Note: Descriptions are shown in the official language in which they were submitted.
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64 Human Secreted Proteins
Field of the Invention
This invention relates to newly identified polynucleotides and the
polypeptides
encoded by these polynucleotides, uses of such polynucleotides and
polypeptides, and
their production.
Background of the Invention
Unlike bacterium, which exist as a single compartment surrounded by a
membrane, human cells and other eucaryotes are subdivided by membranes into
many
functionally distinct compartments. Each membrane-bounded compartment, or
organelle, contains different proteins essential for the function of the
organelle. The cell
uses "sorting signals," which are amino acid motifs located within the
protein, to target
proteins to particular cellular organelles.
One type of sorting signal, called a signal sequence, a signal peptide, or a
leader
sequence, directs a class of proteins to an organelle called the endoplasmic
reticulum
(ER). The ER separates the membrane-bounded proteins from all other types of
proteins. Once localized to the ER, both groups of proteins can be further
directed to
another organelle called the Golgi apparatus. Here, the Golgi distributes the
proteins to
vesicles, including secretory vesicles, the cell membrane, lysosomes, and the
other
organelles.
Proteins targeted to the ER by a signal sequence can be released into the
extracellular space as a secreted protein. For example, vesicles containing
secreted
proteins can fuse with the cell membrane and release their contents into the
extracellular
space - a process called exocytosis. Exocytosis can occur constitutively or
after receipt
of a triggering signal. In the latter case, the proteins are stored in
secretory vesicles (or
secretory granules) until exocytosis is triggered. Similarly, proteins
residing on the cell
membrane can also be secreted into the extracellular space by proteolytic
cleavage of a
"linker" holding the protein to the membrane.
Despite the great progress made in recent years, only a small number of genes
encoding human secreted proteins have been identified. These secreted proteins
include
the commercially valuable human insulin, interferon, Factor VIII, human growth
hormone, tissue plasminogen activator, and erythropoeitin. Thus, in light of
the
pervasive role of secreted proteins in human physiology, a need exists for
identifying
and characterizing novel human secreted proteins and the genes that encode
them. This
knowledge will allow one to detect, to treat, and to prevent medical disorders
by using
secreted proteins or the genes that encode them.
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Summary of the Invention
The present invention relates to novel polynucleotides and the encoded
polypeptides. Moreover, the present invention relates to vectors, host cells,
antibodies,
and recombinant methods for producing the polypeptides and polynucleotides.
Also
provided are diagnostic methods for detecting disorders related to the
polypeptides, and
therapeutic methods for treating such disorders. The invention further relates
to
screening methods for identifying binding partners of the polypeptides.
to Detailed Description
Definitions
The following definitions are provided to facilitate understanding of certain
terms used throughout this specification.
In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is
altered "by the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of matter, or could
be
contained within a cell, and still be "isolated" because that vector,
composition of
matter, or particular cell is not the original environment of the
polynucleotide.
In the present invention, a "secreted" protein refers to those proteins
capable of
being directed to the ER, secretory vesicles, or the extracellular space as a
result of a
signal sequence, as well as those proteins released into the extracellular
space without
necessarily containing a signal sequence. If the secreted protein is released
into the
extracellular space, the secreted protein can undergo extracellular processing
to produce
a "mature" protein. Release into the extracellular space can occur by many
mechanisms, including exocytosis and proteolytic cleavage.
As used herein , a "polynucleotide" refers to a molecule having a nucleic acid
sequence contained in SEQ 1D NO:X or the cDNA contained within the clone
deposited
with the ATCC. For example, the polynucleotide can contain the nucleotide
sequence
of the full length cDNA sequence, including the 5' and 3' untranslated
sequences, the
coding region, with or without the signal sequence, the secreted protein
coding region,
as well as fragments, epitopes, domains, and variants of the nucleic acid
sequence.
Moreover, as used herein, a "polypeptide" refers to a molecule having the
translated
amino acid sequence generated from the polynucleotide as broadly defined.
In the present invention, the full length sequence identified as SEQ ID NO:X
was often generated by overlapping sequences contained in multiple clones
(contig
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3
analysis). A representative clone containing all or most of the sequence for
SEQ m
NO:X was deposited with the American Type Culture Collection ("ATCC"). As
shown in Table l, each clone is identified by a cDNA Clone ID (Identifier) and
the
ATCC Deposit Number. The ATCC is located at 10801 University Boulevard,
Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the
terms of the Budapest Treaty on the international recognition of the deposit
of
microorganisms for purposes of patent procedure.
A "polynucleotide" of the present invention also includes those
polynucleotides
capable of hybridizing, under stringent hybridization conditions, to sequences
contained
in SEQ m NO:X, the complement thereof, or the cDNA within the clone deposited
with
the ATCC. "Stringent hybridization conditions" refers to an overnight
incubation at 42°
C in a solution comprising 50% formamide, Sx SSC (750 mM NaCI, 75 mM sodium
citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran
sulfate, and 20 p.g/ml denatured, sheared salmon sperm DNA, followed by
washing the
filters in O.lx SSC at about 65°C.
Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides of the present invention at lower stringency hybridization
conditions.
Changes in the stringency of hybridization and signal detection are primarily
accomplished through the manipulation of formamide concentration (lower
percentages
of formamide result in lowered stringency); salt conditions, or temperature.
For
example, lower stringency conditions include an overnight incubation at
37°C in a
solution comprising 6X SSPE (20X SSPE = 3M NaCI; 0.2M NaH2P04; 0.02M EDTA,
pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;
followed by washes at 50°C with 1XSSPE, 0.1% SDS. In addition, to
achieve even
lower stringency, washes performed following stringent hybridization can be
done at
higher salt concentrations (e.g. SX SSC).
Note that variations in the above conditions may be accomplished through the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in hybridization experiments. Typical blocking reagents include
Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and
commercially available proprietary formulations. The inclusion of specific
blocking
reagents may require modification of the hybridization conditions described
above, due
to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such
as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or
to a
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complementary stretch of T (or U) residues, would not be included in the
definition of
"polynucleotide," since such a polynucleotide would hybridize to any nucleic
acid
molecule containing a poly (A) stretch or the complement thereof (e.g.,
practically any
double-stranded cDNA clone).
The polynucleotide of the present invention can be composed of any
polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or
DNA
or modified RNA or DNA. For example, polynucleotides can be composed of single-
and double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions, single- and double-stranded RNA, and RNA that is mixture of single-
and
double-stranded regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded or a mixture of single-
and double-
stranded regions. In addition, the polynucleotide can be composed of triple-
stranded
regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also
contain one or more modified bases or DNA or RNA backbones modified for
stability
or for other reasons. "Modified" bases include, for example, tritylated bases
and
unusual bases such as inosine. A variety of modifications can be made to DNA
and
RNA; thus, "polynucleotide" embraces chemically, enzymatically, or
metabolically
modified forms.
The polypeptide of the present invention can be composed of amino acids joined
to each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres, and
may contain amino acids other than the 20 gene-encoded amino acids. The
polypepddes may be modified by either natural processes, such as
posttranslational
processing, or by chemical modification techniques which are well known in the
art.
Such modifications are well described in basic texts and in more detailed
monographs,
as well as in a voluminous research literature. Modifications can occur
anywhere in a
polypeptide, including the peptide backbone, the amino acid side-chains and
the amino
or carboxyl termini. It will be appreciated that the same type of modification
may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a
given polypeptide may contain many types of modifications. Polypeptides may be
branched , for example, as a result of ubiquitination, and they may be cyclic,
with or
without branching. Cyclic, branched, and branched cyclic polypeptides may
result
from posttranslation natural processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation,
covalent
attachment of flavin, covalent attachment of a heme moiety, covalent
attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid
derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine,
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formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI
anchor formation, hydroxylation, iodination, methylation, myristoylation,
oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins
such as arginylation, and ubiquitination. (See, for instance, PROTEINS -
STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.
H. Freeman and Company, New York ( 1993); POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic
Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646
(1990);
Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
"SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ >D NO:Y"
refers to a polypeptide sequence, both sequences identified by an integer
specified in
Table 1.
"A polypeptide having biological activity" refers to polypeptides exhibiting
activity similar, but not necessarily identical to, an activity of a
polypeptide of the
present invention, including mature forms, as measured in a particular
biological assay,
with or without dose dependency. In the case where dose dependency does exist,
it
need not be identical to that of the polypeptide, but rather substantially
similar to the
dose-dependence in a given activity as compared to the polypeptide of the
present
invention (i.e., the candidate polypeptide will exhibit greater activity or
not more than
about 25-fold less and, preferably, not more than about tenfold less activity,
and most
preferably, not more than about three-fold less activity relative to the
polypeptide of the
present invention.)
Polvnucleotides and Polvneutides of the Invention
FEATURES OF PROTEIN ENCODED BY GENE NO: 1
Preferred polypeptides encoded by this gene comprise the following amino acid
sequence:
IRHELGCSWRFRAVi~AASAQGLFLSAPGPAARRCHGVVRCFSTCRALTA
RCTGRVPWEACLYSSEPPLTETVARSVSWTCELALTCYAPRALSGAPVLCRHD
V (SEQ ID NO:155). Also provided are polynucleotides encoding such
polypeptides.
This gene is expressed in human substantia nigra tissue.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
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not limited to, neurological disorders or abnormalities. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the neurological systems,
expression of this
gene at significantly higher or lower levels may be routinely detected in
certain tissues
or cell types (e.g., brain and of the tissue of the nervous system, and
cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder. Preferred epitopes
include
those comprising a sequence shown in SEQ ID N0:83 as residues: Pro-30 to Leu-
35.
The tissue distribution indicates that the protein products of this gene are
useful
for diagnosis and treatment of neurological disorders and abnormalities. Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
NO:11 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 544 of SEQ ID NO:11, b is an integer of 15 to 558,
where
both a and b correspond to the positions of nucleotide residues shown in SEQ
ID
NO:11, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
This gene is expressed primarily in breast and testes.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, tumors, particularly those of the breast or testes. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the breast and
testes, expression
of this gene at significantly higher or lower levels may be routinely detected
in certain
tissues or cell types (e.g., reproductive tissue, and cancerous and wounded
tissues) or
bodily fluids (e.g., breast milk, seminal fluid, serum, plasma, urine,
synovial fluid and
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spinal fluid) or another tissue or cell sample taken from an individual having
such a
disorder, relative to the standard gene expression level, i.e., the expression
level in
healthy tissue or bodily fluid from an individual not having the disorder.
Preferred
epitopes include those comprising a sequence shown in SEQ ID N0:84 as
residues:
Ser-32 to His-37.
The tissue distribution indicates that the protein products of this gene are
useful
for diagnosis and treatment of disorders or abnormalities of breast and testes
such as
tumors of those tissues. Many polynucleotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:12 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 701 of SEQ ID N0:12, b is
an
integer of 1 S to 715, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:12, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 3
This gene is expressed in apoptotic T cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, disorder or abnormalities of T cells. Similarly, polypepddes
and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the immune system, expression
of this gene
at significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cells and tissues of the immune system, developmental tissue, and
cancerous and wounded tissues) or bodily fluids {e.g., lymph seminal fluid,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken
from an individual having such a disorder, relative to the standard gene
expression
level, i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder. Preferred epitopes include those comprising a sequence
shown in
SEQ ID N0:85 as residues: Met-1 to Glu-6, Leu-39 to Lys-46.
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The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of disorders
in T cells
and other immune system disorders such as inflammation. Many polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:13 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 824 of SEQ ID N0:13, b is an integer of 15 to 838, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:13, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 4
The translation product of this gene shares sequence homology with ubiquitin-
conjugating enzyme (UCE) involved in selective protein degradation. Based on
the
sequence similarity, the translation product of this gene is expected to share
biological
activities with UCE proteins. Such activities are known in the art and
described
elsewhere herein. Preferred polypepddes encoded by this gene comprise the
following
amino acid sequence:
FLAIHFPTDFPLKPPKVAFTRMYFPNSNSNGSTCLDILWSQWSPAL (SEQ ID
NO: 156). Also provided are polynucleotides encoding such polypeptides.
This gene is expressed primarily in testes.
Therefore, polynucleotides and poiypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, disorders in testes, particularly cell cycle disorders, (e.g.
testes tumor).
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
testes and reproductive systems, expression of this gene at significantly
higher or lower
levels may be routinely detected in certain tissues or cell types (e.g.,
reproductive
tissues, and cancerous and wounded tissues) or bodily fluids (e.g., seminal
fluid,
serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or
cell sample
taken from an individual having such a disorder, relative to the standard gene
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9
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution and homology to ubiquitin-conjugating enzyme indicates
that the protein product of this gene is useful for diagnosis and treatment of
disorders
in testes and reproductive system such as tumors, as well as the treatment of
tumors of
other origins. Many polynucieotide sequences, such as EST sequences, are
publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:14 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 499 of SEQ ID N0:14, b is an integer
of 1 S to
513, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ ID N0:14, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5
This gene is expressed primarily in testes.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, disorders and abnormalities in the testes. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the reproductive system,
expression of this
gene at significantly higher or lower levels may be routinely detected in
certain tissues
or cell types (e.g., reproductive tissue, and cancerous and wounded tissues)
or bodily
fluids (e.g., seminal fluid, serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, (i.e., the expression level in healthy
tissue or
bodily fluid from an individual not having the disorder). Preferred epitopes
include
those comF~rising a sequence shown in SEQ ID N0:87 as residues: Ser-22 to Thr-
32,
Pro-37 to ~er-42.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of disorders
in testes
and the reproductive system. Many polynucleotide sequences, such as EST
sequences,
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are publicly available and accessible through sequence databases. Some of
these
sequences are related to SEQ ID NO:15 and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are
specifically excluded from the scope of the present invention. To list every
related
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 698 of SEQ ID
N0:15, b is
an integer of 15 to 712, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:15, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
This gene is expressed primarily in thymus, activated monocytes and spleen.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, disorders and abnormalities of immune function and
hematopoiesis, e.g.
leukemia. Similarly, polypeptides and antibodies directed to these
polypeptides are
useful in providing immunological probes for differential identification of
the tissues)
or cell type(s). For a number of disorders of the above tissues or cells,
particularly of
the immune and hematopoietic systems, expression of this gene at significantly
higher
or lower levels may be routinely detected in certain tissues or cell types
(e.g., endocrine
tissue cell and tissue of the immune system, and haematopoietic tissue, and
cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or cell sample taken from an
individual having
such a disorder, relative to the standard gene expression level, i.e., the
expression level
in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution indicates that the protein products of this gene are
useful
for diagnosis and treatment of immunological and hematopoietic disorders such
as
leukemia. Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:16 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 638 of SEQ ID NO:16, b is an integer
of 15 to
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I1
652, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ ID N0:16, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
This gene is expressed in T cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, T cell dysfunction. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cells and tissue of the immune system, and cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of disorders
in T cells
and immune systems. Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:17 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 728 of SEQ m N0:17, b is
an
integer of 15 to 742, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:17, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 8
This gene is expressed primarily in pleural cancer and to a less extent in T
cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
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12
not limited to, pleural cancer. Similarly, polypeptides and antibodies
directed to these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the pleural system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cells and tissue of the immune system, pleural tissue and
cancerous and
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial
fluid
and spinal fluid) or another tissue or cell sample taken from an individual
having such
a disorder, relative to the standard gene expression level, i.e., the
expression level in
healthy tissue or bodily fluid from an individual not having the disorder.
Preferred
epitopes include those comprising a sequence shown in SEQ ID N0:90 as
residues:
Ser-30 to Tyr-37.
The tissue distribution indicates that polynucleotides and poiypeptides
corresponding to this gene are useful for diagnosis and treatment of pleural
cancer.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:18 and may have been publicly available prior to conception of the present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence is cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 1205 of SEQ ID N0:18, b is an integer
of 15
to 1219, where both a and b correspond to the positions of nucleotide residues
shown
in SEQ ID N0:18, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 9
This gene is expressed in endothelial cells that shares the same origin as
hematopoietic cells and in spleen and liver which are hematopoietic tissues.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, disorders of endothelial cells or hematopoiesis. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the endothelial and
hematopoietic
systems, expression of this gene at significantly higher or lower levels may
be routinely
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13
detected in certain tissues or cell types (e.g., endothelial tissue,
haematopoietic cells and
tissue, and cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken
from an individual having such a disorder, relative to the standard gene
expression
level, i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of
disorders in
endothelial or hematopoietic systems. Many polynucleotide sequences, such as
EST
sequences, are publicly available and accessible through sequence databases.
Some of
these sequences are related to SEQ ID N0:19 and may have been publicly
available
prior to conception of the present invention. Preferably, such related
polynucleotides
are specifically excluded from the scope of the present invention. To list
every related
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleoddes comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 860 of SEQ ID
N0:19, b is
an integer of 15 to 874, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:19, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 10
This gene is expressed primarily in breast lymph node and to a lesser extent
in
melanocytes.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, metastatic melanoma. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the lymphatic system, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., reproductive tissue, cells and tissue of the immune system,
cancerous and
wounded tissues) or bodily fluids (e.g., lymph, breast milk, serum, plasma,
urine,
synovial fluid and spinal fluid) or another tissue or cell sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an . individual
not having the
disorder.
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14
The tissue distribution of this gene in melanocytes and lymph node indicates
that
the protein product of this gene is useful for the diagnosis and treatment of
metastatic
melanoma involving lymphatic tissues. Many polynucleotide sequences, such as
EST
sequences, are publicly available and accessible through sequence databases.
Some of
these sequences are related to SEQ ID N0:20 and may have been publicly
available
prior to conception of the present invention. Preferably, such related
polynucleotides
are specifically excluded from the scope of the present invention. To list
every related
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 450 of SEQ ID
N0:20, b is
an integer of 15 to 464, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:20, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11
This gene is believed to reside on chromosome 2. Therefore, this gene is
useful
in linkage analysis as a marker for chromosome 2.
This gene is expressed primarily in infant brain and to a lesser extent in
fetal
liver/spleen.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, neurological disorders. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the central nervous system, expression of
this gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., neural tissue, cells and tissue of the immune system, developing
tissue, and
cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken
from an individual having such a disorder, relative to the standard gene
expression
level, (i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder). Preferred epitopes include those comprising a sequence
shown in
SEQ ID N0:93 as residues: Tyr-59 to Gln-68, His-84 to Leu-90, Ser-105 to Asn-
110,
Leu-112 to Pro-118.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of
neurological
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disorders. Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:21 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
5 from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 623 of SEQ ID N0:21, b is an integer
of 15 to
637, where both a and b correspond to the positions of nucleotide residues
shown in
10 SEQ ID N0:21, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
This gene is expressed primarily in adipose tissue.
Therefore, polynucleotides and polypeptides of the invention are useful as
15 reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, metabolic disorders. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the endocrine system, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., adipose tissue, and cancerous and wounded tissues) or bodily
fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for regulation of fat metabolism and
treatment of
obesity. Many polynucleotide sequences, such as EST sequences, are publicly
available
and accessible through sequence databases. Some of these sequences are related
to SEQ
ID N0:22 and may have been publicly available prior to conception of the
present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence is cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 738 of SEQ ID N0:22, b is an integer
of 15 to
*rB
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16
752, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ ID N0:22, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
The translation product of this gene shares sequence homology with
NADH:ubiquinone oxidoreductase, the first enzyme in the respiratory electron
transport
chain of mitochondria.
This gene is expressed primarily in HSC 172 cells and to a lesser extent in
pineal
gland.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, jet lag. Similarly, polypeptides and antibodies directed to
these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the endocrine system, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., endocrine tissue, metabolic tissue, and cancerous and wounded
tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, i.e., the expression level in healthy
tissue or
bodily fluid from an individual not having the disorder. Preferred epitopes
include those
comprising a sequence shown in SEQ ID N0:95 as residues: Thr-30 to Val-38, Glu-
49
to Ile-54.
The tissue distribution and homology to NADH:ubiquinone oxidoreductase
indicates that polynucleotides and polypeptides corresponding to this gene are
useful for
minimizing the negative effects of travel across time zones by altering the
body's
circadean clock. Many polynucleotide sequences, such as EST sequences, are
publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:23 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 478 of SEQ ID N0:23, b is an integer
of 15 to
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492, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ ID N0:23, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 14
This gene is expressed primarily in synovial IL-1/TNF stimulated cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, arthritis. Similarly, polypeptides and antibodies directed to
these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the skeletal system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., skeletal tissue and cancerous and wounded tissues) or bodily
fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an .
individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for treatment of degenerative conditions
of joints,
including arthritis. Many polynucleodde sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:24 and may have been publiciy available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between l to 518 of SEQ ID N0:24, b is
an
integer of 15 to 532, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:24, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 15
This gene is expressed primarily in 12 Week Old Early Stage Human.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
*rB
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18
not limited to, developmental disorders. Similarly, polypeptides and
antibodies directed
to these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the fetal systems, expression of this gene
at significantly
higher or lower levels may be routinely detected in certain tissues or cell
types (e.g.,
developing tissue, and cancerous and wounded tissues) or bodily fluids (e.g.,
amniotic
fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of some
developmental disorders. Many polynucleotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ >D N0:25 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 906 of SEQ >D N0:25, b is
an
integer of 15 to 920, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:25, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 16
This gene is expressed primarily in thymus and to a lesser extent in
neutrophils.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune disorders. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cells and tissue of the immune system, and cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
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relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an . individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of some
immune
disorders including lupus and other disorders involving thymic dysfunction.
Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:26 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 903 of SEQ ID N0:26, b is an integer of 15 to 917,
where
both a and b correspond to the positions of nucleotide residues shown in SEQ
B7
N0:26, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 17
This gene is expressed primarily in fibrosarcoma and to a lesser extent in 1L1
and IPS induced neutrophils.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, fibrosarcoma. Similarly, polypeptides and antibodies directed
to these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., connective tissue, and cells and tissue of the immune system, and
cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or cell sample taken from an
individual having
such a disorder, relative to the standard gene expression level, i.e., the
expression level
in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred
epitopes include those comprising a sequence shown in SEQ ID N0:99 as
residues:
Gly-6 to Pro-11.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of
fibrosarcoma or
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other immune disorders. Many polynucleotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:27 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
5 excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 648 of SEQ ID N0:27, b is
an
integer of 15 to 662, where both a and b correspond to the positions of
nucleotide
10 residues shown in SEQ ID N0:27, and where the b is greater than or equal to
a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 18
Preferred polypeptides comprise the following amino acid sequence:
MLLTPHFNVANPQNLLAGLWLENEHSFTLMAPER.ARTHHCQPEERKVLFCLFP
15 IVPNSQAQVQPPQMPPFCCAAAKEKTQEEQLQEPLGSQCPDTCPNSLC (SEQ ID
NO: 157}. Polynucleotides encoding such polypeptides are also provided.
This gene is expressed primarily in jurkat T-Cells in S phase, and to a lesser
extent in IL,-1 and LPS induced neutrophils.
Therefore, polynucleotides and polypeptides of the invention are useful as
20 reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune disorders. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., ells and tissue of the immune system, and cancerous and wounded
tissues)
or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, i.e., the expression level in healthy
tissue or
bodily fluid from an individual not having the disorder. Preferred epitopes
include those
comprising a sequence shown in SEQ ID NO:100 as residues: Lys-97 to Gln-106,
Gln-
112 to Pro-118, Pro-123 to Lys-130, Arg-153 to Gly-158.
The tissue distribution indicates that polynucleotides and polypepddes
corresponding to this gene are useful for diagnosis and treatment of immune
disorders
related to jurkat T-cells and induced neutrophils. Many polynucleotide
sequences, such
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21
as EST sequences, are publicly available and accessible through sequence
databases.
Some of these sequences are related to SEQ ID N0:28 and may have been publicly
available prior to conception of the present invention. Preferably, such
related
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
685 of
SEQ ID N0:28, b is an integer of 15 to 699, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:28, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 19
The translation product of this gene shares sequence homology with FK506-
binding protein FKBP-12 and FKBP13, members of a family of proteins which bind
1 S the immunosuppressant drugs FK506 and rapamycin. The homology to FK506
binding
proteins indicates that the protein encoded by this gene has similar activity
to the known
FK506 BP family members. Such activity may be assayed according to methods
known in the art and described elsewhere herein. Preferred polypepddes encoded
by
this gene comprise the following amino acid sequence:
MRLFLWNAVLTLFVTSLIGALIPEPEVK
IEVLQKPFICHRKTKGGDLMLVHYEGYLEKDGSLFHSTHKHNNGQPIWFTLGI
LEALKGWDQGLKGMCVGEKRKLIIPPALGYGKEGKGKIPPESTLIFNIDLLEIR
NGPRSHESFQEMDLNDDWKLSKDEVKAYLKKEFEKHGAVVNESHHDALVEDI
FDKEDEDKDGFISAREFTYKHDEL (SEQ ID NO: 158) or comprise a mature form
of the foregoing polypeptide having the following amino acid sequence:
EV KIEVLQKPFICHRKTKGGDLMLVHYEGYLEKDGSLFHSTHKHNNGQPIWF
TLGILEALKGWDQGLKGMCVGEKRKLIIPPALGYGKEGKGKIPPESTLIFNIDL
LEIRNGPRSHESFQEMDLNDDWKLSKDEVKAYLKKEFEKHGAVVNESHHDAL
VEDIFDKEDEDKDGFISAREFTYKHDEL (SEQ ID NO: 159). . Polynucleotides
encoding these polypeptides are also provided. Particularly preferred is the
following
polynucleotide sequence:
ATGAGGCTITTCTTGTGGAACGCGGTCTTGACTCTGTTCGTCACTTCT
TTGATTGGGGCTITGATCCCTGAACCAGAAGTGAAAATTGAAGTTCTCCAGA
AGCCATTCATCTGCCATCGCAAGACCAAAGGAGGGGATTTGATGTTGGTCC
ACTATGAAGGCTACTTAGAAAAGGACGGCTCCTTATTTCACTCCACTCACAA
ACATAACAATGGTCAGCCCATTTGGTTTACCCTGGGCATCCTGGAGGCTCTC
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AAAGGTTGGGACCAGGGCTTGAAAGGAATGTGTGTAGGAGAGAAGAGAAA
GCTCATCATTCCTCCTGCTCTGGGCTATGGAAAAGAAGGAAAAGGTAAAATT
CCCCCAGAAAGTACACTGATATTTAATATTGATCTCCTGGAGATTCGAAATG
GACCAAGATCCCATGAATCATTCCAAGAAATGGATCTTAATGATGACTGGAA
ACTCTCTAAAGATGAGGTTAAAGCATATTTAAAGAAGGAGTTTGAAAAACAT
GGTGCGGTGGTGAATGAAAGTCATCATGATGCTTTGGTGGAGGATATTZ1'T
GATAAAGAAGATGAAGACAAAGATGGGTTTATATCTGCCAGAGAATTTACAT
ATAAACACGATGAG TTA (SEQ m NO:160), and the portion of it, nucleotide
residues 76 to the 3' end, which encode the mature form shown above.
lp This gene is expressed primarily in fetal heart, ovary, and thymus.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the dssue(s) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune dysfunction such as autoimmune disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
immune system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., cells and tissue of the immune system,
and
cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine,
synovial fluid and spinal fluid) or another tissue or cell sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder.
The tissue distribution and homology to FK506-binding protein FKBP-12 and
FKBP13 indicates that polynucleotides and polypeptides corresponding to this
gene are
useful for identifying immunosuppressant drugs, and may be used in combination
with
immunosuppressant drugs for therapeutic purposes in the treatment of
autoimmune
diseases and organ/tissue transplant rejection. Many polynucleotide sequences,
such as
EST sequences, are publicly available and accessible through sequence
databases.
Some of these sequences are related to SEQ m N0:29 and may have been publicly
available prior to conception of the present invention. Preferably, such
related
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
1623 of
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23
SEQ ID N0:29, b is an integer of 15 to 1637, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:29, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 20
The protein product of this gene is believed to be the human homologue of
yeast
GAAIP which anchors proteins to cell surface membranes by
glycosylphosphatidylinositols (GPIs). See, for example, Yeast Gaalp is
required for
attachment of a completed GPI anchor onto proteins (J Cell Biol. 1995 May;
129(3):
629-639. PMID: 7730400; UI: 95247814).
This gene is expressed primarily in primary breast cancer and to a lesser
extent
in amniotic cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, breast cancer. Similarly, polypeptides and antibodies directed
to these
polypeptides are useful in providing immunological probes for differential
identification of the tissue{s) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the metabolic system, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., reproductive tissue, amniotic cells and tissue and cancerous and
wounded
tissues) or bodily fluids (e.g., breast milk, lymph, amniotic fluid, serum,
plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell sample taken
from an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder. Preferred epitopes include those comprising a sequence shown in SEQ
ID
N0:102 as residues: Pro-113 to Met-123.
The tissue distribution and similarity to GAA1P indicates that polynucleotides
and polypeptides corresponding to this gene are useful for diagnosis and
treatment of
primary breast cancers. Many polynucleotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:30 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
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24
formula of a-b, where a is any integer between 1 to 2128 of SEQ ID N0:30, b is
an
integer of 15 to 2142, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:30, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 21
This gene is expressed primarily in activated T-cells and to a lesser extent
in
prostate cancer and HSC172 cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune dysfunction and/or prostate cancer. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the immune and metabolic
systems,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., cells and tissue of the immune system,
reproductive
tissue, and cancerous and wounded tissues) or bodily fluids (e.g., lymph,
seminal
fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:103 as residues: Ala-17 to Pro-26, Phe-92 to Tyr-
97,
Gly-104 to Glu-111.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of immune
disorders
and/or prostate cancer. Many polynucleotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:31 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleoddes are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 1550 of SEQ ID N0:31, b is
an
integer of 15 to 1564, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:31, and where the b is greater than or equal to a
+ 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 22
This gene maps to chromosome 12, and therefore, may be used as a marker in
linkage analysis for chromosome 12.
This gene is expressed primarily in breast tissues and to a lesser extent in
fetal
5 spleen.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, breast cancer, lactation deficiencies, and other breast
related diseases,
10 including breast cancer. Similarly, polypeptides and antibodies directed to
these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the haemolymphoid and reproductive systems,
expression of this gene at significantly higher or lower levels may be
routinely detected
15 in certain tissues or cell types (e.g., breast tissue, cells and tissue of
the immune
system, and haemolymphoid tissue, cancerous and wounded tissues) or bodily
fluids
(e.g., lymph, breast milk, serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, i.e., the expression level in healthy
tissue or
20 bodily fluid from an individual not having the disorder.
The tissue distribution in tumors of breast origins indicates that
polynucleotides
and polypeptides corresponding to this gene are useful for diagnosis and
intervention of
these tumors, in addition to other tumors where expression has been indicated.
Protein,
as well as, antibodies directed against the protein may show utility as a
tissue-specific
25 marker and/or immunotherapy target for the above listed tissues.
Alternatively, The
tissue distribution indicates that polynucleotides and polypeptides
corresponding to this
gene are . useful for the treatment and diagnosis of hematopoetic related
disorders
such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since
stromal
cells are important in the production of cells of hematopoietic lineages. The
uses include
bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow
reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product
may also
be involved in lymphopoiesis, therefore, it can be used in immune disorders
such as
infection, inflammation, allergy, immunodeficiency etc. In addition, this gene
product
may have commercial utility in the expansion of stem cells and committed
progenitors
of various blood lineages, and in the differentiation and/or proliferation of
various cell
types. Many polynucleotide sequences, such as EST sequences, are publicly
available
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26
and accessible through sequence databases. Some of these sequences are related
to SEQ
n7 N0:32 and may have been publicly available prior to conception of the
present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence is cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 1617 of SEQ ID N0:32, b is an integer
of 15
to 1631, where both a and b correspond to the positions of nucleotide residues
shown
in SEQ ID N0:32, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 23
The translation product of this gene shares sequence homology with mutant or
polymorphic BRCA1 gene which is thought to be important in the diagnosis and
therapy of human breast and ovarian cancer as a predisposing gene (See
Genebank
Accession No T17455).This gene maps to chromosome 18, and therefore, may be
used
as a marker in linkage analysis for chromosome 18.
This gene is expressed primarily in primary dendritic cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, breast, ovarian, and other reproductive related disorders and
cancers.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
haemolymphoid and reproductive systems, expression of this gene at
significantly
higher or lower levels may be routinely detected in certain tissues or cell
types (e.g.,
reproductive tissue, pleural tissue, breast tissue, and cancerous and wounded
tissues)
or bodily fluids (e.g., breast milk, lymph, serum, plasma, urine, synovial
fluid and
spinal fluid) or another tissue or cell sample taken from an individual having
such a
disorder, relative to the standard gene expression level, i.e., the expression
level in
healthy tissue or bodily fluid from an individual not having the disorder.
Preferred
epitopes include those comprising a sequence shown in SEQ m NO:105 as
residues:
Pro-13 to Lys-18, Ala-50 to Leu-58.
The homology to the mutant or polymorphic BRCA 1 gene indicates that
polynucleotides and polypeptides corresponding to this gene are useful for
diagnosis
and intervention of breast and/or ovarian tumors, in addition to other tumors
where
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27
expression has been indicated. Protein, as well as, antibodies directed
against the
protein may show utility as a tissue-specific marker and/or immunotherapy
target for the
above listed tissues. Alternatively, the tissue distribution within dendritic
cells indicates
that the protein product of this gene is useful for the detection/treatment of
neurodegenerative disease states and behavioral disorders such as Alzheimers
Disease,
Parkinsons Disease, Huntingtons Disease, Tourette Syndrome, schizophrenia,
mania,
dementia, paranoia, obsessive compulsive disorder, panic disorder, learning
disabilities, ALS, psychoses , autism, and altered behaviors, including
disorders in
feeding, sleep patterns, balance, and perception. In addition, the gene or
gene product
may also play a role in the treatment and/or detection of developmental
disorders
associated with the developing embryo, sexually-linked disorders, or disorders
of the
cardiovascular system. Many polynucleotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:33 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 964 of SEQ ID N0:33, b is
an
integer of 15 to 978, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:33, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 24
The translation product of this gene shares sequence homology with smaller
hepatocellular oncoprotein which is thought to be important in protein
synthesis (See
Genebank Accession No. R07057).One embodiment of this gene comprises
polypeptides of the following amino acid sequence: LRSVVQDHPGQHGETPSLLKIQ
(SEQ ID N0:161 ). An additional embodiment is the polynucleotides encoding
these
polypeptides.
This gene is expressed primarily in embryonic tissues and to a lesser extent
in
uterine cancer.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, heptocellular tumors, and other disorders characterized by
proliferating
and/or developing tissues. Similarly, polypeptides and antibodies directed to
these
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28
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the haemolymphoid system, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., reproductive tissue, cells and tissue of the immune system, and
cancerous
and wounded tissues) or bodily fluids (e.g., amniotic fluid, serum, plasma,
urine,
synovial fluid and spinal fluid) or another tissue or cell sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder. Preferred epitopes include those comprising a sequence shown in SEQ
ID
N0:106 as residues: Asn-15 to Ser-20, Ile-32 to Asn-37.
The tissue distribution combine with the homology to a conserved human
hepatocellular oncoprotein indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of
cancer and other
proliferative disorders. Expression within embryonic tissue and other cellular
sources
marked by proliferating cells indicates that this protein may play a role in
the regulation
of cellular division. Additionally, the expression also indicates that this
protein may
play a role in the proliferation, differentiation, and/or survival of
hematopoietic cell
lineages. In such an event, this gene may be useful in the treatment of
lymphoproliferative disorders, and in the maintenance and differentiation of
various
hematopoietic lineages from early hematopoietic stem and committed progenitor
cells.
Similarly, embryonic development also involves decisions involving cell
differentiation
and/or apoptosis in pattern formation. Thus this protein may also be involved
in
apoptosis or tissue differentiation and could again be useful in cancer
therapy.
Alternatively, the homology to a hepatocellular protein indicates that the
protein product
of this gene is useful for the detection and treatment of liver disorders and
cancers (e.g.
hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions
that are
attributable to the differentiation of hepatocyte progenitor cells). In
addition the
expression in fetus would suggest a useful role for the protein product in
developmental
abnormalities, fetal deficiencies, pre-natal disorders and various would-
healing models
andlor tissue trauma. Many polynucleotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:34 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
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29
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 884 of SEQ ID N0:34, b is
an
integer of 15 to 898, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:34, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 25
The translation product of this gene shares sequence homology with a gene
encoding for anti-heparanase activity which is thought to be important in
inhibition of
heparin or heparan sulphate degradation. Moreover, this gene was shown to have
homology to the human 3-oxo-5-beta-steroid 4-dehydrogenase, which is known to
be
important in metabolism since it catalyzes the reduction of delta(4) double
bonds of bile
acid intermediates and steroid hormones carrying the delta(4)-3-one structure
in the A/B
Cis configuration (See Genebank Accession No. 228339) One embodiment of this
gene
comprises polypeptides of the following amino acid sequence:
MFYNFVRQLDTVSIEHAGKSKLKMTVGTKLTSGXGPRKSSQSGRIAASITDCQ
QCKA @ (SEQ ID N0:162), andlor
MEAAILPLWLLFLGPXPEVSFVPTVIFNLDFPACSILTVSSCLTKL @ (SEQ D7
N0:163). An additional embodiment is the polynucleotides encoding these
polypeptides.
This gene is expressed primarily in fetal tissues.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, arthritis, metabolic disorders, as well as reproductive
disorders.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
haemolymphoid system, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues or cell types (e.g. reproductive,
metabolic,
cancerous and wounded tissues) or bodily fluids (e.g., bile acid, amniotic
fluid, serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken
from an individual having such a disorder, relative to the standard gene
expression
level, i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of
cancer and other
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proliferative disorders. Expression within embryonic tissue and other cellular
sources
marked by proliferating cells indicates that this protein may play a role in
the regulation
of cellular division. Additionally, the expression indicates that this protein
may play a
role in the proliferation, differentiation, and/or survival of hematopoietic
cell lineages.
5 In such an event, this gene may be useful in the treatment of
lymphoproliferative
disorders, and in the maintenance and differentiation of various hematopoietic
lineages
from early hematopoietic stem and committed progenitor cells. Similarly,
embryonic
development also involves decisions involving cell differentiation andlor
apoptosis in
pattern formation. Thus this protein may also be involved in apoptosis or
tissue
10 differentiation and could again be useful in cancer therapy. Alternatively,
based upon its
homology to a conserved human anti-heparanase gene, mutations of which are
known
to be important in the predisposition of arthritis, may suggest that this
protein may also
be important in the diagnosis or treatment of various autoimmune disorders
such as
rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as
dwarfism,
15 spinal deformation, and specific joint abnormalities as well as
chondrodysplasias i.e.
spondyloepiphyseal dysplasia congenita, familial osteoarthritis,
Atelosteogenesis type
II, metaphyseal chondrodysplasia type Schmid. Moreover, the homology to a
conserved human metabolic gene may suggest that the protein product of this
gene is
useful for the diagnosis, prevention, and/or treatment of various metabolic
disorders
20 such as Tay-Sachs disease, phenylkenonuria, galactosemia, porphyrias, and
Hurler's
syndrome. Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:35 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
25 from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 740 of SEQ ID N0:35, b is an integer
of 15 to
754, where both a and b correspond to the positions of nucleotide residues
shown in
30 SEQ ID N0:35, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 26
The translation product of this gene shares sequence homology with pulmonary
hydrophobic surfactant-associated proteins which is thought to be important in
useful
for normalising pulmonary surface tension (See Genebank Accession No. N80643
).
This gene is expressed primarily in embryonic tissues.
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Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, hyaline membrane disease, developmental and pulmonary
disorders.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
respiratory system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues or cell types (e.g., pulmonary
tissue, developing
tissue and cancerous and wounded tissues) or bodily fluids (e.g., surfactant,
amniotic
fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution and homology to pulmonary hydrophobic surfactant-
associated proteins indicates that polynucleotides and polypepddes
corresponding to
this gene are useful for the treatment and diagnosis of such lung related
diseases such as
hyaline membrane disease which is often characteristic of premature infants -
leading to
significant pulmonary disorders throughout childhood. Alternatively, The
tissue
distribution indicates that polynucleotides and polypeptides corresponding to
this gene
are . useful for the diagnosis and treatment of cancer and other proliferative
disorders.
Expression within embryonic tissue and other cellular sources marked by
proliferating
cells indicates that this protein may play a role in the regulation of
cellular division.
Additionally, the expression indicates that this protein may play a role in
the
proliferation, differentiation, and/or survival of hematopoietic cell
lineages. In such an
event, this gene may be useful in the treatment of lymphoproliferative
disorders, and in
the maintenance and differentiation of various hematopoietic lineages from
early
hematopoietic stem and committed progenitor cells. Similarly, embryonic
development
also involves decisions involving cell differentiation and/or apoptosis in
pattern
formation. Thus this protein may also be involved in apoptosis or tissue
differentiation
and could again be useful in cancer therapy. Many polynucleotide sequences,
such as
EST sequences, are publicly available and accessible through sequence
databases.
Some of these sequences are related to SEQ 117 N0:36 and may have been
publicly
available prior to conception of the present invention. Preferably, such
related
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
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32
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
685 of
SEQ ID N0:36, b is an integer of 15 to 699, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:36, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 27
This gene is expressed primarily in fetal tissue and to a lesser extent in
thymus.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immunological disorders, particularly immunodeficiency.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
haemolymphoid
system, expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., cells and tissue of the
immune system,
developing tissue, and cancerous and wounded tissues) or bodily fluids (e.g.,
amniotic
fluid, lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or
cell sample taken from an individual having such a disorder, relative to the
standard
gene expression level, i.e., the expression level in healthy tissue or bodily
fluid from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:109 as residues: Pro-18 to Trp-24.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of a
variety of
immune system disorders. Expression of this gene product in thymus indicates a
role in
the regulation of the proliferation; survival; differentiation; and/or
activation of
potentially all hematopoietic cell lineages, including blood stem cells. This
gene product
may be involved in the regulation of cytokine production, antigen
presentation, or other
processes that may also suggest a usefulness in the treatment of cancer (e.g.
by
boosting immune responses). Since the gene is expressed in cells of lymphoid
origin,
the natural gene product may be involved in immune functions. Therefore it may
be also
used as an agent for immunological disorders including arthritis, asthma,
immune
deficiency diseases such as AIDS, and leukemia. Protein, as well as,
antibodies
directed against the protein may show utility as a tumor marker andlor
imrnunotherapy
targets for the above listed tumors and tissues. In addition, this gene
product may have
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33
commercial utility in the expansion of stem cells and committed progenitors of
various
blood lineages, and in the differentiation and/or proliferation of various
cell types.
Protein, as well as, antibodies directed against the protein may show utility
as a tumor
marker and/or immunotherapy targets for the above listed tissues. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:37 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 957 of SEQ ID N0:37, b is an integer of 15 to 971, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:37, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 28
This gene is expressed primarily in small intestine and to a lesser extent in
ulcerative colitis.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, ulcers, acid reflux and other gastrointestinal disorders.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
digestive system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., gastrointestinal tissue, and cancerous
and wounded
tissues) or bodily fluids (e.g., bile, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder. Preferred epitopes
include
those comprising a sequence shown in SEQ ID N0:110 as residues: Ile-2 to Ser-
8,
Gln-23 to Ser-31, Lys-61 to Lys-66, Lys-74 to Thr-79, Val-138 to Glu-160, Glu-
178
to Thr-183.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment and diagnosis of
digestive and
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34
gastrointestinal problems such as acid reflux or ulcers. Alternatively, the
tissue
distribution may suggest that the protein product of this gene is useful for
the
diagnosis, prevention, and/or treatment of various metabolic disorders such as
Tay-
Sachs disease, phenylkenonuria, galactosemia, porphyrias, and Hurler's
syndrome.
Protein, as well as, antibodies directed against the protein may show utility
as a tumor
marker and/or immunotherapy targets for the above listed tissues. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:38 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 858 of SEQ ID N0:38, b is an integer of 15 to 872, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:38, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 29
The translation product of this gene was shown to have homology to catalase
(See Genebank Accession No. pirIA550921A55092} which is known to play an
integral
role in the oxidative prophylaxis in eukaryotic and mammalian cells. One
embodiment
of this gene comprises polypeptides of the following amino acid sequence:
NHGHSCFLCEIVIRSQFHTTYEPEA @ (SEQ ID N0:164), and/or
SGRHRVELQLLFPLVRVNFELGVNHGHSCFLCEIVIRSQFHTTYEPEA @ (SEQ
ID NO:I65). An additional embodiment is the polynucleotides encoding these
polypeptides. This gene maps to chromosome 3, and therefore, may be used as a
marker in linkage analysis for chromosome 3.
This gene is expressed primarily in skin and to a lesser extent in human
uterine
cancer.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, uterine cancer; fibrosis; melanoma. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissue{s) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the epidermis and/or
reproductive system,
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expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., integumentary tissue, and cancerous
and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
5 relative to the standard gene expression level, i.e., the expression level
in healthy tissue
or bodily fluid from an individual not having the disorder.
The tissue distribution combined with the homology to catalase indicates that
polynucleotides and poiypeptides corresponding to this gene are useful for the
treatment
and/or diagnosis of human uterine cancer and potentially, all cancers in
general
10 (particularly of the epidermis) due to the fact that diminished catalase
activity has been
shown to lead to significant cellular damage which could predispose cancer.
Likewise,
this gene and/or its protein product may be useful in the treatment and/or
diagnosis of
other disorders of the skin, such as fibrosis or in wound healing. Protein, as
well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
15 immunotherapy targets for the above listed tissues. Many polynucleotide
sequences,
such as EST sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ 1D N0:39 and may have
been
publicly available prior to conception of the present invention. Preferably,
such related
polynucleotides are specifically excluded from the scope of the present
invention. To
20 list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
594 of
SEQ ID N0:39, b is an integer of 15 to 608, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:39, and where the b is
greater
25 than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 30
This gene is expressed primarily in fetal liver/spleen and bone marrow stromal
cells, and to a lesser extent in neural tissues (brain; spinal cord) and
cancers
30 (glioblastoma; chondrosarcoma).
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, cancers, particularly of glial cells and cartilage;
hematopoietic and other
35 immune disorders. Similarly, polypeptides and antibodies directed to these
polypeptides
are useful in providing immunological probes for differential identification
of the
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36
tissues) or cell type{s). For a number of disorders of the above tissues or
cells,
particularly of the hematopoietic/immune system and CNS, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., hematopoietic cells and tissue, and cancerous and wounded
tissues) or
bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, i.e., the expression level in healthy
tissue or
bodily fluid from an individual not having the disorder. Preferred epitopes
include those
comprising a sequence shown in SEQ ID N0:112 as residues: Arg-2 to Asp-7.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the expansion, proliferation, and/or
differentiation of hematopoietic cells. This gene is expressed predominantly
at sites of
hematopoiesis, such as fetal liver and bone marrow, and thus may control the
proliferation and/or differentiation of hematopoietic stem and progenitor
cells. Thus it
could be used for chemoprotection, or for the production of specific blood
cell lineages,
as well as the amplification of stem cells. Additionally, its expression in
neural cells of
the brain and spinal cord suggest that it may also play a role in the
maintenance and
differentiation of neuronal stem cells, or in the treatment of neurological
disorders, such
as Alzheimers Disease, Parkinsons Disease, Huntingtons Disease, Tourette
Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses , autism, and altered
behaviors,
including disorders in feeding, sleep patterns, balance, and perception. In
addition, the
gene or gene product may also play a role in the treatment and/or detection of
developmental disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system. Protein, as well as,
antibodies
directed against the protein may show utility as a tumor marker and/or
immunotherapy
targets for the above listed tissues. Finally, its observed expression in
various cancers
suggest that it may play a role in the control of cell proliferation. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:40 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 841 of SEQ ID N0:40, b is an integer of 15 to 855, where both a
and b
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37
correspond to the positions of nucleotide residues shown in SEQ ID N0:40, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 31
This gene is expressed primarily in placenta, bone marrow, and fetal
liver/spleen and to a lesser extent in brain and CNS.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, hematopoietic disorders; placental insufficiency; neurological
disorders.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
immune system and/or CNS, expression of this gene at significantly higher or
lower
levels may be routinely detected in certain tissues or cell types (e.g.,
hematopoietic cells
and tissue, cells and tissue of the immune system, and neurological tissue,
and
cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken
from an individual having such a disorder, relative to the standard gene
expression
level, i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder. Preferred epitopes include those comprising a sequence
shown in
SEQ ID N0:113 as residues: Arg-53 to Gln-58.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the proliferation, maintenance,
and/or
differentiation of hematopoietic cells. In addition, it may be involved in the
maintenance
and establishment of the vasculature, and may play a role in the regulation of
angiogenesis. Thus, it may play a role in the establishment andlor maintenance
of
tumors. Expression in the CNS indicates that this gene product may also be
useful in
the treatment of neurodegenerative disorders, such as Alzheimers Disease,
Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, schizophrenia, mania,
dementia,
paranoia, obsessive compulsive disorder, panic disorder, learning
disabilities, ALS,
psychoses , autism, and altered behaviors, including disorders in feeding,
sleep
patterns, balance, and perception. In addition, the gene or gene product may
also play a
role in the treatment and/or detection of developmental disorders associated
with the
developing embryo, sexually-linked disorders, or disorders of the
cardiovascular
system. Protein, as well as, antibodies directed against the protein may show
utility as a
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38
tumor marker and/or immunotherapy targets for the above listed tissues. Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:41 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 1028 of SEQ ID N0:41, b is an integer of 15 to 1042,
where
both a and b correspond to the positions of nucleotide residues shown in SEQ
ID
N0:41, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 32
The translation product of this gene was shown to have homology to the ras-
related protein RABB from Dictyostelium discoideum (See Genebank Accession
No.P34142). Members of the Ras family of proteins are known to be essential to
normal cell cycle control and mutations of which have been shown to lead to
the
predisposition of cancer.
This gene is expressed primarily in Jurkat T cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, T cell lymphomas; defects in immune surveillance and/or T cell
activation. Similarly, polypeptides and antibodies directed to these
polypeptides are
useful in providing immunological probes for differential identification of
the tissues)
or cell type(s). For a number of disorders of the above tissues or cells,
particularly of
the immune system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues or cell types (e.g., cells and tissue
of the immune
system, and cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken
from an individual having such a disorder, relative to the standard gene
expression
level, i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
The tissue distribution combined with its homology to a Ras-related protein
indicates that polynucleotides and polypeptides corresponding to this gene are
useful for
the treatment of defects in immune surveillance and cancer. Expression of this
gene
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39
product in T cells indicates that it represents a secreted protein that may
regulate T cells
in an autocrine fashion, thereby impacting on their ability to recognize
antigen and
become activated, or may be involved in immune modulation. Likewise, this may
represent a secreted protein made by T cells that affects other hematopoietic
cells and
regulates their proliferation and/or differentiation.Protein, as well as,
antibodies directed
against the protein may show utility as a tumor marker and/or immunotherapy
targets
for the above listed tissues. Many polynucleotide sequences, such as EST
sequences,
are publicly available and accessible through sequence databases. Some of
these
sequences are related to SEQ ID N0:42 and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are
specifically excluded from the scope of the present invention. To list every
related
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 688 of SEQ ID
N0:42, b is
an integer of 15 to 702, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:42, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 33
The translation product of this gene shares sequence homology with a known
human spliceosome associated protein (See Genebank Accession No. AA523942) as
well as a yeast protein (ORF YBR173c [Saccharomyces cerevisiae]).Preferred
polypeptides comprise the following amino acid sequence:
MNARGLGSELKDSIPVTELSASGPFES
HDLLRKGFSCVKNELLPSHPLELSEKNFQLNQDKMNFSTLRNIQGLFAPLKLQ
MEFKAVQQVQRLPFLSSSNLSLDVLRGNDETIGFEDILNDPSQSEVMGEPHLMV
EYKLGLL (SEQ ID N0:166). Also preferred are the polynucleotides encoding these
polypeptides.
This gene is expressed primarily in bone marrow and stromal cells, and to a
lesser extent in T cells and peripheral blood cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, hematopoietic disorders; leukemias; defects in immune
surveillance;
defects in T cell activation; inflammation; bacterial infections, and other
disorders
characterized by immunodeficiency. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
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identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cells and tissue of the immune system, and cancerous and wounded
5 tissues) or bodily fluids {e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder. Preferred epitopes
include
those comprising a sequence shown in SEQ ID NO:115 as residues: Lys-16 to Thr-
24.
10 The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the proliferation, differentiation,
and/or
activation of hematopoietic cell lineages. Such treatments could involve
chemoprotection or expansion of either progenitor cells or specific mature
blood
lineages. Likewise, this gene product could be involved in immune modulation,
or in
I S affecting T cell activation and antigen recognition. Alternatively, the
protein product of
this gene is useful for the treatment and diagnosis of hematopoetic related
disorders
such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since
stromal
cells are important in the production of cells of hematopoietic lineages. The
uses include
bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow
20 reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product
may also
be involved in lymphopoiesis, therefore, it can be used in immune disorders
such as
infection, inflammation, allergy, immunodeficiency etc. In addition, this gene
product
may have commercial utility in the expansion of stem cells and committed
progenitors
of various blood lineages, and in the differentiation and/or proliferation of
various cell
25 types. Protein, as well as, antibodies directed against the protein may
show utility as a
tumor marker and/or immunotherapy targets for the above tissues. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:43 and
may
have been publicly available prior to conception of the present invention.
Preferably,
30 such related polynucleotides are specifically excluded from the scope of
the present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 628 of SEQ ID N0:43, b is an integer of 15 to 642, where both a
and b
35 correspond to the positions of nucleotide residues shown in SEQ ID N0:43,
and where
the b is greater than or equal to a + 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 34
This gene maps to chromosome 4, and therefore, may be used as a marker in
linkage analysis for chromosome 4.
This gene is expressed primarily in placenta and brain, and to a lesser extent
in
specific tumors and cancers (kidney, colorectal, colon, osteoclastoma).
Therefore, polynucleoddes and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, tumors of the kidney, colon, and bone; neurological disorders;
defects of
the vasculature. Similarly, polypeptides and antibodies directed to these
polypeptides
are useful in providing immunological probes for differential identification
of the
dssue(s) or cell type(s). For a number of disorders of the above tissues or
cells,
particularly of the vasculature and CNS, expression of this gene at
significantly higher
or lower levels may be routinely detected in certain tissues or cell types
(e.g.,
reproductive tissue, cells and tissue of the immune system, neural tissue, and
cancerous
and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum,
plasma,
urine, synovial fluid and spinal fluid) or another tissue or cell sample taken
from an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment of neurological
disorders, such
as Alzheimers or schizophrenia. Expression of this gene in the placenta may
indicate a
role in fetal development, or may simply be a hallmark of expression in the
vasculature.
Expression of this gene product in endothelial cells may indicate secretion of
the protein
product into the circulation, where it may have effects on circulating blood
cells, or on
tissues at distant locations. At such sites, it may control cellular
proliferation and/or
differentiation. In addition, expression in a variety of tumors indicates that
this gene
product may play a role in cellular proliferation. Protein, as well as,
antibodies directed
against the protein may show utility as a tumor marker and/or immunotherapy
targets
for the above listed tissues. Many polynucleotide sequences, such as EST
sequences,
are publicly available and accessible through sequence databases. Some of
these
sequences are related to SEQ ID N0:44 and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are
specifically excluded from the scope of the present invention. To list every
related
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42
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1205 of SEQ ID
N0:44, b
is an integer of 15 to 1219, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:44, and where the b is greater than or
equal
to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 35
This gene is expressed primarily in macrophages, treated with GM-CSF.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune deficiencies; susceptibility to bacterial infections;
improper
stimulation of lymphocyte pools. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cells and tissue of the immune system, and cancerous and wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder. Preferred epitopes
include
those comprising a sequence shown in SEQ ID N0:117 as residues: Arg-48 to Asn-
56.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the regulation and/or stimulation of
hematopoietic cells, particularly the circulating lymphocytes. Macrophages are
one of
the front lines of immune defense, particularly against bacterial pathogens,
and are able
to secrete cytokines and proteins that affect other blood cells. Thus, this
gene product
may be important in regulation of the immune system, in activation of
hematopoietic
cells, such as T cells; and may play a role in antigen recognition. Protein,
as well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Many polynucleotide
sequences,
such as EST sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ ID N0:45 and may have
been
publicly available prior to conception of the present invention. Preferably,
such related
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43
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
423 of
SEQ ID N0:45, b is an integer of 15 to 437, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:45, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 36
This gene is expressed primarily in endothelial cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, inflammation and circulatory system disorders. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the circulatory
system, expression
of this gene at significantly higher or lower levels may be routinely detected
in certain
tissues or cell types (e.g., endothelial cells, cancerous and wounded tissues)
or bodily
fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another
tissue or cell sample taken from an individual having such a disorder,
relative to the
standard gene expression level, i.e., the expression level in healthy tissue
or bodily
fluid from an individual not having the disorder.
The tissue distribution indicates that polynucleoddes and polypeptides
corresponding to this gene are useful for diagnosis and treatment of
inflammatory
disorders involving endothelial cells, such as sepsis, inflammatory bowel
diseases,
psoriasis, and rheumatoid arthritis as well as atherosclerosis, which can lead
to strokes
and heart attacks. Protein, as well as, antibodies directed against the
protein may show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ )D
N0:46 and may have been publicly available prior to conception of the present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence is cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
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a-b, where a is any integer between 1 to 519 of SEQ ID N0:46, b is an integer
of 15 to
533, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ m N0:46, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 37
The translation product of this gene was shown to have homology to the human
transducin(beta}-like 1 protein (See Genebank Accession No P38262).
This gene is expressed primarily in fetal lung and to a lesser extent in adult
lung
and breast.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, respiratory system diseases, and/or disorders. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the respiratory
system, expression
of this gene at significantly higher or lower levels may be routinely detected
in certain
tissues or cell types (e.g., pulmonary tissue, and cancerous and wounded
tissues) or
bodily fluids (e.g., pulmonary surfactant, serum, plasma, urine, synovial
fluid and
spinal fluid) or another tissue or cell sample taken from an individual having
such a
disorder, relative to the standard gene expression level, i.e., the expression
level in
healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and disease affecting the
lung such
as lung cancer, emphysema, pulmonary edema, asthma, cystic fibrosis, and
pulmonary
emboli. Alternatively, the protein product of this gene may be useful in the
detection,
treatment, and/or prevention of various inflammatory conditions afflicting
endothelial
tissue such as the vasculature and cardiovascular systems. Protein, as well
as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Many polynucleotide
sequences,
such as EST sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ LD N0:47 and may have
been
publicly available prior to conception of the present invention. Preferably,
such related
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
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described by the general formula of a-b, where a is any integer between 1 to
1835 of
SEQ ID N0:47, b is an integer of 15 to 1849, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:47, and where the b is
greater
than or equal to a + 14.
5
FEATURES OF PROTEIN ENCODED BY GENE NO: 38
This gene is expressed primarily in prostate cancer and to a lesser extent in
osteoblasts.
Therefore, polynucleotides and polypeptides of the invention are useful as
IO reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, prostate cancer, osteoporosis. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
differential identification of the tissues) or cell type(s). For a number of
disorders of
15 the above tissues or cells, particularly of the reproductive system,
expression of this
gene at significantly higher or lower levels may be routinely detected in
certain tissues
or cell types (e.g., skeletal tissue, reproductive tissue, and cancerous and
wounded
tissues) or bodily fluids (e.g., seminal fluid, lymph, serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or cell sample taken from an
individual having
20 such a disorder, relative to the standard gene expression level, i.e., the
expression level
in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution in tumors of prostate origins indicates that
polynucleotides and polypeptides corresponding to this gene are useful for
diagnosis
and intervention of these tumors, in addition to other tissues where
expression has been
25 indicated. Protein, as well as, antibodies directed against the protein may
show utility as
a tissue-specific marker and/or immunotherapy target for the above listed
tissues. Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:48 and
may have been publicly available prior to conception of the present invention.
30 Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 912 of SEQ ID N0:48, b is an integer of 15 to 926,
where
35 both a and b correspond to the positions of nucleotide residues shown in
SEQ ID
N0:48, and where the b is greater than or equal to a + 14.
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46
FEATURES OF PROTEIN ENCODED BY GENE NO: 39
The translation product of this gene was shown to have homology to the mdkk-
1 protein from Mus musculus (See Genebank Accession No. gi12736292 (AF030433))
which has been shown to be important as a possible homeobox gene inducer
specific to
head development. One embodiment of this gene comprises polypeptides of the
following amino acid sequence: GGNKYQT1DNYQPYP C (SEQ ID N0:167),
PLLGVSATLNSVLNSNAIKN C~ (SEQ ID N0:168), and/or GSAVSAAPGILYPG.
An additional embodiment is the polynucleotides encoding these polypeptides
(SEQ )D
N0:169).
This gene is expressed primarily in placenta and to a lesser extent in smooth
muscle.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, developmental disorders of the fetus, and/or reproductive
disorders,
particularly of the female. Similarly, polypeptides and antibodies directed to
these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of developing tissues in the fetus, expression
of this gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., reproductive tissue, and cancerous and wounded tissues) or bodily
fluids
(e.g., amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid)
or another
tissue or cell sample taken from an individual having such a disorder,
relative to the
standard gene expression level, i.e., the expression level in healthy tissue
or bodily
fluid from an individual not having the disorder.
The tissue distribution combined with the homology to a suspected homeobox
domain inducer indicates that polynucleoddes and polypeptides corresponding to
this
gene are useful for diagnosis and treatment of developmental disorders,
particularly
those involving hematopoesis and pattern formation in the embryo. Protein, as
well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Many polynucleotide
sequences,
such as EST sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ 1D N0:49 and may have
been
publicly available prior to conception of the present invention. Preferably,
such related
polynucleotides are specifically excluded from the scope of the present
invention. To
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47
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
1579 of
SEQ ID N0:49, b is an integer of 15 to 1593, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:49, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 40
When tested against sensory neuron cell lines (PC 12), supernatants removed
from cells containing this gene activated the early growth response gene 1
pathway
(EGR1). Thus, it is likely that this gene activates neuronal cells through the
Jaks-STAT
signal transduction pathway. EGR1 (early growth response gene 1) is a separate
signal
transduction pathway from Jaks-STAT, genes containing the EGR1 promoter are
induced in various tissues and cell types upon activation, leading the cells
to undergo
differentiation and proliferation.
This gene is expressed primarily in neutrophils and to a lesser extent in
pancreatic carcinoma.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, inflammation, gastrointestinal, and immune disorders.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
immune system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., cells and tissue of the immune system,
and
cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine,
synovial fluid and spinal fluid) or another tissue or cell sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder. Preferred epitopes include those comprising a sequence shown in SEQ
ID
N0:122 as residues: Pro-8 to Ala-16.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of a
variety of
immune system disorders. Expression of this gene product in tonsils indicates
a role in
the regulation of the proliferation; survival; differentiation; and/or
activation of
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48
potentially all hematopoietic cell lineages, including blood stem cells. This
gene product
may be involved in the regulation of cytokine production, antigen
presentation, or other
processes that may also suggest a usefulness in the treatment of cancer (e.g.
by
boosting immune responses). Since the gene is expressed in cells of lymphoid
origin,
the natural gene product may be involved in immune functions. Therefore it may
be also
used as an agent for immunological disorders including arthritis, asthma,
immune
deficiency diseases such as AIDS, and leukemia. Protein, as well as,
antibodies
directed against the protein may show utility as a tumor marker and/or
immunotherapy
targets for the above listed tumors and tissues. In addition, this gene
product may have
commercial utility in the expansion of stem cells and committed progenitors of
various
blood lineages, and in the differentiation and/or proliferation of various
cell types.
Protein, as well as, antibodies directed against the protein may show utility
as a tumor
marker and/or immunotherapy targets for the above listed tissues. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID NO:50 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 964 of SEQ ID NO:50, b is an integer of 15 to 978, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID NO:50, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 41
This gene is expressed primarily in breast lymph node and to a lesser extent
in
anergic T-cell.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, inflammation, and immunodeficiency disorders. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the immune system,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues or cell types (e.g., cells and tissue of the immune system, and
cancerous and
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49
wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial
fluid
and spinal fluid) or another tissue or cell sample taken from an individual
having such
a disorder, relative to the standard gene expression level, i.e., the
expression level in
healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of a
variety of
immune system disorders. Expression of this gene product in T-cells indicates
a role in
the regulation of the proliferation; survival; differentiation; and/or
activation of
potentially all hematopoietic cell lineages, including blood stem cells. This
gene product
may be involved in the regulation of cytokine production, antigen
presentation, or other
processes that may also suggest a usefulness in the treatment of cancer (e.g.
by
boosting immune responses). Since the gene is expressed in cells of lymphoid
origin,
the natural gene product may be involved in immune functions. Therefore it may
be also
used as an agent for immunological disorders including arthritis, asthma,
immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory
bowel
disease, sepsis, acne, and psoriasis. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the above
listed tumors and tissues. In addition, this gene product may have commercial
utility in
the expansion of stem cells and committed progenitors of various blood
lineages, and in
the differentiation andlor proliferation of various cell types. Protein, as
well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Many polynucleotide
sequences,
such as EST sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ ID NO:51 and may have
been
publicly available prior to conception of the present invention. Preferably,
such related
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
419 of
SEQ ID NO:51, b is an integer of 15 to 433, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID NO:51, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 42
This gene is expressed primarily in synovium.
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Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, inflammation and diseases of the joints. Similarly,
polypeptides and
5 antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the immune system and
connective tissue,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., skeletal tissue, and cancerous and
wounded
10 tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and
spinal fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, i.e., the expression level in healthy
tissue or
bodily fluid from an individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
15 corresponding to this gene are useful for diagnosis and treatment of
inflammatory
disorders, particularly those involving the joints and skeletal system, such
as
rheumatoid arthritis and in particular the connective tissues (e.g. trauma,
tendonitis,
chrondomalacia). Protein, as well as, antibodies directed against the protein
may show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues. .
20 Many polynucleotide sequences, such as EST sequences, are publicly
available and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:52 and may have been publicly available prior to conception of the present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence is cumbersome.
25 Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 847 of SEQ ID N0:52, b is an integer
of 15 to
861, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ ID N0:52, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 43
This gene maps to chromosome 5, and therefore, may be used as a marker in
linkage analysis for chromosome 5.
This gene is expressed primarily in synovium.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
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51
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, diseases of the joints and connective tissues. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the immune system, expression
of this gene
at significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., skeletal tissue, and cancerous and wounded tissues) or bodily
fluids (e.g.
serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or
cell sample
taken from an individual having such a disorder, relative to the standard gene
expression level; i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of
inflammatory joint
disorders as well as suggest a role in the detection and treatment of
disorders and
conditions affecting the skeletal system, in particular the connective tissues
(e.g.
arthritis, trauma, tendonitis, chrondomalacia and inflammation). Protein, as
well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Many polynucleotide
sequences,
such as EST sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ )D N0:53 and may have
been
publicly available prior to conception of the present invention. Preferably,
such related
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence
described by the general formula of a-b, where a is any integer between 1 to
496 of
SEQ ID N0:53, b is an integer of 15 to 510, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:53, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 44
This gene is expressed primarily in activated T cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, inflammation and other immune system disorders. Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
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52
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
immune system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., cells and tissue of the immune system,
and
cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine,
synovial fluid and spinal fluid) or another tissue or cell sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder. Preferred epitopes include those comprising a sequence shown in SEQ
ID
N0:126 as residues: Met-i to Lys-7.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of a
variety of
immune system disorders. Expression of this gene product in T-cells indicates
a role in
the regulation of the proliferation; survival; differentiation; and/or
activation of
potentially all hematopoietic cell lineages, including blood stem cells. This
gene product
may be involved in the regulation of cytokine production, antigen
presentation, or other
processes that may also suggest a usefulness in the treatment of cancer (e.g.
by
boosting immune responses). Since the gene is expressed in cells of lymphoid
origin,
the natural gene product may be involved in immune functions. Therefore it may
be also
used as an agent for immunological disorders including arthritis, asthma,
immune
deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory
bowel
disease, sepsis, acne, and psoriasis. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the above
listed tumors and tissues. In addition, this gene product may have commercial
utility in
the expansion of stem cells and committed progenitors of various blood
lineages, and in
the differentiation and/or proliferation of various cell types. Protein, as
well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues. Many polynucleotide
sequences,
such as EST sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ ID N0:54 and may have
been
publicly available prior to conception of the present invention. Preferably,
such related
polynucleotides are specifically excluded from the scope of the present
invention. To
list every related sequence is cumbersome. Accordingly, preferably excluded
from the
present invention are one or more polynucleotides comprising a nucleotide
sequence.
described by the general formula of a-b, where a is any integer between 1 to
295 of
SEQ ID N0:54, b is an integer of 15 to 309, where both a and b correspond to
the
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53
positions of nucleotide residues shown in SEQ ID N0:54, and where the b is
greater
than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 45
This gene is expressed primarily in placenta, liver, lung, endometrial stromal
cell and embryo.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions such as
cancers,
immunodeficiency and autoimmune diseases, as well as reproductive and hepatic
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are
useful in providing immunological probes for differential identification of
the tissues)
or cell type(s). For a number of disorders of the above tissues or cells,
particularly of
the immune system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues or cell types (e.g., reproductive
tissue, and
cancerous and wounded tissues) or bodily fluids (e.g., amniotic fluid, bile,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or cell
sample taken
from an individual having such a disorder, relative to the standard gene
expression
level, i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection and treatment of liver
disorders
and cancers (e.g. hepatoblastoma, jaundice, hepatitis, liver metabolic
diseases and
conditions that are attributable to the differentiation of hepatocyte
progenitor cells). In
addition the expression in fetus would suggest a useful role for the protein
product in
developmental abnormalities, fetal deficiencies, pre-natal disorders and
various would-
healing models and/or tissue trauma. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the above
listed tissues. Many polynucleotide sequences, such as EST sequences, are
publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:55 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 1571 of SEQ ID N0:55, b is an integer
of 15
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54
to 1585, where both a and b correspond to the positions of nucleotide residues
shown
in SEQ ID NO:55, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 46
This gene is expressed primarily in placenta, amniotic cells and adrenal gland
tumor.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions such as
cancers, as well
as various reproductive and endocrine disorders. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
differential identification of the tissues) or cell type(s). For a number of
disorders of
the above tissues or cells, particularly of the immune system, expression of
this gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., endocrine tissue, reproductive tissue, and cancerous and wounded
tissues)
or bodily fluids (e.g., amniotic fluid, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder. Preferred epitopes
include
those comprising a sequence shown in SEQ )D N0:128 as residues: Cys-52 to Val-
61.
The tissue distribution within placenta and amniotic tissues indicates that
polynucleotides and polypeptides corresponding to this gene useful for the
treatment
and diagnosis of reproductive disorders, particularly those of the female
reproductive
system, including cancer and tissues characteristic of the developing embryo.
In
addition, the tissue distribution within the adrenal gland indicates that the
protein
product of this gene is useful for the detection, treatment, and/or prevention
of various
endocrine disorders and cancers, particularly Addisonis disease, Cushingis
Syndrome,
and disorders and/or cancers of the pancrease (e.g. diabetes mellitus),
adrenal cortex,
ovaries, pituitary (e.g., hyper-, hypopituitarism), thyroid (e.g. hyper-,
hypothyroidism), parathyroid (e.g. hyper-,hypoparathyroidism) , hypothallamus,
and
testes. Protein, as well as, antibodies directed against the protein may show
utility as a
tumor marker and/or immunotherapy targets for the above listed tissues. Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:56 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
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present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 860 of SEQ ID N0:56, b is an integer of 15 to 874,
where
5 both a and b correspond to the positions of nucleotide residues shown in SEQ
ID
N0:56, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 47
This gene is expressed primarily in melanocyte, melanoma, dendritic cells and
10 fetal brain.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, skin disorders, particularly melanoma, as well as
neurodegenerative
15 disorders and cancer. Similarly, polypeptides and antibodies directed to
these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
20 types (e.g., neural tissue, integumentary tissue, and cancerous and wounded
tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or
another
tissue or cell sample taken from an individual having such a disorder,
relative to the
standard gene expression level, i.e., the expression level in healthy tissue
or bodily
fluid from an individual not having the disorder. Preferred epitopes include
those
25 comprising a sequence shown in SEQ ID N0:129 as residues: Lys-76 to Gly-81.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment, diagnosis, and/or
prevention of
various skin disorders including congenital disorders (i.e. nevi, moles,
freckles,
Mongolian spots, hemangiomas, port-wine syndrome), integumentary tumors (i.e.
30 keratoses, Bowenis disease, basal cell carcinoma, squamous cell carcinoma,
malignant
melanoma, Pagetis disease, mycosis fungoides, and Kaposiis sarcoma), injuries
and
inflammation of the skin (i.e., wounds, rashes, prickly heat disorder,
psoriasis,
dermatitis), atherosclerosis, uticaria, eczema, photosensitivity, autoimmune
disorders
(i.e. lupus erythematosus, vitiligo, dermatomyositis, morphea, scleroderma,
35 pemphigoid, and pemphigus), keloids, striae, erythema, petechiae, purpura,
and
xanthelasma. Moreover, such disorders may predispose increased susceptibility
to viral
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56
and bacterial infections of the skin (i.e. cold sores, warts, chickenpox,
molluscum
contagiosum, herpes zoster, boils, cellulitis, erysipelas, impetigo, tinea,
althletes foot,
and ringworm). Alternatively, the tissue distribution within dendritic cells
and fetal
brain indicates that the protein product of this gene is useful for the
detectionltreatment
of neurodegenerative disease states and behavioral disorders such as
Alzheimers
Disease, Parkinsons Disease, Huntingtons Disease, Tourette Syndrome,
schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic disorder,
learning
disabilities, ALS, psychoses , autism, and altered behaviors, including
disorders in
feeding, sleep patterns, balance, and perception. In addition, the gene or
gene product
may also play a role in the treatment and/or detection of developmental
disorders
associated with the developing embryo, sexually-linked disorders, or disorders
of the
cardiovascular system. Protein, as well as, antibodies directed against the
protein may
show utility as a tumor marker and/or immunotherapy targets for the above
listed
tissues. Many polynucleotide sequences, such as EST sequences, are publicly
available
and accessible through sequence databases. Some of these sequences are related
to SEQ
ID N0:57 and may have been publicly available prior to conception of the
present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence is cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 1155 of SEQ ID N0:57, b is an integer
of 15
to 1169, where both a and b correspond to the positions of nucleotide residues
shown
in SEQ ID N0:57, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 48
This gene maps to chromosome 1, and therefore, may be used as a marker in
linkage analysis for chromosome 1.
This gene is expressed primarily in ovary tumors and to a lesser extent in
breast,
placenta, pineal gland, infant brain, T cell and B cell lymphoma.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, ovary cancer, reproductive disorders, particularly of the
female
reproductive system, as well as disorders of the immune system, including
lymphoma
and immunodeficiencies. Similarly, polypeptides and antibodies directed to
these
polypeptides are useful in providing immunological probes for differential
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57
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system and female reproductive
system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., reproductive, endocrine tissue, cells
and tissue of
the immune system, and cancerous and wounded tissues) or bodily fluids (e.g.,
amniotic fluid, lymph, breast milk, serum, plasma, urine, synovial fluid and
spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder. Preferred epitopes
include
those comprising a sequence shown in SEQ ID N0:130 as residues: Asn-33 to Lys-
38.
The tissue distribution in tumors of the ovary and lymph nodes, as well as, in
breast and placental tissues indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and intervention of such
tumors, in
addition to other tumors where expression has been indicated. Protein, as well
as,
antibodies directed against the protein may show utility as a tissue-specific
marker
and/or immunotherapy target for the above listed tissues. Alternatively, The
tissue
distribution indicates that polynucleotides and polypeptides corresponding to
this gene
are useful for the diagnosis and treatment of a variety of immune system
disorders.
Expression of this gene product in T-cells indicates a role in the regulation
of the
proliferation; survival; differentiation; and/or activation of potentially all
hematopoietic
cell lineages, including blood stem cells. This gene product may be involved
in the
regulation of cytokine production, antigen presentation, or other processes
that may
also suggest a usefulness in the treatment of cancer (e.g. by boosting immune
responses). Since the gene is expressed in cells of lymphoid origin, the
natural gene
product may be involved in immune functions. Therefore it may be also used as
an
agent for immunological disorders including arthritis, asthma, immune
deficiency
diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel
disease,
sepsis, acne, and psoriasis. Protein, as well as, antibodies directed against
the protein
may show utility as a tumor marker and/or immunotherapy targets for the above
listed
tumors and tissues. In addition, this gene product may have commercial utility
in the
expansion of stem cells and committed progenitors of various blood lineages,
and in the
differentiation and/or proliferation of various cell types. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:58 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
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58
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 1052 of SEQ ID N0:58, b is an integer of 15 to 1066, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:58, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 49
This gene is expressed primarily in breast tissue.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the dssue(s) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, breast cancer. Similarly, polypeptides and antibodies directed
to these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., breast tissue and cancerous and wounded tissues) or bodily fluids
(e.g.,
breast milk, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or
cell sample taken from an individual having such a disorder, relative to the
standard
gene expression level, i.e., the expression level in healthy tissue or bodily
fluid from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:131 as residues: Ala-40 to Trp-45.
The tissue distribution in breast tissue indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis and
intervention of
tumors within this tissue. Alternatively, the expression in breast may suggest
that the
protein product of this gene is useful for the diagnosis, treatment, and/or
prevention of
various reproductive system disorders, particularly of the female reproductive
system.
Protein, as well as, antibodies directed against the protein may show utility
as a tissue-
specific marker and/or immunotherapy target for the above listed tissues. Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:59 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
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59
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 758 of SEQ ID N0:59, b is an integer of 15 to 772,
where
both a and b correspond to the positions of nucleotide residues shown in SEQ m
N0:59, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 50
This gene is expressed primarily in fetal liver, retina and to a lesser extent
in
thyroid and pineal gland.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissue{s) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, hepatic and endocrine disorders. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
differential identification of the tissues) or cell type(s). For a number of
disorders of
the above tissues or cells, particularly of the hematopoesis and immune
system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., liver, endocrine tissue, and cancerous
and
wounded tissues) or bodily fluids (e.g., bile, serum, plasma, urine, synovial
fluid and
spinal fluid) or another tissue or cell sample taken from an individual having
such a
disorder, relative to the standard gene expression level, i.e., the expression
level in
healthy tissue or bodily fluid from an individual not having the disorder.
Preferred
epitopes include those comprising a sequence shown in SEQ ID N0:132 as
residues:
Lys-9 to Trp-14.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection and treatment of liver
disorders
and cancers (e.g. hepatoblastoma, jaundice, hepatitis, liver metabolic
diseases and
conditions that are attributable to the differentiation of hepatocyte
progenitor cells). In
addition the expression in fetus would suggest a useful role for the protein
product in
developmental abnormalities, fetal deficiencies, pre-natal disorders and
various would-
healing models and/or tissue trauma. Alternatively, expression in pineal and
thyroid
glands indicates that the protein product of this gene is useful for the
detection,
treatment, and/or prevention of various endocrine disorders and cancers,
particularly
Addisonis disease, Cushingis Syndrome, and disorders and/or cancers of the
pancrease
(e.g. diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-,
hypopituitarism), thyroid (e.g. hyper-, hypothyroidism), parathyroid (e.g.
hyper-
,hypoparathyroidism) , hypothallamus, and testes. Protein, as well as,
antibodies
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directed against the protein may show utility as a tumor marker and/or
immunotherapy
targets for the above listed tissues. Many polynucleotide sequences, such as
EST
sequences, are publicly available and accessible through sequence databases.
Some of
these sequences are related to SEQ ID N0:60 and may have been publicly
available
5 prior to conception of the present invention. Preferably, such related
polynucleotides
are specifically excluded from the scope of the present invention. To list
every related
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1184 of SEQ ID
N0:60, b
10 is an integer of 15 to 1198, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID N0:60, and where the b is greater than or
equal
to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 51
15 The translation product of this gene shares sequence homology with seizure-
related gene product 6 type 2 precursor which is a novel, brain-specific, gene
thought to
be important in the predisposition of seizures (See Genebank Accession
No.gnIlPIDId 1006729). One embodiment of this gene comprises polypeptides of
the
following amino acid sequence: AGIQHELACDNPGLPENGYQILYKRLYLPGESLT
20 FMCYEGFELMGEVTIRCILGQPSHWNGPLPVCKVAE A.AAETSLEGGN @ (SEQ
ID N0:170) QPSHWNGPLPVCKVAEAAAETSLEGGN @ (SEQ ID N0:171), and/or
YETGETREYEVSI (SEQ ID N0:172). An additional embodiment is the
polynucleotides encoding these polypeptides.
This gene is expressed primarily in brain.
25 Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, seizures and other neural and CNS disorders. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
30 probes for differential identification of the tissues) or cell type(s). For
a number of
disorders of the above tissues or cells, particularly of the brain, expression
of this gene
at significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., neural tissue, and cancerous and wounded tissues) or bodily
fluids (e.g.,
serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or
cell sample
35 taken from an individual having such a disorder, relative to the standard
gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
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individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:133 as residues: Cys-26 to Leu-32, Thr-49 to Ile-
55,
Glu-57 to Glu-63.
The tissue distribution in brain combined with its homology to a putative
seizure
gene indicates that polynucleotides and polypeptides corresponding to this
gene are
useful for the detection/treatment of seizures and epilepsy, including
neurodegenerative
disease states and behavioral disorders such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, schizophrenia, mania,
dementia,
paranoia, obsessive compulsive disorder, panic disorder, learning
disabilities, ALS,
psychoses , autism, and altered behaviors, including disorders in feeding,
sleep
patterns, balance, and perception. In addition, the gene or gene product may
also play a
role in the treatment and/or detection of developmental disorders associated
with the
developing embryo, sexually-linked disorders, or disorders of the
cardiovascular
system. Protein, as well as, antibodies directed against the protein may show
utility as a
1 S tumor marker and/or immunotherapy targets for the above listed tissues.
Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:61 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 544 of SEQ ID N0:61, b is an integer of 15 to 558,
where
both a and b correspond to the positions of nucleotide residues shown in SEQ
ID
N0:61, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 52
When tested against PC12 cell lines, supernatants removed from cells
containing this gene activated the EGR1 (early growth response 1) pathway.
Thus, it is
likely that this gene activates sensory neuron cells through the EGR1 signal
transduction pathway. EGRI is a separate signal transduction pathway from Jaks-
STAT, genes containing the EGR1 promoter are induced in various tissues and
cell
types upon activation, leading the cells to undergo differentiation and
proliferation.
This gene is expressed primarily in fetal brain.
Therefore, polynucleotides and polypepddes of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
*rB
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62
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, neurological disorders, particularly of the developing embryo.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the fetal
brain and
CNS, expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues , serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, i.e., the expression level in healthy
tissue or
bodily fluid from an individual not having the disorder. Preferred epitopes
include those
comprising a sequence shown in SEQ ID N0:134 as residues: Arg-16 to Thr-35.
The tissue distribution in fetal brain combined with the detected biological
activity within sensory neurons indicates that polynucleotides and
polypeptides
corresponding to this gene are useful for the detection/treatment of
neurodegenerative
disease states and behavioral disorders such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, schizophrenia, mania,
dementia,
paranoia, obsessive compulsive disorder, panic disorder, learning
disabilities, ALS,
psychoses , autism, and altered behaviors, including disorders in feeding,
sleep
patterns, balance, and perception. In addition, the gene or gene product may
also play a
role in the treatment and/or detection of developmental disorders associated
with the
developing embryo, sexually-linked disorders, or disorders of the
cardiovascular
system. Protein, as well as, antibodies directed against the protein may show
utility as a
tumor marker and/or immunotherapy targets for the above listed tissues. Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:62 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 602 of SEQ ID N0:62, b is an integer of 15 to 616,
where
both a and b correspond to the positions of nucleotide residues shown in SEQ
ll~
N0:62, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 53
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This gene is expressed primarily in frontal cortex, and schizophrenoid brain
tissue.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, Schizophrenia, and other neurodegenerative disorders,
including cancer.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
brain, expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., neural tissue, and cancerous
and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and
spinal fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
to the standard gene expression level, i.e., the expression level in healthy
tissue or
bodily fluid from an individual not having the disorder. Preferred epitopes
include
those comprising a sequence shown in SEQ ID N0:135 as residues: Asp-65 to Asn-
72.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
neurodegenerative
disease states and behavioral disorders such as Alzheimers Disease, Parkinsons
Disease, Huntingtons Disease, Tourette Syndrome, schizophrenia, mania,
dementia,
paranoia, obsessive compulsive disorder, panic disorder, learning
disabilities, ALS,
psychoses , autism, and altered behaviors, including disorders in feeding,
sleep
patterns, balance, and perception. In addition, the gene or gene product may
also play a
role in the treatment and/or detection of developmental disorders associated
with the
developing embryo, sexually-linked disorders, or disorders of the
cardiovascular
system. Protein, as well as, antibodies directed against the protein may show
utility as a
tumor marker and/or immunotherapy targets for the above listed tissues. Many
polynucleotide sequences, such as EST sequences, are publicly available and
accessible
through sequence databases. Some of these sequences are related to SEQ ID
N0:63 and
may have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence is cumbersome. Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is
any integer between 1 to 797 of SEQ ID N0:63, b is an integer of 15 to 811,
where
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both a and b correspond to the positions of nucleotide residues shown in SEQ
ID
N0:63, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 54
This gene is expressed primarily in osteoclastoma, gall bladder, and infant
brain.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, Osteoclastoma, and gastrointestinal, skeletal, and neural
disorders,
particularly cancer. Similarly, polypeptides and antibodies directed to these
poiypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the bone, expression of this gene at
significantly higher
or lower levels may be routinely detected in certain tissues or cell types
(e.g., skeletal
tissue, neural tissue, and cancerous and wounded tissues) or bodily fluids
(e.g., bile,
serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or
cell sample
taken from an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:136 as residues: Gly-23 to His-30.
The tissue distribution in osteoclastoma cells indicates that polynucleotides
and
polypeptides corresponding to this gene are useful for the detection,
treatment, and/or
prevention of various skeletal system disorders, particularly bone cancer.
Moreover, the
tissue distribution within the gall bladder indicates that the protein product
of this gene
is useful for the detection and treatment of liver disorders and cancers (e.g.
hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions
that are
attributable to the differentiation of hepatocyte progenitor cells). In
addition the
expression in fetus would suggest a useful role for the protein product in
developmental
abnormalities, fetal deficiencies, pre-natal disorders and various would-
healing models
and/or tissue trauma. Osteoclastoma, Gall Bladder tumor. Many polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:64 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
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excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 979 of SEQ ID N0:64, b is an integer of 15 to 993, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:64, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 55
The translation product of this gene shares sequence homology with b cell
growth factor which is thought to be important in B cell development.
10 This gene is expressed primarily in breast lymph node and primary dendritic
cells
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
15 not limited to, immune disorders. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system expression of this gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
20 types (e.g., cells and tissue of the immune system, and cancerous and
wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or cell sample taken from an individual having such a
disorder,
relative to the standard gene expression level, i.e., the expression level in
healthy tissue
or bodily fluid from an individual not having the disorder. Preferred epitopes
include
25 those comprising a sequence shown in SEQ ID N0:137 as residues: Ser-39 to
Tyr-46.
The tissue distribution combined with its homology to a B-cell growth factor
indicates that polynucleotides and polypeptides corresponding to this gene are
useful for
the diagnosis and treatment of a variety of immune system disorders.
Expression of this
gene product in lymph nodes indicates a role in the regulation of the
proliferation;
30 survival; differentiation; and/or activation of potentially all
hematopoietic cell lineages,
including blood stem cells. This gene product may be involved in the
regulation of
cytokine production, antigen presentation, or other processes that may also
suggest a
usefulness in the treatment of cancer (e.g. by boosting immune responses).
Since the
gene is expressed in cells of lymphoid origin, the natural gene product may be
involved
35 in immune functions. Therefore it may be also used as an agent for
immunological
disorders including arthritis, asthma, immune deficiency diseases such as
AIDS,
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leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and
psoriasis. Protein, as well as, antibodies directed against the protein may
show utility as
a tumor marker and/or immunotherapy targets for the above listed tumors and
tissues.
In addition, this gene product may have commercial utility in the expansion of
stem
cells and committed progenitors of various blood lineages, and in the
differentiation
and/or proliferation of various cell types. Protein, as well as, antibodies
directed against
the protein may show utility as a tumor marker and/or immunotherapy targets
for the
above listed tissues. Many poiynucieotide sequences, such as EST sequences,
are
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:65 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 675 of SEQ ID N0:65, b is
an
integer of 15 to 689, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:65, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 56
The translation product of this gene shares sequence homology with small
hepatocellular oncoprotein which is thought to be important in liver
development (See
Genebank Accession No. R07057).This gene maps to chromosome 20, and therefore,
may be used as a marker in linkage analysis for chromosome 20.
This gene is expressed primarily in monocytes.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases arid conditions which include,
but are
not linuted to, liver disorders, particularly hepatoma. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the immune and digestive
systems,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types (e.g., haematopoetic cells and tissue, liver,
and cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or cell sample taken from an
individual having
such a disorder, relative to the standard gene expression level, i.e., the
expression level
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in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred
epitopes include those comprising a sequence shown in SEQ ID N0:138 as
residues:
Met-17 to Lys-32.
The homology to a hepatocellular oncogene indicates that polynucleotides and
polypeptides corresponding to this gene are useful for the detection and
treatment of
liver disorders and cancers (e.g. hepatoblastoma, jaundice, hepatitis, liver
metabolic
diseases and conditions that are attributable to the differentiation of
hepatocyte
progenitor cells). The expression also indicates a useful role for the protein
product in
developmental abnormalities, fetal deficiencies, pre-natal disorders and
various would-
healing models and/or tissue trauma. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the above
listed tissues. Alternatively, The tissue distribution indicates that
polynucleotides and
polypeptides corresponding to this gene are . useful for the treatment and
diagnosis of
hematopoetic related disorders such as anemia, pancytopenia, leukopenia,
thrombocytopenia or leukemia since stromal cells are important in the
production of
cells of hematopoietic lineages. The uses include bone marrow cell ex vivo
culture,
bone marrow transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of neoplasia. The gene product may also be involved in
lymphopoiesis,
therefore, it can be used in immune disorders such as infection, inflammation,
allergy,
immunodeficiency etc. In addition, this gene product may have commercial
utility in the
expansion of stem cells and committed progenitors of various blood lineages,
and in the
differentiation and/or proliferation of various cell types. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:66 and
may
have been publicly available prior to conception of the present invention.
Preferably,
such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 928 of SEQ ID N0:66, b is an integer of 15 to 942, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ m N0:66, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 57
The translation product of this gene was shown to have homology to the human
proteins myotubularin related protein 3 and NTII-1 nerve protein (See Genebank
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68
Accession Nos. gi11378120 and 899800, respectively) which are both thought to
play
important roles as growth factors in muscle and nerve tissue,
respectively.Preferred
polypeptides comprise the following amino acid sequence:
DDDGLPFPTDVIQHRLRQIEAGYKQEVEQLRR
QVRDSDEXGHPSLLCPSSRAPMDYEDDFTCLKESDGSDTEDFGSDHSEDCLSEA
SWEPVDKKETEVTRWVPDHMASHCYNCDCEFWLAKRRHHCRNCGNVFCAG
CCHLKLPIPDQQLYDPVLVCNSCYXTHSSLSCQGTHEPTAEETHCYSFQLNAGE
KPVQF (SEQ ID N0:173), SEASWEPVDKKETEVTRWVPDHMASHCY (SEQ ID
N0:174), HHCRNCGNVF (SEQ ID N0:175, and/or RLRQIEAGYKQEVE (SEQ ID
N0:176). Also preferred are the polynucleotides encoding these polypeptides.
This gene is expressed primarily in bone, spleen, brain, apoptotic T cells,
hypothalmus, and other immune cells.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune system, musculoskeletal, and neural disorders.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s). For
a number of disorders of the above tissues or cells, particularly of the
central nervous
and immune systems, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues or cell types (e.g., cells and
tissue of the
immune system, and cancerous and wounded tissues) or bodily fluids (e.g.,
lymph,
serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or
cell sample
taken from an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:139 as residues: Glu-63 to Asp-68.
The tissue distribution in neural tissue combined with the homology to a nerve
growth factor indicates that polynucleotides and polypeptides corresponding to
this
gene are useful for the detection/treatment of neurodegenerative disease
states and
behavioral disorders such as Alzheimers Disease, Parkinsons Disease,
Huntingtons
Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia,
obsessive
compulsive disorder, panic disorder, learning disabilities, ALS, psychoses ,
autism,
and altered behaviors, including disorders in feeding, sleep patterns,
balance, and
perception, as well as neuromuscular disorders such as MS and muscular
dystrophy. In
addition, the gene or gene product may also play a role in the treatment
and/or detection
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69
of developmental disorders associated with the developing embryo, sexually-
linked
disorders, or disorders of the cardiovascular system. Protein, as well as,
antibodies
directed against the protein may show utility as a tumor marker and/or
immunotherapy
targets for the above listed tissues. Many polynucleotide sequences, such as
EST
sequences, are publicly available and accessible through sequence databases.
Some of
these sequences are related to SEQ 117 N0:67 and may have been publicly
available
prior to conception of the present invention. Preferably, such related
polynucleotides
are specifically excluded from the scope of the present invention. To list
every related
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
IO are one or more polynucleotides comprising a nucleotide sequence described
by the
general formula of a-b, where a is any integer between 1 to 2295 of SEQ ID
N0:6?, b
is an integer of IS to 2309, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:67, and where the b is greater than or
equal
to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 58
This gene is expressed primarily in ovary.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, ovarian cancer, and other disorders that afflict the female
reproductive
system. Similarly, polypeptides and antibodies directed to these polypeptides
are useful
in providing immunological probes for differential identification of the
tissues) or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
female reproductive system, expression of this gene at significantly higher or
lower
levels may be routinely detected in certain tissues or cell types (e.g.,
reproductive
tissue, and cancerous and wounded tissues) or bodily fluids (e.g., amniotic
fluid,
serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or
cell sample
taken from an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:140 as residues: Pro-23 to Gly-54.
The tissue distribution in tumors the ovary indicates that polynucleotides and
polypeptides corresponding to this gene are useful for diagnosis and
intervention of
these tumors, in addition to other tumors where expression has been indicated.
Protein,
as well as, antibodies directed against the protein may show utility as a
tissue-specific
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marker and/or immunotherapy target for the above listed tissues. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
sequence databases. Some of these sequences are related to SEQ ID N0:68 and
may
have been publicly available prior to conception of the present invention.
Preferably,
5 such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 800 of SEQ ID N0:68, b is an integer of 15 to 814, where both a
and b
10 correspond to the positions of nucleotide residues shown in SEQ ID N0:68,
and where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 59
This gene is expressed primarily in ovary.
15 Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, ovarian cancer, and other disorders afflicting the female
reproductive
system. Similarly, polypeptides and antibodies directed to these polypeptides
are useful
20 in providing immunological probes for differential identification of the
tissues) or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
reproductive system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues or cell types (e.g., reproductive
tissue, and
cancerous and wounded tissues) or bodily fluids (e.g., amniotic fluid, serum,
plasma,
25 urine, synovial fluid and spinal fluid) or another tissue or cell sample
taken from an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder.
The tissue distribution in ovarian tissue indicates that polynucleotides and
30 polypeptides corresponding to this gene are useful for diagnosis and
intervention of
ovarian tumors, in addition to other tumors of the female reproductive system.
Protein,
as well as, antibodies directed against the protein may show utility as a
tissue-specific
marker and/or immunotherapy target for the above listed tissues. Many
polynucleotide
sequences, such as EST sequences, are publicly available and accessible
through
35 sequence databases. Some of these sequences are related to SEQ ID N0:69 and
may
have been publicly available prior to conception of the present invention.
Preferably,
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such related polynucleotides are specifically excluded from the scope of the
present
invention. To list every related sequence is cumbersome. Accordingly,
preferably
excluded from the present invention are one or more polynucleotides comprising
a
nucleotide sequence described by the general formula of a-b, where a is any
integer
between 1 to 774 of SEQ ID N0:69, b is an integer of 15 to 788, where both a
and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:69, and
where
the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 60
When tested against PC12 cell lines, supernatants removed from cells
containing this gene activated the EGR1 (early growth response 1) pathway.
Thus, it is
likely that this gene activates sensory neuronal cells through the EGR1 signal
transduction pathway. . EGR1 is a separate signal transduction pathway from
Jaks-
STAT, genes containing the EGR1 promoter are induced in various tissues and
cell
types upon activation, leading the cells to undergo differentiation and
proliferation.
This gene is expressed primarily in osteoblast.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, skeletal disorders, particular bone cancer. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the Bone, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., skeletal tissue, and cancerous and wounded tissues) or bodily
fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or cell
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:142 as residues: Tyr-20 to Lys-31.
In addition the expression of this gene product in synovium would suggest a
role in the detection and treatment of disorders and conditions affecting the
skeletal
system, in particular osteoporosis as well as disorders afflicting connective
tissues (e.g.
arthritis, trauma, tendonitis, chrondomalacia and inflammation) as well as in
the
diagnosis or treatment of various autoimmune disorders such as rheumatoid
arthritis,
lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal
deformation,
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andspecific joint abnormalities as well as chondrodysplasias i.e.
spondyloepiphyseal
dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II,
metaphyseal
chondrodysplasia type Schmid. Protein, as well as, antibodies directed against
the
protein may show utility as a tumor marker and/or immunotherapy targets for
the above
listed tissues. Many polynucleotide sequences, such as EST sequences, are
publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:70 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 777 of SEQ ID N0:70, b is an integer
of 15 to
791, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ ID N0:70, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 61
This gene is expressed primarily in adipocyte.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, obesity. Similarly, polypeptides and antibodies directed to
these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type{s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., adipose tissue, and cancerous and wounded tissues) or bodily
fluids (e.g.,
serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or
cell sample
taken from an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred epitopes include those
comprising a
sequence shown in SEQ ID N0:143 as residues: His-2 to Leu-8.
The tissue distribution would suggest that polynucleotides and polypeptides
corresponding to this gene are useful in the diagnosis, treatment, and/or
prevention of
obesity and lipid metabolism disorders. Protein, as well as, antibodies
directed against
the protein may show utility as a tumor marker and/or immunotherapy targets
for the
above listed tissues. Many polynucleotide sequences, such as EST sequences,
are
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73
publicly available and accessible through sequence databases. Some of these
sequences
are related to SEQ ID N0:71 and may have been publicly available prior to
conception
of the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one or
more polynucleotides comprising a nucleotide sequence described by the general
formula of a-b, where a is any integer between 1 to 790 of SEQ ID N0:71, b is
an
integer of 15 to 804, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:71, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 62
When tested against Jurkat T-cell lines, supernatants removed from cells
containing this gene activated the GAS (gamma activation site) pathway. Thus,
it is
likely that this gene activates T-cells through the Jak-STAT signal
transduction
pathway. GAS is a promoter element found upstream in many genes which are
involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a large, signal
transduction pathway involved in the differentiation and proliferation of
cells.
This gene is expressed primarily in synovial tissue.
Therefore, polynucleotides and polypepddes of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, chronic synovitis, immune. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
differential identification of the tissues) or cell type(s). For a number of
disorders of
the above tissues or cells, particularly of the immune system expression of
this gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cells and tissue of the immune system, and skeletal tissue, and
cancerous
and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or cell sample taken from an
individual having
such a disorder, relative to the standard gene expression level, i.e., the
expression level
in healthy tissue or bodily fluid from an individual not having the disorder.
Preferred
epitopes include those comprising a sequence shown in SEQ B7 N0:144 as
residues:
Pro-74 to Lys-82.
The biological assay results indicating activity in Jurkat T-cells for the
protein
product of this gene indicates that polynucleotides and polypeptides
corresponding to
this gene useful for the diagnosis and treatment of a variety of immune system
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74
disorders. This gene product may be involved in the regulation of cytokine
production,
antigen presentation, or other processes that may also suggest a usefulness in
the
treatment of cancer (e.g. by boosting immune responses). Since the gene has
been
shown to activate genes in cells of lymphoid origin, the natural gene product
may be
involved in immune functions. Therefore it may be also used as an agent for
imlnunological disorders including arthritis, asthma, immune deficiency
diseases such
as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis,
acne, and
psoriasis. Protein, as well as, antibodies directed against the protein may
show utility as
a tumor marker and/or immunotherapy targets for the above listed tumors and
tissues.
In addition, this gene product may have commercial utility in the expansion of
stem
cells and committed progenitors of various blood lineages, and in the
differentiation
and/or proliferation of various cell types. In addition the expression of this
gene product
in synovial tissues indicates a role for this protein in the detection and
treatment of
disorders and conditions affecting the skeletal system, in particular the
connective
tissues (e.g. arthritis, trauma, tendonitis, chrondomalacia and inflammation)
as well as
in the diagnosis or treatment of various autoimmune disorders such as
rheumatoid
arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal
deformation, and specific joint abnormalities as well as chondrodysplasias
i.e.
spondyloepiphyseal dysplasia congenita, familial osteoarthritis,
Atelosteogenesis type
II, metaphyseal chondrodysplasia type Schmid. Protein, as well as, antibodies
directed
against the protein may show utility as a tumor marker and/or immunotherapy
targets
for the above listed tissues. . Many polynucleotide sequences, such as EST
sequences,
are publicly available and accessible through sequence databases. Some of
these
sequences are related to SEQ ID N0:72 and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are
specifically excluded from the scope of the present invention. To list every
related
sequence is cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 769 of SEQ ID
N0:72, b is
an integer of 15 to 783, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:72, and where the b is greater than or equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 63
The translation product of this gene shares sequence homology with fetal
troponin which is known to be essential for normal muscular function in all
skeletal and
cardiovascular muscles.
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This gene is expressed primarily in melanocytes, fetal liver, brain,testes,
spleen,
and placenta.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
5 biological sample and for diagnosis of diseases and conditions which
include, but are
not limited to, melanoma, neuromuscular disorders, such as multiple sclerosis,
and
endothelial-related diseases. Similarly, polypeptides and antibodies directed
to these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
10 tissues or cells, particularly of the immune system, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., muscle, endothelial cells and tissue, and cancerous and wounded
tissues)
or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal
fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative
15 to the standard gene expression level, i.e., the expression level in
healthy tissue or
bodily fluid from an individual not having the disorder. Preferred epitopes
include those
comprising a sequence shown in SEQ ID N0:145 as residues: Ala-27 to Leu-36,
Phe-
40 to Leu-50.
The tissue distribution and homology to troponin indicates that
polynucleotides
20 and polypeptides corresponding to this gene are useful for treatment,
diagnosis, and/or
prevention of various neuromuscular disorders such as MS, muscular dystrophy,
cardiomyopathy, myositis, myomas, leiomyomas, rhabdomyosarcomas, and coronary
heart disease. Alternatively, the expression in a variety of fetal immune
tissues indicates
that the protein product of this gene is useful for the diagnosis and
treatment of a
25 variety of immune system disorders. Expression of this gene product in
spleen and fetal
liver indicates a role in the regulation of the proliferation; survival;
differentiation;
and/or activation of potentially all hematopoietic cell lineages, including
blood stem
cells. This gene product may be involved in the regulation of cytokine
production,
antigen presentation, or other processes that may also suggest a usefulness in
the
30 treatment of cancer (e.g. by boosting immune responses). Since the gene is
expressed
in cells of lymphoid origin, the natural gene product may be involved in
immune
functions. Therefore it may be also used as an agent for immunological
disorders
including arthritis, asthma, immune deficiency diseases such as AIDS,
leukemia,
rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis.
Protein,
35 as well as, antibodies directed against the protein may show utility as a
tumor marker
and/or immunotherapy targets for the above listed tumors and tissues. In
addition, this
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76
gene product may have commercial utility in the expansion of stem cells and
committed
progenitors of various blood lineages, and in the differentiation and/or
proliferation of
various cell types. Protein, as well as, antibodies directed against the
protein may show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:73 and may have been publicly available prior to conception of the present
invention. Preferably, such related polynucleotides are specifically excluded
from the
scope of the present invention. To list every related sequence is cumbersome.
Accordingly, preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of
a-b, where a is any integer between 1 to 1509 of SEQ ID N0:73, b is an integer
of 15
to 1523, where both a and b correspond to the positions of nucleotide residues
shown
in SEQ ID N0:73, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 64
The translation product of this gene shares sequence homology with small
hepatocellular oncoprotein gene which is thought to be important in liver
disorders (See
Genebank Accession No. R07057). One embodiment of this gene comprises
polypeptides of the following amino acid sequence:
MSHCARPLFFETFFILLSPRLKCSGTNTVHYSLDLLGSSNSASVPQVGGLTNAQ
HDTWLIFVFCVCVCEPLRRPWAAFLISVTSSIK (SEQ ID N0:177), and/or
VPQVGGLTNAQHDTWLIFVFCVCVCEPLRR (SEQ ID N0:178}. An additional
embodiment is the polynucleotides encoding these polypeptides.
This gene is expressed primarily in neutrophils, hemangiopericytoma, activated
T cells.
Therefore, polynucleoddes and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune and hepatic disorders. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
differential identification of the tissues) or cell type(s). For a number of
disorders of
the above tissues or cells, particularly of the hepatoma and immune system
disorder,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues or cell types {e.g., cells and tissue of the immune system,
and
cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma,
urine,
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77
synovial fluid and spinal fluid) or another tissue or cell sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder. Preferred epitopes include those comprising a sequence shown in SEQ
ID
N0:146 as residues: Arg-20 to Gly-25.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of a
variety of
immune system disorders. Expression of this gene product in neutrophils and T-
cells
indicates a role in the regulation of the proliferation; survival;
differentiation; andlor
activation of potentially all hematopoietic cell lineages, including blood
stem cells. This
gene product may be involved in the regulation of cytokine production, antigen
presentation, or other processes that may also suggest a usefulness in the
treatment of
cancer (e.g. by boosting immune responses). Since the gene is expressed in
cells of
lymphoid origin, the natural gene product may be involved in immune functions.
Therefore it may be also used as an agent for immunological disorders
including
arthritis, asthma, immune deficiency diseases such as AIDS, leukemia,
rheumatoid
arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. Protein,
as well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tumors and tissues. In addition,
this gene
product may have commercial utility in the expansion of stem cells and
committed
progenitors of various blood lineages, and in the differentiation and/or
proliferation of
various cell types. Alternatively, the homology to a hepatic oncogene
indicates that the
protein product of this gene is useful for the detection and treatment of
liver disorders
and cancers (e.g. hepatoblastoma, jaundice, hepatitis, liver metabolic
diseases and
conditions that are attributable to the differentiation of hepatocyte
progenitor cells). In
addition, the expression would suggest a useful role for the protein product
in
developmental abnormalities, fetal deficiencies, pre-natal disorders and
various would-
healing models and/or tissue trauma. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the above
listed tissues. Many polynucleotide sequences, such as EST sequences, are
publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:74 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically excluded
from the scope of the present invention. To list every related sequence is
cumbersome.
Accordingly, preferably excluded from the present invention are one or more
poiynucleotides comprising a.nucleodde sequence described by the general
formula of
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78
a-b, where a is any integer between 1 to 744 of SEQ ID N0:74, b is an integer
of 15 to
758, where both a and b correspond to the positions of nucleotide residues
shown in
SEQ ID N0:74, and where the b is greater than or equal to a + 14.
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79
M V7 N I~ M d' V7 ~
d ~+. ~ O O O TWO M vD Ov Ov M Owt oo ~n
O V ~O N M d' N N M ~-~ N d' '~Y N M N
O~~~N~NNM~N~V~'NMN
N M ~ V1
~ ~ z ~ oM0 0~0 0~0 0~0 0~0 0~0 0~0 ~ Ov Ov pv Ov Ov
w
2 ~ w ~ ~ ~O .-~r ~ .-~.r ~ ~ ~ ~ ~ ~ M N O
...r ...r .-r M .r ..-~ .-r
~ ~ b p ~ ~ ~ ~ ~ ~ ~ ~ ~ M N O
..~r .-.r ~ .-.~ M ~ .., r.,
U
C p~ 00 V7 00 M N N N M tt ~ ~ N d'
V1 ~~ M ~ ~ ~ d' ~~ l~ M V~ V1
M U ~ tn l~ 00 Y7 l~ ~O t~ ~ 00 'Ch ~O C'~ d'
Cn U ~
' 00 ~_ 00 M_ N_ N N ~ ~t ef t~ N N
j t~ t~ OMO ~ t~ ~ ~ ~ 0~0 ~ ~ I~~. due'
z o' o ~ N M ~ V7 ~G l'~ 00 O~ O ~ N M
.-, .-r .--i .--~ .-.~ .-~ ~ ~ N N N N
d ~ d ~ ~ ~ ~ ~ d
~NNNN~,~~N ~''NNNW
'c '~ '~ '~ '~ P~1 0. ~c ~'' '~ 'c 'c
c~ t~ t~ r. ~ c~ ~ ~ t~ r. r
00 ~ oo ~ 00 00 ~ oo ~ oo ~ oo ~ oo ~ 00 00 ~ a4 ~ oo ~ oo ~
U ~ M ~ M w. M ~ M w M .~ M w M ~ M ~ M ~ M ~ M ~ M w" M w"
U O 'r~" ~ M ~ M ~ M ~ M ~-~ M ~ M ~~~ M ~ M ~ M ~ M ~-i M ~ M ~ M
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x x x x x x x x x x x x x
N M d' v~ ~D l~ 00 C~ ~ ~ ~ ,-M,
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
O O M ~ ef'M ~ N N ~ M ~ -r
N vp
b C
w ~ Ov Ov ~ Ov N ~O 00 N O ~ ~O O~ M
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CA 02296815 2000-O1-14
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81
~ M N N N M f M
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CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
82
O ~ d' N ~G -~ 00 ~C M M d' N ~D 00 N
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CA 02296815 2000-O1-14
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O ~ M ~ ~ '~d~~ ~ ~ M O00N N
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OO N ~ ~ ~ ~' ~O N
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c~
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Table 1 summarizes the information corresponding to each "Gene No." described
above. The nucleotide sequence identified as "NT SEQ ID NO:X" was assembled
from
partially homologous ("overlapping") sequences obtained from the "cDNA clone
1D"
identified in Table 1 and, in some cases, from additional related DNA clones.
The
5 overlapping sequences were assembled into a single contiguous sequence of
high
redundancy (usually three to five overlapping sequences at each nucleotide
position),
resulting in a final sequence identified as SEQ ID NO:X.
The cDNA Clone ID was deposited on the date and given the corresponding
deposit number listed in "ATCC Deposit No:Z and Date." Some of the deposits
contain
10 multiple different clones corresponding to the same gene. "Vector" refers
to the type of
vector contained in the cDNA Clone ID.
"Total NT Seq." refers to the total number of nucleotides in the contig
identified
by "Gene No." The deposited clone may contain all or most of these sequences,
reflected by the nucleotide position indicated as "5' NT of Clone Seq." and
the "3' NT
15 of Clone Seq." of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of
the
putative start codon (methionine) is identified as "5' NT of Start Codon."
Similarly ,
the nucleotide position of SEQ ID NO:X of the predicted signal sequence is
identified as
"5' NT of First AA of Signal Pep."
The translated amino acid sequence, beginning with the methionine, is
identified
20 as "AA SEQ ID NO:Y," although other reading frames can also be easily
translated
using known molecular biology techniques. The polypeptides produced by these
alternative open reading frames are specifically contemplated by the present
invention.
The first and last amino acid position of SEQ ID NO:Y of the predicted signal
peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep." The
predicted
25 first amino acid position of SEQ ID NO:Y of the secreted portion is
identified as
"Predicted First AA of Secreted Portion." Finally, the amino acid position of
SEQ 1D
NO:Y of the last amino acid in the open reading frame is identified as "Last
AA of
ORF."
SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and
30 otherwise suitable for a variety of uses well known in the art and
described further
below. For instance, SEQ ID NO:X is useful for designing nucleic acid
hybridization
probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the
cDNA
contained in the deposited clone. These probes will also hybridize to nucleic
acid
molecules in biological samples, thereby enabling a variety of forensic and
diagnostic
35 methods of the invention. Similarly, polypeptides identified from SEQ ID
NO:Y may
be used to generate antibodies which bind specifically to the secreted
proteins encoded
by the cDNA clones identified in Table 1.
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Nevertheless, DNA sequences generated by sequencing reactions can contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or
deletions of nucleotides in the generated DNA sequence. The erroneously
inserted or
deleted nucleotides cause frame shifts in the reading frames of the predicted
amino acid
sequence. In these cases, the predicted amino acid sequence diverges from the
actual
amino acid sequence, even though the generated DNA sequence may be greater
than
99.9% identical to the actual DNA sequence (for example, one base insertion or
deletion
in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide
sequence or the amino acid sequence, the present invention provides not only
the
generated nucleotide sequence identified as SEQ ID NO:X and the predicted
translated
amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid
DNA
containing a human cDNA of the invention deposited with the ATCC, as set forth
in
Table 1. The nucleotide sequence of each deposited clone can readily be
determined by
sequencing the deposited clone in accordance with known methods. The predicted
amino acid sequence can then be verified from such deposits. Moreover, the
amino
acid sequence of the protein encoded by a particular clone can also be
directly
determined by peptide sequencing or by expressing the protein in a suitable
host cell
containing the deposited human cDNA, collecting the protein, and determining
its
sequence.
The present invention also relates to the genes corresponding to SEQ ID NO:X,
SEQ ID NO:Y, or the deposited clone. The corresponding gene can be isolated in
accordance with known methods using the sequence information disclosed herein.
Such methods include preparing probes or primers from the disclosed sequence
and
identifying or amplifying the corresponding gene from appropriate sources of
genomic
material.
Also provided in the present invention are species homologs. Species
homologs may be isolated and identified by making suitable probes or primers
from the
sequences provided herein and screening a suitable nucleic acid source for the
desired
homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood in the art.
The polypeptides may be in the form of the secreted protein, including the
mature form, or may be a part of a larger protein, such as a fusion protein
(see below).
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It is often advantageous to include an additional amino acid sequence which
contains
secretory or leader sequences, pro-sequences, sequences which aid in
purification ,
such as multiple histidine residues, or an additional sequence for stability
during
recombinant production.
The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purified. A recombinantly produced
version of a
polypeptide, including the secreted polypeptide, can be substantially purified
by the
one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
Polypeptides of the invention also can be purified from natural or recombinant
sources
using antibodies of the invention raised against the secreted protein in
methods which
are well known in the art.
Signal Seauences
Methods for predicting whether a protein has a signal sequence, as well as the
cleavage point for that sequence, are available. For instance, the method of
McGeoch,
Virus Res. 3:271-286 (1985), uses the information from a short N-terminal
charged
region and a subsequent uncharged region of the complete (uncleaved) protein.
The
method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the
information
from the residues surrounding the cleavage site, typically residues -13 to +2,
where +1
indicates the amino terminus of the secreted protein. The accuracy of
predicting the
cleavage points of known mammalian secretory proteins for each of these
methods is in
the range of 75-80%. (von Heinje, supra.) However, the two methods do not
always
produce the same predicted cleavage points) for a given protein.
In the present case, the deduced amino acid sequence of the secreted
polypeptide
was analyzed by a computer program called SignalP (Henrik Nielsen et al.,
Protein
Engineering 10:1-6 (1997)), which predicts the cellular location of a protein
based on
the amino acid sequence. As part of this computational prediction of
localization, the
methods of McGeoch and von Heinje are incorporated. The analysis of the amino
acid
sequences of the secreted proteins described herein by this program provided
the results
shown in Table 1.
As one of ordinary skill would appreciate, however, cleavage sites sometimes
vary from organism to organism and cannot be predicted with absolute
certainty.
Accordingly, the present invention provides secreted polypeptides having a
sequence
shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues
(i.e., +
or - 5 residues) of the predicted cleavage point. Similarly, it is also
recognized that in
some cases, cleavage of the signal sequence from a secreted protein is not
entirely
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88
uniform, resulting in more than one secreted species. These polypeptides, and
the
polynucleotides encoding such polypeptides, are contemplated by the present
invention.
Moreover, the signal sequence identified by the above analysis may not
necessarily predict the naturally occurring signal sequence. For example, the
naturally
occurring signal sequence may be further upstream from the predicted signal
sequence.
However, it is likely that the predicted signal sequence will be capable of
directing the
secreted protein to the ER. These polypeptides, and the polynucleotides
encoding such
polypeptides, are contemplated by the present invention.
Polvnucleotide and Poly~entide Variants
"Variant" refers to a polynucleotide or polypeptide differing from the
polynucleotide or polypeptide of the present invention, but retaining
essential properties
thereof. Generally, variants are overall closely similar, and, in many
regions, identical
to the polynucleotide or polypeptide of the present invention.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended that
the nucleotide sequence of the polynucieotide is identical to the reference
sequence
except that the polynucleotide sequence may include up to five point mutations
per each
100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
In other
words, to obtain a polynucleotide having a nucleotide sequence at least 95%
identical to
a reference nucleotide sequence, up to 5% of the nucleotides in the reference
sequence
may be deleted or substituted with another nucleotide, or a number of
nucleotides up to
5% of the total nucleotides in the reference sequence may be inserted into the
reference
sequence. The query sequence may be an entire sequence shown inTable 1, the
ORF
(open reading frame), or any fragement specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleotide
sequence of the presence invention can be determined conventionally using
known
computer programs. A preferred method for determing the best overall match
between
a query sequence (a sequence of the present invention) and a subject sequence,
also
referred to as a global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.
( 1990)
6:237-245). In a sequence alignment the query and subject sequences are both
DNA
sequences. An RNA sequence can be compared by converting U's to T's. The
result
of said global sequence alignment is in percent identity. Preferred parameters
used in a
FASTDB alignment of DNA sequences to calculate percent identiy are:
Matrix=Unitary, k-tuple=4., Mismatch Penalty=1, Joining Penalty=30,
Randomization
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Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window
Size=500 or the lenght of the subject nucleotide sequence, whichever is
shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3'
deletions, not because of internal deletions, a manual correction must be made
to the
results. This is becuase the FASTDB program does not account for 5' and 3'
truncations of the subject sequence when calculating percent identity. For
subject
sequences truncated at the 5' or 3' ends, relative to the the query sequence,
the percent
identity is corrected by calculating the number of bases of the query sequence
that are 5'
and 3' of the subject sequence, which are not matched/aligned, as a percent of
the total
bases of the query sequence. Whether a nucleotide is matched/aligned is
determined by
results of the FASTDB sequence alignment. This percentage is then subtracted
from
the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arnve at a final percent identity score. This corrected score
is what is
used for the purposes of the present invention. Only bases outside the 5' and
3' bases
of the subject sequence, as displayed by the FASTDB alignment, which are not
matched/aligned with the query sequence, are calculated for the purposes of
manually
adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the 5' end of
the subject
sequence and therefore, the FASTDB alignment does not show a
matched/alignement of
the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the
sequence
(number of bases at the 5' and 3' ends not matched/total number of bases in
the query
sequence) so 10% is subtracted from the percent identity score calculated by
the
FASTDB program. If the remaining 90 bases were perfectly matched the final
percent
identity would be 90%. In another example, a 90 base subject sequence is
compared
with a 100 base query sequence. This time the deletions are internal deletions
so that
there are no bases on the 5' or 3' of the subject sequence which are not
matched/aligned
with the query. In this case the percent identity calculated by FASTDB is not
manually
corrected. Once again, only bases 5' and 3' of the subject sequence which are
not
matched/aligned with the query sequnce are manually corrected for. No other
manual
corrections are to made for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that
the amino acid sequence of the subject polypeptide is identical to the query
sequence
except that the subject polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the query amino acid sequence. In
other words,
to obtain a polypeptide having an amino acid sequence at least 95% identical
to a query
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amino acid sequence, up to 5% of the amino acid residues in the subject
sequence may
be inserted, deleted, (indels) or substituted with another amino acid. These
alterations
of the reference sequence may occur at the amino or carboxy terminal positions
of the
reference amino acid sequence or anywhere between those terminal positions,
5 interspersed either individually among residues in the reference sequence or
in one or
more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
95%,
96%, 97%, 98% or 99% identical to, for instance, the amino acid sequences
shown in
Table 1 or to the amino acid sequence encoded by deposited DNA clone can be
10 determined conventionally using known computer programs. A preferred method
for
determing the best overall match between a query sequence (a sequence of the
present
invention) and a subject sequence, also referred to as a global sequence
alignment, can
be determined using the FASTDB computer program based on the algorithm of
Brutlag
et al. (Comp. App. Biosci. ( 1990) 6:237-245). In a sequence alignment the
query and
15 subject sequences are either both nucleotide sequences or both amino acid
sequences.
The result of said global sequence alignment is in percent identity. Preferred
parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
20 Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
If the subject sequence is shorter than the query sequence due to N- or C-
terminal deletions, not because of internal deletions, a manual correction
must be made
to the results. This is becuase the FASTDB program does not account for N- and
C-
terminal truncations of the subject sequence when calculating global percent
identity.
25 For subject sequences truncated at the N- and C-termini, relative to the
the query
sequence, the percent identity is corrected by calculating the number of
residues of the
query sequence that are N- and C-terminal of the subject sequence, which are
not
matched/aligned with a corresponding subject residue, as a percent of the
total bases of
the query sequence. Whether a residue is matched/aligned is determined by
results of
30 the FASTDB sequence alignment. This percentage is then subtracted from the
percent
identity, calculated by the above FASTDB program using the specified
parameters, to
arnve at a final percent identity score. This final percent identity score is
what is used
for the purposes of the present invention. Only residues to the N- and C-
termini of the
subject sequence, which are not matched/aligned with the query sequence, are
35 considered for the purposes of manually adjusting the percent identity
score. That is,
only query residue positions outside the farthest N- and C-terminal residues
of the
subject sequence.
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91
For example, a 90 amino acid residue subject sequence is aligned with a 100
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subject sequence and therefore, the FASTDB alignment does not
show
a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired
residues represent 10% of the sequence (number of residues at the N- and C-
termini
not matched/total number of residues in the query sequence) so 10% is
subtracted from
the percent identity score calculated by the FASTDB program. If the remaining
90
residues were perfectly matched the final percent identity would be 90%. In
another
example, a 90 residue subject sequence is compared with a 100 residue query
sequence.
This time the deletions are internal deletions so there are no residues at the
N- or C-
termini of the subject sequence which are not matched/aligned with the query.
In this
case the percent identity calculated by FASTDB is not manually corrected. Once
again,
only residue positions outside the N- and C-terminal ends of the subject
sequence, as
displayed in the FASTDB alignment, which are not matched/aligned with the
query
sequnce are manually corrected for. No other manual corrections are to made
for the
purposes of the present invention.
The variants may contain alterations in the coding regions, non-coding
regions,
or both. Especially preferred are polynucleotide variants containing
alterations which
produce silent substitutions, additions, or deletions, but do not alter the
properties or
activities of the encoded polypeptide. Nucleotide variants produced by silent
substitutions due to the degeneracy of the genetic code are preferred.
Moreover,
variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or
added in any
combination are also preferred. Polynucleotide variants can be produced for a
variety
of reasons, e.g., to optimize codon expression for a particular host (change
codons in
the human mRNA to those preferred by a bacterial host such as E. coli).
Naturally occurnng variants are called "allelic variants," and refer to one of
several alternate forms of a gene occupying a given locus on a chromosome of
an
organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).)
These
allelic variants can vary at either the polynucleotide and/or polypeptide
level.
Alternatively, non-naturally occurnng variants may be produced by mutagenesis
techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics
of the
polypeptides of the present invention. For instance, one or more amino acids
can be
deleted from the N-terminus or C-terminus of the secreted protein without
substantial
loss of biological function. The authors of Ron et al., J. Biol. Chem. 268:
2984-2988
( 1993), reported variant KGF proteins having heparin binding activity even
after
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deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon
gamma
exhibited up to ten times higher activity after deleting 8-10 amino acid
residues from the
carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (
1988).)
Moreover, ample evidence demonstrates that variants often retain a biological
activity sinular to that of the naturally occurring protein. For example,
Gayle and
coworkers (J. Biol. Chem 268:22105-22111 ( 1993)) conducted extensive
mutational
analysis of human cytokine IL,-la. They used random mutagenesis to generate
over
3,500 individual IL,-la mutants that averaged 2.5 amino acid changes per
variant over
the entire length of the molecule. Multiple mutations were examined at every
possible
amino acid position. The investigators found that "[m]ost of the molecule
could be
altered with little effect on either [binding or biological activity)." (See,
Abstract.) In
fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide
sequences examined, produced a protein that significantly differed in activity
from wild-
type.
Furthermore, even if deleting one or more amino acids from the N-terminus or
C-terminus of a polypeptide results in modification or loss of one or more
biological
functions, other biological activities may still be retained. For example, the
ability of a
deletion variant to induce and/or to bind antibodies which recognize the
secreted form
will likely be retained when less than the majority of the residues of the
secreted form
are removed from the N-terminus or C-terminus. Whether a particular
polypeptide
lacking N- or C-terminal residues of a protein retains such immunogenic
activities can
readily be determined by routine methods described herein and otherwise known
in the
art.
Thus, the invention further includes polypeptide variants which show
substantial biological activity. Such variants include deletions, insertions,
inversions,
repeats, and substitutions selected according to general rules known in the
art so as
have little effect on activity. For example, guidance concerning how to make
phenotypically silent amino acid substitutions is provided in Bowie, J. U. et
al.,
Science 247:1306-1310 (1990), wherein the authors indicate that there are two
main
strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by
natural
selection during the process of evolution. By comparing amino acid sequences
in
different species, conserved amino acids can be identified. These conserved
amino
acids are likely important for protein function. In contrast, the amino acid
positions
where substitutions have been tolerated by natural selection indicates that
these
positions are not critical for protein function. Thus, positions tolerating
amino acid
substitution could be modified while still maintaining biological activity of
the protein.
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The second strategy uses genetic engineering to introduce amino acid changes
at
specific positions of a cloned gene to identify regions critical for protein
function. For
example, site directed mutagenesis or alanine-scanning mutagenesis
(introduction of
single alanine mutations at every residue in the molecule) can be used.
(Cunningham
and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can
then
be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which
amino acid changes are likely to be permissive at certain amino acid positions
in the
protein. For example, most buried (within the tertiary structure of the
protein) amino
acid residues require nonpolar side chains, whereas few features of surface
side chains
are generally conserved. Moreover, tolerated conservative amino acid
substitutions
involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu
and Ile;
replacement of the hydroxyl residues Ser and Thr; replacement of the acidic
residues
Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the
basic
residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and
Trp,
and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Besides conservative amino acid substitution, variants of the present
invention
include (i) substitutions with one or more of the non-conserved amino acid
residues,
where the substituted amino acid residues may or may not be one encoded by the
genetic code, or (ii) substitution with one or more of amino acid residues
having a
substituent group, or (iii) fusion of the mature polypeptide with another
compound,
such as a compound to increase the stability and/or solubility of the
polypeptide (for
example, polyethylene glycol), or (iv) fusion of the polypeptide with
additional amino
acids, such as an IgG Fc fusion region peptide, or leader or secretory
sequence, or a
sequence facilitating purification. Such variant polypeptides are deemed to be
within
the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of
charged amino acids with other charged or neutral amino acids may produce
proteins
with improved characteristics, such as less aggregation. Aggregation of
pharmaceutical
formulations both reduces activity and increases clearance due to the
aggregate's
immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);
Robbins et al., Diabetes 36: 838-845 ( 1987); Cleland et al., Crit. Rev.
Therapeutic
Drug Carrier Systems 10:307-377 (1993).)
Polynucleotide and Poly~eotide Fragments
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In the present invention, a "polynucleotide fragment" refers to a short
polynucleodde having a nucleic acid sequence contained in the deposited clone
or
shown in SEQ ID NO:X. The short nucleotide fragments are preferably at least
about
15 nt, and more preferably at least about 20 nt, still more preferably at
least about 30 nt,
and even more preferably, at least about 40 nt in length. A fragment "at least
20 nt in
length," for example, is intended to include 20 or more contiguous bases from
the
cDNA sequence contained in the deposited clone or the nucleotide sequence
shown in
SEQ 117 NO:X. These nucleotide fragments are useful as diagnostic probes and
primers
as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,
2000
nucleotides) are preferred.
Moreover, representative examples of polynucleotide fragments of the
invention, include, for example, fragments having a sequence from about
nucleotide
number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-
450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900,
901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,
1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-160(?,
1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,
1951-2000, or 2001 to the end of SEQ ID NO:X or the cDNA contained in the
deposited clone. In this context "about" includes the particularly recited
ranges, larger
or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at
both termini.
Preferably, these fragments encode a polypeptide which has biological
activity. More
preferably, these polynucleotides can be used as probes or primers as
discussed herein.
In the present invention, a "polypeptide fragment" refers to a short amino
acid
sequence contained in SEQ ID NO:Y or encoded by the cDNA contained in the
deposited clone. Protein fragments may be "free-standing," or comprised within
a
larger polypeptide of which the fragment forms a part or region, most
preferably as a
single continuous region. Representative examples of polygeptide fragments of
the
invention, include, for example, fragments from about amino acid number 1-20,
21-40,
41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the
coding
region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70,
80, 90,
100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about"
includes the particularly recited ranges, larger or smaller by several (5, 4,
3, 2, or 1)
amino acids, at either extreme or at both extremes.
Preferred polypeptide fragments include the secreted protein as well as the
mature form. Further preferred polypeptide fragments include the secreted
protein or
the mature form having a continuous series of deleted residues from the amino
or the
carboxy terminus, or both. For example, any number of amino acids, ranging
from 1-
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60, can be deleted from the amino terminus of either the secreted polypeptide
or the
mature form. Similarly, any number of amino acids, ranging from 1-30, can be
deleted
from the carboxy terminus of the secreted protein or mature form. Furthermore,
any
combination of the above amino and carboxy terminus deletions are preferred.
5 Similarly, polynucleotide fragments encoding these polypeptide fragments are
also
preferred.
Particularly, N-terminal deletions of the polypeptide of the present invention
can
be described by the general formula m-p, where p is the total number of amino
acids in
the polypeptide and m is an integer from 2 to (p-1), and where both of these
integers (m
10 & p) correspond to the position of the amino acid residue identified in SEQ
ID NO:Y.
Moreover, C-terminal deletions of the polypeptide of the present invention can
also be described by the general formula 1-n, where n is an integer from 2 to
(p-1), and
again where these integers (n & p) correspond to the position of the amino
acid residue
identified in SEQ ID NO:Y.
15 The invention also provides polypeptides having one or more amino acids
deleted from both the amino and the carboxyl termini, which may be described
generally as having residues m-n of SEQ ID NO:Y, where m and n are integers as
described above.
Also preferred are polypeptide and polynucleotide fragments characterized by
20 structural or functional domains, such as fragments that comprise alpha-
helix and alpha-
helix forming regions, beta-sheet and beta-sheet-forming regions, turn and
turn-
forming regions, coil and coil-forming regions, hydrophilic regions,
hydrophobic
regions, alpha amphipathic regions, beta amphipathic regions, flexible
regions, surface-
forming regions, substrate binding region, and high antigenic index regions.
25 Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are
specifically contemplated by the present invention. Moreover, poiynucleotide
fragments encoding these domains are also contemplated.
Other preferred fragments are biologically active fragments. Biologically
active
fragments are those exhibiting activity similar, but not necessarily
identical, to an
30 activity of the polypeptide of the present invention. The biological
activity of the
fragments may include an improved desired activity, or a decreased undesirable
activity.
Epitopes & Antibodies
In the present invention, "epitopes" refer to polypeptide fragments having
35 antigenic or immunogenic activity in an animal, especially in a human. A
preferred
embodiment of the present invention relates to a polypeptide fragment
comprising an
epitope, as well as the polynucleotide encoding this fragment. A region of a
protein
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molecule to which an antibody can bind is defined as an "antigenic epitope."
In
contrast, an "immunogenic epitope" is defined as a part of a protein that
elicits an
antibody response. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci.
USA
81:3998- 4002 ( 1983).)
Fragments which function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985) further described in U.S. Patent No. 4,631,211.)
In the present invention, antigenic epitopes preferably contain a sequence of
at
least seven, more preferably at least nine, and most preferably between about
15 to
about 30 amino acids. Antigenic epitopes are useful to raise antibodies,
including
monoclonal antibodies, that specifically bind the epitope. (See, for instance,
Wilson et
al., Cell 37:767-778 (1984); Sutcliffe, J. G. et al., Science 219:660-666
(1983).)
Similarly, immunogenic epitopes can be used to induce antibodies according to
methods well known in the art. (See, for instance, Sutcliffe et al., supra;
Wilson et al.,
1 S supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle,
F. J. et
al., J. Gen. Virol. 66:2347-2354 (1985).) A preferred immunogenic epitope
includes
the secreted protein. The immunogenic epitopes may be presented together with
a
Garner protein, such as an albumin, to an animal system (such as rabbit or
mouse) or, if
it is long enough (at least about 25 amino acids), without a carrier. However,
immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown
to be
sufficient to raise antibodies capable of binding to, at the very least,
linear epitopes in a
denatured polypeptide (e.g., in Western blotting.)
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is
meant to include intact molecules as well as antibody fragments (such as, for
example,
Fab and F(ab')2 fragments) which are capable of specifically binding to
protein. Fab
and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more
rapidly from
the circulation, and may have less non-specific tissue binding than an intact
antibody.
(Wahl et al., J. Nucl. Med. 24:316-325 (1983).) Thus, these fragments are
preferred,
as well as the products of a FAB or other immunoglobulin expression library.
Moreover, antibodies of the present invention include chimeric, single chain,
and
humanized antibodies.
Fusion Proteins
Any polypeptide of the present invention can be used to generate fusion
proteins. For example, the polypeptide of the present invention, when fused to
a
second protein, can be used as an antigenic tag. Antibodies raised against the
polypeptide of the present invention can be used to indirectly detect the
second protein
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by binding to the polypeptide. Moreover, because secreted proteins target
cellular
locations based on trafficking signals, the polypeptides of the present
invention can be
used as targeting molecules once fused to other proteins.
Examples of domains that can be fused to polypeptides of the present invention
include not only heterologous signal sequences, but also other heterologous
functional
regions. The fusion does not necessarily need to be direct, but may occur
through
linker sequences.
Moreover, fusion proteins may also be engineered to improve characteristics of
the polypeptide of the present invention. For instance, a region of additional
amino
acids, particularly charged amino acids, may be added to the N-terminus of the
polypeptide to improve stability and persistence during purification from the
host cell or
subsequent handling and storage. Also, peptide moieties may be added to the
polypeptide to facilitate purification. Such regions may be removed prior to
final
preparation of the polypeptide. The addition of peptide moieties to facilitate
handling of
polypeptides are familiar and routine techniques in the art.
Moreover, polypeptides of the present invention, including fragments, and
specifically epitopes, can be combined with parts of the constant domain of
immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion
proteins
facilitate purification and show an increased half life in vivo. One reported
example
describes chimeric proteins consisting of the first two domains of the human
CD4-
polypeptide and various domains of the constant regions of the heavy or light
chains of
mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86
(1988).) Fusion proteins having disulfide-linked dimeric structures (due to
the IgG)
can also be more efficient in binding and neutralizing other molecules, than
the
monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J.
Biochem. 270:3958-3964 (1995).)
Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion
proteins comprising various portions of constant region of immunoglobulin
molecules
together with another human protein or part thereof. In many cases, the Fc
part in a
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
for
example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and purified,
would be desired. For example, the Fc portion may hinder therapy and diagnosis
if the
fusion protein is used as an antigen for immunizations. In drug discovery, for
example, human proteins, such as hIL-5, have been fused with Fc portions for
the
purpose of high-throughput screening assays to identify antagonists of hII,-5.
(See, D.
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Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al.,
J. Biol.
Chem. 270:9459-9471 (1995).)
Moreover, the polypeptides of the present invention can be fused to marker
sequences, such as a peptide which facilitates purification of the fused
polypeptide. In
preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide,
such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, CA, 91311), among others, many of which are commercially
available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989),
for
instance, hexa-histidine provides for convenient purification of the fusion
protein.
Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope
derived from the influenza hemagglutinin protein. {Wilson et al., Cell 37:767
(1984).)
Thus, any of these above fusions can be engineered using the polynucleotides
or the polypeptides of the present invention.
Vectors. Host Cells, and Protein Production
The present invention also relates to vectors containing the polynucleotide of
the
present invention, host cells, and the production of polypeptides by
recombinant
techniques. The vector may be, for example, a phage, plasmid, viral, or
retroviral
vector. Retroviral vectors may be replication competent or replication
defective. In the
latter case, viral propagation generally will occur only in complementing host
cells.
The polynucleotides may be joined to a vector containing a selectable marker
for
propagation in a host. Generally, a plasmid vector is introduced in a
precipitate, such
as a calcium phosphate precipitate, or in a complex with a charged lipid. If
the vector is
a virus, it may be packaged in vitro using an appropriate packaging cell line
and then
transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and
tac
promoters, the SV40 early and late promoters and promoters of retroviral LTRs,
to
name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription initiation,
termination,
and, in the transcribed region, a ribosome binding site for translation. The
coding
portion of the transcripts expressed by the constructs will preferably include
a
translation initiating codon at the beginning and a termination codon (UAA,
UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase, 6418 or
neomycin
resistance for eukaryotic cell culture and tetracycline, kanamycin or
ampicillin resistance
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genes for culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast
cells; insect
cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
CHO, COS,
293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums
and
conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9,
available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA,
pNHl6a, pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech,
Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
from Pharmacia. Other suitable vectors will be readily apparent to the skilled
artisan.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAF-dextran mediated transfection, cationic lipid-
mediated
transfection, electroporation, transduction, infection, or other methods. Such
methods
are described in many standard laboratory manuals, such as Davis et al., Basic
Methods
In Molecular Biology ( 1986). It is specifically contemplated that the
polypeptides of the
present invention may in fact be expressed by a host cell lacking a
recombinant vector.
A polypeptide of this invention can be recovered and purified from recombinant
cell cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is employed for
purification.
Polypeptides of the present invention, and preferably the secreted form, can
also
be recovered from: products purified from natural sources, including bodily
fluids,
tissues and cells, whether directly isolated or cultured; products of chemical
synthetic
procedures; and products produced by recombinant techniques from a prokaryotic
or
eukaryotic host, including, for example, bacterial, yeast, higher plant,
insect, and
mammalian cells. Depending upon the host employed in a recombinant production
procedure, the polypeptides of the present invention may be glycosylated or
may be
non-glycosylated. In addition, polypeptides of the invention may also include
an initial
modified methionine residue, in some cases as a result of host-mediated
processes.
Thus, it is well known in the art that the N-terminal methionine encoded by
the
translation initiation codon generally is removed with high efficiency from
any protein
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after translation in all eukaryotic cells. While the N-terminal methionine on
most
proteins also is efficiently removed in most prokaryotes, for some proteins,
this
prokaryotic removal process is inefficient, depending on the nature of the
amino acid to
which the N-terminal methionine is covalently linked.
Uses of the Pol~ucleotides
Each of the polynucleotides identified herein can be used in numerous ways as
reagents. The following description should be considered exemplary and
utilizes
known techniques.
The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers,
since few chromosome marking reagents, based on actual sequence data (repeat
polymorphisms), are presently available. Each polynucleotide of the present
invention
can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be
selected using computer analysis so that primers do not span more than one
predicted
exon in the genomic DNA. These primers are then used for PCR screening of
somatic
cell hybrids containing individual human chromosomes. Only those hybrids
containing
the human gene corresponding to the SEQ ID NO:X will yield an amplified
fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per
day using a single thermal cycler. Moreover, sublocalization of the
polynucleotides can
be achieved with panels of specific chromosome fragments. Other gene mapping
strategies that can be used include in situ hybridization, prescreening with
labeled flow
sorted chromosomes, and preselection by hybridization to construct chromosome
specific-cDNA libraries.
Precise chromosomal location of the polynucleotides can also be achieved using
fluorescence in situ hybridization (FISH} of a metaphase chromosomal spread.
This
technique uses polynucleotides as short as 500 or 600 bases; however,
polynucleotides
2,000-4,000 by are preferred. For a review of this technique, see Verma et
al.,
"Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York
( 1988).
For chromosome mapping, the polynucleotides can be used individually (to
mark a single chromosome or a single site on that chromosome) or in panels
(for
marking multiple sites and/or multiple chromosomes). Preferred polynucleotides
correspond to the noncoding regions of the cDNAs because the coding sequences
are
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more likely conserved within gene families, thus increasing the chance of
cross
hybridization during chromosomal mapping.
Once a polynucleotide has been mapped to a precise chromosomal location, the
physical position of the polynucleotide can be used in linkage analysis.
Linkage
analysis establishes coinheritance between a chromosomal location and
presentation of a
particular disease. (Disease mapping data are found, for example, in V.
McKusick,
Mendelian Inheritance in Man (available on line through Johns Hopkins
University
Welch Medical Library) .) Assuming 1 megabase mapping resolution and one gene
per
20 kb, a cDNA precisely localized to a chromosomal region associated with the
disease
could be one of 50-500 potential causative genes.
Thus, once coinheritance is established, differences in the polynucleotide and
the corresponding gene between affected and unaffected individuals can be
examined.
First, visible structural alterations in the chromosomes, such as deletions or
translocations, are examined in chromosome spreads or by PCR. If no structural
alterations exist, the presence of point mutations are ascertained. Mutations
observed in
some or all affected individuals, but not in normal individuals, indicates
that the
mutation may cause the disease. However, complete sequencing of the
polypeptide and
the corresponding gene from several normal individuals is required to
distinguish the
mutation from a polymorphism. If a new polymorphism is identified, this
polymorphic
polypeptide can be used for further linkage analysis.
Furthermore, increased or decreased expression of the gene in affected
individuals as compared to unaffected individuals can be assessed using
polynucleotides of the present invention. Any of these alterations (altered
expression,
chromosomal rearrangement, or mutation) can be used as a diagnostic or
prognostic
marker.
In addition to the foregoing, a polynucleotide can be used to control gene
expression through triple helix formation or antisense DNA or RNA. Both
methods
rely on binding of the polynucleotide to DNA or RNA. For these techniques,
preferred
polynucleotides are usually 20 to 40 bases in length and complementary to
either the
region of the gene involved in transcription (triple helix - see Lee et al.,
Nucl. Acids
Res. 6:3073 ( 1979); Cooney et al., Science 241:456 ( 1988); and Dervan et
al., Science
251:1360 ( 1991 ) ) or to the mRNA itself (antisense - Okano, J. Neurochem.
56:560
( 1991 ); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression,
CRC
Press, Boca Raton, FL {1988).) Triple helix formation optimally results in a
shut-off
of RNA transcription from DNA, while antisense RNA hybridization blocks
translation
of an mRNA molecule into polypeptide. Both techniques are effective in model
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systems, and the information disclosed herein can be used to design antisense
or triple
helix polynucleotides in an effort to treat disease.
Polynucleotides of the present invention are also useful in gene therapy. One
goal of gene therapy is to insert a normal gene into an organism having a
defective
gene, in an effort to correct the genetic defect. The polynucleotides
disclosed in the
present invention offer a means of targeting such genetic defects in a highly
accurate
manner. Another goal is to insert a new gene that was not present in the host
genome,
thereby producing a new trait in the host cell.
The polynucleotides are also useful for identifying individuals from minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel. In
this technique, an individual's genomic DNA is digested with one or more
restriction
enzymes, and probed on a Southern blot to yield unique bands for identifying
personnel. This method does not suffer from the current limitations of "Dog
Tags"
which can be lost, switched, or stolen, making positive identification
difficult. The
polynucleotides of the present invention can be used as additional DNA markers
for
RFLP.
The polynucleotides of the present invention can also be used as an
alternative to
RFLP, by determining the actual base-by-base DNA sequence of selected portions
of an
individual's genome. These sequences can be used to prepare PCR primers for
amplifying and isolating such selected DNA, which can then be sequenced. Using
this
technique, individuals can be identified because each individual will have a
unique set
of DNA sequences. Once an unique 1D database is established for an individual,
positive identification of that individual, living or dead, can be made from
extremely
small tissue samples.
Forensic biology also benefits from using DNA-based identification techniques
as disclosed herein. DNA sequences taken from very small biological samples
such as
tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc.,
can be
amplified using PCR. In one prior art technique, gene sequences amplified from
polymorphic loci, such as DQa class II HLA gene, are used in forensic biology
to
identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).)
Once
these specific polymorphic loci are amplified, they are digested with one or
more
restriction enzymes, yielding an identifying set of bands on a Southern blot
probed with
DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of
the
present invention can be used as polymorphic markers for forensic purposes.
There is also a need for reagents capable of identifying the source of a
particular
tissue. Such need arises, for example, in forensics when presented with tissue
of
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unknown origin. Appropriate reagents can comprise, for example, DNA probes or
primers specific to particular tissue prepared from the sequences of the
present
invention. Panels of such reagents can identify tissue by species and/or by
organ type.
In a similar fashion, these reagents can be used to screen tissue cultures for
contamination.
In the very least, the polynucleotides of the present invention can be used as
molecular weight markers on Southern gels, as diagnostic probes for the
presence of a
specific mRNA in a particular cell type, as a probe to "subtract-out" known
sequences
in the process of discovering novel polynucleotides, for selecting and making
oligomers
for attachment to a "gene chip" or other support, to raise anti-DNA antibodies
using
DNA immunization techniques, and as an antigen to elicit an immune response.
Uses of the Poly .peptides
Each of the polypeptides identified herein can be used in numerous ways. The
following description should be considered exemplary and utilizes known
techniques.
A polypeptide of the present invention can be used to assay protein levels in
a
biological sample using antibody-based techniques. For example, protein
expression in
tissues can be studied with classical immunohistological methods. (Jalkanen,
M., et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol.
105:3087-
3096 (1987).) Other antibody-based methods useful for detecting protein gene
expression include immunoassays, such as the enzyme linked immunosorbent assay
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are
known
in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine ( 125I, 121I), carbon ( 14C), sulfur (35S), tritium (3H), indium ( 1
l2In), and
technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine,
and
biotin.
In addition to assaying secreted protein levels in a biological sample,
proteins
can also be detected in vivo by imaging. Antibody labels or markers for in
vivo
imaging of protein include those detectable by X-radiography, NMR or ESR. For
X-
radiography, suitable labels include radioisotopes such as barium or cesium,
which emit
detectable radiation but are not overtly harmful to the subject. Suitable
markers for
NMR and ESR include those with a detectable characteristic spin, such as
deuterium,
which may be incorporated into the antibody by labeling of nutrients for the
relevant
hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with
an appropriate detectable imaging moiety, such as a radioisotope (for example,
131I,
1 l2In, 99mTc), a radio-opaque substance, or a material detectable by nuclear
magnetic
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resonance, is introduced (for example, parenterally, subcutaneously, or
intraperitoneally) into the mammal. It will be understood in the art that the
size of the
subject and the imaging system used will determine the quantity of imaging
moiety
needed to produce diagnostic images. In the case of a radioisotope moiety, for
a human
subject, the quantity of radioactivity injected will normally range from about
5 to 20
rriillicuries of 99mTc. The labeled antibody or antibody fragment will then
preferentially accumulate at the location of cells which contain the specific
protein. In
vivo tumor imaging is described in S.W. Burchiel et al.,
"Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The
Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson
Publishing Inc. (1982).)
Thus, the invention provides a diagnostic method of a disorder, which involves
(a) assaying the expression of a polypeptide of the present invention in cells
or body
fluid of an individual; (b) comparing the level of gene expression with a
standard gene
expression level, whereby an increase or decrease in the assayed polypeptide
gene
expression level compared to the standard expression level is indicative of a
disorder.
Moreover, polypeptides of the present invention can be used to treat disease.
For example, patients can be administered a polypeptide of the present
invention in an
effort to replace absent or decreased levels of the polypeptide (e.g.,
insulin), to
supplement absent or decreased levels of a different polypeptide (e.g.,
hemoglobin S
for hemoglobin B), to inhibit the activity of a polypeptide (e.g., an
oncogene), to
activate the activity of a polypeptide (e.g., by binding to a receptor), to
reduce the
activity of a membrane bound receptor by competing with it for free ligand
(e.g.,
soluble TNF receptors used in reducing inflammation), or to bring about a
desired
response (e.g., blood vessel growth).
Similarly, antibodies directed to a polypeptide of the present invention can
also
be used to treat disease. For example, administration of an antibody directed
to a
polypeptide of the present invention can bind and reduce overproduction of the
polypeptide. Similarly, administration of an antibody can activate the
polypeptide, such
as by binding to a polypeptide bound to a membrane (receptor).
At the very least, the polypeptides of the present invention can be used as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the art. Polypeptides
can also
be used to raise antibodies, which in turn are used to measure protein
expression from a
recombinant cell, as a way of assessing transformation of the host cell.
Moreover, the
polypeptides of the present invention can be used to test the following
biological
activities.
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Biological Activities
The polynucleotides and polypeptides of the present invention can be used in
assays to test for one or more biological activities. If these polynucleotides
and
polypeptides do exhibit activity in a particular assay, it is likely that
these molecules
may be involved in the diseases associated with the biological activity. Thus,
the
polynucleotides and polypeptides could be used to treat the associated
disease.
Immune Activity
A polypeptide or polynucleotide of the present invention may be useful in
treating deficiencies or disorders of the immune system, by activating or
inhibiting the
proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
Immune
cells develop through a process called hematopoiesis, producing myeloid
(platelets, red
blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)
cells
from pluripotent stem cells. The etiology of these immune deficiencies or
disorders
may be genetic, somatic, such as cancer or some autoimmune disorders, acquired
(e.g.,
by chemotherapy or toxins), or infectious. Moreover, a polynucleotide or
polypeptide
of the present invention can be used as a marker or detector of a particular
immune
system disease or disorder.
A polynucleotide or polypeptide of the present invention may be useful in
treating or detecting deficiencies or disorders of hematopoietic cells. A
polypeptide or
polynucleotide of the present invention could be used to increase
differentiation and
proliferation of hematopoietic cells, including the pluripotent stem cells, in
an effort to
treat those disorders associated with a decrease in certain (or many) types
hematopoietic
cells. Examples of immunologic deficiency syndromes include, but are not
limited to:
blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia),
ataxia
telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV
infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency
(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or
hemoglobinuria.
Moreover, a polypeptide or polynucleotide of the present invention could also
be used to modulate hemostatic (the stopping of bleeding) or thrombolytic
activity (clot
formation). For example, by increasing hemostatic or thrombolytic activity, a
polynucleotide or polypeptide of the present invention could be used to treat
blood
coagulation disorders (e.g., aflbrinogenemia, factor deficiencies), blood
platelet
disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery,
or other
causes. Alternatively, a polynucleotide or polypeptide of the present
invention that can
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decrease hemostatic or thrombolytic activity could be used to inhibit or
dissolve
clotting. These molecules could be important in the treatment of heart attacks
(infarction), strokes, or scarring.
A polynucleotide or polypeptide of the present invention may also be useful in
treating or detecting autoimmune disorders. Many autoimmune disorders result
from
inappropriate recognition of self as foreign material by immune cells. This
inappropriate recognition results in an immune response leading to the
destruction of the
host tissue. Therefore, the administration of a polypeptide or polynucleotide
of the
present invention that inhibits an immune response, particularly the
proliferation,
differentiation, or chemotaxis of T-cells, may be an effective therapy in
preventing
autoimmune disorders.
Examples of autoimmune disorders that can be treated or detected by the
present
invention include, but are not limited to: Addison's Disease, hemolytic
anemia,
antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic
encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple
Sclerosis,
Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,
Polyendocrinopathies, Purpura, Reiter's Disease, Stiff Man Syndrome,
Autoimmune
Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune
inflammatory eye disease.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic
asthma) or other respiratory problems, may also be treated by a polypeptide or
polynucleotide of the present invention. Moreover, these molecules can be used
to treat
anaphylaxis, hypersensitivity to an antigenic molecule, or blood group
incompatibility.
A polynucleotide or polypeptide of the present invention may also be used to
treat and/or prevent organ rejection or graft-versus-host disease (GVHD).
Organ
rejection occurs by host immune cell destruction of the transplanted tissue
through an
immune response. Similarly, an immune response is also involved in GVHD, but,
in
this case, the foreign transplanted immune cells destroy the host tissues. The
administration of a polypeptide or polynucleotide of the present invention
that inhibits
an immune response, particularly the proliferation, differentiation, or
chemotaxis of T-
cells, may be an effective therapy in preventing organ rejection or GVHD.
Similarly, a polypeptide or polynucleodde of the present invention may also be
used to modulate inflammation. For example, the polypeptide or polynucleotide
may
inhibit the proliferation and differentiation of cells involved in an
inflammatory
response. These molecules can be used to treat inflammatory conditions, both
chronic
and acute conditions, including inflammation associated with infection (e.g.,
septic
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shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-
reperfusion injury, endotoxin lethality, arthritis, complement-mediated
hyperacute
rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory
bowel
disease, Crohn's disease, or resulting from over production of cytokines
(e.g., TNF or
lL-1.)
Hyperproliferative Disorders
A polypepdde or polynucleotide can be used to treat or detect
hyperproliferative
disorders, including neoplasms. A polypeptide or polynucleotide of the present
invention may inhibit the proliferation of the disorder through direct or
indirect
interactions. Alternatively, a polypeptide or polynucleotide of the present
invention
may proliferate other cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing
antigenic qualities of the hyperproliferative disorder or by proliferating,
differentiating,
or mobilizing T-cells, hyperproliferative disorders can be treated. This
immune
response may be increased by either enhancing an existing immune response, or
by
initiating a new immune response. Alternatively, decreasing an immune response
may
also be a method of treating hyperproliferative disorders, such as a
chemotherapeutic
agent.
Examples of hyperproliferative disorders that can be treated or detected by a
polynucleotide or polypeptide of the present invention include, but are not
limited to
neoplasms located in the: abdomen, bone, breast, digestive system, liver,
pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles,
ovary, thymus,
thyroid), eye, head and neck, nervous (central and peripheral), lymphatic
system,
pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative disorders can also be treated or detected
by a
poiynucleotide or polypeptide of the present invention. Examples of such
hyperproliferative disorders include, but are not limited to:
hypergammaglobulinemia,
lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary
Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis,
and
any other hyperproliferative disease, besides neoplasia, located in an organ
system
listed above.
Infectious Disease
A polypeptide or polynucleotide of the present invention can be used to treat
or
detect infectious agents. For example, by increasing the immune response,
particularly
increasing the proliferation and differentiation of B and/or T cells,
infectious diseases
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may be treated. The immune response may be increased by either enhancing an
existing
immune response, or by initiating a new immune response. Alternatively, the
polypeptide or polynucleotide of the present invention may also directly
inhibit the
infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated or detected by a polynucleotide or polypeptide of
the
present invention. Examples of viruses, include, but are not limited to the
following
DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae,
Arterivirus,
Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,
Flaviviridae,
Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes
Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae,
Parvoviridae,
Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g.,
Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,
Rubivirus). Viruses falling within these families can cause a variety of
diseases or
symptoms, including, but not limited to: arthritis, bronchiollitis,
encephalitis, eye
infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome,
hepatitis (A, B, C,
E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS),
pneumonia, Burkitt's Lymphoma, chickenpox , hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually
transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. A
polypeptide
or polynucleotide of the present invention can be used to treat or detect any
of these
symptoms or diseases.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that
can be treated or detected by a polynucleotide or polypeptide of the present
invention
include, but not limited to, the following Gram-Negative and Gram-positive
bacterial
families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,
Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium),
Bacteroidaceae,
Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter,
Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae
(Klebsiella,
Saim;onella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis,
Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter,
Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g., Actinobacillus,
Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae,
Syphilis,
and Staphylococcal. These bacterial or fungal families can cause the following
diseases
or symptoms, including, but not limited to: bacteremia, endocarditis, eye
infections
(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections
(e.g., AIDS
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related infections), paronychia, prosthesis-related infections, Reiter's
Disease,
respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme
Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,
Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria,
Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus,
impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases
(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound
infections.
A polypeptide or polynucleotide of the present invention can be used to treat
or detect
any of these symptoms or diseases.
Moreover, parasitic agents causing disease or symptoms that can be treated or
detected by a polynucleotide or polypeptide of the present invention include,
but not
limited to, the following families: Amebiasis, Babesiosis, Coccidiosis,
Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,
Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
These parasites can cause a variety of diseases or symptoms, including, but
not limited
to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g.,
dysentery,
giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS
related),
Malaria, pregnancy complications, and toxoplasmosis. A polypeptide or
polynucleotide
of the present invention can be used to treat or detect any of these symptoms
or
diseases.
Preferably, treatment using a polypeptide or polynucleotide of the present
invention could either be by administering an effective amount of a
polypeptide to the
patient, or by removing cells from the patient, supplying the cells with a
polynucleotide
of the present invention, and returning the engineered cells to the patient
(ex vivo
therapy). Moreover, the polypeptide or polynucleotide of the present invention
can be
used as an antigen in a vaccine to raise an immune response against infectious
disease.
Regeneration
A polynucleotide or polypeptide of the present invention can be used to
differentiate, proliferate, and attract cells, leading to the regeneration of
tissues. (See,
Science 276:59-$7 (1997).) The regeneration of tissues could be used to
repair,
replace, or protect tissue damaged by congenital defects, trauma (wounds,
burns,
incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,
periodontal
disease, liver failure), surgery, including cosmetic plastic surgery,
fibrosis, reperfusion
injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs
(e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal
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or cardiac), vascular (including vascular endothelium), nervous,
hematopoietic, and
skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,
regeneration occurs
without or decreased scarring. Regeneration also may include angiogenesis.
Moreover, a polynucleotide or polypeptide of the present invention may
increase
regeneration of tissues difficult to heal. For example, increased
tendon/ligament
regeneration would quicken recovery time after damage. A polynucleotide or
polypeptide of the present invention could also be used prophylactically in an
effort to
avoid damage. Specific diseases that could be treated include of tendinitis,
carpal tunnel
syndrome, and other tendon or ligament defects. A further example of tissue
regeneration of non-healing wounds includes pressure ulcers, ulcers associated
with
vascular insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a
polynucleotide or polypeptide of the present invention to proliferate and
differentiate
nerve cells. Diseases that could be treated using this method include central
and
1 S peripheral nervous system diseases, neuropathies, or mechanical and
traumatic
disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease,
and
stoke). Specifically, diseases associated with peripheral nerve injuries,
peripheral
neuropathy (e.g., resulting from chemotherapy or other medical therapies),
localized
neuropathies, and central nervous system diseases (e.g., Alzheimer's disease,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-
Drager syndrome), could all be treated using the polynucleotide or polypeptide
of the
presentinvention.
Chemol.~xis
A polynucleotide or polypeptide of the present invention may have chemotaxis
activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial
cells) to a particular site in the body, such as inflammation, infection, or
site of
hyperproliferation. The mobilized cells can then fight off and/or heal the
particular
trauma or abnormality.
A polynucleodde or polypeptide of the present invention may increase
chemotaxic activity of particular cells. These chemotactic molecules can then
be used to
treat inflammation, infection, hyperproliferative disorders, or any immune
system
disorder by increasing the number of cells targeted to a particular location
in the body.
For example, chemotaxic molecules can be used to treat wounds and other trauma
to
tissues by attractlng immune cells to the injured location. Chemotactic
molecules of the
present invention can also attract fibroblasts, which can be used to treat
wounds.
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It is also contemplated that a polynucleotide or polypeptide of the present
invention may inhibit chemotactic activity. These molecules could also be used
to treat
disorders. Thus, a polynucleotide or polypeptide of the present invention
could be used
as an inhibitor of chemotaxis.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules
that
bind to the polypeptide or for molecules to which the polypeptide binds. The
binding
of the polypeptide and the molecule may activate (agonist), increase, inhibit
(antagonist), or decrease activity of the polypeptide or the molecule bound.
Examples
of such molecules include antibodies, oligonucleoddes, proteins (e.g.,
receptors),or
small molecules.
Preferably, the molecule is closely related to the natural ligand of the
polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand,
a structural
or functional mimetic. (See, Coligan et al., Current Protocols in Immunology
1 (2):Chapter 5 ( 1991 ).) Similarly, the molecule can be closely related to
the natural
receptor to which the polypeptide binds, or at least, a fragment of the
receptor capable
of being bound by the polypeptide (e.g., active site). In either case, the
molecule can
be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate
cells which express the polypeptide, either as a secreted protein or on the
cell
membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coli.
Cells expressing the polypeptide (or cell membrane containing the expressed
polypeptide) are then preferably contacted with a test compound potentially
containing
the molecule to observe binding, stimulation, or inhibition of activity of
either the
polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the polypeptide,
wherein binding is detected by a label, or in an assay involving competition
with a
labeled competitor. Further, the assay may test whether the candidate compound
results
in a signal generated by binding to the polypeptide.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
mixtures. The assay may also simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide, measuring
polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule activity or
binding to a
standard.
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Preferably, an ELISA assay can measure polypeptide level or activity in a
sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
The
antibody can measure polypeptide level or activity by either binding, directly
or
indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
S All of these above assays can be used as diagnostic or prognostic markers.
The
molecules discovered using these assays can be used to treat disease or to
bring about a
particular result in a patient (e.g., blood vessel growth) by activating or
inhibiting the
polypeptide/molecule. Moreover, the assays can discover agents which may
inhibit or
enhance the production of the polypeptide from suitably manipulated cells or
tissues.
Therefore, the invention includes a method of identifying compounds which
bind to a polypeptide of the invention comprising the steps of: (a) incubating
a
candidate binding compound with a polypeptide of the invention; and (b)
determining if
binding has occurred. Moreover, the invention includes a method of identifying
agonists/antagonists comprising the steps of: (a) incubating a candidate
compound with
a polypeptide of the invention, (b) assaying a biological activity , and (b)
determining if
a biological activity of the polypeptide has been altered.
Other Activities
A polypeptide or polynucleotide of the present invention may also increase or
decrease the differentiation or proliferation of embryonic stem cells,
besides, as
discussed above, hematopoietic lineage.
A polypeptide or polynucleotide of the present invention may also be used to
modulate mammalian characteristics, such as body height, weight, hair color,
eye color,
skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,
cosmetic
surgery). Similarly, a polypeptide or polynucleotide of the present invention
may be
used to modulate mammalian metabolism affecting catabolism, anabolism,
processing,
utilization, and storage of energy.
A polypeptide or polynucleotide of the present invention may be used to change
a mammal's mental state or physical state by influencing biorhythms, caricadic
rhythms, depression (including depressive disorders), tendency for violence,
tolerance
for pain, reproductive capabilities (preferably by Activin or Inhibin-like
activity),
hormonal or endocrine levels, appetite, libido, memory, stress, or other
cognitive
qualities.
A polypeptide or polynucleotide of the present invention may also be used as a
food additive or preservative, such as to increase or decrease storage
capabilities, fat
content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other
nutritional
components.
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Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated
nucleic acid molecule comprising a nucleotide sequence which is at least 95%
identical
to a sequence of at least about 50 contiguous nucleotides in the nucleotide
sequence of
SEQ ID NO:X wherein X is any integer as defined in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5'
Nucleotide of the
Clone Sequence and ending with the nucleotide at about the position of the 3'
Nucleotide of the Clone Sequence as defined for SEQ iD NO:X in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5'
Nucleotide of the
Start Codon and ending with the nucleotide at about the position of the 3'
Nucleotide of
the Clone Sequence as defined for SEQ 11? NO:X in Table 1.
Similarly preferred is a nucleic acid molecule wherein said sequence of
contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X
in the
range of positions beginning with the nucleotide at about the position of the
5'
Nucleotide of the First Amino Acid of the Signal Peptide and ending with the
nucleotide
at about the position of the 3' Nucleotide of the Clone Sequence as defined
for SEQ ID
NO:X in Table 1.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least about 150
contiguous
nucleotides in the nucleotide sequence of SEQ 1D NO:X.
Further preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least about 500
contiguous
nucleotides in the nucleotide sequence of SEQ ID NO:X.
A further preferred embodiment is a nucleic acid molecule comprising a
nucleotide sequence which is at least 95% identical to the nucleotide sequence
of SEQ
ID NO:X beginning with the nucleotide at about the position of the 5'
Nucleotide of the
First Amino Acid of the Signal Peptide and ending with the nucleotide at about
the
position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID NO:X
in
Table 1.
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A further preferred embodiment is an isolated nucleic acid molecule comprising
a nucleotide sequence which is at least 95% identical to the complete
nucleotide
sequence of SEQ ID NO:X.
Also preferred is an isolated nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule, wherein said
nucleic acid
molecule which hybridizes does not hybridize under stringent hybridization
conditions
to a nucleic acid molecule having a nucleotide sequence consisting of only A
residues or
of only T residues.
Also preferred is a composition of matter comprising a DNA molecule which
comprises a human cDNA clone identified by a cDNA Clone Identifier in Table 1,
which DNA molecule is contained in the material deposited with the American
Type
Culture Collection and given the ATCC Deposit Number shown in Table 1 for said
cDNA Clone Identifier.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least 50
contiguous
nucleotides in the nucleotide sequence of a human cDNA clone identified by a
cDNA
Clone Identifier in Table 1, which DNA molecule is contained in the deposit
given the
ATCC Deposit Number shown in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said sequence of
at
least 50 contiguous nucleotides is included in the nucleotide sequence of the
complete
open reading frame sequence encoded by said human cDNA clone.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to sequence of at least 150
contiguous
nucleotides in the nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule comprising
a nucleotide sequence which is at least 95% identical to sequence of at least
500
contiguous nucleotides in the nucleotide sequence encoded by said human cDNA
clone.
A further preferred embodiment is an isolated nucleic acid molecule comprising
a nucleotide sequence which is at least 95% identical to the complete
nucleotide
sequence encoded by said human cDNA clone.
A further preferred embodiment is a method for detecting in a biological
sample
a nucleic acid molecule comprising a nucleotide sequence which is at least 95%
identical
to a sequence of at least 50 contiguous nucleotides in a sequence selected
from the
group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any
integer
as defined in Table 1; and a nucleotide sequence encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1; which method
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comprises a step of comparing a nucleotide sequence of at least one nucleic
acid
molecule in said sample with a sequence selected from said group and
determining
whether the sequence of said nucleic acid molecule in said sample is at least
95%
identical to said selected sequence.
Also preferred is the above method wherein said step of comparing sequences
comprises determining the extent of nucleic acid hybridization between nucleic
acid
molecules in said sample and a nucleic acid molecule comprising said sequence
selected
from said group. Similarly, also preferred is the above method wherein said
step of
comparing sequences is performed by comparing the nucleotide sequence
determined
from a nucleic acid molecule in said sample with said sequence selected from
said
group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
A further preferred embodiment is a method for identifying the species, tissue
or
cell type of a biological sample which method comprises a step of detecting
nucleic acid
molecules in said sample, if any, comprising a nucleotide sequence that is at
least 95%
identical to a sequence of at least 50 contiguous nucleotides in a sequence
selected from
the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any
integer as defined in Table 1; and a nucleotide sequence encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table l and contained in the
deposit with
the ATCC Deposit Number shown for said cDNA clone in Table 1.
The method for identifying the species, tissue or cell type of a biological
sample
can comprise a step of detecting nucleic acid molecules comprising a
nucleotide
sequence in a panel of at least two nucleotide sequences, wherein at least one
sequence
in said panel is at least 95% identical to a sequence of at least 50
contiguous nucleotides
in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein
identified in Table 1, which method comprises a step of detecting in a
biological sample
obtained from said subject nucleic acid molecules, if any, comprising a
nucleotide
sequence that is at least 95% identical to a sequence of at least 50
contiguous
nucleotides in a sequence selected from the group consisting of: a nucleotide
sequence
of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a
nucleotide
sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier
in
Table l and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
The method for diagnosing a pathological condition can comprise a step of
detecting nucleic acid molecules comprising a nucleotide sequence in a panel
of at least
two nucleotide sequences, wherein at least one sequence in said panel is at
least 95%
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identical to a sequence of at least 50 contiguous nucleotides in a sequence
selected from
said group.
Also preferred is a composition of matter comprising isolated nucleic acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise a
panel of at least two nucleotide sequences, wherein at least one sequence in
said panel is
at least 95% identical to a sequence of at least 50 contiguous nucleotides in
a sequence
selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X
wherein
X is any integer as defined in Table 1; and a nucleotide sequence encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table l and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. The
nucleic acid molecules can comprise DNA molecules or RNA molecules.
Also preferred is an isolated polypeptide comprising an amino acid sequence at
least 90% identical to a sequence of at least about 10 contiguous amino acids
in the
amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1.
Also preferred is a polypeptide, wherein said sequence of contiguous amino
acids is included in the amino acid sequence of SEQ ID NO:Y in the range of
positions
beginning with the residue at about the position of the First Amino Acid of
the Secreted
Portion and ending with the residue at about the Last Amino Acid of the Open
Reading
Frame as set forth for SEQ ID NO:Y in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence at
least 95% identical to a sequence of at least about 30 contiguous amino acids
in the
amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 100 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid sequence
at least 90% identical to a sequence of at least about 10 contiguous amino
acids in the
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is a polypeptide wherein said sequence of contiguous amino
acids is included in the amino acid sequence of a secreted portion of the
secreted protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and
contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in
Table 1.
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Also preferred is an isolated polypeptide comprising an amino acid sequence at
least. 95% identical to a sequence of at least about 30 contiguous amino acids
in the
amino acid sequence of the secreted portion of the protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table l and contained in the
deposit with
the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence at
least 95% identical to a sequence of at least about 100 contiguous amino acids
in the
amino acid sequence of the secreted portion of the protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table l and contained in the
deposit with
the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence at
least 95% identical to the amino acid sequence of the secreted portion of the
protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and
contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in
Table 1.
Further preferred is an isolated antibody which binds specifically to a
polypeptide comprising an amino acid sequence that is at least 90% identical
to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as
defined in Table l; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table l and
contained in
the deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Further preferred is a method for detecting in a biological sample a
polypeptide
comprising an amino acid sequence which is at least 90% identical to a
sequence of at
least 10 contiguous amino acids in a sequence selected from the group
consisting of: an
amino acid sequence of SEQ D7 NO:Y wherein Y is any integer as defined in
Table 1;
and a complete amino acid sequence of a protein encoded by a human cDNA clone
identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1; which method
comprises a step of comparing an amino acid sequence of at least one
polypeptide
molecule in said sample with a sequence selected from said group and
determining
whether the sequence of said polypepdde molecule in said sample is at least
90%
identical to said sequence of at least 10 contiguous amino acids.
Also preferred is the above method wherein said step of comparing an amino
acid sequence of at least one polypeptide molecule in said sample with a
sequence
selected from said group comprises determining the extent of specific binding
of
polypeptides in said sample to an antibody which binds specifically to a
polypeptide
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comprising an amino acid sequence that is at least 90% identical to a sequence
of at least
contiguous amino acids in a sequence selected from the group consisting of: an
amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1;
and a complete amino acid sequence of a protein encoded by a human cDNA clone
5 identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is the above method wherein said step of comparing sequences is
performed by comparing the amino acid sequence determined from a polypeptide
molecule in said sample with said sequence selected from said group.
10 Also preferred is a method for identifying the species, tissue or cell type
of a
biological sample which method comprises a step of detecting polypeptide
molecules in
said sample, if any, comprising an amino acid sequence that is at least 90%
identical to
a sequence of at least 10 contiguous amino acids in a sequence selected from
the group
consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as
defined in Table 1; and a complete amino acid sequence of a secreted protein
encoded
by a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
Also preferred is the above method for identifying the species, tissue or cell
type
of a biological sample, which method comprises a step of detecting polypeptide
molecules comprising an amino acid sequence in a panel of at least two amino
acid
sequences, wherein at least one sequence in said panel is at least 90%
identical to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
above
group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein
identified in Table 1, which method comprises a step of detecting in a
biological sample
obtained from said subject polypeptide molecules comprising an amino acid
sequence in
a panel of at least two amino acid sequences, wherein at least one sequence in
said panel
is at least 90% identical to a sequence of at least 10 contiguous amino acids
in a
sequence selected from the group consisting of: an amino acid sequence of SEQ
1D
NO:Y wherein Y is any integer as defined in Table 1; and a complete amino acid
sequence of a secreted protein encoded by a human cDNA clone identified by a
cDNA
Clone Identifier in Table l and contained in the deposit with the ATCC Deposit
Number
shown for said cDNA clone in Table 1.
In any of these methods, the step of detecting said polypeptide molecules
includes using an antibody.
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Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a nucleotide sequence encoding a
polypeptide wherein said polypeptide comprises an amino acid sequence that is
at least
90% identical to a sequence of at least 10 contiguous amino acids in a
sequence selected
from the group consisting of: an amino acid sequence of SEQ ID NO:Y wherein Y
is
any integer as defined in Table 1; and a complete amino acid sequence of a
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table
1 and contained in the deposit with the ATCC Deposit Number shown for said
cDNA
clone in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said nucleotide
sequence encoding a polypeptide has been optimized for expression of said
polypeptide
in a prokaryotic host.
Also preferred is an isolated nucleic acid molecule, wherein said polypeptide
comprises an amino acid sequence selected from the group consisting of: an
amino acid
sequence of SEQ ID NO:Y wherein Y is any integer as defined in Table 1; and a
complete amino acid sequence of a secreted protein encoded by a human cDNA
clone
identified by a cDNA Clone Identifier in Table 1 and contained in the deposit
with the
ATCC Deposit Number shown for said cDNA clone in Table 1.
Further preferred is a method of making a recombinant vector comprising
inserting any of the above isolated nucleic acid molecule into a vector. Also
preferred is
the recombinant vector produced by this method. Also preferred is a method of
making
a recombinant host cell comprising introducing the vector into a host cell, as
well as the
recombinant host cell produced by this method.
Also preferred is a method of making an isolated polypeptide comprising
culturing this recombinant host cell under conditions such that said
polypeptide is
expressed and recovering said polypeptide. Also preferred is this method of
making an
isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell
and said
polypeptide is a secreted portion of a human secreted protein comprising an
amino acid
sequence selected from the group consisting of: an amino acid sequence of SEQ
ID
NO:Y beginning with the residue at the position of the First Amino Acid of the
Secreted
Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table 1 and said
position
of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y is defined in
Table 1;
and an amino acid sequence of a secreted portion of a protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1. The
isolated polypeptide produced by this method is also preferred.
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Also preferred is a method of treatment of an individual in need of an
increased
level of a secreted protein activity, which method comprises administering to
such an
individual a pharmaceutical composition comprising an amount of an isolated
polypeptide, polynucleotide, or antibody of the claimed invention effective to
increase
the level of said protein activity in said individual.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
xam es
Fx~ ample 1: Isolation of a Selected cDNA Clone From the De oo sited
Sample
Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector.
Table 1 identifies the vectors used to construct the cDNA library from which
each clone
was isolated. In many cases, the vector used to construct the library is a
phage vector
from which a plasmid has been excised. The table immediately below correlates
the
related plasmid for each phage vector used in constructing the cDNA library.
For
example, where a particular clone is identified in Table 1 as being isolated
in the vector
"Lambda Zap," the corresponding deposited clone is in "pBluescript."
Vector Used to Construct Li_ brarv Corresponding_Deposited Plasmid
Lambda Zap pBluescript (pBS)
Uni-Zap XR pBluescript (pBS)
Zap Express pBK
lafmid BA plafmid BA
pSport 1 pSport 1
pCMVSport 2.0 pCMVSport 2.0
pCMVSport 3.0 pCMVSport 3.0
pCR~2.1 pCR~2.1
Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap
XR (U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic
Acids Res.
i 6:7583-7600 ( 1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res.
17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992))
are
commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey
Pines
Road, La Jolla, CA, 92037. pBS contains an ampicillin resistance gene and pBK
contains a neomycin resistance gene. Both can be transformed into E. coli
strain XL-1
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Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK-, KS+ and
KS.
The S and K refers to the orientation of the polylinker to the T7 and T3
primer
sequences which flank the polylinker region ("S" is for SacI and "K" is for
KpnI which
are the first sites on each respective end of the linker). "+" or "-" refer to
the orientation
of the f l origin of replication ("ori"), such that in one orientation, single
stranded rescue
initiated from the fl on generates sense strand DNA and in the other,
antisense.
Vectors pSportl, pCMVSport 2.0 and pCMVSport 3.0, were obtained from
Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897. All Sport
vectors
contain an ampicillin resistance gene and may be transformed into E. coli
strain
DH10B, also available from Life Technologies. (See, for instance, Gruber, C.
E., et
al., Focus 15:59 (1993).) Vector lafmid BA (Bento Soares, Columbia University,
NY)
contains an ampicillin resistance gene and can be transformed into E. coli
strain XL-1
Blue. Vector pCR~2.l, which is available from Invitrogen, 1600 Faraday Avenue,
Carlsbad, CA 92008, contains an ampicillin resistance gene and may be
transformed
into E. coli strain DH IOB, available from Life Technologies. (See, for
instance, Clark,
J. M., Nuc. Acids Res. 16:9677-9686 ( 1988) and Mead, D. et al.,
Bio/Technology 9:
( 1991 ).) Preferably, a polynucleotide of the present invention does not
comprise the
phage vector sequences identified for the particular clone in Table 1, as well
as the
corresponding plasmid vector sequences designated above.
The deposited material in the sample assigned the ATCC Deposit Number cited
in Table 1 for any given cDNA clone also may contain one or more additional
plasmids,
each comprising a cDNA clone different from that given clone. Thus, deposits
sharing
the same ATCC Deposit Number contain at least a plasmid for each cDNA clone
identified in Table 1. Typically, each ATCC deposit sample cited in Table 1
comprises
a mixture of approximately equal amounts (by weight) of about 50 plasmid DNAs,
each
containing a different cDNA clone; but such a deposit sample may include
plasmids for
more or less than 50 cDNA clones, up to about 500 cDNA clones.
Two approaches can be used to isolate a particular clone from the deposited
sample of plasmid DNAs cited for that clone in Table 1. First, a plasmid is
directly
isolated by screening the clones using a polynucleotide probe corresponding to
SEQ ID
NO:X.
Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized
using an Applied Biosystems DNA synthesizer according to the sequence
reported.
The oligonucleotide is labeled, for instance, with 32P-'y ATP using T4
polynucleotide
kinase and purified according to routine methods. (E.g., Maniatis et al.,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY
(1982).)
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The plasmid mixture is transformed into a suitable host, as indicated above
(such as
XL-1 Blue (Stratagene)) using techniques known to those of skill in the art,
such as
those provided by the vector supplier or in related publications or patents
cited above.
The transformants are plated on 1.5% agar plates (containing the appropriate
selection
agent, e.g., ampicillin) to a density of about 150 transformants (colonies)
per plate.
These plates are screened using Nylon membranes according to routine methods
for
bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A
Laboratory
Manual, 2nd Edit., ( 1989), Cold Spring Harbor Laboratory Press, pages 1.93 to
1.104), or other techniques known to those of skill in the art.
Alternatively, two primers of 17-20 nucleotides derived from both ends of the
SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded by the 5' NT and
the
3' NT of the clone defined in Table 1 ) are synthesized and used to amplify
the desired
cDNA using the deposited cDNA plasmid as a template. The polymerase chain
reaction
is carried out under routine conditions, for instance, in 25 p.l of reaction
mixture with
0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM
MgClz, 0.01 % (w/v) gelatin, 20 ~,M each of dATP, dCTP, dGTP, dTTP, 25 pmol of
each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR
(denaturation
at 94°C for 1 min; annealing at 55°C for 1 min; elongation at
72°C for 1 min) are
performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified
product
is analyzed by agarose gel electrophoresis and the DNA band with expected
molecular
weight is excised and purified. The PCR product is verified to be the selected
sequence
by subcloning and sequencing the DNA product.
Several methods are available for the identification of the 5' or 3' non-
coding
portions of a gene which may not be present in the deposited clone. These
methods
include but are not limited to, filter probing, clone enrichment using
specific probes,
and protocols similar or identical to 5' and 3' "RACE" protocols which are
well known
in the art. For instance, a method similar to 5' RACE is available for
generating the
missing 5' end of a desired full-length transcript. (Fromont-Racine et al.,
Nucleic Acids
Res. 21(7):1683-1684 (1993).)
Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a
population
of RNA presumably containing full-length gene RNA transcripts. A primer set
containing a primer specific to the ligated RNA oligonucleotide and a primer
specific to
a known sequence of the gene of interest is used to PCR amplify the 5' portion
of the
desired full-length gene. This amplified product may then be sequenced and
used to
generate the full length gene.
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This above method starts with total RNA isolated from the desired source,
although poly-A+ RNA can be used. The RNA preparation can then be treated with
phosphatase if necessary to eliminate 5' phosphate groups on degraded or
damaged
RNA which may interfere with the later RNA ligase step. The phosphatase should
then
be inactivated and the RNA treated with tobacco acid pyrophosphatase in order
to
remove the cap structure present at the 5' ends of messenger RNAs. This
reaction
leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can
then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
This modified RNA preparation is used as a template for first strand cDNA
synthesis using a gene specific oligonucleotide. The first strand synthesis
reaction is
used as a template for PCR amplification of the desired 5' end using a primer
specific to
the ligated RNA oligonucleotide and a primer specific to the known sequence of
the
gene of interest. The resultant product is then sequenced and analyzed to
confirm that
the 5' end sequence belongs to the desired gene.
~xam~le 2: Isolation of Genomic Clones Corresponding to a
Polvnucleotide
A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR
using primers selected for the cDNA sequence corresponding to SEQ ID NO:X.,
according to the method described in Example 1. (See also, Sambrook.)
Example 3: Tissue Distribution of Polype tide
Tissue distribution of mRNA expression of polynucleotides of the present
invention is determined using protocols for Northern blot analysis, described
by,
among others, Sambrook et al. For example, a cDNA probe produced by the method
described in Example 1 is labeled with P32 using the rediprimeTM DNA labeling
system
(Amersham Life Science), according to manufacturer's instructions. After
labeling, the
probe is purified using CHROMA SPIN-100TM column (Clontech Laboratories,
Inc.),
according to manufacturer's protocol number PT1200-1. The purified labeled
probe is
then used to examine various human tissues for mRNA expression.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or
human immune system tissues (IM) (Clontech) are examined with the labeled
probe
using ExpressHybTM hybridization solution (Clontech) according to
manufacturer's
protocol number PT1190-1. Following hybridization and washing, the blots are
mounted and exposed to film at -70°C overnight, and the films developed
according to
standard procedures.
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~xamp~le 4~ Chromosomal MaQying of the Polynucleotides
An oligonucleotide primer set is designed according to the sequence at the 5'
end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This
primer set is then used in a polymerase chain reaction under the following set
of
conditions : 30 seconds, 95°C; 1 minute, 56°C; 1 minute,
70°C. This cycle is repeated
32 times followed by one 5 minute cycle at 70°C. Human, mouse, and
hamster DNA
is used as template in addition to a somatic cell hybrid panel containing
individual
chromosomes or chromosome fragments (Bios, Inc). The reactions is analyzed on
either 8% polyacrylamide gels or 3.5 % agarose gels. Chromosome mapping is
determined by the presence of an approximately 100 by PCR fragment in the
particular
somatic cell hybrid.
Example 5: Bacterial Expression of a Poly~eptide
A polynucleotide encoding a polypeptide of the present invention is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the
DNA
sequence, as outlined in Example 1, to synthesize insertion fragments. The
primers
used to amplify the cDNA insert should preferably contain restriction sites,
such as
BamHI and XbaI, at the 5' end of the primers in order to clone the amplified
product
into the expression vector. For example, BamHI and XbaI correspond to the
restriction
enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc.,
Chatsworth,
CA). This plasmid vector encodes antibiotic resistance (Amps, a bacterial
origin of
replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome
binding site
(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment
is ligated into the pQE-9 vector maintaining the reading frame initiated at
the bacterial
RBS. The ligation mixture is then used to transform the E. coli strain
M15/rep4
(Qiagen, Inc.) which contains multiple copies of the piasmid pREP4, which
expresses
the lacI repressor and also confers kanamycin resistance (Kanr). Transfonnants
are
identified by their ability to grow on LB plates and ampicillin/kanamycin
resistant
colonies are selected. Plasmid DNA is isolated and confirmed by restriction
analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid
culture in LB media supplemented with both Amp ( 100 ug/ml) and Kan (25
ug/ml).
The O/N culture is used to inoculate a large culture at a ratio of 1:100 to
1:250. The
cells are grown to an optical density 600 (O.D.6°°) of between
0.4 and 0.6. IPTG
*rB
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(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration
of 1 mM.
IPTG induces by inactivating the lacI repressor, clearing the P/O leading to
increased
gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by
centrifugation (20 rains at 6000Xg). The cell pellet is solubilized in the
chaotropic
agent 6 Molar Guanidine HCI by stirnng for 3-4 hours at 4°C. The cell
debris is
removed by centrifugation, and the supernatant containing the polypeptide is
loaded
onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity resin column
(available from
QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA resin
with high
affinity and can be purified in a simple one-step procedure (for details see:
The
QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCI, pH 8,
the column is first washed with 10 volumes of 6 M guanidine-HCI, pH 8, then
washed
with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is
eluted with
6 M guanidine-HCI, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-
buffered
saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCI. Alternatively,
the
protein can be successfully refolded while immobilized on the Ni-NTA column.
The
recommended conditions are as follows: renature using a linear 6M-1M urea
gradient in
500 mM NaCI, 20% glycerol, 20 mM Tris/HCi pH 7.4, containing protease
inhibitors.
The renaturation should be performed over a period of 1.5 hours or more. After
renaturation the proteins are eluted by the addition of 250 mM immidazole.
lmmidazole
is removed by a final dialyzing step against PBS or SO mM sodium acetate pH 6
buffer
plus 200 mM NaCI. The purified protein is stored at 4° C or frozen at -
80° C.
In addition to the above expression vector, the present invention further
includes
an expression vector comprising phage operator and promoter elements
operatively
linked to a polynucleotide of the present invention, called pHF.~4a. (ATCC
Accession
Number 209645, deposited on February 25, 1998.) This vector contains: 1 ) a
neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of
replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences,
5) a
Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The
origin
of replication (oriC) is derived from pUCl9 (LTI, Gaithersburg, MD). The
promoter
sequence and operator sequences are made synthetically.
DNA can be inserted into the pHEa by restricting the vector with NdeI and
XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and
isolating
the larger fragment (the stuffer fragment should be about 310 base pairs). The
DNA
*rB
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insert is generated according to the PCR protocol described in Example l,
using PCR
primers having restriction sites for NdeI (5' primer) and XbaI, BamHI, XhoI,
or
Asp718 {3' primer). The PCR insert is gel purified and restricted with
compatible
enzymes. The insert and vector are ligated according to standard protocols.
The engineered vector could easily be substituted in the above protocol to
express protein in a bacterial system.
Example 6~ Purification of a Polypeotide from an Inclusion Body
The following alternative method can be used to purify a polypeptide expressed
in E toll when it is present in the form of inclusion bodies. Unless otherwise
specified,
all of the following steps are conducted at 4-10°C.
Upon completion of the production phase of the E. toll fermentation, the cell
culture is cooled to 4-10°C and the cells harvested by continuous
centrifugation at
15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein
per unit
weight of cell paste and the amount of purified protein required, an
appropriate amount
of cell paste, by weight, is suspended in a buffer solution containing 100 mM
Tris, 50
mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The
homogenate is
then mixed with NaCI solution to a final concentration of 0.5 M NaCI, followed
by
centrifugation at 7000 xg for 15 min. The resultant pellet is washed again
using 0.5M
NaCI, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine
hydrochloride (GuHCI) for 2-4 hours. After 7000 xg centrifugation for 15 min.,
the
pellet is discarded and the polypeptide containing supernatant is incubated at
4°C
overnight to allow further GuHCI extraction.
Following high speed centrifugation (30,000 xg) to remove insoluble particles,
the GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract
with 20
volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at 4°C
without mixing
for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential
filtration unit equipped with 0.16 ~.t,m membrane filter with appropriate
surface area
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(e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed.
The
filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50,
Perseptive
Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted
with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCI in the same buffer, in a
stepwise manner. The absorbance at 280 nm of the effluent is continuously
monitored.
Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4 volumes
of water. The diluted sample is then loaded onto a previously prepared set of
tandem
columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns are
equilibrated
with 40 mM sodium acetate, pH 6Ø Both columns are washed with 40 mM sodium
acetate, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted using a 10
column
volume linear gradient ranging from 0.2 M NaCI, 50 mM sodium acetate, pH 6.0
to 1.0
M NaCI, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant
AZBo
monitoring of the effluent. Fractions containing the polypeptide (determined,
for
instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the
above
refolding and purification steps. No major contaminant bands should be
observed from
Commassie blue stained 16% SDS-PAGE gel when 5 p.g of purified protein is
loaded.
The purified protein can also be tested for endotoxin/LPS contamination, and
typically
the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 7: Cloning and Expression of a Polypeptide in a Baculovirus
Expression S-ystem
In this example, the plasmid shuttle vector pA2 is used to insert a
polynucleotide
into a baculovirus to express a polypeptide. This expression vector contains
the strong
polyhedrin promoter of the Autographs californica nuclear polyhedrosis virus
(AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and
Asp718. The polyadenylation site of the simian virus 40 ("SV40") is used for
efficient
polyadenylation. For easy selection of recombinant virus, the plasmid contains
the
beta-galactosidase gene from E. coli under control of a weak Drosophila
promoter in the
same orientation, followed by the polyadenylation signal of the polyhedrin
gene. The
inserted genes are flanked on both sides by viral sequences for cell-mediated
homologous recombination with wild-type viral DNA to generate a viable virus
that
express the cloned polynucleotide.
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Many other baculovirus vectors can be used in place of the vector above, such
as pAc373, pVL941, and pAcIIVII, as one skilled in the art would readily
appreciate, as
long as the construct provides appropriately located signals for
transcription,
translation, secretion and the like, including a signal peptide and an in-
frame AUG as
required. Such vectors are described, for instance, in Luckow et al., Virology
170:31-
39 ( 1989).
Specifically, the cDNA sequence contained in the deposited clone, including
the
AUG initiation codon and the naturally associated leader sequence identified
in Table l,
is amplified using the PCR protocol described in Example 1. If the naturally
occurring
signal sequence is used to produce the secreted protein, the pA2 vector does
not need a
second signal peptide. Alternatively, the vector can be modified (pA2 GP) to
include a
baculovirus leader sequence, using the standard methods described in Summers
et aL,
"A Manual of Methods for Baculovirus Vectors and Insect Cell Culture
Procedures,"
Texas Agricultural Experimental Station Bulletin No. 1555 ( 1987).
The amplified fragment is isolated from a 1 % agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is
digested
with appropriate restriction enzymes and again purified on a 1 % agarose gel.
The plasmid is digested with the corresponding restriction enzymes and
optionally, can be dephosphorylated using calf intestinal phosphatase, using
routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4
DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue
(Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the
ligation
mixture and spread on culture plates. Bacteria containing the plasmid are
identified by
digesting DNA from individual colonies and analyzing the digestion product by
gel
electrophoresis. The sequence of the cloned fragment is confirmed by DNA
sequencing.
Five p,g of a plasmid containing the polynucleotide is co-transfected with 1.0
~.g
of a commercially available linearized baculovirus DNA ("BaculoGoldTM
baculovirus
DNA", Pharmingen, San Diego, CA), using the lipofection method described by
Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One p,g of
BacuIoGoldTM virus DNA and 5 p.g of the plasmid are mixed in a sterile well of
a
microtiter plate containing 50 N,l of serum-free Grace's medium (Life
Technologies
Inc., Gaithersburg, MD). Afterwards, 10 p.l Lipofectin plus 90 p.l Grace's
medium are
added, mixed and incubated for 15 minutes at room temperature. Then the
transfection
mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711 ) seeded in a 35
mm
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tissue culture plate with 1 ml Grace's medium without serum. The plate is then
incubated for 5 hours at 27° C. The transfection solution is then
removed from the plate
and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is
added.
Cultivation is then continued at 27° C for four days.
After four days the supernatant is collected and a plaque assay is performed,
as
described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life
Technologies Inc., Gaithersburg) is used to allow easy identification and
isolation of
gal-expressing clones, which produce blue-stained plaques. (A detailed
description of a
"plaque assay" of this type can also be found in the user's guide for insect
cell culture
and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-
10.)
After appropriate incubation, blue stained plaques are picked with the tip of
a
micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses
is then
resuspended in a microcentrifuge tube containing 200 ~,l of Grace's medium and
the
suspension containing the recombinant baculovirus is used to infect Sf9 cells
seeded in
35 mm dishes. Four days later the supernatants of these culture dishes are
harvested
and then they are stored at 4° C.
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's
medium supplemented with 10% heat-inactivated FBS. The cells are infected with
the
recombinant baculovirus containing the polynucleotide at a multiplicity of
infection
("MOI") of about 2. If radiolabeled proteins are desired, 6 hours later the
medium is
removed and is replaced with SF900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Rockville, MD). After 42 hours, 5 p.Ci
of 35S-
methionine and 5 p.Ci 35S-cysteine (available from Amersham) are added. The
cells are
further incubated for 16 hours and then are harvested by centrifugation. The
proteins
in the supernatant as well as the intracellular proteins are analyzed by SDS-
PAGE
followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified
protein may be used to determine the amino terminal sequence of the produced
protein.
Example 8: Expression of a Polypeptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian cell.
A typical mammalian expression vector contains a promoter element, which
mediates
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the initiation of transcription of mRNA, a protein coding sequence, and
signals required
for the termination of transcription and polyadenylation of the transcript.
Additional
elements include enhancers, Kozak sequences and intervening sequences flanked
by
donor and acceptor sites for RNA splicing. Highly efficient transcription is
achieved
with the early and late promoters from SV40, the long terminal repeats (LTRs)
from
Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the
cytomegalovirus
(CMV). However, cellular elements can also be used (e.g., the human actin
promoter).
Suitable expression vectors for use in practicing the present invention
include,
for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBCI2MI (ATCC 67109),
pCMVSport 2.0, and pCMVSport 3Ø Mammalian host cells that could be used
include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos l,
Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO)
cells.
~ Alternatively, the polypeptide can be expressed in stable cell lines
containing the
polynucleotide integrated into a chromosome. The co-transfection with a
selectable
marker such as dhfr, gpt, neomycin, hygromycin allows the identification and
isolation
of the transfected cells.
The transfected gene can also be amplified to express large amounts of the
encoded protein. The DHFR (dihydrofolate reductase) marker is useful in
developing
cell lines that carry several hundred or even several thousand copies of the
gene of
interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978);
Hamlin,
J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J.
and
Sydenham, M. A., Biotechnology 9:64-68 (199I).) Another useful selection
marker is
the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (
1991);
Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the
mammalian cells are grown in selective medium and the cells with the highest
resistance
are selected. These cell lines contain the amplified genes) integrated into a
chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the
expression vectors pC4 {ATCC Accession No. 209646) and pC6 (ATCC Accession
No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen
et
al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of
the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple cloning sites,
e.g.,
with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate
the
cloning of the gene of interest. The vectors also contain the 3' intron, the
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polyadenylation and termination signal of the rat preproinsulin gene, and the
mouse
DHFR gene under control of the SV40 early promoter.
Specifically, the plasmid pC6, for example, is digested with appropriate
restriction enzymes and then dephosphorylated using calf intestinal phosphates
by
procedures known in the art. The vector is then isolated from a 1% agarose
gel.
A polynucleotide of the present invention is amplified according to the
protocol
outlined in Example 1. If the naturally occurring signal sequence is used to
produce the
secreted protein, the vector does not need a second signal peptide.
Alternatively, if the
naturally occurnng signal sequence is not used, the vector can be modified to
include a
heterologous signal sequence. (See, e.g., WO 96/34891.)
The amplified fragment is isolated from a 1 % agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is
digested
with appropriate restriction enzymes and again purified on a 1% agarose gel.
The amplified fragment is then digested with the same restriction enzyme and
purified on a 1 % agarose gel. The isolated fragment and the dephosphorylated
vector
are then ligated with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells are
then
transformed and bacteria are identified that contain the fragment inserted
into plasmid
pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for
transfection. Five ~.g of the expression plasmid pC6 is cotransfected with 0.5
~,g of the
plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that
confers resistance to a group of antibiotics including 6418. The cells are
seeded in
alpha minus MEM supplemented with 1 mg/ml 6418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha
minus
MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml 6418.
After about 10-14 days single clones are trypsinized and then seeded in 6-well
petri
dishes or 10 ml flasks using different concentrations of methotrexate (50 nM,
100 nM,
200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of
methotrexate are then transferred to new 6-well plates containing even higher
concentrations of methotrexate ( 1 EtM, 2 ~t,M, 5 u,M, 10 mM, 20 mM). The same
procedure is repeated until clones are obtained which grow at a concentration
of 100 -
200 ltM. Expression of the desired gene product is analyzed, for instance, by
SDS-
PAGE and Western blot or by reversed phase HPLC analysis.
Exam~,le 9: Protein Fusions
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The polypeptides of the present invention are preferably fused to other
proteins.
These fusion proteins can be used for a variety of applications. For example,
fusion of
the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and
maltose
binding protein facilitates purification. (See Example 5; see also EP A
394,827;
Traunecker, et al.> Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-
3, and
albumin increases the halflife time in vivo. Nuclear localization signals
fused to the
polypeptides of the present invention can target the protein to a specific
subcellular
localization, while covalent heterodimer or homodimers can increase or
decrease the
activity of a fusion protein. Fusion proteins can also create chimeric
molecules having
more than one function. Finally, fusion proteins can increase solubility
andlor stability
of the fused protein compared to the non-fused protein. All of the types of
fusion
proteins described above can be made by modifying the following protocol,
which
outlines the fusion of a polypeptide to an IgG molecule, or the protocol
described in
Example 5.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using
primers that span the 5' and 3' ends of the sequence described below. These
primers
also should have convenient restriction enzyme sites that will facilitate
cloning into an
expression vector, preferably a mammalian expression vector.
For example, if pC4 (Accession No. 209646) is used, the human Fc portion can
be ligated into the BamHI cloning site. Note that the 3' BamHI site should be
destroyed. Next, the vector containing the human Fc portion is re-restricted
with
BamHI, linearizing the vector, and a polynucleotide of the present invention,
isolated
by the PCR protocol described in Example 1, is ligated into this BamHI site.
Note that
the polynucleotide is cloned without a stop codon, otherwise a fusion protein
will not
be produced.
If the naturally occurring signal sequence is used to produce the secreted
protein, pC4 does not need a second signal peptide. Alternatively, if the
naturally
occurring signal sequence is not used, the vector can be modified to include a
heterologous signal sequence. (See, e.g., WO 96/34891.)
Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAA.ACTCACACATGCCCACCGTGCC
CAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGT
GGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG
GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
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AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCA
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGC
GACGGCCGCGACTCTAGAGGAT (SEQ ID NO:1 )
Example 10: Production of an Antibody from a Poly,~g tp ide
The antibodies of the present invention can be prepared by a variety of
methods.
(See, Current Protocols, Chapter 2.) For example, cells expressing a
polypeptide of
the present invention is administered to an animal to induce the production of
sera
containing polyclonal antibodies. In a preferred method, a preparation of the
secreted
protein is prepared and purified to render it substantially free of natural
contaminants.
Such a preparation is then introduced into an animal in order to produce
polyclonal
antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or protein binding fragments thereof). Such monoclonal
antibodies can be prepared using hybridoma technology. (Kohler et al., Nature
256:495 ( 1975); Kohler et al., Eur. J. Immunol. 6:511 ( 1976); Kohler et al.,
Eur. J.
Immunol. 6:292 ( 1976); Hammerling et al., in: Monoclonal Antibodies and T-
Cell
Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures
involve immunizing an animal (preferably a mouse) with polypeptide or, more
preferably, with a secreted polypeptide-expressing cell. Such cells may be
cultured in
any suitable tissue culture medium; however, it is preferable to culture cells
in Earle's
modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated
at
about 56°C), and supplemented with about 10 g/1 of nonessential amino
acids, about
1,000 U/ml of penicillin, and about 100 ~tg/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the ATCC. After fusion, the resulting hybridoma cells
are
3 S selectively maintained in HAT medium, and then cloned by limiting dilution
as
described by Wands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma
cells
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obtained through such a selection are then assayed to identify clones which
secrete
antibodies capable of binding the polypeptide.
Alternatively, additional antibodies capable of binding to the poiypeptide can
be
produced in a two-step procedure using anti-idiotypic antibodies. Such a
method
makes use of the fact that antibodies are themselves antigens, and therefore,
it is
possible to obtain an antibody which binds to a second antibody. In accordance
with
this method, protein specific antibodies are used to immunize an animal,
preferably a
mouse. The splenocytes of such an animal are then used to produce hybridoma
cells,
and the hybridoma cells are screened to identify clones which produce an
antibody
whose ability to bind to the protein-specific antibody can be blocked by the
polypeptide.
Such antibodies comprise anti-idiotypic antibodies to the protein-specific
antibody and
can be used to immunize an animal to induce formation of further protein-
specific
antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the
antibodies
of the present invention may be used according to the methods disclosed
herein. Such
fragments are typically produced by proteolytic cleavage, using enzymes such
as papain
(to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
Alternatively,
secreted protein-binding fragments can be produced through the application of
recombinant DNA technology or through synthetic chemistry.
For in vivo use of antibodies in humans, it may be preferable to use
"humanized" chimeric monoclonal antibodies. Such antibodies can be produced
using
genetic constructs derived from hybridoma cells producing the monoclonal
antibodies
described above. Methods for producing chimeric antibodies are known in the
art.
(See, for review; Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4:214
(1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496;
Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268
(1985).)
Example 11: Production Of Secreted Protein For High-Throug~~ut
Screeninlr Assa~rs
The following protocol produces a supernatant containing a polypeptide to be
tested. This supernatant can then be used in the Screening Assays described in
Examples 13-20.
First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution
(lmg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker)
for a
working solution of 50ug/ml. Add 200 ul of this solution to each well (24 well
plates)
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and incubate at RT for 20 minutes. Be sure to distribute the solution over
each well
(note: a 12-channel pipetter may be used with tips on every other channel).
Aspirate off
the Poly-D-Lysine solution and rinse with lml PBS (Phosphate Buffered Saline).
The
PBS should remain in the well until just prior to plating the cells and plates
may be
poly-lysine coated in advance for up to two weeks.
Plate 293T cells (do not carry cells past P+20) at 2 x 105 cells/well in .5m1
DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine
(12-604F Biowhittaker))/10% heat inactivated FBS(14-503F Biowhittaker)/lx
Penstrep(17-602E Biowhittaker). Let the cells grow overnight.
The next day, mix together in a sterile solution basin: 300 ul Lipofectamine
( 18324-OI2 GibcoBRL) and 5ml Optimem I (31985070 GibcoBRL)/96-well plate.
With a small volume mufti-channel pipetter, aliquot approximately tug of an
expression
vector containing a polynucleotide insert, produced by the methods described
in
Examples 8 or 9, into an appropriately labeled 96-well round bottom plate.
With a
mufti-channel pipetter, add 50u1 of the Lipofectamine/Optimem I mixture to
each well.
Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about
20
minutes, use a mufti-channel pipetter to add 150u1 Optimem I to each well. As
a
control, one plate of vector DNA lacking an insert should be transfected with
each set of
transfections.
Preferably, the transfection should be performed by tag-teaming the following
tasks. By tag-teaming, hands on time is cut in half, and the cells do not
spend too
much time on PBS. First, person A aspirates off the media from four 24-well
plates of
cells, and then person B rinses each well with .5-lml PBS. Person A then
aspirates off
PBS rinse, and person B, using a12-channel pipetter with tips on every other
channel,
adds the ZOOuI of DNA/Lipofectamine/Optimem I complex to the odd wells first,
then to
the even wells, to each row on the 24-well plates. Incubate at 37°C for
6 hours.
While cells are incubating, prepare appropriate media, either 1%BSA in DMEM
with lx penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L
CuS04 5H20; 0.050 mg/L of Fe(N03)3-9H20; 0.417 mg/L of FeS04 7H20; 311.80
mg/L of Kcl; 28.64 mg/L of MgCl2; 48.84 mg/L of MgS04; 6995.50 mg/L of NaCI;
2400.0 mg/L of NaHC03; 62.50 mg/L of NaH2P04 H20; 71.02 mg/L of NazHP04;
.4320 mg/L of ZnS04 7Hz0; .002 mg/L of Arachidonic Acid ; 1.022 mg/L of
Cholesterol; .070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic
Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic
Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; I00 mg/L of
Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of
D-
*rH
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Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50
mg/ml
of L-Asparagine-HzO; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-
2HCL-HZO; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0
mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-
H20; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-
Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0
mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H20; 99.65 mg/ml of L-
Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of
Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L
of
Niacinamide; 3.00 mglL of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319
mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and
0.680 mg/L, of Vitamin B,2; 25 mM of HEPES Buffer; 2.39 mg/L of Na
Hypoxanthine;
0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of
Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20uM of Ethanolamine; 0.122
mg/L of Fernc Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with
Linoleic
Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10
mg/L
of Methyl-B-Cyclodextrin complexed with Retinal) with 2mm glutamine and lx
penstrep. (BSA (81-068-3 Bayer) 100gm dissolved in 1L DMEM for a 10% BSA stock
solution). Filter the media and collect 50 ul for endotoxin assay in l5ml
polystyrene
conical.
The transfection reaction is terminated, preferably by tag-teaming, at the end
of
the incubation period. Person A aspirates off the transfection media, while
person B
adds l.5ml appropriate media to each well. Incubate at 37°C for 45 or
72 hours
depending on the media used: 1 %BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300u1 multichannel pipetter, aliquot 600u1 in one 1m1
deep
well plate and the remaining supernatant into a 2m1 deep well. The
supernatants from
each well can then be used in the assays described in Examples 13-20.
It is specifically understood that when activity is obtained in any of the
assays
described below using a supernatant, the activity originates from either the
polypeptide
directly (e.g., as a secreted protein) or by the polypeptide inducing
expression of other
proteins, which are then secreted into the supernatant. Thus, the invention
further
provides a method of identifying the protein in the supernatant characterized
by an
activity in a particular assay.
FYample 12~ Construction of GAS Reuorter Construct
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One signal transduction pathway involved in the differentiation and
proliferation
of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-
STATs
pathway bind to gamma activation site "GAS" elements or interferon-sensitive
responsive element ("ISRE"), located in the promoter of many genes. The
binding of a
protein to these elements alter the expression of the associated gene.
GAS and ISRE elements are recognized by a class of transcription factors
called
Signal Transducers and Activators of Transcription, or "STATs." There are six
members of the STATs family. Stat 1 and Stat3 are present in many cell types,
as is
Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and
is not in
many cell types though it has been found in T helper class I, cells after
treatment with
IL-12. StatS was originally called mammary growth factor, but has been found
at
higher concentrations in other cells including myeloid cells. It can be
activated in tissue
culture cells by many cytokines.
The STATs are activated to translocate from the cytoplasm to the nucleus upon
tyrosine phosphoryladon by a set of kinases known as the Janus Kinase ("Jaks")
family. Jaks represent a distinct family of soluble tyrosine kinases and
include Tyk2,
Jakl, Jak2, and Jak3. These kinases display significant sequence similarity
and are
generally catalytically inactive in resting cells.
The Jaks are activated by a wide range of receptors summarized in the Table
below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-
51
(1995).) A cytokine receptor family, capable of activating Jaks, is divided
into two
groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL,-7, IL-
9, IL-11, IL-
12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and
(b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a
conserved cysteine motif (a set of four conserved cysteines and one
tryptophan) and a
WSXWS motif (a membrane proxial region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID
N0:2)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn
activate STATs, which then translocate and bind to GAS elements. This entire
process
is encompassed in the Jaks-STATs signal transduction pathway.
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of
the GAS or the ISRE element, can be used to indicate proteins involved in the
proliferation and differentiation of cells. For example, growth factors and
cytokines are
known to activate the Jaks-STATs pathway. (See Table below.) Thus, by using
GAS
elements linked to reporter molecules, activators of the Jaks-STATs pathway
can be
identified.
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JAKs STATS SiASjelementsl or ISRE
Li 13!k2
and
g
_
IFN famil3r
+ - - 1 ISRE
2
3
~_~ + + + - , GAS (IRF 1 >Lys6>IFP)
IFN-g ,
1
ll-10 + ? ? - 1,3
gp130 family
10IL-6 (Pleiotrohic)+ + + ? 1, 3 GAS (IRF 1 >Lys6>IFP)
II-11 (Pleiotrohic)? + ? ? 1, 3
OnM(Pleiotrohic)? + + ? 1, 3
LIF(Pleiotrohic)? + + ? 1, 3
CNTF(Pleiotrohic)-/+ + + ? 1, 3
15G-CSF(Pleiotrohic)? + ? ? 1,3
IL-12(Pleiotrohic)+ - + + 1,3
1
C2
(ly phocytes) - + - + 1,3,5 GAS
L-
20IL-4 (lymph/myeloid) + - + 6 GAS (IRF1 = IFP Ly6)(IgH)
-
IL-7 (lymphocytes)- + - + 5 GAS
IL-9 (lymphocytes)- + - + 5 GAS
IL-13 (lymphocyte)- + ? ? 6 GAS
IL-15 ? + ? + 5 GAS
25
gp140 family
IL-3 (myeloid) - - + - 5 GAS (IRF 1 >IFPLy6)
IL-5 (myeloid) - - + - 5 GAS
GM-CSF (myeloid)- - + - 5 GAS
30
Growth hormone ily
fam
GH ? - + - 5
PRL ? +/- + - 1,3,5
gp0 ? - + - 5 GAS(B-CAS>IRF1=IFPLy6)
35
rosine K inases
Receptor TTx
_ ? + + - 1,3 GAS (IRF1)
EGF
PDGF ? + + - 1,3
CSF-1 ? + + - 1,3 GAS (not IRF1)
40
* rH~
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To construct a synthetic GAS containing promoter element, which is used in the
Biological Assays described in Examples 13-14, a PCR based strategy is
employed to
generate a GAS-SV40 promoter sequence. The 5' primer contains four tandem
copies
of the GAS binding site found in the IRF1 promoter and previously demonstrated
to
S bind STATs upon induction with a range of cytokines (Rothman et al.,
Immunity
1:457-468 (1994).), although other GAS or ISRE elements can be used instead.
The 5'
primer also contains l8bp of sequence complementary to the SV40 early promoter
sequence and is flanked with an XhoI site. The sequence of the 5' primer is:
5' :GCGCCTCGAGATITCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCG
AAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ Ifl N0:3)
The downstream primer is complementary to the SV40 promoter and is flanked
with a Hind III site: 5':GCGGCAAGCZ"I"I"I'I'GCAAAGCCTAGGC:3' (SEQ ID
N0:4)
PCR amplification is performed using the SV40 promoter template present in
the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment
is
digested with Xho1/Hind III and subcloned into BLSK2-. (Stratagene.)
Sequencing
with forward and reverse primers confirms that the insert contains the
following
sequence:
5' : CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATG
ATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCC
CTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGC
CCCATGGCTGACfiAATTI'TTT"TTATTTATGCAGAGGCCGAGGCCGCCTCGGC
CTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTI'ITTI'GGAGGCCTAGGCTTT
TGCAAAA~G~:3' {SEQ ID NO:S)
With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2
reporter construct is next engineered. Here, the reporter molecule is a
secreted alkaline
phosphatase, or "SEAP." Clearly, however, any reporter molecule can be instead
of
SEAP, in this or in any of the other Examples. Well known reporter molecules
that can
be used instead of SEAP include chloramphenicol acetyltransferase (CAT),
luciferase,
alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any
protein
detectable by an antibody.
The above sequence 'confirmed synthetic GAS-SV40 promoter element is
subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII
and
XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40
promoter
element, to create the GAS-SEAP vector. However, this vector does not contain
a
neomycin resistance gene, and therefore, is not preferred for mammalian
expression
systems.
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Thus, in order to generate mammalian stable cell lines expressing the GAS-
SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using
SaII and NotI, and inserted into a backbone vector containing the neomycin
resistance
gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple
cloning
site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into
mammalian cells, this vector can then be used as a reporter molecule for GAS
binding
as described in Examples 13-14.
Other constructs can be made using the above description and replacing GAS
with a different promoter sequence. For example, construction of reporter
molecules
containing NFK-B and EGR promoter sequences are described in Examples 15 and
16.
However, many other promoters can be substituted using the protocols described
in
these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be
substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-
2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test
reporter
construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-
cell),
Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 13~ High-Throughput Screening Assay for T-cell Activity
The following protocol is used to assess T-cell activity by identifying
factors,
such as growth factors and cytokines, that may proliferate or differentiate T-
cells. T-
cell activity is assessed using the GAS/SEAP/Neo construct produced in Example
12.
Thus, factors that increase SEAP activity indicate the ability to activate the
Jaks-STATS
signal transduction pathway. The T-cell used in this assay is Jurkat T-cells
(ATCC
Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552)
and
Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.
Jurkat T-cells are lymphoblastic CD4+ Thl helper cells. In order to generate
stable cell lines, approximately 2 million Jurkat cells are transfected with
the GAS-
SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure
described below). The transfected cells are seeded to a density of
approximately
20,000 cells per well and transfectants resistant to 1 mg/ml genticin
selected. Resistant
colonies are expanded and then tested for their response to increasing
concentrations of
interferon gamma. The dose response of a selected clone is demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 wells
containing 200 ul of cells. Thus, it is either scaled up, or performed in
multiple to
generate sufficient cells for multiple 96 well plates. Jurkat cells are
maintained in RPMI
+ 10% serum with 1 %Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies)
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with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing SO ul
of DMItIE-C and incubate at room temperature for 15-45 rains.
During the incubation period, count cell concentration, spin down the required
number of cells ( 10' per transfection), and resuspend in OPTI-MEM to a final
concentration of 10' cells/ml. Then add 1 ml of 1 x 10' cells in OPTI-MEM to
T25 flask
and incubate at 37°C for 6 hrs. After the incubation, add 10 ml of
ItPMI + 15% serum.
The Jurkat:GAS-SEAP stable reporter lines are maintained in 1ZPMI + 10%
serum, 1 mg/ml Genticin, and 1 % Pen-Strep. These cells are treated with
supernatants
containing a polypeptide as produced by the protocol described in Example 11.
On the day of treatment with the supernatant, the cells should be washed and
resuspended in fresh 12PMI + 10% serum to a density of 500,000 cells per ml.
The
exact number of cells required will depend on the number of supernatants being
screened. For one 96 well plate, approximately 10 million cells (for 10
plates, 100
million cells) are required.
Transfer the cells to a triangular reservoir boat, in order to dispense the
cells into
a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette,
transfer 200 ul
of cells into each well (therefore adding 100, 000 cells per well).
After all the plates have been seeded, 50 ul of the supernatants are
transferred
directly from the 96 well plate containing the supernatants into each well
using a 12
channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0,
10 ng)
is added to wells H9, H 10, and H 11 to serve as additional positive controls
for the
assay.
The 96 well dishes containing Jurkat cells treated with supernatants are
placed in
an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul
samples
from each well are then transferred to an opaque 96 well plate using a 12
channel
pipette. The opaque plates should be covered (using sellophene covers) and
stored at -
20oC until SEAP assays are performed according to Example 17. The plates
containing the remaining treated cells are placed at 4oC and serve as a source
of material
for repeating the assay on a specific well if desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is
known to activate Jurkat T cells. Over 30 fold induction is typically observed
in the
positive control wells.
Fxamnle 14~ Hi -Throughout Screening, Assail Identifying M a
ct'vit
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The following protocol is used to assess myeloid activity by identifying
factors,
such as growth factors and cytokines, that may proliferate or differentiate
myeloid cells.
Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in
Example 12. Thus, factors that increase SEAP activity indicate the ability to
activate the
Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is
U937,
a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced
in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth &
Differentiation, 5:259-265) is used. First, harvest 2x 10e7 U937 cells and
wash with
PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-
inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin
and 100
mg/ml streptomycin.
Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing
0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCI, 5 mM
KCI, 375 uM Na2HP04.7H20, 1 mM MgCl2, and 675 uM CaCl2. Incubate at 37oC
for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then
resuspend in 10 ml complete medium and incubate at 37oC for 36 hr.
The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400
ug/mI 6418. The 6418-free medium is used for routine growth but every one to
two
months, the cells should be re-grown in 400 ug/ml 6418 for couple of passages.
These cells are tested by harvesting 1x108 cells (this is enough for ten 96-
well
plates assay) and wash with PBS. Suspend the cells in 200 ml above described
growth
medium, with a final density of 5x105 cells/ml. Plate 200 ul cells per well in
the 96-
well plate (or 1x105 cells/well).
Add 50 ul of the supernatant prepared by the protocol described in Example 11.
Incubate at 37oC for 48 to 72 hr. As a positive control, 100 Unit/ml
interferon gamma
can be used which is known to activate U937 cells. Over 30 fold induction is
typically
observed in the positive control wells. SEAP assay the supernatant according
to the
protocol described in Example 17.
Example 15: High-Throughout Screening Assay Identifying Neuronal
ctivi
When cells undergo differentiation and proliferation, a group of genes are
activated through many different signal transduction pathways. One of these
genes,
EGR1 (early growth response gene 1), is induced in various tissues and cell
types upon
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activation. The promoter of EGR1 is responsible for such induction. Using the
EGR1
promoter linked to reporter molecules, activation of cells can be assessed.
Particularly, the following protocol is used to assess neuronal activity in PC
12
cell lines. PC 12 cells (rat phenochromocytoma cells) are known to proliferate
and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl
phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth
factor). The
EGR1 gene expression is activated during this treatment. Thus, by stably
transfecting
PC 12 cells with a construct containing an EGR promoter linked to SEAP
reporter,
activation of PC I 2 cells can be assessed.
The EGR/SEAP reporter construct can be assembled by the following protocol.
The EGR-1 promoter sequence (-633 to +1)(Sakamoto K et al., Oncogene 6:867-871
( 1991 )) can be PCR amplified from human genomic DNA using the following
primers:
S' GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ ID N0:6)
S' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3' (SEQ ID N0:7)
Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplified
product can then be inserted into this vector. Linearize the GAS:SEAP/Neo
vector
using restriction enzymes Xho1/HindIII, removing the GAS/SV40 stuffer.
Restrict the
EGRI amplified product with these same enzymes. Ligate the vector and the EGR1
promoter.
To prepare 96 well-plates for cell culture, two mls of a coating solution (
1:30
dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol
(filter
sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well
plate, and
allowed to air dry for 2 hr.
PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)
containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), S% heat-
inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin
and 100
ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split
is done
every three to four days. Cells are removed from the plates by scraping and
resuspended with pipetting up and down for more than 15 times.
Transfect the EGR/SEAP/Neo construct into PC 12 using the Lipofectamine
protocol described in Example 11. EGR-SEAP/PC12 stable cells are obtained by
growing the cells in 300 ug/ml 6418. The 6418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in 300 ug/ml
6418
for couple of passages.
To assay for neuronal activity, a 10 cm plate with cells around 70 to 80%
confluent is screened by removing the old medium. Wash the cells once with PBS
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(Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-
1640
containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
The next morning, remove the medium and wash the cells with PBS. Scrape
off the cells from the plate, suspend the cells well in 2 ml low serum medium.
Count
the cell number and add more low serum medium to reach final cell density as
5x105
cells/ml.
Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to
1x105 cells/well). Add 50 ul supernatant produced by Example 11, 37oC for 48
to 72
hr. As a positive control, a growth factor known to activate PC 12 cells
through EGR
can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold
induction of SEAP is typically seen in the positive control wells. SEAP assay
the
supernatant according to Example 17.
Examgle 16~ High-Throughout Screening Assax for T-cell Activity
NF-xB (Nuclear Factor xB) is a transcription factor activated by a wide
variety
of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40,
lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by
expression of certain viral gene products. As a transcription factor, NF-xB
regulates
the expression of genes involved in immune cell activation, control of
apoptosis (NF-
xB appears to shield cells from apoptosis), B and T-cell development, anti-
viral and
antimicrobial responses, and multiple stress responses.
In non-stimulated conditions, NF- xB is retained in the cytoplasm with I-xB
(Inhibitor xB). However, upon stimulation, I- xB is phosphorylated and
degraded,
causing NF- xB to shuttle to the nucleus, thereby activating transcription of
target
genes. Target genes activated by NF-1cB include 1L-2, IL-6, GM-CSF, ICAM-l and
class 1 MHC.
Due to its central role and ability to respond to a range of stimuli, reporter
constructs utilizing the NF-xB promoter element are used to screen the
supernatants
produced in Example 11. Activators or inhibitors of NF-kB would be useful in
treating
diseases. For example, inhibitors of NF-xB could be used to treat those
diseases
related to the acute or chronic activation of NF-kB, such as rheumatoid
arthritis.
*rE
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To construct a vector containing the NF-tcB promoter element, a PCR based
strategy is employed. The upstream primer contains four tandem copies of the
NF-tcB
binding site (GGGGACTTTCCC) (SEQ ID N0:8), 18 by of sequence complementary
to the 5' end of the SV40 early promoter sequence, and is flanked with an XhoI
site:
S':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGAC
TTTCCATCCTGCCATCTCAATTAG:3' (SEQ ID N0:9)
The downstream primer is complementary to the 3' end of the SV40 promoter
and is flanked with a Hind III site:
5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID N0:4)
PCR amplification is performed using the SV40 promoter template present in
the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment
is
digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene)
Sequencing with the T7 and T3 primers confirms the insert contains the
following
sequence:
5' :CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCC
ATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCA
TCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT
AATIfiI"TTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTC
CAGAAGTAGTGAGGAGGCZTI'T'I"TGGAGGCCTAGGCTTTTGCAAAAAGCTT:
3' (SEQ ID NO:10)
Next, replace the SV40 minimal promoter element present in the pSEAP2-
promoter plasmid (Clontech) with this NF-xB/SV40 fragment using XhoI and
HindIII.
However, this vector does not contain a neomycin resistance gene, and
therefore, is not
preferred for mammalian expression systems.
In order to generate stable mammalian cell lines, the NF-xB/SV40/SEAP
cassette is removed from the above NF-xB/SEAP vector using restriction enzymes
SaII
and NotI, and inserted into a vector containing neomycin resistance.
Particularly, the
NF-tcB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the
GFP
gene, after restricting pGFP-1 with SaII and NotI.
Once NF-oB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are
created and maintained according to the protocol described in Example 13.
Similarly,
the method for assaying supernatants with these stable Jurkat T-cells is also
described
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in Example 13. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is
added to
wells H9, H10, and H11, with a 5-10 fold activation typically observed.
Example 17~ Assa~~ for SEAP Activity
As a reporter molecule for the assays described in Examples 13-16, SEAP
activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according
to the
following general procedure. The Tropix Phospho-light Kit supplies the
Dilution,
Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.Sx Dilution Buffer and dispense 15 ~,l of 2.Sx
dilution buffer into Optiplates containing 35 111 of a supernatant. Seal the
plates with a
plastic sealer and incubate at 65°C for 30 min. Separate the Optiplates
to avoid uneven
heating.
Cool the samples to room temperature for 15 minutes. Empty the dispenser and
prime with the Assay Buffer. Add 50 ~1 Assay Buffer and incubate at room
temperature S min. Empty the dispenser and prime with the Reaction Buffer (see
the
table below). Add 50 ~,1 Reaction Buffer and incubate at room temperature for
20
minutes. Since the intensity of the chemiluminescent signal is time dependent,
and it
takes about 10 minutes to read 5 plates on luminometer, one should treat 5
plates at each
time and start the second set 10 minutes later.
Read the relative light unit in the luminometer. Set H 12 as blank, and print
the
results. An increase in chemiluminescence indicates reporter activity.
Reaction Buffer Formulation:
_# of platesRxn buffer diluentCSPD (ml)
(ml)
i0 60 3
11 65 3.25
12 70 3.5
I3 75 3.75
14 80 4
15 85 4.25
16 90 4.5
17 95 4.75
18 100 5
19 105 5.25
20 110 5.5
2I 115 5.75
22 120 6
23 125 6.25
24 130 6.5
135 6.75
26 140 7
27 145 7.25
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28 150 7.5
29 155 7.75
30 160 8
31 165 8.25
32 170 8.5
33 175 8.75
34 180
35 185 9.25
36 190 9.5
37 195 9.75
38 200 10
39 205 10.25
40 210 10.5
41 215 10.75
42 220 11
43 225 11.25
44 230 11.5
45 235 11.75
46 240 12
47 245 12.25
48 250 12.5
49 255 12.75
50 260 13
Example 18~ High Throyghuut Screening Assay Identifying Changes in
Small Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of
small
molecules, such as calcium, potassium, sodium, and pH, as well as alter
membrane
potential. These alterations can be measured in an assay to identify
supernatants which
bind to receptors of a particular cell. Although the following protocol
describes an
assay for calcium, this protocol can easily be modified to detect changes in
potassium,
sodium, pH, membrane potential, or any other small molecule which is
detectable by a
fluorescent probe.
The following assay uses Fluorometric Imaging Plate Reader ("FLIPR") to
measure changes in fluorescent molecules (Molecular Probes) that bind small
molecules. Clearly, any fluorescent molecule detecting a small molecule can be
used
instead of the calcium fluorescent molecule, fluo-3, used here.
For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-star
black
96-well plate with clear bottom. The plate is incubated in a COZ incubator for
20 hours.
The adherent cells are washed two times in Biotek washer with 200 ul of HBSS
{Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
A stock solution of 1 mg/ml fluo-3 is made in 10% pluronic acid DMSO. To
load the cells with fluo-3, 50 ul of 12 ug/ml fluo-3 is added to each well.
The plate is
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incubated at 37°C in a COZ incubator for 60 min. The plate is washed
four times in the
Biotek washer with HBSS leaving 100 ul of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are
re-suspended to 2-5x106 cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1
mg/ml
fluo-3 solution in 10% piuronic acid DMSO is added to each ml of cell
suspension.
The tube is then placed in a 37°C water bath for 30-60 min. The cells
are washed twice
with HBSS, resuspended to 1x106 cells/ml, and dispensed into a microplate, 100
ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then
washed once
in Denley CellWash with 200 ul, followed by an aspiration step to 100 ul final
volume.
For a non-cell based assay, each well contains a fluorescent molecule, such as
fluo-3. The supernatant is added to the well, and a change in fluorescence is
detected.
To measure the fluorescence of intracellular calcium, the FLIPR is set for the
following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4
second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is
530 nm; and
(6) Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular
signaling event which has resulted in an increase in the intracellular Ca'~"f'
concentration.
Example 19~ High-Throughout Screening Assail Identifvin~ Tyrosine
Kinase Activity
The Protein Tyrosine Kinases (PTK) represent a diverse group of
transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine
Kinase
RPTK) group are receptors for a range of mitogenic and metabolic growth
factors
including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In
addition there are a large family of RPTKs for which the corresponding ligand
is
unknown. Ligands for RPTKs include mainly secreted small proteins, but also
membrane-bound and extracellular matrix proteins.
Activation of RPTK by ligands involves ligand-mediated receptor dimerizadon,
resulting in transphosphorylation of the receptor subunits and activation of
the
cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include
receptor
associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn)
and non-
receptor linked and cytosolic protein tyrosine kinases, such as the Jak
family, members
of which mediate signal transduction triggered by the cytokine superfamily of
receptors
(e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
Because of the wide range of known factors capable of stimulating tyrosine
kinase activity, the identification of novel human secreted proteins capable
of activating
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tyrosine kinase signal transduction pathways are of interest. Therefore, the
following
protocol is designed to identify those novel human secreted proteins capable
of
activating the tyrosine kinase signal transduction pathways.
Seed target cells {e.g., primary keratinocytes) at a density of approximately
25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased
from
Nalge Nunc {Naperville, IL). The plates are sterilized with two 30 minute
rinses with
100% ethanol, rinsed with water and dried overnight. Some plates are coated
for 2 hr
with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or
polylysine
(50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO)
or
10% Matrigel purchased from Becton Dickinson (Bedford,MA), or calf serum,
rinsed
with PBS and stored at 4oC. Cell growth on these plates is assayed by seeding
5,000
cells/well in growth medium and indirect quantitation of cell number through
use of
alamarBlue as described by the manufacturer Alamar Biosciences, Inc.
(Sacramento,
CA) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford,MA)
are
used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell
culture
plates can also be used in some proliferation experiments.
To prepare extracts; A431 cells are seeded onto the nylon membranes of
Loprodyne plates (20,000/200m1/well) and cultured overnight in complete
medium.
Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-
20
minutes treatment with EGF (60ng/ml) or 50 ul of the supernatant produced in
Example
11, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH
7.5, 0.15 M NaCI, 1% Triton X-100, 0.1% SDS, 2 mM Na3V04, 2 mM Na4P2O7
and a cocktail of protease inhibitors (# 1836170) obtained from Boeheringer
Mannheim
(Indianapolis, IN) is added to each well and the plate is shaken on a rotating
shaker for
5 minutes at 4oC. The plate is then placed in a vacuum transfer manifold and
the extract
filtered through the 0.45 mm membrane bottoms of each well using house vacuum.
Extracts are collected in a 96-well catch/assay plate in the bottom of the
vacuum
manifold and immediately placed on ice. To obtain extracts clarified by
centrifugation,
the content of each well, after detergent solubilization for 5 minutes, is
removed and
centrifuged for 15 minutes at 4oC at 16,000 x g
Test the filtered extracts for levels of tyrosine kinase activity. Although
many
methods of detecting tyrosine kinase activity are known, one method is
described here.
Generally, the tyrosine kinase activity of a supernatant is evaluated by
determining its ability to phosphorylate a tyrosine residue on a specific
substrate (a
biotinylated peptide). Biotinylated peptides that can be used for this purpose
include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34)
and
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PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are
substrates for
a range of tyrosine kinases and are available from Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in
order. First, add 10u1 of 5uM Biotinylated Peptide, then l0ul ATP/Mg2+ (5mM
ATP/50mM MgCl2), then 10u1 of 5x Assay Buffer (40mM imidazole hydrochloride,
pH7.3, 40 mM beta-glycerophosphate, 1mM EGTA, 100mM MgCl2, 5 mM MnCl2,
0.5 mg/ml BSA), then 5ul of Sodium Vanadate(1mM), and then 5u1 of water. Mix
the
components gently and preincubate the reaction mix at 30oC for 2 min. Initial
the
reaction by adding 10u1 of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120mm
EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of
reaction
mixture to a microtiter plate (MTP) module and incubating at 37oC for 20 min.
This
allows the streptavadin coated 96 well plate to associate with the
biotinylated peptide.
Wash the MTP module with 300u1/well of PBS four times. Next add 75 ul of anti-
phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-
POD(0.5u/ml)) to each well and incubate at 37oC for one hour. Wash the well as
above.
Next add 100u1 of peroxidase substrate solution (Boehringer Mannheim) and
incubate at room temperature for at least 5 rains (up to 30 min). Measure the
absorbance of the sample at 405 nm by using ELISA reader. The level of bound
peroxidase activity is quantitated using an ELISA reader and reflects the
level of
tyrosine kinase activity.
Example 20~ High-Throughput Screening Assav Identifying
Phos~horyiation Activity
As a potential alternative and/or compliment to the assay of protein tyrosine
kinase activity described in Example 19, an assay which detects activation
(phosphorylation) of major intracellular signal transduction intermediates can
also be
used. For example, as described below one particular assay can detect tyrosine
phosphorylation of the Erk-l and Erk-2 kinases. However, phosphorylation of
other
molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase,
Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other
phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected
by
substituting these molecules for Erk-1 or Erk-2 in the following assay.
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Specifically, assay plates are made by coating the wells of a 96-well ELISA
plate with O.lml of protein G (lug/ml) for 2 hr at room temp, (RT). The plates
are then
rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G
plates
are then treated with 2 commercial monoclonal antibodies ( 100ng/well) against
Erk-1
and Erk-2 ( 1 hr at RT) (Santa Cruz Biotechnology). (To detect other
molecules, this
step can easily be modified by substituting a monoclonal antibody detecting
any of the
above described molecules.) After 3-5 rinses with PBS, the plates are stored
at 4oC
until use.
A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and
cultured overnight in growth medium. The cells are then starved for 48 hr in
basal
medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the
supernatants
obtained in Example 11 for 5-20 minutes. The cells are then solubilized and
extracts
filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed.
As a
positive control, a commercial preparation of MAP kinase ( lOng/well) is used
in place
of A431 extract. Plates are then treated with a commercial polyclonal (rabbit)
antibody
(lug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1
and
Erk-2 kinases ( 1 hr at RT). This antibody is biotinylated by standard
procedures. The
bound polyclonal antibody is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent in the
Wallac
DELFIA instrument (time-resolved fluorescence). An increased fluorescent
signal over
background indicates a phosphorylation.
Example 21 ~ Method of Determining Alterations in a Gene
Corresponding to a Polynucleotide
RNA isolated from entire families or individual patients presenting with a
phenotype of interest (such as a disease) is be isolated. cDNA is then
generated from
these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA
is
then used as a template for PCR, employing primers surrounding regions of
interest in
SEQ ID NO:X. Suggested PCR conditions consist of 35 cycles at 95°C
for 30
seconds; 60-120 seconds at 52-58°C; and 60-120 seconds at 70°C,
using buffer
solutions described in Sidransky, D., et al., Science 252:706 ( 1991 ).
PCR products are then sequenced using primers labeled at their 5' end with T4
polynucleodde kinase, employing SequiTherm Polymerase. (Epicentre
Technologies).
The intron-exon borders of selected exons is also determined and genomic PCR
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products analyzed to confirm the results. PCR products harboring suspected
mutations
is then cloned and sequenced to validate the results of the direct sequencing.
PCR products is cloned into T-tailed vectors as described in Holton, T.A. and
Graham, M.W., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7
polymerase (United States Biochemical). Affected individuals are identified by
mutations not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining
alterations in a gene corresponding tv a polynucleotide. Genomic clones
isolated
according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5'-
triphosphate (Boehringer Manheim), and FISH performed as described in Johnson,
Cg. et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled
probe is
carried out using a vast excess of human cot-1 DNA for specific hybridization
to the
corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and
propidium iodide, producing a combination of C- and R-bands. Aligned images
for
precise mapping are obtained using a triple-band filter set (Chroma
Technology,
Brattleboro, VT) in combination with a cooled charge-coupled device camera
(Photometrics, Tucson, AZ) and variable excitation wavelength filters.
(Johnson, Cv.
et al., Genet. Anal. Tech. Appl., 8:75 ( 1991 ).) Image collection, analysis
and
chromosomal fractional length measurements are performed using the ISee
Graphical
Program System. (Inovision Corporation, Durham, NC.) Chromosome alterations of
the genomic region hybridized by the probe are identified as insertions,
deletions, and
translocations. These alterations are used as a diagnostic marker for an
associated
disease.
~xamgle 22~ Method of Detecting Abnormal Levels of a Poly~eptide in a
Biological Sample
A polypeptide of the present invention can be detected in a biological sample,
and if an increased or decreased level of the polypeptide is detected, this
polypepdde is
a marker for a particular phenotype. Methods of detection are numerous, and
thus, it is
understood that one skilled in the art can modify the following assay to fit
their
particular needs.
For example, antibody-sandwich ELISAs are used to detect polypeptides in a
sample, preferably a biological sample. Wells of a microtiter plate are coated
with
specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The
antibodies are either
monoclonal or polyclonal and are produced by the method described in Example
10.
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The wells are blocked so that non-specific binding of the polypeptide to the
well is
reduced.
The coated wells are then incubated for > 2 hours at RT with a sample
containing the polypeptide. Preferably, serial dilutions of the sample should
be used to
validate results. The plates are then washed three times with deionized or
distilled water
to remove unbounded polypeptide.
Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a
concentration of 25-400 ng, is added and incubated for 2 hours at room
temperature.
The plates are again washed three times with deionized or distilled water to
remove
unbounded conjugate.
Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl
phosphate (NPP) substrate solution to each well and incubate 1 hour at room
temperature. Measure the reaction by a microtiter plate reader. Prepare a
standard
curve, using serial dilutions of a control sample, and plot polypeptide
concentration on
the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear
scale).
Interpolate the concentration of the polypeptide in the sample using the
standard curve.
xamnip 23~ Formulating a Polypeptide
The secreted polypeptide composition will be formulated and dosed in a fashion
consistent with good medical practice, taking into account the clinical
condition of the
individual patient (especially the side effects of treatment with the secreted
polypeptide
alone), the site of delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The "effective
amount" for
purposes herein is thus determined by such considerations.
As a general proposition, the total pharmaceutically effective amount of
secreted
polypeptide administered parenterally per dose will be in the range of about 1
p,g/kg/day
to 10 mg/kg/day of patient body weight, although, as noted above, this will be
subject
to therapeutic discretion. More preferably, this dose is at least 0.01
mg/kg/day, and
most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
If
given continuously, the secreted polypeptide is typically administered at a
dose rate of
about 1 pg/kg/hour to about 50 wg/kg/hour, either by 1-4 injections per day or
by
continuous subcutaneous infusions, for example, using a mini-pump. An
intravenous
bag solution may also be employed. The length of treatment needed to observe
changes
and the interval following treatment for responses to occur appears to vary
depending
on the desired effect.
Pharmaceutical compositions containing the secreted protein of the invention
are
administered orally, rectally, parenterally, intracistemally, intravaginally,
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intraperitoneally, topically (as by powders, ointments, gels, drops or
transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically acceptable
carrier" refers
to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating
material or
formulation auxiliary of any type. The term "parenteral" as used herein refers
to modes
of administration which include intravenous, intramuscular, intraperitoneal,
intrasternal,
subcutaneous and intraarticular injection and infusion.
The secreted polypeptide is also suitably administered by sustained-release
systems. Suitable examples of sustained-release compositions include semi-
permeable
polymer matrices in the form of shaped articles, e.g., films, or mirocapsules.
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP
58,481 ),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al.,
Biopolymers 22:547-556 (1983)), poly (2- hydroxyethyl methacrylate) {R. Langer
et
al., J. Biomed. Mater. Res. 15:167-277 ( 1981 ), and R. Langer, Chem. Tech.
12:98-
105 {1982)), ethylene vinyl acetate (R. Langer et al.) or poly-D- (-)-3-
hydroxybutyric
acid (EP 133,988). Sustained-release compositions also include liposomally
entrapped
polypeptides. Liposomes containing the secreted polypeptide are prepared by
methods
known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-
3692
(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP
52,322;
EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008;
U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the
liposomes
are of the small (about 200-800 Angstroms) unilamellar type in which the lipid
content
is greater than about 30 mol. percent cholesterol, the selected proportion
being adjusted
far the optimal secreted polypeptide therapy.
For parenteral administration, in one embodiment, the secreted polypeptide is
formulated generally by mixing it at the desired degree of purity, in a unit
dosage
injectable form (solution, suspension, or emulsion), with a pharmaceutically
acceptable
carrier, i.e., one that is non-toxic to recipients at the dosages and
concentrations
employed and is compatible with other ingredients of the formulation. For
example, the
formulation preferably does not include oxidizing agents and other compounds
that are
known to be deleterious to polypeptides.
Generally, the formulations are prepared by contacting the polypeptide
uniformly and intimately with liquid carriers or finely divided solid Garners
or both.
Then, if necessary, the product is shaped into the desired formulation.
Preferably the
carrier is a parenteral carrier, more preferably a solution that is isotonic
with the blood
of the recipient. Examples of such carrier vehicles include water, saline,
Ringer's
solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and
ethyl
oleate are also useful herein, as well as liposomes.
* rE~
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The carrier suitably contains minor amounts of additives such as substances
that
enhance isotonicity and chemical stability. Such materials are non-toxic to
recipients at
the dosages and concentrations employed, and include buffers such as
phosphate,
citrate, succinate, acetic acid, and other organic acids or their salts;
antioxidants such as
ascorbic acid; low molecular weight (less than about ten residues)
polypeptides, e.g.,
polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids,
such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its derivatives,
glucose,
manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or
sorbitol; counterions such as sodium; and/or nonionic surfactants such as
polysorbates,
poloxamers, or PEG.
The secreted polypeptide is typically formulated in such vehicles at a
concentration of about 0.1 mg/ml to 100 mg/mI, preferably 1-10 mg/ml, at a pH
of
about 3 to 8. It will be understood that the use of certain of the foregoing
excipients,
carriers, or stabilizers will result in the formation of polypeptide salts.
Any polypeptide to be used for therapeutic administration can be sterile.
Sterility is readily accomplished by filtration through sterile filtration
membranes (e.g.,
0.2 micron membranes). Therapeutic polypeptide compositions generally are
placed
into a container having a sterile access port, for example, an intravenous
solution bag or
vial having a stopper pierceable by a hypodermic injection needle.
Polypeptides ordinarily will be stored in unit or mufti-dose containers, for
example, sealed ampoules or vials, as an aqueous solution or as a lyophilized
formulation for reconstitution. As an example of a lyophilized formulation, 10-
ml vials
are filled with 5 ml of sterile-filtered 1 % (w/v) aqueous polypeptide
solution, and the
resulting mixture is lyophilized. The infusion solution is prepared by
reconstituting the
lyophilized polypeptide using bacteriostatic Water-for-Injection.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Associated with such containers) can be a
notice in the
form prescribed by a governmental agency regulating the manufacture, use or
sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency of
manufacture, use or sale for human administration. In addition, the
polypeptides of the
present invention may be employed in conjunction with other therapeutic
compounds.
~~~le 24~ Method of Treating Decreased Levels of the Polvoeptide
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It will be appreciated that conditions caused by a decrease in the standard or
normal expression level of a secreted protein in an individual can be treated
by
administering the polypeptide of the present invention, preferably in the
secreted form.
Thus, the invention also provides a method of treatment of an individual in
need of an
increased level of the polypeptide comprising administering to such an
individual a
pharmaceutical composition comprising an amount of the polypeptide to increase
the
activity level of the polypeptide in such an individual.
For example, a patient with decreased levels of a polypepdde receives a daily
dose 0.1-100 ug/kg of the polypeptide for six consecutive days. Preferably,
the
polypeptide is in the secreted form. The exact details of the dosing scheme,
based on
administration and formulation, are provided in Example 23.
Example 25~ Method of Treating Increased Levels of the Polvpeptide
Antisense technology is used to inhibit production of a polypeptide of the
present invention. This technology is one example of a method of decreasing
levels of
a polypeptide, preferably a secreted form, due to a variety of etiologies,
such as cancer.
For example, a patient diagnosed with abnormally increased levels of a
polypeptide is administered intravenously antisense polynucleotides at 0.5,
1.0, 1.5,
2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day
rest period
if the treatment was well tolerated. The formulation of the antisense
polynucleotide is
provided in Example 23.
Example 26 Method of Treatment Using Gene Therapy
One method of gene therapy transplants fibroblasts, which are capable of
expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained
from a
subject by skin biopsy. The resulting tissue is placed in tissue-culture
medium and
separated into small pieces. Small chunks of the tissue are placed on a wet
surface of a
tissue culture flask, approximately ten pieces are placed in each flask. The
flask is
turned upside down, closed tight and left at room temperature over night.
After 24
hours at room temperature, the flask is inverted and the chunks of tissue
remain fixed to
the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS,
penicillin and streptomycin) is added. The flasks are then incubated at
37°C for
approximately one week.
At this time, fresh media is added and subsequently changed every several days
After an additional two weeks in culture, a monolayer of fibroblasts emerge.
The
monolayer is trypsinized and scaled into larger flasks.
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pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)), flanked by the long
terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI
and
HindIII and subsequently treated with calf intestinal phosphatase. The linear
vector is
fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention can be amplified
using PCR primers which correspond to the 5' and 3' end sequences respectively
as set
forth in Example 1. Preferably, the 5' primer contains an EcoRI site and the
3' primer
includes a HindllI site. Equal quantities of the Moloney murine sarcoma virus
linear
backbone and the amplified EcoRI and HindIII fragment are added together, in
the
presence of T4 DNA ligase. The resulting mixture is maintained under
conditions
appropriate for ligation of the two fragments. The ligation mixture is then
used to
transform bacteria HB 101, which are then plated onto agar containing
kanamycin for
the purpose of confirming that the vector has the gene of interest properly
inserted.
The amphotropic pA317 or GP+am 12 packaging cells are grown in tissue
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with
10%
calf serum (CS), penicillin and streptomycin. The MSV vector containing the
gene is
then added to the media and the packaging cells transduced with the vector.
The
packaging cells now produce infectious viral particles containing the gene
(the
packaging cells are now referred to as producer cells).
Fresh media is added to the transduced producer cells, and subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the media
from the producer cells. This media is removed and replaced with fresh media.
If the
titer of virus is high, then virtually all fibroblasts will be infected and no
selection is
required. If the titer is very low, then it is necessary to use a retroviral
vector that has a
selectable marker, such as neo or his. Once the fibroblasts have been
efficiently
infected, the fibroblasts are analyzed to determine whether protein is
produced.
The engineered fibroblasts are then transplanted onto the host, either alone
or
after having been grown to confluence on cytodex 3 microcarrier beads.
Exam~,le 27~ Method of Treatment Using gene Theranv - In Vivo
Another aspect of the present invention is using in vivo gene therapy
methods to treat disorders, diseases and conditions. The gene therapy method
relates to the introduction of naked nucleic acid (DNA, RNA, and antisense
DNA or RNA) sequences into an animal to increase or decrease the expression
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158
of the polypeptide of the present invention. A polynucleotide of the present
invention may be operatively linked to a promoter or any other generic
elements
necessary for the expression of the encoded polypeptide by the target tissue.
Such gene therapy and delivery techniques and methods are known in the art,
see, for example, W090111092, W098111779; U.S. Patent NO. 5693622,
5705151, 5580859; Tabata H. et al. (1997) Cardiovasc. Res. 35(3):470-479,
Chao J et al. (1997) Pharmacol. Res. 35(6):517-522, Wolff J.A. (1997)
Neuromuscul. Disord. 7(5):314-318, Schwartz B. et al. (1996) Gene Ther.
3(5):405-411, Tsurumi Y. et al. (1996) Circulation 94(12):3281-3290
(incorporated herein by reference).
The polynucleotide constructs of the present invention may be delivered
by any method that delivers injectable materials to the cells of an animal,
such
as, injection into the interstitial space of tissues (heart, muscle, skin,
lung, liver,
intestine and the like). These polynucleotide constructs can be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
The term "naked" polynucleotide, DNA or RNA, refers to sequences
that are free from any delivery vehicle that acts to assist, promote, or
facilitate
entry into the cell, including viral sequences, viral particles, liposome
formulations, lipofectin or precipitating agents and the like. However, the
polynucleotides may also be delivered in liposome formulations (such as those
taught in Felgner P.L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and
Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by
methods well known to those skilled in the art.
The poiynucleotide vector constructs of the present invention used in
the gene therapy method are preferably constructs that will not integrate into
the
host genome nor will they contain sequences that allow for replication. Any
strong promoter known to those skilled in the art can be used for driving the
expression of DNA. Unlike other gene therapies techniques, one major
advantage of introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells. Studies have
shown that non-replicating DNA sequences can be introduced into cells to
provide production of the desired polypeptide for periods of up to six months.
The polynucleotide construct of the present invention can be delivered to
the interstitial space of tissues within the an animal, including of muscle,
skin,
brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,
cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary,
uterus, rectum, nervous system, eye, gland, and connective tissue.
Interstitial
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159
space of the tissues comprises the intercellular fluid, mucopolysaccharide
matrix
among the reticular fibers of organ tissues, elastic fibers in the walls of
vessels or
chambers, collagen fibers of fibrous tissues, or that same matrix within
connective tissue ensheathing muscle cells or in the lacunae of bone. It is
similarly the space occupied by the plasma of the circulation and the lymph
fluid
of the lymphatic channels. Delivery to the interstitial space of muscle tissue
is
preferred for the reasons discussed below. They may be conveniently delivered
by injection into the tissues comprising these cells. They are preferably
delivered
to and expressed in persistent, non-dividing cells which are differentiated,
although delivery and expression may be achieved in non-differentiated or less
completely differentiated cells, such as, for example, stem cells of blood or
skin
fibroblasts. In vivo muscle cells are particularly competent in their ability
to take
up and express polynucleotides.
For the naked polynucleotide injection, an effective dosage amount of
DNA or RNA will be in the range of from about 0.05 g/kg body weight to about
50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg
to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg.
Of course, as the artisan of ordinary skill will appreciate, this dosage will
vary
according to the tissue site of injection. The appropriate and effective
dosage of
nucleic acid sequence can readily be determined by those of ordinary skill in
the
art and may depend on the condition being treated and the route of
administration. The preferred route of administration is by the parenteral
route of
injection into the interstitial space of tissues. However, other parenteral
routes
may also be used, such as, inhalation of an aerosol formulation particularly
for
delivery to lungs or bronchial tissues, throat or mucous membranes of the
nose.
In addition, naked polynucleotide constructs can be delivered to arteries
during
angioplasty by the catheter used in the procedure.
The dose response effects of injected polynucleotide in muscle in vivo is
determined as follows. Suitable template DNA for production of mRNA coding
for the polypepdde of the present invention is prepared in accordance with a
standard recombinant DNA methodology. The template DNA, which may be
either circular or linear, is either used as naked DNA or complexed with
liposomes. The quadriceps muscles of mice are then injected with various
amounts of the template DNA.
Five to six week old female and male Balb/C mice are anesthetized by
intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is
made
on the anterior thigh, and the quadriceps muscle is directly visualized. The
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160
template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27
gauge
needle over one minute, approximately 0.5 cm from the distal insertion site of
the
muscle into the knee and about 0.2 cm deep. A suture is placed over the
injection site for future localization, and the skin is closed with stainless
steel
clips.
After an appropriate incubation time (e.g., 7 days) muscle extracts are
prepared
by excising the entire quadriceps. Every fifth 15 um cross-section of the
individual
quadriceps muscles is histochemically stained for protein expression. A time
course for
protein expression may be done in a similar fashion except that quadriceps
from
different mice are harvested at different times. Persistence of DNA in muscle
following
injection may be determined by Southern blot analysis after preparing total
cellular DNA
and HIRT supernatants from injected and control mice. The results of the above
experimentation in mice can be use to extrapolate proper dosages and other
treatment
parameters in humans and other animals using naked DNA of the present
invention.
It will be clear that the invention may be practiced otherwise than as
particularly
described in the foregoing description and examples. Numerous modifications
and
variations of the present invention are possible in light of the above
teachings and,
therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, laboratory manuals, books, or other
disclosures) in the Background of the Invention, Detailed Description, and
Examples is
hereby incorporated herein by reference.
Further, the Sequence Listing submitted herewith in paper and computer
readable forms are herein incorporated by reference in their entireties.
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below relate
to the microorganism referred
to in the description
on page 79 , line N/A
B. IDENTIFICAT10N OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
American Type Culture Collection
Address of depositary institution
(including postal code and country)
10801 University Boulevard
Mantissas, Virginia 20110-2209
United States of America
Date of deposit July 3, 1997 Accession Number 209138
C. ADDIT10NAL INDICATIONS (leave
blank if not applicable) This
information is continued on an
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are not jar all designated states)
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peave blank ijnot applicable)
The indications listed below will
be submitted to the International
Bureau later (specify the general
nature ojthe indications, e.g..
'Accession
Number ojDeposit'~
For receiving Office use only ~,~~.~ r~ For International Bureau use only
~s sheet was received with the international application ~ ~ ~ This sheet was
received by the International Bureau on:
Authorized officer
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below relate
to the microorganism referred
to in the description
on page 80 , line N/A
IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
B.
Name of depositary institution
American Type Culture Collection
Address of depositary institution
(including postal code and country)
10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date of deposit July 3, 1997 Accession Number 209139
C. ADDITIONAL INDICATIONS peave
blank fnor applicable This information
is continued on an additional
sheet
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ljehe indications
are not jot all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ijnor applicable)
The indications listed below will
be submitted to the International
Bureau later (sped the general
nature ojthe indications, e.g..
"Accession
Number ojDeposit'~
C.... L.~e..n.~rinnol rZttrPatt
tteP (lnlV
For receW ng vrnce use onry ~~ r~---= ~a =~».
his sheet was received with the international application ~ ~ ~ This sheet was
received by the International 9uresu on'
Authorized officer Authorized officer
AA.AAA.fL.C~
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below relate
to the microorganism referred
to in the description
on page 82 , line N/A
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an
additional sheet
Name of depositary institution
American Type Culture Collection
Address of depositary institution
(including pasta! code and country)
10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date of deposit July 9, 1997 ~ Accession Number 209141
C. ADDITIONAL INDICATIONS (leave
blank fnot applicable) This information
is continued on an additional
sheet -
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (Irthe indications
are norfor au designated states)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not appltcable)
The indications listed below will
be submitted to the lntemational
Bureau later (sped the general
nature of the indications, e.g.,
'Accession
Number of Deposit's
For receiving Office use only .~~ r~ For lntetnattonal Jiureau use only
his sheet was received with the international application ~ ~ ~ This sheet was
received by the lntemational Bureau on
Authorized officer
CA 02296815 2000-O1-14
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<110> Human Genome Sciences, Inc.
<120> 64 Human Secreted Proteins
<130> PZO11PCT
<140> Unassigned
<141> 1998-07-15
<150> 60/052,870
<151> 1997-07-16
<150> 60/052,871
<151> 1997-07-16
<150> 60/052,872
<151> 1997-07-16
<150> 60/052,661
<151> 1997-07-16
<150> 60/052,874
<151> 1997-07-16
<150> 60/052,873
<151> 1997-07-16
<150> 60/052,875
<151> 1997-0?-16
<150> 60/053,440
<151> 1997-07-22
<150> 60/053,441
<151> 1997-07-22
<150> 60/053,442
<151> 1997-07-22
<150> 60/055,952
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<150> 60/055,725
<151> 1997-08-18
<150> 60/056,359
<151> 1997-08-18
SO <150> 60/055,985
<151> 1997-0$-18
<150> 60/055,724
<151> 1997-08-18
CA 02296815 2000-O1-14
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<150> 60/055,726
<151> 1997-OS-18
<150> 60/056,361
<151> 1997-08-18
<150> 60/055,989
<151> 1997-08-18
<150> 60/055,946
<151> 1997-08-18
<150> 60/055,683
<151> 1997-08-18
<160> 178
<170> PatentIn Ver. 2.0
<210>
1
<211>
733
<212>
DNA
<213> Sapiens
Homo
<400>
1
gggatccggagcccaaatcttctgacaaaactcacacatgcccaccgtgcccagcacctg60
aattcgagggtgcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatga120
tctcccggactcctgaggtcacatgcgtggtggtggacgtaagccacgaagaccctgagg180
tcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcggg240
aggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggact300
ggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccaacccccatcg360
agaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc420
catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttct480
atccaagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaaga540
ccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtgg600
acaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgc660
acaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtgcgacggccgc720
gactctagaggat 733
<210>
2
<211>
5
<212>
PRT
4$ <213> Sapiens
Homo
<220>
<221>
Site
<222>
(3)
$0 <223> equals one twenty ing L-aminoacids
Xaa any of naturally
the ocurr
<400>
2
Trp Ser
Xaa
Trp
Ser
1 5
55
<210>
3
<211>
86
<212>
DTIA
CA 02296815 2000-O1-14
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3
<213> Homo sapiens
<400> 3
gcgcctcgag atttccccga aatctagatt tccccgaaat gatttccccg aaatgatttc 60
cccgaaatat ctgccatctc aattag 86
<210>
4
<211>
27
<212>
DNA
<213> Sapiens
Homo
<400>
4
gcggcaagctttttgcaaagcctaggc 27
<210>
5
<211>
271
<212>
~1P.
<213> Sapiens
Homo
<400>
5
ctcgagatttccccgaaatctagatttccccgaaatgatttccccgaaat gatttccccg60
aaatatctgccatctcaattagtcagcaaccatagtcccgcccctaactc cgcccatccc120
gcccctaactccgcccagttccgcccattctccgccccatggctgactaa ttttttttat180
ttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagt gaggaggctt240
ttttggaggcctaggcttttgcaaaaagctt 271
<210> s
<211> 32
<212> DNA
<213> Homo Sapiens
<400> 6
gcgctcgagg gatgacagcg atagaacccc gg 32
<210> 7
<211> 31
<212> DNA
<213> Homo Sapiens
<400> 7
gcgaagcttc gcgactcccc ggatccgcct c 31
<210> 8
<211> 12
<212> DI3A
<213> Homo Sapiens
<400> 8
12
ggggactttc cc
<210> 9
<211> 73
<212> DNA
()0 <213> Homo sapiens
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<400> 9
gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg 60
ccatctcaat tag 73
<210> 10
<211> 256
<212> DNA
<213> Homo Sapiens
<400> 10
ctcgagggga ctttcccggggactttccggggactttccgggactttcca tctgccatct60
caattagtca gcaaccatagtcccgcccctaactccgcccatcccgcccc taactccgcc120
IS cagttccgcc cattctccgccccatggctgactaattttttttatttatg cagaggccga180
ggccgcctcg gcctctgagctattccagaagtagtgaggaggcttttttg gaggcctagg240
cttttgcaaa aagctt 256
<210> 11
<211> 558
<212> ~1A
<213> Homo Sapiens
<220>
<221> SITE
<222> (546)
<223> n equals a,t,g, or c
<400> 11
gaattcggca cgagctgggctgcagttggcgattccgcgcggtgaaagcagccagtgccc60
agggtctttt cctgagtgcacctgggcctgccgcccggcgatgccatggggtcgtgcgct120
gcttttctac ttgccgcgctctcactgctcggtgtactgggagggtaccctgggaggcgt180
gcctttattc ttccgaaccgccgctcactgagacagtggctagaagtgtctcttggacct240
gtgagttagccttaacctgttatgcccccagagccctcagtggagcgcccgtactttgcc300
ggcatgacgt ttgatttcccggtgataatccgacgagtttgacagattgaggtagtgagc360
aaagttgccc gtcagttggtggccacttgacttcgtgcggaccctggccttgctcttgga420
agagatagtg ttcttagggctggtttcactgtctcttaagactgaarggtggarctggga480
tatagatgtg ttgtttcttttcaaatcaaacctgcttargtcgtcactcgaaggggggcc540
cggtanccaattcgccct 558
<210> 12
<211> 715
<z12> DNA
<213> Homo Sapiens
<400>
12
cggatttcgagtgcttttctccttacctccaccctcccccatgttttaatgcagccctcc60
aaaaatatttactgagtgtggactctagaccagggcctgtgctaggatacaaagatgaat120
gaggcaccacccttatcttcgagtagtatatgttttattttattttatttttttcccctg180
ctgcctcccttgagtagtacatgttttagtaaggggaacagacactaaagagtcctggta240
atgatgagcaaagtactgcatgagtaagtatctggggggcaagtgtccccactaggactc300
ctgtcagatctggaaaaggcctgaggaatctgatacatgacttaatgcagcgtatacttg360
cagcctggaaaactaagtaatgacaaaatagacattcttgtcagtgtgagccattctctg420
agtccmaggggagtacataattcaaaccagaattggtcattttggagtttgcactcttag480
cagtatacagtggagtgaaatttaagaatcaatttaatttcttttcagtttttatgtaca540
taaaacctgcttactacaagagacccagtttattattttgtgttggttaacattcataag600
tatatttcatcataataaggctccgtgaaattagtcattttatcatttgccaataaagac660
()0atatatctgaaaataaatgttcctgaacctgaaaaaaaaaaaaaaaaaaactcga 715
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<210> 13
<211> 838
5 <212> DNA
<213> Homo Sapiens
<400>
13
gaattcggcacgagccaaaacaaaagaaacctttggaggcatgtgtcagaacagagaaag60
tgtcctggttttgcttatagaatcaaatatgttctcattctacctactgttttcattcta120
catagtgttttccttctttatagttttacgtcctcttcctaggaatgagtctattaagaa180
aataggtgttatcttttagctttggcatttgactttcaggataatagagctatctgctac240
tgacagaaaagctttgacaagtgtttaatactctgggattaccttcatcttacttttgca300
atcattatgtgaacattgtcttccgtccacatctayaggctagtawgtaacaccgttgac360
taaatccaaactttaggctagggaaaaagggtatactttctgggtttcggttgtagatta420
tgtttagatctaaycaaaacaggacagtggtccaaacagaaaattgctattttctgtatc480
ttgtaaatctaggatttgagtttttaagatgaatttatggttccctttctgatatcattt540
ctcatctgcagctcctaatgcctggtaccttgggtatggagtgaggagagacaatggaca600
gttttatataagaaatggaagtaatgatactatctttcctggaatatttgcaggccccag660
aggagatgatgagcaaggactgttggcctgtattacacacaacagggttgtagttactat720
cccagcaaggaaagggtgtatctttcttctttcatgcaaattatctatgatgacctaaca780
gtttgattatagtgagtggactaaccacaacaataaaaaaaaaaaaaaaaaactcgaa 838
<210> 14
<211>
513
<212>
DNA
<213> Sapiens
Homo
<400>
14
ctgcaggaattcggcacgagggaacaactccatgtttttgtaaaggcctagagaacatat60
atccagtgcctttcctttttgcctttgtattcatcattttggcaaattactggaagatga120
cggttctggccaaaaggctggttttgtttttgggtcacattttcttgcttctctgcgtta180
gaatcttggattagatgatggacatggtgaagatctcagcaacctcattcactagaagat240
catgtggattggaatcatacaatggggaacaaatggaaaagagtacttttgaaatagtgc300
tggagaccactgtgaccacagaatgtcaagacacgtgctgccattactgttactatttgg360
aaaatacattcttgtaaatgcaaccttagggggtttgagggggaagtctgttgggaaatg420
aattgcaagaaaaatattacaccctgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa480
aaaaaaaaaaaaaaaaaaaaaaaaaaaactcga 513
<210>
15
<211>
712
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
SITE
<222>
(565)
<223> or c
n equals
a,t,g,
<400>
15
gaattcggcacgagctggacaggaccggagaggaccccgcgtaaccgcggaacagacact60
cccggcagcggccgccgccgcggcactgctacgggacgagccggagcgcttggccatggc120
ggcccgatccgcactggcgctgctgctgctgctgccagtcctgctcctgccggtgcagag190
ycgctcagagcccgagaccaccgcgcccacccctaccccaatcccgggtggcaactcgtc240
aktgagcaggcccctgcccagcatcgagctccacgcctgcggcccataccccaaaccagg300
cctgctcatcctgctggccccgctggccctgtggcccattctcctgtagggacgcccagc360
cagccacctctaagtcgccgctgggactggcctgccccattgagcaacagagacgcttga420
cagccgcccgcctccattccttgacttcacccagaaatgggtccagaaaactgaatccca480
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ccagcactgg tttggagcaa ccggacaccg aggtttcacc tccagggrtt ccatggaaga 540
gcctcaatgg agatgccaca tcctnactga gttaaagatg ggctgaggaa cttgggtacc 600
cacaagtytg ccttgggrat caaaagaaaa tatttacctt tagtttggtt cattaaatgc 660
atgaagtcaa aatatgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaactc ga 712
<210>
16
<211>
652
<212>
DNA
<213> sapiens
Homo
<400>
16
gaattcggcacgagcaacagtggggcactctgctcccaggcaggtcccactgggctgagc60
cgcacagcctggctttgggcttccctgactgcaccacccacatcasctgcctctagccct120
IS taamatacaaaacttcccccagtcactggccgccaggctgagttgggggatgtgttacat180
ccctgggtccactggggggcagtgttggccatggtgttggtgctggctctgccgagaggc240
gttggagtggctgtgtggggcggtgagcgccggcccagcctgatggaacccactgtacca300
ggcccaggcctcagcctctgagaaggacttccctgtgtcactcactcatacatgtcctca360
ggacgtgaagacatttcagcagaccaaagtttccttcgaatttccttcgaatcgtccaga420
tacttggagacatctcctcctcacctgtggggtgctggggcagtcctaggcgtgggggca480
gatgggtggacagctgctgctgccctgctgggggtgggcagcccttggagcacacagtgg540
tgaagacattcctgaatatgtctcaggctgtagaaatcttattttgtggaaagattttag600
agaatcatcaaaataaacttttaccaaataaaaaaaaaaaaaaaaaactcga 652
<210> 17
<211> 742
<212> DNA
<213> Homo Sapiens
<400>
17
ggtcgacccacgcgtctgatatgatctccttatccttctccctttgattgatctttttct60
ttgagctgatttgagctttcttcttttctctgtagttggcggaatcagctcagttacatt120
ttttactaagttacccacattctgacactccttgacagtkttaagatcttcttctaacac180
acttgaatagaatggatactggaatctattttgacagctgttgaaaatctattctgttgt240
tacaggaggttaaggaggttatttgtaacactgggattatttaatgaaccttttgaaaag300
gtgtgcagactgttcaggcaaatagtattttttagaattaaatgattttggttttcacag360
ttaaattatcaaatgtaatgcttttaagaattatacacctagtaatatttttcattaatt420
tctccaccagtgtagtaatagtacattacaatgttctcaattaccggtgccttctaaaat480
gcaggtgtagagtcyttaaatacagctagtctatkgccagctgtcccatagataaccttc540
tcyttaaaartgaccttkgrgcaattycataaagaataaatatttctagttttttgttgc600
tgaactgctaaaagatggttctatacatgtaacaggtggctttagttgggttgctttcac660
tgaaatttgattcaaataaagcattgcattattttacctttggaattataaaaaaaaaaa720
aaaaaaaaaaaaaagggcggcc 742
<210>
18
<211>
1219
<212>
DNA
$0 <213> Sapiens
Homo
<400>
18
aacgcactcaatattcagaagtttgaattctaccactctcaaacacagttcaaaagatag60
ctgtttgagaatgcttcttaactaatactagtacaatatcttcaataatgtatgtacctt120
atagaaaatcttgaacagtacaagattttcataattaaggcatgcaaaactgcttgggct180
ctttgattccaggtgtcctcttctcccttctgcttttgccatctatgttcaatataattc240
taacccagtctaagtatggagaaaattcctaccctgcctgcttttatagctcatcaaatt300
tccctgtatcagctatcacttttctggtaggtgtagtctgatttctgtctgtcatgcctt360
tgccacaatcctttctttgaagagtaggtaaaagatctattaaagtgttaatcacattgc420
tctaatatataaagcctccagtggtttcccatatcactctgtaaaatgccccttgccagc480
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
7
ctctcccatc aacctcgctttttctgttcttgtatatgcacatctcttcctgagccttta540
ttgccatcct catgtggggatgtttctgtctcagagatagtctttattcaggtcccactc600
tgcagtcctc tccagaggggctgctttcaccaccccttctaagtaagcctctctaaacac660
ctctatcata ttctatcccttagccagcactaattttttcataatgcttaccactaactg720
aaatttactt tatcatttaatctcttcctcattagaatgtaagctcgtaagggaggggca780
gctctgtagt ttattcattattgtatgtccctcacctaatcctatgagtgtctggcccat840
attagggtat gtaataaatattacttgaggaatgaatgaatttaacatactaccaattct900
ctgagtgact ctttttaaagccttcatcatcattcacactttcttgtctttcatatgggc960
atgtccaatc acccttccatgaatatctgtacctgttacaaagagaggactaggttcctg1020
1~ gagttcatagatgtaaacaacatcctagggktagcaaactggtgggccctgagccaaatc1080
ctggcctgca catgtattttgtttgakttgtacaatgtttgttataaatgaactggctga1140
taatattttt taattggaaatgtttacattaaaaacctaracttctagctgctcttaaaa1200
aataaaaata cggctgggc 1219
20
<210> 19
<211> 874
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (461)
<223> n equals a,t,g, or c
<400>
19
ggtcgacccacgcgtccgagcaattgaatcatctgcccaaggataagctgctggtgagag60
cagagttgggatttgaagtcgagttagaccccagtgatcacagtcttgacgattaaattc120
ttccagctttcatttttcactgagataatggtagtgatagtactgacctctaatgtgtgc180
atttgtgggtatgtggtccattcagctttaatccccagaagacaaggcttattccttttc240
ttatttttggtcatgttttatttttccattgcttttaacaggattaccaaaggcacactc300
agtagtcagtaaacacatttctaggaaaggtgttgtgtcatcatgccacatattcatact360
ttcctgggttggaaaatagatcatcagtaaaaacatacaggaaaaatgaatcttgccaat420
gcaattgttaacctacaaccataatataccttaagtatatntttgcacataagtataaca480
tgcgatttaaaacaataaaccagattgagatctaaggagcattttgtaagtaattactaa540
tgtttattttagagagatcacacaacttcaaataaaaactgacatagattgaacaccttg600
agaataaactttagtgccaaatggaaaataattttttacaagtaaatttgaagaacaatg660
tgaactttctataattatatacagraaatatactgatttgccaaaatgagtaattttgat720
atattaatatttcacttataagaatgcataccacctgatccaggatgggatccaggaaca780
gaaaaagaacattagktaaaaatgacagaaatctgaatatagtatagagtagctaaaaac840
aaaccaaaaaaaaaaaaaaaaaaaaagggcggcc 874
<210> 20
<211> 464
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (21)
<223> n equals a,t,g, or c
<400> 20
5$ caaacccttcagtggatgagnccaagtcgcagaaagcattctgttgacagatgaacagcc60
gaaagctggc cagaccctcctgkatgcactcccwgccccktktatcagaaacacaggcaa120
ggaaattgga actgccacccagcccagcatggtggctcaattggttggttgcgttgtcag180
ttgtctcttc gttttgttaaggtttttaataagtacgtttggcataatgtcttttaatgg240
gtttgtaata tttgtaacggttttagcagcctataacttttcagctggtgcttttactta300
gggaaaaaaa caatttgtaaatacagaacattgtttaaaagacataaccatagaacatag360
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
8
cttcctgttt gtggattttg tttcctatat attcaaagta aaatgactta caggaaaaaa 420
ataaaaaaaa aaaaaaaaaa aaaaaaatcg gggg9ggggc ccgg 464
$ <210> 21
<211> 637
<212> ~1P.
<213> Homo sapiens
<400> 21
60
gattttcctg cttgcatcatttctagcacagagctggaggaaatggcgaggtgcaggtgg
ccgctggccm tgctgttctacatgggagcaagacagctgctaggtgaaggggaatgacca120
ggcagccaca gggaggacatgtggcctcaggaagcctgggtgtgtatcctggttctgcta180
ggaacacgtg tggggctttgtgtgggtgactctctggctccccaagcctccctttcctac240
1$ tgttatatccttaaagtgcctctgaggccaaagcctttgtggcaattgtcaaatgagtcc300
atatgcagtg agtaccgtgttgagggaggacaaggtcaccaagagctgagaatgtttctc360
cgactgatga gacctagatattgggtacatggaggtccccggtccctttgtgattcctgc420
agcctgttgc ctccttgcctggaccccgcctcagctcagaaagccaattccctagattcc480
aaaggccttc ccagaccaattagcatgtcctgcagctgtcagctccctgtgcctagcctg540
gacctcagct catgtctagcacccagtctcccaaccccacacatattcacaaataaaaga600
aaataacaaa tgaaaaaaaaaaaaaaaaaaaaaaaat 637
<210> 22
25 <211> 752
<212> L~NA
<213> Homo Sapiens
<400>
22
gaattcggcacgaggggattacaggcatgagccaccatgcccggccatataaagcattta60
ggatagttagttgctatttttatttatttattattgttgttgttattatattactacttt120
atcccatttcacaaggatggcatgttgccaacattgtctttctaaagaatatctctgatc180
acatccttgttctattaaaaaccttttgaaagctccctcttacctttagaagaaattgga240
acttcatgattcctcatggtctggctccagcactgagtctggaatgctagtgtgagatga300
35 ggccttagaagtcatccagctgaactcctggaatttttatagatgaataaatgtagcatc360
cagacatttttcytgttgcacccctgtamgccatgtcctcttccagactcctggataaga420
ctgrcagacatcaccattctcttaaaccagaactacacttgccttcatccatttgatcac480
ctggttccaggtaactcatgagctttgtagcttcccttctctcagaccttccaaggaaga540
caatggcataattttccccatatgctctaattagcaacctttccctgcccttctgtgggt600
gggcagggccggacacagtgggtcacacctgcaacctgtaatcccagcactttgggaggc660
tgaggtgggcagattgcctgagctcaggagttcaagacagtctgggtaacatggcaaaat720
cctgtctcaaaaaaaaaaaaaaaaaaactcga 752
45 <210> 23
<211> 492
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
<222> (486)
<223> n equals a,t,g, or c
5$ <400>
23
aagctggact cgcgcgcttgcaggtcgacactagtggatccmaaagaattcggcacgagc60
aaggacccag aagtagggttttggcctaggtaacggggcagagatgtggttcgagattct120
ccccggactc tccgtcatgggcgtgtgcttgttgattccaggactggctactgcgtacat180
ccacaggttc actaacgggggcaaggaaaaaagggttgctcattttgggtatcactggag240
tctgatggaa agagataggcgcatctctggagttgatcgttactatgtgtcaaagggttt300
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
9
ggagaacatt gattaaggaa gcattttcct gattgatgaa aaaaataact cagttatggc 360
catctacccc tgctagaagg ttacagtgta ttatgtagca tgcaatgtgt tatgtagtgc 420
ttaataaaaa taaaatgaaa aaaawrmaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 480
492
aaaaanaaaa as
<210>
24
<211>
532
<212>
I7NA
<213> sapiens
Homo
<400>
24 gaaagcagtacgccgcagtaccggtccgaattccgggtcgacccacgcgt60
actcatataa
ccgcccacgcgtccgcacctcccttggctgtggggaggggcttccatgccctgtgtggct120
1$ ctcgggtgggctgtcgcaccacactgctcttcctttctcttcacgaatcacgcaagcctc180
ctagtcagttctgatgagataacctggatatcttggttgccggtgaaggatttacatgct240
tattatggtttttttgttgttgttgttgtttggttttttttttgatgggagcctcagatc300
gccgctgttgctaatcatccatcttggccctgcccccacatttctgcaaatttaaatatg360
agatttgtccccttaggtgcacagtccagaccccatccagtccagctccttttaaagcca420
catggaaagtcagctgagaatggtttgggagcccaggtgcgctgtcttccgccctgccct480
ctccctgaaataaagaacagcttgacagaaaaaaaaaaaaaaaagggcggcc 532
<210>
25
2$ <211>
920
<212>
DNA
<213>
Homo
Sapiens
<220>
<221>
SITE
<222>
(907)
<223>
n equals
a,t,g,
or c
<220>
35 <221>
SITE
<222>
(914)
<223> or c
n equals
a,t,g,
<220>
4O <221>
SITE
<222>
(920)
<223> or c
n equals
a,t,g,
<400>
25
45 gtcggaattcccgggtcgacccacgacgtccgcaaaattaacatcaaaaaggtatatact60
ttttaaaaaaaatttacttttattgatgtgtactcttcctattgatgagttaattccata120
aatctctacttagtttaacttattggatcaaattatcttcagcatgtatatctggggaaa180
aaaggtccgaattttcacatttatatttaaacttcaattttttatatttaaacttcaatt240
ttttagcaacagctgaatagctttgcggaggagtttaatagttacacattcatgctaata300
$O tacatttcctttaaacatccacaaattcttaaaaagrttgaatcagtaaatttcatttca360
gctaaaaatggagtctaatatattgtttcaaaagatacatttttacccaccataaatgtt420
acaatatctgaatatgctttgtcaaactatccctttatgcaatcgtcttcatattgtttt480
tatgattctaatcaagctgtatgtagagactgaatgtgaagtcaagtctgagcacaaaaa540
gataatgcacgatgagattgcctaccattttataggatatttactatgtatttatacgtt600
55 aagacctctatgaatgaatgtatcagagaatgtctttgtaactaactgtttaattcaatc660
tgtaataaaaatctaactaactaactcatttatttctattaaaaaggtattgtcctttag720
gcggggaatgggaatccttgctgcactgttgcagtcattctgaaaggacctttccctgta780
cttacctttcaacatgcttcaatcttatcaacgctacattttgtatttttcaaacaagta840
taaattctgcaataaagagatgtagtttttttttaaacaaaaaaaaaaaaaaaaaaaaaa900
cccccn 920
6O aaaaaanggggggn
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
<210>
26
<211>
917
<212>
I7NA
<213>
Homo
Sapiens
<220>
<221>
SITE
<222>
t434)
<223> or c
n equals
a,t,g,
<400>
26 cgaggtttcattgccctcaacattctctgttcttcaccgaatcatgtctg60
gaattcggca
IS tttcctccaacctctggcaaacactgatcttgttactgtctttgtggttttgcctttttc120
cagaatgtcatatagttggaatcatacaattgtgcagactttttagattgccttctttca180
cttagtaacatttaagtttcctccaccccttttcatggcttgatagttcatttcttttaa240
ttgctcaataataaatattccattatctagatagaacggtttatctacctagtgaaggac300
atctcaattgcctccaagtttaggcaaatataaacaaagctgctatcaggatttttcaca360
gaggaaaagacagtgggatccaaaactgaatggtctatcaataaatgacgcatggtacat420
ctacacccatgranccattgtgcatccatgagaaaaatccagatgtaggaaggtatgtat480
aattttgcagaaaagagtatgtaactggaaacaccaargaaaaaaggaaatggatctata540
tatttaggtggagatatttatgtggctgcagaagaaatatattattattcatactagata600
gttaatgtttgcctttggtgggcaagaaaggtaaaaagggagaagggagcccaaccaaaa660
gaggaagaggaagaaaaaaaaactgcactaagaaaaatcttttaaaagtatgtgatcaca720
gccaggtgcagtggctgacaaatgtaatcccagctacttgggaggctgaggcaggagaat780
cgcttgaacccaggaggctgaagttgcagtgagctgagatcatgccattgcactccagcc840
tggtgacagagactctgtttcaaaaaaaaaaaaaaagtatatgatcacatctgtgttaac900
ttacagactagtctcga 917
<210>
27
<211>
662
<212>
DNA
3$ <213> Sapiens
Homo
<400>
27
gaattcggcacgaggttcccatggcactttatatgtgtgcatagagagccagggagcagt60
ggggttcagggtgggcccatgctatgtgctgcagagctggtgggtcacagtctccccagg120
tgatggtggtgttaataatcatcctaggcccgtggggtggggtgaggattgatgcatgag180
aaagttgaggcgggggccctggcatggagcagggctcaggccgcttgtcacccaggctca240
tgtcagccctccggagcctgtgggtgtataggggaagcgcaggggttcttcagccagagg300
gacaggttcarggcctgctgatgccccttgctggttttgggaccttgagcaagtcccctt360
gccttttggtgctgtgcctcggtttcttcttctataagaaggaggtgatgatgtaaccca420
cccacccagcccctctaccccgcgcatcagggtagcaggcgagctagcactgtggcacca480
ggagtggagctggcccctggcgggcccacgctggagaggcatcgccatctctgctgcccc540
cctgtggcgtcatcatatcaacctgccagtccccctcacctggtgttaatctcccagagg600
atggggactgrttctgcatattctttgctaaacaaagacgctagtttggctgtggctctc660
662
ga
<210>
28
<211>
699
<212>
DNA
<213> Sapiens
Homo
<400>
28
gattcggcacgagaaacttt aatacttaga 60
taaatcttta acacttaaac
gttatttctt
aaaactttac tcaggagaat 120
aaaacaaaag atgacttttt
agcagaataa
ttagatcctt
tttcctaagcacactggacc 180
atagaggaag
accaaaggaa
tgtacagttg
cctgctcctt
CA 02296815 2000-O1-14
WO 99103990 PCT/US98114613
11
cctgacttgc tgtatttgac tctgtcccca ctggtggtgg 240
caatgctatt aaccccacac
tttaacgtgg caaatcccca gaatctgttg gctggtctct 300
ggctagagaa tgagcacagt
ttcaccctta tggctccaga aagagcaaga acacaccact 360
gccagccaga agagagaaaa
gtcttgttct gtctctttcc cattgtccca aatagccaag 420
cacaggttca accaccccaa
atgccaccct tctgctgtgc agcagccaag gaaaagaccc 480
aggaggagca gctccaagaa
cctctgggca gtcagtgccc agatacttgc cccaattctt 540
tgtgtccaag ccacactcag
ctgacaaaag ccaacacttt gtctctcttt tttttttttt 600
cttttttttt gagcagagtt
tcactcttgt cacccaggct ggagtgcaat ggcaggatct 660
tggctcattg caacctccac
ctcccgggtt caagcaattc tcctgtctca gcctctcga 699
1~
<210> 29
<211> 1637
<212> DNA
15 <213> Homo Sapiens
<220>
<221> SITE
<222> (726)
ZO <223> n equals a,t,g, or c
<220>
<221> SITE
<222> (727)
2S <223> n equals a,t,g, or c
<220>
<221> SITE
<222> (728)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (899)
3$ <223> n equals a,t,g, or c
<220>
<221> SITE
<222> (901)
<223> n equals a,t,g, or c
<400> 29
aaatgtgcca cgtcttctaa gaagggggag tcctgaactt 60
gtctgaagcc cttgtccgta
agccttgaac tacgttctta aatctatgaa gtcgagggac 120
ctttcgctgc ttttgtaggg
45 acttctttcc ttgcttcagc aacatgaggc ttttcttgtg 180
gaacgcggtc ttgactctgt
tcgtcacttc tttgattggg gctttgatcc ctgaaccaga 240
agtgaaaatt gaagttctcc
agaagccatt catctgccat cgcaagacca aaggarggga 300
tttgatgttg gtccactatg
aaggctactt agaaaaggac ggctccttat ttcactccac 360
tcacaaacat aacaatggtc
agcccatttg gtttaccctg ggcatcctgg aggctctcaa 420
aggttgggac cagggcttga
SO aaggaatgtg tgtaggagag aagagaaagc tcatcattcc 480
tcctgctctg ggctatggaa
aagaaggaaa aggtaaaatt cccccagaaa gtacactgat 540
atttaatatt gatctcctgg
agattcgaaa tggaccaaga tcccatgaat cattccaaga 600
aatggatctt aatgatgact
ggaaactctc taaagatgag gttaaagcat atttaaagaa 660
ggagtttgaa aaacatggtg
cggtggtgaa tgaaagtcat catgatgctt tggtggagga 720
tatttttgat aaagaagatg
55 aagacnnnta tgggtttata tctgccagag aatttacata ?80
taaacacgat gagttataga
gatacatcta cccttttaat atagcactca tctttcaaga 840
gagggcagtc atctttaaag
aacattttat ttttatacaa tgttctttct tgctttgttt 900
aattattttt atatatttnt
nctgactcct atttaaagaa ccccttaggt ttctaagtac 960
ccatttcttt ctgataagtt
attgggaaga aaaagctaat tggtctttga atagaagact 1020
tctggacaat ttttcacttt
()0cacagatatg aagctttgtt ttactttctc acttataaat 1080
ttaaaatgtt gcaactggga
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
12
atataccacg acatgagaccaggttatagcacaaattagcaccctatatttctgcttccc1140
tctattttct ccaagttagaggtcaacatttgaaaagccttttgcaatagcccaaggctt1200
gctattttca tgttataatgaaatagtttatgtgtaactggctctgagtctctgcttgag1260
gaccagagga aaatggttgttggacctgacttgttaatggctactgctttactaaggaga1320
$ tgtgcaatgctgaagttagaaacaaggttaatagccaggcatggtggctcatgcctgtaa1380
tcccagcact ttgggaggctgaggcgggcggatcacctgaggttgggagttcgagaccag1440
cctgaccaac acggagaaaccctatctctactaaaaatacaaaagtagccgggcgtggtg1500
atgcgtgcct gtaatcccagctacccaggaaggctgaggcggcagaatcacttgaacccg1560
gaggcggagg ttgcggtaagccgagatcacctccagcctggacactctgtctcgaaaaaa1620
1~ aaaaaaaaaaaactcga 1637
<210> 30
<211> 2142
IS <212> ETA
<213> Homo sapiens
<400>
30
aattcggcacagagacgcgggtccccgggtctgacaggagcagcctgtgggcaccgcggc60
ZQ ggtagttggaggcgggagagggtccgtagccgcgccgccctgccccgccatgggcctcct120
gtcggacccggttcgccggcgcgcgctcgcccgcctagtgctgcgcctcaacgcgccgtt180
gtgcgtgctgagctacgtggcgggcatcgcctggttcttggcgctggttttcccgccgct240
gacccagcgcacttacatgtcggagaacgccatgggctccaccatggtggaggagcagtt300
tgcgggcggagaccgtgcccgggcttttgcccgggacttcgccgcccaccgcaagaagtc360
25 gggggctctgccagtggcctggcttgaacggacgatgcggtcagtagggctggaggtcta420
cacgcagagtttctcccggaaactgcccttcccagatgagacccacgagcgctatatggt480
gtcgggcaccaacgtgtacggcatcctgcgggccccgsgtgctgccagcaccgagtcgct540
tgtgctcaccgtgccctgtggctctgactctaccaacagccaggctgtggggctgctgct600
ggcactggctgcccacttccgggggcagatttattgggccaaagatatcgtcttcctggt660
aacagaacatgaccttctgggcactgaggcttggcttgaagcctaccacgatgtcaatgt720
cactggcatgcagtcgtctcccctgcagggccgagctggggccattcaggcagccgtggc780
cctggagctgagcagtgatgtggtcaccagcctcgatgtggccgtggaggggcttaacgg840
gcagctgcccaaccttgacctgctcaatctcttccagaccttctgccagaaagggggcct900
gttgtgcacgcttcagggcaagctgcagcccgaggactggacatcattggatggaccgct960
35 gcagggcctgcagacactgctgctcatggttctgcggcaggcctccggccgcccccacgg1020
ctcccatggcctcttcctgcgctaccgtgtggaggccctaaccctgcgtggcatcaatag1080
cttccgccagtacaagtatgacctggtggcagtgggcaaggctttggagggcatgttccg1140
caagctcaaccacctcctggagcgcctgcaccagtccttcttcctctacttgctccccgg1200
cctctcccgcttcgtctccatcggcctctacatgcccgctgtcggcttcttgctcctggt1260
ccttggtctcaaggctctggaactgtggatgcagctgcatgaggctggaatgggccttga1320
ggagcccgggggtgcccctggccccagtgtaccccttcccccatcacagggtgtggggct1380
ggcctcgctcgtggcacctctgctgatctcacaggccatgggactggccctctatgtcct1440
gccagtgctgggccaacacgttgccacccagcacttcccagtggcagaggctgaggctgt1500
ggtgctgacactgctggcgatttatgcagctggcctggccctgccccacaatacccaccg1560
45 ggtggtaagcacacaggccccagacaggggctggatggcactgaagctggtagccctgat1620
ctacctagcactgcagctgggctgcatcgccctcaccaacttctcactgggcttcctgct1680
ggccaccaccatggtgcccactgctgcgcttgccaagcctcatgggccccggaccctcta1740
tgctgccctgctggtgctgaccagcccggcagccacgctccttggcagcctgttcctgtg1800
gcgggagctgcaggaggcgccactgtcactggccgagggctggcagctcttcctggcagc1860
50 gctagcccagggtgtgctggagcaccacacctacggcgccctgctcttcccactgctgtc1920
cctgggcctctacccctgctggctgcttttctggaatgtgctcttctggaagtgagatct1980
gcctgtccgggctgggacagagactccccaaggaccccattctgcctccttctggggaaa2040
taaatgagtgtctgtttcagcarmaaaaaaaaaaaaaaaa aatgaccctc2100
aaaaaaaaaa
gagggggggcccgggtacccaattggccctatgaagaggc 2142
ga
55
<210>
31
<211>
1564
<212>
DNA
<213> sapiens
Homo
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
13
<400>
31 ttaggggaacgtggctttccctgcagagccggtgtctccgcctgcgtccc60
cc
ggcacgagaccggagctggagtcggatcccgaacgcaccctcgccatggactcggccc120
ca
a
t
gc gcataacggcagtgccgaggcaggcggccccaccaacagcactacgcggc180
g
tgc
tcagcgatcc
cgccttccacgcccgagggcatcgcgctggcctacggcagcctcctgctcatggcgctgc240
tgcccatcttcttcggcgccctgcgctccgtacgctgcgcccgcggcaagaatgcttcag300
acatgcctgaaacaatcaccagccgggatgccgcccgcttccccatcatcgccagctgca360
cactcttggggctctacctctttttcaaaatattctcccaggagtacatcaacctcctgc420
]0 tgtccatgtatttcttcgtgctgggaatcctggccctgtcccacaccatcagccccttca480
tgaataagttttttccagccagctttccaaatcgacagtaccagctgctcttcacacagg540
gttctggggaaaacaaggaagagatcatcaattatgaatttgacaccaaggacctggtgt600
gcctgggcctgagcagcatcgttggcgtctggtacctgctgaggaagcactggattgcca660
acaacctttttggcctggccttctcccttaatggagtagagctcctgcacctcaacaatg720
15 tcagcactggctgcatcctgctgggcggactcttcatctacgatgtcttctgggtatttg780
gcaccaatgtgatggtgacagtggccaagtccttcgaggcaccaataaaattggtgtttc840
cccaggatctgctggagaaaggcctcgaagcaaacaactttgccatgctgggacttggag900
atgtcgtcattccagggatcttcattgccttgctgctgcgctttgacatcagcttgaaga960
agaatacccacacctacttctacaccagctttgcagcctacatyttcggcctggggcytt1020
accatcttcatcatgcacatcttcaagcatgctcagttatgaggagtcaaatcctaagga1080
tccagcggcagtgacagaatccaaagagggaacagaggcatcagcatcgaaggggctgga1140
gaagaaagagaaatgatgcagctggtgcccgagcctctcagggccagaccagacagatgg1200
gggctgggcccacacaggcgtgcaccggtagagggcacaggaggccaagggcagctccag1260
gacagggcagggggcagcaggatacctccagccaggcctctgtggcctctgtttccttct1320
25 ccctttcttggccctcctctgctcctccccacaccctgcaggcaaaagaaacccccagct1380
tcccccctccccgggagccaggtgggaaaagtgggtgtgatttttagattttgtattgtg1440
gactgattttgcctcacattaaaaactcatcccatggcmaaaaaaaaaaaaaaaaaaaaa1500
aaaaaaaaaaaaaaaaaaaaaaaacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggggg1560
1564
gggg
30
<210>
32
<211>
1631
<212>
DNA
35 <213> Sapiens
Homo
<400>
32
cttagggggagccctggtgctacttgcttgaagttttcagtgtaagtaccctgatgcctt60
ttggaccttgggatcagatcaagagttttggagatcaggtaccaaggaaataaggacagt120
40 ctagctgcctcaagtgaggggccctttgcatagctctccttccccctcactgaagctggg180
tagcctattggggttgagagggaaaatgtgaaatctcagaatttatctcccttagaagag240
agccagtaacttatgtacaaggatgaaagaaaggtcgcagcagtagctttggggaaaggg300
aggaagatatggcacttctccaaccccggaaaacattgcttttgaaaactgctgataaaa360
tatgagccggttattacttctgtttgggagactgtgctctctgtggtgcctctcttggct420
45 ctactccacagataccagacctcttctaagaggatgagcagaccagctttgaggttgacc480
tgtttctctttgtctgccttcccaaaacaccagcccccaggaagacattaagcagcctta540
agcttaaattcctactccctcttccaaatttggctcacttgccttagatccaaggcaggg600
aaaggaaaagaaggggggtctctggctttattactcccctaagtctttactctgacttcc660
ccaaacccagaaagattttctccacagtgttcatttgaaagaggagtattttgtcccatt720
50 ttccccttcctcattatcaaacagccccagtcttccttgtctctgctaagaaagtagagg780
catgatgatctgcctctcaactgccctaagtcctagctaagtatcaggggaaaaaaaaaa840
aaaaaaagcctaacaaatgggattagactagggctgcaagtagtgaggattttgttgata900
cctctgctgggatgtgtgctttcccatatcttgccttcaggaattacactgtgccttttc960
cccagggatatgggctctgtctacccagtgctccagtttcccggtaactgctcttgaaca1020
55 ttgtggacaagggcaggtcttcatatttttgatcatccctttctcccagtgaaatcccat1080
agcccttacctagagtctagggcacaaagacttcggggaagatacactgagattgacctg1140
aggagacatctacacacaccagtggcagctgccccagggcctgcttccccttcctaagtc1200
tgtcatcctctggaagggatgggtggtgctccaatctctggtgcctaaaaacccaagttt1260
atttctctcttaacactggcaataaccagtccacaccactgttgccttttaaaacctctt1320
aataatctcatgctgtgtttgttttgattccaatccaattatcaccagggctgtgtgggt1380
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
14
aaatgctttt aaatgctctc tcatcttgtt cttccccctc accccccact cttaggtatg 1440
tatgatgcta atcttgtccc taagtaagtt tcttcctgct ccttttgtat cttcctttct 1500
tgtctttcct cctacctttt gtctcttggt gttttgggac tttttttttt ttttttggcc 1560
ttttgtacaa agattagttt caatgtagtc tgtagcctcc tttgtaaacc aattaaaaag 1620
ttttttaata a 1631
<210> 33
<211> 978
<212> L~NP.
<213> Homo Sapiens
<220>
<221> SITE
15 <222> (2)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (2?)
<223> n equals or c
a,t,g,
<400> 33
angagttgca tgcaagsgtaagttggncccytsgrggatctttagagcggccgccctttt60
25 tttttttttt tgcatgtctgagtttgtggaataagattcatatttactacaagtaatgga120
attggagatt cagaggggagaaagtcacttatcacattagtgtaattttctgatggtagg180
attatggaga gttttaggttttccctttttttccccaacttctctcccctcagtatttta240
aaaataacat tgtgtgggttgtttttttttgtttttgattgttttttgtttttcaaacag300
gtctcactcc tatccatgtaggctagagtgcagtagtgcaatcttggctcactgcagcct360
3O cgacttcctg agctcaggtgatcttcccacctcagtctcctgagtagctgggactccagg420
tgtgtgccac catgcctggctaaatttttgtatttttattagatacagggtctcaccatg480
ttgcccagac tggtcttgaactcctgggcctgcccacctcagcctcccaaagtgctagaa540
ttacaggcat gtgccaccatatccagcctaataacattgtttttaatgttcattaagtca600
tcccaccctc tcagtcttgcagaagcctctcaagagggacagaatcagttgcaaagtacc660
35 atttctgacc ctgagacatggatattatttgttcatttaaatgtcacctgaaaaacccac720
tcactcaaat ggtctgtgaagcttgcaaaaacaggaatgcttaccctcctgggtcctgaa780
tttttggttc tcttggactctttgaaattcttctttctcagaaaggagccctctttctat840
ttcccctcaa agttgtgacttgaccctcacatccctttcttctccagggccccttgataa900
gattctttta aaatttctttggagggcatcccttttaggaagacggacgcgtgggtcgac960
4O cgggaattcc ggacggta 978
<210> 34
<211> 898
4$ <212> DNP.
<213> Homo Sapiens
<220>
<221> SITE
<222> (402)
<223> n equals or c
a,t,g,
<220>
<221> SITE
55 <222> (452)
<223> n equals or c
a,t,g,
<400> 34
gaattcggca cgagattatg 60
tagtagatgt cactagaatt
cttgaaattt gtcttcaagt
catggcagta tttcagtatc 120
gctcctttgg gattgcctga
gtgatactca agagttagac
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
tagttttatc tgggttctttgaagaaccggggacacctcactggcttatgttgaatttct180
gcactgcagg gaccaactataaatggtgtttttggttttttacgtgttaagagctttaaa240
atgtaattct tcctatcattcatgcacaaatgttctcacacaaattgcttcacagattga300
taaaactttg aataatttttccctgaagaaatgttgaacttttctgcaagctgttggaat360
$ kggagcgcgtgttgaaaggcytgaakgggaccgtactgtacngcctawttcttttaaaaa420
aaattawgat ttcyatttttwatycatttacngatgactgaatakgtycaggccagaaaa480
tatcccctta tttcaaaatgcagcaatctataaacaaaatacttgccatttttctaaatg540
acaccttttt ctataatttg~tatagaaaat taagtgcaagggccaggcaccgtgtaacgc600
ctgtaatccc agcactttgggaggccaaggcgggtggatcgcctgaggtcagtagttcaa660
10 gaccaccctggccaacatggcgaaactccatctctactaaaaatacaaaacaattagcca720
ggtgtggtgg cagacgcctgtaatcccagctacttgggaggctgaggcatgagaatcact780
tgaacccagg aggcagaggtggcagtgagctcagatggcgccattgcactccagcctggg840
taacaagagt gaaaactgaagctgtctcaaaaaaaaaaaaaaaaaaaaaaaactcgga 898
20
<210> 35
<211> 754
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (311)
<223> n equals a,t,g, or c
<400>
cagcctcatctcctgttggccccttgtatgtaccctgtgtttgagttgta atgaacccct60
gcttgtccataatctttcttttaactcctgtgcttctctctcatcctttg cagagccttc120
actttctgcttaaagtggaccttgacttctctttatcttgctccatttgc acctgaaact180
30 tgtcctcaactgcagtgctaattccttggtaatgttttataactttgtca ggcagctaga240
cactgtaagtatagaacatgctgggaaatccaaattaaaaatgacagttg gcacaaagct300
gacttctgggnagggaccaaggaaaagtagccagagtggcaggatagctg cttccatcac360
ggattgccagcaatgtaaagcgtagactccagaggaacagtgctaactta aattaactat420
gcaggcatcagtacttctggttctgatggcccggggatttctaagtagta gtgagtctca480
35 gcattatttgttatacagtctactgctagatgaacaaggctaagtctaca gagaaggtaa540
attatagaaattaggccccgtctctgctaagaatacaaaaaattagccgg gcgcggtggt600
ggggtcctgtggtcccagctactcgggaggtgacgcaggagaatggcgtg aacccgggag660
gcggagcttgcggtgggccgagatagcgccactgcagtctggcctgggcg aaagagcgag720
actccgtcttaaaaaaaaaaaaaaaaaactcgta 754
<210>
36
<211>
699
<212>
DT1A
<213> Sapiens
Homo
<220>
<221>
SITE
<222>
(483)
$0 <223> or c
n equals
a,t,g,
<400>
36
gaattcggcacgagcggcacgagccaccttctcagtccagtctatgggta tgacagttta60
tctgctgaaaacccatccttgcttctttgttgcctaccagatgcaggtcg cactcataat120
cctccttcccggactcaggaacagcaagactgttactatgccattgtccc ctgccctcct180
tcccaccctccttttttttccctctcccactcccttctttcacccctttc tttctgtttt240
atgctgcttcaagtattaattttasaattgttctacaagaatgcgattta tcagaaggat300
gtgaaccaagcagaatttcttagtatttctttgccttagggcattcccct tgtgtggktt360
aaaatttgtcccccattcctttttgcctgtggaacttatccttattcttc aagagactcc420
tamtcctaatagcactttga cctggtagttcttctcagcc aaatttcacc480
atttaacctc
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
16
ttnctgaaaa caggattctc tgttctccat gtctggctaa tttttgtatt ttttgtggag 540
acaaagtctc actatgttgc ccaggcaggt ctcaaacacc tggccttaag ccatcctccc 600
accttggcct cccaagtgct gggattataa gcatgtgcca ctggacccag ccagagaccc 660
tgtctcttta aaaaaaaaaa aaaaaaaaaa aaactcgta 699
<210> 37
<211> 971
<212> IkIA
]0 <213> Homo Sapiens
<400> 37
c cgcagttcct gggtcgcgcg gcagctgtga gcgccgaggg 60
caaggcggtg
a
g 120
gccaccg
cca tcctgggcgg cgccatgagc gtggtgtcgg cctgcgtgct
cctgacccag
1$ cagaccg 180
tgcctcaggg atctggcgca accccgacgg ggcgccaaga tgtcggacca
cagggagagg
ctgaggaact cggcctgcgc cgtgtctgaa ggctgcaccc tgctatctca240
ggctttaagg
gagaggtctt cgcccaggac tttaccgcca gtgaattcca attctgtgaa300
ttagcacccc
acccccatac cccttcttcc acccccagac taaaggaaga tacttactct360
ctgcccctct
ccatttatac caaagaaatc ataggtgaaa ccccctaccc tccccaacgt420
taaatgctcg
20 agaggaatct tccacaaggc agggccatgc acgcaacctg cacacgcact480
tggagggccc
aggtgtctct ccaccagccc ccatgcagta gggactggaa gatatgtcat540
ctgctggttg
tgttatcact cccaccccct accccagccc gtsttccgga atttctcaac600
taaatttsat
tattgggcag gaaggaggtc atgggttcat ttcatttttg ttttttgtgt660
ttttaattaa
aagaaaggtt acctcagttt tcactcctta gacatggatg tagctacctt720
tttttgtatg
2$ tctttttttt tttaagcaat cgtgttgaat taggagtata cttggtgtgg780
aaagagtatg
aatttgccat gtgatttgca aatgggggga agctactgtg agcgtgtgtt840
tttttaattt
acactataga gtgatttttt tttcccccaa cgtcaagttt ttaccttgca900
tgtactggag
tatttatttc atctattaaa atgttatgtt tctcagaaaa aaaaaaaaaa960
aaaaaaaaaa
971
aaaaaactcg a
30
<210> 38
<211> 872
<212> DNA
3$ <213> Homo Sapiens
<220>
<221> SITE
<222> (2)
40 <223> n equals a,t,g, or c
<400> 38
tngcagttct ccacaccgaa gaggacggtg ggcgccaaca gacaggcgat60
taatgcggct
cttacccagg caaccaggac tacagtatac attgtggaca ttcaggacat120
agattctgca
4$ gctcgggccc gacctcactc ctacctcgat gcctactttg tcttccccaa180
tgggtcagcc
ctgaccyttg atgagctgag tgtgatgatc cggaatgatc aggactcgct240
gatgcagctg
ctgcagctgg ggctggtggt gctgggctcc caggagagcc aggagtcaga300
cctgtcgaaa
cagctcatca gtgtcatcat aggattggga gtggctttgc tgctggtcct360
tgtgatcatg
accatggcct tcgtgtgtgt gcggaagagc tacaaccgga agcttcaagc420
tatgaaggct
$0 gccaaggagg ccaggaagac agcagcaggg gtgatgccct cagcccctgc480
catcccaggg
actaacatgt acaacactga gcgagccaac cccatgctga acctccccaa540
caaagacctg
ggcttggagt acctctctcc ctccaatgac ytggactctg tcagcgtcaa600
ctccctggac
gacaactctg tggatgtgga caagaacagt caggaaatca aggagcacag660
gccaccacac
acaccaccag agccagatcc agagcccctg agcgtggtcc tgttaggacg720
gcaggcaggc
$$ gcaagtggac agctggaggg gccatcctac accaacgctg gcctggacac780
cacggacctg
tgacaggggc ccccactctt ctggacccct tgaagaggcc ctaccacacc840
ctaactgcac
ctgtctccct ggagatgaaa atatatgacg ct 872
60 <210> 39
CA 02296815 2000-O1-14
WO 99/03990 PCT1US98114613
17
<211> 608
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (10)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (16)
<223> n equals a,t,g, or c
IS <400>
39
ccatacgcanaccgcntctccccgcgcgttggccgattcttatggcagctggcacgacag60
gtttcccgatggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcat120
taggcaccccggctttacactttatgcttccggctcgtatgtkgtgtggaattgtgagcg180
gataacaatttcacacaggaaacagctatgaccatgatttacgccaagctcgaaattaac240
cctcactaaagggaacaaaagctggagctccacgcggtggcggccgctctagaactagtg300
gatcccccgggctgcaggaattcggcacgagtttgggtggagtttccaaggtgaaagttt360
ctgaattggtcaatcagtgacgcctttgtaaagatggctcatgtggtggtcgctcgcaat420
gaatgcctgataagggcttttctgtttcttttgcactgtgtaagtttgctcccatcgcct480
ggggaagttaatatcagacacacactttttacggtagaagagaggttgactactccaagg540
25 gcactgaaactctcactgagccttattgtttctctacacgcgamttgcagaaagcaggag600
tgctcgta 608
<210>
40
3~ <211>
855
<212>
DNA
<213> Sapiens
Homo
<220>
35 <z21>
SITE
<222>
(850)
<223> or c
n equals
a,t,g,
<220>
<221>
SITE
<222>
(851)
<223> or c
n equals
a,t,g,
<400>
40
45 ctgtaatagcacacaactcagaactcttcagcatttgtgtgattccttacctctggctga60
taaaactctaatgggttgtggcttactttgtttccattttctttggctttgtgcaatttt120
tgtgtaactttacttgtacctatattttctgtttacagttctttttaaggggaggggtag180
ggttctaagatcttgttgtttattgtagataaaaattttttcgtgttgtagaaaagcatg240
ggttatgcgtttgactgaaaaagacactgtattatttaccaaaggggtattgtttttgca300
$~ tttgtttataaatgcattattttggtactgtaaatttggacataatttctgagtttatta360
ctactggcattttctttttcccttttttttttttttaaccgtaagtgcacgatgcaggtg420
cataggccccagaccaaactagaccaccagcatgttcatgtccagacctcggcagtggcg480
tgcactgcttgtgcacctcagttcctccagtgttggtttgtttgttttttaattcagcat540
cctgctggttttactttccaagcaagatctgttgcgactcccaaatgcgttttaatgagc600
55 tcatccttatttgcctttcttcttacgtattttgtgtattagattgtgcaggagatattc660
tagaaggcattaatggtttgcattcaaaacgatgtggtttgtccaagttattttctgtct720
ttattactgagacggattaatctccttatttttttcttgatgatttgaagttgtaacagt780
tgtccagctattgcttaataaaattttgcagatcaaaaaaaaaaaaaaaaamctcggggg840
gggccccggnnccca 855
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
18
<210> 41
<211> 1042
<212> L~IA
<213> Homo sapiens
<400> 41
acggcccgta attcccgggtcgayccacgc gkccgtgctt cctagaaggt60
cgtgtcacgt
ggaacctctt aatctcagcatccggagctc caggaaggga aaatttcaag120
tcagatagaa
1~ ttctatatat accatttctttggaaccttc agccctcaag attccaacat180
catgacctca
gtttcaacac agttgtccttagtcctcatg tcactgcttt tggtgctgcc240
tgttgtggaa
gcagtagaag ccggtgatgcaatcgccctt ttgttaggtg tggttctcag300
cattacaggc
atttgtgcct gcttgggggtatatgcacga aaaagaaatg gacagatgtg360
actttgaaag
gcctactgag tcaaacctcaccctgaaaac ctttgcgctt tagaggctaa420
acctgagmtt
IS tggtgtgtga aaggttccaagaatcagtaa ataagggagt ttcacatttt480
tcattgtttc
catgaaatgg caacaaacatacatttataa attgaaaaaa aaatgttttc540
tttacaacaa
ataatgcaca gaaaaatgcagcctataatt tgctagttag gtagtcaaag600
aagtaagatg
gctgaaattt acataagtaatatttcataa tcttagaatt ctctcaaagc660
atgtgaaata
ggaagaagga agttcttgcccagaatctta ggaaatcacc actgttcggt720
tataatcact
gcctcctgaa tcgttgaggagtcttttaaa ttagattttt gttttgttgt780
ctcccaagtt
aatattatat ttagatatcagagagtcagg yaaaaaggaa aacttttatc840
tctagggaaa
aaacatttag aaaaatgtattcagtgtatc taatactgaa atgcggaaaa900
aaatttaatg
ttaaaaaaaa actatagacattgacatgga aaagagattt aatgttttga960
aaaaaaactt
tatattaact gagtaacatcctcctgatga gaagtactat attaaatata1020
aacccattat
25 gttataagtt aaaaaaaaaatt 1042
<210> 42
<211> 702
<212> I7~TA
<213> Homo sapiens
<220>
<221> SITE
35 <222> (515)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (614)
<223> n equals or c
a,t,g,
<220>
<221> SITE
45 <222> (673)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (677)
<223> n equals or c
a,t,g,
<400> 42
gggacaatga actccttctg 60
gtctaagtta
ttggtgctgc
ccctgctggc
tccgctgtcc
55 atggcccgag cctctgcctg 120
tcagagatgg
tagagccacc
aggacatgga
gtcattgctg
acacagggaa acatgagatg 180
tcttaggttt
ggtgtatgtg
aaacatgcat
gagaaataga
ggccaasagt tccactgtgg 240
agcgcagaca
gaatggtctg
aatgctcttg
cagttactac
gtcagtagtt tgtcatctaa 300
tatatattat
acatctataa
cctatgtatt
taccttattg
tgataatact gttttgtttt 360
gttttttttc
taattttgct
ttgtgcaaag
ccaaatccct
()~ ttcagcagca ttgagctaaa 420
aaaaaaaaaa
agtgcatgtt
tagggctggg
cacggtggct
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
19
catgcctata atctcagtac ttcgggaggc cgaggcaggc ggatcacaag480
gtcaggagtt
cgagaccagc ctggccaata tggtgaaatc acgtntctac taaaaataca540
aaaattagct
gggcatggtg gtgggtgcct atagtcccag ctatgcggga ggctgaggca600
ggaaaaaccg
cttgaaccct ggangcggaa attcccagtt gagccaagat cgcgccactg660
cactcccagc
ctggttgaca gancganact cttgtctcca acaaccagca ac 702
<210> 43
<211> 642
<212> I7NA
<213> Homo sapiens
<220>
<221> SITE
<222> (593)
<223> n equals a,t,g, or c
<400> 43
60
aattcggcac gagcggcggg gtcgactgac ggtaacgggg cagagaggct
gttcgcagag
ctgcggaaga tgaatgccag aggacttgga tctgagctaa aggacagtat120
tccagttact
gaactttcag caagtggacc ttttgaaagt catgatcttc ttcggaaagg180
tttttcttgt
gtgaaaaatg aacttttgcc tagtcatccc cttgaattat cagaaaaaaa240
tttccagctc
aaccaagata aaatgaattt ttccacactg agaaacattc agggtctatt300
tgctccgcta
aaattacaga tggaattcaa ggcagtgcag caggttcagc gtcttccatt360
tctttcaagc
tcaaatcttt cactggatgt tttgaggggt aatgatgaga ctattggatt420
tgaggatatt
cttaatgatc catcacaaag cgaagtcatg ggagagccac acttgatggt480
ggaatataaa
cttggtttac tgtaatagtg tgctgttcat ggaaaccgag ggctgcatct540
tgtttatagt
catctttgta ctgtaatttg atgtacacaa cattaaaagt actgacacct600
ganaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaagcggccg ccgaattaag cc 642
<210> 44
<211> 1219
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (25)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (26)
4$ <223> n equals a,t,g, or c
<400> 44
aattcccggg tcgacccacg cgtcnnctaa aatccccaaa ctgacaggta60
aatgtagccc
tcagagctca gcccaaggca gaatctaaat cacactattt tcgagatcat120
gtataaaaag
aaaaaaaaga agtcatgctg tgtggccaat tataattttt ttcaaagact180
ttgtcacaaa
actgtctata ttagacattt tggagggacc aggaaatgta agacaccaaa240
tcctccakct
cttcagtgtg cctgatgtca cctcatgatt tgctgttact tttttaactc300
ctgcgccaag
gacagtgggt tctgtgtcca cctttgtgct ttgcgaggcc gagcccaggc360
atctgctcgc
ctgccacggc tgaccagaga aggtgcttca ggagctctgc cttagacgac420
gtgttacagt
atgaacacac agcagaggca ccctcgtatg ttttgaaagt tgccttctga480
aagggcacag
ttttaaggaa aagaaaaaga atgtaaaact atactgaccc gttttcagtt540
ttaaagggtc
gtgagaaact ggctggtcca atgggattta cagcaacatt ttccattgct600
gaagtgaggt
agcagctctc ttctgtcagc tgaatgttaa ggatggggaa aaagaatgcc660
tttaagtttg
ctcttaatcg tatggaagct tgagctatgt gttggaagtg ccctggtttt720
aatccataca
caaagacggt acataatcct acaggtttaa atgtacataa aaatatagtt780
tggaattctt
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
tgctctactgtttacattgcagattgctataatttcaaggagtgagattataaataaaat840
gatgcactttaggatgtttcctatttttgaaatctgaacatgaatcattcacatgaccaa900
aaattgtgtttttttaaaaatacatgtctagtctgtcctttaatagctctcttaaataag960
ctatgatattaatcagatcattaccagttagcttttaaagcacatttgtttaagactatg1020
tttttggaaaaatacgctacagaatttttttttaagctacaaataaatgagatgctacta1080
attgttttggaatctgttgtttctgccaaaggtaaattaactaaagatttattcaggaat1140
ccccatttgaatttgtatgattcaataaaagaaaacaccaagtaagttatataaaataaa1200
1219
aaaaaaaaaaaaaactcga
15
25
35
<210> 45
<211> 437
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (422)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (423)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (427)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (437)
<223> n equals a,t,g, or c
<400> 45
gaattcggca cgagggcggcaccagggagcctgggcgcccggggctccgccgcgacccca60
tcgggtagac cacagaagctccgggacccttccggcacctctggacagcccaggatgctg120
ttggccaccc tcctcctcctcctccttggaggcgctctggcccatccagaccggattatt180
tttccaaatc atgcttgtgaggaccccccagcagtgctcttagaagtgcagggcacctta240
cagaggcccc tggtccgggacagccgcacctcccctgccaactgcacctggctcacaaaa300
agagtgcaac aaatgcttctattccatagctacggcattgctcagtaagttgaggtcaaa360
aataaaggaa tcatacatctcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa420
437
annaaanaaa aaaaaan
<210> 46
<211> 533
<212> LIP.
<213> Homo Sapiens
<220>
<221> SITE
<222> (305)
<223> n equals or c
a,t,g,
<400> 46
gaattcggca cgaggaccctatcttacaaaaaagaagaagaagaagaaaaccatgacagg60
tgtctttaag ctgcccttgctgttctgggttcatgaagcatctgtgggaggttgcccata120
tgtaaaatta gttgagtttgaagaaatgttaacgttatatggtattcttttaattttgtt180
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
21
ttaaaaataatttttctcattcaaatcctg tgtttggtat 240
aattagaagt aaatattgaa
aattgttgaggggagaatttattcaaagtttaatcatttgctttatctatgttatactta300
gctantagttactggaagtgtcaagttttatttttagatcttaactagagtctaaagtaa360
ttactaaaagctagttttcaaataatatgtaagagtaaagtcctgagttaaaagatttag420
catactgaattaacttagttgactgatgctgtacttacatgggcctcctatttcttgtgg480
ccaagatagcatcaacagaaaaaaaamaaaaaaaaactcgagggggggcccgg 533
<210> 47
<211> 1849
<212> IxdA
<213> Homo Sapiens
<220>
IS <221> SITE
<222> (222)
<223> n equals a,t,g, or c
<220>
2~ <221> SITE
<222> (1300?
<223> n equals a,t,g, or c
<400>
47
25 gtttttaaaaaattaaacaaggctttgtgttcctagaagagcttcatttcagtgaatctg60
gtgacctccatctgcttgctgtcataacccgacacggacttatttttgtcattagcaagg120
gggaaaaggccaaaggacaagggcctcttctcccattggttttcctgtgggcagaagggc180
tgaggaagatggcccagcccgtgggggctgctgggtcaccancagygggtagggtgcaat240
ctggtgtgtgttccagcagtgagacggtgttattgtgaaggtggcattcatctgcggacc300
aaaacccagccatcggggaagggtcagggcttctgtggaacttggaacgtgccaggacca360
cctgcaaaagccagggtgcgttgatcattctcagatcattgattggcctccacttgggta420
tgtgaattattcatgtcccagaagaccaaaaagtgctctggttctgagatgagtatttta480
ttcgtgttctgtttccgaaacacttagcaaagaaggtcacagtgatgtggagtcgccgca540
cccatctttgaagatagccagtgtccctggatgaggtgatgatttcccgtcccaaggact600
35 ctgtgaagtttagagtacagtttgttggggtccaaaagacaccatctctaccccacccaa660
ataaaaatgcactcatctctgtagaacatctgctgtcaaaggccagcctgtcgttagggc720
atggcttatgcttgacaaaccagtaacaactgtgggatggcgatggtgggatgtgtcgca780
agcaattcactagacaatcttcacatgaatgtcggtagccagggtctctcccgagggatg840
gctttagtcttgatgaatgtgaaccatgtcggaattgttaggtagaaacctgggctggga900
ggcctcggaccccaggctccatccctggcttccccagcctgcggccgcaagcaaaaccaa960
gcgcgagatgcagctagcacccttcatatccatccccgttctcagcgggacaacaccatg1020
gacagccgttttcagagcctccagcatttgcacaccactactcaccctctctgctgctgg1080
catgttggtagagtcatccctgtaatcaagaaatggcctgtggaatgttattgttcaacg1140
ttgtttacagctcttaaaacatggtgaggaatgcctaagtcttagtgaccaaacgtgacc1200
45 ttgaaagcagacatagcatgacagaccttcctagagtgtttggtcgggttcacagtgacc1260
gagagtcaggtccagcacacacctgggaaagggatgctgncccaagggggaccaaaaggg1320
ccggacgttacagggtgaaaccctctgacccctcgcgacaccgtaggacttgacttttgt1380
ttagtctttctaagaaatagatcatggagccaagtgaagtgcactttgtcaaatgtaagg1440
gtctgctttgttcttgttgcttttctgttttttaaccttttgttccgccatttaaaaaaa1500
gaaaaaaaaaaagcttatgtttcttgtcaaatgcagaaatgttccttccgccactcactg1560
aagttttgcattctggcttgtgcagtttttattgtctgtgtcagacgtacagccagacat1620
gttctctattggcatttttccgattctgttcagatgacagcgaccgccttttcattcccc1680
ccgccacctgtactcaccctcacgctctttgaagaaaaaaaaaaaaatcaccttgtgtgt1740
tgtagctcatttgtttcaagagagaatcaacagatcatattcagtgtcttgaataaattg1800
55 ctctattttgatattagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1849
<210> 48
<211> 926
<212> DNA
CA 02296815 2000-O1-14
WO 99/03990 PCTlUS98/14613
22
<213> Homo sapiens
<400>
48 actagaatttgcacaatataagcttgaaacgaaattcaaaagtggtttaa60
caca
$ ctcaac cttggctgagaggaaagaacccctccgatgcctaataaagttctctagcc120
atgggagcat
cacatcttctggaagcattgaaatccttagcaccagcgggtattgcagatgctccacttt180
ctccactgctcacttgcatacccaacaagagaatgaattattttaaaattagagataaat240
aagacgtgcgtggtttcttaagcacagctcctccttcttgatattgcacatgcacttcag300
ttcatggctagctgtatagcttccgtctgtaaacttgtattttcaagaatccttggtatt360
1~ gaatttttagaaatgctcacataattgttgggactgattcattcctccacgatatgcctc420
ctctctctgatatcctgctaactgtagccgttgtggcatttgagatgacaggacatatat480
atatatggccccacacttgaccttgagtgcctgaatgctctgaaatcaagcatatggcac540
agcgctcaagacttttgggtttgtgtccttttttctatggctgtctcttctcaattctgg600
agaggtctggttccagtggctggtttcyarggattgattcttaagctctggatcacagag660
1$ agaagcaacaaggaactatactcaactcaaaactttttaggagaatcatgaaattggtct720
attcaaaggatggagttgagtccatwmtgttattgttgcaagaggttgcatatttggtga780
gtcagttatataaaatagtgttcttattgtaaatatgatacttctcataatctattttat840
catgtgtataacattcaaactgacaaatatattgacttatgaataaaggtgtcaaaaaac900
aaaaaaaaaaaaaaaaaaaactcgta 926
20
<210>
49
<211>
1593
<212>
DMA
2$ <213> Sapiens
Homo
<400>
49
gcggacgcgtgggctgtgctccctgcagtcaggactctgggaccgcaggggctcccggac60
cctgactctgcagccgaaccggcacggtttcgtggggacccaggcttgcaaagtgacggt120
cattttctctttctttctccctcttgagtccttctgagatgatggctctgggcgcagcgg180
gagctacccgggtctttgtcgcgatggtagcggcggctctcggcggccaccctctgctgg240
gagtgagcgccaccttgaactcggttctcaattccaacgctatcaagaacctgcccccac300
cgctgggcggcgctgcggggcacccaggctctgcagtcagcgccgcgccgggaatcctgt360
acccgggcgggaataagtaccagaccattgacaactaccagccgtacccgtgcgcagagg420
3$ acgaggagtgcggcactgatgagtactgcgctagtcccacccgcggaggggacgcaggcg480
tgcaaatctgtctcgcctgcaggaagcgccgaaaacgctgcatgcktcamgctatgtgct540
gccccgggaattactgcaaaaatggaatatgtgtgtcttctgatcaaaatcatttccgag600
gagaaattgaggaaaccatcactgaaagctttggtaatgatcatagcaccttggatgggt660
attccagaagaaccaccttgtcttcaaaaatgtatcacaccaaaggacaagaaggttctg720
tttgtctccggtcatcagactgtgcctcaggattgtgttgtgctagacacttctggtcca780
agatctgtaaacctgtcctgaaagaaggtcaagtgtgtaccaagcataggagaaaaggct840
ctcatggactagaaatattccagcgttgttactgtggagaaggtctgtcttgccggatac900
agaaagatcaccatcaagccagtaattcttctaggcttcacacttgtcagagacactaaa960
ccagctatccaaatgcagtgaactccttttatataatagatgctatgaaaaccttttatg1020
4$ accttcatcaactcaatcctaaggatatacaagttctgtggtttcagttaagcattccaa1080
taacaccttccaaaaacctggagtgtaagagctttgtttctttatggaactcccctgtga1140
ttgcagtaaattactgtattgtaaattctcagtgtggcacttacctgtaaatgcaatgaa1200
acttttaattatttttctaaaggtgctgcactgcctatttttcctcttgttatgtaaatt1260
tttgtacacattgattgttatcttgactgacaaatattctatattgaactgaagtaaatc1320
atttcagcttatagttcttaaaagcataaccctttaccccatttaattctagagtctaga1380
acgcaaggatctcttggaatgacaaatgataggtacctaaaatgtaacatgaaaatacta1440
gcttattttctgaaatgtactatcttaatgcttaaattatatttccctttaggctgtgat1500
agtttttgaaataaaatttaacatttaatatcaaaaaaaaaaaaaaaaaaaaaaaaaaaa1560
ctcgaggtcgacggtatcgataagcttgatatc 1593
$$
<210>
50
<211>
978
<212>
DNA
<213>
Homo
Sapiens
CA 02296815 2000-O1-14
WO 99/03990 PCT1US98I14613
23
<400> 50
ca cgagatgagt ttggccacgtgatgcaccagctctgctccc aggtgggtgc60
t
cgg gcaggggcaggggcaggggctgcctgtggt cagcgaggcc120
gaat
c aggggcaggg
c
gggcc ccggctctctctgcacccgtccggtggctc cttcatccaa180
ggg
gcttcggctt
ct
ggg tccctgtcatgcccgtgtcctggggctgcc ccagcaaaac240
caagc
ccaa agatggtgac
tgccac caaggtgcgtgtatttcctcatggtcctag aggctggagt300
acc agggcttaca
accacag gggttggctccctcgargtccctccttggc ttgtggccgc360
cggaggtcac agtgtcagca
caacaacttc ccgcatctcatgtggtcgtccttctgtgtgggtccccaty tygtcttctt420
acrggacccc agtctgccggatccgggcccgcccaacaacctcacttgac ctagtgacct480
ccttagacat ctgtctctaagtagtcacatctgggattacggcgtgagcc atgttcccgc540
ggaatttctt ttttatagtattggataaagtttggtgtttttacagagga gaagcaatgg600
gtcttagctc tttctctattatgttatcatcctcccttttttgtacaata tgttgtttac660
ctgaaaggaa ggtttctattcgttggttgtggacctggacaaagtccaag tctgtggaac720
1$ ttaaaacctt gaaggtctgtcataggactctggacaatctcacaccttag ctattcccag780
ggaaccccag ggggcaactgacattgctccaagatgttctcctgatgtag cttgagatat840
aaaggaaagg ccctgcacaggtggctgtttcttgtctgttatgtcagagg aacagtcctg900
ttcagaaagg ggctcttctgagcagaaatggctaataaactttgtgctga tctggaaaaa960
aaaaaaaaaa aaactcga 978
<210> 51
<211> 433
<212> 1~1A
<213> Homo Sapiens
<220>
<221> SITE
<222> (424)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (430)
3$ <223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (431)
<223> n equals or c
a,t,g,
<400> 51
cggccgctct agaactagtg 60
gatcccccgg gctgcaggaa
ttcggcacga ggcgggaagg
cttattccaa ggtaagaggg 120
gctgtgtgaa ggggcagtgg
gatggaatgg ggggtggcat
gggacaggca caagggaagc 180
ctccagcccc ttttctgcca
caagcaagag gcactcagcc
ctacctgaga tgtgttattt 240
tttagaaata tctttattga
tggtctttgc actcaatata
aaggcagcat atggttgttg 300
caatataaat ggtacagaag
tccacagagc aaaagggcca
gtttctgtcc cctttcctct 360
ctccaggcct ctttctggga
ccccattatt ggatagatta
agacctttcc agaccttgta 420
aaaaaaaaaa aaaaaaactc
ggggggggsc ccggaaacca
attngccccn naa 433
<210> 52
<211> 861
<212> I~1A
<213> Homo Sapiens
<400> 52
gaattcggca cgagcctgag tcaacttgat atccaagctt tttacttcaa ttatctggca 60
()0 agattacata gactgtcaaa gtttgtgaaa gtttagcaag aaaactgtct tactcacaga 120
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
24
ac taactgactg caccatttgcccctatttccaggcgttatg180
aaccacactc
accacagg agtttctaatctgtatagatgtgtagagcatgcctcttccctcttccttt240
gtcaccctgt
cccctccctg ttttcctttcctcttgccctttcttaatgtctgtytctattggcttcttg300
atcttggtct ttaatgttcatccttaagcttgcttctctcttcagactactgattcagcc360
$ tcttgcattttctttcaacttgggccaaaaaaacaggcaacattttcttcctccactacc420
tcatcatcat ccaatttattcctttagtttatattaccacaactctcctaaacgtcccaa480
gtctattatt aagtctaacaacttagcttcgaacctcaatccaagcatctgacaacacac540
tgaaatgtgc aagcaagagtcccwatggccgggtgcagtggctcatgcctgtaatcccag600
cactttggga ggccaaggtgggatcacctgaggtcgggagttcgggaccagcctggccag660
tatggtgaagccatgtctmwactaaaaatacaaaattagccggacattgtggtgcacgtc720
tgtcatccca gcaaggcaggcgaatcgcttgaacccgggaggcggaggttgcggtgagcc780
gggatcgtgc cattgcactccagcctggtcaacagagcgagactccgcctcattaaaaaa840
861
aaaaaaaaaa aaaactcgtag
IS
25
<210> 53
<211> 510
<212> IaNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (380)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (396)
<223> n equals a,t,g, or c
<400> 53
gatcccccgg gctgcaggaattccgcacgagtgaaaaccgcctccaccaacacccccgtt60
tgcctacacc accccccttttacttagtatgtttattttttgtgtgtctcttgccttcct120
cccacgtttt atttcccctcagagctgtgaatgggcaggtctgtctctggtttggcatca180
ctgagttttt cccatgcattggccccagggctgctaggatgtgagacaaatctccctaca240
atgggcttgc tcccattgtctgtacagtttaatagatgctggcatgtcggaggttaccca300
tgagtcaaaa tccgctctccatgcttactcttgacaccccattgaagccactcattgtgt360
gtgcgtctgg gtgtgaagtnccagctccgtgtggtncctgtgcttgtactgyccctgctt420
tgcagttcct ttgcacttactcatcgagtgctgttttgaaatgctgacattatataaacg480
taaaagaaaa aaaaaaaaaaaaaactcgta 510
<210> 54
<211> 309
<212> ETA
<2i3> Homo sapiens
<220>
<221> SITE
50 <222> (301)
<223> n equals or c
a,t,g,
<220>
<221> SITE
55 <222> (305)
<223> n equals or c
a,t,g,
<400> 54
gaattccccg ggataaatttcatttcccaaagatgagtaggtatgaaaaa 60
taatactcag
60 aagagattgt tcttgtgggg ctacaggatctagctttgat 120
agaactgctt tttgtatctt
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/146I3
tcaatctttt taaaatcaac tttaacgaat ttaaacctat tttaagtgta caagtaataa 180
gtttgacaat tgtatgtgac ttctaccaca ataaaatata gaacattttt atcattctat 240
aaaaaaaaaa aaaaaaaaac tcgagggggg gcccggtacc caattcgccc tatagtgagt 300
309
ngtancgtc
5
<210>
55
<211>
1585
<212>
CIA
10 <213> Sapiens
Homo
<400>
55 aaatatgttcatgtataatacttgatcaaaatatttttgggttttttgtt60
ggaatttctt
ttgttttaatgggttagaaaatgtttacaatcttggtcttatatgatcaccaatggaata120
15 gtaacttccaggtttatatcaatatgagctgactttaactgagttgtttgggatagggaa180
gaagcagtccctctacagtatacaactactgcttgccagctggatcaaaataatcatgtt240
ttatgaaaatatctcccttaagcagtgttaaggttggtttgcagtgtgtaagtggcacat300
tgaactggaagttttcttgaaagctgcttcatctattaagaagcaattttcaaattgtag360
cgaattatattatcccctcttttaaagaaacagtcgttatatgctgatgtttcttaaaat420
20 aactaaaatgtkcctcttaatgtgattttaaatggagttatttgtaggtcctttcttagt480
agtaaagaatcttctagagggaaacatttgtgcttttagggataatcttccttgtgcctc540
actacatccctaagtgggtatgactcttgttattaccacatgcttttttagtatatttca600
caaatttacttttaaatattattttagatacggtgtaacatgtgcaattcagaataattt660
tataacaggtcatgaaaaacataactttagttaggattcacaatatttgtv~rctccacata720
25 atgagagaatgaatgagcctttggagatactgatataaggcaattattttttgcaatgtt780
gaatgtgttttttagtttgattctttttttttcccccaatagggcactacctgccatatc840
atcttgtattactttttgatgtaaagcgactaatatttacactatgccatatttttttta900
attatagttgtaaattatgaaagatccttgaattttctacagatctacaactactaatgt960
aacagacaagggcaatcttggtatttaaatctgagcatggcagttctaccataaaaagta1020
ctctatttttctaatttctaggatttttaaaataacatttctgtaagtctgacatactaa1080
tagtcactcaagcagtaccatttattttagtttgcatatattttcactgtttttaattta1140
atgtattgagtctaataggactgttttgcaataattrgaataaagatttatttcttctaa1200
tcaaagatgcataacagctattatctaggggaccmccaaatgtgatttcaaaattttgtt1260
aactattacaaatgtaatccttatatagaaattttaattttgtaaagtagtgtataatat1320
tgtaatattaaattcttgttcttaaattcaaatatgtattgatcttcaatgtgctgtgtt1380
aaatcttgcttctctgaaaagttggagacaagatttgtcttcctttttacagtttgtaat1440
tttcactgttttattcctgttaaaaaaaaaaaaaagtcatttgtaacccatgcagaccat1500
tgtttgatctatgctaacttatcaacttggctattcaataaagttaattgaaaagaaaaa1560
aaaaaaaaaaaaaaaaaaaactcga 1585
<210>
56
<211>
874
<212>
ET1A
<213> Sapiens
Homo
<220>
<221>
SITE
<222> )
(468
<223> or c
n equals
a,t,g,
<220>
<221>
SITE
<222> )
(501
<223> or c
n equals
a,t,g,
<220>
<221>
SITE
<222>
(546)
(70<223> or c
n equals
a,t,g,
* rE3
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
26
<400>
56
aggggaatctcggtgctgcgacgagtgtggggccagccgtggaggctccaggtgttctct60
ctgccccagcagagcccggcaggagccccaacaggaagccagcgcggcatggctgccacc120
$ gacttcgtgcaggagatgcgcgccgtgggcgagaggctgctgctcaagctgcagagactg180
ccccaggctgagcccgtggagatcgtggccttctcagtcatcatccttttcacagctact240
gttctgctgttgctgctgatagcctgcagctgctgctgcactcactgctgctgccctgag300
cggagaggcaggaaggtccaggtgcagccgacaccaccatgacggacgggcgatggctga360
ggagaagctggagaggagatggccaatgccatgacacaggccatcagcctggccctgcag420
cccttacccctcaagaccaggctcccctggccccagctctggcccagncccaggtacctg480
gacactgacaacttgagcccntaccaaggaaacaagggctggtataggtgcaaacctctc540
atctgnccagtggacactgggtgctggggagtcagctgtttcaaagactgggtcaactgc600
ctgggcttcttcgcctacctgcactttttaacaaaacaaggaagtaggggtccccatacc660
ttgatggagaacagtccccacctgtgggcaattggcccttggggctctgctgatacatgc720
1$ caaagaggagcaaggcaatcagaggggctttgtgcaatagcttctgcatccgagctcccg780
ccagagcgtgagcatgtcagtattctagtccagtatttgccagtttccaagtaaaagctt840
ttgtgttaaaaaaaaaaaaaaaaaaaaactcgta 874
<210> 57
<211>
1169
<212>
DNA
<213> sapiens
Homo
2$ <220>
<221>
SITE
<222>
(2)
<223> or c
n equals
a,t,g,
<220>
<221>
SITE
<222>
(9)
<223> or c
n equals
a,t,g,
3$ <400>
57
gngcggccnccctttttttttttttttatattttatcaattttattgaaatattccaagg60
atcccaaccccatttaaaaataaaaattgtaaagcactccattcaataaaagcacataag120
tccccctcaataattagtatgacaattcacgatacagctcttactctgggagagtttatt180
ttaccctttattccaaaaggcacaaagtcatctgaggcctcagatattaaccccactgca240
tgttaatgacacaccactgaggtgcagctcaatgtaattattaaagcttataacacactt300
ccccaagaatttatagattctttctataaataataatttaaaaaatactgcaccttaaga360
ccaatacaggcttaacaaaagacctgaaatttctgcaagggcagttttgtttcttgatag420
aagtacaacttttgaaagtctattcccagcaaaagaaacactagacccagcttggccaaa480
gaaacaaaataaaacaagtgatttctaacacgctaaaagagtacattttcatcagctcca540
4$ aagaaagcagtcctggtcattcagaaggctcctatgatcccaccagtctgcagtcattag600
aaatatatgctttacaggccacaggctgctctggatttggtttcagacaccagtgaccag660
aagaagccagttttgcgtgtgaggggtgtgggcccccgctgccttgggcctgctcaccgg720
ggtggatggacccccgccgggtcacagcctgctgtcacgtctggactgttggcctcttct780
gcatctgggctgttgggctctcctgctctctgtccctcagtcacgtcattgtctggctgt840
$~ ccggtgctggctgcactctcatttgtgaggataaccccttccttcttcttttctcccaat900
acctccagccccatcatcctgagataatgaagccgttcattcttgggcacaaaagttcga960
atggaggcctttccccgccatccgcataagacgatgggacactgcagagcgtctggattc1020
gcagaatctggttcatacttcagcacgatgcttccctttgccaggtcctttgcttgactg1080
taggtctcactgctgagttttctaaaaaagggattttcctgggtcaacagtatcttaaca1140
$$ tcttccattgatacagtaataattctttg 1169
<210> 58
<211> 1066
60 <21z> DNA
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
27
<213> Homo sapiens
<400>
58
gaattcggcacgagcaaatgttgaaccaattatgttttggtggtggtgttcttagctgtt60
gaatcctgaatggtttataaagtgaactagctggcttaatgcagccagcgttctgggcag120
cagaacatattcattcttactgtaaattctatttgctgcttccaaaggtgatgattttca180
agcagacatgttctatatggtctgtgttttaggatctggtgcccagcctctatcagagct240
tgcctacctggcaaagctgcctacccttcaagtgggaaaatataatccactgtttaacaa300
ggctcaccctctccaccctgtcctaacgaccttttgtgaatgtgctgtgatattttcttg360
1~ ctcaatagcaaggtggtagctctgctttcattttaagaaagtggaggctgagggcattgt420
atcaatactgttgcaactccaagaagttttccttgtaaaattaaaggaaagatcttgtta480
ttgattaaccattttcttatgccttgctattgacatattcatgctctttctacgtctagt540
ggctgaaaatgtttgcatttgttcatttgactaatggtgtgatttttgkyycwatattat600
tagacctgtaatgttttaaaatgtattttattaaatttggactggatgtatgkcctctag660
IS caatacgaggtactttctaaactattaagggaggggttgtaycctcatgttgagataaga720
tgatggtcgtttaaattttgcaattttttttggcctgcagggatattttgtgtttatgtg780
tccaaaaaaggaataaattggcattcttgtgccaaaagttgtttttcctgtcaattgtct840
aataagtatgcagtacactgtaatggcaacatacatggttgctttataaaaacagtttcc900
tcagtatgagaaattttacaaagaacagtggaaaaactttgtgtttttaactcttgggtc960
2~ tccctatttttaaaaattgctatttggtatacaattattatgtgtcaattaaaactaaaa1020
taaaacttttaaaaaaraaaaaaaaaaaaaaaaaaaaaaactcgta 1066
<210> 59
25 <211> 772
<212> ~1A
<213> Homo sapiens
<400>
59
gaattcggcacgagctttcctgagcctcagtttctccaacggtgggaggtggtagaaatt60
gatatagtacttaccactgagggtaaaatgagatataacctgtgtaaatactgtacacca120
cagtcattcaatagtggcagcttaaaaaaattattctacgattacccttgcttcagtgat180
tcttcttggtgttattgaagggtgagatctcggtggggatctcccaggtgtttccataat240
cccagcgatcaccccagggagaacctctctccttaggctgctagaggacatgtgccatag300
35 gaccagataggagggaggggcagcggtgggaatgcgttttcagagctacctttggccaag360
ccgtatccttgtggggacctattgcattgctgctgaagtgctgttcccatcagccctggc420
ttcgtgtggccctgtctggcaagggggtgctcctacaaagtcatggcagcctggtgccaa480
aaccatcatcccataggacctgctgtagctttgccagaagcctggcccaaggggtggagg540
cccctggagctctgacccaccacgtggagggtgggaaatgccacagagcaggttctctag600
4~ aagggatttgtcagaagctaaactggggtgccccctgggctcaggcctgcacagtttctc660
cctgaccacccagctgggatggatatagagacaggtgtcatgttgcagaaagcctgccct720
aagaggccctactggtgttttcctttattaaaaaaaaaaaaaaaaaactcga 772
45 <210> so
<211>1198
<212>DNA
<213>Homo sapiens
50 <220>
<221>SITE
<222>(1189)
<223>n equals a,t,g,
or c
55 <2zo>
<221>SITE
<222>(1191)
<223>n equals a,t,g,
or c
<400>60
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
28
tcgacccacgcgtccgattt ccccacagtttaggtatttttcattagtac60
aattcttatt
atcaatttgacacactgaatgcaagactattaaggaagaacgattaaatattattttatt120
ttgtgaagagttggcagcagattacatctcaagaacttgcagagagaggaaggtagatgg180
acaatcctaaattgtaagatgttacaaaaaacagtgaagtaagagtactcctgaagacta240
aaatagagaggctggggtttgagccattttactgagtagcttagctggaacctgatatca300
gaagtagcctttaacaaaaagcctcttggcaattgtatggtactaacaactagagtactg360
aagtgtaagttgaaaccaagttgcagtgggaaatcaaaggtgaggtagcttatttgaaac420
cagcaaatgagacaggttggacagttttaaaatctcttctaacaaagaaactgcacggta480
gcaaggactagcggttctcaaagcccttctttttcagtgttctcattcaccttggcaccc540
aagtatgtttaacaggccatgcattaaaaataaatacaaaaatataaaagccgcttaaag600
ggaacttacaaactgacaatctctcctctgtatttgtgttcatagtggctgggagtttaa660
ttatatgcacaaaagttaggagccacttgtttctgcacagactgtaggagcaagatgagg720
agatgggcaggttttggtaagagcccccagttctggtggacaggcatacttgtggcattg780
ggtgcggcattgctgggaggaccacgtcttgggaggcgattgacttttggtttgtaattt840
IS ccctttaaacaagaagagatggctcacattttccatatatatctcaatgaatgtactgta900
ttactgttttaaaaatttgatgaaataataatgaattggtctccttttgttatctggtcc960
ttgtttaatttgtttaagggtttttgtatacaaaagtttacatttttatgtatatttttc1020
ttgtgtaaaaactgatgtaatatgtgtatgaaacactgtatgtattatctgtatatagtg1080
tgacaaaatcatttttctttctttcttttggatgtattaataaatcttgctgtgaagtaa1140
aaaaaaaaaaaaaaaaactcgagggggggcccggtacccaataaccctntnatgatct 1198
<210> 61
<211> 558
<212> ~.
<213> Homo sapiens
<400> 61
ctgcaggaat tcagcacgagytggcatgtgacaacccagggctgcctgaaaatggatacc60
aaatcctgtacaagcgactctacctgccaggagagtccctcaccttcatgtgctacgaag120
gctttgagct catgggtgaagtgaccatccgctgcatcctgggacagccatcccactgga180
acgggcccct gcccgtgtgtaaagtagcagaagcggcagcagagacgtcgctggaagggg240
ggaacatggc cctggctatcttcatcccggtcctcatcatctccttactgctgggaggag300
cctacattta catcacaagatgtcgctactattccaacctccgcctgcctctgatgtact360
3$ cccacccctacagccagatcaccgtggaaaccgagtttgacaaccccatttacgagacag420
gggaaaccag agagtatgaggtttctatctaaagagagctacacttgagaaggggacttg480
tgaactcaac cacaatctcctcgagggggggccggtacccaattcgscctatagtgagtc540
gtattacaat taatgggc 558
<210> 62
<211> 616
<212> DNA
<213> Homo Sapiens
<400>
62
gaattcggcacgagtcttgacagcctggtcaccaagggtttggaaaaaggttctattgga60
gtggagattgatgggtggaaaaaggagagaggggagttggacctgataccaaagagatgt120
tttcagccatcaaccagctgcaaaacaagatgggcttccttttcctacatattcttccaa180
gcatcataaatactcggtctgctccccaacccacatcctgcaggatgcagccagagcaac240
agccccactccactctgaaaccagtcatcctagggatgatgatcatttcttagcttccct300
gttggaggtcggttggggttggctgatcgctgcttggttcactcctgcactggctgggcg360
ttggctgcatggtaaagctgttccctgtctcatcctgttgggataaacagagtatcctag420
gcatattttctccagagcagtggcagacacaaagggtcaacagaaaccctcaaggttttg480
5$ tcatgcctactcttgcaactagcacattgtcatttcagcctcatgctattgaccaaagca540
agtcacttgaccaaattcaaagccacaaaactcgtgccgaattcgatatcaagcttatcg600
ataccgtcgacctcga 616
<210> 63
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
29
<211> 811
<212> DNA
<213> Homo sapiens
$ <400>
63
gaattcggcacgaggagcttccatcttttctgatgtgagtggtgtcaggaatgactatgg60
tggtggtagtggcagtggcgatggttttctggaggctgaaaggttaaagtcccaatgcag120
aagtgatgtcagggctagtgggtggcggtagcaggtgcagtaaagtcaggttcagatgct180
tcaatggtgactcccttctcgtgttagtcctacagcatcatttcagactttgttcttggt240
gcttagctccaagcctcttcctcctgctgtcctgtcaggttgtgtccactatgatggagc300
aagaccctgtcatctatgatgatgatgacgacttgcctaattatttttctgtttaagcta360
gccatagtggatcctgttatttgtgcctaagagctcttactgacaaagaacgtgttaccg420
gaagtgggatgctacaagtaacaacactaaaagtagaattgactaagtgcagcaggcagg480
cctttgagcaaggaggggacacacattacaggctggaaagctggtgactcttgtaatgca540
1$ gtggcaaaattttgcttcaactactatatacaatacttgaagatgcacactgcaagctga600
gtgaggctgtgataagaggggaaatagtggggagcattcagaatgttggtttacattgat660
gacttcttgctctttcagcagtcttgatagagcagctatacccacaccagagtcctccag720
ctgacaagagaggtaaggagagaaactgctttgccaggaggggccctctgctgcagctgg780
aggtccaagttgaccgagagcccaaattttg 811
<210>
64
<211>
993
<212>
DNA
<213> sapiens
Homo
<220>
<221>
SITE
<222>
(370)
<223> or c
n equals
a,t,g,
<400>
64
ggcacgagcccaaagtgctgggattacagggagttgatgaaagtggagatgtttttagag60
ctacctatgcagcattcagatgttctcctatttctggtctgctggaaagccatgggatcc120
aaaaagtctccatcacatttttscccagaggtaggggggattatscccagttttgggatg180
ttgaatgtcaccctcttaaggagcctcacatgaaacacacgttgagattccaactctctg240
gacaaagcatcgaagcagaaaatgagcctgaaaacgcatgcctttccacggattccctca300
ttaaaatagatcatttagttaagccccgaagacaagctgtgtcagargcttctgctcgca360
tacctgacangcagcttgatgtgactgctcgtggagtttatgccccagaggatgtgtaca420
ggttcctgccgactagtgtgggggaatcacggacacttaaagtcaatctgcgaaataatt480
cttttattacacactcactgaagtttttgagtcccagagagccattctatgtcaaacatt540
ccaagtactctttgagagcccagcattaacatcaacatgcccgtgcagttcaaaccgaag600
tcccgcaggcaaatttgaagctttgcttgtcattcaaacagatgaaggcaagagtattgc660
tattcgactaattggtgaagctcttggaaaaaattaactagaatacatttttgtgtaaag720
taaattacataagttgtattttgttaactttatctttctacactacaattatgcytttgt780
atatatattttgtatgatggatatctataattgtagattttgtttttacaagctaatact840
gaagactcgactgaaatattatgtatctagcccatagtattgtacttaacttttacaggt900
gagaagagagttctgtgtttgcattgattatgatattctgaataaatatggaatatattt960
taaaaaaaaaaaaaaaaaaaaaaaaaaaaaatt 993
<210>
65
<211>
689
<212>
DNA
<213> sapiens
Homo
<400>
gaattcggcacgagctaaggtgggcgggtcacttaagcctcgaactcctggcctcaagca60
atcctcctgcctttccttcccaaagctatgaaattgcagacaggagccaccatgcctggc120
60 tggtttttgggggccatggcaagtgcaggcttgtcagaggaattggagaagcagggatta180
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
gttaggaaaacctctccacttcttgtgtttcatgccaggtagtgtttgtaacttcagaac240
ccgcccttaccttacctacctaccatgttatgctcatttcacctactgtcccctgctgta300
tagggagtgccttgagggcagagatcatgttagttttgttccctcttctgtacagagggt360
ggagcccagtacctggcacagctgaaggaggaatgtgctgctgctgtctctgtatttcca420
ggtactccttgttgacctctagccaagacaaggaacctccttatgagatgtcatcttctg480
agctctcttgatggagggaataccacggtgatgattgaatatgaaaagtcttggcacagt540
ggctcacacctgtaatcccaacactttgggtggccgaggtgggaggattgcttgaagcca600
ggcattgagaccatccttggccaccaaacgagaccccatctctacaaaaaaagaaaaaca660
aaaccaaaaaaaaaaaaaaaaaactcgta 689
10
<210>
66
<211>
942
<212>
CIA
IS <213> Sapiens
Homo
<400>
66
gaattccaggactgctgggaccccctgcacctcctggccacggagagatcctgctcccag60
ggaccagcgtctgggtgggacacagttcactcctctctccacttcatgttctttttcttc120
20 agcagatggctcaagttccttgtttttctccttgctttctgacagccgtagcttctgaaa180
cctgccatttttggtctcctgatgcctgatttcctaattgtcctgactgtgtcttctagg240
aagcattaagtctgaactgacttattagggaacttcagaaagttaaacacacaaaaccct300
ttctttgactcctatcttaaggacatggagatacagttacatatatttatacacaaggat360
attcatatggcaaaaacggggagaaggcacaatttaagagcccaatggggactgggattg420
25 tgtatgcatctgtacaatgacatgttatgaagtcattctgttttttataaaactttttag480
tgacatgggaaaatacaaagaatgtaaagaatttaaaaagcagcgtacaaaacsnatatat540
gtgatccaatttgtggtggaaatattttatctatatatatccattttaaamcaccaarga600
aaatacacagttaacagtagttatctttggaaggcaggattataagtgatcttagttttc660
ttccttccacttttgttaccgatatcagaaaaaaactctgtctctacgaaaataaaataa720
30 aatgaaataaaataaaattagctgggtgcagtggctcatgcctgttgcctcagctcctca780
ggaggctgaggcgggagaatcacttgggcccggcaggtcgaggctgcagtgagctaggat840
cgtgccactgcactctagcctgggtggcagcaagaccttgtctcaaaaaaaaaaaaaaaa900
aaaggaattcgatatcaagcttatcgataccgtcgacctcga 942
<210> 6?
<211> 2309
<212> CIA
<213> Homo Sapiens
45
SO
<220>
<221> SITE
<222> (13)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (652)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (677)
<223> n equals a,t,g, or c
<400> 67
ggtaagagag aangtgtgaa gtcacctgtctgttctagtcattccaatggacattgtact60
ggcccaggag gaaagaacca gatgtggttgtccagtcatccaaagcaagtctctagcaca120
aagcccgttc cactgaactg cccttctccagtgcctcctctgtatttggatgatgatgga180
ctcccctttc ccacggatgt aggttacggcaaatcgaagcagggtacaaa240
gatccagcat
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
31
caagaggtggagcagctacgtcgacaggtgcgtgasttcagatgaggctggacatccgtc300
actgctgtgcccctccagcagagccccccatggactatgaggatgattttacatgtttga360
aggagtcagatggcagtgatactgaggattttggctctgatcacagtgaagactgccttt420
cagaagcaagctgggaacctgttgataagaaagagactgaggtgactcgctgggttccag480
accatatggcatcacactgctataactgtgactgtgaattctggttggccaaacgaagac540
accattgcagaaattgtgggaatgtattttgtgctggatgctgccacctgaagctgccca600
ttcctgatcagcaactctatgacccagttctcgtctgtaactcatgttacgnaacacatt660
caagtctctcgtgccangggaactcatgagccaacagctgaagaaacccattgctacagc720
ttccagttgaatgccggggagaaacctgtccaattttagcaggtttgaagggaggatctt780
10cttcagttgtagtttggaaggttccttggtgtggctcatgaaatcacagagctcagagat840
accatcttgagaaatcctccttggtatcatgaaactggagcagaggaattgcaatttagc900
aggaggtcctctactggtgataccctcaccttggggtaatggtcctaacccagacccagg960
gtctggaaagcttaatgttgagttggtgactccagcctctttctcctggaggtcacaaga1020
tgatgattgcgtagatgttgcctggtgcaaagtgccccaaacagcaatagaaaggcatat1080
ISgtataaccaaactccaagtgataaccagacccatctctcctccaccttgacaaaagcaga1140
ttatagtatacaaggtaggaattcctgtcctatttgagatgaactatatcctgtacctct1200
gtgctctgtgtctgcatgaaggctcagcctttagaggcactccttctagttgcattagta1260
ctgtctttctgtggagtttggtttgaagactggctcagcaagtggaggtttcaatgtatt1320
tttcagttggctcatcagccagcattggtgaatattcagtttaggggaacagttctaggg1380
agtgagacatttttgggagcagaggaaaactctgctgatgttcggtcctggcaaacattg1440
agttattttgagctgtgaaggcagtcgtctctgttacacagtggcagctcttgagttatg1500
cactgtgaagaatgagaagggaaaagcaaaaattatccttgtgaaatatctgctgattgt1560
gccctactctttgcacctgacttttcctagttgtcctggtgctaacacaggagctacacc1620
ttgatcctctcctggcatgaaaataaaacaaaggttttcgttgttgttgttccattgccc1680
25atttcccccatgttgtctttcccttggctgatgcctcctctgggtcacattgcttcttat1740
cctgaacacttgacaccttgagggtagaatttagcgtttggtttttacctcctagcatat1800
gctgtttggtatgtgagggtttcagtacaaatgctgctgtctatttctgtgcacttaaca1860
atggaacccaaacagaagagaataaagccttgataccaaaattgggaaagaacatgtgtc1920
catttggaccaaacgttgttggtttttaaaaaattttattttgtttttttgtttttgttt1980
ttgttttttttcatcttaatatgtaccagtggcacttaaccaaaagatacagtgatatag2040
ccatgtatctgtctacttagcgtggctgttttgagggactgtcccatcagtgaacaaact2100
gcatggccttggagagagactctgggctcttggctcagatgtgttcatcaaatactcctt2160
tcagagctgttgtgggtgtaagtgacatgatgtggccaaaaatccaaactgtgcagttgc2220
gttgtgacaaacatgcaatgtgctgtaaaaattcaatacagtttaaataaaatctctata2280
35ttagtaaaaaaaaaaaaaaaaaactcgag 2309
<210> 68
<211> 814
<212> DNA
<213> Homo sapiens
<220>
<221> SITE
45 <222> (421)
<223> n equals a,t,g, or c
<400>
68
tacgagtttttttttttttttttagccataattaccaaaaacattagtgcaggacaccat60.
$~ tttaaaaaactatttaaaatagtcttcagagaaaaaatattaagtattacagtttaggag120
tatattgactttgggccaacggattccaatattttacaaaaaggcaatatccacgcaaca180
tattccagattcgggttgtggagaagctgcagggcttgaggtgactctatcacaactgct240
ttccgtacggaggagccactgccaactgtgtggacgagaatacttaagcacgtgcttcat300
tgctccactgccacaggtggatatttcaggggaattattattaatttcaaagttttttta360
55 aaargytatgataagtaaataaaagtaatggtaggaktcacggtcggagagcttatcgcc420
naagtctttctatagccttcccccggaagccccagttcaggcatcggtcacccgaagtgt480
caccctctgatctttcccccatcccatctgaggaagttaaagagatccctcacaggtacc540
gtggctctcggtgccctcgcacttccaacagccggttcgggcccaggagactcgctccga600
cctccaccacaatggcggccagtgtgggccgcgcaaccagaagtgcggccgcgcacctga660
60 cccagcttccgcctgcacctagagctcagcgcaccagcccggctcagccagacgaaggca720
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
32
aacgaagaga tgcggatccc tggaggactg gccccaccgt gaacaaaaca ggaagcattc 780
caggaagact gcgggggtgg gctcgtgccg aatc 814
$ <210>
69
<211>
788
<212>
DNA
<213> Sapiens
Homo
<220>
<221>
SITE
<222>
1370)
<223> or c
n equals
a,t,g,
IS <400>
69
gaattcggcacgaggcaattttcaatgaaccttgaatggtaggaagaattgaagaagaaa60
tcagagcatttttgccttgcagaaggcagctgctgtgatggcaggaggctgaaatggaca120
tggcctggcagaagagtattatggggtggttgtgttgtgagccatctggcctgtacaatt180
tggagaaacaatacttttttttttcttctctgcaagctgggcttcctgtgattgtgtcct240
caggctgcacaaaaatagcgtatggctttgctgtgtattcaccttcatcttaaaatagct300
agaacattttccctcttcttttaaaaagtttttaaaatgagggttagactcttgtaggaa360
aaggtagaantcttaataacagtactcatgttgacaaacctttctcgtcaaaattcctat420
gtaatcaagactcttattaaatatgaacaaatgtaatgtatggaaattaatgtttaccct480
caaggtaaaagctgaaatggatttataaagaattattttaaacagcaataatgtttgagg540
ggtgggggaagtgagaaaaatgaaattttaaatcacatgtttatgactatgaagctagac600
tttaaaaataggtcagttagggtatgactcttataatacaaaagtttatttggtatacaa660
aggatttatagctaatgtattttttaattatattcactaatacttgtaaaagatcattca720
atttataaagtttccaaaataaacctgtttaaagtgtcaaaaaaaaaaaaaaaaaaaaaa780
aaactcga 788
<zlo>
70
<211>
791
<212>
DNA
<213> Sapiens
Homo
<400>
70
gaattcggcacgagctcaaggctaaaatcttgatctctcctgaatatgaggaggtgtgtt60
aggcatgttttggggattggattaatagtgttaaaaaatttgtattttcacaaaaatagc120
atgtacccatcacccaaactcagcagctttcaagaagcttttctttttttctttcttatt180
ttaaaaaatcctttaaccttatgtagttagtatatcttttttaaaaagtagaaaatcatg240
taaccttaggatttttagttttaatgtagagtttcacaaatttccatctttagtaagaca300
aaagggtcacatattggctgtctccttcaactatactttcttcagtataaaatatgttta360
ccatggttgtcattatcgagcacgtaactgcatgttagactctatgctaagtgttttaca420
taatcatttaaagctcactaaggccctaggagtaattattatcctcccatcaaaaaggta480
agtgaaatgttaacctgaagtttgactactttaggtctctgagctagtaagtacaatagc540
caggtttcaaaccaagatccttttaactgcagcacctgtgccttatctggtagcgtcatc600
ttggttcatacatttaaaaaagagttatctatgtgccgggtgccctggctcatgcctgta660
atcccagcactttgggaggccgaggagggcggatcaccaggtcaggagtttgagactgac720
caataaggtgaaatcctgtctctactaaaaaaaaaagggggggcccgtacccaatcgccc780
aaaaagatcgt 791
<210> 71
<211> so4
<212> DNP.
<213> Homo Sapiens
<400> 71
gaattcggca cgagcggcac gagcttgaaa tggcgtcttc tgatgaacac tcatccatcc 60
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98114613
33
ttcaaggtct actctctcatcacagcttgtgactcttccactttttgaactggtgtttcc120
cattcccagt tcacagagccctttctcattgaactatttatctgagttccctctgccgga180
acatgagcca tgcctagagtagccacctagtagtgagtgacagctctgtgctggatgcac240
ataaatggtc tcccttaactgccatgagscctaaagaaggtttgctacagctattttaca300
gatggggaaa actgacagagagatattaatgaattgcccacatgcaaatatgtgctgagt360
cttggatttg catctttatcgtgactccacggagacccaccctctaagaccagagccagt420
gtcctattca tcttttgtctctgcagcgttcagcatggcactgtcttggcttacaaaatc480
tgctctatgc ttgctgactgctgaatgaatgaatgaatgaataggtagtcacaaagaatg540
tttagaatgt ttctcagacaggctgagaaaaaacacaacgaaacattatttccgtttgga600
aagtttttttatttttgtgttcagtactgaagtaaaacaaaaatctgaataacagctgca660
ccgttaaaaa tgaaattaccaatatatgaactctaggcatcatgcatatataattttttg720
tagataactt ttcttctcattttccttctcattctcttcatctttttctttttgtttgag780
caaaaaaaaa aaaaaaaaactcga 804
20
<210> 72
<211> 783
<212> I~IA
<213> Homo sapiens
<400> 72
gaattcggca cgagctaaaacttacaatgacatgttgttgcttgctctgtaagctccaag60
gcattttttt tttcagttttaattcaagtgttctaaaaagtattttgggtacaaccagaa120
ctctctctgc tccttggattggagtcagtgtgaaaggaacacagtgggctctggggtcag180
ctagacctgg atgtggatcacagctcacctcttcattgggaggcctcaggcaagttattt240
gccaacctca cctacaaaagcatgatgctaagctcwtttcagtttagttgtggatatcag300
agcatatgta tacaatgcctgccatagtgagtgcctggcccttggcagactgtcaaatgg360
agctatggag cagcagcgggagtaatattattatctagaccttatctgtccttttaaact420
cagttcagat tccttctcctttttaaattactgcaacctgattttacctgcccctgcctc480
caagttgctg~atcagttagcctctgaacaattcatttagcaattttaattatatattgc540
ttcttgacac tgctttgtgatcttaaaaactctgcttcaaatacgtacttggttgctttt600
cctgagtgct gttaattcctgctctaacggactaaagtaatttgaaggcaggactaggtt660
ttatgcatgg cacacagtctggtgccttacatgtaactactcacaaacttttttgatcca720
aaatttagaa acttcacacgcattcataagaaatcaataaaaaaaaaaaaaaaaaactcg780
tag 783
<210> 73
<211> 1523
<212> DNA
<213> Homo Sapiens
<220>
<221> SITE
<222> (1)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (8)
<223> n equals or c
a,t,g,
<220>
<221> SITE
<222> (15)
<223> n equals or c
a,t,g,
<400> 73
nggggggncc ccccntttttttttttttttttttttttttttcagttctactattattta60
tttttttaaa tatttttgaaaaaatataatttttttacaatattttcaacttaaacacta120
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
34
ttcacactgaacacgtatggcagcttaacctacccaaatatgaagtttaagaagccaaaa180
ctgttctagctttgttaaaagttgtgctgcagactctcgtgatggttaacaaagcaagga240
aaagcaccactcaaatcataatgttacagtatctttgttcagctggattatgggttggta300
ttggtcatatgttagactccatacaggcatagctatgatgcagtgaatcccttagaagtt360
acaattctcaaattacatacttcctcagatgtaacattagaactcaatatttctaacaat420
aacataccagaaaaggctggactggcactcatctgctgactaacttgtagcctcagtaat480
atgacatacttgcctttaacaaattatctcaaattaactaacagaccttcagaaaatgga540
gattctttttgatggggacataatcaaatttaagtctgagaaatatgcttaacagttgga600
actcaaattaaatgtactgattttaaagtttagacattaacaagtgatagattagcctca660
aaaaaagacaatttggtaaggtttaggtcttttaatttggtgcttgttcacaacttgact720
ggtgcttctttccttgctgtcttcacatcaagccatggggccaattctattttcagtaaa780
tgtttgacagctttttacttagtaacagtctcagcacttttattaagcatgcaagactaa840
caaaaactttggcaatgcataagtgtaacacagtgacaagagagcttttacaattaagtc900
ttctaatactgccttcacagtgtggaaattgtgctacatccaccaaaagagggccccgtc960
1$ tactcaaatatttccgtacttcaccccaggaacaaactcctttgcatttggattcagatt1020
gctcttgaccacaagatcttccagagaagagccatcactgataacaaggtcattaaactg1080
gtcttggatttggtccatagtttgtgggagatctcgagctggaataaaccattcatgctc1140
ttcttcctcttccagcatttcttggaaacagcgttcaataaattcttcttcccataactc1200
ctcttctatttgtctgttgaattcttcttcattttccatccacatgtactctgcaaatgg1260
attgtcatcttcatgagaatgaccgttaataatcacatcttcattgatgatgcttgggct1320
agtactgctgcgacttggatctttcatggctgatgttggttgtcgtttttaacccaatgc1380
acagcagcggggacggcagccaacgaatcctgtcggcctccgcggatctccacaggcagc1440
gccgctcccccgctcgacgtgcgcttcgcccgccgcctcccttctcccggacgcgtgggc1500
ggacgcgtgggcggacgcgtggg 1523
<210>
74
<211>
758
<212>
D61A
<213> Sapiens
Homo
<400>
74
gaattcggcacgagacasggtttcaccctgttggccaggatggtctcaatctcttgacct60
cgtgatctgcctgcctcggcctcccaaagtgctaggattacaggcatgagccactgtgcc120
cggcctttgttttttgagaccttttttattttgttgtcacccaggctgaagtgcagtggc180
acaaacacagttcactacagccttgacctcctgggctcaagcaattctgcctcagtccca240
caagtaggtgggcttacaaatgcacagcatgacacctggcttatttttgtattttgtgtg300
tgtgtgtgtgagccactgcgcaggccttgggcagctttcttgatctctgttacctcatct360
ataaaatgatgataataatagcttctcccttattggggaattgtaatgattaaatgagat420
aacatgtaaaatgctcagtacaggccaggcatggtggctcacgcttgcaatcccagcact480
ttgggaggctgaggctgctagatctcttgaggccagcagttaagaccagcctggccaata540
tggtgaaaccctgtgtctaccaaaaaatacagaaagtcagccaggcatggtggtgcatgc600
ctgtggtcccagctactcagaggctgaggtgggagaatcacttgagcccgggagacagaa660
gttgaagtgagccaagatggcgccactgcactctagcatgggctacagagtgagagcctc720
tctcaaaaaaaaaaaaaaaaaaaaaaaaaaaactcgta 758
<210> 75
<211> 1096
<21z> ~
<213> Homo Sapiens
<400> 75
ccccacggct cccatggcctcttcctgcgctaccgtgtggaggccctaaccctgcgtggc60
atcaatagcttccgccagtacaagtatgacctggtggcagtgggcaaggctttggagggc120
atgttccgca agctcaaccacctcctggagcgcctgcaccagtccttcttcctctacttg180
ctccccggcc tctcccgcttcgtctccatcggcctctacatgcccgctgtcggcttcttg240
ctcctggtcc ttggtctcaaggctctggaactgtggatgcagctgcatgaggctggaatg300
ggccttgagg agcccgggggtgcccctggccccagtgtaccccttcccccatcacagggt360
gtggggctggcctcgctcgtggcacctctgctgatctcacaggccatgggactggccctc420
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
tatgtcctgc cagtgctgggccaacacgttgccacccagcacttcccagtggcagaggct480
gaggctgtgg tgctgacactgctggcgatttatgcagctggcctggccctgccycacaat540
acccaccggg tggtaagcacacaggccccagacaggggctggatggcactgaagctggta600
gccctgatct acctagcactgcagctgggctgcatcgccctcaccaacttctcactgggc660
5 ttcctgctggccaccaccatggtgcccactgctgcgcttgccaagcctcatgggccccgg720
accctctatg ctgccctgctggtgctgaccagcccggcagccacgctccttggcagcctg780
ttcctgtggc gggagctgcaggaggcgccactgtcactggccgagggctggcagctcttc840
ctggcagcgc tagcccagggtgtgctggagcaccacactacggcgccctgctcttcccac900
tgctgtccct gggcctctacccctgctggctgcttttctggaatgtgctcttctggaagt960
1~ gagatctgcctgtccgggctgggacagagactccccaaggaccccattctgcctccttct1020
ggggaaataa atgagtgtctgtttcagcarmwaaaaaaaaaaaaaaaaaaaaaaaaaaaa1080
aaaaaaaagg gcggcc 1096
I S <210> 76
<211> 1230
<212> 1~1A
<213> Homo Sapiens
20 <400> 76
cacgagtgccgctaaccttcttcatcctttggtggcaaagtagaaagattccagaattaa60
ctcgacctttctaaagacctgggctcagaggcagctggcactgactgagcacccactatg120
tgccaggcactgtgctgaatgcattagatcatcaattatgaatttgacaccaaggacctg180
gtgtgcctgggcctgagcagcatcgttggcgtctggtacctgctgaggaagcactggatt240
25 gccaacaacctttttggcctggccttctcccttaatggagtagagctcctgcacctcaac300
aatgtcagcactggctgcatcctgctgggcggactcttcatctacgatgtcttctgggta360
tttggcaccaatgtgatggtgacagtggccaagtccttcgaggcaccaataaaattggtg420
tttccccaggatctgctggagaaaggcctcgaagcaaacaactttgccatgctgggactt480
ggagatgtcgtcattccagggatcttcattgccttgctgctgcgctttgacatcagcttg540
aagaagaatacccacacctacttctacaccagctttgcagcctacatcttcggcctgggc600
cttaccatcttcatcatgcacatcttcaagcatgctcagcctgccctcctatacctggtc660
cccgcctgcatcggttttcctgtcctggtggcgctggccaagggagaagtgacagagatg720
ttcagttatgaggagtcaaatcctaaggatccagcggcagtgacagaatccaaagaggga780
acagaggcatcagcatcgaaggggctggagaagaaagagaaatgatgcagctggtgcccg840
35 agcctctcagggccagaccagacagatgggggctgggcccacacaggcgtgcaccggtag900
agggcacaggaggccaagggcagctccaggacagggcagggggcagcaggatacctccag960
ccaggcctctgtggcctctgtttccttctccctttcttggccctcctctgctcctcccca1020
caccctgcaggcaaaagaaacccccagcttcccccctccccgggagccaggtgggaaaag1080
tgggtgtgatttttagattttgtattgtggactgattttgcctcacattaaaaactcatc1140
4~ ccatggccagggcgggccactgtgctcctggaaaaaaaaaaaaaaaaaaaaaaaaaaaaa1200
aaaaaaaaaaaaaaaaaaaaggggaggggc 1230
<210> 77
<211> 911
<212> DATA
<213> Homo Sapiens
<400>
77
tcgacccacgcgtccgtcttcctaaaagggatgccctccaaagaaattttaaaagaatct60
tatcaaggggccctggagaagaaagggatgtgagggtcaagtcacaactttgaggggaaa120
tagaaagagggctcctttctgagaaagaagaatttcaaagagtccaagagaaccaaaaat180
tcaggacccaggagggtaagcattcctgtttttgcaagcttcacagaccatttgagtgag240
tgggtttttcaggtgacatttaaatgaacaaataatatccatgtctcagggtcagaaatg300
gtactttgcaactgattctgtccctcttgagaggcttctgcaagactgagagggtgggat360
gacttaatgaacattaaaaacaatgttattaggckggatatggtggcacatgcctgtaat420
tctagcactttgggargctgaggtgggcaggcccargarttcaagaccagtctgggcaac480
atggtgagaccctgtatctaataaaaatacaaaaatttagccaggcatggtggcacacac540
ctggagtcccagctactcaggagactgaggtgggaagatcacctgagctcaggaagtcga600
()~ggctgcagtgagccaagattgcactactgcactctagcctacatggataggagtgagacc660
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
36
tgtttgaaaaacaaaaaacaatcaaaaaca aaaaaaaacaacccacacaatgttattttt720
aaaatactgaggggagagaagttggggaaa aaaagggaaaacctaaaactctccataatc780
ctaccatcagaaaattacactaatgtgata agtgactttctcccctctgaatctccaatt840
ccattacttgtagtaaatatgaatcttatt ccacaaactcagacatgcaaaaaaaaaaaa900
aaagggcggcc 911
<210> 78
<211> 488
]0<212> DNA
<213> Homo
sapiens
<220>
<221> SITE
IS<222> (324)
<223> n
equals
a,t,g,
or c
<220>
<221> SITE
20<222> (438)
<223> n
equals
a,t,g,
or c
<220>
<221> SITE
25<222> (484)
<223> n
equals
a,t,g,
or c
<400> 78
accgcaggggctcccggaccctgactctgc agccgaaccggcacggtttcgtggggaccc60
30aggcttgcaaagtgacggtcattttctctt tctttctccctcttgagtccttctgagatg120
atggctctgggcgcacgggagctacccggg tctttgtcgcgatggtagcggcggctctcg180
gcggccaccctctgctgggagtgagcgcca ccttgaactcggttctcaattccaacgcta240
tcaagaacctgcccccaccgctgggcggcg ctgcggggcacccaaggctctgcagtcagc300
gccgcgccgggaatcctgtacccngggcgg gaataagtaccagaccattgacaactacca360
35gccgtacccsttgcgcaaaagaacraaaga aatttgccgcactgaaataaatttacttgc420
gcctaattccccaccccncccggaaagggg aaacccccggggcgtttttccaaattcttt480
tttnttcc 488
40 <zlo> 79
<211> 753
<212> DT1A
<213> Homo sapiens
45 <220>
<221> SITE
<222> (745)
<223> n equals a,t,g,
or c
$0 <220>
<221> SITE
<222> (752)
<223> n equals a,t,g,
or c
55 <400> 79
gaattcggca cgagcggcgg accccggtgagcaggcccaaggcagcgggg60
gggtccatcc
gcccacaccc ctcacacgca cttctggtcactggtgtctgaaaccaaatc120
aaactggctt
cagagcagcc tgtggcctgt tttctaatgactgcagactggtgggatcat180
aaagcatata
aggagccttc tgaatgacca ctttggagctgatgaaaatgtactctttta240
ggactgcttt
()0gcgtgttaga aatcacttgt ttctttggccaagctgggtctagtgtttct300
tttattttgt
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
37
15
tttgctgggaatagactttc cttctatcaa tgcccttgca360
aaaagttgta gaaacaaaac
gaaatttcaggtcttttgttaagcctgtattggtcttaaggtgcagtattttttaaatta420
ttatttatagaaagaatctataaattcttggggaagtgtgttataagctttaataattac480
attgagctgcacctcagtggtgtgtcattaacatgcagtggggttaatatctgaggcctc540
agatgactttgtgccttttggaataaagggtaaaataaactctcccagagtaagagctgt600
atcgtgaattgtcatactaattattgagggggacttatgtgcttttattgaatggagtgc660
tttacaatttttatttttaaatggggttgggatccttggaatatttcaataaaattgata720
aaatataaaaaaaaaaaaaaagggnggccgcnc 753
<210> 80
<211> 2138
<212> DNA
<213> Homo sapiens
<400>
80
tggatgatgatggactcccctttcccacggatgtgatccagcataggttacggcaaatcg60
aagcagggtacaaacaagaggtggagcagctacgtcgacaggtgcgtgactcagatgagr120
ctggacatccgtcactgctgtgcccctccagcagagcccccatggactatgaggatgatt180
ttacatgtttgaaggagtcagatggcagtgatactgaggattttggctctgatcacagtg240
aagactgcctttcagaagcaagctgggaacctgttgataagaaagagactgaggtgactc300
gctgggttccagaccatatggcatcacactgctataactgtgactgtgaattctggttgg360
ccaaacgaagacaccattgcagaaattgtgggaatgtattttgtgctggatgctgccacc420
tgaagctgcccattcctgatcagcaactctatgacccagttctcgtctgtaactcatgtt480
25 acgraacacattcaagtctctcgtgccagggaactcatgagccaacagctgaagaaaccc540
attgctacagcttccagttgaatgccggggagaaacctgtccaattttagcaggtttgaa600
gggaggatcttcttcagttgtagtttggaaggttccttggtgtggctcatgaaatcacag660
agctcagagataccatcttgagaaatcctccttggtatcatgaaactggagcagaggaat720
tgcaatttagcaggaggtcctctactggtgataccctcaccttggggtaatggtcctaac780
ccagacccagggtctggaagcttaatgttgagttggtgactccagcctctttctcctgga840
ggtcacaagatgatgattgcgtagatgttgcctggtgcaaagtgccccaaacagcaatag900
aaaggcatatgtataaccaaactccaagtgataaccagacccatctctcctccaccttga960
caaaagcagattatagtatacaaggtaggaattcctgtcctatttgagatgaactatatc1020
ctgtacctctgtgctctgtgtctgcatgaaggctcagcctttagaggcactccttctagt1080
35 tgcattagtactgtctttctgtggagtttggtttgaagactggctcagcaagtggaggtt1140
tcaatgtatttttcagttggctcatcagccagcattggtgaatattcagtttaggggaac1200
agttctagggagtgagacatttttgggagcagaggaaaactctgctgatgttcggtcctg1260
gcaaacattgagttattttgagctgtgaaggcagtcgtctctgttacacagtggcagctc1320
ttgagttatgcactgtgaagaatgagaagggaaaagcaaaaattatccttgtgaaatatc1380
tgctgattgtgccctactctttgcacctgacttttcctagttgtcctggtgctaacacag1440
gagctacamottgatcctctcctggcatgaaaataaaacaaaggttttcgttgttgttgt1500
tccattgcccatttcccccatgttgtctttcccttggctgatgcctcctctgggtcacat1560
tgcttcttatcctgaacacttgacaccttgagggtagaatttagcgtttggtttttacct1620
cctagcatatgctgtttggtatgtgagggtttcagtacaaatgctgctgtctatttctgt1680
45 gcacttaacaatggaacccaaacagaagagaataaagccttgataccaaaattgggaaag1740
aacatgtgtccatttggaccaaacgttgttggtttttaaaaaattttattttgttttttt1800
gtttttgtttttgttttttttcatcttaatatgtaccagtggcacttaaccaaaagatac1860
agtgatatagccatgtatctgtctacttagcgtggctgttttgagggactgtcccatcag1920
tgaacaaactgcatggccttggagagagactctgggctcttggctcagatgtgttcatca1980
aatactcctttcagagctgttgtgggtgtaagtgacatgatgtggccaaaaatccaaact2040
gtgcagttgcgttgtgacaaacatgcaatgtgctgtaaaaattcaatacagtttaaataa2100
aatctctatattagtaaaaaaaaaaaaaaaaaactcga 2138
$$ <210> 81
<211> 1327
<212> DNA
<213> Homo sapiens
60 <220>
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
38
<221> SITE
<222> (5)
<223> n equals a,t,g, or c
<220>
<221> SITE
<222> (7)
<223> n equals or c
a,t,g,
1~ <220>
<221> SITE
<222> (9)
<223> n equals or c
a,t,g,
15 <220>
<221> SITE
<222> (10)
<223> n equals or c
a,t,g,
2~ <220>
<22i> SITE
<222> (1205)
<223> n equals or c
a,t,g,
25 <400> sI
aaccnangnn taccggtccggaattcccgggtcggacccacgcgtccgcggcgggcgacg60
cacgtcgagc gggggagcggcgctgcctgtggagatccgcggaggccgacaggattcgtt120
ggctgccgtc cccgctgctgtgcattgggttaaaaacgacaaccaacatcagccatgaaa180
gatccaagtc gcagcagtactagcccaagcatcatcaatgaagatgtgattattaacggt240
3~ cattctcatg aagatgacaatccatttgcagagtacatgtggatggaaaatgaagaagaa300
ttcaacagac aaatagaagaggagttatgggaagaagaatttattgaacgctgtttccaa360
gaaatgctgg aagaggaagaagagcatgaatggtttattccagctcgagatctcccacaa420
actatggacc aaatccaagaccagtttaatgaccttgttatcagtgatggctcttctctg480
gaagatcttg tggtcaagagcaatctgaatccaaatgcaaaggagtttgttcctggggtg540
35 aagtacggaa atatttgagtagacggggccctcttttggtggatgtagcacaatttccac600
actgtgaagg cagtattagaagacttaattgtaaaagctctcttgtcactgtgttacact660
tatgcattgc caaagtttttgttagtcttgcatgcttaataaaagtgctgagactgttac720
taagtaaaaa gctgtcaaacatttactgaaaatagaattggccccatggcttgatgtgaa780
gacagcaagg aaagaagcaccagtcaagttgtgaacaagcaccaaattaaaagacctaaa840
ccttaccaaa ttgtctttttttgaggctaatctatcacttgttaatgtctaaactttaaa900
atcagtacat ttaatttgagttccaactgttaagcatatttctcagacttaaatttgatt960
atgtccccat caaaaagaatctccattttctgaaggtctgttagttaatttgagataatt1020
tgttaaaggc aagtatgtcatattactgaggctacaagttagtcagcagatgagtgccag1080
tccagccttt tctggtatgttattgttagraatattgagttctaatgttacatctgaggr1140
45 agtatgtaat tgagrattgtaacttctaaggggttcactgcatcatrgctatgcctgtat1200
ggrgntctwa ccatatgaccmataccamcccwtaatcccagctgraccaargrtacckgt1260
aaccattwwg gatttgaggggkggcctttcccyggcyttgkttwacccmtccacggagaa1320
tctggca 1327
50
<210> 82
<211> 758
<212> DNA
<213> Homo sapiens
<400> 82
gaattcggca cgagacacgg tttcaccctg ttggccagga tggtctcaat ctcttgacct 60
cgtgatctgc ctgcctcggc ctcccaaagt gctaggatta caggcatgag ccactgtgcc I20
cggcctttgt tttttgagac cttttttatt ttgttgtcac ccaggctgaa gtgcagtggc 180
acaaacacag ttcactacag ccttgacctc ctgggctcaa gcaattctgc ctcagtccca 240
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
39
caagtaggtgggcttacaaatgcacagcatgacacctggcttatttttgtattttgtgtg300
tgtgtgtgtgagccactgcgcaggccttgggcagctttcttgatctctgttacctcatct360
ataaaatgatgataataatagcttctcccttattggggaattgtaatgattaaatgagat420
aacatgtaaaatgctcagtacaggccaggcatggtggctcacgcttgcaatcccagcact480
ttgggaggctgaggctgctagatctcttgaggccagcagttaagaccagcctggccaata540
tggtgaaaccctgtgtctaccaaaaaatacagaaagtcagccaggcatggtggtgcatgc600
ctgtggtcccagctactcagaggctgaggtgggagaatcacttgagcccgggagacagaa660
gttgaagtgagccaagatggcgccactgcactctagcatgggctacagagtgagagcctc?20
tctcaaaaaaaaaaaaaaaaaaaaaaaaaaaactcgta 758
<210> 83
<211> 48
<212> PRT
1$<213> Homo
Sapiens
<220>
<221> SITE
<222> (48)
20<223> Xaa
equals
stop translation
<400> 83
Met Gly Cys Leu Gly
Ser Ala
Ala
Phe
Leu
Leu
Ala
Ala
Leu
Ser
Leu
1 5 10 15
25
Val Leu Gly Asn Arg
Gly Tyr
Pro
Gly
Arg
Arg
Ala
Phe
Ile
Leu
Pro
20 25 30
Arg Ser Arg Ser Xaa
Leu Gln
Trp
Leu
Glu
VaI
Ser
Leu
Gly
Pro
Val
3035 40 45
<210> 84
<211> 38
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (38)
<223> Xaa equals stop translation
<400> 84
Met Asn Glu Ala Pro Pro Leu Ser Ser Ser Ser Ile Cys Phe Ile Leu
1 5 10 15
Phe Tyr Phe Phe Pro Leu Leu Pro Pro Leu Ser Ser Thr Cps Phe Ser
20 25 30
Lys Gly Asn Arg His Xaa
55
<210> es
<211> 53
<212> PRT
()0 <213> Homo Sapiens
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
<220>
<221> SITE
<222> (53)
$ <223> Xaa equals stop translation
<400> 85
Met Cys Gln Asn Arg Glu Ser Val Leu Val Leu Leu Ile Glu Ser Asn
1 5 10 15
Met Phe Ser Phe Tyr Leu Leu Phe Ser Phe Tyr Ile Val Phe Ser Phe
25 30
Phe Ile Val Leu Arg Pro Leu Pro Arg Asn Glu Ser Ile Lys Lys Ile
1$ 35 40 45
Gly Val Ile Phe Xaa
20
<210> 86
<211> 26
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (26)
<223> Xaa equals stop translation
<400> 86
Met Thr Val Leu Ala Lys Arg Leu Val Leu Phe Leu Gly His Ile Phe
1 5 10 15
Leu Leu Leu Cys Val Arg Ile Leu Asp Xaa
20 25
<210> 87
<211> 78
<212> PRT
<213> Homo Sapiens
<220>
<zzl> SITE
<222> (43)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (78)
<223> xaa equals stop translation
<400> 87
Met Ala Ala Arg Ser Ala Leu Ala Leu Leu Leu Leu Leu Pro Val Leu
1 5 10 15
Leu Leu Pro Val Gln Ser Arg Ser Glu Pro Glu Thr Thr Ala Pro Thr
20 25 30
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
41
Pro Thr Pro Ile Pro Gly Gly Asn Ser Ser Xaa Ser Arg Pro Leu Pro
35 40 45
Ser Ile Glu Leu His Ala Cys Gly Pro Tyr Pro Lys Pro Gly Leu Leu
50 55 60
Ile Leu Leu Ala Pro Leu Ala Leu Trp Pro Ile Leu Leu Xaa
65 70 75
<210> 88
<211> 38
<212> PRT
<213> Homo Sapiens
1$
<220>
<221> SITE
<222> (38)
<223> Xaa equals stop translation
<400> as
Met Cys Tyr Ile Pro Gly Ser Thr Gly Gly Gln Cars Trp Pro Trp Cys
1 5 10 15
Trp Cys Trp Leu Gds Arg Glu Ala Leu Glu Trp Leu Cars Gly Ala Val
20 25 30
Ser Ala Gly Pro Ala Xaa
30
<210> 89
<211> 44
<212> PRT
35 <213> Homo Sapiens
<220>
<221> SITE
<222> (40)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (44)
<223> Xaa equals stop translation
<400> 89
Met Leu Leu Arg Ile Ile His Leu Val Ile Phe Phe Ile Asn Phe Ser
1 5 10 15
Thr Ser Val Val Ile Val His Tyr Asn Val Leu Asn Tyr Arg Cys Leu
20 25 30
Leu Lys (.ys Arg Cars Arg Val Xaa Lys Tyr Ser Xaa
35 40
<210> 90
<211> 60
<212> PRT
CA 02296815 2000-O1-14
WO 99/03990 PCT/ETS98/14613
42
<213> Homo sapiens
<220>
<221> SITE
<222> (60)
<223> Xaa equals
stop translation
<400> 90
Met Gln Asn Gars Ser Leu ProGlyVal PheSer
Leu Gly Ile Leu Leu
1 5 10 15
Leu Leu Leu Pro Phe Asn IleLeuThr SerLys
Ser Met Ile Gln Tyr
25 30
IS Gly Glu Asn Ser Ala Cys TyrSerSer AsnPhe
Tyr Pro Phe Ser Pro
35 40 45
Val Ser Ala Ile Leu Val ValValxaa
Thr Phe Gly
50 55 60
20
<210> 91
<211> 55
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (55)
<223> Xaa equals
stop translation
<400> 91
Met Val Val Ile Thr Ser ValCysIle GlyTyr
Val Leu Asn Cys Val
1 5 10 15
Val His Ser Ala Pro Arg GlnGlyLeu LeuPhe
Leu Ile Arg Phe Leu
20 25 30
Phe Leu Val Met Phe Ser AlaPheAsn IleThr
Phe Tyr Ile Arg Lys
35 40 45
Gly Thr Leu Ser Ser Gln Xaa
55
<210> 92
<211> 51
<212> PRT
<213> Homo Sapiens
SO
<220>
<221> SITE
<222> (51)
<223> Xaa equals stop translation
<400> 92
Met Val Ala Gln Leu Val Gly Cys Val Val Ser Cys Leu Phe Val Leu
1 5 10 15
Leu Arg Phe Leu Ile Ser Thr Phe Gly Ile Met Ser Phe Asn Gly Phe
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
43
20 25 30
Val Ile Phe Val Thr Val Leu Ala Ala Tyr Asn Phe Ser Ala Gly Ala
35 40 45
Phe Thr Xaa
10 <210> 93
<211> 156
<212> PRT
<213> Homo sapiens
IS <220>
<221> SITE
<222> (156)
<223> Xaa equals stop translation
20 <400> 93
Met Trp Pro Gln Glu Ala Trp Val Cys Ile Leu Val Leu Leu Gly Thr
1 5 10 15
Arg Val Gly Leu Gars Val Gly Asp Ser Leu Ala Pro Gln Ala Ser Leu
25 20 25 30
Ser Tyr Cys Tyr Ile Leu Lys Val Pro Leu Arg Pro Lys Pro Leu Trp
35 40 45
30 Gln Leu Ser Asn Glu Ser Ile Cys Ser Glu Tyr Arg Val Glu Gly Gly
50 55 60
Gln Gly His Gln Glu Leu Arg Met Phe Leu Arg Leu Met Arg Pro Arg
65 70 75 80
Tyr Txp Val His Gly Gly Pro Arg Ser Leu Cys Asp Ser Cys Ser Leu
85 90 95
Leu Pro Pro Cys Leu Asp Pro Ala Ser Ala Gln Lys Ala Asn Ser Leu
100 105 110
Asp Ser Lys Gly Leu Pro Arg Pro Ile Ser Met Ser Cys Ser Cys Gln
115 120 125
Leu Pro Val Pro Ser Leu Asp Leu Ser Ser Cys Leu Ala Pro Ser Leu
130 135 140
Pro Thr Pro His Ile Phe Thr Asn Lys Arg Lys Xaa
145 150 155
<210> 94
<211> 61
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (61)
<223> Xaa equals stop translation
*rB
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
44
<400> 94
Met Ser His His Ala Arg Pro Tyr Lys Ala Phe Arg Ile Val Ser Cys
1 5 10 15
Tyr Phe Tyr Leu Phe Ile Ile Val Val Val Ile Ile Leu Leu Leu Tyr
20 25 30
Pro Ile Ser Gln Gly Trp His Val Ala Asn Ile Val Phe Leu Lys Asn
1~ 35 40 45
Ile Ser Asp His Ile Leu Val Leu Leu Lys Thr Phe Xaa
50 55 60
<210> 95
<211> 71
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (71)
<223> Xaa equals stop translation
<400> 95
Met Txp Phe Glu Ile Leu Pro Gly Leu Ser Val Met Gly Val Cys Leu
1 5 10 15
Leu Ile Pro Gly Leu Ala Thr Ala Tyr Ile His Arg Phe Thr Asn Gly
20 25 30
Gly Lys Glu Lys Arg Val Ala His Phe Gly Tyr His Trp Ser Leu Met
40 45
Glu Arg Asp Arg Arg Ile Ser Gly Val Asp Arg Tyr Tyr Val Ser Lys
50 55 60
Gly Leu Glu Asn Ile Asp Xaa
65 70
<210> 96
<211> 37
<2I2> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (37)
<223> Xaa equals stop translation
<400> 96
Met Val Phe Leu Leu Leu Leu Leu Phe Gly Phe Phe Phe Asp Gly Ser
1 5 10 15
Leu Arg Ser Pro Leu Leu Leu Ile Ile His Leu Gly Pro Ala Pro Thr
20 25 30
Phe Leu Gln Ile Xaa
CA 02296815 2000-O1-14
WO 99/03990 PCT/13598/14613
35
<210> 97
<211> 60
<212> PRT
e213> Homo Sapiens
<220>
10 <221> SITE
<222> (60)
<223> Xaa equals stop translation
<400> 97
IS Met Leu Cps Gln Thr Ile Pro Leu Cys IleVal
Asn Arg Leu His Phe
1 5 10 15
Met Ile Leu Ile Lys Leu Tyr Val Glu ValLys
Thr Glu Cys Glu Ser
20 25 30
20
Glu His Lys Lys Ile Met His Asp Glu PheIle
Ile Ala Tyr His Gly
35 40 45
Tyr Leu Leu Cys Ile Tyr Thr Leu Arg Pro Leu Xaa
2.$ 50 55 60
<210> 98
<211> 44
30 <212> PRT
<213> Homo Sapiens
<220>
<221> SITE
35 <222> (44)
<223> Xaa equals
stop translation
<400> 98
Met Ser Val Ser Leu Trp Thr Leu Leu Leu
Ser Asn Gln Ile Leu Ser
4O 1 5 10 15
Leu Trp Phe Cys Pro Glu His Ile Gly Ile
Leu Phe Cys Val Ile Gln
20 25 30
45 Leu Cys Arg Leu Leu Pro Phe Thr
Phe Arg Ser Xaa
35 40
<210> 99
<211> 32
<212> PRT
<213> Homo Sapiens
<220>
Jrs<221> SITE
<222> (32)
<223> Xaa equals
stop translation
<400> 99
Met Cys Cys Arg Gly Ser Ser Pro Val Met
Ala Gly Gln Gln Val Val
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
46
1 5 10 15
Leu Ile Ile Ile Leu Gly Pro Trp Gly Gly Val Arg Ile Asp Ala Xaa
20 25 30
10 <210> 100
<211> 180
<212> PRT
<213> Homo Sapiens
<400> 100
Met Tyr Ser Cars Leu Leu Leu Pro Asp Leu Leu Tyr Leu Thr Leu Ser
1 5 10 15
Pro Leu Val Val Ala Met Leu Leu Thr Pro His Phe Asn Val Ala Asn
25 30
Pro Gln Asn Leu Leu Ala Gly Leu Trp Leu Glu Asn Glu His Ser Phe
35 40 45
Thr Leu Met Ala Pro Glu Arg Ala Arg Thr His His Cys Gln Pro Glu
50 55 60
Glu Arg Lys Val Leu Phe Cys Leu Phe Pro Ile Val Pro Asn Ser Gln
65 70 75 80
Ala Gln Val Gln Pro Pro Gln Met Pro Pro Phe Cps Gds Ala Ala Ala
85 90 95
Lys Glu Lys Thr Gln Glu Glu Gln Leu Gln Glu Pro Leu Gly Ser Gln
loo l05 llo
Cyrs Pro Asp Thr Cys Pro Asn Ser Leu Cps Pro Ser His Thr Gln Leu
115 120 125
Thr Lys Ala Asn Thr Leu Ser Leu Phe Phe Phe Phe Ser Phe Phe Leu
130 135 140
Ser Arg Val Ser Leu Leu Ser Pro Arg Leu Glu Cys Asn Gly Arg Ile
145 150 155 160
Leu Ala His Gars Asn Leu His Leu Pro Gly Ser Ser Asn Ser Pro Val
165 170 175
Ser Ala Ser Arg
180
<210> 101
<211> 212
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
()0 <222> (451
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
47
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
<221> SITE
$ <222> (195)
<223> Xaa equals thenaturally L-amino
any one of occurring acids
<220>
<221> SITE
1~ <222> (212)
<223> Xaa equals
stop translation
<400> 101
Met Arg Leu Trp AlaValLeuThrLeu ValThr
Phe Leu Asn Phe Ser
15 1 5 10 15
Leu Ile Gly Ile GluProGluValLys GluVal
Ala Leu Pro Ile Leu
20 25 30
2~ Gln Lys Pro Cys ArgLysThrLysGiy AspLeu
Phe Ile His Xaa Met
35 40 45
Leu Val His Gly LeuGluLysAspGly LeuPhe
Tyr Glu Tyr Ser His
50 55 60
25
Ser Thr His Asn GlyGlnProIleTrp ThrLeu
Lys His Asn Phe Gly
65 70 75 80
Ile Leu Glu Lys TrpAspGlnGlyLeu GlyMet
Ala Leu Gly Lys C'.ys
85 90 95
Val Gly Glu Lys IleIleProProAla GlyTyr
Lys Arg Leu Leu Gly
100 105 110
35 Lys Glu Gly Lys ProProGluSerThr IlePhe
Lys Gly Ile Leu Asn
115 120 125
Ile Asp Leu Ile AsnGlyProArgSer GluSer
Leu Glu Arg His Phe
130 135 140
Gln Glu_Met Asn AspTrpLysLeuSer AspGlu
Asp Leu Asp Lys Val
145 150 155 160
Lys Ala Tyr Lys PheGluLysHisGly ValVal
Leu Lys Glu Ala Asn
45 165 170 175
Glu Ser His Ala ValGluAspIlePhe LysGlu
His Asp Leu Asp Asp
180 185 190
Glu Asp Xaa Phe SerAlaArgGluPhe TyrLys
Tyr Gly Ile Thr His
195 200 205
Asp Glu Leu Xaa
210
<210> loz
<211> 621
<212> PRT
<213> Homo sapiens
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
48
<220>
<221> SITE
<222> (137)
<223> Xaa the naturally acids
equals occurring
any one L-amino
of
<400> 102
Met Gly LeuSer ProVal Arg AlaLeu ArgLeu
Leu Asp Arg Arg Ala
1 5 10 15
Val Leu LeuAsn ProLeu Cys SerTyr AlaGly
Arg Ala Val Leu Val
20 25 30
Ile Ala PheLeu LeuVal Phe LeuThr ArgThr
Trp Ala Pro Pro Gln
IS 35 40 45
Tyr Met GluAsn MetGly Ser ValGlu GlnPhe
Ser Ala Thr Met Glu
50 55 60
2~ Ala Gly AspArg ArgAla Phe AspPhe AlaHis
Gly Ala Ala Arg Ala
65 70 75 80
Arg Lys SerGly LeuPro Val LeuGlu ThrMet
Lys Ala Ala Trp Arg
85 90 95
25
Arg Ser GlyLeu ValTyr Thr PheSer LysLeu
Val Glu Gln Ser Arg
100 105 110
Pro Phe AspGlu HisGlu Arg ValSer ThrAsn
Pro Thr Tyr Met Gly
115 120 125
Val Tyr IleLeu AlaPro Xaa SerThr SerLeu
Gly Arg Ala Ala Glu
130 135 140
35 Val Leu ValPro GlySer Asp AsnSer AlaVal
Thr Cys Ser Thr Gln
145 150 155 160
Gly Leu LeuAla AlaAla His GlyGln TyrTrp
Leu Leu Phe Arg Ile
165 170 175
40
Ala Lys IleVal LeuVal Thr AspLeu GlyThr
Asp Phe Glu His Leu
180 185 190
Glu Ala LeuGlu TyrHis Asp ValThr MetGln
Trp Ala Val Asn Gly
45 195 200 205
Ser Ser LeuGln ArgAla Gly GlnAla ValAla
Pro Gly Ala Ile Ala
210 215 220
J'0Leu Glu SerSer ValVal Thr AspVal ValGlu
Leu Asp Ser Leu Ala
225 230 235 240
Gly Leu GlyGln ProAsn Leu LeuAsn PheGln
Asn Leu Asp Leu Leu
245 250 255
55
Thr Phe GlnLys GlyLeu Leu LeuGln LysLeu
Cys Gly Cys Thr Gly
260 265 270
Gln Pro AspTrp SerLeu Asp LeuGln LeuGln
Glu Thr Gly Pro Gly
60 275 280 285
CA 02296815 2000-O1-14
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49
Thr Leu Leu Leu Met Val Leu Arg Gln Ala Ser Gly Arg Pro His Gly
290 295 300
Ser His Gly Leu Phe Leu Arg Tyr Arg Val Glu Ala Leu Thr Leu Arg
305 310 315 320
Gly Ile Asn Ser Phe Arg Gln Tyr Lys Tyr Asp Leu Val Ala Val Gly
325 330 335
Lys Ala Leu Glu Gly Met Phe Arg Lys Leu Asn His Leu Leu Glu Arg
340 345 350
Leu His Gln Ser Phe Phe Leu Tyr Leu Leu Pro Gly Leu Ser Arg Phe
IS 355 360 365
Val Ser Ile Gly Leu Tyr Met Pro Ala Val Gly Phe Leu Leu Leu Val
370 375 380
Leu Gly Leu Lys Ala Leu Glu Leu Trp Met Gln Leu His Glu Ala Gly
385 390 395 400
Met Gly Leu Glu Glu Pro Gly Gly Ala Pro Gly Pro Ser Val Pro Leu
405 410 415
Pro Pro Ser Gln Gly Val Gly Leu Ala Ser Leu Val Ala Pro Leu Leu
420 425 430
Ile Ser Gln Ala Met Gly Leu Ala Leu Tyr Val Leu Pro Val Leu Gly
435 440 445
Gln His Val Ala Thr Gln His Phe Pro Val Ala Glu Ala Glu Ala Val
450 455 460
Val Leu Thr Leu Leu Ala Ile Tyr Ala Ala Gly Leu Ala Leu Pro His
465 470 475 480
Asn Thr His Arg Val Val Ser Thr Gln Ala Pro Asp Arg Gly Trp Met
485 490 495
Ala Leu Lys Leu Val Ala Leu Ile Tyr Leu Ala Leu Gln Leu Gly Cys
500 505 510
Ile Ala Leu Thr Asn Phe Ser Leu Gly Phe Leu Leu Ala Thr Thr Met
4$ 515 520 525
Val Pro Thr Ala Ala Leu Ala Lys Pro His Gly Pro Arg Thr Leu Tyr
530 535 540
Ala Ala Leu Leu Val Leu Thr Ser Pro Ala Ala Thr Leu Leu Gly Ser
545 550 555 560
Leu Phe Leu Trp Arg Glu Leu Gln Glu Ala Pro Leu Ser Leu Ala Glu
565 570 575
Gly Trp Gln Leu Phe Leu Ala Ala Leu Ala Gln Gly Val Leu Glu His
580 585 590
His Thr Tyr Gly Ala Leu Leu Phe Pro Leu Leu Ser Leu Gly Leu Tyr
595 600 605
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
Pro Cys Trp Leu Leu Phe Trp Asn Val Leu Phe Trp Lys
610 615 620
5
<210> 103
<211> 287
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (263?
<223> Xaa equals any one of the naturally occurring L-amino acids
<400> l03
Met Ala Leu Leu Pro Ile Phe Phe Gly Ala Leu Arg Ser Val Arg Cys
1 5 10 15
Ala Arg Gly Lys Asn Ala Ser Asp Met Pro Glu Thr Ile Thr Ser Arg
20 25 30
Asp Ala Ala Arg Phe Pro Ile Ile Ala Ser Cys Thr Leu Leu Gly Leu
35 40 45
Tyr Leu Phe Phe Lys Ile Phe Ser Gln Glu Tyr Ile Asn Leu Leu Leu
50 55 60
Ser Met Tyr Phe Phe Val Leu Gly Ile Leu Ala Leu Ser His Thr Ile
65 70 75 80
Ser Pro Phe Met Asn Lys Phe Phe Pro Ala Ser Phe Pro Asn Arg Gln
85 90 95
Tyr Gln Leu Leu Phe Thr Gln Gly Ser Gly Glu Asn Lys Glu Glu Ile
100 105 110
Ile Asn Tyr Glu Phe Asp Thr Lys Asp Leu Val Cars Leu Gly Leu Ser
115 120 125
Ser Ile Val Gly Val Trp Tyr Leu Leu Arg Lys His Trp Ile Ala Asn
130 135 140
Asn Leu Phe Gly Leu Ala Phe Ser Leu Asn Gly Val Glu Leu Leu His
145 150 155 160
Leu Asn Asn Val Ser Thr Gly Gars Ile Leu Leu Gly Gly Leu Phe Ile
165 170 175
Tyr Asp Val Phe Trp Val Phe Gly Thr Asn Val Met Val Thr Val Ala
180 185 190
Lys Ser Phe Glu Ala Pro Ile Lys Leu Val Phe Pro Gln Asp Leu Leu
195 200 205
Glu Lys Gly Leu Glu Ala Asn Asn Phe Ala Met Leu Gly Leu Gly Asp
210 215 220
Val Val Ile Pro Gly Ile Phe Ile Ala Leu Leu Leu Arg Phe Asp Ile
225 230 235 240
CA 02296815 2000-O1-14
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51
Ser Leu Lys Lys Asn Thr His Thr Tyr Phe Tyr Thr Ser Phe Ala Ala
245 250 255
Tyr Ile Phe Gly Leu Gly Xaa Tyr His Leu His His Ala His Leu Gln
260 265 270
Ala Cys Ser Val Met Arg Ser Gln Ile Leu Arg Ile Gln Arg Gln
275 280 285
<210> 104
<211> 32
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (32)
<223> Xaa equals stop translation
<400> 104
Met Ser Arg Leu Leu Leu Leu Phe Gly Arg Leu Cys Ser Leu Trp Cys
1 5 10 15
Leu Ser Trp Leu Tyr Ser Thr Asp Thr Arg Pro Leu Leu Arg Gly Xaa
20 25 30
<210> 105
<211> 77
<zlz> PRT
<213> Homo Sapiens
<400> 105
Met Leu Pro Arg Leu Val Leu Asn Ser Trp Ala Cys Pro Pro Gln Pro
1 5 10 15
Pro Lys Val Leu Glu Leu Gln Ala Cys Ala Thr Ile Ser Ser Leu Ile
20 25 30
Thr Leu Phe Leu Met Phe Ile Lys Ser Ser His Pro Leu Ser Leu Ala
35 40 45
Glu Ala Ser Gln Glu Gly Gln Asn Gln Leu Gln Ser Thr Ile Ser Asp
55 60
Pro Glu Thr Trp Ile Leu Phe Val His Leu Asn Val Thr
65 70 75
<210> 106
<211> 45
<212> PRT
<213> Homo Sapiens
<220>
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
52
<221> SITE
<222> (45)
<223> Xaa equals stop translation
$ <400> 106
Met Val Phe Leu Val Phe Tyr Val Leu Arg Ala Leu Lys Cys Asn Ser
1 5 10 15
Ser Tyr His Ser Cys Thr Asn Val Leu Thr Gln Ile Ala Ser Gln Ile
20 25 30
Asp Lys Thr Leu Asn Asn Phe Ser Leu Lys Lys Cys Xaa
35 40 45
IS
<210> I07
<211> 42
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (42)
<223> Xaa equals stop translation
<400> I07
Met Asn Pro Cps Leu Ser Ile Ile Phe Leu Leu Thr Pro Val Leu Leu
1 5 10 15
Ser His Pro Leu Gln Ser Leu His Phe Leu Leu Lys Val Asp Leu Asp
20 25 30
Phe Ser Leu Ser Cars Ser Ile Cys Thr Xaa
40
<zlo> loe
<211> 70
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (70)
<223> Xaa equals stop translation
<400> 108
Met Thr Val Tyr Leu Leu Lys Thr His Pro Cys Phe Phe Val Ala Tyr
1 5 10 15
Gln Met Gln Val Ala Leu Ile Ile Leu Leu Pro Gly Leu Arg Asn Ser
20 25 30
Lys Thr Val Thr Met Pro Leu Ser Pro Ala Leu Leu Pro Thr Leu Leu
5$ 35 40 45
Phe Phe Pro Ser Pro Thr Pro Phe Phe His Pro Phe Leu Ser Val Leu
50 55 60
Cys Cys Phe Lys Tyr Xaa
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
53
65 70
<210> 109
$ <211> 49
<212> PRT
<213> Homo Sapiens
<220>
1~ <221> SITE
<222> (43)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
IS <221> SITE
<222> (49)
<223> Xaa equals stop translation
<400> 109
2~ Met His Ala Thr Cys Thr Arg Thr Trp Arg Ala Gln Val Ser Leu His
1 5 10 15
Gln Pro Pro Cars Ser Arg Asp Trp Lys Ile Cys His Leu Leu Val Val
20 25 30
2$
Leu Ser Leu Pro Pro Pro Thr Pro Ala Arg Xaa Pro Glu Phe Leu Asn
35 40 45
Xaa
<210> 110
<211> 193
3$ <212> PRT
<213> Homo
Sapiens
<220>
<221> SITE
<222> (193)
<223> Xaa equals
stop translation
<400> 110
Met Ile Arg Gln SerLeuMetGlnLeuLeuGlnLeu
Asn Asp Asp Gly
4$ 1 s l0 15
Leu Val Val Ser GluSerGlnGluSerAspLeuSer
Leu Gly Gln Lys
20 25 30
$0 Gln Leu Ile Ile GlyLeuGlyValAlaLeuLeuLeu
Ser Val Ile Val
35 40 45
Leu Val Ile Met PheValCysValArgLysSerTyr
Met Thr Ala Asn
55 60
$$
Arg Lys Leu Met AlaAlaLysGluAlaArgLysThr
Gln Ala Lys Ala
65 70 75 80
Ala Gly Val Ser ProAlaIleProGlyThrAsnMet
Met Pro Ala Tyr
85 90 95
*rB
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
54
Asn Thr Glu Arg Ala Asn Pro Met Leu Asn Leu Pro Asn Lys Asp Leu
100 105 110
Gly Leu Glu Tyr Leu Ser Pro Ser Asn Asp Leu Asp Ser Val Ser Val
115 120 125
Asn Ser Leu Asp Asp Asn Ser Val Asp Val Asp Lys Asn Ser Gln Glu
130 135 140
Ile Lys Glu His Arg Pro Pro His Thr Pro Pro Glu Pro Asp Pro Glu
145 150 155 160
Pro Leu Ser Val Val Leu Leu Gly Arg Gln Ala Gly Ala Ser Gly Gln
IS 165 170 175
Leu Glu Gly Pro Ser Tyr Thr Asn Ala Gly Leu Asp Thr Thr Asp Leu
180 185 190
xaa
<210> 111
<211> 71
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (64)
<223> Xaa equals L-amino
any one of the acids
naturally occurring
<400> 111
Met Ala His Val Ala Arg Glu Cys IleArg Ala
Val Val Asn Leu Phe
1 5 10 15
Leu Phe Leu Leu Val Ser Leu Pro ProGly Glu
His Cys Leu Ser Val
20 25 30
Asn Ile Arg His Phe Thr Glu Glu LeuThr Thr
Thr Leu Val Arg Pro
35 40 45
Arg Ala Leu Lys Leu Ser Leu Ser Leu Ile Val Ser Leu His Ala Xaa
50 55 60
Cyrs Arg Lys Gln Glu Cars Ser
65 70
<210> 112
<211> 36
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (36)
<223> Xaa equals stop translation
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
$5
<400> 112
Met Arg Leu Thr Glu Lys Asp Thr Val Leu Phe Thr Lys Gly Val Leu
1 5 10 I5
$ Phe Leu His Leu Phe Ile Asn Ala Leu Phe Trp Tyr Cys Lys Phe Gly
20 25 30
His Asn Phe Xaa
10
<210> I13
<211> 60
<212> PRT
1$ <213> Homo
Sapiens
<220>
<221> SITE
<222> (60)
2~ <223> Xaa equals
stop translation
<400> 113
Met Thr Ser Thr Gln SerLeuVal Leu Met Ser
Val Ser Leu Leu Leu
1 5 10 15
2$
Leu Val Leu Val Glu ValGluAla Gly Asp Ala
Pro Val Ala Ile Ala
20 25 30
Leu Leu Leu Val Leu IleThrGly Ile Cys Ala
Gly Val Ser Cys Leu
30 35 40 45
Gly Val Tyr Lys Arg GlyGlnMet Xaa
Ala Arg Asn
50 55 60
3$
<210> 114
<211> 29
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (29)
<223> Xaa equals stop translation
4$
<400> 114
Met Asn Ser Phe Trp Ser Lys Leu Leu Val Leu Pro Leu Leu Ala Pro
1 5 10 15
$0 Leu Ser Met Ala Arg Ala Ser Ala Cys Gln Arg Trp Xaa
20 25
<210> 115
$$ <211> 25
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98l14613
56
<222> (25)
<223> Xaa equals stop translation
<400> 115
Met Met Arg Leu Leu Asp Leu Arg Ile Phe Leu Met Ile His His Lys
1 5 10 15
Ala Lys Ser Trp Glu Ser His Thr Xaa
20 25
<210> 116
<211> 35
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (35}
<223> Xaa equals stop translation
<400> 116
Met Pro Leu Ser Leu Leu Leu Ile Val Trp Lys Leu Glu Leu Cys Val
1 5 10 15
Gly Ser Ala Leu Val Leu Ile His Thr Gln Arg Arg Tyr Ile Ile Leu
20 25 30
Gln Val Xaa
<210> 117
<211> 78
35 <212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (78)
<223> Xaa equals stop translation
<400> 117
Met Leu Leu Ala Thr Leu Leu Leu Leu Leu Leu Gly Gly Ala Leu Ala
4$ 1 5 10 15
His Pro Asp Arg Ile Ile Phe Pro Asn His Ala Cys Glu Asp Pro Pro
20 25 30
50 Ala Val Leu Leu Glu Val Gln Gly Thr Leu Gln Arg Pro Leu Val Arg
35 40 45
Asp Ser Arg Thr Ser Pro Ala Asn Cys Thr Txp Leu Thr Lys Arg Val
50 55 60
Gln Gln Met Leu Leu Phe His Ser Tyr Gly Ile Ala Gln Xaa
70 75
<210> 118
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
57
<211> 44
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (44)
<223> Xaa equals stop translation
<400> 118
Met Thr Gly Val Phe Lys Leu Pro Leu Leu Phe Trp Val His Glu Ala
1 5 10 15
Ser Val Gly Gly Cys Pro Tyr Val Lys Leu Val Glu Phe Glu Glu Met
IS 20 25 30
Leu Thr Leu Tyr Gly Ile Leu Leu Ile Leu Phe Xaa
35 40
<210> 119
<211> 46
<212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (46)
<223> Xaa equals stop
translation
<400> 119
Met Gln Leu Ala Pro IleProVal Leu Ser Gly
Phe Ile Ser Thr Thr
1 5 10 15
Pro Trp Thr Ala Val SerSerIle Cys Thr Pro
Phe Arg Ala Leu Leu
20 25 30
Thr Leu Ser Ala Ala ValGluSer Ser Leu Xaa
Gly Met Leu
35 40 45
<210> 120
<211> 29
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (29)
<223> Xaa equals stop
translation
<400> 120
Met Pro Pro Leu Ser LeuThrVal Ala Val Val
Asp Ile Leu Ala Phe
1 5 10 15
Glu Met Thr Gly His TrpProHis Thr Xaa
Ile Tyr Ile
20 25
()0 <210> 121
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
58
<211> 62
<212> PRT
<213> Homo Sapiens
$ <400> 121
Met Glu Leu Pro Cys Asp Cys Ser Lys Leu Leu Tyr Cps Lys Phe Ser
1 5 10 15
Val Trp His Leu Pro Val Asn Ala Met Lys Leu Leu Ile Ile Phe Leu
20 25 30
Lys Val Leu His Cys Leu Phe Phe Leu Leu Leu CSrs Lys Phe Leu Tyr
35 40 45
IS Thr Leu Ile Val Ile Leu Thr Asp Lys Tyr Ser Ile Leu Asn
50 55 60
<210> 122
<211> 87
<212> PRT
<213> Homo sapiens
<220>
2.5 <221> SITE
<222> (68)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
30 <221> SITE
<222> (72)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
35 <221> SITE
<222> (87)
<223> Xaa equals stop translation
<400> 122
40 Met Pro Val Ser Trp Gly Cps Pro Ser Lys Thr Pro Gln Thr Arg Ala
1 5 10 15
Tyr Thr Arg Cys Val Tyr Phe Leu Met Val Leu Glu Ala Gly Val Gly
25 30
Gly His Ser Val Ser Arg Val Gly Ser Leu Glu Val Pro Pro Trp Leu
35 40 45
Val Ala Ala Asn Asn Phe Pro His Leu Met Trp Ser Ser Phe Cys Val
50 55 60
Gly Pro His Xaa Val Phe Leu Xaa Asp Pro Ser Leu Pro Asp Pro Gly
65 70 75 80
Pro Pro Asn Asn Leu Thr Xaa
<210> 123
()0 <211> 64
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
59
<212> PRT
<213> Homo sapiens
<220>
$ <221> SITE
<222> (64)
<223> Xaa equals stop translation
<400> 123
Met Cys Tyr Phe Leu Glu Leu ValPhe Leu
Ile Ser Leu Met Ala Asn
1 5 10 15
Ile Lys Ala Ala Tyr Gly Ile GlyThr Val
Cys Cys Asn Asn Glu His
25 30
1$
Arg Ala Lys Gly Pro Val Phe LeuSer Pro
Ser Val Pro Pro Arg Leu
35 40 45
Ser Gly Thr Pro Leu Leu Arg PheGln Leu
Asp Arg Leu Pro Thr Xaa
20 50 55 60
2$
<210> 124
<211> 36
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (36)
<223> Xaa equals stop translation
3$
<400> 124
Met Pro Leu Pro Ser Ser Phe Pro Leu Pro Val Phe Leu Ser Ser Cys
1 5 10 15
Pro Phe Leu Met Ser Val Ser Ile Gly Phe Leu Ile Leu Val Phe Asn
20 25 30
Val His Pro Xaa
4$
<210> 125
<211> 32
<212> PRT
$0 <213> Homo Sapiens
<220>
<221> SITE
<222> (32)
$$ <223> Xaa equals stop translation
<400> 125
Met Phe Ile Phe Cys Val Ser Leu Ala Phe Leu Pro Arg Phe Ile Ser
1 5 10 15
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
Pro Gln Ser Cars Glu Trp Ala Gly Leu Ser Leu Val Trp His His Xaa
20 25 30
5
<210> 126
<211> 41
10 <212> PRT
<213> Homo Sapiens
<220>
<221> SITE
IS <222> (41)
<223> Xaa equals stop translation
<400> 126
Met Lys Asn Asn Thr Gln Lys Arg Leu Phe Leu Trp Gly Glu Leu Leu
20 1 5 10 15
Leu Gln Asp Leu Ala Leu Ile Leu Tyr Leu Ser Ile Phe Leu Lys Ser
20 25 30
25 Thr Leu Thr Asn Leu Asn Leu Phe Xaa
35 40
<210> 127
30 <211> 28
<212> PRT
<213> Homo sapiens
<220>
35 <221> SITE
<222> (28)
<223> Xaa equals stop translation
<400> 127
40 Met Leu Asn Val Phe Phe Ser Leu Ile Leu Phe Phe Ser Pro Asn Arg
1 5 10 15
Ala Leu Pro Ala Ile Ser Ser Cps Ile Thr Phe Xaa
20 25
<210> lza
<211> 69
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (69)
<223> Xaa equals stop translation
<400> 128
Met Arg Ala Val Gly Glu Arg Leu Leu Leu Lys Leu Gln Arg Leu Pro
1 5 10 15
CA 02296815 2000-O1-14
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61
Gln Ala Glu Pro Val Glu Ile Val Ala Phe Ser Val Ile Ile Leu Phe
20 25 30
Thr Ala Thr Val Leu Leu Leu Leu Leu Ile Ala Cys Ser Cys Cys Cys
$ 35 40 45
Thr His Cys Cys Cys Pro Glu Arg Arg Gly Arg Lys Val Gln Val Gln
50 55 60
1~ Pro Thr Pro Pro Xaa
<210> 129
15 <211> 87
<212> PRT
<213> Homo sapiens
<400> 129
2~ Met Asp Pro Arg Arg Val Thr Ala Cys Cys His Val Txp Thr Val Gly
1 5 10 15
Leu Phe Cps Ile Trp Ala Val Gly Leu Ser Cys Ser Leu Ser Leu Ser
20 25 30
His Val Ile Val Trp Leu Ser Gly Ala Gly Cys Thr Leu Ile Cys Glu
40 45
Asp Asn Pro Phe Leu Leu Leu Phe Ser Gln Tyr Leu Gln Pro His His
50 55 60
Pro Glu Ile Met Lys Pro Phe Ile Leu Gly His Lys Ser Ser Asn Gly
65 70 75 80
35 Gly Leu Ser Pro Pro Ser Ala
<210> 130
<211> 64
<212> PRT
<213> Homo Sapiens
<220>
4$ <221> SITE
<222> (64)
<223> Xaa equals stop translation
<400> 130
Met Phe Tyr Met Val Cys Val Leu Gly Ser Gly Ala Gln Pro Leu Ser
1 5 10 15
Glu Leu Ala Tyr Leu Ala Lys Leu Pro Thr Leu Gln Val Gly Lys Tyr
20 25 30
Asn Pro Leu Phe Asn Lys Ala His Pro Leu His Pro Val Leu Thr Thr
35 40 45
Phe Cys Glu Cps Ala Val Ile Phe Ser C'ys Ser Ile Ala Arg Trp Xaa
50 55 60
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
62
<210> 131
<211> 54
<212> PRT
<213> Homo sapiens
<400> 131
Met Arg Phe Gln Ser Tyr Leu Trp Pro Ser Arg Ile Leu Val Gly Thr
1 5 10 15
Tyr Cys Ile Ala Ala Glu Val Leu Phe Pro Ser Ala Leu Ala Ser Cys
25 30
Gly Pro Val Trp Gln Gly Gly Ala Pro Thr Lys Ser Trp Gln Pro Gly
35 40 45
Ala Lys Thr Ile Ile Pro
25 <210> 132
<211> 41
<212> PRT
<213> Homo Sapiens
30 <220>
<221> SITE
<222> (41)
<223> Xaa equals stop translation
35 <400> 13z
Met Arg Arg Trp Ala Gly Phe Gly Lys Ser Pro Gln Phe Trp Trp Thr
1 5 10 15
Gly Ile Leu Val Ala Leu Gly Ala Ala Leu Leu Gly Gly Pro Arg Leu
40 20 25 30
Gly Arg Arg Leu Thr Phe Gly Leu Xaa
35 40
<210> 133
<211> 69
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (69)
<223> Xaa equals stop translation
<400> 133
Met Ala Leu Ala Ile Phe Ile Pro Val Leu Ile Ile Ser Leu Leu Leu
1 5 10 15
Gly Gly Ala Tyr Ile Tyr Ile Thr Arg Cys Arg Tyr Tyr Ser Asn Leu
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
63
2a z5 30
Arg Leu Pro Leu Met Tyr Ser His Pro Tyr Ser Gln Ile Thr Val Glu
35 40 45
Thr Glu Phe Asp Asn Pro Ile Tyr Glu Thr Gly Glu Thr Arg Glu Tyr
50 55 60
Glu Val Ser Ile Xaa
IO 65
<210> 134
<211> 48
1$ <212> PRT
<213> Homo sapiens
<220>
<221> SITE
2O <222> (48)
<223> Xaa equals
stop translation
<400> 134
Met Gly Phe Leu His Ile ProSer Ile Thr
Phe Leu Leu Ile Asn Arg
25 1 5 to is
Ser Ala Pro Gln Ser Cys MetGln Pro Gln
Pro Thr Arg Glu Gln Pro
20 25 30
3~ His Ser Thr Leu Val Ile GlyMet Met Ser
Lys Pro Leu Ile Ile Xaa
35 40 45
<210> 135
<211> 76
<212> PRT
4O <213> Homo Sapiens
<400> 135
Met Ser Gly Leu Val Gly Gly Gly Ser Arg GSrs Ser Lys Val Arg Phe
1 5 10 15
Arg Cys Phe Asn Gly Asp Ser Leu Leu Val Leu Val Leu Gln His His
20 25 30
Phe Arg Leu Cys Ser Trp Cys Leu Ala Pro Ser Leu Phe Leu Leu Leu
35 40 45
Ser Cys Gln Val Val Ser Thr Met Met Glu Gln Asp Pro Val Ile Tyr
55 60
Asp Asp Asp Asp Asp Leu Pro Asn Tyr Phe Ser Val
70 75
<210> 136
6O <211> 55
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
64
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (32)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (39)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
IS <221> SITE
<222> (55)
<223> Xaa equals stop translation
<400> 136
Met Phe Leu Glu Leu Pro Met Gln His Ser Asp Val Leu Leu Phe Leu
1 5 10 15
Val Cys Trp Lys Ala Met Gly Ser Lys Lys Ser Pro Ser His Phe Xaa
20 25 30
Pro Glu Val Gly Gly Ile Xaa Pro Ser Phe Gly Met Leu Asn Val Thr
40 45
Leu Leu Arg Ser Leu Thr Xaa
30 50 55
<210> 137
<211> 54
35 <212> PRT
<213> Homo Sapiens
<400> 137
Met Leu Val Leu Phe Pro Leu Leu Tyr Arg Gly Trp Ser Pro Val Pro
1 5 10 15
Gly Thr Ala Glu Gly Gly Met Cys Cars Cys Gys Leu Cys Ile Ser Arg
20 25 30
Tyr Ser Leu Leu Thr Ser Ser Gln Asp Lys Glu Pro Pro Tyr Glu Met
35 40 45
Ser Ser Ser Glu Leu Ser
50
<210> 138
<211> 36
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (33)
<223> Xaa equals any one of the naturally occurring L-amino acids
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
<220>
<221> SITE
<222> (36)
<223> Xaa equals stop translation
<400> 138
Met Thr Cys Tyr Glu Val Ile Leu Phe Phe Ile Lys Leu Phe Ser Asp
1 5 10 15
Met Gly Lys Tyr Lys Glu Cps Lys Glu Phe Lys Lys Gln Arg Thr Lys
25 30
Xaa Tyr Met Xaa
15 35
<210> 139
<211> BO
20 <212> PRT
<213> Homo sapiens
<400> 139
Met Lys Ala Gln Pro Leu Glu Ala Leu Leu Leu Val Ala Leu Val Leu
1 5 10 15
Ser Phe Cys Gly Val Trp Phe Glu Asp Trp Leu Ser Lys Trp Arg Phe
20 25 30
Gln Cys Ile Phe Gln Leu Ala His Gln Pro Ala Leu Val Asn Ile Gln
40 45
Phe Arg Gly Thr Val Leu Gly Ser Glu Thr Phe Leu Gly Ala Glu Glu
50 55 60
Asn Ser Ala Asp Val Arg Ser Trp Gln Thr Leu Ser Tyr Phe Glu Leu
65 70 75 80
<210> 140
<211> 67
4$ <212> PRT
<213> Homo sapiens
<400> 140
Met Ala Ala Ser Val Gly Arg Ala Thr Arg Ser Ala Ala Ala His Leu
1 5 10 15
Thr Gln Leu Pro Pro Ala Pro Arg Ala Gln Arg Thr Ser Pro Ala Gln
20 25 30
Pro Asp Glu Gly Lys Arg Arg Asp Ala Asp Pro Trp Arg Thr Gly Pro
35 40 45
Thr Val Asn Lys Thr Gly Ser Ile Pro Gly Arg Leu Arg Gly Trp Ala
50 55 60
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
66
Arg Ala Glu
<210> 141
<211> 51
<212> PRT
<213> Homo sapiens
10 <220>
<221> SITE
<222> (51)
<223> Xaa equals
stop translation
IS <400> 141
Met Gly Trp Leu Glu Pro Leu Tyr Asn Leu
Gars Cys Ser Gly Glu Lys
1 5 10 15
Gln Tyr Phe Phe Ser Leu Gly Leu Pro Val
Phe Ser Gln Ala Ile Val
20 20 25 30
Ser Ser Gly Cys Ile Ala Phe Ala Val Tyr
Thr Lys Tyr Gly Ser Pro
35 40 45
2S ser ser xaa
<210> 142
30 <211> 54
<212> PRT
<213> Homo sapiens
<400> 142
35 Met Arg Arg Cys Val Arg His Val Leu Gly Ile Gly Leu Ile Val Leu
1 5 10 15
Lys Asn Leu Tyr Phe His Lys Asn Ser Met Tyr Pro Ser Pro Lys Leu
20 25 30
Ser Ser Phe Gln Glu Ala Phe Leu Phe Phe Phe Leu Ile Leu Lys Asn
35 40 45
Pro Leu Thr Leu Cys Ser
50
<210> 143
<211> 50
$0 <212> PRT
<213> Homo sapiens
<220>
<221> SITE
<222> (50)
<223> Xaa equals stop translation
<400> 143
Ile His Pro Ser Arg Ser Thr Leu Ser Ser Gln Leu Val Thr Leu Pro
1 5 10 15
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
67
Leu Phe Glu Leu Val Phe Pro Ile Pro Ser Ser Gln Ser Pro Phe Ser
20 25 30
Leu Asn Tyr Leu Ser Glu Phe Pro Leu Pro Glu His Glu Pro Cys Leu
35 40 45
Glu Xaa
10
<210> 144
<211> 87
<212> PRT
15 <213> Homo Sapiens
<220>
<221> SITE
<222> (84)
<223> Xaa equals any one of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (87)
25 <223> Xaa equals stop translation
<400> 144
Met Thr Cys Cys Cys Leu Leu Cys Lys Leu Gln Gly Ile Phe Phe Phe
1 5 10 15
Ser Phe Asn Ser Ser Val Leu Lys Ser Ile Leu Gly Thr Thr Arg Thr
20 25 30
Leu Ser Ala Pro Trp Ile Gly Val Ser Val Lys Gly Thr Gln Trp Ala
35 40 45
Leu Gly Ser Ala Arg Pro Gly Cys Gly Ser Gln Leu Thr Ser Ser Leu
50 55 60
Gly Gly Leu Arg Gln Val Ile Cys Gln Pro His Leu Gln Lys His Asp
65 70 75 80
Ala Lys Leu Xaa Ser Val Xaa
45
<210> 145
<211> 57
<212> PRT
50 <213> Homo Sapiens
<400> 145
Met His Lys Cys Asn Thr Val Thr Arg Glu Leu Leu Gln Leu Ser Leu
1 5 10 15
Leu Ile Leu Pro Ser Gln Gys Gly Asn Cys Ala Thr Ser Thr Lys Arg
20 25 30
Gly Pro Arg Leu Leu Lys Tyr Phe Arg Thr Ser Pro Gln Glu Gln Thr
35 40 45
CA 02296815 2000-O1-14
WO 99/03990 PCT/IIS98/14613
68
Pro Leu His Leu Asp Ser Asp Cys Ser
50 55
<210> 146
<211> 87
<212> PRT
<213> Homo Sapiens
<400> 146
Met Ser His Cys Ala Arg Pro Leu Phe Phe Glu Thr Phe Phe Ile Leu
1 5 10 15
IS Leu Ser Pro Arg Leu Lys Cys Ser Gly Thr Asn Thr Val His Tyr Ser
25 30
Leu Asp Leu Leu Gly Ser Ser Asn Ser Ala Ser Val Pro Gln Val Gly
35 40 45
Gly Leu Thr Asn Ala Gln His Asp Thr Trp Leu Ile Phe Val Phe Cys
50 55 60
Val Cys Val Cys Glu Pro Leu Arg Arg Pro Trp Ala Ala Phe Leu Ile
65 70 75 80
Ser Val Thr Ser Ser Ile Lys
30
<210>
147
<211>
230
<212>
PRT
<213>
Homo
Sapiens
35
<220>
<221>
SITE
<222>
(216)
<223> ofthe L-amino
Xaa equals naturally acids
any one occurring
40
<400>
147
Met Gly Ala Leu ValLeu Val Leu HisVal
Leu Tyr Pro Gly Gln Ala
1 5 10 15
45 Thr Gln Phe Pro AlaGlu Glu Ala LeuThr
His Val Ala Val Val Leu
20 25 30
Leu Ala Tyr Ala GlyLeu Leu Pro ThrHis
Ile Ala Ala His Asn Arg
35 90 45
50
Val Val Thr Gln ProAsp Gly Trp LeuLys
Ser Ala Arg Met Ala Leu
50 55 60
Val Ala Ile Tyr AlaLeu Leu Gly AlaLeu
Leu Leu Gln Cys Ile Thr
55 6s 70 75 80
Asn Phe Leu Gly LeuLeu Thr Thr ProThr
Ser Phe Ala Met Val Ala
85 90 95
60 Ala Leu Lys Pro GlyPro Thr Leu AlaLeu
Ala His Arg Tyr Ala Leu
*rB
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
69
100 105 110
Val Leu Thr Ser Pro Ala Ala Thr Leu Leu Gly Ser Leu Phe Leu Trp
115 120 125
Arg Glu Leu Gln Glu Ala Pro Leu Ser Leu Ala Glu Gly Trp Gln Leu
130 135 140
Phe Leu Ala Ala Leu Ala Gln Gly Val Leu Glu His His Thr Thr Ala
145 150 155 160
Pro Cys Ser Ser His Gds Cys Pro Trp Ala Ser Thr Pro Ala Gly Cys
165 170 175
IS Phe Ser Gly Met Cys Ser Ser Gly Ser Glu Ile Cys Leu Ser Gly Leu
180 185 190
Gly Gln Arg Leu Pro Lys Asp Pro Ile Leu Pro Pro Ser Gly Glu Ile
195 200 205
Asn Glu CSrs Leu Phe Gln Gln Xaa Lys Lys Lys Lys Lys Lys Lys Lys
210 215 220
Lys Lys Lys Lys Gly Gly
225 230
<210> 148
<211> 63
<212> PRT
<213> Homo Sapiens
.<220>
<221> SITE
<222> (63)
<223> Xaa equals stop translation
<400> 148
Gln Pro Ala Leu Leu Tyr Leu Val Pro Ala Cys Ile Gly Phe Pro Val
1 5 to is
Leu Val Ala Leu Ala Lys Gly Glu Val Thr Glu Met Phe Ser Tyr Glu
20 25 30
Glu Ser Asn Pro Lys Asp Pro Ala Ala Val Thr Glu Ser Lys Glu Gly
35 40 45
Thr Glu Ala Ser Ala Ser Lys Gly Leu Glu Lys Lys Glu Lys Xaa
55 60
<210> 149
<211> 18
<212> PRT
<213> Homo Sapiens .
<220>
<221> SITE .
<222> (18)
()0 <223> Xaa equals stop translation
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98114613
5
<400> 149
Gln Leu Ile Leu Ser Leu Leu Arg Gly Phe Gds Lys Thr Glu Arg Val
1 5 10 15
Gly Xaa
10 <210> 150
<211> 16
<212> PRT
<213> Homo Sapiens
15 <220>
<221> SITE
<222> (16)
<223> Xaa equals stop translation
20 <400> 150
Met Ala Leu Gly Ala Arg Glu Leu Pro Gly Ser Leu Ser Arg Trp Xaa
1 5 10 15
<zlo> 151
<211> 22
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (22)
<223> Xaa equals stop translation
<400> 151
Met Tyr Ser Phe Ser Val Leu Glu Ile Thr Cys Phe Ile Leu Phe Leu
1 5 to 15
Txp Pro Ser Trp Val Xaa
45
<210> 152
<211> 25
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (25)
<223> Xaa equals stop translation
<400> 152
Met Lys Ile Lys Gln Arg Phe Ser Leu Leu Leu Phe His Cys Pro Phe
1 5 10 15
Pro Pro GSrs Cys Leu Ser Leu Gly Xaa
CA 02296815 2000-O1-14
WO 99/03990 PCTNS98/14613
71
20 25
<210> 153
$ <211> 40
<2I2> PRT
<213> Homo Sapiens
<400> 153
1~ Met Asn Gly Leu Phe Gln Leu Glu Ile Ser His Lys Leu Trp Thr Lys
1 5 10 15
Ser Lys Thr Ser Leu Met Thr~Leu Leu Ser Val Met Ala Leu Leu Trp
20 25 30
1$
Lys Ile Leu Trp Ser Arg Ala Ile
35 40
<210> 154
<211> 25
<212> PRT
<213> Homo Sapiens
2$ <220>
<221> SITE
<222> (25)
<223> Xaa equals stop translation
<400> 154
Met Thr Pro Gly Leu Phe Leu Tyr Phe Val Gys Val Cys Val Ser His
1 5 10 15
Gys Ala Gly Leu Gly Gln Leu Ser Xaa
3$ 20 25
<210> 155
<211> 103
4fl <212> PRT
<213> Homo Sapiens
<400> 155
Ile Arg His Glu Leu Gly Cys Ser Trp Arg Phe Arg Ala Val Lys Ala
4$ 1 s to 15
Ala Ser Ala Gln Gly Leu Phe Leu Ser Ala Pro Gly Pro Ala Ala Arg
20 25 30
$~ Arg Cys His Gly Val Val Arg Cys Phe Ser Thr Cps Arg Ala Leu Thr
35 40 45
Ala Arg Cys Thr Gly Arg Val Pro Trp Glu Ala Cys Leu Tyr Ser Ser
50 55 60
$$
Glu Pro Pro Leu Thr Glu Thr Val Ala Arg Ser Val Ser Trp Thr Cys
65 70 75 80
Glu Leu Ala Leu Thr Cys Tyr Ala Pro Arg Ala Leu Ser Gly Ala Pro
85 90 95
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
72
Val Leu Cys Arg His Asp Val
100
<210> 156
<211> 46
<212> PRT
<213> Homo Sapiens
<400> 156
Phe Leu Ala Ile His Phe Phe Leu Lys Pro
Pro Thr Asp Pro Pro Lys
1 5 10 15
ISVal Ala Phe Thr Arg Met Asn Asn Ser Asn
Tyr Phe Pro Ser Gly Ser
25 30
Thr Cys Leu Asp Ile Leu Trp Pro Ala Leu
Txp Ser Gln Ser
35 40 45
20
<210> 157
<211> I01
<212> PRT
<213> Homo Sapiens
<400> 157
Met Leu Leu Thr Pro His Phe Asn Val Ala Asn Pro Gln Asn Leu Leu
1 5 10 15
Ala Gly Leu Trp Leu Glu Asn Glu His Ser Phe Thr Leu Met Ala Pro
20 25 30
Glu Arg Ala Arg Thr His His Cys Gln Pro Glu Glu Arg Lys Val Leu
35 40 45
Phe Cys Leu Phe Pro Ile Val Pro Asn Ser Gln Ala Gln Val Gln Pro
50 55 60
Pro Gln Met Pro Pro Phe Cys Cys Ala Ala Ala Lys Glu Lys Thr Gln
65 70 75 80
Glu Glu Gln Leu Gln Glu Pro Leu Gly Ser Gln Cys Pro Asp Thr Cps
85 90 95
Pro Asn Ser Leu Cys
100
<210> 158
<211> 211
<212> PRT
<213> Homo Sapiens
<400> 158
Met Arg Leu Phe Leu Trp Asn Ala Val Leu Thr Leu Phe Val Thr Ser
1 5 10 15
Leu ile Gly Ala Leu Ile Pro Glu Pro Glu Val Lys Ile Glu Val Leu
20 25 30
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
73
Gln Lys Pro Phe Ile Gys His Arg Lys Thr Lys Gly Gly Asp Leu Met
35 40 45
Leu Val His Tyr Glu Gly Tyr Leu Glu Lys Asp Gly Ser Leu Phe His
50 55 60
Ser Thr His Lys His Asn Asn Gly Gln Pro Ile Trp Phe Thr Leu Gly
65 70 75 80
1~
Ile Leu Glu Ala Leu Lys Gly Trp Asp Gln Gly Leu Lys Gly Met Cys
85 90 95
Val Gly Glu Lys Arg Lys Leu Ile Ile Pro Pro Ala Leu Gly Tyr Gly
15 100 105 110
Lys Glu Gly Lys Gly Lys Ile Pro Pro Glu Ser Thr Leu Ile Phe Asn
115 120 125
20 Ile Asp Leu Leu Glu Ile Arg Asn Gly Pro Arg Ser His Glu Ser Phe
130 135 140
Gln Glu Met Asp Leu Asn Asp Asp Txp Lys Leu Ser Lys Asp Glu Val
145 150 155 160
Lys Ala Tyr Leu Lys Lys Glu Phe Glu Lys His Gly Ala Val Val Asn
165 170 175
Glu Ser His His Asp Ala Leu Val Glu Asp Ile Phe Asp Lys Glu Asp
180 185 190
Glu Asp Lys Asp Gly Phe Ile Ser Ala Arg Glu Phe Thr Tyr Lys His
195 200 205
Asp Glu Leu
210
<210> 159
<211> 186
<212> PRT
<213> Homo Sapiens
<400> 159
Glu Val Lys Ile Glu Val Leu Gln Lys Pro Phe Ile Cys His Arg Lys
1 5 10 15
Thr Lys Gly Gly Asp Leu Met Leu Val His Tyr Glu Gly Tyr Leu Glu
20 25 30
Lys Asp Gly Ser Leu Phe His Ser Thr His Lys His Asn Asn Gly Gln
35 40 45
Pro Ile Trp Phe Thr Leu Gly Ile Leu Glu Ala Leu Lys Gly Trp Asp
50 55 60
Gln Gly Leu Lys Gly Met Cys Val Gly Glu Lys Arg Lys Leu Ile Ile
70 75 80
Pro Pro Ala Leu Gly Tyr Gly Lys Glu Gly Lys Gly Lys Ile Pro Pro
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
74
85 90 95
Glu Ser Thr Leu Ile Phe Asn Ile Asp Leu Leu
Glu Ile Arg Asn Gly
100 105 110
Pro Arg Ser His Glu Ser Phe Gln Glu Met Asp
Leu Asn Asp Asp Trp
115 120 125
Lys Leu Ser Lys Asp Glu Val Lys Ala Tyr Leu
Lys Lys Glu Phe Glu
130 135 140
Lys His Gly Ala Val Val Asn Glu Ser His His
Asp Ala Leu Val Glu
145 150 155 160
Asp Ile Phe Asp Lys Glu Asp Glu Asp Lys Asp
Gly Phe Ile Ser Ala
165 170 175
Arg Glu Phe Thr Tyr Lys His Asp Glu Leu
180 185
<210> 160
<211> 633
<212> I7NA
2$ <213> Homo Sapiens
<400> 160
ATGAGGCTTZ' TCTTGTGGAA CGCGGTCTTG ACI'(."TGTTCG60
TCACTTCI"TT GATTGGGGCT
TZGATCCCTG AACCAGAAGT GAAAATTGAA GTTCTCCAGA 120
AGCCATTCAT CTGCCATCGC
AAGACCAAAG GAGGGGATTT GATi~I'IGGTC CACTATGAAG 180
GCTACTTAGA AAAGGACGGC
TCCTTATTTC ACTCCACTCA CAAACATAAC AATGGTCAGC 240
CCATTTGGTT TACCCTGGGC
ATCCTGGAGG CTCTCAAAGG TIGGGACCAG GGCTrGAAAG 300
GAATGTGTGT AGGAGAGAAG
AGAAAGCTCA TCATTCC'!'CC TGCTCTGGGC TATGGAAAAG 360
AAGGAAAAGG TAAAATTCCC
4O CCAGAAAGTA CACTGATATT TAATATTGAT CTCCTGGAGA 420
TTCGAAATGG ACCAAGATCC
CATGAATCAT TCCAAGAAAT GGATCTTAAT GATGACTGGA 480
AACTCTCTAA AGATGAGGTT
AAAGCATATT TAAAG,AAGGA GTTTGAAAAA CATGGTGCGG 540
TGGTGAATGA AAGTCATCAT
GATGCT'ITGG TGGAGGATAT T'TTTGATAAA GAAGATGAAG 600
ACAAAGATGG GTTTATATCT
GCCAGAGAAT TTACATATAA ACACGATGAG TTA 633
<210> 161
<211> 22
<212> PRT
<213> Homo Sapiens
<400> 161
Leu Arg Ser Val Val Gln Asp His Pro Gly Gln
His Gly Glu Thr Pro
1 5 10 15
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
Ser Leu Leu Lys Ile Gln
5 <210> 162
<211> 57
<212> PRT
<213> Homo Sapiens
1~ <220>
<221> SITE
<222> (34)
<223> Xaa equals any one of the naturally occurring L-amino acids
~5 <400> 162
Met Phe Tyr Asn Phe Val Arg Gln Leu Asp Thr Val Ser Ile Glu His
1 5 10 i5
Ala Gly Lys Ser Lys Leu Lys Met Thr Val Gly Thr Lys Leu Thr Ser
20 25 30
Gly Xaa Gly Pro Arg Lys Ser Ser Gln Ser Gly Arg Ile Ala Ala Ser
35 40 45
Ile Thr Asp Cys Gln Gln Cys Lys Ala
50 55
<210> 163
<211> 46
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (16)
<223> Xaa equals any one of the naturally occurring L-amino acids
<400> 163
4~ Met Glu Ala Ala Ile Leu Pro Leu Trp Leu Leu Phe Leu Gly Pro Xaa
1 5 10 15
Pro Glu Val Ser Phe Val Pro Thr Val Ile Phe Asn Leu Asp Phe Pro
20 25 30
Ala Cys Ser Ile Leu Thr Val Ser Ser Cys Leu Thr Lys Leu
35 40 45
<210> 164
<211> 25
<212> PRT
<213> Homo Sapiens
<400> 164
Asn His Gly His Ser Cys Phe Leu Cps Glu Ile Val Ile Arg Ser Gln
1 5 10 15
Phe His Thr Thr Tyr Glu Pro Glu Ala
20 25
CA 02296815 2000-O1-14
WO 99/03990 PCT/I3S98/14613
76
<210> 165
<211> 48
<212> PRT
<213> Homo Sapiens
<400> 165
Ser Gly Arg His Arg Val Glu Leu Gln Leu Leu Phe Pro Leu Val Arg
1 5 10 15
Val Asn Phe Glu Leu Gly Val Asn His Gly His Ser Cys Phe Leu Cys
25 30
15 Glu Ile Val Ile Arg Ser Gln Phe His Thr Thr Tyr Glu Pro Glu Ala
35 40 45
<210> 166
<211> 141
<212> PRT
<213> Homo Sapiens
<400> 166
Met Asn Ala Arg Gly Leu Gly Ser Glu Leu Lys Asp Ser Ile Pro Val
1 5 10 15
Thr Glu Leu Ser Ala Ser Gly Pro Phe Glu Ser His Asp Leu Leu Arg
20 25 30
Lys Gly Phe Ser Gds Val Lys Asn Glu Leu Leu Pro Ser His Pro Leu
35 40 45
Glu Leu Ser Glu Lys Asn Phe Gln Leu Asn Gln Asp Lys Met Asn Phe
50 55 60
Ser Thr Leu Arg Asn Ile Gln Gly Leu Phe Ala Pro Leu Lys Leu Gln
65 70 75 80
Met Glu Phe Lys Ala Val Gln Gln Val Gln Arg Leu Pro Phe Leu Ser
85 90 95
Ser Ser Asn Leu Ser Leu Asp Val Leu Arg Gly Asn Asp Glu Thr Ile
100 105 110
Gly Phe Glu Asp Ile Leu Asn Asp Pro Ser Gln Ser Glu Val Met Gly
115 120 125
Glu Pro His Leu Met Val Glu Tyr Lys Leu Gly Leu Leu
130 135 140
<210> 167
<211> 15
<212> PRT
<213> Homo Sapiens
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
77
<400> 167
Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn Tyr Gln Pro Tyr Pro
1 5 10 15
<210> 168
<211> 20
<212> PRT
<213> Homo Sapiens
<400> 168
Pro Leu Leu Gly Val Ser Ala Thr Leu Asn Ser Val Leu Asn Ser Asn
1 5 10 15
IS Ala Ile Lys Asn
<210> 169
20 <211> 14
<212> PRT
<213> Homo sapiens
<400> 169
Gly Ser Ala Val Ser Ala Ala Pro Gly Ile Leu Tyr Pro Gly
1 5 10
<210> 170
<zll> s1
<212> PRT
<213> Homo Sapiens
<400> 170
3$ Ala Gly Ile Gln His Glu Leu Ala Cys Asp Asn Pro Gly Leu Pro Glu
1 5 10 15
Asn Gly Tyr Gln Ile Leu Tyr Lys Arg Leu Tyr Leu Pro Gly Glu Ser
20 25 30
Leu Thr Phe Met Cys Tyr Glu Gly Phe Glu Leu Met Gly Glu Val Thr
35 40 45
Ile Arg Cys Ile Leu Gly Gln Pro Ser His Trp Asn Gly Pro Leu Pro
50 55 60
Val Cys Lys Val Ala Glu Ala Ala Ala Glu Thr Ser Leu Glu Gly Gly
65 70 75 80
Asn
<210> 171
<211> 27
<212> PRT
<213> Homo Sapiens
<400> 171
Gln Pro Ser His Trp Asn Gly Pro Leu Pro Val Cys Lys Val Ala Glu
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
78
1 5 10 15
Ala Ala Ala Glu Thr Ser Leu Glu Gly Gly Asn
20 25
<210> 172
<211> 13
<212> PRT
1~ <213> Homo Sapiens
<400> 172
Tyr Glu Thr Gly Glu Thr Arg Glu Tyr Glu Val Ser Ile
1 5 10
IS
<210> 173
<211> 195
<212> PRT
<213> Homo Sapiens
<220>
<221> SITE
<222> (40)
2,5 <223> Xaa equals any one of the naturally occurring L-amino acids
<220>
<221> SITE
<222> (161)
3~ <223> Xaa equals any one of the naturally occurring L-amino acids
<400> 173
Asp Asp Asp Gly Leu Pro Phe Pro Thr Asp Val Ile Gln His Arg Leu
1 5 10 15
Arg Gln Ile Glu Ala Gly Tyr Lys Gln Glu Val Glu Gln Leu Arg Arg
20 25 30
Gln Val Arg Asp Ser Asp Glu Xaa Gly His Pro Ser Leu Leu Cys Pro
35 40 45
Ser Ser Arg Ala Pro Met Asp Tyr Glu Asp Asp Phe Thr Cys Leu Lys
50 55 60
Glu Ser Asp Gly Ser Asp Thr Glu Asp Phe Gly Ser Asp His Ser Glu
65 70 75 80
Asp Cps Leu Ser Glu Ala Ser Trp Glu Pro Val Asp Lys Lys Glu Thr
85 90 95
$fl
Glu Val Thr Arg Trp Val Pro Asp His Met Ala Ser His CSrs Tyr Asn
100 105 110
Cys Asp Cys Glu Phe Trp Leu Ala Lys Arg Arg His His Cys Arg Asn
115 120 125
Cars Gly Asn Val Phe Cys Ala Gly Cys Cys His Leu Lys Leu Pro Ile
130 135 140
Pro Asp Gln Gln Leu Tyr Asp Pro Val Leu Val (.ys Asn Ser Cys Tyr
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
79
145 150 155 160
Xaa Thr His Ser Ser Leu Ser Cys Gln Gly Thr His Glu Pro Thr Ala
165 170 175
Glu Glu Thr His Cys Tyr Ser Phe Gln Leu Asn Ala Gly Glu Lys Pro
180 185 190
Val Gln Phe
195
<210> 174
<211> 28
<212> PRT
<213> Homo Sapiens
<400> 174
Ser Glu Ala Ser Trp Glu Pro Val Asp Lys Lys Glu Thr Glu Val Thr
1 5 10 15
Arg Trp Val Pro Asp His Met Ala Ser His Cys Tyr
20 25
<210> 175
<211> 10
<212> PRT
<213> Homo sapiens
<400> 175
His His C.ys Arg Asn Cys Gly Asn Val Phe
1 5 10
<210> 176
<211> 14
<212> PRT
<213> Homo sapiens
<400> 17s
Arg Leu Arg Gln Ile Glu Ala Gly Tyr Lys Gln Glu Val Glu
1 5 10
<210> 177
<211> 87
<212> PRT
<213> Homo Sapiens
<400> 177
Met Ser His Cys Ala Arg Pro Leu Phe Phe Glu Thr Phe Phe Ile Leu
1 5 10 15
Leu Ser Pro Arg Leu Lys Cys Ser Gly Thr Asn Thr Val His Tyr Ser
20 25 30
Leu Asp Leu Leu Gly Ser Ser Asn Ser Ala Ser Val Pro Gln Val Gly
35 40 45
CA 02296815 2000-O1-14
WO 99/03990 PCT/US98/14613
Gly Leu Thr Asn Aia Gln His Asp Thr Trp Leu Ile Phe Val Phe Cys
5p 55 60
Val Cps Val Cps Glu Pro Leu Arg Arg Pro Trp Ala Ala Phe Leu Ile
$ 65 70 75 80
Ser Val Thr Ser Ser Ile Lys
1~
<210> 178
<211> 30
<212> PRT
<213> Homo sapiens
<400> 178
Val Pro Gln Val Gly Gly Leu Thr Asn Ala Gln His Asp Thr Trp Leu
5 10 15
Zfl Ile Phe Val Phe Gds Val Cys Val Gys Glu Pro Leu Arg Arg
25 30
