Note: Descriptions are shown in the official language in which they were submitted.
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GENE EXPRESSION MODULATED IN
GASTROINTESTINAL INFLAMMATION
REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application S.N.
60/138,487,
filed June 10, 1999, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Chronic inflammatory bowel diseases (IBD) are as a persistent problem in
medicine.
The two major types of IBD are Crohn's disease (CD), which can affect the
whole digestive
tract from mouth to anus, and ulcerative colitis (UC), which affects only the
large intestine.
Although CD and UC are both characterized by massive gut damage arising from
intestinal inflammation, these diseases are quite distinct. Macroscopically,
the best
distinguishing features of these diseases are that UC is 1) a continuous
inflammatory
disorder, 2) primarily restricted to the mucosa of the colon and rectum, 3)
usually primarily
vascular, and 4) usually associated with a striking shortening of the colon.
In contrast,
inflammation in CD is nearly always discontinuous and may present anywhere in
the large or
small intestine. With CD the mucosa usually appears 'cobblestoned' and there
is fissuring and
a thickening of the bowel wall with associated stenosis of the lumen and
strictures.
Microscopically, UC is essentially a superficial inflammation of the mucous
membrane and even in chronic disease, the muscularis propria and serosa are
free of
inflammatory infiltration. In contrast, CD presents microscopically as a
transmural
inflammation, spreading through the mucosa and submucosa into the muscle
layers. In UC,
crypt abscesses and destruction of the epithelium are common, whereas in CD, a
valuable
diagnostic feature is the presence of sarcoid type granulomas.
The histological features of both of these diseases suggest that there is a
dysregulation
of the lymphoid tissue in IBD. Whether the immunological dysregulation is a
primary cause
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of IBD or whether the inflammatory response is secondary to another mucosal
insult, such as
a breach in the epithelial barner, remains unclear.
Despite advances in recent years, the precise etiology and pathogenesis of CD
and UC
remain undefined. In order to try and better understand the mechanistic basis
of IBD, much
effort has been directed towards the discovery and development of different
animal models of
inflammatory bowel disease. One of the best characterized systems for studying
intestinal
inflammation is the mouse model of dextran sodium sulfate (DSS)-induced
colitis (I.
Okayasu et al, Gastroenterology 98:694702, 1990; H.S. Cooper et al, Laboratory
Investigation 69:238-249, 1993; L.A. Dielman et al, Gastroenterology 107:1643-
1652, 1994;
C.O. Elson et al, Gastroenterology 109:1344-1367, 1995).
DSS is thought to cause breaches in the epithelial tight junctions (J. Ni et
al, Gut
39:234-241, 1996), thus permitting the huge antigenic load in the gut lumen to
come into
direct contact with the underlying tissues. As a result, a vigorous immune
response directed
against antigens located in the gut lumen is initiated in DSS-treated mice.
The intestinal
inflammation is primarily restricted to the large intestine.
The DSS-induced colitis model system is used to examine (i) the nature of the
earliest
response to epithelial damage, (ii) the mechanisms responsible for recruiting
cells to the sites
of inflammation, (iii) the nature of the protective immune response at the
height of intestinal
inflammation and (iv) the mechanisms that direct recovery and trigger the
repair of damaged
tissues. This model system can be used to examine the mechanisms of induction
and
recovery of IBD and should aid the identification of genes/proteins that may
be able to
modulate or prevent intestinal damage or stimulate recovery of the mucosa.
Given the diversity of factors that may contribute to these processes, a clear
need is
evident for the discovery, identification and elucidation of the roles of new
proteins involved
in the different stages of IBD.
Features of DSS-induced colitis
In the DSS-induced colitis model system, weight loss is apparent in mice
beginning at
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day 4. Histological analysis of the intestine reveals the presence of early
patchy lesions
identifiable by the loss of epithelial cells and goblet cells. By day 8,
weight loss is fairly
severe (approximately 20% reduction) and the mice appear moribund.
Histologically, the gut
epithelium is almost totally destroyed at this stage. There is evidence of a
large mixed
inflammatory cell infiltration into the lamina propria and submucosa. The
inflammatory cell
infiltrate appears to be composed primarily of T cells, B cells and
granulocytes. By day 12,
weight gain is apparent as the mice recover. At this later stage, crypt
recovery and epithelial
regeneration provide histological evidence of the beginning of repair
processes.
DSS-induced colitis and human IBD
The primary event in DSS-induced colitis is epithelial cell damage, with
inflammation
associated with immune activation a secondary event. Whether epithelial cell
damage or
immune dysregulation is the triggering event in human IBD is unclear. The
model of DSS-
colitis is therefore useful in providing an opportunity to study the
development and
consequences of both processes.
SUMMARY OF THE INVENTION
Molecules have been identified that correspond to genes that are regulated by
the
DSS treatment. Such molecules are useful in therapeutic, prognostic and
diagnostic
applications in the treatment of IBD and other gut pathologies. The present
invention
provides novel polynucleotides and the encoded polypeptides. Moreover, the
present
invention relates to vectors, host cells, antibodies, and recombinant methods
for producing
the polynucleotides and the polypeptides. Also provided are diagnostic methods
for detecting
disorders related to the polypeptides and the polynucleotides encoding them,
and therapeutic
methods for treating such disorders. The invention further relates to
screening methods for
identifying binding partners of the polypeptides.
The present invention provides novel polynucleotides and the encoded
polypeptides.
Moreover, the present invention relates to vectors, host cells, antibodies,
and recombinant
methods for producing the polynucleotides and the polypeptides. Also provided
are
diagnostic methods for detecting disorders related to the polypeptides and the
polynucleotides
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encoding them, and therapeutic methods for treating such disorders. The
invention further
relates to screening methods for identifying binding partners of the
polypeptides.
In one embodiment, the invention provides an isolated nucleic acid molecule
comprising a polynucleotide chosen from the group consisting of SEQ ID NO:1,
SEQ >D
N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID
N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ >D N0:13, SEQ
ID N0:14, SEQ ID N0:15, SEQ >D N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID
N0:19,
SEQ >D N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID
N0:25, SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30,
SEQ >D N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID
N0:36, SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41,
SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID
N0:47, SEQ ID N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ )D N0:51, SEQ ID N0:52,
SEQ ID N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID
N0:58, SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61 and SEQ ID N0:62. Another
embodiment comprises an isolated nucleic acid molecule at least 95% identical
to the isolated
nucleic acid molecule of SEQ ID NO:1-62. A further embodiment comprises an
isolated
nucleic acid molecule at least ten bases in length that is hybridizable to the
isolated nucleic
acid molecule of SEQ ID NO:1-62 under stringent conditions.
In another embodiment, the invention provides an isolated polypeptide encoded
by a
polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID N0:2,
SEQ m
N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID N0:13, SEQ ID N0:14,
SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID
N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ B7 N0:25,
SEQ >D N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ >D N0:30, SEQ ID
N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ ID N0:36,
SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ >D
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47,
SEQ ID N0:48, SEQ ID N0:49, SEQ ID N0:50, SEQ ID NO:51, SEQ ID N0:52, SEQ >D
N0:53, SEQ ID N0:54, SEQ ID N0:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58,
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SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61 and SEQ ID N0:62. In another
embodiment, the invention provides an isolated nucleic acid molecule encoding
the
polypeptide of the present invention.
In a further embodiment, the invention provides a substantially pure isolated
DNA
molecule suitable for use as a probe for genes regulated in gastrointestinal
inflammation,
chosen from the group consisting of the DNA molecules identified in Table 1,
having a 5'
partial nucleotide sequence and length as described by their digital address,
and having a
characteristic regulation pattern in gastrointestinal inflammation.
The present invention also provides a system and method for detecting the
presence of
a gene regulated in gastrointestinal inflammation. In one embodiment, the
present invention
provides a kit for suitable for detecting the presence of a gene regulated in
gastrointestinal
inflammation, comprising at least one polynucleotide of the present invention,
or fragment
thereof having at least 10 contiguous bases, in an amount sufficient for at
least one assay;
label means; instructions for use; and suitable packaging material. In one
embodiment, the
polynucleotide is chosen from the group consisting of SEQ ID NO:1, SEQ ID
N0:2, SEQ ID
N0:3, SEQ ID N0:4, SEQ ID NO:S, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID NO:1 l, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14,
SEQ ID NO:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID
N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25,
SEQ ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID
N0:31, SEQ )D N0:32, SEQ ID N0:33, SEQ ID N0:34, SEQ ID N0:35, SEQ B7 N0:36,
SEQ ID N0:37, SEQ ID N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47,
SEQ ID N0:48, SEQ ID N0:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID N0:52, SEQ ID
N0:53, SEQ ID N0:54, SEQ ID NO:55, SEQ ID N0:56, SEQ ID N0:57, SEQ ID N0:58,
SEQ ID N0:59, SEQ ID N0:60, SEQ ID N0:61 and SEQ ID N0:62. Another embodiment
comprises a polynucleotide at least 95% identical to the isolated nucleic acid
molecule of
SEQ ID NO:1-62. A further embodiment comprises a polynucleotide at least ten
bases in
length that is hybridizable to the isolated nucleic acid molecule of SEQ ID
NO:1-62 under
stringent conditions. In yet another embodiment, the polynucleotide is chosen
from the group
consisting of the DNA molecules identified in Table 1, having a 5' partial
nucleotide
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sequence and length as described by their digital address, and having a
characteristic
regulation pattern in gastrointestinal inflammation.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention
will
become better understood with reference to the following description, appended
claims, and
accompanying drawings where:
Figure 1 is a graphical representation of the results of TOGA analysis (TOtal
Gene
expression Analysis) using a 5' PCR primer with parsing bases CGCG, showing
PCR
products produced from mRNA extracted from colons isolated from untreated mice
(Figure
1A, "0 Days"), mRNA extracted from colons isolated from mice receiving DSS for
four days
(Figure 1B, "4 Days"), mRNA extracted from colons isolated from mice receiving
DSS for
eight days (Figure 1 C, "8 Days"), and mRNA extracted from colons isolated
from mice
receiving DSS for twelve days (Figure 1D, "12 Days"), where the vertical index
line indicates
a PCR product of about 458 b.p. that is up-regulated by DSS treatment,
reaching a maximum
at eight days, where the ordinate is in arbitrary units of fluorescence
intensity and the abscissa
is length of PCR product in nucleotides;
Figure 2 is a graphical representation of more detailed analysis of the 458
b.p. PCR
product indicated in Figure 1; Figure 2A shows the PCR product obtained using
an extended
5' primer as described in the text; Figure 2B shows the PCR products obtained
using the
original PCR primers, and in Figure 2C, the traces from Figure 2A and 2B are
overlaid,
demonstrating that the PCR product of the isolated and sequenced clone is the
same length as
the original PCR product, where the ordinate is in arbitrary units of
fluorescence intensity and
the abscissa is length of PCR product in nucleotides;
Figure 3 is a graphical representation of the results of Northern Blot
analysis of clone
IMX 2 46, SEQ ID NO: 10, where an agarose gel containing poly A enriched mRNA
from
the four experimental samples (0, 4, 8 or 12 days DSS treatment) as well as
size standards
was blotted after electrophoresis and probed with radiolabelled IMX 2 46, SEQ
ID NO: 10,
imaged using a phosphorimager and quantified. Quantitative results showing the
relative
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expression levels of the 1.6 kb transcript were: 0 day, 64; 4 days, 53; 8
days, 223; and 12
days, 269. The amount of RNA loaded on the gel was determined by probing for
cyclophilin
(~~cyc~~).
Figure 4 is a graphical representation of the results of RT-PCR of clone IMX2
48.
Figure 5 is a graphical representation of the results of RT-PCR of clone
IMX2_74
Figure 6 is a graphical representation of the results of Northern blot
analysis of clone
IMX 2-17, SEQ ID NO: 3, where an agarose gel containing poly A enriched mRNA
from the
experimental samples and size standards was blotted after electrophoresis,
probed, imaged
using a phosphorimager and quantified. Figure 6A shows the results from
C57BL/6 mice
with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with aCD3
ileitis 0, 6, 30
or 72 hours after treatment, as well as samples from large intestines from
FVB, mdr knock-
out mice without colitis and mdr knock-out mice with colitis. Figure 6B shows
the results
from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c
mice treated
with 0%, 5% and 8% DSS, and Balb/c mice with aCD3 ileitis 0, 6, 30 or 72 hours
after
treatment. The predicted transcript size for IMX2_17 CRP-ductin is 6.6 Kb; the
actual
transcript size found in this study was approximately 6.5 Kb.
Figure 7 is a graphical representation of the results of Northern blot
analysis of clone
IMX 2 22, SEQ ID NO: 4, where an agarose gel containing poly A enriched mRNA
from the
experimental samples and size standards was blotted after electrophoresis,
probed, imaged
using a phosphorimager and quantified. Figure 7A shows the results from
C57BL/6 mice
with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with aCD3
ileitis 0, 6, 30
or 72 hours after treatment, as well as samples from large intestines from
FVB, mdr knock-
out mice without colitis, mdr knock-out mice with colitis and C57BL/6 spleen.
Figure 7B
shows the results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after
treatment, Balb/c
mice with aCD3 ileitis 0, 6, 30 or 72 hours after treatment, and C57BL/6
normal lymphoid
tissue samples (MLM, PP, spleen and thymus). The predicted transcript size for
IMX2 22
HPK1 is 2.7 Kb; the actual transcript size found in this study was
approximately 2.8 Kb.
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Figure 8 is a graphical representation of the results of Northern blot
analysis of clone
IMX 2 28, SEQ ID NO: 5, where an agarose gel containing poly A enriched mRNA
from the
experimental samples and size standards was blotted after electrophoresis,
probed, imaged
using a phosphorimager and quantified. Figure 8A shows the results from
C57BL/6 mice
S with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with aCD3
ileitis 0, 6, 30
or 72 hours after treatment, as well as samples from large intestines from
FVB, mdr knock-
out mice without colitis and mdr knock-out mice with colitis. Figure 8B shows
the results
from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c
mice treated
with 0%, 5% and 8% DSS, and Balb/c mice with aCD3 ileitis 0, 6, 30 or 72 hours
after
treatment. The predicted transcript size for IMX2 28 DRA is 2.6 Kb; the actual
transcript
size found in this study was approximately 3 Kb.
Figure 9 is a graphical representation of the results of Northern blot
analysis of clone
IMX 2 33, SEQ ID N0:21, where an agarose gel containing poly A enriched mRNA
from
the experimental samples and size standards was blotted after electrophoresis,
probed,
imaged using a phosphorimager and quantified. Figure 9A shows the results from
C57BL/6
mice with DSS colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with
aCD3 ileitis 0,
6, 30 or 72 hours after treatment, as well as samples from large intestines
from FVB, mdr
knock-out mice without colitis and mdr knock-out mice with colitis. Figure 9B
shows the
results from Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment,
Balb/c mice
treated with 0%, 5% and 8% DSS, and Balb/c mice with aCD3 ileitis 0, 6, 30 or
72 hours
after treatment. The predicted transcript size for IMX2 33 SLPI is 1.1 Kb; the
actual
transcript size found in this study was 1.1 Kb.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
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
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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:1-62. For example, the polynucleotide can
contain all or
part of 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.
A "polynucleotide" of the present invention also includes those
polynucleotides
capable of hybridizing, under stringent hybridization conditions, to sequences
contained in
SEQ ID NO:1-62, or the complement thereof, or the cDNA. "Stringent
hybridization
conditions" refers to an overnight incubation at 42° C in a solution
comprising 50%
formamide, Sx SSC (750 mM NaCI, 7~ mM sodium citrate), 50 mM sodium phosphate
(pH
7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 ~.g/ml denatured,
sheared salmon
sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides of
the present invention at lower stringency hybridization conditions. Changes in
the stringency
of hybridization and signal detection are primarily accomplished through the
manipulation of
formamide concentration (lower percentages of formamide result in lowered
stringency); salt
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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
NaHzPO:~; 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 lx SSPE, 0.1% SDS. In addition, to
achieve even lower
stringency, washes performed following stringent hybridization can be done at
higher salt
concentrations (e.g. 5x SSC).
Note that variations in the above conditions may be accomplished through the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in
hybridization experiments. Typical blocking reagents include Denhardt's
reagent, BLOTTO,
heparin, denatured salmon sperm DNA, and commercially available proprietary
formulations.
The inclusion of specific blocking reagents may require modification of the
hybridization
conditions described above, due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as
any
3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a
complementary
stretch of T (or U) residues, would not be included in the definition of
"polynucleotide," since
such a polynucleotide would hybridize to any nucleic acid molecule containing
a poly (A)
stretch or the complement thereof (e.g., practically any double-stranded cDNA
clone).
The polynucleotide of the present invention can be composed of any
polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or
DNA or
modified RNA or DNA. For example, polynucleotides can be composed of single-
and
double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions, single-
and double-stranded RNA, and RNA that is mixture of single- and double-
stranded regions,
hybrid molecules comprising DNA and RNA that may be single-stranded or, more
typically,
double-stranded or a mixture of single- and double-stranded regions. In
addition, the
polynucleotide can be composed of triple-stranded regions comprising RNA or
DNA or both
RNA and DNA. A polynucleotide may also contain one or more modified bases or
DNA or
RNA backbones modified for stability or for other reasons. "Modified" bases
include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications can
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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
1 S 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, formation
of pyroglutamate, formulation, gamma-carboxylation, glycosylation, GPI anchor
formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation,
proteolytic
processing, phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-
RNA mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol
182:626-646
(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
"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,
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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.).
The translated amino acid sequence, beginning with the methionine, is
identified
although other reading frames can also be easily translated using known
molecular biology
techniques. The polypeptides produced by the translation of these alternative
open reading
frames are specifically contemplated by the present invention.
SEQ 117 NO:1-62 and the translations of SEQ ID NO:1-62 are sufficiently
accurate
and otherwise suitable for a variety of uses well known in the art and
described further below.
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 the translations of SEQ ID NO:1-62 may be used to
generate
antibodies which bind specifically to the secreted proteins encoded by the
cDNA clones
identified.
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).
The present invention also relates to the genes corresponding to SEQ ID NO:1-
62,
and translations of SEQ ID NO:1-62. 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.
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Also provided in the present invention are species homologues. Species
homologues may be isolated and identified by making suitable probes or primers
from the
sequences provided herein and screening a suitable nucleic acid source for the
desired
homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood in
the art.
The polypeptides may be in the form of the secreted protein, including the
mature
form, or may be a part of a larger protein, such as a fusion protein (see
below). It is often
advantageous to include an additional amino acid sequence which contains
secretory or
leader sequences, pro-sequences, sequences which aid in purification, such as
multiple
histidine residues, or an additional sequence for stability during recombinant
production.
The polypeptides of the present invention are preferably provided in an
isolated form,
and preferably are substantially purified. A recombinantly produced version of
a
polypeptide, including the secreted polypeptide, can be substantially purified
by the one-step
method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of
the
invention also can be purified from natural or recombinant sources using
antibodies of the
invention raised against the secreted protein in methods which are well known
in the art.
Signal Seguences
Methods for predicting whether a protein has a signal sequence, as well as the
cleavage point for that sequence, are available. For instance, the method of
McGeoch, Virus
Res. 3:271-286 (1985), uses the information from a short N-terminal charged
region and a
subsequent uncharged region of the complete (uncleaved) protein. The method of
von
Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the
residues
surrounding the cleavage site, typically residues -13 to +2, where +1
indicates the amino
terminus of the secreted protein. Therefore, from a deduced amino acid
sequence, a signal
sequence and mature sequence can be identified.
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Polvnucleotide and Polvpeptide 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.
"Identity" per se has an art-recognized meaning and can be calculated using
published
techniques. (See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed.,
Oxford University Press, New York, (1988); BIOCOMPUTING: INFORMATICS AND
GENOME PROJECTS, Smith, D.W., ed., Academic Press, New York, (1993); COMPUTER
ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin, H.G., eds.,
Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR
BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE ANALYSIS
PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,
(1991).) While
there exists a number of methods to measure identity between two
polynucleotide or
polypeptide sequences, the term "identity" is well known to skilled artisans.
(Carillo, H., and
Lipton, D., SIAM J Applied Math 48:1073 (1988).) Methods commonly employed to
determine identity or similarity between two sequences include, but are not
limited to, those
disclosed in "Guide to Huge Computers," Martin J. Bishop, ed., Academic Press,
San Diego,
(1994), and Carillo, H., and Lipton, D., SIAM J Applied Math 48:1073 (1988).
Methods for
aligning polynucleotides or polypeptides are codified in computer programs,
including the
GCG program package (Devereux, J., et al., Nucleic Acids Research (1984)
12(1):387
(1984)), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J. Molec. Biol. 215:403
(1990),
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics
Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711
(using
the local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics
2:482-489 ( 1981 ).)
When using any of the sequence alignment programs to determine whether a
particular sequence is, for instance, 95% identical to a reference sequence,
the parameters are
set so that the percentage of identity is calculated over the full length of
the reference
polynucleotide and that gaps in identity of up to S% of the total number of
nucleotides in the
reference polynucleotide are allowed.
A preferred method for determining the best overall match between a query
sequence
(a sequence of the present invention) and a subject sequence, also referred to
as a global
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sequence alignment, can be determined using the FASTDB computer program based
on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990).) The term
"sequence"
includes nucleotide and amino acid sequences. In a sequence alignment the
query and
subject sequences are either both nucleotide sequences or both amino acid
sequences. The
result of said global sequence alignment is in percent identity. Preferred
parameters used in a
FASTDB search of a DNA sequence to calculate percent identity are:
Matrix=Unitary, k-
tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0,
and
Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, and Window Size=500 or
query
sequence length in nucleotide bases, whichever is shorter. Preferred
parameters employed to
calculate percent identity and similarity of an amino acid alignment are:
Matrix=PAM 150, k-
tuple=2, Mismatch Penalty= 1, Joining Penalty=20, Randomization Group
Length=0, Cutoff
Score=1, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query
sequence
length in amino acid residues, whichever is shorter.
1 S As an illustration, a polynucleotide having a nucleotide sequence of at
least 95%
"identity" to a sequence contained in SEQ >D NO:1-62 means that the
polynucleotide is
identical to a sequence contained in SEQ ID NO:1-62 or the cDNA except that
the
polynucleotide sequence may include up to five point mutations per each 100
nucleotides of
the total length (not just within a given 100 nucleotide stretch). In other
words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical to SEQ ID
NO:1-62, up to
5% of the nucleotides in the sequence contained in SEQ ID NO: l-62 or the cDNA
can be
deleted, inserted, or substituted with other nucleotides. These changes may
occur anywhere
throughout the polynucleotide.
Further embodiments of the present invention include polynucleotides having at
least
80% identity, more preferably at least 90% identity, and most preferably at
least 95%, 96%,
97%, 98% or 99% identity to a sequence contained in SEQ ID NO:1-62. Of course,
due to
the degeneracy of the genetic code, one of ordinary skill in the art will
immediately recognize
that a large number of the polynucleotides having at least 85%, 90%, 95%, 96%,
97%, 98%,
or 99% identity will encode a polypeptide identical to an amino acid sequence
contained in
the translations of SEQ ID NO:1-62.
Similarly, by a polypeptide having an amino acid sequence having at least, for
example, 95% "identity" to a reference polypeptide, is intended that the amino
acid sequence
of the polypeptide is identical to the reference polypeptide except that the
polypeptide
sequence may include up to five amino acid alterations per each 100 amino
acids of the total
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length of the reference polypeptide. In other words, to obtain a polypeptide
having an amino
acid sequence at least 95% identical to a reference amino acid sequence, up to
5% of the
amino acid residues in the reference sequence may be deleted or substituted
with another
amino acid, or a number of amino acids up to 5% of the total amino acid
residues in the
reference sequence may be inserted into the reference sequence. 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.
Further embodiments of the present invention include polypeptides having at
least
80% identity, more preferably at least 85% identity, more preferably at least
90% identity,
and most preferably at least 95%, 96%, 97%, 98% or 99% identity to an amino
acid sequence
contained in translations of SEQ ID NO:1-62. Preferably, the above
polypeptides should
exhibit at least one biological activity of the protein.
In a preferred embodiment, polypeptides of the present invention include
polypeptides
having at least 90% similarity, more preferably at least 95% similarity, and
still more
preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence
contained in
translations of SEQ ID NO:1-62.
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
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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 deleting 3, 8, or 27 amino-terminal
amino acid
residues. Similarly, Interferon gamma exhibited up to ten times higher
activity after deleting
8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et
al., J.
Biotechnology 7:199-216 (1988).)
Moreover, ample evidence demonstrates that variants often retain a biological
activity
similar to that of the naturally occurring protein. For example, Gayle and
coworkers (J. Biol.
Chem 268:22105-22111 (1993)) conducted extensive mutational analysis of human
cytokine
IL-1 a. They used random mutagenesis to generate over 3,500 individual IL- 1 a
mutants that
averaged 2.5 amino acid changes per variant over the entire length of the
molecule. Multiple
mutations were examined at every possible amino acid position. The
investigators found that
"[m]ost of the molecule could be altered with little effect on either [binding
or biological
activity]." (See Gayle et al., (1993), Abstract.) In fact, only 23 unique
amino acid sequences,
out of more than 3,500 nucleotide sequences examined, produced a protein that
significantly
differed in activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-
terminus of a polypeptide results in modification or loss of one or more
biological functions,
other biological activities may still be retained. For example, the ability of
a deletion variant
to induce and/or to bind antibodies which recognize the secreted form will
likely be retained
when less than the majority of the residues of the secreted form are removed
from the N-
terminus or C-terminus. Whether a particular polypeptide lacking N- or C-
terminal residues
of a protein retains such immunogenic activities can readily be determined by
routine
methods described herein and otherwise known in the art.
Thus, the invention further includes polypeptide variants which show
substantial
biological activity. Such variants include deletions, insertions, inversions,
repeats, and
substitutions selected according to general rules known in the art so as have
little effect on
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activity. For example, guidance concerning how to make phenotypically silent
amino acid
substitutions is provided in Bowie, J. U. et al., Science 247:1306-1310
(1990), wherein the
authors indicate that there are two main strategies for studying the tolerance
of an amino acid
sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by
natural
selection during the process of evolution. By comparing amino acid sequences
in different
species, conserved amino acids can be identified. These conserved amino acids
are likely
important for protein function. In contrast, the amino acid positions where
substitutions have
been tolerated by natural selection indicates that these positions are not
critical for protein
function. Thus, positions tolerating amino acid substitution could be modified
while still
maintaining biological activity of the protein.
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)
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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).)
Polvnucleotide and Polvpeptide Fragments
In the present invention, a "polynucleotide fragment" refers to a short
polynucleotide
having a nucleic acid sequence contained in that shown in SEQ ID NO:1-62. 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 that shown in SEQ ID NO:l-
62.
These nucleotide fragments are useful as diagnostic probes and primers as
discussed herein.
Of course, larger fragments (e.g., S0, 150, and more 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, to the end
of SEQ 1D
NO:l-62. 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 the translations of SEQ ID NO:1-62. 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
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acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region.
Moreover,
polypeptide fragments can be about 20, 30, 40, 50 or 60, 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. Similarly, polynucleotide fragments
encoding these
polypeptide fragments are also preferred.
Also preferred are polypeptide and polynucleotide fragments characterized by
structural or functional domains, such as fragments that comprise alpha-helix
and alpha-helix
forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-
forming regions,
coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha
amphipathic
regions, beta amphipathic regions, flexible regions, surface-forming regions,
substrate
binding region, and high antigenic index regions. Polypeptide fragments of the
translations
of SEQ ID NO:1-62 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
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defined as a part of a protein that elicits an antibody response. (See, for
instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).)
Fragments which function as epitopes may be produced by any conventional
means.
(See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985)
further
described in U.S. Patent No. 4,631,211.)
In the present invention, antigenic epitopes preferably contain a sequence of
at least
seven, more preferably at least nine, and most preferably between about 15 to
about 30 amino
acids. Antigenic epitopes are useful to raise antibodies, including monoclonal
antibodies,
that specifically bind the epitope. (See, for instance, Wilson et al., Cell
37:767-778 (1984);
Sutcliffe, J. G. et al., Science 219:660-666 (1983).)
Similarly, immunogenic epitopes can be used to induce antibodies according to
methods well known in the art. (See, for instance, Sutcliffe et al., supra;
Wilson et al., supra;
Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et
al., J. Gen.
Virol. 66:2347-2354 (1985).) A preferred immunogenic epitope includes the
secreted
protein. The immunogenic epitopes may be presented together with a carrier
protein, such as
an albumin, to an animal system (such as rabbit or mouse) or, if it is long
enough (at least
about 25 amino acids), without a Garner. 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
Additional embodiments include chimeric antibodies, e.g., humanized versions
of
murine monoclonal antibodies. Such humanized antibodies may be prepared by
known
techniques, and offer the advantage of reduced immunogenicity when the
antibodies are
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administered to humans. In one embodiment, a humanized monoclonal antibody
comprises
the variable region of a murine antibody (or just the antigen binding site
thereof) and a
constant region derived from a human antibody. Alternatively, a humanized
antibody
fragment may comprise the antigen binding site of a murine monoclonal antibody
and a
variable region fragment (lacking the antigen-binding site) derived from a
human antibody.
Procedures for the production of chimeric and further engineered monoclonal
antibodies
include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al.
(PNAS
84:3439, 1987), Larrick et al. (BiolTechnology 7:934, 1989), and Winter and
Hams (TIPS
14:139, May, 1993).
One method for producing an antibody comprises immunizing a non-human animal,
such as a transgenic mouse, with a polypeptide translated from a nucleotide
sequence chosen
from SEQ ID NO:I-62, whereby antibodies directed against the polypeptide
translated from a
nucleotide sequence chosen from SEQ ID NO:1-62 are generated in said animal.
Procedures
have been developed for generating human antibodies in non-human animals. The
antibodies
may be partially human, or preferably completely human. Non-human animals
(such as
transgenic mice) into which genetic material encoding one or more human
immunoglobulin
chains has been introduced may be employed. Such transgenic mice may be
genetically
altered in a variety of ways. The genetic manipulation may result in human
immunoglobulin
polypeptide chains replacing endogenous immunoglobulin chains in at least some
(preferably
virtually all) antibodies produced by the animal upon immunization. Antibodies
produced by
immunizing transgenic animals with a polypeptide translated from a nucleotide
sequence
chosen from SEQ ID NO:l-62 are provided herein.
Mice in which one or more endogenous immunoglobulin genes are inactivated by
various means have been prepared. Human immunoglobulin genes have been
introduced into
the mice to replace the inactivated mouse genes. Antibodies produced in the
animals
incorporate human immunoglobulin polypeptide chains encoded by the human
genetic
material introduced into the animal. Examples of techniques for production and
use of such
transgenic animals are described in U.S. Patents 5,814,318, 5,569,825, and
5,545,806, which
are incorporated by reference herein.
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Monoclonal antibodies may be produced by conventional procedures, e.g., by
immortalizing spleen cells harvested from the transgenic animal after
completion of the
immunization schedule. The spleen cells may be fused with myeloma cells to
produce
hybridomas, by conventional procedures.
A method for producing a hybridoma cell line comprises immunizing such a
transgenic animal with a immunogen comprising at least seven contiguous amino
acid
residues of a polypeptide translated from a nucleotide sequence chosen from
SEQ ID NO:1-
62; harvesting spleen cells from the immunized animal; fusing the harvested
spleen cells to a
myeloma cell line, thereby generating hybridoma cells; and identifying a
hybridoma cell line
that produces a monoclonal antibody that binds a polypeptide translated from a
nucleotide
sequence chosen from SEQ ID NO:1-62. Such hybridoma cell lines, and monoclonal
antibodies produced therefrom, are encompassed by the present invention.
Monoclonal
antibodies secreted by the hybridoma cell line are purified by conventional
techniques.
Antibodies may be employed in an in vitro procedure, or administered in vivo
to
inhibit biological activity induced by a polypeptide translated from a
nucleotide sequence
chosen from SEQ ID NO:1-62. Disorders caused or exacerbated (directly or
indirectly) by
the interaction of such polypeptides of the present invention with cell
surface receptors thus
may be treated. A therapeutic method involves in vivo administration of a
blocking antibody
to a mammal in an amount effective for reducing a biological activity induced
by a
polypeptide translated from a nucleotide sequence chosen from SEQ ID NO:1-62.
Also provided herein are conjugates comprising a detectable (e.g., diagnostic)
or
therapeutic agent, attached to an antibody directed against a polypeptide
translated from a
nucleotide sequence chosen from SEQ ID NO:1-62. Examples of such agents are
well
known, and include but are not limited to diagnostic radionuclides,
therapeutic radionuclides,
and cytotoxic drugs. The conjugates find use in in vitro or in vivo
procedures.
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,
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because secreted proteins target cellular locations based on trafficking
signals, the
polypeptides of the present invention can be used as targeting molecules once
fused to other
proteins.
Examples of domains that can be fused to polypeptides of the present invention
include not only heterologous signal sequences, but also other heterologous
functional
regions. The fusion does not necessarily need to be direct, but may occur
through linker
sequences.
Moreover, fusion proteins may also be engineered to improve characteristics of
the
polypeptide of the present invention. For instance, a region of additional
amino acids,
particularly charged amino acids, may be added to the N-terminus of the
polypeptide to
improve stability and persistence during purification from the host cell or
subsequent
handling and storage. Also, peptide moieties may be added to the polypeptide
to facilitate
purification. Such regions may be removed prior to final preparation of the
polypeptide. The
addition of peptide moieties to facilitate handling of polypeptides are
familiar and routine
techniques in the art.
Moreover, polypeptides of the present invention, including fragments, and
specifically
epitopes, can be combined with parts of the constant domain of immunoglobulins
(IgG),
resulting in chimeric polypeptides. These fusion proteins facilitate
purification and show an
increased half life in vivo. One reported example describes chimeric proteins
consisting of
the first two domains of the human CD4-polypeptide and various domains of the
constant
regions of the heavy or light chains of mammalian immunoglobulins. (EP A
394,827;
Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-
linked dimeric
structures (due to the IgG) can also be more efficient in binding and
neutralizing other
molecules, than the monomeric secreted protein or protein fragment alone.
(Fountoulakis et
al., J. Biochem. 270:3958-3964 (1995).)
Similarly, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion
proteins
comprising various portions of constant region of immunoglobulin molecules
together with
another human protein or part thereof. In many cases, the Fc part in a fusion
protein is
beneficial in therapy and diagnosis, and thus can result in, for example,
improved
pharmacokinetic properties. (EP-A 0 232 262.) Alternatively, deleting the Fc
part after the
3~ 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
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immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have been
fused with Fc portions for the purpose of high-throughput screening assays to
identify
antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-
58 (1995); K.
Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)
Moreover, the polypeptides of the present invention can be fused to marker
sequences, such as a peptide which facilitates purification of the fused
polypeptide. In
preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
CA,
91311), among others, many of which are commercially available. As described
in Gentz et
al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for
convenient purification of the fusion protein. Another peptide tag useful for
purification, the
"HA" tag, corresponds to an epitope derived from the influenza hemagglutinin
protein.
(Wilson et al., Cell 37:767 (1984).)
Thus, any of these above fusions can be engineered using the polynucleotides
or the polypeptides of the present invention.
Vectors. Host Cells, and Protein Production
The present invention also relates to vectors containing the polynucleotide of
the
present invention, host cells, and the production of polypeptides by
recombinant techniques.
The vector may be, for example, a phage, plasmid, viral, or retroviral vector.
Retroviral
vectors may be replication competent or replication defective. In the latter
case, viral
propagation generally will occur only in complementing host cells.
The polynucleotides may be joined to a vector containing a selectable marker
for
propagation in a host. Generally, a plasmid vector is introduced in a
precipitate, such
as a calcium phosphate precipitate, or in a complex with a charged lipid. If
the vector is a
virus, it may be packaged in vitro using an appropriate packaging cell line
and then
transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate
promoter,
such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac
promoters, the SV40
early and late promoters and promoters of retroviral LTRs, to name a few.
Other suitable
promoters will be known to the skilled artisan. The expression constructs will
further contain
sites for transcription initiation, termination, and, in the transcribed
region, a ribosome
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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, Bowes melanoma cells
and plant
cells. Appropriate culture mediums and conditions for the above-described host
cells are
known in the art.
1 S Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-
9,
available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA,
PNHl6a,
pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK223-
3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among
preferred
eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, 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.
Introduction of the construct into the host cell can be effected by calcium
phosphate
transfection, DEAE-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.
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Polypeptides of the present invention, and preferably the secreted form, can
also be
recovered from: products purified from natural sources, including bodily
fluids, tissues and
cells, whether directly isolated or cultured; products of chemical synthetic
procedures; and
products produced by recombinant techniques from a prokaryotic or eukaryotic
host,
including, for example, bacterial, yeast, higher plant, insect, and mammalian
cells.
Depending upon the host employed in a recombinant production procedure, the
polypeptides
of the present invention may be glycosylated or may be non-glycosylated. In
addition,
polypeptides of the invention may also include an initial modified methionine
residue, in
some cases as a result of host-mediated processes. Thus, it is well known in
the art that the
N-terminal methionine encoded by the translation initiation codon generally is
removed with
high efficiency from any protein after translation in all eukaryotic cells.
While the N-
terminal methionine on most proteins also is efficiently removed in most
prokaryotes, for
some proteins, this prokaryotic removal process is inefficient, depending on
the nature of the
amino acid to which the N-terminal methionine is covalently linked.
Uses of the Polvnucleotides
Each of the polynucleotides identified herein can be used in numerous ways as
reagents. The following description should be considered exemplary and
utilizes known
techniques.
The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers, since few
chromosome marking reagents, based on actual sequence data (repeat
polymorphisms), are
presently available. Each polynucleotide of the present invention can be used
as a
chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the sequences shown in SEQ ID NO:I-62. 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:1-62 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
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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 SO-
S00 potential causative genes.
Thus, once coinheritance is established, differences in the polynucleotide and
the corresponding gene between affected and unaffected individuals can be
examined. The
polynucleotides of SEQ ID NO:1-62 can be used for this analysis of individual
humans.
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
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several normal individuals is required to distinguish the mutation from a
polymorphism. If a
new polymorphism is identified, this polymorphic polypeptide can be used for
further linkage
analysis.
Furthermore, increased or decreased expression of the gene in affected
individuals as
compared to unaffected individuals can be assessed using polynucleotides of
the present
invention. Any of these alterations (altered expression, chromosomal
rearrangement, or
mutation) can be used as a diagnostic or prognostic marker.
In addition to the foregoing, a polynucleotide can be used to control gene
expression
through triple helix formation or antisense DNA or RNA. Both methods rely on
binding of
the polynucleotide to DNA or RNA. For these techniques, preferred
polynucleotides are
usually 20 to 40 bases in length and complementary to either the region of the
gene involved
in transcription (triple helix - see Lee et al., Nucl. Acids Res. 6:3073
(1979); Cooney et al.,
Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991) ) or to the
mRNA itself
(antisense - Okano, J. Neurochem. 56:560 ( 1991 ); Oligodeoxy-nucleotides as
Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).) Triple helix
formation
optimally results in a shut-off of RNA transcription from DNA, while antisense
RNA
hybridization blocks translation of an mRNA molecule into polypeptide. Both
techniques are
effective in model systems, and the information disclosed herein can be used
to design
antisense or triple helix polynucleotides in an effort to treat disease.
Polynucleotides of the present invention are also useful in gene therapy. One
goal of
gene therapy is to insert a normal gene into an organism having a defective
gene, in an effort
to correct the genetic defect. The polynucleotides disclosed in the present
invention offer a
means of targeting such genetic defects in a highly accurate manner. Another
goal is to insert
a new gene that was not present in the host genome, thereby producing a new
trait in the host
cell.
The polynucleotides are also useful for identifying individuals from minute
biological
samples. The United States military, for example, is considering the use of
restriction
fragment length polymorphism (RFLP) for identification of its personnel. In
this technique,
an individual's genomic DNA is digested with one or more restriction enzymes,
and probed
on a Southern blot to yield unique bands for identifying personnel. This
method does not
suffer from the current limitations of "Dog Tags" which can be lost, switched,
or stolen,
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making positive identification difficult. The polynucleotides of the present
invention can be
used as additional DNA markers for RFLP.
The polynucleotides of the present invention can also be used as an
alternative to
RFLP, by determining the actual base-by-base DNA sequence of selected portions
of an
individual's genome. These sequences can be used to prepare PCR primers for
amplifying
and isolating such selected DNA, which can then be sequenced. Using this
technique,
individuals can be identified because each individual will have a unique set
of DNA
sequences. Once an unique ID database is established for an individual,
positive
identification of that individual, living or dead, can be made from extremely
small tissue
samples.
Forensic biology also benefits from using DNA-based identification techniques
as
disclosed herein. DNA sequences taken from very small biological samples such
as tissues,
e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be
amplified using PCR.
In one prior art technique, gene sequences amplified from polymorphic loci,
such as DQa
class II HLA gene, are used in forensic biology to identify individuals.
(Erlich, H., PCR
Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are
amplified,
they are digested with one or more restriction enzymes, yielding an
identifying set of bands
on a Southern blot probed with DNA corresponding to the DQa class H HLA gene.
Similarly, polynucleotides of the present invention can be used as polymorphic
markers for
forensic purposes.
There is also a need for reagents capable of identifying the source of a
particular
tissue. Such need arises, for example, in forensics when presented with tissue
of unknown
origin. Appropriate reagents can comprise, for example, DNA probes or primers
specific to
particular tissue prepared from the sequences of the present invention. Panels
of such
reagents can identify tissue by species and/or by organ type. In a similar
fashion, these
reagents can be used to screen tissue cultures for contamination.
In the very least, the polynucleotides of the present invention can be used as
molecular weight markers on Southern gels, as diagnostic probes for the
presence of a
specific mRNA in a particular cell type, as a probe to "subtract-out" known
sequences in the
process of discovering novel polynucleotides, for selecting and making
oligomers for
attachment to a "gene chip" or other support, to raise anti-DNA antibodies
using DNA
immunization techniques, and as an antigen to elicit an immune response.
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Uses of the Polvpeptides
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
('ZSI,'Z'I), carbon
('4C), sulfur (3SS), tritium (3H), indium ("ZIn), and technetium (99"'Tc), and
fluorescent labels,
1 S 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,'3'I, "ZIn,
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
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Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A.
Rhodes,
eds., Masson Publishing Inc. ( 1982).)
Thus, the invention provides a diagnostic method of a disorder, which involves
(a)
assaying the expression of a polypeptide of the present invention in cells or
body fluid of an
individual; (b) comparing the level of gene expression with a standard gene
expression level,
whereby an increase or decrease in the assayed polypeptide gene expression
level compared
to the standard expression level is indicative of a disorder.
Moreover, polypeptides of the present invention can be used to treat disease.
For example, patients can be administered a polypeptide of the present
invention in an effort
to replace absent or decreased levels of the polypeptide (e.g., insulin), to
supplement absent
or decreased levels of a different polypeptide (e.g., hemoglobin S for
hemoglobin B), to
inhibit the activity of a polypeptide (e.g., an oncogene), to activate the
activity of a
polypeptide (e.g., by binding to a receptor), to reduce the activity of a
membrane bound
receptor by competing with it for free ligand (e.g., soluble TNF receptors
used in reducing
inflammation), or to bring about a desired response (e.g., blood vessel
growth).
Similarly, antibodies directed to a polypeptide of the present invention can
also be
used to treat disease. For example, administration of an antibody directed to
a polypeptide of
the present invention can bind and reduce overproduction of the polypeptide.
Similarly,
administration of an antibody can activate the polypeptide, such as by binding
to a
polypeptide bound to a membrane (receptor).
At the very least, the polypeptides of the present invention can be used as
molecular
weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns
using
methods well known to those of skill in the art. Polypeptides can also be used
to raise
antibodies, which in turn are used to measure protein expression from a
recombinant cell, as a
way of assessing transformation of the host cell. Moreover, the polypeptides
of the present
invention can be used to test the following biological activities.
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
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diseases associated with the biological activity. Thus, the polynucleotides
and polypeptides
could be used to treat the associated disease.
Immune Activitiy
A polypeptide or polynucleotide of the present invention may be useful in
treating
deficiencies or disorders of the immune system, by activating or inhibiting
the proliferation,
differentiation, or mobilization (chemotaxis) of immune cells. Immune cells
develop through
a process called hematopoiesis, producing myeloid (platelets, red blood cells,
neutrophils,
and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent
stem cells.
The etiology of these immune deficiencies or disorders may be genetic,
somatic, such as
cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or
toxins), or
infectious. Moreover, a polynucleotide or polypeptide of the present invention
can be used as
a marker or detector of a particular immune system disease or disorder.
A polynucleotide or polypeptide of the present invention may be useful in
treating or
detecting deficiencies or disorders of hematopoietic cells. A polypeptide or
polynucleotide of
the present invention could be used to increase differentiation and
proliferation of
hematopoietic cells, including the pluripotent stem cells, in an effort to
treat those disorders
associated with a decrease in certain (or many) types hematopoietic cells.
Examples of
immunologic deficiency syndromes include, but are not limited to: blood
protein disorders
(e.g. agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common
variable
immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection,
leukocyte
adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,
severe
combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,
thrombocytopenia,
or hemoglobinuria.
Moreover, a polypeptide or polynucleotide of the present invention could also
be used to modulate hemostatic (the stopping of bleeding) or thrombolytic
activity (clot
formation). For example, by increasing hemostatic or thrombolytic activity, a
polynucleotide
or polypeptide of the present invention could be used to treat blood
coagulation disorders
(e.g., 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
3~ treatment of heart attacks (infarction), strokes, or scarnng.
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A polynucleotide or polypeptide of the present invention may also be useful in
treating or detecting autoimmune disorders. Many autoimmune disorders result
from
inappropriate recognition of self as foreign material by immune cells. This
inappropriate
recognition results in an immune response leading to the destruction of the
host tissue.
Therefore, the administration of a polypeptide or polynucleotide of the
present invention that
inhibits an immune response, particularly the proliferation, differentiation,
or chemotaxis of
T-cells, may be an effective therapy in preventing autoimmune disorders.
Examples of autoimmune disorders that can be treated or detected by the
present
invention include, but are not limited to: Addison's Disease, hemolytic
anemia,
antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic
encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple
Sclerosis,
Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,
Polyendocrinopathies, Purpura, Reiter's Disease, Stiff Man Syndrome,
Autoimmune
Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune
inflammatory
eye disease.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic
asthma) or other respiratory problems, may also be treated by a polypeptide or
polynucleotide
of the present invention. Moreover, these molecules can be used to treat
anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group incompatibility.
A polynucleotide or polypeptide of the present invention may also be used to
treat
and/or prevent organ rejection or graft-versus-host disease (GVHD). Organ
rejection occurs
by host immune cell destruction of the transplanted tissue through an immune
response.
Similarly, an immune response is also involved in GVHD, but, in this case, the
foreign
transplanted immune cells destroy the host tissues. The administration of a
polypeptide or
polynucleotide of the present invention that inhibits an immune response,
particularly the
proliferation, differentiation, or chemotaxis of T-cells, may be an effective
therapy in
preventing organ rejection or GVHD.
Similarly, a polypeptide or 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,
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including inflammation associated with infection (e.g., septic shock, sepsis,
or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality,
arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine
induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting
from over
production of cytokines (e.g., TNF or IL-1
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
interactions.
Alternatively, a polypeptide or polynucleotide of the present invention may
proliferate other
cells which can inhibit the hyperproliferative disorder.
For example, by increasing an immune response, particularly increasing
antigenic
qualities of the hyperproliferative disorder or by proliferating,
differentiating, or mobilizing
T-cells, hyperproliferative disorders can be treated. This immune response may
be increased
by either enhancing an existing immune response, or by initiating a new immune
response.
Alternatively, decreasing an immune response may also be a method of treating
hyperproliferative disorders, such as a chemotherapeutic agent.
Examples of hyperproliferative disorders that can be treated or detected by a
polynucleotide or polypeptide of the present invention include, but are not
limited to
neoplasms located in the: abdomen, bone, breast, digestive system, liver,
pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles,
ovary, thymus,
thyroid), eye, head and neck, nervous (central and peripheral), lymphatic
system, pelvic, skin,
soft tissue, spleen, thoracic, and urogenital.
Similarly, other hyperproliferative disorders can also be treated or detected
by a
polynucleotide or polypeptide of the present invention. Examples of such
hyperproliferative
disorders include, but are not limited to: hypergammaglobulinemia,
lymphoproliferative
disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,
Waldenstron's
Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other
hyperproliferative
disease, besides neoplasia, located in an organ system listed above.
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Infectious Disease
A polypeptide or polynucleotide of the present invention can be used to treat
or detect
infectious agents. For example, by increasing the immune response,
particularly increasing
the proliferation and differentiation of B and/or T cells, infectious diseases
may be treated.
The immune response may be increased by either enhancing an existing immune
response, or
by initiating a new immune response. Alternatively, the polypeptide or
polynucleotide of the
present invention may also directly inhibit the infectious agent, without
necessarily eliciting
an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms
that can be treated or detected by a polynucleotide or polypeptide of the
present invention.
Examples of viruses, include, but are not limited to the following DNA and RNA
viral
families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,
Bunyaviridae,
Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae
(Hepatitis),
Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),
Mononegavirus
(e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,
Influenza),
Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or
Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and
Togaviridae
(e.g., Rubivirus). Viruses falling within these families can cause a variety
of diseases or
symptoms, including, but not limited to: arthritis, bronchiollitis,
encephalitis, eye infections
(e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,
C, E, Chronic
Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia,
Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza,
Rabies, the
common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin
diseases (e.g.,
Kaposi's, warts), and viremia. A polypeptide or polynucleotide of the present
invention can
be used to treat or detect any of these symptoms or diseases.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that can
be treated or detected by a polynucleotide or polypeptide of the present
invention include, but
not limited to, the following Gram-Negative and Gram-positive bacterial
families and fungi:
Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),
Aspergillosis,
Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis,
Bordetella,
Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,
Cryptococcosis,
Dermatocycoses, Enterobacteriaceae (Klebsielia, Salmonella, Serratia,
Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria,
Mycoplasmatales,
Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea
Infections
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(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.
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
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(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
scarnng. Regeneration also may include angiogenesis.
Moreover, a polynucleotide or polypeptide of the present invention may
increase
regeneration of tissues difficult to heal. For example, increased
tendon/ligament regeneration
would quicken recovery time after damage. A polynucleotide or polypeptide of
the present
invention could also be used prophylactically in an effort to avoid damage.
Specific diseases
that could be treated include of tendinitis, carpal tunnel syndrome, and other
tendon or
ligament defects. A further example of tissue regeneration of non-healing
wounds includes
pressure ulcers, ulcers associated with vascular insufficiency, surgical, and
traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a
polynucleotide
or polypeptide of the present invention to proliferate and differentiate nerve
cells. Diseases
that could be treated using this method include central and peripheral nervous
system
diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord disorders,
head trauma, cerebrovascular disease, and stroke). Specifically, diseases
associated with
peripheral nerve injuries, peripheral neuropathy (e.g., resulting from
chemotherapy or other
medical therapies), localized neuropathies, and central nervous system
diseases (e.g.,
Alzheimer's disease,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome), could all be treated using the polynucleotide or polypeptide of the
present
invention.
Chemotaxis
A polynucleotide or polypeptide of the present invention may have chemotaxis
activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes,
fibroblasts,
neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial
cells) to a particular
site in the body, such as inflammation, infection, or site of
hyperproliferation. The mobilized
cells can then fight off and/or heal the particular trauma or abnormality.
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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
(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
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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.
Therefore, the invention includes a method of identifying compounds which
bind to a polypeptide of the invention comprising the steps of: (a) incubating
a candidate
binding compound with a polypeptide of the invention; and (b) determining if
binding has
occurred. Moreover, the invention includes a method of identifying
agonists/antagonists
comprising the steps of: (a) incubating a candidate compound with a
polypeptide of the
invention, (b) assaying a biological activity, and (c) determining if a
biological activity of the
polypeptide has been altered.
Other Activities
A polypeptide or polynucleotide of the present invention may also increase or
decrease the differentiation or proliferation of embryonic stem cells,
besides, as discussed
above, hematopoietic lineage.
A polypeptide or polynucleotide of the present invention may also be used to
modulate mammalian characteristics, such as body height, weight, hair color,
eye color, skin,
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percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic
surgery).
Similarly, a polypeptide or polynucleotide of the present invention may be
used to modulate
mammalian metabolism affecting catabolism, anabolism, processing, utilization,
and storage
of energy.
A polypeptide or polynucleotide of the present invention may be used to change
a
mammal's mental state or physical state by influencing biorhythms, circadian
rhythms,
depression (including depressive disorders), tendency for violence, tolerance
for pain,
reproductive capabilities (preferably by activin or inhibin-like activity),
hormonal or
endocrine levels, appetite, libido, memory, stress, or other cognitive
qualities.
A polypeptide or polynucleotide of the present invention may also be used as a
food
additive or preservative, such as to increase or decrease storage
capabilities, fat content, lipid,
protein, carbohydrate, vitamins, minerals, cofactors or other nutritional
components.
Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated
nucleic
acid molecule comprising a nucleotide sequence which is at least 80%,
preferably at least
85%, more preferably at least 90%, most preferably at least 95% identical to a
sequence of at
least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID
NO:1-62.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:1-62 in the
range of
positions beginning with the nucleotide at about the position of the S'
nucleotide of the clone
sequence and ending with the nucleotide at about the position of the 3'
nucleotide of the clone
sequence.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:1-62 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:1-62.
Similarly preferred is a nucleic acid molecule wherein said sequence of
contiguous
nucleotides is included in the nucleotide sequence of SEQ ID NO:1-62 in the
range of
positions beginning with the nucleotide at about the position of the 5'
nucleotide of the first
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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:I-62.
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:1-62.
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:1-62.
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:1-62
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:1-62.
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:1-62.
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.
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:1-62, 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.
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Also preferred is the above method wherein said step of comparing sequences
comprises determining the extent of nucleic acid hybridization between nucleic
acid
molecules in said sample and a nucleic acid molecule comprising said sequence
selected from
said group. Similarly, also preferred is the above method wherein said step of
comparing
sequences is performed by comparing the nucleotide sequence determined from a
nucleic
acid molecule in said sample with said sequence selected from said group. The
nucleic acid
molecules can comprise DNA molecules or RNA molecules.
A further preferred embodiment is a method for identifying the species, tissue
or cell
type of a biological sample which method comprises a step of detecting nucleic
acid
molecules in said sample, if any, comprising a nucleotide sequence that is at
least 95%
identical to a sequence of at least 50 contiguous nucleotides in a sequence
selected from the
group consisting of: a nucleotide sequence of SEQ ID NO: l-62.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene, 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:1-62.
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:1-62. The nucleic acid
molecules
can comprise DNA molecules or RNA molecules.
Also preferred is an isolated polypeptide comprising an amino acid sequence at
least
90% identical to a sequence of at least about 10 contiguous amino acids in an
amino acid
sequence translated from SEQ ID NO:1-62.
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Also preferred is a polypeptide, wherein said sequence of contiguous amino
acids is included in acids in an amino acid sequence translated from SEQ ID
NO:1-62, 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.
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 an
amino acid
sequence translated from SEQ ID NO:1-62.
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 an amino
acid sequence translated from SEQ ID NO:1-62.
Further preferred is an isolated polypeptide comprising an amino acid sequence
at
least 95% identical to acids in an amino acid sequence translated from SEQ >D
NO:1-62.
Further preferred is a method for detecting in a biological sample a
polypeptide
comprising an amino acid sequence which is at least 90% identical to a
sequence of at least
10 contiguous amino acids in a sequence selected from the group consisting of
amino acid
sequences translated from SEQ ID NO:1-62, which method comprises a step of
comparing an
amino acid sequence of at least one polypeptide molecule in said sample with a
sequence
selected from said group and determining whether the sequence of said
polypeptide molecule
in said sample is at least 90% identical to said sequence of at least 10
contiguous amino acids.
Also preferred is the above method wherein said step of comparing an amino
acid
sequence of at least one polypeptide molecule in said sample with a sequence
selected from
said group comprises determining the extent of specific binding of
polypeptides in said
sample to an antibody which binds specifically to a polypeptide comprising an
amino acid
sequence that is at least 90% identical to a sequence of at least 10
contiguous amino acids in a
sequence selected from the group consisting of amino acid sequences translated
from SEQ ID
NO:1-62.
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Also preferred is the above method wherein said step of comparing sequences is
performed by comparing the amino acid sequence determined from a polypeptide
molecule in
said sample with said sequence selected from said group.
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
amino acid sequences translated from SEQ ID NO:1-62.
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, 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 amino acid sequences
translated
from SEQ ID NO:1-62.
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 amino
acid sequences translated from SEQ ID NO:1-62.
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.
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Also preferred is an isolated nucleic acid molecule, wherein said nucleotide
sequence
encodes a polypeptide comprising an amino acid sequence selected from the
group consisting
of amino acid sequences translated from SEQ ID NO:1-62.
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 amino acid sequences translated from SEQ ID NO:1-
62. 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.
EXAMPLE 1
Identification and Characterization of Polynucleotides
Regulated by in vivo DSS Treatment
For the induction of colitis, DSS (MW 40,000) is dissolved in the drinking
water and
given to mice ad libitum for a period of 7 days. The DSS water is then
replaced with regular
drinking water. Distinct mouse strains demonstrate differential susceptibility
to DSS-colitis.
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Three percent DSS is sufficient for the induction of acute colitis in C57BL/6
mice, whereas
5% DSS is required for the induction of acute colitis in BALB/c mice.
For the purpose of this study, samples of colon were harvested from cohorts of
C57BL/6 mice treated with 3% DSS. Tissues were harvested at day 0, day 4, day
8 or day 12
- a schedule designed to encompass the full range of induction of intestinal
damage through
recovery.
Damage to colonic tissue is a hallmark of IBD. The mouse model of DSS-induced
colitis gives rise to damaged colonic tissue and as such it is one of the more
useful models for
studying IBD.
Mice were treated with DSS in their drinking water as described above. At
various
intervals during DSS-treatment, cohorts of mice were sacrificed and the colons
removed by
dissection. Freshly dissected colonic tissue was immediately placed in GT
buffer (4.5M
guanidinium isothiocyanate, SOmM sodium citrate, 0.5%w/v sodium sarcosyl, 2% 2-
beta-
mercaptoethanol) and homogenized. Homogenized lysates were spun briefly to
remove large
debris before being layered onto a CsCI gradient. RNA was extracted using
conventional
methods and was subsequently used for TOGA analysis.
Features of DSS-induced colitis
Weight loss is apparent in mice beginning at day 4. Histological analysis of
the
intestine reveals the presence of early patchy lesions identifiable by the
loss of epithelial cells
and goblet cells. By day 8, weight loss is fairly severe (approximately 20%
reduction) and
the mice appear moribund. Histologically, the gut epithelium is almost totally
destroyed at
this stage. There is evidence of a large mixed inflammatory cell infiltration
into the lamina
propria and submucosa. The inflammatory cell infiltrate appears to be composed
primarily of
T cells, B cells and granulocytes. By day 12, weight gain is apparent as the
mice recover. At
this later stage, crypt recovery and epithelial regeneration provide
histological evidence of the
beginning of repair processes.
Isolated RNA was analyzed using a method of simultaneous sequence-specific
identification of mRNAs known as TOGA (TOtal Gene expression Analysis)
described in
Sutcliffe, J.G., et al Proc Natl Acad Sci U S A 2000 Feb 29; 97(5):1976-1981,
International
published application PCT/US99/23655, U.S. Patent No. 5,459,037, U.S. Patent
No.
5,807,680, and U.S. Patent No. 6,030,784, hereby incorporated herein by
reference.
Preferably, prior to the application of the TOGA method or other methods, the
isolated RNA
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was enriched to form a starting polyA-containing mRNA population by methods
known in
the art. In such a preferred embodiment, the TOGA method further comprises an
additional
Polymerase Chain Reaction ("PCR") step performed using one of four 5' PCR
primers and
cDNA templates prepared from a population of antisense complimentary RNA
("cRNAs").
A final PCR step using one of a possible 256 5' PCR primers and a universal 3'
PCR primer
produced as PCR products, cDNA fragments that corresponded to a 3'-region of
the starting
mRNA population. The produced PCR products were then identified by a) the
sequence of at
least the 5' seven base pairs, preferably the sequence of the entire fragment,
and b) the length
of the fragment. These two parameters, sequence and fragment length, were used
to compare
the obtained PCR products to a database of known polynucleotide sequences.
The method yields Digital Sequence Tags (DSTs), that is, polynucleotides that
are
expressed sequence tags (ESTs) of the 3' end of mRNAs. DSTs that showed
changes in
relative levels following DSS treatment were selected for further study. The
intensities of the
laser-induced fluorescence of the labeled PCR products were compared across
sample
isolated at different time intervals after treatment.
In general, double-stranded cDNA is generated from poly(A)-enriched
cytoplasmic
RNA extracted from the tissue samples of interest using an equimolar mixture
of all 48 5'-
biotinylated anchor primers of a set to initiate reverse transcription. One
such suitable set is
G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-
T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 63), where V is A, C or G and N is A, C, G
or T.
One member of this mixture of 48 anchor primers initiates synthesis at a fixed
position at the
3' end of all copies of each mRNA species in the sample, thereby defining a 3'
endpoint for
each species, resulting in biotinylated double stranded cDNA.
Each biotinylated double stranded cDNA sample was cleaved with the restriction
endonuclease MspI, which recognizes the sequence CCGG. The 3' fragments of
cDNA
were then isolated by capture of the biotinylated cDNA fragments on a
streptavidin-coated
substrate. Suitable streptavidin-coated substrates include microtitre plates,
PCR tubes,
polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass
particles. A
preferred streptavidin-coated substrate is a suspension of paramagnetic
polymer beads
(Dynal, Inc., Lake Success, NY).
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After washing the streptavidin-coated substrate and captured biotinylated cDNA
fragments, the cDNA fragment product was released by digestion with NotI,
which cleaves at
an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-
derived
portion of the cDNAs. The 3' MspI-NotI fragments, which are of uniform length
for each
mRNA species, were directionally ligated into CIaI-, NotI-cleaved plasmid pBC
SK+
(Stratagene, La Jolla, CA) in an antisense orientation with respect to the
vector's T3
promoter, and the product used to transform Escherichia coli SURE cells
(Stratagene). The
ligation regenerates the NotI site, but not the MsnI site. Each library
contained in excess of 5
x 105 recombinants to ensure a high likelihood that the 3' ends of all rriRNAs
with
concentrations of 0.001% or greater were multiply represented. Plasmid preps
(Qiagen) were
made from the cDNA library of each sample under study.
An aliquot of each library was digested with MspI, which effects linearization
by
cleavage at several sites within the parent vector while leaving the 3' cDNA
inserts and their
flanking sequences, including the T3 promoter, intact. The product was
incubated with T3
RNA polymerase (MEGAscript kit, Ambion) to generate antisense cRNA transcripts
of the
cloned inserts containing known vector sequences abutting the MspI and NotI
sites from the
original cDNAs.
At this stage, each of the cRNA preparations was processed in a three-step
fashion. In
step one, 250 ng of cRNA was converted to first-strand cDNA using the 5' RT
primer (A-G-
G-T-C-G-A-C-G-G-T-A-T-C-G-G, (SEQ ID NO: 64). In step two, 400 pg of cDNA
product
was used as PCR template in four separate reactions with each of the four 5'
PCR primers of
the form G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 65), each paired with a
"universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO: 66).
In step three, the product of each subpool was further divided into 64
subsubpools
(2ng in 20p1) for the second PCR reaction, with 100 ng each of the
fluoresceinated "universal"
3' PCR primer, the oligonucleotide (SEQ ID NO: 66) conjugated to 6-FAM and the
appropriate S' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID
NO:
67), using a program that included an annealing step at a temperature X
slightly above the Tm
of each S' PCR primer to minimize artifactual mispriming and promote high
fidelity copying.
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Each polymerase chain reaction step was performed in the presence of TaqStart
antibody
(Clonetech).
The products from the final polymerase chain reaction step for each of the
tissue
samples were resolved on a series of denaturing DNA sequencing gels using the
automated
ABI Prizm 377 sequencer. Data were collected using the GeneScan software
package (ABI)
and normalized for amplitude and migration. Complete execution of this series
of reactions
generated 64 product subpools for each of the four pools established by the 5'
PCR primers of
the first PCR reaction, for a total of 256 product subpools for the entire 5'
PCR primer set of
the second PCR reaction.
The mRNA samples from each timepoint after DSS treatment as described above
were analyzed. Table 1 is a summary of the expression levels of 414 mRNAs
determined
from cDNA. These cDNA molecules are identified by their digital address, that
is, a partial
5' terminus nucleotide sequence coupled with the length of the molecule, as
well as the
relative amount of the molecule produced at different time intervals after
treatment. The 5'
terminus partial nucleotide sequence is determined by the recognition site for
MspI and the
nucleotide sequence of the parsing bases of the 5' PCR primer used in the
final PCR step. The
digital address length of the fragment was determined by interpolation on a
standard curve
and, as such, may vary ~ 1-2 b.p. from the actual length as determined by
sequencing.
For example, the entry in Table 1 that describes a DNA molecule identified by
the
digital address MspI AGTG 244, is further characterized as having a 5'
terminus partial
nucleotide sequence of CGGAGTG and a digital address length of 244 b.p. The
DNA
molecule identified as MsnI AGTG 244 is further described as being expressed
at increasing
levels at days 0, 4 and 8 with a moderate decline at day 12. However, the
treatment results
in a different pattern of expression of MspI AGTA 187, which declines on days
4 and 8 from
the relatively high level seen at day 0, but increases at day 12.
Similarly, the other 412 DNA molecules identified in Table 1 by their MspI
digital
addresses are further characterized by the pattern of the level of gene
expression on days 0,
4, 8 and 12 following the end of the DSS treatment. Many of the isolated
clones were further
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characterized in Tables 2 and 3. Their nucleotide sequences are provided as
SEQ ID NO:1-
62 in the Sequence Listing below.
The data shown in Figure 1 were generated with a 5'-PCR primer (C-G-A-C-G-G-T
A-T-C-G-G-C-G-C-G, SEQ ID NO: 68) paired with the "universal" 3' primer (SEQ
ID NO:
66) labeled with 6-carboxyfluorescein (6FAM, ABI) at the S' terminus. PCR
reaction
products were resolved by gel electrophoresis on 4.5% acrylamide gels and
fluorescence data
acquired on ABI377 automated sequencers. Data were analyzed using GeneScan
software
(Perkin-Elmer).
The results of TOGA analysis using a 5' PCR primer with parsing bases CGCG
(SEQ
~ NO: 68) are shown in Figure l, which presents the results of TOGA analysis
using a 5'
PCR primer with parsing bases CGCG, showing PCR products produced from mRNA
extracted from (top to bottom panels) colons isolated from mice 0 (Figure 1A),
4 (Figure 1B)
, 8 (Figure 1C) or 12 days (Figure 1D) after a seven day course of treatment
with DSS. The
vertical index line indicates a PCR product of about 458 b.p. that is present
on day 0, reduced
on day 4, much increased on day 8 and whose expression relatively decreases
but is still
elevated on day 12.
Some products, which were also differentially represented, appeared to migrate
in
positions that suggest that the products were novel based on comparison to
data extracted
from GenBank. In these cases, the PCR product was isolated, cloned into a TOPO
vector
(Invitrogen) and sequenced on both strands. Examples are found in Table 4,
below. In order
to verify that the clones isolated are from the same peak, PCR primers were
designed based
on the determined sequence and PCR was performed using the cDNA produced in
the first
PCR reaction as substrate. For example, for the 458 b.p. product disclosed
above,
oligonucleotides were synthesized using the universal 3' PCR primer and a 5'
PCR primer
corresponding to the 5' PCR primer in the second PCR step extended at the 3'
end with
additional nucleotides from the clone sequence 3' to the parsing bases (in
this case, CGCG).
This extended 5' PCR primer had the sequence: GATCGAATCC GGCGCGCACG
GGGACCAGAC (SEQ ID NO: 78).
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The products were separated by electrophoresis and the length of the clone was
compared to the length of the original PCR product as shown in Figure 2.
Figure 2A shows
the length (as peak position) of the PCR product derived as described above.
Figure 2B
shows the PCR products produced using the original PCR primers, SEQ ID NO: 68
and SEQ
ID NO: 66 (compare to Figure 1A). In Figure 2C, the traces from the top and
middle panels
are overlaid, demonstrating that the PCR product using the sequence of the
isolated clone is
the same length as the original PCR product.
The same procedure was used to verify candidate matches to database entries.
The
results are shown in Table 4, below. In each case, oligonucleotides were
synthesized using
the universal 3' PCR primer and a S' PCR primer corresponding to the 5' PCR
primer in the
second PCR step extended at the 3' end with additional nucleotides from the
sequences
adjacent to the terminal MspI sites in the identified corresponding GenBank
sequences. In
three cases (IMX2_33, SEQ ID N0:21, IMX2 34, SEQ ID N0:61 and IMX2 70, SEQ >D
N0:62) the DST sequence listed was obtained from the verified database match
sequence in
GenBank.
Figure 3 is a graphical representation of the results of Northern Blot
analysis of clone
IMX 2 46, SEQ ID NO: 10, where an agarose gel containing poly A enriched mRNA
from
the four experimental samples (0, 4, 8 or 12 days post-treatment) as well as
size standards
was blotted after electrophoresis and probed with radiolabelled IMX 2 46, SEQ
ID NO: 10,
imaged using a phosphorimager and quantified. Quantitative results showing the
relative
expression levels of the 1.6 kb transcript were: 0 day, 64; 4 days, 53; 8
days, 223; and 12
days, 269. The amount of RNA loaded on the gel was determined by probing for
cyclophilin
("cyc").
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TABLE
1
Seq ID Clone Digital Address0 Days4 Days8 12 Days
ID (Mspl) Days
AAAA 157 405 247 181 459
AAAA 316 42 34 47 174
51 IMX2 65 AAAC 186 3722 1457 4314 5810
50 IMX2 1 AAAC 407 95 106 345 281
AAAG 449 214 121 249 299
AACA 377 84 50 197 107
59 IMX2 2 AACG 299 79 108 366 91
AATC 318 326 174 36 72
60 IMX2 3 AATG 114 210 136 1059 514
ACAA 227 110 496 1568 564
ACAA 381 51 57 120 69
ACAC 283 131 69 212 143
1 1MX2 4 ACAC 320 28 38 284 106
26 IMX2 5 ACAC 398 29 37 94 70
ACAG 288 69 67 264 101
ACAT 335 767 362 231 449
ACCA 359 652 134 227 452
ACCC 493 128 332 316 398
ACCG 387 218 60 149 570
ACCT 225 851 189 650 1106
27 IMX2 6 ACCT 364 173 92 530 142
ACGA 171 761 192 909 790
ACGA 206 618 984 2071 1168
ACGA 235 623 489 1511 1276
ACGA 326 514 1217 805 722
ACGC 361 455 97 227 299
ACGG 198 1483 2094 5210 3294
52 1MX2 66 ACGG 238 1576 537 1476 1734
ACGG 409 405 172 473 800
ACGG 449 102 66 69 152
ACGT 174 743 401 265 615
ACTA 333 555 181 305 468
ACTC 111 468 488 1773 1095
28 IMX2 7 ACTC 171 55 242 1326 651
ACTG 184 1889 742 596 924
ACTG 319 67 130 22 91
ACTT 292 51 39 180 177
AGAR 104 325 215 319 546
AGAR 263 423 840 1050 1197
AGAC 487 1568 826 354 1594
AGAG 110 1692 2388 3206 1620
AGAG 205 710 300 547 453
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AGAG 318 283 166 139 452
AGAG 329 300 125 234 400
AGAG 372 158 94 193 272
AGCA 251 1125 353 659 1187
29 IMX2 8 AGCA 285 314 2300 1575 1050
AGCA 316 158 656 179 174
AGCC 379 439 148 366 544
AGCG 258 278 176 543 239
AGCG 284 163 39 25 87
AGCG 396 476 57 261 605
AGCT 175 920 1790 1538 1268
AGCT 230 179 192 33 132
AGGA 163 691 924 1919 830
AGGA 190 1099 473 638 791
AGGA 266 354 106 74 133
AGGA 332 63 192 130 58
AGGA 381 20 88 49 33
AGGC 367 599 165 273 723
AGGC 410 4628 1660 5080 4468
AGGT 101 316 524 211 133
AGGT 282 169 55 30 64
AGGT 304 47 33 39 133
AGGT 485 403 181 292 421
AGTA 187 1331 412 457 753
AGTA 209 194 331 365 412
AGTA 285 62 257 269 164
AGTC 288 101 354 197 163
AGTC 324 665 319 66 112
30 IMX2 11 AGTC 476 51 150 661 373
31 IMX2 12 AGTG 244 165 338 2558 1077
AGTT 101 92 235 234 70
AGTT 248 70 164 303 124
ATAA 170 410 116 241 315
41 IMX2 39 ATAA 226 38 44 174 69
ATAA 261 485 282 891 766
32 IMX2 13 ATAC 226 174 303 598 221
ATAC 275 954 756 1600 1133
ATAG 110 4836 1250 1558 3142
ATAG 151 1774 630 919 1402
ATAG 223 513 584 1488 1078
ATAG 323 1044 599 1180 1349
ATAG 403 187 79 110 225
ATCA 87 1018 1221 1588 664
ATCA 121 671 263 160 314
ATCA 466 376 150 251 298
ATCC 224 89 175 382 232
ATCC 304 489 245 553 537
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ATCC 347 229 106 283 291
ATCC 451 309 112 187 295
ATCG 137 399 111 147 135
ATCG 205 67 240 341 76
ATCG 362 61 44 165 94
ATCG 464 393 139 330 394
ATCT 392 208 72 123 236
ATGA 203 743 1154 1968 1196
ATGA 275 425 255 680 395
ATGA 286 460 167 143 330
ATGC 126 1310 551 609 711
ATGG 196 284 470 636 361
ATGG 349 481 588 178 439
ATTA 105 432 233 67 94
ATTA 162 568 201 235 399
ATTA 263 104 48 51 69
ATTG 140 1744 870 1490 2200
CAAA 267 94 77 244 127
CAAA 310 405 145 404 441
CAAA 359 1777 461 594 1572
CAAC 267 868 129 357 710
CAAC 338 469 131 342 468
CAAC 347 378 174 230 486
CAAC 362 289 125 620 369
CAAG 464 162 42 112 198
CAAG 479 87 42 59 105
CAAT 132 983 727 1005 1669
CART 188 2436 1137 2640 3409
CART 279 374 449 123 377
CART 311 108 102 41 110
CACA 392 150 55 41 137
CACC 86 682 1597 1849 916
CACC 360 85 92 280 89
CACC 386 242 65 133 222
CACC 394 45 47 135 39
CACG 92 2188 4464 5216 2074
CAGA 276 43 49 145 129
CAGC 116 3026 6760 2728 3430
CAGG 315 299 84 145 335
CAGG 421 55 79 185 163
CATC 162 1182 2890 1216 1262
CATC 334 389 332 196 702
CATC 368 176 57 103 162
CATG 257 532 157 326 468
CATG 353 2449 321 1050 1948
42 IMX2 40 CATT 90 299 2530 3750 823
CATT 331 52 127 194 33
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GATT 347 449 72 261 393
CCAA 126 2480 589 976 1713
CCAA 166 1119 1451 960 545
43 IMX2 42 CCAA 201 242 1101 645 304
8 IMX2 43 CCAA 272 70 439 261 75
CCAA 309 319 87 203 458
CCAA 344 185 1389 992 201
CCAC 178 85 295 222 122
CCAC 248 1237 3609 891 1472
CCAC 291 345 141 179 291
CCAC 354 198 95 47 189
CCAG 274 238 112 165 248
CCAG 424 181 69 94 167
CCAT 85 160 192 198 80
CCAT 141 265 613 424 447
CCAT 177 6480 4379 1011 1817
CCCA 78 1043 1200 2301 1018
CCCA 220 1438 773 454 463
CCCA 255 29 27 23 79
CCCG 102 2353 1931 3666 4085
CCCG 211 1290 512 169 678
13 IMX2 55 CCCG 473 3711 1320 23 34
CCCT 314 430 222 154 171
CCCT 462 46 138 57 65
CCGA 188 536 1180 948 647
CCGA 207 297 803 357 340
CCGC 114 1184 515 251 817
CCGC 257 206 135 77 82
53 IMX2 68A CCGC 266 286 77 289 435
54 IMX2 68B CCGC 266 286 77 289 435
CCGC 496 78 29 95 77
55 IMX2 69 CCGT 151 1603 264 440 1074
CCGT 177 2085 836 411 338
CCGT 214 1054 2000 260 512
CCGT 252 682 1085 2385 2117
CCGT 444 47 35 109 79
CCGT 484 72 262 151 78
CCTA 356 45 30 38 306
CCTC 142 451 509 262 946
CCTC 197 684 472 246 443
CCTC 379 164 215 80 81
CCTC 446 45 45 47 153
CCTG 116 250 722 485 182
CCTG 164 627 77 764 46
CCTT 272 225 484 179 98
CCTT 356 76 82 146 53
CCTT 445 238 114 47 376
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CCTT 490 100 11 37 17
CGAA 83 5360 4861 1634 4700
CGAA 164 124 317 258 270
CGAC 166 540 1253 1829 1039
CGAC 267 503 79 680 609
CGAC 364 169 65 415 336
CGAG 262 23 208 146 600
CGAG 302 373 49 217 335
CGAG 352 120 49 155 185
15 IMX2 57 CGAT 176 1458 722 192 188
CGCA 209 431 161 233 251
CGCA 221 787 381 113 138
IMX2 46 CGCG 458 195 108 1280 793
CGCG 460 230 89 1136 732
47 IMX2 58 CGCG 472 846 115 44 45
CGCG 491 148 70 145 285
CGGA 205 41 276 112 291
48 IMX2 59 CGGC 323 1873 1563 344 544
CGGG 123 296 431 648 521
CGGG 245 486 162 280 364
CGGT 468 29 31 50 80
CGTC 310 134 365 399 217
CGTG 153 482 238 157 245
CGTG 374 340 152 88 216
CGTT 90 231 1068 1139 306
CTAA 87 2094 817 719 2495
CTAA 98 722 470 272 296
CTAC 336 431 249 517 653
CTAC 452 28 189 87 40
CTAG 406 457 149 307 479
CTAT 175 313 295 71 75
CTAT 348 353 327 525 737
CTCA 223 2614 726 2346 2193
CTCA 242 66 74 172 146
CTCA 329 143 63 84 150
CTCA 347 179 62 57 178
CTCA 391 3031 972 1564 2427
21 IMX2 33 CTCC 198 151 210 1699 2347
CTCC 287 110 38 45 129
CTCC 308 440 188 493 622
12 IMX2 48 CTCC 386 35 43 233 103
CTCG 188 1648 225 1027 1373
CTCG 428 157 56 220 262
49 IMX2 60 CTCG 472 486 28 15 20
CTCT 117 111 370 213 161
CTCT 407 59 37 107 91
CTGC 147 300 763 888 484
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CTGC 257 908 711 2519 1665
CTGC 414 463 70 223 550
CTGG 200 1421 332 1230 1459
CTGG 272 624 214 572 638
CTGG 414 119 58 109 149
CTTA 395 81 27 73 186
CTTC 247 562 239 377 431
CTTC 404 82 111 85 243
CTTG 205 363 607 283 230
CTTG 258 299 86 121 158
CTTG 297 450 160 630 472
CTTG 354 236 95 162 300
CTTG 366 507 170 434 717
CTTT 400 71 30 101 283
CTTT 410 41 48 67 138
CTTT 453 74 20 64 187
GAAA 305 106 111 210 126
GAAC 279 75 49 199 217
GAAG 95 628 604 1402 449
GALA 386 300 123 132 362
GACA 400 802 295 412 1007
GACC 119 217 771 217 202
GACC 129 1056 3122 2325 1367
GACG 89 1849 741 474 324
GACG 169 502 538 464 179
GACG 283 150 111 223 328
GACG 381 43 44 161 53
GACT 83 371 878 473 299
GACT 315 894 554 823 1160
GAGA 220 198 561 395 297
GAGA 368 149 71 66 146
GAGC 88 275 782 236 166
GAGC 262 1703 474 511 1530
GAGC 419 97 56 165 227
GAGG 88 1550 2018 736 639
GAGG 112 304 701 1046 788
GAGG 253 143 59 75 107
GAGT 197 1309 3415 1817 860
GATA 339 200 131 32 42
GATC 156 483 1229 764 551
GATC 253 167 152 293 133
GATC 450 434 180 389 477
24 IMX2 49 GATG 285 49 40 301 106
GATT 126 3424 1173 2935 4890
GATT 214 2246 692 1808 2796
GCAA 226 173 755 823 572
GCAA 333 177 41 116 152
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GCAG 118 337 434 654 1602
GCAT 242 1111 1093 115 369
62 IMX2_70 GCAT 276 2425 1784 183 770
56 IMX2 71 GCAT 361 261 65 168 476
GOAT 447 93 73 94 176
GCCC 444 145 77 228 156
GCCC 452 162 60 209 149
GCCG 269 770 28 359 512
33 IMX2 15 GCCG 364 72 120 378 206
34 IMX2 16 GCGA 190 169 539 392 140
GCTA 82 1049 3530 2007 819
GCTA 269 1387 490 598 1485
GCTA 452 41 261 150 42
GCTC 157 1515 467 659 1151
3 IMX2_17 GCTC 245 258 864 1457 514
57 IMX2 72 GCTC 425 460 67 208 650
GCTG 160 1171 2011 1804 726
GGAA 416 1601 1067 553 2083
GGAC 270 62 231 162 93
GGAC 327 527 145 409 727
GGAG 274 309 83 398 472
GGAG 283 266 67 215 306
GGAG 440 73 38 73 98
GGAT 276 1016 639 118 259
GGAT 362 140 35 55 152
GGCA 341 155 94 56 168
GGCA 349 214 63 88 192
GGCC 170 920 103 257 467
GGCC 327 86 345 470 87
GGCT 445 447 79 403 606
GGGA 430 817 139 777 1212
GGGC 142 1375 565 529 389
GGGC 355 288 61 205 271
GGGC 418 53 44 64 99
GGGG 241 126 389 812 371
35 IMX2 20 GGGT 150 138 190 461 328
61 IMX2 34 GGGT 177 622 1213 3329 2197
GGTC 117 1171 2648 2360 1133
GGTC 374 104 72 295 285
GGTT 268 73 41 22 129
GTAG 117 204 331 299 1780
GTAG 437 57 66 146 118
GTAT 242 407 356 18 73
17 IMX2 61 GTAT 276 896 608 39 189
GTAT 362 184 67 106 257
GTAT 382 132 25 30 121
GTAT 409 40 32 27 116
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GTCA 92 555 813 1400 722
GTGA 88 968 2297 1809 1928
GTGC 293 565 160 147 541
GTGC 442 730 204 705 1245
GTGT 240 31 24 22 160
GTGT 276 65 59 97 143
GTTC 167 104 172 323 318
GTTC 490 35 24 72 33
GTTT 198 70 86 203 64
TAAC 92 144 162 500 167
TAAC 331 122 45 68 63
TAAC 395 47 52 104 117
TAAG 143 88 120 335 162
23 IMX2 21 TAAG 192 129 46 92 594
TAAG 201 59 120 193 150
TAAG 244 278 118 107 133
TAAG 266 44 26 37 90
58 IMX2 73 TAAG 302 4180 1203 490 1807
TART 267 59 131 88 178
TACC 343 54 91 242 49
TACC 415 206 218 446 439
TACG 284 1286 411 984 1079
TACT 160 1169 944 656 533
40 IMX2 35 TACT 338 235 316 850 781
TAGA 121 329 139 52 157
TAGC 219 1774 2833 577 4624
44 IMX2 51 TAGG 80 392 2299 2373 469
TAGG 175 2013 846 314 489
TAGT 177 858 886 199 141
4 IMX2 22 TAGT 236 261 118 1534 1085
TATC 314 379 292 115 241
TATT 268 80 153 112 238
TCAG 223 3378 931 1638 3793
36 IMX2 23 TCAT 161 314 800 1426 849
TCAT 176 1442 802 372 371
6 IMX2 36 TCAT 188 99 856 1141 921
TCCA 312 430 51 267 553
TCCC 314 468 74 316 519
TCCG 472 388 54 24 43
TCCT 176 1100 1002 2127 1547
25 IMX2_62 TCCT 259 618 175 281 347
20 IMX2 74 TCTC 232 1700 164 230 653
37 IMX2 24 TCTC 365 94 118 819 435
TCTC 392 27 62 199 126
TCTG 120 907 1918 925 586
TCTG 154 219 437 813 345
TCTG 263 138 301 612 292
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TCTG 381 113 15 17 28
TCTT 197 396 1276 779 660
TGAC 175 365 45 54 36
TGAC 283 340 175 157 71
38 IMX2 25 TGAC 376 9 38 394 448
TGAC 416 2068 581 787 2274
TGAT 279 193 129 286 664
19 IMX2 63 TGAT 476 2230 1330 42 139
TGCA 79 1129 2391 2202 689
TGCA 124 1783 1512 1373 982
TGCA 186 203 147 1013 628
TGCA 217 471 163 66 44
TGCA 414 35 29 52 120
TGCC 301 312 267 35 135
39 IMX2 26 TGCC 344 372 598 2223 563
TGCG 120 186 215 683 581
TGCG 410 2776 258 1208 2951
TGCT 89 1267 2754 2523 928
TGCT 403 61 105 280 208
TGGA 95 1917 1923 1911 839
TGGC 80 755 1969 2468 1069
TGGC 121 185 1280 1415 455
22 IMX2 64 TGGC 175 1747 262 427 73
TGGC 219 2529 5287 5480 5240
TGGC 402 185 36 128 104
TGGT 175 946 288 215 213
IMX2 28 TGTA 166 194 554 102 157
TGTC 154 1177 918 3439 3334
TGTC 264 5701 4480 1159 2280
45 IMX2 52 TGTC 343 38 26 296 32
TGTG 266 67 664 294 187
TGTG 362 1800 943 370 1050
TTAA 313 140 288 281 340
46 IMX2 53 TTCC 320 29 515 354 109
TTCG 346 37 255 100 32
TTCT 267 59 88 98 146
TTGA 161 161 239 803 467
TTGC 353 54 92 220 109
TTGC 383 37 201 573 255
TTGG 289 139 116 541 324
TTGT 264 144 264 465 325
TTTC 149 287 1262 979 147
TTTT 268 93 150 130 256
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EXAMPLE 2
RT-PCR Validation
RT-PCR validation of cloned DSTs was performed, and results are presented in
Table
2. The starting amount of template was chosen based on a control curve that
was generated to
accurately define the linear range of implication for the given cloned DST.
Based upon the
intensity of the peak cloned from the TOGATM panel, the following amounts of
template were
chosen:
TOGA peak intensity Low f cDNA] H~i h [cDNAl
0 - 400 2000 pg 5000 pg
300-1000 400 pg 2000 pg
> 1000 50 pg 250 pg
The PCR primers used for validation of cloned DSTs are listed in Table 5.
Duplicate
reaction mixtures were assembled using the appropriate low and high
concentration of cDNA
template chosen from the time point sample showing the strongest TOGATM
signal. The
reaction mixtures were cycled for 23, 26, 29, 32, 35, and 38 cycles. The
resulting
amplification products from the duplicate reactions were quantitated and
plotted against the
cycle number to generate a standard curve. From these data, the cycle number
and cDNA
concentration combination which yielded acceptable levels of PCR product
within the linear
range of amplification were chosen for RT-PCR validation across the various
time-points.
The RT-PCR validation consisted of assembling triplicate reactions using the
chosen
concentration of cDNA cycled to the defined cycle number. For example, the
data in Table 2
for IMX2-55 (SEQ >D N0:13) were generated using 50 pg cDNA template and 29
cycles.
An internal control primer pair amplified under the same conditions was also
performed to
provide the basis for normalizing any differences between the cDNA templates.
EXAMPLE 3
RT-PCR Analysis Using Fluorimetry
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Two DSTs were validated using an alternative protocol. The primers used for RT-
PCR are listed in Table 5. For each DST examined, the optimal annealing
temperature and
reagent conditions were determined for the corresponding set of primers (see
Table 5) based
on results from a preliminary experiment. In eight separate reactions, each
set of primers was
assayed to find the optimal conditions by adjusting the following four
parameters: primer
concentration, dNTP concentration, MgCh concentration, and Taq polymerase.
Once
optimal conditions were determined, each DST was run in duplicate multiple
simultaneous
reactions which usually included at least four dilutions of template, plus
control reactions
lacking template, and six sequential data points for numbers of cycles.
Reactions were performed using "Hot Start" PCR with the Clontech TaqStart
antibody system (Cat. #5400-1). Each reaction contained 1~l of the cDNA
library dilution as
template, determined amounts of AmpliTaq DNA polymerase (cat. #N808-0156),
MgClz,
dNTPs (GibcoBRL cat. #10297-018), primer, and Clontech TaqStart Antibody in a
201
reaction volume using l Ox Taq buffer II (without MgCIZ). Typically, a master
mix
containing all components except the template was prepared and aliquoted.
Various
templates were then added to these master mix samples and 20 p,l volumes were
subsequently
dispensed into individual reaction tubes. During the PCR run, tubes were
removed
sequentially on a predetermined schedule in order to quantitate expression of
the target DST
over a "window" of cycles. After amplification, the samples were quantified
via fluorimetry.
PCR was performed at annealing temperatures about 5 degrees above the lowest
melting temperature of each primer pair using the following program: 1) 95
degrees Celsius,
3 minutes; 2) 95 degrees Celsius, 30 seconds; 3) Tnt+5 degrees Celsius, 30
seconds; 4) 72
degrees Celsius, for a time dependent on target length at 16 bp/second; 5)
repeat steps 2-4 33
more cycles; 6) 72 degrees Celsius, 3 minutes; 7) 14 degrees Celsius, forever.
Following temperature cycling, 2p1 of the PCR reaction was added to 1401 of a
1:280 dilution of PicoGreen (Molecular Probes cat. #P-11495 (1Ox100~1)) in TE
pH 7.5 in a
96-well Costar IIV microtiter plate (Fisher cat. #07-200623). The samples were
mixed
gently for 1.5 minutes and allowed to equilibrate at room temperature in the
dark for 1 S
minutes. The concentration of the PCR products was quantified by fluorimetry
using a
PerSeptive Biosystems CytoFluor series 4000 mufti-well plate reader.
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Background fluorescence was determined by using duplicate control samples that
were cycled with all reaction components except the template. The mean value
from these
duplicate background control samples was subtracted from the corresponding
experimental
values prior to analyzing results. The sensitivity of the PicoGreen dsDNA
assay is reported
to be 250pg/ml (SOpg dsDNA in a 200p1 assay volume) using a fluorescence
microplate
reader such as was used in these measurements.
The results of the quantified RT-PCR of IMX 2 48 for a 1:2000 dilution of the
library
are shown in Figure 4 (in arbitrary fluorescence units) and in Table 6
(normalized to the
control value at each time point). The results of the quantified RT-PCR of IMX
2 74 for a
1:500 dilution of the library are shown in Figure ~ (in arbitrary fluorescence
units) and in
Table 6 (normalized to the control value at each time point).
Table 6
I
i IMX2 48 I IMX2
74
~'C cle i ~ I ~ c cle
~
1:2000 i 1:2000 1:2000 1:2000 1:5001:500 1:500 1:500
' Od 4d 8d 12d
Od ~ 4d 8d 12d
24 1.01 0.9 1.0 1.0
26 1.0 2.4 2.0 1.4 26 1.00 0.17 0.12 0.38
28 1.0I 3.3 3.4 3.4
I 29 1.00 0.25 0.33 0.44
30 1.01 4.51 6.7. 6.11
32 i 1.01 3.9 ~ 10.2 11.3 32 1.00 0.60 0.56 0.70
i
34 ~ 1.01 15.5 i 36.0 36.5
I
I i
TOGA ~ 1.01 1.61 7.3 2.8I TOGA 1.00 0.10 0.14 0.38
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
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CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
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EXAMPLE 4
Extended Se_guence Clone for IMX 2 4
The clone was isolated from a D4/D8 DSS colon library using the Clonecapture
procedure from Edge Biosystems. The library was constructed by Edge Biosystems
for
Immunex from RNA isolated from D4 and D8 inflamed colons from DSS treated
C57BL/6
mice. The clone is in an Edge vector pEAKl2.
IMX2 04 DST (SEQ ID NO:1) matches extended sequence for IMX2 04 from base
688 to 957 (SEQ ID N0:2) (See Table 7, above). There is an open reading frame
starting at
base 1 that may encode a partial gene product. The amino acid sequence of the
encoded
partial protein is given in SEQ ID N0:129.
When the extended sequence was compared to NCBI nr database wBLAST, linear
segments of the nucleotide sequence for bases 1-936 show about 70% identity
with segments
of the nucleotide sequence of a cosmid clone from human chromosome 19,
AC007565.1
(See Table 7, above). This indicates that the human homologue of IMX2 4 is, at
least in part,
found on the cosmid. However, several exons predicted on the cosmid (by GRAIL
program
or by some homology to mouse ESTs) are skipped by the linear sequence of IMX2-
4-
ElO.seq. It appears that one predicted, yet skipped exon, is real in that a
perfect match is
found in the DERWENT database. The Derwent entry is for a "secreted" molecule
with the
protein fragment in the Derwent protein entry being a signal peptide
containing amino acid
sequence not found on the cosmid (or the nucleotide Derwent entry). However,
the Derwent
nucleotide entry also has a match to another more 5' segment of the cosmid
which does show
a match with the 5' end of IMX2-4-ElO.seq at the 75% identity level. These
forms may
represent different splice variants of a secreted protein.
EXAMPLE 5
Extended Sequence Clone for IMX 2_36
The IMX2 36.EXT sequence information was derived from the clone
IMX2 36pT7T3-2.seq which is an EST clone derived from FVB/N mouse proximal
colon
obtained from IMAGE consortium. The accession number for the EST is AA529850,
and the
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clone id is IMAGE:921608. The EST is in the pT7T3 vector. The IMX2 36 DST (SEQ
ID
N0:6) matches the extended sequence for IMX2 36.EXT (SEQ ID N0:7) from base
293 to
427 (See Table 7, above). Blast of the EST to GenBank gives hits to 'mouse KAR
(killer
activating receptor)' and 'DAP12 protein' (See Table 7, above).
S
EXAMPLE 6
Extended Sequence Clone for IMX 2 43
The clone was isolated from a D4/D8 DSS colon library using the Clonecapture
procedure from Edge Biosystems. The library was constructed by Edge Biosystems
for
Immunex from RNA isolated from pooled D4 and D8 inflamed colons from DSS
treated
C57BL/6 mice. The clone is in the Edge vector pEAKl2. The IMX2 43 DST (SEQ ID
N0:8) matches the IMX2._43 extended sequence (SEQ ID N0:9) from base 1079 to
1298
(See Table 7, above).
EXAMPLE 7
Extended Seguence Clone for IMX 2 46
The IMX2 46.EXT sequence information was derived from the two clones
IMX2 46pT7T3-6.seq and IMX2_46pT7T3-7.seq, which are EST clones in the pT7T3
vector
that were obtained from IMAGE consortium. IMX2 46pT7T3-6.seq (accession number
AA290194, clone id IMAGE:750847) was derived from C57BL/6 mouse lymph node.
IMX2 46pT7T3-7.seq (accession number AA174968, clone id IMAGE: 617717) was
derived from C57BL/6 mouse spleen. The IMX2,_46 DST (SEQ ID NO:10) aligns with
the
IMX2 46.EXT extended sequence (SEQ ID NO:11) from base 157 to 561 (See Table
7,
above).
Blast of ESTs against GenBank gives hits to human TOSO: regulator of fas-
induced
apoptosis (See Table 7, above).
EXAMPLE 8
Extended Sequence Clone for IMX 2 55
The IMX2 SS.EXT extended sequence information was derived from the two clones
IMX2 SSpT7T3-8.seq and IMX2-S~pT7T3-24.seq, which are EST clones that were
obtained
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from IMAGE consortium. IMX2 55pT7T3-8.seq (accession number AA823573, clone id
IMAGE: 1079189) was derived from FVB irradiated mouse colon. IMX2 55pT7T3-
24.seq
(accession number AA690372, clone id IMAGE:1164692) was derived from from FVB
mouse proximal colon.
The accession number for the EST for IMX2_55pT7T3-8.seq is AA823573, and the
clone id is IMAGE:1079189. The accession number for the EST for IMX2 55pT7T3-
24.seq
is AA690372, and the clone id is IMAGE:l 164692. The both ESTs are in the
pT7T3 vector
and were obtained from IMAGE consortium. IMX2 55pT7T3-8.seq clone was derived
from
FVB irradiated mouse colon and IMX2 55pT7T3-24.seq clone was derived from FVB
mouse proximal colon. The IMX2 55 DST (SEQ ID N0:13) aligns bases of 322 to
744 with
the extended sequence IMX2-55.EXT (SEQ ID N0:14) (See Table 7, above).
Blast of the extended IMX2_55 sequence to GenBank disclosess ESTs with
homology to C reactive protein (See Table 7, above).
EXAMPLE 9
Extended Sequence Clone for IMX 2_57
PCR primers were designed for IMX2 57 based on sequence information obtained
from the sequences of two EST clones that were commercially unavailable. The
EST clones
used for deriving sequence information were (1) an EST with accession number
AA240177
and clone id IMAGE:679264 derived from mouse liver and (2) an EST with
accession
number AV005227 and clone id 0910001608 derived from C57BL/6 spleen.
The primers used were forward primer IMX2 57-FP and reverse primer IMX2 57-RP
which prime off the sequence obtained from electronically assembling the DST
and the EST
AV005227. The PCR reaction was performed on cDNA derived from RNA prepared
from
C57BL/6 mouse colon. The PCR product was cloned into the pGEM vector resulting
in the
clone IMX2_57PCR1. The IMX2-57 DST, (SEQ ID N0:15) aligns with bases 283 to
408 of
the extended sequence IMX2-57.EXT (SEQ ID N0:16) (See Table 7, above).
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Blast of assembled ESTs to Genbank gives hits to 'human chymotrypsin-like
(CTRL)
mRNA' and 'human proteosome-like subunit (MECL-1 ), chymotrypsin-like protease
(CTRL-
1) and protein-serine kinase (PSK-H1) last exon (See Table 7, above).
EXAMPLE 10
Extended Sequence Clone for IMX 2 61
The IMX2 61 extended sequence information was derived from the EST clone
IMX2 6lpBS-47.seq, an EST clone that was obtained from IMAGE consortium. The
accession number for the EST for IMX2-6lpBS-47.seq is AA981092, and the clone
ID is
fVIAGE:1279287. The IMX2 6lpBS-47.seq clone was derived from WEHI3 mouse
macrophage cells. The IMX2 61 DST (SEQ ID N0:17) aligns with bases 204 to 425
of the
IMX2 61 extended sequence (SEQ ID N0:18) (See Table 7, above).
EXAMPLE 11
Extended Sequence Clone for IMX 2_17:
Crypt-ductin alpha scavenger receptor (CRP-ductin)
TOGA analysis indicated that IMX2-17 (SEQ ID N0:3) corresponds to Mus
musculus CRP-ductin-alpha mRNA, accession number U37438 (Table 3). CRP-ductin
localizes to the apical portion of crypt cells in the small intestine. In the
colon, it is seen
predominantly in surface epithelial cells (EC). It is also seen in the apical
portion of the EC
lining pancreatic and larger hepatic ducts. The CRP-ductin-alpha sequence
predicts a mosaic
protein with a short cytoplasmic region, a transmembrane domain and a large
extracellular
region composed of many repeats. The extracellular region contains 21
potential N-
glycosylation sites in the C-terminal half of the protein. There are also two
potential
phosphorylation sites in the cytoplasmic domain.
Forward primer mCRP.S 179-FP and reverse primer mCRP.6106-RP were used to
PCR a 936 by product from a cDNA template derived from RNA isolated from
C57BL/6
colon. The PCR product was subcloned into the pGEM vector and the sequence was
verified
before being used as a probe for Northern blot analysis.
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Figure 6 presents the results of Northern blot analysis of clone IMX 2_17, SEQ
ID
NO: 3, where an agarose gel containing poly A enriched mRNA from the
experimental
samples from and size standards was blotted after electrophoresis, probed with
a labeled
probe corresponding to U37438 bases 5179-6106, imaged using a phosphorimager
and
quantified. Figure 6A shows the results from C57BL/6 mice with DSS colitis 0,
4, 8, or 12
days after treatment, C57BL/6 mice with aCD3 ileitis 0, 6, 30 or 72 hours
after treatment, as
well as samples from large intestines from FVB, mdr knock-out mice without
colitis and mdr
knock-out mice with colitis. Figure 6B shows the results from Balb/c mice with
DSS colitis
0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8%
DSS, and Balb/c
mice with aCD3 ileitis 0, 6, 30 or 72 hours after treatment. The predicted
transcript size for
IMX2-17 CRP-ductin is 6.6 Kb; the actual transcript size found in this study
was
approximately 6.5 Kb.
Northern blots were performed on mRNA samples from several models of IBD.
Analysis of samples from C57BL/6 mice with DSS colitis showed high levels of
constitutive
expression in the large intestine, maximal at day 8 of DSS colitis (Figure
6A), consistent with
the results of the initial TOGA analysis (Table 1). Analysis of samples from
Balb/c mice with
DSS colitis showed similar results (Figure 6B).
Analysis of samples from C57BL/6 mice with aCD3 ileitis showed low levels of
constitutive expression in the small intestine that increases early in
inflammation (Figure 6A)
reaching a maximum at six hours and declining thereafter. Analysis of samples
from Balb/c
mice with aCD3 ileitis showed similar, though less intense, results (Figure
6B).
Constitutive expression was seen in FVB large intestine samples (Figure 6A).
Increased expression in healthy mdr knock-out mice with no signs of colitis,
with little
further increase in expression in mdr knock-out mice with active colitis
(Figure 6A).
The expression shown on the Northern blot of Figure 6 was quantified and
normalized
to the amount of G3PDH in each lane. The normalized results are shown in Table
8, below.
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Table 8
uantitation of Fi ure
6
IBD Model C57BL/6 BALB/c
DSS induced colitis
Dav 0 27740 11455
Dav 4 30853 21103
Dav 8 43857 28838
Dav 12 22622 26248
DSS Concentration Effect
0 % Not Done 11832
% Not Done 21278
8 % Not Done 12717
Anti CD3 Induced Ileitis
0 Hours 2519 2491
6 Hours 14088 7870
30 Hours 11144 1287
72 Hours ~ 9000 2238
Constituitive Ex ression
FVB Mdr Knock Out Mdr Knock Out + Colitis
14427 ~ 29166 15988
EXAMPLE 12
Extended Sequence Clone for IMX 2 22
IMX2 22 Hematopoietic Progenitor Kinase HPK1
5
TOGA analysis indicated that IMX2 22 corresponds to Mus musculus mRNA for
serine/threonine kinase, accession number Y09010 (Table 3). HPK1 is a
hematopoietic
protein kinase activating the SAPK/JNK pathway.
Forward primer mHPK1.1640-FP and reverse primer mHPKI .2420-RP were used to
PCR a 780 by product from a cDNA template was derived from RNA isolated from
C57BL/6 colon. The PCR product was subcloned into the pGEM vector and was
sequence
verified before being used as a probe for Northern blot analysis. The
predicted transcript size
for IMX2 22 HPK1 was 2.7 Kb and published transcript sizes are 2.8 Kb and 3.6
Kb. The
actual transcript size found in this study was 2.8Kb.
Figure 7 presents the results of Northern blot analysis of clone IMX 2 22, SEQ
ID
NO: 4, where an agarose gel containing poly A enriched mRNA from the
experimental
samples and size standards was blotted after electrophoresis, probed with a
labeled probe
corresponding to bases 1640-2420 of Y09010, imaged using a phosphorimager and
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quantified. Figure 7A shows the results from C57BL/6 mice with DSS colitis 0,
4, 8, or 12
days after treatment, C57BL/6 mice with aCD3 ileitis 0, 6, 30 or 72 hours
after treatment, as
well as samples from large intestines from FVB, mdr knock-out mice without
colitis, mdr
knock-out mice with colitis and C57BL/6 spleen. Figure 7B shows the results
from Balb/c
mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice with
aCD3 ileitis 0, 6,
30 or 72 hours after treatment and C57BL/6 normal lymphoid tissue samples
(MLM, PP,
spleen and thymus).
Northern blots were performed on mRNA samples from several models of IBD.
Analysis of samples from C57BL/6 mice with DSS colitis showed low levels of
constitutive
expression in the large intestine, maximal at day 12 of DSS colitis (Figure
7A), not very
consistent with the results of the initial TOGA analysis (Table 1). Analysis
of samples from
Balb/c mice with DSS colitis showed similar results with a maximum at day 8
(Figure 7B).
Analysis of samples from C57BL/6 mice with aCD3 ileitis showed constitutive
expression in the small intestine that decreases at six hours (Figure 7A).
Analysis of samples
from Balb/c mice with aCD3 ileitis showed similar results (Figure 7B).
Increased expression in mdr knock-out mice with active colitis (Figure 7A), in
contrast to little change in corresponding TOGA analysis (data not shown).
Strong expression was found in lymphoid tissues in MLN, PP, thymus, especially
the
spleen (Figure 7A & B).
The expression shown on the Northern blot of Figure 7 was quantified and
normalized
to the amount of G3PDH in each lane. The normalized results are shown in Table
9, below.
Table 9
Quantitation of Fi ure 7
IBD Model C57BL/6 BALB/c
DSS induced colitis
i Dav 0 ~ -50 271
i Dav 4 64 143
Dav 8 I 82 502
i Dav 12 I 419 I 341
I
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Anti CD3 Induced
Ileitis
0 Hours 614 369
6 Hours 133 -47
30 Hours 465 341
72 Hours 168 149
I
Constituitive
Ex ression
FVB Mdr Knock Mdr
Out Knock
Out
+
Colitis
59 146 739
Normal Tissue
MLN PP S leen Thvmus
10008 4883 33260 8826
EXAMPLE 13
Extended Sequence Clone for IMX 2,28
Down-Regulated in Adenoma protein (DRAB
TOGA analysis indicated that IMX2 28 corresponds to Mus rnusculus DRA down-
regulated in adenoma protein, accession number AF136751 (Table 3).
Forward primer Dra.1551-FP and reverse primer Dra.2390-RP were used to PCR an
840 by product from a cDNA template derived from RNA isolated from C57BL/6
colon. The
PCR product was subcloned into the pGEM vector and was sequence verified
before being
used as a probe for Northern blot analysis. The predicted transcript size for
IMX2 28 DRA is
2.6 Kb; the actual transcript size found in this study was approximately 3 Kb.
Figure 8 presents the results of Northern blot analysis of clone IMX 2 28, SEQ
ID
NO: 5, where an agarose gel containing poly A enriched mRNA from the
experimental
samples and size standards was blotted after electrophoresis, probed with a
labeled probe
corresponding to bases 1551-2390 of AF136751, imaged using a phosphorimager
and
quantified. Figure 8A shows the results from C57BL/6 mice with DSS colitis 0,
4, 8, or 12
days after treatment, C57BL/6 mice with aCD3 ileitis 0, 6, 30 or 72 hours
after treatment, as
well as samples from large intestines from FVB, mdr knock-out mice without
colitis and mdr
knock-out mice with colitis. Figure 8B shows the results from Balb/c mice with
DSS colitis
0, 4, 8, or 12 days after treatment, Balb/c mice treated with 0%, 5% and 8%
DSS, and Balb/c
mice with aCD3 ileitis 0, 6, 30 or 72 hours after treatment.
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Northern blots were performed on mRNA samples from several models of IBD.
Analysis of samples from C57BL/6 mice with DSS colitis showed high levels of
constitutive
expression in the large intestine that decrease with inflammation, minimal at
day 8 of DSS
colitis (Figure 8A), consistent with the results of the initial TOGA analysis
(Table 1 ).
Analysis of samples from Balb/c mice with DSS colitis showed the same pattern
(Figure 8B).
Analysis of samples from C57BL/6 mice with aCD3 ileitis showed levels of
constitutive expression in the small intestine lower than that seen in the
large intestine with
DSS colitis. Expression decreases with inflammation rapidly by 6 hours and
remains low
(Figure 8A). Analysis of samples from Balb/c mice with aCD3 ileitis showed the
same
pattern (Figure 8B). TOGA analysis in this model (data not shown) did not show
a
corresponding peak.
Constitutive expression was seen in FVB large intestine samples (Figure 8A).
Constitutive expression was seen in healthy mdr knock-out mice with no signs
of colitis, with
a dramatic decrease in expression seen in mdr knock-out mice with active
colitis (Figure 8A).
TOGA analysis in this model (data not shown) did not show a corresponding
peak.
The expression shown on the Northern blot of Figure 8 was quantified and
normalized
to the amount of G3PDH in each lane. The normalized results are shown in Table
10, below.
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Table 10
nnantitatinn nf' Fianrr R
I IBD Model C57BL/6 BALB/c
DSS induced colitis
Dav 0 25541 34441
Dav 4 20316 35270
Dav 8 7569 22711
Dav 12 10218 22481
DSS Concentration Effect
0 % Not Done 22463
% Not Done 19861
8 % Not Done 17548
Anti CD3 Induced Ileitis
0 Hours 5937 3534
6 Hours 2211 1275
30 Hours I 2192 2051
72 Hours 5300 4142
Constituitive Ex ression
FVB Mdr Knock Out Mdr Knock Out + Colitis
55232 28854 5046
EXAMPLE 14
5 Extended Sequence Clone for IMX2 33
Secretory Leukocyte Protease Inhibitor (SLPI)
TOGA analysis indicated that IMX2 33 corresponds to Mus musculus secretory
leukocyte protease inhibitor, accession number U73004 (Table 3). Secretory
leukocyte
protease inhibitor is an epithelial cell and macrophage derived inhibitor of
leukocyte serine
proteases. SLPI expression is suppressed by gamma-IFN. SLPI is an LPS induced
gamma-
IFN suppressible phagocyte product that serves to inhibit LPS responses.
Forward primer mSLPL447-FP and reverse primer mSLPL800-RP were used to PCR
a 350 by product from a cDNA template was derived from RNA isolated from
C57BL/6
colon. The PCR product was subcloned into the pGEM vector and the sequence was
verified
before being used as a probe for Northern blot analysis. The probe used
corresponded to
bases 447-800 of U73004. The predicted transcript size for IMX2 33 SLPI is 1.1
Kb; the
actual transcript size found in this study was 1.1 Kb.
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Figure 9 presents the results of Northern blot analysis of clone IMX 2 33, SEQ
ID
N0:21, where an agarose gel containing poly A enriched mRNA from the
experimental
samples and size standards was blotted after electrophoresis, probed, imaged
using a
phosphorimager and quantified. Figure 9A shows the results from C57BL/6 mice
with DSS
colitis 0, 4, 8, or 12 days after treatment, C57BL/6 mice with aCD3 ileitis 0,
6, 30 or 72
hours after treatment, as well as samples from large intestines from FVB, mdr
knock-out
mice without colitis and mdr knock-out mice with colitis. Figure 9B shows the
results from
Balb/c mice with DSS colitis 0, 4, 8, or 12 days after treatment, Balb/c mice
treated with 0%,
5% and 8% DSS, and Balb/c mice with aCD3 ileitis (0, 6, 30, 72 hours).
Northern blots were performed on mRNA samples from several models of IBD.
Analysis of samples from C57BL/6 mice with DSS colitis showed minimal
constitutive
expression in the large intestine that increases significantly with
inflammation, maximal at
day 8 of DSS colitis and still high at day 12 (Figure 9A), consistent with the
results of the
initial TOGA analysis (Table 1 ). Analysis of samples from Balb/c mice with
DSS colitis
showed the same pattern (Figure 9B).
Analysis of samples from C57BL/6 mice with aCD3 ileitis showed low to moderate
levels of constitutive expression in the small intestine with less regulation
than seen in the
large intestine with DSS colitis. There was a significant increase with
inflammation maximal
at 30 hours, then decreasing (Figure 9A). Analysis of samples from Balb/c mice
with aCD3
ileitis showed the same pattern (Figure 9B). The pattern was consistent with
that seen in
TOGA analysis in this model (data not shown).
Minimal constitutive expression was seen in healthy mdr knock-out mice with no
signs of colitis, with an increase in expression seen in mdr knock-out mice
with active colitis
(Figure 9A). The pattern was consistent with that seen in TOGA analysis in
this model (data
not shown).
The expression shown on the Northern blot of Figure 9 was quantified and
normalized
to the amount of G3PDH in each lane. The normalized results are shown in Table
11, below.
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Table 1 l
I uantitation of Fi
ure 9
IBD Model C57BL/6 BALB/c
DSS induced colitis
Dav 0 78 197
Dav 4 1580 2237
Dav 8 7491 8170
Dav 12 6313 4390
DSS Concentration Effect
0 % Not Done -54
% Not Done 3201
8 % Not Done -688
Anti CD3 Induced Ileitis I
0 Hours 658 180
6 Hours 832 1100
30 Hours 1465 2572
72 Hours 526 1245
Constituitive Ex ression
FVB Mdr Knock Out Mdr Knock
Out + Colitis I
965 14505 30085
EXAMPLE 15
Extended Sequence Clone for IMX 2 48
IMX2 48 macrophage inflammator~protein 2 (MIP2)
TOGA analysis indicated that IMX2 48 corresponds to Mus musculus MIP-2,
macrophage inflammatory protein 2, accession number X53798 (Table 3).
Forward primer MIP2.61-FP and reverse primer MIP2.345-RP were used to PCR a
284 by product from a cDNA template was derived from RNA isolated from C57BL/6
colon.
The PCR product was subcloned into the pGEM vector and the sequence was
verified. The
predicted transcript size for IMX2 48 MIP2 is 1.1 Kb.
CA 02376667 2001-12-10
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1
SEQUENCE LISTING
S <110> Viney, Joanne L.
Sims, John E.
DuBose, Robert F.
Hasel, Karl W.
Hilbush, Brian S.
Buchner, Robert R.
<120> Gene Expression Modulated In Gastrointestinal Inflammation
<130> 99,104-A
<140> 60/138,487
<141> 1999-06-12
<150>
<151> 2000-06-09
<160> 129
<170> PatentIn Ver. 2.0
<210> 1
<211> 270
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_4
<400> 1
cggacaccca gtcaggcaca agaggtctac attctctaac tcctttccag tgcttcccca 60
acggtcactt acttccagac tgcgtgtttt atttttagga gagatgtgta tattttttgt 120
tgctgttgtt gtttctagat agggtctcac tgtgtagccc tggcttttct ggaactcact 180
gtgtagagca agccagcctc aaactcatag atccacctgc ctctgcctcc agatcgccag 240
aattaaagtt actgccataa cacccaaaaa 270
<210> 2
<211> 964
<212> DNA
<213> Mus musculus
<220>
4$ <223> IMX2_4 ExtendedSequence
<400> 2
ggctggcagggagccccagatccccgtggc cttggccagctttcccagccctacatggga60
ggagagatgccctggaccatcctgctgttt gcatctgtccccacctggatcttggcactc120
tccctgagcctggctggagctgtgctgttc tcagggctggtggccatcacagtgctggtg180
agaaaagctaaagccaaaaacttacagaag cagagagagcgtgaatcctgctgggctcag240
atcaacttcaccaatacagacatgtccttt gataactctctgtttgctatctccacgaaa300
atgactcaggaagactcagtggcaacccta gactcagggcctcggaagaggcccacctct360
gcatcatcctctccggagccccctgagttc agcactttccgggcctgccagtgaggctga420
cgaatgaggaccactttatccagttccttc cctcccactgccagaggctgcacatctgtc480
5$ cagagacttggcagtggaggtagggtgggg gtgggaatcaagccatagctttcttaggga540
agcactggccaaaggaaggggactcctaga gttgtaaccttcctcacagaagacaagaaa600
atgagttggggtatcagcctcaggctagac agagagccagaacctcttcacagattccca660
gatcaccggagaagtcactattgaatccgg acacccagtcaggcacaagaggtctacatt720
ctctaactcctttccagtgcttccccaacg gtcacttacttccagactgcgtgttttatt780
tttaggagagatgtgtatattttttgttgc tgttgttgtttctagatagggtctcactgt840
gtagccctggcttttctggaactcactgtg tagagcaagccagcctcaaactcatagatc900
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,..
cacctgcctc tgcctccaga tcgctagaat taaagttact gccataacac ctaaaaaaaa 960
aaaa 964
<210> 3
S <211> 192
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_17
<400> 3
cgggctctgg gtctattgtt ctggatgacg tggcctgtac aggacacgag gactatctgt 60
ggagctgctc tcaccgaggc tggctctctc ataactgtgg acaccatggg gatgctggag 120
tcatctgttc agatgcccaa atccagagca caaccaggcc agatctgtgg cctactacta 180
IS ctaccccaaa as 192
<210> 4
<211> 183
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_22
<400> 4
2S cggtagtggt ggagacaagg cccacggatg accctacggc ccccagcaac ctctacatcc 60
aggaatgagc cattgagagg gcatgggaaa cggatgcctg cagactccta acagacgcac 120
tagtggtcat gacatgacct tatctcccaa taaacttgac tttagtcttg tcatcctgaa 180
aaa 183
<210> 5
<211> 115
<212> DNA
<213> Mus musculus
3S <2zo>
<223> IMX2_28
<400> 5
cggtgtactc cacaaagact tttggagagg agtttaagaa gacgcacaga catcacaagg 60
cattcctgga ccatctcaaa gggtgttgta gctgctcctc acagaaggcc aaaaa 115
<210> 6
<211> 135
<212> DNA
<213> Mus musculus
4S
<220>
<223> IMX2_36
<400> 6
cggtcattcc agatgcctac tcaacaagcc ctctctggga tcaggactcc cgttggaata 60
S0 cagatccaca gggtacctcc ctgagatatc tgacattgta ccatttctgt ccccaaataa 120
aagacagagc aaaaa 135
<210> 7
<211> 474
SS <212> DNA
<213> Mus musculus
<220>
<223> IMX2_36 Extended sequence
60 <400> ~
ctttcccaag atgcgactgt tcttccgtga gccctggtgt actggctggg attgttctgg 60
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3
gtgacttggt gttgactctg ctgattgccc tggctgtgta ctctctgggc cgcctggtct 120
cccgaggtca agggacagcg gaagggaccc ggaaacaaca cattgctgag actgagtcgc 180
cttatcagga gcttcagggt cagagaccag aagtatacag tgacctcaac acacagaggc 240
aatattacag atgagcccac tctatgccca tcagcggcct gatgcccgga tccggtcatt 300
ccagatgcct actcaacaag ccctctctgg gatcaggact cccgttggaa tacagatcca 360
cagggtacct ccctgagata tctgacattg taccatttct gtccccaaat aaaagacaga 420
gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagggcggc cgca 474
<210> 8
1~ <211> 221
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_43
<400> 8
cggccaaacc tactcaggtt gcaaaggact tatgtgactt atgtgactgt aggaaaaaga 60
gaaatgagtg atcatcctgt ggctactagc agatttccac tgtgcccaga ccagtcggca 120
ggttttgaag gaagtatatg aaaactgtgc ctcagaagcc aatgacagga cacatgactt 180
tttttttcta agtcaaataa acaatatatt gaacagaaaa a 221
<210>
9
<211>
1377
<212>
DNA
<213>
Mus musculus
<220>
<223> 43 Extendedsequence
IMX2_
<400>
9
ccgctccttgcttccacacc tgggactgttcctgtgcctggctctgcacttatccccctc60
cctctctgccagtgataatg ggtcctgcgtggtccttgataacatctacacctccgacat120
cttggaaatcagcactatgg ctaacgtctctggtggggatgtaacctatacagtgacggt180
ccccgtgaacgattcagtca gtgccgtgatcctgaaagcagtgaaggaggacgacagccc240
agtgggcacctggagtggaa catatgagaagtgcaacgacagcagtgtctactataactt300
gacatcccaaagccagtcgg tcttccagacaaactggacagttcctacttccgaggatgt360
gactaaagtcaacctgcagg tcctcatcgtcgtcaatcgcacagcctcaaagtcatccgt420
gaaaatggaacaagtacaac cctcagcctcaacccctattcctgagagttctgagaccag480
ccagaccataaacacgactc caactgtgaacacagccaagactacagccaaggacacagc540
caacaccacagccgtgacca cagccaataccacagccaataccacagccgtgaccacagc600
caagaccacagccaaaagcc tggccatccgcactctcggcagccccctggcaggtgccct660
ccatatcctgcttgtttttc tcattagtaaactcctcttctaaagaaaactggggaagca720
gatctccaacctccaggtca tcctcccgagctcatttcaggccagtgcttaaacataccc780
gaatgaaggttttatgtcct cagtccgcagctccaccaccttggaccacagacctgcaac840
actagtgcacttgagggata caaatgcttgcctggatctttcagggcacaaattccgctt900
cttgtaaatacttagtccat ccatcctgcgtgtaacctgaagttctgactctcagtttaa960
cctgttgacagccaatctga acttgtgtttcttgccaaaggtattcccatgagcctcctg1020
ggtgtgggggtggggaggga atgatccttctttactttcaaactgatttcagatttctgg1080
ccaaacctactcaggttgca aaggacttatgtgacttatgtgactgtaggaaaaagagaa1140
atgagtgatcatcctgtggc tactagcagatttccactgtgcccagaccagtcggtaggt1200
tttgaaggaagtatatgaaa actgtgcctcagaagccaatgacaggacacatgacttttt1260
ttttctaagtcaaataaaca atatattgaacaagaaaaaaaaaaaaaaaaaaaaaaaaaa1320
aaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1377
<210> 10
<z11> 407
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_46
<400> 10
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cggcgcgcac ggggaccagacagcttgggtccagcggaggctccgctcctcaacgcccca60
gcctcagcgt cccccgcttctccgcaggtacttgaagctccttggccccacaccccatct120
ctgaagatga gctgtgaatacgtgagcttgggctaccagcctgctgtcaacctggaagac180
cctgattcag atgattacatcaatattcctgacccatctcatctccctagctatgcccca240
$ gggcccagatcttcatgccaatgagttctgcctgtttgctgatgtctagcacgttttcct300
tataggatcc ctgtcatggcgtatgtcctataccctaagtcgactctcacctgactatct360
gaatgccttg agaatgatcaattacaggctaatttttcacccaaaaa 407
<210> 11
ID <211> 655
<212> DNA
<213> Mus musculus
<220>
1$ <223> IMX2_46 Extended sequence
<400> 11
gctcttttct tggtgacctc ttgaagcctc ctccagacgt gcgggccgac tagcgatgag 60
gaggcgaggc cggggggctt cccgcccgtt ccccacacag cgccgggatg cctcgcagag 120
gccgcgctcg cagaacaacg tctacagcgc ctgcccccgg cgcgcacggg gaccagacag 180
20 cttgggtcca gcggaggctc cgctcctcaa cgccccagcc tcagcgtccc ccgcttctcc 240
gcaggtactt gaagctcctt ggccccacac cccatctctg aagatgagct gtgaatacgt 300
gagcttgggc taccagcctg ctgtcaacct ggaagaccct gattcagatg attacatcaa 360
tattcctgac ccatctcatc tccctagcta tgccccaggg cccagatctt catgccaatg 420
agttctgcct gtttgctgat gtctagcacg ttttccttat aggatccctg tcatggcgta 480
2$ tgtcctatac cctaagtcga ctctcacctg actatctgaa tgccttgaga atgatcaatt 540
acaggctaat ttttcacccc attgaagccc cctgcattca tttgcgagag ttctggataa 600
gacgtgcaga acattcaaaa aaaaaaaaaa aaaaaaaaaa aaaaagtatg cggcc 655
<210> 12
<211> 337
<212> DNA
<213> Mus musculus
<220>
3$ <223> IMX2_48
<400> 12
cggctcctca gtgctgcact ggtcctgctg ctgmtgctgg ccaccaacca ccaggctaca 60
ggggctgttg tggccagtga actgcgctgt caatgcctga agaccctgcc aagggttgac 120
ttcaagaaca tccagagctt gagtgtgacg cccccaggac cccaytgcgc ccagacagaa 180
4~ gtcatagcca ctctcaaggg cggtcaaaaa gtttgccttg accctgaagc ccccctggtt 240
cagaaaatca tccaaaagat wctgaacaaa ggcaaggcta actgacctgg aaaggaggag 300
cctgggctgc tgtccctcaa cggaagaacc ataaaaa 337
<210> 13
4$ <211> 414
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_55
<400> 13
cggcccgtat ctgtgtgaac tgggagtctg gctctgggat tgcagaattc tggctgaatg 60
gaaaaccact ggggaggaaa ggcttgaaga agggatacac tgtggggggt gatgcaatga 120
tcactctagg acaagagcag gattcctatg ggggaaattt tgatgcaaag caatcctttg 180
$$ ttggggagat atgggatgtt tccttgtggg accatgtggt ccccctagaa aaggtatcag 240
acagctgtaa caatggcaac cttataaact ggcaagctct taattatgaa gacaatggct 300
atgtggtgac taagcccaaa ctgtggcctt aagctaattg ctctatgaaa tataagtctg 360
cttttggttc tgttaaaatg ataatgtgca ttgcattaaa aaagcaaaga aaaa 414
(7~ <210> 14
<211> 797
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WO 00/77166 PCT/US00/15973
<212> DNA
<213> Mus musculus
<220>
<223> IMX2-55 ExtendedSequence
<400> 14
gcacaatggagaagcttattgtgggcatcc tgtttctctctgttctttcaggaagtgtag60
cacaaacagacatgaaggggaaggcattta ttttccctcaagaatcatccactgcctagt120
gtccctgataccgaaggtgaggaagtcact gcagaacttcactctgtgtatgaaggcctt180
10cacagacctgacacgcccttacagcatctt ctcctacaacacaagaactaaggacaatga240
gattcttctctttgtggaaaatataggaga atacatgttctatgttgggaatttggtagc300
cattttcaaagcacccacaaatcttcctga tccagtccgtatctgtgtgaactgggagtc360
tgtctctgggattgcagaattctggctgaa tggaaaaccactggggaggaaaggtttgaa420
taagggatacacggtggggggtgatgcaat gatcattataggacaagagcaggattcctt480
15tgggggaaattttgatgcaaagcaatcctt tgttggggagatatgggatgtttccttgtg540
ggaccatgtggtccccctagaaaaggtatc agacagctgtaacaatggcaaccttataaa600
ctggcaagctcttaattatgaagacaatgg ctatgtggtgattaagcccaaactgtggcc660
ttaagctaattgctctatgaaatataagtc tgcttttggctctgttaaaatgataatgtg720
cattgcattaaaaaagcaaagaaatgagaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa780
20aaaaaaaaagggcggcc 797
<210> 15
<211> 125
<212> DNA
25<213> Mus
musculus
<220>
<223> IMX2_57
<400> 15
30 cggcgatgta cactcgggtc agcaagttca gtacctggat caaccaagtc atggcctaca 60
actaaactgt ccacagatcc gttccccatc tcaatctaat aaacatactc gtctcagttc 120
aaaaa 125
<210> 16
35 <211> 440
<212> DNA
<213> Mus musculus
<220>
4~ <223> IMX2-57 Extended sequence
<400> 16
cnttnggggc tcacctgtgt caccactggc tggggccgaa tcagtggngt gggcaatgtg 60
acaccagctc gcctgcagca agtngttcta cccctggtca ctgtgaatca gtgtcggcag 120
tactggggtg cacgcattac cgatgccatg atatgtgcag gtggctcagg cgcctcctca 180
45 tgtcngggtg actcaggagg ccctcttgtc tgccagaagg gaaacacctg ggtgcttatt 240
gggattgtct cctggggcac taagaactgc aacatacaag caccggccat gtacactcgg 300
gtcagcaagt tcagtacctg gatcaaccaa gtcatggcct acaactaaac tgtccacaga 360
tccgttcccc atctcaatct aataaacata ctcgtctcaa aaaaaaaaaa aaaaaaaaaa 420
aaaaaaaaaa aaaaaaaaaa 440
5~
<210> 17
<211> 223
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_61
<400> 17
cgggtatggc agggatctgg agctcctggg atggcgcgct ctctctcctt tcatttgtga 60
ccagcatgtc agtctgtaaa gctccaaccc catgctcaga aggcaggagg gccacatagt 120
gaagacacca gcccaaaacc actggctgcc tcttatgtgt ggctaggggt ggggtccagt 180
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
gagcttccca tcaaatctct gtacaacacc atcccctcaa aaa 223
<210> 18
<211> 1225
$ <212> DNA
<213> Mus musculus
<220>
<223> IMX2_61 Extendedsequence
<400> 18
aggaattcggcacgaggcatcctactcctg tgttggcaatggaagcagtacaaagctgac60
tcccacacgaccacgtcactcaccgttgct ggtatctgcacacaccagggtcctgtgctc120
cttggtttattctccatccctacactacac tgggactctatgccaggcgatgagctagct180
atgctcgccttccttgtgctcctgagtatg gcagggatctggagctcctgggatggcgcg240
1$ ctctctctcctttcatttgtgaccagcatg tcagtctgtaaagctccaaccccatgctca300
gaaggcaggagggccacatagtgaagacac cagcccaaaaccactggctgcctcttatgt360
gtggctaggggtggggtccagtgagcttcc catcaaatctctgtacaacaccatcccctc420
aaaaaaaagctatccccactgtaagggacc cagacctcacattcaggaacaggtcacagg480
tggctatgaacaaaattatatgttgtttct tgttctgttggtttttttttttacatctag540
aataaattatttaaattatttcatagcaag ggagagggatatttgtcatctttttttttc600
ttttgaagattttgtcatatttttttaaga ttatgtttttatgttcttgggctaatggag660
caacactgccccctgacacagtgaccaccc aagcagcaaagccgccctcggctccttcct720
tcttgccttgggagctttctttctgatgac tcaggaactttgtgtgaatgagggagaacg780
cttggagatgagcttgtacccaccttagct ctacaataattctgcttcctagaacaaaac840
2$ ttgaggttgtatcccagagggaaacgggaa tcaagatacggacctatgcttttcatatga900
aaccgtgcctgaagccgtttgagtgattgt ttgaatgtttcttaaattccttgtaccttt960
gtaaaaaagtaaataaaaaataattaagaa ataaaagttaaaatagacacagaatcgtgc1020
aatgtaagaatatgacaatctactgtgggt ggtaattcctgcctgtaatcccagttcatg1080
gaaggctgaggcaggaagattgaaaattcc agaccagcttgggcaaaggagtctaagact1140
ctgcctcaaccaaaataataataaataata acaccagactcgaaaaaaaaaaaaaaaaaa1200
aaactcgagggggggccggtaccca 1225
<210> 19
3$ <211> 427
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_63
<400> 19
cggtgataag agcaacttcgcacgttggcggtaccaggtgactgtcaccctgtctggaca 60
gaaggtcact gggcacattctagtttctttgtttggaaatggaggaaactctaaacagta 120
tgaagttttc aagggctctctgcagccaggtacttctcacgtcaatgaattcgactctga 180
4$ tgtggatgttggagatttgcagaaggttaaatttatttggtacaacaatgtgatcaaccc 240
aactctaccc aaagtgggagcatcaaggatcacagtggaaagaaatgatggcagagtgtt 300
caacttctgt agtcaagagacagtgagggaagacgtcctgctcacactgtctccatgtta 360
ggaggctgct gctgtgtgaccaccaagtcccactgttgtaataaaagtctagtattaaag 420
ccaaaaa 427
$0
<210> 20
<211> 180
<212> DNA
<213> Mus musculus
$$
<220>
<223> IMX2_74
<400> 20
cggtctcaga gattagcatg gtgggacaag ggcttctggt ctccgtgttc actctacaat 60
60 cctttctggt actccccttc cctctcattg tcttaaacag caatgcttaa caagctagaa 120
atgtgctttc ttgactactg cgtctctgtc aaaccagtaa agttttggag ccaacaaaaa 180
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
<210> 21
<211> 147
<212> DNA
$ <213> Mus musculus
<220>
<223> IMX2_33
<400> 21
cggctccctg tatcccaggc ttggatcctg tggaccaggg ttactgtttt accactaaca 60
tctccttttg gctcagcatt caccgatctt tagggaaatg ctgttggaga gcaaataaat 120
aaacgcattc atttctctat gcaaaaa 147
1$ <210> 22
<211> 124
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_64
<400> 22
cggtggccta acgaaagagg gagccgtcta aggtaggaca gatgattggg gttaagtcgt 60
aacaaggtat ccctacgaga acgtggggat ggatcacctc ctttctaagg agaaaaacga 120
~$ aaaa 124
<210> 23
<211> 140
30 <212> DNA
<213> Mus musculus
<220>
<223> IMX2_21
3$ <400> 23
cggtaagtga aagcgggagg ggcatggcag tatccagagt accacgagac acgtggaacc 60
ttgtgggaat gagcggggac caccccgtaa ggctaaatac tactcagtga ccgatagtgc 120
acagtactgt gaaggaaaaa 140
<210> 24
<211> 233
<212> DNA
<213> Mus musculus
4$
<220>
<223> IMX2_49
<400> 24
cgggatgtgggaaggttagaaacgttctttggactgataataggcacatgtatcgggata 60
$0 acatgatggaggaatgtgattcgtcaaaagtttgtcctgcggtaaagaagaaagagaaaa 120
tcctcaaatcaagctgcatggactagtttgtggcttcattgaggatttcacatggtcacg 180
ttggccccatttttttcaagaggaaaatggggatctttcctaatgcagaaaaa 233
<210> 25
$$ <211> 209
<212> DNA
<213> Mus musculus
<220>
60 <223> IMX2_62
<400> 25
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
cggtcctggc agacagacat gctcattggt tcagctttgc atcagcacag acttcttgta 60
acgaagaaga ttgtgtatac gaagctttat tgaacactgc cattgacgta gattcattag 120
aagcaaactc acatttagat gaagtctgga tcaaagaagt tataaagaag gcaggaatga 180
agctgaaatg gagcaaatta aaacaaaaa 209
<210>
26
<211>
348
<212>
DNA
<213>
Mus musculus
1O
<220>
<223> 5
IMX2
<400>
26
cggacaccatagagaccctgatctgtggcctgggcctggttctcggccttatgggctgcc60
tcctgggcaccgtgctcatgatcacaggcacacgcaggcccagtatccgcaggtaacttc120
tcttctgagaaacccttgagagatgattcctggcggacttctggaagcttctgcgtgctc180
agcggcagcctgtgacagtgttgacctcgagtggcatcaacctctgttcaccaaatccca240
ggagaacattgtgggcgcagtctcctgccctggtaccccatttcactcacagctccagtg300
ccatccacagctctggcagccgcactaaattctcttaagagtcaaaaa 348
<210>
27
<211>
310
<212>
DNA
<213> musculus
Mus
<220>
<223> _6
IMX2
<400>
27
cggacctcaccgaccagcccatcccagacgccgaccacacctggtataccgatgggagca60
gctttttgcaagaaggacagcgaaaggctggggcagcagtgacgactgagaccgaggtaa120
tctgggcgagggccctgccagctggaacgtcagcccagcgagccgaactgatcgcactca180
cccaagccctgaaaatggcagaaggtaagaagctaaatgtttatactgacagccgatatg240
ctttcgccacggcccatgtccatggagaaatctataggaggcgagggttgctgacctcag300
3$ agggcaaaaa 310
<210>
28
<211>
117
<212>
DNA
<213> musculus
Mus
<220>
<223> _7
IMX2
<400>
28
cggactcggcaaatgtgaagaagctgatgaaagaatgggaaaagaagatcagccaaaaga60
aaaagcaaaagagggggaaaaacatcaaaagaacatgaaaaacagaaaacccaaaaa 117
<210> 29
<211> 234
SO <212> DNA
<213> Mus musculus
<220>
<223> IMX2 8
<400> 29
cggagcacca catcgatcta agagtgagca acgacgcgca atcgggagaa acaagcgaga 60
taggaatgtc ttacacgcgg ggcaagacag ttactgatac gggcagacac agaacaagtg 120
aacacaacga gcgactgcca caaaaaaaaa agtgcactcg ggatgcacgt ggcatgaaca 180
cttggacacc gcagacagga gtgaagtact cgggactctc cacctcccca aaaa 234
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
<210> 30
<211> 421
<212> DNA
<213> Mus musculus
<220>
<223> IMX2 11
<400> 30
cggagtcgctatgtgtccaagccgagctaaccancatagagctgttgnatgattttgatg60
agtaccccat gccatccagcaggtcatcaagtcaggctcagatgaggtgcaggcagggca120
gcaacgcaag ttcatcagccacatcaagtgcagaaacgccctgaagctgcagaaagggaa180
gaagtacctc atgtggggcctctcctctgacctctggggagaaaagcccaacaccagcta240
catcattggg aaggacacgtgggtggagcactggcctgaggcggaagaatgccaggatca300
1$ gaagtaccagaaacagtgcgaagaacttggggcattcacagaatctatggtggtttatgg360
ttgtcccaac tgactacagcccagccctctaataaagcttcagttgtatttcacacaaaa420
a 421
<210> 31
<211> 191
<212> DNA
<213> Mus musculus
<220>
2$ <223> IMX2 12
<400> 31
cggagtggca aagaccccaa ccacttccga cctgctggcc tgcctaaaag atactgagtt 60
ttctcttcct gttgttccca gtcatgctgc cccccgagaa gaggagcaac tactgggttg 120
30 agatattttc taaaatctgg atccctaaac atcccaatgt gctgaataaa tacttgtgaa 180
atgcagaaaa a 191
<210> 32
3$ <211> 173
<212> DNA
<213> Mus musculus
<220>
40 <223> IMX2 13
<400> 32
cggatacagc agcagctggg ccagctgacc ctggaaaatc tccagatgct acccgagagc 60
gaggatgagg agagctatga cacggagtca gaattcacag aggatgagct gccctatgat 120
4$ gactgtgtgt ttggaggcca gcgtctgaca ttataagtgg aaagtggcaa aaa 173
<210> 33
<211> 311
<212> DNA
$0 <213> Mus musculus
<220>
<223> IMX2 15
$$ <400> 33
cgggccgatg atgctaacgtggttcgtgaccgtgaccttgaggtggacaccaccctcaag60
agcctgagtc agcagattgagaacatccgcagccccgaaggcagccgcaagaaccctgcc120
cgcacatgcc gcgacctcaagatgtgccactctgactggaagagcggagagtactggatc180
gaccctaacc aaggctgcaacctggacgccatcaaggtctactgcaacatggagacaggt240
60 cagacctgtgtgttccctactcagccgtctgtgcctcagaagaactggtacatcagcccg300
aaccccaaaa a 311
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
(O
<210> 34
<211> 138
<212> DNA
$ <213> Mus musculus
<220>
<223> IMX2 16
1~ <400> 34
cgggcgatgg tggtgtatgc ctttaatccc agcacttggg aggcagaggc agttggattt 60
ctgagttcga ggccagtctg gtctataaag tgagttccag gtcagccagg gctatacaga 120
gaaattctgt cccaaaaa 138
1$ <210> 35
<211> 99
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_20
<400> 35
cgggggtgcc aggtgtgagg ccttaggact ctggctctct gagctcagct cagggttagg 60
gcctcactgg attagaggct ctgctctaca ggataaaaa 99
<210> 36
<211> 109
<212 >
DNA
<213> Mus
musculus
<220>
<223> IMX223
3$<400> 36 _
cggtcatgggaactcagtattattaatagtcacaacatgatttcagaactagatagccct 60
cccacaccaagaagaatgtgagaggaagtaaggtcactttatgcaaaaa 109
<210> 37
<211> 313
<212> DNA
<213> Mus musculus
<220>
4$<223> IMX2_24
<400> 37
cggtctccatggcctgccactagtgtgttcgccatgttgggataccttcttcccttgaac 60
caaagggagagatgtggaaatctgctcctctgttctcctttttcagaaaagcacagaaca 120
aatctacttcagtaaatctctcatctgcccagccaagtgagggtctgagctcagccaacc 180
JOcctactgtctctcgagacctcctactctacttgaagggtagagctgttccttcttgggac 240
tgtccactccacctgccagtcaggacccgatccatagcaaatggaagatacagctctctt 300
gcttacccaaaaa 313
<210> 38
5$<211> 325
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_25
<400> 38
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
cggtgaccat cgagaacaaaggatccacaccccaaacctacaaggtcataagcacactta60
ccatctctga aatcgactggctgaacctgaatgtgtacacctgccgtgtggatcacaggg120
gtctcacctt cttgaagaacgtgtcctccacatgtgctgccagtccctccacagacatcc180
taaccttcac catccccccctcctttgccgacatcttcctcagcaagtccgctaacctga240
cctgtctggtctcaaacctggcaacctatgaaaccctggatatctcctgggcttctcaaa300
gtggtgaacc actggaaaccaaaaa 325
<210> 39
<211> 294
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_26
<400> 39
cggtgccctg tctgctctga gcgacctgca tgcccacaag ctgcgtgtgg atcccgtcaa 60
cttcaagctc ctgagccact gcctgctggt gaccttggct agccaccacc ctgccgattt 120
cacccccgcg gtgcatgcct ctctggataa attccttgcc tctgtgagca ccgtgctgac 180
ctccaagtac cgttaagctg ccttctgcgg ggcttgcctt ctggccatgc ccttcttctc 240
tcccctgcac ctgtacctct tggtctttga ataaagcctg agtaggaata aaaa 294
<210> 40
<211> 288
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_35
<400> 40
cggtactggg gaggcacagg caggcggatc cctgtgagtt cagggccagc ctgggctaca 60
gagtgagttg caggacagcc agggctacac aaagaagccc tgtcttgaga gaccaaaacc 120
ccaatctaac caaacaaaac caaaaacaaa ccaaaaaaca aaacccaaac aaaacaggtt 180
tttgggaatg ggttgtagtt cagaacactt gtctaatatg ggcaatgctc tgggttccat 240
ctcagcatta cagaaattaa taaaaaacta ttttgggcat aataaaaa 288
<210> 41
<211> 172
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_39
<400> 41
cggataacag tatgtgtatg tgctgcatgc caatgagcca agtcctggag agggagacag 60
caattgtgtg accaggattt accactccca tgttgatgct ccaaaagata ttgcatcagg 120
actcatagga cctctaatac tctgtaaaaa aggttctcta tataaggaaa as 172
<210> 42
<211> 39
SO <212> DNA
<213> Mus musculus
<220>
<223> IMX2_40
SS <400> 42
cggcattgta gaacagtgta tatcaatgag ttacaaaaa 39
<210> 43
<211> 150
60 <212> DNA
<213> Mus musculus
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
la
<220>
<223> IMX2_42
<400> 43
$ cggccaaact ctcaattacc atagatggag aaaccaaagt attccacgac aaaaccaaat 60
tcacacatta tatttccaag aatccagccc ttcaaaggat aataacagga aaaaaaacaa 120
tacaaggaca gaaatcatgc cctagaaaaa 150
<210> 44
1~ <211> 39
<212> DNA
<213> Mus musculus
<220>
1$ <223> IMX2_51
<400> 44
cggtagggta gagtgtcgcc aaggaaaaa 39
<210> 45
<211> 291
<212> DNA
<213> Mus musculus
2$ <220>
<223> IMX2_ 52
<400> 45
cggtgtcctg tctgctctgagcgacctgcatgcccacaagctgcgtgtggatcccgtcaa60
cctcaagctc ctgagccactgcctgctggtgaccttggctagccaccaccctgccgattt120
30 cacccccgcggtgcatgcctctctggataaattccttgcctctgtgagcaccgtgctgac180
ctccaagtac cgttaagctgccttctgcggggcttgccttctggccatgcccttcttctc240
tcccttgcac ctgtacctcttggtctttgaataaagcctgagtaggaaaaa 291
<210> 46
3$ <211> 283
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_ 53
<400> 46
cggttcccat atctttgagggccctgggaccgagggcccgatgacccgttttttggcaca60
tcagttgatt gactatcaggtgggtgaaggactctgccctttatatccctcacagagcga120
cactggtcag ctctatgataacccttgccacacttagagcaaagagtgagagtccctccc180
4$ tgtttatctggagctctgcaatctttcttaaaatgcccaggctttccgcaattaaaacat240
gtcctctgat catttctgctcatggagcggttctgagattgga 283
<210> 47
<211> 421
$~ <212> DNA
<213> Mus musculus
<220>
<223> IMX2 58
_
$$ <400> 47
cggcgcgtat ctgtgtgaactgggagtctggctctgggattgcagaattctggctgaatg 60
gaaaaccact ggggaggaaaggcttgaagaagggatacactgtggggggtgatgcaatga 120
tcactctagg acaagagcaggattcctatgggggaaattttgatgcaaagcaatcctttg 180
ttggggagat atgggatgtttccttgtgggaccatgtggtccccctagaaaaggtatcag 240
60 acagctgtaacaatggcaaccttataaactggcaagctcttaattatgaagacaatggct 300
atgtggtgac taagcccaaactgtggccttaagctaattgctctatgaaatataagtctg 360
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
13
cttttggttc tgttaaaatg ataatgtgca ttgcattaaa aaagcaaaga aatgtgaaaa 420
a 421
<210> 48
$ <211> 271
<212> DNA
<213> Mus musculus
<220>
<223> 59
IMX2_
<400> 48
cggcggcgat atccagtctggctgcaacggtgactctggaggacccctcaactgtcccgc60
tgacaatggc acctggcaggtccacggtgtgaccagctttgtgtcctccttgggctgcaa120
caccctgagg aagcccacagtgttcacccgtgtctcagccttcattgactggattgagga180
1$ gaccattgccaacaactagatccaaggttcggctggcagagaggacccccaggtcctcta240
aagaataaag acctttctgaaagcctaaaaa 271
<210> 49
<211> 418
<212> DNA
<213> Mus musculus
<220>
<223> IMX2 60
_
25 <400>
49
cggctcgtat ctgtgtgaactgggagtctggctctgggattgcaagaattctggctgaat60
ggaaaaccac tggggaggaaaggcttgaagaagggatacactgtggggggtgatgcaatg120
atcactctag gacaagagcaggattcctatgggggaaattttgatgcaaagcaatccttt180
gttggggaga tatgggatgtttccttgtgggaccatgtggtccccctagaaaaggtatca240
30 gacagctgtaacaatggcaaccttataaactggcaagctcttaattatgaagacaatggc300
tatgtggtga ctaagcccaaactgtggccttaagctaattgctctatgaaatataagtct360
gcttttggtc tgttaaaatgataatgggcattgcattaaaaaagcaaagaaataaaaa 418
<210> 50
3$ <211> 352
<212> DNA
<213> Mus musculus
<220>
40 <223> IMX2_1
<400> 50
cggaaacggg gaccgctggt ggctgcggtg ctgttcatca cgggaattat cattctcact 60
agtgggaagt gtaggcagtt gtctcaattt tgcctgaatc gccacaggtg agtgcgggcc 120
agcaccctga tgggcacccc agctggagcc tccaaactac accaactcac caccccctgc 180
4$ ctcctccctc taccccaaga gcctacagag tgatcaacat gaaagaatcc tgaaaggaag 240
aggccactgg agggagtcag gcttaaggct aatggtcttc ccaccctggg gagagaggtc 300
tccctaggca ctgctgtggc tgttcagata aatccacatg gtctctcaaa as 352
<210> 51
$0 <211> 135
<212> DNA
<213> Mus musculus
<220>
$$ <223> IMX2_65
<400> 51
cggaaacccc gaaaccaaac gagctaccta aaaacaattt tatgaatcaa ctcgtctatg 60
tggcaaaata gtgagaagat ttttaggtag aggtgaaaag cctaacgagc ttggtgatag 120
ctggttaccc aaaaa 135
<210> 52
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
<211> 186
<212> DNA
<213> Mus musculus
$ <220>
<223> IMX2_66
<400> 52
cggacggagg accacccgtg ccagaagtgt ggccacaagg aggcagtgtt ctttcagtca 60
cacagtgccc gagctgagga cgccatgcgc ctgtactatg tttgcactgc cccacactgc 120
ggccaccgct ggactgagtg atcgttcctt cttccacctg taataaatgc cagtttctac 180
taaaaa
186
<210> 53
<211> 216
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_68A
<400> 53
cggccgccac ccaacaactt tgtacatttc tcattctgta gcgtttgtca tgaaattgct 60
tctccagtct aacccgcctg atgtacatct actatttcca ggagagtctg ctcccagaca 120
ctctgccttt ccctccaaaa ccctctcact cccagctcgt gcaaactggt tacacagcag 180
aaacgcaaaa taaagaggtg gctttcgcgg caaaaa 216
<210> 54
<211> 216
<212> DNA
<213> Mus musculus
<220>
<223> IMX2_68B
<400> 54
cggccgcccg cagaggtccg aaagaagccg agtgagggtg aagaggaggc agcctcagct 60
ggaggacccc aggttaaccc aatgccagtg acagatgagg tcgtgtgacc ttcagtggct 120
gtctacagct cctgcttgag tttctgtgga gttgtccccc cccccccagg gtggtgttgc 180
tcactgtaat aaacatgatt aatagctggc taaaaa 216
<210> 55
<211> loo
<212> DNA
<213> Mus musculus
<220>
4$ <223> IMX2_69
<400> 55
cggccgtgtg tgccgtagga gtgggaaact ttgcatttct ctctccttat ccttcttgta 60
agacatccat ttaataaagt ctcatgctga gagccaaaaa 100
$0 <210> 56
<211> 312
<212> DNA
<213> Mus musculus
55 <220>
<223> IMX2_71
<400> 56
cgggcatcca tgggttccaa ctgccactgc cccagtcttg gccagagata cccctcctgc 60
ctgactggaa gctgcacatc tgcccactga gctttggtga aaggtccaga ggctt.tgggg 120
60 acctctgttc ctgggccacc ctgcccgtgg gcaccctcta ccttggggca cgttctagca 180
ccccattcct gactcctgga agatgcactt gccccgacag ctgggcagca cggctgtcct 240
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
IS
ctgcagagac tgcctggtcc tcattgtact ttggtggctc aactgaataa agccttgtgg 300
gaagcacaaa as 312
<210> 57
$ <211> 374
<212> DNA
<213> Mus musculus
<220>
10<223> IMX2_72
<400> 57
cgggctcaaccgcgtgaaggtttcccaggcagctgcagacttgaaacagttctgtccgca60
gaatgctcaacatgaccctctgctgactggagtgtcttcaagtacgaatcccttcagacc120
ccagaaagtctgctcctttttgtagtcatctatcttgaggtttctcaaaccacttttcat180
15gaaccagtgaatattcaagagaactaaatttgaagtctgtacaaaagcttctctttaaca240
cgtgccataatacactatcttctgctcgtcagtccttaacatctacctctctgaatttca300
tggatttctgtctcacaaggtttaactattttatatacactggctgtagcatacaataaa360
gcatcatccaaaaa 374
20<210> 58
<211> 251
<212> DNA
<213> Mus
musculus
25<220>
<223> IMX2_73
<400> 58
cggtaagcatggcaagacccgcaagttcaccgcgggttcttaccctcgcctggaagagta60
ccgcaaaggcatctttggagactggtccgactccatctctgccctctactgcaagtgcta120
30ttgatgccttgaggctctgtctacccagcctggccttgggaattgctgtagctccaagag180
ccaggaggcaagatgaccccacgacctgctctcatagcttccctgtaatacagccctttc240
aaaggtaaaaa 251
<210> 59
35<z11> 248
<212> DNA
<213> Mus musculus
<220>
40 <223> 2
IMX2_
<400> 59
cggaacgcca aggaggcagatgtgtcactcacagccttcgtcctcatcgcactgcaggaa60
gccagggaca tctgtgaggggcaggtcaatagccttcctgggagcatcaacaaggcaggg120
gagtatattg aagccagttacatgaacctgcagagaccatacacagtggccattgctggg180
45 tatgccctggccctgatgaacaaactggaggaaccttacctcggcaagtttctgaacaca240
gccaaaaa 248
<210> 60
50 <211> 64
<212> DNA
<213> Mus musculus
<220>
55 <223> IMX2 3
<400> 60
cggaatggga gcggggccgt gacacccagc tagggcacaa taaagttata cttacgctga 60
aaaa 64
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
~~0
<210> 61
<211> 121
<212> DNA
<213> Mus musculus
<220>
<223> IMX2 34
<400> 61
cgggggtgcc aggtgtgagg ccttaggact ctggctctct gagctcagct cagggtcagg 60
gcctcgctgg atgaggggct ctgctctaca gggtaaataa aagaaaagct ttttgacagc 120
c 121
<210> 62
<211> 219
<212> DNA
<213> Mus musculus
<220>
<223> IMX2 70
<400> 62
cgggcatcta atggccagtg gcaggtgcat ggcatcgtga gcttcggctc ctctctgggc 60
tgcaactacc cccgcaagcc atccgtcttc accagggtct ccaactacat tgactggatc 120
aactcggtga tggcaaggaa ctaactgaag acattactgc cactgtcccc ctggaaatgc 180
catagaaaag aaatagtaat aaagtaatta aagaatcac 219
<210> 63
<211> 49
<212> DNA
<213> ArtificialSequence
35<223> Descriptionof ArtificialSequence: synthetic
primer
<400> 63
gaattcaact ggaagcggcc cgcaggaatttttttttttt ttttttvnn 49
<210> 64
<211> 16
<212> DNA
<213> ArtificialSequence
<223> Descriptionof ArtificialSequence: synthetic
primer
<400> 64
50aggtcgacgg tatcgg 16
<210> 65
<211> 16
<212> DNA
$$<213> ArtificialSequence
<223> Descriptionof ArtificialSequence: synthetic
primer
()0<400> 65
ggtcgacggt atcggn 16
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
<210> 66
<211> 15
<212> DNA
<213> Artificial Sequence
<223> Description of ArtificialSequence: synthetic
primer
10<400> 66
gagctccacc gcggt 15
<210> 67
<211> 16
15<212> DNA
<213> Artificial Sequence
<223> Description of ArtificialSequence: synthetic
primer
20
<400> 67
cgacggtatc ggnnnn 16
<210> 68
25<211> 16
<212> DNA
<213> Artificial Sequence
<223> Description of ArtificialSequence: synthetic
30primer
<400> 68
cgacggtatc ggcgcg 16
35<210> 69
<211> 30
<212> DNA
<213> Artificial Sequence
40<223> Description of ArtificialSequence: synthetic
primer
<400> 69
gatcgaatcc ggatacagca gcagctgggc 30
45
<210> 70
<211> 30
<212> DNA
<213> Artificial Sequence
$0
<223> Description of ArtificialSequence: synthetic
primer
<400> 70
55gatcgaatcc gggctctggg tctattgttc 30
<210> 71
<211> 30
<212> DNA
60<213> Artificial Sequence
CA 02376667 2001-12-10
WO 00/77166 PCT/LTS00/15973
Ig
<223> Description of Artificial synthetic
Sequence:
primer
<400> 71
gatcgaatcc gggggtgcca ggtgtgaggc 30
<210> 72
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 72
gatcgaatcc ggtcatggga actcagtatt 30
<210> 73
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 73
gatcgaatcc ggtgccctgt ctgctctgag 30
<210> 74
<z11> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 74
gatcgaatcc ggctccctgt atcccaggct 30
<210> 75
<211> 30
<212> DNA
<213> Artificial Sequence
4$ <223> Description of Artificial synthetic
Sequence:
primer
<400> 75
gatcgaatcc gggggtgcca ggtgtgaggc 30
<210> 76
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 76
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
~q
gatcgaatcc ggataacagt atgtgtatgt 30
<210> 77
<211> 30
S <212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 77
gatcgaatcc ggccaaactc tcaattacca 30
<210> 78
IS <211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 78
gatcgaatcc ggcgcgcacg gggaccagac 30
ZS <210> 79
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 79
gatcgaatcc ggtgtcctgt ctgctctgag 30
3S
<210> 80
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 80
4S gatcgaatcc ggaaaccccg aaaccaaacg 30
<210> 81
<211> 30
<212> DNA
S0 <213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
SS <400> 81
gatcgaatcc ggacggagga ccacccgtgc 30
<210> 82
60 <211> 30
<212> DNA
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
o~D
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 82
gatcgaatcc ggccgtgtgt gccgtaggag 30
<210> 83
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 83
gatcgaatcc gggcatctaa tggccagtgg 30
<210> 84
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 84
gatcgaatcc gggcatccat gggttccaac 30
<210> 85
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
primer
<400> 85
gatcgaatcc
ggacaccata
gagaccctga
30
<210> 86
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
primer
<400> 86
gatcgaatcc
ggagcaccac
atcgatctaa
30
<210> 87
<211> 30
<212> DNA
<213> Artificial Sequence
(70<220>
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
<223> Description of ArtificialSequence: synthetic
primer
<400> 87
S gatcgaatcc gggcgatggt ggtgtatgcc 30
<210> 88
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 88
gatcgaatcc ggtactgggg aggcacaggc 30
<210> 89
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 89
gatcgaatcc gggatgtggg aaggttagaa 30
<210> 90
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 90
gatcgaatcc ggtagggtag agtgtcgcca 30
<210> 91
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 91
gatcgaatcc ggttcccata tctttgaggg 30
<210> 92
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
o~
primer
<400> 92
gatcgaatcc ggcgatgtac actcgggtca 30
J
<210> 93
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
15<400> 93
gatcgaatcc ggcgcgtatc tgtgtgaact 30
<210> 94
<211> 30
20<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
25primer
<400> 94
gatcgaatcc ggcggcgata tccagtctgg 30
30<210> 95
<211> 30
<212> DNA
<213> Artificial Sequence
3$<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 95
40gatcgaatcc ggtcctggca gacagacatg 30
<210> 96
<211> 30
<212> DNA
45<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
50
<400> 96
gatcgaatcc ggtgataaga gcaacttcgc 30
<210> 97
55<211> 30
<212> DNA
<213> Artificial Sequence
<220>
60<223> Description of ArtificialSequence: synthetic
primer
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
o~
<400> 97
gatcgaatcc ggccgccacc caacaacttt30
$ <210> 98
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 98
1$ gatcgaatcc ggccgcccgc agaggtccga30
<210> 99
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
2$
<400> 99
gatcgaatcc ggtaagcatg gcaagacccg30
<210> 100
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
3$ <223> Description of ArtificialSequence: synthetic
primer
<400> 100
cacagccttc gtcctcatcg cactg 25
<210> 101
<211> 25
<212> DNA
<213> Artificial Sequence
4$
<220>
<223> Description of ArtificialSequence: synthetic
primer
$0 <400> lol
ttgttcatca gggccagggc atacc 25
<210> 102
<211> 25
$$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
60 primer
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
a~
<400> 102
tctgaagccc cgtgctccac ccact 25
<210> 103
$ <211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 103
tcacggcccc gctcccattc c 21
1$
<210> 104
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
2$ <400> 104
ccaagtccca ggcctgtctg tt 22
<210> 105
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
3$ primer
<400> 105
tggtctccac tgtagaaccc ccaaaa 25
<210> 106
<211> 25
<212> DNA
<213> Artificial Sequence
4$ <220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 106
$0 acatagagct gttggatgat toga 25
<210> 107
<211> 25
<212> DNA
$$ <213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
60
<400> 107
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
caagttcttc gcactgtttc tggta 25
<210> 108
<211> 24
$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 108
cgacctcaag atgtgccact ctga 24
1$ <210> 109
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 109
2$ accagttctt ctgaggcaca gacgg 25
<210> 110
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
3$
<400> 110
gaacaaagga tccacacccc aaacc 25
<210> 111
<z11> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 111
gcacatgtgg aggacacgtt cttca 25
$0
<210> 112
<211> 25
<212> DNA
<213> Artificial Sequence
$$
<220>
<223> Description of ArtificialSequence: synthetic
primer
60 <400> 112
atgaaaaata tggaaaatga taaaa 25
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
o~~
<210> 113
<211> 24
<212> DNA
$ <213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 113
ctaaaatgtt ctacagtgtg gttt 24
<210> 114
1$ <211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 114
gcccagacag aagtcatagc cactc 25
<210> 115
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 115
tttatggttc ttccgttgag ggaca 25
<210> 116
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 116
gagtctggct ctgggattgc agaa 24
<210> 117
<211> 24
<212> DNA
<213> Artificial Sequence
$5 <220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 117
cccccatagg aatcctgctc ttgt 24
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
aZ
<210> 118
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 118
ccactgggga ggaaaggctt gaa 23
<210> 119
<211> 25
1$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 119
ccacatggtc ccacaaggaa acatc 25
2$ <210> 120
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
<400> 120
3$ gcaggtgcat ggcatcgtga 20
<210> 121
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: synthetic
primer
4$
<400> 121
ggggacagtg gcagtaatgt cttca 25
<210> 122
$~ <211> 23
<212> DNA
<213> Artificial Sequence
<220>
$$ <223> Description of ArtificialSequence: synthetic
primer
<400> 122
tcagagatta gcatggtggg aca 23
60
<210> 123
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
a8
<211> 25
<212> DNA
<213> Artificial Sequence
$ <220>
<223> Description of Artificial synthetic
Sequence:
primer
<400> 123
ctggtttgac agagacgcag tagtc 25
<210> 124
<211> 30
<212> DNA
IS <213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 124
gatcgaatcc ggaaacgggg accgctggtg 30
<210> 125
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 125
gatcgaatcc ggacctcacc gaccagccca 30
<210> 126
<211> 30
<212> DNA
<213> Artificial Sequence
<223> Description of Artificial synthetic
Sequence:
primer
<400> 126
gatcgaatcc ggagtggcaa agaccccaac 30
<zlo> 127
<211> 25
<212> DNA
<213> Artificial Sequence
$0
<223> Description of Artificial Sequence: synthetic
primer
<400> 127
$$ cagtgtggag gaagcctggg aggtg 25
<210> 128
<211> 24
<212> DNA
60 <213> Artificial Sequence
CA 02376667 2001-12-10
WO 00/77166 PCT/US00/15973
<223> Description of Artificial Sequence: synthetic
primer
<400> 128
cacatcgggg gcaggcagac tttc 24
<210> 129
<211> 137
<212> PRT
<213> Mus
musculus
<220>
<223> Translation IMX2-4 Extended Sequences, bases
of 688-947
<400> 129
Gly Trp GlyAla ProAspPro ArgGlyLeu GlyGln LeuSerGln
Gln
1 5 10 15
Pro Tyr GlyGly GluMetPro TrpThrIle LeuLeu PheAlaSer
Met
20 25 30
Val Pro TrpIle LeuAlaLeu SerLeuSer LeuAla GlyAlaVal
Thr
35 40 45
Leu Phe GlyLeu ValAlaIle ThrValLeu ValArg LysAlaLys
Ser
50 55 60
Ala Lys LeuGln LysGlnArg GluArgGlu SerCys TrpAlaGln
Asn
65 70 75 80
Ile Asn ThrAsn ThrAspMet SerPheAsp AsnSer LeuPheAla
Phe
85 90 95
Ile Ser Thr Lys Met Thr Gln Glu Asp Ser Val Ala Thr Leu Asp Ser
100 105 110
Gly Pro Arg Lys Arg Pro Thr Ser Ala Ser Ser Ser Pro Glu Pro Pro
115 120 125
Glu Phe Ser Thr Phe Arg Ala Cys Gln
130 135
