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CA 02298852 2000-O1-28
WO 99/Ob423 PCTIUS98/15949
83 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
CA 02298852 2000-O1-28
WO 99/06423 PCT/US98/15949
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.
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.
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 ID 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"
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WO 99/06423 PCT/US98115949
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
analysis). A representative clone containing all or most of the sequence for
SEQ ID
NO:X was deposited with the American Type Culture Collection ("ATCC"). As
shown in Table 1, 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 ID 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, 5x SSC
(750 mM NaCI, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6}, 5x
Denhardt's solution, 10% dextran sulfate, and 20 pglml denatured, sheared
salmon
sperm DNA, followed by washing the filters in 0.1 x 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 IXSSPE, 0.1%
SDS. In addition, to achieve even lower stringency, washes performed following
stringent hybridization can be done at higher salt concentrations (e.g. 5X
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
CA 02298852 2000-O1-28
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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
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 polypeptides 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
CA 02298852 2000-O1-28
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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,
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 ID 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 mast preferably, not more than about three-fold less activity
relative to
the polypeptide of the present invention.)
Pol~~nucleotides and Poly~e~tides of the Invention
FEATURES OF PROTEIN ENCODED BY GENE NO: 1
This gene is expressed in a broad variety of tissues and cell types.
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, liver cancer. Similarly, polypeptides and antibodies
directed to
CA 02298852 2000-O1-28
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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 hepatic system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or
cell types (e.g., hepatic, cancerous and wounded tissues) or bodily fluids
(e.g.,
bile, 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 N0:99 as residues: Ser-34 to Arg-39,
Leu-50 to Ser-55.
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 1747 of SEQ ID NO:11, b is
an
integer of 15 to 1761, 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
Preferred polypeptides encoded by this gene comprise the following amino
acid sequence:
MEQTWTRDYFAEDDGEMVPRTSHTAAFLSDTKDRGPPVQSQIWRSGEKVP
FVQTYSLRAFEKPPQVQTQALRDFEKHLNDLKKENFSLKLXIYFLEERMQQ
KYEASREDIYKRNTELKVEVESLKRELQDKKQHLDKTWADVENLNSQNEA
ELRRQFEERHXETEHVYELLENKXQLLQEESRLAKNEAARMAALVEAEKEC
NLELSEKLKGVTKNWEDVPGDQVKPDQYTEALAQRDK (SEQ ID N0:188) or
MVPRTSHTAAFLSDTKDRGPPVQSQIWRSGEKVPFVQTYSLRAFEKPPQVQ
TQALRDFEKHLNDLKKENFSLKLXIYFLEERMQQKYEASREDIYKRNTELK
VEVESLKRELQDKKQHLDKTWADVENLNSQNEAELRRQFEERHXETEHVY
ELLENKXQLLQEESRLAKNEAARMAALVEAEKECNLELSEKLKGVTKNWE
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7
DVPGDQVKPDQYTEALAQRDK (SEQ ID N0:187). Polynucleotides encoding
these polypeptides are also provided.
This gene is expressed primarily in infant brain and to a lesser extent in a
large variety of other tissues, organs and cell types.
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, various forms of congentital mental retartation.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunoiogical 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., developmental,
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 N0:100 as residues: Met-1 to Arg-22, Leu-46 to Arg-52, Asn-64 to Gln-
70.
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 I to 1505 of SEQ ID N0:12, b is
an
integer of 15 to 1519, 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
Preferred polypeptide encoded by this gene comprise the following amino
acid sequence:
IRHELLPALHLQAHDAAYNLLFFASGGGKFNYQGTKRWLEDNLDHTGERP
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RVGVGVPRWWCRGEAXRPRGCHGGSQEAQREGRGPLPGPHPPRQLS VSC
RLQPASGQCGLRAVPGHRGPGQQPAPAXVRPXREGTLQHAFXRELETVAA
HQFPEVRFSMVHKRINLAEDVLAWEHERFAIRRLPAFTLSHLESHRDGQRS
SIMDVRSRVDSKTLIRLPQPPKVLGLRV (SEQ ID N0:189). Polynucleotides
encoding such polypeptides are also provided. This gene is believed to reside
on
chromosome 19. Thus, polynucleotides related to this gene are useful as
chromosome markers in linkage analysis for chromosome 19.
This gene is expressed primarily in microvascular endothelial cells and to a
lesser extent in in a variety of other cell types including 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, blood circulatory 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 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., vascular, and blood, 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.
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 1057 of SEQ ID N0:13, b is
an
integer of 15 to 1071, 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
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The gene encoding the disclosed cDNA sequence is believed to reside on
chromosome 22. Accordingly, polynucleotides related to this invention are
useful in
linkage analysis as markers for chromosome 22.
This gene is expressed primarily in a variety of cell types of muscle and
bone origin.
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, osteoporosis or any of a variety of disease that involve
wasting to
bone or muscle. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing irnmunological 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 and muscular systems,
expression of this
gene at significantly higher or lower levels may be routinely detected in
certain
tissues or cell types (e.g., musculoskeletal, 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 N0:102 as residues: Lys-81
to Thr-92, Arg-168 to Tyr-176, Gly-199 to Ser-216.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:14 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 941 of SEQ ID N0:14, b is
an
integer of 15 to 955, 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 the brain.
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WO 99/06423 PCT/US98115949
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, various brain disorders including mood disorders, memory
5 disorders, depression, and seizures. 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
10 cell types {e.g., neural, 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
1 S comprising a sequence shown in SEQ ID N0:103 as residues: Ser-62 to Cys-
67.
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: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 1494 of SEQ ID NO:15, b is
an
integer of 15 to 1508, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID NO:15, and where the b is greater than or equal to a
+
14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
When tested against PC12 cell Lines, supernatants removed from cells
containing this gene activated the EGR 1 (early growth response gene 1 )
pathway.
Thus, it is likely that this gene activates sensory neuron cells through the
EGR 1
signal transduction pathway. EGR1 is a separate signal transduction pathway
from
Jaks-STAT, genes containing the EGR 1 promoter are induced in various tissues
and cell types upon activation, leading the cells to undergo differentiation
and
proliferation. This gene maps to chromosome 1, and therefore, may be used as a
marker in linkage analysis for chromosome 1.
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WO 99/06423 PCT/US98/15949
This gene is expressed primarily in small intestine, and to a lesser extent,
in
fetal liver 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, intestinal cancers, premalignancies, and development
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 (e.g.gastrointesinal, developing, or
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 diagnosis and treatment of
intestinal
cancers and premalignancies, or ulcers, intestinal infections or other
conditions
arising from disorders of the gastrointesinal system. Alternatively, based
upon the
detected EGR activity in sensory neurons may suggest that the protein product
of
this gene is useful for the detection/treatment of neurodegenerative disease
states
and behavioural 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 preception. 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 andlor 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 N0:16 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
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12
or more polynucleotides comprising a nucleotide sequence described by the
general
formula of a-b, where a is any integer between 1 to 1992 of SEQ ID N0:16, b is
an
integer of 15 to 2006, 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
The translation product of this gene was shown to have homology to the
human LAK-4p which is thought to be involved in T-cell activation as this gene
is
specifically expressed during such a response (See Genebank Accession
No.gnIIPIDId1025089 (AB002405)). Preferred polypeptides comprise the
following amino acid sequence:
IYLNIQVVRGQRKVICLLKEQISNEGEDKIFLINKLHSIY (SEQ ID N0:190),
ERKEREERSRVGTTEEAAAPPALLTDE (SEQ I D N0:191 ), and/or RHEMENT
{SEQ ID N0:192),. Also preferred are the polynucleotides encoding these
polypeptides.
This gene is expressed primarily in several types of leukocytes, thymus,
bone marrow, and spleen.
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, immune system disorders, particularly of the leukocytes.
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 (e.g.immune, hematopoietic, or
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:105 as residues: Gln-38 to Asp-45, Glu-58 to Arg-67.
The protein product of the gene, based upon its homology to the human
immune-specific LAK-4p protein, in addition to its tissue distribution in
leukocytes,
is likely to be a modulator of the immune system and could be used in a
variety of
CA 02298852 2000-O1-28
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13
theraputic situations which require modulation of immune cell production such
as
leukemias and in protection of hematoprogenitors during chemotherapy.
Additionally, the protein product of this gene is useful for the diagnosis and
treatment of a variety of immune system disorders. Expression of this gene
product
in thymus and bone marrow 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 innmunological 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 N0:17 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 531 of SEQ ID N0:17, b is an
integer of
15 to 545, 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 it lymphocytes.
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
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14
are not limited to, diseases of the immune system, particularly of the
lymphocytes.
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 (e.g.immune, hematopoietic, or
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 a
variety of
immune system disorders. Expression of this gene product in lymphocytes
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 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
588 of
CA 02298852 2000-O1-28
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SEQ ID N0:18, b is an integer of 15 to 602, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID NO: i 8, and where the b is
greater than or equal to a + 14.
5
FEATURES OF PROTEIN ENCODED BY GENE NO: 9
This gene is expressed primarily in the human embryo.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
10 biological sample and for diagnosis of diseases and conditions, which
include, but
are not limited to, dvelopmental 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 reproductive system,
expression of
15 this gene at significantly higher or lower levels may be routinely detected
in certain
tissues (e.g.developing, differentiating, or 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 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. 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. 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 N0:19 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 polynucleotides comprising a nucleotide sequence
described by the general formula of a-b, where a is any integer between 1 to
573 of
CA 02298852 2000-O1-28
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16
SEQ ID N0:19, b is an integer of 15 to 587, 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 the human 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, which include,
but
are not limited to, reproductive and developmental disorders. 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
reproductive system, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues (e.g.developing, differentiating,
or
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 N0:108 as residues: Asn-6 to Ser-13.
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. 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. 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 polynucIeotide 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
CA 02298852 2000-O1-28
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17
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
630 of
SEQ ID N0:20, b is an integer of 15 to 644, 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 expressed primarily in the human embryo and the prostate.
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 and reproductive disorders, particularly
with
prostate cancer. Similarly, polypeptides and antibodies directed to these
I S 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 and urogenital systems,
expression
of this gene at significantly higher or lower levels may be routinely detected
in
certain tissues (e.g.developmental, reproductive, or cancerous and wounded
tissues) or bodily fluids (e.g.Iymph, 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 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. 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, expression within the
prostate
indicates that polynucleotides and poiypeptides corresponding to this gene are
useful for the diagnosis, treatment, and/or prevention of prostate cancer, and
related
reproductive 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
publicly
available and accessible through sequence databases. Some of these sequences
are
CA 02298852 2000-O1-28
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18
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 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 1243 of SEQ ID N0:21, b is
an
integer of 15 to 1257, where both a and b correspond to the positions of
nucleotide
residues shown in 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 the human 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, which include,
but
are 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 reproductive system,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues (e.g.developmental, or 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 NO:110 as residues: Trp-6
to Arg-13.
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. 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. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the
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19
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 N0:22 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
527 of
SEQ ID N0:22, b is an integer of 15 to 541, 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
This gene is expressed primarily in the human 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, which include,
but
are not limited to, reproductive and 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
reproductive system, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues (e.g.developmental, or 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 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. 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. Protein, as well as, antibodies directed
against the
CA 02298852 2000-O1-28
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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 N0:23 and may have been publicly available
prior
5 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
553 of
10 SEQ ID N0:23, b is an integer of 15 to 567, 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.
15 FEATURES OF PROTEIN ENCODED BY GENE NO: 14
This gene maps to chromosome 11, and therefore, may be used as a marker
in linkage analysis for chromosome 11.
This gene is expressed primarily in immune cells, particularly T cells and
dendritic cells.
20 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, particularly immunodeficiences such as
AIDS.
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 (e.g.immune, hematopoietic, or
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 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
CA 02298852 2000-O1-28
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21
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 N0:24 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 I to
572 of
SEQ ID N0:24, b is an integer of 15 to 586, 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 maps to chromosome 1, and therefore, may be used as a marker
in linkage analysis for chromosome 1.
This gene is expressed primarily in the brain and, to a lesser extent, in
prostate and kidney.
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 the brain and CNS, such as Alzheimer's and
Parkinson's disease. Similarly, polypeptides and antibodies directed to these
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22
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 and central nervous system,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues (e.g.neural, urogenital, or 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.
The tissue distribution indicates that polynucieotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
neurodegenerative disease states and behavioural 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
bahaviors,
including disorders in feeding, sleep patterns, balance, and preception. 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. Alternatively, the
tissue
distribution in kidney indicates that this gene or gene product could be used
in the
treatment and/or detection of kidney diseases including renal failure,
nephritus,
renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis,
hydronephritis,
nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic
and kidney stones, in addition to Wilms Tumor Disease, and congenital kidney
abnormalities such as horseshoe kidney, polycystic kidney, and Falconi'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 N0:25 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 1496 of SEQ ID N0:25, b is an
integer
of 15 to 1510, where both a and b correspond to the positions of nucleotide
CA 02298852 2000-O1-28
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23
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 was found to have homology to both the human ni06c07.s1 and
mouse Mpgc60 cDNAs which are specifically expressed in intestinal tissue (See
Genebank Accession Nos AA526969 and gbIY115051MMMPGC60, respectively).
As such, it is probable that the translation product of this gene is useful
for the
diagnosis, treatment, and/or prevention of various gastrointestinal disorders
and
afflictions.
This gene is expressed primarily in multiple tissues, including the brain,
breast, and kidney.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissue{s) or cell type{s)
present in a
biological sample and for diagnosis of diseases and conditions, which include,
but
are not lin>ited to, disorders involving the brain and central nervous system,
such as
Alzheimer s and Parkinson's, and reproductive and gastrointestinal disorders.
Also
disorders of the breast and kidney, 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 and central
nervous
system also the urogenital system, expression of this gene at significantly
higher or
lower levels may be routinely detected in certain tissues {e.g.neural,
endothelial,
hepatic, and mammary, and cancerous and wounded tissues) or bodily fluids
(e.g.lymph, bile, 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 N0:114 as residues: Pro-3
to Pro-9.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
neurodegenerative disease states and behavioural 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
bahaviors,
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24
including disorders in feeding, sleep patterns, balance, and preception. In
addition,
the gene or gene product may also play a role in the treatment andlor
detection of
developmental disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system. Alternatively,
considering the
homology to intestinal-specific proteins may suggest that the translation
product of
this gene is useful for the diagnosis, treatment, and/or prevention of various
gastrointestinal 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
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 521 of SEQ ID N0:26, b is
an
integer of 1 S to 535, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:26, and where the b is greater than or equal to a
+
14.
FEATURES OF PROTEIN ENCODED BY GENE NO: I7
This gene maps to chromosome 19, and therefore, may be used as a marker
in linkage analysis for chromosome 19.
This gene is expressed in multiple tissues, particularly brain 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
biological sample and for diagnosis of diseases and conditions, which include,
but
are not limited to, disorders of the brain and central nervous system, such as
Alzheimer's and Parkinson's, in addition to reproductive and developmental
disorders. Also disorders of the reproductive system 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 brain, central
nervous
system, and the reproductive system, expression of this gene at significantly
higher
or lower levels may be routinely detected in certain tissues (e.g.neural,
reproductive, or cancerous and wounded tissues) or bodily fluids (e.g.amniotic
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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
5 comprising a sequence shown in SEQ ID NO:115 as residues: Pro-6 to Glu-35,
Ser-47 to Glu-52, Gly-67 to Trp-73, Arg-85 to Asn-90, Asn-114 to Asn-119, Thr-
134 to Ser-141, Asn-250 to Glu-260.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
10 neurodegenerative disease states and behavioural 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
bahaviors,
including disorders in feeding, sleep patterns, balance, and preception. In
addition,
15 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. Alternatively, the
tissue
distribution in placenta indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of
cancer and
20 other proliferative disorders since development relies on decisions
involving cell
differentiation andlor apoptosis in pattern formation. Thus this protein may
also be
involved in apoptosis or tissue differentiation and could again be useful in
cancer
therapy. Protein, as well as, antibodies directed against the protein may show
utility
as a tumor marker andlor immunotherapy targets for the above listed tissues.
Many
25 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 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 1259 of SEQ ID N0:27, b is an
integer
of 15 to 1273, where both a and b correspond to the positions of nucleotide
residues shown in SEQ ID N0:27, and where the b is greater than or equal to a
+
14.
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26
FEATURES OF PROTEIN ENCODED BY GENE NO: 18
The translation product of this gene was found to have homology to several
collagen proteins.
This gene is expressed primarily in cells of the immune system, including
monocytes and neutrophils.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissue{s} or cell type{s)
present in a
biological sample and for diagnosis of diseases and conditions, which include,
but
are not limited to, disorders affecting the immune systems such as AIDS 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 (e.g.immune,
hematopoietic, or cancerous and wounded tissues) or bodily fluids (e.g.EGE,
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 in immune cells combined with its homology to
collagen would 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, spinal deformation, and
specific joint abnormalities as well as chondrodysplasias ie.
spondyloepiphyseal
dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II,
metaphyseal
chondrodysplasia type Schmid, and would healing disorders. 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 neutrophils and monocytes
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
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27
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 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
766 of
SEQ ID N0:28, b is an integer of 15 to 780, 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
This gene is expressed primarily in hepatocellular 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, which include,
but
are not limited to, hepatoma, and other disorders of the liver. 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
liver,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g.hepatic, or 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
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28
comprising a sequence shown in SEQ ID N0:117 as residues: Glu-33 to Glu-56,
Thr-75 to Cys-81.
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). The tissue distribution in hepatic tumors 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 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 N0:29 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 805 of SEQ ID N0:29, b is an integer of 15 to 819, 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
This gene is expressed primarily in apoptotic 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, hematopoietic and immune diseases, or 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
immune
system, expression of this gene at significantly higher or lower levels may be
routinely detected in certain tissues (e.g.hematopoietic, immune or 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
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29
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
irmnune 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 Iineages, 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 N0:30 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 formula of a-b, where a is any integer between 1 to
594 of
SEQ ID N0:30, b is an integer of 15 to 60$, 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
The translation product of this gene shares sequence homology with mouse
erythroid ankyrin protein which is thought to be important in linking the
spectrin-
based membrane skeleton to the plasma membrane in red blood cells. As such,
the
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translation product of this gene may show utility in the treatment and/or
diagnosis of
various hematopoietic disorders involving structural anomalies such as
thalassemia
and sickle-cell anemia syndromes {See Genebank Accession No. gi1311822). When
tested against K562 cell lines, supernatants removed from cells containing
this gene
5 activated the ISRE (interferon-sensitive responsive element ) pathway. Thus,
it is
likely that this gene activates kindey cells through the Jaks-STAT signal
transduction pathway. The ISRE 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
10 of cells. Therefore, activation of the Jaks-STATs pathway, reflected by the
binding
of the ISRE element, can be used to indicate proteins involved in the
proliferation
and differentiation of cells.
This gene is expressed primarily in colon cancer cells and, to a lesser
extent,
in pancreatic and testical tumors.
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, cancers and tumors of the urogenital, hematopoietic, or
endocrine
systems. Similarly, polypeptides and antibodies directed to these polypeptides
are
20 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 and repruductive system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues
(e.g.hematopoietic, urogenital, or cancerous and wounded tissues) or bodily
fluids
25 (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:119 as residues: Met-1 to Gly-6, Lys-
30 13 to Tyr-18, Asp-23 to Asp-28, Leu-55 to Glu-60, Pro-148 to Gly-155.
The tissue distribution combined with the observed ISRE activity indicates
that polynucleotides and polypeptides corresponding to this gene are useful
for the
diagnosis and treatment of cancer and other proliferative disorders.
Expression
within tumor tissues and other cellular sources marked by proliferating cells
indicates that this protein may play a role in the regulation of cellular
division.
Additionally, the homology to a structural protein in hematopoietic cells and
tissues
indicates that this protein may play a role in the proliferation,
differentiation, and/or
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31
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 tissue distribution
indicates that
polynucleotides and polypeptides corresponding to this gene are 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
irnmunotherapy 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 N0:31 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 1203 of SEQ ID N0:31, b is an integer of 15 to 1217, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 22
This gene maps to chromosome 19, and therefore, may be used as a marker
in linkage analysis for chromosome 19.
This gene is expressed primarily in umbilical vein endothelial cells and, to a
lesser extent, in human adipose.
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:reproductive or
metabolic disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for differential
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32
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
(e.g.reproductive, or 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 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. 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, expression in adipose tissue
indicates that polynucleotides and polypeptides corresponding to this gene are
useful for the diagnosis, prevention, and/or treatment of various metabolic
disorders
such as Tay-Sachs disease, phenylkenonuria, galactosemia, porphyrias, Hurler's
syndrome, or disorders related to lipid metabolism. 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 N0:32 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 751 of SEQ ID N0:32, b is an integer of 15 to 765, 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
This gene is expressed primarily in bone marrow stromal cells, and, to a
lesser extent, in epithelial-TNF alpha induced cells.
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33
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, integumentary and hematopoietic 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 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 (e.g.immune, hematopoietic, or 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 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. Alternatively, expression in cells induced by epithelial TNF-alpha
indicates
that polynucleotides and polypeptides corresponding to this gene are useful
for the
diagnosis and treatment of cancer and other proliferative disorders.
Expression
within differentiating 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 in hematopoietic cells and tissues indicates that
this
protein may play a role in the proliferation, differentiation, andlor 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
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34
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 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 738 of SEQ ID N0:33, b is an integer of 15 to 752, 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
IS This gene maps to chromosome 1, and therefore, may be used as a marker
in linkage analysis for chromosome I .
This gene is expressed primarily in brain, and, to a lesser extent, in ovary
and activated 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:immune
deficiencies
and brain degenerative diseases, in addition to 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
immune
and neural system, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues (e.g.neural, and reproductive, or
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 N0:122 as residues: Glu-2 to Glu-13, Pro-23 to Cys-36, Glu-47
to Ser-56, Val-64 to Pro-69, Val-106 to Asn-113, Ser-128 to Ala-134, Ser-155
to
Thr-163, Lys-176 to Phe-188, Leu-192 to Asp-207, Leu-209 to GIy-232, Glu-262
to Asn-269, Thr-274 to Lys-279, Lys-284 to Gly-294, Pro-309 to Cys-314, Phe-
318 to Lys-337.
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The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of .
neurodegenerative disease states and behavioural disorders such as Alzheimers
Disease, Parkinsons Disease, Huntingtons Disease, Tourette Syndrome,
5 schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,
panic
disorder, learning disabilities, ALS, psychoses , autism, and altered
bahaviors,
including disorders in feeding, sleep patterns, balance, and preception. In
addition,
the gene or gene product may also play a role in the treatment andlor
detection of
developmental disorders associated with the developing embryo, sexually-linked
10 disorders, female reproductive 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
15 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 more
polynucleotides comprising a nucleotide sequence described by the general
formula
20 of a-b, where a is any integer between 1 to 2251 of SEQ ID N0:34, b is an
integer
of 15 to 2265, 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
mouse fat-specific protein FSP27 which is thought to be important in adipose
differentiation (See Genebank Accession No. pirIA424451A42445). One
embodiment of this gene comprises polypeptides of the following amino acid
sequence:
RKLSTGPFSACKPRATCCFTSCYLQQLLDATEDGHPPKGKASSLIPTCLKIL
Q (SEQ ID N0:193), TSCYLQQLLDATEDGHPPKGKASSLIPTC (SEQ 117
N0:194), and/or CCGAKRIMKEALHWALFSMQATGHV (SEQ ID N0:195). 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.
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36
This gene is expressed primarily in adipose and to a lesser extent in small
intestine and a few other tissues.
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, adipose related disorders, including lipid metabolism
disorders,
and 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 adipose tissue, expression of this gene at significantly
higher or
lower levels may be routinely detected in certain tissues (e.g.adipose and
gastrointestinal, or cancerous, or 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 N0:123 as residues: Arg-30 to Gln-41.
The tissue distribution in adipose tissue combined with the homology to
ASP27 indicates that polynucleotides and polypeptides corresponding to this
gene
are useful for the diagnosis, treatment, and/or prevention of adipose related
disorders, particularly hyper- and hypolidemias, Tay-Sachs, atherosclerosis,
and
obesity. 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 N0:35 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 629 of SEQ ID N0:35, b is an
integer of
15 to 643, where both a and b correspond to the positions of nucleotide
residues
shown in SEQ ID N0:35, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 26
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37
The translation product of this gene was shown to have homology to a the
human KIAA0427 protein, novel, brain-specific protein that may be important in
brain development (See Genebank Accession No.gnIIPIDId1025779 (AB007887)).
One embodiment of this gene comprises polypeptides of the following amino acid
sequence:
PPAGATSPGRIIXPXSAVLIPSPVKSYRGWLVMGEPSREEYKIQSFDAETQQ
LLKTALKDPGAVDLEKVA
NVIVDHSLQDCVFSKEAGRMXYAIIQAESKQAGQSV
FRRGLLNRLQQEYQAREQLXARSLQGWVCYVTFICNIFDYLRVNNMPMM
ALVNPVYDCLFRLAQPDSLSKEEEVDCLVLQLHRVGEQLEK (SEQ ID
N0:196), PGRIIXPXSAVLIPSPVKSYRGWL (SEQ ID N0:197)
KQAGQSVFRRGLL NRLQQEYQAREQ (SEQ ID N0:198), and/or
YDCLFRLAQPDSLSKEEEVDC (SEQ ID N0:199),. An additional embodiment is
the polynucleotides encoding these polypeptides.
This gene is expressed primarily in hematopoiesis related tissues and cell
types and to a lesser extent in brain and a few cancer cell lines and 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 linvted to, immune, neural, andinflammatory 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 (e.g.neural, immune, cancerous, or
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 N0:124 as
residues: Met-1 to Met-6, Lys-50 to Arg-59.
The tissue distribution in brain combined with the homology to a novel
brain-specific human protein indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
neurodegenerative disease states and behavioural disorders such as Alzheimers
Disease, Parkinsons Disease, Huntingtons Disease, Tourette Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic
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38
disorder, learning disabilities, ALS, psychoses , autism, and altered
bahaviors,
including disorders in feeding, sleep patterns, balance, and preception. 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. Alternatively, the
tissue
distribution in hematopoetic tissue 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; 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. Protein, as well as, antibodies directed
against
the protein may show utility as a tumor marker andlor 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 N0:36 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 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
1288
of SEQ ID N0:36, b is an integer of 15 to 1302, 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.
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39
FEATURES OF PROTEIN ENCODED BY GENE NO: 27
The translation product of this gene was shown to have homology to the rat
mitochondria) brown-fat uncoupling protein which is an uncoupling protein
specific
to mitochondria) brown fat and is thought to play an integral role in the
thermogenesis of this tissue (See Genebank Accession No.P04633 ). One
embodiment of this gene comprises polypeptides of the following amino acid
sequence:
MKRTSVNPQTLCEARPAGXSQQPLSLDSEAPRGGVAPPRLQGPPPHQRVHL
TLECTTHPT V GKAS V
LGPCLLLLSCPRAPAGPPPPPHSRVRAGGCRPWARREGH
CRPLGADTDTSRICHGRRPFSL (SEQ ID N0:200),
MSLPAAPAGRLSPLYWRSS
NTRSQLSLLWELGHFFTRCCRRPHPNPHLPALSVCRCHILHKIMLWEPS
SPLLPALP (SEQ ID N0:201 ), and/or
MTSPGQGRAGRRGDEGSHNMILCKIWQR
HTLRAGRWGLGWGRRQHRVKKCPSSHSKESCDRVFELLQYKGES
RPAGAA GRDIIWFP (SEQ ID N0:202). An additional embodiment is the
polynucleotides encoding these polypeptides.
This gene is expressed primarily in hematopoietic tissues and neuronal
tissues and to a lesser extent in some cancer and other 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, immune, neural, and/or lipid metabolism disorders and/or
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 tissues or
cells,
particularly of the immune and neuronal tissues, expression of this gene at
significantly higher or lower levels may be routinely detected in certain
tissues
(e.g.neural, adipose, hematopoietic, and cancerous, or 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 N0:125 as residues: Glu-11 to Ser-17.
The tissue distribution in neural tissue combined with the homology to a
protein specific to adipose tissue indicates that polynucleotides and
polypeptides
CA 02298852 2000-O1-28
WO 99I064Z3 PCT/US98115949
corresponding to this gene are useful for the detection/treatment of
neurodegenerative disease states and behavioural disorders such as Alzheimers
Disease, Parkinsons Disease, Huntingtons Disease, Tourette Syndrome,
schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic
5 disorder, learning disabilities, ALS, psychoses , autism, and altered
bahaviors,
including disorders in feeding, sleep patterns, balance, preception, and
particularly
neural disorders involving anomylous lipid metabolism. 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
10 disorders of the cardiovascular system. Alternatively, considering the
tissue
distribution in hematopoetic tissue, 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
15 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
20 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
25 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
30 polynucleotides comprising a nucleotide sequence described by the general
formula
of a-b, where a is any integer between 1 to 991 of SEQ ID N0:37, b is an
integer of
15 to 1005, 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
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41
The translation product of this gene was shown to have homology to the
serine protease PfSP6 N-terminal fragment (See Genebank Accession No.
W01189) which may show utility in treatment and/or prevention of various
insect or
worm infestations, and/or diseases. One embodiment of this gene comprises
polypeptides of the following amino acid sequence: PSLRGPKAGAPPRWRPL
(SEQ ID N0:203), NLVDPPXCRNSARETLKLGRVEVSI (SEQ ID N0:204),
KAGAPPR (SEQ ID N0:205), and/or CRNSAR (SEQ ID N0:206). An
additional embodiment is the polynucleotides encoding these polypeptides.
This gene is expressed primarily in breast lymph node and to a lesser extent
in some other 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, immune, and 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 breast lymph
node,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g.immune, reproductive, cancerous, or 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.
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 breast lymph nodes
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 ilnmunological 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
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42
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 andlor 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
594 of
SEQ ID N0:38, b is an integer of 15 to 608, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:38, and where the b is
greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 29
This gene is expressed primarily in breast lymph node, and to a lesser
extent, in other 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, immune, and reproductive 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 tissues or cells, particularly of the breast lymph
node,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g.immune, reproductive, cancerous, or 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. Preferred epitopes include those comprising a sequence shown in SEQ
ID
N0:127 as residues: Pro-32 to Gly-39.
*rB
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43
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 breast lymph nodes
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 N0:39 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 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
911 of
SEQ ID N0:39, b is an integer of 15 to 925, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:39, and where the b is
greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 30
The translation product of this gene was shown to have homology to the
unc-50 related protein of Rattus norvegicus (See Genebank Accession No.
gi12735550) which is thought to be a novel RNA-binding protein that regulates
neuronal nicotinic receptor expression.Preferred polypeptides comprise the
following amino acid sequence: QDSRKMLPSTSVNSLVQGNG
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44
VLNSRDAARHTAGAKRYKYLRRLFRFRQMDF
EFAAWQMLYLFTSPQRVYRNFHYRKQTKDQWARDDPAFLVLLSIWLCV
STI GFGFVLD (SEQ 113 N0:207 ) NXQSRDYDVEWGYAFDVHLNAFYPLLV
ILHFIQLFFINHVILTDTFIGYLVGNTLWLVAVGYYIYVTFLGYSALPFLKNT
VIL LYPFAPLILLYGLSLALGWNFTHTLCSFYKYRVK (SEQ ID N0:208),
SVNS LVQGNGVLNSRDAARHTAGAKRYKYLRRLFRFRQMDFEFAA (SEQ
ID N0:209), VILTDTFIGYLVGNTLWLVAVGY (SEQ ID N0:210), and/or
GWNFT HTLCSFYKYRV (SEQ ID N0:211). Also preferred are the
polynucleotides encoding these polypeptides.
This gene is expressed primarily in hematopoietic tissues and to a lesser
extent in prostate, placenta, and other 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, immune, neural, and inflammatory 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
and neural systems, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues (e.g.neural, immune, cancerous,
or
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 the homology to a putative, brain
specific transcription factor indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
neurodegenerative disease states and behavioural 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
bahaviors,
including disorders in feeding, sleep patterns, balance, and preception. In
addition,
the gene or gene product may also play a role in the treatment andlor
detection of
developmental disorders associated with the developing embryo, sexually-linked
disorders, or disorders of the cardiovascular system. 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
CA 02298852 2000-O1-28
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disorders. Expression of this gene product in hematopoeitic tissue 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
5 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
10 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
15 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 N0:40 and may have been publicly available
prior
20 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
25 of SEQ ID N0:40, b is an integer of 15 to 1219, where both a and b
correspond to
the positions of nucleotide residues shown in SEQ ID N0:40, and where the b is
greater than or equal to a + 14.
30 FEATURES OF PROTEIN ENCODED BY GENE NO: 31
This gene is expressed primarily in proliferating tissues and tumors, and to a
lesser extent, in other tissues.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
35 biological sample and for diagnosis of diseases and conditions, which
include, but
are not limited to, growth related diseases and cancers. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological
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46
probes for differential identification of the tissues) or cell type{s). For a
number of
disorders of the above tissues or cells, particularly of tumors such as breast
cancer,
colon cancer, and many other common cancers expression of this gene at
significantly higher or lower levels may be routinely detected in certain
tissues
(e.g.differentiating, or 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
cancer and
other proliferative disorders. Expression within tumor tissues and other
cellular
sources marked by proliferating cells indicates that this protein may play a
role in
the regulation of cellular division. 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. 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 N0:41 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
1710
of SEQ ID N0:41, b is an integer of 15 to 1724, 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
This gene is expressed primarily in placenta, lung, and to a lesser extent in
other tissues.
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47
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, reproductive, 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
lung and placenta, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues (e.g.reproductive, pulmonary, or
cancerous and wounded tissues) or bodily fluids (e.g.pulmonary 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. Preferred epitopes include
those
comprising a sequence shown in SEQ ID N0:130 as residues: Met-1 to Trp-7, Ala-
37 to Arg-48.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis, treatment, and/or
prevention
of developmental and pulmonary disorders, particularly of cancer since
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 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 784 of SEQ ID N0:42, b is an integer of 15 to 798, 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.
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48
FEATURES OF PROTEIN ENCODED BY GENE NO: 33
The translation product of this gene was shown to have homology to the
human mitosis-associated nuclear antigen RMSA-1 which may be useful as an
antisense therapy for blocking the onset of mitosis (See Genebank Accession
No.Q72501).
This gene is expressed primarily in spleen of chronic lymphocytic leukemia.
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, disorders of immune 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 hematopoetic and
immune
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues (e.g. blood cells, and hematopoetic,
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 a
variety of
immune system disorders. Expression of this gene product in leukemia cells
combined with its homology to a mitotic regulatory factor indicates a role in
the
regulation of the proliferation; survival; differentiation; and/or activation
of
potentially all hematopoietic cell Iineages, 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
*rB
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49
proliferation of various cell types. Protein, as well as, antibodies directed
against
the protein rnay 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 N0:43 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
679 of
SEQ ID N0:43, b is an integer of 1 S to 693, where both a and b 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 34
The sequence of this gene was shown to have homology to the guinea pig
platelet activating factor (PAF) receptor which is a unique phospholipid
mediator,
possesses potent proinflammatory, smooth-muscle contractile and hypotensive
activities, and appears to be crucial in the pathogenesis of bronchial asthma
and in
the lethality of endotoxin and anaphylactic shock. Sequence analysis indicates
that
the receptor belongs to the superfamily of G protein-coupled receptors. (See
Genebank Accession No.gbIX567361CCPAFREC).
This gene is expressed primarily in human 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, disorders related to central nervous system, as well as
the
hematopoetic 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 brain expression of this gene at
significantly
higher or lower levels may be routinely detected in certain tissues
(e.g.neural,
hematopeotic, or 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
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from an individual not having the disorder. Preferred epitopes include those
comprising a sequence shown in SEQ ID N0:132 as residues: Pro-25 to Thr-31.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
5 neurodegenerative disease states and behavioural 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
bahaviors,
including disorders in feeding, sleep patterns, balance, and preception. In
addition,
10 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. Considering the
homoology to
a platelet activating factor, in addition the tissue distribution in brain,
indicates that
the protein product of this gene may show utility in the diagnosis, treatment,
and/or
15 prevention of stroke, amnesia, and other neural disorders related to
vascular trauma
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
20 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
sequence is
cumbersome. Accordingly, preferably excluded from the present invention are
one
or more polynucleotides comprising a nucleotide sequence described by the
general
25 formula of a-b, where a is any integer between I to 1344 of SEQ ID N0:44, b
is an
integer of 15 to 1358, 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 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, skin disorders. Similarly, polypeptides and antibodies
directed to
these polypeptides are useful in providing immunological probes for
differential
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51
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the epithelial cells of skin, expression of
this gene at
significantly higher or lower levels may be routinely detected in certain
tissues
(e.g.integumentary, cancerous and wounded tissues) or bodily fluids
(e.g.Iymph,
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 N0:133 as residues: His-106 to Ser-117.
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.
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, pemphigoid, and pemphigus), keloids, striae, erythema,
petechiae, purpura, and xanthelasma. Moreover, such disorders may predispose
increased susceptibility to viral and bacterial infections of the skin (i.e.
cold sores,
warts, chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis,
erysipelas, impetigo, tines, althletes foot, and ringworm). 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 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 951 of SEQ ID N0:45, b is an integer of 15 to 965, 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.
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52
FEATURES OF PROTEIN ENCODED BY GENE NO: 36
This gene is expressed primarily in macrophages.
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, hematopoetic, and/or immune disorders and afflictions,
particularly bacterial infections. 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 immunesystem, expression of this gene at
significantly higher or lower levels may be routinely detected in certain
tissues (e.g.
blood cells, and hematopoetic, immune, or 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 N0:134 as residues: Ser-12
to Trp-19, Val-51 to Thr-57, Ser-103 to Glu-116, His-123 to Leu-130, Gln-138
to
Gly-I43.
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
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
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53
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 N0:4b 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 N0:46, b is an integer of 15 to 791, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:46, and where the b is
greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 37
This gene is expressed primarily in human microvascular 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, vascular diseases, particularly stroke. 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 blood vescle
system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g.vascular, or 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, treatment, and/or
prevention
of vascular diseases, such as vasculitis, varicose veins, stroke, aneurysm, in
addition to disorders involving vasodilation and constriction. 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 N0:47 and may have
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54
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 756 of SEQ ID N0:47, b is an integer of 15 to 770, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 38
This gene is expressed primarily in human rhabdomyosarcoma.
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, neuromuscular disorders, particularly 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
muscular
system, expression of this gene at significantly higher or lower levels may be
routinely detected in certain tissues (e.g.muscle, neural, or 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 N0:136 as
residues: Ser-82 to Val-87, Pro-103 to Gly-110.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection, treatment, andlor
prevention
of various muscle disorders, such as muscular dystrophy, cardiomyopathy,
fibroids, myomas, and rhabdomyosarcomas. 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 N0:48 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
*rB
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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 861 of SEQ ID N0:48, b is an integer of 15 to 875, where both a
and
5 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 39
10 This gene is expressed primarily in spleen metastatic melanoma.
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, hematopoetic, and immune disorders. Similarly,
polypeptides and
15 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 hematopoeitic
system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g.hematopoetic, immune, cancerous and wounded
20 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 N0:137 as
25 residues: Met-1 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 tonsils indicates
a role
in the regulation of the proliferation; survival; differentiation; and/or
activation of
30 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.
35 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
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56
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 andlor 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 N0:49 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 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
600 of
SEQ ID N0:49, b is an integer of 15 to 614, 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
The translation product of this gene was shown to have homology to a zinc
finger protein of Rattus norvegicus which is known to be testis-specific and,
as
such, may suggest that the protein would have utility as a transcription
factor (See
Genebank Accession No. gi157504). One embodiment of this gene comprises
polypeptides of the following amino acid sequence:
PMVLKLKDWPPGEDFRDMMP (SEQ ID N0:212), YFVRPDLGPKMYNAYG
(SEQ ID N0:213), NSAREDGQP (SEQ ID N0:214), andlor LNLASRLP (SEQ
ID NO:215). An additional embodiment is the polynucleotides encoding these
polypeptides.
This gene is expressed primarily in bone marrow cells.
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, immune and hematopoetic 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,
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57
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g.hematopoetic, immune, or 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 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. Alternatively, based upon the homology to a testis-specific zinc finger
protein
may suggest that the protein product of this gene is useful in the diagnosis,
treatment, and/or prevention of various male reproductive 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 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 542 of SEQ m NO:50, b is an integer of 15 to 556,
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 bone marrow cells.
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58
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 and bone marrow disorders, particularly of the
hematopoetic system. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell types}. 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
(e.g.hematopoetic, or 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: Arg-52 to Asn-60,
Asn-b5 to Ala-73, Ala-81 to Ser-89.
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, immunodeflciency 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. 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 989 of SEQ ID N0:5 l, b is an
integer of
CA 02298852 2000-O1-28
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59
15 to 1003, where both a and b correspond to the positions of nucleotide
residues
shown in SEQ ID N0:51, 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 teratocarcinoma cells, and to a lesser
extent in human amygdala.
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, neural disorders, particularly 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 or CNS,
expression
of this gene at significantly higher or lower levels may be routinely detected
in
certain tissues (e.g.neural, or 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 N0:140 as residues: Pro-20 to Cys-26.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the detection/treatment of
neurodegenerative disease states and behavioural 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
bahaviors,
including disorders in feeding, sleep patterns, balance, preception, and
particularly
cancer. 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 N0:52 and
may
have been publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of
CA 02298852 2000-O1-28
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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 872 of SEQ ID N0:52, b is an integer of 15 to 886,
5 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
10 When tested against promyelocyctic cell lines, supernatants removed from
cells containing this gene activated the GAS (gamma activation site} pathway.
Thus,
it is likely that this gene activates myeloid cells through the Jaks-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,
15 signal transduction pathway involved in the differentiation and
proliferation of cells.
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of
the
GAS element, can be used to indicate proteins involved in the proliferation
and
differentiation of cells.
This gene is expressed primarily in human neutrophil.
20 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, hematopoetic disorders. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
25 differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the hematopoetic system.
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues (e.g. blood cells, and immune, or cancerous and wounded tissues) or
bodily
fluids (e.g.lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another
30 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 N0:141 as residues: Gly-11 to Ser-18,
Thr-26 to Lys-36.
35 The tissue distribution combined with the biological activity within
myeloid
cells indicates that polynucleotides and polypeptides corresponding to this
gene are
useful for the diagnosis and treatment of a variety of immune system
disorders.
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61
Expression of this gene product in neutrophils 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 immunoIogical 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
I S proliferation of various cell types. Protein, as well as, antibodies
directed against
the protein may show utility as a tumor marker andlor 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 N0:53 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
550 of
SEQ ID N0:53, b is an integer of 15 to 564, 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 human neutrophil.
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, hematopoetic and immune disorders. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunologicai
probes for differential identification of the tissues) or cell type(s). For a
number of
CA 02298852 2000-O1-28
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62
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 (e.g. blood cells, and immune, or 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: Leu-41 to Glu-48.
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
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
andlor
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 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
919 of
SEQ ID N0:54, b is an integer of 15 to 933, where both a and b correspond to
the
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63
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
When tested against K562 cell lines, supernatants removed from cells
containing this gene activated the ISRE (interferon-sensitive responsive
element ).
Thus, it is likely that this gene activates kidney and endothelial cells
through the
Jaks-STAT signal transduction pathway. ISRE is also a promoter element found
i 0 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. Therefore, activation of the Jaks-
STATs
pathway, reflected by the binding of the ISRE element, can be used to indicate
proteins involved in the proliferation and differentiation of cells.
This gene is expressed primarily 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 and hematopoetic disorders. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunoIogical
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 disorders
relating to
hemopoietic system, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues (e.g. blood cells, and immune, or
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 N0:143 as residues: Met-1 to His-6, Cys-29 to Ser-49, Pro-72 to Gly-
77.
The tissue distribution combined with the biological activity in stimulating
the interferon-sensitive responsive element 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 indicates a role in the regulation of the proliferation; survival;
differentiation; and/or activation of potentially all hematopoietic cell
lineages,
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64
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 N0:55 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 I to 583 of SEQ ID N0:55, b is an integer of 15 to 597, where both a
and
b correspond to the positions of nucleotide residues shown in SEQ ID N0:55,
and
where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 46
The translation product of this gene was shown to have homology to the
human thromboxane A2 receptor which is known to be a potent stimulator of
platelet aggregation (See Genebank Accession No. P21731).This gene maps to
chromosome 19, and therefore, may be used as a marker in linkage analysis for
chromosome 19.
This gene is expressed primarily 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, neutropenia and neutrophia, and other immunological and
*rB
CA 02298852 2000-O1-28
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hematopoetic 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 diseases relating to hemopoietic system,
5 expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g. blood cells, and immune, or 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
10 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 neutrophils
indicates
a role in the regulation of the proliferation; survival; differentiation;
and/or activation
15 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.
20 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
25 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,
30 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 present invention. To list every
related
sequence is cumbersome. Accordingly, preferably excluded from the present
35 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
759 of
SEQ ID N0:56, b is an integer of 15 to 773, where both a and b correspond to
the
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66
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 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:neutropenia and
other hemopoetic or 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 disorders relating to
hemopoietic
system, expression of this gene at significantly higher or lower levels may be
routinely detected in certain tissues (e.g. blood cells, and hemopoetic, or
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
N0:145 as residues: Pro-14 to Pro-28.
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
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
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67
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 N0:57 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
719 of
SEQ ID N0:57, b is an integer of 15 to 733, 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 is expressed primarily 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, neutropenia, and other hemopoetic 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
diseases relating to hemopoietic system, expression of this gene at
significantly
higher or lower levels may be routinely detected in certain tissues (e.g.
blood cells,
and immune, or 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 N0:146 as residues: Pro-23 to Tyr-28.
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
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
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68
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 N0:58 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 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
517 of
SEQ ID N0:58, b is an integer of 15 to 531, 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
The translation product of this gene was shown to have homology to the
human cathepsin E which is thought to play a role in modulation of the immune
system (See Genebank Accession No.P14091). 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 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, hemopoetic or immune disorders. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunological
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69
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 (e.g. blood cells, and immune, or 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 conserved human
cathepsin gene 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 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 N0:59 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 comprising a nucleotide sequence
described by the general formula of a-b, where a is any integer between 1 to
838 of
SEQ ID N0:59, b is an integer of 15 to 852, where both a and b correspond to
the
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positions of nucleotide residues shown in SEQ ID N0:59, and where the b is
greater than or equal to a + 14.
5 FEATURES OF PROTEIN ENCODED BY GENE NO: 50
The translation product of this gene was shown to have homology to the
human uridine 5' monophosphate synthase which is known to be involved in
purine
biosynthesis (See Genebank Accession No. P11172). This gene maps to
chromosome 3, and therefore, may be used as a marker in linkage analysis for
10 chromosome 3.
This gene is expressed primarily in neutrophils.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell type{s)
present in a
biological sample and for diagnosis of diseases and conditions, which include,
but
15 are not limited to, hemopoetic or 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 and
hemopoetic
systems, expression of this gene at significantly higher or lower levels may
be
20 routinely detected in certain tissues (e.g. blood cells, and immune, or
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.
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
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
30 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
35 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
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71
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 N0:60 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
666 of
SEQ ID N0:60, b is an integer of 15 to 680, 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: SI
This gene is expressed primarily 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, neurotropenia, and other hemopoetic or 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
hemopoietic system, expression of this gene at significantly higher or Lower
levels
may be routinely detected in certain tissues (e.g. blood cells, and immune, or
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 N0:149 as residues: Gln-73 to GIn-82.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment and diagnosis of
CA 02298852 2000-O1-28
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72
hematopoetic related disorders such as anemia, pancytopenia, leukopenia,
thrombocytopenia or leukemia since stromal cells are important in the
production of
cells of hematopoietic Iineages. 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
IO 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 N0:61 and may have been publicly available prior to conception of the
present
I5 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 880 of SEQ ID N0:61, b is an
integer of
20 15 to 894, 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
25 The translation product of this gene was shown to have homology to the
enhancer-trap-locus-1 of Mus musculus (See Genebank Accession No. gi150866)
which is thought to be involved in gene regulation pathways during mouse
development, particularly in the regulation of homeotic genes. As such, it can
be
suggested that the protein product of this gene would play a similiar role in
humans.
30 One embodiment of this gene comprises polypeptides of the following amino
acid
sequence:
V KPDPPRAPGENEDS S VPETPDNERKAS IS YFKNQRGIQYIDLS S DSED V V SP
N
CSNTVQEKTFNKDTVIIVSEPSEDEESQGLPTMARRNDDISELEDLSELEDLK
35 DAKLQTLKELFPQRSDN DLLKVIFIGYCSCNDDKISPAFSAIVSSG (SEQ ID
N0:216), KDAKLQTLKELFPQRSD (SEQ ID N0:217), KDTVIIVSEPSEDEES
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73
(SEQ ID N0:218), and/or EDSSVPETPDNERKAS (SEQ ID N0:219}. An
additional embodiment is the polynucleotides encoding these polypeptides.
This gene is expressed primarily 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:hemopoietic or
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
I O 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 (e.g.
blood cells, and immune, or 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:150 as residues: Lys-38 to Gln-46.
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
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. Alternatively, considering the homology
to a
conserved horneobox protein, would suggest that the protein product of this
gene is
useful in the detection, treatment, and/or prevention of developmental
disorders,
particularly those involving the immune system (e.g. immunodeficiencies
secondary
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74
to congentital defects or loss of immune organs). 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 677 of SEQ ID N0:62, b is an integer of IS to 691, where both a
and
b correspond to the positions of nucleotide residues shown in SEQ ID N0:62,
and
where the b is greater than or equal to a f 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 53
When tested against PC12 cell lines, supernatants removed from cells
containing this gene activated the EGR 1 (early growth response gene 1 )
pathway.
Thus, it is likely that this gene activates sensory neuron cells through the
EGR1
signal transduction pathway. EGR1 is a separate signal transduction pathway
from
Jaks-STAT, genes containing the EGRl 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 human B cell lymphoma and
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, particularly of B cell related diseases,
and
disorders related to hemopoiesis. 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 (e.g.
blood cells, and immune, hemopoietic, 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
CA 02298852 2000-O1-28
WO 99106423 PCTIUS98115949
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:151 as residues: Met-1 to Asp-12.
The tissue distribution indicates that polynucleotides and polypeptides
5 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
indicates
a role in the regulation of the proliferation; survival; differentiation;
andlor activation
of potentially all hematopoietic cell lineages, including blood stem cells.
Considering the expression in B-cell lymphomas, this gene product may be
10 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,
15 asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid
arthritis,
inflammatory bowel disease, sepsis, lymphomas, acne, and psoriasis. Protein,
as
well as, antibodies directed against the protein may show utility as a tumor
marker
andlor 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
20 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 rnay 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
25 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
30 described by the general formula of a-b, where a is any integer between 1
to 877 of
SEQ ID N0:63, b is an integer of 15 to 891, where 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
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76
This gene is expressed primarily 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, neutropenia, and other hemopoietic or 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
hemopoietic system, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues {e.g. blood cells, and immune, or
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 N0:152 as residues: Ser-32 to Cys-37.
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
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 N0:64 and may have been publicly available
prior
CA 02298852 2000-O1-28
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77
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
944 of
SEQ ID N0:64, b is an integer of 15 to 958, 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
When tested against PC12 cell lines, supernatants removed from cells
containing this gene activated the EGR 1 (early growth response gene 1 )
pathway.
Thus, it is likely that this gene activates sensory neuron cells through the
EGR1
l 5 signal transduction pathway. EGR 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.
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:neutropenia,
and
other immune or hemopoietic 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 hemopoietic system,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues (e.g. blood cells, and immune, hemopoietic, or 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 a
variety of
immune system disorders. Expression of this gene product in neutrophils
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
CA 02298852 2000-O1-28
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78
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 immunoIogical 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 andlor 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 N0:65 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
788 of
SEQ ID N0:65, b is an integer of 15 to 802, 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
This gene is expressed primarily in fetal liver.
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, hepatoblastoma, hepatitis, liver metabolic diseases, and
conditions that are attributable to the differentiation of hepatocyte
progenitor cells.
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
liver, expression of this gene at significantly higher or lower levels may be
routinely
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79
detected in certain tissues {e.g.hepatic, developing, or cancerous and wounded
tissues) or bodily fluids (e.g.bile, 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
N0:154 as residues: His-27 to Arg-34.
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
imrnunotherapy 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 N0:66 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 gresent 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 1078 of SEQ ID N0:66, b is an integer of 15 to 1092, where both a
and b correspond to the positions of nucleotide residues shown in SEQ ID
N0:66,
and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 57
This gene is expressed primarily in IL-1 and LPS 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, neutropenia, and other immune or hemopoietic disorders,
particularly bacterial infections. 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
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tissues or cells, particularly of the diseases relating to hemopoietic system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g. blood cells, and immune, or cancerous and
wounded tissues) or bodily fluids (e.g.lymph, serum, plasma, urine, synovial
fluid
5 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
10 immune system disorders. Expression of this gene product in neutrophils
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
15 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
20 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
25 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 N0:67 and may have been publicly available
prior
to conception of the present invention. Preferably, such related
polynucleotides are
30 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
720 of
5EQ ID N0:67> b is an integer of 15 to 734, where both a and b correspond to
the
35 positions of nucleotide residues shown in SEQ ID N0:67, and where the b is
greater than or equal to a + 14.
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81
FEATURES OF PROTEIN ENCODED BY GENE NO: 58
This gene is expressed primarily in IL-1 and LPS 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, neutropenia, and other hemopoietic or immune disorders,
particularly bacterial infections. 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 disorders relating to hemopoietic
system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g. blood cells, and immune, or 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 a
variety of
immune system disorders. Expression of this gene product in neutrophils
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 andlor immunotherapy targets
for
the above listed tissues. Many polynucleotide sequences, such as EST
sequences,
CA 02298852 2000-O1-28
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82
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, 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
692 of
SEQ ID N0:68, b is an integer of 15 to 706, where both a and b 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 IL-I and LSP 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, infection, inflammation, in addition to disorders of the
immune or
hemopoietic systems. Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunoIogical 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 (e.g.
blood cells, and immune, 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 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
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.
*rB
CA 02298852 2000-O1-28
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83
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 N0:69 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
422 of
SEQ ID N0:69, b is an integer of 15 to 436, 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
This gene is expressed primarily in IL-1 and LSP treated neutrophils.
Therefore, polynucIeotides 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, bacterial infections, inflammation, in addition to
disorders of the
hemopoietic or immune systems. 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 immune systems, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues (e.g.
blood cells, and immune, 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
CA 02298852 2000-O1-28
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84
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 neutrophils
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 rnay show utility as a tumor
marker andlor
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 N0:70 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
707 of
SEQ ID N0:70, b is an integer of 15 to 721, 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 IL-1 and LSP treated neutrophils.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell type{s)
present in a
*rB
CA 02298852 2000-O1-28
WO 99/06423 PCTIUS98115949
biological sample and for diagnosis of diseases and conditions, which include,
but
are not limited to, bacterial infection, inflammation, in addition to
disorders of the
hemopoietic or immune systems. Similarly, polypeptides and antibodies directed
to
these polypeptides are useful in providing immunological probes for
differential
5 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 (e.g.
blood cells, and immune, or cancerous and wounded tissues) or bodily fluids
(e.g.lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue
10 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 N0:159 as residues: Glu-36 to Lys-46.
The tissue distribution indicates that polynucleotides and polypeptides
15 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
indicates
a role in the regulation of the proliferation; survival; differentiation;
and/or activation
of potentially all hematopoietic cell Iineages, including blood stem cells.
This gene
product may be involved in the regulation of cytokine production, antigen
20 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
25 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
30 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 N0:71 and rnay have been publicly available
prior
35 to conception of the present invention. Preferably, such related
polynucIeotides are
specifically excluded from the scope of the present invention. To list every
related
sequence is cumbersome. Accordingly, preferably excluded from the present
CA 02298852 2000-O1-28
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86
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
779 of
SEQ ID N0:71, b is an integer of 15 to 793, 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
This gene is expressed primarily in IL-1 and LSP treated 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, bacterial infection, inflammation, in addition to immune
or
hemopoietic 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 {e.g.
blood cells, and immune, 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 N0:160 as residues: Gly-18 to Lys-29,
Pro-45 to Gly-51, Pro-53 to Lys-58, Pro-72 to Gly-79, Pro-88 to Leu-108, AIa-
124 to Ser-134, Ser-138 to Lys-148.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of a
variety of
inunune system disorders. Expression of this gene product in neutrophils
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
*rB
CA 02298852 2000-O1-28
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87
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 N0:72 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
747 of
SEQ ID N0:72, b is an integer of 15 to 761, 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
This gene is expressed primarily in IL.-1 and LSP treated 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 or hemopoietic 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 (e.g. blood cells, and immune, or 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 N0:161
as residues: Asp-6 to Glu-15, Pro-76 to Ser-87.
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88
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
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
innmunotherapy 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 Iineages, 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
659 of
SEQ ID N0:73, b is an integer of 15 to 673, 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
This gene is expressed primarily in IL-1 and LSP treated neutrophils.
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, immune or hemopoietic disorders. Similarly, polypeptides
and
CA 02298852 2000-O1-28
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89
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 (e.g. blood cells, and immune, or 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 a
variety of
immune system disorders. Expression of this gene product in neutrophils
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 N0:74 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 polynucleotides comprising a nucleotide sequence
described by the general formula of a-b, where a is any integer between 1 to
569 of
SEQ ID N0:74, b is an integer of 15 to 583, where both a and b correspond to
the
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positions of nucleotide residues shown in SEQ ID N0:74, and where the b is
greater than or equal to a + 14.
5 FEATURES OF PROTEIN ENCODED BY GENE NO: 65
This gene maps to chromosome 19, and therefore, may be used as a marker
in linkage analysis for chromosome 19.
This gene is expressed primarily in activated neutrophils.
Therefore, polynucleotides and polypeptides of the invention are useful as
10 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, Inflammatory bowel disease, chronic neutropenia
(Kostmann's
syndrome), chemotherapy induced neutropenia, AIDS, and other immunodefiencicy
disorders. Similarly, polypeptides and antibodies directed to these
polypeptides are
15 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 hemopoietic systems, expression of this gene at
significantly higher or lower levels may be routinely detected in certain
tissues (e.g.
blood cells, and immune, or cancerous and wounded tissues) or bodily fluids
20 (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 N0:163 as residues: Gly-17 to GIy-23.
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
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
30 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
35 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
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91
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 andlor 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 N0:75 and may have been publicly available
prior
to conception of the present invention. Preferably, such related
polynucIeotides 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
787 of
SEQ ID N0:75, b is an integer of 15 to 801, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:75, and where the b is
greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 66
This gene is expressed primarily in activated 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 linuted to, chronic and acute neutropenia, inflammatory bowel disease,
neutrophil related multiple organ failure, and other immune or hemopaietic
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 (e.g. blood cells,
and
hemopoietic, or 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 N0:164 as residues: Met-35 to Glu-51.
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92
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
potentially all hematopoietic cell lineages, including blood stern 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.
i0 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 andlor 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 N0:76 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
968 of
SEQ ID N0:76, b is an integer of 15 to 982, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:76, and where the b is
greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 67
This gene is expressed primarily in activated 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, acute and chronic neutropenia, inflammatory bowel disease,
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93
neutrophil-related multiple organ failure, and other immune or hemopoietic
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 (e.g. blood cells,
and
hemopoietic, or 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 N0:165 as residues: Asp-21 to His-26,
Leu-31 to His-39, Arg-64 to Thr-70.
The tissue distribution of this gene specifically in neutrophils indicates a
possible role in the treatment and/or detection of disease states in which
either a lack
or excess of neutrophils plays a role in the pathophysiology of the disease
state.
Targetting this protein could provide a mechanism to inhibit the role of
neutrophils
in Inflammatory bowel disease and neutrophil releted multiple organ failure.
The
protein encoded by this gene could be important in the treatment of
neutropenia,
such as the chronic neutropenic Kostmann's syndronme, AIDS related
neutropenia,
chemotherapy induced neutropenia, in addition to juvenile periodontis and
other
states which are caused by decreased neutrophil chemotaxis. 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 N0:77 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 987 of SEQ ID N0:77, b is an integer of 15 to 1001, where both a
and
b correspond to the positions of nucleotide residues shown in SEQ ID N0:77,
and
where the b is greater than or equal to a + 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 68
This gene is expressed primarily in activated 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, neutropenia, inflammatory bowel disease, neutrophil
related
multiple organ failure, and other 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 or
hemopoietic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues (e.g. blood cells, and hemopoietic,
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
N0:166 as residues: Ile-26 to Ala-34, Thr-81 to Asp-88.
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
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. 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,
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are publicly available and accessible through sequence databases. Some of
these
sequences are related to SEQ ID N0:78 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
5 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
734 of
SEQ ID N0:78, b is an integer of I S to 748, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:78, and where the b is
10 greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 69
This gene is expressed primarily in adipocytes.
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, obesity, and diabetes. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
20 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 (e.g.endocrine, metabolic, or cancerous and wounded tissues) or bodily
fluids (e.g. serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue
25 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 N0:167 as residues: Ser-26 to Lys-36.
The tissue distribution predominantly in adipose tissue, indicates a role in
30 the treatment and/or detection of adipofibrosarcoma, adiponecrosis, obesity
and
diabetes. 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
35 ID N0:79 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.
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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 572 of SEQ ID N0:79, b is an
integer of
15 to 586, where both a and b correspond to the positions of nucleotide
residues
shown in SEQ ID N0:79, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 70
This gene is expressed primarily in kidney.
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, kidney diseases, particularly nephritis 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
renal and
urogenital systems, expression of this gene at significantly higher or lower
levels
may be routinely detected in certain tissues (e.g.urogenital, or 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 in kidney indicates that this gene or gene product
could be used in the treatment andlor detection of kidney diseases including
renal
failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema,
pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome,
glomerulonephritis, hematuria, renal colic and kidney stones, in addition to
Wilms
Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney,
polycystic kidney, and Falconi'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 N0:80 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
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sequence described by the general formula of a-b, where a is any integer
between 1
to 532 of SEQ ID N0:80, b is an integer of 15 to 546, where both a and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:80, and
where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7I
This gene is expressed primarily in T-cells and hepatocytes.
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 and hepatic disorders. Similarly, poiypeptides 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
hepatic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues (e.g. blood cells, and immune, hepatic,
or
cancerous and wounded tissues) or bodily fluids (e.g.lyrnph, 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 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
*rB
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98
committed progenitors of various blood lineages, and in the differentiation
and/or
proliferation of various cell types. Alternatively, considering the expression
in
hepatocytes 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 N0:81 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 694 of SEQ ID N0:81, b is an integer of 15 to 708, where both a and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:81, and
where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 72
The translation product of this gene was shown to have homology to the
human KIAA0213 which is thought to be a serine/threonine protein kinase which
may implicate this gene as playing an integral role in signal transduction,
particularly in cell cycle regulation (See Genebank Accession No. P25390).
When
tested against K562 cell lines, supernatants removed from cells containing
this gene
activated the ISRE (interferon-sensitive responsive element ) pathway. Thus,
it is
likely that this gene activates kidney cells through the Jak-Stat signal
transduction
pathway. ISRE 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.
Therefore, activation of the Jaks-STATs pathway, reflected by the binding of
the
ISRE element, can be used to indicate proteins involved in the proliferation
and
differentiation of cells.
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This gene is expressed primarily in rhabdomyosarcoma.
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, rhabdomyosarcoma, and other 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 muscular system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues (e.g.proliferating, muscle, or 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 N0:170 as
residues: Ser-24 to Ala-30.
The tissue distribution in rhabdomyosarcoma tissue combined with its
homology to a putative cell cycle modulating protein indicates that
polynucleotides
and polypeptides corresponding to this gene are useful for the diagnosis and
treatment of cancer and other proliferative disorders, particularly of muscle
tissue.
Expression within tumor tissue and other cellular sources marked by
proliferating
cells indicates that this protein may play a role in the regulation of
cellular division.
Alternatively, considering its expression in muscle tissue may suggest
indicates that
polynucleotides and polypeptides corresponding to this gene are useful for the
detection, treatment, and/or prevention of various muscle disorders, such as
muscular dystrophy, cardiomyopathy, fibroids, myomas, and rhabdomyosarcomas.
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 N0:82 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 810 of SEQ ID N0:82, b is an
integer of
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15 to 824, where both a and b correspond to the positions of nucleotide
residues
shown in SEQ ID N0:82, and where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 73
When tested against fibroblast cell lines, supernatants removed from cells
containing this gene activated the EGRl (early growth response gene 1 pathway.
Thus, it is likely that this gene activates cells through the EGR1 signal
transduction
pathway. EGR1 is a separate signal transduction pathway from 3aks-STAT, genes
containing the EGR 1 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 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, immune disorders and disease states. 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
hemopoietic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues (e.g. blood cells, and immune, or
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 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
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101
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 N0:83 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
775 of
SEQ ID N0:83, b is an integer of 15 to 789, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:83, and where the b is
greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 74
The translation product of this gene was shown to have homology to the
human zinc finger protein 7 which is thought to a play a role as a
transcriptional
modulator (See Genebank Accesion No. P17097). This gene maps to the
chromosome X, and therefore, may be used as a marker in linkage analysis for
the
chromosome X.
This gene is expressed primarily 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, immune disorders and disease states. Sinularly,
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
hemopoietic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues {e.g. blood cells, and immune, or
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
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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 N0:172
as residues: Glu-4 to Arg-12, Glu-63 to Arg-69.
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 alI 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 N0:84 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 N0:84, b is an integer of 15 to 811, where both a and b correspond to
the
positions of nucleotide residues shown in SEQ ID N0:84, and where the b is
greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 75
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This gene is expressed primarily in anergic 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, leukemias, lymphomas, auto-immunities, immunodeficiencies,
immuno-supressive conditions and hematopoeitic disorders. 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 immune and
hematopoeitic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues (e.g. blood cells, and immune,
hematopoietic,
or 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
immune
disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies
(e.g. AIDS), immuno-supressive conditions (transplantation) and hematopoeitic
disorders. In addition this gene product may be applicable in conditions of
general
nucrobial infection, inflammation or cancer. 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 N0:85 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 1056 of SEQ ID N0:85, b is an integer of 15 to 1070, where both a and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:85, and
where the b is greater than or equal to a + 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 76
This gene maps to chromosome 11, and therefore, may be used as a marker
in linkage analysis for chromosome 11.
This gene is expressed primarily in T-cells (resting and anergic).
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell type{s)
present in a
biological sample and for diagnosis of diseases and conditions, which include,
but
are not limited to, leukemias, lymphomas, auto-immunities, immunodeficiencies,
immuno-supressive conditions and hematopoeitic 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 and
hematopoeitic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues (e.g. blood cells, and immune,
hematopoietic,
or 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 N0:174 as residues: Thr-25 to Asp-38.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of
immune
disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies
(e.g. AIDS), immuno-supressive conditions (transplantation) and hematopoeitic
disorders. In addition this gene product may be applicable in conditions of
general
microbial infection, inflammation or cancer. Protein, as well as, antibodies
directed
against the protein may show utility as a tumor marker andlor 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 N0:86 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 713 of SEQ ID N0:86, b is an integer of 15 to 727, where both a and b
CA 02298852 2000-O1-28
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105
correspond to the positions of nucleotide residues shown in SEQ ID N0:86, and
where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 77
This gene maps to chromosome 8, and therefore, may be used as a marker
in linkage analysis for chromosome 8.
This gene is expressed primarily in anergic 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, leukemias, lymphomas, auto-immunities, immunodeficiencies,
immuno-supressive conditions and hematopoeitic 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 and
hematopoeitic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues (e.g. blood cells, and immune,
hematopoietic,
or 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 N0:175 as residues: Glu-8 to Lys-17, Val-42 to Trp-51.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the diagnosis and treatment of
immune
disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies
(e.g. AIDS), immuno-supressive conditions (transplantation) and hematopoeitic
disorders. In addition this gene product may be applicable in conditions of
general
microbial infection, inflammation or cancer. 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 N0:87 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
CA 02298852 2000-O1-28
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106
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 676 of SEQ ID N0:87, b is an integer of 15 to 690, where both a and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:87, and
where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 78
This gene is expressed primarily in anergic 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, leukemias, lymphomas, auto-immunities, immunodeficiencies,
immuno-supressive conditions and hematopoeitic 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 and
hematopoeitic
systems, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues {e.g. blood cells, and immune,
hematopoietic,
or 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
immune
disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies
(e.g. AIDS), immuno-supressive conditions (transplantation) and hematopoeitic
disorders. In addition this gene product may be applicable in conditions of
general
microbial infection, inflammation or cancer. Protein, as well as, antibodies
directed
against the protein may show utility as a tumor marker andlor 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 N0:88 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
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107
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 882 of SEQ ID N0:88, b is an integer of 15 to 896, where both a and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:88, and
where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 79
The translation product of this gene was shown to have homology to the
human clathrin light chain B which is the major protein for the polyhedral
coat of
clathrin coated pits and vesicles (See Genebank Accession No. P09497).
This gene is expressed primarily in the spinal cord.
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, meningitis, spina bifida, spinal tumors and neoplasms as
well as
other developmental and neurodegenerative conditions. 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 spinal cord and
central
nervous system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues (e.g.neural, or 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 in spinal cord combined with the homology to human
clathrin indicates that polynucleotides and polypeptides corresponding to this
gene
are useful for the treatment and diagnosis of trauma, meningitis, spins
bifida, spinal
tumors and neoplasms as well as other developmental and neurodegenerative
conditions of the spinal cord and central nervous system, particularly those
neural
disorders involving cell-cell signalling. 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 N0:89 and may have been publicly
available prior to conception of the present invention. Preferably, such
related
*rB
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108
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 843 of SEQ ID N0:89, b is an integer of 15 to 857, where both a and b
correspond to the positions of nucleotide residues shown in SEQ ID N0:89, and
where the b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 80
This gene is expressed primarily in spinal cord.
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, meningitis, spins bifida, spinal tumors and neoplasms as
well as
other developmental and neurodegenerative conditions. 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 spinal cord and
central
nervous system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues (e.g.neural, or 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 the treatment and diagnosis of
trauma,
meningitis, spins bifida, spinal tumors and neoplasms as well as other
developmental and neurodegenerative conditions of the spinal cord and central
nervous system, 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 bahaviors, including disorders in feeding, sleep patterns,
balance, and preception. Protein, as well as, antibodies directed against the
protein
may show utility as a tumor marker andlor 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
*rB
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109
related to SEQ ID N0:90 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 547 of SEQ ID N0:90, b is
an
integer of 15 to 561, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:90, and where the b is greater than or equal to a
+
14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 81
This gene is expressed primarily in spinal cord.
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, meningitis, spina biflda, spinal tumors and neoplasms as
well as
other developmental and neurodegenerative conditions. Similarly, polypeptides
and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell types}. For a
number of
disorders of the above tissues or cells, particularly of the spinal cord and
central
nervous system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues (e.g.neural, or 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 N0:179 as residues: Met-1
to Arg-6, Ser-98 to Met-104.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment and diagnosis of
trauma,
meningitis, spina bifida, spinal tumors and neoplasms as well as other
developmental and neurodegenerative conditions of the spinal cord and central
nervous system, 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 bahaviors, including disorders in feeding, sleep patterns,
CA 02298852 2000-O1-28
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110
balance, and preceptiori. 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 N0:9I 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 641 of SEQ ID N0:91, b is
an
integer of 15 to 655, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:9I, and where the b is greater than or equal to a
+
I4.
FEATURES OF PROTEIN ENCODED BY GENE NO: 82
This gene is expressed primarily in spinal cord.
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, meningitis, spina bifida, spinal tumors and neoplasms as
well as
other developmental and neurodegenerative conditions. 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 spinal cord and
central
nervous system, expression of this gene at significantly higher or lower
levels may
be routinely detected in certain tissues (e.g.neural, or 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 N0:180 as residues: Asn-9
to Tyr-14, Ala-30 to Va1-39.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for the treatment and diagnosis of
trauma,
meningitis, spina bifida, spinal tumors and neoplasms as well as other
developmental and neurodegenerative conditions of the spinal cord and central
CA 02298852 2000-O1-28
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111
nervous system, 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 bahaviors, including disorders in feeding, sleep patterns,
balance, and preception. 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 N0:92 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 834 of SEQ ID N0:92, b is
an
integer of 15 to 848, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:92, and where the b is greater than or equal to a
+
14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 83
This gene is expressed primarily in flbrosarcoma, and tonsils.
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, tosilitis, and other muscle or 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 and musculoskeletal systems, expression of this gene at significantly
higher or lower levels may be routinely detected in certain tissues
(e.g.immune,
muscle, or 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 a
variety of
CA 02298852 2000-O1-28
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112
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
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 expression in
fibrosarcoma
indicates that polynucleotides and polypeptides corresponding to this gene are
useful for the detection, treatment, and/or prevention of various muscle
disorders,
such as muscular dystrophy, cardiomyopathy, fibroids, myomas, and
rhabdomyosarcomas. 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 N0:93 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 598 of SEQ ID N0:93, b is
an
integer of 15 to 612, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID N0:93, and where the b is greater than or equal to a
+
14.
CA 02298852 2000-O1-28
WO 99/06423 PCT/US98/15949
113
Q ~ o
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CA 02298852 2000-O1-28
WO 99/06423 PCT/US98/15949
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CA 02298852 2000-O1-28
WO 99/06423 PCT/US98/15949
115
_ O
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CA 02298852 2000-O1-28
WO 99/06423 PCTlUS98/15949
116
w p~ ~D ~ ~n oo ~ .-w p ..,.,
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CA 02298852 2000-O1-28
WO 99/06423 PCTIUS98/15949
117
'+. ~1 V~ N ~t N
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CA 02298852 2000-O1-28
WO 99/06423 PCTIUS98/15949
118
w M ~ 00 N O M N
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a ~ ~ a ~ ~ a
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U ~ ' ~ i ~ i ~ i~ ~ i ~ i ~ i ~ i
v,.~ o~.~ a~..~v,.-~ .~ ~ .~ ~, a,
o o g o o
z ~ ~' ~ ~ x ~ ~ ~ w
z z
x x ~ ~ x x
O M M ~fi ~' ~ d' ~' ~Y
z
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119
d' N M M M ~ pMp
airp
0o O oo v0
N N M
...
p ~ ~
N ~ M N N M ti
w
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G. N N M ~ N N N
p ~ N ~ ~ ~
N N N
'~"'p O ~ C1 ~ I~ M M 1n 000 G~1
V~ V~ l~ I~ ~ 00 ~O 00
z
U ...~ .-..~ .-. N .-, ._.
~n
z
z ~ ~ z ~c
x ~ ~ ~ x x ~ x
Q Q Q a Q a a Q
j N N N N N N N N
U
U ~ 'ti 'ti ~
~
_ _ _ _
Q ~ z O ~ O '~ O~ O ~ O ~ O ~ O ~ O
N ~ N ~ Np N Q N ~ N ~ N ~ N
A ~ ~ ~ ~ O~ O ~ ti
Q ~ ~ ~ ~ ~ A 3
A _o ~ Ca7 ~ ~ CA7 C~7
C7 x c~
U x ~ x x x x x
a~
c
*rB
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120
ct ~ M ~ o~O N dN'
_
Q ~ o ~ _ M M
V ~ d'
....,
(%~~.~.,d' ~t M M ~ N
... .--, .,... ~..~
O ~ N M ~t t W p
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b_0~ ~ M M
Q",-, ._.. .~ .~ N O~ N
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N N O
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M C/,1 C ~- ~ ~ ~ M O M
.~ l~ l~ d'
z o o ~ ~ ~ ~ ~ o
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C~ ~n O ~' M ~ M
z w~ ~ z x
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O p. C1, fs. 0.. v 0.~ 0.. 0.
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N N N N ~ N N N
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U 'n'~ ~ ~ ~ o~,~ ~ ~ ~ ~ ~ ~
U ~.~ ~ ~ti ~ti ~ i ~ i ~ i ~ i ~ i d.i
ov o,~. ov-
d C~z A o 0 0 o a o 0 0 0 0 0 0 0 0 0 0
M ~ M N Q1
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A o Cw7 ~ ~ ~ ~ ~4 c~a w
v x x x x
x x x x x x x x
~ z
~ ;
CA 02298852 2000-O1-28
WO 99/06423 PCT/US98/15949
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M ~ ~ ~ ~ ~
_
U '~ M O ~ M ~ ~ N
Ar
4..~A~ .~ Q\ ~ O ~
M N --~ M ~G M N h
_~ ~ ~ ~
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w ~ o ~ ~ ~ .~ .o o M
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d ~ U ~ ~ r~n > ~ a. wi
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~ ~ ~ ~ ~
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122
O 00 M M M V7
Q .~ o _
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O C/~per.,~ N N OM ~ N M N
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o C7 U ~ W W
A o x a a x x
x x x Q
x x x x x
0 00 0\ O
CA 02298852 2000-O1-28
WO 99/06423 PCT/US98/15949
123
!f' ~ M iN ~t ~ M N
~ ..
U
~ N due' ~ N N ~ M
[/~ar
N v1 vD --~ M
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Q ~ : z ~ M N g' r
x Q
A ~ ~ A w
x x x x x
x x x
~ z
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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
ID"
identified in Table 1 and, in some cases, from additional related DNA clones.
The
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
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
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
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
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 ID
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
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
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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.
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 occurnng polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
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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).
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 Sequences
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
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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
uniform, resulting in more than one secreted species. These polypeptides, and
the
polynucleotides encoding such polygeptides, 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
occurnng 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.
Polynucleotide and Polypeptide 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 polynucleotide 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
polypepdde 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
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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
Group Length=0, Cutoff Score=1, 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 arrive 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
*rB
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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
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,
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
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 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=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
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.
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 matchedlaligned 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
arrive 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
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130
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.
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 matchedlaligned 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 S-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. roll).
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 occurring 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
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131
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
deleting 3, 8, or 27 amino-terminal an>ino 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 similar 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 II,-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-ternunus 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 andlor 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 fox protein function. In contrast, the amino acid
positions
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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.
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 Carner Systems 10:307-377 ( 1993).)
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Polynucleotide and Polypeptide Fragments
In the present invention, a "polynucleotide fragment" refers to a short
polynucleotide 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
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
10 SEQ ID 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
15 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, 65I-700, 701-750, 751-800, 800-850, 851-900,
901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, l I51-1200, 1201-1250,
1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600,
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 polypeptide 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
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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-
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.
Similarly, polynucleotide fragments encoding these polypeptide fragments are
also
preferred.
Also preferred are polypeptide and polynucleotide fragments characterized by
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. '
Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are
specifically contemplated by the present invention. Moreover, polynucleotide
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
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
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
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,b31,211.)
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
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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,
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
carrier 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
poIypeptide of the present invention can be used to indirectly detect the
second protein
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
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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 hIL-5.
(See, D. _
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).)
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Thus, any of these above fusions can be engineered using the polynucleotides
or the polypeptides of the present invention.
Vectors. Host Ceils. 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 --
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, pNHlBA, 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
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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 _
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 Polynucleotides
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,
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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 1 S-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
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.
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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
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
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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 ID 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
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.
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Uses of the PolYpeptides
Each of the polypeptides identiEed 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 (I25I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium
(112In), 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,
13II,
1 l2In, 99mTc), a radio-opaque substance, or a material detectable by nuclear
magnetic
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
millicuries 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).)
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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.
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
*rB
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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
hernatopoietic
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
hemogIobinuria.
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., afibrinogenemia, 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 -
decrease hemostatic or thromboiytic activity could be used to inhibit or
dissolve
clotting. These molecules could be important in the treatment of heart attacks
(infarction), strokes, or scarnng.
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 chernotaxis of T-cells, may be an effective therapy in
preventing
autoimmune disorders.
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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 polynucleotide 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
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
IL-1.)
Hyper~roliferative Disorders
A polypeptide 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
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interactions. Alternatively, a polypeptide or polynucleotide of the present
invention
may proliferate other cells which can inhibit the hyperproiiferative 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
polynucleotide 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 Macroglobulinenva, 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
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: Arbovilus, Adenoviridae, Arenaviridae,
Arterivirus,
Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,
Flaviviridae,
Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes
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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,
J O 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,
Salmonella, 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
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.
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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 276:59-87 (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
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
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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
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
present invention.
Chemotaxis
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 polynucleotide 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 attracting immune cells to the injured location. Chemotactic
molecules of the
present invention can also attract fibroblasts, which can be used to treat
wounds.
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
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(antagonist), or decrease activity of the polypeptide or the molecule bound.
Examples
of such molecules include antibodies, oligonucleotides, 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 S (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.
Preferably, an ELISA assay can measure polypeptide level or activity in a
sample (e.g., biological sample) using a monoclonal or polyclonai 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.
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.
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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.
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 >D 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
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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 def ned 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 ID 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 ID 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.
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
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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
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.
*rB
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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 l; 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.
The method for identifying the species, tissue or cell type of a biological
sample
I O 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
15 associated with abnormal structure or expression of a gene encoding a
secreted protein
identified in Table I, which method comprises a step of detecting in a
biological sample
obtained from said subject nucleic acid rnolecuIes, 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
20 of SEQ ID NO:X wherein X is any integer as defined in Table l; 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
25 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 composition of matter comprising isolated nucleic acid
30 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
35 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
nucleic acid molecules can comprise DNA molecules or RNA molecules.
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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 117 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.
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 1 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 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 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 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.
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 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 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 polypeptide 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
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 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 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.
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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 l, 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
ID
NO:Y wherein Y is any integer as defined in Table 1; and a complete anuno 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.
In any of these methods, the step of detecting said polypeptide molecules
includes using an antibody.
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.
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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 l 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.
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.
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Examples
Example 1: Isolation of a Selected cDNA lone From the Deposited
am le
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 Library Corresponding Deposited Plasmid
Lambda Zap pBluescript (pBS)
Uni-Zap XR pBluescript (pBS)
1S 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.
16:7583-7600 (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
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 fl 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
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DH10B, also available from Life Technologies. (See, for instance, Gruber, C.
E., et
al., Focus 15:59 (I993).) 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~2.1, which is available from Invitrogen, 1600 Faraday Avenue,
CarIsbad, CA 92008, contains an ampicillin resistance gene and may be
transformed
into E. coli strain DH l OB, 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 IZ7
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 3zP-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).)
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.
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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 Tag 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.
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
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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.
S
Example 2~ Isolation of Genomic Clones CorrespondinE to a
Polynucleotide
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 Polypentide
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 P;'- 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.
Example 4: Chromosomal Mapping 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
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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.
Examule 5: Bacterial Expression of a PolYpeptide
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 l, 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 (Ampr), 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
MlS/rep4
(Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which
expresses
the IacI repressor and also confers kanamycin resistance (Kanr). Transformants
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 (OIN) 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
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration
of 1 mM.
1PTG 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
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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/HCl pH 7.4, containing protease
inhibitors.
The renaturation should be performed over a period of l .S hours or more.
After
renaturation the proteins are eluted by the addition of 250 mM immidazole.
Immidazole
is removed by a final dialyzing step against PBS or 50 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 pHE4a. (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 TS 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 pI-IEa 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
insert is generated according to the PCR protocol described in Example 1,
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.
Examule 6: Purification of a Poly~eptide from an Inclusion Bodv
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The following alternative method can be used to purify a polypeptide expressed
in E coli 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. coli 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 pm membrane filter with appropriate surface
area
(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,
Perceptive
Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted
with 250 mM, 500 mM, 1~0 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
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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 i0
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
AZgo
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 endotoxinlLPS contamination, and
typically
the LPS content is less than 0.1 ng/ml according to LAL assays.
Examule 7: Cloninsz and Expression of a Polvueptide in a Baculovirus
Expression System
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 Autographa 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
poiyadenylation. 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.
Many other baculovirus vectors can be used in place of the vector above, such
as pAc373, pVL941, and pAcIMI, 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 1,
is amplified using the PCR protocol described in Example 1. If the naturally
occurring
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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 ("GenecIean" 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 ~g of a plasmid containing the polynucleotide is co-transfected with 1.0
pg
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 ~,g of
BaculoGoldTM virus DNA and 5 ~g of the plasnud are mixed in a sterile well of
a
microtiter plate containing 50 ~1 of serum-free Grace's medium {Life
Technologies
Inc., Gaithersburg, MD). Afterwards, 10 ~,l Lipofectin plus 90 ~,1 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
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 Snuth, 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
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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 p.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 ~Ci
of ;SS-
methionine and 5 pCi ~5S-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 Polype~tide in Mammalian ells
The polypeptide of the present invention can be expressed in a mammalian cell.
A typical mammalian expression vector contains a promoter element, which
mediates
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 C 127 cells,
Cos 1,
Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO)
cells.
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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-I43 (1990); Page, M. J.
and
Sydenham, M. A., Biotechnology 9:64-68 ( 1991 ).) 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-I75 (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
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 occurring 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.
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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 p,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 ~tM, 2 p,M, 5 p,M, 10 mM, 20 mM). The same
procedure is repeated until clones are obtained which grow at a concentration
of 100 -
200 pM. Expression of the desired gene product is analyzed, for instance, by
SDS-
PAGE and Western blot or by reversed phase HPLC analysis.
Example 9: Protein Fusions
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-l, 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
and/or 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 polypepdde to an IgG molecule, or the protocol
described in
Example 5.
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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 Barnl-iI
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
occurnng 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:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCC
CAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGT
GGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG
GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCA
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGC
GACGGCCGCGACTCTAGAGGAT (SEQ ID NO:1 )
Example 10: Production of an Antibody from a PolJrpe to 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
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172
containing poIyclonal 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
1 S about 56°C), and supplemented with about 10 gII of nonessential
anuno acids, about
1,000 U/ml of penicillin, and about 100 p,glml 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
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
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.
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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).)
Examule 11: Production Of Secreted Protein For High-Throughput
Screening Asst
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
(lmglml in PBS) 1:20 in PBS (wlo calcium or magnesium 17-516F Biowhittaker)
for a
working solution of SOug/ml. Add 200 ul of this solution to each well (24 well
plates)
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 .Sml
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
(18324-012 GibcoBRL) and Sm1 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
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174
Examples 8 or 9, into an appropriately labeled 96-well round bottom plate.
With a
mufti-channel pipetter, add SOuI 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 200u1 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 mglL of CaCl2 (anhyd); 0.00130 mg/L
CuS04-SHzO; 0.050 mglL of Fe(NO~}~-9H20; 0.417 mg/L of FeSOa-7Hz0; 311.80
mglL of Kcl; 28.64 mg/L of MgClz; 48.84 mg/I. of MgSO,~; 6995.50 mglL of NaCl;
2400.0 mglL of NaHC03; 62.50 mglL of NaH2P04 H,O; 71.02 mg/L of NazHP04;
.4320 mg/L of ZnS04 7H20; .002 rng/L of Arachidoruc 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 mglL of Myristic Acid; 0.010 mg/L of
Oleic
Acid; 0.010 mg/L of PaImitric Acid; 0.010 mg/L of Palmitic Acid; 100 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-
Glucose; 130.85 mg/ml of L- Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50
mg/ml
of L-Asparagine-H20; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/mi of L-Cystine-
2HCL-HZO; 31.29 mg/m1 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/m1 of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H20; 99.65 mg/m1 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-InositoI; 3.02 mg/L
of
Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mglL 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;
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0.105 mg/L of Lipoic Acid; 0.081 mglL 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 Ferric Citrate; 41.70 mglL 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 cornplexed with Retinal} with 2mm glutamine and 1 x
penstrep. (BSA (81-068-3 Bayer) 100gm dissolved in 1L DMEM for a IO% BSA stock
solution). Filter the media and collect 50 ul for endotoxin assay in 15m1
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.SmI appropriate media to each well. Incubate at 37°C for 45 or
72 hours
depending on the media used: I %BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300u1 multichannel pipetter, aliquot 600u1 in one lml
deep
well plate and the remaining supernatant into a 2ml 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.
Example 12: Construction of GAS Reporter Construct
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 ("IS1ZE"), 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. Statl 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.
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The STATs are activated to translocate from the cytoplasm to the nucleus upon
tyrosine phosphorylation 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-
I2, 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 GASlelements) or IS F
Li t~rk2J 1 Ja Jak3
c2
IFN family
IFN-aB + + - - 1,2,3 ISRE
IFN-g + + - 1 GAS (IRF1>Lys6>IFP}
Il-10 + ? ? - 1,3
gp 130 family
IL-6 (Pleiotrohic)+ + + ? 1,3 ~ GAS (IRF 1 >Lys6>IFP)
Il-11(Pleiotrohic)? + ? ? 1,3
OnM(Pleiotrohic)? + + ? 1,3
LIF(Pleiotrohic)? + + ? 1,3
CNTF(Pleiotrohic)-/+ + + ? 1,3
G-CSF(Pleiotrohic}? + ? ? 1,3
IL-12(Pleiotrohic)+ - + + 1,3
g-C family
IL-2 (lymphocytes)- + - + 1,3,5 GAS
IL-4 (lymph/myeloid)- + - + b GAS (IRF1 = IFP Lyb)(IgH)
IL-7 (lymphocytes)- + - + 5 GAS
IL-9 (lymphocytes)- + - + 5 GAS
IL-13 (lymphocyte)- + ? ? 6 GAS
IL-15 ? + ? + 5 GAS
gp 140 family
IL-3 (myeloid) - - + - 5 GAS (IRF1>IFPLyb)
IL-5 (myeloid) - - + - 5 GAS
GM-CSF (myeloid)- - + - 5 GAS
Growth hormone ly
fami
GH ? - + - 5
PRL ? +/- + - 1,3,5
EPO ? - + - 5 GAS(B-CAS>IRF1=IFPLy6)
Receptor Tyrosineases
Kin
EGF ? + + - 1, 3 GAS (IRF 1 )
PDGF ? + + - 1,3
CSF-1 ? + + - 1,3 GAS (not IRF1) -
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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-S V40 promoter sequence. The 5' primer contains four tandem
copies
of the GAS binding site found in the IRF I promoter and previously
demonstrated to
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' :GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCG
AAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3' (SEQ ID N0:3)
The downstream primer is complementary to 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 B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment
is
digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.)
Sequencing
with forward and reverse primers confirms that the insert contains the
following
sequence:
5' :~GAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATG
ATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCC
CTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGC
CCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGC
CTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTITTTGGAGGCCTAGGCTTT
TGCAAAAAGCTT: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, II-
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. T1B-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 I 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 50 ul
of DMRIE-C and incubate at room temperature for 15-45 mins.
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 RPMI
+ 15% serum.
The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI + 10%
serum, 1 mglml 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 RPMI + 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 I O 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 I 1 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, 1(>fl Unit/ml interferon gamma can be used which is
known tv activate Jurkat T cells. Over 30 fold induction is typically observed
in the
positive control wells.
Example 14: High-Throughput Screening Assay Identifying Myeloid
Ac i '
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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 KG 1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced
in Example 12, a DEAF-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/mI 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/ml 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 mI 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 Asst Identifying l~euronal
'v't .
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 EGRI is responsible for such induction. Using the
EGRI
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 12 cells can be assessed.
The EGR/SEAP reporter construct can be assembled by the following protocol.
The EGR-1 promoter sequence (-633 to +I)(Sakamoto K et al., Oncogene 6:867-871
( 1991 )) can be PCR amplified from human genomic DNA using the following
primers:
5' GCGCTCGAGGGATGACAGCGATAGAACCCCGG -3' (SEQ ID N0:6)
5' 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 XhoI/HindIII, removing the GAS/SV40 stuffer.
Restrict the
EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1
promoter.
To prepare 96 well-plates far 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), 5% 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 PC12 using the Lipofectamine
protocol described in Example 11. EGR-SEAP/PC I 2 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
Sx 105
cells/ml.
Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to
1 x 105 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.
Examule 16: High-Throughput Screening Assav for T-cell Activity
NF-oB (Nuclear Factor xB) is a transcription factor activated by a wide
variety
of agents including the inflammatory cytokines IL-l 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-
~cB 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 phosphoryiated and
degraded,
causing NF- xB to shuttle to the nucleus, thereby activating transcription of
target
genes. Target genes activated by NF- xB include IL-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-1cB 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-oB could be used to treat those
diseases
related to the acute or chronic activation of NF-kB, such as rheumatoid
arthritis.
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To construct a vector containing the NF-xB promoter element, a PCR based
strategy is employed. The upstream primer contains four tandem copies of the
NF-xB
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:
5':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 Xhol 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
AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTC
CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:
3' (SEQ ID NO:10)
Next, replace the SV40 minimal promoter element present in the pSEAP2-
promoter plasmid (Clontech) with this NF-1cB/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-xB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the
GFP
gene, after restricting pGFP-1 with SaII and NotI.
Once NF-xB/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: Assay for SEAP Activitx
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 2.5x Dilution Buffer and dispense 15 p,l of 2.Sx
dilution buffer into Optiplates containing 35 p.l 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 p,l Assay Buffer and incubate at room
temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see
the
table below). Add 50 ~t.l 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 I2 as blank, and print
the
results. An increase in chenuluminescence indicates reporter activity.
Reaction Buffer Formulation:
_# of-platesRxn buffer diluent CSPD (ml)
(ml)
10 60 3
11 65 3.25
12 70 3.5
13 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
21 115 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 9
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 I 2.5
49 255 12.75
50 260 13
Example 18: High-Throughput 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 cellslweh 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 u1 of buffer after the final wash.
A stock solution of 1 mg/ml fluo-3 is made in 10°lo 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/mI
S fluo-3 solution in 10% pluronic 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'~"~'
concentration.
~xamnle 19: High-Throughput Screening Assay Identifying 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 dimerization,
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 axe sterilized with two 30 minute
rinses with
I00% 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 mglml), gelatin (2%) or
polylysine
(50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO)
or
IO 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
a.lamarBlue as described by the manufacturer Alamar Biosciences, Inc.
(Sacramento,
CA) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford,MA)
are
I S 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
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
25 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 S minutes, is
removed and
30 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
35 biotinylated peptide). Biotinylated peptides that can be used for this
purpose include
PSKI (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34)
and
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PSKZ (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 l0ul of SuM Biotinylated Peptide, then lOul ATP/Mg2+ (SnnM
ATP/SOmM MgCl2), then 10u1 of Sx Assay Buffer (40mM imidazole hydrochloride,
pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100mM MgCI2, 5 mN1 MnCl2~
0.5 mg/ml BSA), then Sul of Sodium Vanadate{1mM), and then Sul 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 transfernng 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(O.Su/rnl)) 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 mins (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-Throughout Screening Assay Identifying
Phosphorylation Activity
As a potential alternative andlor 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-1 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.
*rB
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Specifically, assay plates are made by coating the wells of a 96-well ELISA
plate with 0.1 ml of protein G ( 1 ug/ml) for 2 hr at room temp, (RT). The
plates are then
rinsed with PBS and blocked with 3% BSA/PBS for I 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 fllterplate 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 nunutes. 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 ( IOng/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
Gorres on nding_ 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
polynucleotide 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
poiymerase (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 to 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 B iol. 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.
Example 22: Method of Detecting Abnormal Levels of a Polypeptide 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
polypeptide 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.
Example 23: Formulating a Polype tp ide
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
~,g/kg/day -
to IO mglkg/day of patient body weight, although, as noted above, this will be
subject
to therapeutic discretion. More preferably, this dose is at least 0.0I
mg/kglday, 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 ~g/kglhour to about 50 Pg/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
transdenmal
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
for 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.
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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 polygeptide is typically formulated in such vehicles at a
concentration of about 0.1 mg/ml to 100 mglml, 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.
Example 24~ Method of Treating Decreased Levels of the Poly~eptide
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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 polypeptide receives a daily
dose 0.1-100 uglkg 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 Polypeptide
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 Fl2 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 (I988)), 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 HindIII 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+aml2 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.
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
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disclosures) in the Background of the Invention, Detailed Description, and
Examples is
hereby incorporated herein by reference. Further, the hard copy of the
sequence listing
submitted herewith and the corresponding computer readable form are both
incorporated herein by reference in their entireties.
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rute l3bisJ
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. .__~.___~~ ~ ~ Authorircdolticcr
1T
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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 136is)
A. The indications made below relate
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B. IDENTIFICATION OF DEPOSIT Further
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sheet
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American Type Culture Collection
Address of depositary institution
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10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date of deposit 3uly 17, 1997 Accession Number 209145
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nature oJrhe indications, e.g..
'Accession
Number of Depostt'~
For receiving Office use only ~ ~ For International Bureau use only
This sheet was roceived with the international application ~ ~ ~ This sheet
was received by the International Bureau on:
....
Authorized officer ~~ ~ ~ Authorized officer
CA 02298852 2000-O1-28
WO 99/Ob423 PCT/US98115949
200
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 113 , line N/A
B. IDENTIFICATION OF DEPOSIT Further
deposits arc 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
Manassas, Virginia 20110-2209
United States of America
Date of deposit July 17, 1997 Accession Number 209148
C. ADDITIONAL INDICATIONS (leave
blank fnor applicable) This information
is continued on an additional
sheet
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (ijthe indieatios
are not jot all designated States)
E. SEPARATE FURNISHING OF INDICATIONS
(leave blank ijnot applicable)
The indications listed below will
be submitted to the International
Bureau later (sped the general
nature ojthe indications, e.g..
'Accession
Number ojDepostt'~
For receiving Office use only ~ ~~ For International Bureau use only
This sheet was received with the international application ~ ~ ~ This sheet
was received by the International Bureau on:
officerAuthorized officer
7~0~,906~1~1T
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTS PART1E DE CETTE DEMANDS OU CE BREVET
COMPREND PLUS D'UN TOME.
CECt EST LE TOME / DE
NOTE: Pour les tomes additionels, veuiilez contacter !e Bureau canadien des
brevets
JUMBO APPL~CAT10NS/PATE1VTS
THIS SECTION OF THE APPL1CAT10N/PATENT CONTAINS MORE
THAN ONE VOLUME
THtS tS VOLUME OF
NOTE. For additional voiumes-pi~ase contact the Canadian Patent Office .
