Note: Descriptions are shown in the official language in which they were submitted.
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10
SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of the following applications: Ser.
No.
60/XXX,XXX (converted to a provisional application from non-provisional
application
08/769,192), filed December 18, 1996; and Ser. No. 08/783,401, filed January
13, 1997; all
of which are incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by
such polynucleotides, along with therapeutic, diagnostic and research
utilities for these
polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and interleukins) has matured rapidly
over the
past decade. The now routine hybridization cloning and expression cloning
techniques
3 0 clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein (i.e., partial DNA / amino acid sequence of
the protein
in the case of hybridization cloning; activity of the protein in the case of
expression
cloning). More recent "indirect" cloning techniques such as signal sequence
cloning, which
isolates DNA sequences based on the presence of a now well-recognized
secretory leader
3 5 sequence motif, as well as various PCR-based or low stringency
hybridization cloning
techniques, have advanced the state of the art by making available large
numbers of
DNA / amino acid sequences for proteins that are known to have biological
activity by
virtue of their secreted nature in the case of leader sequence cloning, or by
virtue of the
cell or tissue source in the case of PCR-based techniques. It is to these
proteins and the
4 0 polynucleotides encoding them that the present invention is directed.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:2;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:2 from nucleotide 41 to nucleotide 760;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CBI07_1 deposited under accession
number ATCC 98279;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CBI07_1 deposited under accession number ATCC 98279;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CB107_1 deposited under accession number
ATCC 98279;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CB107_1 deposited under accession number ATCC 98279;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:3;
2 0 (h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:3 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
2 5 of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in {a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:2 from nucleotide 41 to nucleotide 760; the nucleotide sequence of the full-
length
3 0 protein coding sequence of clone CB107_1 deposited under accession number
ATCC
98279; or the nucleotide sequence of the mature protein coding sequence of
clone CB107_l
deposited under accession number ATCC 98279. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CB107_1 deposited under accession number ATCC 98279. In yet other
preferred
2
_._~.~__..__ _.___ .
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embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:3 from amino acid 127 to amino
acid
240.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:2, SEQ ID NO:1 or SEQ ID N0:4 .
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:3;
(b) the amino acid sequence of SEQ ID N0:3 from amino acid 127 to
amino acid 240;
(c) fragments of the amino acid sequence of SEQ ID N0:3; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CB107_1 deposited under accession number ATCC 98279;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:3 or the amino acid
sequence
of SEQ ID N0:3 from amino acid 127 to amino acid 240.
1n one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 374 to nucleotide 1108;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:5 from nucleotide 500 to nucleotide 1108;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1 to nucleotide 387;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CG300_3 deposited under accession
3 0 number ATCC 98279;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CG300 3 deposited under accession number ATCC 98279;
3
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(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CG300 3 deposited under accession number
ATCC 98279;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CG300 3 deposited under accession number ATCC 98279;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleoHde capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 374 to nucleotide 1108; the nucleotide sequence of SEQ ID
N0:5
from nucleotide 500 to nucleotide 1108; the nucleotide sequence of SEQ ID N0:5
from
nucleotide 1 to nucleotide 387; the nucleotide sequence of the full-length
protein coding
2 0 sequence of clone CG300 3 deposited under accession number ATCC 98279; or
the
nucleotide sequence of the mature protein coding sequence of clone CG300 3
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CG300_3 deposited under accession number ATCC 98279. In yet other
preferred
2 S embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:6 from amino acid 23 to amino
acid
57.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
3 0 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
4
_.._._ .~_____,._ ..~_.~ __~__.w~.~.r.~.__. ~._._ . . _..__~_ .
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(b) the amino acid sequence of SEQ ID N0:6 from amino acid 23 to
amino acid 57;
(c) fragments of the amino acid sequence of SEQ ID N0:6; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CG300_3 deposited under accession number ATCC 98279;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:6 or the amino acid
sequence
of SEQ ID N0:6 from amino acid 23 to amino acid 57.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 126 to nucleotide 3053;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 180 to nucleotide 3053;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 49 to nucleotide 382;
(e) a polynucleotide comprising the nucleotide sequence of the full-
2 0 length protein coding sequence of clone CJ145_1 deposited under accession
number ATCC 98279;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CJ145_1 deposited under accession number ATCC 98279;
(g) a polynucleotide comprising the nucleotide sequence of the mature
2 5 protein coding sequence of clone CJ145_1 deposited under accession number
ATCC 98279;
(h) a polynucieotide encoding the mature protein encoded by the
cDNA insert of clone CJ145_1 deposited under accession number ATCC 98279;
(i) a polynucleotide encoding a protein comprising the amino acid
- 3 0 sequence of SEQ ID N0:8;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
5
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(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleodde capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 126 to nucleotide 3053; the nucleotide sequence of SEQ ID
N0:7
from nucleotide 180 to nucleotide 3053; the nucleotide sequence of SEQ ID N0:7
from
nucleotide 49 to nucleotide 382; the nucleotide sequence of the full-length
protein coding
sequence of clone CJ145_1 deposited under accession number ATCC 98279; or the
nucleotide sequence of the mature protein coding sequence of clone CJ145_2
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CJ145_1 deposited under accession number ATCC 98279. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:8 from amino acid 1 to amino
acid 87.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
2 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) the amino acid sequence of SEQ ID N0:8 from amino acid 1 to
amino acid 87;
(c) fragments of the amino acid sequence of SEQ ID N0:8; and
2 5 (d) the amino acid sequence encoded by the cDNA insert of clone
CJ145_l deposited under accession number ATCC 98279;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:8 or the amino acid
sequence
of SEQ ID N0:8 from amino acid 1 to amino acid 87.
3 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
6
___....._ ___ ~_._~~..._._ __-
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(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 40 to nucleotide 342;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 127 to nucleotide 342;
S (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 11 to nucleotide 181;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CJ160_ll deposited under accession
number ATCC 98279;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CJ160_11 deposited under accession number ATCC 98279;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CJ160_11 deposited under accession number
ATCC 98279;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CJ160_ll deposited under accession number ATCC 98279;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:10;
(j) a polynucleotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID N0:10 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
2 S {m) a polynucleotide capable of hybridizing under stringent condirions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 40 to nucleotide 342; the nucleotide sequence of SEQ ID
N0:9 from
nucleotide 127 to nucleotide 342; the nucleotide sequence of SEQ ID N0:9 from
3 0 nucleotide 11 to nucleotide 181; the nucleotide sequence of the full-
length protein coding
sequence of clone CJ160_11 deposited under accession number ATCC 98279; or the
nucleotide sequence of the mature protein coding sequence of clone CJ160_11
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
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clone CJ160_11 deposited under accession number ATCC 98279. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:10 from amino acid 7 to amino
acid
48.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 7 to
amino acid 48;
(c) fragments of the amino acid sequence of SEQ ID NO:10; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CJ160_l l deposited under accession number ATCC 98279;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid
sequence
of SEQ ID NO:10 from amino acid 7 to amino acid 48.
In one embodiment, the present invention provides a composition comprising an
2 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 180 to nucleotide 467;
2 5 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:I1 from nucleotide 267 to nucleotide 467;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone C020_1 deposited under accession
number ATCC 98279;
3 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone C020_1 deposited under accession number ATCC 98279;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone C020_1 deposited under accession number
ATCC 98279;
8
___._. . _ .._ _. _ __~..~_....T_._ r.._._.~_ . ._.__ .. i
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(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone C020_1 deposited under accession number ATCC 98279;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:11 from nucleotide 180 to nucleotide 467; the nucleotide sequence of SEQ ID
NO:11
from nucleotide 267 to nucleotide 467; the nucleotide sequence of the full-
length protein
coding sequence of clone C020_1 deposited under accession number ATCC 98279;
or the
nucleotide sequence of the mature protein coding sequence of clone C020_1
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
2 0 clone C020_1 deposited under accession number ATCC 98279. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:12 from amino acid 1 to amino
acid
37.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 5 ID N0:11 or SEQ ID N0:13.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:12;
3 0 (b) the amino acid sequence of SEQ ID N0:12 from amino acid 1 to
amino acid 37;
(c) fragments of the amino acid sequence of SEQ ID N0:12; and
(d) the amino acid sequence encoded by the cDNA insert of clone
C020 1 deposited under accession number ATCC 98279;
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the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:12 or the amino acid
sequence
of SEQ ID N0:12 from amino acid 1 to amino acid 37.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 176 to nucleotide 520;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 317 to nucleotide 520;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 118 to nucleotide 413;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone C0223_3 deposited under accession
number ATCC 98291;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone C0223_3 deposited under accession number ATCC 98291;
(g) a polynucleotide comprising the nucleotide sequence of the mature
2 0 protein coding sequence of clone C0223_3 deposited under accession number
ATCC 98291;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone C0223 3 deposited under accession number ATCC 98291;
(i) a polynucleotide encoding a protein comprising the amino acid
2 5 sequence of SEQ ID N0:15;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:15 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
3 0 (1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
_. . ________...~_..__.~~... _._
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:14 from nucleotide 176 to nucleotide 520; the nucleotide sequence of SEQ ID
N0:14
from nucleotide 317 to nucleotide 520; the nucleotide sequence of SEQ ID N0:14
from
nucleotide 118 to nucleotide 413; the nucleotide sequence of the full-length
protein coding
sequence of clone C0223 3 deposited under accession number ATCC 98291; or the
nucleotide sequence of the mature protein coding sequence of clone C0223_3
deposited
under accession number ATCC 98291. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone C0223_3 deposited under accession number ATCC 98291. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:15 from amino acid 1 to amino
acid
80.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:14.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:15;
(b) the amino acid sequence of SEQ ID N0:15 from amino acid 1 to
2 0 amino acid 80;
(c) fragments of the amino acid sequence of SEQ ID N0:15; and
(d) the amino acid sequence encoded by the cDNA insert of clone
C0223 3 deposited under accession number ATCC 98291;
the protein being substantially free from other mammalian proteins. Preferably
such
2 5 protein comprises the amino acid sequence of SEQ ID N0:15 or the amino
acid sequence
of SEQ ID N0:15 from amino acid 1 to amino acid 80.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 303 to nucleotide 542;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 1 to nucleotide 435;
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(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone C0310 2 deposited under accession
number ATCC 98279;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone C0310 2 deposited under accession number ATCC 98279;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone C0310_2 deposited under accession number
ATCC 98279;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone C0310_2 deposited under accession number ATCC 98279;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:17;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:17 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
2 0 to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:16 from nucleotide 303 to nucleotide 542; the nucleotide sequence of SEQ ID
N0:16
from nucleotide 1 to nucleotide 435; the nucleotide sequence of the full-
length protein
coding sequence of clone C0310 2 deposited under accession number ATCC 98279;
or the
2 5 nucleotide sequence of the mature protein coding sequence of clone C0310 2
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone C0310_2 deposited under accession number ATCC 98279. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
3 0 comprising the amino acid sequence of SEQ ID N0:17 from amino acid 1 to
amino acid
44.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:16.
12
_.__..~.~ . Y......~. _. ..
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:17;
(b) the amino acid sequence of SEQ ID N0:17 from amino acid 1 to
amino acid 44;
(c) fragments of the amino acid sequence of SEQ ID N0:17; and
(d) the amino acid sequence encoded by the cDNA insert of clone
C0310 2 deposited under accession number ATCC 98279;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:17 or the amino acid
sequence
of SEQ ID N0:17 from amino acid 1 to amino acid 44.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18 from nucleotide 40 to nucleotide 455;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:18 from nucleotide 85 to nucleotide 455;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18 from nucleotide 265 to nucleotide 515;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CP258_3 deposited under accession
2 5 number ATCC 98279;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CP258_3 deposited under accession number ATCC 98279;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CP258 3 deposited under accession number
3 0 ATCC 98279;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CP258_3 deposited under accession number ATCC 98279;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:19;
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(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:19 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:18 from nucleotide 40 to nucleotide 455; the nucleotide sequence of SEQ ID
N0:18
from nucleotide 85 to nucleotide 455; the nucleotide sequence of SEQ ID N0:18
from
nucleotide 265 to nucleotide 515; the nucleotide sequence of the full-length
protein coding
sequence of clone CP258 3 deposited under accession number ATCC 98279; or the
nucleotide sequence of the mature protein coding sequence of clone CP258 3
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CP258_3 deposited under accession number ATCC 98279. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:19 from amino acid 64 to amino
acid
2 0 138.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:18.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
2 5 consisting of:
(a) the amino acid sequence of SEQ ID N0:19;
(b) the amino acid sequence of SEQ ID N0:19 from amino acid 64 to
amino acid 138;
(c) fragments of the amino acid sequence of SEQ ID N0:19; and
3 0 (d) the amino acid sequence encoded by the cDNA insert of clone
CP258 3 deposited under accession number ATCC 98279;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:19 or the amino acid
sequence
of SEQ ID N0:19 from amino acid 64 to amino acid 138.
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In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20 from nucleotide 105 to nucleotide 1007;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20 from nucleotide 801 to nucleotide 1007;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20 from nucleotide 1 to nucleotide 352;
(e) a polynucieotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CW1155 3 deposited under accession
number ATCC 98279;
(f) a polynucleotide encoding the full-length protein encoded by the
15. cDNA insert of clone C W 1155 3 deposited under accession number ATCC
98279;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CW1155_3 deposited under accession number
ATCC 98279;
(h) a polynucleotide encoding the mature protein encoded by the
2 0 cDNA insert of clone CW 1155 3 deposited under accession number ATCC
98279;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:21;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:21 having biological activity;
2 5 (k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
3 0 to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:20 from nucleotide 105 to nucleotide 1007; the nucleotide sequence of SEQ
ID N0:20
from nucleotide 801 to nucleotide 1007; the nucleotide sequence of SEQ ID
N0:20 from
nucleotide 1 to nucleotide 352; the nucleotide sequence of the full-length
protein coding
CA 02274732 1999-06-14
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sequence of clone CW 1155 3 deposited under accession number ATCC 98279; or
the
nucleotide sequence of the mature protein coding sequence of clone CW1155 3
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CW1155 3 deposited under accession number ATCC 98279. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:21 from amino acid 1 to amino
acid
83.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:20.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:21;
(b) the amino acid sequence of SEQ ID N0:21 from amino acid 1 to
amino acid 83;
(c) fragments of the amino acid sequence of SEQ ID N0:21; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CW1155 3 deposited under accession number ATCC 98279;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:21 or the amino acid
sequence
of SEQ ID N0:21 from amino acid 1 to amino acid 83.
1n one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:22;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:22 from nucleotide 11 to nucleotide 1699;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:22 from nucleotide 1682 to nucleotide 1699;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:22 from nucleotide 737 to nucleotide 1134;
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(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CZ247_2 deposited under accession
number ATCC 98279;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CZ247_2 deposited under accession number ATCC 98279;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CZ247_2 deposited under accession number
ATCC 98279;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CZ247_2 deposited under accession number ATCC 98279;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:23;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:23 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
2 0 to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:22 from nucleotide 11 to nucleotide 1699; the nucleotide sequence of SEQ ID
N0:22
from nucleotide 1682 to nucleotide 1699; the nucleotide sequence of SEQ ID
N0:22 from
nucleotide 737 to nucleotide 1134; the nucleotide sequence of the full-length
protein
2 5 _coding sequence of clone CZ247_2 deposited under accession number ATCC
98279; or the
nucleotide sequence of the mature protein coding sequence of clone CZ247_2
deposited
under accession number ATCC 98279. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CZ247_2 deposited under accession number ATCC 98279. In yet other
preferred
3 0 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:23 from amino acid 298 to
amino acid
374.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:22.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:23;
(b) the amino acid sequence of SEQ ID N0:23 from amino acid 298 to
amino acid 374;
(c) fragments of the amino acid sequence of SEQ ID N0:23; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CZ247_2 deposited under accession number ATCC 98279;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:23 or the amino acid
sequence
of SEQ ID N0:23 from amino acid 298 to amino acid 374.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
2 0 (a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
{b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention. Preferred embodiments include those in which the protein produced
by such
2 5 process is a mature form of the protein.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present
invention.
Methods are also provided for preventing, treating or ameliorating a medical
3 0 condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
18
_r.___~. ~. _.__._v~_ .. i
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BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature) can then be determined from such nucleotide sequence. The amino
acid
sequence of the protein encoded by a particular clone can also be determined
by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable
host
cell, is transported across or through a membrane, including transport as a
result of signal
sequences in its amino acid sequence. "Secreted" proteins include without
limitation
2 0 proteins secreted wholly (e.g., soluble proteins) or partially {e.g. ,
receptors) from the cell
in which they are expressed. "Secreted" proteins also include without
limitation proteins
which are transported across the membrane of the endoplasmic reticulum.
Clone "CB107 1"
2 5 A polynucleotide of the present invention has been identified as clone
"CB107_1".
CB107_1 was isolated from a human fetal brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins {see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CB107_1 is a full-
length
3 0 clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CB107_1 protein")
The nucleotide sequence of the 5' portion of CB107_1 as presently determined
is
reported in SEQ ID N0:1. An additional internal nucleotide sequence from
CB107_1 as
presently determined is reported in SEQ ID N0:2. What applicants believe is
the proper
19
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reading frame and the predicted amino acid sequence encoded by such internal
sequence
is reported in SEQ ID N0:3. Additional nucleotide sequence from the 3' portion
of
CB107_l, including the polyA tail, is reported in SEQ ID N0:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CB107_1 should be approximately 3300 bp.
The nucleotide sequence disclosed herein for CBi07_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CB107_1 demonstrated at least some similarity with
sequences
identified as AA121485 (zn80a02.s1 Stratagene lung carcinoma 937218 Homo
Sapiens
cDNA clone 564458 3'), AA428192 (zw51b08.s1 Soares total fetus Nb2HF8 9w Homo
sapiens cDNA clone 773559 3'), D83018 (Human mRNA for nel-related protein 2,
complete
cds), F10919 (H. sapiens partial cDNA sequence; clone c-31g01), H15375
(ym28d09.r1
Homo sapiens cDNA clone 49527 5' similar to SP A54105 A54105 FIBRILLIN-2
PRECURSOR), U48245 (Rattus norvegicus protein kinase C-binding protein Nel
mRNA,
complete cds), U59230 (Mus musculus mel (MEL91) mRNA, complete cds), and
W28387
(46c5 Human retina cDNA randomly primed sublibrary Homo sapiens cDNA). The
predicted amino acid sequence disclosed herein for CB107_1 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted CB107_1 protein demonstrated at least some similarity to
sequences
2 0 identified as D83018 (nel-related protein 2 [Homo sapiens]), 805222
(Antigen GX5401FL
encoded by Eimeria tenella genomic DNA), 879964 (Connective tissue growth
factor),
U48245 (RNU48245_1 protein kinase C-binding protein Nel [Rattus norvegicus]),
and
U59230 (mel [Mus musculus]). Based upon sequence similarity, CB107_1 proteins
and
each similar protein or peptide may share at least some activity.
Clone "CG300 3"
A polynucleotide of the present invention has been identified as clone
"CG300_3".
CG300_3 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
3 0 identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CG300_3 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CG300_3 protein").
~ ~ _ .__~___
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The nucleotide sequence of CG300 3 as presently determined is reported in SEQ
ID N0:5. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CG300_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:6. Amino acids 30 to 42 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 43, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CG300 3 should be approximately 1800 bp.
The nucleotide sequence disclosed herein for CG300 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CG300 3 demonstrated at least some similarity with
sequences
identified as N40185 (yy44d08.s1 Homo Sapiens cDNA clone 276399 3') and W01791
(za72d06.r1 Soares fetal lung NbHLI9W Homo sapiens cDNA clone 298091 5').
Based
upon sequence similarity, CG300 3 proteins and each similar protein or peptide
may
share at least some activity. The TopPredII computer program predicts four
potential
transmembrane domains within the CG300 3 protein sequence, centered around
amino
acids 34, 98, 151, and 179 of SEQ ID N0:6, respectively.
Clone "CT145 I"
2 0 A polynucleotide of the present invention has been identified as clone
"CJ145_l".
CJI45_1 was isolated from a human fetal brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CJ145_1 is a full-
length clone,
2 5 including the entire coding sequence of a secreted protein (also referred
to herein as
"CJ145_I protein").
The nucleotide sequence of CJ145_1 as presently determined is reported in SEQ
ID N0:7. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CJ145_l protein corresponding to the
foregoing
3 0 nucleotide sequence is reported in SEQ ID N0:8. Amino acids 6 to 18 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 19, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CJ145_1 should be approximately 3600 bp.
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The nucleotide sequence disclosed herein for CJ145_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CJ145_1 demonstrated at least some similarity with
sequences
identified as 843655 (yc86b04.s1 Homo sapiens cDNA clone 22829 3'), 850995
(yg63f06.s1
Homo sapiens cDNA clone 37377 3' similar to contains MER22 repetitive
element), and
W92748 (zd92h03.s1 Soares fetal heart NbHHI9W Homo sapiens cDNA clone 356981
3').
Based upon sequence similarity, CJ145 1 proteins and each similar protein or
peptide may
share at least some activity. The nucleotide sequence of CJ145_1 indicates
that it may
contain a CA simple repeat element.
Clone"CT160 11"
A polynucleotide of the present invention has been identified as clone
"CJ160_11".
CJ160_ll was isolated from a human fetal brain cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CJ160_ll is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CJ160_11 protein")
The nucleotide sequence of CJ160 11 as presently determined is reported in SEQ
2 0 ID N0:9. What applicants presently believe to be the proper reading frame
and the
predicted amino acid sequence of the CJ160_ll protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID NO:10. Amino acids 17 to 29 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 30, or are a transmembrane domain.
2 5 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CJ160_l l should be approximately 1700 bp.
The nucleotide sequence disclosed herein for CJ160_11 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CJ160_11 demonstrated at least some similarity with
sequences
3 0 identified as AA024511 (ze76e04.s1 Soares fetal heart NbHHI9W Homo sapiens
cDNA
clone 364926 3') and AC000074 (00884; HTGS phase 3, complete sequence). Based
upon
sequence similarity, CJ160_11 proteins and each similar protein or peptide may
share at
least some activity.
22
_..~ _.. ~_..
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Clone "C020 1"
A polynucleotide of the present invention has been identified as clone
"C020_1".
C020_1 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. C020_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"C020_1 protein")
The nucleotide sequence of the 5' portion of C020_1 as presently determined is
reported in SEQ ID NO:11. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID N0:12. The predicted amino acid
sequence
of the C020_1 protein corresponding to the foregoing nucleotide sequence is
reported in
SEQ ID N0:12. Amino acids 17 to 29 are a predicted leader/signal sequence,
with the
predicted mature amino acid sequence beginning at amino acid 30, or are a
transmembrane domain. Additional nucleotide sequence from the 3' portion of
C020_l,
including the polyA tail, is reported in SEQ ID N0:13.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
C020_l should be approximately 2400 bp.
The nucleotide sequence disclosed herein for C020_1 was searched against the
2 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. C020_1 demonstrated at least some similarity with
sequences
identified as AA045770 (z168b10.s1 Stratagene colon (#937204) Homo sapiens
cDNA clone
509755 3' similar to SW:R13A_HUMAN P40429 60S RIBOSOMAL PROTEIN L13A),
AA070899 (zm66c01.s1 Stratagene neuroepithelium (#937231) Homo Sapiens cDNA
clone
2 5 530592 3' similar to contains Alu repetitive element), AA325205 (EST28155
Cerebellum II
Homo sapiens cDNA 5' end), N22253 (yw36a08.s1 Homo Sapiens cDNA clone 254294
3'
similar to SP S29539 S29539 BASIC PROTEIN, 23K), 801933 (ye85g07.s1 Homo
Sapiens
cDNA clone 124572 3' similar to SP:S29539 S29539 BASIC PROTEIN, 23K), 812008
(yf51f04.r1 Homo Sapiens cDNA clone 25456 5'), 839848 (yf51f04.s1 Homo Sapiens
cDNA
3 0 clone 25456 3' similar to contains Alu repetitive element;contains PTR5
repetitive element),
856565 (yg91c12.r1 Homo sapiens cDNA clone 40891 5'), T19487 (Human gene
signature
HUMGS00543), T30988 (EST25695 Homo Sapiens cDNA 5' end similar to None),
U37026
(Rattus norvegicus brain sodium channel beta 2 subunit (SCNB2) mRNA, complete
cds),
and X56932 (H.sapiens mRNA for 23 kD highly basic protein). The predicted
amino acid
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sequence disclosed herein for C020_1 was searched against the GenPept and
GeneSeq
amino acid sequence databases using the BLASTX search protocol. The predicted
C020_1
protein demonstrated at least some similarity to sequences identified as
U37026 (sodium
channel beta 2 subunit [Rattus norvegicus]), U58658 (unknown [Homo sapiensj),
and
X56932 (23 kD highly basic protein [Homo sapiens]). The sodium channel beta 2
subunit
is a glycoprotein with an extracellular domain containing an immunoglobulin-
like fold
with similarity to the neural cell adhesion molecule contactin. Based upon
sequence
similarity, C020_1 proteins and each similar protein or peptide may share at
least some
activity. The nucleotide sequence of C020_1 indicates that it may contain an
Alu
repetitive element.
Clone "C0223 3"
A polynucleotide of the present invention has been identified as clone
"C0223_3".
C0223_3 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. C0223_3 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "C0223 3 protein").
2 0 The nucleotide sequence of C0223 3 as presently determined is reported in
SEQ
ID N0:14. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the C0223 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:15. Amino acids 35 to 47 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
2 5 amino acid 48, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing
clone
C0223 3 should be approximately 700 bp.
The nucleotide sequence disclosed herein for C0223_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
3 0 FASTA search protocols. C0223 3 demonstrated at least some similarity with
sequences
identified as AA004498 (zh87b06.r1 Soares fetal liver spleen 1NFLS S1 Homo
sapiens
cDNA clone 428243 5' similar to gb M62505 C5A ANAPHYLATOXIN CHEMOTACTIC
RECEPTOR (HUMAN);contains Ll.t1 L1 repetitive element) and U47924 (Human
chromosome 12p13 gene cluster, surface antigen CD4 (CD4), A, B, G-protein beta-
3
24
T_ , __. __.
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subunit (GNB3), isopeptidase T (ISOT) and triosephosphate isomerase (TPI)
genes,
complete cds). _Based upon sequence similarity, C0223_3 proteins and each
similar
protein or peptide may share at least some activity.
The 3' end of the C0223_3 polynucleotide sequence contains a 54-by sequence
that
is repeated three times in the clone; these repeats begin at positions 314,
368, and 422 of
SEQ ID N0:14 and encode amino acids 47 to 64, 65 to 82, and 83 to 99 of SEQ ID
N0:15,
respectively.
Clone "C0310 2"
A polynucleotide of the present invention has been identified as clone "C0310
2".
C0310 2 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding ~ secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. C0310 2 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "C0310 2 protein").
The nucleotide sequence of C0310 2 as presently determined is reported in SEQ
ID N0:16. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the C0310_2 protein corresponding to the
foregoing
2 0 nucleotide sequence is reported in SEQ ID N0:17.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
C0310 2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for C0310_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. No hits were found in the database. The nucleotide
sequence
of C0310_2 indicates that it may contain an L1 repetitive element.
Clone "CP258 3"
A polynucleotide of the present invention has been identified as clone "CP258
3".
3 0 CP258 3 was isolated from a human adult salivary gland cDNA library using
methods
which are selective for eDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or
was identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CP258_3 is a full-
length clone,
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including the entire coding sequence of a secreted protein (also referred to
herein as
"CP258_3 protein").
The nucleotide sequence of CP258 3 as presently determined is reported in SEQ
ID N0:18. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CP258 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:19. Amino acids 3 to 15 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 16, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CP258_3 should be approximately 560 bp.
The nucleotide sequence disclosed herein for CP258_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. No hits were found in the database.
Clone "CW1155 3"
A polynucleotide of the present invention has been identified as clone
"CW1155 _3". CW1155 3 was isolated from a human fetal brain cDNA library using
methods which are selective for cDNAs encoding secreted proteins (see U.S.
Pat. No.
5,536,637), or was identified as encoding a secreted or transmembrane protein
on the basis
2 0 of computer analysis of the amino acid sequence of the encoded protein. CW
1155 3 is a
full-length clone, including the entire coding sequence of a secreted protein
(also referred
to herein as "CW1155 3 protein").
The nucleotide sequence of CW1155_3 as presently determined is reported in SEQ
ID N0:20. What applicants presently believe to be the proper reading frame and
the
2 5 predicted amino acid sequence of the CW1155 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:21. Amino acids 220 to 232 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 233, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing
clone
3 0 CW 1155 3 should be approximately 1170 bp.
The nucleotide sequence disclosed herein for CW1155_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CW1155 3 demonstrated at least some similarity with
sequences
identified as AA169043 (ms36h08.r1 Stratagene mouse heart (#937316) Mus
musculus
26
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WO 98127205 PCT/US97/23330
cDNA clone 613695 5'), D86145 (Rat mRNA), and H29261 (ym32b03.s1 Homo Sapiens
cDNA clone 49733 3'). Based upon sequence similarity, CW1155_3 proteins and
each
similar protein or peptide may share at least some activity.
Clone "CZ247 2"
A polynucleotide of the present invention has been identified as clone
"CZ247_2".
CZ247_2 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CZ247_2 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CZ247_2 protein").
The nucleotide sequence of CZ247_2 as presently determined is reported in SEQ
ID N0:22. What applicants presently believe to be the proper reading frame and
the
1 S predicted amino acid sequence of the CZ247_2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:23. Amino acids 545 to 557 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 558, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 0 CZ247_2 should be approximately 2300 bp.
The nucleotide sequence disclosed herein for CZ247_2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CZ247_2 demonstrated at least some similarity with
sequences
identified as T09256 (Human ara Kb beta-galactosidase fusion protein coding
sequence),
2 5 W27222 (26h9 Human retina cDNA randomly primed sublibrary Homo Sapiens
cDNA),
and W72736 {zd71e02.s1 Soares fetal heart NbHHI9W Homo sapiens cDNA clone
346106
3'). The predicted amino acid sequence disclosed herein for CZ247_2 was
searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
search protocol. The predicted CZ247_2 protein demonstrated at least some
similarity to
3 0 sequences identified as 888069 {Human ara Kb beta-galactosidase fusion
protein). Based
upon sequence similarity, CZ247_2 proteins and each similar protein or peptide
may share
at least some activity.
27
CA 02274732 1999-06-14
WO 98/27205 PCT/US97123330
Deposit of Clones
Clones CB107_l, CG300 3, CJ145_l, CJ160_ll, C020_l, C0223_l, C0310_2,
CP258 3, CW1155_3 and CZ247_2 were deposited on December 17, 1996 with the
American Type Culture Collection as an original deposit under the Budapest
Treaty and
were given the accession number ATCC 98279, from which each clone comprising a
particular polynucleotide is obtainable. Clone C0223_3 was deposited on
January 9,1997
with the American Type Culture Collection as an original deposit under the
Budapest
Treaty and were given the accession number ATCC 98291. All restrictions on the
availability to the public of the deposited material will be irrevocably
removed upon the
granting of the patent, except for the requirements specified in 37 C.F.R. ~
1.808(b).
Each clone has been transfected into separate bacterial cells (E. coli) in
this
composite deposit. Each clone can be removed from the vector in which it was
deposited
by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to
produce the
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from
pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman
et al.,
1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from
pMT2
(Kaufman et nl., 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR
sequences,
insertion of a new polylinker, and insertion of the M13 origin of replication
in the CIaI site.
2 0 In some instances, the deposited clone can become "flipped" (i.e., in the
reverse
orientation) in the deposited isolate. In such instances, the cDNA insert can
still be
isolated by digestion with EcoRI and NotI. However, NotI will then produce the
5' site
and EcoRI will produce the 3' site for placement of the cDNA in proper
orientation for
expression in a suitable vector. The cDNA may also be expressed from the
vectors in
2 5 which they were deposited.
Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
3 0 provided herein, or from a combination of those sequences. The sequence of
the
oligonucleotide probe that was used to isolate each full-length clone is
identified below,
and should be most reliable in isolating the clone of interest.
28
CA 02274732 1999-06-14
WO 98127205 PCT/US97/23330
Clone Probe Seauence
CB107 1 SEQ ID N0:24
CG300 3 SEQ ID N0:25
CJ145_ 1 SEQ ID N0:26
CJ160_11 SEQ ID N0:27
C020 1 SEQ ID N0:28
C0223 3 SEQ ID N0:29
C0310 2 SEQ ID N0:30
CP258 3 SEQ ID N0:31
CW1155 3 SEQ ID N0:32
CZ247 2 SEQ ID N0:33
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research) cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
2 0 (a} It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming
2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g-;'P ATP {specific
activity 6000
2 5 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques
for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
3 0 probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 ul of the stock used to inoculate a sterile culture flask
containing 25 ml
of sterile L-broth containing ampicillin at 100 lxg/ml. The culture should
preferably be
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
29
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
a concentration greater than or equal to 1e+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
filter is then preferably dried and subjected to autoradiography for
sufficient time to
visualize the positives on the X-ray film. Other known hybridization methods
can also
be employed.
2 0 The positive colonies are picked, grown in culture, and plasmid DNA
isolated
using standard procedures. The clones can then be verified by restriction
analysis,
hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the
2 S protein may be in linear form or they may be cyclized using known methods,
for example,
as described in H.U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and
in R.S.
McDowell, et al., J. Amer. Chem. Soc.114, 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency of protein
binding
3 0 sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. For a bivalent form of the protein, such
a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes
may also
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
.~_~_.._ _ .... _..____~__..T._..___ ~ _ _ ..___ .
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WO 98/27205 PCT/US97/23330
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature form
of such protein may be obtained by expression of the disclosed full-length
polynucleotide
(preferably those deposited with ATCC) in a suitable mammalian cell or other
host cell.
The sequence of the mature form of the protein may also be determinable from
the amino
acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
are transcribed to produce the mItNAs from which cDNA polynucleotide sequences
are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene" is
a gene that
has been separated from the adjacent coding sequences, if any, present in the
genome of
2 0 the organism from which the gene was isolated.
Organisms that have enhanced, reduced, or modified expression of the genes)
corresponding to the polynucleotide sequences disclosed herein are provided.
The
desired change in gene expression can be achieved through the use of antisense
polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from
the
2 5 gene (Albert and Morris,1994, Trends Pharrnacol. Sci. 15(7): 250-254;
Lavarosky et nl., 1997,
Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res.
Mol. Biol. 58: 1-
39; all of which are incorporated by reference herein). Transgenic animals
that have
multiple copies of the genes) corresponding to the polynucleotide sequences
disclosed
herein, preferably produced by transformation of cells with genetic constructs
that are
3 0 stably maintained within the transformed cells and their progeny, are
provided.
Transgenic animals that have modified genetic control regions that increase or
reduce
gene expression levels, or that change temporal or spatial patterns of gene
expression, are
also provided (see European Patent No. 0 649 464 Bl, incorporated by reference
herein).
In addition, organisms are provided in which the genes) corresponding to the
31
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of all or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays I4(9): 629-633; Zwaal et
nl.,1993, Proc. Nafl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Scf.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by posifive/negafive genetic selection strategies (Mansour
et al., 1988,
Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
the present invention also provides for soluble forms of such protein. In such
forms part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
2 0 transmembrane domains of proteins of the invention can be identified in
accordance with
known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
2 5 identity (more preferably, at least 75% identity; most preferably at least
90% or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
3 0 (more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
Species homologs of the disclosed polynucleotides and proteins are also
provided
by the present invention. As used herein, a "species homologue" is a protein
or
32
__ . ___._ _.~ ~ ~
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide, as determined by those of skill in the art. Species homologs
may be
isolated and identified by making suitable probes or primers from the
sequences provided
herein and screening a suitable nucleic acid source from the desired species.
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide
which also encode proteins which are identical, homologous, or related to that
encoded
by the polynucleotides .
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing
under
reduced stringency conditions, more preferably stringent conditions, and most
preferably
highly stringent conditions, to polynucleotides described herein. Examples of
stringency
2 5 conditions are shown in the table below: highly stringent conditions are
those that are at
least as stringent as, for example, conditions A-F; stringent conditions are
at least as
stringent as, for example, conditions G-L; and reduced stringency conditions
are at least
as stringent as, for example, conditions M-R.
33
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)= Buffer' and Buffer'
A DNA:DNA >- 65C; lxSSC -or- 65C; 0.3xSSC
50 42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC Tg*; lxSSC
C DNA:RNA s 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC) 50/ formamide
D DNA:RNA <50 T~*; lxSSC Tp"; lxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF"; IxSSC TF*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <SO T"*; 4xSSC T"*; 4xSSC
1 DNA:RNA > 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T~"; 4xSSC
K RNA:RNA s 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~*; 2xSSC Ti *; 2xSSC
M DNA:DNA s 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA s 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
P DNA:RNA <50 T,,*; 6xSSC T,,*; 6xSSC
Q RNA:RNA s 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
2 R RNA:RNA <50 TK"; 4xSSC TR*; 4xSSC
0
I: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleoHdes. When
hybridizing a poiynucleotide to a target polynucleotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaH~POa, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 *TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length)
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,o[Na']) + 0.41(%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
34
. ___.~__ _. _ ._ ~~~ ~___ _ _ _~_
CA 02274732 1999-06-14
WO 98/27205 PCT/US97123330
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biolog~,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991 ), in order to produce
the protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 185 537-566 (1990). As defined herein "operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
S enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
i 0 e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and
such methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (1987), incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
15 The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 InVitrogen, respectively. The protein can also be tagged with an epitope
and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
36
__ n__..._ _....~_____r. . ~ _ _ _. _ _
CA 02274732 1999-06-14
WO 98/Z7205 PCT/US97/23330
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and / or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 0 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
2 5 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
3 0 expected to retain protein activity in whole or in part and may thus be
useful for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
presentinvention.
37
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutiveiy or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to rr~ap related gene positions; to
compare
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
2 0 known sequences in the process of discovering other novel polynucleotides;
for selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
2 5 binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays {such as, for
example, that
described in Gyuris et al., Cell 75:792-803 (1993)) to identify
polynucleotides encoding the
other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
3 0 The proteins provided by the present invention can similarly be used in
assay to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
levels of the protein (or its receptor) in biological fluids; as markers for
tissues in which
38
1
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
the corresponding protein is preferentially expressed (either constitutively
or at a
particular stage of tissue differentiation or development or in a disease
state); and, of
course, to isolate correlative receptors or ligands. Where the protein binds
or potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Bergen S.L. and A.R. Kimmel
eds.,1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
2 0 or amino acid supplement, use as a carbon source, use as a nitrogen source
and use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
2 5 can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation
(either
inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
3 0 induce production of other cytokines in certain cell populations. Many
protein factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
39
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and
CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.I-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and / or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Cacrrent Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon ~y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
2 0 Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
2 5 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto. 1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11 - Berinett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
3 0 in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. VoI
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
_.. ~___.. _ r ..__._T_... _.j __._......
CA 02274732 1999-06-14
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Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512) 1988.
Immune Stimulating or Suppressing_Activity
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and / or B
lymphocytes, as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
2 0 infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
2 5 be useful where a boost to the immune system generally may be desirable,
i.e., in the
treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
3 0 Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma {particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
41
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
~n~hich requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
the tolerizing agent has ceased. Operationally, tolerance can be demonstrated
by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.~>.,
preventing
high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
exampla,
blockage of T cell function should result in reduced tissue destruction in
tissue
2 0 transplantation. Typically, in tissue transplants, rejection of the
transplant is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
2 5 monomeric form of a peptide having an activity of another B lymphocyte
antigen (e.g., B7-
l, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
3 0 immunosuppressant. Moreover, the lack of costimulation may also be
sufficient to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
42
_T.._. ~
CA 02274732 1999-06-14
WO 9$/27205 PCT/US97/23330
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 {1992) and Turka et nl.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
2 0 activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
2 5 human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid
mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York, 1989, pp. 840-856).
3 0 Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
43
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
2 0 For example, tumor cells obtained from a patient can be transfected ex
vivo with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
2 5 to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
3 0 molecules, or which fail to reexpress sufficient amounts of MHC class i or
MHC class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
biz
microglobulin protein or an MHC class II a chain protein and an MHC class II
(3 chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
44
....._~ _._ .__ . . . _ _ . _ _ .__.T.._. _...~..__.. .. . _..__._
CA 02274732 1999-06-14
WO 98/27205 PCT/US97123330
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
S as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;
Handa et al.,
J. Immunol. 135:1564-1572,1985; Takai et al., J. Immunol. 137:3494-3500,1986;
Takai et al.,
J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
2 0 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolii et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
2 5 (which will identify, among others, proteins that modulate T-cell
dependent antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Cicrrent Protocols in
Imrnnnology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
3 0 Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Th1 and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-
260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival / apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
2 0 development include, without limitation, those described in: Antica et
al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Re ug lating Activity
2 5 A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
3 0 thereby indicating utility, for example, in treating various anemias or
for use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
46
_____..~._..,... 1
CA 02274732 1999-06-14
WO 98127205 PCT/US97123~30
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-~3ivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietac
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Keller et
2 0 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,
Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In
Cultc~re of
2 S Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-
Liss, Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece)
LK. and
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology
22:353-359,
3 0 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hentatopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay,
Sutherland,
47
CA 02274732 1999-06-14
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li.J. In Culture of Hematopoietic Cells. R.I. Freshney, et nl. eds. Vol pp.
139-162, Wiley-Liss,
Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and / or nerve tissue growth or
regeneration, as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
2 0 differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
2 5 Another category of tissue regeneration activity that may be attributable
to the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
3 0 other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
48
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congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
2 0 invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
2 5 non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invenrion may also exhibit
activity for
generation or regeneration of other tissues, such as organs (including, for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
3 0 and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
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A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095 / 16035 (bone, cartilage,
tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers) Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Actiyin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
2 0 activities. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
invention, alone or in heterodimers with a member of the inhibin a family, may
be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
2 5 and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
~3 group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
3 0 United States Patent 4,798,885. A protein of the invention may also be
useful for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782,
1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
1 S against the tumor or infecting agent.
A protein or peptide has chemoiactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
2 0 of cells can be readily determined by employing such protein or peptide in
any known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
2 5 chemotaxis) consist of assays that measure the ability of a protein to
induce the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
3 0 Publishing Associates and Wiley-Interscience {Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,
1995; Lind et al.
APMIS 103:140-146, 1995; Muller et ai Eur. J. Immunol. 25: 1744-1748; Gruber
et al. J. of
Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
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Hemostatic and Thrombolvtic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et ai., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
Receptor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
2 0 such receptors and ligands include, without limitation, cytokine receptors
and their
ligands, receptor kinases and their Iigands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
2 5 development of cellular and humoral immune responses). Receptors and
ligands are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
3 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
52
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WO 98/27205 PCTIUS97123330
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68) 1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as 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
2 0 from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
2 5 roles during development, particularly in defining specific cell types.
Loss or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
3 0 The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
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CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
2 0 in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
2 5 polynucleotides of the present invention encoding such proteins, can used
to generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
3 0 cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
54
_. __ ___.___.____._ __ .____..__ . ~.._._.~.~___._.._ .:
CA 02274732 1999-06-14
WO 98127205 PCTIUS97/23330
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038,
1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
or inhibiting factors, agents or cell types which promote tumor growth.
2 0 Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
2 5 weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
3 0 carbohydrate, vitamins, minerals, cofactors or other nutritional factors
or component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
CA 02274732 1999-06-14
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lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF) TNF, IL-1, IL-2, iL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
2 0 cell factor, and erythropoietin. The pharmaceutical composition may
further contain other
agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
2 5 in formulations of the particular cytokine, lymphokine, other
hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g.,
heterodimers
3 0 or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
56
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CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
The pharmaceutical composition of the invention rnay be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; L;.S.
2 0 Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
2 5 amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
3 0 In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
57
CA 02274732 1999-06-14
WO 98/27205 PCTlUS97/23330
or other hematopoietic factors. When co-administered with one or more
cytokines,
lyrnphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder) solution or elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid carrier such as
a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
2 0 When administered in liquid form, a liquid Garner such as water,
petroleum, oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
2 5 form, the pharmaceutical composition contains from about 0.5 to 90% by
weight of protein
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
3 0 invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
58
_ . . ~~ .._ .. ~._._.~. _ .
CA 02274732 1999-06-14
WO 98/27205 PCT/US97/23330
Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention may be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 ug
to about 100
mg (preferably about O.lng to about 10 mg, more preferably about 0.1 lZg to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
2 0 contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
2 5 polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
3 0 R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky,
et al., FEBS Lett.
211, 10 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
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WO 98/27205 PCT/US97/23330
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and / or cartilage formation,
the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and / or cartilage damage, providing a
structure for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
2 0 applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
2 5 sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,
polyglycolic acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
3 0 ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
_. _ . ~ _ _. ....r_ ..T _ _.. i
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Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylceliulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined wah other
agents beneficial to the treatment of the bone and / or cartilage defect,
wound, or tissue in
2 0 question. These agents include various growth factors such as epidermal
growth factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors {TGF-
a and
TGF-(3), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
2 5 humans, are desired patients for such treatment with proteins of the
present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
3 0 a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
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the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and / or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
S polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation) in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
62
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie) Edward R.
Racie, Lisa A.
Merberg, David
Treacy, Maurice
Spaulding) Vikki
Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 33
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0) Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
NO:1:
AATCGCACCT GTCCAACTTG CAATGACTTC TGCAGAAAAT CATGGAGCTA60
CATGGACTTG
CAGGATATTT TAGCCAAAAC ATCAGCCAAG CTGAACAGCG AATGAATAGA120
CTGTCTCGAG
TTGGATCAGT GCTATTGTGA AAGGACTTGC GAACCACCTA CCGAGAATTT180
ACCATGAAGG
GAGTCCTGGA TAGACGGCTG TAAGAACTGC ATGGAACCAT CCAGTGTGAA240
ACATGCCTGA
ACTCTAATCT GCCCAAATCC TGACTGCCCA CTCTTGCGTA TGTGGATGGC300
CTTAAGTCCG
AAATGCTGTA AGGAATGCAA ATCGATATTC GACGAACCTA CTTTGAAGGA360
CAATTTCAAG
GAAAGAAATA CA 372
(2) INFORMATION FOR SEQ ID N0:2:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 761 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION.
SEQ ID
N0:2:
TTCTGGAGTATGTGTTCTCTATGAGTGCAAGGACCAGACC ATGAAATTTGTTGAGAGTTC60
AGGCTGTCCAGCTTTGGATTGTCCAGAGTCTCATCAGATA ACCTTGTNTCACAGCTGTTG120
CAAAGTTTGTAAAGGTTATGATTTTTGTTTTGAAAGGCAT AACTGCATGGAGAATTCCAT180
CTGCAGAAATNTGAATGACAGGGCTGTTTGTAGCTGTCGA GATGGTTTTAGGGTTTTTCG240
AGAGGATAATGCCTACTGTGAAGACATNGATGAGTGTGCT GAAGGGCGCCATTACTGTNG30C
TGAAAATACAATGTGTGTCAACACCCCGGGTTCTTTTATG TGCATCTGCAAAACTGGATA360
CATCAGAATTGATGATTATTCATGTACAGAACATGATGAG TGTATCACAAATCAGCACAG420
CTGTGATGAAAATGCTTTATGCTTCAACACTGTTGGAGGA CACAACTGTGTTTGCAAGCC480
GGGCTATACAGGGAATGGAACGACATGCAAAGCATTTTGC AAAGATGGCTGTAGGAATGG540
AGGAGCCTGTATTGCCGCTAATGTGTGTGCCTGCCCACAA GGCTTCACTGGACCCAGCTG600
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TGAAACGGAC ATTGATGAAT GCTCTGATGG TTTTGTTCAA TGTGACAGTC GTGCTAATTG 660
CATTAACCTG CCTGGATGGT ACCACTGTGA GTGCAGAGAT GGCTACCATG ACAATGGGAT 720
GTTTTCACCA AGTGGAGAAT CGTGTGAAGA TATTGATGAG T 761
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Met Lys Phe Val Glu Ser Ser Gly Cys Pro Ala Leu Asp Cys Pro Glu
1 5 10 15
Ser His Gln Ile Thr Leu Xaa His Ser Cys Cys Lys Val Cys Lys Gly
20 25 30
Tyr Asp Phe Cys Phe Glu Arg His Asn Cys Met Glu Asn Ser Ile Cys
35 40 45
Arg Asn Xaa Asn Asp Arg Ala Val Cys Ser Cys Arg Asp Gly Phe Arg
50 55 60
Val Phe Arg Glu Asp Asn Ala Tyr Cys Glu Asp Xaa Asp Glu Cys Ala
65 70 75 80
Glu Gly Arg His Tyr Cys Xaa Glu Asn Thr Met Cys Val Asn Thr Pro
85 90 95
Gly Ser Phe Met Cys Ile Cys Lys Thr Gly Tyr Ile Arg Ile Asp Asp
100 105 110
Tyr Ser Cys Thr Glu His Asp Glu Cys Ile Thr Asn Gln His Ser Cys
115 120 125
Asp Glu Asn Ala Leu Cys Phe Asn Thr Val Gly Gly His Asn Cys Val
130 135 140
Cys Lys Pro Gly Tyr Thr Gly Asn Gly Thr Thr Cys Lys Ala Phe Cys
145 150 155 160
Lys Asp Gly Cys Arg Asn Gly Gly Ala Cys Ile Ala Ala Asn Val Cys
165 170 175
Ala Cys Pro Gln Gly Phe Thr Gly Pro Ser Cys Glu Thr Asp Ile Asp
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180 185 190
Glu Cys Ser Asp Gly Phe Val Gln Cys Asp Ser Arg Ala Asn Cys Ile
195 200 205
Asn Leu Pro Gly Trp Tyr His Cys Glu Cys Arg Asp Gly Tyr His Asp
210 215 220
Asn Gly Met Phe Ser Pro Ser Gly Glu Ser Cys Glu Asp Ile Asp Glu
225 230 235 240
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A; LENGTH: 342 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GCAGAAAATT TTCCTCTAGA TCAGAATCTT CAAGAATCAG TTAGGTTCCT6G
CACTGCAAGA
AATAAAATGT CAGGCAGTGA ATGAATTATA TTTTAAGAAG TAAAGCAAAG120
AAGCTATAAC
ATGTTATGTA CAGTACACTC TGAAAAGAAA TCTGAAACAA GTTATTGTAA180
TGATAAAAAT
AATGCACAGG CATGGTTACT TAATATTTTC TAACAGGAAA AGTCATCCCT240
ATTTCCTTGT
TTTACTGCAC TTAATATTAT TTGGTTGAAT TTGTTCAGTA TAAGTTCGTT300
CCTTGTGCAA
AATTAAATAA ATATTTTTCT TACCTTAAAA P,AAAAAAAAA AA 342
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1445 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GTGCGCATGG GGACGCTATA GCAATTCGTT TGCTGTCCTT CCTCTCCTTC GAAGATGACA 60
66
,._ .. ._ _ _. ~ _ ... ~._ .
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AGGCCTACCATCGTTTCTTCCTGCCTTTGGGCCGTCAGGCAGTTGGTTGG GACCCGCTCC120
AACCCTCGGTTCTTCCTGCAATACAGTGGATACAATTTGTCATGGCTACT CTGAGATAAG180
ACCACTTTTTTATCTGAGCTTCTGTGACCTGCTCCTGGGACTTTGCTGGC TCACGGAGAC240
ACTTCTCTATGGAGCTTCAGTAGCAAATAAGGACATCATCTGCTATAACC TACAAGCAGT300
TGGACAGATATTCTACATTTCCTCATTTCTCTACACCGTCAATTACATCT GGTATTTGTA360
CACAGAGCTGAGGATGAAACACACCCAAAGTGGACAGAGCACATCTCCAC TGGTGATAGA420
TTATACTTGTCGATTTTGTCAAATGGCCTTTGTTTTCTCAAGCCTGATAC CTCTGCTATT480
GATGACACCTGTATTCTGTCTGGGAAATACTAGTGAATGTTTCCAAAACT TCAGTCAGAG540
CCACAATTGTATCTTGATGCACTCACCACCATCAGCCATGGCTGAACTTC CACCTTCTGC600
CAACACATCTGTCTGTAGCACACTTTATTTTTATGGTATCGCCATTTTCC TGGGCAGCTT660
TGTACTCAGCCTCCTTACCATTATGGTCTTACTTATCCGAGCCCAGACAT TGTATAAGAA720
GTTTGTGAAGTCAACTGGCTTTCTGGGGAGTGAACAGTGGGCAGTGATTC ACATTGTGGA780
CCAACGGGTGCGCTTCTACCCAGTGGCCTTCTTTTGCTGCTGGGGCCCAG CTGTCATTCT840
AATGATCATAAAGCTGACTAAGCCACAGGACACCAAGCTTCACATGGCCC TTTATGTTCT900
CCAGGCTCTAACGGCAACATCTCAGGGTCTACTCAACTGTGGAGTATATG GCTGGACGCA960
GCACAAATTCCACCAACTAAAGCAGGAGGCTCGGCGTGATGCAGATACCC AGACACCATT1020
ATTATGCTCACAGAAGAGATTCTATAGCAGGGGCTTAAATTCACTGGAAT CCACCCTGAC1080
TTTTCCTGCCAGTACTTCTACCATTTTTTGAAACTACAATACTGGAACAT CCAGGAACTG1140
GAGTTATTCTACGCTAATGGATTGGAAAGAATGTTGGGAAAGGACATCTT AAATCTTTTC2200
TAACTATGCCCTAAACTGCAGAACTCAAAGGAAATATAGTGCCATTGTTA GTAGTCATTC1260
TAGATGAATTGGGAGTATCTCTCCAGTTATTCCCAGATTCACTAGTGATC CTTAAAGTCT1320
CTATTCAGGGAGAGGAAGACACTTTCCATCTCAGAGATAGACTCGTGTTA CCTTGATGGA1380
TATTGGATTTGTCTAAGTCTCTTCTAGAAAAAATAAATTCTAGATTATTA P,~~,~AAAAAAA1440
AAAAA 1445
(2) INFORMATION EQ ID
FOR N0:6:
S
(i) SEQUENCE RACTERISTICS:
CHA
(A) LENGTH:245 aminoacids
(B) TYPE:mino acid
a
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:6:
Met LysHisThr GlnSerGlyGln SerThrSerPro LeuValI1e Asp
1 5 10 15
Tyr ThrCysArg PheCysGlnMet AlaPheValPhe SerSerLeu Ile
20 25 30
Pro LeuLeuLeu MetThrProVal PheCysLeuGIy AsnThrSer Glu
35 40 45
Cys PheGlnAsn PheSerGlnSer HisAsnCysIie LeuMetHis Ser
50 55 60
Pro ProSerAla MetAlaGluLeu ProProSerAla AsnThrSer Val
65 70 75 80
Cys SerThrLeu TyrPheTyrGly IleAlaIlePhe LeuGlySer Phe
85 90 95
Val LeuSerLeu LeuThrIleMet ValLeuLeuIle ArgAlaGln Thr
100 105 110
Leu TyrLysLys PheValLysSer ThrGlyPheLeu GlySerGlu Gln
115 120 125
Trp AlaValIle HisIleValAsp GlnArgValArg PheTyrPro Val
130 135 140
Ala PhePheCys CysTrpGlyPro AlaValIleLeu MetIleIle Lys
145 150 155 160
Leu ThrLysPro GlnAspThrLys LeuHisMetAla LeuTyrVal Leu
165 170 175
Gln AlaLeuThr AlaThrSerGln GlyLeuLeuAsn CysGlyVal Tyr
180 185 190
Gly TrpThrGln HisLysPheHis GlnLeuLysGln GluAlaArg Arg
195 200 205
Asp AlaAspThr G1nThrProLeu LeuCysSerGln LysArgPhe Tyr
210 215 220
Ser ArgGlyLeu AsnSerLeuGlu SerThrLeuThr PheProAla Ser
225 230 235 240
Thr SerThrIle Phe
245
68
__.~___.~.~..~. _. ~
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(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3550 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:7:
CCCCTCGATAATGGATTACTAAATGGGATACACGCTGTACCAGTTCGCTCCGAGCCCCGG60
CCGCCTGTCCGTCGATGCACCGAAAAGGGTGAAGTAGAGAAATAAAGTCTCCCCGCTGAA120
CTACTATGAGGTCAGAAGCCTTGCTGCTATATTTCACACTGCTACACTTTGCTGGGGCTG180
GTTTCCCAGAAGATTCTGAGCCAATCAGTATTTCGCATGGCAACTATACAAAACAGTATC240
CGGTGTTTGTGGGCCACAAGCCAGGACGGAACACCACACAGAGGCACAGGCTGGACATCC300
AGATGATTATGATCATGAACGGAACCCTCTACATTGCTGCTAGGGACCATATTTATACTG360
TTGATATAGACACATCACACACSGAAGAAATTTATTGTAGCAAAAAACTGACATGGAAAT420
CTAGACAGGCCGATGTAGACACATGCAGAATGAAGGGAAAACATAAGGATGAGTGCCACA480
ACTTTATTAAAGTTCTTCTAAAGAAAAACGATGATGCATTGTTTGTCTGTGGAACTAATG540
CCTTCAACCCTTCCTGCAGAAACTATAAGATGGATACATTGGAACCATTCGGGGATGAAT600
TCAGCGGAATGGCCAGATGCCCATATGATGCCAAACATGCCAACGTTGCACTGTTTGCAG660
ATGGAAAACTATACTCAGCCACAGTGACTGACTTCCTTGCCATTGACGCAGTCATTTACC720
GGAGTCTTGGAGAAAGCCCTACCCTGCGGACCGTCAAGCACGATTCAAAATGGTTGAAAG780
AACCATACTTTGTTCAAGCCGTGGATTACGGAGATTATATCTACTTCTTCTTCAGGGAAA840
TAGCAGTGGAGTATAACACCATGGGAAAGGTAGTTTTCCCAAGAGTGGCTCAGGTTTGTA900
AGAATGATATGGGAGGATCTCAAAGAGTCCTGGAGAAACAGTGGACGTCGTTCCTGAAGG960
CGCGCTTGAACTGCTCAGTTCCTGGAGACTCTCATTTTTATTTCAACATTCTCCAGGCAG2020
TTACAGATGTGATTCGTATCAACGGGCGTGATGTTGTCCTGGCAACGTTTTCTACACCTT1080
ATAACAGCATCCCTGGGTCTGCAGTCTGTGCCTATGACATGCTTGACATTGCCAGTGTTT1140
TTACTGGGAGATTCAAGGAACAGAAGTCTCCTGATTCCACCTGGACACCAGTTCCTGATG1200
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AACGAGTTCCTAAGCCCAGGCCAGGTTGCTGTGCTGGCTCATCCTCCTTAGAAAGATATG1260
CAACCTCCAATGAGTTCCCTGATGATACCCTGAACTTCATCAAGACGCACCCGCTCATGG1320
ATGAGGCAGTGCCCTCCATCTTCAACAGGCCATGGTTCCTGAGAACAATGGTCAGATACC1380
GCCTTACCAAAATTGCAGTGGACACAGCTGCTGGGCCATATCAGAATCACACTGTGGTTT1440
TTCTGGGATCAGAGAAGGGAATCATCTTGAAGTTTTTGGCCAGAATAGGAAATAGTGGTT1500
TTCTAAATGACAGCCTTTTCCTGGAGGAGATGAGTGTTTACAACTCTGAAAAATGCAGCT1560
ATGATGGAGTCGAAGACAAAAGGATCATGGGCATGCAGCTGGACAGAGCAAGCAGCTCTC1620
TGTATGTTGCGTTCTCTACCTGTGTGATAAAGGTTCCCCTTGGCCGGTGTGAACGACATG1680
GGAAGTGTAAAAAAACCTGTATTGCCTCCAGAGACCCATATTGTGGATGGATAAAGGAAG1740
GTGGTGCCTGCAGCCATTTATCACCCAACAGCAGACTGACTTTTGAGCAGGACATAGAGC2800
GTGGCAATACAGATGGTCTGGGGGACTGTCACAATTCCTTTGTGGCACTGAATGGAGTGA1860
TTCGGGAAAGTTACCTCAAAGGCCACGACCAGCTGGTTCCCGTCACCCTCTTGGCCATTG1920
CAGTCATCCTGGCTTTCGTCATGGGGGCCGTCTTCTCGGGCATCACCGTCTACTGCGTCT1980
GTGATCATCGGCGCAAAGACGTGGCTGTGGTGCAGCGCAAGGAGAAGGAGCTCACCCACT2040
CGCGCCGGGGCTCCATGAGCAGCGTCACCAAGCTCAGCGGCCTCTTTGGGGACACTCAAT2100
CCAAAGACCCAAAGCCGGAGGCCATCCTCACGCCACTCATGCACAACGGCAAGCTCGCCA2160
CTCCCGGCAACACGGCCAAGATGCTCATTAAAGCAGACCAGCACCACCTGGACCTGACGG2220
CCCTCCCCACCCCAGAGTCAACCCCAACGCTGCAGCAGAAGCGGAAGCCCAGCCGCGGCA2280
GCCGCGAGTGGGAGAGGAACCAGAACCTCATCAATGCCTGCACAAAGGACATGCCCCCCA2340
TGGGCTCCCCTGTGATTCCCACGGACCTGCCCCTGCGGGCCTCCCCCAGCCACATCCCCA2400
GCGTGGTGGTCCTGCCCATCACGCAGCAGGGCTACCAGCATGAGTACGTGGACCAGCCCA2460
AAATGAGCGAGGTGGCCCAGATGGCGCTGGAGGACCAGGCCGCCACACTGGAGTATAAGA2520
CCATCAAGGAACATTTCAGCAGCAAGAGTCCCAACCATGGGGTGAACCTTGTGGAGAACC2580
TGGACAGCCTGCCCCCCAAAGTTCCACAGCGGGAGGCCTCCCTGGGTCCCCCGGGAGCCT2640
CCCTGTTTCAGACCGGTTTAAGCAAGCGGCTGGAAATGCACCACTCCTTTTCCTACGGGG2700
TTGACTATAAGAGGAGCTACCCCACGAACTCGCTCACGAGAAGCCACCAGGCCACCACTC2760
TCAAAAGAAACAACACTAACTCCTCCAATTCCTCTCACCTCTCCAGAAACCAGAGCTTTG2820
GCAGGGGAGACAACCCGCCGCCCGCCCCGCAGAGGGTGGACTCCATCCAGGTGCACAGCT2880
_ T ~
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CCCAGCCATCTGGCCAGGCC GTGACTGTCTCGAGGCAGCCCAGCCTCAAC GCCTACAACT2940
CACTGACAAGGTCGGGGCTG AAGCGTACGCCCTCGCTAAAGCCGGACGTA CCCCCCAAAC3000
CATCCTTTGCTCCCCTTTCC ACATCCATGAAGCCCAATGATGCGTGTACA TAATCCCAGG3060
GGGAGGGGGTCAGGTGTCGA ACCAGCAGGCAAGGCGAGGTGCCCGCTCAG CTCAGCAAGG3120
TTCTCAACTGCCTCGAGTAC CCACCAGACCAAGAAGGCCTGCGGCAGAGC CGAGGACGCT3180
GGGTCCTCCTCTCTGGGACA CAGGGGTACTCACGAAAACTGGGCCGCGTG GTTTGGTGAA3240
GGTTTGCAACGGCGGGGACT CACCTTCATTCTCTTCCTTCACTTTCCCCC ACACCCTACA3300
ACAGGTCGGACCCACAAAAG ACTTCAGTTATCATCACAAACATGAGCCAA AAGCACATAC3360
ATACCCCATCCCCCACCCCC ACACACACACACACATGCACACAACACATA CACACACACG3420
CACAGAGGTGAACAGAAACT GAAACATTTTGTCCACAACTTCACGGGACG TGGCCAGACT3480
GGGTTTGCGTTCCAACCTGC AAAACACAAATACATTTTTTAAAATCAAGA AAATTTAAAA3540
3550
(2) INFORMATION
FOR SEQ
ID N0:8:
(i) S EQUENCE CHARACTERISTICS:
(A) LENGTH: 975 adds
aminc
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
linear
(ii) MOLECULE
TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
Met Arg Ser Glu Ala Leu Leu Leu Tyr Phe Thr Leu Leu His Phe Ala
1 S 10 15
Gly Ala Gly Phe Pro Glu Asp Ser Glu Pro Ile Ser Ile Ser His Gly
20 25 30
Asn Tyr Thr Lys Gln Tyr Pro Val Phe Val Gly His Lys Pro Gly Arg
35 40 45
Asn Thr Thr Gln Arg His Arg Leu Asp Ile Gln Met Ile Met Ile Met
50 55 60
Asn Gly Thr Leu Tyr Ile Ala Ala Arg Asp His Ile Tyr Thr Val Asp
65 70 75 80
Ile Asp Thr Ser His Thr Glu Glu Ile Tyr Cys Ser Lys Lys Leu Thr
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85 90 95
Trp Lys Ser Arg Gln Ala Asp Val Asp Thr Cys Arg Met Lys Gly Lys
100 105 110
His Lys Asp Glu Cys His Asn Phe Ile Lys Val Leu Leu Lys Lys Asn
115 120 125
Asp Asp Ala Leu Phe Val Cys Gly Thr Asn Ala Phe Asn Pro Ser Cys
130 135 140
Arg Asn Tyr Lys Met Asp Thr Leu Glu Pro Phe Gly Asp Glu Phe Ser
145 150 155 160
Gly Met Ala Arg Cys Pro Tyr Asp Ala Lys His Ala Asn Val Ala Leu
165 170 175
Phe Ala Asp Gly Lys Leu Tyr Ser Ala Thr Val Thr Asp Phe Leu Ala
180 185 190
Ile Asp Ala Val Ile Tyr Arg Ser Leu Gly Glu Ser Pro Thr Leu Arg
195 200 205
Thr Val Lys His Asp Ser Lys Trp Leu Lys Glu Pro Tyr Phe Val Gln
210 215 220
Ala Val Asp Tyr Gly Asp Tyr Ile Tyr Phe Phe Phe Arg Glu Ile Ala
225 230 235 240
Val Glu Tyr Asn Thr Met Gly Ly.s Val Val Phe Pro Arg Va1 Ala Gln
245 250 255
Val Cys Lys Asn Asp Met Gly Gly Ser Gln Arg Val Leu Glu Lys G1n
260 265 2'70
Trp Thr Ser Phe Leu Lys Ala Arg Leu Asn Cys Ser VaI Pro Gly Asp
275 280 285
Ser His Phe Tyr Phe Asn Ile Leu Gln Ala Val Thr Asp Val Ile Arg
290 295 300
Ile Asn Gly Arg Asp Val Val Leu Ala Thr Phe Ser Thr Pro Tyr Asn
305 310 315 320
Ser Ile Pro Gly Ser Ala Val Cys Ala Tyr Asp Met Leu Asp Ile Ala
325 330 335
Ser Val Phe Thr Gly Arg Phe Lys Glu Gln Lys Ser Pro Asp Ser Thr
340 345 350
Trp Thr Pro Val Pro Asp Glu Arg Val Pro Lys Pro Arg Pro Gly Cys
355 360 365
Cys Ala Gly Ser Ser Ser Leu Glu Arg Tyr Ala Thr Ser Asn Glu Phe
370 375 380
72
__.__.____. ..__...._.._ T_ ..__.~____._... _._____.
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Pro Asp Asp Thr Leu Asn Phe Ile Lys Thr His Pro Leu Met Asp Glu
385 390 395 400
Ala Val Pro Ser Ile Phe Asn Arg Pro Trp Phe Leu Arg Thr Met Val
405 410 415
Arg Tyr Arg Leu Thr Lys Ile Ala Val Asp Thr Ala Ala Gly Pro Tyr
420 425 430
Gln Asn His Thr Val Val Phe Leu Gly Ser Glu Lys Gly Ile Ile Leu
435 440 445
Lys Phe Leu Ala Arg Ile Gly Asn Ser Gly Phe Leu Asn Asp Ser Leu
450 455 460
Phe Leu Glu Glu Met Ser Val Tyr Asn Ser Glu Lys Cys Ser Tyr Asp
465 470 475 480
Gly Val Glu Asp Lys Arg Ile Met Gly Met Gln Leu Asp Arg Ala Ser
485 490 495
Ser Ser Leu Tyr Val Ala Phe Ser Thr Cys Val Ile Lys Val Pro Leu
500 505 510
Gly Arg Cys Glu Arg His Gly Lys Cys Lys Lys Thr Cys Ile Ala Ser
515 520 525
Arg Asp Pro Tyr Cys Gly Trp Ile Lys Glu Gly Gly Ala Cys Ser His
530 535 540
Leu Ser Pro Asn Ser Arg Leu Thr Phe Glu Gln Asp Ile Glu Arg Gly
545 550 555 560
Asn Thr Asp Gly Leu Gly Asp Cys His Asn Ser Phe Val Ala Leu Asn
565 570 575
Gly Val Ile Arg Glu Ser Tyr Leu Lys Gly His Asp Gln Leu Val Pro
580 585 590
Val Thr Leu Leu Ala Ile Ala Val Ile Leu Ala Phe Val Met Gly Ala
595 600 605
Val Phe Ser Gly Ile Thr Val Tyr Cys Val Cys Asp His Arg Arg Lys
610 615 620
Asp Val Ala Val Val Gln Arg Lys Glu Lys Glu Leu Thr His Ser Arg
625 630 635 640
Arg Gly Ser Met Ser Ser Val Thr Lys Leu Ser Gly Leu Phe Gly Asp
645 650 655
Thr Gln Ser Lys Asp Pro Lys Pro Glu Ala Ile Leu Thr Pro Leu Met
660 665 670
His Asn Gly Lys Leu Ala Thr Pro Gly Asn Thr Ala Lys Met Leu Ile
73
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675 680 685
Lys Ala Asp Gln His His Leu Asp Leu Thr Ala Leu Pro Thr Pro Glu
690 695 700
Ser Thr Pro Thr Leu Gln Gln Lys Arg Lys Pro Ser Arg Gly Ser Arg
705 710 725 720
Glu Trp Glu Arg Asn Gln Asn Leu Ile Asn Ala Cys Thr Lys Asp Met
725 730 735
Pro Pro Met Gly Ser Pro Val Ile Pro Thr Asp Leu Pro Leu Arg Ala
740 745 750
Ser Pro Ser His Ile Pro Ser Val Val Val Leu Pro Ile Thr Gln Gln
755 760 765
Gly Tyr Gln His Glu Tyr Val Asp Gln Pro Lys Met Ser Glu Val Ala
770 775 780
Gln Met Ala Leu Glu Asp Gln Ala A1a Thr Leu Glu Tyr Lys Thr Ile
785 790 795 800
Lys Glu His Phe Ser Ser Lys Ser Pro Asn His G1y Val Asn Leu Val
805 810 815
Glu Asn Leu Asp Ser Leu Pro Pro Lys Val Pro Gln Arg Glu Ala Ser
820 825 830
Leu Gly Pro Pro Gly Ala Ser Leu Phe Gln Thr Gly Leu Ser Lys Arg
835 840 845
Leu Glu Met His His Ser Phe Ser Tyr Gly Val Asp Tyr Lys Arg Ser
850 855 860
Tyr Pro Thr Asn Ser Leu Thr Arg Ser His Gln Ala Thr Thr Leu Lys
865 870 875 880
Arg Asn Asn Thr Asn Ser Ser Asn Ser Ser His Leu Ser Arg Asn Gln
885 890 895
Ser Phe Gly Arg Gly Asp Asn Pro Pro Pro Ala Pro Gln Arg Val Asp
900 905 910
Ser Ile Gln Val His Ser Ser Gln Pro Ser Gly Gln Ala Val Thr Val
915 920 925
Ser Arg Gln Pro Ser Leu Asn Ala Tyr Asn Ser Leu Thr Arg Ser Gly
930 935 940
Leu Lys Arg Thr Pro Ser Leu Lys Pro Asp Val Pro Pro Lys Pro Ser
945 950 955 960
Phe Ala Pro Leu Ser Thr Ser Met Lys Pro Asn Asp Ala Cys Thr
965 970 975
74
_ . ._..~_._...~..._.~...~...._.... T
CA 02274732 1999-06-14
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(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1723 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:9:
CTGCAGACTTTGGGGTCACCGGCCAGCCACACAGGCACCGTTTTCAGATGTCCACTTCTC60
ATTGGGTACATCAATCTTTTAACTTTGGGGGTCACAGTTTTAGCCACCTTTCGGGGGGTG120
ACTGGAGCAGTAGGAGGTGTGGGGTCATTTTATGAATATAATAAAATGGAGCTGACTATG180
GACRRRGACTWAGTGTGGGGGAGAGGGGACGATACAGGGTGTGTGTCTGGGAGTGCCTGG240
GGGACAGGGACCCCCCGGTGGTCCTATGGCAGGATGAGAARGGAGGGACTTGGCTCCCCC300
AGAGCCCGGTGGAAGCTACTGTTCTCTCCAGTGTCTCGAGCGTAGCCAAAATAAGGTTGG360
GAGGCTCCCGGCCTGTCTGCTGTGGTCTGAGCTGGCTGCAAGCCCAGGTGGGGGAGCGAG420
TCTGGGAAGATTGGCTTTGACTCTCTGTTGCCAGAGGAGATGCCATCCCAGCACGGCCCC480
CACTGTAGTCCAGGCTCGTGGTGGCAGCGGGGGCAAGGGGAGGGGCAAGGCTGCCCCCAC540
CCCACGCACCAAGTCACGCCAAGTCTCAGCAGGTAAAAGCACGTGAGCCTAGGGCGAGCG600
GAGGGAGTCCTGGTGGCCCCGCAGGTCAGGAGGGAAAGCAGGGCTCAGAGGGCATCGTGG660
CCCCAGGGCAGGGTCCTACCTGGGGGTCAGGAGCACCTTGGTCTTGATGATTGATTGATT720
GATAGAATGGAGCTGGGTCTGAGCCTCCCAGGCTTGAGCTCCTGGGAGTTCTTGTGCGGT780
GAGCTGGGCAGCTCCTGGGTAGGTCCGGGCACCAAGCAGGCCCTGATGTGGACAGAGTCC840
CATCAGAGGGAGCTGATGAAGAATGGTCCCTGTAAGTAAGTCACTAGGTTCAACAACTGC900
CTGGCCGAGCACTCAGCCCGTGGAGCTCAGGCCAACACCAGAGCCCCGGTTTTAGGGGCC960
AGGAGAGCAGGTGACCAATTATTTGGGGAGTCTTGGGTAGAATTTCCGCCACACATTCTC1020
CCCAGGGCTGCAGGGGTCTTCCGAGGCAGGGCGGTGGAGCAGGATTCAGGATGTGGTGGG1080
AATAGAGTGAGGGGCAGTGGGTGGGCAGACCTGGGCGTCAGAGGTCCTGATGGGAAAGGA1140
GGCAGGGGCTACCCAGAGAGGGGGGCTCGTGTGGCACAGCCCCCACCGACTCCGCCGTCC1200
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CCCTCCCCTG TGAGCCCCGG GGGCTGTACA CATCCCCTTG TCCATCCCTG1260
TACTCTACTC
AGACCACCCC CGCCGCCCTT GCGTCGACTT TCATAGGCCC ACCCACCTCG1320
AGCAACCACC
GGATCCGAGC CAACCATCCC ACATCACAAA GGGGACTTTA CGTTCGTTTA1380
CTTTGGTTTG
ATTTCTCATT TTGTACGGAG AAATATTCTT TCTTTTGACT GAAGTAACTT1440
TTCAAAAGCG
TCCTGGTGCT GTTGTTAACT CGTTCCTTTT TCCCCCACCC CAGGCAGCCC1500
TTTAATTTAT
TCCTGGTTCC TACTCACCCT CCCCCCCTCC GTCCCATCTG AACCATTTGT1560
CCCACCCTCC
TTCTTTTCTT TCCGTCAGAT TTTGGAAAAA CTCCCCGCCC CCTCCACACC1620
TTCTCCTCTC
ATCCTCCCSG ATTTAAATAT AGTCACTGCT GATGCACTGT GAAGATTCCA1680
ACAAGTAACA
GTATTAATAA AGGTGTACTG TAATTAACAA AAA 1723
P,AAAAAAAAA
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 101 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Val Phe Arg Cys Pro Leu Leu Ile Gly Tyr Ile Asn Leu Leu Thr Leu
1 5 10 15
Gly VaI Thr Val Leu Ala Thr Phe Arg Gly Val Thr Gly Ala Val Gly
20 25 30
Gly Val Gly Ser.Phe Tyr Glu Tyr Asn Lys Met Glu Leu Thr Met Asp
35 40 45
Xaa Asp Xaa Val Trp Gly Arg Gly Asp Asp Thr Gly Cys Val Ser Gly
50 55 60
Ser Ala Trp Gly Thr Gly Thr Pro Arg Trp Ser Tyr Gly Arg Met Arg
65 70 75 80
Xaa Glu Gly Leu Gly Ser Pro Arg Ala Arg Trp Lys Leu Leu Phe Ser
85 90 95
Pro Val Ser Arg Ala
100
(2) INFORMATION FOR SEQ ID N0:11:
~s
T ~
CA 02274732 1999-06-14
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 469 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
GCAACATACA AGCCGGCCAT ATTAGAGAGA TGGAAATAAA TGTTGTATAT60
GCTTCCTTAA
GTCTTTGAAG TACATCCGTG CATTTTTTTT TAGCATCCAA CTTGTAGTTC120
CCATTCCTCC
TCGCCCCCTC AAATCACCCT CTCCCGTAGC CCACCCGACT TCTCTGAAAA180
AACATCTCAG
TGCACAGAGA TGCCTGGCTA CCTCGCCCTG CCTTCAGCCT AGTCTCTTTT240
CACGGGGCTC
TCTCTTTGGT GCCACCAGGA CGGAGCATGG AGGTCACAGT CTCAACGTCC300
ACCTGCCACC
TCAATGGCTC TGACGCCCGC CTGCCCTGCA CCTTCAACTC GTGAACCACA360
CTGCTACACA
AACAGTTCTC CCTGAACTGG ACTTACCAGG AGTGCAACAA GAGATGTTCC420
CTGCTCTGAG
TCCAGTTCCG CATGAAGATC ATTAACCTGA AGCTGGAGCG 469
GTTTCAAGA
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:12:
MetHis ArgAspAlaTrp LeuProArg ProAla Ser LeuThr Gly
Phe
1 5 10 15
LeuSer LeuPhePheSer LeuValPro ProGly Ser MetGlu Val
Arg
20 25 30
ThrVal ProAlaThrLeu AsnValLeu AsnGly Asp AlaArg Leu
Ser
35 40 45
ProCys ThrPheAsnSer CysTyrThr ValAsn Lys GlnPhe Ser
His
50 55 60
77
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Leu Asn Trp Thr Tyr Gln Glu Cys Asn Asn Cys Ser Glu Glu Met Phe
65 70 75 80
Leu Gln Phe Arg Met Lys Ile Ile Asn Leu Lys Leu Glu Arg Phe Gln
85 90 95
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 454 base pairs
(B} TYPE: nucleic acid
(C} STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:13:
TGGCTTTTGG CTACAGAGAG GGAAGGGAAA GGCATAAGGG GAGGCCTTGG60
GCCTGAGGCC
AACCTGAGCT GCCAATGCCA GCCCTGTCCC ACGATACTCG CTCCTCTCCC120
ATCTGCGGCC
AACAACTCCT TTGGTGGGGA CAAAAGTGAC CAGGCACAGT GGCTCACGCC180
AATTGTAGGC
TGTAATCCCA GCACTTTGGG AGGCCAAGGC CCTCCATCTG TTTAGTAGAA240
GGGTGGATTA
ATGGGCAAAA CCCCATTTTT ACTAAAAATA CTGGGCGTGG TGGCGTGTGC300
CAAGAATTAG
CTGTAATCCC AGCTATTTGG GAGGCTGAGG GCTTGAGCCC GGGAAGCAGA360
CAGGAGAATC
GGTTGCAGTG AACTGAGATA GTGATAGTGC CAGCCTGGGT GACATAGAGA420
CACTGCAATT
GACTCCATCT CPS AAAAAAAAAA AAAA 454
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 736 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GTTTTTAAAC ATTATGTTCT ACATGATAAA TACATATAAT AGTATGTCTA TTTAAATAAT 60
7g
_.____.~..__r._______. _ .r.. _ ~
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TAAATTTGAA AAAAACTAAT CAAATATTATCATAAGTAATGATAAAAACC ACAATTTCTT120
TTGCAGCAAA CTAATAACAC CTGGATTTCTCAATTTATTAAGTTGTACTT ACCTGATGCT180
GATGATGATT ACTGTATTTA CACATTGTCTCAGAGCTCACTCTTGCGGAG GTTGTGGCCT240
CGAAAATGCC TTGTTGTCCC TCTGGAATCTGTCTTTTCAGCTTCATCTCC TCCTCCTCAC300
CTCCTGCTGT GGTGCACAGA TACCTATAGGCAGGCTCCATCTCCTCCTCC CCAGCTCCTC360
CCCTAGTGCA CAGATACCTA TAGGCAGGCTTCATCTCCTCCTCCCCAGCT TCTCCCCTAG420
TGCACAGATA CCTATAGGCA GGCTCCATCTCCTCCTCCCCAGCTCCTCCC CTARTGCACA480
GACACCTATA GGCAAGCTCC ATCTCCTCCTCTTTAGCTAGCCTCCCCATC TCATCACAAC540
GCATGTCTGT GACCTTTGGT AATCATTTACAGTGCCACACGGAACCCTGT ATTTTGCACA600
CAGCAAAACA AACAATGTTT AGCTTTATTTATGGTATTTGATGACTGTAA ATGGAAATAA660
ATATTGTTCT TTATTTTTTN AAAAAAAAAAPO~AAAAAAAAAAANAA.AAAA AAAAAAAAAA720
A,Fu~AAAAAAA AAA.AAA 7
3
6
(2) INFORMATION FOR SEQ
ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 114 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Leu Met Met Ile Thr Val Phe Thr His Cys Leu Arg Ala His Ser
1 5 10 15
Cys Gly Gly Cys Gly Leu Glu Asn Ala Leu Leu Ser Leu Trp Asn Leu
20 25 30
Ser Phe Gln Leu His Leu Leu Leu Leu Thr Ser Cys Cys Gly Ala Gln
35 40 45
Ile Pro Ile Gly Arg Leu His Leu Leu Leu Pro Ser Ser Ser Pro Ser
50 55 60
Ala Gln Ile Pro Ile Gly Arg Leu His Leu Leu Leu Pro Ser Phe Ser
65 70 75 80
Pro Ser Ala Gln Ile Pro Ile Gly Arg Leu His Leu Leu Leu Pro Ser
79
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85 90 95
Ser Ser Pro Xaa Ala Gln Thr Pro Ile Gly Lys Leu His Leu Leu Leu
100 105 110
Phe Ser
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1427 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:16:
GTAGTTACTAACTCCAACACCTAATAGCATTGGTAGAAAG CTTATAAATGCAGTTATTTA60
GCCTCGACTAAGATTTTTCTGATACCTAGTTTCACTTTTT AATGCCCTCTGAAAGTTTTT120
TGATCAGTTGTTTAATGGGAGATCTGAAATGTTAAACTCA GACCAGAAAGAAGAGAACCT180
GTTTTCTAGAAATTAGGTTTTTAATCCAAGTAAGATGCAA GCTTTTGCTTTTTTAATAAC240
TTGTATAGCTAAAAACTTGACGGTGAAAAGCTCTCAGATC AAAGCTGATCCTTCTGTCAG300
TAATGATTCTAAAAATAAGCAAGATTTTAATGGGGAATAT ATTTTATTTCATTCTTATCT360
CAAACCTAGGTACTGTGGTCGTTTTGAGTTCATTTCGAGG CATTTTCAATGTGCCTCAGG420
CCACATCCAACCTCTYCCCAGGGCCAGATTTAATGTTCAG CCTCATAAAGGTTATCATAG480
TTTTAACATTTAAGTACTATTTTGCAGTGGGTATATACCA AAATTTGCTAATAGTAAGAT540
AACCTTAGTTATATATCATTCACGTTAGTTCTATCTTGGA GGCAATAAACATTTCTTGTT600
CAAGAAATTCATGTTCTATCTTGGAGGCAATAAACAAACA TTTTTTGTTCAAAATTAGGG660
CTACCCTATTGTCCTTATGTCTTTTCCTGATCTGTGGTCA AACATTTTTCTTAGTCATTT720
AGAAATTTTCTATGTTGTTTTAAATTTTCTTTAAATCTAG AATGGAGTATGTGACCAATA780
CTTTCCTTTGGAATGGTATGGACATTTGAAATAGAGCCCA TTCTTTATAAAGTATAAAAT840
ATGTTTAATGCTAGTATTTTTAACTAAACTTTTGAGAAAC TAGATTCACATGCTGTTGTA900
AGAAATAATACAGAGACCTCTTTCGTGTACCTTTCACTTT GTTTCCCACACAGTGAACAT960
1
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CTTTCAAAAC TGTCATACAA TATCATACCCAGGATACTGACACTGGTATA GCTAAGATAG1020
AGAACGTTTC CACACAGAAC TTTTTCTAGCACAGGGATCCCTCATCTTGC TTTTGATGAC1080
CATACCCACT TCACTCCCAT CCCTACTCCCTTCTTAACCCTTGGCAACCA TAATCTGTTC1140
TCCATTTTTA TAGTTTTTTT TTTTTCATTTCAATAAAGCTGTATAACTGG AATCATAATA1200
ATATGTAACC TTTTGGGATT GGCTTTTTTTCATTTAGCATGATTTTCTGG AGGTTAATCC1260
AGCTTATTAT GTGTATCAAG TCTATTGACAGGTACTTTTTAGTGTGAATA GAATCCCATA1320
GTATAGATGT ACCACAGTTT GTTTAACTGTTCACCTGCTGAGAGACATTG GGCCAGTTTT1380
TGGCTACTAT AAATAAAGTT GCTATAAACAP~~AAAAAAAAAAAAAAA 1427
(2) INFORMATION FOR SEQ
ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids
{B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Ile Leu Lys Ile Ser Lys Ile Leu Met Gly Asn Ile Phe Tyr Phe
1 5 10 15
Ile Leu Ile Ser Asn Leu Gly Thr Val Val Val Leu Ser Ser Phe Arg
20 25 30
Gly Ile Phe Asn Val Pro Gln Ala Thr Ser Asn Leu Xaa Pro Gly Pro
35 40 45
Asp Leu Met Phe Ser Leu Ile Lys Val Ile Ile Val Leu Thr Phe Lys
50 55 60
Tyr Tyr Phe Ala Val Gly Ile Tyr Gln Asn Leu Leu Ile Val Arg
65 70 75
(2) INFORMATION FOR SEQ ID NO:1B:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 572 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
81
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(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:18:
TGCAGATTCT GTGGTTATAC TCACTCCTCATCCCAAAGAA TGAAATTTACCACTCTCCTC60
TTCTTGGCAG CTGTAGCAGG GGCCCTGGTCTATGCTGAAG ATGCCTCCTCTGACTCGACG120
GGTGCTGATC CTGCCCAGGA AGCTGGGACCTCTAAGCCTA ATGAAGAGATCTCAGGTCCA180
GCAGAACCAG CTTCACCCCC AGAGACAACCACAACAGCCC AGGAGACTTCGGCGGCAGCA240
GTTCAGGGGA CAGCCAAGGT CACCTCAAGCAGGCAGGAAC TAAACCCCCTGAAATCCATA300
GTGGAGAAAA GTATCTTACT AACAGAACAAGCCCTTGCAA AAGCAGGAAAAGGAATGCAC360
GGAGGCGTGC CAGGTGGAAA ACAATTCATCGAAAATGGAA GTGAATTTGCACAAAAATTA420
CTGAAGAAAT TCAGTCTATT AAAACCATGGGCATGAGAAG CTGAAAAGAATGGGATCATT480
GGACTTAAAG CCTTAAATAC CCTTGTAGCCCAGAGYTATT AAAACGAAAGCATCCAAAAA540
A,~~AAAAAAAA A,~~A P~~.AAAAAAAAAA 5
7
2
(2) INFORMATION FOR SEQ
ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:19:
Met Lys Phe Thr Thr Leu Leu Phe Leu Ala Ala Val Ala Gly Ala Leu
1 5 10 15
Val Tyr Ala Glu Asp Ala Ser Ser Asp Ser Thr Gly Ala Asp Pro Ala
20 25 30
Gln Glu Ala Gly Thr Ser Lys Pro Asn Glu Glu Ile Ser Gly Pro Ala
35 40 45
Glu Pro Aia Ser Pro Pro Glu Thr Thr Thr Thr Ala Gln G1u Thr Ser
50 55 60
Ala Ala Ala Val Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu
65 70 75 80
82
T' f Z
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Leu Asn Pro Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu
85 90 95
Gln Ala Leu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly
100 105 110
Gly Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu
115 120 125
Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala
130 135
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1223 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:20:
CCTTGTTCCACGTAGCTGGCAAGGTCTTCATTCACTTGCCACTGCTAGTCTTCCAACCCT60
TCTGGACTTTCTTTGCTCTTGTCTTGTTTTGGGTGTACTGGATCATGACACTTCTTTTTC120
TTGGCACTACCGGCAGTCCTGTTCAGAATGAGCAAGGCTTTGTGGAGTTCAAAATTTCTG180
GGCCTCTGCAGTACATGTGGTGGTACCATGTGGTGGGCCTGATTTGGATCAGTGAATTTA240
TTCTAGCATGTCAGCAGATGACAGTGGCAGGAGCTGTGGTAACATACTATTTTACTAGGG300
ATAAAAGGAATTTGCCATTTACACCTATTTTGGCATCAGTAAATCGCCTTATYCGTTACC360
ACCTAGGTACGGTGGCAAAAGGATCTTTCATTATCACATTAGTCAAAATTCCGCGAATGA420
TCCTTATGTATATTCACAGTCAGCTCAAAGGAAAGGAAAATGCTTGTGCACGATGTGTGC480
TGAAATCTTGCATTTGTTGCCTTTGGTGTCTTGAAAAGTGCCTAAATTATTTAAATCAGA540
ATGCATACACAGCCACAGCTATCAACAGCACCAACTTCTGCACCTCAGCAAAGGATGCCT600
TTGTCATTCTGGTGGAGAATGCTTTGCGAGTGGCTACCATCAACACAGTAGGAGATTTTA660
TGTTATTCCTTGGCAAGGTGCTGATAGTCTGCAGCACAGGTTTAGCTGGGATTATGCTGC720
TCAACTACCAGCAGGACTACACAGTATGGGTGCTGCCTCTGATCATCGTCTGCCTCTTTG780
CTTTCCTAGTCGCTCATTGCTTCCTGTCTATTTATGAAATGGTAGTGGATGTATTATTCT840
83
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KGKGTTTTGC CATTGAWACA AAATACAATG ATGGGMGCCC TGGCAGAGAA TTCTATATGG 900
ATAAAGTGCT GATGGAGTTT GTGGAAAACA GTAGGAAAGC AATGAAAGAA GCTGGTAAGG 960
GAGGCGTCGC TGATTCCAGA GAGCTAAAGC CGATGCTGAA GAAAAGGTGA CTGGTCTCAT 1020
GAGCCCTGAA GAATGAACTC AGAGGAGGTT GTTTACATGA GGTTCTCCCA CTCACCAGCT 1080
GTTGAGAGTC TGCGATTATG AAGAGCAGGA TCTTATTACT TCAATGAAAG CATGTAACAA 1140
GTTTCTCAAA CCACCAACAG CCAAGTGGAT TTGGTACAGT GCGGCTGTCT AATAAATAAT 1200
CAAAAGCAAA AAAAAAAAAA AAA 1223
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 301 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Met Thr Leu Leu Phe Leu Gly Thr Thr Gly Ser Pro Val Gln Asn Glu
1 5 10 15
Gln Gly Phe Val Glu Phe Lys Ile Ser Gly Pro Leu Gln Tyr Met Trp
20 25 30
Trp Tyr His Val Val Gly Leu Ile Trp Ile Ser Glu Phe Ile Leu Ala
35 40 45
Cys Gln Gln Met Thr Val Ala Gly Ala Val Val Thr Tyr Tyr Phe Thr
SO 55 60
Arg Asp Lys Arg Asn Leu Pro Phe Thr Pro Ile Leu Ala Ser Val Asn
65 70 75 80
Arg Leu Ile Arg Tyr His Leu Gly Thr Val Ala Lys Gly Ser Phe Ile
85 90 95
Ile Thr Leu Val Lys Ile Pro Arg Met Ile Leu Met Tyr Ile His Ser
100 105 110
Gln Leu Lys Gly Lys Glu Asn Ala Cys Ala Arg Cys Val Leu Lys Ser
115 120 125
Cys Ile Cys Cys Leu Trp Cys Leu Glu Lys Cys Leu Asn Tyr Leu Asn
130 135 240
84
__...._ __._. ___. . ._.... . .. ~ ._ _.....~ ..
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Gln Asn Ala Tyr Thr Ala Thr Ala Ile Asn Ser Thr Asn Phe Cys Thr
145 150 155 160
Ser Ala Lys Asp Ala Phe Val Ile Leu Val Glu Asn Ala Leu Arg Val
165 170 175
Ala Thr Ile Asn Thr Val Gly Asp Phe Met Leu Phe Leu Gly Lys Val
180 185 190
Leu Ile Val Cys Ser Thr Gly Leu Ala Gly Ile Met Leu Leu Asn Tyr
195 200 205
Gln Gln Asp Tyr Thr Val Trp Val Leu Pro Leu Ile Ile Val Cys Leu
210 215 220
Phe Ala Phe Leu Val Ala His Cys Phe Leu Ser Ile Tyr Glu Met Val
225 230 235 240
Val Asp Val Leu Phe Xaa Xaa Phe Ala Ile Xaa Thr Lys Tyr Asn Asp
245 250 255
Gly Xaa Pro Gly Arg Glu Phe Tyr Met Asp Lys Val Leu Met Glu Phe
260 265 270
Val Glu Asn Ser Arg Lys Ala Met Lys Glu Ala Gly Lys Gly Gly Val
275 280 285
Ala Asp Ser Arg Glu Leu Lys Pro Met Leu Lys Lys Arg
290 295 300
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2460 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
GAGAAGCGCG ATGACGGCTA CGAGGCCGCT GCCAGCTCCA AAACTAGCTC AGGCGATGCC 60
TCCTCACTCA GCATCGAGGG AGACTAACAA ACTCCGGGCA AAGTTGGGGC TGAAACCCTT 120
GGAGGTTAAT GCCATCAAGA AGGAGGCGGG CACCAAGGAG GAGCCCGTGA CAGCTGATGT 180
CATCAACCCT ATGGCCTTGC GACAGCGAGA GGAGCTGCGG GAGAAGCTGG CGGCTGCCAA 240
GGAGAAGCGC CTGCTGAACC AAAAGCTGGG GAAGATAAAG ACCCTAGGAG AGGATGACCC 300
g5
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CTGGCTGGACGACACTGCAGCCTGGATCGAGAGGAGCCGGCAGCTGCAGAAGGAGAAGGA360
CCTGGCAGAGAAGAGGGCCAAGTTACTGGAGGAGATGGACCAAAAGTTTGGTGTCAGCAC420
TCTGGTGGAGGAGGAGTTCGGGCAGAGGCGGCAGGACCTGTACAGTGCCCGGGACCTGCA480
GGGCCTCACTGTGGAGCATGCCATTGATTCCTTCCGAGAAGGGGAGACAATGATTCTTAC540
CCTCAAGGACAAAGGCGTGCTGCAGGAGGAGGAGGACGTGCTGGTGAACGTGAACCTGGT600
GGATAAGGAGCGGGCAGAGAAAAATGTGGAGCTGCGGAAGAAGAAGCCTGACTACCTGCC660
CTATGCCGAGGACGAGAGCGTGGACGACCTGGCGCAGCAAAAACCTCGCTCTATCCTGTC720
CAAGTATGACGAAAAGCTTGAAGGGGAGCGGCCACATTCCTTCCGCTTGGAGCAGGGCGG780
CACGGCTGATGGCCTGCGGGAGCGGGAGCTGGAGGAGATCCGGGCCAAGCTGCGGCTGCA840
GGCTCAGTCCCTGAGCACAGTGGGGCCCCGGCTGGCCTCCGAATACCTCACGCCTGAGGA900
GATGGTGACCTTTAAAAAGACCAAGCGGAGGGTGAAGAAAATCCGCAAGAAGGAGAAGGA960
GGTAGTAGTGCGGGCAGATGACTTGCTGCCTCTCGGGGACCAGACTCAGGATGGGGACTT1020
TGGTTCCAGACTGCGGGGACGGGGTCGCCGCCGAGTGTCCGAAGTGGAGGAGGAGAAGGA1080
GCCTGTGCCTCAGCCCCTGCCGTCGGACGACACCCGAGTGGAGAACATGGACATCAGTGA1140
TGAGGAGGAAGGTGGAGCTCCACCGCCGGGGTCCCCGCAGGTGCTGGAGGACGACGAGGC1200
GGAGCTGGAGCTGCAGAAGCAGCTGGAGAAGGGACGCCGGCTGCGACAGTTACAGCAGCT1260
ACAGCAGCTGCGAGACAGTGGCGAGAAGGTGGTGGAGATTGTGAAGAAGCTGGAGTCTCG1320
CCAGCGGGGCTGGGAGGAGGATGAGGATCCCGAGCGGAAGGGGGCCATCGTGTTCAACGC1380
CACGTCCGAGTTCTGCCGCACCTTGGGGGAGATCCCCACCTACGGGCTGGCTGGCAATCG1440
CGAGGAGCAGGAGGAGCTCATGGACTTTGAACGGGATGAGGAGCGCTCAGCCAACGGTGG1500
CTCCGAATCTGACGGGGAGGAGAACATCGGCTGGAGCACGGTGAACCTGGACGAGGAGAA1560
GCAGCAGCAGGATGTGAGGGCCACGCCGCTGGGGGGTGGGCGTTTGGGGGTGCTCAAGCT1620
GGAGATGAGCACCGGGCTCGGTGTCCAGAGCCTCAGCCTCCTCATCCAGAGTGGGCTCTG1680
CAGACCTCCCAGGGCGATCTGAGGAGTAAATGAGGAAATTAAATGTTGTGGAGGGCTGGT1740
GCCTGGCAGGTGGTGACCAGTGGGTGGGGCTGAGAAGAGCCGGTATGGCCTGCTAACCAC1800
CCCCGCCACGTGTCCCGTAGTTCTCTGCTTCCTCCACCACCATCCTGGACGAGGAACCGA1860
TCGTGAATAGGGGGCTGGCAGCTGCCCTGCTCCTGTGTCAGAACAAAGGGCTGCTGGAGA1920
CCACAGTGCAGAAGGTGGCCCGGGTGAAGGCCCCCAACAAGTCGCTGCCCTCAGCCGTGT1980
86
.._...._.. __...___._.____.~_.
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ACTGCATCGA GGATAAGATGGCCATCGATGACAAGTACAG CCGGAGGGAG GAATACCGAG2040
GCTTCACACA GGACTTCAAGGAGAAGGACGGTACAAACCC GACGTTAAGA TCGAATACGT2100
GGATGAGACG GGCCGGAAACTCACACCCAAGGAGGCTTTC CGGCAGCTGT CGCACCGCTT2160
CCATGGCAAG GGCTCAGGCAAGATGAAGACAGAGCGGCGG ATGAAGAAGC TGGACGAGGA2220
GGCGCTCCTG AAGAAGATGAGCTCCAGSGACACGCCCYTG GGCACCGTGG CCCTGYTCCA2280
GGAGAAGCAG AAGGCTCAGAAGACCCCCTACATYGTGTTC AGCGGCAGCG GCAAGAGCAT2340
GAACGCGAAC ACCATCACCAAGTGACAGCGCCCTCCCGCC CCGGCCCTGC CTCAACCTTC2400
ATATTAAATA AAGCTCCCTCCTTAAAAAAAF,~~,AAAAAAAA AAAAAAAAAA 2
P~~,AAAAAAAA 4
6
0
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 563 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Met Thr Ala Thr Arg Pro Leu Pro Ala Pro Lys Leu Ala Gln Ala Met
1 5 10 15
Pro Pro His Ser Ala Ser Arg Glu Thr Asn Lys Leu Arg Ala Lys Leu
20 25 30
Gly Leu Lys Pro Leu Glu Val Asn Ala Ile Lys Lys Glu Ala Gly Thr
35 40 45
Lys Glu Glu Pro Val Thr Ala Asp Val Ile Asn Pro Met Ala Leu Arg
50 55 60
Gln Arg Glu Glu Leu Arg Glu Lys Leu Ala Ala Ala Lys Glu Lys Arg
65 70 75 80
Leu Leu Asn Gln Lys Leu Gly Lys Ile Lys Thr Leu Gly Glu Asp Asp
85 90 95
Pro Trp Leu Asp Asp Thr Ala Ala Trp Ile Glu Arg Ser Arg Gln Leu
100 105 110
Gln Lys Glu Lys Asp Leu Ala Glu Lys Arg Ala Lys Leu Leu Glu Glu
115 120 125
87
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Met Asp Gln Lys Phe Gly Val Ser Thr Leu Val Glu Glu Glu Phe Gly
130 135 140
Gln Arg Arg Gln Asp Leu Tyr Ser Ala Arg Asp Leu Gln Gly Leu Thr
145 150 155 160
Val Glu His Ala Ile Asp Ser Phe Arg Glu Gly Glu Thr Met Ile Leu
165 170 175
Thr Leu Lys Asp Lys Gly Val Leu Gln Glu Glu Glu Asp Val Leu Val
180 185 190
Asn Val Asn Leu Val Asp Lys Glu Arg Ala Glu Lys Asn Val GIu Leu
195 200 205
Arg Lys Lys Lys Pro Asp Tyr Leu Pro Tyr Ala Glu Asp Glu Ser Val
210 225 220
Asp Asp Leu Ala Gln Gln Lys Pro Arg Ser Ile Leu Ser Lys Tyr Asp
225 230 235 240
G1u Lys Leu Glu Gly Glu Arg Pro His Ser Phe Arg Leu Glu Gln Gly
245 250 255
Gly Thr Ala Asp Gly Leu Arg Glu Arg Glu Leu Glu Glu Ile Arg Ala
260 265 270
Lys Leu Arg Leu Gln Ala Gln Ser Leu Ser Thr Val Gly Pro Arg Leu
275 280 285
Ala Ser Glu Tyr Leu Thr Pro Glu Glu Met Val Thr Phe Lys Lys Thr
290 295 300
Lys Arg Arg Val Lys Lys Ile Arg Lys Lys Glu Lys Glu Val Val Val
305 310 315 320
Arg Ala Asp Asp Leu Leu Pro Leu Gly Asp Gln Thr Gln Asp Gly Asp
325 330 335
Phe Gly Ser Arg Leu Arg Gly Arg Gly Arg Arg Arg Val Ser Glu Val
340 345 350
Glu Glu Glu Lys Glu Pro Val Pro Gln Pro Leu Pro Ser Asp Asp Thr
355 360 365
Arg Val Glu Asn Met Asp Ile Ser Asp Glu Glu Glu Gly Gly Ala Pro
370 375 380
Pro Pro Gly Ser Pro Gln Val Leu Glu Glu Asp Glu Ala Glu Leu Glu
385 390 395 400
Leu Gln Lys Gln Leu Glu Lys Gly Arg Arg Leu Arg Gln Leu Gln Gln
405 410 415
Leu Gln Gln Leu Arg Asp Ser Gly Glu Lys Val Val Glu Ile Val Lys
gg
. . . . ._._. _.._ .__ _ ..._ ___._.__T__...__.~_. ..
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420 425 430
Lys Leu Glu Ser Arg Gln Arg Gly Trp Glu Glu Asp Glu Asp Pro Glu
435 440 445
Arg Lys Gly Ala Ile Val Phe Asn Ala Thr Ser Glu Phe Cys Arg Thr
450 455 460
Leu Gly Glu Ile Pro Thr Tyr Gly Leu Ala Gly Asn Arg Glu Glu Gln
465 470 475 480
Glu Glu Leu Met Asp Phe Glu Arg Asp Glu Glu Arg Ser Ala Asn Gly
485 490 495
Gly Ser G1u Ser Asp Gly Glu Glu Asn Ile Gly Trp Ser Thr Val Asn
500 505 510
Leu Asp Glu Glu Lys Gln Gln Gln Asp Val Arg Ala Thr Pro Leu Gly
515 520 525
Gly Gly Arg Leu Gly Val Leu Lys Leu Glu Met Ser Thr Gly Leu Gly
530 535 540
Val Gln Ser Leu Ser Leu Leu Ile Gln Ser Gly Leu Cys Arg Pro Pro
545 550 555 560
Arg Ala Ile
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: ldesc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
ANAGGCTCCTC CATTCCTACA GCCATCTT 29
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
89
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(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
CNGTCCAACTG CTTGTAGGTT ATAGCAGA 29
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
TNCTCTACTTC ACCCTTTTCG GTGCATCG 29
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
TNCATAAAAT GACCCCACAC CTCCTACTG 29
(2} INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
~ ~. i
_. ._._ .___,.._ _.
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
GNGAGGTAGCC AGGCATCTCT GTGCATTT 29
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
ANCACAGCAGG AGGTGAGGAG GAGGAGAT 29
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
CNTTGATCTGA GAGCTTTTCA CCGTCAAG 29
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
91
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
ANCTGCCAAGA AGAGGAGAGT GGTAAATT 29
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
ANGAAAAAGAA GTGTCATGAT CCAGTACA 29
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
TNATACTTGGA CAGGATAGAG CGAGGTTT 29
92
