Note: Descriptions are shown in the official language in which they were submitted.
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20 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 ali other types of
proteins. Once localized to the ER, both groups of proteins can be further
directed to
another organelle called the Golgi apparatus. Here, the Golgi distributes the
proteins to
vesicles, including secretory vesicles, the cell membrane, lysosomes, and the
other
organelles.
Proteins targeted to the ER by a signal sequence can be released into the
extracellular space as a secreted protein. For example, vesicles containing
secreted
proteins can fuse with the cell membrane and release their contents into the
extracellular
space - a process called exocytosis. Exocytosis can occur constitutively or
after receipt
of a triggering signal. In the latter case, the proteins are stored in
secretory vesicles (or
secretory granules) until exocytosis is triggered. Similarly, proteins
residing on the cell
membrane can also be secreted into the extracellular space by proteolytic
cleavage of a
"linker" holding the protein to the membrane.
Despite the great progress made in recent years, only a small number of genes
encoding human secreted proteins have been identified. These secreted proteins
include
the commercially valuable human insulin, interferon, Factor VIII, human growth
hormone, tissue plasminogen activator, and erythropoeitin. Thus, in light of
the
pervasive role of secreted proteins in human physiology, a need exists for
identifying
and characterizing novel human secreted proteins and the genes that encode
them. This
knowledge will allow one to detect, to treat, and to prevent medical disorders
by using
secreted proteins or the genes that encode them.
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Summary of the Invention
The present invention relates to novel polynucleotides and the encoded
polypeptides. Moreover, the present invention relates to vectors, host cells,
antibodies,
and recombinant methods for producing the polypeptides and polynucleotides.
Also
provided are diagnostic methods for detecting disorders related to the
polypeptides, and
therapeutic methods for treating such disorders. The invention further relates
to
screening methods for identifying binding partners of the polypeptides.
to Detailed Description
Definitions
The following definitions are provided to facilitate understanding of certain
terms used throughout this specification.
In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is
altered "by the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of matter, or could
be
contained within a cell, and still be "isolated" because that vector,
composition of
matter, or particular cell is not the original environment of the
polynucleotide.
In the present invention, a "secreted" protein refers to those proteins
capable of
being directed to the ER, secretory vesicles, or the extracellular space as a
result of a
signal sequence, as well as those proteins released into the extracellular
space without
necessarily containing a signal sequence. If the secreted protein is released
into the
extracellular space, the secreted protein can undergo extracellular processing
to produce
a "mature" protein. Release into the extracellular space can occur by many
mechanisms, including exocytosis and proteolytic cleavage.
As used herein , a "polynucleotide" refers to a molecule having a nucleic acid
sequence contained in SEQ 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" refers to a molecule having the
translated
amino acid sequence generated from the polynucleotide as broadly defined.
In the present invention, the full length sequence identified as SEQ ID NO:X
was often generated by overlapping sequences contained in multiple clones
{contig
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3
analysis). A representative clone containing all or most of the sequence for
SEQ 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,
S 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, Sx SSC (750 mM NaCI, 75 mM sodium
citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran
sulfate, and 20 ~glml denatured, sheared salmon sperm DNA, followed by washing
the
filters in O.lx SSC at about 65°C.
Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides of the present invention at lower stringency hybridization
conditions.
Changes in the stringency of hybridization and signal detection are primarily
accomplished through the manipulation of formamide concentration (lower
percentages
of formamide result in lowered stringency); salt conditions, or temperature.
For
example, lower stringency conditions include an overnight incubation at
37°C in a
solution comprising 6X SSPE (20X SSPE = 3M NaCI; 0.2M NaHzP04; 0.02M EDTA,
pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;
followed by washes at 50°C with 1XSSPE, 0.1% SDS. In addition, to
achieve even
lower stringency, washes performed following stringent hybridization can be
done at
higher salt concentrations (e.g. SX SSC).
Note that variations in the above conditions may be accomplished through the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in hybridization experiments. Typical blocking reagents include
Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and
commercially available proprietary formulations. The inclusion of specific
blocking
reagents may require modification of the hybridization conditions described
above, due
to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such
as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or
to a
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4
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 processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation,
covalent
attachment of flavin, covalent attachment of a heme moiety, covalent
attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid
derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine,
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formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI
anchor formation, hydroxylation, iodination, methylation, myristoylation,
oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins
5 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 most
preferably, not more than about three-fold less activity relative to the
polypeptide of the
present invention.)
Po~nacleotides and Polypeptides of the Invention
FEATURES OF PROTEIN ENCODED BY GENE NO: 1
This gene is expressed primarily in brain and CD34 positive 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, central nervous system (CNS) and immune-system 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 CNS and
inunune
system, expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues and cell types (e.g., brain and other tissue of
the nervous
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6
system, blood cells, and cells and tissue of the immune system, and cancerous
and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid
or 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 N0:31 as residues: Asp-44 to Gly-
49,
Val-84 to Lys-90.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosing and treating CNS and
immune
system diseases.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
The translation product of this gene shares sequence homology with LIM
domain proteins which are thought to be important in regulating cellular
functions such
as cell proliferation and differentiation. In particular, it is believed that
this gene encodes
the human ortholog of mouse testin. See, for example, Gene 156{2):283-286 (
1995),
which is incorporated herein by reference. LIM proteins are described in Proc.
Natl.
Acad. Sci. U.S.A. 90:4404-4408{ 1993}, which is incorporated herein by
reference.
Based on the sequence similarity to other members of the LIM family,
polypeptides
encoded by this gene are expected to share certain biological activities with
other LIM
polypeptides, in particular mouse Testin. Preferred polypeptides encoded by
this gene
comprise the following amino acid sequence (LIM domain):
CAGCDELIFSNEYTQAENQNWHLKHFCCFDCDSIL (SEQ ID NO:S 1 ). Especially
preferred polypeptides encoded by this gene comprise the following amino acid
sequence: ARGFVCSTCHELLVDMIYFWKNEKLYCGRHYCD
SEKPRCAGCDELIFSNEYTQAENQNWHLKHFCCFDCDSILAGEIYVMVNDKPV
CKPCYVKNHAVVCQGCHNAIDPEVQRVTYNNFSWHASTECFLCSCCSKCLIG
QKFMPVEGMVFCSVECKKRMS (SEQ ID N0:52). Polynucleotides encoding these
polypeptides are also encompassed by the invention.
This gene is expressed primarily in testis and to a lesser extent in Hodgkin's
lymphoma, T cell and adrenal gland tumor.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune diseases, reproductive disorders and cancers.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
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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
immune systems, expression of this gene at significantly higher or lower
levels may be
routinely detected in certain tissues and cell types (e.g., testis and other
reproductive
S tissue, lymphoid tissue, tissue and cells of the immune system, blood cells,
and adrenal
gland, and cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell sample taken
from an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder.
The tissue distribution and homology to LIM proteins indicates that
polynucleotides and polypeptides corresponding to this gene are useful for
treating
diseases of the immune system and male reproductive system.
FEATURES OF PROTEIN ENCODED BY GENE NO: 3
This gene is expressed primarily in infant brain, prostate, embryo and to a
lesser
extent in parathyroid, adrenal gland tumor, thymus.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, immune system and central nervous system (CNS) 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
and CNS, expression of this gene at significantly higher or lower levels may
be
routinely detected in certain tissues and cell types (e.g., brain and other
tissue of the
nervous system, tissue and cells of the immune system, differentiating tissue,
parathyroid, adrenal gland, and thymus, and cancerous and wounded tissues) or
bodily
fluids (e.g., serum, plasma, urine, synovial fluid or 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 treating and diagnosis of immune
system and
CNS diseases.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 4
This gene maps to chromosome 4 (Chr.4, D4S395-D4S414) and therefore
polynucleotides of the present invention can be used in linkage analysis as a
marker for
chromosome 4.
This gene is expressed primarily in infant brain, embryo, parathyroid tumor
and
melanocyte and to a lesser extent in testis, chondrosarcoma, epididyma,
placenta,
endothelia'1 cells and many other cell types, tissues and organs.
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 nervous system, developmental related defects
and
abnormalities. Similarly, polypeptides and antibodies directed to these
polypeptides are
useful in providing immunological probes for differential identification of
the tissues)
or cell type(s). For a number of disorders of the above tissues or cells,
particularly of
the nervous system and immune system, expression of this gene at significantly
higher
or lower levels may be routinely detected in certain tissues or cell types
(e.g., brain and
other tissue of the nervous system, embryonic or differentiating cells or
tissue,
parathyroid, melanocytes, chondrocytes, testis and other reproductive tissue,
placenta,
and endothelial cells, and cancerous and wounded tissues) or bodily fluids
(e.g.,
serum, plasma, urine, synovial fluid or 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 N0:34 as residues: Glu-28 to Thr-35.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of disorders
of the
nervous system, such as congenital malformations, degenerative diseases,
trauma,
inflammatory diseases, neoplasia, metabolic disorders, and immune diseases,
particularly with T-cell involvement. The abundant expression in the
parathyroid tumor
indicates that protein products of this gene are useful in modulating calcium
metabolism.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5
The translation product of this gene shares sequence homology with ancient
ubiquitous 46 kDa protein AUP46 precursor [Mus musculus] which is thought to
be
important in tissue and organ development.
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This gene is expressed primarily in testes and placenta and to a lesser extent
in
fetal liver, brain, and 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, liver diseases and immunological 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 digestive
system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues and cell types (e.g., testes and other reproductive tissue,
placenta,
liver, brain and other tissue of the nervous system, and T-cells and other
blood cells,
and cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,
urine,
synovial fluid or 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
N0:35 as residues: Pro-4 to Pro-9, Asp-14 to Gly-20, Arg-78 to His-87, Glu-161
to
Gly-I70, Leu-252 to Arg-258, Lys-269 to Pro-293, Asp-344 to Thr-349, Ser-379
to
Gln-391, Arg-399 to Asp-410.
The tissue distribution and homology to AUP46 indicates that polynucleotides
and polypeptides corresponding to this gene are useful for diagnosis and
treatment of
diseases in testes, placenta, liver, brain and activated T-cells, particularly
diseases
related to development of the organs associated with the foregoing tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
The translation product of this gene shares sequence homology with ATP7
region hypothetical protein which is thought to be important in development.
This gene
maps to chromosome 17 (D17S849-D17S796) and therefore polynucleotides of the
present invention can be used in linkage analysis as a marker for chromosome
17.
This gene is expressed primarily in breast, brain and liver and to a lesser
extent
in prostate and thymus.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, breast cancer, brain tumor and liver cancer. Similarly,
polypeptides and
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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 nerve system,
expression
of this gene at significantly higher or lower levels may be routinely detected
in certain
5 tissues (e.g., developing tissue, mammary tissue, brain and other tissue of
the nervous
system, liver, prostate, and thymus, and cancerous and wounded tissues) or
bodily
fluids (e.g., serum, plasma, urine, synovial fluid or 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
10 individual not having the disorder.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
This gene is expressed primarily in liver, spleen, bone marrow and to a lesser
extent in 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, liver, spleen 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 digestive systems, expression
of this
gene at significantly higher or lower levels may be routinely detected in
certain tissues
(e.g., liver, spleen, bone marrow, cells and tissue of the immune system, and
amygdala
and cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma,
urine,
synovial fluid or 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 8
Preferred mature polypeptides encoded by this gene comprise the following
amino acid sequence:
GVARGHRDRGQASRRWLQEGGQECECKDWFLRAPRRKFMTVSGL
PKKQCPCDHFKGNVKKTRHQRHHRKPNKHSRACQQFLKQCQLRSFALPL
(SEQ ID N0:53).
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This gene is expressed primarily in lung and to a lesser extent in pancreatic
carcinoma and gall bladder.
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, lung diseases and pancreatic carcinoma. 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, pulmonary and
digestive
systems, expression of this gene at significantly higher or lower levels may
be routinely
detected in certain tissues (e.g., lung, pancreas, and gall bladder, and
cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid
or 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 N0:38 as residues: Gly-31 to Gln-
37.
FEATURES OF PROTEIN ENCODED BY GENE NO: 9
This gene is expressed primarily in rhabdomyosarcoma and pituitary, and, to a
lesser extent, in fetal lung and keratinocytes.
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, rhabdomyosarcoma and disorders of the endocrine system or
other
endocrinopathies, including, but not limited to, endocrine polyglandular
syndrome,
endocrinoma, endocrine ophthalmopathy, and any of the great number of disease
states
and disorders which are caused by or relate to the abnormal secretion of
factors
originating in the endocrine gland. 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 disorders of
the above
tissues or cells, particularly of the immune and musculoskeletal system,
expression of
this gene at significantly higher or lower levels may be routinely detected in
certain
tissues and cell types (e.g., skeletal muscle, pituitary, endocrine glands,
lung and
keratinocytes, and cancerous and wounded tissues) or bodily fluids (e.g.,
serum,
plasma, urine, synovial fluid or 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.,
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12
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
N0:39 as residues: Pro-34 to Phe-40.
FEATURES OF PROTEIN ENCODED BY GENE NO: 10
This gene is expressed primarily in endometrial tumor, osteoblasts, and smooth
muscle, and, to a lesser extent, in osteoclastoma, heart, and lung.
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, endometrial tumor, osteoclastoma, and other bone remodeling
disorders,
and heart and lung 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 bone systems, expression of this gene at
significantly
higher or lower levels may be routinely detected in certain tissues (e.g.,
endometrium,
bone, heart and other cardiovascular tissue, lung and other pulmonary tissue,
and
cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial
fluid or 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 N0:40 as
residues:
Thr-33 to Arg-40.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11
This gene is expressed primarily in meniingima and dermatofibrosarcoma.
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, fibrotic and neoplastic conditions of skin, connective tissue
and other
mesenchymal organs. 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,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues (e.g., meninx, liver, skin, and vascular tissue, and
cancerous and
wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid
or spinal
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I3
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 N0:41 as residues: Gln-50 to Met-
56.
Tissue distribution of this gene indicates that it may be useful in the study,
- treatment, and diagnosis of fibrotic disorders and neoplasms of skin, liver,
and other
tissues.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
This gene is expressed primarily in placenta, and colon cancer, and to a
lesser
extent in adult lung and brain frontal cortex.
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, 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 and neural system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues (e.g.,
placenta, colon, cells and tissue of the immune system, lung and other
pulmonary
tissue, brain and other tissue of the nervous system, and cancerous and
wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
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 N0:42 as residues: Met-1 to His-9, Arg-
31 to
Gly-38, Gly-102 to Trp-108.
The tissue distribution indicates that the protein product of this gene is
useful for
the treatment of neoplasia.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
This gene is expressed primarily in thymus, bone marrow, T-cells,
macrophages, and, to a lesser extent, in breast and testes.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
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14
not limited to, cancer, autoimmune diseases, bone 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 and
cell types (e.g., thymus, bone, T-cells and other blood cells, mammary tissue,
and
testes and other reproductive tissue, and cancerous and wounded tissues) or
bodily
fluids (e.g., serum, plasma, urine, synovial fluid or 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 N0:43 as residues: Pro-36 to Trp-42, Arg-48 to Trp-
56,
Ser-58 to Ser-67.
FEATURES OF PROTEIN ENCODED BY GENE NO: 14
This gene is expressed primarily in breast cancer, pituitary, and activated T-
cells, and, to a lesser extent, in frontal cortex and breast.
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, breast cancer, growth, and immune disorders. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the immune and neural
system,
expression of this gene at significantly higher or lower levels may be
routinely detected
in certain tissues and cell types (e.g., mammary tissue, pituitary, T-cells
and other
blood cells, cells and tissue of the immune system, brain and other tissue of
the nervous
system, and cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma,
urine, synovial fluid or spinal fluid) or another tissue or cell sample taken
from an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder.
The tissue distribution indicates that the protein product of this gene is
useful for
diagnosis or treatment of breast cancer and growth disorders.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 15
This gene is primarily expressed exclusively 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
5 biological sample and for diagnosis of diseases and conditions which
include, but are
not limited to, lymphoma and bacterial infection. Similarly, polypeptides and
antibodies
directed to these polypeptides are useful in providing immunological probes
for
differential identification of the tissues) or cell type(s). For a number of
disorders of
the above tissues or cells, particularly of the immune system, and more
particularly, in
10 neutrophils, expression of this gene at significantly higher or lower
levels may be
routinely detected in certain tissues and cell types (e.g., lymphoid tissue,
hematopoietic
tissue, neutrophils and other blood cells, and cancerous and wounded tissues)
or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) or another
tissue or
cell sample taken from an individual having such a disorder, relative to the
standard
15 gene expression level, i.e., the expression level in healthy tissue or
bodily fluid from an
individual not having the disorder.
The tissue distribution indicates that the protein product of this gene is
useful for
treatment of lymphomas and various hematopoietic disorders, as well as in the
diagnosis and treatment of bacterial infections, sepsis, and in disorders
which
particularly involve neutrophils.
FEATURES OF PROTEIN ENCODED BY GENE NO: 16
In specific embodiments, polypeptides of the invention comprise the sequence:
HTQVEFIPRMQC (SEQ ID N0:54), LKIRKPINVIYHINRL {SEQ ID N0:55),
RKMGIERNFHQSGKGI (SEQ ID N0:56), KVPTANIILNGERLNAFPIRT (SEQ ID
N0:57), MYFLSSLLIHEHVISVIFSIL (SEQ ID N0:60), and/or
IFSSVLHSFQYTNPV PFFFRFTPSTLFF (SEQ ID N0:58). Polynucleotides
encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in neutrophils only.
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, lymphomas. 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
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16
lower levels may be routinely detected in certain tissues (e.g., lymphoid
tissue,
hematopoietic tissue, neutrophils and other blood cells, and cancerous and
wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative to
the standard gene expression level, i.e., the expression level in healthy
tissue or bodily
fluid from an individual not having the disorder.
The tissue distribution indicates that the protein product of this gene is
useful for
treatment of lymphomas and a variety of hematopoietic disorders.
FEATURES OF PROTEIN ENCODED BY GENE NO: 17
This gene maps to chromosome 19 and therefore polynucleotides of the present
invention can be used in linkage analysis as a marker for chromosome 19.
This gene is expressed primarily in prostate cancer, adult lung and adult
pulmonary and to a lesser extent in prostate and adrenal gland tumor.
Therefore, polynucleotides and polypeptides of the invention are useful as
reagents for differential identification of the tissues) or cell types)
present in a
biological sample and for diagnosis of diseases and conditions which include,
but are
not limited to, prostate cancer and endocrine disorders. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological probes
for differential identification of the tissues) or cell type(s). For a number
of disorders
of the above tissues or cells, particularly of the endocrine system,
expression of this
gene at significantly higher or lower levels may be routinely detected in
certain tissues
(e.g., prostate, lung and other pulmonary tissue, endocrine tissue, adrenal
gland,
cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial
fluid or 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 N0:47 as
residues:
Gly-2 to Gly-9, Thr-39 to Arg-47.
The tissue distribution indicates that the protein product of this gene is
useful for
diagnosis or treatment of prostate cancer, prostate disorders and endocrine
disorders.
FEATURES OF PROTEIN ENCODED BY GENE NO: 18
This sequence shares high degree homology with the UFO oncoprotein. This
protein is a tyrosine kinase receptor. While the functions of this UFO
receptor are
unknown, it is known that the receptor plays a role in tumorigenesis.
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17
This gene is expressed primarily in L8 cell line.
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, 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., cancerous and
wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
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 N0:48 as residues: Ala-37 to Ser-49.
The tissue distribution and homology with the UFO oncoprotein indicates that
the protein product of this gene is useful for the treatment of cancer.
FEATURES OF PROTEIN ENCODED BY GENE NO: 19
This gene is expressed primarily in L8 cell line.
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, cancer and the 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 immune system), expression of
this gene at
significantly higher or lower levels may be routinely detected in certain
tissues (e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial
fluid or spinal fluid) or another tissue or cell sample taken from an
individual having
such a disorder, relative to the standard gene expression level, i.e., the
expression level
in healthy tissue or bodily fluid from an individual not having the disorder.
The tissue distribution indicates that the protein product of this gene is
useful for
the treatment of cancer.
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18
FEATURES OF PROTEIN ENCODED BY GENE NO: 20
In specific embodiments, polypeptides of the invention comprise the sequence
EDGSAPREGETSAPRLPEVVRITSAGIC (SEQ ID N0:61),. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in A 1 and A 14 cell lines.
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, 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., cancerous and
wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or
spinal fluid) or
another tissue or cell sample taken from an individual having such a disorder,
relative to
the standard gene expression level, i.e., the expression level in healthy
tissue or bodily
fluid from an individual not having the disorder.
The tissue distribution indicates that the protein product of this gene is
useful for
the diagnosis and treatment of cancer.
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19
N ~ h- i N
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CA 02286303 1999-10-12
WO 98145712 PCT/US98/06801
O ~ M ~O ~ ~ ~ O
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CA 02286303 1999-10-12
WO 98/45712 PCTIUS98/06801
21
~~ o~ ~ ~ ~ '
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CA 02286303 1999-10-12
WO 98/45712 PCTlUS98/06801
22
oo v o
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23
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
S 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
methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y
may
be used to generate antibodies which bind specifically to the secreted
proteins encoded
by the cDNA clones identified in Table 1.
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24
Nevertheless, DNA sequences generated by sequencing reactions can contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or
deletions of nucleotides in the generated DNA sequence. The erroneously
inserted or
deleted nucleotides cause frame shifts in the reading frames of the predicted
amino acid
sequence. In these cases, the predicted amino acid sequence diverges from the
actual
amino acid sequence, even though the generated DNA sequence may be greater
than
99.9% identical to the actual DNA sequence (for example, one base insertion or
deletion
in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide
sequence or the amino acid sequence, the present invention provides not only
the
generated nucleotide sequence identified as SEQ ID NO:X and the predicted
translated
amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid
DNA
containing a human cDNA of the invention deposited with the ATCC, as set forth
in
Table 1. The nucleotide sequence of each deposited clone can readily be
determined by
sequencing the deposited clone in accordance with known methods. The predicted
.
amino acid sequence can then be verified from such deposits. Moreover, the
amino
acid sequence of the protein encoded by a particular clone can also be
directly
determined by peptide sequencing or by expressing the protein in a suitable
host cell
containing the deposited human cDNA, collecting the protein, and determining
its
sequence.
The present invention also relates to the genes corresponding to SEQ ID NO:X,
SEQ ID NO:Y, or the deposited clone. The corresponding gene can be isolated in
accordance with known methods using the sequence information disclosed herein.
Such methods include preparing probes or primers from the disclosed sequence
and
identifying or amplifying the corresponding gene from appropriate sources of
genomic
material.
Also provided in the present invention are species homologs. Species
homologs may be isolated and identified by making suitable probes or primers
from the
sequences provided herein and screening a suitable nucleic acid source for the
desired
homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood in the art.
The polypeptides may be in the form of the secreted protein, including the
mature farm, or may be a part of a larger protein, such as a fusion protein
(see below).
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It is often advantageous to include an additional amino acid sequence which
contains
secretory or leader sequences, pro-sequences, sequences which aid in
purification ,
such as multiple histidine residues, or an additional sequence for stability
during
recombinant production.
5 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
10 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
15 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
20 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
25 was analyzed by a computer program called SignalP (Henrik Nielsen et al.,
Protein
Engineering 10:1-6 (1997)), which predicts the cellular location of a protein
based on
the amino acid sequence. As part of this computational prediction of
localization, the
methods of McGeoch and von Heinje are incorporated. The analysis of the amino
acid
sequences of the secreted proteins described herein by this program provided
the results
shown in Table 1.
As one of ordinary skill would appreciate, however, cleavage sites sometimes
vary from organism to organism and cannot be predicted with absolute
certainty.
Accordingly, the present invention provides secreted polypeptides having a
sequence
shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues
(i.e., +
or - 5 residues) of the predicted cleavage point. Similarly, it is also
recognized that in
some cases, cleavage of the signal sequence from a secreted protein is not
entirely
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26
uniform, resulting in more than one secreted species. These polypeptides, and
the
polynucleotides encoding such polypeptides, are contemplated by the present
invention.
Moreover, the signal sequence identified by the above analysis may not
necessarily predict the naturally occurring signal sequence. For example, the
naturally
occurring signal sequence rnay 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 Polyne~~tide 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
polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleotide
sequence of the presence invention can be determined conventionally using
known
computer programs. A preferred method for determing the best overall match
between
a query sequence (a sequence of the present invention) and a subject sequence,
also
referred to as a global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci.
( 1990)
6:237-245). In a sequence alignment the query and subject sequences are both
DNA
sequences. An RNA sequence can be compared by converting U's to T's. The
result
of said global sequence alignment is in percent identity. Preferred parameters
used in a
FASTDB alignment of DNA sequences to calculate percent identiy are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization
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27
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
S 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 I O% is subtracted from the percent identity score calculated by
the
FASTDB program. If the remaining 90 bases were perfectly matched the final
percent
identity would be 90%. In another example, a 90 base subject sequence is
compared
with a 100 base query sequence. This time the deletions are internal deletions
so that
there are no bases on the 5' or 3' of the subject sequence which are not
matched/aligned
with the query. In this case the percent identity calculated by FASTDB is not
manually
corrected. Once again, only bases 5' and 3' of the subject sequence which are
not
matched/aligned with the query sequnce are manually corrected for. No other
manual
corrections are to made for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that
the amino acid sequence of the subject polypeptide is identical to the query
sequence
except that the subject polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the query amino acid sequence. In
other words,
to obtain a polypeptide having an amino acid sequence at least 95 % identical
to a query
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28
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. Prefers
ed parameters
used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch
Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window
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 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 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
considered for the purposes of manually adjusting the percent identity score.
That is,
only query residue positions outside the farthest N- and C-terminal residues
of the
subject sequence.
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For example, a 90 amino acid residue subject sequence is aligned with a 100
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subject sequence and therefore, the FASTDB alignment does not
show
a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired
residues represent 10% of the sequence (number of residues at the N- and C-
termini
not matched/total number of residues in the query sequence) so 10% is
subtracted from
the percent identity score calculated by the FASTDB program. If the remaining
90
residues were perfectly matched the final percent identity would be 90%. In
another
example, a 90 residue subject sequence is compared with a 100 residue query
sequence.
This time the deletions are internal deletions so there are no residues at the
N- or C-
termini of the subject sequence which are not matched/aligned with the query.
In this
case the percent identity calculated by FASTDB is not manually corrected. Once
again,
only residue positions outside the N- and C-terminal ends of the subject
sequence, as
displayed in the FASTDB alignment, which are not matched/aligned with the
query
sequnce are manually corrected for. No other manual corrections are to made
for the
purposes of the present invention.
The variants may contain alterations in the coding regions, non-coding
regions,
or both. Especially preferred are polynucleotide variants containing
alterations which
produce silent substitutions, additions, or deletions, but do not alter the
properties or
activities of the encoded polypeptide. Nucleotide variants produced by silent
substitutions due to the degeneracy of the genetic code are preferred.
Moreover,
variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or
added in any
combination are also preferred. Polynucleotide variants can be produced for a
variety
of reasons, e.g., to optimize codon expression for a particular host (change
codons in
the human mRNA to those preferred by a bacterial host such as E. coli).
Naturally occurring 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
deleted from the N-terminus or C-terminus of the secreted protein without
substantial
loss of biological function. The authors of Ron et al., J. Biol. Chem. 268:
2984-2988
(1993), reported variant KGF proteins having heparin binding activity even
after
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deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon
gamma
exhibited up to ten times higher activity after deleting 8-10 amino acid
residues from the
carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (
1988).)
Moreover, ample evidence demonstrates that variants often retain a biological
5 activity similar to that of the naturally occurring protein. For example,
Gayle and
coworkers {J. Biol. Chem 268:22105-221 I 1 ( 1993)) conducted extensive
mutational
analysis of human cytokine IL-la. They used random mutagenesis to generate
over
3,500 individual IL-la mutants that averaged 2.5 amino acid changes per
variant over
the entire length of the molecule. Multiple mutations were examined at every
possible
10 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.
15 Furthermore, even if deleting one or more amino acids from the N-terminus
or
C-terminus of a polypeptide results in modification or loss of one or more
biological
functions, other biological activities may still be retained. For example, the
ability of a
deletion variant to induce and/or to bind antibodies which recognize the
secreted form
will likely be retained when less than the majority of the residues of the
secreted form
20 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
25 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
30 strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by
natural
selection during the process of evolution. By comparing amino acid sequences
in
different species, conserved amino acids can be identified. These conserved
amino
acids are likely important for protein function. In contrast, the amino acid
positions
where substitutions have been tolerated by natural selection indicates that
these
positions are not critical for protein function. Thus, positions tolerating
amino acid
substitution could be modified while still maintaining biological activity of
the protein.
T... ........._~. .~_._........
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The second strategy uses genetic engineering to introduce amino acid changes
at
specific positions of a cloned gene to identify regions critical for protein
function. For
example, site directed mutagenesis or alanine-scanning mutagenesis
(introduction of
single alanine mutations at every residue in the molecule} can be used.
(Cunningham
and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can
then
be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which
amino acid changes are likely to be permissive at certain amino acid positions
in the
protein. For example, most buried (within the tertiary structure of the
protein) amino
acid residues require nonpolar side chains, whereas few features of surface
side chains
are generally conserved. Moreover, tolerated conservative amino acid
substitutions
involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu
and Ile;
replacement of the hydroxyl residues Ser and Thr; replacement of the acidic
residues
Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the
basic
residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and
Trp,
and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Besides conservative amino acid substitution, variants of the present
invention
include (i) substitutions with one or more of the non-conserved amino acid
residues,
where the substituted amino acid residues may or may not be one encoded by the
genetic code, or (ii) substitution with one or more of amino acid residues
having a
substituent group, or (iii} fusion of the mature polypeptide with another
compound,
such as a compound to increase the stability and/or solubility of the
polypeptide (for
example, polyethylene glycol), or (iv) fusion of the polypeptide with
additional amino
acids, such as an IgG Fc fusion region peptide, or leader or secretory
sequence, or a
sequence facilitating purification. Such variant polypeptides are deemed to be
within
the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of
charged amino acids with other charged or neutral amino acids may produce
proteins
with improved characteristics, such as less aggregation. Aggregation of
pharmaceutical
formulations both reduces activity and increases clearance due to the
aggregate's
immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);
Robbins et al., Diabetes 36: 838-845 ( 1987); Cleland et al., Crit. Rev.
Therapeutic
Drug Carrier Systems 10:307-377 (1993).)
Pol~nucieotide and Polypeptide Fragments
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32
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
15 nt, and more preferably at least about 20 nt, still more preferably at
least about 30 nt,
and even more preferably, at least about 40 nt in length. A fragment "at least
20 nt in
length," for example, is intended to include 20 or more contiguous bases from
the
cDNA sequence contained in the deposited clone or the nucleotide sequence
shown in
SEQ 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
number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-
450, 451-500, 501-550, 551-600, 651-700, or 701 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.
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
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.
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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-
s 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. 4,631,211.)
In the present invention, antigenic epitopes preferably contain a sequence of
at
least seven, more preferably at least nine, and most preferably between about
15 to
about 30 amino acids. Antigenic epitopes are useful to raise antibodies,
including
' monoclonal antibodies, that specifically bind the epitope. (See, for
instance, Wilson et
al., Cell 37:767-778 ( 1984); Sutcliffe, J. G. et al., Science 219:b60-666 (
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
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34
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
polypeptide 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
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.
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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 poiypeptides. These fusion
proteins
facilitate purification and show an increased half life in vivo. One reported
example
5 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
10 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 imrnunoglobulin
molecules
together with another human protein or part thereof. In many cases, the Fc
part in a
15 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
20 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
25 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
30 instance, hexa-histidine provides for convenient purification of the fusion
protein.
Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope
derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767
( 1984).)
Thus, any of these above fusions can be engineered using the polynucleotides
or the polypeptides of the present invention.
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Vectors Host Cells and Protein Production
The present invention also relates to vectors containing the polynucleotide of
the
present invention, host cells, and the production of polypeptides by
recombinant
techniques. The vector may be, for example, a phage, plasmid, viral, or
retroviral
vector. Retroviral vectors may be replication competent or replication
defective. In the
latter case, viral propagation generally will occur only in complementing host
cells.
The polynucleotides may be joined to a vector containing a selectable marker
for
propagation in a host. Generally, a plasmid vector is introduced in a
precipitate, such
as a calcium phosphate precipitate, or in a complex with a charged lipid. If
the vector is
a virus, it may be packaged in vitro using an appropriate packaging cell line
and then
transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and
tac
promoters, the SV40 early and late promoters and promoters of retroviral LTRs,
to
name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription initiation,
termination,
and, in the transcribed region, a ribosome binding site for translation. The
coding
portion of the transcripts expressed by the constructs will preferably include
a
translation initiating codon at the beginning and a termination codon (UAA,
UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase, 6418 or
neomycin
resistance for eukaryotic cell culture and tetracycline, kanamycin or
ampicillin resistance
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, pXTI
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.
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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.
Poiypeptides 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-ternunal 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.
~.Jses of the Pol~nucleotides
Each of the polynucleotides identified herein can be used in numerous ways as
reagents. The following description should be considered exemplary and
utilizes
known techniques.
The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers,
since few chromosome marking reagents, based on actual sequence data (repeat
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38
polymorphisms), are presently available. Each polynucleotide of the present
invention
can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be
selected using computer analysis so that primers do not span more than one
predicted
exon in the genomic DNA. These primers are then used for PCR screening of
somatic
cell hybrids containing individual human chromosomes. Only those hybrids
containing
the human gene corresponding to the SEQ ID NO:X will yield an amplified
fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per
day using a single thermal cycler. Moreover, sublocalization of the
polynucleotides can
be achieved with panels of specific chromosome fragments. Other gene mapping
strategies that can be used include in situ hybridization, prescreening with
labeled flow-
sorted chromosomes, and preselection by hybridization to construct chromosome
specific-cDNA libraries.
Precise chromosomal location of the polynucleotides can also be achieved using
fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread.
This
technique uses polynucleotides as short as 500 or 600 bases; however,
polynucleotides
2,000-4,000 by are preferred. For a review of this technique, see Verma et
al.,
"Human Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York
( 1988).
For chromosome mapping, the polynucleotides can be used individually {to
mark a single chromosome or a single site on that chromosome) or in panels
(for
marking multiple sites and/or multiple chromosomes). Preferred polynucleotides
correspond to the noncoding regions of the cDNAs because the coding sequences
are
more likely conserved within gene families, thus increasing the chance of
cross
hybridization during chromosomal mapping.
Once a polynucleotide has been mapped to a precise chromosomal location, the
physical position of the polynucleotide can be used in linkage analysis.
Linkage
analysis establishes coinheritance between a chromosomal location and
presentation of a
particular disease. (Disease mapping data are found, for example, in V.
McKusick,
Mendelian Inheritance in Man (available on line through Johns Hopkins
University
Welch Medical Library) .) Assuming 1 megabase mapping resolution and one gene
per
20 kb, a cDNA precisely localized to a chromosomal region associated with the
disease
could be one of 50-500 potential causative genes.
Thus, once coinheritance is established, differences in the polynucleotide and
the corresponding gene between affected and unaffected individuals can be
examined.
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39
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
poiypeptide 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
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
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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.
5 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
10 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
15 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
20 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
25 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
contamlnarion.
30 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
35 DNA immunization techniques, and as an antigen to elicit an immune
response.
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41
T~ses of the Po>~neptides
Each of the polypeptides identified herein can be used in numerous ways. The
following description should be considered exemplary and utilizes known
techniques.
A polypeptide of the present invention can be used to assay protein levels in
a
biological sample using antibody-based techniques. For example, protein
expression in
tissues can be studied with classical immunohistological methods. (Jalkanen,
M., et
al., J. Cell. Biol. 101:976-985 ( 1985); Jalkanen, M., et al., J. Cell . Biol.
105:3087-
3096 (1987).) Other antibody-based methods useful for detecting protein gene
expression include immunoassays, such as the enzyme linked immunosorbent assay
{ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are
known
in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine ( 125I, 121I), carbon ( 14C), sulfur (35S), tritium (3H), indium ( 1
l2In), and
technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine,
and
biotin.
In addition to assaying secreted protein levels in a biological sample,
proteins
can also be detected in vivo by imaging. Antibody labels or markers for in
vivo
imaging of protein include those detectable by X-radiography, NMR or ESR. For
X-
radiography, suitable labels include radioisotopes such as barium or cesium,
which emit
detectable radiation but are not overtly harmful to the subject. Suitable
markers for
NMR and ESR include those with a detectable characteristic spin, such as
deuterium,
which may be incorporated into the antibody by labeling of nutrients for the
relevant
hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with
an appropriate detectable imaging moiety, such as a radioisotope (for example,
131I,
112In, 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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42
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 poiynucleotide of the present invention may be useful in
treating deficiencies or disorders of the immune system, by activating or
inhibiting the
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43
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 polynucieotide or poiypeptide of the present invention may be useful in
treating or detecting deficiencies or disorders of hematopoietic cells. A
polypeptide or
polynucleotide of the present invention could be used to increase
differentiation and
proliferation of hematopoietic cells, including the pluripotent stem cells, in
an effort to
treat those disorders associated with a decrease in certain (or many) types
hematopoietic
cells. Examples of immunologic deficiency syndromes include, but are not
limited to:
blood protein disorders (e.g. agammaglobulinemia, dysgammaglobuiinemia},
ataxia
telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV
infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency
(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or
hemoglobinuria.
Moreover, a polypeptide or polynucleotide of the present invention could also
be used to modulate hemostatic (the stopping of bleeding) or thrombolytic
activity (clot
formation). For example, by increasing hemostatic or thrombolytic activity, a
polynucleotide or polypeptide of the present invention could be used to treat
blood
coagulation disorders (e.g., 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 thrombolytic activity could be used to inhibit or
dissolve
clotting. These molecules could be important in the treatment of heart attacks
(infarction), strokes, or scarring.
A polynucleotide or polypeptide of the present invention may also be useful in
treating or detecting autoimmune disorders. Many autoimmune disorders result
from
inappropriate recognition of self as foreign material by immune cells. This
inappropriate recognition results in an immune response leading to the
destruction of the
host tissue. Therefore, the administration of a polypeptide or polynucleotide
of the
present invention that inhibits an immune response, particularly the
proliferation,
differentiation, or chemotaxis of T-cells, may be an effective therapy in
preventing
autoimmune disorders.
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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-l.)
Hyperproliferative 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 hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing
antigenic qualities of the hyperproliferative disorder or by proliferating,
differentiating,
5 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.
10 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,
15 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
20 Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,
histiocytosis, and
any other hyperproliferative disease, besides neoplasia, located in an organ
system
listed above.
Infectious Disease
25 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
30 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
35 DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae,
Arterivirus,
Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,
Flaviviridae,
Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes
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4b
Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae,
Parvoviridae,
Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g.,
Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,
Rubivirus). Viruses falling within these families can cause a variety of
diseases or
symptoms, including, but not limited to: arthritis, bronchiollitis,
encephalitis, eye
infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome,
hepatitis (A, B, C,
E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS),
pneumonia, Burkitt's Lymphoma, chickenpox , hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually
transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. A
polypeptide
or polynucleotide of the present invention can be used to treat or detect any
of these
symptoms or diseases.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that
can be treated or detected by a polynucleotide or polypeptide of the present
invention
include, but not limited to, the following Gram-Negative and Gram-positive
bacterial
families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,
Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium),
Bacteroidaceae,
Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter,
Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae
(Klebsiella,
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
poiypeptide 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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48
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,
flbroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial andlor
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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49
(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 5 ( 1991 ).) Similarly, the molecule can be closely related to
the natural
receptor to which the polypeptide binds, or at least, a fragment of the
receptor capable
of being bound by the polypeptide (e.g., active site). In either case, the
molecule can
be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate
cells which express the polypeptide, either as a secreted protein or on the
cell
membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coLi.
Cells expressing the polypeptide (or cell membrane containing the expressed
polypeptide) are then preferably contacted with a test compound potentially
containing
the molecule to observe binding, stimulation, or inhibition of activity of
either the
polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the polypeptide,
wherein binding is detected by a label, or in an assay involving competition
with a
labeled competitor. Further, the assay may test whether the candidate compound
results
in a signal generated by binding to the polypeptide.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
mixtures. The assay may also simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide, measuring
polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule activity or
binding to a
standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a
sample (e.g., biological sample} using a monoclonal or polyclonal antibody.
The
antibody can measure polypeptide level or activity by either binding, directly
or
indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
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
5 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
10 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,
15 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
20 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.
25 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.
30 Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated
nucleic acid molecule comprising a nucleotide sequence which is at least 95%
identical
to a sequence of at least about 50 contiguous nucleotides in the nucleotide
sequence of
SEQ ID NO:X wherein X is any integer as defined in Table 1.
35 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 defined for SEQ ID NO:X in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5'
Nucleotide of the
Start Codon and ending with the nucleotide at about the position of the 3'
Nucleotide of
the Clone Sequence as defined for SEQ ID NO:X in Table I.
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 I.
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 S'
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 I.
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 l; 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.
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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 1; and a nucleotide sequence encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table l and contained in the
deposit with
the ATCC Deposit Number shown for said cDNA clone in Table 1.
The method for identifying the species, tissue or cell type of a biological
sample
can comprise a step of detecting nucleic acid molecules comprising a
nucleotide
sequence in a panel of at least two nucleotide sequences, wherein at least one
sequence
in said panel is at least 95% identical to a sequence of at least 50
contiguous nucleotides
in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein
identified in Table 1, which method comprises a step of detecting in a
biological sample
obtained from said subject nucleic acid molecules, if any, comprising a
nucleotide
sequence that is at least 95% identical to a sequence of at least 50
contiguous
nucleotides in a sequence selected from the group consisting of: a nucleotide
sequence
of SEQ ID NO:X wherein X is any integer as defined in Table 1; and a
nucleotide
sequence encoded by a human cDNA clone identified by a cDNA Clone Identifier
in
Table l and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
The method for diagnosing a pathological condition can comprise a step of
detecting nucleic acid molecules comprising a nucleotide sequence in a panel
of at least
two nucleotide sequences, wherein at least one sequence in said panel is at
least 95%
identical to a sequence of at least 50 contiguous nucleotides in a sequence
selected from
said group.
Also preferred is a composition of matter comprising isolated nucleic acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise a
panel of at least two nucleotide sequences, wherein at least one sequence in
said panel is
at least 95% identical to a sequence of at least 50 contiguous nucleotides in
a sequence
selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X
wherein
X is any integer as defined in Table 1; and a nucleotide sequence encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table I 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 ID NO:Y in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence at
least 95% identical to a sequence of at least about 30 contiguous amino acids
in the
amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 100 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an anvno 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
5 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
10 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.
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
15 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
20 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
25 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
30 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
35 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 I 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 I, 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 I; and a complete amino acid
sequence of a secreted protein encoded by a human cDNA clone identified by a
cDNA
Clone Identifier in Table I and contained in the deposit with the ATCC Deposit
Number
shown for said cDNA clone in Table I.
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 Clone From the Deposited
Sample
Each cDNA clone in a cited ATCC deposit is contained in a plasmid vector.
Table 1 identifies the vectors used to construct the cDNA library from which
each clone
was isolated. In many cases, the vector used to construct the library is a
phage vector
from whicl' 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)
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 f 1 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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59
DH IOB, also available from Life Technologies. (See, for instance, Gruber, C.
E., et
al., Focus 15:59 ( 1993).) Vector lafmid BA (Bento Soares, Columbia
University, NY)
contains an ampicillin resistance gene and can be transformed into E. coli
strain XL-1
Blue. Vector pCR°2.1, which is available from Invitrogen, 1600 Faraday
Avenue,
Carlsbad, CA 92008, contains an ampicillin resistance gene and may be
transformed
into E. coli strain DH10B, 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 I . First, a plasmid is
directly
isolated by screening the clones using a polynucleotide probe corresponding to
SEQ ID
NO:X.
Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized
using an Applied Biosystems DNA synthesizer according to the sequence
reported.
The oligonucleotide is labeled, for instance, with ~ZP-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
5 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
MgCI2, 0.01 % (w/v) gelatin, 20 ~.M each of dATP, dCTP, dGTP, dTTP, 25 pmol of
each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR
(denaturation
at 94°C for 1 min; annealing at 55°C for 1 min; elongation at
72°C for 1 min) are
10 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
15 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
20 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
25 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
30 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.
35 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.
Example 2~ Isolation of Genomic Clones Corresponding to a
Polynucleotide
A human genomic Pl 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 Polypeptide
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'2 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 Manning-, 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 °lo agarose gels. Chromosome
mapping is
determined by the presence of an approximately 100 by PCR fragment in the
particular
somatic cell hybrid.
Example 5' Bacterial Expression of a PolXpeptide
A polynucleotide encoding a polypeptide of the present invention is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the
DNA
sequence, as outlined in Example 1, to synthesize insertion fragments. The
primers
used to amplify the cDNA insert should preferably contain restriction sites,
such as
BamHI and XbaI, at the 5' end of the primers in order to clone the amplified
product
into the expression vector. For example, BamHI and XbaI correspond to the
restriction
enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc.,
Chatsworth,
CA): This plasmid vector encodes antibiotic resistance (Ampr), a bacterial
origin of
replication (ori), an IPTG-regulatable promoterloperator (P/O), a ribosome
binding site
(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment
is ligated into the pQE-9 vector maintaining the reading frame initiated at
the bacterial
RBS. The ligation mixture is then used to transform the E. coli strain
M15/rep4
(Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which
expresses
the lacI 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 (O/N) in liquid
culture in LB media supplemented with both Amp ( 100 ug/ml) and Kan (25
uglml).
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.G°°) of between
0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration
of 1 mM.
IPTG induces by inactivating the lacI repressor, clearing the P/O leading to
increased
gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by
centrifugation (20 mins at 6000Xg). The cell pellet is solubilized in the
chaotropic
agent 6 Molar Guanidine HCl by stirring 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 1.5 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 pUC 19 {LTI, Gaithersburg, MD). The
promoter
sequence and operator sequences are made synthetically.
DNA can be inserted into the pHEa by restricting the vector with NdeI and
XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and
isolating
the larger fragment {the stuffer fragment should be about 310 base pairs). The
DNA
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.
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Example 6~ Purification of a Polyoeutide from an Inclusion Bodv
The following alternative method can be used to purify a polypeptide expressed
in E toll when it is present in the form of inclusion bodies. Unless otherwise
specified,
all of the following steps are conducted at 4-10°C.
Upon completion of the production phase of the E. toll fermentation, the cell
culture is cooled to 4-10°C and the cells harvested by continuous
centrifugation at
15,000 rpm (Heraeus Sepatech}. On the basis of the expected yield of protein
per unit
weight of cell paste and the amount of purified protein required, an
appropriate amount
of cell paste, by weight, is suspended in a buffer solution containing 100 mM
Tris, 50
mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The
homogenate is
then mixed with NaCI solution to a final concentration of 0.5 M NaCI, followed
by
centrifugation at 7000 xg for 15 min. The resultant pellet is washed again
using O.SM
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 NaCl, 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 ~tm 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,
Perseptive
Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted
with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCI in the same buffer, in a
stepwise manner. The absorbance at 280 nm of the effluent is continuously
monitored.
Fractions are collected and further analyzed by SDS-PAGE.
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Fractions containing the polypeptide are then pooled and mixed with 4 volumes
of water. The diluted sample is then loaded onto a previously prepared set of
tandem
columns of strong anion {Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns are
equilibrated
5 with 40 mM sodium acetate, pH 6Ø Both columns are washed with 40 mM sodium
acetate, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted using a 10
column
volume linear gradient ranging from 0.2 M NaCI, 50 mM sodium acetate, pH 6.0
to 1.0
M NaCI, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant
AZgo
monitoring of the effluent. Fractions containing the polypeptide (determined,
for
10 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 pg of purified protein is
loaded.
The purified protein can also be tested for endotoxin/LPS contamination, and
typically
15 the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 7~ Cloning and Expression of a Polypeptide in a Baculovirus
Expression System
In this example, the plasmid shuttle vector pA2 is used to insert a
polynucleotide
20 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
polyadenylation. For easy selection of recombinant virus, the plasmid contains
the
25 beta-galactosidase gene from E. toll 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.
30 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-
35 39 ( 1989).
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6b
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
signal sequence is used to produce the secreted protein, the pA2 vector does
not need a
second signal peptide. Alternatively, the vector can be modified (pA2 GP) to
include a
baculovirus leader sequence, using the standard methods described in Summers
et al.,
"A Manual of Methods for Baculovirus Vectors and Insect Cell Culture
Procedures,"
Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1 % agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is
digested
with appropriate restriction enzymes and again purified on a 1 % agarose gel.
The plasmid is digested with the corresponding restriction enzymes and
optionally, can be dephosphorylated using calf intestinal phosphatase, using
routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.).
The fragment and the dephosphorylated plasmid are ligated together with T4
DNA ligase. E. coli HB 101 or other suitable E. coli hosts such as XL-1 Blue
{Stratagene Cloning Systems, La Jolla, CA) cells are transformed with the
ligation
mixture and spread on culture plates. Bacteria containing the plasmid are
identified by
digesting DNA from individual colonies and analyzing the digestion product by
gel
electrophoresis. The sequence of the cloned fragment is confirmed by DNA
sequencing.
Five p.g of a plasmid containing the polynucleotide is co-transfected with 1.0
p.g
of a commercially available linearized baculovirus DNA ("BaculoGoldTM
baculovirus
DNA", Pharmingen, San Diego, CA), using the lipofection method described by
Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 ( 1987). One p.g of
BaculoGoldTM virus DNA and 5 p.g of the plasmid are mixed in a sterile well of
a
microtiter plate containing 50 p,l of serum-free Grace's medium (Life
Technologies
inc., Gaithersburg, MD). Afterwards, 10 p.l Lipofectin plus 90 pl 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 Smith, supra. An agarose gel with "Blue Gal" (Life
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Technologies Inc., Gaithersburg) is used to allow easy identification and
isolation of
gal-expressing clones, which produce blue-stained plaques. (A detailed
description of a
"plaque assay" of this type can also be found in the user's guide for insect
cell culture
and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-
10.)
After appropriate incubation, blue stained plaques are picked with the tip of
a
micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses
is then
resuspended in a microcentrifuge tube containing 200 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 p.Ci
of ~SS-
methionine and 5 p.Ci ;SS-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 Pol~e_ptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian cell.
A typical mammalian expression vector contains a promoter element, which
mediates
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
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include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1,
Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary
(CHO)
cells.
Alternatively, the polypeptide can be expressed in stable cell lines
containing the
polynucleotide integrated into a chromosome. The co-transfection with a
selectable
marker such as dhfr, gpt, neomycin, hygromycin allows the identification and
isolation
of the transfected cells.
The transfected gene can also be amplified to express large amounts of the
encoded protein. The DHFR (dihydrofolate reductase) marker is useful in
developing
cell lines that carry several hundred or even several thousand copies of the
gene of
interest. {See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978);
Hamlin,
J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J.
and
Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection
marker is
the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (
1991 );
Bebbington et al., Bioffechnology 10:169-175 ( 1992). Using these markers, the
mammalian cells are grown in selective medium and the cells with the highest
resistance
are selected. These cell lines contain the amplified genes) integrated into a
chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the
production of proteins.
Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the
expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession
No.209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen
et
al., Molecular and Cellular Biology, 438-447 (March, 1985)) pius 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 I % 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.)
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The amplified fragment is isolated from a 1 % agarose gel using a commercially
available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The fragment then is
digested
with appropriate restriction enzymes and again purified on a 1 % agarose gel.
The amplified fragment is then digested with the same restriction enzyme and
purified on a 1 % agarose gel. The isolated fragment and the dephosphorylated
vector
are then ligated with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells are
then
transformed and bacteria are identified that contain the fragment inserted
into plasmid
pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for
transfection. Five ~,g of the expression plasmid pC6 is cotransfected with 0.5
pg 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 p.M, 2 p.M, 5 ~M, 10 mM, 20 mM). The same
procedure is repeated until clones are obtained which grow at a concentration
of 100 -
200 ~,M. 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-1, IgG-
3, and
albumin increases the halflife time in vivo. Nuclear localization signals
fused to the
polypeptides of the present invention can target the protein to a specific
subcellular
localization, while covalent heterodimer or homodimers can increase or
decrease the
activity of a fusion protein. Fusion proteins can also create chimeric
molecules having
more than one function. Finally, fusion proteins can increase solubility
and/or stability
of the fused protein compared to the non-fused protein. All of the types of
fusion
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proteins described above can be made by modifying the following protocol,
which
outlines the fusion of a polypeptide to an IgG molecule, or the protocol
described in
Example 5.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using
primers that span the 5' and 3' ends of the sequence described below. These
primers
also should have convenient restriction enzyme sites that will facilitate
cloning into an
expression vector, preferably a mammalian expression vector.
Far 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
10 destroyed. Next, the vector containing the human Fc portion is re-
restricted with
BamHI, linearizing the vector, and a polynucleotide of the present invention,
isolated
by the PCR protocol described in Example 1, is ligated into this BamHI site.
Note that
the polynucleotide is cloned without a stop codon, otherwise a fusion protein
will not
be produced.
15 If the naturally occurring signal sequence is used to produce the secreted
protein, pC4 does not need a second signal peptide. Alternatively, if the
naturally
occurring signal sequence is not used, the vector can be modified to include a
heterologous signal sequence. (See, e.g., WO 96/34891
20 Human IgG Fc region:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCC
CAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGT
GGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG
25 GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT
30 GACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCA
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGC
35 GACGGCCGCGACTCTAGAGGAT (SEQ ID NO:1 )
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Example 10: Production of an Antibody from a Polypeptide
The antibodies of the present invention can be prepared by a variety of
methods.
(See, Current Protocols, Chapter 2.) For example, cells expressing a
polypeptide of
the present invention is administered to an animal to induce the production of
sera
containing polyclonal antibodies. In a preferred method, a preparation of the
secreted
protein is prepared and purified to render it substantially free of natural
contaminants.
Such a preparation is then introduced into an animal in order to produce
polyclonal
antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or protein binding fragments thereof). Such monoclonal
antibodies can be prepared using hybridoma technology. (Kohler et al., Nature
256:495 ( 1975); Kohler et al., Eur. J. Immunol. 6:51 I ( 1976); Kohler et
al., Eur. J.
Immunol. 6:292 ( 1976); Hammerling et al., in: Monoclonal Antibodies and T-
Cell
Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In general, such procedures
involve immunizing an animal (preferably a mouse) with polypeptide or, more
preferably, with a secreted polypeptide-expressing cell. Such cells may be
cultured in
any suitable tissue culture medium; however, it is preferable to culture cells
in Earle's
modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated
at
about 56°C), and supplemented with about 10 g/1 of nonessential amino
acids, about
1,000 U/ml of penicillin, and about 100 p.g/ml of streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the ATCC. After fusion, the resulting hybridoma cells
are
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 polypeptide 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.
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Such antibodies comprise anti-idiotypic antibodies to the protein-specific
antibody and
can be used to immunize an animal to induce formation of further protein-
specific
antibodies.
It will be appreciated that Fab and F(ab')2 and other fragments of the
antibodies
of the present invention may be used according to the methods disclosed
herein. Such
fragments are typically produced by proteolytic cleavage, using enzymes such
as papain
(to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
Alternatively,
secreted protein-binding fragments can be produced through the application of
recombinant DNA technology or through synthetic chemistry.
For in vivo use of antibodies in humans, it may be preferable to use
"humanized" chimeric monoclonal antibodies. Such antibodies can be produced
using
genetic constructs derived from hybridoma cells producing the monoclonal
antibodies
described above. Methods for producing chimeric antibodies are known in the
art.
(See, for review, Morrison, Science 229:1202 ( 1985); Oi et al., BioTechniques
4:214
( 1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496;
Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Nature 312:643 { 1984); Neuberger et al., Nature
314:268
( 1985).)
Example 11 ~ Production Of Secreted Protein For High-Throughput
Screening Assavs
The following protocol produces a supernatant containing a polypeptide to be
tested. This supernatant can then be used in the Screening Assays described in
Examples 13-20.
First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution
(lmg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker)
for a
working solution of 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)/Ix
Penstrep( 17-602E Biowhittaker). Let the cells grow overnight.
__ _ ~ r ......~..~. ..__... ~.__
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The next day, mix together in a sterile solution basin: 300 ul Lipofectamine
(18324-012 Gibco/BRL) and Sml Optimem I (31985070 GibcoBRL)/96-well plate.
With a small volume multi-channel pipetter, aliquot approximately tug of an
expression
vector containing a polynucleotide insert, produced by the methods described
in
Examples 8 or 9, into an appropriately labeled 96-well round bottom plate.
With a
mufti-channel pipetter, add 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 1 x penstrep, or CHO-5 media ( 116.6 mg/L of CaCI-2 (anhyd); 0.00130 mg/L
CuS04-SH20; 0.050 mg/L of Fe(N03),-9H~0; 0.417 mg/L of FeS04-7H20; 311.80
mg/L of Kcl; 28.64 mglL of MgCl2; 48.84 mg/L of MgS04; 6995.50 mg/L of NaCI;
2400.0 mg/I. of NaHCOj; 62.50 mglL of NaHZP04 H20; 71.02 mg/L of Na2HP04;
.4320 mg/L of ZnS04-7H20; .002 mg/L of Arachidonic Acid ; 1.022 mg/L of
Cholesterol; .070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic
Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic
Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 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-HZO; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-
2HCL-H20; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0
mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-
H,O; 106.97 mglml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-
Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0
mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;
19.22
mg/ml of L-Tryptophan; 91.79 mglml of L-Tryrosine-2Na-2H20; 99.65 mg/ml of L-
Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of
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Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L
of
Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319
mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and
0.680 mg/L of Vitamin B,Z; 25 mM of HEPES Buffer; 2.39 mg/L of Na
Hypoxanthine;
0.105 mglL 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 mg/L of Methyl-B-Cyclodextrin complexed with
Linoleic
Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; and 10
mg/L
of Methyl-B-Cyclodextrin complexed with Retinal) with 2mm glutamine and lx
penstrep. (BSA (81-068-3 Bayer) 100gm dissolved in 1L DMEM for a 10% BSA stock
solution). Filter the media and collect 50 ul for endotoxin assay in 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.5ml appropriate media to each well. Incubate at 37°C for 45 or
72 hours
depending on the media used: 1 %BSA for 45 hours or CHO-5 for 72 hours.
On day four, using a 300u1 multichannel pipetter, aliquot 600u1 in one lml
deep
well plate and the remaining supernatant into a 2m1 deep well. The
supernatants from
each well can then be used in the assays described in Examples 13-20.
It is specifically understood that when activity is obtained in any of the
assays
described below using a supernatant, the activity originates from either the
polypeptide
directly (e.g., as a secreted protein) or by the polypeptide inducing
expression of other
proteins, which are then secreted into the supernatant. Thus, the invention
further
provides a method of identifying the protein in the supernatant characterized
by an
activity in a particular assay.
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 ("ISRE"), located in the promoter of many genes. The
binding of a
protein to these elements alter the expression of the associated gene.
GAS and ISRE elements are recognized by a class of transcription factors
called
Signal Transducers and Activators of Transcription, or "STATs." There are six
members of the STATs family. 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
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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.
5 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 cataiytically inactive in resting cells.
10 The Jaks are activated by a wide range of receptors summarized in the Table
below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-
51
( 1995).) A cytokine receptor family, capable of activating Jaks, is divided
into two
groups: {a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-
12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and
15 (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-Trg-Ser (SEQ ID
N0:2)).
Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn
20 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
25 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.
i n
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JAKs STATS GAS(elements) or ISRE
L~ t~k2 Jak Jak2Jak3
1
,
IF_N family
IFN-aB + + - - 1,2,3 ISRE
IFN-g + + - 1 GAS (IRF 1 >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)? + ? ? I,3
IL-12(Pleiotrohic)+ - + + 1,3
g-C family
IL-2 (lymphocytes)- + - + 1,3,5 GAS
IL-4 (lymphlmyeloid)- + - + 6 GAS (IRFI = IFP Ly6)(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 (IRF 1 >IFPLy6)
IL-5 (myeloid) - - + - S GAS
GM-CSF (myeloid)- - + - 5 GAS
Growth hormone
family
GH ? - + - 5
PRL ? +/- + - 1,3,5
EPO ? - + - 5 GAS(B-CAS>IRFI=IFPLy6)
Receptor Tyrosine
Kinases
EGF ? + + - 1,3 GAS (IRF1)
PDGF ? + + - 1, 3
CSF-1 ? + + - 1,3 GAS (not IRFI)
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To construct a synthetic GAS containing promoter element, which is used in the
Biological Assays described in Examples 13-14, a PCR based strategy is
employed to
generate a GAS-SV40 promoter sequence. The 5' primer contains four tandem
copies
of the GAS binding site found in the IRF1 promoter and previously demonstrated
to
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 S'
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
1 S 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' : CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCC
GAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTC
CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATT
CTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGC
CTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT
AGGCTTTTGCAAAAAGCTT: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 Assav for T-cell Activity.
The following protocol is used to assess T-cell activity by identifying
factors,
such as growth factors and cytokines, that may proliferate or differentiate T-
cells. T-
cell activity is assessed using the GAS/SEAP/Neo construct produced in Example
12.
Thus, factors that increase SEAP activity indicate the ability to activate the
Jaks-STATS
signal transduction pathway. The T-cell used in this assay is Jurkat T-cells
{ATCC
Accession No. TIB-152), although Molt-3 cells {ATCC Accession No. CRL-1552)
and
Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.
3urkat T-cells are lymphoblastic CD4+ Th i helper cells. In order to generate
stable cell lines, approximately 2 million Jurkat cells are transfected with
the GAS-
SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure
described below). The transfected cells are seeded to a density of
approximately
20,000 cells per well and transfectants resistant to 1 mg/ml genticin
selected. Resistant
colonies are expanded and then tested for their response to increasing
concentrations of
interferon gamma. The dose response of a selected clone is demonstrated.
Specifically, the following protocol will yield sufficient cells for 75 wells
containing 200 ui 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 mg/ml Genticin, and 1 % Pen-Strep. These cells are treated with
supernatants
containing a polypeptide as produced by the protocol described in Example 11.
On the day of treatment with the supernatant, the cells should be washed and
resuspended in fresh 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 10 million cells (for 10
plates, 100
million cells) are required.
Transfer the cells to a triangular reservoir boat, in order to dispense the
cells into
a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette,
transfer 200 ul
of cells into each well (therefore adding 100, 000 cells per well).
After all the plates have been seeded, 50 ul of the supernatants are
transferred
directly from the 96 well plate containing the supernatants into each well
using a 12
channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0,
10 ng)
is added to wells H9, H 10, and H 11 to serve as additional positive controls
for the
assay.
The 96 well dishes containing Jurkat cells treated with supernatants are
placed in
an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul
samples
from each well are then transferred to an opaque 96 well plate using a 12
channel
pipette. The opaque plates should be covered (using sellophene covers) and
stored at -
20oC until SEAP assays are performed according to Example 17. The plates
containing the remaining treated cells are placed at 4oC and serve as a source
of material
for repeating the assay on a specific well if desired.
As a positive control, 100 Unit/ml interferon gamma can be used which is
known to activate Jurkat T cells. Over 30 fold induction is typically observed
in the
' positive control wells.
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Example 14~ High-Throughput Screening Assav Identifying Myeloid
Activity
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.
5 Myeloid cell activity is assessed using the GAS/SEAPINeo construct produced
in
Example 12. Thus, factors that increase SEAP activity indicate the ability to
activate the
Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is
U937,
a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.
To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced
10 in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth
&
Differentiation, 5:259-265) is used. First, harvest 2x10e7 0937 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.
15 Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer
containing
0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAPZ plasmid DNA, 140 mM NaCI, 5 mM
KCI, 375 uM Na2HP04.7H20, 1 mM MgCl2, and 675 uM CaCl2. Incubate at
37°C
for 45 min.
Wash the cells with RPMI 1640 medium containing 10% FBS and then
20 resuspend in 10 ml complete medium and incubate at 37oC for 36 hr.
The GAS-SEAP/LJ937 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
25 plates assay) and wash with PBS. Suspend the cells in 200 ml above
described growth
medium, with a final density of 5x105 cells/ml. Plate 200 ul cells per well in
the 96-
well plate (or 1x105 cells/well).
Add 50 ul of the supernatant prepared by the protocol described in Example 11.
Incubate at 37oC for 48 to 72 hr. As a positive control, 100 Unitlml
interferon gamma
30 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.
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Example 15: High-Throughput Screening Assay Identifying Neuronal
Activity.
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
activation. The promoter of EGRI is responsible for such induction. Using the
EGR1
promoter linked to reporter molecules, activation of cells can be assessed.
Particularly, the following protocol is used to assess neuronal activity in PC
12
cell lines. PC 12 cells (rat phenochromocytoma cells) are known to proliferate
and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl
phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth
factor). The
EGRI 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 +1)(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 for cell culture, two mls of a coating solution (
1:30
dilution of collagen type I (Upstate Biotech Inc. Cat#08- I I S) 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 12 stable cells are obtained by
growing the cells in 300 ug/ml 6418. The 6418-free medium is used for routine
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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
{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
1x105 cells/well). Add 50 ul supernatant produced by Example 1 l, 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.
Example 16~ High-Throughput Screening Assay for T-cell Activity
NF-xB {Nuclear Factor oB) is a transcription factor activated by a wide
variety
of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40,
lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by
expression of certain viral gene products. As a transcription factor, NF-xB
regulates
the expression of genes involved in immune cell activation, control of
apoptosis (NF-
1cB 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 tcB). However, upon stimulation, I- oB is phosphorylated and
degraded,
causing NF- oB to shuttle to the nucleus, thereby activating transcription of
target
genes. Target genes activated by NF- tcB include IL-2, IL-6, GM-CSF, ICAM-1
and
class 1 MHC.
Due to its central role and ability to respond to a range of stimuli, reporter
constructs utilizing the NF-oB promoter element are used to screen the
supernatants
produced in Example 11. Activators or inhibitors of NF-kB would be useful in
treating
.._.-_-.__-___~.-.._T._....._. ...... - .....
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diseases. For example, inhibitors of NF-xB could be used to treat those
diseases
related to the acute or chronic activation of NF-kB, such as rheumatoid
arthritis.
To construct a vector containing the NF-tcB promoter element, a PCR based
strategy is employed. The upstream primer contains four tandem copies of the
NF-oB
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 XhoI and Hind III and subcloned into BLSK2-. (Stratagene)
Sequencing with the T7 and T3 primers confirms the insert contains the
following
sequence:5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGA
CTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCC
GCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGC
TGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGC
TATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTT'TTGCAAAAA
GCTT:3' (SEQ ID NO:10)
Next, replace the SV40 minimal promoter element present in the pSEAP2-
promoter plasmid (Clontech) with this NF-xBISV40 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-~cB/SV40/SEAP
cassette is removed from the above NF-tcB/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-~cB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are
created and maintained according to the protocol described in Example 13.
Similarly,
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the method for assaying supernatants with these stable Jurkat T-cells is also
described
in Example 13. As a positive control, exogenous TNF alpha (0.1,1, 10 ng) is
added to
wells H9, H 10, and H I I , with a 5-10 fold activation typically observed.
Example 17~ Assay for SEAP Activity
As a reporter molecule for the assays described in Examples 13-16, SEAP
activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according
to the
following general procedure. The Tropix Phospho-light Kit supplies the
Dilution,
Assay, and Reaction Buffers used below.
Prime a dispenser with the 2.Sx Dilution Buffer and dispense 15 ~.l of 2.Sx
dilution buffer into Optiplates containing 35 E.tl 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 I S minutes. Empty the dispenser and
prime with the Assay Buffer. Add 50 ~tl Assay Buffer and incubate at room
temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see
the
table below). Add 50 ~tl 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 H12 as blank, and print
the
results. An increase in chemiluminescence 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 I 15 5.75
22 I20 6
23 125 6.25
24 130 6.5
135 6.75
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26 140 7
27 145 7.25
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 I1
43 225 11.25
44 230 1 1.5
45 235 1 1.75
46 240 12
47 245 12.25
48 250 12.5
49 255 12.75
50 260 13
xamnle 18: High-Throu~hnut Screening Assay Identifvin~ Changes in
Small Molecule Concentration and Membrane Permeability
Binding of a ligand to a receptor is known to alter intracellular levels of
small
5 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
10 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.
I 5 For adherent cells, seed the cells at 10,000 -20,000 cells/well in a Co-
star black
96-well plate with clear bottom. The plate is incubated in a COZ incubator for
20 hours.
The adherent cells are washed two times in Biotek washer with 200 ul of HBSS
(Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
A stock solution of 1 mg/ml fluo-3 is made in 10°70 pluronic acid
DMSO. To
20 load the cells with fluo-3, 50 ul of 12 ug/ml fluo-3 is added to each well.
The plate is
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incubated at 37°C in a COZ incubator for 60 min. The plate is washed
four times in the
Biotek washer with HBSS leaving 100 ul of buffer.
For non-adherent cells, the cells are spun down from culture media. Cells are
re-suspended to 2-5x106 cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1
mg/ml
fluo-3 solution in 10% 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'i"f'
concentration.
Example 19: High-Throughout Screening Assav Identif~ring 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 iigand-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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87
tyrosine kinase signal transduction pathways are of interest. Therefore, the
following
protocol is designed to identify those novel human secreted proteins capable
of
activating the tyrosine kinase signal transduction pathways.
Seed target cells (e.g., primary keratinocytes) at a density of approximately
25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased
from
Nalge Nunc (Naperville, IL). The plates are sterilized with two 30 minute
rinses with
100% ethanol, rinsed with water and dried overnight. Some plates are coated
for 2 hr
with 100 ml of cell culture grade type I collagen (50 mglml); gelatin (2%) or
polylysine
(50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, MO)
or
10% Matrigel purchased from Becton Dickinson (Bedford,MA}, or calf serum,
rinsed
with PBS and stored at 4oC. Cell growth on these plates is assayed by seeding
5,000
cells/well in growth medium and indirect quantitation of cell number through
use of
alamarBlue as described by the manufacturer Alamar Biosciences, Inc.
(Sacramento,
CA) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford,MA)
are
used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell
culture
plates can also be used in some proliferation experiments.
To prepare extracts, A431 cells are seeded onto the nylon membranes of
Loprodyne plates (20,000/200m1/well) and cultured overnight in complete
medium.
Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-
20
minutes treatment with EGF (60ng/ml) or 50 ul of the supernatant produced in
Example
11, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH
7.5, 0.15 M NaCI, i% Triton X-100, 0.1% SDS, 2 mM Na3V04, 2 mM Na4P2O7
and a cocktail of protease inhibitors (# 1836170) obtained from Boeheringer
Mannheim
(Indianapolis, IN) is added to each well and the plate is shaken on a rotating
shaker for
5 minutes at 4oC. The plate is then placed in a vacuum transfer manifold and
the extract
filtered through the 0.45 mm membrane bottoms of each well using house vacuum.
Extracts are collected in a 96-well catch/assay plate in the bottom of the
vacuum
manifold and immediately placed on ice. To obtain extracts clarified by
centrifugation,
the content of each well, after detergent solubilization for 5 minutes, is
removed and
centrifuged for 15 minutes at 4oC at 16,000 x g.
Test the filtered extracts for levels of tyrosine kinase activity. Although
many
methods of detecting tyrosine kinase activity are known, one method is
described here.
Generally, the tyrosine kinase activity of a supernatant is evaluated by
determining its ability to phosphorylate a tyrosine residue on a specific
substrate (a
biotinylated peptide). Biotinylated peptides that can be used for this purpose
include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34)
and
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88
PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are
substrates for
a range of tyrosine kinases and are available from Boehringer Mannheim.
The tyrosine kinase reaction is set up by adding the following components in
order. First, add l0ul of 5uM Biotinylated Peptide, then lout ATP/Mg2+ (5mM
ATP/50mM MgCl2), then 10u1 of 5x Assay Buffer (40mM imidazole hydrochloride,
pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, I OOmM MgCl2, 5 mM MnCl2,
0.5 mg/ml BSA), then 5u1 of Sodium Vanadate(1mM), and then 5ul of water. Mix
the
components gently and preincubate the reaction mix at 30oC for 2 min. Initial
the
reaction by adding l0ul of the control enzyme or the filtered supernatant.
The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120mm
EDTA and place the reactions on ice.
Tyrosine kinase activity is determined by transferring 50 ul aliquot of
reaction
mixture to a microtiter plate (MTP) module and incubating at 37oC for 20 min.
This
allows the streptavadin coated 96 well plate to associate with the
biotinylated peptide.
Wash the MTP module with 300u1/well of PBS four times. Next add 75 ul of anti-
phospotyrosine antibody conjugated to horse radish peroxidase(anti-P-Tyr-
POD(0.5u/ml)) to each well and incubate at 37oC for one hour. Wash the well as
above.
Next add 100u1 of peroxidase substrate solution (Boehringer Mannheim) and
incubate at room temperature for at Ieast 5 rains (up to 30 min). Measure the
absorbance of the sample at 405 nm by using ELISA reader. The level of bound
peroxidase activity is quantitated using an ELISA reader and reflects the
level of
tyrosine kinase activity.
Example 20: Hi:zh-Throughout Screening Assay Identifying
Phosphorylation Activity
As a potential alternative and/or compliment to the assay of protein tyrosine
kinase activity described in Example 19, an assay which detects activation
(phosphorylation) of major intracellular signal transduction intermediates can
also be
used. For example, as described below one particular assay can detect tyrosine
phosphorylation of the Erk-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.
_ -. ,
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Specifically, assay plates are made by coating the wells of a 96-well ELISA
plate with O.lml of protein G (lug/ml) for 2 hr at room temp, (RT). The plates
are then
rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G
plates
are then treated with 2 commercial monoclonal antibodies (100ng/well) against
Erk-1
and Erk-2 ( 1 hr at RT) (Santa Cruz Biotechnology). (To detect other
molecules, this
step can easily be modified by substituting a monoclonal antibody detecting
any of the
above described molecules.) After 3-5 rinses with PBS, the plates are stored
at 4oC
until use.
A431 cells are seeded at 20,0001we11 in a 96-well Loprodyne filterplate and
cultured overnight in growth medium. The cells are then starved for 48 hr in
basal
medium (DMEM) and then treated with EGF (6ng/well) or 50 ul of the
supernatants
obtained in Example 11 for 5-20 minutes. The cells are then solubilized and
extracts
filtered directly into the assay plate.
After incubation with the extract for 1 hr at RT, the wells are again rinsed.
As a
positive control, a commercial preparation of MAP kinase ( l Ong/well) is used
in place
of A431 extract. Plates are then treated with a commercial polyclonal (rabbit)
antibody
(lug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1
and
Erk-2 kinases ( 1 hr at RT). This antibody is biotinylated by standard
procedures. The
bound polyclonal antibody is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent in the
Wallac
DELFIA instrument (time-resolved fluorescence). An increased fluorescent
signal over
background indicates a phosphorylation.
Example 21: Method of Determining Alterations in a Gene
Corresponding to a Polvnucleotide
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 S' 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
5 polymerase (United States Biochemical). Affected individuals are identified
by
mutations not present in unaffected individuals.
Genomic rearrangements are also observed as a method of determining
alterations in a gene corresponding to a polynucleotide. Geriomic clones
isolated
according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5'-
10 triphosphate (Boehringer Manheim), and FISH performed as described in
Johnson,
Cg. et al., Methods Cell Biol. 35:73-99 ( 1991 ). Hybridization with the
labeled probe is
carried out using a vast excess of human cot-1 DNA for specific hybridization
to the
corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and
I5 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
20 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 Polypeutide 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.
_ _...~__ , r __
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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 Polvpeptide
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 adnunistered parenteraliy per dose will be in the range of about 1
p.g/kg/day
to 10 mg/kg/day of patient body weight, although, as noted above, this will be
subject
to therapeutic discretion. More preferably, this dose is at least 0.01
mg/kg/day, and
most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
If
given continuously, the secreted polypeptide is typically administered at a
dose rate of
about 1 p,g/kg/hour to about 50 p,g/kglhour, either by 1-4 injections per day
or by
continuous subcutaneous infusions, for example, using a mini-pump. An
intravenous
bag solution may also be employed. The length of treatment needed to observe
changes
and the interval following treatment for responses to occur appears to vary
depending
on the desired effect.
Pharmaceutical compositions containing the secreted protein of the invention
are
administered orally, rectally, parenterally, intracistemally, intravaginally,
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intraperitoneally, topically (as by powders, ointments, gels, drops or
transdermal
patch}, bucally, or as an oral or nasal spray. "Pharmaceutically acceptable
carrier" refers
to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating
material or
formulation auxiliary of any type. The term "parenteral" as used herein refers
to modes
of administration which include intravenous, intramuscular, intraperitoneal,
intrasternal,
subcutaneous and intraarticular injection and infusion.
The secreted polypeptide is also suitably administered by sustained-release
systems. Suitable examples of sustained-release compositions include semi-
permeable
polymer matrices in the form of shaped articles, e.g., films, or mirocapsules.
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP
58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al.,
Biopolymers 22:547-556 ( 1983}), poly (2- hydroxyethyl methacrylate) (R.
Langer et
al., J. Biomed. Mater. Res. 15:167-277 ( 1981 ), and R. Langer, Chem. Tech.
12:98-
105 (1982}), ethylene vinyl acetate (R. Langer et al.) or poly-D- (-)-3-
hydroxybutyric
acid (EP 133,988). Sustained-release compositions also include liposomally
entrapped
polypeptides. Liposomes containing the secreted polypeptide are prepared by
methods
known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-
3692
( 1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 ( 1980); EP
52,322;
EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008;
U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the
liposomes
are of the small (about 200-800 Angstroms) unilamellar type in which the lipid
content
is greater than about 30 mol. percent cholesterol, the selected proportion
being adjusted
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 nat 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 carriers
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
immunoglo>?ulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids,
such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its derivatives,
glucose,
manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or
sorbitol; counterions such as sodium; and/or nonionic surfactants such as
polysorbates,
poloxamers, or PEG.
The secreted polypeptide is typically formulated in such vehicles at a
concentration of about 0.1 mg/ml to 100 mg/ml, 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.
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Example 24~ Method of Treating, Decreased Levels of the PolYpeptide
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 ug/kg of the polypeptide for six consecutive days. Preferably,
the
polypeptide is in the secreted form. The exact details of the dosing scheme,
based on
administration and formulation, are provided in Example 23.
Example 25: Method of Treating Increased Levels of the 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 F12 media, with 10% FBS,
penicillin and streptomycin) is added. The flasks are then incubated at
37°C for
approximately one week.
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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.
pMV-7 (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)), flanked by the long
5 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
10 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
15 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
20 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,
25 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
30 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.
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It will be clear that the invention may be practiced otherwise than as
particularly
described in the foregoing description and examples. Numerous modifications
and
variations of the present invention are possible in light of the above
teachings and,
therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, laboratory manuals, books, or other
disclosures) in the Background of the Invention, Detailed Description, and
Examples is
hereby incorporated herein by reference.
... , _ ..__~.
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SEQUENCE LISTING
(11 GENERAL INFORMATION:
(i) APPLICANTS: Human Genome Sciences, Inc. et al.
(ii) TITLE OF INVENTION: 20 Human Secreted Proteins
(iii) NUMBER OF SEQUENCES: 61
' 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Human Genome Sciences, Inc.
(B) STREET: 9410 Key West Avenue
(C) CITY: Rockville
IS (D) STATE: Maryland
(E) COEJNTRY: USA
(F) ZIP: 20850
2O (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.50 inch, l.4Mb storage
(B) COMPUTER: HP VeCtra 486/33
(C) OPERATING SYSTEM: MSDOS version 6.2
(D) SOFTWARE: ASCII Text
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: April 07, 1998
3O (C} CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
3S (B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: A. Anders Brookes
40 (B) REGISTRATION NUMBER: 36,373
(C) REFERENCE/DOCKET NUMBER: PZOOSPCT
(vi) TELECOMMUNICATION INFORMATION:
4$ (A) TELEPHONE: (301) 309-8504
(B) TELEFAX: (301) 309-8439
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
' (A) LENGTH: 733 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GGGATCCGGA GCCCAAATCT TCTGACAAAA CTCACACATG CCCACCGTGC CCAGCACCTG 60
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AATTCGAGGG TGCACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC ACCCTCATGA 120
TCTCCCGGAC TCCTGAGGTC ACATGCGTGG TGGTGGACGT AAGCCACGAA180
GACCCTGAGG
S TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA240
AAGCCGCGGG
AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG300
CACCAGGACT
GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA360
ACCCCCATCG
AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTAC420
ACCCTGCCCC
CATCCCGGGA TGAGCTGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTC480
AAAGGCTTCT
IS ATCCAAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAAC540
AACTACAAGA
CCACGCCTCC CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAG600
CTCACCGTGG
ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT660
GAGGCTCTGC
ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAATGAGTG720
CGACGGCCGC
GACTCTAGAG GAT 733
2S
(2) INFORMATION FOR SEQ ID NO: 2:
3O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
3S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Trp Ser Xaa Trp Ser
1 5
(2) INFORMATION FOR 5EQ ID NO: 3:
4S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
S0
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
GCGCCTCGAG ATTTCCCCGA AATCTAGATT TCCCCGAAAT GATTTCCCCG AAA2GATTTC 60
SS CCCGAAATAT CTGCCATCTC AATTAG 86
GO (2) INFORMATION FOR SEQ ID NO: 4:
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(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 27 base pairs
iB) TYPE: nucleic acid
S (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
~ IO GCGGCAAGCT TTTTGCAAAG CCTAGGC 27
IS (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 base pairs
(B) TYPE: nucleic acid
20 (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ZS CTCGAGATTT CCCCGAAATC TAGATTTCCC CGAAATGATT TCCCCGAAAT GATTTCCCCG 60
AAATATCTGC CATCTCAATT AGTCAGCAAC CATAGTCCCG CCCCTAACTC CGCCCATCCC 120
GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT 180
TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT 240
TTTTGGAGGC CTAGGCT'ITT GCAAAAAGCT T 271
3S
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
4S
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GCGCTCGAGG GATGACAGCG ATAGAACCCC GG 32
SO
(2) INFORMATION FOR SEQ ID NO: 7:
_ SS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GCGAAGCTTC GCGACTCCCC GGATCCGCCT C 31
S
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: I2 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
IS (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GGGGACTTTC CC 12
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 73 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GCGGCCTCGA GGGGACTTTC CCGGGGACTT TCCGGGGACT TTCCGGGACT TTCCATCCTG 60
3S
CCATCTCAAT TAG 73
(2) INFORMATION FOR SEQ ID N0: 10:
(i) SEøUENCE CHARACTERISTICS: '
(A) LENGTH: 256 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
SO
CTCGAGGGGA CTTTCCCGGG GACTTTCCGG GGACTTTCCG GGACTTTCCA TCTGCCATCT 60
CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC 120
SS CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA 180
GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG 240
CTTTTGCAAA AAGCTT 256
__.__._ , .- r _. ._ _.._.
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(2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 919 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
AATTCGGCAG AGGTCCAATT CAGTTTTTCA GGCCAGTGCA NCTTGATACC60
AAAACCAATA
AAACAAACAA ACAAACAAAA AACATAAAGC TATAGACCAA AGTCTCATAG120
ATTTAGATGC
AAAATCCTAA AATTVAAAAA AAAAGTCTAG TCATATCCAT AAACTGTATC180
ATCACCAAGA
2O GATGTTTATT AGGGCAATCA AAAGATGATT TATTATTTTT TAAAAAATCA240
ATGTGGCCTT
CCCTTCCTCT TTCTTTTGAT TCCCCTCTTT GAGTTTTTAT GTGTCTCTTT300
TGCCTTCCCT
TCCCAGAGTG GAGGAGTTAG ACCTGCATTG TGGGATGAGA GGAGTTGTGG360
CTATGTGTCT
GCTGGCACCA AGAGGGCTGA GGGTGAGGTG TGGAAGGGAC AGGGGGAGGA420
GATGGGCAGC
ATTGTTAAGA GATTGGTACC ACTGAGCAAA TATGTTGAGA ATGATGATGG480
CAAGGTTTCT
3 O CCCTGTTAGA GAAGGTATTT GTAGAAATAG GAATGAGGAG AGCTAGAAAA540
CCTGGAGTGT
GGGATTAGAA TAGAACTCAT ATCTTTTAAA TACATAGGAA CAATAGAGAA600
ATTGTTGGGT
GTGCCCATAT ACATATATTT TGTGATTCAT TCTACCGAGA GGACATAAAT660
GCAGTCACAG
CTCAGTAACA GTAAACACAC CAACTGCCAA GTTATTATTT CCTAAATACT720
ATCCACAAAA
AAGGGGACCA GGGATGATTC CTAGTCGGAG ATTGGGAGAA AAAGAAGATG780
AGCCTGAATC
4O ATTTCATGTA CCTAACAGAA AGAAAATACT CTGGCTGGGC TCAGTGGCTC840
ATGTTTGTAA
TTCTAGCATG TTAGGARGTC SARGTGGGTG TGTTGCTTGA GCCCAGGART900
TTGAGACCAG
CCCAGGCAAC ATGGCAATA 919
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1026 base pairs
{B) TYPE: nucleic acid
(C} STRANDEDNESS: double
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GGCACGAGGT TTTGTCTGCA GCACCTGCCA TGAACTCCTG GTTGACATGA TTTATTTTTG 60
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GAAGAAZGAG AAGCTATACT GTGGCAGACA TTACTGTGAC AGCGAGAAAC CCCGATGTGC 120
TGGCTGTGAC GAGCTGATAT TCAGCAATGA GTATACCCAG GCAGAAAACC180
AGAATTGGCA
S CCTGAAACAC TTCTGCTGCT T'IGACTGTGA TAGCATTCTA GCTGGGGAGA240
TATACGTGAT
GGTCAATGAC AAGCCCGTGT GCAAGCCCTG CTATGTGAAG AATCACGCTG300
TGGTGTGTCA
AGGAT'GCCAC AATGCCATCG ACCCAGAAGT GCAGCGCGTG ACCTATAACA360
ATTTCAGCTG
GCATGCATCC ACAGAGTGCT TTCTGTGCTC TTGCTGCAGC AAATGCCTCA420
TTGGGCAGAA
GTTCATGCCA GTAGAAGGGA TGGTTTTCTG TTCAGTGGAA TGTAAGAAGA480
GGATGTCTTA
IS GGAGGAGGGC ACCCAGAAGT ATCGAGCCAT AGCTATCCAA AGTGGTCTGC540
ATTTCTACTG
TAAAATGCAA TTTGAAAAAA ATAAAACGCA AAAP.AAGAAA CTGTAAAGGA600
AACCAAGAGA
TTTTGTTTAA TTTTTT'PGGC CATTTTTTCT TCATCAATTT TTTTTCGGTC660
TCAACTTTTA
AACTTGGTTT AAGCATTT'GA TTTGTAAAAC AGTAAATAAT TGTATCTTTC720
CATAGCTTTT
CAAATGTGAA ATCATTTTTG GAAGCT'IGGA TCTCATTAAA CTTCATGTCT780
CTATTCCATT
2S TGTGCCACAC ACTTAAAAGT TAGTGTACTG AATGGAAAGA TGAGCATTCC840
TAGTTCTACA
CTTCTTTTTT CCCCCTCATG TGTAAAATGA AAAGAAAACT AAATTTGCCC900
TAATACCAAG
GCGCTACGTT TATTGCCTCG TCTTATTCAC TGACCTTTTG TAATGATACA960
CAGTGAATTC
TTTTTTGACA AAGNGGAATG CGGTTTGGTA TGCAGAGCTG CTGGTTTTAA1020
-NGCCCATGGC
ATTNAC 1026
3S
(2) INFORMP.TION FOR SEQ ID NO: 13:
4O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2067 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
4S
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
AATTCGGCAC GAGCTTTTAA TAGGAAGTAA TGTCTCACCC AAGAGAAATG AAGAGCAGGG 60
SO AAGAGTGACT TTCTCCTTCT CCCTCCCTCT CCCCTGGATA TGGAACTCAA CCATTATGCA 120
CTGCTTCTTT TTGTGGTTGC TGCTTTTTGG ACTTCTZGGA ATTAGTGGTT TCCTTGGTTA 280
TATTTCAGTG GCTGGTARCA GTATATATGT CATGTGGAAG GTGGARAAGG AAATGAATAC 240
SS
TTAGGTCTCA AAGACCCACT CTCCATGGCT GCTTTAGCAG ATGGCTGTTT CTTTCTCTCC 300
CTTGCAGGTT GGGGATAGGA TTGTCACCAT CTG'TGGCACA TCCACTGAGG GCATGACTCA 360
C)O CACCCAAGCA GTTAACCTAC TGAAAAATGC ATCTuGCTCC ATTGAAATGC AGGTGGTTGC 420
__.~ . , r. .___ _ .._
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TGGAGGAGAC GTGAGTGTGG TCACAGGTCA TCAGCAGGAG CCTGCAAGTT480
CCAGTCTTTC
TTTCACTGGG CTGACGTCAA GCAGTATATT TCAGGATGAT TTAGGACCTC540
CTCAATGTAA
GTCTATTACA CTAGAGCGAG GACCAGATGG CTTAGGCTTC AGTATAGTTG600
GGAGGATATG
GGCAGCCCTC ATGGGAGACT TACCCATTTA TGTTAAAACA GTGTTTTGCA660
AAGGGGAGCA
" 10 GCCTCTGGAA GACGGACGTC TTGAAAAGGG GGGCGATTCC AGATCATTGC720
TGTCAATGGG
CAGAGTCTAG AWGGAGTCAC CCATGAAGAA GCTKTTGCCA TCCTTAAACG780
GACAAAAGGG
CACTGTCACT TTGATGGTTC TCTCTTGAAT TGGCTGCCAG AATTGAACCA840
ACCCAACCCC
IS
TAGCTCACCT CCTACTGTAA AGAGAATGCA CTGGTCCTGA CAATTTTTAT900
GCTGTGTTCA
GCCGGGTCTT CAAAACTGTA GGGGGGAAAT AACACTTAAG TTTCTTTTTC960
TCATCTAGAA
20 ATGCTTTCCT TACTGACAAC CTAACATCAT TTTTCTTTTC TTCTTGCATT1020
TTGTGAACTT
AAAGAGAAGG AATATTTGTG TAGGTGAATC TCGTTTTTAT TTGTGGAGAT1080
ATCTAATGTT
TTGTAGTCAC ATGGGCAAGA ATTATTACAT GCTAAGCTGG TTAGTATAAA1140
GAAAGATAAT
25
TCTAAAGCTA ACCAAAGAAA ATGGCTTCAG TAAATTAGGA TG,AAAAATGA1200
AAATATAAAA
TAAAGAAGAA AATCTCGGGG AGTTTAAAAA AAATGCCTCA ATTTGGCAAT1260
CTACCTCCTC
30 TCCCCACCCC AAACTAAAAA AARAAAAAAA GGTTTTCTAA TGAAAATCTT1320
TAAAAATACT
GTCAGTATTT TAAAATTTTC AACAGTATTA TAAAAACATT GCATCTCCCC1380
ACCTCTAATA
TGCATATATA TTTTTCCTGC TAAAATTGGT TTCTACAATT GAGTAAATGG1440
CAAATACATG
35
AAGCAATGTC CCTAAATTTT ATAAAGAAAT TATATTTAAT GCACATTTCA1500
ATTTTCATTC
TTATTTTTGA CCTTTTGTAA AATATTTTCA TGTTGCTATA AGTAAATGAT1560
GATGCCACCC
4O CAKGTTGACT ATGGKTTTTC TAGAAAGCAA CTATGCTGCT AACCATAGAG1620
GAACATAGAA
GGGTTCCAGA ATCTTTAGTG CTGGTTTTAA CAACCGATGC AACATTAAAA1680
ATGTGTTAGT
GTGCTGTGCA ATTGGTTTTC AATTCATATT AATCTTAATG ACAGAGAACA1740
ATGTGTTACT
45
AATTATTTTG GTTGTATGCC ATTAGTAAAT TGATAGAAAA ATTAAGGGGA1800
TTAACATAAC
TTCATTTCAT TGCCTTATAT TAACATCTTA TAATACAATA GTTTAAGACT1860
AAGGGAAACA
SO GATGGAGCTG TTTATTGAGA CAACTGGTGA GGAATTATCA TGTGTTCATT1920
CCCATTTTAG
AGCGTGAAAC TCCTACATTA GAATATATAA AGTCACTTTA AATATCTATA1980
TTTGTAACAG
AAGTAGTGTA CAGATATTTT ATTACAGCAT TTTTGTGTAA ATGCAGAATT2040
AAAGTGAATA
_ 55
AATAAGAATT TTCAGTGGTG CACAAAT 2067
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(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1341 base pairs
S (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GGCACGAGCT GGCCTGGACG AACGGGAAGC CGGGAGCTCG GCCACGGGTG60
GCGAGGCTGC
GGTGAGGCCT GGTCTCCGGC TGCCAGACCA TGCTGAGTGG AGCACGCTGC120
AGGCTCGCCT
IS CAGCGC'IGCG GGGAACGCGC GCGCCGCCGT CCGCGGTCGC CCGTAGTGCC180
TGCACGCGTC
GGGGTCGCGG CCTTTGGCAC CGGGGCAAGA AGACTGAGGA GCCGCCCCGC240
GACTTCGATC
CGGCGCTGCT GGAGTTCCTG GTGTGCCCGC TCTCCAAGAA GCCGCTCAGA300
TATGAAGCAT
CAACAAACGA ATTGATTAAT GAAGAGTTGG GAATAGCTTA TCCAATCATT36D
GATGC~GATCC
CTAATATGAT ACCACAGGCA GCTAGGATGA CACGTCAAAG TAAGAAGCAA420
GAAGAAGTGG
2S AGCAGCGCTA GTTCATAATT T~TT AAAAAAACGC AACAGCCAAC 480
TTTTCTTAAT
ACCATATACC TTTTAAAACA CAGTGGCAGG TAATAAGTGG AAGAGAAGAA540
TGTTTCTGTC
TCTTCCTACG TTGACTGTTC TTATTCCACT GGTTTCTTTA GCAGGACTGT600
TCTACTCAGC
CTCTGTGGAA GAAAACTTCC CACAGGGCTG CACTAGCACA GCCAGCCTTT660
GCTTTTACAG
CCTGCTCTTG CCTATTACCA TACCAGTGTA TGTATTCTTC CACCTTTGGA720
CTTGGATGGG
3S TATTAAACTC TTCAGGCATA ATTGATGCAA CTAGAGTCAA TATGCTGTAT780
ATATTAATGA
TAGCTCTTGG GCATCGATCT CTGAAAGCTC AAA'i'GGATGG AATTTAGTTT840
GCGGGAAAGA
GGCTTTGCTT TGCGCATATC AGGCTTAGGA CTGTGGGAGG CTTAAGTTGC900
AGATGCTTCT
TTTATTGTAC TCT'I'GTTCTG CCCTTGTTTT TTGAAGGCTC TGACTTATAA960
CTGCTGTATC
AGAAGAAACA TTTTGACAGT GTCTTGGTTG GAGATGAACA TCCCTAATTG ACATGTGATG 1020
4S ACTATTTCTT ATTCCATTCA TCTAAGAGTC ATTGAAATTT TGTTTTGCTT1080
GTTTGTTTAG
CTTCAAGGTC TTTGGTAAAG TCACATGTTA AGGATGACTG AAATAATTCC1140
AAAGGAGTGA
TGTTGGAATA GTCCCTCTAA GGGAGAGAAA TGCATTTGAA CGAATGTGAT1200
ATAAAACCAC
SO
ATAATCAAAT AGAAACTTCA TGTACTTACA AAAACTGAGT TTGTAAAATT1260
ACCTTCATTT
CTTTGACATT AAATGCTTAT ATTAGCAATA AACATGTTGA CACT'PTCCTA1320
TAAAAAAWAA
SS AAAAAAAAAA AAAAAAAAAT T 1341
C)0 (2) INFORMATION FOR SEQ ID NO: 15:
_.._.._ __ _._._r.._. __.. T ___ _ _..
CA 02286303 1999-10-12
WO 98/45712 PCT/US98/06801
105
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1443 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
' 10 GGCCGGGCGC TCCTGAAGCA GCAGTTATGG AGCTTCCCTC AGGGCCGGGG60
CCGGAGCGGC
TCTTTGACTC GCACCGGCTT CCGGGTGACT GCTTCCTACT GCTCGTGCTG120
CTGCTCTACG
CGCCAGTCGG GTT'CTGCCTC CTCGTCCTGC GCCTCTTTCT CGGGATCCAC180
GTCTTCCTGG
TCAGCTGCGC GCTGCCAGAC AGCGTCCTTC GCAGATTCGT AGTGCGGACC240
ATGTGTGCGG
TGCTAGGGCT CGTGGCCCGG CAGGAGGACT CCGGACTCCG GGATCACAGT300
GTCAGGGTCC
2O TCATTTCCAA CCATGTGACA CCTTTCGACC ACAACATAGT CAATTTGCTT360
ACCACCTGTA
GCACCCCTCT ACTCAATAGT CCCCCCAGCT TTGTGTGCTG GTCTCGGGGC420
TTCATGGAGA
TGAATGGGCG GGGGGAGTTG GTGGAGTCAC TCAAGAGATT CTGTGCTTCC480
ACGAGGCTTC
CCCCCACTCC TCTGCTGCTA TTCCCTGAGG AAGAGGCCAC CAATGGCCGG540
GAGGGGCTCC
TGCGCTTCAG TTCCTGGCCA TTTTCTATCC AAGATGTGGT ACAACCTCTT600
ACCCTGCAAG
3O TTCAGAGACC CCTGGTCTCT GTGACGGTGT CAGATGCCTC CTGGGTCTCA660
GAACTGCTGT
GGTCACTTTT CGTCCCTTTC ACGGTGTATC AAGTAAGGTG GCTTCGTCCT720
GTTCATCGCC
AACTAGGGGA AGCGAATGAG GAGTTTGCAC TCCGTGTACA ACAGCTGGTG780
GCCAAGGAAT
TGGGCCAGAC AGGGACACGG CTCACTCCAG CTGACAAAGC AGAGCACATG840
AAGCGACAAA
GACACCCCAG ATTGCGCCCC CAGTCAGCCC AGTCTTCTTT CCCTCCCTCC900
CCTGGTCCTT
CTCCTGATGT GCAAC2'CGCA ACTCTGGCTC AGAGAGTCAA GGAAGTTTTG960
CCCCATGTGC
CATTGGGTGT CATCCAGAGA GACCTGGCCA AGACTGGCTG TGTAGACTTG1020
ACTATCACTA
ATCTGCTTGA GGGGGCCGTA GCTTTCATGC CTGAAGACAT CACCAAGGGA1080
ACTCAGTCCC
TACCCACAGC CTCTGCCTCC AAGTTTCCCA GCTCTGGCCC GGTGACCCCT1140
CAGCCAACAG
CCCTAACATT TGCCAAGTCT TCCTGGGCCC GGCAGGAGAG CCTGCAGGAG1200
CGCAAGCAAG
JO CACTATATGA ATACGCAAGA AGGAGATTCA CAGAGAGACG AGCCCAGGAG1260
GCTGACTGAG
' CTCAAAGGAA CAGGATGGCA CCCAGAGCCG CAGGACGGAG ACTGGGGGCA1320
GCCCTCACCC
AACTCACAAC AGGCTGGATG GGTGGGTGGT AAAAAGGGAA GGATGAGGCT1380
CCCCCAATGT
CACATTAAAT TCATGGTTTT CATTCAAAAA AA,AAAAAAAA AAACTTCCGG1440
GGGGNGGCCC
CGT 1443
CA'02286303 1999-10-12
WO 98/45712 PCT/US98/06801
106
(2) INFORMATION FOR SEQ ID NO: 16:
S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 654 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
{xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GCGAATTCAT TTCCGANCTG AGGAAGCAAA AAACCCCGCC AACAATGTCA60
TTCCACCCAC
IS CAGTGCCACA ATGGGTCAGC TGTACCAGGA ACACCATGAA GAAGACTTCT120
TTCTCTACAT
TGCCTACAGT GACGAAAGTG TCTACGGTCT GTGAAGCTGC TGCCCCTGAG180
CTGGAGGGGG
GTCTCATTCT ACAAAGAGAG AGGTGGCCCC CCTTTCTTGA CCTCCTCCTC240
CTTCAAGCTC
AAACACCACC TCCCTTATTC AGGACCGGCA CTTCTTAATG TTTGTGGCTT300
TCTCTCCAGC
CTCTCTTAGG AGGGGTAATG GTGGAGTTGG CATCTTGTAA CTCTCCTTTC360
TCCTTTCTTC
2S CCCTTTCTCT GCCCGCCTTT CCCATCCTGC TGTAGACTTC TTGATTGTCA420
GTCTGTGTCA
CATCCAGTGA TTGTTTTGGT TTCTGTTCCC TTTCTGACTG CCCAAGGGGC480
TCAGAACCCC
AGCAATCCCT TCCTTTCACT ACCTTCTTTT TTGGGGGTAG TTGGAAGGGA540
CTGAAATTGT
GGGGGGAAGG TAGGAGGCAC ATCAATAAAG AGGAAACCAC CAAGCTGAAA600
p~~
AAP.AACTCGA GGGGGGGCCC GGTACCCATT GGCCCTAAGG GGGGGGNTTA654
NANT
3S
(2) INFORMATION FOR SEQ ID NO: 17:
4O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 749 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
4S
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
AAAGATGGCT GCGCCAGGTA ATTTGAGCAA AGGCCACAGT GAACTCCGGC GTGGCTGAGG 60
SO AAGGAGGAGG CACCCACAGG CTGCTGGGAG GAGAGCATAA GGCTCAAAAT GGAAAATCAT 120
AAATCCAATA ATAAGGAAAA CATAACAATT GTTGATATAT CCAGAAAAAT TAACCAGCTT 180
CCAGAAGCAG AAAGGAATCT ACTTGAAAAT GGATCGGTTT ATGTTGGATT AAATGCTGCT 240
SS
CTTTGTGGCC TCATAGCAAA CAGTCTTTTT CGACGCATCT TGAATGTGAC AAAGGCTCGC 300
ATAGCTGCTG GCTTACCAAT GGCAGGGATA CCTTTTCTTA CAACAGACTT AACTTACAGA 360
E)0 TGTTTTGTAA GTTTTCCTTT GAATACAGGT GATTTGGATT GTGAAACCTG TACCATAACA 420
CA 02286303 1999-10-12
WO 98/45712 PCT/US98106801
107
CGGAGTGGAC TGACTGGTCT TGTZ'ATTGGT GGTCTATACC CTGZ'T'M'CTT 480
GGCTATACCT
GTAAATGGTG GTCTAGCAGC CAGGTATCAA TCAGCTCTGT TACCACACAA 540
AGGGAACATC
S
. TTAAGTTACT GGATTAGAAC TTCTAAGCCT GTCTTTAGAA AGATGTTATT 600
TCCTATTTTG
CTCCAGACTA TGTTTTCAGC ATACCTTGGG TCTGAACAAT ATAAACTACT 660
TATAAAGGCC
' IO CTTCAGTTAT CTGAACCTGG CAAAGAAATT CACTGATTTT AAACAAATAT720
GTAAACAAAA
ATAAAATGGT AAAAACARAA AAAAAAAAA 749
1S
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 511 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
?S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
AATTCGGCAC GAGCCAGATT CCCATAAAGC ACATGGTCTA ATCTGTTACG60
TAACAGCAAG
ACAGCGTCAC CTCACCTGTT CTCGCCCTCA AATGGGAACG CTGGCCTGGG120
ACTAAAGCAT
30
AGACCACCAG GCTGAGTATC CTGACCTGAG TCATCCCCAG GGATCAGGAG180
CCTCCAGCAG
GGAACCTTCC ATTATATTCT TCAAGCAACT TACAGCTGCA CCGACAGTTG240
CGATGAAAGT
3S TCTAATCTCT TCCCTCCTCC TGTTGC'IGCC ACTAATGCTG ATGTCCATGG300
TCTCTAGCAG
CCTGAATCCA GGGGTCGCCA GAGGCCACAG GGACCGAGGC CAGGSTTCTA360
GGAGATGGCT
CCAGGAAGGC GGCCAAGAAT GTRAGTGCAA AGATTGGTTC CTGAGAGCCC420
GAGAAGAAAA
40
TTCATGACAG TGTCTGGGCT GCCAAAGAAR CARTGCCCNT GTGATTCTTT480
CAAGGGCATG
TGAAGAAAAC AAGNCACCAA AGGCACCACA G 511
4S
(2) INFORMATION FOR SEQ ID NO: 19:
SO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 689 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
SS
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GGCACGAGGA GGCGATGGCC CACGGGGCTG CTAGCCGTGC TGCGGCCCCT GCTCACCTGC 60
C)0 CGGCCCCTGC AAGGCACGAC GCTGCAACGG GATGTGCTGC TCTTTGAGCA TGATCGGGGC 220
CA'02286303 1999-10-12
WO 98/45712 PCT/US98/06801
108
CGCTTCTTCA CCATCCTCGG GCTGTTCTGC GCGGGCCAGG GCGTCTTCTG GGCTTCCATG 180
GCTG2GGCAG CCGTGTCCCG GCCCCCGGTT CCGGTGCAGC CTCTGGATGC240
GGAGGTCCCA
S
AATCG'I'GGCC CCTTCGACCT GCGCTCCGCG YTCTGGCGCT NACGGTCTGG300
CCGTCGGCTG
CGGCGCCATC GGAGCCCTCG TACTCGGTGC TGGTCTTCTC TTCTCTCTCC360
GGTCTG2GCG
IO CTCAGTGGTG CTTCGAGCTG GAGGGCAGCA GGTGACCCTC ACCACTCATG420
CCCCCTTTGG
a
CTTGC'~GGGCC CATTTCACAG TTCCTTTGAA GCAGGTATCT TGCATGGCCC480
ACCGGGGTGA
AGTCCCTGCC ATGCTACCTC TGAAAFCKCAA AGGCCGACGC TTCTATTTCC540
TCT'IGGACAA
1S
AACTGGACAC TTCCCYTAAC ACAAAACTYT TTGACAATAC TGTGGGTGCC600
TACCGGAGCT
TGTGAAGAAA TGACCTCAAG TCACTCACCT CTCCAAGAGG AGGATAAAAA660
CTGAACCTYG
Z0 GGGAGCCAGG TGTGTTGGTT CACACCTGT 689
?S (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1147 base pairs
(B) TYPE: nucleic acid
30 (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20.
3S TCGACCCACG CGTCCGGGCG GGCCTGTTTC CGGGAGGCGC GTGGGGCTTG60
AGGCCGAGAA
CGGCCNTTGC TGCCACCAAC ATGGAGACTT TGTACCGTGT CCCGTTCTTA120
GTGCTCGAAT
GTCCCAACCT GAAGCTGAAG AAGCCGCCCT GGTTGCACAT GCCGTCGGCC180
ATGACTGTGT
40
ATGCTCTGGT GGTGGTGTCT TACTTCCTCA TCACCGGAGG AATAATTTAT240
GATGTTATTG
TTGAACCTCC AAGTGTCGGT TCTATGACTG ATGAACATGG GCATCAGAGG300
CCAGTAGCTT
4S TCTI'GGCCTA CAGAGTAAAT GGACAATATA TTATGGAAGG ACTTGCATCC360
AGCTTCCTAT
TTACAATGGG AGGTTTAGGT TTCATAATCC TGGACCGATC GAATGCACCA420
AATATCCCAA
AACTCAATAG ATTCCTTCTT CTGTTCATTG GATTCGTCTG TGTCCTATTG480
AGTTTT2TCA
50
TGGCTAGAGT ATTCATGAGA ATGAAACTGC CGGGCTATCT GATGGGTTAG540
AGTGCCTTTG
AGAAGAAATC AGTGGATACT GGATTTGCTC CTGTCAATGA AGTTTTAAAG600
GCTGTACCAA
SS TCCTCTAATA TGAAATGTGG AAAAGAATGA AGAGCAGCAG TAAAAGAAAT660
ATCTAGTGAA
AAAACAGGAA GCGTATTGAA GCTTGGACTA GAATTTCTTC TTGGTATTAA720
AGAGACAAGT
TTATCACAGA ATTTTTTTTC CTGCTGGCCT ATTGCTATAC CAATGATGTT780
GAGTGGCATT
60
~...__ _..~_.._r.T. ,.. T.__~ ~__.._
CA 02286303 1999-10-12
WO 98/45712 PCT/US98/06801
109
TTCTTTTTAG TTTTTCATTA AAATATATTC CATATCTACA ACTATAATAT 840
CAAATAAAGT
GATTATTTTT TACAACCCTC TTAACATTTT TTGGAGATGA CATTTCTGAT 900
TTTCAGAAAT
S TAACATAAAA TCCAGAAGCA AGATTCCGTA AGCTGAGAAC TCTGGACAGT 960
TGATCAGCTT
TACCTATGGT GCTTTGCCTT TAACTAGAGT GTGTGATGGT AGATTATTTC 1020
AGATATGTAT
GTAAAACTGT TTCCTGAACA ATAAGATGTA TGAACGGAGC AGAAATAAAT 1080
ACTTTTTCTA
ATTAAAAAAA AAAAAAAAAA F~~AAAAAAAA F~AAAAP.AAAA AAAAAAAAAA1140
AAAAAAAAAA
AAAAAtVN 1147
1S
{2) INFORMATION FOR SEQ ID NO: 21:
2O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
{D) TOPOLOGY: linear
2S
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CTTTGTTCTC TTTCTTCTAC ACCAGCAGTT TTTATTTGCC TGTTTCCCAG AGTAATGTGG 60
3O GCCATGGAGT CAGGCCACCT CCTCTGGGCT CTGCTGTTCA TGCAGTCCTT GTGGCCTCAA 120
CTGACTGATG GAGCCACTCG AGTCTACTAC CTGGGCATCC GGGATGTGCA GTGGAACTAT 180
GCTCCCAAGG GAAGAAATGT CATCACGAAC CAGCCTCTGG ACAGTGACAT GTAGGTTTAA 240
3S
TTTCTTGTGG TATTTGAGGG GAAGTTATGG GAGCACTCTT GAGGTCAGGA AGTAGCCTCT 300
TGAGGCCCCT TTCCCARGGT GTGGTAGCAG CCAGCTCCTG ATTGCTCCGA GCTGTACATA 360
4O CTCAGTGGCA GATTTCCTGG GAAGAAGCTA GTTGAGTCAG AASCCAGCAT TTCATCTGGA 420
GTTTGSCGTA ACATTTTTAG AGTCCTAAAG ARAATTCCAT ATTTGCTGTT TTCTAATCTC 480
ATACCCACAA TGCTACTTAT TTAATAACAA CTGTTTGACT TTP,AAAAAAA AA 532
4S
(2) INFORMATION FOR SEQ ID N0: 22:
SO
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2743 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
SS (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
TGAACCCGCC CCTCTCCCAG AGTGGAGCTG CGGGGGGCGG GAACAGGCAC GGAGAAAATA 60
CAI02286303 1999-10-12
WO 98/45712 PCTIUS98/06801
110
AACAAGACTA AAAAGTCCTGAGTAGCGCTG TGTGGCCGCA AACCTGAACC 120
CACCTTTTGC
ACCACGCGGG ACCCGGCACTCTTCCTGCCA CCCACCCCTG AGAGGG~GCG 180
CGGCCGACCC
S CAGTACTAGA AAACACTCGTCACCTCACTC AAGACGGGTA CGAAGGCCAA 240
CGGACGCCTT
CCTTTAGAAC GCTCAGCACACAGAGCAACT TCTCACGCCT ACTCTCAAAT 300
GGCGTACTCC
AAACTAGCAC TCCCGACGTCCAGCTGTGAA CCCAGAGCGG CGGAAAGCCC 360
TGGAACCCAG
CGCCCGGGCA TGYGCAGACGCGTTGTTGTG GTGGGCGTGG CTCCCTCCGG 420
ACCCGGCGCC
CCGGCCTGCC GCCCCGTGTCCGCATGCGCG ACTGAGCCGG GTGGATGGTA 480
CTGCTGCATC
IS CGGGTGTCTG GAGGCTGTGG CCGTTTTGTT TTCTTGGCTA AAATCGGGGG540
AGTGAGGCGG
GCCGGCGCGG CGCGACACCG GGCTCCGGAA CCACTGCACG ACGGGGCTGG600
ACTGACCTGA
AAAPu~P.TGTC TGGATTTCTA GAGGGCTTGA GATGCTCAGA 660
ATGCATTGAC TGGGGGGAAA
AGCGCAATAC TATTGCTTCC ATTGCTGCTG GTGTACTATT TTTTACAGGC720
TGGTGGATTA
TCATAGATGC AGCTGTTATT TATCCCACCA TGAAAGATTT CAACCACTCA780
TACCATGCCT
2S GTGGT"TT'AT AGCAACCATA GCCTTCCTAA TGATTAATGC AGTATCGAAT840
GGACAAGTCC
GAGGTGATAG TTACAGTGAA GGTTGTCTGG GTCAAACAGG Tv~CTCGCATT900
TGGCTTTTCG
TTGGTTTCAT GTTGGCCTTT GGATCTCTGA TTGCATCTAT GTGGATTCTT960
TTTGGAGGTT
ATGTTGCTAA AGAAAAAGAC ATAGTATACC CTGGAATTGC TGTATTTTTC1020
CAGAATGCCT
TCATCTTTTT TGGAGGGCTG GTTTTTAAGT TTGGCCGCAC TGAAGACTTA1080
TGGCAGTGAA
3S CACATCTGAT TTCCCACAGC ACAACAGCCC TGCATGGGTT TGTTTGTTTT1140
TTTACTGCTC
ACTCCCAACC TTTTGTAATG CCATTTTCTA AACTTATTTC TGAGTGTAGT1200
CTCAGCTTAA
AGTTGTGTAA TACTAAAATC ACGAGAACAC CTAAACAACA ACCAAAAATC1260
TATTGTGGTA
TGCACTTGAT TAACTTATAA AATGTTAGAG GAAACTTTCA CATGAATAAT1320
TTTTGTCAAA
TTTTATCATG GTATAATTTG TAAAAATAAA AAGAAATTAC AAAAGAAATT1380
ATGGATTTGT
4S CAATGTAAGT ATTTGTCATA TCTGAGGTCC AAAACCACAA TGAAAGTGCT1440
CTGAAGATTT
AATGTGTTTA TTCAAATGTG GTCTCTTCTG TGTCAAATGT TAAATGAAAT1500
ATAAACATTT
TTTAGTTTTfi AAAATATTCC GTGGTCAAAA TTCTTCCTCA CTATAATTGG1560
TATTTACTTT
TACCAAAAAT TCTGTGAACA TGTAATGTAA CTGGCTTTTG AGGGTCTCCC1620
AAGGGGTGAG
TGGACGTGTT GGAAGAGAGA AGCACCATGG TCCAGCCACC AGGCTCCCTG1680
TGTCCCTTCC
SS ATGGGAAGGT CTTCCGCTGT GCCTCTCATT CCAAGGGCAG GAAGATGTGA1740
CTCAGCCATG
ACACGTGGTT CTGGTGGGAT GCACAGTCAC TCCACATCCA CCATTGAAGG1800
AAAGGAAAAA
AGGGCAGAGA CTTGACACTC CAGTCTTAGA CAGGGGACAA TTTCTTTGTA1860
GTTGTTCTGA
CA 02286303 1999-10-12
WO 98/45712 PCT/US98/06801
111
TAATAAACTG GGTZ'CCATGT TGCATTTCAT CTGTGTACTG AACCACTAGC1920
CTAATAGACC
AGCACGGTCA ACTAGAATGA CAAACATTAG CTACTGGGAA CTCTTGTTGT1980
CTCTTCCTCT
S TCAGAACTCT TGTCCCCCCA GGCTCCCCAC CTCTGCAAGA TGGGATACAC2040
TCTTCAGGAC
CACAGTTTGA AATGTCTTTC ATAAAATGTG CTACAATTAG CCTTCGGAAT2100
ATTTGCTGGG
TGATTTAAAT GTGTGGGTCT CATTTCCATG CTAGCCATGG TCATCTGACA2160
GTCTCTACRC
TGTGAATATT GCCTGGTGAT CAAGACTCTC CTCAAAGAAA TGACTTGCTG2220
TCATCCCACA
TGAACTCCTG ATGTTTTTTC TACAAAAGTC CATAAAATGT GAAAACTGGA2280
GAAGATCTTA
IS GAGGTTGAAG CCCACCTTCT CTTTTCACAT AGGAGGGAAC AGACCATGGA2340
AATTTAAACG
ACTTCCTCAC GGTCACAGAA CTAGTTTTTT AATCCTCAGG CAGTGCATCC2400
CCCCACCCTA
CAACTGAGCA CAACCTCTTT CCCCACAGTG CAATTCAGAA TAT~uCTCAGG2460
GAATGCCAGC
CACCTTGTAA AACTGCTGGG AGAAAAGCAT GATTCCCACA AGGACTAAGT2520
ATCAGTGATT
TGTAATTTTC CTGTTTTGTA TTATCTGCTT TGCTGATGTA GACAAGAGTT2580
AACTGAGTAG
2S CATGCTTTAT TAAGCATGAG AAAGAATCTT AAGAATTGTC AATAAAATTA2640
ACCCAAAACT
TTAATAATGT GTCTGTAACC AAGAAAATAT TGATAGCATC ATCCTAATGA2700
AACTAAACAT
TTATZ'I'TAAA CTTATTAAAT TGACTCTTAA ACTAAAAP.AA 2743
AAA
(2) INFORMATION FOR SEQ ID NO: 23:
3S
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 820 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
AATTCGGCAC GAGATCACAA GCTACTAGCG CTTCCATTAA CAATTCAAAT60
CCATCTACCT
4S
CTGAGCAGGC CTCTAATACT RCTTCAGCTG TCACCAGTAG CCAACCTTCC120
ACAGTGTCAG
AGACTTCAGC TACTCTTACA AGCAATAGTA CCACTGGCAC TTCTATAGGA180
GATGACTCAA
JO GGAGAACTAC ATCTAGTGCT GTAACGGAAA CTGGCCCTCC TGCAATGCCA240
AGGTTACCTT
" CCTGCTGTCC CCAGCACTCA CCATGTGGAG GGTCGTCACA GAACCACCAT300
GCATTAGGAC
ATCCTCATAC AAGTTGCTTT CAGCAGCATG GTCACCATTT TCAACATCAT360
CACCACCACC
_ SS
ACCATACTCC CCACTCAGAC CGCCGCCGCG CCGCCATCAT GGACACCAGC420
CGTGTGCAGC
CTATCAAGCT GGCCAGGGTC ACCAAGGTCC TGGGCAGGAC CGGTTCTCAG480
GGACAGTGCA
GO CGCAGGTGCG CGTGGAATTC ATGGACGACA CGAGCCGATC CATCATCCGC540
AATGTAAAAG
CA'02286303 1999-10-12
WO 98145712 PCT/US98/06801
112
GCCCCGTGCG CGAGGGCGAC GTGCTCACCC TTTTGGAGTC AGAGCGAGAA600
GCCCGGAGGT
TGCGCTGAGC TTGGCTGCTC GCTGGGTCTT GGATGTCGGG TTCGACCACT660
TGGCCGATGG
S
GAATGGTCTG TCACARTCTG CTCCTTTTTT TTGTCCGCCA CACGTAACTG720
AGATGCTCCT
TTAAATAAAG CGTTTGTGTT TCAAGTTAAA F,F~AAA~AA AAAAAAAAAA780
ACYCCGGGGG
IO GGGNCCCGGT ACCCATTGGC CCTfnFI'AGTGG GTCGTTTTTC 820
IS (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 995 base pairs
(B) TYPE: nucleic acid
20 (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
ZS ATTATGAATT CGGACGCAGA GGTGACAGTG GGAGCTGGCC TGGGCCAGGA60
CGGCAGGTGG
CCCTGGAGAT GGGAAAGTGT CTGTGTCGAG GCGCTGAGCT CTCTCTCTGT120
TTCTCCTTTT
TTCCTCTACT CCTTCCCCTT CACACCCCCG TGGCTGGAAG GAACCTCGGC180
TTCCCTGAAA
30
GCTTGGGGGT CCCACCCTTC TTACCCCACC CGGGAGGAAC GCCCAGGGCC240
CCGGGCTTGT
TTCTCCTCTT GTTTTCCTTT TGGGCAGTTT GATCACTGAT CGAGTAAGGA300
ATGACCTTTA
3S GATTGTGCGA CTTTTGTTTT TGTTTTTTTA AATTTTTTTA AACCAAGAAT360
GATTTCTCCT
GCTTCCTTCT CCTCACCATC TTCCCAGACG GAGTTCAAAG GCCACTTCTC420
AAGCAGCTTT
TGGCACCTTC AGCCTCAGAG TGGAATCTTT TAAAGACAGG ACCCCTATGT480
CCAGGAAAGG
40
GGAAAAGGAA CTTTGCCAAT GATAGTGACC ACAGCAAAAG CAAATAATAA540
TAATATTAAT
AATAATAAAG AGAAATAAAA TAATAAAATA AAARACAATA GCACAGCCCT600
TGTTGAGGTC
4S AGCAGGGAGG AGGGGCTGCC CGGAGTTGGG TCCTTGCCTG GATTTTGACA660
CAGCAACTTC
CTGTAGTGAG CACTTTGTAT GAATCGTGGA CTTCCTGTTC TCAAGGCGCA720
GGTATTTATT
CTGTATCTGT CTAGAGCACA CACCAAAATC CAACCTTCTA ATAAACATGA780
TGGCGCAGTC
SO
CCACTCCCTG CCTCGCCTGT NCCCCTATCC CCCCCAGGCC TGGGATCTTC840
AGGNGTCGGT
GTGGGGAGGG GCCCCTGCCC TCCTTGCCTT GATTTTGCTC CCCTGGGTCC900
AGCTGGTTCC
SS AGGCCTGTGA ATGTCAGTTC GTCGGGCACT GACTCCGTCT GCTCTTGGGC960
CTTGGGGTCA
TTTGACAAAT ATrTGCCCAA GGGCTCCCAA GNCCA 995
__ ___.____~._.r.~__.___ . r _ __
CA 02286303 1999-10-12
WO 98/45712 PCT/US98/06801
113
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
S (A) LENGTH: 649 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
IO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
GAATTCGGCA CGAGCACTTT AAGCACTATT CATTTTCCCT AATTTYTTCC60
ACTCACCGGG
AATTATTCAT TGTTCTCCTT CACTCCCTTG CTCAGACATG TKTCCCATGT120
CACCCCAGGG
1S
GAGGCTGTYA TGTCACAAGA TGTTGTTACT GATGATCTTG GAATTWTTTC180
CCTGCCCCCA
GCCTGGGACT AATGTAATTA TTATTTCAAT GTGCTTTTTC TTAAGCCATA240
GCAATGCAAT
2O TTATCTTCAA CATTATCATT TTTAAACATC TGTTAATTAT TAACAATTTA300
CTGCTYCTYC
TTGCACAAAA TGCTATTCCA GTAACATTTA TTAATTAAGT TATGTWCACA360
TACCAAAGAT
TTTACAGGCT TGTAAAATAG CAGGCCATTY CAAGGATTTC TCTCTTGGTA420
RAGAMATTTG
2S
TuJGGGAAAGA GTTATATAAT CACTAAATTA CATTCATATC AAGAACbCTT480
TTCCTGAGTG
AAATTAGTCT AGGTTTGCTT AAGTGTCTCT TTTTTATTTA ACTAAGNAAA540
TATCATGCCA
3O TATCTGTCTT ATATTGCTAT TATCTCTCCC TTCCGAGGAC CACATCTTCT600
GTTACAAGAG
GGAGACTGTG CTTCAAGGGA GTAGAAGAGA T~vGTTTCAGT ATATTTNNA649
3S
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
40 (A) LENGTH: 979 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
4S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
AGTTTCTTTG CCTTTCCTTG TAAATTTTAG AATAACCTTG TCTATATCTA60
CAGATCTTGC
TGGGATTTTG ATAAGAATTG CATTGATCCT TTATACCAAT TTGAGGAGGA120
TTTATGTACT
~0
TTTTGTCGAG TCTTTTAATC CATGAACATG TTATATCTGT TATATTTAGC180
ATTTTGATTT
TTTCCTCAGT GTTGCATAGT TTTCAGTATA CAAATCCTGT ACCCTTTTTT240
TTTAGATTTA
SS CACCTAGTAC TTTATTTTTT GAGCAATTGT AAATGGTATT GTATTTTAAA300
TTTCATTGCC
CATGTGTTCC ATTGCTAATA TACTGAAATA ARATTGGCTT TTGTATGTTT360
ATCTTGTATC
CCACAATCTT GCTGAACTCM CTTGTTCTAA RACTTTTTGT ARATTACGGG420
GAATTTCYAC
60
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ATARACAGTT ATGTCATCTG CAAATGGGGA TAGTTTTGTT TTTTCCTTTA480
CAAACTGTAT
ACTTTTTTTA TCTGTTTCTT GCTTTATTCC GAGAACTTCT AGAGCTGTGT540
TGAGTRATAG
S TGGATATCTT TGCCTTGTTC ATGTTCTTAT AGGGAAAGCA TTCAGTCTTT600
CACCATTTAG
TATAATGTTA GCTGTAGGGA CTTTTTAGAT CCCTTTACCA GATTGATGAA660
AGTTCCTCTC
TATTCCCATT TTTCTGAGAG GCTTTTTAAA AAAAGAATGA ATGGGTGTTC720
GATTTTGTCA
AATGCATTTC TGTGTCAGTT GCTATGAACA NGTGTTTTTC TTATTTAGCC780
TGTTAATATG
GTAGATTACA TTGATTGGCT TTCTGATCTT GAGTCAGCAT TGCATCCTTG840
GAATAAACTC
IS CACCTGGGTG TGGAGAANAA ATCTTTTTTT TTTTT~"I"I'TT 900
TTTANGAGAT GGAGTCTCGC
TCTGTCACCC AGGCTGGAGT GCAGTGGTGT GATTGTGGCT CACGGCAAGT960
TCCTCCTCCT
GGGTTCCCAT CATTCTCAA 979
(2) INFORMATION FOR SEQ ID NO: 27:
2S
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 905 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
CGACGGTTTC ACCTTGTTGG CCAGGCTGGT TTTGTTGGCC AATTGTCTCT60
AAACTGCTGT
CAAAAAAAGG AATGGATCAG ATTGTCTTGA ATAGGGCAGA GCTAACCTGT120
AATCACCTGT
GTGATGAGAA ASAGCTTTGA CTGCATTTTA CTCCTGACCT GGCCTAAGCT180
TTCTGTTTAC
4O ATAAGATTTT TCAAGAATTC AACTTCAAGT AGCAGCCGAG AGAGCTGCCT240
CAGGATTCTC
TCAAAAACTG GGAATAATAT GGGAACATTT GTTTCTTCTA AAAATAAGGC300
AAATGTTACA
TTGAATGATT TGGGGGGTGA GGTTTAATTG GAAATGGTCT CTGGGGACTG360
AAAACTGATG
4S
TTTTTGCAGA TTACCTCAGG GAAACGGAGG TTTGTTGAGT TTACAGACAC420
ATTAAACCAA
AGGCCGTGGG AAAACCCCTC TCCAGCTCCA GGGGAT'1'GGT 480
CAGGACCACC CACTAACCAG
SO TGCC'I"PCCTT CTTAACATTC ACTTTTAGCA GCTTGTGTTT 540
ATTTTACATG GGCAGTTTTG
ATGGGAAATT GCCATGACCA CAGGGGTTTG GAGTTCTGCT TTTTTTTTTT600
CTTCTTCTTT
TTCGGGGGAC TGGGGGACTC CTCCCAAGAT CACATTTTAG CATCTTTCTC660
TCCTACTCCA
SS
TTTAGAAAAA TAAGTAACAG GTGAAATGTG GTCTCAGTGT TAACGGGATA720
ATTCTGCTAC
CGGCTCCTCC CTGATGATTC TGAAATACAC TACT'GAACGA GCTCTGGCTG780
GTCCTTTCTA
6O TCCTGGATGT GGTTCTTCTG TGTAGCAATT CCTTGATGTC CAGTTTGGAA890
AGATGTACTC
_~___ ~ r. _..~ _~... _..._ _._
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TTCTCAACAA GAAAAACTTA AATCCGTCGT GCCCAAAAAA AAAAAAAAAA AAAAAAAAAA 900
CTCGA 905
S
(2) INFORMATION FOR SEQ ID NO: 28:
' 10
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 299 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
IS (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
GAATTCTTTT TTfTTTTTTT TTTTTTTGAC AGAGTCTCAC TGTGTCTCCC60
AGGCTGGAGT
20
GCAGCAGTAC AATCTCGGCT CTCTGCAACA TCTTCCTTCC AGGTTCAAGC120
GATTCTCATG
CCTCAGCCCC ACAAGTAGCT GGGATTACAG GCATGCATCA CCACACCCTG180
CTAATTTTTG
2S TATTTTTAGT AGAGACGGCG TTTCACCATC TTGGCCAGAC TGGTCTCAAA240
CTCCTGGCCT
CAAGTGATTC GTCTGCCTCA GCCTCCCAAA AAAAAAAAAA P~AAAAAAAAA299
AAGAATTCC
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
3S {A) LENGTH: 338 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
GAATTCTTCT TTTTTTTTTT TTYT GAGACAGGGG TCTTCCTCTG TTGCTCAGGC 60
TAAAGTGCAG TGGTGCAATC ATAGCTCACT GTGGCCTCTA CTTCCTGGTC TCAAGTGATC 120
ATCYTCCCTC AGCYTCCTAA GTACCTAGGA GTCATGCACC AACATGCCCA GCTAAGTATT 180
TTATTTTTGG TAGAGATAAG GTCTTGCTGT GTTGCCCAGG CTAGTCTCAA ATTCCTGGCC 240
SO TCAAGCAATC CTTCTGCCTT GGCCTCCCAA ATTGTTGGGT TTKACAGGCA TTAGCCKTTA 300
TGCTTGGSCC CCAGGTCCTT TTTTTTTAAA ANNZNAAA 338
SS
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
60 (A) LENGTH: 500 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
GAATTCTTTT TTTTTTTTTT TTTTCTCAGT ACCCACTACA GCATTATCCT60
TGTCTGCAGG
TTTGAGGATG GATCTGCTCC CAGGGAGGGC GAGACAAGTG CACCTAGGTT120
GCCGGAAGTT
GTACGCATCA CTTCTGCTGG TATCTGTTAG CCCAACCCAG GCCACGTGGA180
CTCTCCCAGA
TGCCAGAGAC CATGAGAAGA AGAAGAGAAA GGGCTTGGAG AAGGATGGAC240
CACTCACCAT
IS TTGCTGGAAT AAACAATACT GCAGTCCTTG TTTTAACACT TCATTTTCAT300
CATGCCACAC
TGTCGGTAAC TGAATAACGG CCACCCACAG ATGTCAGGGC TTATCCCTGG360
AACCCGGAAA
TGGTGTTTGC AGATGGGAGT GAAAGCAGGT CTTTGYAGAA GGGATCAAGT420
TAGGGATCTT
GAGATGGGAA GAATTTCCTG GATTGTCCTG CTAGACACTA P~AAAAAAAAA480
AAAAAAAAAA
GAATTCAAAA AGCTTCTCGA 500
(2) INFORMATION FOR SEQ ID NO: 31:
3O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Met Ile Tyr Tyr Phe Leu Lys Asn Gln Cys Gly Leu Pro Phe Leu Phe
1 5 10 15
Leu Leu Ile Pro Leu Phe Glu Phe Leu Cys Vai Ser Phe Ala Phe Pro
20 25 30
Ser Gln 5er Gly Gly Val Arg Pro Ala Leu Trp Asp Glu Arg Ser Cys
35 40 45
Gly Tyr Val Ser Ala Gly Thr Lys Arg Ala Glu Gly Glu Val Trp Lys
55 60
Gly Gln Gly Glu Glu Met Gly Ser Ile Val Lys Arg Leu Val Pro Leu
65 70 75 80
Ser Lys Tyr Val Glu Asn Asp Asp Gly Lys Val Ser Pro Cys
85 90
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
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(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met His Pro Gln Ser Ala Phe Cys Ala Leu Ala Ala Ala Asn Ala Ser
1 5 la 15
Leu Gly Arg Ser Ser Cys Gln
1~
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
IS (A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
2~ Met His Cys Phe Phe Leu Trp Leu Leu Leu Phe Gly Leu Leu Gly Ile
1 5 10 15
Ser Gly Phe Leu Gly Tyr Ile Ser Val Ala Gly Xaa Ser Ile Tyr Val
20 25 30
Met Trp Lys Val Glu Lys Glu Met Asn Thr
40
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 amino acids
(B) TYPE: amino acid
(Dl TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: 5EQ ID NO: 34:
Met Phe Leu Ser Leu Pro Thr Leu Thr Val Leu Ile Pro Leu Val Ser
1 5 10 15
Leu Ala Gly Leu Phe Tyr Ser Ala Ser Val Glu Glu Asn Phe Pro Gln
20 25 30
Gly Cys Thr Ser Thr Ala Ser Leu Cys Phe Tyr Ser Leu Leu Leu Pro
35 40 45
Ile Thr Ile Pro Val Tyr Val Phe Phe His Leu Trp Thr Trp Met Gly
55 60
Ile Lys Leu Phe Arg His Asn
65 70
$5
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 410 amino acids
(B) TYPE: amino acid
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(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Met Glu Leu Pro Ser Gly Pro Gly Pro Glu Arg Leu Phe Asp Ser His
$ 1 5 10 15
Arg Leu Pro Gly Asp Cys Phe Leu Leu Leu Val Leu Leu Leu Tyr Ala
20 25 30
Pro Val Gly Phe Cys Leu Leu Val Leu Arg Leu Phe Leu Gly Ile His
35 40 45
Val Phe Leu VaI Ser Cys Ala Leu Pro Asp Ser Val Leu Arg Arg Phe
50 55 60
IS
Val Val Arg Thr Met Cys Ala Val Leu Gly Leu Val Ala Arg Gln Glu
65 70 75 80
Asp Ser Gly Leu Arg Asp His Ser Val Arg Val Leu Ile Ser Asn His
2,0 85 90 95
Val Thr Pro Phe Asp His Asn Ile Val Asn Leu Leu Thr Thr Cys Ser
100 105 110
2 5 Thr Pro Leu Leu Asn Ser Pro Pro Ser Phe Val Cys Trp Ser Arg Gly
115 120 125
Phe Met Glu Met Asn Gly Arg Gly Glu Leu Val Glu Ser Leu Lys Arg
130 135 140
Phe Cys Ala Ser Thr Arg Leu Pro Pro Thr Pro Leu Leu Leu Phe Pro
145 150 155 160
Glu Glu Glu Ala Thr Asn Gly Arg Glu Gly Leu Leu Arg Phe Ser Ser
165 170 175
Trp Pro Phe Ser Ile Gln Asp Val Val Gln Pro Leu Thr Leu G1n Val
180 185 190
Gln Arg Pro Leu Val Ser Val Thr Val Ser Asp Ala Ser Trp Val Ser
195 200 205
Glu Leu Leu Trp Ser Leu Phe Val Pro Phe Thr Val Tyr Gln Val Arg
210 215 220
Trp Leu Arg Pro Val His Arg Gln Leu Gly Glu Ala Asn Glu Glu Phe
225 230 235 240
Ala Leu Arg Val Gln Gln Leu Val Ala Lys Glu Leu Gly Gln Thr Gly
245 250 255
Thr Arg Leu Thr Pro Ala Asp Lys Ala Glu His Met Lys Arg Gln Arg
260 265 270
5 5 His Pro Arg Leu Arg Pro Gln Ser Ala Gln Ser Ser Phe Pro Pro Ser
275 280 285
Pro Gly Pro Ser Pro Asp Val Gln Leu Ala Thr Leu Ala Gln Arg Val
290 295 300
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Lys Glu Val Leu Pro His Val Pro Leu Gly Val Ile Gln Arg Asp Leu
305 310 315 320
Ala Lys Thr Gly Cys Val Asp Leu Thr Ile Thr Asn Leu Leu Glu Gly
325 330 335
Ala ValAla Phe ProGluAspIle LysGlyThr Ser
Met Thr Gln Leu
340 345 350
1~ Pro ThrAla Ser SerLysPhePro SerGlyPro Thr
Ala Ser Val Pro
355 360 365
Gln ProThr Ala ThrPheAlaLys SerTrpAla Gln
Leu Ser Arg Glu
370 375 380
Ser LeuGln Glu LysGlnAlaLeu GluTyrAla Arg
Arg Tyr Arg Arg
385 390 395 400
Phe ThrGlu Arg AlaGlnGluAla
Arg Asp
2~ 405 410
(2) INFORMATION FOR 5EQ ID N0: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
3U (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Met Val Glu Leu Ala Ser Cys Asn Ser Pro Phe Ser Phe Leu Pro Leu
1 5 10 15
Ser Leu Pro Ala Phe Pro Ile Leu Leu
20 25
4O (2I INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Met Leu Phe Pro Ile Leu Leu Gln Thr Met Phe Ser A1a Tyr Leu Gly
1 5 10 15
Ser Glu Gln Tyr Lys Leu Leu Ile Lys Ala Leu Gln Leu Ser Glu Pro
~ 20 25 30
Gly Lys Glu Ile His
35
(2) INFORMATION FOR SEQ ID NO: 38:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
Met Lys Val Leu Ile Ser Ser Leu Leu Leu Leu Leu Pro Leu Met Leu
1 5 10 15
Met Ser Met Val Ser Ser Ser Leu Asn Pro Gly Val Ala Arg Gly His
25 30
Arg Asp Arg Gly Gln Xaa Ser Arg Arg Trp Leu Gln Glu Gly Gly Gln
35 40 45
1S
Glu Cys Xaa Cys Lys Asp Trp Phe Leu Arg Ala Arg Glu Glu Asn Ser
50 55 60
(2) INFORMATION FOR SEQ ID NO: 39:
2S
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
3O (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 39:
Met Cys Cys Ser Leu Ser Met Ile Gly Ala Ala Ser Ser Pro Ser Ser
1 5 10 15
3S Gly Cys Ser Ala Arg Ala Arg Ala Ser Ser Gly Leu Pro Trp Leu Trp
20 25 30
Gln Pro Cys Pro Gly Pro Arg Phe Arg Cys Ser Leu Trp Met Arg Arg
35 40 45
Ser Gln Ile Val Ala Pro Ser Thr Cys Ala Pro Arg Ser Gly Ala Xaa
55 60
Gly Leu Ala Val Gly Cys Gly Ala Ile Gly Ala Leu Val Leu Gly Ala
4S 65 70 75 BO
Gly Leu Leu Phe Ser Leu Arg Ser Val Arg Ser Val Val Leu Arg Ala
85 90 95
J0 Gly Gly Gln Gln Val Thr Leu Thr Thr His Ala Pro Phe Gly Leu Gly
100 105 110
Ala His Phe Thr Val Pro Leu Lys Gln Val Ser Cys Met Ala His Arg
115 120 125
SS
Gly Glu Val Pro Ala Met Leu Pro Leu Lys Xaa Lys Gly Arg Arg Phe
130 135 140
Tyr Phe Leu Leu Asp Lys Thr Gly His Phe Pro
145 150 155
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(2) INFORMATION FOR SEQ ID NO: 40:
_ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
IO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
Met Thr Val Tyr Ala Leu Val Val Val Ser Tyr Phe Leu Ile Thr Gly
1 5 10 15
IS Gly Ile Ile Tyr Asp Val Ile Val Glu Pro Pro Ser Val Gly Ser Met
20 . 25 30
Thr Asp Glu His Gly His Gln Arg Pro Val Ala Phe Leu Ala Tyr Arg
35 40 45
Val Asn Gly Gln Tyr Ile Met Glu Gly Leu Ala 5er Ser Phe Leu Phe
50 55 60
Thr Met Gly Gly Leu Gly Phe Ile Ile Leu Asp Arg Ser Asn Ala Pro
65 70 75 BO
Asn Ile Pro Lys Leu Asn Arg Phe Leu Leu Leu Phe Ile Gly Phe Val
85 90 95
Cys Val Leu Leu Ser Phe Phe Met Ala Arg Val Phe Met Arg Met Lys
100 105 110
Leu Pro Gly Tyr Leu Met Gly
115
(2) INFORMATION FOR SEQ ID NO: 41:
4O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
Met Glu Ser Gly His Leu Leu Trp Ala Leu Leu Phe Met Gln Ser Leu
1 5 10 15
Trp Pro Gln Leu Thr Asp Gly Ala Thr Arg Val Tyr Tyr Leu Gly Ile
'JO 20 25 30
" Arg Asp Val Gln Trp Asn Tyr Ala Pro Lys Gly Arg Asn Val Ile Thr
35 40 45
Asn Gln Pro Leu Asp Ser Asp Met
55
GO (2) INFORMATION FOR SEQ ID NO: 42:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Met Lys Asp Phe Asn His Ser Tyr His Ala Cys Gly Val Ile A1a Thr
1 5 10 15
Ile Ala Phe Leu Met Ile Asn Ala Val Ser Asn Gly Gln Val Arg Gly
25 30
Asp Ser Tyr Ser Glu Gly Cys Leu Gly Gln Thr Gly Ala Arg Ile Trp
IS 35 40 45
Leu Phe Va2 Gly Phe Met Leu Ala Phe Gly Ser Leu Ile Ala Ser Met
50 55 60
20 Trp Ile Leu Phe Gly Gly Tyr Val Ala Lys Glu Lys Asp Ile Val Tyr
65 70 75 80
Pro Gly Ile Ala Val Phe Phe Gln Asn Ala Phe Ile Phe Phe Gly Gly
85 90 95
Leu Val Phe Lys Phe Gly Arg Thr Glu Asp Leu Trp Gln
100 105
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Met Val Thr Ile Phe Asn Ile Ile Thr Thr Thr Thr Ile Leu Pro Thr
1 5 10 15
Gln Thr Ala Ala Ala Pro Pro Ser Txp Thr Pro Ala Val Cys Ser Leu
20 25 30
Ser Ser Trp Pro Gly Ser Pro Arg Ser Trp Ala Gly Pro Val Leu Arg
35 40 45
Asp Ser Ala Arg Arg Cys Ala Trp Asn Ser Trp Thr Thr Arg Ala Asp
55 60
Pro Ser Ser Ala Met
55
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(70 (B) TYPE: amino acid
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(D) TOPOLOGY: linear
(xi1 SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Met Gly Lys Cys Leu Cys Arg Gly Ala Glu Leu Ser Leu Cys Phe Ser
S 1 5 10 15
Phe Phe Pro Leu Leu Leu Pro Leu His Thr Pro Val Ala Gly Arg Asn
20 25 30
Leu Gly Phe Pro Glu Ser Leu Gly Val Pro Pro Phe Leu Pro His Pro
35 40 45
Gly Gly Thr Pro Arg Ala Pro Gly Leu Phe Leu Leu Leu Phe Ser Phe
50 55 60
Trp Ala Val
20
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
2S (B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Met Leu Leu Leu Met Ile Leu Glu Xaa Phe Pro Cys Pro Gln Pro Gly
30 1 5 10 15
Thr Asn Val Ile Ile Ile Ser Met Cys Phe Phe Leu Ser His Ser Asn
20 25 30
3S Ala Ile Tyr Leu Gln His Tyr His Phe
35 40
40 (2) INFORMATION FOR SEQ ID NO: 46.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
4S (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Met Leu Tyr Leu Leu Tyr Leu Ala Phe
1 5
S0
(2) INFORMATION FOR SEQ ID NO: 47:
SS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
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Met Gly Asn Cys His Asp His Arg Gly Leu Glu Phe Cys Phe Phe Phe
1 s is i5
Phe Phe Phe Phe Phe Gly Gly Leu Gly Asp Ser Ser Gln Asp His Ile
20 25 30
Leu Ala Ser Phe Ser Pro Thr Pro Phe Arg Lys Ile Ser Asn Arg
35 40 45
(2} INFORMATION FOR SEQ ID N0: 48:
(i) SEQUENCE CHARACTERISTICS:
IS (A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Met His His His Thr Leu Leu Ile Phe Val Phe Leu Val Glu Thr Ala
1 5 10 15
Phe His His Leu Gly Gln Thr Gly Leu Lys Leu Leu Ala Ser Ser Asp
20 25 30
2S
Ser Ser Ala Ser Ala Ser Gln Lys Lys Lys Lys Lys Lys Lys Lys Asn
35 40 45
Ser
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Met His Gln His Ala Gln Leu Ser Ile Leu Phe Leu Val Glu Ile Arg
1 5 10 15
4S Ser Cys Cys Val Ala Gln Ala Ser Leu Lys Phe Leu Ala Ser Ser Asn
20 25 30
Pro Ser Ala Leu Ala Ser Gln Ile Val Gly Phe Xaa Arg His
35 40 45
(2) INFORMATION FOR SEQ ID NO: 50:
SS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
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Met Asp His Ser Pro Phe Ala Gly Ile Asn Asn Thr Ala Val Leu Val
1 5 10 15
Leu Thr Leu His Phe His His Ala Thr Leu Ser Val Thr Glu
S 20 25 30
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
IS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
Cys Ala Gly Cys Asp Glu Leu Ile Phe Ser Asn Glu Tyr Thr Gln Ala
1 5 10 15
Glu Asn Gln Asn Trp His Leu Lys His Phe Cys Cys Phe Asp Cys Asp
20 25 30
Ser Ile Leu
2S
(2) INFORMATION FOR SEQ ID NO: 52:
3O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 159 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
3S
Ala Arg Gly Phe VaI Cys Ser Thr Cys His Glu Leu Leu Val Asp Met
1 5 10 15
Ile Tyr Phe Trp Lys Asn Glu Lys Leu Tyr Cys Gly Arg His Tyr Cys
20 25 30
Asp Ser Glu Lys Pro Arg Cys Ala Gly Cys Asp Glu Leu Ile Phe Ser
35 40 45
4S Asn Glu Tyr Thr Gln Ala Glu Asn Gln Asn Trp His Leu Lys His Phe
SO 55 60
Cys Cys Phe Asp Cys Asp Ser Ile Leu Ala Gly Glu Ile Tyr Val Met
65 70 75 80
SO
Val Asn Asp Lys Pro Val Cys Lys Pro Cys Tyr Val Lys Asn His Ala
85 90 95
Val Val Cys Gln Gly Cys His Asn Ala Ile Asp Pro Glu Val Gln Arg
SS 100 105 110
Val Thr Tyr Asn Asn Phe Ser Trp His Ala Ser Thr Glu Cys Phe Leu
115 120 125
60 Cys Ser Cys Cys Ser Lys Cys Leu Ile Gly Gln Lys Phe Met Pro Val
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130 135 140
Glu Gly Met Val Phe Cys Ser Val Glu Cys Lys Lys Arg Met Ser
145 150 155
S
{2) INFORMATION FOR SEQ ID N0: 53:
(i) SEQUENCE CHARACTERISTICS.
{A) LENGTH: 93 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Gly Val Ala Arg Gly His Arg Asp Arg Gly Gln Ala Ser Arg Arg Trp
1 5 10 15
Leu Gln Glu Gly Gly Gln Glu Cys Glu Cys Lys Asp Trp Phe Leu Arg
20 25 30
Ala Pro Arg Arg Lys Phe Met Thr Val Ser Gly Leu Pro Lys Lys Gln
35 40 45
ZS Cys Pro Cys Asp His Phe Lys Gly Asn Val Lys Lys Thr Arg His Gln
50 55 60
Arg His His Arg Lys Pro Asn Lys His Ser Arg Ala Cys Gln Gln Phe
65 70 75 80
Leu Lys Gln Cys Gln Leu Arg Ser Phe Ala Leu Pro Leu
85 90
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
His Thr Gln Val Glu Phe Ile Pro Arg Met Gln Cys
1 5 10
(2) INFORMATION FOR SEQ ID NO. 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
SS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
Leu Lys Ile Arg Lys Pro Ile Asn Val Ile Tyr His Ile Asn Arg Leu
1 5 10 25
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127
(2) INFORMATION FOR SEQ ID NO: 56:
S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
Arg Lys Met Gly Ile Glu Arg Asn Phe His Gln Ser Gly Lys Gly Ile
1 5 10 15
1S
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
2S Lys Val Pro Thr Ala Asn Ile Ile Leu Asn Gly Glu Arg Leu Asn Ala
1 5 10 15
Phe Pro Ile Arg Thr
30
(2) INFORMATION FOR SEQ ID NO: 58:
3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Ile Phe Ser Ser Val Leu His Ser Phe Gln Tyr Thr Asn Pro Val Pro
1 5 10 15
Phe Phe Phe Arg Phe Thr Pro Ser Thr Leu Phe Phe
4S 20 25
(2) INFORMATION FOR SEQ ID NO: 59:
SO
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
SS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
Lys Val Pro Thr Ala Asn Ile Ile Leu Asn Gly Glu Arg Leu Asn Ala
1 5 10 15
60 Phe Pro Ile Arg Thr
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128
S (2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
10 (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Met Tyr Phe Leu Ser Ser Leu Leu Ile His Glu His Val Ile Ser Val
1 5 10 15
1$
Ile Phe Ser Ile Leu
20
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: 5EQ ID NO: 61:
Glu Asp Gly Ser Ala Pro Arg Glu Gly Glu Thr Ser Ala Pro Arg Leu
1 5 10 15
Pro Glu Val Val Arg Ile Thr Ser Ala Gly Ile Cys
20 25
