Note: Descriptions are shown in the official language in which they were submitted.
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PROTEINS ASSOCIATED WITH CELL DIFFERENTIATION
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteins
involved in cell
differentiation and to the use of these sequences in the diagnosis, treatment,
and prevention of cell .
proliferative, developmental, and neurological disorders.
BACKGROUND OF THE INVENTION
Multicellular organisms are comprised of diverse cell types that differ
dramatically both in
structure and function, despite the fact that each cell is like the others in
its hereditary endowment.
Cell differentiation is the process by which cells come to differ in their
structure and physiological
function. The cells of a multicellular organism all arise from mitotic
divisions of a single-celled
zygote. The zygote is totipotent, meaning that it has the ability to give rise
to every type of cell in the
adult body. During development the cellular descendants of the zygote lose
their totipotency and
become determined. Once its prospective fate is achieved, a cell is said to
have differentiated. All
descendants of this cell will be of the same type.
Human growth and development requires the spatial and temporal regulation of
cell
differentiation, along with cell proliferation and regulated cell death. These
processes coordinately
control reproduction, aging, embryogenesis, morphogenesis, organogenesis, and
tissue repair and
maintenance. The processes involved in cell differentiation are also relevant
to disease states such as
cancer, in which case the factors regulating normal cell differentiation have
been altered, allowing the
cancerous cells to proliferate in an anaplastic, or undifferentiated state.
The mechanisms of differentiation involve cell-specific regulation of
transcription and
translation, so that different genes are selectively expressed at different
times in different cells.
Genetic experiments using the fruit fly Drosophila melano ag ster have
identified regulated cascades of
transcription factors which control pattern formation during development and
differentiation. These
include the homeotic genes, which encode transcription factors containing
homeobox motifs. The
products of homeotic genes determine how the insect's imaginal discs develop
from masses of
undifferentiated cells to specific segments containing complex organs. Many
genes found to be
involved in cell differentiation and development in Drosophila have homologs
in mammals. For
example, human homologs have recently been found for the Drosophila ash2 gene.
The ash2 gene
product is a transcriptional regulator of homeotic selector genes and is
implicated in early
development and formation of various disc patterns in the fruit fly (Ikegawa,
S. (1999) Cytogenet.
Cell Genet. 84:167-172). The ariadne-2 protein, a retinoic-acid inducible
protein with a RING finger
transcription factor motif, also has a human homolog (GenBank Entry g5453556,
Homo Sapiens
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ariadne-2 (D. melano ag ster) homology.
There is evidence in some cases that the human genes have equivalent
developmental roles as
their Drosophila homologs. The human homolog of the Drosophila eyes absent
gene (eya) underlies
branchio-oto-renal syndrome, a developmental disorder affecting the ears and
kidneys (Abdelhak, S.
et al. ( 1997) Nat. Genet. 15:157-164). The Drosophila slit gene encodes a
secreted leucine-rich
repeat containing protein expressed by the midline glial cells and required
for normal neural
development. Two mammalian homologs, SLIT1 and SLTT 2, have recently been
identified in both
humans and mice. In mice both genes are expressed during CNS development in
the floor plate (the
vertebrate equivalent of midline glial cells), roof plate and developing motor
neurons, suggesting a
conservation of protein function between Drosophila and mammals (Holmes, G. P.
( 1998) Mech.
Dev. 79:57-72).
At the cellular level, growth and development are governed by the cell's
decision to enter into
or exit from the cell cycle and by the cell's commitment to a terminally
differentiated state. The
schlafen family of genes, a novel family of at least 7 members, are involved
in maintenance of T cell
quiescence. These genes are differentially regulated during thymocyte
maturation and are
preferentially expressed in lymphoid tissues. Expression of schlafen genes in
fibroblasts or thyoma
cells either retards or ablates cell growth, indicating that the schlafen
proteins probably participate in
regulation of the cell cycle (Schwarz, D. A. (1998) Immunity 9:657-668).
Differential gene expression within cells is triggered in response to
extracellular signals and
other environmental cues. Such signals include growth factors and other
mitogens such as retinoic
acid; cell-cell and cell-matrix contacts; and environmental factors such as
nutritional signals, toxic
substances, and heat shock. Candidate genes that may play a role in
differentiation can be identified
by altered expression patterns upon induction of cell differentiation in
vitro. For example, the REX
genes display reduced expression during retinoic acid induced differentiation
of murine
teratocarcinoma cells (Faria et al. (1998) Mol. Cell Endocrinol. 143:155-166).
The murine embryonal
carcinoma cell line P19 responds to retinoic acid by differentiating into
neuronal cell types. The shyc
gene was isolated from differentiating P19 cells and found to be predominantly
expressed in the
developing and embryonic nervous system, as well as the olfactory pathway of
the adult mouse brain
(Koster, F. et al. (1998) Neurosci. Lett. 252:69-71). Similarly, the Bdml gene
is upregulated during
differentiation of P19 cells to neuronal cells by retinoic acid, and was
widely expressed in the
olfactory bulb, cerebral cortex, hippocampus, cerebellum, thalamus, and
medulla oblongata
(Yamauchi, Y. et al. ( 1999) Brain Res. Mol. Brain Res. 68:149-58). These
proteins therefore appear
to play a role in the differentiation and later function of neuronal cells.
The final step in cell differentiation results in a specialization that is
characterized by the
production of particular proteins, such as contractile proteins in muscle
cells, serum proteins in liver
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cells and globins in red blood cell precursors. The expression of these
specialized proteins depends at
least in part on cell-specific transcription factors. For example, the homobox-
containing transcription
factor PAX-6 is essential for early eye determination, specification of ocular
tissues, and normal eye
development in vertebrates. PAX-6 is also involved in regulating the
expression of crystalline, the
dominant structural proteins of the eye lens. Defects in crystallin proteins
can cause formation of
cataracts, the most common cause of visual impairment world-wide (Francis, P.
J. et al. (1999) Trends
Genet. 15:191-196).
In the case of epidermal differentiation, the induction of differentiation-
specific genes occurs
either together with or following growth arrest and is believed to be linked
to the molecular events
that control irreversible growth arrest. Irreversible growth arrest is an
early event which occurs when
cells transit from the basal to the innermost suprabasal layer of the skin and
begin expressing
squamous-specific genes. These genes include those involved in the formation
of the cross-linked
envelope, such as transglutaminase I and III, involucrin, loricin, and small
proline-rich repeat (SPRR)
proteins. The SPRR proteins are 8-10 kDa in molecular mass, rich in proline,
glutamine, and
cysteine, and contain similar repeating sequence elements. The SPRR proteins
may be structural
proteins with a strong secondary structure or metal-binding proteins such as
metallothioneins.
(Jetten, A. M. and Harvat, B. L. (1997) J. Dermatol. 24:711-725; PRINTS Entry
PR00021 PRORICH
Small proline-rich protein signature.)
The discovery of new proteins involved in cell differentiation 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, developmental, and
neurological disorders.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, proteins involved in cell
differentiation,
referred to collectively as "CDIFF" and individually as "CDIFF-1," "CDIFF-2,"
"CDIFF-3," "CDIFF-
4," "CDIFF-5," "CDIFF-6," "CDIFF-7," "CDIFF-8," "CDIFF-9," "CDIFF-10," "CDIFF-
11,"
"CDIFF-12," "CDIFF-13," "CDIFF-14," "CDIFF-15," "CDIFF-16," "CDIFF-17," "CDIFF-
18,"
"CDIFF-19," "CDIFF-20," "CDIFF-21," "CDIFF-22," "CDIFF-23," "CDIFF-24," "CDIFF-
25,"
"CDIFF-26," "CDIFF-27," and "CDIFF-28." In one aspect, the invention provides
an isolated
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28, 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:1-28, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:I-28. In one
alternative, the
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invention provides an isolated polypeptide comprising the amino acid sequence
of SEQ ID NO:1-28.
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:1-28, 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:1-28, c) a biologically active fragment of an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid
sequence selected
from the group consisting of SEQ ID NO:1-28. In one alternative, the
polynucleotide encodes a
polypeptide selected from the group consisting of SEQ )D NO:1-28. In another
alternative, the
polynucleotide is selected from the group consisting of SEQ ID N0:29-56.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide comprising
an amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO: I-28, 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:1-28,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-28. In one alternative, the invention provides
a cell transformed
with the recombinant polynucleotide. In another alternative, the invention
provides a transgenic
organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an
amino acid
sequence selected from the group consisting of a) an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-28, 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:1-28,
c) a biologically active fragment of an amino acid sequence selected from the
group consisting of
SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence
selected from the
group consisting of SEQ ID NO:I-28. The method comprises a) culturing a cell
under conditions
suitable for expression of the polypeptide, wherein said cell is transformed
with a recombinant
polynucleotide comprising a promoter sequence operably linked to a
polynucleotide encoding the
polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO: I-28, 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:1-28, c) a biologically active fragment of an
amino acid sequence
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selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28.
The invention further provides an isolated polynucleotide comprising a
polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:29-56, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:29-56, c) a polynucleotide sequence complementary to a), d) a
polynucleotide sequence
complementary to b), and e) an RNA equivalent of a)-d). In one alternative,
the polynucleotide
comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:29-56, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:29-56, 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 N0:29-56, b) a naturally occurring polynucleotide
sequence having at least
90% sequence identity to a polynucleotide sequence selected from the group
consisting of SEQ 1D
N0:29-56, 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 composition comprising an effective amount of
a
polypeptide comprising an amino acid sequence selected from the group
consisting of a) an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a
naturally occurring amino
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acid sequence having at least 90% sequence identity to an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an
amino acid sequence
selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28, and a
pharmaceutically
acceptable excipient. In one embodiment, the composition comprises an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-28. The invention
additionally provides a
method of treating a disease or condition associated with decreased expression
of functional CDIFF,
comprising administering to a patient in need of such treatment the
composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-28,
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:1-28, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-28. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
agonist activity in the sample. In one alternative, the invention provides a
composition comprising an
agonist compound identified by the method and a pharmaceutically acceptable
excipient. In another
alternative, the invention provides a method of treating a disease or
condition associated with
decreased expression of functional CDIFF, comprising administering to a
patient in need of such
treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide comprising an amino acid sequence selected from
the group consisting
of a) an amino acid sequence selected from the group consisting of SEQ 117
NO:1-28, 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:1-28, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-28. The
method comprises a) exposing a sample comprising the polypeptide to a
compound, and b) detecting
antagonist activity in the sample. In one alternative, the invention provides
a composition comprising
an antagonist compound identified by the method and a pharmaceutically
acceptable excipient. In
another alternative, the invention provides a method of treating a disease or
condition associated with
overexpression of functional CDIFF, comprising administering to a patient in
need of such treatment
the composition.
The invention further provides a method of screening for a compound that
specifically binds
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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:1-28, 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:1-28, c) a biologically active fragment of
an amino acid sequence
selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28. The method
comprises a)
combining the polypeptide with at least one test compound under suitable
conditions, and b)
detecting binding of the polypeptide to the test compound, thereby identifying
a compound that
specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide comprising an amino acid sequence selected from the
group consisting of a)
an amino acid sequence selected from the group consisting of SEQ ID NO:1-28,
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:1-28, c) a biologically active
fragment of an amino
acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an
immunogenic
fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-28. 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 N0:29-56, the method
comprising a)
exposing a sample comprising the target polynucleotide to a compound, and b)
detecting altered
expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide comprising a polynucleotide
sequence selected from the
group consisting of i) a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:29-56, ii) a naturally occurnng polynucleotide sequence having at least 90%
sequence identity to
a polynucleotide sequence selected from the group consisting of SEQ ID N0:29-
56, iii) a
polynucleotide sequence complementary to i), iv) a polynucleotide sequence
complementary to ii),
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and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions
whereby a specific
hybridization complex is formed between said probe and a target polynucleotide
in the biological
sample, said target polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of i) a polynucleotide sequence selected from the group consisting
of SEQ ID N0:29-56,
ii) a naturally occurring polynucleotide sequence having at least 90% sequence
identity to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:29-56,
iii) a
polynucleotide sequence complementary to i}, iv) a polynucleotide sequence
complementary to ii),
and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide
comprises a fragment of
a polynucleotide sequence selected from the group consisting of i)-v) above;
c) quantifying the
amount of hybridization complex; and d) comparing the amount of hybridization
complex in the
treated biological sample with the amount of hybridization complex in an
untreated biological
sample, wherein a difference in the amount of hybridization complex in the
treated biological sample
is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs),
clone identification numbers (clone )Ds), cDNA libraries, and cDNA fragments
used to assemble full-
length sequences encoding CDIFF.
Table 2 shows features of each polypeptide sequence, including potential
motifs, homologous
sequences, and methods, algorithms, and searchable databases used for analysis
of CDIFF.
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 CDIFF were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is understood
that this invention is not limited to the particular machines, materials and
methods described, as these
may vary. It is also to be understood that the terminology used herein is for
the purpose of describing
particular embodiments only, and is not intended to limit the scope of the
present invention which
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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
"CDIFF" refers to the amino acid sequences of substantially purified CDIFF
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
CDIFF. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of CDIFF either by
directly interacting with
CDIFF or by acting on components of the biological pathway in which CDIFF
participates.
An "allelic variant" is an alternative form of the gene encoding CDIFF.
Allelic variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding CDIFF include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as CDIFF or a
polypeptide with at least one functional characteristic of CDIFF. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding CDIFF, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
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CDIFF. 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 CDIFF. 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 CDIFF 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 CDIFF. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of CDIFF either by
directly interacting with CDIFF or by acting on components of the biological
pathway in which
CDIFF 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 CDIFF polypeptides can be prepared using intact
polypeptides or using
fragments containing small peptides of interest as the immunizing antigen. The
polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to immunize
the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
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which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or biochemical
functions of a naturally occurring molecule. Likewise, "immunologically
active" or "irrimunogenic"
refers to the capability of the natural, recombinant, or synthetic CDIFF, 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 CDIFF or fragments
of CDIFF may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(PE Biosystems,
Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from
one or more overlapping cDNA, EST, or genomic DNA fragments using a computer
program for
fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison
WI) or Phrap
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(University of Washington, Seattle WA). Some sequences have been both extended
and assembled to
produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide sequence can include, for example,
replacement of
hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative
polynucleotide encodes a
polypeptide which retains at least one biological or immunological function of
the natural molecule.
A derivative polypeptide is one modified by glycosylation, pegylation, or any
similar process that
retains at least one biological or immunological function of the polypeptide
from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
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measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
A "fragment" is a unique portion of CDIFF or the polynucleotide encoding CDIFF
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 N0:29-56 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ ID N0:29-56, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID N0:29-56 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ ID N0:29-56 from related polynucleotide sequences. The precise length of a
fragment of SEQ
ID N0:29-56 and the region of SEQ ID N0:29-56 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:1-28 is encoded by a fragment of SEQ ID N0:29-56. A
fragment
of SEQ ID NO:1-28 comprises a region of unique amino acid sequence that
specifically identifies
SEQ ID NO:1-28. For example, a fragment of SEQ ID NO:1-28 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ ID NO:1-28.
The precise length of
a fragment of SEQ ID NO:1-28 and the region of SEQ ID NO:1-28 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "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
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in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST 2
Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Cap: 2 penalties
Gap x drop-off:' S0
Expect: l0
Word Size: 1l
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
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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: 1I and Extension Gap: 1 penalties
Gap x drop-off:' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length, for
example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
CA 02384324 2002-02-28
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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 lrg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.,
1989, Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring
Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
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
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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 CDIFF
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 CDIFF 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 CDIFF. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of CDIFF.
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
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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 CDIFF 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 CDIFF.
"Probe" refers to nucleic acid sequences encoding CDIFF, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
DNA strand by a DNA polymerise enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerise chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may also
be employed, such as probes and primers that comprise at least 20, 25, 30, 40,
50, 60, 70, 80, 90, 100,
or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers
may be considerably longer than these examples, and it is understood that any
length supported by the
specification, including the tables, figures, and Sequence Listing, may be
used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Cloning: A Laborat~ Manual,
2"° ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et
al. ( 1990) PCR
Protocols, A Guide to Methods and Annlications, 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
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purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MIT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently, a
recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
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(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 CDIFF, or fragments thereof, or CDIFF itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least SEQ ID N0:29-56
free from other
components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" refers to the collective pattern of gene expression by a
particular cell
type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a recipient
cell. Transformation may occur under natural or artificial conditions
according to various methods
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well known in the art, and may rely on any known method for the insertion of
foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method for
transformation is selected based
on the type of host cell being transformed and may include, but is not limited
to, bacteriophage or
viral infection, electroporation, heat shock, lipofection, and particle
bombardment. The term
"transformed" cells includes stably transformed cells in which the inserted
DNA is capable of
replication either as an autonomously replicating plasmid or as part of the
host chromosome, as well
as transiently transformed cells which express the inserted DNA or RNA for
limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transgenic techniques
well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. The term genetic manipulation does not include classical
cross-breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. The
transgenic organisms contemplated in accordance with the present invention
include bacteria,
cyanobacteria, fungi, plants, and animals. The isolated DNA of the present
invention can be
introduced into the host by methods known in the art, for example infection,
transfection,
transformation or transconjugation. Techniques for transferring the DNA of the
present invention
into such organisms are widely known and provided in references such as
Sambrook, J. et al. (1989),
supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
95% or at least 98% or
greater sequence identity over a certain defined length. A variant may be
described as, for example,
an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may
have significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
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
21
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
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 proteins involved in cell
differentiation (CDIFF), the polynucleotides encoding CDIFF, and the use of
these compositions for
the diagnosis, treatment, or prevention of cell proliferative, developmental,
and neurological
disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide
sequences encoding
CDIFF. Columns I and 2 show the sequence identification numbers (SEQ ID NOs)
of the
polypeptide and nucleotide sequences, respectively. Column 3 shows the clone
IDs of the Incyte
clones in which nucleic acids encoding each CDIFF were identified, and column
4 shows the cDNA
libraries from which these clones were isolated. Column 5 shows Incyte clones
and their
corresponding cDNA libraries. Clones for which cDNA libraries are not
indicated were derived from
pooled cDNA libraries. In some cases, GenBank sequence identifiers are also
shown in column S.
The Incyte clones and GenBank cDNA sequences, where indicated, in column 5
were used to
assemble the consensus nucleotide sequence of each CDIF'F 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 along with relevant
citations, all of
which are expressly incorporated by reference herein in their entirety; and
column 7 shows analytical
methods and in some cases, searchable databases to which the analytical
methods were applied. The
methods of column 7 were used to characterize each polypeptide through
sequence homology and
protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or
conditions
associated with nucleotide sequences encoding CDIFF. The first column of Table
3 lists the
nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of
column 1. These
22
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
fragments are useful, for example, in hybridization or amplification
technologies to identify SEQ ID
N0:29-56 and to distinguish between SEQ ID N0:29-56 and related polynucleotide
sequences. The
polypeptides encoded by these fragments are useful, for example, as
immunogenic peptides. Column
3 lists tissue categories which express CDIFF as a fraction of total tissues
expressing CDIFF.
Column 4 lists diseases, disorders, or conditions associated with those
tissues expressing CDIFF as a
fraction of total tissues expressing CDIFF. 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 CDIFF were isolated. Column 1 references the
nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were
isolated, and column 3
shows the tissue origins and other descriptive information relevant to the
cDNA libraries in column 2.
SEQ ID N0:32 maps to chromosome 1 within the interval from 152.2 to 157.4
centiMorgans,
to chromosome 3 within the interval from 157.4 to 158.0 centiMorgans, and to
the X chromosome
within the interval from 104.9 to 150.3 centiMorgans. The interval on
chromosome 1 from 152.2 to
157.4 centiMorgans also contains genes associated with leukemia,
hypothyroidism, and adrenal
hyperplasia. The interval on the X chromosome from 104.9 to 150.3 centiMorgans
also contains
genes associated with X-linked lissencephaly, leiomyomatosis with Alport
syndrome,
lymphoproliferative syndrome, Bruton agammaglobulinemia, and diffuse
angiokeratoma. SEQ ID
N0:37 maps to chromosome 11 within the interval from 19.6 to 23.2
centiMorgans. SEQ ID N0:39
maps to chromosome 16 within the interval from 109.1 to 130.8 centiMorgans,
and to chromosome
22 within the interval from 45.5 to 58.1 centiMorgans. The interval on
chromosome 16 from 109.1 to
130.8 centiMorgans also contains a gene associated with gastric cancer
susceptibility. SEQ ID
N0:45 maps to chromosome 7 within the interval from 105.2 to 109.0
centiMorgans, to chromosome
17 within the interval from 65.0 to 90.2 centiMorgans, and to chromosome 20
within the interval
from 50.2 to 54.9 centiMorgans. The interval on chromosome 7 from 105.2 to
109.0 centiMorgans
also contains a gene associated with osteogenesis imperfecta. The interval on
chromosome 17 from
65.0 to 90.2 centiMorgans also contains genes associated with breast cancer,
hepatic leukemia,
myeloperoxidase deficiency, muscular dystrophy, periodic paralysis, and
placental growth. SEQ ID
N0:54 maps to chromosome 12 within the interval from 21.3 to 36.1
centiMorgans. SEQ ID N0:55
maps to chromosome 1 within the interval from 22.9 to 39.9 centiMorgans and to
chromosome 3
within the interval from 30.9 to 43.0 centiMorgans.
The invention also encompasses CDIFF variants. A preferred CDIFF 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 CDIFF amino acid sequence, and which contains at
least one functional or
structural characteristic of CDIFF.
23
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
The invention also encompasses polynucleotides which encode CDIFF. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:29-56, which encodes CDIFF. The
polynucleotide
sequences of SEQ ID N0:29-56, 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
CDIFF. In
particular, such a variant polynucleotide sequence will have at least about
80%, or alternatively at
least about 90%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding CDIFF. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:29-56 which has at least about 80%, or alternatively at least about 90%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
of SEQ ID N0:29-56. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of CDIFF.
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 CDIFF, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring CDIFF, and all such variations
are to be considered as
being specifically disclosed.
Although nucleotide sequences which encode CDIFF and its variants are
generally capable of
hybridizing to the nucleotide sequence of the naturally occurring CDIFF under
appropriately selected
conditions of stringency, it may be advantageous to produce nucleotide
sequences encoding CDIFF 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 CDIFF and its derivatives without altering the encoded amino
acid sequences
include the production of RNA transcripts having more desirable properties,
such as a greater
half-life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode CDIFF
and
CDIFF derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the
24
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
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 CDIFF 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
N0:29-56 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods
Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise
(PE
Biosystems, Foster City CA), thermostable T7 polymerise (Amersham Pharmacia
Biotech,
Piscataway NJ), or combinations of polymerises and proofreading exonucleases
such as those found
in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably,
sequence preparation is automated with machines such as the MICROLAB 2200
liquid transfer
system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA)
and ABI
CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then carried out
using either the
ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA
sequencing
system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
The resulting
sequences are analyzed using a variety of algorithms which are well known in
the art. (See, e.g.,
Ausubel, F.M. (1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons,
New York NY, unit
7.7; Meyers, R.A. (1995) Molecular Biolo~v and Biotechnology, Wiley VCH, New
York NY, pp.
856-853.)
The nucleic acid sequences encoding CDIFF may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unknown
sequence from a circularized template. The template is derived from
restriction fragments comprising
a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et
al. (1988) Nucleic Acids
Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments
adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
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
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 CDIFF may be cloned in recombinant DNA molecules that direct
expression of CDIFF,
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 CDIFF.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter CDIFF-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
26
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
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 CDIFF, such as its biological or
enzymatic activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding CDIF'F may be synthesized, in whole
or in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic Acids
Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-
232.) Alternatively,
CDIFF itself or a fragment thereof may be synthesized using chemical methods.
For example,
peptide synthesis can be performed using various solution-phase or solid-phase
techniques. (See, e.g.,
Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated
synthesis may be achieved
using the ABI 431 A peptide synthesizer (PE Biosystems). Additionally, the
amino acid sequence of
CDIFF, 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 CDIFF, the nucleotide sequences
encoding CDIFF 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
27
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
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 CDIFF. Such elements may vary in their strength and specificity.
Specific initiation signals
may also be used to achieve more efficient translation of sequences encoding
CDIFF. Such signals
include the ATG initiation codon and adjacent sequences, e.g. the Kozak
sequence. In cases where
sequences encoding CDIFF 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 CDIFF 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
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 CDIFF. 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. 6:307-31 I; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
( 1984) Science 224:838-843; Winter, J. et al. ( 1991 ) Results Probl. Cell
Differ. 17:85-105; The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York
NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-
3659; and Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from
retroviruses,
28
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
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 CDIFF. For
example, routine cloning,
subcloning, and propagation of polynucleotide sequences encoding CDIFF 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 CDIFF 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 CDIFF are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of CDIFF may be used.
For example, vectors
containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of CDIFF. 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
nastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, su ra; and Scorer, supra.)
Plant systems may also be used for expression of CDIFF. Transcription of
sequences
encoding CDIFF may be driven viral promoters, e.g., the 35S and 19S promoters
of CaMV used alone
or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, su ra;~Broglie, su ra; 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 CDIFF
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
29
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses CDIFF 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 CDIFF in cell lines is preferred. For example, sequences encoding CDIFF can
be transformed into
cell lines using expression vectors which may contain viral origins of
replication and/or endogenous
expression elements and a selectable marker gene on the same or on a separate
vector. Following the
introduction of the vector, cells may be allowed to grow for about 1 to 2 days
in enriched media
before being switched to selective media. The purpose of the selectable marker
is to confer resistance
to a selective agent, and its presence allows growth and recovery of cells
which successfully express
the introduced sequences. Resistant clones of stably transformed cells may be
propagated using
tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. (1981)
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech),13 glucuronidase and its substrate f3-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
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also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding CDIFF is inserted within a marker gene sequence, transformed
cells containing
sequences encoding CDIFF can be identified by the absence of marker gene
function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding CDIFF 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 CDIF'F
and that express
CDIF'F 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 CDIFF
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 CDIFF is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding CDIFF
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding CDIFF, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially available,
and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA polymerase
such as T7, T3, or SP6 and labeled nucleotides. These procedures may be
conducted using a variety
of commercially available kits, such as those provided by Amersham Pharmacia
Biotech, Promega
(Madison WI), and US Biochemical. Suitable reporter molecules or labels which
may be used for
ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic
agents, as well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
Host cells transformed with nucleotide sequences encoding CDIFF may be
cultured under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
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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 CDIFF may be designed to contain
signal sequences which
direct secretion of CDIF'F 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 CDIFF 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 CDIFF protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of CDIFF
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 CDIFF encoding sequence and the
heterologous protein
sequence, so that CDIFF may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel (1995, su ra, 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 CDIFF 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
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precursor, for example, 35S-methionine.
CDIFF of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to CDIFF. At least one and up to a plurality of test
compounds may be screened
for specific binding to CDIFF. 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
CDIFF, e.g., a ligand or fragment thereof, a natural substrate, a structural
or functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunoloay 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which CDIFF
binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express CDIFF,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosonhila, or
E. coli. Cells expressing CDIFF or cell membrane fractions which contain CDIFF
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either CDIFF or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
CDIFF, either in
solution or affixed to a solid support, and detecting the binding of CDIFF 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.
CDIFF of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of CDIFF. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
CDIFF activity, wherein CDIFF is combined with at least one test compound, and
the activity of
CDIFF in the presence of a test compound is compared with the activity of
CDIFF in the absence of
the test compound. A change in the activity of CDIFF in the presence of the
test compound is
indicative of a compound that modulates the activity of CDIFF. Alternatively,
a test compound is
combined with an in vitro or cell-free system comprising CDIFF under
conditions suitable for CDIFF
activity, and the assay is performed. In either of these assays, a test
compound which modulates the
activity of CDIFF 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.
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In another embodiment, polynucleotides encoding CDIFF 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 CDIFF may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
( 1998) Science 282:1145-1147).
Polynucleotides encoding CDIFF 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 CDIFF 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 CDIFF, e.g., by secreting CDIFF
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 CDIFF and proteins involved in cell differentiation. In
addition, the expression of
CDIFF is closely associated with cell proliferative disorders (including
cancer) as well as
reproductive and nervous tissue disorders. Therefore, CDIFF appears to play a
role in cell
proliferative, developmental, and neurological disorders. In the treatment of
disorders associated
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with increased CDIFF expression or activity, it is desirable to decrease the
expression or activity of
CDIFF. In the treatment of disorders associated with decreased CDIFF
expression or activity, it is
desirable to increase the expression or activity of CDIFF.
Therefore, in one embodiment, CDIFF 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 CDIFF. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis,
inflammatory disorders, 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; 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; and a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis;
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis; mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD);
akathesia, amnesia,
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia.
In another embodiment, a vector capable of expressing CDIFF 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 CDIFF including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
CDIFF 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 CDIFF
including, but not limited to,
those provided above.
In still another embodiment, an agonist which modulates the activity of CDIFF
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of CDIFF including, but not limited to, those listed above.
In a further embodiment, an antagonist of CDIFF may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of CDIFF.
Examples of such
disorders include, but are not limited to, those cell proliferative,
developmental, and neurological
disorders described above. In one aspect, an antibody which specifically binds
CDIFF 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 CDIFF.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding CDIFF may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of CDIFF 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 CDIFF may be produced using methods which are generally known
in the
art. In particular, purified CDIFF may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind CDIFF.
Antibodies to CDIFF 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
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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 CDIFF or with any fragment or
oligopeptide thereof
which has immunogenic properties. Depending on the host species, various
adjuvants may be used to
increase immunological response. Such adjuvants include, but are not limited
to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among
adjuvants used in
humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
CDIFF 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 CDIFF 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 CDIFF may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. ( 1975) Nature 256:495-497; Kozbor,
D. et al. ( 1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
chain antibodies may be adapted, using methods known in the art, to produce
CDIFF-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 CDIFF may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin
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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
CDIFF and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering CDIFF 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 CDIFF.
Affinity is expressed as an
association constant, Ka, which is defined as the molar concentration of CDIFF-
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 CDIFF epitopes, represents the average affinity, or
avidity, of the antibodies for
CDIFF. The Ka determined for a preparation of monoclonal antibodies, which are
monospecific for a
particular CDIFF epitope, represents a true measure of affinity. High-affinity
antibody preparations
with K~ ranging from about 109 to 10'2 L/mole are preferred for use in
immunoassays in which the
CDIFF-antibody complex must withstand rigorous manipulations. Low-affinity
antibody preparations
with Ka ranging from about 106 to 10' L/mole are preferred for use in
immunopurification and similar
procedures which ultimately require dissociation of CDIFF, preferably in
active form, from the
antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies,
John Wiley & Sons,
New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of CDIFF-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, sera, and Coligan et al., s_ upra.)
In another embodiment of the invention, the polynucleotides encoding CDIFF, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
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WO 01/19860 PCT/US00/25435
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 CDIFF. 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 CDIFF. (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 vital
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 Morris, M.C. et al. (1997)
Nucleic Acids Res.
25( 14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding CDIFF may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470-475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal,
R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia ( 1997) Nature
389:239-242)), (ii)
express a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated
cell proliferation), or (iii) express a protein which affords protection
against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency virus (HIV)
(Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans
and Paracoccidioides
brasiliensis; and protozoan parasites such as Plasmodium falciparum and
Trypanosoma cruzi). In the
case where a genetic deficiency in CDIFF expression or regulation causes
disease, the expression of
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CDIFF 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
CDIFF are treated by constructing mammalian expression vectors encoding CDIFF
and introducing
these vectors by mechanical means into CDIFF-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 CDIFF include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen,
Carlsbad CA),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). CDIFF may be
expressed
using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV),
Rous sarcoma virus
(RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an
inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998)
Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the
ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the
FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible
promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native
promoter of the endogenous
gene encoding CDIFF from a normal individual.
Cornmercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. 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 CDIFF expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding CDIFF 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
CA 02384324 2002-02-28
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required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. ( 1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding CDIFF to cells which have one or more genetic
abnormalities with respect
to the expression of CDIFF. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. ( 1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors
for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding CDIFF to target cells which have one or more genetic
abnormalities with
respect to the expression of CDIFF. The use of herpes simplex virus (HSV)-
based vectors may be
especially valuable for introducing CDIFF 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
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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 CDIFF to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full-length
genomic RNA,
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
CDIFF into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
CDIFF-coding RNAs and the synthesis of high levels of CDIFF in vector
transduced cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of CDIFF 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.
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and B.I. Can, Molecular and Immunologic 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 CDIFF.
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 CDIFF. Such DNA sequences may be incorporated into a wide
variety of vectors
with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these
cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can be
introduced into cell lines,
cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding CDIFF.
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
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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
CDIFF expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding CDIFF may be therapeutically useful, and in the
treament of disorders
associated with decreased CDIFF expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding CDIFF 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 CDIFF 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
CDIFF 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 CDIFF. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
pombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Amdt, 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
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taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of CDIFF, antibodies to CDIFF, and mimetics, agonists, antagonists, or
inhibitors of CDIFF.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton,
J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage
of administration
without needle injection, and obviates the need for potentially toxic
penetration enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising CDIFF or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, CDIFF 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).
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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
CDIFF or fragments thereof, antibodies of CDIFF, and agonists, antagonists or
inhibitors of CDIFF,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDSO (the dose therapeutically effective in 50% of the
population) or LDSo (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed,
the sensitivity of the
patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of the
active moiety or to maintain the desired effect. Factors which may be taken
into account include the
severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
or biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0. I ~g to 100,000 fig, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
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 CDIFF may be used
for the
diagnosis of disorders characterized by expression of CDIFF, or in assays to
monitor patients being
treated with CDIFF or agonists, antagonists, or inhibitors of CDIFF.
Antibodies useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic assays
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for CDIFF include methods which utilize the antibody and a label to detect
CDIFF 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 CDIF'F, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
CDIFF expression. Normal
or standard values for CDIFF expression are established by combining body
fluids or cell extracts
taken from normal mammalian subjects, for example, human subjects, with
antibody to CDIFF under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, such as photometric means. Quantities of
CDIF'F 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 CDIFF 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 CDIFF
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of CDIFF, and to monitor regulation of CDIFF levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding CDIFF or closely related
molecules may be used
to identify nucleic acid sequences which encode CDIFF. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding CDIFF, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the CDIFF encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:29-56 or from
genomic sequences including promoters, enhancers, and introns of the CDIFF
gene.
Means for producing specific hybridization probes for DNAs encoding CDIFF
include the
cloning of polynucleotide sequences encoding CDIFF or CDIFF 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 3'-P or 35S,
or by enzymatic labels,
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such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding CDIFF may be used for the diagnosis of
disorders
associated with expression of CDIFF. Examples of such disorders include, but
are not limited to, a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis,
hepatitis, inflammatory disorders, 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; 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 Syndenharri s chorea
and cerebral palsy,
spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract,
and sensorineural
hearing loss; and a neurological disorder such as epilepsy, ischemic
cerebrovascular disease, stroke,
cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,
dementia, Parkinson's
disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and
other motor neuron
disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary ataxias, multiple
sclerosis and other demyelinating diseases, bacterial and viral meningitis,
brain abscess, subdural
empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis
and radiculitis, viral
central nervous system disease, prion diseases including kuru, Creutzfeldt-
Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional
and metabolic diseases
of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous
system disorders, cranial
nerve disorders, spinal cord diseases, muscular dystrophy and other
neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and polymyositis;
inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis; mental
disorders including
mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD);
akathesia, amnesia,
catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid
psychoses, postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial
frontotemporal dementia. The polynucleotide sequences encoding CDIFF may be
used in Southern or
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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 CDIFF expression. Such qualitative or quantitative methods are
well known in the art.
In a particular aspect, the nucleotide sequences encoding CDIFF may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding CDIFF 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 CDIFF 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
CDIFF, 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 CDIFF, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained from
successive assays may be used to show the efficacy of treatment over a period
ranging from several
days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding CDIFF
may involve the use of PCR. These oligomers may be chemically synthesized,
generated
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enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a polynucleotide
encoding CDIFF, or a fragment of a polynucleotide complementary to the
polynucleotide encoding
CDIFF, 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 CDIFF 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
CDIFF are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory preparation
of DNA and sequencing errors using statistical models and automated analyses
of DNA sequence
chromatograms. In the alternative, SNPs may be detected and characterized by
mass spectrometry
using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego CA).
Methods which may also be used to quantify the expression of CDIFF 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. ( I 993) 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
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used to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, to
monitor progression/regression of disease as a function of gene expression,
and to develop and
monitor the activities of therapeutic agents in the treatment of disease. In
particular, this information
may be used to develop a pharmacogenomic profile of a patient in order to
select the most appropriate
and effective treatment regimen for that patient. For example, therapeutic
agents which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, antibodies specific for CDIFF, or CDIFF or fragments
thereof may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and at
a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurnng environmental
compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-I 13:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they contain
expression information from a large number of genes and gene families.
Ideally, a genome-wide
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measurement of expression provides the highest quality signature. Even genes
whose expression is
not altered by any tested compounds are important as well, as the levels of
expression of these genes
are used to normalize the rest of the expression data. The normalization
procedure is useful for
comparison of expression data after treatment with different compounds. While
the assignment of
gene function to elements of a toxicant signature aids in interpretation of
toxicity mechanisms,
knowledge of gene function is not necessary for the statistical matching of
signatures which leads to
prediction of toxicity. (See, for example, Press Release 00-02 from the
National Institute of
Environmental Health Sciences, released February 29, 2000, available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample are
separated by isoelectric focusing in the first dimension, and then according
to molecular weight by
sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
supra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
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comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for CDIFF
to quantify
the levels of CDIFF expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-11 l; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed
by a variety of methods known in the art, for example, by reacting the
proteins in the sample with a
thiol- or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer ( 1997) Electrophoresis 18:533-537), so proteome
toxicant signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
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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 CDIFF
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences may
be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a multi-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial
chromosomes
(BACs), bacterial 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 CDIFF 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 11 q22-23,
any sequences mapping to that area may represent associated or regulatory
genes for further
investigation. (See, e.g., Gatti, R.A. et al. ( 1988) Nature 336:577-580.) The
nucleotide sequence of
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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, CDIFF, 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 CDIFF and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. ( 1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with CDIFF,
or fragments thereof,
and washed. Bound CDIFF is then detected by methods well known in the art.
Purified CDIFF 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 CDIFF specifically compete with a test compound
for binding CDIFF.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with CDIFF.
In additional embodiments, the nucleotide sequences which encode CDIFF 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, 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
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monophasic solution of phenol and guanidine isothiocyanate. The resulting
lysates were centrifuged
over CsCI cushions or extracted with chloroform. RNA was precipitated from the
lysates with either
isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A+) RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, 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),
peDNA2.1 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
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using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200
thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or
the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were
prepared using reagents
provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such
as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoretic
separation of cDNA sequencing reactions and detection of labeled
polynucleotides were carried out
using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373
or 377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base
calling software; or other sequence analysis systems known in the art. Reading
frames within the
cDNA sequences were identified using standard methods (reviewed in Ausubel,
1997, supra, unit
7.7). Some of the cDNA sequences were selected for extension using the
techniques disclosed in
Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and
analyzed
using a combination of software programs which utilize algorithms well known
to those skilled in the
art. Table 5 summarizes the tools, programs, and algorithms used and provides
applicable
descriptions, references, and threshold parameters. The first column of Table
5 shows the tools,
programs, and algorithms used, the second column provides brief descriptions
thereof, the third
column presents appropriate references, all of which are incorporated by
reference herein in their
entirety, and the fourth column presents, where applicable, the scores,
probability values, and other
parameters used to evaluate the strength of a match between two sequences (the
higher the score, the
greater the homology between two sequences). Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software
(DNASTAR). Polynucleotide and polypeptide sequence alignments were generated
using the default
parameters specified by the clustal algorithm as incorporated into the
MEGALIGN multisequence
alignment program (DNASTAR), which also calculates the percent identity
between aligned
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,
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and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire
annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences
were assembled
into full length polynucleotide sequences using programs based on Phred,
Phrap, and Consed, and
were screened for open reading frames using programs based on GeneMark, BLAST,
and FASTA.
The full length polynucleotide sequences were translated to derive the
corresponding full length
amino acid sequences, and these full length sequences were subsequently
analyzed by querying
against databases such as the GenBank databases (described above), SwissProt,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family
databases such
as PFAM. HMM is a probabilistic approach which analyzes consensus primary
structures of gene
families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were also used to identify polynucleotide sequence
fragments from SEQ ID
N0:29-56. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization and
amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel,
1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
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
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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 CDIFF occurred. Analysis involved the categorization
of cDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive, and urologic. The disease/condition categories included cancer,
inflammation, trauma,
cell proliferation, neurological, and pooled. For each category, the number of
libraries expressing the
sequence of interest was counted and divided by the total number of libraries
across all categories.
Percentage values of tissue-specific and disease- or condition-specific
expression are reported in
Table 3.
V. Chromosomal Mapping of CDIFF Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:29-56 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 N0:29-56 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.
Map locations are represented by ranges, or intervals, or human chromosomes.
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 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:32 maps to chromosome 1 within the interval from
152.2 to
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157.4 centiMorgans, to chromosome 3 within the interval from 157.4 to 158.0
centiMorgans, and to
the X chromosome within the interval from 104.9 to 150.3 centiMorgans. The
interval on
chromosome 1 from 152.2 to 157.4 centiMorgans also contains genes associated
with leukemia,
hypothyroidism, and adrenal hyperplasia. The interval on the X chromosome from
104.9 to 150.3
centiMorgans also contains genes associated with X-linked lissencephaly,
leiomyomatosis with
Alport syndrome, lymphoproliferative syndrome, Breton agammaglobulinemia, and
diffuse
angiokeratoma. SEQ ID N0:37 maps to chromosome 11 within the interval from
19.6 to 23.2
centiMorgans. SEQ ID N0:39 maps to chromosome 16 within the interval from
109.1 to 130.8
centiMorgans, and to chromosome 22 within the interval from 45.5 to 58.1
centiMorgans. The
interval on chromosome 16 from 109.1 to 130.8 centiMorgans also contains a
gene associated with
gastric cancer susceptibility. SEQ ID N0:45 maps to chromosome 7 within the
interval from 105.2
to 109.0 centiMorgans, to chromosome 17 within the interval from 65.0 to 90.2
centiMorgans, and to
chromosome 20 within the interval from 50.2 to 54.9 centiMorgans. The interval
on chromosome 7
from 105.2 to 109.0 centiMorgans also contains a gene associated with
osteogenesis imperfecta. The
interval on chromosome 17 from 65.0 to 90.2 centiMorgans also contains genes
associated with
breast cancer, hepatic leukemia, myeloperoxidase deficiency, muscular
dystrophy, periodic paralysis,
and placental growth. SEQ ID N0:54 maps to chromosome 12 within the interval
from 21.3 to 36.1
centiMorgans. SEQ ID NO:55 maps to chromosome 1 within the interval from 22.9
to 39.9
centiMorgans and to chromosome 3 within the interval from 30.9 to 43.0
centiMorgans.
More than one map location is reported for SEQ ID N0:32, SEQ ID N0:39, SEQ ID
N0:45,
and SEQ ID NO:55, indicating that sequences having different map locations
were assembled into a
single cluster. This situation occurs, for example, when sequences having
strong similarity, but not
complete identity, are assembled into a single cluster.
VI. Extension of CDIFF Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID N0:29-56 were produced by
extension of
an appropriate fragment of the full length molecule using oligonucleotide
primers designed from this
fragment. One primer was synthesized to initiate 5'extension of the known
fragment, and the other
primer, to initiate 3' extension of the known fragment. The initial primers
were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
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was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg2+, (NH4)ZS04,
and (3-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerise (Stratagene), with the following
parameters for primer
pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, 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 ~cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose mini-gel to determine which reactions were
successful in extending
the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise
(Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
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BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:29-56 are used to
obtain 5'
regulatory sequences using the procedure above, along with oligonucleotides
designed for such
extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:29-56 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
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
VIII. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. ( 1995) Science 270:467-470; 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
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comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in the
biological sample are conjugated to a fluorescent label or other molecular tag
for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are
removed, and a
fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT)
primer (2lmer), 1X
first strand buffer, 0.03 units/pl RNase inhibitor, 500 NM dATP, 500 liM dGTP,
500 NM dTTP, 40 _
liM dCTP, 40 I.dVI 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 p1 SX SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with
extensive distilled water
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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
I10°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array
element DNA, at an average
concentration of 100 ng/pl, is loaded into the open capillary printing element
by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60
°C followed by washes in
0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg
each of Cy3 and
Cy5 labeled cDNA synthesis products in 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 p1 of SX SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60 °C. The arrays are washed for 10
min at 45 °C in a first wash
buffer ( 1X SSC, 0.1 % SDS), three times for 10 minutes each at 45 °C
in a second wash buffer (0.1 X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores. Appropriate
filters positioned between the array and the photomultiplier tubes are used to
filter the signals. The
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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 CDIFF-encoding sequences, or any parts thereof,
are used to
detect, decrease, or inhibit expression of naturally occurring CDIFF. Although
use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of CDIFF. 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
CDIF'F-encoding
transcript.
X. Expression of CDIFF
Expression and purification of CDIFF is achieved using bacterial or virus-
based expression
systems. For expression of CDIFF in bacteria, cDNA is subcloned into an
appropriate vector
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containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21 (DE3).
Antibiotic resistant bacteria express CDIFF upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of CDIFF in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autogranhica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding CDIFF 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 Svodoptera fru~iperda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, CDIFF 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 japonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
CDIFF 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 CDIFF obtained by these methods can be used
directly in the assays
shown in Examples XI and XV.
XI. Demonstration of CDIFF Activity
CDIFF activity is demonstrated by measuring the induction of terminal
differentiation or cell
cycle progression when CDIFF is expressed 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 SPORTTM
(Life Technologies, Gaithersburg, MD) and pCRTM 3.1 (Invitrogen, Carlsbad,
CA), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, preferably of endothelial or hematopoietic origin, using
either liposome
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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, Palo Alto, CA), CD64, or a CD64-GFP fusion protein. Flow cytometry
detects and
quantifies the uptake of fluorescent molecules that diagnose events preceding
or coincident with cell
cycle progression or terminal differentiation. 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; up or 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
C try, Oxford, New York, NY.
XII. Functional Assays
CDIFF function is assessed by expressing the sequences encoding CDIFF at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid
(Invitrogen), both of which
contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are
transiently transfected into
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
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discussed in Ormerod, M.G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of CDIFF on gene expression can be assessed using highly
purified populations
of cells transfected with sequences encoding CDIFF 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 CDIFF and other genes of interest can be analyzed
by northern
analysis or microarray techniques.
XIII. Production of CDIFF Specific Antibodies
CDIFF substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used to
immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the CDIFF 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 KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with
the oligopeptide-
KLH complex in complete Freund's adjuvant. Resulting antisera are tested for
antipeptide and anti-
CDIFF activity by, for example, binding the peptide or CDIFF 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 CDIFF Using Specific Antibodies
Naturally occurring or recombinant CDIFF is substantially purified by
immunoaffinity
chromatography using antibodies specific for CDIFF. An immunoaffinity column
is constructed by
covalently coupling anti-CDIFF 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 CDIFF are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of CDIFF (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/CDIFF binding (e.g., a buffer of pH 2 to pH 3, or a high
concentration of a chaotrope, such
68
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
as urea or thiocyanate ion), and CDIFF is collected.
XV. Identification of Molecules Which Interact with CDIFF
CD1FF, or biologically active fragments thereof, are labeled with 'z5I Bolton-
Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled CDIFF, washed,
and any wells with labeled CDIFF complex are assayed. Data obtained using
different concentrations
of CDIFF are used to calculate values for the number, affinity, and
association of CDIFF with the
candidate molecules.
CDIFF may also be used in the PATHCALLING process (CuraGen Corp., New Haven
CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101 ).
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
69
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
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SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom
HILLMAN, Jennifer L.
YUE, Henry
REDDY, Roopa
LAL, Preeti
SHAH, Purvi
AZIMZAI, Yalda
BAUGHN, Mariah R.
LU, Dyung Aina M.
BANDMAN, Olga
SHIH, Leo L.
PATTERSON, Chandra
<120> PROTEINS ASSOCIATED WITH CELL DIFFERENTIATION
<130> PF-0741 PCT
<140> To Be Assigned
<141> Herewith
<150> 60/154,140; 60/169,155
<151> 1999-09-15; 1999-12-06
<160> 56
<170> PERL Program
<210> 1
<211> 367
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1681724CD1
<400> 1
Met Ala Thr Pro Asn Asn Leu Thr Pro Thr Asn Cys Ser Trp Trp
1 5 10 15
Pro Ile Ser Ala Leu Glu Ser Asp Ala Ala Lys Pro Ala Glu Ala
20 25 30
Pro Asp Ala Pro Glu Ala Ala Ser Pro Ala His Trp Pro Arg Glu
35 40 45
Ser Leu Val Leu Tyr His Trp Thr Gln Ser Phe Ser Ser Gln Lys
50 55 60
Val Arg Leu Val Ile Ala Glu Lys Gly Leu Val Cys Glu Glu Arg
65 70 75
Asp Val Ser Leu Pro Gln Ser Glu His Lys Glu Pro Trp Phe Met
80 85 90
Arg Leu Asn Leu Gly Glu Glu Val Pro Val Ile Ile His Arg Asp
95 100 105
Asn Ile Ile Ser Asp Tyr Asp Gln Ile Ile Asp Tyr Val Glu Arg
110 115 120
Thr Phe Thr Gly Glu His Val Val Ala Leu Met Pro Glu Val Gly
125 130 135
Ser Leu Gln His Ala Arg Val Leu Gln Tyr Arg Glu Leu Leu Asp
140 145 150
Ala Leu Pro Met Asp Ala Tyr Thr His Gly Cys Ile Leu His Pro
155 160 165
1 /43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
Glu Leu Thr Thr Asp Ser Met Ile Pro Lys Tyr Ala Thr Ala Glu
170 175 180
Ile Arg Arg His Leu Ala Asn Ala Thr Thr Asp Leu Met Lys Leu
185 190 195
Asp His Glu Glu Glu Pro Gln Leu Ser Glu Pro Tyr Leu Ser Lys
200 205 210
Gln Lys Lys Leu Met Ala Lys Ile Leu Glu His Asp Asp Val Ser
215 220 225
Tyr Leu Lys Lys Ile Leu Gly Glu Leu Ala Met Val Leu Asp Gln
230 235 240
Ile Glu Ala Glu Leu Glu Lys Arg Lys Leu Glu Asn Glu Gly Gln
245 250 255
Lys Cys Glu Leu Trp Leu Cys Gly Cys Ala Phe Thr Leu Ala Asp
260 265 270
Val Leu Leu Gly Ala Thr Leu His Arg Leu Lys Phe Leu Gly Leu
275 280 285
Ser Lys Lys Tyr Trp Glu Asp Gly Ser Arg Pro Asn Leu Gln Ser
290 295 300
Phe Phe Glu Arg Val Gln Arg Arg Phe Ala Phe Arg Lys Val Leu
305 310 315
Gly Asp Ile His Thr Thr Leu Leu Ser Ala Val Ile Pro Asn Ala
320 325 330
Phe Arg Leu Val Lys Arg Lys Pro Pro Ser Phe Phe Gly Ala Ser
335 340 345
Phe Leu Met Gly Ser Leu Gly Gly Met Gly Tyr Phe Ala Tyr Trp
350 355 360
Tyr Leu Lys Lys Lys Tyr Ile
365
<210> 2
<211> 102
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1718047CD1
<400> 2
Met Ala Leu Leu Lys Ala Asn Lys Asp Leu Ile Ser Ala Gly Leu
1 5 10 15
Lys Glu Phe Ser Val Leu Leu Asn Gln Gln Val Phe Asn Asp Pro
20 25 30
Leu Val Ser Glu Glu Asp Met Val Thr Val Val Glu Asp Trp Met
35 40 45
Asn Phe Tyr Ile Asn Tyr Tyr Arg Gln Gln Val Thr Gly Glu Pro
50 55 60
Gln Glu Arg Asp Lys Ala Leu Gln Glu Leu Arg Gln Glu Leu Asn
65 70 75
Thr Leu Ala Asn Pro Phe Leu Ala Lys Tyr Arg Asp Phe Leu Lys
80 85 90
Ser His Glu Leu Pro Ser His Pro Pro Pro Ser Ser
95 100
<210> 3
<211> 205
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1980323CD1
<400> 3
Met Ala Glu Pro Leu Gln Pro Asp Pro Gly Ala Ala Glu Asp Ala
2/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
1 5 10 15
Ala Ala Gln Ala Val Glu Thr Pro Gly Trp Lys Ala Pro Glu Asp
20 25 30
Ala Gly Pro Gln Pro Gly Ser Tyr Glu Ile Arg His Tyr Gly Pro
35 40 45
Ala Lys Trp Val Ser Thr Ser Val Glu Ser Met Asp Trp Asp Ser
50 55 60
Ala Ile Gln Thr Gly Phe Thr Lys Leu Asn Ser Tyr Ile Gln Gly
65 70 75
Lys Asn Glu Lys Glu Met Lys Ile Lys Met Thr Ala Pro Val Thr
80 85 90
Ser Tyr Val Glu Pro Gly Ser Gly Pro Phe Ser Glu Ser Thr Ile
95 100 105
Thr Ile Ser Leu Tyr Ile Pro Ser Glu Gln Gln Phe Asp Pro Pro
110 115 120
Arg Pro Leu Glu Ser Asp Val Phe Ile Glu Asp Arg Ala Glu Met
125 130 135
Thr Val Phe Val Arg Ser Phe Asp Gly Phe Ser Ser Ala Gln Lys
140 145 150
Asn Gln Glu Gln Leu Leu Thr Leu Ala Ser Ile Leu Arg Glu Asp
155 160 165
Gly Lys Val Phe Asp Glu Lys Val Tyr Tyr Thr Ala Gly Tyr Asn
170 175 180
Ser Pro Val Lys Leu Leu Asn Arg Asn Asn Glu Val Trp Leu Ile
185 190 195
Gln Lys Asn Glu Pro Thr Lys Glu Asn Glu
200 205
<210> 4
<211> 120
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1990956CD1
<400> 4
Met Glu Ser Lys Glu Glu Leu Ala Ala Asn Asn Leu Asn Gly Glu
1 5 10 15
Asn Ala Gln Gln Glu Asn Glu Gly Gly Glu Gln Ala Pro Thr Gln
20 25 30
Asn Glu Glu Glu Ser Arg His Leu Gly Gly Gly Glu Gly Gln Lys
35 40 45
Pro Gly Gly Asn Ile Arg Arg Gly Arg Val Arg Arg Leu Val Pro
50 55 60
Asn Phe Arg Trp Ala Ile Pro Asn Arg His.Ile Glu His Asn Glu
65 70 75
Ala Arg Asp Asp Val Glu Arg Phe Val Gly Gln Met Met Glu Ile
80 85 90
Lys Arg Lys Thr Arg Glu Gln Gln Met Arg His Tyr Met Arg Phe
95 100 105
Gln Thr Pro Glu Pro Asp Asn His Tyr Asp Phe Cys Leu Ile Pro
110 115 120
<210> 5
<211> 108
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2009069CD1
3/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<400> 5
Met Ala Lys Val Thr Ser Glu Pro Gln Lys Pro Asn Glu Asp Val
1 5 10 15
Asp Glu His Thr Pro Ser Thr Ser Ser Thr Lys Gly Arg Lys Lys
20 25 30
Gly Lys Thr Pro Arg Gln Arg Arg Ser Arg Ser Gly Val Lys Gly
35 40 45
Leu Lys Thr Thr Arg Lys Ala Lys Arg Pro Leu Arg Gly Ser Ser
50 55 60
Ser Gln Lys Ala Gly Glu Thr Asn Thr Pro Ala Gly Lys Pro Lys
65 70 75
Lys Ala Arg Gly Pro Ile Leu Arg Gly Arg Tyr His Arg Leu Lys
80 85 90
Glu Lys Met Lys Lys Glu Glu Ala Asp Lys Glu Gln Ser Glu Thr
95 100 105
Ser Val Leu
<210> 6
<211> 308
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2009435CD1
<400> 6
Met Ala Lys Met Glu Leu Ser Lys Ala Phe Ser Gly Gln Arg Thr
1 5 10 15
Leu Leu Ser Ala Ile Leu Ser Met Leu Ser Leu Ser Phe Ser Thr
20 25 30
Thr Ser Leu Leu Ser Asn Tyr Trp Phe Val Gly Thr Gln Lys Val
35 40 45
Pro Lys Pro Leu Cys Glu Lys Gly Leu Ala Ala Lys Cys Phe Asp
50 55 60
Met Pro Val Ser Leu Asp Gly Asp Thr Asn Thr Ser Thr Gln Glu
65 70 75
Val Val Gln Tyr Asn Trp Glu Thr Gly Asp Asp Arg Phe Ser Phe
80 85 90
Arg Ser Phe Arg Ser Gly Met Trp Leu Ser Cys Glu Glu Thr Val
95 100 105
Glu Glu Pro Ala Leu Leu His Pro Gln Ser Trp Lys Gln Phe Arg
110 115 120
Ala Leu Arg Ser Ser Gly Thr Ala Ala Ala Lys Gly Glu Arg Cys
125 130 135
Arg Ser Phe Ile Glu Leu Thr Pro Pro Ala Lys Arg Gly Glu Lys
140 145 150
Gly Leu Leu Glu Phe Ala Thr Leu Gln Gly Pro Cys His Pro Thr
155 160 165
Leu Arg Phe Gly Gly Lys Arg Leu Met Glu Lys Ala Ser Leu Pro
170 175 180
Ser Pro Pro Leu Gly Leu Cys Gly Lys Asn Pro Met Val Ile Pro
185 190 195
Gly Asn Ala Asp His Leu His Arg Thr Ser Ile His Gln Leu Pro
200 205 210
Pro Ala Thr Asn Arg Leu Ala Thr His Trp Glu Pro Cys Leu Trp
215 220 225
Ala Gln Thr Glu Arg Leu Cys Cys Cys Phe Leu Cys Pro Val Arg
230 235 240
Ser Pro Gly Asp Gly Gly Pro His Asp Val Phe Thr Ser Leu Pro
245 250 255
Ser Asp Cys Gln Leu Gly Ser Arg Arg Leu Glu Thr Thr Cys Leu
260 265 270
4/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
Glu Leu Trp Leu Gly Leu Leu His Gly Leu Ala Leu Leu His Leu
275 280 285
Leu His Gly Val Gly Cys His His Leu Gln His Val His Gln Asp
290 295 300
Gly Ala Gly Val Gln Val Gln Ala
305
<210> 7
<211> 116
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2027937CD1
<400> 7
Met Ser Phe Ser Glu Gln Gln Cys Lys Gln Pro Cys Val Pro Pro
1 5 10 15
Pro Cys Leu Pro Lys Thr Gln Glu Gln Cys Gln Ala Lys Ala Glu
20 25 30
Glu Val Cys Leu Pro Thr Cys Gln His Pro Cys Gln Asp Lys Cys
35 40 45
Leu Val Gln Ala Gln Glu Val Cys Leu Ser Gln Cys Gln Glu Ser
50 55 60
Ser Gln Glu Lys Cys Pro Gln Gln Gly Gln Glu Pro Tyr Leu Pro
65 70 75
Pro Cys Gln Asp Gln Cys Pro Pro Gln Cys Ala Glu Pro Cys Gln
80 85 90
Glu Leu Phe Gln Thr Lys Cys Val Glu Val Cys Pro Gln Lys Val
95 100 105
Gln Glu Lys Cys Ser Ser Pro Gly Lys Gly Lys
110 115
<210> 8
<211> 1253
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2722347CD1
<400> 8
Met Thr Thr His Val Thr Leu Glu Asp Ala Leu Ser Asn Val Asp
1 5 10 15
Leu Leu Glu Glu Leu Pro Leu Pro Asp Gln Gln Pro Cys Ile Glu
20 25 30
Pro Pro Pro Ser Ser Ile Met Tyr Gln Ala Asn Phe Asp Thr Asn
35 40 45
Phe Glu Asp Arg Asn Ala Phe Val Thr Gly Ile Ala Arg Tyr Ile
50 55 60
Glu Gln Ala Thr Val His Ser Ser Met Asn Glu Met Leu Glu Glu
65 70 75
Gly His Glu Tyr Ala Val Met Leu Tyr Thr Trp Arg Ser Cys Ser
80 85 90
Arg Ala Ile Pro Gln Val Lys Cys Asn Glu Gln Pro Asn Arg Val
95 100 105
Glu Ile Tyr Glu Lys Thr Val Glu Val Leu Glu Pro Glu Val Thr
110 115 120
Lys Leu Met Lys Phe Met Tyr Phe Gln Arg Lys Ala Ile Glu Arg
125 130 135
Phe Cys Ser Glu Val Lys Arg Leu Cys His Ala Glu Arg Arg Lys
140 145 150
Asp Phe Val Ser Glu Ala Tyr Leu Leu Thr Leu Gly Lys Phe Ile
5/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
155 160 165
Asn Met Phe Ala Val Leu Asp Glu Leu Lys Asn Met Lys Cys Ser
170 175 180
Val Lys Asn Asp His Ser Ala Tyr Lys Arg Ala Ala Gln Phe Leu
185 190 195
Arg Lys Met Ala Asp Pro Gln Ser Ile Gln Glu Ser Gln Asn Leu
200 205 210
Ser Met Phe Leu Ala Asn His Asn Arg Ile Thr Gln Cys Leu His
215 220 225
Gln Gln Leu Glu Val Ile Pro Gly Tyr Glu Glu Leu Leu Ala Asp
230 235 240
Ile Val Asn Ile Cys Val Asp Tyr Tyr Glu Asn Lys Met Tyr Leu
245 250 255
Thr Pro Ser Glu Lys His Met Leu Leu Lys Val Met Gly Phe Gly
260 265 270
Leu Tyr Leu Met Asp Gly Asn Val Ser Asn Ile Tyr Lys Leu Asp
275 280 285
Ala Lys Lys Arg Ile Asn Leu Ser Lys Ile Asp Lys Phe Phe Lys
290 295 300
Gln Leu Gln Val Val Pro Leu Phe Gly Asp Met Gln Ile Glu Leu
305 310 315
Ala Arg Tyr Ile Lys Thr Ser Ala His Tyr Glu Glu Asn Lys Ser
320 325 330
Lys Trp Thr Cys Thr Gln Ser Ser Ile Ser Pro Gln Tyr Asn Ile
335 340 345
Cys Glu Gln Met Val Gln Ile Arg Asp Asp His Ile Arg Phe Ile
350 355 360
Ser Glu Leu Ala Arg Tyr Ser Asn Ser Glu Val Val Thr Gly Ser
365 370 375
Gly Leu Asp Ser Gln Lys Ser Asp Glu Glu Tyr Arg Glu Leu Phe
380 385 390
Asp Leu Ala Leu Arg Gly Leu Gln Leu Leu Ser Lys Trp Ser Ala
395 400 405
His Val Met Glu Val Tyr Ser Trp Lys Leu Val His Pro Thr Asp
410 415 420
Lys Phe Cys Asn Lys Asp Cys Pro Gly Thr Ala Glu Glu Tyr Glu
425 430 435
Arg Ala Thr Arg Tyr Asn Tyr Thr Ser Glu Glu Lys Phe Ala Phe
440 445 450
Val Glu Val Ile Ala Met Ile Lys Gly Leu Gln Val Leu Met Gly
455 460 465
Arg Met Glu Ser Val Phe Asn Gln Ala Ile Arg Asn Thr Ile Tyr
470 475 480
Ala Ala Leu Gln Asp Phe Ala Gln Val Thr Leu Arg Glu Pro Leu
485 490 495
Arg Gln Ala Val Arg Lys Lys Lys Asn Val Leu Ile Ser Val Leu
500 505 510
Gln Ala Ile Arg Lys Thr Ile Cys Asp Trp Glu Gly Gly Arg Glu
515 520 525
Pro Pro Asn Asp Pro Cys Leu Arg Gly Glu Lys Asp Pro Lys Gly
530 535 540
Gly Phe Asp Ile Lys Val Pro Arg Arg Ala Val Gly Pro Ser Ser
545 550 555
Thr Gln Leu Tyr Met Val Arg Thr Met Leu Glu Ser Leu Ile Ala
560 565 570
Asp Lys Ser Gly Ser Lys Lys Thr Leu Arg Ser Ser Leu Asp Gly
575 580 585
Pro Ile Val Leu Ala Ile Glu Asp Phe His Lys Gln Ser Phe Phe
590 595 600
Phe Thr His Leu Leu Asn Ile Ser Glu Ala Leu Gln Gln Cys Cys
605 610 615
Asp Leu Ser Gln Leu Trp Phe Arg Glu Phe Phe Leu Glu Leu Thr
620 625 630
6/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
Met Gly Arg Arg Ile Gln Phe Pro Ile Glu Met Ser Met Pro Trp
635 640 645
Ile Leu Thr Asp His Ile Leu Glu Thr Lys Glu Pro Ser Met Met
650 655 660
Glu Tyr Val Leu Tyr Pro Leu Asp Leu Tyr Asn Asp Ser Ala Tyr
665 670 675
Tyr Ala Leu Thr Lys Phe Lys Lys Gln Phe Leu Tyr Asp Glu Ile
680 685 690
Glu Ala Glu Val Asn Leu Cys Phe Asp Gln Phe Val Tyr Lys Leu
695 700 705
Ala Asp Gln Ile Phe Ala Tyr Tyr Lys Ala Met Ala Gly Ser Val
710 715 720
Leu Leu Asp Lys Arg Phe Arg Ala Glu Cys Lys Asn Tyr Gly Val
725 730 735
Ile Ile Pro Tyr Pro Pro Ser Asn Arg Tyr Glu Thr Leu Leu Lys
740 745 750
Gln Arg His Val Gln Leu Leu Gly Arg Ser Ile Asp Leu Asn Arg
755 760 765
Leu Ile Thr Gln Arg Ile Ser Ala Ala Met Tyr Lys Ser Leu Asp
770 775 780
Gln Ala Ile Ser Arg Phe Glu Ser Glu Asp Leu Thr Ser Ile Val
785 790 795
Glu Leu Glu Trp Leu Leu Glu Ile Asn Arg Leu Thr His Arg Leu
800 805 810
Leu Cys Lys His Met Thr Leu Asp Ser Phe Asp Ala Met Phe Arg
815 820 825
Glu Ala Asn His Asn Val Ser Ala Pro Tyr Gly Arg Ile Thr Leu
830 835 840
His Val Phe Trp Glu Leu Asn Phe Asp Phe Leu Pro Asn Tyr Cys
845 850 855
Tyr Asn Gly Ser Thr Asn Arg Phe Val Arg Thr Ala Ile Pro Phe
860 865 870
Thr Gln Glu Pro Gln Arg Asp Lys Pro Ala Asn Val Gln Pro Tyr
875 880 885
Tyr Leu Tyr Gly Ser Lys Pro Leu Asn Ile Ala Tyr Ser His Ile
890 895 900
Tyr Ser Ser Tyr Arg Asn Phe Val Gly Pro Pro His Phe Lys Thr
905 910 915
Ile Cys Arg Leu Leu Gly Tyr Gln Gly Ile Ala Val Val Met Glu
920 925 930
Glu Leu Leu Lys Ile Val Lys Ser Leu Leu Gln Gly Thr Ile Leu
935 940 945
Gln Tyr Val Lys Thr Leu Ile Glu Val Met Pro Lys Ile Cys Arg
950 955 960
Leu Pro Arg His Glu Tyr Gly Ser Pro Gly Ile Leu Glu Phe Phe
965 970 975
His His Gln Leu Lys Asp Ile Ile Glu Tyr Ala Glu Leu Lys Thr
980 985 990
Asp Val Phe Gln Ser Leu Arg Glu Val Gly Asn Ala Ile Leu Phe
995 1000 1005
Cys Leu Leu Ile Glu Gln Ala Leu Ser Gln Glu Glu Val Cys Asp
1010 1015 1020
Leu Leu His Ala Ala Pro Phe Gln Asn Ile Leu Pro Arg Val Tyr
1025 1030 1035
Ile Lys Glu Gly Glu Arg Leu Glu Val Arg Met Lys Arg Leu Glu
1040 1045 1050
Ala Lys Tyr Ala Pro Leu His Leu Val Pro Leu Ile Glu Arg Leu
1055 1060 1065
Gly Thr Pro Gln Gln Ile Ala Ile Ala Arg Glu Gly Asp Leu Leu
1070 1075 1080
Thr Lys Glu Arg Leu Cys Cys Gly Leu Ser Met Phe Glu Val Ile
1085 1090 1095
Leu Thr Arg Ile Arg Ser Tyr Leu Gln Asp Pro Ile Trp Arg Gly
7/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
1100 1105 1110
Pro Pro Pro Thr Asn Gly Val Met His Val Asp Glu Cys Val Glu
1115 1120 1125
Phe His Arg Leu Trp Ser Ala Met Gln Phe Val Tyr Cys Ile Pro
1130 1135 1140
Val Gly Thr Asn Glu Phe Thr Ala Glu Gln Cys Phe Gly Asp Gly
1145 1150 1155
Leu Asn Trp Ala Gly Cys Ser Ile Ile Val Leu Leu Gly Gln Gln
1160 1165 1170
Arg Arg Phe Asp Leu Phe Asp Phe Cys Tyr His Leu Leu Lys Val
1175 1180 1185
Gln Arg Gln Asp Gly Lys Asp Glu Ile Ile Lys Asn Val Pro Leu
1190 1195 1200
Lys Lys Met Ala Asp Arg Ile Arg Lys Tyr Gln Ile Leu Asn Asn
1205 1210 1215
Glu Val Phe Ala Ile Leu Asn Lys Tyr Met Lys Ser Val Glu Thr
1220 1225 1230
Asp Ser Ser Thr Val Glu His Val Arg Cys Phe Gln Pro Pro Ile
1235 1240 1245
His Gln Ser Leu Ala Thr Thr Cys
1250
<210> 9
<211> 98
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2759876CD1
<400> 9
Met Ser Val Asp Met Asn Ser Gln Gly Ser Asp Ser Asn Glu Glu
1 5 10 15
Asp Tyr Asp Pro Asn Cys Glu Glu Glu Glu Glu Glu Glu Glu Asp
20 25 30
Asp Pro Gly Asp Ile Glu Asp Tyr Tyr Val Gly Val Ala Ser Asp
35 40 45
Val Glu Gln Gln Gly Ala Asp Ala Phe Asp Pro Glu Glu Tyr Gln
50 55 60
Phe Thr Cys Leu Thr Tyr Lys Glu Ser Glu Gly Ala Leu Asn Glu
65 70 75
His Met Thr Ser Leu Ala Ser Val Leu Lys Val Ser Ser Val Val
80 85 90
Asn Ser Ser Val Ile Pro Pro Ser
<210> 10
<211> 524
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2763735CD1
<400> 10
Met Glu Glu Glu Gln Asp Leu Pro Glu Gln Pro Val Lys Lys Ala
1 5 10 15
Lys Met Gln Glu Ser Gly Glu Gln Thr Ile Ser Gln Val Ser Asn
20 25 30
Pro Asp Val Ser Asp Gln Lys Pro Glu Thr Ser Ser Leu Ala Ser
35 40 45
Asn Leu Pro Met Ser Glu Glu Ile Met Thr Cys Thr Asp Tyr Ile
50 55 60
8/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
Pro Arg Ser Ser Asn Asp Tyr Thr Ser Gln Met Tyr Ser Ala Lys
65 70 75
Pro Tyr Ala His Ile Leu Ser Val Pro Val Ser Glu Thr Ala Tyr
80 85 90
Pro Gly Gln Thr Gln Tyr Gln Thr Leu Gln Gln Thr Gln Pro Tyr
95 100 105
Ala Val Tyr Pro Gln Ala Thr Gln Thr Tyr Gly Leu Pro Pro Phe
110 115 120
Ala Ser Ser Thr Asn Ala Ser Leu Ile Ser Thr Ser Ser Thr Ile
125 130 135
Ala Asn Ile Pro Ala Ala Ala Val Ala Ser Ile Ser Asn Gln Asp
140 145 150
Tyr Pro Thr Tyr Thr Ile Leu Gly Gln Asn Gln Tyr Gln Ala Cys
155 160 165
Tyr Pro Ser Ser Ser Phe Gly Val Thr Gly Gln Thr Asn Ser Asp
170 175 180
Ala Glu Ser Thr Thr Leu Ala Ala Thr Thr Tyr Gln Ser Glu Lys
185 190 195
Pro Ser Val Met Ala Pro Ala Pro Ala Ala Gln Arg Leu Ser Ser
200 205 210
Gly Asp Pro Ser Thr Ser Pro Ser Leu Ser Gln Thr Thr Pro Ser
215 220 225
Lys Asp Thr Asp Asp Gln Ser Arg Lys Asn Met Thr Ser Lys Asn
230 235 240
Arg Gly Lys Arg Lys Ala Asp Ala Thr Ser Ser Gln Asp Ser Glu
245 250 255
Leu Glu Arg Val Phe Leu Trp Asp Leu Asp Glu Thr Ile Ile Ile
260 265 270
Phe His Ser Leu Leu Thr Gly Ser Tyr Ala Gln Lys Tyr Gly Lys
275 280 285
Asp Pro Thr Val Val Ile Gly Ser Gly Leu Thr Met Glu Glu Met
290 295 300
Ile Phe Glu Val Ala Asp Thr His Leu Phe Phe Asn Asp Leu Glu
305 310 315
Glu Cys Asp Gln Val His Val Glu Asp Val Ala Ser Asp Asp Asn
320 325 330
Gly Gln Asp Leu Ser Asn Tyr Ser Phe Ser Thr Asp Gly Phe Ser
335 340 345
Gly Ser Gly Gly Ser Gly Ser His Gly Ser Ser Val Gly Val Gln
350 355 360
Gly Gly Val Asp Trp Met Arg Lys Leu Ala Phe Arg Tyr Arg Lys
365 370 375
Val Arg Glu Ile Tyr Asp Lys His Lys Ser Asn Val Gly Gly Leu
380 385 390
Leu Ser Pro Gln Arg Lys Glu Ala Leu Gln Arg Leu Arg Ala Glu
395 400 405
Ile Glu Val Leu Thr Asp Ser Trp Leu Gly Thr Ala Leu Lys Ser
410 415 420
Leu Leu Leu Ile Gln Ser Arg Lys Asn Cys Val Asn Val Leu Ile
425 430 435
Thr Thr Thr Gln Leu Val Pro Ala Leu Ala Lys Val Leu Leu Tyr
440 445 450
Gly Leu Gly Glu Ile Phe Pro Ile Glu Asn Ile Tyr Ser Ala Thr
455 460 465
Lys Ile Gly Lys Glu Ser Cys Phe Glu Arg Ile Val Ser Arg Phe
470 475 480
Gly Lys Lys Val Thr Tyr Val Val Ile Gly Asp Gly Arg Asp Ala
485 490 495
Ala Lys Gln His Asn Met Pro Phe Trp Arg Ile Thr Asn His Gly
500 505 510
Asp Leu Val Ser Leu His Gln Ala Leu Glu Leu Asp Phe Leu
515 520
<210> 11
9/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<211> 628
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2848676CD1
<400> 11
Met Ala Ala Ala Gly Ala Gly Pro Gly Gln Glu Ala Gly Ala Gly
1 5 10 15
Pro Gly Pro Gly Ala Val Ala Asn Ala Thr Gly Ala Glu Glu Gly
20 25 30
Glu Met Lys Pro Val Ala Ala Gly Ala Ala Ala Pro Pro Gly Glu
35 40 45
Gly Ile Ser Ala Ala Pro Thr Val Glu Pro Ser Ser Gly Glu Ala
50 55 60
Glu Gly Gly Glu Ala Asn Leu Val Asp Val Ser Gly Gly Leu Glu
65 70 75
Thr Glu Ser Ser Asn Gly Lys Asp Thr Leu Glu Gly Ala Gly Asp
80 85 90
Thr Ser Glu Val Met Asp Thr Gln Ala Gly Ser Val Asp Glu Glu
95 100 105
Asn Gly Arg Gln Leu Gly Glu Val Glu Leu Gln Cys Gly Ile Cys
110 115 120
Thr Lys Trp Phe Thr Ala Asp Thr Phe Gly Ile Asp Thr Ser Ser
125 130 135
Cys Leu Pro Phe Met Thr Asn Tyr Ser Phe His Cys Asn Val Cys
140 145 150
His His Ser Gly Asn Thr Tyr Phe Leu Arg Lys Gln Ala Asn Leu
155 160 165
Lys Glu Met Cys Leu Ser Ala Leu Ala Asn Leu Thr Trp Gln Ser
170 175 180
Arg Thr Gln Asp Glu His Pro Lys Thr Met Phe Ser Lys Asp Lys
185 190 195
Asp Ile Ile Pro Phe Ile Asp Lys Tyr Trp Glu Cys Met Thr Thr
200 205 210
Arg Gln Arg Pro Gly Lys Met Thr Trp Pro Asn Asn Ile Val Lys
215 220 225
Thr Met Ser Lys Glu Arg Asp Val Phe Leu Val Lys Glu His Pro
230 235 240
Asp Pro Gly Ser Lys Asp Pro Glu Glu Asp Tyr Pro Lys Phe Gly
245 250 255
Leu Leu Asp Gln Asp Leu Ser Asn Ile Gly Pro Ala Tyr Asp Asn
260 265 270
Gln Lys Gln Ser Ser Ala Val Ser Thr Ser Gly Asn Leu Asn Gly
275 280 285
Gly Ile Ala Ala Gly Ser Ser Gly Lys Gly Arg Gly Ala Lys Arg
290 295 300
Lys Gln Gln Asp Gly Gly Thr Thr Gly Thr Thr Lys Lys Ala Arg
305 310 315
Ser Asp Pro Leu Phe Ser Ala Gln Arg Leu Pro Pro His Gly Tyr
320 325 330
Pro Leu Glu His Pro Phe Asn Lys Asp Gly Tyr Arg Tyr Ile Leu
335 340 345
Ala Glu Pro Asp Pro His Ala Pro Asp Pro Glu Lys Leu Glu Leu
350 355 360
Asp Cys Trp Ala Gly Lys Pro Ile Pro Gly Asp Leu Tyr Arg Ala
365 370 375
Cys Leu Tyr Glu Arg Val Leu Leu Ala Leu His Asp Arg Ala Pro
380 385 390
Gln Leu Lys Ile Ser Asp Asp Arg Leu Thr Val Val Gly Glu Lys
395 400 405
10/43
Ala Lys Gln His Asn Met Pro Phe Trp Arg Ile Thr Asn H
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
Gly Tyr Ser Met Val Arg Ala Ser His Gly Val Arg Lys Gly Ala
410 415 420
Trp Tyr Phe Glu Ile Thr Val Asp Glu Met Pro Pro Asp Thr Ala
425 430 435
Ala Arg Leu Gly Trp Ser Gln Pro Leu Gly Asn Leu Gln Ala Pro
440 445 450
Leu Gly Tyr Asp Lys Phe Ser Tyr Ser Trp Arg Ser Lys Lys Gly
455 460 465
Thr Lys Phe His Gln Ser Ile Gly Lys His Tyr Ser Ser Gly Tyr
470 475 480
Gly Gln Gly Asp Val Leu Gly Phe Tyr Ile Asn Leu Pro Glu Asp
485 490 495
Thr Glu Thr Ala Lys Ser Leu Pro Asp Thr Tyr Lys Asp Lys Ala
500 505 510
Leu Ile Lys Phe Lys Ser Tyr Leu Tyr Phe Glu Glu Lys Asp Phe
515 520 525
Val Asp Lys Ala Glu Lys Ser Leu Lys Gln Thr Pro His Ser Glu
530 535 540
Ile Ile Phe Tyr Lys Asn Gly Val Asn Gln Gly Val Ala Tyr Lys
545 550 555
Asp Ile Phe Glu Gly Val Tyr Phe Pro Ala Ile Ser Leu Tyr Lys
560 565 570
Ser Cys Thr Val Ser Ile Asn Phe Gly Pro Cys Phe Lys Tyr Pro
575 580 585
Pro Lys Asp Leu Thr Tyr Arg Pro Met Ser Asp Met Gly Trp Gly
590 595 600
Ala Val Val Glu His Thr Leu Ala Asp Val Leu Tyr His Val Glu
605 610 615
Thr Glu Val Asp Gly Arg Arg Ser Pro Pro Trp Glu Pro
620 625
<210> 12
<211> 259
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2956153CD1
<400> 12
Met Asn Leu Val Asp Leu Trp Leu Thr Arg Ser Leu Ser Met Cys
1 5 10 15
Leu Leu Leu Gln Ser Phe Val Leu Met Ile Leu Cys Phe His Ser
20 25 30
Ala Ser Met Cys Pro Lys Gly Cys Leu Cys Ser Ser Ser Gly Gly
35 40 45
Leu Asn Val Thr Cys Ser Asn Ala Asn Leu Lys Glu Ile Pro Arg
50 55 60
Asp Leu Pro Pro Glu Thr Val Leu Leu Tyr Leu Asp Ser Asn Gln
65 70 75
Ile Thr Ser Ile Pro Asn Glu Ile Phe Lys Asp Leu His Gln Leu
80 85 90
Arg Val Leu Asn Leu Ser Lys Asn Gly Ile Glu Phe Ile Asp Glu
95 100 105
His Ala Phe Lys Gly Val Ala Glu Thr Leu Gln Thr Leu Asp Leu
110 115 120
Ser Asp Asn Arg Ile Gln Ser Val His Lys Asn Ala Phe Asn Asn
125 130 135
Leu Lys Ala Arg Ala Arg Ile Ala Asn Asn Pro Trp His Cys Asp
140 145 150
Cys Thr Leu Gln Gln Val Leu Arg Ser Met Ala Ser Asn His Glu
155 160 165
Thr Ala His Asn Val Ile Cys Lys Thr Ser Val Leu Asp Glu His
11 /43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
170 175 180
Ala Gly Arg Pro Phe Leu Asn Ala Ala Asn Asp Ala Asp Leu Cys
185 190 195
Asn Leu Pro Lys Lys Thr Thr Asp Tyr Ala Met Leu Val Thr Met
200 205 210
Phe Gly Trp Phe Thr Met Val Ile Ser Tyr Val Val Tyr Tyr Val
215 220 225
Arg Gln Asn Gln Glu Asp Ala Arg Arg His Leu Glu Tyr Leu Lys
230 235 240
Ser Leu Pro Ser Arg Gln Lys Lys Ala Asp Glu Pro Asp Asp Ile
245 250 255
Ser Thr Val Val
<210> 13
<211> 380
<212> PRT
<213> Homo sapiens
<220>
<221> misC_feature
<223> Incyte ID No: 3333139CD1
<400> 13
Met Ala Ala Pro Trp Trp Arg Ala Ala Leu Cys Glu Cys Arg Arg
1 5 10 15
Trp Arg Gly Phe Ser Thr Ser Ala Val Leu Gly Arg Arg Thr Pro
20 25 30
Pro Leu Gly Pro Met Pro Asn Ser Asp Ile Asp Leu Ser Asn Leu
35 40 45
Glu Arg Leu Glu Lys Tyr Arg Ser Phe Asp Arg Tyr Arg Arg Arg
50 55 60
Ala Glu Gln Glu Ala Gln Ala Pro His Trp Trp Arg Thr Tyr Arg
65 70 75
Glu Tyr Phe Gly Glu Lys Thr Asp Pro Lys Glu Lys Ile Asp Ile
80 85 90
Gly Leu Pro Pro Pro Lys Val Ser Arg Thr Gln Gln Leu Leu Glu
95 100 105
Arg Lys Gln Ala Ile Gln Glu Leu Arg Ala Asn Val Glu Glu Glu
110 115 120
Arg Ala Ala Arg Leu Arg Thr Ala Ser Val Pro Leu Asp Ala Val
125 130 135
Arg Ala Glu Trp Glu Arg Thr Cys Gly Pro Tyr His Lys Gln Arg
140 145 150
Leu Ala Glu Tyr Tyr Gly Leu Tyr Arg Asp Leu Phe His Gly Ala
155 160 165
Thr Phe Val Pro Arg Val Pro Leu His Val Ala Tyr Ala Val Gly
170 175 180
Glu Asp Asp Leu Met Pro Val Tyr Cys Gly Asn Glu Val Thr Pro
185 190 195
Thr Glu Ala Ala Gln Ala Pro Glu Val Thr Tyr Glu Ala Glu Glu
200 205 210
Gly Ser Leu Trp Thr Leu Leu Leu Thr Ser Leu Asp Gly His Leu
215 220 225
Leu Glu Pro Asp Ala Glu Tyr Leu His Trp Leu Leu Thr Asn Ile
230 235 240
Pro Gly Asn Arg Val Ala Glu Gly Gln Val Thr Cys Pro Tyr Leu
245 250 255
Pro Pro Phe Pro Ala Arg Gly Ser Gly Ile His Arg Leu Ala Phe
260 265 270
Leu Leu Phe Lys Gln Asp Gln Pro Ile Asp Phe Ser Glu Asp Ala
275 280 285
Arg Pro Ser Pro Cys Tyr Gln Leu Ala Gln Arg Thr Phe Arg Thr
290 295 300
12/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
Phe Asp Phe Tyr Lys Lys His Gln Glu Thr Met Thr Pro Ala Gly
305 310 315
Leu Ser Phe Phe Gln Cys Arg Trp Asp Asp Ser Val Thr Tyr Ile
320 325 330
Phe His Gln Leu Leu Asp Met Arg Glu Pro Val Phe Glu Phe Val
335 340 345
Arg Pro Pro Pro Tyr His Pro Lys Gln Lys Arg Phe Pro His Arg
350 355 360
Gln Pro Leu Arg Tyr Leu Asp Arg Tyr Arg Asp Ser His Glu Pro
365 370 375
Thr Tyr Gly Ile Tyr
380
<210> 14
<211> 130
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3432292CD1
<400> 14
Met Ser Cys Gln Gln Asn Gln Gln Gln Cys Gln Pro Pro Pro Lys
1 5 10 15
Cys Pro Pro Lys Cys Pro Pro Lys Cys Pro Pro Lys Cys Arg Pro
20 25 30
Gln Cys Pro Ala Pro Cys Pro Pro Pro Val Ser Ser Cys Cys Gly
35 40 45
Pro Ser Ser Gly Gly Cys Cys Gly Ser Ser Ser Gly Gly Cys Cys
50 55 60
Ser Ser Gly Gly Gly Gly Cys Cys Leu Ser His His Arg Pro Arg
65 70 75
Leu Phe His Arg His Arg His Gln Ser Pro Asp Cys Cys Glu Ser
80 85 90
Glu Leu Leu Gly Ala Leu Ala Ala Ser Thr Ala Leu Gly Thr Ala
95 100 105
Ala Asp Gln Thr Ser Asn Ile Thr Glu Gln Ala Phe Met Glu Lys
110 115 120
Thr Cys Lys Arg Gly Thr Cys Pro Gln Glu
125 130
<210> 15
<211> 761
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3478571CD1
<400> 15
Met Ser Leu Arg Ile Asp Val Asp Thr Asn Phe Pro Glu Cys Val
1 5 10 15
Val Asp Ala Gly Lys Val Thr Leu Gly Thr Gln Gln Arg Gln Glu
20 25 30
Met Asp Pro Arg Leu Arg Glu Lys Gln Asn Glu Ile Ile Leu Arg
35 40 45
Ala Val Cys Ala Leu Leu Asn Ser Gly Gly Gly Ile Ile Lys Ala
50 55 60
Glu Ile Glu Asn Lys Gly Tyr Asn Tyr Glu Arg His Gly Val Gly
65 70 75
Leu Asp Val Pro Pro Ile Phe Arg Ser His Leu Asp Lys Met Gln
80 85 90
Lys Glu Asn His Phe Leu Ile Phe Val Lys Ser Trp Asn Thr Glu
13/43
CA 02384324 2002-02-28
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95 100 105
Ala Gly Val Pro Leu Ala Thr Leu Cys Ser Asn Leu Tyr His Arg
110 115 120
Glu Arg Thr Ser Thr Asp Val Met Asp Ser Gln Glu Ala Leu Ala
125 130 135
Phe Leu Lys Cys Arg Thr Gln Thr Pro Thr Asn Ile Asn Val Ser
140 145 150
Asn Ser Leu Gly Pro Gln Ala Ala Gln Gly Ser Val Gln Tyr Glu
155 160 165
Gly Asn Ile Asn Val Ser Ala Ala Ala Leu Phe Asp Arg Lys Arg
170 175 180
Leu Gln Tyr Leu Glu Lys Leu Asn Leu Pro Glu Ser Thr His Val
185 190 195
Glu Phe Val Met Phe Ser Thr Asp Val Ser His Cys Val Lys Asp
200 205 210
Arg Leu Pro Lys Cys Val Ser Ala Phe Ala Asn Thr Glu Gly Gly
215 220 225
Tyr Val Phe Phe Gly Val His Asp Glu Thr Cys Gln Val Ile Gly
230 235 240
Cys Glu Lys Glu Lys Ile Asp Leu Thr Ser Leu Arg Ala Ser Ile
245 250 255
Asp Gly Cys Ile Lys Lys Leu Pro Val His His Phe Cys Thr Gln
260 265 270
Arg Pro Glu Ile Lys Tyr Val Leu Asn Phe Leu Glu Val His Asp
275 280 285
Lys Gly Ala Leu Arg Gly Tyr Val Cys Ala Ile Lys Val Glu Lys
290 295 300
Phe Cys Cys Ala Val Phe Ala Lys Val Pro Ser Ser Trp Gln Val
305 310 315
Lys Asp Asn Arg Val Arg Gln Leu Pro Thr Arg Glu Trp Thr Ala
320 325 330
Trp Met Met Glu Ala Asp Pro Asp Leu Ser Arg Cys Pro Glu Met
335 340 345
Val Leu Gln Leu Ser Leu Ser Ser Ala Thr Pro Arg Ser Lys Pro
350 355 360
Val Cys Ile His Lys Asn Ser Glu Cys Leu Lys Glu Gln Gln Lys
365 370 375
Arg Tyr Phe Pro Val Phe Ser Asp Arg Val Val Tyr Thr Pro Glu
380 385 390
Ser Leu Tyr Lys Glu Leu Phe Ser Gln His Lys Gly Leu Arg Asp
395 400 405
Leu Ile Asn Thr Glu Met Arg Pro Phe Ser Gln Gly Ile Leu Ile
410 415 420
Phe Ser Gln Ser Trp Ala Val Asp Leu Gly Leu Gln Glu Lys Gln
425 430 435
Gly Val Ile Cys Asp Ala Leu Leu Ile Ser Gln Asn Asn Thr Pro
440 445 450
Ile Leu Tyr Thr Ile Phe Ser Lys Trp Asp Ala Gly Cys Lys Gly
455 460 465
Tyr Ser Met Ile Val Ala Tyr Ser Leu Lys Gln Lys Leu Val Asn
470 475 480
Lys Gly Gly Tyr Thr Gly Arg Leu Cys Ile Thr Pro Leu Val Cys
485 490 495
Val Leu Asn Ser Asp Arg Lys Ala Gln Ser Val Tyr Ser Ser Tyr
500 505 510
Leu Gln Ile Tyr Pro Glu Ser Tyr Asn Phe Met Thr Pro Gln His
515 520 525
Met Glu Ala Leu Leu Gln Ser Leu Val Ile Val Leu Leu Gly Phe
530 535 540
Lys Ser Phe Leu Ser Glu Glu Leu Gly Ser Glu Val Leu Asn Leu
545 550 555
Leu Thr Asn Lys Gln Tyr Glu Leu Leu Ser Lys Asn Leu Arg Lys
560 565 570
14/43
CA 02384324 2002-02-28
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Thr Arg Glu Leu Phe Val His Gly Leu Pro Gly Ser Gly Lys Thr
575 580 585
Ile Leu Ala Leu Arg Ile Met Glu Lys Ile Arg Asn Val Phe His
590 595 600
Cys Glu Pro Ala Asn Ile Leu Tyr Ile Cys Glu Asn Gln Pro Leu
605 610 615
Lys Lys Leu Val Ser Phe Ser Lys Lys Asn Ile Cys Gln Pro Val
620 625 630
Thr Arg Lys Thr Phe Met Lys Asn Asn Phe Glu His Ile Gln His
635 640 645
Ile Ile Ile Asp Asp Ala Gln Asn Phe Arg Thr Glu Asp Gly Asp
650 655 660
Trp Tyr Gly Lys Ala Lys Phe Ile Thr Gln Thr Ala Arg Asp Gly
665 670 675
Pro Gly Val Leu Trp Ile Phe Leu Asp Tyr Phe Gln Thr Tyr His
680 685 690
Leu Ser Cys Ser Gly Leu Pro Pro Pro Ser Asp Gln Tyr Pro Arg
695 700 705
Glu Glu Ile Asn Arg Val Val Arg Asn Ala Gly Pro Ile Ala Asn
710 715 720
Tyr Leu Gln Gln Val Met Gln Glu Ala Arg Gln Asn Pro Pro Pro
725 730 735
Asn Leu Pro Pro Gly Ser Leu Val Met Leu Tyr Glu Pro Lys Trp
740 745 750
Ala Gln Gly Cys Pro Arg Gln Leu Arg Asp Tyr
755 760
<210> 16
<211> 197
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3495166CD1
<400> 16
Met Ser Ser Ala Pro Ala Ser Gly Pro Ala Pro Ala Ser Leu Thr
1 5 10 15
Leu Trp Asp Glu Glu Asp Phe Gln Gly Arg Arg Cys Arg Leu Leu
20 25 30
Ser Asp Cys Ala Asn Val Cys Glu Arg Gly Gly Leu Pro Arg Val
35 40 45
Arg Ser Val Lys Val Glu Asn Gly Val Trp Val Ala Phe Glu Tyr
50 55 60
Pro Asp Phe Gln Gly Gln Gln Phe Ile Leu Glu Lys Gly Asp Tyr
65 70 75
Pro Arg Trp Ser Ala Trp Ser Gly Ser Ser Ser His Asn Ser Asn
80 85 90
Gln Leu Leu Ser Phe Arg Pro Val Leu Cys Ala Asn His Asn Asp
95 100 105
Ser Arg Val Thr Leu Phe Glu Gly Asp Asn Phe Gln Gly Cys Lys
110 115 120
Phe Asp Leu Val Asp Asp Tyr Pro Ser Leu Pro Ser Met Gly Trp
125 130 135
Ala Ser Lys Asp Val Gly Ser Leu Lys Val Ser Ser Gly Ala Trp
140 145 150
Val Ala Tyr Gln Tyr Pro Gly Tyr Arg Gly Tyr Gln Tyr Val Leu
155 160 165
Glu Arg Asp Arg His Ser Gly Glu Phe Cys Thr Tyr Gly Glu Leu
170 175 180
Gly Thr Gln Ala His Thr Gly Gln Leu Gln Ser Ile Arg Arg Val
185 190 195
Gln His
15/43
CA 02384324 2002-02-28
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<210> 17
<211> 339
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3554748CD1
<400> 17
Met Pro Glu Cys Trp Asp Gly Glu His Asp Ile Glu Thr Pro Tyr
1 5 10 15
Gly Leu Leu His Val Val Ile Arg Gly Ser Pro Lys Gly Asn Arg
20 25 30
Pro Ala Ile Leu Thr Tyr His Asp Val Gly Leu Asn His Lys Leu
35 40 45
Cys Phe Asn Thr Phe Phe Asn Phe Glu Asp Met Gln Glu Ile Thr
50 55 60
Lys His Phe Val Val Cys His Val Asp Ala Pro Gly Gln Gln Val
65 70 75
Gly Ala Ser Gln Phe Pro Gln Gly Tyr Gln Phe Pro Ser Met Glu
80 85 90
Gln Leu Ala Ala Met Leu Pro Ser Val Val Gln His Phe Gly Phe
95 100 105
Lys Tyr Val Ile Gly Ile Gly Val Gly Ala Gly Ala Tyr Val Leu
110 115 120
Ala Lys Phe Ala Leu Ile Phe Pro Asp Leu Val Glu Gly Leu Val
125 130 135
Leu Val Asn Ile Asp Pro Asn Gly Lys Gly Trp Ile Asp Trp Ala
140 145 150
Ala Thr Lys Leu Ser Gly Leu Thr Ser Thr Leu Pro Asp Thr Val
155 160 165
Leu Ser His Leu Phe Ser Gln Glu Glu Leu Val Asn Asn Thr Glu
170 175 180
Leu Val Gln Ser Tyr Arg Gln Gln Ile Gly Asn Val Val Asn Gln
185 190 195
Ala Asn Leu Gln Leu Phe Trp Asn Met Tyr Asn Ser Arg Arg Asp
200 205 210
Leu Asp Ile Asn Arg Pro Gly Thr Val Pro Asn Ala Lys Thr Leu
215 220 225
Arg Cys Pro Val Met Leu Val Val Gly Asp Asn Ala Pro Ala Glu
230 235 240
Asp Gly Val Val Glu Cys Asn Ser Lys Leu Asp Pro Thr Thr Thr
245 250 255
Thr Phe Leu Lys Met Ala Asp Ser Gly Gly Leu Pro Gln Val Thr
260 265 270
Gln Pro Gly Lys Leu Thr Glu Ala Phe Lys Tyr Phe Leu Gln Gly
275 280 285
Met Gly Tyr Met Pro Ser Ala Ser Met Thr Arg Leu Ala Arg Ser
290 295 300
Arg Thr Ala Ser Leu Thr Ser Ala Ser Ser Val Asp Gly Ser Arg
305 310 315
Pro Gln Ala Cys Thr His Ser Glu Ser Ser Glu Gly Leu Gly Gln
320 325 330
Val Asn His Thr Met Glu Val Ser Cys
335
<210> 18
<211> 109
<212> PRT
<213> Homo sapiens
<220>
16/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<221> misc_feature
<223> Incyte ID No: 3555629CD1
<400> 18
Met Glu Arg Gln Gln Gln Gln Gln Gln Gln Leu Arg Asn Leu Arg
1 5 10 15
Asp Phe Leu Leu Val Tyr Asn Arg Met Thr Glu Leu Cys Phe Gln
20 25 30
Arg Cys Val Pro Ser Leu His His Arg Ala Leu Asp Ala Glu Glu
35 40 45
Glu Ala Cys Val Pro Ser Cys Ala Gly Lys Leu Ile His Ser Asn
50 55 60
His Arg Leu Met Ala Ala Tyr Val Gln Leu Met Pro Ala Leu Val
65 70 75
Gln Arg Arg Ile Ala Asp Tyr Glu Ala Ala Ser Ala Val Pro Gly
80 85 90
Val Ala Ala Glu Gln Pro Gly Val Ser Pro Ser Gly Ser Ser Asp
95 100 105
Unk Unk Unk Unk
<210> 19
<211> 131
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 639636CD1
<400> 19
Met Thr Lys Lys Lys Val Ser Gln Lys Lys Gln Arg Gly Arg Pro
1 5 10 15
Ser Ser Gln Pro Arg Arg Asn Ile Val Gly Cys Arg Ile Ser His
20 25 30
Gly Trp Lys Glu Gly Asp Glu Pro Ile Thr Gln Trp Lys Gly Thr
35 40 45
Val Leu Asp Gln Leu Leu Asp Asp Tyr Lys Glu Gly Asp Leu Arg
50 55 60
Ile Met Pro Glu Ser Ser Glu Ser Pro Pro Thr Glu Arg Glu Pro
65 70 ' 75
Gly Gly Val Val Asp Gly Leu Ile Gly Lys His Val Glu Tyr Thr
80 85 90
Lys Glu Asp Gly Ser Lys Arg Ile Gly Met Val Ile His Gln Val
95 100 105
Glu Ala Lys Pro Ser Val Tyr Phe Ile Lys Phe Asp Asp Asp Phe
110 115 120
His Ile Tyr Val Tyr Asp Leu Val Lys Lys Ser
125 130
<210> 20
<211> 194
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 902218CD1
<400> 20
Met Gly Ala Asn Gln Leu Val Val Leu Asn Val Tyr Asp Met Tyr
1 5 10 15
Trp Met Asn Glu Tyr Thr Ser Ser Ile Gly Ile Gly Val Phe His
20 25 30
Ser Gly Ile Glu Val Tyr Gly Arg Glu Phe Ala Tyr Gly Gly His
17/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
35 40 45
Pro Tyr Pro Phe Ser Gly Ile Phe Glu Ile Ser Pro Gly Asn Ala
50 55 60
Ser Glu Leu Gly Glu Thr Phe Lys Phe Lys Glu Ala Val Val Leu
65 70 75
Gly Ser Thr Asp Phe Leu Glu Asp Asp Ile Glu Lys Ile Val Glu
80 85 90
Glu Leu Gly Lys Glu Tyr Lys Gly Asn Ala Tyr His Leu Met His
95 100 105
Lys Asn Cys Asn His Phe Ser Ser Ala Leu Ser Glu Ile Leu Cys
110 115 120
Gly Lys Glu Ile Pro Arg Trp Ile Asn Arg Leu Ala Tyr Phe Ser
125 130 135
Ser Cys Ile Pro Phe Leu Gln Ser Cys Leu Pro Lys Glu Trp Leu
140 145 150
Thr Pro Ala Ala Leu Gln Ser Ser Val Ser Gln Glu Leu Gln Asp
155 160 165
Glu Leu Glu Glu Ala Glu Asp Ala Ala Ala Ser Ala Ser Val Ala
170 175 180
Ser Thr Ala Ala Gly Ser Arg Pro Gly Arg His Thr Lys Leu
185 190
<210> 21
<211> 184
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1360522CD1
<400> 21
Met Ala Thr Ala Leu Ala Leu Arg Ser Leu Tyr Arg Ala Arg Pro
1 5 10 15
Ser Leu Arg Cys Pro Pro Val Glu Leu Pro Trp Ala Pro Arg Arg
20 25 30
Gly His Arg Leu Ser Pro Ala Asp Asp Glu Leu Tyr Gln Arg Thr
35 40 45
Arg Ile Ser Leu Leu Gln Arg Glu Ala Ala Gln Ala Met Tyr Ile
50 55 60
Asp Ser Tyr Asn Ser Arg Gly Phe Met Ile Asn Gly Asn Arg Val
65 70 75
Leu Gly Pro Cys Ala Leu Leu Pro His Ser Val Val Gln Trp Asn
80 85 90
Val Gly Ser His Gln Asp Ile Thr Glu Asp Ser Phe Ser Leu Phe
95 100 105
Trp Leu Leu Glu Pro Arg Ile Glu Ile Val Val Val Gly Thr Gly
110 115 120
Asp Arg Thr Glu Arg Leu Gln Ser Gln Val Leu Gln Ala Met Arg
125 130 135
Gln Arg Gly Ile Ala Val Glu Val Gln Asp Thr Pro Asn Ala Cys
140 145 150
Ala Thr Phe Asn Phe Leu Cys His Glu Gly Arg Val Thr Gly Ala
155 160 165
Ala Leu Ile Pro Pro Pro Gly Gly Thr Ser Leu Thr Ser Leu Gly
170 175 180
Gln Ala Ala Gln
<210> 22
<211> 528
<212> PRT
<213> Homo Sapiens
<220>
18/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<221> misc_feature
<223> Incyte ID No: 1400678CD1
<400> 22
Met Ala Ser Met Arg Glu Ser Asp Thr Gly Leu Trp Leu His Asn
1 5 10 15
Lys Leu Gly Ala Thr Asp Glu Leu Trp Ala Pro Pro Ser Ile Ala
20 25 30
Ser Leu Leu Thr Ala Ala Val Ile Asp Asn Ile Arg Leu Cys Phe
35 40 45
His Gly Leu Ser Ser Ala Val Lys Leu Lys Leu Leu Leu Gly Thr
50 55 60
Leu His Leu Pro Arg Arg Thr Val Asp Glu Met Lys Gly Ala Leu
65 70 75
Met Glu Ile Ile Gln Leu Ala Ser Leu Asp Ser Asp Pro Trp Val
80 85 90
Leu Met Val Ala Asp Ile Leu Lys Ser Phe Pro Asp Thr Gly Ser
95 100 105
Leu Asn Leu Glu Leu Glu Glu Gln Asn Pro Asn Val Gln Asp Ile
110 115 120
Leu Gly Glu Leu Arg Glu Lys Val Gly Glu Cys Glu Ala Ser Ala
125 130 135
Met Leu Pro Leu Glu Cys Gln Tyr Leu Asn Lys Asn Ala Leu Thr
140 145 150
Thr Leu Ala Gly Pro Leu Thr Pro Pro Val Lys His Phe Gln Leu
155 160 165
Lys Arg Lys Pro Lys Ser Ala Thr Leu Arg Ala Glu Leu Leu Gln
170 175 180
Lys Ser Thr Glu Thr Ala Gln Gln Leu Lys Arg Ser Ala Gly Val
185 190 195
Pro Phe His Ala Lys Gly Arg Gly Leu Leu Arg Lys Met Asp Thr
200 205 210
Thr Thr Pro Leu Lys Gly Ile Pro Lys Gln Ala Pro Phe Arg Ser
215 220 225
Pro Thr Ala Pro Ser Val Phe Ser Pro Thr Gly Asn Arg Thr Pro
230 235 240
Ile Pro Pro Ser Arg Thr Leu Leu Arg Lys Glu Arg Gly Val Lys
245 250 255
Leu Leu Asp Ile Ser Glu Leu Asp Met Val Gly Ala Gly Arg Glu
260 265 270
Ala Lys Arg Arg Arg Lys Thr Leu Asp Ala Glu Val Val Glu Lys
275 280 285
Pro Ala Lys Glu Glu Thr Val Val Glu Asn Ala Thr Pro Asp Tyr
290 295 300
Ala Ala Gly Leu Val Ser Thr Gln Lys Leu Gly Ser Leu Asn Asn
305 310 315
Glu Pro Ala Leu Pro Ser Thr Ser Tyr Leu Pro Ser Thr Pro Ser
320 325 330
Val Val Pro Ala Ser Ser Tyr Ile Pro Ser Ser Glu Thr Pro Pro
335 340 345
Ala Pro Ser Ser Arg Glu Ala Ser Arg Pro Pro Glu Glu Pro Ser
350 355 360
Ala Pro Ser Pro Thr Leu Pro Ala Gln Phe Lys Gln Arg Ala Pro
365 370 375
Met Tyr Asn Ser Gly Leu Ser Pro Ala Thr Pro Thr Pro Ala Ala
380 385 390
Pro Thr Ser Pro Leu Thr Pro Thr Thr Pro Pro Ala Val Ala Pro
395 400 405
Thr Thr Gln Thr Pro Pro Val Ala Met Val Ala Pro Gln Thr Gln
410 415 420
Ala Pro Ala Gln Gln Gln Pro Lys Lys Asn Leu Ser Leu Thr Arg
425 430 435
Glu Gln Met Phe Ala Ala Gln Glu Met Phe Lys Thr Ala Asn Lys
19/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
440 445 450
Val Thr Arg Pro Glu Lys Ala Leu Ile Leu Gly Phe Met Ala Gly
455 460 465
Ser Arg Glu Asn Pro Cys Gln Glu Gln Gly Asp Val Ile Gln Ile
470 475 480
Lys Leu Ser Glu His Thr Glu Asp Leu Pro Lys Ala Asp Gly Gln
485 490 495
Gly Ser Thr Thr Met Leu Val Asp Thr Val Phe Glu Met Asn Tyr
500 505 510
Ala Thr Gly Gln Trp Thr Arg Phe Lys Lys Tyr Lys Pro Met Thr
515 520 525
Asn Val Ser
<210> 23
<211> 298
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1435556CD1
<400> 23
Met Thr Thr Ile Tyr Asp Leu Lys Lys Gln Lys Asp Lys Leu Leu
1 5 10 15
Lys Phe Tyr Ala Glu Ser Asp Glu Gln Ile Leu Met Lys Asn Arg
20 25 30
Lys Thr Leu~His Lys Ala Lys Asn Glu Asp Leu Asp Arg Val Leu
35 40 45
Lys Glu Trp Ile Arg Gln Arg Arg Ser Glu His Met Pro Leu Asn
50 55 60
Gly Met Leu Ile Met Lys Gln Ala Lys Ile Tyr His Asn Glu Leu
65 70 75
Lys Ile Glu Gly Asn Cys Glu Tyr Ser Thr Gly Trp Leu Gln Lys
80 85 90
Phe Lys Lys Arg His Gly Ile Lys Phe Leu Lys Thr Cys Gly Asn
95 100 105
Lys Ala Ser Ala Gly His Glu Ala Thr Glu Lys Phe Thr Gly Asn
110 115 120
Phe Ser Asn Asp Asp Glu Gln Asp Gly Asn Phe Glu Gly Phe Ser
125 130 135
Met Ser Ser Glu Lys Lys Ile Met Ser Asp Leu Leu Thr Tyr Thr
140 145 150
Lys Asn Ile His Pro Glu Thr Val Ser Lys Leu Glu Glu Glu Asp
155 160 165
Ile Lys Asp Val Phe Asn Ser Asn Asn Glu Ala Pro Val Val His
170 175 180
Ser Leu Ser Asn Gly Glu Val Thr Lys Met Val Leu Asn Gln Asp
185 190 195
Asp His Asp Asp Asn Asp Asn Glu Asp Asp Val Asn Thr Ala Glu
200 205 210
Lys Val Pro Ile Asp Asp Met Val Lys Met Cys Asp Gly Leu Ile
215 220 225
Lys Gly Leu Glu Gln His Ala Phe Ile Thr Glu Gln Glu Ile Met
230 235 240
Ser Val Tyr Lys Ile Lys Glu Arg Leu Leu Arg Gln Lys Ala Ser
245 250 255
Leu Met Arg Gln Met Thr Leu Lys Glu Thr Phe Lys Lys Ala Ile
260 265 270
Gln Arg Asn Ala Ser Ser Ser Leu Gln Asp Pro Leu Leu Gly Pro
275 280 285
Ser Thr Ala Ser Asp Ala Ser Ser His Leu Lys Ile Lys
290 295
20/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<210> 24
<211> 630
<212> PRT
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1546633CD1
<400> 24
Met Pro Gln Gln Gln His Lys Val Ser Pro Ala Ser Glu Ser Pro
1 5 10 15
Phe Ser Glu Glu Glu Ser Arg Glu Phe Asn Pro Ser Ser Ser Gly
20 25 30
Arg Ser Ala Arg Thr Val Ser Ser Asn Ser Phe Cys Ser Asp Asp
35 40 45
Thr Gly Cys Pro Ser Ser Gln Ser Val Ser Pro Val Lys Thr Pro
50 55 60
Ser Asp Ala Gly Asn Ser Pro Ile Gly Phe Cys Pro Gly Ser Asp
65 70 75
Glu Gly Phe Thr Arg Lys Lys Cys Thr Ile Gly Met Val Gly Glu
80 85 90
Gly Ser Ile Gln Ser Ser Arg Tyr Lys Lys Glu Ser Lys Ser Gly
95 100 105
Leu Val Lys Pro Gly Ser Glu Ala Asp Phe Ser Ser Ser Ser Ser
110 115 120
Thr Gly Ser Ile Ser Ala Pro Glu Val His Met Ser Thr Ala Gly
125 130 135
Ser Lys Arg Ser Ser Ser Ser Arg Asn Arg Gly Pro His Gly Arg
140 145 150
Ser Asn Gly Ala Ser Ser His Lys Pro Gly Ser.Ser Pro Ser Ser
155 160 165
Pro Arg Glu Lys Asp Leu Leu Ser Met Leu Cys Arg Asn Gln Leu
170 175 180
Ser Pro Val Asn Ile His Pro Ser Tyr Ala Pro Ser Ser Pro Ser
185 190 195
Ser Ser Asn Ser Gly Ser Tyr Lys Gly Ser Asp Cys Ser Pro Ile
200 205 210
Met Arg Arg Ser Gly Arg Tyr Met Ser Cys Gly Glu Asn His Gly
215 220 225
Val Arg Pro Pro Asn Pro Glu Gln Tyr Leu Thr Pro Leu Gln Gln
230 235 240
Lys Glu Val Thr Val Arg His Leu Lys Ile Lys Leu Lys Glu Ser
245 250 255
Glu Arg Arg Leu His Glu Arg Glu Ser Glu Ile Val Glu Leu Lys
260 265 270
Ser Gln Leu Ala Arg Met Arg Glu Asp Trp Ile Glu Glu Glu Cys
275 280 285
His Arg Val Glu Ala Gln Leu Ala Leu Lys Glu Ala Arg Lys Glu
290 295 300
Ile Lys Gln Leu Lys Gln Val Ile Glu Thr Met Arg Ser Ser Leu
305 310 315
Ala Asp Lys Asp Lys Gly Ile Gln Lys Tyr Phe Val Asp Ile Asn
320 325 330
Ile Gln Asn Lys Lys Leu Glu Ser Leu Leu Gln Ser Met Glu Met
335 340 345
Ala His Se-r Gly Ser Leu Arg Asp Glu Leu Cys Leu Asp Phe Pro
350 355 360
Cys Asp Ser Pro Glu Lys Ser Leu Thr Leu Asn Pro Pro Leu Asp
365 370 375
Thr Met Ala Asp Gly Leu Ser Leu Glu Glu Gln Val Thr Gly Glu
380 385 390
Gly Ala Asp Arg Glu Leu Leu Val Gly Asp Ser Ile Ala Asn Ser
21/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
395 400 405
Thr Asp Leu Phe Asp Glu Ile Val Thr Ala Thr Thr Thr Glu Ser
410 415 420
Gly Asp Leu Glu Leu Val His Ser Thr Pro Gly Ala Asn Val Leu
425 430 435
Glu Leu Leu Pro Ile Val Met Gly Gln Glu Glu Gly Ser Val Val
440 445 450
Val Glu Arg Ala Val Gln Thr Asp Val Val Pro Tyr Ser Pro Ala
455 460 465
Ile Ser Glu Leu Ile Gln Ser Val Leu Gln Lys Leu Gln Asp Pro
470 475 480
Cys Pro Ser Ser Leu Ala Ser Pro Asp Glu Ser Glu Pro Asp Ser
485 490 495
Met Glu Ser Phe Pro Glu Ser Leu Ser Ala Leu Val Val Asp Leu
500 505 510
Thr Pro Arg Asn Pro Asn Ser Ala Ile Leu Leu Ser Pro Val Glu
515 520 525
Thr Pro Tyr Ala Asn Val Asp Ala Glu Val His Ala Asn Arg Leu
530 535 540
Met Arg Glu Leu Asp Phe Ala Ala Cys Val Glu Glu Arg Leu Asp
545 550 555
Gly Val Ile Pro Leu Ala Arg Gly Gly Val Val Arg Gln Tyr Trp
560 565 570
Ser Ser Ser Phe Leu Val Asp Leu Leu Ala Val Ala Ala Pro Val
575 580 585
Val Pro Thr Val Leu Trp Ala Phe Ser Thr Gln Arg Gly Gly Thr
590 595 600
Asp Pro Val Tyr Asn Ile Gly Ala Leu Leu Arg Gly Cys Cys Val
605 610 615
Val Ala Leu His Ser Leu Arg Arg Thr Ala Phe Arg Ile Lys Thr
620 625 630
<210> 25
<211> 339
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1794031CD1
<400> 25
Met Asp Glu Asp Leu Ser Ala Ser Gln Asp His Ser Gln Ala Val
1 5 10 15
Thr Leu Ile Gln Glu Lys Met Thr Leu Phe Lys Ser Leu Met Asp
20 25 30
Arg Phe Glu His His Ser Asn Ile Leu Leu Thr Phe Glu Asn Lys
35 40 45
Asp Glu Asn His Leu Pro Leu Val Pro Pro Asn Lys Leu Glu Glu
50 55 60
Met Lys Arg Arg Ile Asn Asn Ile Leu Glu Lys Lys Phe Ile Leu
65 70 75
Leu Leu Glu Phe His Tyr Tyr Lys Cys Leu Val Leu Gly Leu Val
80 85 90
Asp Glu Val Lys Ser Lys Leu Asp Ile Trp Asn Ile Lys Tyr Gly
95 100 105
Ser Arg Glu Ser Val Glu Leu Leu Leu Glu Asp Trp His Lys Phe
110 115 120
Ile Glu Glu Lys Glu Phe Leu Ala Arg Leu Asp Thr Ser Phe Gln
125 ' 130 135
Lys Cys Gly Glu Ile Tyr Lys Asn Leu Ala Gly Glu Cys Gln Asn
140 145 150
Ile Asn Lys Gln Tyr Met Met Val Lys Ser Asp Val Cys Met Tyr
22/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
155 160 165
Arg Lys Asn Ile Tyr Asn Val Lys Ser Thr Leu Gln Lys Val Leu
170 175 180
Ala Cys Trp Ala Thr Tyr Val Glu Asn Leu Arg Leu Leu Arg Ala
185 190 195
Cys Phe Glu Glu Thr Lys Lys Glu Glu Ile Lys Glu Val Pro Phe
200 205 210
Glu Thr Leu Ala Gln Trp Asn Leu Glu His Ala Thr Leu Asn Glu
215 220 225
Ala Gly Asn Phe Leu Val Glu Val Ser Asn Asp Val Val Gly Ser
230 235 240
Ser Ile Ser Lys Glu Leu Arg Arg Leu Asn Lys Arg Trp Arg Lys
245 250 255
Leu Val Ser Lys Thr Gln Leu Glu Met Asn Leu Pro Leu Met Ile
260 265 270
Lys Lys Gln Asp Gln Pro Thr Phe Asp Asn Ser Gly Asn Ile Leu
275 280 285
Ser Lys Glu Glu Lys Ala Thr Val Glu Phe Ser Thr Asp Met Ser
290 295 300
Val Glu Leu Pro Glu Asn Tyr Asn Gln Asn Ile Lys Ala Gly Glu
305 310 315
Lys His Glu Lys Glu Asn Glu Glu Phe Thr Gly Gln Leu Lys Val
320 325 330
Ala Lys Asp Val Glu Lys Leu Ile Gly
335
<210> 26
<211> 189
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2060563CD1
<400> 26
Met Leu Gly Met Ile Lys Asn Ser Leu Phe Gly Ser Val Glu Thr
1 5 10 15
Trp Pro Trp Gln Val Leu Ser Lys Gly Asp Lys Glu Glu Val Ala
20 25 30
Tyr Glu Glu Arg Ala Cys Glu Gly Gly Lys Phe Ala Thr Val Glu
35 40 45
Val Thr Asp Lys Pro Val Asp Glu Ala Leu Arg Glu Ala Met Pro
50 55 60
Lys Val Ala Lys Tyr Ala Gly Gly Thr Asn Asp Lys Gly Ile Gly
65 70 75
Met Gly Met Thr Val Pro Ile Ser Phe Ala Val Phe Pro Asn Glu
80 85 90
Asp Gly Ser Leu Gln Lys Lys Leu Lys Val Trp Phe Arg Ile Pro
95 100 105
Asn Gln Phe Gln Ser Asp Pro Pro Ala Pro Ser Asp Lys Ser Val
110 115 120
Lys Ile Glu Glu Arg Glu Gly Ile Thr Val Tyr Ser Met Gln Phe
125 130 135
Gly Gly Tyr Ala Lys Glu Ala Asp Tyr Val Ala Gln Ala Thr Arg
140 145 150
Leu Arg Ala Ala Leu Glu Gly Thr Ala Thr Tyr Arg Gly Asp Ile
155 160 165
Tyr Phe Cys Thr Gly Tyr Asp Pro Pro Met Lys Pro Tyr Gly Arg
170 175 180
Arg Asn Glu Ile Trp Leu Leu Lys Thr
185
<210> 27
<211> 530
23/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2573955CD1
<400> 27
Met Leu Leu Trp Pro Leu Leu Leu Leu Leu Leu Leu Leu Pro Thr
1 5 10 15
Leu Ala Leu Leu Arg Gln Gln Arg Ser Gln Asp Ala Arg Leu Ser
20 25 30
Trp Leu Ala Gly Leu Gln His Arg Val Ala Trp Gly Ala Leu Val
35 40 45
Trp Ala Ala Thr Trp Gln Arg Arg Arg Leu Glu Gln Ser Thr Leu
50 55 60
His Val His Gln Ser Gln Gln Gln Ala Leu Arg Trp Cys Leu Gln
65 70 75
Gly Ala Gln Arg Pro His Cys Ser Leu Arg Arg Ser Thr Asp Ile
80 85 90
Ser Thr Phe Arg Asn His Leu Pro Leu Thr Lys Ala Ser Gln Thr
95 100 105
Gln Gln Glu Asp Ser Gly Glu Gln Pro Leu Ala Pro Thr Ser Asn
110 115 120
Gln Asp Leu Gly Glu Ala Ser Leu Gln Ala Thr Leu Leu Gly Leu
125 130 135
Ala Ala Leu Asn Lys Ala Tyr Pro Glu Val Leu Ala Gln Gly Arg
140 145 150
Thr Ala Arg Val Thr Leu Thr Ser Pro Trp Pro Arg Pro Leu Pro
155 160 165
Trp Pro Gly Asn Thr Leu Gly Gln Val Gly Thr Pro Gly Thr Lys
170 175 180
Asp Pro Arg Ala Leu Leu Leu Asp Ala Leu Arg Ser Pro Gly Leu
185 190 195
Arg Ala Leu Glu Ala Gly Thr Ala Val Glu Leu Leu Asp Val Phe
200 205 210
Leu Gly Leu Glu Thr Asp Gly Glu Glu Leu Ala Gly Ala Ile Ala
215 220 225
Ala Gly Asn Pro Gly Ala Pro Leu Arg Glu Arg Ala Ala Glu Leu
230 235 240
Arg Glu Ala Leu Glu Gln Gly Pro Arg Gly Leu Ala Leu Arg Leu
245 250 255
Trp Pro Lys Leu Gln Val Val Val Thr Leu Asp Ala Gly Gly Gln
260 265 270
Ala Glu Ala Val Ala Ala Leu Gly Ala Leu Trp Cys Gln Gly Leu
275 280 285
Ala Phe Phe Ser Pro Ala Tyr Ala Ala Ser Gly Gly Val Leu Gly
290 295 300
Leu Asn Leu Gln Pro Glu Gln Pro His Gly Leu Tyr Leu Leu Pro
305 310 315
Pro Gly Ala Pro Phe Ile Glu Leu Leu Pro Val Lys Glu Gly Thr
320 325 330
Gln Glu Glu Ala Ala Ser Thr Leu Leu Leu Ala Glu Ala Gln Gln
335 340 345
Gly Lys Glu Tyr Glu Leu Val Leu Thr Asp Arg Ala Ser Leu Thr
350 355 360
Arg Cys Arg Leu Gly Asp Val Val Arg Val Val Gly Ala Tyr Asn
365 370 375
Gln Cys Pro Val Val Arg Phe Ile Cys Arg Leu Asp Gln Thr Leu
380 385 390
Ser Val Arg Gly Glu Asp Ile Gly Glu Asp Leu Phe Ser Glu Ala
395 400 405
Leu Gly Arg Ala Val Gly Gln Trp Ala Gly Ala Lys Leu Leu Asp
24/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
410 415 420
His Gly Cys Val Glu Ser Ser Ile Leu Asp Ser Ser Ala Gly Ser
425 430 435
Ala Pro His Tyr Glu Val Phe Val Ala Leu Arg Gly Leu Arg Asn
440 445 450
Leu Ser Glu Glu Asn Arg Asp Lys Leu Asp His Cys Leu Gln Glu
455 460 465
Ala Ser Pro Arg Tyr Lys Ser Leu Arg Phe Trp Gly Ser Val Gly
470 475 480
Pro Ala Arg Val His Leu Val Gly Gln Gly Ala Phe Arg Ala Leu
485 490 495
Arg Ala Ala Leu Ala Ala Cys Pro Ser Ser Pro Phe Pro Pro Ala
500 505 510
Met Pro Arg Val Leu Arg His Arg His Leu Ala Gln Cys Leu Gln
515 520 525
Glu Arg Val Val Ser
530
<210> 28
<211> 356
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3404792CD1
<400> 28
Met Ala Gly Leu Gly Ser Asp Pro Trp Trp Lys Lys Thr Leu Tyr
1 5 10 15
Leu Thr Gly Gly Ala Leu Leu Ala Ala Ala Ala Tyr Leu Leu His
20 25 30
Glu Leu Leu Val Ile Arg Lys Gln Gln Glu Ile Asp Ser Lys Asp
35 40 45
Ala Ile Ile Leu His Gln Phe Ala Arg Pro Asn Asn Gly Val Pro
50 55 60
Ser Leu Ser Pro Phe Cys Leu Lys Met Glu Thr Tyr Leu Arg Met
65 70 75
Ala Asp Leu Pro Tyr Gln Asn Tyr Phe Gly Gly Lys Leu Ser Ala
80 85 90
Gln Gly Lys Met Pro Trp Ile Glu Tyr Asn His Glu Lys Val Ser
95 100 105
Gly Thr Glu Phe Ile Ile Asp Phe Leu Glu Glu Lys Leu Gly Val
110 115 120
Asn Leu Asn Lys Asn Leu Gly Pro His Glu Arg Ala Ile Ser Arg
125 130 135
Ala Val Thr Lys Met Val Glu Glu His Phe Tyr Trp Thr Leu Ala
140 145 150
Tyr Cys Gln Trp Val Asp Asn Leu Asn Glu Thr Arg Lys Met Leu
155 160 165
Ser Leu Ser Gly Gly Gly Pro Phe Ser Asn Leu Leu Arg Trp Val
170 175 180
Val Cys His Ile Thr Lys Gly Ile Val Lys Arg Glu Met His Gly
185 190 195
His Gly Ile Gly Arg Phe Ser Glu Glu Glu Ile Tyr Met Leu Met
200 205 210
Glu Lys Asp Met Arg Ser Leu Ala Gly Leu Leu Gly Asp Lys Lys
215 220 225
Tyr Ile Met Gly Pro Lys Leu Ser Thr Leu Asp Ala Thr Val Phe
230 235 240
Gly His Leu Ala Gln Ala Met Trp Thr Leu Pro Gly Thr Arg Pro
245 250 255
Glu Arg Leu Ile Lys Gly Glu Leu Ile Asn Leu Ala Met Tyr Cys
260 265 270
25/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
Glu Arg Ile Arg Arg Lys Phe Trp Pro Glu Trp His His Asp Asp
275 280 285
Asp Asn Thr Ile Tyr Glu Ser Glu Glu Ser Ser Glu Gly Ser Lys
290 295 300
Thr His Thr Pro Leu Leu Asp Phe Ser Phe Tyr Ser Arg Thr Glu
305 310 315
Thr Phe Glu Asp Glu Gly Ala Glu Asn Ser Phe Ser Arg Thr Pro
320 325 330
Asp Thr Asp Phe Thr Gly His Ser Leu Phe Asp Ser Asp Val Asp
335 340 345
Met Asp Asp Tyr Thr Asp His Glu Gln Cys Lys
350 355
<210> 29
<211> 1364
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1681724CB1
<400> 29
gagagagccg ccgcgccgga gcctccttct ttcctgcctc tgattccggg ctgtcatggc 60
gacccccaac aatctgaccc ccaccaactg cagctggtgg cccatctccg cgctggagag 120
cgatgcggcc aagccagcgg aggcccccga cgctcccgag gcggccagcc ccgcccattg 180
gcccagggag agcctggttc tgtaccactg gacccagtcc ttcagctcgc agaaggtgcg 240
gctggtgatc gccgagaagg gcctggtgtg cgaggagcgg gacgtgagcc tgccacagag 300
cgagcacaag gagccctggt tcatgcggct caacctgggc gaggaggtgc ccgtcatcat 360
ccaccgcgac aacatcatca gtgactatga ccagatcatt gactatgtgg agcgcacctt 420
cacaggagag cacgtggtgg ccctgatgcc cgaggtgggc agcctgcagc acgcacgggt 480
gctgcagtac cgggagctgc tggacgcact gcccatggat gcctacacgc atggctgcat 540
cctgcatccc gagctcacca ccgactccat gatccccaag tacgccacgg ccgagatccg 600
cagacattta gccaatgcca ccacggacct catgaaactg gaccatgaag aggagcccca 660
gctctccgag ccctaccttt ctaaacaaaa gaagctcatg gccaagatct tggagcatga 720
tgatgtgagc_tacctgaaga agatcctcgg ggaactggcc atggtgctgg accagattga 780
ggcggagctg gagaagagga agctggagaa cgaggggcag aaatgcgagc tgtggctctg 840
tggctgtgcc ttcaccctcg ctgatgtcct cctgggagcc accctgcacc gcctcaagtt 900
cctgggactg tccaagaaat actgggaaga tggcagccgg cccaacctgc agtccttctt 960
tgagagggtc cagagacgct ttgccttccg gaaagtcctg ggtgacatcc acaccaccct 1020
gctgtcggcc gtcatcccca atgctttccg gctggtcaag aggaaacccc catccttctt 1080
cggggcgtcc ttcctcatgg gctccctggg tgggatgggc tactttgcct actggtacct 1140
caagaaaaaa tacatctagg gccaggcctg gggcttggtg tctgactgtc ggtgtctctg 1200
tgctgtgtga ttccccgtga gctctcagta actcactgtc tcatgaacac ttggacagcc 1260
ctccccgccc ttcgttctga gtaataatac cgtcagtgtg aaaacattcc gtagtttaga 1320
agtagacgtt gccaatgctg tgactcaagg ccagggttca atta 1364
<210> 30
<211> 505
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1718047CB1
<400> 30
cggctcgagg agagattcac gcaccctcaa gagtgtgggt gagacatata cagcctgtta 60
gacctgaagg cagatggctc ttcttaaggc caataaggat ctcatttccg caggattgaa 120
ggagttcagc gttctgctga atcagcaggt cttcaatgat cctctcgtct ctgaagaaga 180
catggtgact gtggtggagg actggatgaa cttctacatc aactattaca ggcagcaggt 240
gacaggggag ccccaagagc gagacaaggc tctgcaggag cttcggcaag agctgaacac 300
tctggccaac cctttcctgg ccaagtacag ggacttcctg aagtctcatg agctcccgag 360
tcacccaccg ccctcctcct agctcaggga cccagccccc cctctctgag aaactctgac 420
26/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
cttcatgtcc ttaggctgtg ctcctgccac tctaccctga cacctcaata aagaccagtg 480
ctggttttgt tggaaaaaaa aaaaa 505
<210> 31
<211> 926
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1980323CB1
<400> 31
gtccggggtc gtgagccggc cccgccttgg tggcggcgcc ccctcgcggt ccagaggcag 60
acgcatcggg tgggctcggg tctccagccc ggccgggagg agggaccggg tctgcggagc 120
ggggactcgg ggcctcggcg gggcgcgcac acgcaggcgg ggcggcccgg ggtgcggggc 180
ctctgcgcgg ctgaccaggc tcccagagcg tcacgccgcc catggccgag ccgctccagc 240
cagaccccgg ggcggccgag gacgcggcgg cccaagctgt ggagacgccg ggctggaagg 300
ccccggagga cgccggcccc cagcccggaa gttatgagat ccgacactat ggaccagcca 360
agtgggtcag cacgtccgtg gagtctatgg actgggattc agccatccag acgggcttta 420
cgaaactgaa cagctacatt caaggcaaaa acgagaaaga gatgaaaata aagatgacag 480
ctccagtgac aagctacgtg gagcctggtt caggtccttt tagtgagtct accattacca 540
tttccctgta tattccctct gaacagcaat ttgatccacc caggccttta gagtcagatg 600
tcttcattga agatagagcc gaaatgactg tgtttgtacg gtctttcgat ggattttcta 660
gtgcccaaaa gaatcaagaa caacttttga cattagcaag cattttaagg gaagatggaa 720
aagttttcga tgagaaggtt tactacactg caggctacaa cagtcctgtc aaattgctta 780
atagaaataa tgaagtgtgg ttgattcaaa aaaatgaacc caccaaagaa aacgaatgag 840
aaaaatgaaa ggaagttctg ctgtcagagg caaaacatct gtttatcata gacatcaaca 900
tgacctataa gtaaaaaaaa aaaaaa 926
<210> 32
<211> 1364
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1990956CB1
<400> 32
gacaacagcc ccacgtgacc ggccaacact gagtgttgtc tcgctctggc gtcagagccg 60
tcgtggctcg ttccattctc ggcggtggta cctgctcccg gtggccctga ggacgtgtgg 120
gccaggggcg gccccgaaat taggaagcgg agggggagca gtctgcaggt ctgcggggct 180
aagtgtcgcg gcggcgcacc tcgcgtcaag aatccggagg aggagactgc aaggataggc 240
ccaggagtaa tggagtccaa agaggaacta gcggcaaaca atctcaacgg ggaaaatgcc 300
caacaagaaa acgaaggagg ggagcaggcc cccacgcaga atgaagaaga atcccgccat 360
ttgggagggg gtgaaggcca gaagcctgga ggaaatatca ggcgggggcg agttaggcga 420
cttgtcccta attttcgatg ggccatacct aataggcata ttgagcacaa tgaagcgaga 480
gatgatgtag aaaggtttgt agggcagatg atggaaatca agagaaagac tagggaacag 540
cagatgaggc actatatgcg cttccaaact cctgaacctg acaaccatta tgacttttgc 600
ctcatacctt gaatcctaaa agttttcgct gaggttaatg tgaacactgc tttacaagct 660
tgtatttttg tgatttactt tttctgtaag ccttttggtg tttacactta ccagtttcta 720
atggaaatta gaattctaat tgaatattgt tttgtctcag cctaaaagtt acggtcagca 780
tggcaattca cctattttag gaaaaatact cttttcataa tatgaaatgc ataaagcagt 840
tcaaaaagca gtctgtattc catcatcttc ctttttcatt ccagtcctta tttttgtaag 900
tattactttt cctcctccgg ctacctggac tcaaaatctc agttgtcttt gacagttttt 960
ttcttgtccc tgaccaaaaa agaatgatca tacccagaat tcaatgtttg atattttaag 1020
aatgtatgtt ctagtgtttt tcagagtgag tctaccatct gtataaaaac accttggggg 1080
caggcagggg catttaaaaa tgtaggacct atcgtccaga ctcacagagt ggggctccag 1140
aatctccatt tttaacaaac tctcttaagt aattctgatg tgtaccaaaa tcagtgccat 1200
tggtgtgtgt gtacgtaact atatacatat gtgtgtgtgt gtatatatat aatgtgtcat 1260
aaccgtaaac aataaacaat atcaagataa atctgacttt gatgggcaag taattaaaaa 1320
agaaaagtat gagaccttaa aaaaaaaaaa aaaaaaaaaa aaaa 1364
27/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<210> 33
<211> 464
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2009069CB1
<400> 33
gagcaggaat tcggcacgag gaattcagta tggcaaaggt gaccagtgag ccacagaagc 60
ctaatgaaga tgtggacgaa cacaccccat caacctcaag taccaaaggg aggaagaagg 120
ggaagacacc ccgtcaacga aggtccagaa gcggcgttaa gggcctaaag accaccagga 180
aggcgaaaag accccttcga gggagctcga gccaaaaagc cggtgaaact aacacccctg 240
caggaaaacc taagaaagct agaggaccaa tactgcgtgg tcgttatcac cggctgaaag 300
aaaaaatgaa gaaagaagag gccgacaaag agcaaagcga gacctcagtt ctgtgatgtc 360
tctagaggtc cgccactgaa aagtcatcaa tcatacagtc agtgaattct acaccaacag 420
gttaaaacca tgaaaataaa atcaacctga atcgaaaaaa aaaa 464
<210> 34
<211> 1549
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2009435CB1
<400> 34
ttccctgggt cgacccacgc gtccgggaag acagtttgca ttcttgcaac attaaaccaa 60
agggacttgg agtgcagatg gcatccttcg gttcttccag acaagctgca agacgctgac 120
catggccaag atggagctct cgaaggcctt ctctggccag cggacactcc tatctgccat 180
cctcagcatg ctatcactca gcttctccac aacatccctg ctcagcaact actggtttgt 240
gggcacacag aaggtgccca agcccctgtg cgagaaaggt ctggcagcca agtgctttga 300
catgccagtg tccctggatg gagataccaa cacatccacc caggaggtgg tacaatacaa 360
ctgggagact ggggatgacc ggttctcctt ccggagcttc cggagtggca tgtggctatc 420
ctgtgaggaa actgtggaag aaccagcact gctccatccc cagtcctgga aacaatttag 480
agcccttcgg tccagtggta cagcggcagc aaaaggggag aggtgccgaa gtttcattga 540
acttacacca ccagccaaga gaggtgagaa aggactactg gaatttgcca cgttgcaagg 600
cccatgtcac cccactctcc gatttggagg gaagcggttg atggagaagg cttccctccc 660
ctcccctccc ttggggcttt gtggcaaaaa tcctatggtt atccctggga acgcagatca 720
cctacatcgg acttcaattc atcagcttcc tcctgctact aacagacttg ctactcactg 780
ggaaccctgc ctgtgggctc aaactgagcg cctttgctgc tgtttcctct gtcctgtcag 840
gtctcctggg gatggtggcc cacatgatgt attcacaagt cttccaagcg actgtcaact 900
tgggtccaga agactggaga ccacatgttt ggaattatgg ctgggccttc tacatggcct 960
ggctctcctt cacctgctgc atggcgtcgg ctgtcaccac cttcaacacg tacaccagga 1020
tggtgctgga gttcaagtgc aagcatagta agagcttcaa ggaaaacccg aactgcctac 1080
cacatcacca tcagtgtttc cctcggcggc tgtcaagtgc agcccccacc gtgggtcctt 1140
tgaccagcta ccaccagtat cataatcagc ccatccactc tgtctctgag ggagtcgact 1200
tctactccga gctgcggaac aagggatttc aaagaggggc cagccaggag ctgaaagaag 1260
cagttaggtc atctgtagag gaagagcagt gttaggagtt aagcgggttt ggggagtagg 1320
cttgagccct accttacacg tctgctgatt atcaacatgt gcttaagcca aaaagctctg 1380
gagctatttc cagattaaat agtttttcta aaactttctg tccttcttta ctgggggcct 1440
gtcagcatca ctgatgaata tttcttgcca cagaggtttt tcttgttttt cccggattcc 1500
tttggatgtg gatcaacttt aaaatatcct gggtgactcc ggttcacca 1549
<210> 35
<211> 1205
<212> DNA
<213> Homo Sapiens
<220>
<221> misc feature
28/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<223> Incyte ID No: 2027937CB1
<400> 35
ctttgttgag tcttgttgaa gatcaggctc tgcaaatcac cctaggatgt ccttcagtga 60
gcagcagtgc aagcagccat gtgtgccccc tccatgcctc ccaaagaccc aggagcagtg 120
ccaagcaaag gctgaggagg tgtgcctccc cacatgccag cacccctgcc aagataagtg 180
tctagtgcag gcccaggagg tatgtctttc tcagtgccag gaatcaagtc aagaaaaatg 240
cccacagcaa ggccaagagc catacctacc tccatgccaa gaccagtgtc cacctcagtg 300
tgcagagcca tgccaggagc tattccagac aaaatgtgtg gaggtttgcc cacagaaagt 360
tcaggagaag tgctcatccc ctggcaaggg aaagtagctg ctcatatgtc atctgggttc 420
aagaagatgg ccagcagatg aaaccctgac cccagcccac gctctggtga ccttcttctg 480
tgggtacctc tgtgtgcaat gtaccttctt gcctcctggc ttccttagca ttccaggact 540
tggtctgtgt ctctgaagac agttctttct gtatttcatc accctctgtg aataagcatt 600
gttctcagca gtctgatgga aggtctcaaa tgtaggaatg gtgtggttgt cagggaagac 660
cacagaagcc tagcacagct tccttggtgc aaaaattcac ccagctctgg gtgtgtaaca 720
gccaaggata ccttcattca tctttcagag tttcaggctc tcaaataagc ctcaaaacaa 780
actgaattct gatggacttt ccacttatca ccaccaccac cacctccacc accaccacca 840
ccacaggtgt tgagacaaag cggcttgcgg tgtttcaaaa atcaaaaatt tggtgatttt 900
gtctctgtac actaaaaaga acagcaaatt attgaacttt gaaatgttgg ttgtgctttt 960
ttaaaatgaa ccgaaagtat gtaaacgatt gaatccaaac aaccagaagg gaaagataag 1020
atggatgttt ggtgcccttg tcaatctctg tgcttcatag ctggtctaat gtgggccctt 1080
agttctcacc atgtacactc tttagagatg tgatttgtgt ctgtgtgatg caggctggtc 1140
tgttctccag cttccttgcc ttcttctccc tggtgaaggt aaaatacata ataaagctga 1200
tcctg 1205
<210> 36
<211> 4061
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2722347CB1
<400> 36
ccggcgcggg gccctgcggt agcctcaggc Ccctcccctg gacccgccgc agagccagtg 60
cagaatacag aaactgcagc catgaccacg cacgtcaccc tggaagatgc cctgtccaac 120
gtggacctgc ttgaagagct tcccctcccc gaccagcagc catgcatcga gcctccacct 180
tcctccatca tgtaccaggc taactttgac acaaactttg aggacaggaa tgcatttgtc 240
acgggcattg caaggtacat tgagcaggct acagtccact ccagcatgaa tgagatgctg 300
gaggaaggac atgagtatgc ggtcatgctg tacacctggc gcagctgttc ccgggccatt 360
ccccaggtga aatgcaacga gcagcccaac cgagtagaga tctatgagaa gacagtagag 420
gtgctggagc cggaggtcac caagctcatg aagttcatgt attttcagcg caaggccatc 480
gagcggttct gcagcgaggt gaagcggctg tgccatgccg agcgcaggaa ggactttgtc 540
tctgaggcct acctcctgac ccttggcaag ttcatcaaca tgtttgctgt cctggatgag 600
ctaaagaaca tgaagtgcag cgtcaagaat gaccactctg cctacaagag ggcagcacag 660
ttcctgcgga agatggcaga tccccagtct atccaggagt cgcagaacct ttccatgttc 720
ctggccaacc acaacaggat cacccagtgt ctccaccagc aacttgaagt gatcccaggc 780
tatgaggagc tgctggctga cattgtcaac atctgtgtgg attactacga gaacaagatg.840
tacctgactc ccagtgagaa acatatgctc ctcaaggtga tgggctttgg cctctaccta 900
atggatggaa atgtcagtaa catttacaaa ctggatgcca agaagagaat taatcttagc 960
aaaattgata aattctttaa gcagctgcag gtggtgcccc ttttcggcga catgcagata 1020
gagctggcca gatacattaa gaccagtgct cactatgaag agaacaagtc caagtggacg 1080
tgcacccaga gcagcatcag cccccagtac aatatctgcg agcagatggt tcagatccgg 1140
gatgaccaca tccgcttcat ctccgagctc gctcgctaca gcaacagtga ggtggtgacg 1200
ggctcagggc tggacagcca gaagtcagac gaggagtatc gcgagctctt cgacctagcc 1260
ctgcggggtc tgcagcttct atccaagtgg agcgcccacg tcatggaggt gtactcttgg 1320
aagctggttc atcccacaga caagttctgc aacaaggact gtcctggcac cgcggaggaa 1380
tatgagagag ccacacgcta caattacacc agtgaggaaa aatttgcctt cgttgaggtg 1440
atcgccatga tcaaaggcct gcaggtgctc atgggcagga tggagagcgt cttcaaccag 1500
gccatcagga acaccatcta cgcggcattg caggacttcg cccaggtgac gctgcgtgag 1560
cccctgcggc aggcggtacg gaagaagaag aatgtcctca tcagcgtcct acaggcaatt 1620
cgaaagacca tctgtgactg ggagggaggg cgagagcccc ctaatgaccc atgcttgaga 1680
29/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
ggggagaagg accccaaagg tggatttgat atcaaggtgc cccggcgtgc tgtggggcca 1740
tccagcacac agctgtacat ggtgcggacc atgcttgaat cactcattgc agacaaaagc 1800
ggctccaaga agaccctgag gagcagcctg gatggaccca ttgtcctcgc catagaggac 1860
tttcacaaac agtccttctt cttcacacat ctgctcaaca tcagtgaagc cctgcagcag 1920
tgttgtgacc tctcccagct ctggttccga gaattcttcc tggagttaac catgggccga 1980
cgaatccagt tccccatcga gatgtccatg ccctggattc taacggacca tatcctggaa 2040
accaaagaac cttccatgat ggagtatgtc ctctaccctc tggatctgta caacgacagc 2100
gcctactatg ctctgaccaa gtttaaaaag cagttcctgt acgatgagat agaagctgag 2160
gtgaacctgt gttttgatca gtttgtctac aagctggcag accagatctt tgcttactac 2220
aaagccatgg ctggcagtgt cctgttggat aaacgttttc gagctgagtg taagaattat 2280
ggcgtcatca ttccgtatcc accgtccaat cgctatgaaa cactgctgaa gcagagacac 2340
gtccagctgt tgggtagatc aattgacttg aacagactca ttacccagcg catctctgcc 2400
gccatgtata aatccttgga ccaagctatc agccgctttg agagtgagga cctgacctcc 2460
attgtggagc tggagtggct gctggagatt aaccggctca cgcatcggct gctctgtaag 2520
catatgacgc tggacagctt cgatgccatg ttccgagagg ccaatcacaa tgtgtccgcc 2580
ccctatggcc gtatcaccct gcatgtcttc tgggaactga actttgactt tctccccaac 2640
tactgctaca atgggtccac taaccgtttt gtgcggactg ccattccttt Cacccaagaa 2700
ccacaacgag acaaacctgc caacgtccag ccttattacc tctatggatc caagcctctc 2760
aacattgcct acagccacat ctacagctcc tacaggaatt tcgtggggcc acctcatttc 2820
aagactatct gcagactcct gggttatcag ggcatcgctg tggtcatgga ggaactgcta 2880
aagattgtga agagcttgct ccaaggaacc attctccagt atgtgaaaac actgatagag 2940
gtgatgccca agatatgccg cttgccccga catgagtatg gctccccagg gatcctggag 3000
ttcttccacc accagctgaa ggacatcatt gagtacgcag agctcaaaac agacgtgttc 3060
cagagcctga gggaagtggg caatgccatc ctcttctgcc tcctcataga gcaagctctg 3120
tctcaggagg aggtctgcga tttgctccat gccgcaccct tccaaaacat cttgcctaga 3180
gtctacatca aagaggggga gcgcctggag gtccggatga aacgtctgga agccaagtat 3240
gccccgctcc acctggtccc tctgatcgag cggctgggga cccctcagca aatcgccatt 3300
gctcgcgagg gtgacctcct gaccaaggag cggctgtgct gtggcctgtc catgttcgag 3360
gtcatcctga cccgcattcg gagctacctg caggacccca tctggcgggg cccaccgccc 3420
accaatggcg tcatgcacgt cgatgagtgt gtggagttcc accggctgtg gagcgccatg 3480
cagttcgtgt actgcatccc tgtgggaacc aacgagttca cagctgagca gtgtttcggc 3540
gatggcttga actgggctgg ttgctccatc attgtcctgc tgggccagca gcgtcgcttt 3600
gacctgttcg acttctgtta ccacctgcta aaagtgcaga ggcaggacgg gaaggatgaa 3660
atcattaaga atgtgcccct gaagaagatg gccgaccgga tcaggaagta tcagatcttg 3720
aacaatgagg tttttgccat cctgaacaaa tacatgaagt ccgtggagac agacagttcc 3780
actgtggagc atgtgcgctg cttccagcca cccatccacc agtccttggc caccacttgc 3840
taagcagaag atcctgcaga cccttatctg gaggaggaag agaagcagga gagagaaagc 3900
cacagccagc ctgccatagg atccaactgg acaacgtgtg ggatggacct ggaaacaagc 3960
acctccccaa acacatcacc actccctagg gcggggcctg tgcatgctct cccatgacat 4020
ctccatgctg gtttctccat agcataaatg aaaaaaaaaa a 4061
<210> 37
<211> 773
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2759876CB1
<400> 37
gcagttctcc tgcctcagcc tcctgagtag ctgggattac agacaaaaat actaatgcat 60
ttgagaaagc ggtagttttg gttggagggg gaaaaatcaa ctgctttcct gatctgcaac 120
ttggctggat gctaagatgt cagtggacat gaatagccag gggtctgaca gcaatgaaga 180
ggactatgac ccaaattgtg aggaagagga agaagaagaa gaagacgacc ctggggacat 240
agaggactat tacgtgggag tagccagcga tgtggagcag cagggggctg atgcctttga 300
tcccgaggag taccagttca cttgcttgac ctacaaggaa tctgagggtg ccctcaatga 360
gcacatgacc agcttagctt ctgtcctaaa ggtgagcagt gttgtaaact ccagtgtaat 420
cccccccagt taaatctaag gatgagctgt tgttaattaa tgtctcctcc agactaattg 480
aatggccttg tagcttgaaa ccaaatttaa gtgtatgtca acatcatttg gacattttgt 540
tacatttact ttgttttcta atatatgcat gtctaaggtc ctcagattcc caaattagta 600
agaattagaa agtaggggtg ggacagattc aagaaagatg taccatacca gaaatgtgta 660
gtagcagaat tgcacaactt tgcacgtttc aggaagtgag ttttcagaat tttatgaggt 720
30/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
tgatacaata agcatcagaa tcaccatctt ctgtacgact gtgagtgact gat 773
<210> 38
<211> 2116
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2763735CB1
<400> 38
ctcgattggt tctactgtgg gtctggactg atctccatgt cctgttgtgg ggcttttaca 60
gcctttggat tgtgaaaact gctgagagag acttgcaatc cagtcacata agtataataa 120
agaaatattg gtcctcatgg aagaagagca agatttacca gagcaaccag tgaaaaaagc 180
caagatgcag gaatcaggag agcaaactat aagtcaagta agcaatccag atgtcagtga 240
tcagaagcct gaaacatcaa gccttgcttc aaaccttccc atgtcagagg aaattatgac 300
atgcaccgat tacatccctc gctcatccaa tgattatacc tcacaaatgt attctgcaaa 360
accttatgca catattctct cagttcctgt ttcggaaact gcttaccctg gacagactca 420
ataccagaca ctacagcaga ctcaacccta tgctgtctac cctcaggcaa cccaaacgta 480
tggactacct ccttttgctt caagcacaaa tgccagcctg atatctactt cttctacaat 540
tgccaatatt ccagcagcag cagtagccag catctcaaac caggattatc ccacctatac 600
tattcttggt cagaatcagt accaggcctg ctaccccagc tccagctttg gagtcacagg 660
tcagactaac agtgatgcag agagcaccac attagcagca accacatacc agtcggagaa 720
gcctagtgtc atggcgcctg cacctgcagc acagagactt tcctctggag acccttctac 780
aagtccatct ttgtcccaga ctacaccaag taaagatact gatgatcagt ccaggaaaaa 840
catgactagc aagaaccggg gcaagaggaa agctgatgcc acttcttccc aagacagtga 900
attagaacgg gtatttctgt gggacttgga tgaaaccatc atcatcttcc actcacttct 960
tactggatcc tatgcccaga aatatggaaa ggacccaaca gtagtgattg gctcaggttt 1020
aacaatggaa gaaatgattt ttgaagtggc tgatactcat ctatttttca atgacttaga 1080
ggagtgtgac caggtacatg tggaagatgt ggcttctgat gacaatggcc aagacttgag 1140
caactacagt ttctcaacag atggtttcag tggctcagga ggtagtggca gccatggttc 1200
atctgtgggt gttcagggag gtgtggactg gatgaggaaa ctagctttcc gctaccggaa 1260
agtgagagaa atctatgata agcataaaag caacgtgggt ggtctcctca gtccccagag 1320
gaaggaagca ctgcagagat taagagcaga aattgaagtt ttaacagatt cctggttagg 1380
aactgcatta aagtccttac ttctcatcca gtccagaaag aattgtgtga atgttctgat 1440
cactaccacc cagctggttc cagccctggc caaggttctc ctatatggac taggagaaat 1500
atttcctatt gagaacatct atagtgctac caaaattggt aaggagagct gctttgagag 1560
aattgtgtca aggtttggaa agaaagtcac atatgtagtg attggagatg gacgagatgc 1620
agccaaacag cacaacatgc ctttctggag gatcacaaac catggagacc tagtatccct 1680
tcaccaggct ttagagcttg attttctcta agaactggaa tgaggagcct tccccttgag 1740
ctccttttca ctcctgaagg gagctggaga ctggaaccaa ctgagaactt tctctgtctg 1800
tctctctctg tgtctctgtc tctatctctc tctctttctc tctttctctc tctccctctc 1860
tccctccctc ccttcctctc tccctccctc tctgcctctc tctctctctc tctctctgtc 1920
tttatccatg gaatgctggc gagaacacaa tcagaaccaa cagctgcaat ttttgctaag 1980
agtgagctgc agccccgtgt tcatctccat acagaagcag ggacagttgg atagagagaa 2040
caatggactc actgcagcta cagtgctttt aatttttctg tgttttgttt ttgttttttt 2100
gttttcaaaa aaaaaa 2116
<210> 39
<211> 2556
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2848676CB1
<400> 39
cgagaagtcc aggggtggcc gtgatggcgg cggcaggagc aggacctggc caggaagcgg 60
gtgccgggcc tggcccagga gcggtcgcaa atgcaacagg ggcagaagag ggggagatga 120
agccggtggc agcgggagca gccgctcctc ctggagaggg gatctctgct gctccgacag 180
ttgagcccag ttccggggag gctgaaggcg gggaggcaaa cttggtcgat gtaagcggtg 240
31 /43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
gcttggagac agaatcatct aatggaaaag atacactaga aggtgctggg gatacatcag 300
aggtgatgga tactcaggcg ggctccgtgg atgaagagaa tggccgacag ttgggtgagg 360
tagagctgca atgtgggatt tgtacaaaat ggttcacggc tgacacattt ggcatagata 420
cctcatcctg tctacctttc atgaccaact acagttttca ttgcaacgtc tgccatcaca 480
gtgggaatac ctatttcctc cggaagcaag caaacttgaa ggaaatgtgc cttagtgctt 540
tggccaacct gacatggcag tcccgaacac aggatgaaca tccgaagaca atgttctcca 600
aagataagga tattatacca tttattgata aatactggga gtgcatgaca accagacaga 660
gacctgggaa aatgacttgg ccaaataaca ttgttaaaac aatgagtaaa gaaagagatg 720
tattcttggt aaaggaacac ccagatccag gcagtaaaga tccagaagaa gattacccca 780
aatttggact tttggatcag gaccttagta acattggtcc tgcttatgac aaccaaaaac 840
agagcagtgc tgtgtctact agtgggaatt taaatggggg aattgcagca ggaagcagcg 900
gaaaaggacg aggagccaag cgcaaacagc aggatggagg gaccacaggg accaccaaga 960
aggcccggag tgaccctttg ttttctgctc agcgccttcc ccctcatggc tacccattgg 1020
aacacccgtt taacaaagat ggctatcggt atattctagc tgagcctgat ccgcacgccc 1080
ctgaccccga gaagctggaa cttgactgct gggcaggaaa acctattcct ggagacctct 1140
acagagcctg cttgtatgaa cgggttttgt tagccctaca tgatcgagct ccccagttaa 1200
agatctcaga tgaccggctg actgtggttg gagagaaggg ctactctatg gtgagggcct 1260
ctcatggagt acggaagggt gcctggtatt ttgaaatcac tgtggatgag atgccaccag 1320
ataccgctgc cagactgggt tggtcccagc ccctaggaaa ccttcaagct cctttaggtt 1380
atgataaatt tagctattct tggcggagca aaaagggaac caagttccac cagtccattg 1440
gcaaacacta ctcttctggc tatggacagg gagacgtcct gggattttat attaatcttc 1500
ctgaagacac agagacagcc aagtcattgc cagacacata caaagataag gctttgataa 1560
aattcaagag ttatttgtat tttgaggaaa aagactttgt ggataaagca gagaagagcc 1620
tgaagcagac tccccatagt gagataatat tttataaaaa tggtgtcaat caaggtgtgg 1680
cttacaaaga tatttttgag ggggtttact tcccagccat ctcactgtac aagagctgca 1740
cggtttccat taactttgga ccatgcttca agtatcctcc gaaggatctc acttaccgcc 1800
ctatgagtga catgggctgg ggcgccgtgg tagagcacac cctggctgac gtcttgtatc 1860
acgtggagac agaagtggat gggaggcgca gtcccccatg ggaaccctga ccaggtccct 1920
cttttctgtc aaggactttc tgggaataat actgggggtt ttgtttttgt ttttgaactg 1980
tctcaaatgt tctcccaaag atgctaaaaa cacagcctct ccttttagca agttaaaagg 2040
ctgggtagga ctgcgggaga ctgcctgcct ttcaccattt tctccccact tccagtgact 2100
gctcttattt tgtgtaccat aagccaacaa ccgctgactc caggattgca taagccccct 2160
gtgaaatcgg tgctgtactg cataccctgc cagctgtgac ttgttatcct actatatttt 2220
ctaaggagtg aataatattg tccgagtaac taacttattt aaaagacatt tccttctgtg 2280
ggcattgact gtatcccacc tgttttccaa ggaaatggta acctgtttct gagaacacct 2340
gaaatcaatg gctatacatt ccaaaccaat ctaaacgcta tttccttttg gtgtgggttt 2400
ggttttgttc attttgaaat acacttttga acactgagat ccgtaaaact actagatctc 2460
tggaagtgta attgtgaaag aaacttgctt gcagctttaa caaaatgaga aacttcccaa 2520
ataaaacttg ttttgaagtt taaaaaaaaa aaaaaa 2556
<210> 40
<211> 1394
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2956153CB1
<400> 40
gcccagacga attcccttgc ggccgctgca gccctggcag gcgcacaccc ctaagcatac 60
gcacccaacg cctcctccct gagccacgag gatggagcag ccaccccggc ccgggactgg 120
cgcaaggtgc ccaagcaagg aaagaaataa tgaagagaca catgtgttag ctgcagcctt 180
ttgaaacacg caagaaggaa atcaatagtg tggacagggc tggaaccttt accacgcttg 240
ttggagtaga tgaggaatgg gctcgtgatt atgctgacat tccagcatga atctggtaga 300
cctgtggtta acccgttccc tctccatgtg tctcctccta caaagttttg ttcttatgat 360
actgtgcttt cattctgcca gtatgtgtcc caagggctgt ctttgttctt cctctggggg 420
tttaaatgtc acctgtagca atgcaaatct caaggaaata cctagagatc ttcctcctga 480
aacagtctta ctgtatctgg actccaatca gatcacatct attcccaatg aaatttttaa 540
ggacctccat caactgagag ttctcaacct gtccaaaaat ggcattgagt ttatcgatga 600
gcatgccttc aaaggtgtag ctgaaacctt gcagactctg gacttgtccg acaatcggat 660
tcaaagtgtg cacaaaaatg ccttcaataa cctgaaggcc agggccagaa ttgccaacaa 720
cccctggcac tgcgactgta ctctacagca agttctgagg agcatggcgt ccaatcatga 780
32/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
gacagcccac aacgtgatct gtaaaacgtc cgtgttggat gaacatgctg gcagaccatt 840
cctcaatgct gccaacgacg ctgacctttg taacctccct aaaaaaacta ccgattatgc 900
catgctggtc accatgtttg gctggttcac tatggtgatc tcatatgtgg tatattatgt 960
gaggcaaaat caggaggatg cccggagaca cctcgaatac ttgaaatccc tgccaagcag 1020
gcagaagaaa gcagatgaac ctgatgatat tagcactgtg gtatagtgtc caaactgact 1080
gtcattgaga aagaaagaaa gtagtttgcg attgcagtag aaataagtgg tttacttctc 1140
ccatccattg taaacatttg aaactttgta tttcagtttc ttttgaatta tgccactgct 1200
gaacttttaa caaacactac aacataaata atttgagttt aggtgatcca ccccttaatt 1260
gtacccccga tggtatattt ctgagtaagc tactatctga acattagtta gatccatctc 1320
actatttaat aatgaaattt atttttttaa tttaaaagca aataaaagct taactttgaa 1380
ccatgaaaaa aaaa 1394
<210> 41
<211> 1376
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3333139CB1
<400> 41
gtcgcaggcc ggagggaaga tggcggcgcc ctggtggcga gccgcgctgt gcgagtgtcg 60
gagatggcgg ggcttcagca cctcggccgt cctgggccgc cggacacccc cgctggggcc 120
gatgcccaac agtgacatcg acttgagcaa cctggagcgg ctggagaagt accggagctt 180
cgaccgctac cggcgccggg cagagcagga ggcgcaggcc ccgcactggt ggcggaccta 240
ccgagagtat ttcggggaga agacagatcc caaagagaag attgatattg ggctgcctcc 300
acccaaagtc tcccggaccc aacagctact ggaacggaaa caggccatcc aggagcttcg 360
ggccaatgtg gaagaggagc gggctgcccg cctccgcaca gccagtgtcc cgctggatgc 420
cgtgcgggcc gagtgggaga ggacctgtgg cccctaccac aagcagcgtc tggctgagta 480
ttacggcctc taccgagacc tgttccacgg tgccaccttt gtgccccgag tccccctgca 540
cgtggcctac gctgtgggtg aggatgacct gatgcctgtg tactgtggca atgaggtgac 600
tccaaccgag gctgcccaag cgccagaggt gacctatgag gcagaagagg gctccttgtg 660
gacgttgcta ctcactagct tggatgggca cctgctggag ccagatgctg agtacctcca 720
ctggctgcta accaacatcc cgggtaaccg ggtggctgaa ggacaggtga cgtgtcccta 780
cctccccccc ttccctgccc gaggctccgg catccaccgt cttgccttcc tgctcttcaa 840
gcaggaccag ccgattgact tctctgagga cgcacgcccc tcaccctgct atcagctggc 900
ccagcggacc ttccgcactt ttgatttcta caagaaacac caagaaacca tgactccagc 960
cggcttgtcc ttcttccagt gccgctggga tgactccgtc acctacatct tccaccagct 1020
tctggacatg cgggagccgg tgtttgagtt cgtgcggccg cccccttacc accccaagca 1080
gaagcgcttc ccccaccggc agcccctgcg ctacctggac cggtacaggg acagtcatga 1140
gcccacctat ggcatctact aaggagccag agtgtgcgca tttcagagca tgggattgat 1200
cggcagcaag agtaaagaca cagctccaga ggcccacact gtggggtctg ggccctgcct 1260
taggcagccc ccctctttgg ccccctcccg tcaggcccag ggcttggagt gaaagtgact 1320
ctcaggtggt ggggtgggga atgtgaataa acatgatttc ttgccgggaa aaaaaa 1376
<210> 42
<211> 526
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3432292CB1
<400> 42
caggacgtgt ctgtgctcct gtgtgtgacc agggttgaaa aagtcgcact gagatgtcct 60
gccagcaaaa ccagcagcag tgccagcccc ctcccaagtg ccccccaaaa tgcccaccca 120
agtgtcctcc aaagtgccga cctcagtgcc cagccccatg cccacctcca gtctcttcct 180
gctgtggtcc cagctctggg ggctgctgcg gctccagctc tgggggctgc tgcagctctg 240
ggggtggcgg ctgctgcctg agccaccaca ggccccgtct cttccaccgg caccggcacc 300
agagccccga ttgttgtgag tctgaacttc tgggggctct ggctgctagc acagctctgg 360
ggactgctgc tgaccaaacc tcgaacatca cagagcaagc ctttatggag aaaacttgca 420
33/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
aacgaggaac ctgtccccaa gagtgatagc ttcttcctga ccccttgttg tctccttatc 480
ccctgggggc tcgacaacac ctttgttgag agttgttttg gctctc 526
<210> 43
<211> 2431
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3478571CB1
<400> 43
gcctgctccc ggaggagacg cgctgccgag gagaacccag cgggagaaca tttcaggata 60
ggaataggcc aagtgctgag aagatgagtc ttaggattga tgtggataca aactttcctg 120
agtgtgttgt agatgcagga aaagtcaccc ttgggactca gcagaggcag gagatggacc 180
ctcgcctgcg ggagaaacag aatgaaatca tcctgcgagc agtatgtgct ctgctgaatt 240
ctggtggggg cataatcaag gctgagattg agaacaaagg ctacaattat gaacgtcatg 300
gagtaggatt ggatgtgcct ccaattttca gaagccattt agataagatg cagaaggaaa 360
accacttttt gatttttgtg aaatcatgga acacagaggc tggtgtgcca cttgctacct 420
tatgctccaa tttgtaccac agagagagaa catccaccga tgtcatggat tctcaggaag 480
ctctggcatt cctcaaatgc aggactcaga ctccaacgaa tattaatgtt tccaattcat 540
taggtccaca ggcagctcag ggtagtgtac aatatgaagg taacataaat gtgtcagctg 600
ctgctttatt tgatagaaag cggcttcagt atctggaaaa actcaacctt cctgagtcca 660
cacatgttga atttgtaatg ttctcgacag acgtgtcaca ctgtgttaaa gacagacttc 720
cgaagtgtgt ttctgcattt gcaaatactg aaggaggata tgtatttttt ggtgtgcatg 780
atgagacttg tcaagtgatt ggatgtgaaa aagagaaaat agaccttacg agcttgaggg 840
cttctattga tggctgtatt aagaagctac ctgtccatca tttctgcaca cagaggcctg 900
agataaaata tgtccttaac ttccttgaag tgcatgataa gggggccctc cgtggatatg 960
tctgtgcaat caaggtggag aaattctgct gtgcggtgtt tgccaaagtg cctagttcct 1020
ggcaggtgaa ggacaaccgt gtgagacaat tgcccacaag agaatggact gcttggatga 1080
tggaagctga cccagacctt tccaggtgtc ctgagatggt tctccagttg agtttgtcat 1140
ctgccacgcc ccgcagcaag cctgtgtgca ttcataagaa ttcggaatgt ctgaaagagc 1200
agcagaaacg ctactttcca gtattttcag acagagtggt atatactcca gaaagcctct 1260
acaaggaact cttctcacaa cataaaggac tcagagactt aataaataca gaaatgcgcc 1320
ctttctctca aggaatattg attttttctc aaagctgggc tgtggattta ggtctgcaag 1380
agaagcaggg agtcatctgt gatgctcttc taatttccca gaacaacacc cctattctct 1440
acaccatctt cagcaagtgg gatgcggggt gcaagggcta ttctatgata gttgcctatt 1500
ctttgaagca gaagctggtg aacaaaggcg gctacactgg gaggttatgc atcaccccct 1560
tggtctgtgt gctgaattct gatagaaaag cacagagcgt ttacagttcg tatttacaaa 1620
tttaccctga atcctataac ttcatgaccc cccagcacat ggaagccctg ttacagtccc 1680
tcgtgatagt cttgcttggg ttcaaatcct tcttaagtga agagctgggc tctgaggttt 1740
tgaacctact gacaaataaa cagtatgagt tgctttcaaa gaaccttcgc aagaccagag 1800
agttgtttgt tcatggctta cctggatcag ggaagactat cttggctctt aggatcatgg 1860
agaagatcag gaatgtgttt cactgtgaac cggctaacat tctctacatc tgtgaaaacc 1920
agcccctgaa gaagttggtg agtttcagca agaaaaacat ctgccagcca gtgacccgga 1980
aaaccttcat gaaaaacaac tttgaacaca tccagcacat tatcattgat gacgctcaga 2040
atttccgtac tgaagatggg gactggtatg ggaaagcaaa gttcatcact cagacagcaa 2100
gggatggccc aggagttctc tggatctttc tggactactt tcagacctat cacttgagtt 2160
gcagtggcct cccccctccc tcagaccagt atccaagaga agagatcaac agagtggtcc 2220
gcaatgcagg tccaatagct aattacctac aacaagtaat gcaggaagcc cgacaaaatc 2280
ctccacctaa cctcccccct gggtccctgg tgatgctcta tgaacctaaa tgggctcaag 2340
ggtgtcccag gcaacttaga gattattgaa gacttgaact tggaggagat actgatctat 2400
gtagcgaata aatgccgttt tctcttgcgg g 2431
<210> 44
<211> 714
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3495166CB1
34/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<400> 44
gcgcaccgtc gcaccggtcc atttccctcg cgtgccgcgc tcacccaccc gcggcatgag 60
cagcgccccc gcgtcgggcc cggcgcccgc cagcctcacg ctctgggacg aggaggactt 120
ccagggccgt cgctgtcggc tgctaagcga ctgtgcgaac gtctgcgagc gcggaggcct 180
gcccagggtg cgctcggtca aggtggaaaa cggcgtttgg gtggcctttg agtaccccga 240
cttccaggga cagcagttca ttctggagaa gggagactat cctcgctgga gcgcctggag 300
tggcagcagc agccacaaca gcaaccagct gctgtccttc cggccagtgc tctgcgcgaa 360
ccacaatgac agccgtgtga cactgtttga gggggacaac ttccaaggct gcaagtttga 420
cctcgttgat gactacccat ccctgccctc catgggctgg gccagcaagg atgtgggttc 480
cctcaaagtc agctccggag cgtgggtggc ctaccagtac ccaggctacc gaggctacca 540
gtatgtgttg gagcgggacc ggcacagcgg agagttctgt acttacggtg agctcggcac 600
acaggcccac actgggcagc tgcagtccat ccggagagtc cagcactagg ctccacggcc 660
ccagacacct tccctgagga cactcaataa aggttcctga atcttcctgc caaa 714
<210> 45
<211> 3154
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3554748CB1
<400> 45
accatctccc actcgagctg cccccgccct ctggacccga gtgactcagg cctttgtttg 60
tccttcctgg tagaggcggg ttccctccct cggcaagatg ccggagtgct gggatgggga 120
acatgacatc gagacaccct acggccttct gcatgtagtg atccggggct cccccaaggg 180
gaaccgccca gccatcctca cctaccatga tgtgggcctc aaccacaaac tatgcttcaa 240
caccttcttc aacttcgagg acatgcagga gatcaccaag cactttgtgg tgtgtcacgt 300
ggatgcccct ggacaacagg tgggggcgtc gcagtttcct caggggtacc agttcccctc 360
catggagcag ctggctgcca tgctccccag cgtggtgcag catttcgggt tcaagtatgt 420
gattggcatc ggagtgggcg ccggagccta tgtgctggcc aagtttgcac tcatcttccc 480
cgacctggtg gaggggctgg tgctggtgaa catcgacccc aatggcaaag gctggataga 540
ctgggctgcc accaagctct ccggcctaac tagcacttta cccgacacgg tgctctccca 600
cctcttcagc caggaggagc tggtgaacaa cacagagttg gtgcagagct accggcagca 660
gattgggaac gtggtgaacc aggccaacct gcagctcttc tggaacatgt acaacagccg 720
cagagacctg gacattaacc ggcctggaac ggtgcccaat gccaagacgc tccgctgccc 780
cgtgatgctg gtggttgggg ataatgcacc cgctgaggac ggggtggtgg agtgcaactc 840
caaactggac ccgaccacta cgaccttcct gaagatggca gactctggag ggctgcccca 900
ggtcacacag ccagggaagc tgactgaagc cttcaaatac ttcctgcaag gcatgggcta 960
catgccctca gccagcatga cccgcctggc acgctcccgc actgcatccc tcaccagtgc 1020
cagctcggtg gatggcagcc gcccacaggc ctgcacccac tcagagagca gcgaggggct 1080
gggccaggtc aaccacacca tggaggtgtc ctgttgaagc ccttgatccc gctgacgacg 1140
cccacgtcga ggccccaccg ccatccttgc gccggctcat gttcccttta gtttattttt 1200
gtgagggcaa aggggaggaa atggggttct gtttgaaaaa aatgagggga tcttagatgc 1260
tgcagcagaa cagtctccag gtgttttaag gggctcagtc ctcctcatcc catctcactc 1320
tccgtggtaa cttagccaac ttgacccctc tcatcccact cccggcggcc caggcacaga 1380
agggcagggc catagggagg gagattcgct acggatccag gccattcctg ggtgagccct 1440
tgggcaggca tgtttggaga tgagagaggc ttcgagaggg tgggtgctgg gccacagggg 1500
tgcggggcca gctcaggcac tggcgtggga gccctgggag accccttccc ccaccctcca 1560
ccaagcacac ctgtttctgt ctcatagcac atgtgacaat catctggaca acagccacaa 1620
gggggcgctc ggaccaggca gccactttcc tggtgctctc tgggcccagc tggtgctgta 1680
gggccacgca ggcaggggcg tcaaggggtt tctctgccca aggaagacag aacatggaga 1740
accgtcaggg caggaacccc acagactgtc ccttccagcc cacactctgc cacctcctgg 1800
ccctgtccca attctgagcc aaggcctccc cgaggcagaa gttgcctggt cctctgtccc 1860
cacagtgacc tgactggggg tgagggagaa ggaggagaga gcccatgtgt ggtgtgtgtg 1920
cccctgagaa cttcgtggtg actgcctttg ggagcccgca ggtggccaga ggcaggggta 1980
gctgagttcc tggagacccc ttttttgccc ccaggttccc cagagggcaa cgccatcagt 2040
agcagtgtgg tgtttcaggc agagctctgg ccaggctgtg ccagtgtgtc ccggacgcat 2100
cactaaggaa gagagagttt atttagtcaa ctggcccaag gcagcgaggc ttctacagtc 2160
ccacacccca tagccgcctg ggctggggct tactgggggc tgaaggttct ggacatgaac 2220
aagggtcagg tagaagagaa aggcttcccc tacaccccag cctcctgctg tcccctgaag 2280
cccaggactg cgttgtatgc tttccatcca ctcaccttac cccatagcat cttgcggccc 2340
35/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
agaaaccaga gccatttgtc tcagacccta aatcaataat cacaaacccc aaaacgggag 2400
agagcagtga aaacatgcag ggctgtggac gggggaaggg ttgtggcggg tgttctgagg 2460
ctgagaggac acctatatgc gtatttcctc tacacacatc accccccttc tataatctta 2520
agccatgact agcctggtgg cgtgttagtt tctgcccagt tctaccccct catgtgcttc 2580
ttctgaatac tgaatgtgac tgtttgaaag ctggtagaat tcatccctct tactgtagat 2640
aacactgcaa atcttggaat tttgtttttt gctgtttcca gatgtatcta taaatatcta 2700
tacattatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtacatcggg tcctcccatg 2760
tgtggtgttc ttctggaggt tgtctctttg gtcaaggtga acttttaatg tttattattt 2820
tcttctccgc acaaagtaaa gagcctaatt ttgtgtattc tggtggctgc tgtcatgaga 2880
tgataaaatg taaaacaaaa ctctagtcaa cgtagaaaga gttaactgtg ctgaaaaact 2940
aataaagaac ctaagaagaa ttccagtgtg gtgatgccat gcccatcatg ggaggctttt 3000
ggagaaacag aatgtttggg caggggctgc tggtgctgct tgggttttgg gttgagggtg 3060
ctaggagagg atggtctcca cccatctttc tatttccagt acacgtcaca ttattttacc 3120
ggtgagatga gaatgtcaca aacattaaaa gcct 3154
<210> 46
<211> 2204
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3555629CB1
<400> 46
agggcatgcg ccggtggcgt gatggagcgg cagcagcagc agcaacagca actgcgaaac 60
ctgcgtgact tcctgttggt ctacaatcgg atgacagaac tctgcttcca gcgctgtgtg 120
cccagcttgc accaccgagc tctggacgct gaggaggagg cctgtgtgcc cagctgtgct 180
gggaagctga tccattccaa ccaccgcctc atggccgctt acgtgcagct catgcctgcc 240
ctggtacagc gccgcatcgc agactacgag gctgcctcgg ctgtgccagg cgttgctgct 300
gaacagcctg gggtctctcc atcaggcagc tagccatacc caaccccagg aaggaaggcc 360
ttggatggac cctcagattg aaggacccgg tggaccttgg ggttggtgaa tcctaaacag 420
agagaattcg aggttgcctg aaagctgggt gtccttgctc cttttcctgg agccaatata 480
cccagttttt actcagtttg atttatattc tgggcaagga agctttgcct actttattgg 540
cacaatccgt tgttctgtcg tttagtgcat atctgctggc ttcagccctg gcagctgaga 600
aattgttttc tatatgtaga aggaaaacct gagcatttgc aggcatctgg ttaaagcagg 660
gtctgtgtgt acaattttaa aacgggtaat atgtcatgct cttagttcat cttcacaaca 720
aaactatgag taagcggtat tagcctcact taacagatga ggaagcaaga ttccagaaag 780
taccagaagg tcattttata caacaggaga ttggttcctg cccagatgac agaaaatggg 840
agctctgtct agttgtcctt aagtctgact gacttcagtg gctcataacc gtgagccaag 900
tatttgttgg ttcataactg ttgttttgtg aactatgtct tacatgtcta gagttctgct 960
ggatctaggg aaaggaggag ctatcgaagt acaacggatc aaaaaaccac agggcttttg 1020
ggcactgcct ccttgggaag ttagtggcca cagaagagag atgaaacctg taagaagtct 1080
ggagtctttt ggaacttcag ccatttcccc aggttgttac tttcttagta tgtacagtct 1140
tctcaggatg agcagtaaaa cctttgaaca aaggtctgtg tggttgtctt cacgggcaat 1200
caggaaggga gagagctggg gaccatattc tgcaatgcag ccaaatccga ggaagagaaa 1260
ctgaagggag aagtagatgg caatggttat gataaaaagg gataaaacta aatcttcggg 1320
acttctttaa tgctacgtta atgtttcact gctcgtctag aaactcctaa atccagcttt 1380
ctatcatctg ccccacattg gtcccattga gtacattctg tgatttctaa ttccagcctc 1440
tccattcttt tctcattatt gcctcccccg ccccccaact ttgtgtaatt tacttctgta 1500
ttcagcagcc tggatagcat atcattccat caccccattt tcttgccacc attggccatc 1560
tttttgtatc attccactta ttctgtcttt tccattcctt cattcaaact gctagagaaa 1620
aacagttgtg taatgaatgc cactaaatat tcaaggcctc caacctcagc caagtcctca 1680
caccaacacg cagtcacacc aacacacacc tttatgggtt cctggtccat ttccttctct 1740
aattaccatg gcagttattt tacacctcta ctgctgtcct taatcccata ccccaccctc 1800
atcaggtgac cctgtttcct tttttagaga aattgaagct cttagacatt ggtttccaca 1860
gtaataattt taaaacttct tacaactacc tacaaagcag atgttctatc ctatctacag 1920
agcagaaaat tgaaattctc aagtagcaga cccggtatta aagtgcagat ctgactttaa 1980
agtccatgtt cattttacac agcaggctgc ctcttaagat agtatttatt gagcacacac 2040
tttgtgtagg ttctgatttt ggtagatgtc atgctttata ttaatcttta caacaactat 2100
aagtaagagg tattaacctc acttaacaga tgaggaagca agaatccaga atatgccaga 2160
aggcacattc tgcagatttc gtgcaaacat ttatacacag cttc 2204
36/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<210> 47
<211> 863
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 639636CB1
<400> 47
cggctcgagg gctgaggggc cgccaccgcc agcagggagg cggagggcta gcgagccaag 60
cggtggggac gccgctgcct tcctctttgc ctgtctcgcc gccctcctag acacgctcct 120
catctacgga tcctaccctc cccgctcttg caagcattca ctcggccggt cgcctgctga 180
ccctccttcg ccacaggctc gtagcggagg cagcagcgag gcatgaagac ccccaacgca 240
caggaagccg aagggcaaca aaccagggca gctgcaggac gggccactgg gtctgcaaac 300
atgacaaaga aaaaagtctc ccaaaagaag cagagaggcc gaccttcatc ccagccccgc 360
aggaacatcg tgggctgcag aatttctcat ggatggaagg aaggagatga gcccatcacg 420
cagtggaaag gaaccgttct ggatcagctt ctagatgatt ataaggaagg tgacctccgc 480
atcatgccag aatccagtga gtctcctcca acagagaggg agccaggagg agttgtagat 540
ggcctaatag gtaagcatgt ggaatatacc aaagaagatg gctccaaaag gatcggcatg 600
gtcattcacc aagtggaagc caaaccctct gtgtatttca tcaagtttga tgatgatttc 660
catatctatg tctacgattt ggtgaaaaag tcctaactgt tagggtaaaa tttggcacat 720
gtgtggaaac aaatgtataa tttgtagaca tgcaaaaaat gttgcctttc agtgtattga 780
aagcttatgg aatccctgat aactaaacat ctttgccagc attaactgtt gttttgctct 840
aaaaaataca aacttaatga aat 863
<210> 48
<211> 3860
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 902218CB1
<400> 48
taagcttgcg gccgcgggga ggaggatggg ggctaaccag ttagtggtgc tcaacgtgta 60
cgacatgtat tggatgaacg aatatacctc atccattgga attggagttt ttcattcagg 120
aattgaagtc tatggcagag aatttgctta tggtggccat ccttacccct tttctggaat 180
atttgaaatt tccccaggaa atgcttctga actaggagaa acatttaaat ttaaagaagc 240
tgttgtttta gggagcacgg acttcctaga agatgatata gaaaaaattg tagaagaact 300
gggaaaagaa tacaaaggca atgcttatca tttaatgcat aaaaactgca atcatttttc 360
ttcagcttta tcagagattc tttgtgggaa agagattcct cgctggatca atcgacttgc 420
ctacttcagc tcctgtatac cctttctaca gagttgcctc ccgaaggagt ggctcacgcc 480
cgcagccctg cagtctagtg tcagccaaga actccaggat gaactggagg aagcagagga 540
tgctgccgca tccgcttccg tggcaagcac tgcagcaggc tccagacccg ggcgccacac 600
taaactataa atgtctccaa agtcacacat tcagaactgt ctctggcagt cgaatatcac 660
tagagaaaag taaacagaga agcatccttt agatattttg tatgcaaaga tggctctccc 720
ccaaatccca gtttttcagc tcaggattat atttgtaatc aaaaaaaaaa aatcacttgg 780
cgcaggaggg agaactttgt aagaagctgc cctctgtttt ttttatccac tcgtaaatct 840
ggatttattt cttctgtttt atacaagctc tgttaagtta tgtttacagt atcttgtatc 900
gctgtttaca aatcttgcat ggactcctgc cacagtgaaa gaagaaaatc ttcatgtctt 960
caaaaattag gcaggtaatc tttgtatact tctgacaatt gccagatcta tggcataaat 1020
aggcacacaa aaaggtactt aaacagttat agtcaccatc acctgcttca gaatggtctt 1080
ttagatttgt gtttgttttg ttaaagttgt tggcaccagg atgcagagaa tcagactggc 1140
ctgaggtgaa ggagcacaca gccctgaggg cttggaaccc tgggtccagt tcctcttcac 1200
acccccttcc actctgagta gcacatctcc ccaggtgccc atggaacacc tgctttcatc 1260
ccaaatatcc gtccacctag gcggggtggt atgttcttac gtctctctga ctttgatgcc 1320
actcattcta tagtttagct ggttttcgtt caagatattc ttggtagtaa ctgacaagta 1380
tgttgcacat gtattggggg aggcgcttca tttttatttt aatacacatg tatttcctcc 1440
ttgcacagga ttttgatggt gtgggaatat cctaagtggt agccttccaa gtagcagtga 1500
gttgacattc agctgctttt aactattcag gctacctttt atactaaacc ttgaaaacta 1560
gaatctaatg tctaccccaa aaaagtagtt ctttgatatt ttatactttt tatgtaccat 1620
37/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
gtcagaaaga gtatgttggc gtttgtcatg ggactcattt cacataatag aatgcctagt 1680
ctcattgacc aatcgttaaa aaatcatatt tgtgtgtctt aagattcata tttatatgtt 1740
ctctcaaatg tatgtctctt acgtaacata ctctaagaat gaaactgtca ccacaggtaa 1800
atccttgtta gcaaggaatc tgtctgctcc agtctactcc tagtttgatc cttggatgta 1860
agaaccaagt cattacatgt caaattcaat ttttctgcct taagaatgaa tgtccttcat 1920
aaaatattgg atgcagtgta acactatcca aggcagtgac ttcagcttta tatacatata 1980
aaatatagtt agttttaaaa ttattgacat ttatttaaac ttttagattg gattgtttgc 2040
tattgctctg tgtgaggata cataatcttt cagtaaactg tatttttaac ttttccataa 2100
gctgattttg gttcatttta tcaacgtaag cacaccctgt tcatagggaa aataaacctt 2160
gggttataag cattagcctg aggacaatga agccacttaa cctaatttat gctttcgact 2220
gttctgtttc cagagaggaa agcctttaca aattactctc agttctttag gggcagaagg 2280
cttgtttcaa gaggtttgac agaagaaagg aatatatgaa cttaatgaga tgtcgacttg 2340
gttcaggtct aaaaatgagg gcaaaacact aaggctctag cagtgacttg ttcactaaaa 2400
agagagagtc ctgtccccag acggttagta caaagccttg gatacagttt gcttgtaata 2460
tttttaataa tgtgaggagt acagtgtttt ctaattcatt caagtatata tgatttaaac 2520
ctgggctact gacacacaca cagtagccat tagttagact cttcttagtg aatatcagga 2580
acatcccatc tgtgcttaac cagaatccag caagtcagca cacaagtgat tttattgtta 2640
ttttgttgta tttacttgca tttgttgtat ttactttcat ctgcagcatt tggagtttaa 2700
aaataatgta aagggttcta gtagaaatag tgtcctaagg ccaattacct accatactaa 2760
caatcagcag ataaaattct ggacgtgaga ttccttataa tctaattata cctgaggttg 2820
agcaagaaat gtcttccttt agaaaatctc attcaagtca ggttcttctc tacagttcaa 2880
aattgagaat ggatttaatt aactagcatt tagccagctt tttcttgccc ttggagaaaa 2940
agaatcattc tcaacctgat aatctgttaa gaaaaatccc atatgaacaa tctggtcatt 3000
aacatacata tgatacggag tctctttgtt gtcaccaagt gaacatactt ctcatggtgg 3060
gttggacagt aatacatgtt acagggtcag aagcttctgg tttctgctgt ttgctttaaa 3120
tacccttggg gttttttttt aaacccttac aaggggagca tcagctttgg aaagtgtgac 3180
tctgtaggag tgtagaaggc agtggtgtat gatcttagcc tcgtcctgat gcctgaatcc 3240
agccagctgt tgctctgacc cacagcaata gagcaagtta cccatcacca gcatttgtac 3300
agagcaggga attctggttt tagtccattg gtagcattgt gtgtatgagg agattcaaca 3360
ccacagacag ctgcaggact cgatatccat ggcttctttc catcacaaaa cgggtagaaa 3420
cacattcact gcttcagggt tctaatctgt gtgtctcctt atgactccat ttctgtaagc 3480
tactctgtaa ctttgatata tgctgtattt tctttcttta aaagatttag atgttttttc 3540
agcaagctag ccatacaacc attgtatctc tttctcttca gtatggttta gagcccagat 3600
cagttagtag gctttcgttg tcttctcttt caatacatgt acatctttac tgtttgaaaa 3660
gtgttacagc tgtcaaagaa tcttcatgga cctgaagata atttcttgtg aagttgaatg 3720
caagtgtact gtcattcata gtgtttatat caaaatacca ggaatcttca cttttgctac 3780
cttgatatag cattgggcta tcatgttaca acattgaaat acattgattt attaaaaaat 3840
acttttataa gaaaaaaaaa 3860
<210> 49
<211> 726
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1360522CB1
<400> 49
cccggggact aacggcgccg gtgacgactt cgccgcgcgt tggtcagcca tggccaccgc 60
tctcgcgcta cgtagcttgt accgagcgcg accctcgctg cgctgtccgc ccgttgagct 120
tccctgggcc ccgcggcgag ggcatcggct ctcgccggcg gatgacgagc tgtatcagcg 180
gacgcgcatc tctctgctgc aacgcgaggc cgctcaggca atgtacatcg acagctacaa 240
cagccgcggc ttcatgataa acggaaaccg cgtgctcggc ccctgcgctc tgctcccgca 300
ctcggtggtg cagtggaacg tgggatccca ccaggacatc accgaagaca gcttttccct 360
cttctggttg ctggagcccc ggatagagat cgtggtggtg gggactggag accggaccga 420
gaggctgcag tcccaggtgc ttcaagccat gaggcagcgg ggcattgctg tggaagtgca 480
ggacacgccc aatgcctgtg ccaccttcaa cttcctgtgt catgaaggcc gagtaactgg 540
agctgctctc atccctccac caggagggac ttcacttaca tctttgggcc aagctgctca 600
atgaaccgcc aggaactgac ctgctgactg cactctgcca ggcttcccaa tgctttcact 660
cttatctacc ctttggcact tatcttgctt atcaacataa taatttatac acttctaaaa 720
aaaaaa 726
38/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<210> 50
<211> 2196
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1400678CB1
<400> 50
gatggcgtcc atgcgggaga gcgacacggg cctgtggctg cacaacaagc tgggggccac 60
ggacgagctg tgggcgccgc ccagcatcgc gtccctgctc acggccgcgg tcatcgacaa 120
catccgtctc tgcttccatg gcctctcgtc ggcagtgaag ctcaagttgc tactcgggac 180
gctgcacctc ccgcgccgca cggtggacga gatgaagggc gccctaatgg agatcatcca 240
gctcgccagc ctcgactcgg acccctgggt gctcatggtc gccgacatct tgaagtcctt 300
tccggacaca ggctcgctta acctggagct ggaggagcag aatcccaacg ttcaggatat 360
tttgggagaa cttagagaaa aggtgggtga gtgtgaagcg tctgccatgc tgccactgga 420
gtgccagtac ttgaacaaaa acgccctgac gaccctcgcg ggacccctca ctcccccggt 480
gaagcatttt cagttaaagc ggaaacccaa gagcgccacg ctgcgggcgg agctgctgca 540
gaagtccacg gagaccgccc agcagttgaa gcggagcgcc ggggtgccct tccacgccaa 600
gggccggggg ctgctgcgga agatggacac caccacccca ctcaaaggca tcccgaagca 660
ggcgcccttc agaagcccca cggcgcccag cgtcttcagc cccacaggga accggacccc 720
catcccgcct tccaggacgc tgctgcggaa ggaacgaggt gtgaagctgc tggacatctc 780
tgagctggat atggttggcg ctggccgaga ggcgaagcgg agaaggaaga ctctcgatgc 840
ggaggtggtg gagaagccgg ccaaggagga aacggtggtg gagaacgcca ccccggacta 900
cgcagccggc ctggtgtcca cgcagaaact tgggtccctg aacaatgagc ctgcgctgcc 960
ctccacgagc taccttccct ccacgcccag cgtggttccc gcctcctcct acatccccag 1020
ctccgagacg cccccagccc catcttcccg ggaagccagc cgcccaccag aggagcccag 1080
cgccccgagc cccacgttgc cagcgcagtt caagcagcgg gcgcccatgt acaacagcgg 1140
cctgagccct gccacaccca cgcctgcggc gcccacctcg cctctgacac ccaccacacc 1200
tccggctgtc gcccctacca ctcagacacc cccggttgcc atggtggccc cgcagaccca 1260
ggcccctgct cagcagcagc ctaagaagaa cctgtccctc acgagagagc agatgttcgc 1320
tgcccaggag atgttcaaga cggccaacaa agtcacgcgg cccgagaagg ccctcatcct 1380
gggcttcatg gccggctccc gagagaaccc gtgccaggag cagggggacg tgatccagat 1440
caagctgagc gagcacacgg aggacctgcc caaggcggac ggccagggta gcacaaccat 1500
gctggtggac acagtgtttg agatgaacta tgccacgggc cagtggacgc gcttcaagaa 1560
gtacaagccc atgaccaatg tgtcctagaa ccacctgcct cacagctggc cgtcacttgt 1620
gggggtccac gggacgatgg ctttgccagc ttaaagtaac cggatggcgg acacctggcc 1680
cccgaggtcc cccggccgcc gccctgctgc tgacccagcc tgttttaagt tctggatgca 1740
tttctctggg gtatttgggg cttattttta aaattttaat atgggttctt ttttgtgtga 1800
tttaagacac tttttggact caacgttaca tttttgaatg tagtaagtaa attaaccaaa 1860
aaagttacaa cttcctaatt ttagtgacag ctctgcctgt tagactctta ctttttaaaa 1920
tcttttctat tttccctcgc tggggcagtg ccctcctacc cccagggttg aggggaccaa 1980
ggtggcacgg tggtactggg ggtgcggcag ggacacccga ccacaccaga gcgtgggaga 2040
cggtgggcct tgtcccctgc ctgtgcctgc ctgggagttt tgtattcatc ttttgtatag 2100
ttgtggacat ttaagacagt ctttgggtac ctattttcat tgtaaaacta tctgaaccat 2160
taaagtcgag cttttctaaa gaaaaaaaaa aaaaaa 2196
<210> 51
<211> 1495
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1435556CB1
<400> 51
gagcagaaat tcggcacgag gaaaaatctg aaatctgaaa tgctccaaaa tcctaaactt 60
tttgagtgct gacattatgc cacaaatgga aaatttcata cctgacctta tgtgagttgc 120
agtcaaaaca caggtgcaca acacccagtt catgcaacat ccccaatggg aaaaaagacc 180
cccccagctc tcttctgctg cagtttttct gctcacacct ggattcccca tgcattccca 240
caaaaagtaa ttaaatggca tgcgtgcagg ctggacacgc caacaacagg tttcccacaa 300
39/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
tgccccacat gggcgaagac ctgtgtgcat tactcattgc atttttttgc ttattctctg 360
ctgtgtggta taaatatatt gttgaaaatg tcaaaaagac ctaaagatac ccctgtgaat 420
atcagtgata agaaaaagag gaagcattta tgtttatcta tagcacagaa agtcaagttg 480
ttggagaaac tggacagtgg tgtaagtgtg aaacatctta cagaagagta tggtgttgga 540
atgaccacca tatatgacct gaagaaacag aaggataaac tgttgaagtt ttatgctgaa 600
agtgatgagc agatattaat gaaaaataga aaaacacttc ataaagctaa aaatgaagat 660
cttgatcgtg tattgaaaga gtggatccgt cagcgtcgca gtgaacacat gccacttaat 720
ggtatgctga tcatgaaaca agcaaagata tatcacaatg aactaaaaat tgaggggaac 780
tgtgaatatt caacaggctg gttgcagaaa tttaagaaaa gacatggcat taaattttta 840
aagacttgtg gcaataaagc atctgctggt catgaagcaa cagagaagtt tactggcaat 900
ttcagtaatg atgatgaaca agatggtaac tttgaaggat tcagtatgtc aagtgagaaa 960
aaaataatgt ctgacctcct tacatataca aaaaatatac atccagagac tgtcagtaag 1020
ctggaagaag aggatatcaa agatgttttt aacagtaata atgaggctcc agttgttcat 1080
tcattgtcca atggtgaagt aacaaaaatg gttctgaatc aagatgatca tgatgataat 1140
gataatgaag atgatgttaa cactgcagaa aaagtgccta tagacgacat ggtaaaaatg 1200
tgtgatgggc ttattaaagg actagagcag catgcattca taacagagca agaaatcatg 1260
tcagtttata aaatcaaaga gagacttcta agacaaaaag catcattaat gaggcagatg 1320
actctgaaag aaacatttaa aaaagccatc cagaggaatg cttcttcctc tctacaggac 1380
ccacttcttg gtccctcaac tgcttctgat gcttcttctc acctaaaaat aaaataaaat 1440
acagtgtaca gtaacctttt agtcaaaaca gcatcatact tggaaactga aagcc 1495
<210> 52
<211> 2794
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1546633CB1
<220>
<221> unsure
<222> 2705, 2709, 2711, 2713-2714, 2717, 2719, 2731, 2735-2736, 2745, 2747,
2750, 2757, 2763-2764, 2766, 2770, 2782, 2785, 2787
<223> a, t, c, g, or other
<400> 52
ccgcgagagc aaggagcaca gagtgcagca tcatgacaag gagatttctc gaagccgaat 60
tccccggttg attcttcggc cccatatgcc ccaacaacag cacaaagtgt ccccagcctc 120
tgagtctcct ttctctgagg aagagagcag agagttcaac cccagcagct ctgggcgctc 180
agcgaggacc gttagcagca acagcttctg ctcagatgac acaggctgtc ctagcagcca 240
gtcagtgtct cctgtgaaga caccctcaga tgctggaaac agccccattg gcttttgccc 300
tggaagtgat gaaggcttca ccagaaagaa atgcacgatt ggaatggttg gtgaaggaag 360
cattcagtcc tctcgatata agaaggaatc aaagtcaggc cttgtgaaac caggtagtga 420
agctgatttt agctcctcga gcagcacagg cagcatttcc gctcctgagg tccatatgtc 480
gactgcggga agcaagcggt cttcttcttc acgcaatcga ggtcctcatg ggcggagtaa 540
tggagcttcg tcacacaagc ctggcagcag cccatcatcc ccgcgggaaa aggaccttct 600
gtccatgctg tgcaggaatc agctgagccc tgtcaatatc catcccagtt atgcaccttc 660
ttccccaagc agtagcaact caggctccta caaaggaagc gactgtagcc ccatcatgag 720
gcgttctgga aggtacatgt cttgcggtga aaatcatggt gtcagacccc caaacccaga 780
gcagtatttg actccactgc agcagaaaga ggtgacagtg agacacctca aaatcaagct 840
gaaggaatct gagcgccgac tccatgaaag ggaaagtgaa atcgtggagc ttaagtccca 900
gctggcccgc atgcgagagg actggattga ggaggagtgt caccgggtag aggcccagtt 960
ggcactcaaa gaagccagga aagagattaa acagctcaaa caggtcatcg aaaccatgcg 1020
gagcagcttg gctgataaag ataaaggcat tcagaaatat tttgtggaca taaacatcca 1080
aaacaagaag ctggagtctc tccttcagag catggagatg gcacacagtg gctctctgag 1140
ggacgaactg tgcctagact ttccatgtga ttccccagag aagagcttaa ccctcaaccc 1200
ccctcttgac acaatggcag atgggttatc tctggaagag caggtcacgg gggaaggggc 1260
tgacagggag ctactggtag gagatagcat agccaacagc acagatttgt tcgatgagat 1320
agtgacagcc accaccacag aatctggtga cctggagctt gtgcattcca cccctggggc 1380
taacgtcctg gagctgctgc ccatagtcat gggtcaggag gagggcagtg tggtggtgga 1440
gcgagccgtt cagaccgacg tggtgcccta cagcccagcc atctcagagc tcattcagag 1500
tgtgctgcag aagctccagg acccctgtcc ctcgagcttg gcgtcccctg atgagtctga 1560
40/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
accagactcg atggagagct tcccagagtc cctctctgcc ttagtggttg atttaactcc 1620
aagaaatcca aactcagcca tccttttgtc tcccgtggag accccctacg ccaatgtgga 1680
tgcagaagtt catgcaaacc gcctcatgag agagctggat tttgcagcct gcgtggaaga 1740
gaggttggat ggtgtcatcc cactggctcg cgggggcgtc gtgaggcagt actggagcag 1800
cagcttcctg gtggatctcc tggctgtggc tgcccccgtg gtccccacgg ttctgtgggc 1860
attcagtact cagagagggg gaacggatcc tgtgtataac atcggggcct tgctcagggg 1920
ctgttgcgtg gttgccctgc attcgctccg ccgcaccgcc ttccgtatca aaacctaaat 1980
agaagttgtt gttaccgtgt gccaatgtgt cccatgtggg ttgtgccagg tagagaaaca 2040
ggaagtcaat catctgtgac agtctctatt ctgtcgtttt gctccttggt atttgatttg 2100
cactatattt agttgaagcc tgttcactgt ttaaaaccgg aggtatcttc aaaggcatgg 2160
agacctggtt ccagtaaatg tcccaccagt ggggtataga aagcatgctc atgaccctgc 2220
cgtgtcgtct gaggtacccg ttcttatcct agtggttcag gaagagaaaa cgcagtttgc 2280
actttcaaga cagcttctct aaggctggca tgttatctcc ttgctttgct ttttgccgtt 2340
ttaaaatgtg taattgttcc agcattccaa tggtcttgtg catagcaggg gactgtaacc 2400
aaaaataaac atgtatttgt gtaattggtt tgaagaagtc ttgaatagct ctttactgtc 2460
ttacttgggg ttgataagat ttgagtgttt gcaatttttt actaaatgta gctccaaagt 2520
cttaaatggc ttgtttgttc ttaaactgtt aattgatgaa actgtgcata agtttacaat 2580
gtactaactt attttgctta ttatatatag tgttttattg gaaattgtaa ccacacactt 2640
cagcatgatg aaaataaaga ttagtgtttc catttaaata aatgttttat cctccccata 2700
aaatnaagna ngnnttnang ggggaggttt ncaannttgg gttgnanaan atttagngcc 2760
ttnngntgtn tcgggggttt gngcnangca aaaa 2794
<210> 53
<211> 1516
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1794031CB1
<400> 53
acgggatctg gatgagctgg acaaggatca tttacagttg agagaagcct gggatggcct 60
cgatcaccag attaatgcat ggaaaataaa gctaaattat gccttgcccc cacccctcca 120
tcaaactgaa gcttggctcc aggaggtaga agagcttatg gatgaagatt tgtcagcctc 180
ccaggatcac tctcaagccg tgactctgat acaagagaaa atgactttat tcaagagcct 240
gatggataga tttgagcatc attcgaacat tctccttacc tttgaaaata aggatgaaaa 300
tcacttgcca ttggtaccac ctaacaaatt ggaggaaatg aaaagacgaa tcaacaacat 360
tttggagaaa aaatttattc tacttctaga atttcattac tacaagtgct tagttcttgg 420
tttggtagat gaagtgaaat caaaattgga tatttggaac attaaatatg ggagcagaga 480
atctgtggaa ttattgctgg aagactggca taaatttatt gaagaaaaag aattcctagc 540
tcgacttgat acttcttttc aaaaatgtgg agaaatttat aagaatttgg ctggagaatg 600
tcagaatatt aataaacagt atatgatggt gaaatctgat gtttgtatgt atagaaaaaa 660
tatatataat gtgaagtcca ctctacaaaa agtgctggca tgttgggcta cttatgtgga 720
aaaccttcgc ttactaaggg cttgctttga ggagacaaag aaggaagaaa ttaaagaggt 780
accctttgag acactagccc agtggaatct agaacacgct actttaaatg aagcaggaaa 840
tttcttagtc gaagtcagca atgatgtggt tggatcatct atttctaaag aactgagaag 900
gctgaataaa agatggagaa agttggtttc aaaaactcaa cttgaaatga acctgccact 960
gatgataaaa aaacaggatc agcccacttt tgacaattct ggaaatattc tatctaaaga 1020
agagaaagca actgttgagt tttcaacaga tatgtcagta gaacttcctg aaaattataa 1080
tcaaaatata aaggctggag agaaacatga aaaagaaaat gaagaattca cagggcaact 1140
aaaagtggct aaagatgttg aaaaactcat tggataagtg gaaatctggg aggcagaagc 1200
caaatctgtt ttggatcaag atgatgtgga cacctcaatg gaagaatctt tgaaggtatg 1260
tgtgtaaaag tattaagagg gtactttcat ggttgtgcat ttatgtttta agttaaataa 1320
gaagttttaa agtaagtagt aataagccta cagttttaat tttctttgtt gggagtttta 1380
aaaatgaatg gattttatcc ctggatcatt tgctgttatt ttgcttgaca gcagaggata 1440
gattaggaga ccactgataa tacctatgaa tgttaagctc ttggacttat tttcttagct 1500
ataacatggg ggttaa 1516
<210> 54
<211> 1146
<212> DNA
<213> Homo sapiens
41/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
<220>
<221> misc_feature
<223> Incyte ID No: 2060563CB1
<400> 54
tgccgccctg ccaccctgcc gccctgccgc cctgccgccc tgccgccctg ccgccggtgg 60
tcgctgcccg tggtgctccg tcgcccccgc cacctcacgt cctcccgtgc gtcgggagcg 120
tctcggctac aacatgttgg gcatgatcaa gaactcgctg ttcggaagcg tagagacgtg 180
gccttggcag gtcctaagca aaggggacaa ggaagaagtt gcctatgaag aaagggcctg 240
tgaaggcggc aaatttgcca cagtagaagt gacagataag cctgtggatg aggctctacg 300
ggaagcaatg cccaaggtcg caaagtatgc ggggggcacc aatgacaagg gaattgggat 360
ggggatgaca gtccctattt cctttgctgt gttccccaat gaagatggct ctctgcagaa 420
gaaattaaaa gtctggttcc ggattccaaa ccaatttcaa agcgacccac cagctcccag 480
tgacaaaagc gttaagattg aggaacggga aggcatcact gtctattcca tgcagtttgg 540
tggttatgcc aaggaagcag actacgtagc acaagccacc cgtctgcgtg ctgccctgga 600
gggcacagcc acctaccggg gggacatcta cttctgcacg ggttatgacc ctcccatgaa 660
gccctacgga cggcgcaatg agatctggct gttgaagaca tgagtgaccc actgaaccaa 720
gaacttactg gaagtgtgcc tctgtgtctc cttcctcggg ggtaaggagg ggacagtgct 780
tcccaagttc cagctgcaag tccaacttaa ccaactttcc ttcaaagtca gttactgcca 840
attttctgaa aaaagcatgt tccatatact aagtctcttt tctcacagta ggaaataata 900
cagccaagat atgcagcatc cttctcattg atgtagaaaa ttctgcgata gaccagaaaa 960
atcctggcag cttttctcca ggcatctggg tcactaaaaa ctgattttct aaaattattg 1020
gatttgtatt ttgttattaa gggggaaaat gtgatttgtg cctgatcttt catctgtgat 1080
tcttataaga gctttgtctt cagaaaaact aaaaataaaa ggcattgact taaaaaaaaa 1140
aaaaaa 1146
<210> 55
<211> 2761
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2573955CB1
<400> 55
cggctcgagg ctgagaccca gagtcaccca ggggtctccg tcacgtgcca ggagtaggca 60
gaagtgggct gtgacagatc aggaaacaga gctcagtgca gcccactaaa ttgctcaggg 120
ccctacagct aacaagcggc agaggcagga tctgcactca ggagctgctt ggagatgctg 180
ctgtggccac tgctgctgct gctgctgctg ctgccaacat tggccctgct caggcagcag 240
cggtcccagg atgccaggct gtcctggctt gctggcctcc agcaccgagt ggcatggggg 300
gccctggtct gggcagccac ctggcagcgg cggaggctgg agcagagcac gctccatgtg 360
caccagagcc agcagcaggc cctgaggtgg tgtctacagg gagcccagcg cccccactgt 420
tccctcagaa ggagcacgga cataagcacc ttccggaatc atctccctct gaccaaggcc 480
agccagaccc agcaggaaga cagtggagag cagccactgg ccccgacctc aaaccaggac 540
cttggggagg cctctctgca ggccaccttg ctgggtctgg cagccctaaa caaggcctac 600
ccagaagtgc tggctcaggg acgcactgcc cgtgtgacgc ttacatcccc ttggccccga 660
cccctgcctt ggcctgggaa taccctgggc caggtgggca cccctggaac caaggaccct 720
agggccctgc tgctggacgc actgaggtcc ccagggctga gggcactgga ggctgggacg 780
gctgtcgaac ttctggatgt tttcttgggc ctggagactg atggtgaaga gctagctggg 840
gcgatagctg ccgggaaccc tggagcgcct ctccgtgaac gggcagctga gctccgggag 900
gccctagagc aggggccacg gggactggcc cttcggctct ggccaaagct gcaggtggtg 960
gtgactctgg atgcaggagg ccaggccgag gctgtggctg ccctcggggc cttgtggtgc 1020
caaggactag ccttcttctc tcctgcttat gctgcctcgg gaggggtgct gggcctaaac 1080
ctacagccag agcagcccca tgggctctac cttctgcccc ctggggcccc ctttatcgag 1140
ctgctcccag tcaaggaagg cacccaggag gaagctgcct ccaccctcct tttggccgag 1200
gcccagcagg gcaaggagta tgagctggtg ctgacggacc gcgccagcct caccaggtgc 1260
cgcctgggtg atgtggtgcg agtggttggt gcctacaatc agtgtccagt cgtcaggttc 1320
atctgcaggc tggaccagac cctgagtgtg cgaggggaag atattggtga agacctgttc 1380
tctgaggccc tgggccgggc agtggggcag tgggcggggg ccaagctgct ggaccatggc 1440
tgtgtggaga gcagcattct ggattcctct gcgggctctg ctccccacta cgaggtgttt 1500
gtggcgctga gggggctgag gaatctgtca gaggaaaatc gagacaagct ggaccactgc 1560
cttcaggaag cctctccccg ctacaagtcc ctgcggttct ggggcagcgt gggccctgcc 1620
42/43
CA 02384324 2002-02-28
WO 01/19860 PCT/US00/25435
agagtccacc tggtggggca gggagccttc cgagcactcc gggcagccct cgctgcctgc 1680
ccctcctccc ccttcccccc tgcgatgccc cgggtccttc ggcacaggca cctggcccag 1740
tgtctgcagg agagggtggt gtcctgagtc aagtcctgcc ccaccgccca gctcccccca 1800
gaggccacct cgcccctccc tctgggacct ctccggatgg ggagtccttg gccagggtct 1860
ctgactctgt gtcacctgac atttgcccat gagagccgct gggccttaga gaggccttgg 1920
cccagctgac cggttctgaa gtatgggcct ccggggttag cagatgccag cagtgcctgc 1980
ccgtgtcccc atgtcccggc atgaaggaca ctgctagaga gttaccatgc acaccgatgg 2040
tttcctgtat cacagcccaa agaggttctc tggtggccac agctgtgtgc tcagtcagtg 2100
cactgggcaa gctagaagtg ttggggggtt aatgtcccca ggagcagcaa ccctgagtca 2160
ataaggagca ggacctcagc ttcattgtcc ttgagcagga caattctgaa gtgtattcta 2220
cataaactct cagaggatgc ccagcaggat ggagtcccag ttgcccgcag cagtaaccca 2280
ctcattcatg tacttcctgc gggggctctc ccttccctct cttccccact cccccgcctt 2340
gggcttcctg ggatggctcc caaataaacc tcttgcaccc agaaaaaaaa aaaaaaaaaa 2400
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaagg 2460
gggggccgct ctaggggttc caggtttagg tacgggtgca tgggaggtca tagctcttct 2520
aaggtgtccc ctaatttcat ttcacgggcg gtggttttaa aaggtcgtga ctgggaaaac 2580
cctggggtta cccaatttaa tcgctttgag gaaattcccc ttttggcaag ttggggtaat 2640
agcgaagggg cccgcacggt tggcctttcc aaaaatttgg gccctctgat tggcgattgg 2700
gacgcgcctg taggggggtt ttaagccggc ggttttggtg gttacgccgg tgacccgtta 2760
c 2761
<210> 56
<211> 1164
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 3404792CB1
<400> 56
atcatggcag ggctgggctc cgatccctgg tggaagaaaa ccctttactt gaccggggga 60
gctttgctgg ccgcagctgc gtatctgctc cacgaactcc tggtcattag gaaacagcaa 120
gagattgact ctaaagatgc tattattttg catcagtttg caagacctaa caatggtgtt 180
ccaagtttat ctcctttctg tttaaagatg gaaacttatt taaggatggc tgacttaccg 240
tatcagaact attttggtgg aaaactctct gctcaaggga aaatgccttg gattgaatat 300
aatcatgaaa aagtttctgg cacagaattc ataattgact ttctggaaga gaagcttgga 360
gtgaatttaa acaaaaacct tggccctcat gaaagagcca tctccagagc ggtgaccaag 420
atggtggagg agcacttcta ctggacgtta gcttattgcc agtgggtgga caatctcaat 480
gagacccgga agatgctctc tcttagtggt ggtggtccct tcagcaacct gctgaggtgg 540
gttgtgtgcc acataacgaa aggaattgtg aaacgcgaga tgcacggcca cggcattggc 600
cgcttctccg aggaagagat ttacatgctg atggagaagg acatgcggtc tttagcaggg 660
cttttgggtg ataagaagta catcatgggg cccaagcttt ccactcttga cgccactgtc 720
tttggacact tggcacaggc aatgtggacc ttaccaggga caagacccga acggctgatc 780
aaaggtgagc tgatcaacct tgccatgtac tgtgagagga taaggaggaa attttggcca 840
gagtggcacc acgatgatga caataccatc tatgagtctg aggagagcag cgaaggcagc 900
aaaacccaca ccccgctgct ggattttagc ttttactcaa ggacagagac ctttgaagat 960
gagggagcag aaaacagttt ttccagaacc ccagacacag attttactgg acactcactc 1020
tttgattcgg atgtggacat ggatgactat acagaccacg aacagtgcaa gtgacgtcca 1080
gcctcactga ccctcttcct tgggacctgc cactccctgg gtcggtccat tttcccaggt 1140
agcaatccat ccgagctggg agga 1164
43/43
