccctc gcggtccctg 1920
cagcccaaga tgggactcag accctgtgcc ccagagctcc cctgccgcag aatggggccc 1980
cagccggccc cgaccgggtc caggagcact gctcgcctgt acatactgtt gccctagccc 2040
acctggtgcc gtgggagcca cccccaggtg ctgggggcac agcccctccc cactccggcc 2100
acgcccccac ccaccccgcg tgtttctgcc ctgtgactcc tggaacctgc gtcctcccca 2160
aagccatggg aggggtgtcc tcctcagacc atgcccccag atgatttttt taaataaaga 2220
aacaaatgca cctgcaaaaa aaaaaaaaaa 2250
<210> 42
<211> 1082
<212> DNA
36/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No. 1736129CB1
<400> 42
ggcaaatccg ggatctcggc tccgagaggc tcatctggca aaagggcgcg gaaaccacgg 60
gggccctgag actgagtggt ccctatccca cttctctctg gggcggcgct gtagccagcg 120
gctgacaggc gcacgaatag gcagccctct gctgtaagga ggaaaactga ggcctgggag 180
caggaacctg taggcagcgc ttgagggtag cgggatagca gctgcaagcg cgcgtgggag 240
gcgggggctc tgggcggaac aaaaatcaca ggatgtcaga ggatgtttcc cgggaagaac 300
tgggataaag gaagggtccc agcaccatgg aggacccgaa ccctgaagag aacatgaagc 360
agcaggattc acccaaggag agaagtcccc agagcccagg aggcaacatc tgccacctgg 420
gggccccgaa gtgcacccgc tgcctcatca ccttcgcaga ttccaagttc caggagcgtc 480
acatgaagcg ggagcaccca gcggacttcg tggcccagaa gctgcagggg gtcctcttca 540
tctgcttcac ctgcgcccgc tccttcccct cctccaaagc cctaatcacc caccagcgca 600
gccacggtcc agccgccaag cccaccctgc cggttgcaac cactactgcc cagcccacct 660
tcccttgtcc tgactgtggc aagacctttg ggcaggctgt ttctctgagg cggcaccgcc 720
agatgcatga ggtccgtgcc cctcctggca ccttcgcctg cacagagtgc ggtcaggact 780
ttgctcagga agcagggctg catcaacact acattcggca tgcccggggg gagctctgag 840
tgcagcttaa gcctctccac ggtgacgggt ggctctgtgg ctggtaggac tcacccatga 900
tatggggtgc aggaactctg ggggccctga aggatttgct tccctcccct gggaaggcag 960
agggctctta ataaagagga cccagaagat tcttatttag agcttcagtc tttggagcac 1020
acagggcctt cgtgagacag tgaaatcaga taataatgag atcttttgtt aaaaaaaaaa 1080
as 1082
<210> 43
<211> 570
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1874312CB1
<400> 43
gttccgctgt gctgtttttc cgtcatggct cgcactaagc aaactgctcg gaagtctact 60
ggtggcaagg cgccacgcaa acagttggcc actaaggcag cccgcaaaag cgctccggcc 120
accggcggcg tgaaaaagcc ccaccgctac cggccgggca ccgtggctct gcgcgagatc 180
cgccgttatc agaagtccac tgaactgctt attcgtaaac tacctttcca gcgcctggtg 240
cgcgagattg cgcaggactt taaaacagac ctgcgtttcc agagctccgc tgtgatggct 300
ctgcaggagg cgtgcgaggc ctacttggta gggctatttg aggacactaa cctgtgcggc 360
atccaacgcc aagcgcgtca ctatcatgcc caaggacatc ccactcaccc gccagcatcc 420
gcggaagaga gggcggtgat tactgtgggt ctctcttgcc ggtccaagca aagggtcttt 480
ttcagggcca ccaccttttc caaataaagt tgctgtaaga aaacccattt atggcccaaa 540
agggattcct ttggggggca tttttctttt 570
<210> 44
<211> 1007
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1969301CB1
<400> 44
cccacgcgtc cgcccacgcg tccggtttgg gtttcttcgc ggctgctcaa gatgaaccga 60
ctcttcggga aagcgaaacc caaggctccg ccgcccagcc tgactgactg cattggcacg 120
gtggacagta gagcagaatc cattgacaag aagatttctc gattggatgc tgagctagtg 180
aagtataagg atcagatcaa gaagatgaga gagggtcctg caaagaatat ggtcaagcag 240
aaagccttgc gagttttaaa gcaaaagagg atgtatgagc agcagcggga caatcttgcc 300
caacagtcat tcaacatgga acaagccaat tataccatcc agtctttgaa ggacaccaag 360
accacggttg atgctatgaa actgggagta aaggaaatga agaaggcata caagcaagtg 420
aagatcgacc agattgagga tttacaagac cagctagagg atatgatgga agatgcaaat 480
gaaatccaag aagcactgag tcgcagttat ggcaccccag aactggatga agatgattta 540
37/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
gaagcagagt tggatgcact aggtgatgag cttctggctg atgaagacag ttcttatttg 600
gatgaggcag catctgcacc tgcaattcca gaaggtgttc ccactgatac aaaaaacaag 660
gatggagttc tggtggatga atttggattg ccacagatcc ctgcttcata gatttgcatc 720
attcaagcat atcttgtaaa acaaacacat attatgggac taggaaatat ttatctttcc 780
aaatttgcca taacagattt aggtttcttt cctttctttg aaggaaagtt taattacatt 840
gctcttttat tttttccatt aagagactca ttgcttggga aatgctttct tcgtactaaa 900
atttgattcc tttttttctt atgaaaaacg aactcagttt aaaagtattt ttagctcgta 960
tgacttgttt tcattcatta ataataattt gagataacca agggaat 1007
<210> 45
<211> 1622
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1986873CB1
<400> 45
ttaaccaaat gatgcaaaac agcacctttt ttcatagcac gacagcaaca aattttgcaa 60
taattactgt ccttgtccac gcttcatgta ttttgcaatc tcctcttcgg agataaaagc 120
ttctttttct tcagtaaaat ggacatgact agtccagagc agtctagaaa tgtgctacag 180
tttactgaag aaaaagaagc ttttatctct gaagaggaga ttgcaaaata catgaagcgt 240
ggaaaaggaa agtattattg caaaatttgt tgctgtcgtg ctatgaaaaa aggtgctgtt 300
ttgcatcatt tggttaataa gcataatgtt catagccctt acaaatgcac aatctgtgga 360
aaggcttttc ttttggaatc tctccttaaa aatcatgtag cagcccatgg gcaaagttta 420
cttaaatgtc cacgttgtaa ttttgaatca aatttcccaa gaggttttaa gaaacattta 480
actcattgtc aaagccggca taatgaagag gcaaataaaa agctaatgga agctcttgaa 540
ccgccactgg aggagcagca aatttgataa cacagtgtga atatttgttc tacaaaggtg 600
tttgttggaa ccattctttg taagtatagc ttatcagata gcatagttgg atcagtagat 660
gacatgtatg gtgtaccgtg tttcactgtc tcagttgtgt tactaagaat gagcatttga 720
tcattttttt ctggtctctg tctatgtgac tatcttgtaa gtcaataaat ttctgtatag 780
tccagatgga ttaaacttct catttctttt aaatatgtat gaataataat acaaggaagt 840
aggcattcca tttaataatc aagagcaagt tgtactcaaa gcattcagtt aaagtgtatc 900
tgtgtgtgga actaatttca gacaatagaa aatattagtt gaaatgttta agaattaggc 960
atgaaaaata aatttgagaa attttgtttc cttacatgta tttttaaatc ataagagtta 1020
ttttctatct gatgtaaaat tagtttataa atcttaatca gcttctagat gtttattagc 1080
ttttatgtca tgaaatgttg gagtctcagg gttgctgatt ttctgctaat gggaaaaatt 1140
gactaagtct ttaaaatagt ttgcagcctt ctcccacagg agacaagtga aagataagtg 1200'
tgattttaga tctttcttgt ccatagttgt tttcagtgga gtcttccatt ctgtatctta 1260
ccctaagatc tggttcttcc ctccccatcc ccacccccca cccaccgcct gccagctcac 1320
actaatagat gattcttaat tgccaaatgt gttagagttt gtatatccta ctcctgggcc 1380:-
ttacatgtcg cctgttgggg cttaagacca ggttgataag taggaactga aagtcttcca 1440
gattcacagt agaaaatttt atagacattt ctgttaaaga aatatatcga ttttatgttt 1500
ttcaattatg ttactgtaaa taccttgtac ctgttcatgg attattttat tctaaaatat 1560
tttgtcaaat gtgtatcaac caaattaaaa agaaaggttt tcatgtcaaa aaaaaaaaaa 1620
as 1622
<210> 46
<211> 2047
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2010820CB1
<400> 46
tgttgaaaga ctactccaca tcattataga tgataataag agaacacaaa ttgttacaga 60
aattatctca gagattcggg cgcccattgt tactgttggt gttaataacg atccagctga 120
tgtaagatag aaagaactca agatggctga aataaaagtt aagcttatcg aagccaaaga 180
agctttggaa aattgcatta ccttacagga ttttaatcgg gcatcagaat taaaagaaga 240
aataaaagca ttagaagatg ccagaataaa ccttttgaaa gagacagagc aacttgaaat 300
taaagaagtc cacatagaga agaatgatgc tgaaacattg cagaaatgtc ttattttatg 360
ctatgaactg ttgaagcaga tgtccatttc aacaggctta agtgcaacca tgaatggaat 420
catcgaatct ttgattcttc ctggaataat aagtattcat cctgttgtaa gaaacctggc 480
tgttttatgc ttgggatgct gtggactaca gaatcaggat tttgcaagga aacacttcgt 540
38/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
attactattg caggttttgc aaattgatga tgtcacaata aaaataagtg ctttaaaggc 600
aatctttgac caactgatga cgttcgggat tgaaccattt aaaactaaaa aaatcaaaac 660
acttcattgt gaaggtacag aaataaacag tgatgatgag caagaatcaa aagaagttga 720
agagactgct acagctaaga atgttctgaa actcctttct gatttcttag atagtgaggt 780
atctgaactt aggactggag ctgcagaagg actagccaag ctgatgttct ctgggctttt 840
ggtcagcagc aggattcttt ctcgtcttat tttgttatgg tacaatcctg tgactgaaga 900
ggatgttcaa cttcgacatt gcctaggcgt gttcttcccc gtgtttgctt atgcaagcag 960
gactaatcag gaatgctttg aagaagcttt tcttccaacc ctgcaaacac tggccaatgc 1020
ccctgcatct tctcctttag ctgaaattga tatcacaaat gttgctgagt tacttgtaga 1080
tttgacaaga ccaagtggat taaatcctca ggccaagact tcccaagatt atcaggcctt 1140
aacagtacat gacaatttgg ctatgaaaat ttgcaatgag atcttaacaa gtccgtgctc 1200
gccagaaatt cgagtctata caaaagcctt gagttcttta gaactcagta gccatcttgc 1260
aaaagatctt ctggttctat tgaatgagat tctggagcaa gtaaaagata ggacatgtct 1320
gagagctttg gagaaaatca agattcagtt agaaaaagga aataaagaat ttggtgacca 1380
agctgaagca gcacaggatg ccaccttgac tacaactact ttccaaaatg aagatgaaaa 1440
gaataaagaa gtatatatga ctccactcag gggtgtaaaa gcaacccaag catcaaagtc 1500
tactcagcta aagactaaca gaggacagag aaaagtgaca gtttcagcta ggacgaacag 1560
gaggtgtcag actgctgaag ccgactctga aagtgatcat gaagttccag aaccagaatc 1620
agaaatgaag atgagactac caagacgagc caaaaccgca gcactagaaa aaagtaaact 1680
taaccttgcc caatttctca atgaagatct aagttaggaa agacgatgga ggtggaatcc 1740
tttaagatta tgtccagtta tttgctttaa taaagaagaa gttacccttg tcaaaatcag 1800
aacaaacctg atgtctttct gaagattttc tgctgtgcgc ttccacgtta ctttggcctg 1860
tattaaagca gtagagcagc atcagttatt atagtccaga aaaagtgtgc atcagtcagt 1920
cacacagatt tatcccaatc tgaaggtggg ctaggaatct catttttaaa tagtctctcc 1980
aagtgattcc tatggactct ttaagtttaa atcatgtcct tatggaaaac ttacagtgtt 2040
actagct 2047
<210> 47
<211> 1817
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2013818CB1
<400> 47
caacaggaag catcagcaga tgttgctact cctaagatgc cagggcagtc agtcaggaag 60
aaaactagga aggcaaaaga aatttctgaa gcttctgaaa acatctattc tgatgtcaga 120
ggactatctc agaaccagca aatacctcaa aattctgtta cgcctaggag aggaaggaga 180
aagaaagaag ttaatcagga catactagaa aacaccagtt ctgtggaaca agaattacag 240
atcactacag gtagggaatc aaaaagatta aaatcatctc agctgttgga accagcagtt 300
gaagaaacta ctaaaaaaga agttaaggtt tcatctgtta caaaaaggac tcctagaaga 360
attaaaagat ctgtagaaaa tcaggaaagt gttgaaatta taaatgatct aaaagttagt 420
acggtaacaa gtcctagcag aatgatcaga aaattgagaa gtactaattt agatgcttct 480
gaaaatacag gaaataagca agatgataaa tccagtgaca agcagctgcg tattaaacat 540
gttagaaggg tcagagggag agaagttagt ccatcagatg tgagagaaga ctccaacctt 600
gagtcatctc agttgactgt tcaagcagaa tttgatatgt ctgccatacc tagaaaacgt 660
ggtagaccaa gaaaaatcaa tccatctgaa gatgtaggat ctaaggctgt taaggaagag 720
agaagcccca agaagaaaga agctcccagc attagaagga gatctacaag aaatacccca 780
gctaaaagtg aaaatgttga tgttggaaaa ccagctttag gaaaatccat tttagtgcca 840
aacgaggaac tttcgatggt gatgagctct aagaaaaaac ttacaaaaaa gactgaaagt 900
caaagccaaa aacgttcatt gcactcagta tcagaagaac gcacagatga aatgacacat 960
aaagaaacaa atgagcagga agaaagattg ctcgccacag cttccttcac taaatcatcc 1020
cgcagcagca ggactcggtc tagcaaggcc atcttgttgc cggacctttc tgaaccaaac 1080
aatgagcctt tattttctcc agcgtcagaa gttccaagga aagcaaaagc taaaaaaata 1140
gaggttcctg cacagctgaa agaattagtt tcggatttat cttctcagtt tgtcatctca 1200
cctcctgctt taaggagcag acaaaaaaac acatccaata agaacaagct tgaagatgaa 1260
ctgaaagatg atgcacaatc agtagaaact ctgggaaagc caaaagcgaa acgaatcagg 1320
acgtcaaaaa caaaacaagc aagcaaaaac acagaaaaag aaagtgcttg gtcacctcct 1380
cccatagaaa ttcggctgat ttcccccttg gctagcccag ctgacggagt caagagcaaa 1440
ccaagaaaaa ctacagaagt gacaggaaca ggtcttggaa ggaacagaaa gaaactgtct 1500
tcctatccaa agcaaatttt acgcagaaaa atgctgtaat ttcttgggaa gattttaatg 1560
tacacctatt tgtaaagtca tcagaatagt gtggattatt aaatatctag tttggaagaa 1620
aataatttat ataaattatt gtaaattttt atgtaaacag aaggtcttca ataagtaaag 1680
taactccata tggagtgatt gtttcagtcc aggcaatttt tctattttat attaagactt 1740
catacattta tatatgtaaa tatggcttat taatggaatg ttaaataaaa tgtatacttc 1800
39/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
acagtcaaaa aaaaaaa 1817
<210> 48
<211> 700
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2302032CB1
<400> 48
cccgcctcgc agagttggga gaaggcaggg tggggggtgt ggaaaaataa aaggaaaagt 60
ccttgcacca tgtagatcag cgtcccccac tttggcatcc cggccggccg gggacctccc 120
agtctgcggc catgaacgcg agcagcgagg gcgagagctt cgcgggctcg gtgcaaattc 180
caggtggcac aacggtgctg gtggagctga ctcccgacat ccatatctgc ggcatctgca 240
agcagcagtt taacaacctg gatgcctttg tagctcacaa gcaaagtggc tgccagctga 300
caggcacatc cgcagcagcc cccagcacgg tccagtttgt atcggaggaa acagtgcctg 360
ccacccagac tcagaccacc accagaacca tcacctcgga gacccagaca atcacaggta 420
cagctggagc atgggggagt cggccagaat tggcctggct gtgtctcaaa cacgtccatg 480
gaacttgtta aaaatataga ttcccagagc ccacccccta gggttgctga ttcattattt 540
ttaacaagct ccaccaggaa gccagctgcc cagccaggtt tgggagctgc taggttaaaa 600
cattaactgt tagcagttat aaatgttgtc acgttacaaa aaaaacagac aaaaaagaga 660
tttctgaaac aaaaatctat atactatagg aaaaaaaaaa 700
<210> 49
<211> 2704
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2326109CB1
<400> 49
cggggcggag gcggaggcag aggtggaggc gctttgaaag ccgtcattgt gtggcactgg 60
acggggaatc aagagacccg gtttctaatc cctgctctgt cactaacaag ctgcgtgatc 120
ttcggaaaaa agtaatcctc ttggaaagct ggaaacatag tccgaagctt aagtgaaaat 180
gatacatgtt agaagacatg aaacaaggag aaattctaag agtcacgtgc ctgagcagaa 240
atctcgagtt gattggaggc gaactaaaag aagtagtatc tcacaattac ttgatagtga 300
tgaagagctt gatagtgaag aatttgatag tgatgaagag cttgatagtg atgaaagttt 360
tgaaaatgat gaagagcttg atagtaacaa gggacctgat tgtaataaaa caccaggaag 420
tgaaagagag ctcaacttaa gtaaaattca aagtgaagga aatgacagta agtgtctcat 480
taactctggc aacggttcaa catatgaaga agaaacgaac aaaatcaaac ataggaatat 540
tgacttacaa gatcaggaaa aacatttaag tcaagaggat aatgatctca acaaacaaac 600
tggacaaata atagaggatg atcaggaaaa acatttaagt caagaggata atgatctcaa 660
caaacaaact ggacaaataa tagaggatga tttagaagaa gaagacatca agcgaggaaa 720
aagaaaaagg ctatcctctg tgatgtgtga cagtgatgag agtgatgaca gcgatatcct 780
agttagaaaa gtaggtgtta aacgtccccg tagagtggtt gaagatgaag gttcttcagt 840
ggaaatggag caaaagactc ctgaaaaaac attagctgca caaaagcgag aaaaacttca 900
gaagctcaaa gaactctcaa aacaaagatc tcgtcagaga cgcagtagtg gtagagattt 960
tgaggactct gaaaaggaat cttgcccaag cagtgatgaa gttgatgagg aggaagaaga 1020
ggataattat gaatctgatg aagatggaga tgattatatt atcgatgact ttgtagtgca 1080
agatgaggag ggtgatgaag agaataaaaa ccaacaagga gaaaaattga ctacatcaca 1140
actgaaatta gtaaaacgga attctcttta ttcttttagt gaccactata ctcattttga 1200
aagagttgtg aaggctcttc tgatcaacgc tttagatgaa tcttttctgg gaacattata 1260
tgatggcaca aggcaaaaat catatgcaaa agatatgcta acatctcttc attatttgga 1320
taaccgcttt gttcagcctc gtctagagag cttggtatct agaagtcgtt ggaaagagca 1380
atataaggag cgagtagaaa attattctaa tgtaagtatt catttgaaga atcctgaaaa 1440
ctgttcctgc caggcttgtg gactgcatcg ctactgtaaa tattcagtgc atttatcagg 1500
agagttgtat aacaccagga ccatgcaaat agataatttc atgtcacatg ataaacaggt 1560
gttcactgtt ggcagaattt gtgccagccg taccagaatt tatcataaac tgaaacattt 1620
taaattcaaa ctataccagg aatgttgcac cattgcaatg acagaagaag ttgaagatga 1680
acaagttaaa gaaacagtgg aaagaatttt caggcggtca aaagaaaatg gctggattaa 1740
ggagaaatat ggtcaacttg aagaatatct caattttgcg gattactttc aagaagagaa 1800
gtttgagttg taacacattt ccatcagaga agatttttta aattcctgta aatgtgaaga 1860
tcatgattct tgtttttctg tatcatgtga catgtttgta cattttatct atatcttcat 1920
40/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
ggcaaaatat tttgtttaaa acatgattat cttaattcct gtgaagtggc actctacact 1980
ctaattaaac ttttatctga tgtacataaa agcattatct taatttttaa gtctgtaaat 2040
atattttaaa gttatataat aatggcttta taacagatga ctgtcaagtg aatgagctgt 2100
tgatatcctg tcagtttagt caaaatatat tgtatcttaa aaatgtattt agactaacgt 2160
ctacatgtat ttttacggaa ttcctgtcca gatctgttta ttcttttaca gtattaaatg 2220
attttcacta atatattttt tactgctatc atctaaatca gtgggccatg gaagaaacct 2280
catatatcat tcagttcaat cccattgctt cagaatcaga tcatagcata tctattctaa 2340
gtagaggaat gcttttttcc ttttcttgaa agtttctctt tcaaatagat ttcaggcctc 2400
tagagtcttt caaccctcac attcaggaat aattttatgt aaattttcat gctttataat 2460
gttcttactt ttttctattc aattttgtct atatattgat gattaagatg tattttattt 2520
atttttatcc atagcttttt ccatataagt atgtatctta gggtcagaac tgctgaaaga 2580
gacaaactca gccaaaaaca cttggaaagc atattttggt atctgcattg ctttgcatat 2640
ctaaattttc ccataaagta ataaagtaaa atggttgcag agtgaacaaa aaaaaaaaaa 2700
aagg 2704
<210> 50
<211> 3086
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2354751CB1
<400> 50
cgccggcgtc cgcccagccg acccctctgg gccgcggccg acggctcccg gaactgggca 60
gccgcgggac agaagtcggt cctaggcccc ccaggctctg accttctttc ccaggatgag 120
gtggggccac catttgccca gggcctcttg gggctctggt tttagaagag cactccagcg 180
accagatgat cgtatcccct tCCtgatCCa ctggagttgg ccccttcaag gggagcgtcc 240
ctttgggccc cctagggcct ttatacgcca ccacggaagc tcggtagata gcgctccccc 300
acccgggagg catggacggc tgttccccag cgcctctgca actgaagcta tacagcggca 360
ccgccggaac ctggctgagt ggttcagccg gctgcccagg gaggagcgcc agtttggccc 420
aacctttgcc ctagacacgg tccacgttga ccctgtgatc cgcgagagta cccctgatga 480
gctacttcgc ccacccgcgg agctggccct ggagcatcag ccaccccagg ccgggctccc 540
cccactggcc ttgtctcagc tctttaaccc ggatgcctgt gggcgccggg tgcagacagt 600
ggtgctgtat gggacagtgg gcacaggcaa gagcacgctg gtgcgcaaga tggttctgga 660
ctggtgttat gggcggctgc cggccttcga gctgctcatc cccttctcct gtgaggacct 720
gtcatccctg ggccctgccc cagcctccct gtgccaactt gtggcccagc gctacacgcc 780
cctgaaggag gttctgcccc tgatggctgc tgctgggtcc cacctcctct ttgtgctcca 840
tggcttagag catctcaacc tcgacttccg gctggcaggc acgggacttt gtagtgaccc 900
ggaggaaccg caggaaccag ctgctatcat cgtcaacctg ctgcgcaaat acatgctgcc 960
tcaggccagc attctggtga ccactcggcc ctctgccatt ggccgtatcc ccagcaagta 1020.
cgtgggccgc tatggtgaga tctgcggttt ctctgatacc aacctgcaga agctctactt 1080
ccagctccgc ctcaaccagc cgtactgcgg gtatgccgtt ggcggttcag gtgtctctgc 1140
cacaccagct cagcgtgacc acctggtgca gatgctctcc cggaacctgg aggggcacca 1200
ccagatagcc gctgcctgct tcctgccgtc ctattgctgg ctcgtttgtg ccaccttgca 1260
cttcctgcat gcccccacgc ctgctgggca gacccttaca agcatctata ccagcttcct 1320
gcgcctcaac ttcagcgggg aaaccctgga cagcactgac ccctccaatt tgtccctgat 1380
ggcctatgca gcccgaacca tgggcaagtt ggcctatgag ggggtgtcct cccgcaagac 1440
ctacttctct gaagaggatg tctgtggctg cctggaggct ggcatcagga cggaggagga 1500
gtttcagctg ctgcacatct tccgtcggga tgccctgagg tttttcctgg ccccatgtgt 1560
ggagccaggg cgtgcaggca ccttcgtgtt caccgtgccc gccatgcagg aatacctggc 1620
tgccctctac attgtgctgg gtttgcgcaa gacgaccctg caaaaggtgg gcaaggaagt 1680
ggctgagctc gtgggccgtg ttggggagga cgtcagcctg gtactgggca tcatggccaa 1740
gctgctgcct ctgcgggctc tgcctctgct cttcaacctg atcaaggtgg ttccacgagt 1800
gtttgggcgc atggtgggta aaagccggga ggcggtggct caggccatgg tgctggagat 1860
gtttcgagag gaggactact acaacgatga tgttctggac cagatgggcg ccagtatcct 1920
gggcgtggag ggcccccggc gccacccaga tgagccccct gaggatgaag tcttcgagct 1980
cttccccatg ttcatggggg ggcttctctc tgcccacaac cgagctgtgc tagctcagct 2040
tggctgcccc atcaagaacc tggatgccct ggagaatgcc caggccatca agaagaagct 2100
gggcaagctg ggccggcagg tgctgccccc atcagagctc cttgaccacc tcttcttcca 2160
ctatgagttc cagaaccagc gcttctccgc tgaggtgctc agctccctgc gtcagctcaa 2220
cctggcaggt gtgcgcatga caccagtcaa gtgcacagtg gtggcagctg tgctgggcag 2280
cggaaggcat gccctggatg aggtgaactt ggcctcctgc cagctagatc ctgctgggct 2340
gcgcacactc ctgcctgtct tcctgcgtgc ccggaagctg ggcttgcaac tcaacagcct 2400
gggccctgag gcctgcaagg acctccgaga cctgttgctg catgaccagt gccaaattac 2460
cacactgcgg ctgtccaaca acccgctgac ggaggcaggt gttgccgtgc taatggaggg 2520
41/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
gctggcagga aacacctcag tgacgcacct gtccctgctg cacacgggcc ttggggacga 2580
aggcctggag ctgctggctg cccagctgga ccgcaaccgg cagctgcagg agctgaacgt 2640
ggcgtacaac ggtgctggtg acacagcggc cctggccctg gccagagctg cccgggagca 2700
cccttccctg gaactgctac agtgagtcct gtccctggtc ccattgcccc cagcccttca 2760
gactacttcc ctctctcaac tgtgctcctc caatcctagg gagtgcttct gggcctgggc 2820
ctggtgcctg tcctttattc ctggtttctg actgagcctt gtcaacctag ctctgagcca 2880
gcccaccccc tctgtgactg gcgcttaaac tgcaccaatt ccctaccacc atttctccta 2940
aagccctact gtctctggtc ccactgaatt ctagtatcag ggtcggctgt ggtggctcac 3000
gtctgtaatc ccagcacttc ggggggccaa ggcgggcaca tcacctgagg tcaggagttc 3060
aagaccagtc tggccaacat ggtgaa 3086
<210> 51
<211> 676
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2378058CB1
<400> 51
ttcgtttttg tcgcgcgagg ttttggtttg tgaggatcgg cgagtggcgc ccaccatctc 60
tgcttgctga aaagctgcag aggccgccag gagcccacgc acctgagact tttaccttta 120
cccagaaagg aataaaagag gtcagatgat gacagctgtg tccttaacaa ccaggcccca 180
ggaatcagtg gcttttgagg acgtggctgt gtacttcact acgaaggaat gggccatcat 240
ggtgcctgcc gagagggcct tgtacaggga tgtgatgctg gagaactatg aggctgtggc 300
ctttgtagtg ccacccactt ccaaaccagc tttggtctct catctggagc aagggaaaga 360
gtcctgtttc acccagccac agggagtcct aagcaggaat gactggagag caggctggat 420
aggatacttg gaactaagaa gatatactta cttagctaaa gcagtgttac gtagaatagt 480
atcaaaaatt tttcgaaatc gtcaatgctg ggaagacagg agaaaagctt aattcttgac 540
atttaaatac cagttttcca agtaaggagt tgatgtaaga gccaccttaa acgatgtcaa 600
atacacattt tctttttctg tagactggta gatttaatgt ttttcattca ttaaaataac 660
tcattttgat gtacaa 676
<210> 52
<211> 2135
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 1913
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 2595747CB1
<400> 52
agtcccagcc ttggaagcac actgtgtaga atttctcacc aaacatctta gggcagataa 60
tgcctttatg ttacttactc aggctcgatt atttgatgaa cctcagcttg ctagtctttg 120
tctagataca atagacaaaa gcacaatgga tgcaataagt gcagaagggt ttactgatat 180
tgatatagat acactctgtg cagttttaga gagagacaca ctcagtattc gagaaagtcg 240
actttttgga gctgttgtac gctgggcaga agcagaatgt cagagacaac aattacctgt 300
gacttttggg aataaacaaa aagttctagg aaaagcactt tccttaatcc ggttcccact 360
gatgacaatt gaggaatttg cagcaggtcc tgctcaatct ggaattttgt cagatcgtga 420
agtggtaaac ctctttcttc attttactgt caaccctaaa ccccgagttg aatacattga 480
ccgaccaaga tgctgtctca ggggaaagga atgctgcatc aatagattcc agcaagtaga 540
aagccgctgg ggttacagtg ggacgagtga tcgaatcaga ttcacagtta atagaaggat 600
ctctatagtt ggatttggct tgtatggatc tattcatggc cctacagatt atcaagtgaa 660
tatacagatc attgaatatg agaaaaagca aaccctggga cagaatgata ccggctttag 720
ttgtgatggg acagctaaca cattcagggt catgttcaag gaacccatag agatcctgcc 780
caatgtgtgc tacacagcat gtgcaacact caaaggtcca gattcccact atggcacaaa 840
aggattgaag aaagtagtgc atgagacacc tgctgcaagc aagactgttt ttttcttttt 900
tagttcccct ggcaataata atggcacttc aatagaagat ggacaaattc cagaaatcat 960
attttataca taatttagca ttataataca tcttggctaa ataataccat acaatctagt 1020
gtcaaaaaca taaatggcca caaaaaagta gtttgagtgt tatgaatatt taaaattgta 1080
42/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
agataagaaa cagtttctta gagcagatag aaaaatgctt atttaaatct ttgcatgatt 1140
taaaaacaga ttttccattt tcttacaact ttaagagaaa agaactgggt ttaatggttt 1200
aaaaaaaagc acagcttttt caccttcatc ttgtataatt tcatagattg gctgacttag 1260
ggtctttcaa tagtttggga attgaaagat tcttgttata tatagctagt ttgggtttgt 1320
ttttgtttta actattttga aggttaggtg agatgggcaa ataggcttaa ctattttgaa 1380
ggttggatga aaagagatgg gtcagtattc ctacagaatt cttattaact caaataacta 1440
aatttcagaa aattaagaag ctgactttat atttggtggt ttgaagtatc ttgttgttag 1500
catttgtaat aatgctaaaa aaggcctaat aaaatgccca agaaaatatt cagtgcattt 1560
atagagaagg atattttgta gtagtatagt aatgtgttat gtagtacagt tttaaagcta 1620
taaatggaat tttgtgtaaa ttcacaaaaa tgtgatataa acaggatcta agactggatt 1680
ccctgtcact aaactgcaca actatacctg tctctctgtg tgggggacac tgctgatgat 1740
tcccaagatt taggatgatg aacggtgatg aacgcttggg tgaacagcca tcaactttaa 1800
acattgggga ttaatccttt caacaggcaa ggagaacaag gatttaaaat tgactaacac 1860
aatttcttaa acaaacattg cttcatgacc aaatttgata agggaggatt canaacaggg 1920
tagttaaatt ccgggcttgg aaaagataaa cgagttttca tgaccaacca ttttctgagg 1980
gggggggaac acggttaaca caatttcgac cattttggag ggggccggtt tacaatttaa 2040
atggccggtg ggtttaaaac gcggtgactg gggaaccatg ggggtaccaa cattggattt 2100
tttgaggaaa tccccttttt gcaagggggg tgatt 2135
<210> 53
<211> 1385
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2634391CB1
<400> 53
gccaaccatt ccaagtcagg ggctcccaac aaatgataga ccaggcttcc ctgtaccagt 60
attctccaca gaaccagcat gtagagcagc agccacacta cacccacaaa ccaactctgg 120
aatacagtcc ttttcccata cctccccagt cccccgctta tgaaccaaac ctctttgatg 180
gtccagaatc acagttttgc ccaaaccaaa gcttagtttc ccttcttggt gatcaaaggg 240
aatctgagaa tattgctaat cccatgcaga cttcctccag tgttcagcag caaaatgatg 300
ctcacttgca cagcttcagc atgatgccca gcagcgcctg tgaggccatg gtggggcacg 360
agatggcctc tgactcttca aacacttcac tgccattctc aaacatggga aatccaatga 420
acaccacaca gttagggaaa tcactttttc agtggcaggt ggagcaggaa gaaagcaaat 480
tggcaaatat ttcccaagac cagtttcttt caaaggatgc agatggtgac acgttccttc 540
atattgctgt tgcccaaggg agaagggcac tttcctatgt tcttgcaaga aagatgaatg 600
cacttcacat gctggatatt aaagagcaca atggacagag tgcctttcag gtggcagtgg 660
ctgccaatca gcatctcatt gtgcaggatc tggtgaacat cggggcacag gtgaacacca 720
cagactgctg gggaagaaca cctctgcatg tgtgtgctga gaagggccac tcccaggtgc 780
ttcaggcgat tcagaaggga gcagtgggaa gtaatcagtt tgtggatctt gaggcaacta 840
actatgatgg cctgactccc cttcactgtg cagtcatagc ccacaatgct gtggtccatg 900
aactccagag aaatcaacag cctcattcac ctgaagttca ggagctttta ctgaagaata 960
agagtctggt tgataccatt aagtgcctaa ttcaaatggg agcagcggtg gaagcgaagg 1020
cttacaatgg caacactgcc ctccatgttg ctgccagctt gcagtatcgg ttgacacaat 1080
tagatgctgt ccgcctgttg atgaggaagg gagcagaccc aagtactcgg aacttggaga 1140
acgaacagcc agtgcatttg gttcccgatg gccctgtggg agaacagatc cgacgtatcc 1200
tgaagggaaa gtccattcag cagagagctc caccgtatta gctccattag cttggagcct 1260
ggctagcaac actcactgtc agttaggcag tcctgatgta tctgtacata gaccatttgc 1320
cttatattgg caaatctaag ttgtttctat gacacaaaca tatttagttc actattatat 1380
acagt 1385
<210> 54
<211> 1500
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2637522CB1
<400> 54
cacagagtga acaagagaga gtcatttggg aaacaaaagg agaattttac agagagagag 60
ggatagctaa aactacgtga gcctggcgag ggtgcagagc agaaagtaga gactgtccga 120
agactgctat ctgggacgag acaagttgtt aaagggacag gagagaaagc agagctattt 180
43/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
caagagtgag ccacagaagg gaatccagag gccatctaag cgaggaaggg tctacaggca 240
gtgagtgaag gccaggagca gggcccaggc caggcacgac caccgagggg atgaacttca 300
cagtgggttt caagccgctg ctaggggatg cacacagcat ggacaacctg gagaagcagc 360
tcatctgccc catctgcctg gagatgttct ccaaaccagt ggtgatcctg ccctgccaac 420
acaacctgtg ccgcaaatgt gccaacgacg tcttccaggc ctcgaatcct ctatggcagt 480
cccggggctc caccactgtg tcttcaggag gccgtttccg ctgcccatcg tgcaggcatg 540
aggttgtcct ggacagacac ggtgtctacg gcctgcagcg aaacctgcta gtggagaaca 600
ttatcgacat ttacaagcag gagtcatcca ggccgctgca ctccaaggct gagcagcacc 660
tcatgtgcga ggagcatgaa gaagagaaga tcaatattta ctgcctgagc tgtgaggtgc 720
ccacctgctc tctctgcaag gtcttcggtg cccacaagga ctgtgaggtg gccccactgc 780
ccaccattta caaacgccag aagagtgagc tcagcgatgg catcgcgatg ctggtggcag 840
gcaatgaccg cgtgcaagca gtgatcacac agatggagga ggtgtgccag actatcgagg 900
acaatagccg gaggcagaag cagttgttaa accagaggtt tgagagcctg tgcgcagtgc 960
tggaggagcg caacggtgag ctgctgcagg cgctggcccg ggaacaagca ggacaagctt 1020
caacgcgatc cgacgggact cattccggtc agtaatgggc gaagcaactt gggaaggccc 1080
tccctgccta aaagcctggt ttggagggtc actgtccaaa tcccaagttt ccacatttgg 1140
atataccaga gcccaacaga aaattgtgtg cggacactta gtaaaatgcc cgtccaagag 1200
gccaagggtg cccaccaatg ggggagaacc tttgtacact ccacaggttt aaaaaagtgt 1260
tcccccctga ggggccccac tatttttttc ccacataagc gagttgtggg aaaaaaactt 1320
tttgtgtcca cagagggggc cctcgggacc cccctgggga aaagttccac agaggcgctc 1380
ttaattttgt aatagaaggg gtccaaattt gtgcgaaaag ctccaaaaag tttcccaaac 1440
acccgggggt tataataccg aggggctttc gggggaaaag gggccacccc cccttttttg 1500
<210> 55
<211> 2051
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2650980CB1
<400> 55
gcggacgccg aggctcgcgt tccgccgctg cttccgtcgc cggggcgggc gccgcggtgt 60
ccctgccgag cgtgaccccg aactgtcacc gcgccgagcc ccagcctcat ccgtcggtgt 120
ccgcggttga ttcttcacca cactgaaacc attaggaaaa atccttgtgg ttaacagcag 180
aggcttcaga gtgtaacctg tactcgggcc tagaaattat ttaaaatggc gactgatacg 240
tctcaaggtg aactcgtcca tcctaaggca ctcccactta tagtaggagc tcagctgatc 300
cacgcggaca agttaggtga gaaggtagaa gatagcacca tgccgattcg tcgaactgtg 360
aattctaccc gggaaactcc tcccaaaagc aagcttgctg aaggggagga agaaaagcca 420
gaaccagaca taagttcaga ggaatctgtc tccactgtag aagaacaaga gaatgaaact 480
ccacctgcta cttcgagtga ggcagagcag ccaaaggggg aacctgagaa tgaagagaag 540
gaagaaaata agtcttctga ggaaaccaaa aaggatgaga aagatcagtc taaagaaaag 600
gagaagaaag tgaaaaaaac aattccttcc tgggctaccc tttctgccag ccagctagcc 660
agggcccaga aacaaacacc gatggcttct tccccacgtc ccaagatgga tgcaatctta 720
actgaggcca ttaaggcatg cttccagaag agtggtgcat cagtggttgc tattcgaaaa 780
tacatcatcc ataagtatcc ttctctggag ctggagagaa ggggttatct ccttaaacaa 840
gcactgaaaa gagaattaaa tagaggagtc atcaaacagg ttaaaggaaa aggtgcttct 900
ggaagttttg ttgtggttca gaaatcaaga aaaacacctc agaaatccag aaacagaaag 960
aataggagct ctgcagtgga tccagaacca caagtaaaat tggaggatgt cctcccactg 1020
gcctttactc gcctttgtga acctaaagaa gcttcctaca gtctcatcag gaaatatgtg 1080
tctcagtatt atcctaagct tagagtggac atcaggcctc agctgttgaa gaacgctctg 1140
cagagagcag tagagagggg ccagttagaa cagataactg gcaaaggtgc ttcggggaca 1200
ttccagctga agaaatcagg ggagaaaccc ctgcttggtg gaagcctgat ggaatatgca 1260
atcttgtctg ccattgctgc catgaatgag ccgaagacct gctctaccac tgctctgaag 1320
aagtatgtcc tagagaatca cccaggaacc aattctaact atcaaatgca tttgctgaaa 1380
aaaaccctgc agaaatgcga aaagaatggg tggatggaac agatctctgg gaaagggttc 1440
agtggcacct tccagctctg ttttccctat tatcccagcc caggagttct gtttccgaag 1500
aaagagccag atgattctag agatgaggat gaagatgaag atgagtcatc agaagaagac 1560
tctgaggatg aagagccgcc acctaagaga aggttgcaga agaaaacccc agccaagtcc 1620
ccagggaagg ccgcatctgt gaagcagaga gggtccaaac ctgcacctaa agtctcagct 1680
gcccagcggg ggaaagctag gcccttgcct aagaaagcac ctcctaaggc caaaacgcct 1740
gccaagaaga ccagaccctc atccacagtc atcaagaaac ctagtggtgg ctcctcaaag 1800
aagcctgcaa ccagtgcaag aaaggaagta aaattgccgg gcaagggcaa atccaccatg 1860
aagaagtctt tcagagtgaa aaagtaaatt ttataggaaa aaagggtatc atgatgaaat 1920
tcaaaatctt attttctaag gtcagtgtgc atttgtttag ttttgatgct tttcaaatta 1980
cattattttc ctcccctatg aacattgtgg ggagggactc taaataaacc agtttaggca 2040
44/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
aaaaaaaaaa a 2051
<210> 56
<211> 1694
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 239
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 2939607CB1
<400> 56
gaattttgtg tttgcatcgt catcatgcgc gtgttgagcc ctttagtaat gtttttgtgt 60
attagcttct tatttctagg tagacggtaa tttcgtcaat gaaaaaattt aacggtgatc 120
gtcacccctt gcctcagtca ctttctgtcc ctcgcgcggc cccggggttc accgcggacg 180
ttcctgttct gtggagaata acggctgcac ctgtatattg acgcatattt ggcggcggng 240
gtgctttgtc tcctcagcac tctgctgtca ctcaaggaag tatcatcaag aacaaggagg 300
gcatggatgc taagtcacta actgcctggt cccggacact ggtgaccttc aaggatgtat 360
ttgtggactt caccagggag gagtggaagc tgctggacac tgctcagcag atcgtgtaca 420
gaaatgtgat gctggagaac tataagaacc tggtttcctt gggttatcag cttactaagc 480
cagatgtgat cctccggttg gagaagggag aagagccctg gctggtggag agagaaattc 540
accaagagac ccatcctgat tcagagactg catttgaaat caaatcatca gtttccagca 600
ggagcatttt taaagataag caatcctgtg acattaaaat ggaaggaatg gcaaggaatg 660
atctctggta tttgtcatta gaagaagtct ggaaatgtag agaccagtta gacaagtatc 720
aggaaaaccc agagagacat ttgaggcaag tggcattcac ccaaaagaaa gtacttactc 780
aggagagagt ctctgaaagt ggtaaatatg ggggaaactg tcttcttcct gctcagctag 840
tactgagaga gtatttccat aaacgtgact cacatactaa aagtttaaaa catgatttag 900
ttcttaatgg tcatcaggac agttgtgcaa gtaacagtaa tgaatgtggt caaactttct 960
gtcaaaacat tcaccttatt cagtttgcaa gaactcacac aggtgataaa tcctacaaat 1020
gccctgataa tgacaactct cttactcatg gttcatctct tggtatatca aagggcatac 1080
atagagagaa accctatgaa tgtaaggaat gtggaaaatt cttcagctgg cgctctaatc 1140
ttactaggca tcagcttatt catactggag aaaaacccta tgagtgtaaa gaatgtggaa 1200
agtcttacag ccagagatct caccttgttg tgcatcatag aattcacact ggactaaaac 1260
cttttgagtg taaggattgt ggaaaatgtt ttagtcgaag ctctcacctt tattcacatc 1320
aaagaaccca cactggagag aaaccatatg agtgtcatga ttgtggaaaa tctttcagcc 1380
agagttctgc ccttattgtg catcagagga tacacactgg agagaaacca tatgaatgct 1440
gtcagtgtgg gaaagccttc atccggaaga atgacctcat taagcaccag agaattcatg 1500
ttggagaaga gacctataaa tgtaatcaat gtggcattat cttcagccag aactctccat 1560
ttatagttca tcaaatagct cacactggag agcagttctt aacatgcaat caatgtggga 1620
cagcgcttgt taatacctct aaccttattg gataccagac aaatcatatt agagaaaaag 1680
cttactaata aata 1694
<210> 57
<211> 1177
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3098421CB1
<400> 57
gacctggccc ccgaatcagg aaaaggcagg tattggaacc cacggcctgg gtgttagtcc 60
agccgcttag tattctgact ctattctgcc tttcttgtct cctaagaata actgtgcttg 120
aagaagaaaa ttcccaacat ggacaaacca cgcaaagaaa atgaagaaga gccgcagagc 180
gcgcccaaga ccgatgagga gaggcctccg gtggagcact ctcccgaaaa gcagtccccc 240
gaggagcagt cttcggagga gcagtcctcg gaggaggagt tctttcctga ggagctcttg 300
cctgagctcc tgcctgagat gctcctctcg gaggagcgcc ctccgcagga gggtctttcc 360
aggaaggacc tgtttgaggg gcgccctccc atggagcagc ctccttgtgg agtaggaaaa 420
cataagcttg aagaaggaag ctttaaagaa aggttggctc gttctcgccc gcaatttaga 480
ggggacatac atggcagaaa tttaagcaat gaggagatga tacaggcagc agatgagcta 540
gaagagatga aaagagtaag aaacaaactg atgataatgc actggaaggc aaaacggagc 600
45/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
cgtccttatc ctatttaatg tgttcggcct ttaattctgt tttgcctgct aatagtattg 660
ccattgccac ctggactttc tgtttgcatt ttcttaatgc cttttcccat attctgaatt 720
ttaacttttt gtgaggcttt attttagatg tttagcatgt aactcgctta aagttgaggt 780
ttccccctaa aatctacaag tttccctctt tcagtcatga gccctacaca tttgcatgaa 840
agatgtacat tatatattgt gaaacgaaaa aagcaatttt caaatggtat atattgtatc 900
ccatttttgt aaaaaaaatg tatatttata tattaatatg caaagaaaaa gctaaaagta 960
tagacttcaa aggcataaca gtggttgtgt ggtaagataa taggtgattt tttaaatttt 1020
tgttttatct gaatttctca ttttttcagg acaaacgttt tacttgtgtt gcaaaaatat 1080
ataatgaaaa aatcacacaa ttttgaagaa aactgtcaat cagcttataa cgacaatgtg 1140
gcacttaata aatacttgtc agaactttaa aaaaaaa 1177
<210> 58
<211> 1219
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> 1139
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte ID No: 3296650CB1
<400> 58
gctcggaatt cggctcgagg tagccagtgc cagccaatga aggctctgtt caagcatgaa 60
tctctgggat cccagccctt acacgataga gttctccagg ttcctgggct tgcccaggga 120
gggtgctgca gagaagatgc aatggtagct tccaggctca ctccagggtc ccagggtttg 180
ctgaaaatgg aagatgtggc cctgaccctc actcctgggt ggacacagct ggattcatct 240
caggtgaacc tctacagaga tgaaaagcag gagaaccata gcagcctggt ctcccttggt 300
ggtgaaatac agactaagag cagggacttg cctccagtca agaagcttcc agaaaaggag 360
catgggaaga tatgccacct gagggaagac attgcccaga ttcctacaca tgcagaagct 420
ggtgaacagg agggcaggtt acaaagaaag cagaaaaatg ccatagggag taggcgacat 480
tattgccatg aatgtggaaa gagttttgct caaagttcag gcctgactaa acacaggaga 540
atccacactg gtgagaaacc ctatgaatgt gaagactgtg gaaagacctt cattgggagc 600
tctgcccttg tcattcatca gagagtccac actggtgaga agccatatga gtgtgaagaa 660
tgtggtaagg tcttcagtca cagctcaaac cttatcaaac accagagaac ccacactggg 720
gagaagccct atgagtgtga tgactgtggg aagaccttca gccagagctg cagcctcctt 780
gaacatcaca aaattcatac tggggagaag ccataccagt gcaatatgtg tggcaaagcc 840
tttaggcgga attcacatct cctgagacat cagaggattc atggtgataa aaatgttcag 900
aatcctgagc acggggagtc ctgggaaagt cagggtagga cggaaagcca gtgggaaaat 960
actgaggctc ccgtgtctta taaatgtaat gagtgtgaga gaagtttcac acggaataga 1020
agtcttattg aacatcagaa aatccacact ggtgacaaac cctatcagtg tgacacatgt 1080
ggaaaaggtt tcactcgaac ttcatacctt gttcaacatc agagaagcca tgtagggana 1140
aaaactcttt cacagtgacc catggttatc atgccaacat tggtgctcat ttgtcactga 1200
actgaagcaa cccttggag 1219
<210> 59
<211> 1309
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3687719CB1
<400> 59
ctggaagctg agcctgaccc agcaggtccC agcagcctgg cagcccggcc agcatgcagc 60
agcagcctct gcccgggcct ggcgccccca caactgagcc aaccaagcct ccctacagct 120
acatcgccct tattgctatg gccatccaga gctcaccggg gcagcgggcc accctcagtg 180
gcatctaccg ctacatcatg ggccgattcg ccttctaccg ccacaaccgg cccggctggc 240
agaacagcat ccgccacaat ctgtcactca acgagtgctt tgtcaaggtg ccccgcgatg 300
accgcaagcC aggcaagggc agctactgga cgctggaccc tgactgccac gacatgtttg 360
agcacggcag cttcctacgc cgccgccgcc gcttcacccg gcagacaggt gctgagggca 420
cccggggccc cgccaaggca cgccgtggac ccctcagggc gaccagccag gacccaggag 480
tccccaacgc cacgaccggc aggcagtgct cattcccacc agagctgcca gatcccaagg 540
46/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
gcctaagctt tgggggtctg gtgggggcca tgccagccag tatgtgccca gcaaccactg 600
atggcaggcc tcggccaccc atggagccca aagagatttc cacgcccaag cctgcatgcc 660
caggggagct ccccgtggcc acctcatctt cctcatgccc agcgtttggc tttcctgccg 720
gcttctcaga ggctgagagt tttaataagg cccctacgcc cgtcttgtcc ccggaatcag 780
gcatcgggag cagctaccag tgtcggctgc aggcactgaa tttttgcatg ggggctgacc 840
caggccttga gcacctcttg gcctcagcag ccccctcccc tgcaccaccc acccctccag 900
gctcactccg ggccccactg cccctgccaa ctgaccacaa ggaaccctgg gttgcaggtg 960
gcttccctgt ccagggaggc tccggctacc cattggggct gaccccctgc ctataccgga 1020
cgccaggaat gttcttcttt gagtaaaggc agcctcacct cgggcagtcc ctgcaggtcc 1080
ctcaccctcc gggctgagcc tggctctagg acctgaagaa ctctcgaagg acccagccct 1140
ggtgagccaa ggacaaccac acagaaagcc aggattgaag cggtgctcag ccaggcccct 1200
ggggcctccg gacaaacttg ggggtgaggg gaagcagggc ctcttgggat ttactctgtg 1260
gctctcaggg ccaataaagc catgtgatga tgaaaaaaaa aaaaaaagg 1309
<210> 60
<211> 1326
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3774188CB1
<400> 60
gaggagctca actgatctgt tttctttcgc ccagccaaaa tcacagaatg aaggcggtga 60
agagcgaacg ggagcgaggg agccggcgaa gacaccggga cggggacgtg gtgctgccgg 120
cgggggtggt ggtgaagcag gagcgtctca gcccagaagt cgcacctccc gcccaccgcc 180
gtccggacca ctccggtggt agcccgtctc cgccgaccag cgagccggcc cgctcgggcc 240
accgcgggaa ccgagcccga ggagttagcc ggtccccacc caaaaagaaa aacaaggcct 300
cagggagaag aagcaagtct cctcgcagta agagaaaccg aagtcctcac cactcaacag 360
tcaaagtgaa gcaggagcgt gaggatcatc cccggagagg acgggaggat cggcagcaca 420
gggaaccatc agaacaggaa cacaggagag ctaggaacag tgaccgggac agacaccggg 480
gccattccca ccaaaggaga acgtctaacg agaggcctgg gagtgggcag ggtcagggac 540
gggatcgaga cactcagaac ctgcaggctc aggaagaaga gcgggagttt tataatgcca 600
ggcgacggga gcatcgccag aggaatgacg ttggtggtgg cggcagtgag tctcaggagt 660
tggttcctcg gcctggtggc aacaacaaag aaaaagaggt gcccgctaaa gaaaaaccaa 720
gctttgaact ttctggggca cttcttgagg acaccaacac tttccggggt gtagtcatta 780
aatatagtga gcccccagaa gcacgtatcc ccaaaaaacg gtggcgtctc tacccattta 840
aaaatgatga ggtgcttcca gtcatgtaca tacatcgaca gagtgcgtac ctactgggtc 900
gacaccgccg cattgcagac attccaattg atcacccgtc ttgttcaaag cagcatgcgg 960
tctttcaata tcggcttgtg gaatataccc gtgctgatgg cacagttggc cgaagagtga 1020
agccctacat cattgacctt ggctcaggca atggaacctt cttaaacaac aaacgtattg 1080.
agccacagag atactatgaa ctaaaagaaa aggatgtact caaatttgga ttcagtagca 1140
gagaatacgt cttgctccat gagtcgtcgg acacttctga aatagacagg aaagatgacg 1200
aggatgagga ggaggaggaa gaagtgtctg acagctagca aactaagaac ccaaactatt 1260
gatacacggt ttccttcttg gaagtctttg attgactcag agagcactat ggtggtgggt 1320
ccagca 1326
<210> 61
<211> 1097
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 4349106CB1
<400> 61
gcggcttccg ggatttggcg gtggcctttg ttggctgcag taagagctca gtctcttcac 60
caggggctcc cagtccttcc atctgggagg ccaaggcggc ttcgcgttct gagaatagac 120
agaacctctg ttactctgtg accggcaggc accgggagat ccgtagctca gacgccagga 180
catcccggaa gctgggaaat gggactgttg acattcaggg atgtggccat agaattctct 240
cgggaggagt gggaacacct ggactcagat cagaagcttt tatatgggga tgtgatgtta 300
gagaactacg gaaacctggt ctctctgggt ctcgctgtct ctaagccgga cctgatcacc 360
tttttggagc aaaggaaaga gccctggaat gtgaagagtg cagagacagt agccatccag 420
ccagatatct tttctcatga tactcaaggc ctcttaagaa agaagcttat agaagcatca 480
ttccaaaaag tgatattgga tggatatggg agctgtggcc ctcagaattt aaacttaagg 540
47/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
aaagagtggg aaagtgaggg caaaataatc ctatggtgaa aaaaaatcaa caagataatc 600
tctgcatgag aaaaaggacc aaagacaagt gaattttctg agggtgatga aagtgttgct 660
tcgaaaaggg gtgaagttta tatgggtcta tttatttgtc taacatgtac agttaaggtt 720
tatgccttgc aatgtatgta catcttcaca aaaaaaatct taaaaaaatt aaatgggtgg 780
gggtagggaa agggttgaag tatagatgaa gcagaagtgg tacatgatta gtagttgaag 840
ctgggggcag gtctatatat tttattttta tgtctttaat agcatttgta taaatgtaca 900
atattcgttt acaatgttag ctcaggatct tgtttacctt tggacaggga gggagggaga 960
aatttatttt ctgggtagga gtaaagctgg ttctattcct cagttatgta aattatctgt 1020
tataatccaa aaagctttac atgaatgttt ctgtatgtaa tgtggtatat caataaaaaa 1080
ttaaatatta aaaaaaa 1097
<210> 62
<211> 2404
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No. 4834217CB1
<400> 62
ctcgctggct agtaggagag actggtgctt gccccgcccg gtggactaac tcgcttaatt 60
ttaaataaaa agtcgaggac acggcggtcg ttttcccgaa gacatgggcc ctcccatggg 120
ccatttgctc cctggaggcc ctcgcgtctt gctgagcccg gggagttagg atgacgcgag 180
cggtgaggga gcccggaacg attccttcgc ggaacaattg aggcgaggcc tttgggagta 240
ctttgtggga cggaccctgg cgggccctgc cagacgcaca gggatggcgg cggaggcggc 300
cgatttgggg ctgggggccg ccgtccccgt ggagctgagg cgggagcgac gcatggtgtg 360
cgtggagtac ccgggagtgg tgcgtgatgt ggctaagatg ctgccgactc tgggcggcga 420
ggaaggcgtc tcccggatct acgcagaccc caccaagagg ctggagctgt acttccggcc 480
caaggaccca tactgccacc cagtgtgcgc caaccgcttc agtaccagca gcctgctgct 540
ccgcatcagg aagagaacga ggcggcagaa aggggtgctg ggcactgagg cccactccga 600
ggtcacattt gacatggaga tccttggcat catctccacc atttacaaat ttcaggggat 660
gtctgacttc cagtacttgg ctgtgcatac ggaagcaggc ggcaagcata cgtcaatgta 720
tgacaaggtg ctcatgctcc ggcccgagaa ggaggccttt ttccaccagg agctgccgct 780
ctacatcccc ccacccatct tctcccggct ggacgccccg gtggactact tctaccgacc 840
agagacccag caccgggaag gctacaacaa tccccccatc tcaggtgaga atctgattgg 900
cctgagcaga gcccggcgcc cccacaatgc catctttgtc aactttgagg atgaggaggt 960
gcccaagcag ccactggagg ctgcagccca gacgtggagg agagtctgca ctaaccccgt 1020,
ggaccggaag gtggaggagg agctgaggaa gctgtttgac atccgtccca tctggtcccg 1080%
aaatgctgtc aaggccaaca tcagcgtcca cccagacaag ctcaaggtct tgcttccctt 1140
catagcctat tacatgataa caggcccctg gcgcagccta tggattcgat ttgggtatga 1200
cccccgaaaa aacccagatg ccaagattta tcaagtcctc gatttccgaa tccgttgtgg 1260
aatgaaacac ggttacgccc ccagtgactt gccggtcaaa gcaaagcgca gcacctacaa 1320
ctacagcctc cccatcaccg tcaagaagac atccagccag cttgtcacca tgcatgacct 1380
gaagcagggc ctgggcccgt cggggacgag tggtgctcgg aaaccagctt ccagcaagta 1440
caagctcaag gactctgtct acatcttccg ggaaggggcc ttgccaccct atcggcagat 1500
gttctaccag ttatgcgact tgaatgtgga agagttgcag aagatcattc accgcaatga 1560
cggggcagag aattcctgca cagaacggga tgggtggtgc ctccccaaga ccagcgacga 1620
gctcagggac accatgtccc tcatgatccg gcagaccatc cgctccaaga ggcctgctct 1680
cttttccagc tcagccaagg ctgatggcgg aaaagagcag ctgacgtacg agtctgggga 1740
agacgaggag gatgaggagg aggaggaaga ggaggaggag gacttcaagc catccgacgg 1800
cagtgaaaac gaaatggaga cagagattct ggactacgtg tgacagggcc caaggctggg 1860
cctccctgac ccggccagac tggtgtctgg cctaatgagg gagccggggc tccccattgc 1920
cacccacagt gcccggaatg gccctaggag gccctctgag gagagctaga gtcccagcaa 1980
agggtgcagc tgaccctagc actggctgtg acatgctgct tggtgctgcc tctggtcctg 2040
aggggttagg gacatcccca aagggtatac cctggctctg ccacccatga accagcccag 2100
catccagcca gtgagtgggc acccaatgcc tctcaggatg agaccagtaa atgccggagg 2160
tggagctggg cagctgtgga gccccaggcc acaggccagt ctcgcttggc tctcatgact 2220
gtggtggtgg agatagcgtg gggagcctcg cccatggtct cacgtggcaa gaagtgcctt 2280
tagctctgga tcccaaccgt ttggcacagc tttggccaca gccaggcccc tctggaattg 2340
tccttattaa accagtttcc cgagaaaaaa aagaataaaa acagcagaaa aaataaaaaa 2400
aaaa 2404
<210> 63
<211> 1900
<212> DNA
<213> Homo sapiens
48/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
<220>
<221> misc_feature
<223> Incyte ID No: 5156094CB1
<400> 63
agctggagca ccgtgaggaa gaagcgaggt tctttttaag agttcagctg cgaggctgta 60
acagaggagg aaatgatttt gaatagcctc tctctgtgtt accataataa gctaatcctg 120
gccccaatgg ttcgggtagg gactcttcca atgaggctgc tggccctgga ttatggagcg 180
gacattgttt actgtgagga gctgatcgac ctcaagatga ttcagtgcaa gagagttgtt 240
aatgaggtgc tcagcacagt ggactttgtc gcccctgatg atcgagttgt cttccgcacc 300
tgtgaaagag agcagaacag ggtggtcttc cagatgggga cttcagacgc agagcgagcc 360
cttgctgtgg ccaggcttgt agaaaatgat gtggctggta ttgatgtcaa catgggctgt 420
ccaaaacaat attccaccaa gggaggaatg ggagctgccc tgctgtcaga ccctgacaag 480
attgagaaga tcctcagcac tcttgttaaa gggacacgca gacctgtgac ctgcaagatt 540
cgcatcctgc catcgctaga agataccctg agccttgtga agcggataga gaggactggc 600
attgctgcca tcgcagttca tgggaggaag cgggaggagc gacctcagca tcctgtcagc 660
tgtgaagtca tcaaagccat tgctgatacc ctctccattc ctgtcatagc caacggagga 720
tctcatgacc acatccaaca gtattcggac atagaggact ttcgacaagc cacggcagcc 780
tcttccgtga tggtggcccg agcagccatg tggaacccat ctatcttcct caaggagggt 840
ctgcggcccc tggaggaggt catgcagaaa tacatcagat acgcggtgca gtatgacaac 900
cactacacca acaccaagta ctgcttgtgc cagatgctac gagaacagct ggagtcgccc 960
cagggaaggt tgctccatgc tgcccagtct tcccgggaaa tttgtgaggc ctttggcctt 1020
ggtgccttct atgaggagac cacacaggag ctggatgccc agcaggccag gctctcagcc 1080
aagacttcag agcagacagg ggagccagct gaagatacct ctggtgtcat taagatggct 1140
gtcaagtttg accggagagc atacccagcc cagatcaccc ctaagatgtg cctactagag 1200
tggtgccgga gggagaagtt ggcacagcct gtgtatgaaa cggttcaacg ccctctagat 1260
cgcctgttct cctctattgt caccgttgct gaacaaaagt atcagtctac cttgtgggac 1320
aagtccaaga aactggcgga gcaggctgca gccatcgtct gtctgcggag ccagggcctc 1380
cctgagggtc ggctgggtga ggagagccct tccttgcaca agcgaaagag ggaggctcct 1440
gaccaagacc ctgggggccc cagagctcag gagctagcac aacctgggga tctgtgcaag 1500
aagccctttg tggccttggg aagtggtgaa gaaagccccc tggaaggctg gtgactactc 1560
ttcctgcctt agtcacccct ccatgggcct ggtgctaagg tggctgtgga tgccacagca 1620
tgaaccagat gccgttgaac agtttgctgg tcttgcctgg cagaagttag atgtcctggc 1680
aggggccatc agcctagagc atggaccagg ggccgcccag gggtggatcc tggccccttt 1740
ggtggatctg agtgacaggg tcaagttctc tttgaaaaca ggagcttttc aggtggtaac 1800
tccccaacct gacattggta ctgtgcaata aagacacccc ctaccctcac ccacggctgg 1860
ctgcttcagc cttgggcatc ttcataaaaa aaaaaaaaaa 1900
<210> 64
<211> 2901
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 5665139CB1
<400> 64
accacagcgc agtctcactc tgtccttcag gctgtgagtg cagtctcatg atcttggctc 60
actgcaacct ctgtctcctg ggttcaagtg attcttgtgc ctcagcctac caagtagctg 120
agatacagga ttgacttcta aagactcttg gtacctgagg aagaaaccca gaagaggaag 180
aggaaagcaa aggagtcggg gatggctctt tctcagggtc tattgacatt cagggacgtg 240
gccatagaat tctctcagga ggagtggaaa tgcctggacc ctgctcagag gactctatac 300
agagacgtga tgctggagaa ttataggaac ctggagtctg tggggtctat tgacattcag 360
ggatgtggcc atagaattct ctcaggagga gtggaaatgc ctggaccctg ctcagaggac 420
tctatacaga gatgtgatgt tggagaatta taggaacctg gtctccctgg gtattttttc 480
taaatgtgag atcaaggaat taccaccaaa aaaggagagt aatacaggag aaatattcca 540
gacagtaatg ttggaaagac atgaaagcca cgacatacaa gatttttgct tcagagaaac 600
ccagaaaaat gtacatgact ctcagtgtct gtggaaacat gattgaagac attataagcg 660
agtgcgtgtg acctataagg aaagtctcat tggtagaaga gacatgcatg gtagaaagga 720
tgatgcacaa aagcagcctg ttaaaaatca gcttggatta aacccgcagt cacatctacc 780
agaactgcag ctatttcaag ctgaagggaa aatatataaa tatgatcaca tggaaaaatc 840
tgtcaacagt agttccttag tttccccacc ccaacgtatt tcttctactg tcaaaaccca 900
catttctcat acatatgaat gtaattttgt ggattcatta ttcacacaaa aagagaaagc 960
aaatattggg acagaacact acaaatgtaa tgagcgtggc aaggcctttc atcaaggctt 1020
acattttact atacatcaaa taatccatac taaagagacg caatttaaat gtgatatatg 1080
tggcaagatc ttcaataaaa aatcaaacct tgcaagtcat caaagaattc atactggaga 1140
49/50
CA 02375414 2001-11-28
WO 00/78954 PCT/US00/16766
gaagccatat aaatgtaatg aatgtggcaa ggtcttccat aatatgtcac accttgcaca 1200
gcatcgcagg attcatactg gagagaaacc atataaatgt aatgaatgtg gcaaggtctt 1260
taatcaaatt tcacaccttg cacaacatca aagaattcat accggagaga aaccttataa 1320
atgtaatgaa tgtggaaagg tcttccatca aatttcacac cttgcacaac atcggacaat 1380
tcatactgga gaaaaacctt acgaatgtaa caaatgtggc aaggtgttca gtcgcaattc 1440
ctaccttgta caacatctga tcattcatac tggagagaaa ccttacagat gtaatgtatg 1500
tggaaaggtc ttccatcata tttcacacct tgcacaacat cagagaatcc acactggaga 1560
gaaaccttac aaatgtaatg agtgtggcaa ggtcttcagt cacaagtcat ccctagtaaa 1620
tcactggaga attcatactg gagagaaacc ttacaaatgt aatgagtgtg gcaaggtctt 1680
cagtcacaag tcatccctag taaatcactg gagaatccac actggagaga aaccttacaa 1740
atgtaatgaa tgtggcaagg tcttcagtcg caattcatac cttgcccaac atctgataat 1800
tcatgccggt gagaaacctt ataagtgtga tgaatgtgac aaagcattca gtcaaaattc 1860
acatcttgta caacatcaca gaatccatac tggagagaaa ccttacaaat gtgatgaatg 1920
tggcaaagtc ttcagtcaaa attcatacct tgcatatcat tggagaattc atactggaga 1980
aaaagcttat aaatgtaatg aatgtgggaa ggtcttcggt ctaaactcat ccctagcaca 2040
tcatcggaaa attcacactg gagagaaacc tttcaaatgt aatgaatgtg gcaaagcttt 2100
tagtatgcgt tcaagcctca ctaatcatca tgcgatccac actggagaga aacatttcaa 2160
atgtaatgaa tgtggcaaac tcttccgcga caattcatat cttgtacgtc atcagagatt 2220
tcatgccgga aagaaatcta acacatgtaa ttaatgtggc agagtgttca gttagcatta 2280
aagccttgta agacatacaa taatttatac tggagaaaaa ctttgcaagt ataatgaatg 2340
tagcagagcc tttagttttt gttcaaggct taataaccgt tagctagacc atagaggaca 2400
gaaactttac taatgtactg aatgtggcaa ggtcttaagg taaaatctga gaccaggatt 2460
tttcaaagaa ttcttgctgg tgagaaacct aacaaatgta atgaatgtgg caaggtcttc 2520
tggcacaatt ctcacattgt acaatattgc aaaaattcat gcttgagaga aacaaaaaca 2580
ctgagagtgg gaaaccatta tgacttcaaa cattcatcaa catcagagaa tccatactaa 2640
agagcattta taataattat atgtgataga gattttccgc aggccaaagt ctcactaggc 2700
atcaaaaact tttttgatga aaccatacaa atgtaacgtg catgcttaag cttttaccca 2760
ggcatcaaaa ccggaacatc acagggttta tactggagag taactacaca aagataatgt 2820
aataagcctt tcagtgtaat attcatgatt ttgtcgtgag agatccactc aataaaaacc 2880
aggcaaatgt aaaaaaaaaa a - 2901
SO/S~
